Biosynthetic Engineering for the Assembly of Better Drugs
Vikramaditya Ganapati Yadav
B. A. Sc. (Hons. Co-op.) Chemical Engineering, University of Waterloo (2007)
Submitted to the Department of Chemical Engineering
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Chemical Engineering
at the Massachusetts Institute of Technology
ARCHIVES
May 2013
MASSACHUSETTS INST - lfE
Massachusetts Institute of Technology @2013
All rights reserved
OF TECHN~OLOGY
JUN
2913
L! F R ES
Signature of the author
6oao
Certified by
F
IF
Ce e by
-
nt of Chemical Engineering
28 May 2013
.
Gregory N. Stephanopoulos
W.H.Dow Professor of Chemical Engineering & Biotechnology
Thesis Advisor
Accepted by
Patrick S.Doyle
Professor of Chemical Engineering
Chairman, Committee for Graduate Students
a
Biosynthetic Engineering for the Assembly of Better Drugs
Vikramaditya Ganapati Yadav
Submitted to the Department of Chemical Engineering on May 28, 2013
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Chemical Engineering
The declining prospects of innovator pharmaceutical companies have been attributed to their
inability to discover leads that bind to novel protein domains. All of the 21,000-odd drug products
that have ever been approved by the US FDA bind to just 130 of the over 10,000 functional protein
domains that exist in the human body. The high degree of drug redundancy, in turn, stems from the
industry's persistence with combinatorial chemistry to synthesize drug candidates. Not only is the
chemical space being screened for biological activity usually one that has been previously
interrogated, but the products of combinatorial chemistry too are simply not drug-like. So dire are
the odds that launching a single synthetic compound necessitates screening as many as 100,000
molecules from a combinatorial library.
In comparison, as many as 20 molecules of the 7,000-odd polyketide natural products that have
been screened thus far have made it to the market. Nevertheless, drug companies remain
underwhelmed as synthetic inaccessibility has heretofore stymied drug prospection in natural
product space. However, recent applications of genetic approaches to decipher the biosynthetic code
that guides the assembly of several natural product families have revealed that living systems
methodically and repetitively combine carbohydrate-derived building blocks in metabolic unit
reactions to generate unprecedented molecular diversity. By clustering combinations, permutations
and mutations of these biosynthetic genes into operons to be expressed as heterologous metabolic
pathways in tractable microbial hosts, one can readily generate pharmacophores that are more likely
to bind to novel targets. Ordered combinations of known genes generate pharmacophores that are
natural products in toto, whereas permutations and mutations produce molecules that possess a
scaffold that is an unnatural isomer of a natural product. The atom economy of microbial synthesis
of pharmaceutical intermediates and active ingredients also far exceeds those of conventional
schemes.
We recently demonstrated the feasibility of this new paradigm for drug discovery by integrating two
of the four metabolic unit reactions involved in taxane assembly into the metabolism of E. coli to
produce an unsubstituted, 20-carbon drug scaffold at 1 g/L titers. The taxane family of molecules
includes, among others, the blockbuster anti-cancer drug, taxol. We then proceeded to express the
first of many cytochrome P450 monooxygenases that oxidize the taxane scaffold in the third
metabolic unit reaction. However, plant cytochrome P450s such as those involved in taxane
biosynthesis are notoriously promiscuous and non-selective. A likely candidate that meets the
synthetic requirements must first be selected, following which it must be customized to enhance its
substrate specificity, product selectivity and activity. The use of structure-driven approaches is
preferred herein as the structure-activity insights gained from redesigning one active site could
potentially be extended to re-engineering
downstream homologues. However, conventional
methodologies such as X-ray crystallography are fairly lengthy and laborious. Ergo, we combined
phylogeny-guided mutagenesis with a model-driven assessment of the active site of taxadiene-5ahydroxylase, the cytochrome P450 that catalyzes proto-oxidation of taxane scaffold, to investigate its
catalytic mechanism. The reactive topography of the active site was then charted to relate the impact
of structural features to activity and selectivity, opening up the future possibilities to specifically tune
the reactivity of cytochrome P4 50s to catalyze the regio- and stereospecific oxidation of the taxane
scaffold for prospection of targeted regions of natural product chemical space.
Thesis advisor: Gregory N.Stephanopoulos
W.H.Dow Professor of Chemical Engineering & Biotechnology
To my parents, Gautam & Manali
r
Ci4
lr irtel
4-Flk
WT
:T M
10fr wu
I
I|
a
ACKNOWLEDGEMENTS
Isaac Newton remarked "If I have seen further, it is by standing on the shoulders of giants". I have
been privileged to learn the art and science of research from the very best. Thank you, Professor
Stephanopoulos. You have been a phenomenal guru, a fantastic mentor and a great well-wisher. To
Professor Prather, my academic mother - your humour, energy, advice and support have been
instrumental in the completion of this document.
I still remember the day when I received my high school graduation certificate. It's funny how
people develop selective amnesia when all of their friends have performed better than them on a test,
the state examinations no less! I guess all those years of trying my level best to skip homework had
finally taken their toll. All I remember is that sinking feeling when a school teacher who was a friend
of the family quipped, "What happened? Your parents might not be too pleased" when I told him
my final grades. Nonchalant until that precise moment, I was suddenly overcome with the fear of
having deeply disappointed my parents. After all, in India, a son's state examination grades are as
much a status symbol as the car one drives or the salary one draws. Mummy and Daddy - thank you
for the advice you gave me at that moment when I was looking for the biggest hole in the ground to
hide myself. You have been my best friends, my teachers, my role-models and my guides for all my
life. Were it not for your love and support, I would not have made it this far.
Daddy - thank you for being the best friend and confidante a lad could ask for. You have always
been my beacon, guiding me through all waters. Your poise has been a source of strength. Your wit
and humour have always dispelled the dark clouds away. You are my Charioteer. But I must confess
- I have kept aside a bigger slice of the cake for Mummy. You and I both know why. Mummy - you
are my Architect. You gave me the strongest of foundations in mathematics. You helped me with my
science projects in school. You made learning history and geography stimulating. You kept me
company at 3 am on practically every examination night. Even in university, you were my alarm
clock, making sure I woke up on time for classes, examinations and theses defences. This degree
belongs more to you than to me.
Gautam - thank you for your camaraderie and love. Your counsel has been a source of great
strength. Your mischief has been a source of great enjoyment and entertainment. You have always
brought calm over me when I was beset by tempests. I thank Lord Ganapati for blessing me with the
privilege of having you by my side. You are my General. Manali - thank you for your limitless love
and patience. Your passion energizes me. Your support is my castle. Your serenity is infectious and
has helped me sail through the turbulence. Like Mummy, you too sat by my side as I worked
through the night to complete my thesis. I am truly blessed. But you do owe me some cake. You
have robbed me of many 'bachelorship is bliss' jokes.
Lastly, to all my too-many-to-list-by-name friends and well-wishers at MIT - thank you for the
hours of fun and learning. You have made this journey a memorable one.
TABLE OF CONTENTS
Preface
19
1
The Target-Rich, Lead-Poor Imbalance
1. 1. Drug discovery in the pre-Hatch-Waxman era
1. 2. Deficiencies in the old paradigm
1. 3. The industrialization of drug discovery
1. 4. New paradigm, same old problems
1. 5. The inadequacies of combinatorial chemistry
1. 6. Effect of the needle in a haystack paradigm on the industry's business model
23
23
24
24
27
28
29
2
Natural Products &the Pharmaceutical Industry
2. 1. The historical use of natural products as therapies
2. 2. What makes natural products good drugs?
2. 3. Why is industry interest in natural products declining?
2. 4. Alternatives for probing natural product space
31
31
32
34
37
3
Biosynthetic Engineering for Terpenoid Synthesis
3. 1. The terpenoid superfamily
3. 2. Modularity of terpenoid biosynthesis
3. 3. Metabolic & enzyme engineering for targeted & diversity-oriented biosynthesis
3. 4. Summary of recent progress
3. 5. Possible challenges moving forward
39
39
40
44
46
47
4
Mapping the Taxane Biosynthetic Pathway Using Cheminformatics
4. 1. Current understanding of the taxane pathway
4. 2. Description of the cartography methodology
4. 3. Constraints for ensuring accuracy of the cartographic methodology
4. 4. Computational assessment of the reactivity landscape of the early taxane pathway
4. 4. 1. Homology modeling
4. 4. 2. Substrate docking
4. 5. Results
4. 5. 1. Pathway map
4. 5. 2. The reactive landscape
4. 5. 2. 1. Comments about computational assessment of the P450 reaction
mechanism
4. 5. 2. 2. Using the diffusion of oxygen into the active site as a constraint
4. 5. 2. 3. Method validation
4. 5. 2. 4. Results of the reactivity landscape investigations
51
52
54
58
59
59
61
62
62
63
63
67
68
70
4. 6. Implications of the computational investigations on metabolic engineering
71
5 Re-engineering Taxadien-5a-hydroxylase
5. 1. Background about P450 monooxygenases
5. 2. Materials & methods
5. 2. 1.Strains &plasmids
5. 2. 2. Culturing conditions
5. 2. 3. GC-MS analysis of hydroxylated taxanes
5. 2. 4. Protein engineering for optimal expression of taxadiene 5a-hydroxylase
in E.coli
5. 2.5. Site-directed mutagenesis
5. 3. Results
5. 3. 1.Taxadiene 5a-hydroxylase is a promiscuous enzyme
5. 3. 2. Temporal profile of taxadiene proto-oxidation
5. 3. 3. The utility of traditional mutagenic practices to improve P450 reactivity
5. 3. 3. 1. Phylogeny-guided mutagenesis of taxadiene 5a-hydroxylase
5. 3. 3. 2. Mutations to taxadiene 5a-hydroxylase based on
structural inferences
5. 3. 4. Computational assessment of the taxadiene 5a-hydroxylase
catalytic cycle
5. 3. 5. Computational assessment of taxadiene 5a-hydroxylase mutants
5. 4. Conclusions & perspectives
5. 4. 1.Comparing the methodologies
5. 4. 2. The possibility of multiple substrates in a single active site
5. 4. 3. Directed evolution versus in silico modeling
5. 4. 4. Implications for metabolic engineering
94
98
99
99
100
102
102
6 Technoeconomic Analysis of Microbial Paclitaxel
6. 1. Background & assumptions
6. 2. Comparing the economics of the two competing approaches
6. 3. Summary of the economic assessment
6. 4. Providing unprecedented access to inaccessible chemical space
105
106
108
113
113
7 Putting It All Together: AVision for Drug Discovery
7. 1.What is biosynthonics?
7. 2. The informed design of superior pharmacophores
7. 3. Cheminformatics: An integral part of pharmacophore design
7. 4. Mining the natural world
7. 5. Continuous manufacturing as a tool for discovery
7. 6. Metabolic engineering The ultimate demonstration of continuous
manufacturing
7. 7. Final words
117
118
119
122
124
125
Chapter References
75
76
77
77
78
79
79
80
81
81
83
87
87
90
126
128
129
Appendix A: List of taxanes analyzed for mapping the taxane biosynthetic pathway
141
Appendix B: Taxus cDNA library
177
Appendix C: Deduced taxane metabolic pathway
193
Appendix D: Sample PDB files for heme moiety of P450 HI & docking code
197
Appendix E: NMR raw data
207
a
LIST OF FIGURES
1. 1. The target-rich, lead-poor imbalance
28
2. 1. The disparity between natural products and synthetic molecules
2. 2. Decline in the approval of natural product-based pharmaceuticals
34
35
3. 1.
3.2.
3. 3.
3. 4.
Modularity of terpenoid biosynthesis
Diversity-oriented biosynthesis
Modularization and multivariate analysis
The Jones-Firn Hypothesis
43
45
49
50
4.1.
4. 2.
4. 3.
4.4.
4. 5.
4. 6.
4. 7.
4. 8.
4. 9.
Overview of the pattern-finding algorithm
Summary of the taxane biosynthetic pathway
The P450 catalytic cycle
The 'snapshot' methodology
Substrate docking
Diffusion of oxygen as a constraint
Method validation via assessing docking of Vitamin D3 into active site of CYP1 05Al
Taxane biosynthetic reactivity landscape
TOS of paclitaxel
57
62
65
66
67
68
69
71
73
5. 1.
5.2.
5. 3.
5.4.
5. 5.
5. 6.
Map of plasmid expressing the P450 system
Overview of the strain and enzyme engineering protocols used in the study
Promiscuity & poor selectivity of taxadiene 5a-hydroxylase
Characterized structures of some of the products
Daily variation in eicosanoid production titers
Assessment of the daily variation in the rate of change in the concentrations of the
16 eicosanoids
Speculative reaction scheme
Targets for phylogeny-guided mutagenesis
Activity and selectivity comparisons between the mutant and parental P450
Differentiating the two types of selectivity improvements
Grouping the selectivity improvements
The lysine-aspartate dyad
Influence of mutations to the dyad and amino acids at the periphery of the active
site
Improving reaction selectivity is a non-trivial challenge
The oxygen rebound mechanism
Overview of the competing proton abstractions from taxadiene
78
80
82
83
85
5. 7.
5. 8.
5. 9.
5.10.
5.11.
5. 12.
5. 13.
5. 14.
5.15.
5. 16.
86
87
88
89
90
91
92
93
94
95
96
5. 17. Hypothesized reaction mechanism
5. 18. Comparing mutation efficacy using the computational methodology
5.19. Ageneralized algorithm for in silico enzyme engineering
97
99
101
6. 1. Semi-synthesis of paclitaxel from plant-derived 10-deacetylbaccatin Ill (10-DAB)
6. 2. Total synthesis of taxadiene
6. 3. Biosynthesis of taxadiene
107
114
115
7. 1.
7.2.
7.3.
7.4.
120
121
123
127
Fragment-based pharmacophore discovery
Overview of the biosynthonics paradigm
Two scaffolds, one binding site
Metabolic engineering, the ultimate demonstration of reaction intensification
LIST OF TABLES
3. 1. Titers &yields for conversion of glycerol to taxadiene by the engineered E.coli
strain
46
4. 1. Truncations & estimated pocket volumes of the 16 P450 monooxygenases
investigated
64
6.1. Raw material costs for reaction scheme in Figure 6.1
6.2. Raw material costs for reaction scheme in Figure 4.9.
6. 3. Total manufacturing costs for plant-based and microbial-based manufacturing
schemes
107
110
112
Preface
A BRIEF HISTORY OF THE AMERICAN DRUG ENTERPRISE
Every commentary that one reads about Big Pharma these days paints the same, grim picture1 . So
bleak is the outlook in these reports that it seems implausible that innovator pharmaceutical
companies were once amongst the most profitable and envied corporations in America2 . Even
addressing the Pfizers and Mercks of this world as 'innovator' drug companies seems misplaced
nowadays considering the extent to which they outsource drug discovery and clinical trials to
contract research organizations (CROs)3 5 . Yet, such was the hegemony of these companies over the
markets that, in the 1960s, a prominent senator even advocated passing reforms to curtail the drugmakers' burgeoning profits6' -.
So how did Big Pharma land itself in this current predicament?
Prior to the 1930s, the United States Food & Drug Administration (FDA) was nothing more than a
titular regulatory agency whose powers extended to merely inspecting drugs for possible
adulterations and seizing adulterated products7' 8. In fact, so lenient was regulation during this era
that drugs could not only be marketed without testing their safety, but they could also be directly
purchased by consumers without a prescription from a doctor or physician. This perilous situation
19
continued uninterrupted for many years, until finally in 1937, it all came to a head when several
individuals
succumbed
to
chemical
poisoning
after
consuming a liquid
formulation
of
sulphanilamide that had been prepared using diethylene glycol as the solvent. That diethylene
glycol's toxic effects on humans were fully known and well understood at the time aptly sums up the
FDA's alarming lack of regulatory oversight.
Duly pressed, the government enacted the Federal Food, Drug, and Cosmetic Act (FFDCA) a year
later, and, it then became mandatory for drugs to be tested for their safety by the FDA prior to
market release. Other than this change, however, the provisions of the new act did not really expand
the powers of the FDA. Clauses pertaining to the effectiveness of marketed drugs were still absent
from the FFDCA, and, the FDA remained restricted to functioning as a passive regulatory body that
could only object to the sale of unsafe drugs (as opposed to approving its release into the market).
Moreover, the issue of consumers being able to purchase medication without a doctor's prescription
also remained unaddressed.
It wasn't until the passage of the Humphrey-Durham Amendment in 1951 that it finally became
mandatory for drugs to be sold only by prescription. While some older drugs such as aspirin were
exempted from its provisions, the Humphrey-Durham Amendment to the FFDCA successfully
managed to stratify the drug demand-supply chain, thereby making it considerably easier for drug
manufacturers to market their products. In this new drug market, pharmaceutical companies were
only required to sell their products to consumers through 'agents' - the doctors, and, instead of
20
manipulating the preferences of the average consumer, drug manufacturers would only have to focus
their marketing efforts on swaying the opinions of doctors. In due course, the practice of 'detailing' aggressively promoting a drug-maker's products to a doctor, mainly via personal visits to the clinic
by the company's sales representatives - would take root 7 .
The ballooning profits of the pharmaceutical companies in the decade that followed would once
again catch the attention of the federal government. This time around, the influential senator from
Tennessee, Estes Kefauver, was one among those who saw a profound flaw in the dichotomous
structure of pharmaceutical sales and even advocated implementing reforms to curb the rapidly
growing profits of drug companies. While this demand failed to materialize, the horrific thalidomide
disaster that coincided with these debates eventually led to the passage of the Kefauver-Harris
Amendment to the FFDCA that greatly expanded the role of the FDA to actively approving the
release of a drug into the market only following validation of its safety and efficacy through extensive
clinical testing. In a way, Senator Kefauver did get his wish - by mandating that a drug be
extensively tested for safety and efficacy in lengthy clinical trials, the FDA was prolonging the time it
took for the drug to enter the market, thus reducing its effective duration of patent protection and
curtailing the drug-maker's profits6 . Patent protection and profit maximization would now become
buzzwords in drug discovery, development and marketing.
The very mention of patents is a veiled reference to the threat that innovator pharmaceutical
companies constantly face from generic or copycat drug manufacturers - a threat that had now
21
escalated with the passage of the Kefauver-Harris Amendment. However, drugs manufactured by
generic drug producers were still required to undergo extensive testing prior to marketing and this
requirement often placed generic manufacturers - who tended to be much smaller than their
innovative counterparts - at a significant disadvantage. Unsurprisingly, by the 1980s, riding the
wave of their considerable competitive advantage, innovator drug producers continued to account
for roughly 85% of the American drug market6 .
Suspecting a lack of competitiveness in the pharmaceutical industry, the government would
intervene yet again - this time, by passing the Hatch-Waxman Act in 1984. Formulated with the
specific aim of remedying the competitive disparity in the drug markets, the Hatch-Waxman Act
greatly simplified the regulatory requirements for the manufacture and marketing of off-patent drugs
by generic manufacturers, and, by 1996 - now amidst fiercer competition than before - the share of
innovator pharmaceutical companies in the American pharmaceutical market had eroded to 50%.
To counter what had now become a clear and present danger from generic drug-makers, innovator
pharmaceutical companies would adopt two paradigms that would eventually become the defining
traits of the pharmaceutical industry as we know it today - direct-to-consumer (DTC) advertising
and the needle in a haystack model of drug discovery and development. While one strategy
emphasized the 'industrialization' of drug discovery to reduce R&D timelines and lengthen the
duration of market exclusivity granted by patent protection, the other would, in due course, earn
innovator drug companies roughly $4.20 in sales for every dollar it invested in advertising7 .
22
Chapter 1
THE TARGET-RICH, LEAD-POOR IMBALANCE
1. 1. Drug discovery in the pre-Hatch-Waxman era
Prior to implementation of the Hatch-Waxman Act, drug discovery was a team-based exercise and
each team would usually focus on a specific therapeutic class. Biochemical or cell-based assays were
generally unavailable at the time and 'lead molecules' were selected from a library of molecules
synthesized by a team of chemists based on their ability to elicit desired effects in whole animal
models9 . More often than not, lead molecules were natural products or their synthetic analogues.
While team sizes would vary from project to project, each chemist in a team was expected to
contribute between 50-150 molecules annually' 1. Each molecule in the library would be uniquely
designed and synthesized individually in gram quantities and at high purities. Once a lead had been
identified, 'lead optimization' followed suit. Herein, analogues would be synthesized in order to
explore structure-activity relationships (SARs) to possibly modify the molecule in order to improve
its pharmacological and drug metabolism and pharmacokinetic (DMPK) properties. Once a lead had
been sufficiently improved, the drug discovery team would then prepare pre-formulations for
preclinical testing.
23
1. 2. Deficiencies in the old paradigm
The candidates that emerged from such a discovery pipeline, in general, fared quite well in clinical
testing. The probability of clinical approval was sufficiently high, and, once a drug candidate gained
clinical approval, it assured a company sizable revenues for several years. In fact, in most cases, these
earnings comfortably exceeded the company's original R&D investments. Yet, despite these
successes, many in the industry were concerned with what they viewed to be glaring deficiencies in
this paradigm of drug discovery. Then, lead generation, identification and optimization were viewed
as inseparable functions of the same drug discovery team, implying that drug discovery was
sequential and slowl". Also, the 'black box'-like nature of the in vivo animal models meant that
exclusive reliance on such models for evaluating DMPK properties such as absorption, distribution,
metabolism and excretion (ADME) increased the risk of selecting a drug candidate that might work
in the animal it was tested in, but not in humans'. Escalation in competition from generic drugmakers that followed the passage of the Hatch-Waxman Act merely accentuated these concerns.
1. 3. The industrialization of drug discovery
At about the same time as the passage of the Hatch-Waxman Act, the use of biochemical and cellbased assays and computational tools in drug discovery was rapidly gaining popularity. In vitro assays
greatly aided medicinal chemists to understand the molecular mechanisms of action of the drugs that
they synthesized, and, as a result, allowed them to design superior drugs against molecularly well-
24
defined targets. A clear understanding of a drug's molecular mechanism of action was also beneficial
from a regulatory perspective. Cognizant of these benefits, innovator drug companies responded by
mandating in vitro and in silico assessments of the drug's pharmacological and DMPK properties
prior to preclinical testing, and, soon, the use of biochemical and cell-based assays as well as in silico
tools for drug discovery became pervasive across the industry0 .
As part of the new paradigm, molecules that gave a positive result in an in vitro screening assay were
referred to as 'primary hits', and if the activity of a primary hit and its degree of specificity for the
target could be repeatedly confirmed, it was designated as a 'confirmed hit"0 . When the activity of a
confirmed hit was corroborated in more elaborate assays such as organ bath assays, its selectivity
towards a range of closely-related targets had been determined, and its chemical structure had been
unambiguously proven by re-synthesis, it was then called a 'validated hit'. If the in vivo
pharmacological properties and pharmacokinetic profile of the validated hit were deemed to be
acceptable and it met a set of business criteria, the molecule would then become a lead compound,
which, following numerous rounds of lead optimization, would later become a 'drug candidate'. In
comparison, in the pre-Hatch-Waxman
era, a lead compound rarely underwent standalone
pharmacokinetic profiling since these characteristics were (presumably) satisfactorily addressed by
the whole animal models9 .
Despite their obvious benefits, exhaustively assessing potential leads in silico" and in vitro prior to in
vivo pharmacology and DMPK studies significantly prolonged discovery timelines, diminishing
25
R&D productivity. Faced with ever-increasing competition from generic drug-makers, it soon
dawned that the throughput of chemical synthesis had to rise in order to compensate for the
increased testing molecules now underwent prior to in vivo testing. The scale and throughput of
drug discovery had to rise by several orders of magnitude and this necessitated greatly accelerating
lead generation and optimization by multiplexing and automating chemical synthesis and screening.
Accordingly, the next wave of technological adoption across the industry duly focused on massively
parallelizing chemical synthesis and screening via miniaturization of reaction systems and assays, all
under robotic control. In keeping with this new vision, combinatorial chemistry soon became central
to drug discovery'". Combinatorial chemistry generates molecular diversity by multiplicatively
combining sets of precursors to yield a vast collection of discrete compounds which, depending on
their functional groups, could be further reacted combinatorially to produce a larger library of
molecules. This library can then be rapidly screened and pruned to yield smaller, more focused
libraries whose members can then be selectively manipulated to improve their physicochemical
and/or pharmacological properties"-".
Information technology, too, became pervasive throughout the drug discovery pipeline - right from
improving the quality of lead generation through use of virtual screens17
18,
'bar-coding' molecules"
viz. storing data about a molecule's synthesis and its performance in an assay, to retrieving and
utilizing information about a molecule in real-time. Aided by sterling developments in robotics and
26
21
20
instrumentation 9, the omics revolution , as well as new developments such as the lab-on-a-chip , 22
technology, the speed, quality and information density of assays also improved by leaps and bounds.
1. 4. New paradigm, same old problems
For all the exponential improvements in workflow efficiency, the ensemble of available reactions in
the synthetic chemistry toolbox remained unchanged. If anything, the number of reactions in the
toolbox may have reduced on account of the kinetic incompatibility of several reactions with the
timescales imposed by automation. Unsurprisingly, in the three decades following passage of the
Hatch-Waxman Act, R&D expenditures by innovator drug companies have ballooned six-fold.
Yet, new drug approvals by the FDA have remained relatively unchanged24 , and candidate attrition
rates and development times have risen markedly during this period 2
25, 26.
In fact, all of the 21,000-
odd drug products that have ever been approved by the FDA act on no more than 324 unique
molecular targets27 and, to make matters worse, this statistic has not changed appreciably following
the transition to multiplexed, automated chemical synthesis and screening. Even if one were to
generously assume that proteins sharing >35% identity exhibit related pharmacology, the entire
pharmacopoeia is estimated to perturb only 1,048 genes - a mere 3.5% of the entire human genome
or 130 unique functional protein domains from the more than 10,000 folds and 16,000 protein
families. In fact, over 50% of all drugs target just 4 gene families, and, of the new molecular entities
that were approved in the past three decades, only 6% targeted a previously undrugged protein
domain (Figure 1.1). Drug discovery in its present multiplexed, automated form, it appears, is
27
highly redundant, and, the industry's declining prospects rest solely on the inability of innovator
drug companies to discover leads that bind to novel protein domains - and not throughput, as was
initially deduced.
Undrugged 10(000
Drugged
130
Protein domains
Inthe human body
NME targets
Inthe last 3 decades
Figure 1.1. The target-rich, lead-poor imbalance: Of the roughly 10,000 unique protein domains in the human body,
only as many as 130 (or 1.3%) have been successfully drugged over the course of the past century. Implementation of the
multiplexed, automated paradigm for drug discovery, which is now in its fourth decade, did not alter the synthetic
capabilities of the industry. Instead, it merely made the industry more efficient at what it had been doing for close to a
century. As a consequence, while there is unanimity in which targets the industry should be pursuing to reverse its
declining fortunes, the absence of revolutionary new synthetic techniques implies that only 6% of all NMEs approved
during the last three decades targeted domains from the undrugged 98.7%.
1. 5. The inadequacies of combinatorial chemistry
The high degree of drug redundancy in small-molecule drug discovery - which some experts have
labeled as a 'target-rich, lead-poor' imbalance 28 - stems from the industry's use of combinatorial
28
chemistry for generating compound libraries. In the absence of innovation in chemical syntheses, the
chemical space being screened for biological activity is usually one that has been previously
interrogated29 and, as such, is unlikely to yield hits bearing steric and electronic features capable of
binding novel protein domains. Even on the off chance that combinatorial chemistry outputs a lead
that binds to a new target, it is highly probable that the candidate will exhibit clinically relevant
polypharmacology30 .
Furthermore, not only are leads generated by combinatorial chemistry promiscuous, but since the
precursors used in their syntheses are predominantly aromatic, they bind to their intended targets
quite weakly. Aromaticity introduces planarity into the molecules, which imparts them with
rotational flexibility. This often has detrimental entropic consequences for strong and specific
binding". That combinatorial chemistry yields only a single NME for every 100,000 molecules that
are screened points to how stacked the odds are against it.
1. 6. Effect of the needle in a haystack paradigm on the industry's business model
The low probability that a drug candidate had of gaining clinical approval, allied with the exorbitant
costs3 2 associated with installing and maintaining the technological base required for automated,
high-throughput lead generation, made it imperative for an approved drug to achieve exceptionally
high sales in order to recoup its significant development costs33'
31.
In fact, so enormous were these
costs that only a third of all drugs approved in the first half of the 1990s were actually profitable.
29
As a consequence, innovator drug companies began targeting only those drugs that were capable of
becoming therapeutic standards for chronic diseases that affected a large section of the population,
or, in other terms, drugs that stood a good chance of possibly earning millions to even billions of
dollars each year - the so-called 'blockbuster' drugs.
The shift in companies' business models is best illustrated by the noticeable rise in market prevalence
of blockbuster drugs during the past few years. In 1995, for example, only 17 drugs were
blockbusters3 5 , whereas in 1999 and 2000, those figures rose to 35 and 44 respectively. The rise in
market concentration of blockbuster drugs coincided with a similar surge in Big Pharma's profits,
and, by 2001, despite constituting only 20% of the global marketplace, blockbuster drugs accounted
for nearly 45% of total sales - or 70% of the industry's profits10 .
The innovator companies' decision to pursue quantity over quality drastically altered their technical
capabilities. Their chemistry know-how - the very knowledge that formed the basis of their existence
and success - more or less stagnated. Perhaps what hurt the most was that the pursuit of quantity
forced these companies to forego natural products - a rich source of therapeutic molecules that
contributed roughly half of the 877 small-molecule NMEs approved by the FDA between 1981 and
2002.
30
Chapter 2
NATURAL PRODUCTS & THE PHARMACEUTICAL INDUSTRY
2. 1.The historical use of natural products as therapies
Natural products, particularly those from plants, have been used to treat human ailments since
antiquity". In fact, the plant-based traditional medicine systems of India, China and Egypt date as
far back as 4000 years3 7 . So economical and effective are these remedies that it comes as no surprise
that plant-based medicine forms the bedrock of healthcare in much of the developing world37 . In
developed nations too, plant products continue to be extensively used as food additives and
nutritional supplements. This widespread popularity of plant products for their perceived medicinal
properties ultimately prompted the scientific community to elucidate their pharmacological
mechanisms and, in due course, several secondary metabolites exhibiting potent therapeutic
properties would be characterized and isolated.
The latter half of the
2 0 th
century witnessed several additions to the collection of bioactive plant
compounds, prominently vinblastine, an anti-cancer agent isolated from the Madagascar periwinkle,
paclitaxel, an anti-cancer compound produced by the Pacific yew, and digitalis, a compound isolated
31
from the purple foxglove and used to treat heart conditions38 ; and several of these compounds would
eventually become blockbuster molecules. These developments inevitably attracted the interest of
several large pharmaceutical corporations and by the 1970s, bioprospecting - the process of
screening, isolating and identifying bioactive secondary metabolites - became the dominant
paradigm for drug discovery.
2. 2. What makes natural products good drugs?
Most small molecule drugs elicit their therapeutic effect by selectively inhibiting or augmenting
enzyme activity. In fact, the more selective the interaction, the more efficacious the drug is. It follows
that 'drug-like' molecules are those that can selectively interact with specific biological targets,
possess physical properties that facilitate transport by biological fluids across biological membranes,
and achieve the right balance between retention time and binding equilibrium.
As natural products play vital roles in reproduction, development, defence and symbiosis - especially
in sessile plants 9 - the strength and selectivity of their interactions with their designated targets
largely determines the evolutionary fate of their hosts. As a result, it is believed that the architecture
and physicochemical and physiological properties of these molecules have been stringently selected
by evolutionary pressures to bear diverse ring systems and chiral centres, exhibit a high degree of
oxygenation, and contain a large number of solvated hydrogen-bond donors and acceptors40 , which,
32
in turn, allows these molecules to precisely localize, selectively bind and be efficiently transported
within biological systems".
The limitations of combinatorial chemistry, which yields a single NME for every 100,000 molecules
that are screened, are in stark contrast to the fecundity of natural product chemical space". For
instance, 20 or so of the 7,000-odd polyketides to have ever been screened for bioactivity have made
it to the market, translating to an approval rate of 1 in 350. In fact, whereas Nature has provided the
lion's share of the leads that bind to previously undrugged protein domains, de novo combinatorial
chemistry has yielded just one drug in the past three decades that is linked with a novel target
(Section 1. 4)
The disparity44 between the drug-likeness4 5 of natural products and compounds generated via
combinatorial chemistry (Figure 2.1), in turn, can be attributed to the ensemble of reactions that is
used to generate the latter. A good number of chemical transformations such as enantioselective
synthesis and oxidation, to name a couple, that are kinetically or technologically incompatible with
the timescales imposed by high-throughput synthesis and screening have been largely done away
with, severely curtailing access to some of the very structural features that are believed to make
natural products more drug-like.
Nevertheless, drug prospectors aren't migrating to the natural product space with any particular
urgency (Figure 2.2).
33
Cobihm*irayNatural
-5
i
products
- 3,287)
.1ssos;-5(a
Figure 2.1. The disparity between natural products and synthetic molecules: Plotting the two principal components
derived from the orthogonal transformation of a set variables that include, among others, (1) molecular weight, (2)
number of heavy atoms, (3) number of chiral centers, (4) number of rotatable bonds, (5) chain length, (6) degree of
unsaturation, (7) number of rings, (8) ratio of aromatic atoms to ring atoms, (9) ratio of in-ring to out-of-ring heavy
atoms, (10) number of ring systems, (11) ring fusion degree, (12) number of carbon, nitrogen, oxygen, sulfur and
1
halogen atoms, (13) ratio of carbon atoms to all heavy atoms, (14) number of Lipinski-type acceptors" , (15) number of
Lipinski-type donors, and (16) number of C-C, C-N and
C-O bonds reveals the differences between the chemical space
traversed by natural products and combinatorial chemistry (from Ref. 44). Typically, the products of combinatorial
chemistry bear fewer chiral centers, rings, oxygenated substituents, and solvated hydrogen-bond
donors and acceptors
than natural products.
2. 3.Why isindustry interest innatural products declining?
Bioprospecting poses several scientific and non-scientific challenges that make it unappealing for
pharmaceutical companies in the current business climate. On account of the great strength and
selectivity with which natural products bind to their designated targets, these molecules are almost
never produced in copious quantities by their hosts. For example, six fully developed yew trees
(Taxus spp.), which take about 200 years to grow, cumulatively produce no more than a single dose
34
of the blockbuster anticancer drug, paclitaxe
6
. Additionally, as natural products are generally
synthesized via highly modular secondary metabolic pathways, most pharmacoactive natural
products have several inactive analogues. Paclitaxel, for instance, is just one of hundreds of closelyrelated taxanes that are synthesized by the yew species. As a consequence, isolation and structural
characterization of natural products continues to be encumbered, slow and not necessarily
unambiguous. Natural product extracts aren't nearly as 'screen friendly' as synthetic compounds.
Synthetic NMEs
Natural Product NMEs
NMEs derived from natural products
M
m
M
25
Number 20
of NMEs
approved 15
by the FDA 10
5
0
Total approved
1992
1995
1998
2001
2007
45
42
42
41
20
Figure 2.2. Decline in the approval of natural product-based pharmaceuticals: The total number of natural product
and natural product-derived NMEs approved by the US FDA has been in steady decline since the late 1990s. The
number of approvals in 2012 was not any greater than 2007 levels. However, if one excludes the number of repurposed
NMEs, namely, molecules that were once approved for activity against a particular target but are now approved for
activity against a newer target, the statistics are much worse.
47
On the manufacturing front, total synthesis of natural products, for all its exquisiteness , remains a
cottage industry, and, synthetic routes for large-scale production of a majority of these molecules
35
either do not exist or are commercially infeasible. A synthon is defined as a structural unit, or
building block, within a molecule that is the product of a specific synthetic unit operation", and, the
combinatorial conjugation of synthons generates vast populations of diverse molecular structures,
which are then screened to identify hits49' 1O.Despite their best efforts, synthetic chemists have been
unable to effectively de-construct natural products into synthons, stymying natural product
prospection as well as lead identification and optimization. In fact, since process development does
not normally commence until the latter half of Phase II clinical trials", bioprospecting frequently
yielded hits that had to be disregarded as late as the latter stages of Phase II clinical trials owing to
insurmountable difficulties associated with their synthesis or procurement considerable
amounts had already been
spent on their development.
all this after
While commercial
considerations and material sourcing difficulties accounted for only 5% of candidate attritions from
clinical trials conducted in 1991, this number had risen to 28% in 2001 52. Process development
continues to remain a major stumbling block in the manufacture of natural products and only those
products that were extractable in generous quantities from relatively easy-to-culture organisms have
ever realized commercialization.
The supply of source material from which natural products are extracted is also often unreliable and
prone to seasonal and environmental variations. In fact, it is not uncommon for promising leads to
be forsaken since their native hosts are critically endangered or large-scale production of the drug
could be detrimental to the biodiversity of the host's natural habitat. Take the case of medicinal
36
plants, a source that has yielded about a fifth of all clinically approved natural products". According
to one estimate54 , roughly a quarter of medicinal plant species now face the threat of extinction.
2. 4. Alternatives for probing natural product space
Recent applications of genetic approaches to decipher the biosynthetic code that guides the assembly
of polyketides have revealed that living systems methodically and repetitively combine building
blocks produced by a handful of enzyme-catalyzed unit reactions to generate unprecedented
molecular diversity. On the back of these developments, and aided by sterling advances in microbial
genetics and industrial biotechnology, the recent explosion in the volume of gene and protein data,
the development of more precise techniques for studying cellular metabolism and genetic regulation
and the exponentially declining costs of genome sequencing and oligonucleotide synthesis, microbial
metabolic engineering is poised to become the fulcrum of new technologies for probing natural
product space. Already, significant strides in developing novel microbial strains for the production of
artemisinic acid, a precursor to the antimalarial drug, artemisinin55 ; the nutraceutical, lycopene56 ; the
production of several antibiotics 57 ; levopimaradiene, a gateway precursor to the ginkgolide family of
pharmaceuticals and health supplements58; and taxadiene, the first dedicated metabolite in the
biosynthesis of taxol, a blockbuster anticancer drug59 . In principle, all that is required is to transplant
the genes of the biosynthetic pathway yielding the target product into the host, eliminate
unnecessary or physiologically less significant native reactions, and lastly, re-tool the cellular
regulatory networks via rational or random approaches to ensure optimal production6 0 . This strategy
37
offers numerous advantages over de novo chemical synthesis and plant cell cultures in the production
of plant secondary metabolites as it is readily scalable, consumes lower quantities of resources, is
benign to the environment and uses inexpensive sugar-based carbon inputs38 . A synergism of
bioprocess optimization and metabolic engineering promises high titre production of these
therapeutic molecules and offers a commercially viable alternative to the production of bioactive
plant-derived chemicals.
38
Chapter 3
BIOSYNTHETIC ENGINEERING FOR TERPENOID SYNTHESIS
3. 1. The terpenoid superfamily
Natural products are estimated to total about 170,000 molecules, of which, a vast majority are plant
secondary metabolites 61, and, hard as it is to believe, this immense chemical diversity is the product
of just 3 metabolic pathways. Nearly 60% of all natural products are believed to synthesized by the
terpenoid (or isoprenoid) pathway, 30% are contributed by the related polyphenol, phenylpropanoid
and polyketide pathways, and about 10% originate from the alkaloid pathway.
Terpenoids are interesting from several perspectives - not only are they the most abundant and
ancient natural product family, but several terpenoids have been characterized as anti-cancer, antimicrobial, anti-allergenic, anti-inflammatory, and immunomodulatory drugs6 1,
62.
Some such as
menthol, a counterirritant harvested in large quantities from the peppermint herb, Mentha piperita;
artemisinin, an anti-malarial compound produced by the plant, Artemisia annua; abietic acid, an
acidic resin extracted from conifers and used as a feedstock; and paclitaxel, a blockbuster anti-cancer
drug isolated from the bark of the Pacific yew, Taxus brevifolia6 2- 64 have already been commercialized
by pharmaceutical companies.
39
3. 2. Modularity of terpenoid biosynthesis
Despite being a potential treasure chest of blockbuster molecules, interest from metabolic engineers
in terpenoid biosynthesis, especially for pharmaceutical production, has been modest in comparison
to the polyketide family, a substantially smaller class of molecules. This is explained, in part, by the
fact that a great number of bioactive polyketides are products of multimodular megasynthases called
type I polyketide synthases (PKSs), which made their study and manipulation less encumbered than
the distributed terpenoid pathways. However, as our understanding of terpenoid biosynthesis has
greatly matured over the past two decades, it is now apparent that re-engineering terpenoid pathways
to meet a discovery program's synthetic requirements
is arguably easier than modulating
multienzyme PKSs that shuttle metabolic intermediates from one active site to the other.
In addition to modifying the reaction mechanism and substrate specificity of the pathway enzymes,
PKS re-engineering also incurs the additional task of altering the interactions between its constituent
modules as minor perturbations to the contact interfaces of the domains could detrimentally lower
the activity of the entire enzyme complex. The enzymes that participate in terpenoid assembly, in
contrast, act individually.
Terpenoid construction (Figure 3.1) commences with the head-to-tail condensation reaction
between two C5 molecules, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate
40
(DMAPP). The latter is the smallest member of a homologous series of metabolites known as the
allylic pyrophosphates. The pyrophosphate moiety of the allylic pyrophosphates is a strong leaving
group and the transition state n-conjugated
carbocation is very stable. This makes allylic
pyrophosphates strong alkylating agents". IPP combines with DMAPP to generate a 10-carbon
allylic pyrophosphate, which itself then alkylates IPP in situ to produce a 15-carbon species.
Successive repetitions of the same head-to-tail condensation in the same active site generate longer
allylic pyrophosphates in Cs increments. The number of repetitions is pre-programmed and enzymespecific 66 . Incidentally, IPP is synthesized by either the mevalonate or non-mevalonate pathways.
The latter commences with the condensation of glyceraldehyde-3-phosphate with pyruvate to yield
2-methyl-(D)-erythritol-4-phosphate (MEP). In contrast, the mevalonate pathway commences with
the co-condensation of acetyl-CoA to produce acetoacetyl-CoA 67 . The non-mevalonate pathway,
with a few exceptions, is specific to prokaryotes and utilizes carbon and energy sources more
efficiently than the eukaryotic-based mevalonate pathway 68.
The head-to-tail condensation that elongates the terpenoid chain is re-implemented in the second
stage in a different configuration to convert the linear allylic pyrophosphate to a dephosphorylated,
polycyclic molecule. Cyclization proceeds via an intra-chain Markovnikov addition reaction between
any one of the several alkenyl units in the polymer chain and the allylic carbocation that is formed at
the tail end of the molecule following the departure of the pyrophosphate group66, 69. Since the
alkenyl units are quite close to one another, repeating at 5-carbon intervals within the chain,
41
addition to any one alkene initiates a cascade of proton abstractions and bond migrations that
culminates in the formation of a predominantly saturated polycycle.
The catalogue of terpenoid structures synthesized by an organism can be expanded by evolving
enzymes that favor coupling between two allylic pyrophosphates over electrophilic alkylation of IPP,
thereby yielding branched chains 66 70. Varying the number of elongation events that occur during the
first stage or altering the configuration of the electrophilic attack during the second stage also adds to
terpenoid diversity7 1 . Crucially - and, perhaps unique to terpenoid biosynthesis - addition of the
allylic carbocation to the carbon-carbon double bond in IPP can be cis/trans. While organisms
generally evolve enzymes that selectively form one geometric isomer over the other, combinatorial
biocatalysis offers the potential to literally exponentiate the number of synthesizable structures by
introducing geometric isomerism each time the enzymatic reaction occurs70 .
In the third stage, the polycycle is oxidized by a cytochrome P450 monooxygenase. The P450s
constitute a large family of highly conserved, heme-thiolate enzymes that are present in all forms of
life72 . They usually act in concert with a flavoprotein redox partner - a cytochrome P450 reductase that shuttles electrons and protons derived from NADPH to the oxygenase's active site, whereat
molecular oxygen is protonated and heterolytically cleaved to form water and a reactive iron-oxygen
species that then inserts the second oxygen atom into the substrate73 . Oxidation of the hydrocarbon
scaffold activates it for further functionalization in the fourth stage of terpenoid synthesis.
42
Chain elongation via head-to-tall condensations:
+OPP
OPP
IPP
1 -
OPP
GPP
DMAPP
P
Opp
OPP
FPP
General formula for head-to-tall condensation:
'Op p
-n
OPP+
'PP
-n
-
Scaffold formation via intra-chain condensation:
OPP
Taxadiene
GGPP
Scaffold activation by oxidation:
+
02
+
NADPH -
I-OH?
>it
+
NADP*
Taxadien-5a-ol
Core diversification:
Jt
I.OH
+
+
S
CoA-SH
Taxadien-5a-ethanoate
Figure 3.1. Modularity of terpenoid biosynthesis: De novo total synthesis of the C20 taxane scaffold involves in excess
of 20 steps. It biosynthetic assembly and structural core diversification, in comparison, is considerably more
straightforward.
The nucleophilic oxygen atom of the hydroxyl group is either directly attacked by strong
electrophiles or forms a more nucleophilic alkoxide species that then attacks weaker electrophiles.
Acylations are the most commonly observed products during this stage of terpenoid assembly. The
hydroxylated
scaffolds can
also undergo hydroxyl
hydrogen substitution
reactions such as
43
esterifications, oxidations, halogenations, hydrosulphonations, glycosylations and even protein
conjugations in vitro. Being oxidized, such scaffolds also allow the ready addition and replacement of
bioisosteres for faster and more direct lead optimization.
3. 3. Metabolic & enzyme engineering for targeted & diversity-oriented biosynthesis
Ordered combinations of known genes into biosynthetic operons to be expressed as heterologous
metabolic pathways in tractable microbial hosts - a strategy that is referred to as biosynthetic
engineering - generates pharmacophores that are natural products in toto, whereas permutations and
mutations produce molecules that possess a scaffold that is an unnatural isomer of a natural product.
Conversely, clustering combinations, permutations and mutations of the biosynthetic genes could
generate libraries of natural product-like molecules (Figure 3.2).
Once a combination of enzymatic reactions that can or could produce the molecule of interest has
been identified, individual active sites must then be re-engineered in order to enhance substrate
specificity, product selectivity and activity. This is especially true for targeted synthesis using
enzymes from secondary metabolic pathways that are known to be promiscuous and poorly
selective 74 . The mutagenized enzymes must then be combined into a pathway and enzyme expression
must be suitably toggled in order to adjust their concentrations. Discriminatory expression equalizes
enzyme turnovers and elevates flux through the pathway.
44
Cyclization
Tailoring
C
e.g. Esleification Sub-type
Sub-type A
Polynerization
Sub-type 8B3
Metagenomics will add to our repository of
natural product biosynthetic pathways
Oxidation
Sub-type A
Cataloguing the reactive proclivities of the enzymes,
including the sub-types of the reactions they catalyze
Generating molecular diversity using
combinatorial biocatalysis and chemistry
~,
~
-us~Al
~K~
Using a microbial chassis for expressing
the enzyme combinations, permutations
and mutations
High-throughPut screeningdicvr
Anti-inHective
Figure 3.2. Diversity-oriented biosynthesis: Akin to the concept of 'combinatorial biosynthesis' that had been
espoused by advocates of polyketide-based pharmaceuticals, controlled expression of combinations, permutations and
mutations of terpenoid biosynthetic genes, when perfected, can potentially generate focused libraries of natural productlike molecules.
The engineered pathway must later be integrated into a retrofitted microbial metabolic network.
Interventions to microbial metabolism can occur at one or many of the following levels: (1)
enhancing the rate of substrate uptake, (2) reducing the flux to competing native pathways, (3)
enhancing the flux of precursors and cofactors to the engineered pathway, (4) exporting the product
to the extracellular medium in order to shift equilibrium towards product formation. The choice of
45
the microbial host is guided by its propensity for growth, carbohydrate substrate preference,
potential toxicity of products and by-products, permissible metabolic load, and adherence to
desirable bioprocessing requirements.
3. 4. Summary of recent progress
Our research group recently demonstrated the feasibility of biosynthetic engineering to successfully
synthesize taxadiene, the first intermediate in the paclitaxel biosynthetic pathway that bears the
unique taxane scaffold, using glycerol as the primary carbon feedstock at unprecedented titers of
0.30 and 1.00 g/L in 2 mL fed-batch and 1 L batch fermentations5 9 . For the 1 L fermentations, the
optical density of the E. coli cultures reached a peak of ~40 after 5 days. Assuming an equivalency
between an optical density of 1.0 and a dry biomass titer of 0.3 g/L, the overall yield of taxadiene on
a dry cell weight basis is estimated to be 0.0925 g/g.
Table 3.1. Titers & yields for conversion of glycerol to taxadiene by the engineered E. coli strain
Actual yield as a %
of theoretical
glycerol)
(g/g
Yield
(g/L)
titer
Highest taxadiene
Scale & configuration
maximum yield
2 mL fed-batch
0.30
0.02
5.88
1 L batch
1.00
0.03
8.82
46
This work marks an important conceptual advance as 2 of the 4 biosynthetic modules of terpenoid
synthesis - namely, chain elongation and cyclization - appear to function quite robustly in a simple
and easily manipulable host such as E coli.
3. 5. Possible challenges moving forward
The next logical goal is demonstration of microbial-based stereo- and regiospecific scaffold oxidation
for the production of activated molecules for structural core diversification, either by enzymatic or
chemical approaches, to corroborate the claims of biosynthetic engineering for drug discovery. To
this end, we have targeted the oxidation of taxadiene, which is presumed to first undergo
hydroxylation at the 5a-position. Unfortunately, the exact sequence of substitutions following 5ahydroxylation of the taxane scaffold is unknown and deciphering the precise order of the
biotransformations has been the subject of intense research over the past two decades. Yet, despite
their best efforts, researchers have simply been unable to map the taxane metabolic pathway. In fact,
a large majority of the biosynthetic pathways that produce potentially valuable terpenoids are
uncharacterized.
Moreover, even if a pathway has been completely catalogued, its regulatory mechanisms are often
poorly understood 75 . This implies that construction and optimization of expression of the pathway,
especially balancing the catabolic, primary metabolic and secondary metabolic fluxes to ensure
47
equitable distribution of resources between growth, maintenance and product formation 76 are fairly
long-drawn. For instance, strain engineering and optimization for the production of taxadiene at the
yield and productivity stated earlier (which, as shall be demonstrated in Chapter 6, ensure costcompetitiveness of taxol production using microbial metabolic engineering) consumed an equivalent
number of cloning hours as work conducted to demonstrate production of amorphadiene, another
unsubstituted terpenoid scaffold, in E. coli. Here, the methodology of assessing and eliminating
regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct
modules whose constitution and overall concentrations can be efficiently adjusted using a variety of
transcriptional, post-transcriptional and translational mechanisms could maximize pathway turnover
with respect to resource expenditure. The practice of re-casting metabolic networks as a collection of
interacting modules makes the analysis of a very complicated system considerably more tractable and
allows easy assessment of the performance of module configurations using multivariate statistics
(Figure 3.3).
Additionally, as evidenced from the gibberellin biosynthetic pathway 77, being components of the
adaptive immune system in plants78-80, secondary metabolic pathways comprise several promiscuous
and non-selective enzymes that maximize the chemical diversity produced within the pathway,
which, in turn, allows the plant to respond to a greater number of environmental stimuli without
expending additional energy or increasing the information content of its genome (Figure 3.4).
Although useful from the perspective of diversity-oriented synthesis, this hypothesis - called the
48
I, "-'I
i IVII
Jones-Firn Selection
Hypothesis - presents some interesting challenges for the targeted production of
molecules using metabolic engineering.
ioer
EWIM
-
Ad1
Strength of RIBS
I
Source of
enzpmPromoter
strength
Figure 3.3. Modularization and multivariate analysis: The flux through a linear pathway is the reaction rate of the
pathway's slowest step. This implies that, at steady state, the observed turnovers of several enzymes of the pathway are
below their maximum values. By grouping enzymes with similar turnovers into a subset, or module, and later equalizing
the turnovers of the different subsets by adjusting variables such as module constitution, promoter strength, plasmid
copy numbers, RBS strength, among others, one can assess pathway turnover using multivariate statistical operations
such as principal component analysis. This approach has already found extensive use in analysis of molecular events in
signal transduction pathways"1.
Once a likely combination of enzymatic reactions that could produce the molecule of interest has
been identified, they will necessitate customization or active site re-engineering to enhance substrate
49
specificity, product selectivity and activity to minimize flux dissipation away from the molecule of
interest. Consequently, enzyme engineering becomes another critical endeavor in the goal to produce
specific natural products in microbial hosts.
Central
metabolism
IPP
I
DMAPP
-
A
Figure 3.4. The Jones-Firn Hypothesis: According to Clive Jones and Richard Firn, if every secondary metabolite can
be thought of as being a unique response to an environmental stimulus, it can be argued that the enzymes of secondary
metabolic pathways may have evolved to become promiscuous. Substrate promiscuity or poor product selectivity
maximises the structural and functional diversity of secondary metabolites produced by the host, which, in turn, allows it
to respond to a greater diversity of environmental stimuli, thereby improving its evolutionary fitness. In the two
hypothetical scenarios depicted here, the plant in B can respond to the same number of environmental stimuli by
incurring a roughly 30% lower metabolic load compared to the plant in A.
50
Chapter 4
MAPPING THE TAXANE BIOSYNTHETIC PATHWAY
USING CHEMINFORMATICS
Paclitaxel is manufactured via a semi-synthetic process that commences with extraction of its
precursor,
10-deacetylbaccatin
III (10-DAB),
from plant cell cultures of Taxus spp.
As
metabolomics investigations have revealed that paclitaxel is one of several hundred closely related
taxanes produced by the Taxus cell cultures, a significant proportion of the bulk price of paclitaxel,
which, in 2012 was estimated to be roughly $190,000 per kg, is attributed to separation costss2
Development of a microbial-route that can produce 10-DAB, paclitaxel or another precursor at high
titers and very few by-products could drastically lower the price of the drug, thereby increasing access
to more patients.
However, as discussed in Section 3.5, the paucity of information regarding taxane and several other
terpenoid biosynthetic pathways has greatly restricted access to these valuable molecules through
microbial metabolic engineering. For the case of the taxane pathway, all that is known is that it
commences with the cyclization of the linear molecule, geranylgeranyl pyrophosphate (GGPP) to
produce taxadiene8 3. It has been presumed that taxadiene is then substituted at the 5a-position with
51
a hydroxyl group. The exact sequence of substitutions following 5ct-hydroxylation of the taxane
scaffold is unknown and deciphering the precise order of the biotransformations has been the subject
of intense research over the past two decades. Yet, despite their best efforts, researchers have simply
been unable to map the taxane metabolic pathway.
Demonstrating the successful expression of the third chemical module in terpenoid biosynthesis namely, scaffold oxidation - in E. coli necessitates metabolic cartography to determine the identities
of the genes for their eventual expression in the microbe. To this end, we developed a generalizable
methodology to map the biosynthetic pathway by analyzing chemical substitution patterns in
naturally occurring taxane metabolites as well as investigating taxane biochemistry via molecular
modeling and docking studies. Identifying the sequential order of metabolic reactions in the taxane
biosynthetic allows one to study the biochemistry of the pathway by correctly pairing enzymes with
their substrates, providing valuable insights for protein engineering to improve enzyme activity and
selectivity. This new intellectual framework for elucidating biosynthetic routes and the biochemical
mechanisms of their constituent reactions could open up opportunities for investigating several more
terpenoid pathways.
4. 1. Current understanding of the taxane pathway
While the exact sequence of reactions in the pathway has been hitherto elusive, significant progress
has nonetheless been made in the study of taxane metabolism. Among these, metabolomics
52
investigations on T. cuspidata cell cultures have been able to successfully isolate and characterize 128
taxane metabolites (Appendix A), and several putative taxane biosynthetic genes have also been
sequenced (Appendix B) 83-86 . However, the absence of a blueprint or map of taxane metabolism has
prevented the identification of metabolite-enzyme pairs, which in turn has hampered efforts to study
the pathway's biochemical and regulatory mechanisms in greater detail.
Graph theory and atom mapping have been previously utilized to discover novel or non-standard
metabolic pathways 87, 88. Herein, genome-scale models and information about atom conservation
within known pathways are utilized to design novel metabolic pathways for the production of
desired molecules from defined precursors can be designed and tested. Our situation is a direct
contrast to these methodologies. Additionally, since secondary metabolic pathways proceed using a
structural core formation-diversification synthesis plan, atom mapping is unlikely to shed any light
on pathway topology.
Some recent studies have attempted to decipher the sequence of reactions in the taxane biosynthetic
pathway by systematically evaluating the reactivity of individual enzymes towards different substrates
in vitro83'89. For example, if an enzyme that is presented with taxadiene-5a-ol, taxadiene-1Op-ol and
taxadiene-5ct, 10jp-diol as substrates yields taxadiene- 1 0p,2a-diol as the major product, it is annotated
as taxadiene-1Op-ol 2a-hydroxylase. The non-reacting or partially reacting substrates are then
presented to another enzyme to determine its identity and this evaluation continues until all enzymesubstrate pairs have been identified.
53
All that is known about the taxane metabolic scheme thus far, including annotations of genes of the
pathway, has been achieved using the aforementioned methodology. However, such a method for
pairing enzymes to their corresponding substrates suffers from a glaring shortcoming that assumes
that taxol is the major end-product of a linear pathway. This might not be the case. As suggested by
the Jones-Firn Screening Hypothesis (Section 3.5), enzymes of the taxane pathway may very well be
promiscuous and non-selective, implying that the pathway could be highly branched.
4. 2. Description of the cartography methodology
The methodology utilizes the chemical structures of intermediates and products, and the putative
sequences of the pathway's genes (which is used to derive homology models of the pathway's
enzymes) as inputs and then outputs the map or sequence of reactions in the metabolic pathway.
The analysis is unique in its integration of data across three strata of the metabolic hierarchy, namely
metabolites, genes, and proteins and makes novel use of tools and techniques that are otherwise
commonly applied to the study of chemical or biological problems9". Moreover, the algorithmic
architecture of the analytical procedure ensures that it is easily programmable for use as a software
package for the analysis of poorly characterized metabolic pathways.
The analysis commences by cataloguing the chemical structures of as many naturally-derived taxanes
and then recasting this pictorial information into a mathematically interpretable format. Examples of
54
mathematically interpretable formats include the SMILES' (simplified molecular input line entry
specification) nomenclature that is frequently used to catalogue molecules in chemical databases, or
the more generic, binary vector representation wherein the length, column numbers and elements of
the vector represent the total number of carbon atoms in the molecule, their assigned IUPAC
designations, and the type of substitution they bear, respectively. It is, however, essential that a single
format be used for all the molecules to preserve consistency and allow easy analysis of metabolite
structural data. Also, using mathematically interpretable formats makes programming the analysis
into a software package more amenable. Structure cataloguing revealed that all naturally-derived
taxanes bear a subset of the following substitutions:
p-substituted
with either a hydroxyl group or an ester group.
i.
Carbon 1 is exclusively
ii.
Carbon 2 is exclusively a-substituted with either a hydroxyl group or an ester group.
iii.
Carbon 3, which is a tertiary carbon, is never substituted. Substitution at this position is
sterically hindered.
iv.
A double bond is mostly observed between carbons 4 and 20. In some cases, carbon 4 is part
of a 4-member ring known as the oxetane ring wherein it is directly bonded to carbon 20
and carbon 5, which, in turn, are connected to each other via an ether bond. Carbon 4 is also
a-substituted with an ester group when it is part of the oxetane ring. In rare cases, carbon 4
55
and 20 form an epoxide ring, while in even rarer cases, carbon 20 is hydroxylated by
hydration of the carbon 4-carbon 20 double bond.
v.
Carbon 5 is generally a-substituted with either a hydroxyl group or an ester group. In some
cases, it is also a part of the oxetane ring.
vi.
Carbons 6 and 8 are never substituted.
vii.
Carbon 7 is exclusively
viii.
Carbon 9 is exclusively a-substituted with either a hydroxyl group or an ester group. Carbon
P-substituted with
either a hydroxyl group or an ester group.
9 is also ketonated in many taxanes.
p-substituted with either a hydroxyl group
ix.
Carbon 10 is exclusively
x.
Carbons 11 and 12 are never substituted and are connected to each other by a double bond.
xi.
Carbon 13 is exclusively a-substituted with either a hydroxyl group or an ester group.
or an ester group.
Carbon 13 is also ketonated in many taxanes.
xii.
Carbon 14 is exclusively n-substituted with either a hydroxyl group or an ester group.
56
xiii.
Carbon 15 is very rarely connected to carbon 13 via an ether bond. Otherwise, it is
unsubstituted.
xiv.
Carbons 16, 17, 18 and 19 are never substituted.
Next, a Bayesian probability-based algorithm is used to infer relationships between the taxane
metabolites. The conceptual foundations of this approach, called 'substitution pattern-finding', are
similar to those of applying Bayesian probability to establish phylogenetic relationships between
organisms in evolutionary biology (Figure 4.1)9'.
R
R
R
R
R
n= Number of metabolites with
/
R
R R
R
R
R
hydroxylation at 5a position
OH
R
OH
R
R
R
n2
Number of metabolites with
hydroxylation at 70 position
n
Number of metabolites with hydroxylations
at both, 5a and 70 positions
R
RR R
R
If n, > n2 and n2 = n, hydroxylation at 5a precedes that at 7P position
Figure 4.1. Overview of the pattern-finding algorithm
57
4. 3. Constraints for ensuring accuracy of the cartographic methodology
Consider a hypothetical sequence of reactions that commences with the conversion of taxadiene to
taxadiene-5a-ol. Taxadiene-5ct-ol is then converted to taxadiene- Taxadiene-5a, 10p-diol, which then
produces taxadiene-5a-acetate-10p-ol following esterification of the 5a-hydroxyl group. If one was
only provided with the chemical structures of these metabolites without any information regarding
the sequence of the reactions that connect them, evaluating the substitution patterns of the
metabolites can yield the putative sequence of reactions. However, merely relying on substitution
occurrence frequencies could lead to confounding conclusions. Accordingly, chemical feasibility
constraints have to be imposed on the pattern finding exercise for deducing meaningful and nonconfounding relationships between the taxane metabolites. These constraints include inviolable rules
in organic chemistry as well as information about putative reaction mechanisms. One example of an
inviolable rule in organic chemistry is the impossible occurrence of ester formation prior to
hydroxylation. Also, understanding the mechanism of a reaction can provide insights into aspects
such as transient species stabilization via steric or electronic modifications in the molecule.
Amongst the metabolites listed above, it is evident that taxadiene is the least substituted molecule
and hence must precede all the other species in the hypothetical pathway. Similarly, since the 5aacetylation must succeed the 5c-hydroxylation, it can be concluded that taxadiene-5a-acetate-10p-ol
can be produced by one of three possible routes: (1) taxadiene-5ax-ol to taxadiene-5c-acetate to
taxadiene-5a-acetate-10p-ol, (2) taxadiene-5a-ol to taxadiene-5at,10p-diol to taxadiene-5a-acetate58
1OP-ol, or (3) taxadiene-10p-ol to taxadiene-5a,10P-diol to taxadiene-5a-acetate-10p-ol. However,
the separate occurrence of taxadiene-5a-ol and taxadiene-5c,10p-diol suggests that the likeliest
pathway from taxadiene to taxadiene-5a-acetate- 10fp-ol is via path 2.
The map of taxane metabolism as outputted by the constraints-based substitution pattern finding is
then validated in the second stage of the theoretical framework - computational enzyme catalysis.
Herein, bioinformatics and computational chemistry are utilized to study enzyme catalysis. To
eliminate bias, we ignore enzyme annotations as they appear in GenBank.
4. 4. Computational assessment of the reactivity landscape of the early taxane pathway
4. 4. 1. Homology modeling
Since crystallographic structures of none of the taxane biosynthetic enzymes have been hitherto
characterized, homology or comparative modeling has to be utilized in order to obtain precise
representations of the enzymes and their active sites. Briefly, homology modeling of an enzyme, or
target, commences with template selection, and a wide variety of open-source fold-recognition or
threading software can be utilized for this purpose". These programs score and rank templates based
on the similarity of their structural folds to those predicted for the target enzyme. While the topranked templates as suggested by the fold-recognition or threading programs generally produce fairly
accurate homology models, it is recommended that they be manually checked for similarity with the
59
target enzyme by comparing predictions for physicochemical parameters such as their secondary
structures, solvent accessibility, membrane-binding domains, ordered-disordered regions as well as
phenomena such as distance bonding. In the rare event that a suitable template is unavailable, one
has to resort to de novo enzyme modeling. Fortunately, the likelihood of such a scenario arising for
enzymes of plant secondary metabolic pathways is quite slim.
After suitable templates have been selected, their sequences are carefully aligned with that of the
target using the compatibility between the aforementioned physicochemical parameters as a
constraint. Open-source software is also available for this purpose". Incidentally, most foldrecognition software also produce alignments between the top-ranked templates and the targets, but
these are quite crude and have to be substantially refined if they are to be used for model building.
The target-template alignment is then utilized to build homology models. Many software packages,
each differing in their optimization criteria, are freely available for this task, and the quality of
models that they output is fairly comparable for most families of proteins. Once the homology
models have been built, they are checked for structural correctness using gauges such as a
Ramchandran plot and further, if necessary.
The Taxus cDNA library from which the sequences were derived contains 16 P450 and 10
acyltransferase (9 acetyl- and 1 benzoyltransferase) sequences"3 ' 84, 86,
95.
Homology models of the
taxane biosynthetic enzymes were constructed using MODELLER96 . Target sequences that exceeded
60
their templates in length were suitably truncated in order to ensure that the homology modeling
program is able to capture the folds of the templates. As the cytochrome P450 monooxygenases
contain heme prosthetic groups, their active sites were first located using the pocket searching
algorithm, POCASSA97 , following which the heme groups were manually docked and their
geometries optimized
using the BFGS energy minimization algorithm. The structural database,
DALI 98, was also searched for crystallographically solved homologues to validate the output of the
pocket searching algorithm.
4. 4. 2. Substrate docking
Next, computational models of an enzyme's putative substrate and product, as suggested by the
consensus map, as well as a broad selection of other taxane molecules were docked into the active site
of the enzyme model using an open-source software package, AUTODOCK, to predict the preferred
orientation of the ligand as well as its binding affinity (or energy of interaction) with the active site.
It was hypothesized that docking a large collection of molecules into the active site of the enzyme
could potentially offer several insights into its promiscuity towards substrates. The docking exercise
is repeated for all enzymes, and upon its completion, one can finally compare the energies of
interaction for several enzyme-metabolite pairs, thereby confirming or rejecting those suggested by
the consensus map.
61
4. 5. Results
4. 5. 1. Pathway map
Extending the pair-wise comparisons to encompass all substituted positions produced a putative map
of taxane metabolism, whose early stages almost exclusively comprise of P450-mediated oxidations
(pathway summary in Figure 4.2, entire pathway is been presented in Appendix C).
32.5%
24.5%
Sa-OH
-
1O0-OH -- 9u-OH -
13a-OH -
2a-OH
Variation in the order between
9a, 13o and 2a is probable
13-oxo
9-oxo - -
Paclitaxel
% 9a-acetate
18.8%
13G-aCetate
5.4%
7p-OH
Figure 4.2. Summary of the taxane biosynthetic pathway: Interestingly, studies on the production of paclitaxel by
plant cell cultures of T cuspidatahave revealed that paclitaxel, on a weight basis, accounts for about a fifth of all taxanes
produced. Accounting for variations in molecular weights, the map outputted by the substitution pattern finding exercise
appears to mirror actual production of paclitaxel.
While substitutions at most carbon atoms on the taxane scaffold can be definitively ordered, the
temporal relation between some functionalizations such as hydroxylations at the 2a, 9a and 13a
positions cannot be deduced. Also, substitution patterns suggest that oxetane ring formation is
among the last modifications to occur on the scaffold.
62
The suggestion that the early pathway almost exclusively consists of P450-mediated reactions is not
all that surprising as this biosynthetic strategy has also been observed in other natural product
biosynthetic pathways. As P450 monooxygenases catalyze C-H bond functionalization instead of
acylation of an O-H bond that is mediated by acyltransferases, the former are more prone to
promiscuity and non-selectivity, increasing chemical diversity within the pathway. Single amino acid
mutations within the active site of P450 monooxygenases could make these reaction sites 'plastic',
which, according to the Jones-Firn Selection Hypothesis, is a more robust and reliable way to
generate diversity compared to the significant alterations that might be required to re-orient a
polyoxidized molecule in order to drive acylation at one hydroxyl group over another. Accordingly,
the reactive landscape was subsequently investigated by assuming that the hydroxylations at the 5aand 10p-positions precede the others and assuming all possible permutations of the 2a-, 9a- and
13a-hydroxylations.
4. 5. 2. The reactive landscape
4. 5. 2. 1. Comments about computational assessment of the P450 reaction mechanism
Homology models of all 16 P450 monooxygenases in the Taxus cDNA library were constructed
using MODELLER. The enzymes were randomly annotated as HI through H16 in order to
eliminate bias. The reductase domain was excluded from this analysis. The A-chains of the P450
enzymes, 2CIB, 3EQM, 3GW9, 3DBG, 3LD6 and 2VE3 were used as templates. The homology
63
models were then augmented with the heme prosthetic group. The thiolate linkage between the
heme iron and cysteinyl sulfur was first created, following which, geometry of the heme moiety was
optimized using the BFGS energy minimization algorithm9 9 . The number of iterations in the BFGS
algorithm was left uncapped in order to ensure convergence to the optimal geometry. Accuracy of
the optimized geometry was verified by comparing the bond length of the thiolate linkage in the
homology model to those that are typically observed in solved crystal structures of other P450s. The
average bond length of the thiolate linkage for the 16 homology models was 2.56
quite favorably with the average thiolate bond length of 2.47
A, which compares
A for the 6 templates.
Table 4.1. Truncations & estimated pocket volumes of the 16 P450 monooxygenases investigated
Annotation
Truncation for modeling
(# of amino acids)
Untruncated length
(# of amino acids)
Pocket volume (A3)
H1
10
484
256
H2
40
484
422
H3
50
501
601
H4
50
500
748
H5
40
492
395
H6
50
500
387
H7
40
483
400
H8
50
503
239
H9
45
497
610
H1O
50
507
559
HI
50
509
286
H12
35
485
250
H13
40
493
382
H14
45
498
250
H15
45
495
477
H16
50
499
474
64
The heme prosthetic group proceeds through 4 states during the P450 catalytic cycle (Figure 4.3) the ground, activated, peroxyheme and oxyferyl states. As the modeling approach cannot capture nor
predict the dynamics of substrate orientation within the active site, a 'snapshot' methodology
(Figure 4.4) that assesses the substrate pose at each stage of the P450 cycle and its eventual influence
on the reactive pose viz. the pose that the substrate adopts just prior to reaction with the oxyferyl
group, is implemented.
NADPH --
Figure 4.3. The P450 catalytic
Monooxygenase
Reductase
NADP+
2e-+H*
Ground
R-H
R-H
From NADPH
Fe
FeI
FAD + 2e + H*+ H* From
intracellular
environment
S-Cys
R-OH
FADH2
cycle: Substrate binding within the
H*
2
state
F$
S-Cys
5cs
(
02
P450 active site activates the heme
H*,e
H', e(occurs only
fter
HO
subst,.te
R-H
iron. Binding of molecular oxygen
has bound)
R-OH
FADH2
2
FAD
FADH'
FADH AD
FAD*
FADH'
Fe-,
FDH*
5-Cys
FMN
FMNH.-
FMN
H*,e-
FMNH~ FMN
monooxygenase
H, eShuttled to
monooxygenase
0
0
H+.eShuttled to
follows suit, and electrons supplied
S y
-Cys
Fe '
I ,
OH
H2O R-H
R'
.Fe5-Cys
-
H2O
-
Fe"
*
by
NADPH
are
shuttled to the
active site shortly after. Transition of
the heme iron from the peroxy to
the oxyferyl state reductively cleaves
the oxygen molecule to form water. The oxyferyl species then oxidizes the substrate to typically form a hydroxylated
product.
65
0
Ground state
0
/ Zt+/
Fe3
S-Cys
H
8-Cys
Activated heme
S-Cys
Peroxyheme
8-Cys
Oxyferyl heme
Initiates ORM
Figure 4.4. The 'snapshot' methodology: As the in silico models cannot capture dynamic changes in the active site of
the P450 monooxygenases, the binding affinity between the substrate and each successive heme state in the P450
catalytic cycle is assessed in order to draw rational conclusions regarding the reactive proclivities of the enzymes.
In the snapshot methodology, owing to our use of the BFGS energy minimization algorithm to
optimize the geometry of heme in the active site, AUTODOCK was unable to distinguish between
the ground and activated states of heme, and peroxy- and oxyferyl heme. The energy trajectory for
the oxidation of taxadiene, therefore, was estimated by comparing the binding affinity of taxadiene
in the active site that includes heme iron in its activated state to its affinity to the oxyferyl hemecontaining active site. Also, as AUTODOCK typically outputs a series of closely related poses from a
single input, it was executed iteratively using the most energetically favorable output of one run as
the input for the program's next implementation, until an unchanging set of substrate poses was
obtained (Figure 4. 5).
66
POSOX,
The docking algoithm
outputs so poses
ft, X2
8ufX
2
Figure 4.5. Substrate docking: As AUTODOCK outputs a series of the most energetically-favorable poses (which is
significantly different from the series of likely poses), the exhaustive list of 9 poses is first culled to a smaller set of 2-3
poses by using an iterative algorithm that recasts the docking exercise as an unconstrained optimization. The
'optimization' loop is terminated when 3 successive iterations output a common set of poses with the same binding
affinity.
Accuracy of the docking program was assessed by evaluating the closeness of fit of the
computationally predicted pose of androstenedione in the active site of human aromatase
cytochrome P450 (CYP19A1) with the crystal structure of their association10.
4. 5. 2. 2. Using the diffusion of oxygen into the active site as a constraint
The diffusional trajectory of oxygen into the activated heme-containing active site greatly limits the
translational and rotational degrees of freedom the now-destabilized substrate has in order that it
may assume a more stable position. This constraint was utilized to discriminate between the
67
numerous poses outputted by AUTODOCK in order to identify the single likeliest pose of
taxadiene in both the activated and oxyferyl heme-containing active sites (Figure 4.6). The likeliest
path that oxygen takes to diffuse into the active site was calculated using CAVER 0 1 .
Pose B:
PoseA:
4.8 Walmol
-.
kalmo
4.1
kcallmoI
4A kcaI/moI
Figure 4.6. Diffusion of oxygen as a constraint: The modeling methodology identifies two possible reaction
trajectories for the oxidation of taxadiene by the P450 hydroxylase. The first panel for both schemes represents binding
of taxadiene to the activated heme-containing active site. The diffusion path of oxygen is highlighted in red in the second
panel, and the reactive conformation that leads to substrate oxidation is depicted in the third panel. Both trajectories,
however, predict competition between abstractions of the same 3 protons by the oxyferyl heme iron. These are, in their
order of favorability, the protons attached to 3-, 13a- and 20-positions.
This program re-casts the atom centers in the enzyme model as a Voronoi diagram, whose edges and
vertices are then converted to a weighted graph. The Delaunay triangulation is later applied to the
weighted graph and the likeliest path of oxygen diffusion is calculated using a modified Djikstra's
algorithm.
4. 5. 2. 3. Method validation
The computational methodology for studying P450-mediated substrate oxidation was validated by
predicting the reaction trajectories for the dihydroxylation of vitamin D 3 by CYP105A1 (Figure
68
4.7). Vitamin D 3 is first hydroxylated at either the la- or 20-position, following which, the
monohydroxylated product -
la-hydroxy or 20-hydroxy vitamin D 3
-
undergoes a second
hydroxylation to form 1at,20-dihydroxy vitamin D 3. The modeling methodology's outputs compare
favorably to the crystallographically determined substrate poses in both, the activated heme and
oxyferyl heme active sites of CYP105A1 (PDB codes: 2ZBZ and 3CV9).
The 5-10% difference between the binding affinities of the substrate to the activated heme and
oxyferyl heme states of the P450 was also identified as an important constraint to identify likely
poses from the other outputs of the docking program.
VDs -+ 25-OH1-D
3
25-OH-D, -.
1a,25-(OH)rDa
la-OH-D
-
1a,25-(OH)rD
0
-2
Binding affinity (kcallimol)
-10
4
4
-4
-12
VDs In
CYPIOA1
2"-H-D3
In CYP10SA1
Activated home
-10 kcallmol
Activated home
-10A kcailmoI
4.1 kcallmol
Oxyferyl heme
-7.9 kcallmol
Oxyferyl heme
Oxyferyl heme
4.1 kcallmol
4.9 kcallmol
34.5*6.9 min.maiCYP
Activated heme
75.7 129.2
m.
a-OH-O,
In
CYPIONGAI
anActivated
sOxyfaryl
C
Figure 4.7. Method validation via assessing docking of Vitamin
D3
into active site of CYP105A1: The binding
position of vitamin D 3 within the active site of CYP105A1 has been determined using X-ray crystallography for both, its
activated and oxyferyl heme forms. Model validation proceeded by comparing its predictions of the reaction trajectories
with the crystallographic structures of CYP105A1 bound to its substrate.
69
4. 5. 2. 4. Results of the reactivity landscape investigations
The enzyme that has been annotated as taxadiene 5a-hydroxylase in the sequences derived from the
Taxus cDNA library is re-annotated as H16 in the computational assessment described herein. It is
evident that taxadiene 5a-hydroxylase is a very promiscuous enzyme (Figure 4.8).
Interestingly, none of the other 16 P450s exhibit 5a-hydroxylase activity. H6 is predicted to exhibit
10p-hydroxylase activity, and the computational methodology corroborates it annotation in
GenBank. H13, on the other hand, appears to catalyze stereo- and regiospecific 7p-hydroxylation of
taxadiene but is annotated as a 1Op-hydroxylase in GenBank. This dissonance merits further
investigation but has not been pursued in this study.
H9, on the other hand, is predicted to be a taxadiene-5a-ol 1Op-hydroxylase, and, H4 is predicted to
catalyze the 2a-hydroxylation of taxadiene-5a,10p,13a-triol. Additionally, the size of the active site
(in A3) does necessarily correlate with promiscuity. For instance, H9, which is estimated to possess
the second largest active site among the 16 P450 monooxygenases does not exhibit any activity (as
predicted by our computational methodology) to substrates bearing substitutions at more than a
single carbon atom on the taxane scaffold.
70
H1
H2
H3
H44
H5
H6
H7
HS
H9
H10
H11
H12
H13
H14
H15
H16
TaxadleneI
Taxadlen4a-Wo
Taxadlen-100-ol
5a,100-di
2a,Ga,100-trol
a91003-triol
6aa,100,13a-ttro1
2a,Sa,9a,10-%trol
I
9
Ol0"o
2a,6a10,13a-tetrol
2a,a,9a,10p,13a-pentol
2a,a,10,13a-tetol--one
2a,6a,9a,100-tetrol-13-one
6a-ethanoate
-10 kcalimol
-10 kcallmol
10%
-10%
Activated home
Oxyferyl home
acnergy diference between
activ a OxyfeIl stat..
Figure 4.8. Taxane biosynthetic reactivity landscape: All 16 P450 enzymes from the Taxus cDNA library were
included in the analysis. The enzymes were designated as H1-H16. The reactivity of an enzyme towards a substrate is
affirmed using the 5-10% difference in binding affinities between the activated and oxyferyl states of the enzymes, as was
previously established for the dihydroxylation of vitamin D 3 by CYP 10 5AL.
4. 6. Implications of the computational investigations on metabolic engineering
An important consideration in pharmacophore design is its potential use as an input for
conventional target-oriented synthesis (TOS)"10, an exercise that aims to access precise regions of
chemical space - either a single molecule or a small assortment of molecules exhibiting minor
variations on a chemical theme. Natural product biosynthetic pathways typically consist of a large
71
number of reactions that are controlled by fairly complex regulatory mechanisms. Manipulating the
metabolism of simple microorganisms to accommodate the expression of every single enzyme of the
paclitaxel biosynthetic pathway is a prohibitively long process and is perhaps infeasible owing to the
physiological stress that the expression of such a large set of enzymes might induce within the host.
Instead, synthesizing an advanced intermediate that acts as a gateway molecule for TOS via the
expression of a vastly smaller set of enzymes takes advantage of the core competencies of both,
metabolic engineering and synthetic chemistry.
Based on our computational analysis and bio- and retrosynthetic planning for the production of
paclitaxel (Figure 4.9), we have identified (1,2a,5a,7p,10p)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax4,11 -diene as a potential product of microbial metabolic engineering.
Our analysis suggests that
expressing H16, H9 and H6 in a strain expressing taxadiene should, in theory, yield taxadiene2a,5a,10s-triol. Of these enzymes, H16 might have to be significantly re-engineered in order to
minimize its promiscuity towards taxanes other than taxadiene.
H13, which catalyzes 7p-hydroxylation of taxadiene, and H4, which hydroxylates taxadiene5a,103,13a-triol at the 1-carbon, will also need to be sufficiently re-engineered to alter their
substrate preference to taxadiene-2a,5a,100-triol and its 7p- or 1-hydroxy derivative. Target yields
and productivities to ensure cost-competitiveness of a microbial-based process manufacturing
(1,2a, 5a,7p, 100)-5-acetyloxy- 1,2,7,1 0-tetrahydroxy-tax-4, 11 -diene have been calculated in Chapter
6.
72
O
AcO
0
AcO
H
b
NH
H
OTES
0
HO
0
C
d
NH
0
f
N
N
H
og
_
NH2
OH
HO
H
0
HO
h
0
9
O0
0
0
OH
OTES
O
OH
O0-
A
O
O
0
OTES
OTES
n
k
OH
O
O )(
0
O
0
0
0
OTES
0
OTES
0
O0OH OTES
OH OTES
OTES
S
t
0
0
O O-
O-
H
O0
00UO
0
HO
0
C 0
00OTES
0
0
HO
HO
HO
O
0
OTES
+
0
/
HO
O
0
0
OTES
OH
00
HOt 0
HO0
0
-
H0
OTES
H~
0
Paclitaxel
Figure 4.9. TOS of paclitaxel: The synthesis of paclitaxel in our scheme commences from (1,2a,5a,7p,10p)-5-acetyloxy-1,2,7,10tetrahydroxy-tax-4,1 1-diene (A). The synthesis scheme comprises of 22 steps and the yield of paclitaxel is 1.9%. Key: a.CH 2Cl2
(yield 99%); b. Et 3N, 2-acetoxyacetyl chloride, CH 2Cl 2 (65%); c. (NH 4) 2Ce(NO 3)6, CH 3CN (80%); d. MeOH, Na 2CO 3 (82%); e.
TESCI, pyridine (89%); f 4-DMAP, Et 3N, benzoyl chloride, CH 2Cl 2 (82%); g. CDI, NaH, DMF (8 1%); h. TESCl, pyridine
(85%); i. H 5 10 6 , CH 3CN, PCC (97%); j. dioxane, MeOH, KOH (99%); k. quinuclidine, NMO, tBuOH, NaBH 4, Os04, THF
(64%); L.4-DMAP, CH 3 COCl, CH 2Cl 2 (79%); m. 4-DMAP, MsCl, CH 2 Cl 2 (84%); n. K2 CO 3 , MeOH (80%); o. HMPA, DIEA
(77%); p. 4-DMAP, (CH 3 CO)2 0, CH 2 Cl 2 (70%); q. PhLi, CH 3COOH, THF, cyclohexane (70%); r. tBuOK, (PhSeO) 20, THF
(90%); S. 4-DMAP, (CH 3CO)2 0, pyridine (50%); t. CH 3COONa, PCC, NaBH4, benzene, MeOH (8 1%); u. NaH, THF (68%);
10
v. MeOH, 32% HC (90%). SciFinder SciPlanner, Reaxys, ARChem and published literature 3-113 were referenced to conceive this
original synthetic scheme.
73
Chapter 5
RE-ENGINEERING TAXADIEN-5a-HYDROXYLASE
As was presented in Chapter 4, H16, or taxadien-5a-hydroxylase, is a supposedly a highly
promiscuous cytochrome P450 monooxygenase, and, as set out in that chapter, the reactivity of this
enzyme would have to be altered in order to minimize flux dissipation away from the eventual
product of interest, (1,2c,5a,7p,1Op)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax-4,11-diene.
Traditional
enzyme engineering methodologies such as directed evolution and site-directed mutagenesis each
have their merits. However, as P450 homologs share ~15% amino acid sequence identity and -30%
sequence similarity with one another
14,
the use of structure-guided approaches is preferred since the
insights gained from redesigning one active site could easily be extended to manipulating the reactive
proclivities of another P450, drastically reducing strain development times for the production of
secondary metabolites. This is particularly true for the semi-synthetic manufacturing scheme
proposed in Figure 4.9, which bears a preponderance of P450-catalyzed reactions in the
biosynthetic stage. Approaches that are more rapid and more economical than conventional
techniques such as X-ray crystallography and NMR, though, are required.
75
In this chapter, we build on the modeling methodology prescribed in Chapter 4 to formulate a
robust and generalizable methodology to assess the mechanistic basis for the promiscuity and lack of
selectivity of plant P450 enzymes. The methodology populates one's understanding of the
biocatalytic mechanism using well-established experimental protocols such as phylogeny-driven and
site-directed mutagenesis, and computational techniques such as homology modeling and substrate
docking. The insights that are gained from this exercise can be applied to chart the reactive
topography of the active site and identify the impact of individual structural features on stability,
activity and selectivity, potentially opening up possibilities to specifically tune the reactivity of plant
P 4 50s. Such tunable production of oxidized polycyclic compounds using microbial metabolic
engineering represents a significant step towards realizing more exhaustive prospection and
sustainable production of natural products and their analogues for drug discovery and development.
5. 1. Background about P450 monooxygenases
P450 monooxygenases constitute a large family of highly conserved, heme-thiolate enzymes that are
present in all forms of life7 2. They usually act in concert with a flavoprotein redox partner - a
cytochrome P450 reductase
-
that shuttles electrons and protons derived from NADPH to the
oxygenase's active site, whereat molecular oxygen is protonated and heterolytically cleaved to form
water and a reactive iron-oxygen species that then inserts the second oxygen atom into the substrate 7 3
(Figure 4.3). In mammals, mitochrondrial P450s play vital roles in the steroid biosynthetic pathway
and also orchestrate the breakdown of drugs and xenobiotics. In plants, microsomal P450s are an
76
integral component of the secondary metabolic apparatus that plays vital roles in communication,
defense, development and reproduction.
5. 2. Materials & methods
5. 2. 1. Strains & plasmids
Our research group had previously engineered E coli K12 MG1655 lrecA iendA to synthesize
taxadiene at titers of about 300 mg/L and 1 g/L in 2 mL batch cultures and 1 L fed-batch cultures,
respectively 59 . In this strain, additional copies of the rate-limiting steps of the non-mevalonate
pathway dxs, ispD, ispF and idi were integrated into the chromosome of the aforementioned E coli
strain using the FRT system under the control of the Trc promoter, and, the taxadiene biosynthetic
apparatus, which consists of ggps (GGPP synthase) and txs (taxadiene synthase) was expressed on a 5copy, pACYCDUET-1
vector under the control of a T 7 promoter. For production of the
hydroxylated taxanes, the taxadiene biosynthetic apparatus, along with the T 7 promoter that controls
its expression, was also integrated onto the chromosome of the E. coli strain and the operon was
localized to the arabinose operon with a kanamycin antibiotic marker. This strain was then electrotransformed to express the P450 apparatus consisting of P450 monooxygenase and its redox partner,
also on a 5-copy, pACYCDUET-1 vector (Figure 5.1). Plasmid construction and sub-cloning was
performed using chemically competent DH5a cells (purchased from Invitrogen). XL10-Gold
ultracompetent cells (purchased from Agilent) were used to select the P450 mutants.
77
t24A-CYP
Bovine 17a
rrnB antitermination signa
P(trc)
rrnB TT
lacO
P(lac) transcription start poin
6xHis tag
inker
aadAI
CPR
repA
lacl ACYCDuetUPI Primer #71178-3
pCL-Trc09-t24CYP-CPR
97
28bp
Figure 5.1. Map of plasmid expressing the P450 system
5. 2. 2. Culturing conditions
Biosynthesis of taxadien-5a-ol by the engineered E. coli strain was investigated in 2 mL batch
cultures. Pre-cultures propagated in 20 pg/mL chloramphenicol-supplemented LB medium were
used to inoculate the 2 mL production cultures at a starting OD of 0.1. The production medium
comprised of 5 g/L yeast extract, 20 ptg/mL chloramphenicol, 0.66 g/L 6-aminolevulinic acid, 13.3
g/L KH 2PO 4, 4 g/L (NH4) 2HPO 4, 1.7 g/L citric acid, 0.0084 g/L EDTA, 0.0025 g/L CoCl2,
0.015 g/L MnC 2, 0.0015 g/L CuCl 2 , 0.003 g/L H 3BO 3 , 0.0025 g/L Na 2 MoO 4 , 0.008 g/L
(CH 3COO) 2Zn, 0.06 g/L Fe(III) citrate, 0.0045 g/L thiamine, 1.3 g/L MgSO4, 10 g/L glycerol
and 0.024 g/L IPTG. The starting pH was set to 7.0 and 20 vol.% dodecane was added to the
production medium to scavenge the nascent taxane. The cultures were agitated at 200 rpm at 220C
and production was ceased after 5 days by extracting the product into 1 mL of a 80-20 vol.%
78
solution of hexane and diethyl ether. The organic phase was later separated and analyzed for its
taxane content using GC-MS.
5. 2. 3. GC-MS analysis of hydroxylated taxanes
1 mL of the batch cultures was combined with an equal volume of a 80-20 vol.% hexane-diethyl
ether solution and vortexed for 30-45 minutes. The vortexed mixture was then centrifuged at 3000
rcf at room temperature. The organic layer so-formed was then analyzed in a Varian Saturn 3800
GC 2000 MS system housing an Agilent HP-5ms column (30 m length, 0.25 mm ID, 0.25 pm film
thickness). Ultrapure helium was used as the carrier gas (flow rate of 1 mL/min). Each analysis lasted
30 minutes, wherein the oven temperature was maintained at 50C for 1 minute, ramped up at a
rate of 1OOC/min until it reach 2200C, and then held at 2200C for 12 minutes). The injector and
transfer line temperatures were maintained at 2000C and 250 oC respectively.
5. 2. 4. Protein engineering for optimal expression of taxadiene Sa-hydroxylase inE. coli
The amino-terminal transmembrane binding regions of the P450 monooxygenase (GenBank
AY289209) and it redox partner (GenBank AY571340) were eliminated in order to favor
expression in E. coli. 24 and 74 amino acids were excised from the P450 and reductase, respectively.
An 8-amino acid (MALLIAVF) leader sequence of a modified 1
5
bovine microsomal steroid 17a-
hydroxylase (GenBank NM_174304) was subsequently appended to the truncated P450 to
79
improve expression and stability. The chimeric P450 and the truncated reductase were then fused
together using a 5-amino acid (GSTGS) linker' 1 6 (Figure 5.2).
A. Biosynthetic pathway
G3P
co, NADP
1
- DOXP
NADPH
Pyruvate
chm
PP
ADP
MEP
2
IapE
1p0
Native pathway
(N
H20
I
MECPP
ATP
CTP
AIpC
CMP
CDP-MEP
CDP-ME
H201
HMBPP
2e-
Jap
Id
GGPP
Taxediene
3 DP
DP NADPH+ 02
ggpe
t0
2e
ApG
IepF
NADP'+ H20
IPP L*DMAPPj-.
cy
Taxadien-So.oi
pr
Heterologous pathway
Rate-iknimngsteps)
B. Strain construction
*
X--~.Ai,,&-* 1*
+
Non-mevalonste pathway is
expressed natively
Extra copies of rate-limiting
enzymes on the chromosome
0
X+
+
Texadiene biosynthetic
genes Integrated Into the
chromosome
Monooxygenamereductase fusion protein
expressed on a plasmid
C. Fusion protein construction
Leader
sequence
froma
binding region (TBR)
N
C
C
Monooxygensee
s
N
CC W
TBRexcision
C
r"-
N
C
Un
ethmpiMrcy
acustitoonsureopumal
electron shuttng
i7artydroxyles,
N
C
EnzymeUitilng
.
Figure 5.2. Overview of the strain and enzyme engineering protocols used in the study
5. 2. 5. Site-directed mutagenesis
The Quick Change II Site-Directed Mutagenesis kit was used to mutagenize taxadiene 5ahydroxylase in the P450-reductase complex. The mutants were constructed using a protocol
recommended by the kit manufacturer, Agilent, and the mutagenesis primers were also designed
using a software package hosted on the manufacturer's website.
(http://www.genomics.agilent.com/primerDesignProgram.jsp).
5. 3. Results
5. 3. 1. Taxadiene 5a-hydroxylase is a promiscuous enzyme
As predicted by the modeling methodology outlined in Chapter 4, instead of detecting a single peak
that corresponds to taxadien-5a-ol, 15 species are consistently identified in the gas chromatograms
of the samples upon expression of taxadiene 5a-hydroxylase (Figure 5.3), lending credence to the
hypothesis that P450s that catalyze transformations in the early stages of plant secondary metabolic
pathways are promiscuous. In the case of taxadiene 5a-hydroxylase, it appears that the enzyme is also
very poorly selective. The taxadiene (species B) content in the samples is negligible, suggesting that it
is completely converted to downstream products.
Intriguingly, taxadien-5a-ol (K) is not the major product (Figure 5.4). That distinction goes to the
cyclic ether, 5(12)-oxa-3(1 1)-cyclotaxane (OCT, I), a structural isomer of taxadien-5ac-ol. Mass
spectral analysis of the remaining 13 products confirms that they are eicosanoids, 7 of which are
monooxygenated (species A, C, G, H,
J, K, M and
P), 3 are dioxygenated (L, N and 0) and 3 are
unsubstituted (D, E and F). Species G is a structural isomer of OCT and it is evident that D, E and
F are non-taxanes. However, the low abundance and instability of these molecules has hitherto
precluded elucidation of their structures. It is uncertain how many of the remaining 9 species are
taxanes.
81
A. Gas chromatogran
-
Kilocounts
400-
A
B
C D
E F G
H
GGPS-TXS strain
J K
I
L
GGPS-TXS-CYP-CPR strain
N O
M
Kilocounts
P
2M0
350
300
1600
2000
250
200
1000
100
-
-.-l
0
10. I1
21.00
23.30
22.20
0
24.58
Retention tke (minutes)
B. Mass spectra
Spectracolor coding: black - non-hydroxylated ecosanode. red - monohydroxylated elcosanode, blue - dihydroxylated elcosanolds
100%
48.
140.6
249.5
34.5
mix
49
149.6
249.6
349.5
M11
100%
100%
149.5
249.6
349.6
m/x
406
240.6
30.6
40.
Species I
149.5
249.5
340.6
40.5
20.6
249.1
340.5
49.6
249.6
349.5
nVZ
00
1 %r
140.5
240.6
34.5
48.
143.6
249.5
348.5
nVZ
Species H
100%
149.6
24.5
3495
48.6
Species K
100%
149.6
SpeclesD
100%
149.6
40.6
34.6
m/x
Species 0
SpeciesJ
100%
149.5
100%
100%
140.5
100%
Species C
Species F
Species E
40.
49.1
Species B: Taxedlen.e
SpeciesA
48.1
100%
100%
149.6
248.5
34
Species L
100%
100%
100%
4.
148.5
240.
SpeclesM
3465
ms
4.
140.5
24.6
SpeciesN
340.5
4.
14.
249.6
Speces 0
343.5
msx
4.
148.6
24.
34A
SpeclesP
Figure 5.3. Promiscuity & poor selectivity of taxadiene 5a-hydroxylase: (A) Typical gas chromatograms of the
organic extracts from fermentation broths populated by strains expressing only the taxadiene synthesis module (black)
and the taxadiene-P450 modules (red) reveal the presence of as many as 16 eicosanoids (labeled A-P), all of which elute
from the column after approximately 20 minutes of the analysis has been completed. The katharometric signal strengths
of the species (y-axis) have been expressed in kilocounts and the retention times have been reported in minutes (x-axis).
(B) Fragments with the highest m/z ratio were normalized to an intensity of 100% in the mass spectral plots of the 16
eicosanoids (spectral plots in black - non-hydroxylated C 2os, red - monooxygenated C 20s, and blue - dioxygenated C 20s).
82
-2500
400
350
2000
300
250
1500
1000
150H
100
500
50
0
19.81
0
21.00
22.20
23.39
24.58
Retention time (minutes)
Figure 5.4. Characterized structures of some of the products: OCT, a cyclic ether, is the major product and elutes a
little after 21 minutes in the 30-minute GC-MS run. Taxadiene elutes after roughly 20 minutes and the less-volatile
taxadien-5c-ol is detected close to 21.5 minutes. The katharometric signal strengths of the species (y-axis) have been
expressed in kilocounts and the retention times have been reported in minutes (x-axis).
5. 3. 2. Temporal profile of taxadiene proto-oxidation
The monooxygenase catalytic cycle proceeds under tight thermodynamic control, wherein molecular
oxygen is reductively cleaved to form water and the oxidized substrate. In some instances, the
catalytic cycle is shunted and produces hydrogen peroxide instead of water. In situ substrate
dihydroxylation, therefore, is neither mechanistically nor thermodynamically feasible for P450
monooxygenases such as taxadiene 5a-hydroxylase.
The re-diffusion of a nascent monooxygenated product into the P450 active site, followed by its
participation in a second oxidation cycle appears to be the only likely explanation for formation of
the dioxygenated eicosanoids. Moreover, not only is this interpretation in agreement with the
83
hypothesis regarding P450 promiscuity, but adding polymeric bead-conjugated silyl-based scavengers
similar to the packing material of reversed-phase HPLC columns to the culture medium greatly
reduces eicosanoid production, more so the production of the dioxygenated compounds'
17.
Accordingly, daily production of the 16 eicosanoids was tracked (Figure 5.5) in order to derive
qualitative temporal relationships between the species. The absence of taxadiene on all days of the
production run points to high activity of the P450. Of the remaining 15 species, only G, H, OCT,
taxadien-5t-ol, M and N are detected after 1 day of culturing. The remaining eicosanoids
accumulate only after 2 days. These trends possibly suggest that G, H, OCT, taxadien-5a-ol and M
are oxidized derivatives of taxadiene. The concentrations of D and C exhibit similar trends to that of
H. Species A, C, P and
J, therefore,
appear to be oxidized derivatives of D, while C could be the
source of L and 0.
Interestingly, assessing the rates of change in the concentration of all species clusters the species into
3 groups - one comprising of G, OCT, J, taxadien-5a-ol, L, P and 0; another which includes A, E,
F, M and N; and a third group consisting of C, D and H (Figure 5.5.). The rate of change in the
concentrations of a species is simply the sum of the rate at which it is formed, the rate at which it is
consumed by a downstream metabolic reaction, the rate at which it inhibits host growth or the
activity of the enzyme that synthesizes it, and its volatilization rate. The similarity in the variation in
rates of change in the concentrations between species from one group but the difference in the
variation in rates between species from different groups provides a qualitative snapshot of their
84
propensities to participate in another P450 catalytic cycle, as well as their volatilities and inhibitory
effects.
Daily production titers for all eicosanoids
mg/L
mg/L
12.00
6.00
2.50
mg/L
2.00-
1.25
100
0,00
0.00
0.00
0
2
1
3
4
5 Days
0
2
1
3
4
0
5 Days
mgIL
2.50
mgIL
60.00 -
1.75
1.25
30.00 -
0.00
0.00
1
2
3
4
0.00
0
5Days
1
2
3
4
0
5Days
mg/L
25.00
mg/L
80.00
4.00
12.50
40.00
1
2
4
3
5 Days
0
1
2
3
4
5 Days
0
Species I
Species H
5 Days
2
3
4
5Days
1
2
3
4
5 Days
Species J
mg/L
16.00-
mg/L
7.50
mg/L
30.00
4
0.00
0.00
0.00
3
Species G
mg/L
8.00
0
'
1
Species F
Species E
2
Species D
mg/L
3.50
0
1
Species C
SpeciesA
5.00
8.00-
15.00
2.50
0.00
0.00
0.00
0
1
2
3
4
5 Days
0
1
2
3
4
Species L
Species K
mg/L
mg/L
750
20.00
3.75
1
2
3
4
Species N
5Days
1
2
3
4
5 Days
Species M
mg/L
20.00
10.00
0.00
0.00
0.00
0
0
5 Days
0
1
2
3
Species 0
4
SDays
0
1
2
3
4
5 Days
Species P
Figure 5.5. Daily variation in eicosanoid production titers: Concentrations of all 16 eicosanoids (in mg/L) were
measured on each day of the 5-day runs. The concentrations were calculated from the katharometric signal strengths
using a calibration curve for taxadiene. Error bars represent standard deviations (n = 3). Of the 16 species, taxadiene
(species B) is undetectable on all 5 days and only G, H, J, K, M and N can be detected after 24 hours of the run have
been completed.
85
-.- G -+-1 -+-J -+-K -+-L -+-P - -o
-*-A -wE .oF +M -N
100
k
-C
-eD oH
15
s
150
10
-
50
5
30
(nM/day)
(nWday) 20
(nday) 0
-10
-20
-100
0
1
2
3
Day
4
5
-10
0
1
2
3
Day
4
5
0
1
3
2
4
5
Day
Figure 5.6. Assessment of the daily variation in the rate of change in the concentrations of the 16 eicosanoids:
Daily variations in the forward derivative of the concentrations of the species with respect to time (in mM/day) have
been plotted for 15 eicosanoid species - A, C-P. Trends suggest that the compounds fall into three groups. G, OCT (I),
J, taxadien-5a-ol (K), L, P and 0 comprise one group. The second group includes A, E, F, M and N. C, D and H form
a third group. Species from two groups do not appear to react further, but that members of the first group are more
volatile and/or exert a greater inhibitory effect than those of the latter. The variation in the rate of the change of C and
H suggests that they are likeliest sources of the dioxygenated molecules.
The variation in their rates of change suggests that species from the first two groups do not appear to
react further, but. that members of the first group are more volatile and/or exert a greater inhibitory
effect than those of the latter. The variation in the rate of the change of D suggests that it reacts with
the P450 to yield several oxygenated non-taxanes, whereas C and H appears to be the likeliest
sources of the 3 dioxygenated molecules (Figure 5.7). The formation of D, E and F is particularly
intriguing. We speculate that these molecules are formed in response to the concerted effect of
inhibition by the oxygenated taxanes and a high flux through the prenyl pyrophosphate biosynthetic
pathway.
86
H
GGPP -
Taxadiene(B)
-N
I
K
+E
F
-- M
-+A
-C
-D
-
-
L& O
+P
Figure 5.7. Speculative reaction scheme
5. 3. 3. The utility of traditional mutagenic practices to improve P450 reactivity
5. 3. 3. 1. Phylogeny-guided mutagenesis of taxadiene 5a-hydroxylase
As mentioned in Section 4.1, the Taxus cDNA library from which the sequence of taxadiene 5ahydroxylase was obtained contains an additional 15 P450 monooxygenases. It was reasoned that
replacing amino acid residues within the active site of taxadiene 5a-hydroxylase with similarly
positioned residues in its paralogs could perhaps shed light on the reactive contributions of each
residue. Consequently, all amino acids located at the periphery of the active site cavity predicted
from the modeling methodology were replaced using a multiple sequence alignment of all P450
sequences in the Taxus cDNA library that was then utilized to identify substitutions for the selected
amino acids (Figure 5.8).
87
Bits
4
2
1c2 2I3
2
1
L52
61
1
N1
Figure 5.8. Targets for phylogeny-guided mutagenesis: Amino acid residues that fall within 3.5
A of the
periphery of
the active site cavity of taxadiene 5a-hydroxylase are compared to its homologs in this multiple sequence alignment.
Amino acids located at the periphery of the active site are labeled with a red circle. Numbers below the red circles denote
the number of mutations investigated at that site. The multiple sequence alignment was created in WebLogo, a webbased service. The use of bits instead of probabilities is a feature of the program. 3.5 bits corresponds to a probability of
1, suggesting complete conservation across all the homologs.
Many phylogeny-derived mutants exhibit vastly improved activities compared to the parental P450
(Figure
5.9).
The activity is calculated by summing the molarities of all oxygenated eicosanoids
produced by the enzyme. Selectivity, on the other hand, is simply the ratio between the molarity of
taxadien-5ca-ol and the activity of the enzyme.
88
nM of al
nM of all hydroxylated elcosanolds
droxylte elcosanoda
150% -
E
0%
0
-150% Mutation: 5124E Q128A
0128S
K131L K131E
S301F
T14OG T140A
T140V R141K
R1411 L225F
L225V
V374A
V374L T377S
T377N
T377Y T377C
S302A S302V S302L
150%
r75%-
0%
0%
-150%
Mutation: S302M S302G S302F L303M D300E
V3741
T3771
L483V
L483F
P434A
Figure 5.9. Activity and selectivity comparisons between the mutant and parental P450: Percent changes in the
selectivities (gray) and activities (black) of the 32 mutants are compared to those of the parental enzyme. Selectivity is
defined as the ratio of the concentration of taxadien-5a-ol to all hydroxylated eicosanoids. Activity is defined as the sum
of the concentrations of all hydroxylated eicosanoids. Concentrations have been converted to nM. Many phylogenyderived mutants exhibit vastly improved activities and selectivities compared to the parental P450.
Amino acid mutations that improve enzyme selectivity can be distributed into 3 categories. If one
assumes that product formation depends on the regiospecificity of the proton that is abstracted by
the oxyferyl species of the heme group in the P450 active site, competing proton abstractions could
explain the distribution of products formed by a single catalytic cycle. Consequently, the first class
amino acid mutations include those that improve product selectivity by directly affecting the
probability of regiospecific proton abstraction viz. improvements in 'reaction selectivity'. Product
89
selectivity can also be improved by disfavoring the association between the enzyme and its other
substrates, viz. controlling the promiscuity of the enzyme. Mutations that achieve this result fall into
the second category - improvements in 'overall selectivity'. The third group includes amino acid
mutations that affect both, the probability of regiospecific proton abstraction and enzyme
promiscuity.
E
SA
E
PAI +PA2+PA
S -
E'PB1+
Pa2
Desired product
Activity = [PA1] + [Pa] + [PA] + [Pml + [Pe21
Overall selectivity =
Reaction selectivity =
[PA1
[PA11 + [PA2l + [PA! + [PMl + IP621
[Al]
[PA1l
+
[Pu]
+
[Pu]
Figure 5.10. Differentiating the two types of selectivity improvements
Apparently, nearly all phylogeny-guided mutations investigated herein fall in the second category
(Figures 5.10 and 5.11). The product distribution of T377S is particularly surprising as it appears
to favor oxygenation of the non-taxane, C, to produce the dioxygenated eicosanoid, 0.
5. 3. 3. 2. Mutations to taxadiene 5u-hydroxylase based on structural inferences
Of the amino acid mutations exhibiting improvements in activity and/or selectivity, K131E and
90
D309E were investigated in further detail. Not only are these residues highly conserved, but K131
and D309 form a dyad that extends laterally across the roof of the active site, parallel to the heme
moiety below. It was hypothesized that modifications to these residues could potentially affect the
orientation in which taxadiene and the other unsubstituted eicosanoids bind within the active site
(Figure 5.12).
*A
AC
eG
mH
x1
+J
AL
*K
oM
oN
-O
xP
8
7
-
A
6
A
A
.C
U'
Vl
S
2
A
A
A
+
+.
A,
4.
4.
I
*
2
1
U
0
0
Mutation: S124E C1128A Q1283
A
K131L K131E T140G T140A T140V
R141K
R1411 L225F
I
a
L225V
301F S302A
S302V S302L
10
9
a
7
8
A
2
1
0
Mutation:
S302M
3302G 3302F L303M D30BE V374A
V3741 V374L T377S
T377N
T3771
T377Y T377C
L483V L483F
P434A
Figure 5.11. Grouping the selectivity improvements: The fold change of the concentration of a particular eicosanoid
is simply the ratio of the concentration of the species produced by the mutant to that produced by the parental enzyme.
Significant variations between the fold changes of products of the same catalytic cycle is indicative of direct
manipulations to the catalytic cycle viz. improvements in reaction selectivities. D309 is the only mutation that exhibits
quantifiable differences in the selectivity towards taxadien-5a-ol (K) that can be attributed to possible differences in the
91
probability of abstracting a specific proton (species G, I, K, which potentially are products of the same catalytic cycle, are
colored red).
Accordingly, a series of mutants combining site-saturation mutagenesis at both locations of the dyad
and other phylogeny-guided mutants were constructed and evaluated for their influence on the
activity and selectivity of taxadiene 5ct-hydroxylase (Figure 5.13). The number of mutations varied
from a single-site mutation to as many as 6 simultaneous mutations. The mutation frequency
seemed to have a deleterious effect on enzyme function. The production of oxygenated compounds
was all but abolished for enzymes bearing 4 mutations in their amino acid sequence.
Figure 5.12. The lysine-aspartate dyad: The high
D309V
degree
of conservation
functional
groups
and
of
their
these
residues,
relative
sizes
their
were
instrumental in their selection as mutation targets.
A double-mutant combining substitutions to K131 and D309 with arginine exhibited the greatest
improvement in selectivity. However, as before, the increase in selectivity is attributable to
disfavoring the formation of the mono- and dioxygenated eicosanoids (Figure 5.14). It is evident
that phylogeny-guided
and site-directed mutageneses do not alter any proton abstraction
92
probabilities in the catalytic cycle that yields, among others, taxadien-5ct-ol. A double-mutant
combining substitutions to K131 and D309 with arginine exhibited the greatest improvement in
selectivity.
0 K131-D309 mitiagenesis series
0 Phylogeny-guided mutagenesis
% change inactivity over
150% - parental enzyme
3
0
100% -
-m
C
50f-
0
1
0
m
LI
%change inoverall
0
selectivity over
U
U
parental enzyme
Eman
I
V
U
-_U
IV
0 V0
-50%
-100%
0
0
0
0o
*} 4substitutions
M
0
*
50%
b*"0
Eb
3
-100%
-
100%
0
0
odp 10
0
0 K131R + 0309R
Figure 5.13. Influence of mutations to the dyad and amino acids at the periphery of the active site: Selectivity is
defined as the ratio of the concentration of taxadien-5a-ol to all hydroxylated eicosanoids. Activity is defined as the sum
of the concentrations of all hydroxylated eicosanoids. Concentrations have been converted to nM. Percent changes in the
selectivities (gray) and activities (black) of the 32 mutants are compared to those of the parental enzyme. The phylogenyguided mutants were single-site mutants. Single- and multi-site mutations were investigated in the K131-D309 study.
Increasing the number of mutations appears to have a deleterious effect on enzyme function. Enzyme constructs bearing
>4 mutations formed very little product.
However, as before, the increase in selectivity is attributable to disfavoring the formation of the
mono- and dioxygenated eicosanoids (Figure 5.14). It is evident that phylogeny-guided and sitedirected mutageneses do not alter any proton abstraction probabilities in the catalytic cycle that
yields, among others, taxadien-5a-ol.
*A
AC
*G
EH
+J
Xi
OK
AL
OM
ON
-O
XP
4.5
4
3.5
3
A
2.5
.o
I
2
+
1.5
0.5
0
a
M
I
V
U
4
A
A
S
U
a
A
N
D
U
F
D309 srirs
Y
A
B
I
a
K131 series
a
M
A
A
I
A
F
V
Y
K131R+ D306 series
R
F
W
N
E
K131R + P484A + D309 series
Figure 5.14. Improving reaction selectivity is a non-trivial challenge: Like in Figure 5.11, the fold change in the
concentration of particular species simply refers to the ratio of its concentration as produced by a given mutant to the
concentration obtained by expressing the parental enzyme. None of the mutants constructed in the study thus far
improve product selectivity by directly affecting the probability of regiospecific proton abstraction. Instead, selectivity
improvements have been achieved by disfavoring the association between the P450 and its other substrates.
5. 3. 4. Computational assessment of the taxadiene 5a-hydroxylase catalytic cycle
The preponderance of amino acid mutations that improve product selectivity by destabilizing
binding of competing substrates over mutations that alter the orientation of taxadiene within the
94
active site germinated computational investigations to understand the mechanistic basis for the
enzyme's peculiar product distribution. The computational methodology discussed in Section 4. 5.
2 was utilized for this purpose.
Oxidation of the substrate typically proceeds via the oxygen rebound mechanism (Figure 5.15)'18.
Proton abstraction by the oxygen atom of oxyferyl heme forms a radical intermediate and a hydroxyiron species, the latter which then dissociates via a rebound mechanism to form the carbon-oxygen
bond. The heme group simultaneously returns to its pre-reaction, activated state, and, radical
stabilization, where possible, determines the abundance of the oxygenated products.
R-
Re
Re
H
H
0
-
S-Cys
0
0
F03+
F03+
S-Cys
S-Cys
e-
S-Cys
Figure 5.15. The oxygen rebound mechanism: The oxygen rebound mechanism involves the formation of a hydroxyiron species, which then dissociates to insert oxygen into the substrate. Bond-breaking and bond-forming electron
migrations are depicted in red and blue, respectively.
Analysis of the average position of the two likeliest poses of taxadiene in the oxyferyl state of
taxadiene 5a-hydroxylase suggests that proton abstractions occur at as many as 3 positions on the
taxane scaffold (Figure 5.16). Abstraction of any one of the three protons attached at the 20position produces allyl-stabilized radical species at the 20- and 5-positions (Figure 5.17.), which
95
then yield taxadien-20-ol (4) and taxadien-5a-ol (5), respectively. Taxadien-5a-ol is more abundant
than taxadien-20-ol as the 5-position secondary carbon radical is the more stable of the two allylstabilized radical intermediates.
Figure
A
13"
~F
5.16.
abstractions
Overview of the competing
from
taxadiene:
The
proton
energy-averaged
structure of the two likeliest orientations of taxadiene in the
active site (poses A and B in Figure 4.6.) suggests that as
many as 3 proton abstractions might be occurring. In the
B
order of preference, these are at the 3-, 13a-, and 5aEnergy-averapd pose
positions.
In comparison, abstraction of the sole proton that is attached at the 3-position produces a tertiary
carbon radical. However, as insertion of oxygen at this position appears to be sterically hindered, the
lone electron shifts to the tertiary carbon at the 12-position. This transfer does not mitigate the steric
hindrance to the oxygen rebound mechanism either. Instead, we speculate that the close proximity of
the alkenyl moiety to the hydroxy-iron species and the tertiary carbon radical facilitates a concerted
sequence of electron exchanges that results in the addition of the oxygen atom to the carbons at the
5- and 12-positions to form OCT (8). This explanation for ether formation is different from that of
a previous study on the OCT biosynthesis" 9 . However, oxidation of taxadiene in the reaction
mechanism detailed therein does not proceed via the oxygen rebound mechanism and neither can it
explain formation of, among others, taxadien-5a-ol and OCT in the same active site.
96
Y
3
12
11
c
136
b
6
3
a
20
1 Taxadlene
) D?
6
9I
I I
I
9
10
H
4 Taxad on)-20-ol
5 Taxadien-5a-ol
Fe
8 OCT
Ether formation
mechanism
9
11
Ether formation
mechanism
I
14o
12 Taxadien-13ool
13
Figure 5.17. Hypothesized reaction mechanism: In silico models of the binding of taxadiene in the active site of taxadiene 5ahydroxylase predict competition between the abstraction of protons from the (a) 20-, (b) 3- and (c) 13a-positions. Radical species
formed via proton abstractions from the 20- and 13a-positions are allyl-stabilized. Insertion of oxygen to the 3- and 12-positions
is sterically inhibited. Formation of OCT following proton abstraction from the 3-position is hypothesized to proceed via the
constrained Markovnikov-like addition of oxygen to the 5- and 12-positions.
Lastly, proton abstraction from the carbon at the 13a-position produces taxadiene-13a-ol (12) and
yet
another
cyclic
ether
species,
5(13)-oxa-3(11)-cyclotaxane
(13).
Preliminary
NMR
characterization of the oxygenated eicosanoids appears to support this hypothesis and has also
identified species G to be 5(13)-oxa-3(11)-cyclotaxane
0
.
5. 3. 5. Computational assessment of taxadiene 5a-hydroxylase mutants
We applied our computational methodology to investigate the higher incidence of phylogeny-guided
and dyad mutants that improve product selectivity by destabilizing binding of competing substrates
over mutants that alter the reaction trajectory of taxadiene oxidation (Figure 5.18).
Of all the mutants that were constructed previously, K131R, S302A and V374L exhibited deducible
differences in the selectivity towards 5(13)-oxa-3(1 1)-cyclotaxane (G), H, OCT (I), taxadien-5c-ol
(K) and M. Not only are the orientations of taxadiene in the active sites of the parental P450 enzyme
and its 3 mutants visibly different, but the proton abstraction lengths predicted by the model
correlate well with the differences observed in the relative proportion of the monooxygenated
taxadiene derivatives produced by each enzyme. To a degree of approximation, the abstraction
lengths and observed product distributions correlate quite well, underscoring the previous suspicion
of the limitations of traditional mutagenic (or 'structure-blind') approaches to truly identify mutants
that alter the reaction selectivity of the enzyme.
98
aH
0.5
0e G
0.4
0.3
01
0
0
0
0
0
0.2
AM
A
A
0.10~
Parental
V374L
K131R
S302A
Distance of proton to oxyfei oxygen (A)
4.6
4.6
5.6
3-position
13a-position
20-position
4.7
5.0
5.6
4.5
4.5
5.6
4.8
5.3
5.4
Difference In bindingaffinities of taxadiene to activated and oxyferyl states of the enzyme (kcallmol)
2
% change In the production
of taxadien-6a-ol compared to parental enzyme
% change In the total production of oxygenated
eIcosanolds to compared to parental enzyme
0.85
0.8
0.6
24.4
-23.4
-53.5
-11.6
13.5
1.1
Figure 5.18. Comparing mutation efficacy using the computational methodology: The computational methodology
successfully predicts variations in product selectivities of the 3 mutants that were investigated. Electron densities of the
average structures of the two likeliest poses predicted by the model (represented as dot models) exhibit visible differences
between the 4 enzymes.
5. 4. Conclusions & perspectives
5. 4. 1. Comparing the methodologies
Phylogeny-guided
and site-directed mutageneses are efficient methodologies to improve the
throughput of the enzyme. However, the selectivity improvements that are achieved through the use
of these techniques often result from destabilizing binding of competing substrates rather than
99
improving the selectivity of the catalytic mechanism. In the case of promiscuous and notoriously
non-selective nodal enzymes such as taxadiene 5a-hydroxylase, the benefit of being able to rapidly
probe enzyme reactivity using phylogeny-guided mutagenesis is soon lost over the inability of the
methodology to identify desirable amino acid substitutions without having to construct an
exhaustive collective of enzyme mutants. In comparison, the computational methodology we have
prescribed in Chapter 4 can conclusively identify the mechanistic basis for non-selectivity. It is
robust, generalizable and manifold more rapid than other structure-guided enzyme engineering
approaches. Not only can the insights that it provides be applied to chart the reactive topography of
the active site and identify the impact of individual structural features on stability, activity and
selectivity, one can even possibly extend the methodology to prescribe mutational targets within the
active site. For this to occur, though, NMR characterization of all interacting substrates and their
oxidation products is required. The algorithms utilized in the methodology can then multiplicatively
compare the binding affinities and orientations of all characterized substrates within the active site to
guide the construction of mutants that favor only specific orientations of desired substrates (Figure
5. 19).
5. 4. 2. The possibility of multiple substrates ina single active site
Formulation of the computational methodology assumes that only a single taxadiene substrate docks
into the active site of taxadiene 5a-hydroxylase. While this assumption is well-grounded in literature,
100
questions still remain as this enzyme is somewhat of a novelty compared to some of the P450
monooxygenases studied in literature.
EE
SA -
P1 + PA2 + PAa
so -
PS1 + PE2
Desired product
Step 1: NMR characterization of PA1,
L
P PB
1 & PB2
Step 2: Dock PA1, PA2, PAa, P9 i &PB2 inE sites
Step 3: Informed mutation of E
Step 4: Dock PA1, PA2, PAa, P9 1 & P82 inbinding sites of mutant
Step 5: Assessment of pose coordinates & binding affinities
Figure 5.19. A generalized algorithm for in silico enzyme engineering
The diffusion of two molar equivalents of taxadiene into the active site of the enzyme, if it were to
occur, would not be constrained by space as the active site of taxadiene 5a-hydroxylase is roughly
474
A' (Table 4.1.).
Evaluation of this hypothesis necessitates isolation and purification of taxadiene
5ax-hydroxylase, followed by an in vitro assessment of the spectral shift that is induced by substrate
binding. Comparing the spectral shift to P450 standards known to bind to single or multiple
substrate molecules simultaneously would provide the much-needed constraint that is required to
include or eliminate the possibility of multiple substrates binding simultaneously within the active
site. As the computational methodology can account for the simultaneous binding of several
substrates within the active site, this constraint could improve the predictions of the model.
101
5. 4. 3. Directed evolution versus in silicomodeling
Given that most biological experimentation still proceeds with a 'black box' mentality and each
assumption that one makes in the formulation of a utilizable model diminishes the closeness of fit of
the model with the phenomenon it describes, there is no argument that directed evolution is the
superior of the two approaches. However, the absence of a screen that can efficiently couple the
selectivity of the enzyme to gene expression or platforms such as phage-display or the yeast twocomponent bait-prey system preclude the use of directed evolution 2 1 . The most realistic
demonstration of directed evolution of a selective taxane P450 hydroxylase would utilize phagedisplay to assess the reactivity of the enzyme in vitro (and possibly high-throughput) using taxanebinding antibodies. Paclitaxel-binding antibodies are presently available commercially and generating
antibodies capable of binding to the hydroxylated products of taxadiene 5a-hydroxylase is not a farfetched goal.
5. 4. 4. Implications for metabolic engineering
Consider a metabolic pathway comprising of 3 enzymes - A, B and C. Each enzyme catalyzes a
substitution at a unique position on a chemical scaffold. If only enzymes A and C of the pathway are
expressed in the microbial host to produce a unique, di-substituted product, it is vital that enzyme C
be suitably re-engineered in order to ensure that it exhibits the same specificity and activity towards
the mono-substituted substrate as it does towards the di-substituted substrate in the unabridged
102
pathway, failing which, conversion and throughput of the pathway will be low. If the enzymes are
promiscuous, as is the case for the early taxane pathway, many more copies of a suitably reengineered enzyme A would have to be expressed relative to the re-engineered enzyme C to minimize
flux dissipation away from the pathway of interest. One can also maximize conversion to the product
of interest by temporally inducing the expression of the respective enzymes viz. induce expression of
enzyme A to produce the preferred intermediate and only induce the expression of C after sufficient
time has been allowed for enzyme A to act. The permutations are numerous and serve to underscore
the mutual dependence of enzyme engineering and metabolic engineering for the heterologous
production of secondary plant metabolites and molecules that are variations of a new chemical
theme.
103
Chapter 6
TECHNOECONOMIC ANALYSIS OF MICROBIAL PACLITAXEL
One of the principal criticisms leveled at microbial metabolic engineering is the seeming inability of
simple microbial hosts such as E. coli to support the elaborate enzymatic machinery that is required
to synthesize molecules as complex as paclitaxel. However, as demonstrated in Chapter 5, E. coli is
more than able to accommodate the expression of plant secondary metabolic enzymes. An E coli
strain expressing a mutant of the cytochrome P450 monooxygenase-reductase complex described
earlier produced nearly 0.45 g/L of mono- and dioxygenated taxanes in 2 mL batch cultures, which,
as shall be demonstrated herein, could be sufficient to ensure cost-competitive production of
paclitaxel using the microorganism.
Instead, the biggest hurdle in realizing the production of (1,2ct,5ct,7p,10p)-5-acetyloxy-1,2,7,10tetrahydroxy-tax-4, 11 -diene, which, in Chapter 4, was identified as a promising 'gateway' molecule
for synthesis of paclitaxel and its taxane analogues, is the notorious promiscuity and poor selectivity
of the pathway enzymes. Remedying these traits to minimize flux dissipation away from the
molecule of interest is arguably the biggest challenge facing microbial metabolic engineering and
protein engineering.
105
Presently, we do not possess a methodology that permits directed evolution of plant secondary
metabolic enzymes and rational techniques for enzyme engineering such as the methodology
presented in Chapter 5 are far from maturity. Nevertheless, using a technoeconomic comparison
between the existing plant-based semi-synthetic process and the one that has been proposed in
Figure 4.9, we identify target titers and productivities for a strain expression an optimal
combination of P450 monooxygenases and acyltransferases. The titers calculated herein serve as
benchmarks for future protein and metabolic engineering activities.
6. 1. Background & assumptions
Paclitaxel is currently produced via a 4-step synthetic scheme from 10-DAB that has been extracted
from plant cell cultures of T. baccata (Figure 6.1). The 10-DAB yield is roughly 160 mg/g DCW12 '
123
and the paclitaxel yield from 10-DAB is 44.7%11, which translates to an overall semi-synthetic
yield (assuming a plant water content of 80 wt.%) of 2.25 wt.%. This figure represents a >200-fold
increase over the paclitaxel content in the bark needles of T. baccata, underscoring the importance of
semi-synthesis in the production of therapeutic natural products that are otherwise too miniscule to
be directly extracted from their native hosts.
In comparison, production of (1,2a,5a,7p,1OP)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax-4,11-diene
(for simplicity, hereinafter this molecule will be abbreviated as ATHTAX) by the previously
described taxadiene-producing E coli strain necessitates the expression of 6 additional enzymes (5
106
cytochrome P450 monooxygenase-reductase complexes and a single acetyltransferase), all of whose
identities are already established' 4 . Significantly, the use of chemically-inducible promoters for the
expression of the taxane biosynthetic apparatus couples production with growth, thereby enabling
continuous chemical synthesis through continuous cultivation.
0
AcO
0
0
Ib
NH
NH2
NH
__
HO
0
ACO
d
NH
0
OTES
0
ef
N.
O~
HO
HO
0OH
QOTES
H
~~OL~oHo
H
0
OOTES
.H
_H
HO
H-
0
OV
O
10-Deacetylbaccatin III
0
OTES
+OTES
0
He/7
,
+
OH
H
-
OH 001O
0
OTES
H0
0>~t
e
N
H
0
OTES
H
H
N
H
INHO
Paclitaxel
Figure 6.1. Semi-synthesis of paclitaxel from plant-derived 10-deacetylbaccatin III (10-DAB): The yield of
paclitaxel from 10-DAB is 44.7%. Key: a.CH 2C 2 (yield 99%); b. Et 3N, 2-acetoxyacetyl chloride, CH 2 Cl 2 (65%); c.
(NH 4 )2 Ce(NO 3 )6 , CH 3CN (80%); d. MeOH, Na 2 CO 3 (82%); e. TESCl, pyridine (89%); f 4-DMAP, Et 3N, benzoyl
chloride, CH 2Cl 2 (82%); g. TESC, pyridine (85%); h. CH 3 COCl, pyridine (85%); i. NaH, THF (68%);j. 32% HC1,
MeOH (90%)
107
The global paclitaxel supply chain is populated by little over a dozen API producers who supply the
bulk drug to numerous formulators such as BMS or Mylan that then produce finished doses of the
drug for distribution to various consumer channels. The API manufacturers either produce 10-DAB
internally or source the material from external providers and typically produce 50-100 kg of
paclitaxel annually 2 . Accordingly, in the economic assessment that follows, the annual production
volume for the manufacturing facility is also assumed to be 100 kg. As bulk chemical prices for many
of the reagents, catalysts and solvents that are required by the two semi-synthetic schemes are not
readily available, SciFinder Scholar was queried to identify the lowest price for all raw materials
(Tables 6.1 & 6.2). The price of ATHTAX was estimated by comparing the economics of plant
and microbial cultivation. The optical density of the taxadiene producing E coli cultures reaches a
peak of -40 in 5-day, 1 L fed-batch cultures. Assuming an equivalency between an optical density of
1.0 and a dry biomass titer of 0.3 g/L, the overall yield of taxadiene on a dry cell weight basis is
estimated to be 0.0925 g/g. A 30% reduction in pathway flux was factored for every additional
enzyme that is expressed within the pathway, which translates to a dry basis yield of 0.016 g/g for
ATHTAX. The doubling time of the engineered E. coli strains is estimated to be about 12 hours,
which is nearly 12-fold lower than the doubling time of T. baccata suspension cultures.
6. 2. Comparing the economics of the two competing approaches
Despite having a dry basis yield that is 10 times lower than that of 10-DAB, microbial production of
ATHTAX is nearly 275-fold more productive than the plant cultures that produce 10-DAB.
108
Another important distinction between the two cultures is that the E. coli strains utilize glycerol as
the principal carbon source whereas the plant cells are cultivated using a combination of sucrose,
glucose and fructose as carbohydrate feedstocks. Although consumption of glycerol by the E. coli
cultures is estimated to be roughly 4 times greater than the mono- and disaccharide demand by the
suspension cultures of T. baccata, the media formulation costs for plant cell cultivation are greater
owing to the higher unit price of the sugars as well as higher nutrition supplementation costs.
Consequently, the productivity of the microbial cultures is conservatively estimated to be 300-fold
greater than that of the plant suspension cultures, which amounts to each kg of ATHTAX costing
about $36,600. The raw material costs for synthesizing paclitaxel from 10-DAB are, therefore, a
little over 6% greater than the competing semi-synthetic process that commences from ATHTAX.
Table 6.1. Raw material costs for reaction scheme in Figure 6.1
Total cost
Unit cost
Chemical Amount
Benzaldehyde
66 kg
$12/kg
$818
Anisidine
77 kg
$32/kg
$2423
10548 L
$8/L
$84553
Triethylamine
265 kg
$15/kg
$3975
2-Acetoxyacetylchloride
126 kg
$1950/kg
$244725
Cerium ammonium nitrate
657 kg
$95/kg
$62429
Acetonitrile
3560 L
$16/L
$56960
13455 L
$7/L
$89747
3 kg
$2/kg
$7
832 kg
$440/kg
$366282
19489 L
$30/L
$578835
3 kg
$88/kg
$251
44 kg
$13/kg
Dichloromethane
Methanol
Sodium carbonate
Triethylsilylchloride
Pyridine
4-Dimethylaminopyridine
Benzoylchloride
$578
6
143 kg
$10.98 x 10 /kg
$1.56 x 109
Acetyl chloride
87 kg
$47/kg
$4064
Sodium hydride
13 kg
$101/kg
$1266
Tetrahydrofuran
2683 L
$10/kg
$27903
50 L
$1/L
$73
10-Deacetylbaccatin III
32% Hydrochloric acid
Total raw material costs = $15.7 x
106/kg
109
The capital costs of the two processes can be related using the six-tenths power law as follows
6
V
Cp
:
(6.1)
M
CM
12 6
Cp and Cm are the capital costs for the plant (P) and microbial (M) processes, respectively. VP and
VM are the total material flows associated with the two processes.
Table 6.2. Raw material costs for reaction scheme in Figure 4.9.
Chemical Amount
Unit cost
Total cost
Benzaldehyde
66 kg
$12/kg
$815
76 kg
1020451 L
$32/kg
$2416
$8/L
$8179934
Triethylamine
264 kg
$15/kg
$3963
2-Acetoxyacetylchloride
125 kg
$1950/kg
$244335
Cerium ammonium nitrate
656 kg
$95/kg
$62341
37500 L
$16/L
$600000
338329 L
$7/L
$2256656
3 kg
$2/kg
$7
Triethylsilylchloride
15023 kg
$440/kg
$6610208
Pyridine
4-Dimethylaminopyridine
398154 L
3065 kg
$30/L
$88/kg
$11825163
44 kg
$13/kg
$579
2431 kg
$36,660/kg
$89.12 x 106
25064 kg
$75/kg
$1879800
249 kg
$101/kg
$25124
205438 L
$5/L
$1022054
Periodic acid
1012 kg
$238/kg
$240737
Pyridinium chlorochromate
4287 kg
$250/kg
Anisidine
Dichloromethane
Acetonitrile
Methanol
Sodium carbonate
Benzoylchloride
(1,2a,5a,7p,100)-5-acetyloxy1,2,7,1 0-tetrahydroxy-tax-4, 11 -diene
Carbonyldiimidazole
Sodium hydride
Dimethylformamide
$269746
257258 L
$28/L
$1071650
$7095176
Potassium hydroxide
1873 kg
$6/kg
$11236
Quinuclidine
1057 kg
$64000/kg
$67616000
N-Methylmorpholine-N-oxide
1675 kg
$75/kg
$125633
2222783 L
$8/L
$17782262
12249 kg
$276/kg
$3380818
606 kg
$65000/kg
$39364000
3879647 L
$10/L
$40348329
423 kg
$47/kg
$19682
Dioxane
t-Butyl alcohol
Sodium borohydride
Osmium tetroxide
Tetrahydrofuran
Acetyl chloride
110
2356 kg
$40/kg
$94249
1723 kg
$7/kg
$12408
1892373 L
$148/L
$280071204
13975 kg
17520 kg
$35/kg
$489118
$20/kg
$345492
Phenyllithium
324 kg
$2272/kg
$735219
Acetic acid
925 kg
$4/kg
$3513
3606 L
7641 kg
$6/L
$22300
$120/kg
$916944
54504 kg
$15948/kg
$869223413
2940 kg
$6/kg
$18816
298530 L
$51/L
$15174280
50 L
$1/L
$73
Mesyl chloride
Potassium carbonate
Hexamethylphosphoramide
N,N-Diisopropylethylamine
Acetic anhydride
Cyclohexane
Potassium tert butoxide
Benzeneseleninic acid anhydride
Sodium acetate
Benzene
32% Hydrochloric acid
Total raw material costs = $14.7 x 106 /kg
The E-factor for the semi-synthesis of paclitaxel from 10-DAB - defined as the ratio of the mass of
the product to the mass of all raw materials (both in kg) - is approximately 200-fold greater than the
microbial semi-synthetic process, implying that the ratio of VM to VP is 200. As a consequence, the
capital costs for the microbial process are 24 times greater. Additionally, capital costs for processes
handling material flows in the range of 50,000 kg/yr are roughly $40 million. Capital depreciation
(D) over the lifetime of the manufacturing facility can be related by:
k
D = S.N
(3.2)
where S is the annual output of the manufacturing plant (100 kg) and N is the lifetime of the project
(10 years). Accordingly, capital depreciation costs for the botanical process too are 24-fold lower.
Operation of the 3 GMP-compliant steps that are common to both processes is assumed to be
supervised by 1 personnel each, and 2 personnel oversee operation of the fermenter. As both
111
manufacturing schemes implement chemistries that are close to being or already have been adapted
to the flow configuration, the personnel required to monitor the remaining synthetic steps are
assumed to scale according to the six-tenths power law. Consequently, the botanical and microbial
processes are assumed to be operated by 10 and 14 personnel, respectively. Calculation of other fixed
costs follows rules of thumb that are commonly applied by process engineers. Additionally, waste
and treatment costs (W) are often a fifth of the raw material costs for small-to-medium volume
chemical processes, and, bioprocessing costs are not itemized as these have already been factored into
the material costs for 10-DAB and ATHTAX.
Table 6.3. Total manufacturing costs for plant-based and microbial-based manufacturing schemes
Itemized costs
Number of operations
Identities of GMP-compliant steps
Number of personnel (n)
Total capital investment (C)
Plant
Microbial
11
23
3 (f i, j)
3 (f u, v)
10
14
$40 x 106
$960 x 106
6
$15.7 X 10 /kg
$14.7 X 106 /kg
10 years
10 years
$40,000/kg
$960,000/kg
$3050/kg
$2754300/kg
Operating labor costs (Lo = 300,000.n)
$3 x 106/yr
$4.2 x 106/yr
Non-operating labor costs (LN = 0.6-L)
$1.8 x 106 /yr
$2.52 x 106/yr
$2.7 x 106 /yr-
$3.78 x 106/yr
Supplies (P = 0.3.L)
6
$0.9 x 10 /yr
$1.26 x 106 /yr
Maintenance (M = 0.02-C)
$0.8 x 106 /yr
$19.2 x 106 /yr
Utilities (U = 0.01-C)
$0.4 x 106 /yr
$9.6 x 10 6 /yr
Miscellaneous (Ms = 0.01-C)
$0.4 x 106 /yr
$9.6 x 106/yr
6
$10 x 10 /yr
$50.16 x 106 /yr
$1 x 106/kg
$5 x 10 6/kg
16.74 x 106 /kg
23.41 x 106 /kg
Raw material costs (R)
Project lifetime
Depreciation costs (D)
Waste & treatment costs (W)
Administration/overhead (0 = 0.9-L)
Annual fixed costs (A = Lo + LN
+
0
+ P + M + U + Ms)
Fixed costs (F = A / S)
Total manufacturing costs (= R + D + W + F)
112
6. 3. Summary of the economic assessment
The 40% difference between the unit price of paclitaxel produced using both schemes (Table 6.3) is
simply an artifact of the high cost of benzeneseleninic acid anhydride, which individually accounts
for 60% of the raw material cost for microbial semi-synthetic process. In fact, a 50% reduction in
the price of this forbiddingly expensive reagent makes microbially semi-synthesized paclitaxel only
10% more expensive than 10-DAB-derived paclitaxel, whereas a >60% reduction makes the
microbial-based process more economical than the plant-based one. Nevertheless, it appears that
producing (1,2a,5a,7p,10p)-5-acetyloxy-1,2,7,10-tetrahydroxy-tax-4,11-diene
at 0.0032 g/g DCW
per day is not only technically feasible given the tools currently at our disposal, but is also costcompetitive with the semi-synthetic plant-based process.
6. 4. Providing unprecedented access to inaccessible chemical space
Besides competing quite favorably with plant-based systems, microbial metabolic engineering offers
another significant benefit, best illustrated by the example of taxadiene. Though not pharmacoactive
itself, taxadiene is of interest to medicinal chemists as it is the first intermediate in the paclitaxel
biosynthetic pathway that bears the unique, C2 0 taxane scaffold. Unfortunately, like many
therapeutic natural products, the taxadiene content in its native host is prohibitively low. As 9 of
113
paclitaxel's 11 stereocenters are located on its scaffold, total synthesis of taxadiene involves as many
as 18 transformations and has an overall yield of 0.21% (Figure 6.2)127
0
~
O
OH
OH
2
TBSO
OTBS
OTBS
TBSO
9
X
O~n
nE15
TBSO
OTBS
5
0
OBn
11
HO
OBn
12
R, = R2 = CH(Ph)
R,=H, R2= Bn
OBn
14 X=OH
OTBS
OR2
8 R1=R2=H
E: 10
o E
TBSO
4
R10
h
7
O
OTBS
3
6
13
O)K!yH)
~
O
OTBS
1
00
0
17
OH
18
19 Taxadiene
X= OC(S)OPh
16 x-H
Figure 6.2. Total synthesis of taxadiene: The scheme involves 18 steps and has an overall yield of 0.21%. Key: a. [1]
Me 2tBuSiCl, DMF, [2] H 2 , Pd/C (yield 62%); b. [1] m-CPBA, CH 2 Cl 2 , [2] NaOMe, MeOH (77%); c. Me 2tBuSiCl,
imidazole, Et 2 O (70%); d. [1] Me 2tBuSiCl, DMF, [2] DIBAH, toluene (71%); e. [1] H 2C=CMeMgBr, THF, [2] DessMartin periodinane, CH 2Cl 2 (65%);f Me 2C(SeMe) 2, n-BuLi, THF (100%); g. [1] P13, Et 3N, CH 2Cl 2 , [2] HF, MeCN,
THF (64%); h. PhCH(OMe) 2, PPTS, CHC 3 (88%); i. LiAlH 4, AiCl 3 , Et 2 0, CH 2Cl 2 (100%); j. Dess-Martin
periodinane, CH 2 Cl 2 (48%); k. CH 2CHMgBr, THF (100%); 1. BF 3 0Et3, toluene (28%); m. LiAIH 4, THF (100%); n.
PhOCSCl, NaN(SiMe) 2, THF (100%); o. Bu3SnH, VAZO, toluene (53%); p. [1] Na/NH 3, THF, [2] Dess-Martin
periodinane, CH2Cl2 (56%); q. MeMgBr, CeCl 3, THF (100%); r. MeO 2 CNSO 2 NEt 3 , toluene (60%)
The biosynthesis of taxadiene (Figure 6.3), however, fares much better in comparison. As was
presented earlier, taxadiene assembly in E. coli commences with the head-to-tail condensation of
isopentenyl pyrophosphate with dimethylallyl pyrophosphate (DMAPP, 21) to produce geranyl
pyrophosphate (GPP, 22), which then condenses with IPP in situ to form farnesyl pyrophosphate
(FPP, 23). Yet another in situ condensation reaction follows suit and IPP combines with FPP to
114
form geranylgeranyl pyrophosphate (GGPP, 24). Cyclization of GGPP via intra-chain condensation
subsequently yields taxadiene (19).
a
Opp
OPP
20
21
b
'N
PP
Opp
22
21
20
b
app+
'Opp
PP
23
22
20
' pp
Op
oPP
PP
20
24
23
+
20
'N
PP
OPP
21
24
C
S.pp
24
Figure 6.3. Biosynthesis of taxadiene: The pathway commences with the isomerization of IPP (20) by an isomerase to
form DMAPP (21) (step a). Condensation between IPP with DMAPP forms GPP (22), which then re-condenses with
IPP to form FPP (23) in situ. FPP further condenses with IPP to form GGPP (24). All condensation reactions occur in
the active site of a single enzyme, GGPP synthase (step b). GGPP is then cyclized by taxadiene synthase to form
taxadiene (19) (step c).
The sequence of 3 consecutive condensation reactions involving IPP is catalyzed by the enzyme,
GGPP synthase, whereas DMAPP is an isomerization product of IPP formed by the enzyme, IPP
isomerase. Taxadiene formation is catalyzed by taxadiene synthase. IPP is synthesized by the nonmevalonate pathway, which commences with the condensation of glyceraldehyde-3-phosphate with
115
pyruvate to yield 2-methyl-(D)-erythritol-4-phosphate (MEP). Our demonstrations of taxadiene
synthesis with E. coli consuming glycerol as the primary carbon source achieved yields of 0.68% and
0.85% in 2 mL batch cultures and 1 L fed-batch fermentations, respectively5 9 .
The biosynthetic atom economy is also several orders of magnitude higher than that of total
synthesis. Significantly, considering that the theoretical yield of taxadiene on glycerol is 11.625%
(for strains utilizing glucose as the primary carbon feedstock, the theoretical yield is 20%) and that
the batch and fed-batch fermentations were yet to be optimized and the metabolism of E coli could
be further manipulated to improve the flux to taxadiene, the improvement in overall yield of
microbial-aided synthesis over total synthesis could be even more marked. Our group has already
successfully elevated the yield of taxadiene at the 2 mL scale by as much as 50% by expressing a
downstream cytochrome P450 monooxygenase-reductase complex that oxidizes taxadiene to form
taxadien-5a-o 1 2 8 .
116
Chapter 7
PUTTING IT ALL TOGETHER: A VISION FOR DRUG DISCOVERY
In Chapter 1, we elaborated how deficiencies in combinatorial chemistry were contributing to the
pharmaceutical industry's 'target-rich, lead-poor' imbalance. The decision to pursue quantity over
quality, how it contributed to the decline of the industry's synthetic capabilities and how these
lamentable decisions led to companies foregoing natural product space as a source of new drugs were
discussed at length in Chapter 2. In the three chapters that followed, a case was made for microbial
metabolic engineering as a viable platform for the discovery of natural products and some of the
technical challenges stymying the field were addressed therein. In this chapter, we shall elaborate a
unifying vision that could translate unprecedented numbers of small-molecule drugs to the bedside.
Called 'biosynthonics', our vision for re-inventing drug discovery is driven by microbial metabolic
engineering and recent developments in analytics, genome sequencing, flow chemistry, informatics,
oligonucleotide synthesis and retrosynthetic analysis, as well as exponential improvements in the raw
computing power that is now available for drug research. Many of its individual elements are already
in place and if metabolic and enzyme engineering are able to address some of the challenges that
were discussed in Chapter 5, biosynthonics could become a broad paradigm for generating focused
libraries of natural product-like molecules.
117
7. 1.What isbiosynthonics?
Biosynthonics comprises four principal domains - design, synthesis, exploration and integration.
Design encompasses the selection of electronic features and their steric optimization onto a rigid
molecular framework in order to ensure optimal binding to a specific biological target. The
conceptual underpinning of this exercise is rooted in fragment-based pharmacophore modeling
29 3
1 1
and all steps are completed in silico. The rigidity of the molecular framework, or scaffold, translates
to a minimization of the substituents' rotational entropy 132 . The pharmacophores so-designed are
then synthesized in a microbial chassis that has been carefully constructed via metabolic
engineering 3 3 '3 5 . This activity constitutes the second domain of biosynthonics - synthesis.
Although secondary metabolic enzyme homologues usually combine metabolic building blocks via a
common reaction mechanism, they often differ from one another in their preferences for substrates.
Even homologues combining the same building blocks frequently yield products that are stereo- and
regiochemically unique. The third domain of biosynthonics - exploration - expands the biosynthetic
ensemble for metabolic engineering through a systematic search of Nature's metabolic landscape 3'
136, 137.
Another important consideration in pharmacophore design is its potential use as an input for
conventional target-oriented synthesis (TOS)102, an exercise that aims to access precise regions of
chemical space - either a single molecule or a small assortment of molecules exhibiting minor
variations on a chemical theme. Accordingly, the fourth principal activity of biosynthonics 118
integration - is a modern, biosynthetic take on retrosynthesis and targets the synthesis of an
advanced intermediate that can act as a gateway molecule for TOS via the expression of a vastly
smaller set of enzymes takes advantage of the core competencies of both metabolic engineering and
synthetic chemistry.
7. 2. The informed design of superior pharmacophores
Lead selection by forward chemical genetics generally involves high-throughput screening of a
combinatorial library comprising of >106 compounds, and, compounds that are typically selected for
lead optimization exhibit Ka values of <1 gM. Fragment-based pharmacophore design (Figure 7.1.),
in comparison, commences by screening only a few thousand compounds for Ka values that fall in
high pM to mM range138 . High-throughput 2-D isotope-edited NMR is commonly utilized to
screen the compounds", and, the interactions of their molecular appendages - or fragments - with
residues within the target's binding pocket are then characterized using X-ray crystallography.
Several fragments are then incorporated within a single molecular entity.
The amalgamation of multiple fragments is rationalized by the additive effect that their individual
interactions have on the binding affinity of the chimeric molecule, and, the likelihood of generating
a hit from a compound library synthesized using fragment-based approaches is s3 orders of
magnitude higher than it is for high-throughput screening of combinatorial libraries.
119
B
A
HN
H2 NH
"
H2 N
H2N
N
1
2
3
26% at 1 mM (SPR)
IC. - 130 pM (FRET aay)
LE - 0.28
ICN w 0.08 pM (FRET aay)
LE = 0.37
LE < 0.37
Figure 7.1. Fragment-based pharmacophore discovery: Inhibition of
p-secretase has been shown to be an effective
strategy for treating Alzheimer's disease. Species 1 was identified by 1-D NMR screening of a fragment library. Its
binding affinity was determined by surface plasmon resonance (SPR)"0. X-ray crystallography was then utilized to
1
unearth insights regarding the structural features of 1 that interact with the binding site of p-secretase" , which led to the
synthesis of species 2. The use of pharmacophore and structure-based drug design approaches was then implemented to
derive species 312
The design methodology (Figure 7.2) we envision builds upon fragment-based pharmacophore
discovery and commences with comparative modeling of the putative target14. However, as only a
third of all protein sequences can be accurately modeled in silico144 , high-throughput X-ray
crystallography is used either to determine the structures of targets comprising of unknown or poorly
characterized folds145 , or, in conjunction with NMR, electron microscopy and X-ray scattering,
supply restraints that can aid model generation".
The target itself is discovered and validated using omics-based or clinical approaches14 , or, more
recently, by metabolic control analysis 48 '149. Omics technologies, in particular, rapidly generate vast
120
amounts of relevant data that can be compiled into systems-wide models for simulations to prioritize
targets, accelerate hypothesis generation and testing in disease models, and design clinical trials 20.
A
~~ omic.
lncreeena
set
B
arehnologies
*
In targe iecovey
0a02....0 V
(bdo)eynetse
Oncethetopology
of thebining eshas
beencharectered,
vlUM end t4MR-sed
s-nig
identllen
I silca scaffold
screeningand
-fragments
C
106am suit
D
I
=j
Figure 7.2. Overview of the biosynthonics paradigm: The proposed design methodology builds upon fragment-based
drug discovery. The application of cheminformatics features prominently, and, casting the steric and electronic effects of
the fragments as a three-dimensional free energy relationship for informed screening of a scaffold database by
minimizing the cost function is a unique addition to the approach. Panel A highlights the workflow for the entire
methodology. Panel B describes how omics-derived targets are analyzed to identify optimally binding fragments (also
Panel C) whose three-dimensional positioning within the active site guide scaffold design (also Panel D).
Once the topographies of the target's binding pockets have been charted, fragments that might
interact with residues in the binding pockets are screened virtually using approaches such as
similarity searching 4 2 . The catalogue of in silico fragment 'hits' is subsequently refined using high121
throughput 2-D NMR, and interactions between the fragments and residues in the binding pocket
are validated using high-throughput X-ray crystallography.
7. 3. Cheminformatics: An integral part of pharmacophore design
Though an improvement over compounds generated using combinatorial chemistry, synthetic
molecules that incorporate multiple fragments still possess rotational degrees of freedom that have
detrimental entropic consequences for binding. Instead, pharmacophores consisting of fragments
whose coordinates within the binding site have been greatly restricted by steric effects induced by
stereo- and regiospecifically linking them to privileged, natural product-like scaffolds are more likely
to interact strongly and selectively with the target. Accordingly, the next phase of pharmacophore
design involves mining natural product-like scaffolds that meet the spatial constraints of the
interacting fragments. The steric and electronic effects of the fragments are cast as a threedimensional free energy relationship" 0 '
15,
following which, a scaffold database"
is screened for
candidates that minimize the cost function.
A database similar to the National Cancer Institute's ChemBank" 3 would have to be created for
natural products. Estimates suggest that such a database could contain in excess of 170,000 entries".
Software that allows virtual screening of the structural database and analysis of the scaffolds will also
be needed" 4 . As the pharmacophores are intended to be gateway molecules for TOS, integration of
computer-aided synthetic planning"'' 56 into the screening software is desired. Additionally, the
122
software package would also include National Center for Biotechnology Information (NCBI)
database search and analyses capabilities that append the cheminformatics output with information
about the biosynthesis of the molecule. For instance, paclitaxel and epothilone B, a 16-membered
macrolide produced by the myxobacteria, Sorangium cellulosum157 , arrest the cancerous cell cycle in
its metaphase by inhibiting tubulin depolymerization. Both molecules also share a common binding
58
pocket, albeit they interact quite differently therein' . Pharmacophore design for the discovery of
more potent and safer tubulin-binding drugs according to the methodology outlined herein would,
therefore, yield structures based on the scaffolds of both paclitaxel and epothilone B (Figure 7.3).
However, based on an assessment of the biosynthetic information that is presently available for both
molecules 83' 159 and assuming that the TOS schemes do not exhibit significant differences in their
coverage of local chemical space, one is more likely to select the pharmacophore based on the
epothilone scaffold. Gateway molecules, thus, can be identified through synthetic and biosynthetic
analyses involving candidates that have been objectively screened from a natural product database.
O
1H
H HH
HOH
NN
4 Paclitaxel
5 Epothllone B
Figure 7.3. Two scaffolds, one binding site: Structural elements of paclitaxel and epothilone B that share a common
interaction site within the binding pocket have been highlighted with the same color.
123
It is evident that cheminformatics is central to the success of the biosynthonics drug discovery
platform. Individual software elements already exist but a single program in the mold of Accelrys,
Inc.'s Discovery Studio, that accesses databases of the kind described previously and supports
rigorous scientific analysis of the retrieved data as discussed does not exist. The development of such
tools could not be more urgent. Once available, they could transform drug discovery.
7. 4. Mining the natural world
Information about biosynthetic gene sequences encoding individual assembly steps and structural
characterization of the intermediates and products that various enzyme combinations produce is
crucial to the success of the biosynthonics platform. To this end, high-throughput whole genome
sequencing, assembly and bioinformatics analysis will greatly expand the biosynthetic ensemble that
is available for metabolic engineering136'137,
160,161.
The vast majority of recently uncovered bacterial
terpenoids were discovered through genome mining. Warp Drive Bio, a biotechnology start-up in
Cambridge, MA has adopted a similar approach for discovering new drug candidates by leveraging
its competencies in whole genome sequencing, assembly, mining and gene synthesis to produce
natural products for Sanofi Aventis' drug discovery programs
2
.
Additionally, NMR-based comparative metabolomics should aid rapid molecular characterization,
and, the development of in silico tools and methodologies for correlating the sequence and domain
124
63 69
organization of PKSs will accelerate annotation of more orphan PKS -' . Predictive capabilities
for characterizing novel terpenoid biosynthetic enzymes would also have to expanded8 6'170.
7. 5. Continuous manufacturing as a tool for discovery
It has been proposed that continuous manufacturing could potentially benefit drug discovery by
allowing investigations of reaction conditions, scale-up from gram-scales to ton-scales to meet
material requirements for clinical trials, evaluation of supply chain constraints, and rapid transfer of
the technology to production 1 7 1. Click chemistry", DNA-templated organic synthesis17 2 and a very
recent, multidimensional experimentation technique to evaluate reactions catalyzed by transition
metal complexes17 3 offer improved and high-throughput prospection of chemical space. However,
none of these techniques can consistently generate molecular architectures capable of rivaling the
complexity of natural products. Among other platforms, automated synthesizers have lately gained
acceptance as robust tools for natural product syntheses0 4 . However, as these units predominantly
utilize batch chemistries to synthesize target molecules,
their integration with continuous
manufacturing faces many technical challenges.
Microreactors, on the other hand, appear to be much more promising. Linearly-linked flow reactors
have been
successfully
used for the multistep
syntheses
of the cytotoxic alkaloid,
(±)-
oxomaritidine174 , as well as a small library of piperazines targeting a novel chemokine receptor linked
to several inflammations and allergies175 . In the case of (±)-oxomaritidine, synthesis was achieved
125
using packed columns containing immobilized reagents, catalysts and scavengers. The [5+2]
cycloaddition of quinone monoketals and styrene derivatives to produce the bicyclo[3.2.1]octanoid
scaffolds of neolignan natural products has also been demonstrated in flow. Yet, these successes are
but a few examples of the microreactor-facilitated traversal of natural product space1 7 6 . Adapting
batch chemistries to the flow format is fairly protracted and technically challenging, thereby
rendering the design of chemical processes involving six to eight synthetic steps - as is often the case
for small-molecule drugs - impractical for drug discovery.
7. 6. Metabolic engineering _ The ultimate demonstration of continuous manufacturing
Microbial synthesis of natural products intensifies and replaces an otherwise complicated synthetic
scheme comprising of scaffold assembly and multiple functional group transformations with a single
unit operation wherein a carbohydrate feedstock is converted to the product of interest under
considerably milder operating conditions by a microbial host (Figure 7.4). Moreover, it is unlikely
that the microbial-aided synthesis of APIs and pharmaceutical intermediates will face many of the
challenges that flow synthetic schemes encounter during work-up. As the routine use of perfusion
bioreactors by biopharmaceutical companies to manufacture protein products that rapidly degrade in
the culture broth would suggest, continuous biomanufacturing and cultivation are well-established
industrial unit operations177'
178.
This versatility makes it an important value addition to drug
discovery. Also, whereas isolation of the API and its synthetic intermediates typically incur
considerable material and energy costs in traditional, batch-based manufacturing, microbial
126
production of an API entirely does away with the need to isolate synthetic intermediates, greatly
shrinking the footprint of the manufacturing facility. For an industry that typically generates
between 25 to 100 kg of waste for every kg of product it manufactures, adoption of metabolic
engineering for chemical synthesis promises quantum leaps in atom and energy economies 1 79. The
API itself can be extracted from the culture broth through use of continuous countercurrent
chromatography17 8, followed by purification and crystallization to produce the bulk API.
Solvent
R1
S,
R2
S2
RN3
3-+ -+N-3 --
2
1
W
RN-2
SN3
[4
-
SN2
N-2
N --
W-N-1
s
WN 1W
WN2
WN-
W2
Crystallizer
RN
Bulk
API
RN-
1
2
E2
3- -+N-3-
RN
R2
EN3
N-2
EN-
Dryer
EN
N-i
N
Solvent
Solvent
Crystallizer
N
Carbohydrate feedstock
Chromatography
Fermenter
1
Waste
LLE
Bulk
API
Waste
Dryer
Figure 7.4. Metabolic engineering, the ultimate demonstration of reaction intensification: E and G denote a
biosynthetic enzyme and gene, respectively. R represents the reactant, S denotes the solvent and W represents the waste
stream emerging from the unit operation.
127
7. 7. Final words
Any technology is only as good as its weakest link and much remains to be done before
biosynthonics crystallizes into a viable drug discovery platform. However, many of its elements are
already in place. Future advances and the convergence of analytical biochemistry, bioinformatics,
computation, flow chemistry and metabolic & protein engineering will drive this revolution in drug
discovery. One sincerely hopes this is the case. After all, the price of the status quo for discovering
and developing a drug has already exceeded a billion dollars180 .
128
Chapter References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
in The Economist, Vol. 385(8552) 88London; 27 October 2007).
Booth, B. & Zemmel, R. Prospects for productivity. Nature Reviews Drug Discovery 3, 451456 (2004).
Clark, D.E. & Newton, C.G. Outsourcing lead optimization: The quiet revolution. Drug
Discovery Today 9, 492-500 (2004).
Sinha, G. Outsourcing drug work. Scientfic American 291, 24-25 (2004).
Scott, A. Pharma outsourcing to exceed $26 billion by 2011. Chemical Week 168, 27 (2006).
Ferraindiz, J.M. The impact of generic goods in the pharmaceutical industry. Health
Economics 8, 599-612 (1999).
Finkelstein, S. & Temin, P. Reasonable Rx: Solving the Drug Price Crisis. (Pearson
Education, Inc., Upper Saddle River; 2008).
Last accessed: February 6, 2011).
Lombardino, J.G. & Lowe, J.A. The role of the medicinal chemist in drug discovery - Then
and now. Nature Reviews Drug Discovery 853, 853-862 (2004).
Rang, H.P. Drug Discovery & Development: Technology in Transition. (Churchill
Livingstone, Edinburgh; 2006).
Lipinski, C.A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discovery
Today: Technologies 1, 337-341 (2004).
Hogan, J.C. Combinatorial chemistry in drug discovery. Nature Biotechnology 15, 328 - 330
(1997).
Ramstram, 0. & Lehn, J.-M. Drug discovery by dynamic combinatorial libraries. Nature
Reviews Drug Discovery 1, 26-36 (2002).
Geysen, H.M., Schoenen, F., Wagner, D. & Wagner, R. A guide to drug discovery:
Combinatorial compound libraries for drug discovery: an ongoing challenge. Nature Reviews
Drug Discovery 2, 222-230 (2003).
Kolb, H.C. & Sharpless, K.B. The growing impact of click chemistry on drug discovery.
Drug Discovery Today 8, 1129-1137 (2003).
Schreiber, S.L. Target-oriented and diversity-oriented organic synthesis in drug discovery.
Science 287, 1964-1969 (2000).
Bleicher, K.H., Bohm, H.J., Muller, K. & Alanine, A.I. Hit and lead generation: beyond
high-throughput screening. Nat Rev Drug Discov 2, 369-378 (2003).
Rauwerda, H., Roos, M., Hertzberger, B.O. & Breit, T.M. The promise of a virtual lab in
drug discovery. DrugDiscovery Today 11, 228-236 (2006).
Wu, X. & Schultz, P.G. Synthesis at the interface of chemistry and biology. J. Am. Chem.
Soc. 131, 12497-12515 (2009).
Butcher, E.C., Berg, E.L. & Kunkel, E.J. Systems biology in drug discovery. Nature
Biotechnology 22, 1253-1259 (2004).
129
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Cooper, M.A. Optical biosensors in drug discovery. Nature Reviews Drug Discovery 1, 515528 (2002).
Dittrich, P.S. & Manz, A. Lab-on-a-chip: microfluidics in drug discovery. Nature Reviews
Drug Discovery 5, 210-218 (2006).
Pharmaceutical Research & Manufacturers of America Pharmaceutical Industry Profile
2012. (PhRMA, Washington, DC; 2012).
Mullard, A. 2011 FDA drug approvals. Nature Reviews DrugDiscovery 11, 91-94 (2012).
Pammolli, F., Magazzini, L. & Riccaboni, M. The productivity crisis in pharmaceutical
R&D. Nature Reviews Drug Discovery 10, 428-438 (2011).
Kola, I. & Landis, J. Can the pharmaceutical industry reduce attrition rates? Nature Reviews
Drug Discovery 3, 711-715 (2004).
Overington, J.P., Al-Lazikani, B. & Hopkins, A.L. How many drug targets are there? Nature
Reviews Drug Discovery 5, 993-996 (2006).
Pang, Y.P. In silico drug discovery: Solving the 'target-rich' and 'lead-poor' imbalance using
the genome-to-drug-lead paradigm. ClinicalPharmacolog & Therapeutics81, 30-34 (2007).
Feher, M. & Schmidt, J.M. Property distributions: Differences between drugs, natural
products, and molecules from combinatorial chemistry. Journalof Chemical Information and
Modeling 43, 218-227 (2003).
Paolini, G.V., Shapland, R.H.B., van Hoorn, W.P., Mason, J.S. & Hopkins, A.L. Global
mapping of pharmacological space. Nature Biotechnology 24, 805-815 (2006).
Oprea, T.I. & Gottfries, J. Chemography: The art of navigating in chemical space. Journal
of CombinatorialChemistry 3, 157-166 (2001).
Ashburn, T.T. & Thor, K.B. Drug repositioning: Identifying and developing new uses for
existing drugs. Nature Reviews Drug Discovery 3, 673-683 (2004).
DiMasi, J.A., Hansen, R.W. & Grabowski, H.G. The price of innovation: new estimates of
drug development costs. J Health Econ 22, 151-185 (2003).
Adams, C.P. & Brantner, V.V. Estimating the cost of new drug development: Is it really 802
million dollars? Health Aff (Millwood)25, 420-428 (2006).
Grabowski, H., Vernon, J. & DiMasi, J.A. Returns on research and development for 1990s
new drug introductions. Pharmacoeconomics20, 11-29 (2002).
Koehn, F. & Carter, G. The evolving role of natural products in drug discovery. Nature
Reviews Drug Discovery 4, 206-220 (2005).
Newman, D.J., Cragg, G.M. & Snader, K.M. The influence of natural products upon drug
discovery NaturalProductReports 17, 215-234 (2000).
Chang, M. & Keasling, J. Production of isoprenoid pharmaceuticals by engineered microbes.
Nature Chemical Biology 2, 674 - 681 (2006).
Bohlmann, J. & Keeling, C.I. Terpenoid biomaterials. PlantJ54, 656-669 (2008).
Koehn, F.E. & Carter, G.T. The evolving role of natural products in drug discovery. Nat
Rev Drug Discov 4, 206-220 (2005).
Firn, R.D. & Jones, C.G. The evolution of secondary metabolism - a unifying model. Mol
Microbiol37, 989-994 (2000).
130
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
Li, J.W. & Vederas, J.C. Drug discovery and natural products: end of an era or an endless
frontier? Science 325, 161-165 (2009).
Newman, D.J. & Cragg, G.M. Natural products as sources of new drugs over the last 25
years. JournalofNaturalProducts 70, 461-477 (2007).
Feher, M. & Schmidt, J.M. Property distributions: Differences between drugs, natural
products, and molecules from combinatorial chemistry. Journalof Chemical Information and
Computer Science 43, 218-227 (2003).
Leeson, P.D. & Springthorpe, B. The influence of drug-like concepts on decision-making in
medicinal chemistry. Nature Reviews Drug Discovery 6, 881-890 (2007).
Goodman, J. & Walsh, V. The Story of Taxol: Nature and Politics in the Pursuit of an AntiCancer Drug (Cambridge University Press, Cambridge, UK; 2001).
Nicolaou, K.C. & Montagnon, T. Molecules That Changed the World. (Wiley-VCH,
Weinheim; 2008).
Corey, E.J. & Cheng, X.-M. The Logic of Chemical Synthesis. (John Wiley & Sons, Inc.,
New York; 1995).
Gallop, M.A., Barrett, R.W., Dower, W.J., Fodor, S.P.A. & Gordon, E.M. Applications of
combinatorial technologies to drug discovery. 1. Background and peptide combinatorial
libraries. J Med. Chem. 37, 1233-1251 (1994).
Gordon, E.M., Barrett, R.W., Dower, W.J., Fodor, S.P.A. & Gallop, M.A. Applications of
combinatorial technologies to drug discovery. 2. Combinatorial organic synthesis, library
screening strategies, and future directions. J.Med. Chem. 37, 1385-1401 (1994).
Federsel, H.J. Logistics of process R&D: transforming laboratory methods to manufacturing
scale. Nat Rev DrugDiscov 2, 654-664 (2003).
Kola, I. & Landis, J. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug
Discov 3, 711-715 (2004).
Zhu, F., Qin, C., Tao, L., Liu, X., Shi, Z., Ma, X., Jia, J., Tan, Y., Cui, C., Lin, J., Tan, C.,
Jiang, Y. & Chen, Y. Clustered patterns of species origins of nature-derived drugs and clues
for future bioprospecting. Proceedings of the NationalAcademy of Sciences of the United States
ofAmerica 108, 12943-12948 (2011).
Brower, V. Back to Nature: Extinction of medicinal plants threatens drug discovery. Journal
ofthe National CancerInstitute 100, 838-839 (2008).
Ro, D.-K., Paradise, E.M., Ouellet, M., Fisher, K.J., Newman, K.L., Ndungu, J., Ho, K.,
Eachus, R., Ham, T., Kirby, J., Chang, M.C.Y., Withers, S., Shiba, Y., Sarpong, R. &
Keasling, J.D. Production of the antimalarial drug precursor artemisinic acid in engineered
yeast. Nature, 940-943 (2006).
Stephanopoulos, G., Alper, H. & Moxley, J. Exploiting biological complexity for strain
improvement through systems biology. Nat Biotechnol22, 1261-1267 (2004).
Pfeifer, B.A., Admiraal, S.J., Gramajo, H., Cane, D.E. & Khosla, C. Biosynthesis of complex
polyketides in a metabolically engineered strain of E. coli. Science 291, 1790-1792 (2001).
Leonard, E., Ajikumar, P.K., Thayer, K., Xiao, W.H., Mo, J.D., Tidor, B., Stephanopoulos,
G. & Prather, K.L. Combining metabolic and protein engineering of a terpenoid
131
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
biosynthetic pathway for overproduction and selectivity control. Proc NatI Acad Sci U S A
107, 13654-13659 (2010).
Ajikumar, P.K., Xiao, W.H., Tyo, K.E., Wang, Y., Simeon, F., Leonard, E., Mucha, 0.,
Phon, T.H., Pfeifer, B. & Stephanopoulos, G. Isoprenoid pathway optimization for Taxol
precursor overproduction in Escherichia coli. Science 330, 70-74 (2010).
Klein-Marcushamer, D., Ajikumar, P.K. & Stephanopoulos, G. Engineering microbial cell
factories for biosynthesis of isoprenoid molecules: Beyond lycopene. Trends in Biotechnology,
417-424 (2007).
Firn, R. Nature's Chemicals: The Natural Products that Shaped Our World. (Oxford
University Press, Oxford, UK; 2010).
Ajikumar, P.K., Tyo, K., Carlsen, S., Mucha, 0., Phon, T.H. & Stephanopoulos, G.
Terpenoids: Opportunities for biosynthesis of natural product drugs using engineered
microorganisms. Molecular Pharmaceutics,167-190 (2008).
Bohlmann, J. & Keeling, C.I. Terpenoid biomaterials. The PlantJournal,656-669 (2008).
Breitmaier, E. Terpenes. (Wiley-VCH, Weinheim, Germany; 2006).
Barkovich, R. & Liao, J.C. Metabolic engineering of isoprenoids Metabolic Engineering 3,
27-39 (2001).
Thulasiram, H.V., Erickson, H.K. & Poulter, C.D. Chimeras of two isoprenoid synthases
catalyze all four coupling reactions in isoprenoid biosynthesis. Science 316, 73-76 (2007).
Ajikumar, P.K., Tyo, K., Carlsen, S., Mucha, 0., Phon, T.H. & Stephanopoulos, G.
Terpenoids: opportunities for biosynthesis of natural product drugs using engineered
microorganisms. MolPharm 5, 167-190 (2008).
Dugar, D. & Stephanopoulos, G. Relative potential of biosynthetic pathways for biofuels
and bio-based products. Nature Biotechnology 29, 1074-1078 (2011).
Kaksal, M., Jin, Y., Coates, R.M., Croteau, R. & Christianson, D.W. Taxadiene synthase
structure and evolution of modular architecture in terpene biosynthesis. Nature 469, 116120 (2011).
Poulter, C.D. Farnesyl diphosphate synthase: A paradigm for understanding structure and
function relationships in E-polyprenyl diphosphate synthases. Phytochemistry Reviews 5, 1726 (2006).
Yoshikuni, Y., Ferrin, T.E. & Keasling, J.D. Designed divergent evolution of enzyme
function. Nature 440, 1078-1082 (2006).
Meunier, B., de Visser, S.P. & Shaik, S. Mechanism of oxidation reactions catalyzed by
cytochrome P450 enzymes. Chemical reviews 104, 3947-3980 (2004).
Groves, J.T. in Cytochrome P450: Structure, Mechanism, and Biochemistry. (ed. P. Ortiz
de Montellano) 1-43 (Kluwer, New York; 2005).
Firn, R.D. & Jones, C.G. The evolution of secondary metabolism - A unifying model.
Molecular Microbiology 37, 989-994 (2000).
Yadav, V.G. & Stephanopoulos, G. Reevaluating synthesis by biology. Curr Opin Microbiol
13, 371-376 (2010).
132
76.
77.
Ajikumar, P.K., Xiao, W.H., Tyo, K.E., Wang, Y., Simeon, F., Leonard, E., Mucha, 0.,
Phon, T.H., Pfeifer, B. & Stephanopoulos, G. Isoprenoid pathway optimization for Taxol
precursor overproduction in Escherichia coli. Science 330, 70-74.
Fischbach, M.A. & Clardy, J. One pathway, many products. Nature Chemical Biology 3,
353-355 (2007).
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
Maplestone, R.A., Stone, M.J. & Williams, D.H. The evolutionary role of secondary
metabolites - A review. Gene 115, 151-157 (1992).
Dixon, R.A. Natural products and plant disease resistance. Nature 411, 843-847 (2001).
Gershenzon, J. & Dudareva, N. The function of terpene natural products in the natural
world. Nature ChemicalBiology 3, 408-414 (2007).
Janes, K.A., Albeck, J.G., Gaudet, S., Sorger, P.K., Lauffenburger, D.A. & Yaffe, M.B. A
systems model of signaling identifies a molecular basis set for cytokine-induced apoptosis.
Science 310, 1646-1653 (2005).
Frost & Sullivan New York, NY; 2001).
Walker, K. & Croteau, R. Taxol biosynthetic genes. Phytochemistry 58, 1-7 (2001).
Croteau, R., Schoendorf, A. & Jennewein, S. Cytochrome P450 oxygenases and their uses.
US PatentApp. 20,040/236,089 (2004).
Ketchum, R.E.B. & Croteau, R.B. in Plant Metabolomics. (eds. K. Saito, R.A. Dixon & L.
Willmitzer) 291-309 (Springer Verlag, Heidelberg; 2006).
Jennewein, S., Wildung, M.R., Chau, M.-D., Walker, K. & Croteau, R. Random
sequencing of an induced Taxus cell cDNA library for identification of clones involved in
Taxol biosynthesis. Proceedings of the National Academy of Sciences of the United States of
America 101, 9149-9154 (2004).
Blum, T. & Kohlbacher, 0. Using atom mapping rules for an improved detection of
relevant routes in weighted metabolic networks. Journal of Computational Biology 15, 565576 (2008).
Heath, A.P., Bennett, G.N. & Kavraki, L.E. Finding metabolic pathways using atom
tracking. Bioinformatics26, 1548-1555 (2010).
Floss, H.G. & Mocek, U. in Taxol: Science & Applications. (ed. M. Suffness) 191-208
(CRC Press, Boca Raton, FL; 1995).
Gasteiger, J. & Engel, T. Chemoinformatics. (Wiley-VCH, Weinheim; 2003).
http://www.daylight.com/dayhtiml/doc/theory/theory.similes.htill.
Ronquist, F. & Huelsenbeck, J.P. MrBayes 3: Bayesian phylogenetic inference under mixed
models. Bioinformatics 19, 1572-1574 (2003).
Orry, A.J.W. & Abagyan, R. Homology Modeling: Methods and Protocols. (Springer, New
York; 2012).
94.
http://www.cgdl.ucsf.edu/chiminera/docs/ContribLtedSoftware/mlIItaligiviewer/frame
95.
nav.httml.
Schoendorf, A., Rithner, C.D., Williams, R.M. & Croteau, R. Molecular cloning of a
cytochrome P450 taxane 10p-hydroxylase cDNA from Taxus and functional expression in
133
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
yeast. Proceedingsof the NationalAcademy ofSciences of the United States ofAmerica 98, 15011506 (2001).
Eswar, N., M. A. Marti-Renom, Webb, B., Madhusudhan, M.S., Eramian, D., Shen, M.,
Pieper, U. & Sali, A. Comparative protein structure modeling with MODELLER. Current
Protocols in Bioinformatics 15, 5.6.1-5.6.30 (-2006).
Yu, J., Zhou, Y., Tanaka, I. & Yao, M. Roll: A new algorithm for the detection of protein
pockets and cavities with a rolling probe sphere. Bioinformatics26, 46-52 (2010).
http://ekhidna.biocenter.helsinki.fi/dali server/.
Mordecai, A. Nonlinear Programming: Analysis and Methods. (Dover Publications,
Mineola, New York; 2003).
Ghosh, D., Griswold, J., Erman, M. & Pangborn, W. Structural basis for androgen
specificity and oestrogen synthesis in human aromatase. Nature 457, 219-223 (2009).
Chovancovi, E., Pavelka, A., Benes, P., Strnad, 0., Brezovskf, J., Kozlikovai, B., Gora, A.,
Sustr, V., Klvana, M., Medek, P., Biedermannovai, L., Sochor, J. & Damborskf, J. Eva
Chovancovai, Antonin Pavelka, Petr Benes, Ondrej Strnad, Jan Brezovskf, Barbora
Kozlikovai, Artur Gora, Vilem 9ustr, Martin Klvana, Petr Medek, Lada Biedermannovai, Jiff
Sochor & Jifi Damborskf: CAVER 3.0: A Tool for the Analysis of Transport Pathways in
Dynamic Protein Structures, PLoS Computational Biology 8: e1002708,. PLoS
ComputationalBiology (2012).
Burke, M.D. & Schreiber, S.L. A planning strategy for diversity-oriented synthesis.
Angewandte Chemie InternationalEdition 43, 46-58 (2004).
Doi, T., Fuse, S., Miyamoto, S., Nakai, K., Sasuga, D. & Takahashi, T. A formal total
synthesis of taxol aided by an automated synthesizer. Chemistry - An Asian Journal 1, 370383 (2006).
Fuse, S., Machida, K. & Takahashi, T. in New Strategies in Chemical Synthesis and
Catalysis. (ed. B. Pignataro) 33-45 (Wiley-VCH, Weinheim; 2012).
Nicolaou, K.C., Nantermet, P.G., Ueno, H., Guy, R.K., Couladouros, E.A. & Sorensen,
E.J. Total synthesis of taxol. 1. Retrosynthesis, degradation, and reconstitution. Journalofthe
American Chemical Society 117, 624-633 (1995).
Nicolaou, K.C., Liu, J.-J., Yang, Z., Ueno, H., Sorensen, E.J., Claiborne, C.F., Guy, R.K.,
Hwang, C.-K., Nakada, M. & Nantermet, P.G. Total synthesis of taxol. 2. Construction of
A and C ring intermediates and initial attempts to construct the ABC ring system. Journalof
the American Chemical Society 117, 634-644 (1995).
Nicolaou, K.C., Yang, Z., Liu, J.-J., Nantermet, P.G., Claiborne, C.F., Renaud, J., Guy,
R.K. & Shibayama, K. Total synthesis of taxol. 3. Formation of taxol's ABC ring skeleton.
Journalofthe American Chemical Society 117, 645-652 (1995).
Nicolaou, K.C., Ueno, H., Liu, J.-J., Nantermet, P.G., Yang, Z., Renaud, J., Paulvannan, K.
& Chadha, R. Total synthesis of taxol. 4. The final stages and completion of the synthesis.
Journalofthe American Chemical Society 117, 653-659 (1995).
134
109.
Nicolaou, K.C., Yang, Z., Liu, J.J., Ueno, H., Nantermet, P.G., Guy, R.K., Claiborne, C.F.,
Renaud, J., Couladouros, E.A., Paulvannan, K. & Sorensen, E.J. Total synthesis of taxol.
Nature 367, 630-634 (1994).
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
Danishefsky, S.J., Masters, J.J., Young, W.B., Link, J.T., Snyder, L.B., Magee, T.V., Jung,
D.K., Isaacs, R.C.A., Bornmann, W.G., Alaimo, C.A., Coburn, C.A. & M. J. Di Grandi
Total synthesis of baccatin III and taxol. Journalof the American Chemical Society 118, 28432859 (1996).
Kleemann, A., Engel, J., Kutscher, B. & Reichert, D. Pharmaceutical Substances: Syntheses,
Patents, Applications, Vol. 1 & 2. (Georg Thieme Verlag, New York, NY; 2001).
Greene, T.W. & Wuts, P.G.M., Vol. 3 (John Wiley & Sons, Inc., New York, NY; 1991).
Kocienski, P. Protecting Groups, 3rd Ed. (Georg Thieme Verlag, New York, NY; 2005).
Munro, A.W., Leys, D.G., McLean, K.J., Marshall, K.R., Ost, T.W.B., Daff, S., Miles, C.S.,
Chapman, S.K., Lysek, D.A., Moser, C.C., Page, C.C. & Dutton, P.L. P450 BM3: The very
model of a modern flavocytochrome. Trends in Biochemical Sciences 27, 250-257 (2002).
Barnes, H.J., Arlotto, M.P. & Waterman, M.R. Expression and enzymatic activity of
recombinant cytochrome P450 17t-hydroxylase in Escherichia coli. Proceedings of the
NationalAcademy of Sciences of the United States ofAmerica 88, 5597-5601 (1991).
Leonard, E. & Koffas, M.A.G. Engineering of artificial plant cytochrome P450 enzymes for
synthesis of isoflavones by Escherichia coli. Applied & Environmental Microbiology 73, 72467251 (2007).
Zhou, K. & Stephanopoulos, G. Unpublished results.
Hamdane, D., Zhang, H. & Hollenberg, P. Oxygen activation by cytochrome P450
monooxygenase. Photosynthesis Research 98, 657-666 (2008).
Rontein, D., Onillona, S., Herbette, G., Werck-Reichhart, D., Sallauda, C. & Tissier, A.
Oxidation of taxadiene to oxacyclotaxane by the yew CYP725A4 previously described as a
taxadiene 5a-hydroxylase. JournalofBiological Chemistry 283, 6067-6075 (2008).
White, C. & Stephanopoulos, G. Unpublished results.
Smith, G.P. Filamentous fusion phage: Novel expression vectors that display cloned antigens
on the virion surface. Science 228, 1315-1317 (1985).
Zocher, R., Weckwerth, W., Hacker, C., Kammer, B., Hornbogen, T. & Ewald, D.
Biosynthesis of taxol: Enzymatic acetylation of 10-deacetylbaccatin-III to baccatin-III in
crude extracts from roots of Taxus baccata. Biochemical and Biophysical Research
Communications 229, 16-20 (1996).
Zarek, M. & Wallg6rski, P. Determination of 10-deacetylbaccatine III in Taxus baccata
needles by micellar electrokinetic chromatography. Herba Polonica 55, 25-35 (2009).
Yadav, V.G. & Stephanopoulos, G. Elucidation of the early taxane biosynthetic pathway for
biosynthesis of a gateway molecule for target-oriented chemical synthesis of paclitaxel.
Submitted (2013).
Wickremesinhe, E.R.M. & Arteca, R.N. Taxus cell suspension cultures: Optimizing growth
and production of taxol. JournalofPlant Physiology 144, 183-188 (1994).
135
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
Anderson, J. Determining manufacturing costs. Chemical Engineering Progress 105, 27-31
(2009).
Rubenstein, S.M. & Williams, R.M. Studies on the biosynthesis of taxol: Total synthesis of
taxa-4(20),111( 2)-diene and taxa-4(5),111(2 )-diene. The first committed biosynthetic
intermediate. Journalof Organic Chemistry 60, 7215-7223 (1995).
Yadav, V.G., Edgar, S.M. & Stephanopoulos, G. Investigations into the selectivity and
activity of a promiscuous Taxus P450 monooxygenase. Submitted (2013).
Erlanson, D.A., McDowell, R.S. & O'Brien, T. Fragment-based drug discovery. Journalof
Medicinal Chemistry 47, 3463-3482 (2004).
Yang, S.-Y. Pharmacophore modeling and applications in drug discovery: Challenges and
recent advances. Drug Discovery Today 15, 444-450 (2010).
Rees, D.C., Congreve, M., Murray, C.W. & Carr, R. Fragment-based lead discovery. Nature
Reviews Drug Discovery 3, 660-672 (2004).
Murray, C.W. & Verdonk, M.L. The consequences of translational and rotational entropy
lost by small molecules on binding to proteins. Journalof Computer-Aided Molecular Design
16, 741-753 (2002).
Bailey, J.E. Toward a science of metabolic engineering. Science 252, 1668-1675 (1991).
Stephanopoulos, G.N. Metabolic fluxes and metabolic engineering. Metabolic Engineering 1,
1-11 (1999).
Keasling, J.D. Manufacturing molecules through metabolic engineering. Science 330, 13551358 (2010).
Schloss, P.D. & Handelsman, J. Biotechnological prospects from metagenomics. Current
Opinion in Biotechnology 14, 303-310 (2003).
Cane, D.E. & Ikeda, H. Exploration and mining of the bacterial terpenome. Accounts of
Chemical Research 45, 463-472 (2012).
Hajduk, P.J. & Greer, J. A decade of fragment-based drug design: Strategic advances and
lessons learned. Nature Reviews Drug Discovery 6, 211-219 (2007).
Hajduk, P.J., Gerfin, T., Boehlen, J.-M., Hiberli, M., Marek, D. & Fesik, S.W. Highthroughput nuclear magnetic resonance-based screening. JournalofMedicinal Chemistry 42,
2315-2317 (1999).
Geschwindner, S., Olsson, L.L., Albert, J.S., Deinum, J., Edwards, P.D., Beer, T. & Folmer,
R.H. Discovery of a novel warhead against beta-secretase through fragment-based lead
generation. JournalofMedicinal Chemistry 50, 5903-5911 (2007).
Murray, C.W., Callaghan, 0., Chessari, G., Cleasby, A., Congreve, M., Frederickson, M.,
Hartshorn, M.J., McMenamin, R., Patel, S. & Wallis, N. Application of fragment screening
by X-ray crystallography to beta-secretase. Journal of Medicinal Chemistry 50, 1116-1123
(2007).
Edwards, P.D., Albert, J.S., Sylvester, M., Aharony, D., Andisik, D., Callaghan, 0.,
Campbell, J.B., Carr, R.A., Chessari, G., Congreve, M., Frederickson, M., Folmer, R.H.,
Geschwindner, S., Koether, G., Kolmodin, K., Krumrine, J., Mauger, R.C., Murray, C.W.,
Olsson, L.L., Patel, S., Spear, N. & Tian, G. Application of fragment-based lead generation
136
143.
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency,
cellular activity, and high ligand efficiency. Journal of Medicinal Chemistry 50, 5912-5925
(2007).
Cavasotto, C.N. & Phatak, S.S. Homology modeling in drug discovery: Current trends and
applications. Drug Discovery Today 14, 676-683 (2009).
Marti-Renom, M.A., Stuart, A., Fiser, A., Sanchez, R., Melo, F. & Sali, A. Comparative
protein structure modeling of genes and genomes. Annual Review of Biophysics &
BiomolecularStructure 29, 291-326 (2000).
Blundell, T.L., Jhoti, H. & Abell, C. High-throughput crystallography for lead discovery in
drug design. Nature Reviews DrugDiscovery 1, 45-54 (2002).
Ward, A.B., Sali, A. & Wilson, I.A. Structural biology unleashed through use of restraints.
Science In press (2013).
Lindsay, M.A. Target Discovery. Nature Reviews Drug Discovery 2, 831-838 (2003).
Cascante, M., Boros, L.G., Comin-Anduix, B., P. de Atauri, Centelles, J.J. & Lee, P.W.-N.
Metabolic control analysis in drug discovery and disease. Nature Biotechnology 20, 243-249
(2002).
Metallo, C.M., Gameiro, P.A., Bell, E.L., Mattaini, K.R., Yang, J., Hiller, K., Jewell, C.M.,
Johnson, Z.R., Irvine, D.J., Guarente, L., Kelleher, J.K., M. G. Vander Heiden, Iliopoulos,
0. & Stephanopoulos, G. Reductive glutamine metabolism by IDHI mediates lipogenesis
under hypoxia. Nature481, 380-384 (2012).
Harper, K.C. & Sigman, M.S. Three-Dimensional Correlation of Steric and Electronic Free
Energy Relationships Guides Asymmetric Propargylation. Science 333, 1875-1878 (2011).
Harper, K.C., Bess, E.N. & Sigman, M.S. Multidimensional steric parameters in the analysis
of asymmetric catalytic reactions. Nature Chemistry 4, 366-374 (2012).
Welsch, M.E., Snyder, S.A. & Stockwell, B.R. Privileged scaffolds for library design and
drug discovery. CurrentOpinion in Chemical Biology 14, 1-15 (2010).
Strausberg, R.L. & Schreiber, S.L. From knowing to controlling: A path from genomics to
drugs using small molecule probes. Science 300, 294-295 (2003).
Wetzel, S., Klein, K., Renner, S., Rauh, D., Oprea, T.I., Mutzel, P. & Waldmann, H.
Interactive exploration of chemical space with Scaffold Hunter. Nature Chemical Biology 5,
581-583 (2009).
Cook, A., Johnson, A.P., Law, J., Mirzazadeh, M., Ravitz, 0. & Simon, A. Computer-aided
synthesis design: 40 years on. Wiley Interdisciplinary Reviews: Computational Molecular
Science 2, 79-107 (2012).
Ravitz, 0. Data-driven computer aided synthesis design. Drug Discovery Today: Technologies
In press (2013).
157.
H6fle, G., Bedorf, N., Steinmetz, H., Schomburg, D., Gerth, K. & Reichenbach, H.
Epothilone A and B - Novel 16-membered macrolides with cytotoxic activity: Isolation,
crystal structure, and conformation in solution. Angewandte Chemie InternationalEdition 35,
1567-1569 (1996).
137
158.
159.
160.
161.
162.
163.
164.
165.
Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollubuck, B.,
Poy, G., Sackett, D., Nicolaou, K.C. & Fojo, T. A common pharmacophore for epothilone
and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human
cancer cells. Proceedings of the NationalAcademy of Sciences of the United States ofAmerica 97,
2904 (2000).
Tang, L., Shah, S., Chung, L., Carney, J., Katz, L., Khosla, C. & Julien, B. Cloning and
heterologous expression of the epothilone gene cluster. Science 287, 640-642 (2000).
Baker, M. De novo genome assembly: What every biologist should know. Nature Methods 9,
333-337 (2012).
Kircher, M. & Kelso, J. High-throughput DNA sequencing - Concepts and limitations.
BioEssays 32, 524-536 (2010).
Waldholz, M. Biology's master programmers. MIT Technolog Review July/August, 56-60
(2012).
Forseth, R.R. & Schroeder, F.C. NMR-spectroscopic analysis of mixtures: From structure to
function. Current Opinion in Chemical Biolog 15, 38-47 (2011).
Molinski, T.F. NMR of natural products at the 'nanomole-scale'. NaturalProduct Reports
27, 321-329 (2010).
Yadav, G., Gokhale, R.S. & Mohanty, D. Computational approach for prediction of domain
organization and substrate specificity of modular polyketide synthases. Journalof Molecular
Biolog 328, 335-363 (2003).
166.
167.
168.
169.
170.
171.
172.
Medema, M.H., Blin, K., Cimermancic, P., Jager, V.d., Zakrzewski, P., Fischbach, M.,
Weber, T., Takano, E. & Breitling, R. antiSMASH: Rapid identification, annotation and
analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome
sequences. Nucleic Acids Research 39, W339-W346 (2011).
Challis, G.L., Ravel, J. & Townsend, C.A. Predictive, structure-based model of amino acid
recognition by nonribosomal peptide synthetase adenylation domains. Chemistry 6- Biology
7, 211-224 (2000).
Challis, G.L. & Ravel, J. Ceolichelin, a new peptide siderophore encoded by the Streptomyces
coelicolor genome: Structure prediction from the sequence of its non-ribosomal peptide
synthetase. FEMS Microbiology Letters 187, 111-114 (2000).
Lautru, S., Deeth, R.J., Bailey, L.M. & Challis, G.L. Discovery of a new peptide natural
product by Streptomyces coelicolor genome mining. Nature Chemical Biolog 1, 265-269
(2005).
Dietrich, J.A., Yoshikuni, Y., Fisher, K.J., Woolard, F.X., Ockey, D., McPhee, D.J.,
Renninger, N.S., Chang, M.C., Baker, D. & Keasling, J.D. A novel semi-biosynthetic route
for artemisinin production using engineered substrate-promiscuous P450(BM3). ACS
Chemical Biolog 4, 261-267 (2009).
Sach, N. in Optimizing Organic ReactionsVancouver, BC; 2008).
Kanan, M.W., Rozenman, M.M., Sakurai, K., Snyder, T.M. & Liu, D.R. Reaction discovery
enabled by DNA-templated synthesis and in vitro selection. Nature431, 545-549 (2004).
138
173.
174.
175.
176.
177.
178.
179.
180.
Robbins, D.W. & Hartwig, J.F. A simple, multidimensional approach to high-throughput
discovery of catalytic reactions. Science 333, 1423-1427 (2011).
Baxendale, I.R., Deeley, J., Griffiths-Jones, C.M., Ley, S.V., Saaby, S. & Tranmer, G.K. A
flow process for the multi-step synthesis of the alkaloid natural product oxomaritidine: A
new paradigm for molecular assembly. Chemical Communications, 2566-2568 (2006).
Petersen, T.P., Ritzen, A. & Ulven, T. A multistep continuous-flow system for rapid ondemand synthesis of receptor ligands. OrganicLetters 11, 5134-5137 (2009).
Goodell, J.R., McMullen, J.P., Zaborenko, N., Maloney, J.R., Ho, C.-X., Jensen, K.F., J. A.
Porco, J. & Beeler, A.B. Development of an automated microfluidic reaction platform for
multidimensional screening: Reaction discovery employing bicyclo[3.2.1]octanoid scaffolds.
Journalof Organic Chemistry 74, 6169-6180 (2009).
Koller, M.R., Emerson, S.G. & Palsson, B.O. Large-scale expansion of human stem and
progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures.
Blood 82, 378-384 (1993).
Jungbauer, A. & Peng, J. Continous bioprocessing: An interview with Konstantin
Konstantinov from Genzyme. BiotechnologyJournal6, 1431-1434 (2011).
Trost, B.M. The Atom Economy - The Search for Synthetic Efficiency. Science 254, 14711477 (1991).
Rawlins, M.D. Cutting the cost of drug development? Nature Reviews Drug Discovery 3,
360-364 (2004).
139
Appendix A: List of taxanes analyzed for mapping the taxane biosynthetic pathway
141
[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
1alpha,12alpha,1 2aalpha,1
2balpha]]-beta4290260
C47H51 N
(benzoylamino)-alphaView in
014
hydroxy-benzenepropanoic
Reaxys
acid, 6,12b-bis(acetyloxy)12-(benzoyloxy)2a4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 1-
33069-624; 10545404-4;
119323-97
6; 14755712-8;
148553-70
2; 14919723-9
C47H51014 heterocyclic
N
853.92
Journal; Dong, Huiru; Luo, Lina;
Zhou, Shan; Bi, Pengyu; Liu, Kailu;
Zhao, Jingcheng; Chemistry of
Natural Compounds; vol. 43; 4;
(2007); p. 478 - 480; Khimiya
Prirodnykh Soedinenii; 4; (2007); p.
392 - 393; View in Reaxys
RCINICONZ
NJXQFMZXODVAD
SA-N
dihydroxy-4a,8,13,a.13-1
f2aR[2alpha,4beta,4abeta,6bet
a,9alphaelphaR*,betaS*),1
1alpha,12alpha,I2aalpha,1
Z2
(benzoylamino)-alpha-
hydroxy-benzenepropanoic
acid, 6,12b-bis(acetyloxy)-
,Journal;
11923-97.
4290260 2balpha]-betaC47H51N
S (benzoylamino)-alpha014
e
hydroxy-benzenepropanoic
Rexsacid, 6,12b-bis(acetyloxy)12-(benzoyloxy)2a,3,44a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 1-239wc
dihydroxy-4a,8,13,13-III
[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
1alpha,12alpha,12aalpha,1
2balpha]]-beta-
V
I bnv
HI
306-2
433069 62-
C47H51014
N
RCINICONZ
hrcNJXQFMZXODVAD
1-;SA-N
ReansReax8;
2349-
ln
33069-62Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
1;Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
.mJournal;
00545413-57
C47H51N
014
853.92
C47H51014
N
heterocyclic
RCINICONZ
NJXQF-
yung dmr
ofTawin
MZXODVAD
SA-N
12-(benzoyloxy)-148553-7
2a.3,44a,5,6,9,10,11,2,12
2; 149197-
a, 12b-dodecahydro-4,1 1-
23-9
dihydroxy-4a,8,13,13[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
1alpha,12alpha,12aapha,1
1; (2004); p. 58 - 63; View in Reaxys
(a) washing
a raw
material
with
044RM)
119323-97
853.92
6147557- 85.2
12-8with
148553;70
2; 149197-.
23-9
2
4751014heterocyclic
N
heeoylcMZODVAD
RCINICONZ
NJXQF-
water; (b)
extracting
SA-N
organic
solvent a wet
RM; (c)
contacting
the wet RMI
33069-624; 105454-
6;14755714757
148553-70
2; 149197-
Patent; Chaichem Pharmaceuticals
International; US6759539; 831;
(2004); View in Reaxys
Joumal; Ketchum, Raymond E. B.;
0454;
1alpha,l2alpha,12aalpha,1
4290260 2balpha]]-betaView
(benzoylamino)-alphaC47H51N
hydroxy-benzenepropanoic
014
Reaxys
acid, 6,12b-bis(acetyloxy)12-(benzoyloxy)2a,3,4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 1I_ _
_ dihvdroxy-4a,8,13,13-
Journal of Natural Products; vol. 67;
33069-624 105454-
4290260 2balpha]]-betaC4751N
(benzoyamino)-apha04
view in hyrx-eznpoaoc
Reaxys hydroxy-benzenepropanoic
014
acid, 6,12b-bis(acetyloxy)12-(benzoyloxy)2a,3,4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 1dihvdroxy-4a,8,13,13[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
4290260 2balpha]]-betaC47H51N
View in (benzoylamino)-alpha014
hydroxy-benzenepropanoic
acid, 6,12b-bis(acetyloxy)12-(benzoyloxy)2a,3,4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,11_dihvdroxy-4a,8,13.13[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
1alpha,12alpha,12aalpha,1
Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
853.92
C47H51014 heterocyclic
N
RCINICONZ
NJXQFMZXODVAD
SA-N
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
23-9
33069-624
044-
119323-97
6; 14755712-8;
148553-70
RCINICONZ
853.92
C47H51014 heterocyclic
NMXDA
NM
SA-N
2; 14919723-9
I
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys 8 is a trademark owned and protected byElsevier Properties SA and used under license.
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
V4ewin
Reaxys
33069-624; 10454-
04~
1 2a]]-beta.
,2b
42902
2bapha]]-beta-119323-97.
4290260
14755C47H51N
(benzoylamino)-alpha014
hydroxy-benzenepropanoic
hyrx-ezeerp
0e14y 148553-70
12-8;
acid,
6,12b-bis(acetyloxy)-i
12-(benzoyloxy)2a,3,4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 1idihvdroxy-4a,8,13.13[2aR[2aalpha,4beta,4abeta,6bet
a,9alpha(alphaR*,betaS*),1
4290260
View n
SRexs
9
--
-
.
RCINICONZ
NJXQFMZXODVAD
SA-N
C4751014
N
heterocyclic
RCINICONZ
NJXQFMZXODVAD
ihvdroxv-4a.8.13.13-
[2aR33069-62[2aalpha,4beta,4abeta,6bet
4; 105454a,9alpha(alphaR*,betaS*),1
1alpha,12alpha,12aalpha,1
4;
05454
2balpha]]-beta119323-97
42n9y0mi26al0a
4H 1192 97
C4751N 6; 147557- 853.92
benzoylamino)-alphaV
hydroxy-benzenepropanoic
014
12-8;
acid, 6,12b-bis(acetyloxy)148553-70
2 14919712-(benzoyloxy)232a3,4,4a,5,6,9,10,11,12,12
23-9
a,12b-dodecahydro-4,1113dihvdroxy-4a.8.13
[2aR[2aalpha,4beta,4abeta,6bet
33069-62a,9alpha(alphaR*,betaS*),1
4; 10545404-4;
1alpha,12alpha,12aalpha,1
19~-'
2balpha]]-betaC47H51N
(benzoylamino)-alphaView0in hydroxy-benzenepropanoic
014
6; 147557- 853.92
12-8;
Reaxys
148553-70
acid, 6,12b-bis(acetyloxy)Ra
12-(benzoyloxy)2; 1491972a,3,4,4a,5,6,9,10,11,12,12
23-9
a,12b-dodecahydro-4,11dihvdroxy-4a8 13.13[2aR[2aalpha,4beta,4abeta,6bet
33069-62a,9alpha(alphaR*,betaS*),1
4 1054544;stems
1alpha,12alpha,12aalpha,1
04-4,
2balpha]]-betaC47H51N 119323-97
(benzoylamino)-alphaView in
6; 147557- 853.92
Reaxys acid, 6,12b-bis(acetyloxy)12-8;
OH
HO
HO
H
Reaxys
0
0
o
benzoyloxy-5,20-epoxy-9oxo-1,7,10,131-ene;
10P-deacetylbaccatin Ill;
10-0-deacetylbaccatin 111
~tetrahydroxytax-1
Journal; Kawamura, Fumio; Kikuchi,
Yoshinari; Ohira, Tatsuro; Yatagai,
Mitsuyoshi; Journal of Natural
Products; vol. 62; 2; (1999); p. 244 247; View in Reaxys
6aN of
Taws
0uspidta
Zdc
nelso
Japanese
C471-51
014
N
heterocyclic
C47H51014
N
heterocyclic
RCINICONZ
NJXQF-Sa
Joumal; Murakami, Ryo; Shi, Qingwen; Oritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580; View in
(199)
Reaxys
-
tZ=
MZXODVAD
SA-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
RCINICONZ
NJXQFXO ADvl4;2(19)p.11-13;
SA-N
C47H51014
N
heterocyclic
RCINICONZ
NJXQFMZXODVAD
SA-N
dihvdrox-4a8.1313HO
plde
oft
j
y#WTV ',Shigemori,
t
2 14919723-9
2-
12-(benzoyloxy)2a,3,4,4a,5,6,9,10,11,12,12
a,12b-dodecahydro-4,1 110-deacetyl baccatin Ill;
10-desacetylbaccatin
10-deacetylbaccatin 1ll;
10-Deacetyl baccatin;
1445248 (1S,2S,3R,4S,5R,7S,8S,10
View in R,13S)-4-acetoxy-2-
Journal; Aoyagi, Hideki; DiCosmo,
Frank; Tanaka, Hideo; Planta
Medica; vol. 68; 5; (2002); p. 420 424; View in Reaxys
slwfo
2; 14919723-9
33069-624; 10545404-4;
1alpha,12alpha,12aalpha,1
119323-97
2balpha]]-betaC47H51N 6; 147557- 853.92
(benzoylamino)-alpha014
hydroxy-benzenepropanoic
acid, 6,12b-bis(acetyloxy)148553-70S
12-(benzoyloxy)2; 1491972a,3,4,4a,5,6,9,10,11,12,12
23-9
a,12b-dodecahydro-4,11-
_
C47H51014
N
Eamlt f
Taxane-
containing
.ll; material is
w n inig chipped into
maerlpiW
small pieces
Patent; Phytogen Life Sciences Inc.;
and
inc;e
extracted (8h US2005/288520; Al; (2005); View in
Reaxys
3days) with
'
v
-edlibis
0
32981-865; 7162992
92993-544.599
4; 11550992-7
C29H36010 heterocyclic
YWLXLRUD
GLRYDRZHPRIASZS
A-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Kobayashi, Junichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
species of
organic
Yow plants
solvent,
cmpn
preferably
the-r
]methanol, (6-,
Ho
OH
H
Ho
0
H
H
O
Ho
OH
H
HO
HO
H;115509-
0
10-deacety baccatin 1ll;
10-desacetylbaccatin III;
10-deacetylbaccatin 111;
32981-8610-Deacetyl baccatin;
5; 716291445248 (1S,2S,3R,4S,5R,7S,8S,10 C29H360
92-010544.599
View in R,13S)-4-acetoxy-2Reaxys benzoyloxy-5,20-epoxy-94; 115509oxo-1,7,10,1392-7
tetrahydroxytax- 11-ene;
100-deacetylbaccatin IlIl;
10-0-deacetylbaccatin III
10-deacetyl baccatin III;
10-desacetylbaccatin li1;
10-deacetylbaccatin 111;
32981-8610-Deacetyl baccatin;
5;716291445248 (1S,2S,3R,4S,5R,7S,8S,10 C29H360
92-0View in R,13S)-4-acetoxy-210
92999-93Reaxys benzoyloxy-5,20-epoxy-94;
0
oxo-1,7,10,13tetrahydroxytax-1 1-ene;
1OP-deacetylbaccatin Ill;
10-0-deacetylbaccatin III
-\
0
baccatine Ill;
4732442 baccatin Ill;
47
VOoe
baccatin-il;
Reaxys
baccatin;
Baccatin I1;
YWLXLRUD
C29H36010
.3GLRYDRheterocyclic
A-N
5
C29H36010
.
heterocyclic
YWLXLRUD
GLRYDRZHPRIASZS
A-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
OVMSOCFB
DVBLFWVHLOTGQH
SA-N
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62: 6; (2003); p. 901 910; View in Reaxys
OVMSOCFB
DVBLFWVHLOTGQH
SA-N
Joumal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol. Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
92-7
27548-93C311H380 2 107711
81-3
586.636
C31H38011
heterocyclic
BACCIII
0
baccatine III;
baccatin I1;
4732442
o 473442baccatin-Ill;
View in baccatin;
Reaxys
ReaysBaccatin IlIl;
BACC Ill
27548-93C31 H380 27548-932;3107781-3
586.636
C31H38011
heterocyclic
27548-93C31H380 2; 31077- 586.636
81-3VHLOTGQH
11
C31H38011
heterocyclic
baccatine 1ll;
Oo
4732442 baccatin 1i;
View in baccati-ill;
Reaxys baccatin;
Baccatin Ill;
BACC Ill
10-deacetyl taxol(7a-OH);
7-epi-10-deacetyltaxol;
4290167 10-deacetyl-paclitaxel;
-ect
View in 10-deacetylpaclitaxel;
Reaxys 10-deacetyltaxol A;
10-deacetyl taxol;
10-deacetyltaxol
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
78432-776; 7845417-8;
125354-13
C
-8183C45H49N01
C45H49N
013
4; 133577- 811.883
3
34-1;
133577-35
2; 13550118-7
OVMSOCFB
DVBLFWSA-N
heterocyclic
TYLVGQKN
NUHXIPMHHARFCS
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Journal; Kawamura, Fumio; Kikuchi,
Yoshinari; Ohira, Tatsuro; Yatagai,
Mitsuyoshi; Journal of Natural
Products; vol. 62; 2; (1999); p. 244 247; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.;Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
4290167
View in
Reaxys
10-deacetyl taxol(7a-OH);
7-epi-1O-deacetyltaxol;
10-deacetyl-paclitaxel;
10-deacetylpaclitaxel;
10-deacetyltaxol A;
1O-deacetyl taxol;
10-deacetyltaxol
78432-776; 7845417-8;
C45H49N 125354-13
013
4; 133577- 811.883
34-1;MSA-N
133577-35
2; 13550118-7
C45H49NO1
3
heterocyclic
3'-N-(trans-2-methyl-2butenoyi)-3'-N8032151
V ew in
Reaxvs
debenzoylpaclitaxel;
chephalomannine;
caphalomannine;
taxol B;
cephalomannine
C45H53N 71610-009
014
831.914
C45H53014 heterocyclic
N
stems of
Japanese
yew Taxus
cuspiata
Sol.47
ZUcc.
TYLVGQKN
NUHXIPMHHARFCS
Identification(14)
DBXFAPJCZ Physical Data(1)
ABTDR- Spectra(12)
WBYYIXQIS Us/pl0ains
Bioactivity/Ecotox(40)
Use/Application(5)
A-N
35
Journal; Dong, Huiru; Luo, Lina;
Zhou, Shan; Bi, Pengyu; Liu, Kailu;
Zhao, Jingcheng; Chemistry of
vol. 43; 4; p.
Natural Compounds;
Compound; 4;o(2007;
Pauroyal
(2007); p. 478 - 480; Khimiya
Prodnykh Soedinenii; 4; (2007); p.
392 - 393; View in Reaxvs
40
3'-N-(trans-2-methyl-2butenoyl)-3'-N8032151 debenzoylpaclitaxel;
chephalomannine;
View in Cephalomannine;
caphalomannine;
C45H53N
014
71610-009
831.914
C45H53014
N
heterocyclic
DBXFAPJCZ
ABTDRWBYYlXQlS
A-N
n
taxol B;
cephalomannine
3'-N-(trans-2-methyl-2butenoyl)-3'-N8032151debenzoylpaclitaxel;
V032151 chephalomannineCephalomannine;
Reaxys ophalomannine;
C45H53014
N
C45H53N 71610-009
014
.
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
DBXFAPJCZ
ABTDRWBYYlXQIS
A-N
taxol B;
cephalomannine
3'-N-(trans-2-methyl-2butenoyl)-3'-Ndebenzoylpaclitaxel;
V 802 1chephalomannine;
VeinCephalomannine;
oA-N
C
Reaxy s caphalomannine;
taxol B;
Joumal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
Reaxys
2; e
Rki
C45H53N 71610-009
014
831.914
8194
C45H53014 heterocyclic
heeoycWBYlXlS
N
DBXFAPJCZ
ABTDR-
Journal; Murakami, Ryo; Shi, Qing5
4
A-N
emity (Elsevier);
(Elsev i; vol. 52; 8;
P
Phytochemistry
(1999); p. 1577 - 1580; View in
Reaxys
cephalomannine
3'-N-(trans-2-methyl-2butenoyl)-3'-Ndebenzoylpaclitaxel;
8032151 chephalomannine;
View in
Cephalomannine;
Reaxys caphalomannine;
C45H53N
04
014
71610-009
9
831.914
C45H53014
N
heeocci
heterocyclc
DBXFAPJCZ
ABTDRWBYYIXQISoHieyk;
WBYYlXQIS
A-N
taxol B;
cephalomannine
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
SMo
Japan p
yeShigemori,
cusphata
Sieb.et
u-
Journal; Kobayashi, Jun'ichi;
Hideyuki; Heterocycles;
eerccls
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Journal; Oshige, Masahiko;
Takenouchi, Mika; Kato, Yasutaro;
Kamisuki, Sinji; Takeuchi, Toshifumi;
Kuramochi, Kouji; Shiina, Isamu;
Suenaga, Yoshihito; Kawakita, Yoichi; Kuroda, Kazufumi; Sato,
Noriyuki; et al.; Bloorganic &
Medicinal Chemistry; vol. 12; 10;
(2004); p. 2597 - 2602; View in
Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
Journal; Murakami, Ryo; Shi, Qingwen; Oritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580; View in
Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
Journal; Kondo; Takahashi;
Yakugaku Zasshi; vol. 45; (1925); p.
861,874; dtsch. Ref. Nr. 524, S. 2;
Chem.Abstr.; (1926); p. 767; View in
Reaxys
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @is a trademark owned and protected byElsevier Properties SA and used under license.
2a,5a,1 00,140-tetraacetoxy
4(20),1-taxadiene;
taxuyunnanine C;
dark brown
callus
of TaKus
KFFHSFCO
05 02a,510p4-tetraacetoxy
ViewView
in
134in taxa-4(20),11(12)-diene;
Reaxys 2a,5a,10P,14p-
156127-34
C28H400
3
8
4(20),11(12)-diene;
2a,5a,10p,14p-tetra-c
acetoxy-4(20),11taxadiene;
2a,5a,10s,140-tetraacetoxy
4(20),1 1-taxadiene;
taxuyunnanine C:
sinenxan A;
2a,5a,1Op,14p-tetraacetoxy
7059440 taxa-4(20),11(12)-diene
C28H400 156127-34
view in 2a,5a,10p,14p8
3
Reaxys tetraacetoxytaxa4(20),11(12)-diene;c
20,50,10p,140-tetraacetoxy-4(20),1 1taxadiene_
2a,5a,10p, 14p-tetraacetoxy
4(20),1 1-taxadiene;
taxuyunnanine C;
sinenxan A;
2a,5a,10p,14p-tetraacetoxy
C28H400 156127-34
7059440 taxa-4(20),11(12)-diene;
View in 2a,5a,10p,14p8
3
Reaxys tetraacetoxytaxa-
54621
5461
C2814008
C840
lic
schcVCPDXWRA
KCGBBWSA-N
2a-deacetoxytaxinine J;
2-desacetoxytaxinine J;
6637340 2-deacetoxytaxinine J;
Viewin 2-desacetyltaxinine J;
Reaxys 2-deacetoxytaxinin J;
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
aosse)
-_--,-
Sendai
w
iduced from
young 3tems
of Tla
caus
504.621
C28H4008
isocychc
KFFHSFCO
KCGBBWVCPDXWRA
SA-N
cuspidta
Seb. et
d
Sendai,
504.621
C28H4008
.
.
isocychc
KFFHSFCO
KCGBBWVCPDXWRA
SA-N
cetf mitdme
Of Tgus
apid-*
(P93i) )rnf
Tutx
cy,
media
4(20),11(12)-diene;
2a,5a,10p,14p-tetraacetoxy-4(20),1 1taxadiene_
n
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
C37H460 119347-14
10
7
650.766
C37.46010
lic
socyci
MIJTXBNFQ
DJTPLPXORYUGN
SA-N
isocyclic
MIJTXBNFQ
DJTPLPXORYUGN
SA-N
sns o
Jaanese
asShigemori,
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
cinnamate 5
2a-deacetoxytaxinine J;
68373402-desacetoxytaxinine J;
6837340 2-deacetoxytaxinine J;
view in 2-desacetyltaxinine J;
Rea xys 2-deacetoxytaxinin J;
stems gi the
C37H460 119347-14.
10
650.766
7
C37H46010
cinnamate 5
yerTau
Aldb, 6t
&c:Reaxys
cafus O
e
of1ausn
3115696 (+)-taxusin;
View in taxusin
Reaxys Taxusin
C28H400 19605-80~
8
2; 11600225-6
504.621
C28H4008
isocyclic
SKJSIVQEP
KBFTJHUWILPJBS
A-N
Copyright @2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Joumal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
Journal; Miyazaki,M. et al.; Chemical
and Pharmaceutical Bulletin; vol. 16;
(1968); p. 546 - 548;;: Journal;
Sugiyama, Takeyoshi; Oritani,
Takayuki; Oritani, Takashi;
Bioscience, Biotechnology, and
Biochemistry; vol. 58; 10; (1994); p.
1923 - 1924; View in Reaxys
Joumal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Journal; Murakami, Ryo; Shi, Qingwen; Oritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580; View in
Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemor, Hideyuki; Heterocycles;
1111
vol. 47; 2; (1998); p.
- 1134;
View in Reaxys
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
0
stemsd the,
Japanese
yew Taxus
mapitt
Sieb. at
\
6853366
View in
Reaxys
taxinine A
C26H360 18530-098
1
476.567
C26H3608
OQXJKHGIA
ZCMBXSOIIJPRYSA
N
isocyclic
Zucc.
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemor, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
H0
0
dark brow
-i
6952531
695253
Reaxys
3'-N-hexanoyl-3'-Ndebenzoylpaclitaxel;
debenuyunasite0;
taxuyunnanin;
taxoi C
C46H57N 153415-45
14847.957
C46H57NO1
heterocyclic
BEHTXUBG
UDGCNQIEAAAIHOS
A-N
Identification(11)
Physical Data(3)
Spectra(9)
Bioactivity/Ecotox(12)
2of
15
15
u
16
16
-Yw
ca"us OAbM
inducedIfiiJournal;
flem
young
6952531
View in
Reaxys
3'-N-hexanoyl-3'-Ndebenzoylpaclitaxel;153415-45
847.957
taxuyunnansine;
3
014
taxuyunnanin;
taxol C
C46H57N1
heterocyclic
4IEAAAIH0S
of'T1;U
BEHTXUBG
UDGNA-N
t
Ccoeedin
Sennds
c
3'-N-hexanoyl-3'-N6952531 debenzoylpaclitaxel;
View in taxuyunnansine;
Reaxys taxuyunnanin;
taxol C
6952531
View in
3'-N-hexanoyl-3'-Ndebenzoypaclitaxel;
taxuyunnansine;
taxuyunnanin;
taxol C
C46H57N 153415-45
014
3
C46H57NO1
4
3'-Nhexnoy-3'NC46H57N 153415-45
C46H57NO1
01495
4
4Reaxys 3
.
.
heterocyclic
BEHTXUBG
UDGCNQIEAAAIHOS
A-N
(
BEHTXUBG
BHTXBG
UDGCNQEAAAHOS
A-N
a
6wlf
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
Natural Products; vol. 68; 4;
(2005); p.497 - 501; View n Reaxys
Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihimo;
Tsuruo Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
TOrval; Zamir, Lolita o.;Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
stefm of
6952531
View in
Reaxys
3'-N-hexanoyl-3'-Ndebenzoylpaclitaxel;
taxuyunnansine;
taxuyunnanin;
taxol C
C46H57N 153415-45
1
3
BEHTXUBG
UDGCNQ-
C46H57NO1
847.957
4
heterocyclic
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
IEAAAIHOS
A-Nieb
|
|~
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
2a,5a,10-triacetoxy-143(2'-methyl)-butysyloxy4(20),11-taxadiene;
2a,5a,1 00-triacetoxytaxa755733 4(20),11(12)-dien-14p-y 2C31 H460
methylbutyrate;
View in~5,0-ractx-43
Rexs2a,5a,100I-triacetoxy-14pReaxys
(2'-methyl)butyryloxy-
4(20),1 1-taxadiene;
2a,5a,100-triacetoxy-140(2-methyl-butyryloxy)14(20)1 1-taxadiene;
2a,5a,1 00-triacetoxy-14p(2'-methyl)-butysyoxy4(20),1 1-taxadiene;
2a,5a,100-triacetoxytaxa4(20),11(12)-dien-14p-yl 2-
7557334
View in methylbutyrate;
;
eays2a,5a,100-triacetoxy-14p-
546.701
C31H4608
isocyclic
A-N
546.701
C311H4608
isocyclic
8
1
Bioactivity/Ecotox(44)
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
USNOOZXS
USNOOZXSt;C3
AYFE
ALYFCE-S
ALLYKNSAS
A-N
(2'-methyl)butyryloxy4(20),1 1-taxadiene;
2a,5a,100i-triacetoxy-14p(2-methyl-butyryloxy)-
Y
20
ALLYKNSAS Spectra(8)
8
H6
C31H460
USNOOZXS Identification(9)
Phcal Data(S)
a2
ALYFCEAIYFCEPhysical
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
4(20)1 1-taxadiene;
2a,5a,10si-triacetoxy-140(2'-methyl)-butysyloxy4(20),1 1-taxadiene;
2a,5a,1 00-triacetoxytaxa7557334 4(20),11(12)-dien-140-yl 2C31H460
methylbutyrate;
V;ew
8
2a,5a,10-triacetoxy-14p(2'-methyl)butyryloxy1-taxadiene;
4(20),1
2a 5,5a,1
00-triacetoxy-140(2-methyl-butyryloxy)4(20) 1-taxadiene;
4633976
View in
Reaxys
1p-hydroxybaccatin
1p-hydroxybaccatin-l;
10-Hydroxybaccatin I
6173928
View in
10-deacetyl-7-epipaclitaxel;
10-deacetyl-7-epi-taxol A;
10-deacetyl 7-epi-taxol;
10-deacetyl-7-epi-taxol
Reaxys
14
C32H440 30244-372
14
546.701
C31H4608
socyclic
USNOOZXS
AIYFCEALLYKNSAS
A-N
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View inReaxys
652.693
C32H44014
heterocyclic
LUTPIRPNUI
NHFEVMBMCFSISS
A-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles:
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
TYLVGQKN
NUHXIP-
C45H49N1
013
811.883
3
heterocyclic
7-epi-10-deacetyl taxol,
10-deacetyl-7-epitaxol;
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
DIYBZAJCS
-N
-
7-epi-10-deacetyltaxol
Sa-hydroxy2a,7p,9a,10p,1 3apentaacetoxy-4(20),1 1gotaxadiene;
0
-a-hydroxy-
5
View in
Reaxys
C30H420 84652-332a,70,9a,100,13a5
11
pentaacetoxytaxa-4(20),1 1diene;
5a-decinnamoyltaxinine J;
2-decinnamoyltaxinine J;
decinnamoyl-taxinine J;
decinnamoyltaxinine J
578.657
C30H42011
isocyclic
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
NTRHFSRA
AXRZLCFJANQRQL
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
1I
o36
46
0~-.,pentaacetoxytaxa-4(20),11-
ddiene;
o
View
32-ir
Reaxys
5a-hydroxy2a,7P,9a,10p,13apentaacetoxy-4(20),1 1taxadiene;
dRHFSR135
279
p
9a ,10p ,1a -
C30H420 84652-338 5 2 -3
C3- H4 2
11
5
578 .657
Rsdcinn nSA-Naxinene
5a-decinnamoyltaxinine J;
2-decinnamoyltaxinine J;
decinnamoyi-taxinine J;
I
decinnamovltaxinine J
5a-hydroxy2a,70,9a,10p,13apentaacetoxy-4(20), 11taxadiene;
a-hydroxy60326335-ydoC30H420 84652-33- 578.657
view in 2a,7,9o,10P,13a5
11
Viewin pentaacetoxytaxa-4(20),11eaxys diene;
_______________________decinnamoyl-taxinine
C30 H4 20 11
A.X RZ LC
FJANQRQL
SA-N
iso cyclic
itthd
thTracy
t h e Ta xu
'~Journal
Se.e et
Zucc.
I
stemO
NTRHFSRA
C30H4201.1
icclic
C37H44011
isocyclic
AXRZLCFJANQRQL
SA-N
lata
Sieb. at
a
C37H440
11
664.75
Reaxs
SLJNSLIEGI
NNSE
UWUO- JI
UWUOQOJI
SA-N
SAN
yeJournal;
ol
taxinin B
.
C37H440
11
NNEE-
.
,fthe
ya
UWUOQQJI
SA-N
.
cc
2,9,1 0-triacetyl-5-0cinnamoyltaxicin-1;
5-cinnamoyltaxicin I
triacetate;
i
13452-36O-cinnamoyltaxicin I
C35H420 3; 148090triacetate;
72-6
10
5-cinnamoyltracetyltaxicin
I;
0-cinnamoytaxicin;
1-hydroxytaxinine;
5-Cinnamoyltaxicin-l 2,9,10-
622.712
C35H42010
5843093
View
O-cinnamoyltaxicin;
1-hydroxytaxinine;
5-Cinnamoyltaxicin-I 2,9,10triacetate
isocyclic
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
-
-
_____
____triacetate
2,9,10-triacetyl-5-0cinnamoyltaxicin-I;
5-cinnamoyltaxicin I
triacetate;
0-cinnamoyltaxicin I
triacetate;
Vein3148090cinnamyltriacetyltaxicin
.Reaxys
VBLNERPS
NINUBRFRS
A-N R
Murakami, Ryo; Shi, Qingwen; Oritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580; View in
-Reaxys
4W.
\4SLJNSLIEGI
taxinine B;
V7
5843093
View in
Reaxys
R"
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
-deinamoyaineJ
2decinnamoyltaxinine J;
decinnamoyltaxinine J;
J;
6837456
View in taxinine B;
RexstaxinineB
Rainns
iew
Reaxys
Journal; Shi, Qing-Wen; Petzke,
L.; Sauriol, Francoise; Mamer,
Orva l; Z amir, Lolita 0 .; Cana d ian
o.8;1
Jua
81; 1;
Chemiatry; vol.
offCeity
(2003); p. 64 - 74; View in Reaxys
C35H420 13452-3610
7
622.712
VBLNERPS
GWCFQJNINUBRFRS
NINUBRFRS
A-N
C35H42010
I
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
stems of
Japanese
yew Taxs
cdpidata
Slab.
Zucc.
Joumal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
2,q,1u-tnacetyl-5-O-
cinnamoyltaxicin-1;
5-cinnamoyltaxicin I
triacetate;
O-cinnamoyltaxicin I
5843093
triacetate;
View in
5-cinnamoyltriacetyltaxicin
Reaxys
--
1;
13452-36C35H420
3; 14809010
72-6
622.712 1C35H42010
isocyclic
VBLNERPS
GWCFQJNINUBRFRS
A-N
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
isocyclic
NNPJVZHKK
MTATLGQZIMJKVS
A-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
isocyclic
NNPJVZHKK
MTATLGQZIMJKVS
A-N
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
O-cinnamoyltaxicin;
1-hydroxytaxinine;
5-Cinnamoyltaxicin- 2,9,10
triacetate
2-deacetoxy-5-(3'dimethylamino-3'phenylpropionoxy)taxinine
6041004
View in
Vew
Reaxs
~L.
~-JL6041004
2-pdeacetoxyaustrospicatine; C39H53N 119777-80. 695.851
2'010
9
desacetoxyaustrospicatine;
2desacetoxyaustrospicatine;
desacetoxy-2'
austrospicatine
2-deacetoxy-5-(3'dimethylamino-3'phenylpropionoxy)taxinine
J;
2'p-
View in deacetoxyaustrospicatine;
Reaxys 2'desacetoxyaustrospicatine;
2desacetoxyaustrospicatine;
desacetoxy-2'
austrospicatine
---
I
C39H53N01
0
10pdeacetylcephalomannine;
7244187 10View in Deacetylcephalomannine;
Reaxys 10-deacetyltaxol B;
10deacetylcephalomannine
695.851
C39H53N 119777-80
9
010
C39H53NO1
0
II
C43H51N
013
76429-85-
1
789.877
C43H51NO1
3
.
heterocyclic
ADDGUHVE
43H51N 76429-851
013
10-
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
heterocyclicJPNWQZGJKAWTKTS
A-N
3
10p-hydroxy-2a,5a,14ptriacetoxy-4(20),1 1taxadiene;
10s-hydroxy-2a,5a,14p-
"o
-
0
Deacetylcephalomannine;
10-deacetyltaxol B;
10deacetylcephalomannine
GJKIWTKTS
A-N
10s3deacetylcephalomannine;
View in
Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
ADDGUHVE
JPNWQZ-
o
o
T
7553628 triacetoxytaxa-4(20),1 1View in diene;
10-desacetyl
taxuyunnanine C;
10-deacetyltaxuyunnanine
C;
10-deacetyltaxuyunnanin
I
C;
C26H380 157056-97- 462.584
7
8
C26H3807
isocyclic
RAKDXBHP Identification(9)
GCOTQG- Physical Data(3)
SFPMZPPX Spectra(5)
SA-N
Bioactivity/Ecotox(8)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
10
10
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
dark brown
2a,5a,100-triacetoxy-14p(2'-methyl-3'-hydroxybutyryoxy)-(20),11 7558016 taxadiene; -JGMCYView J 2a,5a,10s-triacetoxy-14ptaxaReaxys (3-hydroxy-2methylbutyryloxy)-taxa4(20),11-diene;
calus culure
of Taxus
FMPIEMVVE
Slb et
C31H460
9
562.701
C311H4609
isocyclic
IRWPHO
SA-N
Yew'
collected hn
-ryunanaxane;
Y 1-r
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
yunnanxane
2a,5a,1 Op-thacetoxy-143(2-methyl-t-hydroxy-14pbutyryloxy)-4(20), 1utaxadiene;
7558016 2axa,10-tnaetoxy-14View in
methylbutyryloxy)taxaReaxys
methylbryoxy-taxa-POL
I*i
from
stams
yo
O(Taggs
FMPIEMVVE
JGMCY-
031H460
562.701
C31H4609
isocyclic
cdita
IRWPHO
et
Zucr.,
colleteden
Sndal
SA-N
4(20),11-diene;
yunanaxane;
yunnanxane
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View n Reaxys
dirk brown
2a-acetoxytaxusin;
6553639 2-acetoxytaxusin;
View in 2o,5a,9a,100,13aReaxys pentaacetoxy-20(4),11taxadiene
C30H420
10
562.657
C30H42010
isocyciic
CNWPSHA
WQVTMLNRQHYBHEC
SA-N
Identification(5)
Physical Data(1)
Spectra(4)
Bioactivity/Ecotox(6)
2
9
codected In
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p.497 - 501; View in Reaxys
andel
youtems
of 2 eiyus
6553639
View in
Reaxys
2a-acetoxytaxusin;
2-acetoxytaxusin0
2a,5a,9a,10p.13a20YBE
So 0 113 30
pentaacetoxy-20(4),1 1taxadiene
C31140
10
562.657
C30H-42010
isocyclic
CNWPSHA
WQVTMLNRHBE
SA-N
Sieb. at
uc
colectd in
Jawgin
(stimulated
6553639
View in
Reaxys
0
0
?
"*0H
0
OH
-.
2a-acetoxytaxusin;
2-acetoxytaxusin;
2a,5a,9a,10p,13apentaacetoxy-20(4),11taxadiene
C30H420
10
1-hydroxyltaxinine A;
2a,9a,1 0p-triacetyl taxicin-1;
triacetyl-5-decinnamoyl
6832866 taxicin I;
Viw
triacetyl-5C26H360 158137-46
View in decinnamoyltaxicine 1;
9
3
Reaxys triacetyl-5decinnamoyltaxicin I;
5-hydroxytriacetyltaxicin I;
5-hydroxytriacetyltaxicin-l
562.657
492.566
C30H42010
isocyclic
C26H3609
.
.
isocyclic
CNWPSHA
WQVTMLNRQHYBHEC
SA-N
HMVNDOFA
GCGZSAOPHIHEFFS
A-N
and used under license.
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA
Japage
yOghuMg
cusi
aa
the Taxus
Slab. et
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Tomida, Akihiro;
Takao;
Yamori,
aoi
aa;TmdAiio
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Saurio, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
-V(
;
1
7842874
View in
Reaxys
0oH
5-hydroxytaxinine B;
taxuspine F;
taxuspin F
r
C28H380
10
*i
T
534.604
r
T
C28H380110 isocyclic
1
1
1
1
L~:i
CWMUWKW
ZNUFEDYLCFONYPQ
SA-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
H
1p-dehydroxybaccatine VI;
8247724 1p-dehydroxybaccatin VI;
View in 1-dehydroxybaccatin VI;
Reaxys 1-deoxybaccatin VI;
baccatin VI
10-dehydroxybaccatine VI;
8247724 10-dehydroxybaccatin VI;
View in 1-dehydroxybaccatin VI;
Reaxys 1-deoxybaccatin VI;
baccatin VI
C37H460
13
698.764 | C37H46013
I heterocyclic
JHPBZGSKE
Identification(8)
SVROCPhysical Data(1)
IGIVFXLZSABioactivity/Ecotox(2)
N
10
9
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; H-irose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View inReaxys
1$
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
JHPBZGSKE
3H46
13
698.764
C37H4601-3 heterocyclic
SVROC-
IGIVFXLZSA
N
I
8247724
View in
Reaxys
10-dehydroxybaccatine VI;
1p-dehydroxybaccatin VI;
C37H460
1-dehydroxybaccatin VI;
13
1-deoxybaccatin VI;
baccatin VI
698.764
C37H46013 heterocyclic
JHPBZGSKE
SVROCIGIVFXLZSA
N
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
RIVHKUMN
QRHVLQ-
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
5a-cinnamoyloxy9a,100,1 3a-triacetoxytaxa4(20),11-diene;
5a-cinnamyloxy115810-14
4775260 9a, 100,13a-triacetoxytaxa592.73
C35H440 5; 136031View in 4(20),11-diene;
157WPPRULKU
15-7
88y
J;
Rexs2,7-deacetoxytaxinine J;
2-desacetoxytaxinine E;
2-desacetoxytaxinine;
9a, 10p,13a-triacetoxy-5acinnamoyoxytaxa-4(20),1 1-
C35H4408
isocyclic
SA-N
diene
5a-cinnamoyloxy9a,1 0s,1 3a-triacetoxytaxa4(20),11-diene;
5a-cinnamyloxy115810-14
4775260 9a, 100,1 3a-triacetoxytaxaC35H440 5; 1
View in 4(20),11-diene;
Reaxys 2,7-deacetoxytaxinine J;
8
12-desacetoxytaxinine E;
2-desacetoxytaxinine;
9a,10p,13a-triacetoxy-5acinnamoyoxytaxa-4(20),1 1diene
592.73
C35H4408
isocyclic
RIVHKUMN
QRHVLQWPPRULKU
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; at al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxys
2a,5a-dihydroxy70,9a,10p,1 3a-tetraacetoxy
4(20),1 1-taxadiene;
2a,5a-dihydroxyo2437p,9a,100,,13a-tetraacetyl6027 3 taxa-4(20),11-diene;
Reeaxys 2a-deacetyl-5adecinnamoyltaxinine J;
1DOR
C28H400 119777-84
3
10
536.62
C28H40010
isocyclic
BACKFCRA
HXFXCHLNLYLEBQS
A-N
2-deacetoxy-5decinnamoytaxinin J;
2-deacetyl-5_decinnamoyltaxinine J:
2a,5a-dihydroxy7p,9a,1 06,13a-tetraacetoxy
4(20),1 1-taxadiene;
2a,5a-dihydroxy0106027483
View in
Rewxin
-
Reaxy
7j,9a,10),13o-tetraacetytaxa-4(20), 11-diene;
2a-deacetyl-5adecinnamoyltaxinine J;
BACKFCRA
.
C28H400 119777-84
3
10
.
HXFXCH.
LNLYLEBQS
A-N
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita .; Canadian
Journal of Chemistry; vol. 81; 1;
2003); p. 64 - 74; View in Reaxys
thTa
40M
Sieb. e(
sms of
Japanese
yew Taus
Journal; Kobayashi, Jun'ichi;
Sieoi
ieui
-eeoyls
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Sieb. ot
2c
2-deacetoxy-5-
decinnamoyltaxinin J;
2-deacetyl-5decinnamoltaxinine J_
8179958 baccatine IV;
View in baccatin IV
Reaxys
C32H440
14
652.693
C32H44014 heterocychc
CCJGGIKEF
AWRENWBPIOOJS
SA-N
(P9MF) W
Taxus
"
PG
8179958
View in baccatine IV;
Reaxvs baccatin IV
C32H440
14
652.693
C32H44014
heterocyclic
eaxysSA-N(2003);
CCJGGKEF
AWRENWBPIOOJS
~
~910; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
footed
the
p. 64 - 74; View in Reaxys
(a) washing
0
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 -
ce4iultpes
o axp
a raw
~material
r OOS
c.
14282586
.
C31 H400
View in 9-dihydro-13-acetylbaccatin
Viwi
-iyr-310
Reaxyis
RexsSA-N
572.653
IWYSOZLAG
HRRNQVGTKHVQN
SAN
C31H40010
- .water;
.
(RM) with
(b)
Patent; Chaichem Pharmaceuticals
extracting1
Inentinl
Intemational; US6759539; 61;
an
with
(04;Ve
nRay
Reaxys
View in
ranc(2004);
organic
solvent a wet
RM; (c)
contacting
the wet RM
*
.Reaxys
10-0-Acetyl-5-0cinnamoyltaxicine I;
5-cinnamoyl-1 010
cin
5840749 5View ir acetyltaxicin I;
5-0-cinnamoyltaxicin I;
cinmt ;WANJournal
Rx
5-Cinnamoyl-1 0-
C31H380 139126-35
8
5
538.638
C31H3808
isocyclic
NQSA
NGEIXLIYAS
WDWXCWQ
WWCQJua
acetyltaxicin-1
Copyright @2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
tooted
utnso
tns
the
&
. t2003);
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita O.; Canadian
o.8;1
81; 1;
offCeity
Chemistry; vol.
(AS p. 64 - 74; View in Reaxys
2a,5a,10s-triacetoxy-14ppropionyloxy-4(20),1 17556128
C29H420
taxadiene;
View in
8
2a,5a,1 0p-triacetoxy-14pReaxys
propionyloxytaxa-4(20), 11diene
2a,5a,100-triacetoxy-14ppropionyloxy-4(20), 117556128
C29H420
taxadiene;
View in
8
2a,5a, 10p-triacetoxy-14pReaxvs
propionyloxytaxa-4(20),1 1diene
2a,5a,10s-triacetoxy-14ppropionyloxy-4(20),1 17556128
C29H420
taxadiene;
View in
8
2a,5a,100-triacetoxy-140Reaxys
propionyloxytaxa-4(20), 11diene
7685130
View in baccatin VI
Reaxys
C37H460
14
518.648 | C29H4208
518.648
518.648
714.764
isocyclic
ZWQAAAFH
Identification(7)
PAEGHUSpectra(3)
WUJYLJESS
Bioactivity/Ecotox(5)
A-N
7
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
isocyclic
ZWQAAAFH
PAEGHUWUJYLJESS
A-N
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamor, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
isocyclic
ZWQAAAFH
PAEGHUWUJYLJESS
A-N
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
C37H46014 heterocyclic
UJFKTEIDO
RFVQSSKNQRQHZ
SA-N
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Saunol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
C29H4208
C29H4208
7685130
View in baccatin VI
Vew ibc14
ReaxysSAN
C37H460
714.764
C37H46014 heterocyclic
UJFKTEIDO
RFVQSSKNQRQHZ
SA-N
7685130
View in baccatin VI
Reaxys
C37H460
14
714.764 | C37H46014 heterocyclic
UJFKTEIDO
RFVQSSKNQRQHZ
SA-N
Identification(6)
Physical Data(1)
Spectra(3)
Bioactivity/Ecotox(1)
Copyright @2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
1
7
2514274 taxinine;
5-0-Cinnamoyl-taxicin-IlView in
acetat;3835-52-7
Re
Taxinin
C35H420
socyclic
.
BAYHEZUZ
RPMUDMKMEJJDELS
A-N
the brancs
OfTxus
Identification(8)
Physical Data(2)
Spectra(4)
Bioactivity/Ecotox(2)
cupdata
Sieb et Zucc.
taxa-4(20),11-diene2a,5a,70,9a,100,13ahexanol hexaacetate;
2a,5a,70,9a,10p,13aVOUULHDF Identification(7)
PJAWEH- Physical Data(1)
Spectra(6)
Bioactivity/Ecotox(3)
SA-N
6036879 hexaacetoxytaxa-4(20),1 1C32H440 27854-046036879 diene;
Vew in hexaacetoxy12
2
2a,5o,7P,9a,100,13a
taxadiene-4(20),1 1;
taxa-4(20),1 1-dien2a,5a,7p,9a,10P,13ahexaacetate'____
cWI
06bres
Ofaltas
6
2
226KYYDYRBA
6
spida
P93AF
Taog
mej0tcv.
Tax= 3Journal;
C1I00128-2162489-62
C31H400
5
11
7455839
View in taxuspine L
Reaxys
C32N460
3
13
588.652
C31H40011
heterocyclic
YLAVPGTYT Identification(5)
Physical Data(4)
CSGNICSI4
(2
UFJLMRSIS Spectra(10)
A-N
Bioactivity/Ecotox(2)
638.709
C32H46013
isocyclic
6
-~b
Z
taxuspine L
C32H460
13
638.709
C32H46013
isocyclic
NSFKWNXC
HYPDITPNQAOHGV
SA-N
7455839
View in taxuspine L
C32H460
13
638.709
C32H46013
isocyclic
NSFKWNXC
HYPDITPNQAOHGV
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
t
.
Morita, Hiroshi; Wei, Lan;
Gonda, Akira; Takeya, Koichi;
Itokawa, Hideji; Phytochemistry
(Elsevier): vol. 46; 3; (1997); p. 583 586; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
HYP
CIT
HYPDITPNQAOHGV
KSA-N
7455839
Reaxys
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
_____
7157364 10-deacetyl-9-dihydro-13ybacai II;;
view Vie acetylbaccatin
Reaxys 13-Acetyl-10-deacetyl-9dihydrobaccatin III
View in
Reaxys
Journal; Konda, Yaeko; Sasaki,
Tomomitsu; Sun, Xue-Long; Li, Xian;
Onda, Masayuki; et al.; Chemical &
Pharmaceutical Bulletin; vol. 42; 12;
(1994); p. 2621 - 2624; View in
Reaxys
y*
Journal; Kobayashi, Jun'ichi;
Shigemor, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayshi, Jun'ichi;
Tetrahedron; vol. 52; 7; (1996); p.
2337 - 2342; View in Reaxys
8024907 9a-deacetyltaxinine;
View in 2,9-dideacetyltaxinine;
Reaxys 9-deacetyltaxinine;
taxinine NN-3
ye
C33H400
8
9a-deacetyltaxinine;
8024907 2,9-dideacetyltaxinine;
Rewxy
isocyclic
C33H400
8
9-deacetyltaxinine;
taxinine NN-3
8024907 9a-deacetyltaxinine;
2,9-dideacetyltaxinine;
84
ein
564.676 1 C33H4008
8
9-deacetyltaxinine;
taxinine NN-3
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
CZLWREWI
XSHVJDAYFFSUKU
SA-N
Journal; Sakai, Jun-ichi; Sasaki,
Hirofumi; Kosugi, Katsuhiko; Zhang,
Shujun; Hirata, Naonori; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi;
Heterocycles; vol. 54; 2; (2001); p.
999 - 1010; View in Reaxys
SA-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
isocyclic
NQINDQGQ
JLYFLPBRGCZQG
SA-N
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
isocyclic
NQINDOGQ
NIDG
JLIYFLPBRGCZQG
SA-N
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxvs
564676
C334008
.
isocyclicAYFFSUKU
608.729
C35H4409
C35H4409
C33H400
CZLWREWI
XSHVJDAYFFSUKU
SA-N
CZLWREWI
XSHVJD-
9,100,13a-triacetoxy-5a-
-Reaxys
8125199 (cinnamoyloxy)taxaView in 4(20),11-dien-2a-ol;
taxezopidine G;
taxezopidin G;
taxinine NN-1
C35H440 205440-22
8
9
3a-triacetoxy-5a9,100,131
(cinnamoyloxy)taxa8125199
View in 4(20),11-dien-2a-ol;
Reaxys taxezopidine G;
taxezopidin G;
taxinine NN-1
-hydroxytaxusin;
8812346
e23i6 5,9a,1003,13oa
dien-140-ol
C35H440 205440-22
8
9
608.729
CKKORGV
1-
C28H400
520.62
C28H4009
isocyclic
Identification(6)
WCAXDRR- Physical Data(2)
SA-N
Bioactivity/Ecotox(3)
Copyright 1 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
2
6
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
4
a
yunnanxane;
2a,5a,1 0s-tracetoxy-14p[3(S)-hydroxy-2(R)-methyl9459194 butyryl]oxytaxa-4(20),11View in diene;
Reaxys 2a,5a,10s3-triacetoxy-14p(3-hydroxy-2methyi)butyryloxytaxa4(20),11-diene
C31H460 139713-81
9
8
562.701
C31H4609
uspensen
cel utures
o Taxus
FMPIEMVVE
JGMCYYYURHUSP
SA-NTaus
isocyclic
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901910; View in Reaxys
(P9A) *n
media
Hk i
dark br3wn
C32H440 30244-35baccatin 1
1678159
16ew8in baccatin1
Reaxys 1p-hydroxybaccatiniwinbacctin13
0SA-
636.694
C32H44013
.6ey
heterocyclic
NHMSEMKT
ntification(4)
DAYSGWHIQOLQNM Physical Data()
Spectra(2)
heterocyclic
NHMSEMKT
DAYSGWHIQOLQNM
SA-N
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Joumal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
et
5
stgems
View in baccatin 1;
Rewys in-baccatin
cti13
C32H440 30244-350
636.694
C32.44013
cesata-
Sjjab,
c
6
i
Joumal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
Si'
1678159 baccatin 1;
View in baccatin
Reaxys 11-hydroxybaccatin I(
7060991
View in
Reaxys
View in
Reaxys
2a,5a,10p-tracetoxy-14p[(S)-2-methylbutyryloxy]4(20),11-taxadiene
N-butyryl-Ndebenzoylpaclitaxel;
taxcultine;
taxol D
C32H440 30244-3513
0
C31H460
6
C44H53N
014
.
546.701
819.903
C31H4608
isocyclic
C44H53NO1
4
heterocychc
T
NHMSEMKT
DAYSGWHIQOLQNM
SA-N
USNOOZXS
AYFCEXQOWVITF
SA-N
SA-N
SXPIMOCR
RJUHJYMNLIZOKAS
A-N
1
T U
.
11923
Identification(3)
Physical Data(1)
Spectra(7)
Bioactivity/Ecotox(4)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Japane
yew Takus
5
5
ddts
S1064
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Journal; Sugiyama, Takeyoshi;
Oritani, Takayuki; Oritani, Takashi;
Bioscience, Biotechnology, and
Biochemistry; vol. 58; 10; (1994); p.
- 1924; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
00
7613305
View3in taxuspin V;
in taxuspine V
Reaxys
""ew
0
00r
C30H420
13
610.656
C30142013
heterocyclic
610.656
C30142013
heterocyclic
KLIDFAOXP Identification(5)
CGARX- Physica at()
RRHJOLCQ Physical Data(4)
SA-N
Spectra(8)
1
5
HO
0
"""
7
3
V7ewin taxuspin V;
7613305
Reaxys
7613305
taxuspin V;
View in taxuspin v
taxuspine V
C30H420
C301-420
C3
13
610.656
C30H42013
heterocyclic
KLIDFAOXP
CGARXRRHJOLCQ
SAN
Joumal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
1111
vol. 47; 2; (1998); p.
- 1134;
View in Reaxys
KLIDFAOXP
CGARXCRXRRHJOLCQ
SA-N
Joumal; Hosoyama, Hirokazu;
Inubushi, Akiko; Katsui, Takie;
Shigemori, Hideyuki; Kobayashi,
Jun'ichi; Tetrahedron; vol. 52; 41;
(1996); p. 13145 - 13150; View in
Reaxys
2a,5a,100-tracetoxy-14p7672134 isobutyryloxy-4(20),1 1C30H440
View in taxadiene;
8
Rexs2a,5a,10s-triacetoxy-140Reaxys
isobutyryloxytaxa-4(20), 11diene
UXTLOYZKA
532.675
C30H4408
isocyclic
VGZJOldentification(6)
ATYYVORY Bioactivity/Ecotox(5)
SA-N
532.675
C30H4408
isocyclic
Joumal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Joumal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View in Reaxys
c
UXTLOYZKA
VGZJOATYYVORY
SA-N
Joumal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
diene
2a,5a,10p3-triacetoxy-14p7672134 sobutyryloxy-4(20),1 1C30H440
View in taxadiene;
2asa,5 0-triacetoxy-14p8
Reaxys isobutyryloxytaxa-4(20),1 1diene
532.675
C30H440
5
Joumal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Joumal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
UXTLOYZKA
VGZJOATYYVORY
SA-N
2a,5a,1 0s-triacetoxy-14p7672134 isobutyryloxy-4(20),1 1C30H440
View in taxadiene;
8
Vewy'~2a,5a,10p-triacetoxy-14pReaxys isobutyryloxytaxa-4(20),1 1-
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
ic
Copyright @2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Rem of
IoWABUTQVF
Vie in taxuspine G
Reaxys
0
-
C24H340 163979-22.
7434.53
C24H3407
isocyclic
cuspiata
Sieb. at
IIO
H
uc
H
OH
H
2a,5a,1 0p-tracetoxy-taxa4(20),11-diene;
2a,5a,10s-triacetoxy9449731 4(20),11-taxadieneView in 2a,5a,10p-triacetoxytaxaReax
420)11-diene;
0
C26H380 444307-63
6
5
446.584
C26H3806
isocyclic
SWONFRKO
CZLVGRFIXWLCJPS
A-N
identification(8)
Physical Data(2)
Spectra(6)
Bioactivity/Ecotox(10)
2a,5a,9a,100,140p
1IDOQ-Pyia
7655 pentaacetoxy-taxa-4(20),1
dKim,
7675957
C301-420
View in diene;
10
2a,5a,9a,10p,140Reaxys pentaacetoxytaxa-4(20), 11-
ousodat
25
5
(P93AF
Taxus x
media
ev.
suspenion
ceu *uues
562.657
C301-42010
isocyclic
MDPMMMY Identification(5)
aa3
R YMJH- Pscal(9) ()
ZARNYMJH Spectra(9)
Bioactivity/Ecotox(1)
SA-N
Hid
Te
0
o
o
5a-hydroxy-70,9a,10pView in
Reaxys
triacetoxy-4(20),11(12)taxadien-13-one;
taxuspinanane K
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Ta
z.s
moda eg
diene
0
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901910; View in Reaxys
ofTaus
4_
deacetoxytaxuyunnanine C
oo
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Japanese
Identification(5)
TDOH- Physical Data(2)
YCPIXCPXS Spectra(1)
A-N
Bioactivity/Ecotox(8)
C26H360 198631-40
8
2
476.567
C26H3608
isocyclic
NRJCBPQE
LYLDFNVCTMJJASS
A-N
Identification(6)
Physical Data(1)
Spectra(6)
Bioactivity/Ecotox(2)
0Journal;
14
Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; et al.;
Phytochemistry (Elsevier); vol. 48; 5;
(1998); p. 857 - 862; Viewin Reaxys
T
4
'OH
H
Ji
8028189
View in
Reaxys
taxinine E
8028189
View in taxinine E
Reaxye
C37H460
10
C37H460
10
650.766
650.766
C37H46010
C37H46010
isocyclic
isocyclic
XBKVZMYB Identification()
DASNNMX Physical Data(1)
Bioactivity/Ecotox(1)
SA-N
XBKVZMYB
DANNSuAp-dat
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
.
1
4
4
yew Tas
-Journal;
Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
of Chemistry; vol. 81;1;
Journal
(2003); p. 64 - 74; View in Reaxys
Kobayashi, Jun'ichi;
Journal;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
0
H
2a,9a,100,13a-tetraacetoxy
8094274
taxa-4(20),11 -dien-5o-ol;
C28H400 218937-151 520.62
View in
5-decinnamoyltaxinine E;
Reaxys
2-acetyltaxinine E
C28H4009
isocyclic
UVSUZWKQ
LMVQVCTSKXOI-Z
SA-N
heterocyclic
OUZGCGZM
ZBOCBHBTJWADHW
SA-N
Journal; Shi, Qing-Wen; Oritani,
Takayuki; Horiguchi, Tohru;
Sugiyama, Takeyoshi; Murakami,
Ryo; Yamada, Teiko; Bioscience,
Biotechnology, and Biochemistry;
vol. 63; 5; (1999); p. 924 - 929; View
in Reaxys
HK
0
[8
6
6826
View in
Reaxys
7-epi-10deacetyltaxuyunnanine
7152991
View in
Reaxys
2-deacetyl-5-decinnamoyltaxinine E;
C26H380
28
deacetydecinnamoyltaxinin
e
7403605
Vew in
Reaxys
Reaxys
C44H55N
013
7,2'didesacetoxyaustrospicatin
37H51N
72
C37H510
e
805.92
478.583
C44H55NO1
3
C26H3808
isocyclic
C37H51N8
socyclic
E
637.814
Identification(3)
Physical Data(2)
Spectra(6)
Bioactivity/Ecotox(1)
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; et al.
Planta Medica; vol. 64; 2; (1998); p.
183 - 186; View n Reaxvs
3
JTBBGPIYD
SKLLQIdentification (5)
FECRNSTJS Physical Data(2)
A-N
Spectra(5)
A-N
3
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
CBAKLDDY Identification(5)
QIIIJTPhysical Data(5)
CODWPZBD Spectra(3)
3
Shigemori, Hideyuki; Heterocycles;
SA-N
Journal; Kobayashi, Jun'ichi;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Bioactivity/Ecotox(7)
I
7455224
View in taxuspine K
"e
Reaxys
View in
Reaxys
taxuspine K
C30H420
201YBASNLMA
7455224GTJOVEHIH
130420
13
610.656
C30H42013 heterocyclic
610.656
C30H42013 heterocyclic
GTJOVEHIH Identification(4)
MYRMR- Physical Data(1)
Spectra(6)
SA-N
Bioactivity/Ecotox(7)
MYRMR-A
YBASNLMA
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
3
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayshi, Jun'ichi;
Tetrahedron; vol. 52; 7; (1996); p.
2337 - 2342; View in Reaxys
Journal; Bai, Jiao; Ito, Nobuhide;
Sakai, Junichi; Kitabatake, Mutumi;
Fujisawa, Hajime; Bai, Liming; Dai,
Jungui; Zhang, Shujun; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Ando, Masayoshi; Journal
of Natural Products; vol. 68; 4;
(2005); p. 497 - 501; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Tetrahedron; vol. 52; 37; (1996); p.
12159 - 12164; View in Reaxys
Journal; Kosugi, Katsuhiko; Sakai,
Jun-ichi; Zhang, Shujun; Watanabe,
Yusuke; Sasaki, Hirofumi; Suzuki,
Toshio; Hagiwara, Hisahiro; Hirata,
Naonori; Hirose, Katutoshi; Ando,
Masayoshi; Tomida, Akihiro; Tsuruo,
Takashi; Phytochemistry (Elsevier);
vol. 54; 8; (2000); p. 839 - 846; View
in Reaxys
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bloorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View ?n
Reaxys
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxys
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
0
O
Ao o k
8046312 taxezopidine A;
View in taxezopidine
Reaxys taxezopidine
C26H360
9
492.566 | C26H3609
OFABTNFZ
WMLARJFDRWJUCO
SA-N
isocyclic
ldentification(4)
Physical Data(2)
Spectra(4)
Bioactivity/Ecotox(1)
1I
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
3
O
i
0
.OFABTNFZ
8046312
View in taxezopidine A;
Reaxys taxezopidine
C26H360
9
492.566
C26H3609
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Tetrahedron Letters; vol. 38; 43;
(1997); p. 7587 - 7588; View in
Reaxys
WMLARJFDRWJUCO
SA-N
isocyclic
H
e
N-mety -paclitaxel;
C481-153N
8797C48H53NO1 heeoccicJTWBC
Reaxys
)
8300796 4-DAB;
Il:
View in 10-deacetylbaccatin Il
Reaxys 4-deacetylbaccatin III
5a-hydroxytaxa9428505 4(20),11(12)-diene;
View in 5o-hydroxytaxa-4(20),1 1.
=
UATZAEJDB Identification(5)
Pyscl 1Data(3)
SA-N
C29H36O 189006-03
10
9
C20H320
~
544.599
288.473
2H61
eeoylcUHFPHEC29H36010 heterocyclic
C20H320
2
Journal; Kawamura, Fumio; Kikuchi,
Yoshinari; Ohira, Tatsuro; Yatagai,
Mitsuyoshi; Journal of Natural
Products; vol. 62; 2; (1999); p. 244 247; View in Reaxys
KRLGUZQQ
ZHPRIASZS
A-N
QHDGSWA
QHGWA Identification(6)
ZNWaP
Spectra(3)
v
iatvy/coxl
A-N
isocyclic
Reaxys diene;
Htaxa-4(20),11(12)-dien-5-ol
OH
3
Bioactivity/Ecotox(1)
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; et al.;
Phytochemistry (Elsevier); vol. 48; 5;
(1998); p. 857 - 862; View in Reaxys
3
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.;Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
3
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
H
I
9460766 9-dihydrobaccatine 111;
Ri60ax s7r6xat6l
Reaxys hydroxybaccatin 11
NIAII
NEAUUILPX
C31H400
588.652
C31H40011
heterocyclic
FOJJES Identification(5)
A-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
2
C37H49N
09
6837816
View in taxine II
Reaxys
2a,5a,9a-trihydroxy7150289 10,1 3a-diacetyl-taxaC24H360
view ii 4(20),11-diene;
Re7DvF 2",
10P,1 3a-diacetoxy-4(20),11
diene
oH
0
O
H
651.797
436.546
C37H49NO9
C24H3607
isocyclic
isocyclic
UJARNTWL
SVMUJF- Identification(5)
QVKFSLQV Bioactivity/Ecotox(6)
SA-N
QBIXMFOV Identification(5)
MVUJGG- Physical Data(1)
Spectra(5)
SA-N
2
stems of the
Japamse
yew Taxus
cuspdata
Sieb. et
Zucc.
Journal; Kobayashi, Jun'ichi;
Ogiwara, Aya; Hosoyama, Hirokazu;
Shigemori, Hideyuki; Yoshida,
Naotoshi; et al.; Tetrahedron; vol. 50;
25; (1994); p. 7401 - 7416; View in
Reaxvs
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
2003); p. 64 - 74; View in Reaxys
2
Si(bet
Zucc.
H
OH
OH
7238395 70-acetoxytaxinine;
View in taxuspine F
Reaxys
C28H380
10
C37H440
7242009
View in taxinine B
Reaxys
7788786
77878
View in taxuspine ZLTHGSNFJS
Reaxys
C37H51N
534.604
664.75
60
653.813
C28H38010
isocyclic
SLJNSLIEGI
NNEEYIRYCUEJS
A-N
C3744011
C37H51N09
isocyclic
Ohe
Japanese
yew Tagte
cusidta
CWMUWKW
ZNUFEDYLZBAJJJLSA
N
Identification(4)
Physical Data(2)
Spectra(4)
Bioactivity/Ecotox(1)
fI
cullidala.
stemKof X B
jXiao-xia;
Yew
CKXBHBNB
EFREKTTGNJWo
A-N
Journal; Kobayashi, Jun'ichi;
Inubushi, Akiko; Hosoyama,
Hirokazu; Yoshida, Naotoshi; Sasaki,
Takuma; Shigemori, Hideyuki;
Tetrahedron; vol. 51; 21; (1995); p.
5971 - 5978; View in Reaxys
Journal; Konda, Yaeko; Sasaki,
Tomomitsu; Sun, Xue-Long; Li, Xian;
Onda, Masayuki; et al.; Chemical &
Pharmaceutical Bulletin; vol. 42; 12;
(1994); p. 2621 - 2624; View in
Reaxys
Journal; Shigemori, Hideyuki; Wang,
Yoshida, Naotoshi;
Kobayashi, Jun'ichi; Chemical &
Pharmaceutical Bulletin;8 vol. 45; 7;
(1997); p. 1205 - 120 ; View in
Reaxys
7844183
VCewin taxuspinanane D
Reaxys
(
11 RGHsAN
586.636
C31H38011
heterocyclic
GERGUKHE ldentification(3)
ZFJMEB- Physical Data( )
pactivity /Eoox1
A-N
Bioactivity/Ecotox(1)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
T
-us
2
Zuc.R
r
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; et al.;
Planta Medica; vol. 64; 2; (1998); p.
183 - 186; View in Reaxys
View in
Reaxys
HO
786822C29H360
taxuspinane C
7963663
View in 2-deacetyl-taxinine J
Reaxvs
10
10
544.599
VGYGMYOF Identification(3)
CJDRPE- Physical Data(2)
UCLEH PScra()2
UANCLEHB Spectra(5)
SA-N
Bioactivity/Ecotox(2)
Journal; Morita, Hiroshi; Wei, Lan;
Gonda, Akira; Takeya, Koichi;
Itokawa, Hideji; Phytochemistry
(Elsevier); vol. 46; 3; (1997); p. 583 586; View in Reaxys
isocyclic
DACUFYMH
APJQQBVUPYCHFM
SA-N
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; et al.;
Planta Medica; vol. 64; 2; (1998); p.
183 - 186; View in Reaxys
C22H13206
isocyclic
DFVNOLZAL
KWQCQM
MGMIPAKD
SA-N
C22H3206
isocyclic
C29H36010
heterocyclic
C37H460
666.766
C37H46011
OH0
H
8013066
View in Vie in6
Reaxys
C22H320
6392.492
lPK
Identification(3)
Physical Data(1)
Spcr()2
Spectra(5)
Bioactivity/Ecotox(1)
OH
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
H
OH
OH
0
8013086
Vew in
Reaxys
C22H320
6
392.492
Journal; Wang, Xiao-xia; Shigemon,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxys
DFVNOLZAL
M QCQ
MGMIPAKD
-N
'OH
H
H
OH
8023236
View in
C31H3808
isocyclic
LACLKGVXI
BAENIUDMFJDJZS
UDFDZ
A-N
C31 H3808
isocyclic
LACLKGVXI
BAENIUDMFJDJZS
A-N
C31H380
V8
f
8538.638
Reaxys
1
"8023236
View in
Reaxys
031 H380
8
6
538.638
ldentification(3)
Physical Data(1)
Spectra(6)
pcr()2
Bioactivity/Ecotox(1)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxys
8024272
View in
C28H400
10
8024272
in
Reaxys
C28H400
1Y
536.62
536.62
C28H40010
C28H40010
Identification(3)
Physical Data(1)
Spectra(6)
Bioactivity/Ecotox(1)
FUBMOVYE
QAHKMAWDKHEYHY
SA-N
isocyclic
FUBMyew
A KView
A
SA-N
as
U4L "
bt
zutc,
YCZNPMNC
GXWNH- Identification(3)
UFJLMRSIS Spectra(3)
A-N
Bav/
t2
JapantoB
ycccJalSI
2
O
HO
HOW-
stems of
8025976
View in
Reaxys
baccatin VI;
taxuspine E
C31H400
11
taxine liI0
Reaxys
C37H49N
09
588.652
C31H40011
heterocyclic
8
---
Japanese
yew Taxus
isocyclic
8092
View in
Reaxys
8032220
ewin
8098426
View in
Reaxys
taxol
deacetyl-2-
6
651.797
C37H49NO9
C47H53N
C47H53NO1
013
3
isocychic
2
cu
1
608.729
C35H4409
YLUDIAFITL
RBKTIdentification(4)
ATRXLRSR Bioactivity/Ecotox(6)
isocyclic
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bloorganic
Medicinal Chemistry Letters; vol.8;
(1998); p. 1555 - 1558; View in
Reaxys
SA-N812;
C35H440
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
14
vL4;2
19) p..11
1111-1134;
47; 2; (1998);
View in Reaxys
UJARNTWL
SVMUJFHHNKTFFCCvol.
SA-N
SOSLCGUO
SOSLCGUO Identification(4)
OOQMEN- Physical Data(2)
Spectra(9)
SA-N
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bloorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
Japaese
yew Tus
1
2
cussidol
S10b.4a
Journal; Wang, Xiao-xia; Shigemori,
Hideyuki; Kobayashi, Jun'ichi;
Journal of Natural Products; vol. 61;
4; (1998); p. 474 - 479; View in
Reaxys
View in
baccatin Il 13-cinnamate;
taxuspinanane J
C40H440
12
716782
heterocyclic
.044012
Reaxys
View in
Reaxys
taxezopidine J
10
622.712
C35H42010
isocyclic
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Wakabayashi, Hideki; at al.
Phytochemistry (Elsevier); vol. 48; 5;
(1998); p. 857 - 862; View in Reaxys
PPZXDJGR
UOJEBODILUHENS
A-N
CEAAWQZV Identification(5)
SFASVNLVS Spectra(7)
A-N
1
2
Bioactivity/Ecotox(1)
Journal; Shi, Qing-wen; Oritani,
Takayuki; Zhao, Ding; Murakami,
Ryo; Oritani, Takashi; Planta Medica
vol. 66; 3; (2000); p. 294 - 299; View
n Reaxys
8246897CEAQV
Vie2in68972
C3 1140
824889 taxezopidine J
9888672 2-deacetyltaxine B;
View in 1-extxn
Reaxys
9973850taxinine NN-11;
V i
5a,13
acet
9Reaxys cinnamoyloxy-4(20),
taxadien-1013-al
C35H42010
isocyclicSF
A-N
9434037 5a-acetoxytaxaView in 4(20),11(12)-diene;
Reaxys 5a-acetoxytaxa-4(20),1 1diene
-_\
622.712
Journal; Shigemori, Hideyuki;
Sakurai, Cecilia A.; Hosoyama,
Hirokazu; Kobayashi, Akio; Kajiyama
Shinichiro; Kobayashi, Jun'ichi;
Tetrahedron; vol. 55; 9; (1999); p.
2553 - 2558; View in Reaxys
CEAAWQZV
SISBFIVN V
APIZAZFFQ
C22H340
2
330.511
C22H3402
C33H45N
75773C34N7
567.723
C33H45NO7
isocyclic
socyclic
ioylcQRQAICDM
BVSJAIdeniatiiation(4)
ZJDLJICXSA Bioactivity/Ecotox(1)
N
2
BJUSJVLOC
QRPldentification(4)
Spectra(3)2
2
SA-N
GJHAFDSY
-
C33H420
7SA-N
550.692
C33H4207
isocyclic
CIJBTOSA-N
Identification(5)
Physical Data(2)
Bictit/ox4
Bioactivity/Ecotox(44)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
1
2
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Joumal; Morita, Hiroshi; Machida,
Izumi; Hirasawa, Yusuke; Kobayashi,
Junichi; Journal of Natural Products;
vol. 68; 6; (2005); p. 935 - 937; View
in Reaxys
Journal; Dai, Jungui; Bai, Jiao;
Hasegawa, Toshiaki; Nishizawa,
Shigenori; Sakai, Junichi; Oka,
Seiko; Kiuchi, Miwa; H-irose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Li, Minjie; Ando, Masayoshi;
Chemical & Pharmaceutical Bulletin;
vol. 54; 3; (2006); p. 306 - 309; View
in Reaxys
Journal; Bai, Jiao; Kitabatake,
Mutumi; Toyoizumi, Kazuki; Fu,
Liwei; Zhang, Shujun; Dai, Jungui;
Sakai, Junichi; Hirose, Katutoshi;
Yamori, Takao; Tomida, Akihiro;
Tsuruo, Takashi; Ando, Masayoshi;
Journal of Natural Products; vol. 67;
1; (2004); p. 58 - 63; View inReaxys
Journal; Kobayashi, Jun'ichi;
Inubushi, Akiko; Hosoyama,
Hirokazu; Yoshida, Naotoshi; Sasaki,
Takuma; Shigemori, Hideyuki;
Tetrahedron; vol. 51; 21; (1995); p.
5971 - 5978; View in Reaxys
Journal; Kobayashi, Jun'ichi;
Inubushi, Akiko; Hosoyama,
Hirokazu; Yoshida, Naotoshi; Sasaki,
Takuma; Shigemori, Hideyuki;
Tetrahedron; vol. 51; 21; (1995); p.
5971 - 5978; View in Reaxys
Journal; Konda, Yaeko; Sasaki,
Tomomitsu; Sun, Xue-Long; Li, Xian;
Onda, Masayuki; et al.; Chemical &
Pharmaceutical Bulletin; vol. 42; 12;
(1994); p. 2621 - 2624; View in
Reaxys
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Katsui, Takie;
Yoshida, Naotoshi; Shigemori,
Hideyuki; Tetrahedron; vol. 52; 15;
(1996); p. 5391 - 5396; View in
Reaxys
Journal; Morita, Hiroshi; Gonda,
Akira; Wei, Lan; Yamamura,
Yukinori; Takeya, Koichi; Itokawa,
Hideji; Journal of Natural Products;
vol. 60; 4; (1997); p. 390 - 392; View
in Reaxys
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
View in
Reaxys
C26H360
492.566
C26H3609
isocyclic
HMVNDOFA
GCGZSA- Identification(3)
SZMFXVPE Bioactivity/Ecotox(3)
SA-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles;
vol. 47; 2; (1998); p. 1111- 1134;
View in Reaxys
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuki; Heterocycles:
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxvs
Reaxys9
No
-f
8068in
View in
Reaxys
C31H400
10
8028577
View in
Reaxys
C37H51N
09
572.653
C31H40010
heterocyclic
BKASSTPDS
ANLBHIdentification(3)
QDIXYBMO Bioactivity/Ecotox(1)
SA-N
653.813
C37H51NO9
isocyclic
CKXBHBNB
EFREKT- Identification(3)
WOZIFEKR Bioactivity/Ecotox(3)
SA-N
N'
yI
8ea81
80ewin
Reaxys
C37H4606
8029363
View in
Reaxys
C37H49N
10
11A-N
666.766
C37H4601
667.797
C37H49NO1
0
C47H61N
View in
Reaxys
C47H61NO1
48
isocyclic
isocyclic
NOMGXQG
PVDHIQV- Identification(3)
LUAYAUSYS Bioactivity/Ecotox(1)
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bloorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
GODKREDP
NCAIKTIdenatiiation(3))
GLACRUIJS Bioactivity/Ecotox(3)
A-N
Journal; Kobayashi, Jun'ichi;
Shigemori, Hideyuk; Heterocycles;
vol. 47; 2; (1998); p. 1111 - 1134;
View in Reaxys
CXIWJTIMC
KBSGLIdentificaion(3)
heterocyclicYSRBHRHJ Bioactivity/Ecotox()
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, Xiaoxia; Shigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
8032473sxia;
SC49H57N83.99
4
014
Reaxys
8375198
View in
Journal; Kobayashi, Jun'ichi;
Hosoyama, Hirokazu; Wang, XiaoShigemori, Hideyuki; Sudo,
Yojiro; Tsuruo, Takashi; Bioorganic &
Medicinal Chemistry Letters; vol. 8;
812; (1998); p. 1555 - 1558; View in
Reaxys
YPZNPHSH
heterocyclic
eGBZQRDBJ
VAAKID-
dentification(3)
Bioactivity/Ecotox(1)
Taxus
cupIAta
S-
10-deacetyl taxinine
C33H400
8
564.676
C33H4008
isocyclic
NReaxys
8380704 2a,7p,9a,10-13aView i
pentaacetopox5aReaxys
taxa-11-en-1p-ol
8529978
View in 2'-hydroxytaxinine
Reaxys
8530690
V ew in 5-cinnamoyltaxicinR
triacetate
Reaxys
C39H480
C
0
740.802
622.712
C39H48014 heterocyclic
C35H42010
isocyclic
Journal; Shi, Qing-Wen; Oritani,
Takayuki; Horiguchi, Tohru;
Sugiyama, Takeyoshi; Murakami,
Ryo; Yamada, Teiko; Bioscience,
Biotechnology, and Biochemistry;
vol. 63; 5; (1999); p. 924 - 929; View
in Reaxys
MQONWRW Identification(4)
Physical Data(1)
WlLKJHV
AYFFSUKU Spectra(5)
SA-N
Identification(4)
GJVCWSHM Physical Data(1)
SHM Spectra(4)
SA-N
ILYLIAD
SXVIQA-N
1
Ywvol.
Journal; Murakami, Ryo; Shi, Qingwen; Oritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580: View in
Reaxys
Identification1(3)
AQFTYVHS
C37H440
11
664.75
C37H44011
isocyclic
Journal; Shi, Qing-Wen; Oritani,
Takayuki; Horiguchi, Tohru;
Sugiyama, Takeyoshi; Murakami,
Ryo; Yamada, Teiko; Bioscience,
Biotechnology, and Biochemistry;
63; 5; (1999); p. 924 - 929; View
in Reaxys
-Ywen;
ESKJCL- Identification(3)
PXCVUNBY
SA-N
1
Journal; Murakami, Ryo; Shi, QingOritani, Takayuki;
Phytochemistry (Elsevier); vol. 52; 8;
(1999); p. 1577 - 1580; View in
Reaxys
Seeds Of
Tm
8602102 2o,9a,10s-triacetoxy-5aView in cinnamoxy-taxa-4(20),11Reaxys dien-13a-ol
C35H44
C
608.729
C35H4409
QG SG
Identification(4)
A-N
Physical Data(1)
Spectra(6)
isocyclic
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
1
Journal; Shi, Qing-wen; Oritani,
Takayuki; Zhao, Ding; Murakami,
Ryo; Oritani, Takashi; Planta Medica;
vol. 66; 3; (2000); p. 294 - 299; View
2a,7,9a,10p-tetraacetoxy- C39H51N
86047095a2RSN,N-dimethyl-3
View in phenylisoseryloxy]-taxa012
Reaxys phnlssrlx]tx02Spectra(7)
4(20),11-dien-13-one
8667257 5a-cinnamoyl-2a,9a,100View in triacetoxy-taxa-4(20),11Reaxys diene-10,70-diol
C351420
11
8668050 5a-cinnamoyl-2a,70,9a,100 C37H440
Viewin tetraacetoxy-taxa-4(20)C,1112
Reaxys diene-1p-ol
0
725.833
C39H51NO1
2
isocyclic
SA-N
638.712
680.749
C35H42011
C37H44012
562.657
C30H42010
Journal; Shi, Qing-wen; Oritani,
Takayuki; Zhao, Ding; Murakami,
Ryo; Oritani, Takashi; Planta Medica;
vol. 66; 3; (2000); p. 294 - 299; View
in Reaxys
isocyclic
NTSFDQOX Identification(4)
BNSSLPPKS Physical Data(1)
A-N
Spectra(6)
Journal; Cheng, Qian; Oritani,
Takayuki; Horiguchi, Tohru;
Bioscience, Biotechnology, and
Biochemistry; vol. 64; 4; (2000); p.
894 - 898; View in Reaxys
isocyclic
YMFGFUWV
CZQVCH- Identification(4)
WMHRXNFP Physical Data(1)
Spectra(6)
SA-N
Journal; Cheng, Qian; Oritani,
Takayuki; Horiguchi, Tohru;
Bioscience, Biotechnology, and
Biochemistry; vol. 64; 4; (2000); p.
894 - 898; View in Reaxys
,
C301-1420
9457945 2o,709a.100,13aView in pentaacetoxytaxa-4(20),1110
Reaxys diene
ASLJYTZPFI
AOCTIdentification(4)
KGCHRQTP Physical Data(1)
isocyclic
Identification(4)
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
Identification(4)
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
YMXDEIJJS
LDGPB- Identification(4)
FVKBLHKFS Spectra(3)
A-N
Journal; Ketchum, Raymond E. B.;
Rithner, Christopher D.; Qiu, Deyou;
Kim, You Sun; Williams, Robert M.;
Croteau, Rodney B.; Phytochemistry
(Elsevier); vol. 62; 6; (2003); p. 901 910; View in Reaxys
CHNIJRUMA
IIJKNKTXPPAN
SA-N
H
WPPPFZJN
9-dihydro-13-acetylbaccatine 1i1
R
ReaxysSAN
9462524 5a,70,9a,100,13aView in pentaacetoxy-2abenzoyloxytaxa-4(20),1 1eaxys diene
C33H420
12
C37H460
12
630.689
682.765
C33H42012 heterocyclic
C37H46012
isocyclic
KLMYBWXHMZMNHP
SA-N
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
\
9536547 2a,5a,9a,100-tetraacetoxyView in 13a-(Z)-cinnamoyloxy-taxa4(20),11-diene
9539252 taxa-4(20),1 -diene-2a,5adiacetate-14p-(2'S,3'R)-3'Venhydroxy-2'-methyl-butyrateReaxys hdo--me
s10-p-D-glucoside
37H460
0Rexys
0SAN
650.766
C37H46010
C35H540
1368.0
682.806COURTPG3H41
2283 7--xylosyl)-10954223
View in
Ra
deacetyltaxol
dect
o
C50H57N
01
9
23
7-(p-xylosyl)-10\5
deacetyltaxol C
Viewxin9803
C49H63N
017
isocyclic
heeoylcYPST
VPIDEXLSMI
Identification(4)
AAAD
BUVRCVSP Physical Data(1)
Spectra(4)
utiTngs
1
oJournal;
B
M
MNAQWBY Identification(4)
P hsiaDta)
Spectra(7)
AY
A-N
e
-
ArooNted
4.9
C50H57NO1
eeoylcAQKP
AGABNGYO Identification(4)
EYYERMpcr()Orval;
1vZ
0SA-N
C49H63NO1
eactyltxolC
V ewin 93.03
7
07
heeroccli
eercci
[PJournal;
MLIMHVPG
XGHTNR- Identification(4)
Spctr(4)Orval;
GPYCBB
GPTYCBBU Spectra(4)
C33H420
550.692
C33H4207
isocyclic
d tShigenori;
Physicat(4)
O
DL
)
scra(
ARASHVSR
Bioactivity/Ecotox(4)
SA-N
niesdes of
Tas
10632922 2a,9a,10s-triacetoxytaxaC32H460
View in 4(20),11-dien-13-one-5a-013
Reaxys p-D-glucopyranoside
638.709
C32H46013
heterocyclic
OUZIYNFF
WIKDCK- Identification(4)
MSFHKKQR Physical Data(1)
SA-N
S
yae
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected byElsevier Properties SA and used under license.
Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita .; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
Journal; Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
nd
roa
Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
Shi, Qing-Wen; Petzke,
Tracy L.; Sauriol, Francoise; Mamer,
Zamir, Lolita 0.; Canadian
81; 1;
offCeity
Chemistry; vol.
Journal
Jua
o.8;1
(2003); p. 64 - 74; View in Reaxys
0RLP
10404510 5a,13a-diacetoxy-1 OViewin cinnamoyloxy-4(20),11Reaxys taxadien-9a-ol
Journal; Shi, Qing-Wen; Petzke,
Mf
Tracy L.; Sauriol, Francoise; Mamer,
Orval; Zamir, Lolita 0.; Canadian
Journal of Chemistry; vol. 81; 1;
(2003); p. 64 - 74; View in Reaxys
414
Spectra(3)
Journal; Dai, Jungui; Bai, Jiao;
Hasegawa, Toshiaki; Nishizawa,
Sakai, Junichi; Oka,
Seiko; Kiuchi, Miwa; Hirose,
Katutoshi; Tomida, Akihiro; Tsuruo,
Takashi; Li, Minjie; Ando, Masayoshi;
Chemical & Pharmaceutical Bulletin;
vol. 54; 3; (2006); p. 306 - 309; View
Journal; Li, Ligeng; Cao, Congmei;
Huo, Changhong; Zhang, Manli; Shi,
Qingwen; Kiyota, Hiromasa;
Magnetic Resonance in Chemistry;
vol. 43; 6; (2005); p. 475 - 478; View
in Reaxys
11195631 2a,14p-diacetoxy-1001OH
Viewin ethoXytaxa-11,4(20)-dienReaxys 5a-ol
H
C26H400
448.6
C26H4006
CCONFRZG Identification(4)
SFPMZPPX Physical Data(1)
SA-N
Spectra(4)
1
Journal; Dong, Mei; Zhang, Man-Li;
Wang, Yu-Fang; Zhang, Xi-Ping; Li,
Cun-Fang; Shi, Qing-Wen; Cong,
Bin; Kiyota, Hiromasa; Bioscience,
Biotechnology, and Biochemistry;
vol. 71; 8; (2007); p. 2087 - 2090;
View in Reaxvs
1
Journal; Dong, Mei; Zhang, Man-Li;
Wang, Yu-Fang; Zhang, Xi-Ping; Li,
Cun-Fang; Shi, Qing-Wen; Cong,
Bin; Kiyota, Hiromasa; Bioscience,
Biotechnology, and Biochemistry;
vol. 71; 8; (2007); p. 2087 - 2090;
View in Reaxys
o
-o
SSLBZFDBG ldnilain4
"
OH
H
H
11195632 2a,14p-diacetoxy-100C
View in methoxytaxa-11,4(20)-dienReaXys Sa-ol
Identification(4)
8LPC6
434.573
C25H3806
6QTYJYIDZS
AYIPCA
Physical Data(1)
Pyia aa1
Spectra(2)
oy
0
F- 0
OH
11195633
111956332a-acetoxy-10p-ethoxytaxa. C24H380
View in 11,4(20)-diene-5a,14-diol
Reaxy
C
'A-N H
Rexys
406.563
C24H3805
BNHGKUO
WYJNWNFXHNBIOFNS
A-N
ldentification(4)
Pdniicat(4)
Physical Data(1)
Spectra(4)
Journal; Dong, Mei; Zhang, Man-Li;
Wang, Yu-Fang; Zhang, Xi-Ping; Li,
Cun-Fang; Shi, Qing-Wen; Cong,
Bin; Kiyota, Hiromasa; Bioscience,
Biotechnology, and Biochemistry;
vol. 71; 8; (2007); p. 2087 -2090;
View in Reaxys
C22H3407
ZRWDUMZ
SA-N
Identification(4)
Physical Data(1)
Spectra(4)
1
Journal; Huo, Changhong; Su,
Xiaohui; Li, Xing; Zhang, Xiping; Li,
Cunfang; Wang, Yufang; Shi,
Qingwen; Kiyota, Hiromasa;
Magnetic Resonance in Chemistry;
vol. 45; 6; (2007); p. 527 - 530; View
inReaxys
1
Journal; Morita, Hiroshi; Machida,
Izumi; Hirasawa, Yusuke; Kobayashi,
Jun'ichi; Joumal of Natural Products;
vol. 68; 6; (2005); p. 935 - 937; View
in Reaxys
OH
0
o
OH
OH
11296427 2a,5a,7p,9a,13aC23OW
View in pentahydroxy-100C22H340
Reaxys acetoxytaxa-4(20),11-diene
H
410.508
OH
15840365
View in 2-0-deacety taxine II
Reaxys
15840366
* 10
taxezopidine M
ieax
i010
Reaxys
C35H47N
08
609.76
C35H47NO8
YDXFVDIGL
VDFPLd
DYDWKSLW Identification(3)
SA-N
C37H49N
667.797
C37H49NO1
0
GOESCWO Identification(4)
GMSFPPK- Physical Data(1)
CFGDUKOB Spectra()
SA-N
BioactivityfEcotox(2)
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Morita, Hiroshi; Machida,
Izumi; Hirasawa, Vusuke; Kobayashi,
Junichi; Journal of Natural Products;
vol. 68; 6; (2005); p. 935 - 937; View
in Reaxys
'-'4
15882421
View in taxine NA-13
Reaxys
C37H47N
010
010
C37H47NO1
665.781
0
OGLVJVZHL
Identification(4)
DUNAZPhysical Data(2)
DUNAZDKPOKLLXS Spectra(3)
1
the needles
and young
stemsof
Taxus
cupdate
coected in
Abaym-a,
Sendai,
Japan
Journal; Wang, Liyan; Bai, Liming;
Tokunaga, Daisuke; Watanabe,
Yuusuke; Sakai, Jun-ichi; Tang,
Wanxia; Bai, Yuhua; Hirose,
Katsutoshi; Ando, Masayoshi;
Heterocycles; vol. 75; 4; (2008); p.
911 - 917; View in Reaxys
seeds of
Taxus
>
c2
-13817
19381179
View in taxezopidine 0
Reaxys
C36H49N
6976C64N9QOAXJO639.786 C36H49NO9
NPGFWXCZ
Identification(3)
LBYALOOD Physical Data(2)
Spectra(9)
1
cuspkfata
Sieb. et
Zuc4
cofectedat
Sapporo,
Hokkaido,
janw
Copyright @ 2010 Elsevier Properties SA. All rights reserved. Authorized use only. Reaxys @ is a trademark owned and protected by Elsevier Properties SA and used under license.
Journal; Ishiyama, Haruaki;
Kakuguchi, Yuka; Arita, Ed;
Kobayashi, Jun'ichi; Heterocycles;
vol. 73; c; (2007); p. 341 - 348; View
in Reaxys
Appendix B: Taxus cDNA library
I. Cytochrome P450s
a. H1
MDTFIQHESSPLLLSLTLAVILGTILLLILSGKQYRSSRKLPPGNMGFPLIGETIALISDTPR
KFIDDRVKKFGLVFKTSLIGHPAVVICGSSANRFLLSNEEKLVRMSLPNAVLKLLGQDCV
MGKTGVEHGIVRTALARALGPQALQNYVAKMSSEIEHHINQKWKGKDEVKVLPLIRSL
VFSISTSLFFGINDEHQQKRLHHLLETVAMGLVSIPLDFPGTRFRKALYARSKLDEIMSS
VIERRRSDLRSGAASSDQDLLSVLVTFKDERGNSFADKEILDNFSFLLHALYDTTISPLT
LIFKLLSSSPECYENIAQEQLEILGNKKDREEISWKDLKDMKYTWQAVQETLRMFPPVY
GYIREALTDIDYDGYTIPKGWRILCSPHTTHSKEEYFDEPEEFRPSRFEDQGRHVAPYT
FIPFGGGLRICAGWEFAKMEILLFMHHFVKTFSHFIPVDPNEKISRDPLPPIPVKGFSIKP
FPRS
b. H2
MLIEMDTFVQLESSPVLLSLTLTLILLFIFCSKQYRSSLKLPPGNMGFPLIGETIALASQTP
DKFFGDRMKKFGKVFKTSLIGHPTIVLCGSSGNRFLLSNEEKLVRMFPPNSSSKLLGQ
DSVLGKIGEEHRIVRTALARCLGPQALQNYVSKMSSEIQRHINQKWKGKGEVKMLPLIR
SLVFSIATSLFFGITDEQQQERLHHLLETVVTGLLCIPLDFPGTTFRKALHARSKLDEIMS
177
SVIERRRNDLRLGAASSDQDLLSVLLTFKDERGNPFADKEILDNFSFLLHALYDTTISPL
TLVFKLVSSNPECYENIAQEQLEILRNKKDGEDISWADLKDMKYTWQAVQETLRMCPP
VYGNFRKALTDIHYDGYTIPKGWRILCSPYTTHSKEEYFDDPEKFRPSRFEEQGRDVA
PYTFIPFGGGLRICPGREFAKMEILVFMHHFVKAFSSFIPVDPNEKISTDPLPSIPVNGFS
INLVPRS
c. H3
MDSFSFLKSMEAKFGQVIHRDQSSSTALLSLAFTAAVAIFLVLLFRFKSRPSTNFPPGN
FGFPFIGETIQFLRALRSESPHMFFDERLKKFGRVFKTSLTGHPTAVFCGPAGNRFIYS
NEHKLVQSSGPNSFVKLVGQQSIVTKTGEEHRIFLGVLNEFLGPHALQSYTPKMSSKIQ
ENINKHWKGKDEVNMLPSIRQLVFSISSSLFFDINDEDQQEQLKTLLETILVGTLSVPLDI
PGSNFRKALRARSKLDEILSRLIESRRKDMRSGIASTSKNLLSVLLAFKDERGNPLTDTE
ILDNFSFMLHASYDTTVSPTVCIFKLLSANPECYEKVVQEQLGILGNKKDGEEMCWNDL
KAMKYTWQAAQETMRLFPPAFGSFRKVIADIHHDGYI IPKGWKAMVTNYSTSRKEEYF
DEPDNFKPSRFGDGKYVAPYTFLPFGAGIRICPGWEFAKLEMLLFIHHFVKNFSGYLPL
DTKEKISGDPFPPLPKNGFPIKLFPRT-
d. H4
MDALSLVNSTVAKFNEVTQLQASPAILSTALTAIAGIIVLLVITSKRRSSLKLPPGKLGLPF
IGETLEFVKALRSDTLRQFVEEREGKFGRVFKTSLLGKPTVILCGPAGNRLVLSNEEKLL
HVSWSAQIARILGLNSVAVKRGDDHRVLRVALAGFLGSAGLQLYIGKMSALIRNHINEK
178
WKGKDEVNVLSLVRDLVMDNSAILFFN IYDKERKQQLHEILKIILASHFGIPLNIPGFLYR
KALKGSLKRKKILSALLEKRKDELRSRLASSNQDLLSVLLSFRDERGKPLSDEAVLDNC
FAMLDASYDTTTSQMTLILKMLSSNPECFEKVVQEQLEIASNKKEGEEITMKDIKAMKY
TWQVLQESLRMLSPVFGTLRKTMNDINHDGYTIPKGWQVVWTTYSTHQKDIYFKQPD
KFMPSRFEEEDGHLDAYTFVPFGGGRRTCPGWEYAKVEILLFLHHFVKAFSGYTPTDP
HERICGYPVPLVPVKGFPIKLIARS
e. H5
MDTIRASFGEVIQPEYSPLIISVALAAFLGIVIFSIFSSTRRSYVNLPPGNLGLPFIGETIQF
LGALQSEKPHTFFDERVKKFGKVFKTSLIGDPTVVLCGPAGNRLVLSNEDKLVQSAGP
KSFLKLFGEDSVAAKREESHRILRSALGRFLGPHALQNYIGKMNSEMQRHFDDKWKG
KDEVKVLPLVRGLIFSIATSLFFNINDDRQREQLHGLLDTILVGSMTIPLNIPGTLFRKAVK
ARAKLDEILFALIENRRRELRSGLNSGNQDLLSSLLTFKDEKGNPLTDKEILDNFSVMLH
ASYDTTVSPTVLI LKLLASNPECYEKVVQEQLGILASKKEGEEVNWKDLKAMPYTWQAI
QEPLRMPPQLLECFEELSLIFSWKAIQFQKDGQLCGQLIVNGREEFFNEPDKFKPSRFE
EGKPLDPYTFIPFGAGVRICAGWEFAKAELLLFVHPFVKNFSGCIIIDPNEKISGDPFPPL
PTSGQLMKLIPRS
f. H6
MDSFNFLRGIGADFGGFIQFQSSPAVLSLSLITTILGVLLLWFFLHKNGSSVTLPPGNLG
FPFIGETIPFLRALRSETPQTFFDERVKKFGVVFKTRIVGHPTVVLCGPEGNRFLLSNED
179
KLVQASLPNSSEKLIGKYSILSKRGEEHRILRAALARFLRPQALQGYVAKMSSEIQHHIK
QKWKGNDEVKVLPLIRTLIFNIASSLFFGINDEHQQEQLHHLLEAIVLGSLSVPLDFPGT
RFRKALDARSKLDEILSSLMESRRRDLRLGTASENQDLLSVLLTFKDERGNPLTDKEIF
DNFSFMLHASYDTTVSPTGLMLKLLFSSPDCYEKLVQEQLGIVGNKKEGEEISWNDLK
AMKYTCKVVQESMRMLPPVFGSYRKAITYIHYDGYTIPKGWNIFWSPYTTHGKEEYFN
EADKFMPSRFEEGKYVAPYTFLPFGAGLRVCPGWEFAKTEILLFVHHFITTFSSYIPIDP
KDKISGDPFPPLPTNGFSMKLFTRS-
g. H7
MDTLIQIQSSPDFLSFTLTAFLGVVVLLIFRYKHRSALKLPPGNLGLPFIGETITFASQPP
QKFLNERGKKFGPVFKTSLIGHPTVVLCGSSGNRFLLSNEEKLVRMSLPNSYMKLLGQ
DSLLGKTGQEHRIVRTALGRFLGPQELQNHVAKMSSDIQHHINQKWKGNDEVKVLPLI
RNLVFSIATSLFFGINDEHQQERLHLLLETIVMGAVCIPLAFPGSGFRKALQARSELDGIL
ISLMKIRRSDLRSGAASSNQDLLSVLLTFKDERGNPLTDKEILDNFSVLLHGLYDTTISPL
TLIFKLMSSNTECYENVVQEQLEILSHREKGEEIGWKDLKSMKYTWQAIQETLRMFPPV
YGNFRKALTDIHYDGYTIPKGWRVLCSPFTTHSNEEYFNEPDEFRPSRFEGQGKNVPS
YTFIPFGGGLRICPGWEFAKTEMLLFIHYFVKTFSSYVPVDPNEKISADPLASFPVNGFS
VKLFPRS
h. H8
180
MDAFNILKGPAAKLNGVVQLGSYTDRILSITVVAFITILLLLMLRWKSQSSVKLPPGNFG
FPLIGETLQLLRAFRSNTTQQFFDERQKKFGCVFKTSLVGERTVVLCGPSGNRLVLAN
QNKVVESSWPSAFIKLIGEDSIANTNGEKHRILRAALLRYLGPGSLQNYVGKMRSEIEH
HINEKWKGKDEVKVLDLVRKNVFSVATALFFGVNDEERKRIRPPSILRKLHFAGSFSIPL
DFPGTSYRRALEARLKLDKILSSLIERRRSDLRSGLASGNEDLVSVLLTFKDEGGNPLT
DKEILDNFSGLLHASYDTTTSALTLTFKLMSSSAECYDKVVQEQLRIVSNKKEGEEISLK
DLKDMKYTWQVVQETLRMFPPLFGSFRKTIADIQYDGYTIPKGWKVLWATYTTHGRDE
YFSEPQKFRPSRFEEGGKHVAPYTFLPFEGGERTCPGYEFSKTHILLFIHQFVKTFTGY
IPLDPNESISANPLPPLPANGFPVKLFQRS-
i. H9
MDSFIFLRSIGTKFGQLESSPAILSLTLAPILAI ILLLLFRYNHRSSVKLPPGKLGFPLIGETI
QLLRTLRSETPQKFFDDRLKKFGPVYMTSLIGHPTVVLCGPAGNKLVLSNEDKLVEME
GPKSFMKLIGEDSIVAKRGEDHRILRTALARFLGAQALQNYLGRMSSEIGHHFNEKWK
GKDEVKVLPLVRGLIFSIASTLFFDVNDGHQQKQLHHLLETILVGSLSVPLDFPGTRYRK
GLQARLKLDEILSSLIKRRRRDLRSGIASDDQDLLSVLLTFRDEKGNSLTDQGILDNFSA
MFHASYDTTVAPMALIFKLLYSNPEYHEKVFQEQLEIIGNKKEGEEISWKDLKSMKYTW
QAVQESLRMYPPVFGIFRKAITDIHYDGYTIPKGWRVLCSPYTTHLREEYFPEPEEFRP
SRFEDEGRHVTPYTYVPFGGGLRTCPGWEFSKIEILLFVHHFVKNFSSYIPVDPNEKVL
SDPLPPLPANGFSIKLFPRS
181
j. H10
METKFGQLMQLEFLPFILTPILGALVLLHLFRHRNRSSVKLPPGKLGFPVIGETIQFLRAL
RSQTPQKFFDDRVQKFGGVFKTSLIGNPLVVMCGPAGNRLVLSNEDKLVQLEAPNSL
MKLMGQDSLLAKRQEDHRTLRAALARFLGPQALQNYMTKISSRTEHHMNEKWKGKD
EVRTLPLIRELIFSNASSLFFDINDEHQQERLHHLLEAVVVGSMSIPLDFPGTRLRKALQ
ARSKLDEILSSLIKSRRKDLVSGIASDDQDLLSVLLTFKDERGNPLTDKEILDNFSLLLHA
SYDTTVSPMVLTLKLLSSN PECYEKVVQEQLGIVANKRIGEEISWKDLKAMKYTWQVV
QETLRMFPPLFGSFRKAMVDIDYDGYTIPKGWMILWTTYGTHLREEYFNEPLKFRPSR
FEEDGRVTPYTFIPFGGGARTCPGWEFSKTEILLFIHHFVRTFSSYLPVDSNEKISADPF
PPLPANGFSIKLSADPFPPLPANGFSIKLFPRSQSN
k. H1l
MDVFYPLKSTVAKFNECFPAILFIVLSAVAGIVLPLLLFLRSKRRSSVGLPPGKLGYPFIG
ESLLFLKALRSNTVEQFLDERVKNFGNVFKTSLIGHPTVVLCGPAGNRLILANEEKLVQ
MSWPKSSMKLMGEKSITAKRGEGHMIIRSALQGFFSPGALQKYIGQMSKTIENHINEK
WKGNDQVSVVALVGDLVFDISACLFFNINEKHERERLFELLEIIAVGVLAVPVDLPGFAY
HRALQARSKLNAILSGLIEKRKMDLSSGLATSNQDLLSVFLTFKDDRGNPCSDEEILDN
FSGLLHGSYDTTVSAMACVFKLLSSNPECYEKVVQEQLGILSNKLEGDEITWKDVKSM
KYTWQVVQETLRLYPSIFGSFRQAITDIHYNGYIIPKGWKLLWTPYTTHPKEMYFSEPE
182
KFLPSRFDQEGKLVAPYTFLPFGGGQRSCPGWEFSKMEILLSVHHFVKTFSTFTPVDP
AEIIARDSLCPLPSNGFSVKLFPRSYSLHTGNQVKKI
1. H12
MDALKQLEVSPSILFVTLAVMAGIILFFRSKRHSSVKLPPGNLGFPLVGETLQFVRSLGS
STPQQFIEERMSKFGDVFKTSIIGHPTVVLCGPAGNRLVLSNENKLVQMSWPSSMMKL
IGEDCLGGKTGEQHRIVRAALTRFLGPQALQNHFAKMSSGIQRHINEKWKGKDEATVL
PLVKDLVFSVASRLFFGITEEHLQEQLHNLLEVILVGSFSVPLNIPGFSYHKAIQARATLA
DIMTHLIEKRRNELRAGTASENQDLLSVLLTFTDERGNSLADKEILDNFSMLLHGSYDS
TNSPLTMLIKVLASHPESYEKVAQEQFGILSTKMEGEEIAWKDLKEMKYSWQVVQETL
RMYPPIFGTFRKAITDIHYNGYTIPKGWKLLWTTYSTQTKEEYFKDADQFKPSRFEEEG
KHVTPYTYLPFGGGMRVCPGWEFAKMETLLFLHHFVKAFSGLKAIDPNEKLSGKPLPP
LPVNGLPIKLYSRS
m. H13
MDSFTFVTIKMGKIWQVIQVEYILSLTLTAILLFFFRYRNKSSHKLPPGNLGFPFIGETIQF
LRSLRSQTPEFFFDERVKKFGPVFKTSLIGAPTVIFCGAAGSRLVLSNEDKLVQMESPS
SLKKLMGENSILYKREEEHRILRSALSRFLGPQALQTYIAKMSTEIERHINEKWKGKEEV
KTLPLIRGLVFSIASSLFFDINDEPQQERLHHHLESLVAGSMAVRLDFPGTRFRKAVEA
RSKLDEALHSLIKSRRSDLLSGKASSNQDLLSVLLSFKDERGNPLRDEEILDNFSLILHA
SYDTTISPMVLTLKLLSSN PECYDKVVQEQFG ILANKKEGEEISWKDLKAMKYTWQVV
183
QETLRMFPPLFGSFRKAMVDINYDGYTIPKGWIVLWTTYSTHVKEEYFNEPGKFRPSR
FEHDGRHVAPYTFLPFGGGLRTCPGWEFSKTEILLFIHHFVKTFGSYLPVDPNEKISAD
PFPPLPANGFSIKLFPRS
n. H14
MDALYKSTVAKFNEVTQLDCSTESFSIALSAIAGILLLLLLFRSKRHSSLKLPPGKLGIPFI
GESFIFLRALRSNSLEQFFDERVKKFGLVFKTSLIGHPTVVLCGPAGNRLILSNEEKLVQ
MSWPAQFMKLMGENSVATRRGEDHIVMRSALAGFFGPGALQSYIGKMNTEIQSHINE
KWKGKDEVNVLPLVRELVFNISAILFFNIYDKQEQDRLHKLLETILVGSFALPIDLPGFGF
HRALQGRAKLNKIMLSLIKKRKEDCSLDRQQPRRICSLFCSLSEMTKGLPHPMDEILDN
FSSLLHASYDTTTSPMALIFKLLSSNPECYQKVVQEQLEILSNKEEGEEITWKDLKAMK
YTWQVAQETLRMFPPVFGTFRKAITDIQYDGTNSKRGKLLWTTYSTHPKDLYFNEPEK
FMPSRFDQEGKHVAPYTFLPFGGGQRSCVGWEFSKMEILLFVH HFVKTFSSYTPVDP
DEKISGDPLPPLPSKGFSIKLFPRP
o. H15
MDAMDLTVAKFKEFTQLQSSAILLTVVSGIIVIVILLLRSKRRSSLKLPPGKLGLPLIGESL
SFLWALRSNTLEQFVDKRVKKYGNVFKTSLLGQPTVVLCGAAGNRLILSNQEKLLSRT
VSDRVAKLTGDTSISVIAGDSHRIIRAAVAGFLGPAGLKIHIGEMSAHIRNHINQVWKGK
DEVNVLSLARELVFAMSASLFLNINDREEQHQLHKTLETILPGYFSVPINFPGFAFRKAL
EGNSKRRKHFSVLQEKRRRDLSVGLASRTQDLLSVLLAYEDDKGNPLTDEEVLDNISA
184
LIDGSYESTSSQMAMLLKLLSDHPECYEKVVQEQLEIASHKKEGEEITWKDVKAMRYT
WQVMQETLRMFAPVFGPRGKAITDIHYDGYTIPKGWQLSWATYSTHQNDTYFNEPDK
FMPSRFDEEGGRLAPYTFVPFGGGRRKCPGWEFAKTEILLFVHHFVKTFSAYTPIDPH
ESIWGRPLPPVPANGFPIKLISRS
p. H16
MDALYKSTVAKFNEVTQLDCSTESFSIALSAIAGILLLLLLFRSKRHSSLKLPPGKLGIPFI
GESFIFLRALRSNSLEQFFDERVKKFGLVFKTSLIGHPTVVLCGPAGNRLILSNEEKLVQ
MSWPAQFMKLMGENSVATRRGEDHIVMRSALAGFFGPGALQSYIGKMNTEIQSHINE
KWKGKDEVNVLPLVRELVFNISAILFFNIYDKQEQDRLHKLLETILVGSFALPIDLPGFGF
HRALQGRAKLNKIMLSLIKKRKEDLQSGSATATQDLLSVLLTFRDDKGTPLTNDEILDNF
SSLLHASYDTTTSPMALIFKLLSSNPECYQKVVQEQLEILSNKEEGEEITWKDLKAMKY
TWQVAQETLRMFPPVFGTFRKAITDIQYDGYTIPKGWKLLWTTYSTHPKDLYFNEPEK
FMPSRFDQEGKHVAPYTFLPFGGGQRSCVGWEFSKMEILLFVHHFVKTFSSYTPVDP
DEKISGDPLPPLPSKGFSIKLFPRP
II. Acyltransferases
a. Al
MGRFNVDMIERVIVAPCLQSPKNILH LSPIDNKTRGLTNILSVYNASQRVSVSADPAKTI
REALSKVLVYYPPFAGRLRNTENGDLEVECTGEGAVFVEAMADN
DLSVLQDFNEYDP
185
SFQQLVFNLREDVNIEDLHLLTVQVTRFTCGGFVVGTRFHHSVSDGKGIGQLLKGMGE
MARGEFKPSLEPIWNREMVKPEDIMYLQFDHFDFIHPPLNLEKSIQASMVISFERINYIK
RCMMEECKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNSFDSPLPKGYYGN
AIGNACAMDNVKDLLNGSLLYALMLIKKSKFALNENFKSRILTKPSTLDANMKHENVVG
CGDWRNLGFYEADFGWGNAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIKILMF
MPASMVKPFKIEMEVTINKYVAKICNSKL
b.A2
MEKTDLHVNLIEKVMVGPSPPLPKTTLQLSSIDNLPGVRGSIFNALLIYNASPSPTMISA
DPAKPI REALAKI LVYYPPFAGRLRETENGDLEVECTGEGAM FLEAMADN ELSVLGDF
DDSNPSFQQLLFSLPLDTNFKDLSLLVVQVTRFTCGGFVVGVSFHHGVCDGRGAAQF
LKGLAEMARGEVKLSLEPIWNRELVKLDDPKYLQFFHFEFLRAPSIVEKIVQTYFIIDFET
INYIKQSVMEECKEFCSSFEVASAMTWIARTRAFQIPESEYVKILFGMDMRNSFNPPLP
SGYYGNSIGTACAVDNVQDLLSGSLLRAIMIIKKSKVSLNDNFKSRAVVKPSELDVNMN
HENVVAFADWSRLGFDEVDFGWGNAVSVSPVQQQSALAMQNYFLFLKPSKNKPDGI
KILMFLPLSKMKSFKIEMEAMMKKYVAKV
c.A3
MAGSTEFVVRSLERVMVAPSQPSPKAFLQLSTLDNLPGVRENIFNTLLVYNASDRVSV
DPAKVIRQALSKVLVYYSPFAGRLRKKENGDLEVECTGEGALFVEAMADTDLSVLGDL
DDYSPSLEQLLFCLPPDTDIEDIHPLVVQVTRFTCGGFVVGVSFCHGICDGLGAGQFLI
186
AMGEMARGEIKPSSEPIWKRELLKPEDPLYRFQYYHFQLICPPSTFGKIVQGSLVITSET
INCIKQCLREESKEFCSAFEVVSALAWIARTRALQIPHSENVKLIFAMDMRKLFNPPLSK
GYYGNFVGTVCAMDNVKDLLSGSLLRVVRIIKKAKVSLNEHFTSTIVTPRSGSDESINYE
NIVGFGDRRRLGFDEVDFGWGHADNVSLVQHGLKDVSVVQSYFLFIRPPKNNPDGIKI
LSFMPPSIVKSFKFEMETMTNKYVTKP
d.A4
MEKSGSADLHVNIIERVVVAPCQPTPKTILQLSSIDKMGGGFANVLLVFGASHGVSADP
AKTIREALSKTLVFYFPFAGRLRKKEDGDIEVECIEQGALFVEAMADNDLSVVRDLDEY
NPLFRQLQSSLSLDTDYKDLHLMTVQVTPFTCGGFVMGTSVHQSICDGNGLGQFFKS
MAEIVRGEVKPSIEPIWNRELVKPEDYIHLQLYVSEFIRPPLVVEKVGQTSLVISFEKINHI
KRCIMEESKESFSSFEIVTAMVWLARTRAFQIPHNEDVTLLLAMDARRSFDPPIPKGYY
GNVIGTTYAKDNVHNLLSGSLLHALTVIKKSMSSFYENMTSRVLVNPSTLDLSMKYENV
VSLSDWSRLGHNEVDFGWGNAINVSTLQQQWENEVAIPTFFTFLQTPKNIPDGIKILMF
MPPSREKTFEIEVEAMIRKYLTKVSHSKL
e.A5
MKKTGSFAEFHVNMIERVMVRPCLPSPKTILPLSAIDNMARAFSNVLLVYAANMDRVSA
DPAKVI REALSKVLVYYYPFAGRLRN KENGELEVECTGQGVLFLEAMADSDLSVLTDL
DNYNPSFQQLIFSLPQDTDIEDLHLLIVQVTRFTCGGFVVGANVYGSACDAKGFGQFL
QSMAEMARGEVKPSIEPWNRELVKLEHCMPFRMSHLQIIHAPVIEEKFVQTSLVINFEII
187
NHIRRRIMEERKESLSSFEIVAALVWLAKIKAFQIPHSENVKLLFAMDLRRSFNPPLPHG
YYGNAFGIACAMDNVHDLLSGSLLRTIMIIKKSKFSLHKELNSKTVMSSSVVDVNTKFED
VVSISDWRHSIYYEVDFGWGDAMNVSTMLQQQEHEKSLPTYFSFLQSTKNMPDGIKM
LMFMPPSKLKKFKIEIEAMIKKYVTKVCPSKL
f.A6
MEKAGSTDFHVKKFDPVMVAPSLPSPKATVQLSVVDSLTICRGIFNTLLVFNAPDNISA
DPVKIIREALSKVLVYYFPLAGRLRSKEIGELEVECTGDGALFVEAMVEDTISVLRDLDD
LNPSFQQLVFWHPLDTAIEDLHLVIVQVTRFTCGGIAVGVTLPHSVCDGRGAAQFVTAL
AEMARGEVKPSLEPIWNRELLNPEDPLHLQLNQFDSICPPPMLEELGQASFVINVDTIE
YMKQCVMEECNEFCSSFEVVAALVWIARTKALQIPHTENVKLLFAMDLRKLFNPPLPN
GYYGNAIGTAYAMDNVQDLLNGSLLRAIMIIKKAKADLKDNYSRSRVVTNPYSLDVNKK
SDNILALSDWRRLGFYEADFGWGGPLNVSSLQRLENGLPMFSTFLYLLPAKNKSDGIK
LLLSCMPPTTLKSFKIVMEAMIEKYVSKV
g.A7
MEKGNASDVPELHVQICERVMVKPCVPSPSPN LVLQLSAVDRLPGMKFATFSAVLVYN
ASSHSI FANPAQI I RQALSKVLQYYPAFAGRI RQKEN EELEVECTGEGALFVEALVDNDL
SVLRDLDAQNASYEQLLFSLPPNIQVQDLHPLILQVTRFTCGGFVVGVGFHHGICDARG
GTQFLQGLADMARGETKPLVEPVWNRELIKPEDLMHLQFHKFGLIRQPLKLDEICQAS
FTINSEIINYIKQCVIEECNEIFSAFEVVVALTWIARTKAFQIPHNENVMMLFGMDARKYF
188
NPPLPKGYYGNAIGTSCVIENVQDLLNGSLSRAVMITKKSKIPLIENLRSRIVANQSGVD
EEIKHENVVGFGDWRRLGFHEVDFGSGDAVNISPIQQRLEDDQLAMRNYFLFLRPYKD
MPNGIKILMFMDPSRVKLFKDEMEAMIIKYMPKA
h.A8
MEKLHVDIIERVKVAPCLPSSKEILQLSSLDNILRCYVSVLFVYDRVSTVSANPAKTIREA
LSKVLVYYSPFAGRLRNKENGDLEVECSGEGAVFVEAMADNELSVLQDLDEYCTSLK
QLIFTVPMDTKIEDLHLLSVQVTSFTCGGFVVGISFYHTICDGKGLGQFLQGMSEISKGA
FKPSLEPVWNREMVKPEHLMFLQFNNFEFVPHPLKFKKIVKASIEINFETINCFKQCMM
EECKENFSTFEIVAALIWLAKTKSFQIPDSENVKLMFAVDMRTSFDPPLPKGYYGNVIGI
AGAIDNVKELLSGSILRALIIIQKTIFSLKDNFISRRLMKPSTLDVNMKHENVVLLGDWRN
LGYYEADCGCGNLSNVIPMDQQIEHESPVQSRFMLLRSSKNMQNGIKILMSMPESMA
KPFKSEMKFTIKKYVTGACFSEL
i.A9
MEKAGSSTEFHVKISDPVMVPPCIPSPKTILQLSAVDNYPAVRGNILDCLLVYNASNTIS
ADPATVIREALSKVLVYYFPFAGRMRNKGDGELEVDCTGEGALFVEAMADDNLSVLG
GFDYHNPAFGKLLYSLPLDTPIHDLHPLVVQVTRFTCGGFVVGLSLDHSICDGRGAGQ
FLKALAEMARGEAKPSLEPIWNRELLKPEDLIRLQFYHFESMRPPPIVEEIVQASIIVNSE
TISNIKQYIMEECKESSFAFEVVAALAWLARTRAFQIPHTENVKLLFAVDTRRSFDPPLP
KGYYGNAAGNACAMDNVQDLLNGSLLRAVMIIKKSKVSLNENIRAKTVMRPSAIDVNM
189
KHESTVGLSDLRHLGFNEVDFGWGDALNASLVQHGVIQQNYFLFLQPSKNMNGGIKIA
MFMPQSKVKPFKIEMEALISKYATKV
j. B (benzoyltransferase)
MGRFNVDMIERVIVAPCLQSPKNILHLSPIDNKTRGLTNILSVYNASQRVSVSADPAKTI
REALSKVLVYYPPFAGRLRNTENGDLEVECTGEGAVFVEAMADNDLSVLQDFNEYDP
SFQQLVFNLREDVNIEDLHLLTVQVTRFTCGGFVVGTRFHHSVSDGKGIGQLLKGMGE
MARGEFKPSLEPIWNREMVKPEDIMYLQFDHFDFIHPPLNLEKSIQASMVISFERINYIK
RCMMEECKEFFSAFEVVVALIWLARTKSFRIPPNEYVKIIFPIDMRNSFDSPLPKGYYGN
AIGNACAMDNVKDLLNGSLLYALMLIKKSKFALNENFKSRILTKPSTLDANMKHENVVG
CGDWRNLGFYEADFGWGNAVNVSPMQQQREHELAMQNYFLFLRSAKNMIDGIKILMF
MPASMVKPFKIEMEVTINKYVAKICNSKL
III. Cytochrome P450 reductase
a. CPR1
MQANSNTVEGASQGKSLLDISRLDHIFALLLNGKGGDLGAMTGSALILTENSQNLMILT
TALAVLVACVFFFVWRRGGSDTQKPAVRPTPLVKEEDEEEEDDSAKKKVTIFFGTQTG
TAEGFAKALAEEAKARYEKAVFKVVDLDNYAADDEQYEEKLKKEKLAFFMLATYGDGE
PTDNAARFYKWFLEGKEREPWLSDLTYGVFGLGNRQYEHFNKVAKAVDEVLIEQGAK
190
RLVPVGLGDDDQCIEDDFTAWREQVWPELDQLLRDEDDEPTSATPYTAAIPEYRVEIY
DSVVSVYEETHALKQNGQAVYDIHHPCRSNVAVRRELHTPLSDRSCIHLEFDISDTGLI
YETGDHVGVHTENSIETVEEAAKLLGYQLDTIFSVHGDKEDGTPLGGSSLPPPFPGPC
TLRTALARYADLLNPPRKAAFLALAAHASDPAEAERLKFLSSPAGKDEYSQWVTASQR
SLLEI MAEFPSAKPPLGVFFAAIAPRLQPRYYSISSSPRFAPSRI HVTCALVYGPSPTGRI
HKGVCSNWMKNSLPSEETHDCSWAPVFVRQSNFKLPADSTTPIVMVGPGTGFAPFR
GFLQERAKLQEAGEKLGPAVLFFGCRNRQMDYIYEDELKGYVEKGILTNLIVAFSREGA
TKEYVQHKMLEKASDTWSLIAQGGYLYVCGDAKGMARDVHRTLHTIVQEQESVDSSK
AEFLVKKLQMDGRYLRDIW
191
Appendix C Deduced taxane metabolic pathway
193
g
{
t
4~y~
"~
.1
-
/
4
4
4
-b
4
~x~I~A
4
-K
4
~<0
4
44
-K
4
-K
4
'L
.
~c-~
-K
Appendix D: Sample PDB files for heme moiety of P450 HI & docking code
Whereas MODELER successfully generates PDB files for the polypeptide sequence of the enzymes,
the heme moiety has to docked, energy-optimized and then linked to the polypeptide chain
manually. The algorithm utilized in Chapter 4 is wholly novel and atomic coordinates of the heme
group have been provided herein as a reference.
a. Ground state
-13.705 -7.698 59.813 1.00 56.34
HETATM 3795 CBB HEM 600
HETATM 3796 CBC HEM 600 -21.741 -4.086 60.876 1.00 54.16
-17.839 -7.090 64.655 1.00 55.01
HETATM 3797 FE HEM 600
-13.943 -8.656 60.714 1.00 54.29
HETATM 3798 CAB HEM 600
HETATM 3799 CAC HEM 600 -20.430 -4.080 60.621 1.00 53.50
-14.057 -8.795 63.248 1.00 53.56
HETATM 3800 C2B HEM 600
HETATM 3801 C2C HEM 600 -18.476 -5.703 60.617 1.00 53.67
-16.583 -7.878 68.595 1.00 53.10
HETATM 3802 C2A HEM 600
HETATM 3803 C3D HEM 600 -20.340 -4.930 67.231 1.00 54.61
HETATM 3804 C2D HEM 600 -20.826 -4.472 66.012 1.00 54.14
HETATM 3805 C3A HEM 600 -15.499 -8.438 67.935 1.00 53.10
HETATM 3806 C3B HEM 600 -14.554 -8.364 62.030 1.00 54.03
-19.490 -5.021 61.270 1.00 53.94
HETATM 3807 C3C HEM 600
HETATM 3808 CMA HEM 600 -14.555 -9.479 68.479 1.00 52.53
HETATM 3809 CMD HEM 600 -22.035 -3.604 65.798 1.00 53.79
HETATM 3810 CMB HEM 600 -12.835 -9.641 63.472 1.00 52.87
-18.147 -5.623 59.153 1.00 52.76
HETATM 3811 CMC HEM 600
HETATM 3812 CHA HEM 600 -18.447 -6.389 67.900 1.00 53.35
HETATM 3813 CHB HEM 600 -14.755 -8.521 65.595 1.00 53.12
-16.564 -7.081 61.342 1.00 53.29
HETATM 3814 CHC HEM 600
-20.099 -4.703 63.658 1.00 53.36
HETATM 3815 CHD HEM 600
-17.028 -8.260 69.989 1.00 53.34
HETATM 3816 CAA HEM 600
HETATM 3817 CAD HEM 600 -20.994 -4.737 68.577 1.00 55.31
-17.265 -7.104 67.652 1.00 52.88
HETATM 3818 CIA HEM 600
-14.908 -8.296 64.226 1.00 52.92
HETATM 3819 C1B HEM 600
HETATM 3820 C1C HEM 600 -17.742 -6.364 61.593 1.00 53.10
HETATM 3821 C1D HEM 600 -19.967 -4.953 65.031 1.00 53.77
HETATM 3822 C4A HEM 600 -15.581 -8.029 66.611 1.00 53.03
HETATM 3823 C4D HEM 600 -19.213 -5.698 66.947 1.00 54.08
HETATM 3824 C4B HEM 600 -15.714 -7.638 62.305 1.00 53.89
-19.345 -5.294 62.632 1.00 53.55
HETATM 3825 C4C HEM 600
HETATM 3826 CBA HEM 600 -17.758 -9.609 69.999 1.00 55.72
-21.969 -5.882 68.860 1.00 58.09
HETATM 3827 CBD HEM 600
-18.221 -9.945 71.386 1.00 58.55
HEM
600
HETATM 3828 CGA
HETATM 3829 CGD HEM 600 -22.655 -5.679 70.176 1.00 60.08
-16.641 -7.190 66.442 1.00 52.37
HETATM 3830 NA HEM 600
C
C
FE+3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
197
HETATM
HETATM
HETATM
HETATM
HETATM
3831
3832
3833
3834
3835
NB HEM
ND HEM
NC HEM
HETATM
HETATM
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3836
3837
3347
3797
3797
3797
3795
3796
3798
02A HEM
CONECT 3799
CONECT 3800
CONECT
CONECT
CONECT
CONECT
CONECT
600
600
600
01A HEM 600
01D HEM 600
600
02D HEM 600
-15.919 -7.593 33.649 1.00 53.29
-18.991 -5.703 65.605 1.00 53.53
-18.293
-17.423
-22.112
-19.447
-6.139
-9.757
-6.139
-10.375
62.818 1.00 52.75
72.413 1.00 60.38
71.281 1.00 59.70
71.585 1.00 58.24
-23.781 -5.004 70.236 1.00 60.88
N
N
N
0
0
0
0
3797
3347
3830 3831 3832
3833
3798
3799
37953806
37963807
3806 3810
3807 3811
3805 3816
3804 3817
3801
3802
3803
3804 3803 3809
3805 3802 3808
CONECT 3806 3798 3800
CONECT 3807 3799 3801
CONECT 3808 3805
3819
3820
3818
3823
3821
3822
3824
3825
CONECT 3809 3804
CONECT 3810 3800
CONECT
CONECT
CONECT
CONECT
CONECT
3811
3812
3813
3814
3815
CONECT 3816
CONECT 3817
CONECT 3818
CONECT
CONECT
CONECT
CONECT
3801
38183823
38193822
3820 3824
3821 3825
3802 3826
3803 3827
3802 3812 3830
3800 3813 3831
3801 3814 3833
3819
3820
3821 3804 3815 3832
3822 3805 3813 3830
CONECT 3823 3803 3812 3832
CONECT 3824 3806 3814 3831
CONECT 3825 3807 3815
CONECT 3826 38163828
CONECT 3827 3817 3829
CONECT 3828 3826 3834
CONECT 3829 3827 3835
CONECT 3830 3797 3818
CONECT 3831 3797 3819
CONECT 3832 3797 3821
CONECT 3833 3797 3820
CONECT 3834 3828
CONECT 3835 3829
CONECT 3836 3828
CONECT 3837 3829
END
3833
3836
3837
3822
3824
3823
3825
198
b. Activated state
-13.705 -7.698 59.813 1.00 56.34
HETATM 3795 CBB HEM 600
HETATM 3796 CBC HEM 600 -21.741 -4.086 60.876 1.00 54.16
HETATM 3797 FE HEM 600 -17.464 -6.649 64.627 1.00 55.01
HETATM 3798 CAB HEM 600 -13.943 -8.656 60.714 1.00 54.29
HETATM 3799 CAC HEM 600 -20.430 -4.080 60.621 1.00 53.50
-14.057 -8.795 63.248 1.00 53.56
HETATM 3800 C2B HEM 600
HETATM 3801 C2C HEM 600 -18.476 -5.703 60.617 1.00 53.67
-16.583 -7.878 68.595 1.00 53.10
HETATM 3802 C2A HEM 600
HETATM 3803 C3D HEM 600 -20.340 -4.930 67.231 1.00 54.61
HETATM 3804 C2D HEM 600 -20.826 -4.472 66.012 1.00 54.14
HETATM 3805 C3A HEM 600 -15.499 -8.438 67.935 1.00 53.10
HETATM 3806 C3B HEM 600 -14.554 -8.364 62.030 1.00 54.03
HETATM 3807 C30 HEM 600 -19.490 -5.021 61.270 1.00 53.94
HETATM 3808 CMA HEM 600 -14.555 -9.479 68.479 1.00 52.53
HETATM 3809 CMD HEM 600 -22.035 -3.604 65.798 1.00 53.79
HETATM 3810 CMB HEM 600 -12.835 -9.641 63.472 1.00 52.87
HETATM 3811 CMC HEM 600 -18.147 -5.623 59.153 1.00 52.76
HETATM 3812 CHA HEM 600 -18.447 -6.389 67.900 1.00 53.35
HETATM 3813 CHB HEM 600 -14.755 -8.521 65.595 1.00 53.12
-16.564 -7.081 61.342 1.00 53.29
HETATM 3814 CHC HEM 600
-20.099 -4.703 63.658 1.00 53.36
HETATM 3815 CHD HEM 600
-17.028 -8.260 69.989 1.00 53.34
HETATM 3816 CAA HEM 600
HETATM 3817 CAD HEM 600 -20.994 -4.737 68.577 1.00 55.31
HETATM 3818 C1A HEM 600 -17.265 -7.104 67.652 1.00 52.88
HETATM 3819 C1B HEM 600 -14.908 -8.296 64.226 1.00 52.92
HETATM 3820 CC HEM 600 -17.742 -6.364 61.593 1.00 53.10
HETATM 3821 ClD HEM 600 -19.967 -4.953 65.031 1.00 53.77
-15.581 -8.029 66.611 1.00 53.03
HETATM 3822 C4A HEM 600
HETATM 3823 C4D HEM 600 -19.213 -5.698 66.947 1.00 54.08
-15.714 -7.638 62.305 1.00 53.89
HETATM 3824 C4B HEM 600
HETATM 3825 C4C HEM 600 -19.345 -5.294 62.632 1.00 53.55
HETATM 3826 CBA HEM 600 -17.758 -9.609 69.999 1.00 55.72
HETATM 3827 CBD HEM 600 -21.969 -5.882 68.860 1.00 58.09
HETATM 3828 CGA HEM 600 -18.221 -9.945 71.386 1.00 58.55
-22.655 -5.679 70.176 1.00 60.08
HETATM 3829 CGD HEM 600
HETATM 3830 NA HEM 600 -16.641 -7.190 66.442 1.00 52.37
HETATM 3831 NB HEM 600 -15.919 -7.593 63.649 1.00 53.29
HETATM 3832 ND HEM 600 -18.991 -5.703 65.605 1.00 53.53
HETATM 3833 NC HEM 600 -18.293 -6.139 62.818 1.00 52.75
-17.423 -9.757 72.413 1.00 60.38
HETATM 3834 01A HEM 600
HETATM 3835 01D HEM 600 -22.112 -6.139 71.281 1.00 59.70
HETATM 3836 02A HEM 600 -19.447 -10.375 71.585 1.00 58.24
HETATM 3837 02D HEM 600 -23.781 -5.004 70.236 1.00 60.88
CONECT 3347 3797
CONECT 3797 3347
CONECT 3797 3830 3831 3832
CONECT 3797 3833
CONECT 3795 3798
CONECT 3796 3799
CONECT 3798 3795 3806
CONECT 3799 3796 3807
C
C
FE+3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
0
0
0
0
199
CONECT 3800 3806 3810 3819
CONECT 3801 3807 3811 3820
CONECT 3802 3805 3816 3818
CONECT
CONECT
CONECT
CONECT
3803
3804
3805
3806
3804 3817
3803 3809
3802 3808
3798 3800
3823
3821
3822
3824
CONECT 3807 3799 3801 3825
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3808
3809
3810
3811
3812
3813
3814
3805
3804
3800
3801
3818 3823
3819 3822
3820 3824
CONECT
CONECT
CONECT
CONECT
3815
3816
3817
3818
3821
3802
3803
3802
3825
3826
3827
3812 3830
CONECT 3819 3800 3813 3831
CONECT 3820 3801 3814 3833
CONECT 3821 3804 3815 3832
CONECT 3822 3805 3813 3830
CONECT 3823 3803 3812 3832
CONECT 3824 3806 3814 3831
CONECT 3825 3807 3815 3833
CONECT 3826 3816 3828
CONECT 3827 3817 3829
CONECT 3828 3826 3834 3836
CONECT 3829 3827 3835 3837
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3830
3831
3832
3833
3834
3835
3797
3797
3797
3797
3828
3829
3818
3819
3821
3820
3822
3824
3823
3825
CONECT 3836 3828
CONECT 3837 3829
END
c. Peroxyheme state
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
3795
3796
3797
3798
3799
3800
3801
3802
3803
3804
3805
CAA HEM
CAB HEM
CAC HEM
CAD HEM
NA HEM
CBA HEM
CBB HEM
CBC HEM
CBD HEM
NB HEM
CGA HEM
600 -17.028 -8.260 69.989 1.00 53.34
600 -13.943 -8.656 60.714 1.00 54.29
600 -20.430 -4.080 60.621 1.00 53.50
600 -20.994 -4.737 68.577 1.00 55.31
600 -16.641 -7.190 66.442 1.00 52.37
600 -17.758 -9.609 69.999 1.00 55.72
600 -13.705 -7.698 59.813 1.00 56.34
600
-21.741 -4.086 60.876 1.00 54.16
600 -21.969 -5.882 68.860 1.00 58.09
600 -15.919 -7.593 63.649 1.00 53.29
600 -18.221 -9.945 71.386 1.00 58.55
C
C
C
C
N
C
C
C
C
N
C
200
HETATM 3806 CGD HEM 600
HETATM 3807 ND HEM 600
HETATM 3808 CHA HEM 600
HETATM 3809 CHB HEM 600
HETATM 3810 CHC HEM 600
HETATM 3811 CHD HEM 600
HETATM 3812 CMA HEM 600
HETATM 3813 CMB HEM 600
HETATM 3814 CMC HEM 600
HETATM 3815 CMD HEM 600
HETATM 3816 CIA HEM 600
HETATM 3817 C1B HEM 600
HETATM 3818 C1C HEM 600
HETATM 3819 ClD HEM 600
HETATM 3820 01 HEM 600
HETATM 3821 01A HEM 600
HETATM 3822 01D HEM 600
HETATM 3823 C2A HEM 600
HETATM 3824 C2B HEM 600
HETATM 3825 C2C HEM 600
HETATM 3826 C2D HEM 600
HETATM 3827 02 HEM 600
HETATM 3828 02A HEM 600
HETATM 3829 02D HEM 600
HETATM 3830 C3A HEM 600
HETATM 3831 C3B HEM 600
HETATM 3832 C3C HEM 600
HETATM 3833 C3D HEM 600
HETATM 3834 C4A HEM 600
HETATM 3835 C4B HEM 600
HETATM 3836 C4C HEM 600
HETATM 3837 C4D HEM 600
HETATM 3838 NC HEM 600
HETATM 3839 FE HEM 600
HETATM 3840 HI HEM 600
CONECT 3827 3840
CONECT 3840 3827
CONECT 3347 3839
CONECT 3795 38003823
CONECT 3796 3801 3831
CONECT 3797 3802 3832
CONECT 3798 38033833
CONECT 3799 3816
CONECT 3799 3834
CONECT 3799 3839
CONECT 3800 37953805
CONECT 3801 3796
CONECT 3802 3797
CONECT 3803 37983806
CONECT 3804 3817
CONECT 3804 3835
CONECT 3804 3839
CONECT 3805 3800
CONECT 3805 3821
CONECT 3805 3828
-22.655 -5.679 70.176 1.00 60.08
-18.991 -5.703 65.605 1.00 53.53
-18.447 -6.389 67.900 1.00 53.35
-14.755 -8.521 65.595 1.00 53.12
-16.564 -7.081 61.342 1.00 53.29
-20.099 -4.703 63.658 1.00 53.36
-14.555 -9.479 68.479 1.00 52.53
-12.835 -9.641 63.472 1.00 52.87
-18.147 -5.623 59.153 1.00 52.76
-22.035 -3.604 65.798 1.00 53.79
-17.265 -7.104 67.652 1.00 52.88
-14.908 -8.296 64.226 1.00 52.92
-17.742 -6.364 61.593 1.00 53.10
-19.967 -4.953 65.031 1.00 53.77
-16.413 -4.844 64.539 1.00 30.00
-17.423 -9.757 72.413 1.00 60.38
-22.112 -6.139 71.281 1.00 59.70
-16.583 -7.878 68.595 1.00 53.10
-14.057 -8.795 63.248 1.00 53.56
-18.476 -5.703 60.617 1.00 53.67
-20.826 -4.472 66.012 1.00 54.14
-16.659 -4.287 63.318 1.00 30.00
-19.447 -10.375 71.585 1.00 58.24
-23.781 -5.004 70.236 1.00 60.88
-15.499 -8.438 67.935 1.00 53.10
-14.554 -8.364 62.030 1.00 54.03
-19.490 -5.021 61.270 1.00 53.94
-20.340 -4.930 67.231 1.00 54.61
-15.581 -8.029 66.611 1.00 53.03
-15.714 -7.638 62.305 1.00 53.89
-19.345 -5.294 62.632 1.00 53.55
-19.213 -5.698 66.947 1.00 54.08
-18.293 -6.139 62.818 1.00 52.75
-17.464 -6.649 64.627 1.00 55.01
-15.917 -3.654 63.002 1.00 53.51
C
N
C
C
C
C
C
C
C
C
C
C
C
C
0
0
0
C
C
C
C
0
0
0
C
C
C
C
C
C
C
C
N
FE+3
H
201
CONECT 3806 3803
CONECT 3806 3822
CONECT 3806 3829
CONECT
CONECT
CONECT
CONECT
3807
3807
3807
3808
3819
3837
3839
3816
CONECT 3808 3837
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3809
3809
3810
3810
3811
3811
3812
3813
3817
3834
3818
3835
3819
3836
3830
3824
CONECT
CONECT
CONECT
CONECT
CONECT
3814
3815
3816
3816
3816
3825
3826
3799
3808
3823
CONECT 3817 3804
CONECT 3817 3809
CONECT 3817 3824
CONECT 3818 3810
CONECT 3818 3825
CONECT 3818 3838
CONECT 3819 3807
CONECT 3819 3811
CONECT 3819 3826
CONECT
CONECT
CONECT
CONECT
3820
3821
3822
3823
3827 3839
3805
3806
3795
CONECT 3823 3816
CONECT
CONECT
CONECT
CONECT
3823
3824
3824
3824
3830
3813
3817
3831
CONECT 3825 3814
CONECT 3825 3818
CONECT 3825 3832
CONECT 3826 3815
CONECT 3826 3819
CONECT 3826 3833
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3827
3828
3829
3830
3830
3830
3831
3831
3831
3832
3820
3805
3806
3812
3823
3834
3796
3824
3835
3797
202
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
END
3832
3832
3833
3833
3833
3834
3834
3834
3835
3835
3835
3836
3836
3836
3837
3837
3837
3838
3838
3838
3839
3825
3836
3798
3826
3837
3799
3809
3830
3804
3810
3831
3811
3832
3838
3807
3808
3833
3818
3836
3839
3347 3799 3804 3807 3820 3838
d. Oxyferyl state
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
3795
3796
3797
3798
3799
3800
3801
3802
3803
3804
3805
3806
3807
3808
3809
3810
3811
3812
3813
3814
3815
3816
3817
3818
3819
3820
CAA HEM
CAB HEM
CAC HEM
CAD HEM
NA HEM
CBA HEM
CBB HEM
CBC HEM
CBD HEM
NB HEM
CGA HEM
CGD HEM
ND HEM
CHA HEM
CHB HEM
CHC HEM
CHD HEM
CMA HEM
CMB HEM
CMC HEM
CMD HEM
CIA HEM
C1B HEM
C1C HEM
ClD HEM
01 HEM
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
600
-17.028 -8.260 69.989 1.00 53.34
-13.943 -8.656 60.714 1.00 54.29
-20.430 -4.080 60.621 1.00 53.50
-20.994 -4.737 68.577 1.00 55.31
-16.641 - 7.190 66.442 1.00 52.37
-17.758 -9.609 69.999 1.00 55.72
-13.705 -7.698 59.813 1.00 56.34
-21.741 -4.086 60.876 1.00 54.16
-21.969 -5.882 68.860 1.00 58.09
-15.919 - 7.593 63.649 1.00 53.29
-18.221 -9.945 71.386 1.00 58.55
-22.655 -5.679 70.176 1.00 60.08
-18.991 -5.703 65.605 1.00 53.53
-18.447 -6.389 67.900 1.00 53.35
-14.755 -8.521 65.595 1.00 53.12
-16.564 -7.081 61.342 1.00 53.29
-20.099 -4.703 63.658 1.00 53.36
-14.555 -9.479 68.479 1.00 52.53
-12.835 -9.641 63.472 1.00 52.87
-18.147 -5.623 59.153 1.00 52.76
-22.035 -3.604 65.798 1.00 53.79
-17.265 -7.104 67.652 1.00 52.88
-14.908 -8.296 64.226 1.00 52.92
-17.742 -6.364 61.593 1.00 53.10
-19.967 -4.953 65.031 1.00 53.77
-16.644 -5.148 64.446 1.00 30.00
C
C
C
C
N
C
C
C
C
N
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
0
203
HETATM
HETATM
HETATM
HETATM
HETATM
HETATM
3821
3822
3823
3824
3825
3826
01A
01D
C2A
C2B
C2C
C2D
HEM
HEM
HEM
HEM
HEM
HEM
600
600
600
600
600
600
-17.423
-22.112
-16.583
-14.057
-18.476
-20.826
-9.757
-6.139
-7.878
-8.795
-5.703
-4.472
72.413
71.281
68.595
63.248
60.617
66.012
1.00 60.38
1.0059.70
1.00 53.10
1.00 53.56
1.00 53.67
1.00 54.14
0
0
C
C
C
C
HETATM 3827 02A HEM 600
HETATM 3828 02D HEM 600
HETATM 3829 C3A HEM 600
-19.447 -10.375 71.585 1.00 58.24
-23.781 -5.004 70.236 1.00 60.88
-15.499 -8.438 67.935 1.00 53.10
0
0
C
HETATM 3830 C3B HEM 600
-14.554 -8.364 62.030 1.00 54.03
C
HETATM 3831 C3C HEM 600
-19.490 -5.021 61.270 1.00 53.94
C
HETATM 3832 C3D HEM
600
-20.340 -4.930 67.231 1.00 54.61
C
HETATM 3833 C4A HEM 600
HETATM 3834 C4B HEM 600
HETATM 3835 C4C HEM 600
-15.581 -8.029 66.611 1.00 53.03
-15.714 -7.638 62.305 1.00 53.89
-19.345 -5.294 62.632 1.00 53.55
C
C
C
HETATM 3836 C4D HEM
600
-19.213 -5.698 66.947 1.00 54.08
C
HETATM 3837 NC HEM 600
-18.293 -6.139 62.818 1.00 52.75
N
HETATM
CONECT
CONECT
CONECT
CONECT
-17.464 -6.649 64.627 1.00 55.01
FE
3838
3347
3795
3796
3797
FE HEM 600
3838
3800 3823
3801 3830
3802 3831
CONECT 3798 3803 3832
CONECT 3799 3816
CONECT 3799 3833
CONECT 3799 3838
CONECT 3800 3795 3805
CONECT 3801 3796
CONECT 3802 3797
CONECT 3803 3798 3806
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3804
3804
3804
3805
3805
3805
3806
3817
3834
3838
3800
3821
3827
3803
CONECT 3806 3822
CONECT 3806 3828
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3807
3807
3807
3808
3808
3809
3809
3810
3819
3836
3838
3816
3836
3817
3833
3818
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3810
3811
3811
3812
3813
3814
3815
3816
3834
3819
3835
3829
3824
3825
3826
3799
204
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3816
3816
3817
3817
3817
3818
3818
3818
3808
3823
3804
3809
3824
3810
3825
3837
CONECT 3819 3807
CONECT 3819 3811
CONECT 3819 3826
CONECT 3820 3838
CONECT 3821 3805
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3822
3823
3823
3823
3824
3824
3825
3825
3825
3826
3806
3795
3816
3829
3813
3830
3814
3818
3831
3815
CONECT 3826 3819
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3826
3827
3828
3829
3829
3829
3830
3830
3830
3831
3831
3832
3832
3832
3805
3806
3812
3823
3833
3796
3824
3834
3797
3825
3798
3826
CONECT 3832 3836
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
CONECT
3833
3833
3833
3834
3834
3834
3835
3835
3835
3836
3836
3836
3837
3837
3837
3838
3838
3799
3809
3829
3804
3810
3830
3811
3831
3837
3807
3808
3832
3818
3835
3838
3347 3799 3804 3807
3820
205
CONECT 3838 3837
END
e. Sample code for substrate docking
receptor = taxadiene5ahydroxylase.pdbqt
ligand = taxadiene.pdbqt
out = output iteration_1.pdbqt
center_x = -22.519
centery = -19.74
center_z = -10.502
sizex = 40
size_y = 40
sizez = 40
exhaustiveness = 9
206
Appendix E: NMR raw data
207
OH
HH
Taxadien-5a-ol
Species K
.
.4
SA
400
300
200
100
0
19.81
52
5.0
4.B
4.b
4.4
4.2
CD
.
l
34
L.2
3.D
2-a
fi 1vpmno
2.6
2.4
2.Z
2.
1.1,
1.5
1.4
12
1.0
D.5
i-i
r \.-&.
21.00
22.20
Retention time (minutes)
23.39
-
A
24.58
GOk
0.4
3000
2250
1500
750
0
Oxacyclotaxane (OCT)
Species I
4.6
4.4
400
300 200 100
0198
4.2
4.0
a.9
3.6
3.4
.2
LO
LB
2.6
L4
ft I ga
2-2
Z.
L's
1.6
14
L2
o.6
A
L'0
L
19.81
21.00
22.20
Retention time (minutes)
23.39
24.58
A
3000
2250
1500
750
0
Cyclic ether
Species G
i-
k
4.-4
42
400 300 200 100
0 -19.81
I
I-
4.0
.4
1-
-
1.2
3-0
2-
Z.6.
2.4
fl
22
wPPM)
Z.0
1.8
IA.
I
1.2
1-4
1.0
-8
0.b
-.
21.00
22.20
Retention time (minutes)
23.39
24.58
0.4
0-2
3000
2250
1500
750
0