THE CREATION OF GLYCODENDRIMERS FOR THE INHIBITION OF HIV USING
THE DISACCHARID MALTOSE AND TRIS 2-(AMINOETHYL)AMINE
Rachel Ann Blackeye
B.A., California State University, Sacramento, 2008
THESIS
Submitted in partial satisfaction of the
requirements of the degree of
MASTER OF SCIENCE
in
CHEMISTRY
(Biochemistry)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
FALL
2011
THE CREATION OF GLYCODENDRIMERS FOR THE INHIBITION OF HIV USING
THE DISACCHARIDE MALTOSE AND TRIS 2-(AMINOETHYL)AMINE
A Thesis
by
Rachel Ann Blackeye
Approved by:
__________________________________________, Committee Chair
Katherine Dawn McReynolds, Ph.D.
__________________________________________, Committee Member
Mary McCarthy-Hinz, Ph.D.
__________________________________________, Committee Member
Cynthia Kellen-Yuen, Ph.D
___________________________
Date
ii
Student: Rachel Ann Blackeye
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credits
is to be awarded for the thesis.
__________________________, Graduate Coordinator ___________________
Susan Crawford, Ph.D.
Date
Department of Chemistry
iii
Abstract
of
THE CREATION OF GLYCODENDRIMERS FOR THE INHIBITION OF HIV USING
THE DISACCHARIDE MALTOSE AND TRIS 2-(AMINOETHYL)AMINE
by
Rachel Ann Blackeye
The human immunodeficiency virus (HIV) affects people around the globe.
According to the National Institutes of Health, around 33.3 million individuals worldwide
are infected with HIV-1, and within this group more than 1 million people are found in
the United States. There are therapeutic treatments used to fight HIV before it develops
into AIDS (acquired immunodeficiency syndrome), but they are not without drawbacks.
Viral resistance and harmful side effects are problems that have led to new research
directed towards fighting this virus. Most of the drugs created thus far attack the virus
from within the host cell. Scientists are now looking at targets on the viral surface that
would prevent the virus from entering the cell.
Dendrimers, which have been around since the mid 1980s, have the potential to
block viral entry through the multivalent effect. Dendrimers are branched
macromolecules that are globular in shape, and structurally well-defined with multivalent
reactive terminal groups. As these molecules branch out from the core, new generations
iv
of compounds may be added to double the number of reactive ends. When the end groups
on a dendrimer are carbohydrates, it is known as a glycodendrimer. There are two
processes used to formulate glycodendrimers called the divergent and convergent
methods. In the divergent method, dendrimers are synthesized from the core outwards.
With the convergent technique, the outer portions are synthesized first and then added to
the core. Dendrimers have been utilized in a variety of ways since their creation in 1984.
In some studies, glycodendrimers have demonstrated anti-HIV properties.
The goal in this research was to create two glycodendrimers using both the
convergent and divergent processes. The hexavalent maltose amino-oxime
glycodendrimer was synthesized (66% yield) using the convergent method and the
trivalent maltose amino-amide glycodendrimer was created (3.4% yield) via the divergent
method. A hexavalent amino core (16% yield) was synthesized as well, which will be
used in later research to create other glycodendrimers.
In the end, once these glycodendrimers are sulfated, they will be evaluated in a
competitive gp120 binding assays for binding affinity. If strong affinity between gp120
and the glycodendrimers is established, then these glycodendrimers will be further
v
assessed in an inhibition of viral infectivity assay. This assay will involve HIV in vitro
with active viral particles. In the end, this research could lead to the prevention of HIV
infection.
________________________________________, Committee Chair
Katherine Dawn McReynolds, Ph.D.
______________________________
Date
vi
ACKNOWLEDGMENTS
First I have to thank God, because if it were not for all the signs from above, I
would not have completed this difficult task to begin with. There were many times when
I needed the inspiration to continue on this journey and my prayers never went
unanswered.
Next, I must thank my friend and mentor, Dr. Katherine McReynolds, who has
challenged, supported, and guided me through this whole process. Without her constant
guidance through the Masters program, I believe I surely would have pulled out all of my
hair by now. I also want to thank my committee, Dr. Mary McCarthy and Dr. Cynthia
Kellen-Yuen, their effort and patience during this tedious writing process has been
greatly appreciated.
I want to thank all the people in Dr. McReynolds’ research group. Some group
members that I particularly want to thank are: Michelle Watterson, Russ Clayton, and
Carolyn Lozo, for being my lab buddy when working late nights in the lab. I also want to
thank, Vince Trapani, Jon Patane, Careena Cary, and Alex Keith for aiding me in the lab
when I needed some extra work done. I also want to thank Janee′ Hardman for testing
my sulfated molecule as quick as she could, so that I could mention the results at my
thesis defense. I want to say that it has been a pleasure working with each and every one
of you.
Next, I need to thank the organizations and programs that have aided or led me to
vii
this point in my education and in research. This includes my tribe, the Duckwater
Shoshones. Without their financial support during this time, my advancement in this
program would have been incredibly difficult. The Science Transfer Project (STP) and
Science Education Equity (SEE) program have provided opportunities that basically led
me towards this research. The support the NIH NIAID AREA supplement grant program
provided during this time was very much appreciated.
I want to acknowledge my parents, Henry and Jeri, whom I love so much. Thank
you both for passing on virtues that have helped me in all areas of my life. Finally, I
must express my love and gratitude to my husband, Steve Guay. It was my husband that
first encouraged me to go back to school and find my niche. Although my major had
changed quite a few times along this road, his love, support, and encouragement has
always been there. Although hair loss research might have served him better.
viii
TABLE OF CONTENTS
Pages
Acknowledgments…………………………………………………………………….... vii
List of Figures…………………………………………………………………………… xi
List of Schemes…………………………………………………………………….…... xiv
Chapter
1. BACKGROUND ………………………………………………………………….......1
Introduction to HIV …………………………………………….…………..…....1
The Structure of HIV-1………………………………………….…………......... 2
Viral Entry and Replication …………………………..…………………..…….. 4
Available Treatments for HIV………..……………….….……..……………..... 8
The Effects of HIV Treatment………………………….…………………….... 13
Developing Research and Treatment for HIV…………………………………. 14
Anionic Sulfated Compounds ……………………..…………..……………..... 18
The Multivalent Effect and Dendrimers ………………………………………. 19
Overview of the Current Project …..…………………………………………... 27
2. RESULTS AND DISCUSSION…………………………………………………….. 32
3. CONCLUSION AND FUTURE WORK ...………………………………………… 84
4. EXPERIMENTAL…………………………………………………………………... 86
Materials............................................................................................................... 86
Instrumentation………………………………………………………………......87
ix
Characterization………………………………………………………………….87
Methods..................................................................................................................88
Appendix A. 1H NMR Spectra ........................................................................................102
Appendix B. 13C NMR Spectra .......................................................................................115
Appendix C IR Spectra……………………………………………………………...…128
Appendix D Mass Spectra………………………………………………………..……132
References ……………………………………………………………………………...140
x
LIST OF FIGURES
Page
1.
Illustration of the structure of HIV-1 ...…………………………………………….. 3
2.
HIV-1 viral particle seen through electron microscopy…………………………...... 4
3.
Mechanism between HIV and the host cell ……………. …………………………. 5
4.
Heparan sulfated syndecans and tyrosine sulfated coreceptor CCR5 ……………… 6
5.
Overview of HIV replication ………………………………………………………. 8
6.
Zidovudine and didanosine …………………………..…………………………….. 9
7.
Efavirenz and etravirine ……………….………………………………………….. 10
8.
Ritonavir and amprenavir ………….………………………………………...…… 11
9.
Enfuvirtide ……………..…………………………………………………………. 12
10. Mechanism of enfuvirtide ………………………………………………..……….. 12
11. Gag assembly that leads to virion maturation ……………………………….……. 16
12. Bevirimat ……………..……….…………………………………………………... 16
13. Heparin sulfate and Dextran sulfate ………………………………………...…….. 19
14. Colominic acid ……………………………………………………………………. 19
15. Divergent and convergent methods ………………………………………………. 21
16. Generations of dendrimers………………………………………………………… 21
17. A generation 2 PAMAM dendrimer …………………………………………….... 22
18. Dendrimer mimic of glutathione peroxidase ………………………………...…….23
19. Generation 4 PAMAM dendrimer………………………………………………… 24
xi
20. (G3- G5) polypropylenimine (DAB-Am) dendrimers………………….…………. 25
21. The dendrimer SPL7013 ………………………………………………….………. 27
22. Hexavalent maltose amino-oxime glycodendrimer ...………….……………….… 29
23. Trivalent maltose amino-amide glycodendrimer ..…...………….…………..……. 30
24. Hexavalent amino core …………….……………………………………………... 31
25.
1
H NMR of Compound 1 …..…………………………………………………...….37
26.
1
H NMR of Compound 3 ……………………………………………………….….40
27.
1
H NMR of Compound 4 ……………………………………………………….… 44
28. An illustration of T1 and T2 ……………………………………………………….. 45
29.
IR of Compound 4 ……………………………………...………………...……….46
30.
IR of Compound 5….…………...…………………………………………………49
31.
32.
1
H NMR of Compound 6 …………………..…………………………….…..….... 51
Maltose isomers……………………………………………………………………55
33.
1
H NMR of Compound 8 ………………………………………………………..…56
34.
1
H NMR of Compound 9 ……………………………….………………………….58
35.
1
H NMR of Compound 10 ………………..……….……………………………….62
36. Comparing spectra………………………………………………………………… 63
37. MAIDI TOF of Compound 10 ……………………………………..…………….. 66
38.
1
H NMR of Compound 12 …………………..………………………………….….71
39.
1
H NMR of Compound 13 ………………..………………………….…………….73
40.
1
H NMR of Compound 14 …………………..………………………………….….75
41.
IR of Compound 15 ……………………………………...………………………..78
xii
42.
43.
44.
IR of Compound 16 .………………………………………...………………..….. 80
1
H NMR of Compound 17 .………………………………………………..……… 82
Mass spectrum of Compound 17 ..………………………………………..……… 83
xiii
LIST OF SCHEMES
Page
1.
Synthesis of 3-(3-hydroxypropoxy)propanenitrile, Compound 1 ………..………. 34
2.
Example of the Michael reaction …………………………………………………. 34
3.
Synthesis of 3-(2-cyanoethoxy)propyl p-toluenebenzene, Compound 3 ………... 38
4.
Synthesis of hexavalent nitrile core, Compound 4 ………………...……………... 41
5.
Synthesis of hexavalent carboxy core, Compound 6 …………………………….. 47
6.
Synthesis of E,Z oxime sugar-linker, Compound 9 ……...…...…….……………..53
7.
Synthesis of hexavalent maltose amino-oxime glycodendrimer, Compound 10 .... 60
8.
Proposed ring closing mechanism …………………………………………………64
9.
Conditions for sulfated glycodendrimer ………………………………………….. 67
10. Synthesis of trivalent amino-amide core, Compound 13 ………….…...…………. 69
11. Synthesis of maltonic acid, Compound 14 ………..……………………………… 74
12. Synthesis of trivalent maltose amino-amide glycodendrimer, Compound 15 ..........76
13. Synthesis of hexavalent amino core, Compound 17 ………..…………...………... 79
xiv
1
Chapter 1
BACKGROUND
Introduction to HIV
The human immunodeficiency virus (HIV) is a virus that affects millions of
people worldwide. In 2009, it was estimated that 33.3 million people were infected with
HIV/AIDS (acquired immune deficiency syndrome).1 Within this group there were 2.6
million newly infected cases and 1.8 million deaths as a result of the virus. HIV attacks
the immune system and ultimately results in AIDS. The progression from HIV infection
to AIDS varies among the infected persons, depending on how each responds to
treatments that are available. One available treatment is HAART (highly active antiretroviral therapy).2 In this therapy, a combination of HIV inhibitors are taken together.
There are 25 different drugs that are used in various combinations in the HAART
regimen. These drugs fall into 5 major categories: protease inhibitors, fusion inhibitors,
non-nucleoside reverse transcriptase inhibitors (NNRTIs), and nucleoside reverse
transcriptase inhibitors (NRTIs).3 When taken in combination, these drugs have helped
many individuals to live with the virus and slow the progression to AIDS. There are some
drawbacks though, with the HAART regimen, such as toxic side effects and viral
resistance.4 Because of this, more research is needed to overcome these problems. A
new area of research to fight against the virus involves attacking it before it enters the
2
host cell.5-7 To achieve this goal, all processes and components of the virus must be
evaluated.
The Structure of HIV-1
The structural composition of HIV (Figures 1 and 2) has been determined
through various studies.8,9 Its appearance is spherical in shape with protruding protein
spikes on the outer surface. The protruding components covering the outer membrane
consist of a complex of two trimeric proteins, gp120 (glycoprotein, 120 kDa) and gp41
(glycoprotein, 41 kDa). Other components of the viral envelope are lipids and proteins,
with the lipids predominating. Within the outer membrane lies the viral matrix, which is
composed of viral p17 (protein, 17 kDa). Further inside the virion there is a cone-shaped
capsid formed by two thousand copies of protein p24 (a 24 kDa protein). Within the
capsid, there are two single strands of viral genomic RNA. There are a total of nine genes
in each strand of viral RNA; gag, pol, env, tat, rev, nef, vif, vpr, and vpu. There are also
three enzymes in the capsid: reverse transcriptase (RT), protease (PR), and integrase (IN).
These collective components of the virion are essential for the invasion and replication of
the virus in the host cell.8,9
3
Figure 1. Illustration of one HIV-1 viral particle and its components shown in ribbon
form; surface gp120 (SU), transmembrane gp41 (TM), matrix p17 (MA), capsid p24
(CA), nuleocapsid p7 (NC), protease (PR), reverse transcriptase (RT), integrase (IN), and
accessory protein (Nef) .8
4
100 nm scale
100 nm scale
Figure 2. Ultrathin sectioning was used to visualize this HIV-1 viral particle through
electron microscopy.9
Viral Entry and Replication
The HIV virus must gain entry into a host cell to replicate. Researchers have
discovered that there are multiple entry mechanisms.11 The most common starts with
molecular recognition between the virion and the host cell (CD4 lymphocytes, dendritic
cells, and macrophages).11 The viral gp120 initially binds to the CD4 receptor on the
host cell (Figure 3).12 The binding of these proteins creates a conformational change on
the cell surface, which exposes a coreceptor (CCR5 or CXCR4). The co-receptor then
binds to a region within the gp120 called the 3rd variable (V3) loop. When this
interaction occurs, there is another conformational change within the gp120/gp41
complex and the gp120 dissociates from gp41, allowing the N terminal part of the gp41
(i.e., the “fusion peptide”) to insert into the host cell membrane.10 Following this event,
5
more conformational changes occur and a six-helix bundle is formed within the gp41,
causing the viral envelope and the host cell membrane to merge together, resulting in
fusion.10-13
(a)
(b)
(c)
Figure 3. The binding mechanism which causes membrane fusion between the HIV
virion and the host cell membrane.12 (a) The binding between the viral gp120/41
complex and the host receptor CD4. (b) The interaction between viral gp120 to the CD4
receptor and coreceptor (CCR5 or CXCR4), and the insertion of the N-terminal region of
gp41 into the host cell membrane. (c) Gp41 forms a six helix bundle after gp120
dissociates; this process brings the viral and host cell membranes together and fusion
occurs .11-14
Another viral entry mechanism involves cell surface glycoproteins. It has been
proposed that heparin sulfate proteoglycans (HSPGs) aid in viral entry.15,16 HSPGs are
6
also known as syndecans and are found on all host cell surfaces. Syndecans bind to HIV
in the same manner as CCR5 (Figure 4).12 These syndecans are covered with linear
polyanionic glycosaminoglycan (GAG) branches which have sulfated sites that interact
with the virus. It is thought that the 6-O sulfate groups within the GAG branches are the
sites that bind with the viral gp120/V3 loop. Once this binding event occurs there is a
second interaction with gp120 within its conserved region (Arg 298).15,16 This process
ultimately leads to membrane fusion, allowing viral contents to empty into the host cell
so that the replication process can begin.9,14
Figure 4. Syndecans and CCR5 bind to HIV in a similar fashion. The heparan sulfated
syndecan (left) has sulfated sugars (top left) that interact with the viral gp120/V3 loop.
The CD4 receptor (right) interact with gp120, while the CCR5 coreceptor (right) interacts
with the viral gp120/V3 loop with the support of sulfated tyrosine residues (top right)
found in CCR5.12,15
7
Viral replication (Figure 5) is a process with numerous steps.11 Once fusion (vide
supra) occurs between the target cell and the virus, the viral capsid is released into the
cytoplasm of the host cell.9,14 Next, the capsid is uncoated and viral reverse transcriptase
starts copying the single stranded viral RNA into double-stranded viral DNA. When this
process is complete, the viral DNA is transferred into the nucleus and inserted into the
host chromosome by the viral integrase. Following this, transcription begins and viral
RNA is created, which then leads to the translation of the RNA to viral polyproteins. The
long strands of proteins are cleaved by host and viral proteases, forming viable structural
and functional proteins for the new virion. Once a substantial accumulation of these viral
components has occurred near the host cell membrane, the budding process begins,
forming new virions.9,14 Each time a virion buds, it carries away some of the host cell
membrane. Figure 5 demonstrates the replication of one virion.17 In reality, many
copies are formed simultaneously. As this phase continues over and over again the host
cell membrane is consumed, ultimately killing the host cell. Eventually, the immune
system is compromised due to the loss of CD4 positive cells.9 However, scientists have
discovered ways to block viral replication before it completely destroys the immune
system through drug therapy.
8
Figure 5. An overview of HIV replication.
First, viral HIV binds to the host CD4
receptor/coreceptor. Once fusion occurs, the
capsid is released into the cytoplasm of the
host cell and uncoated. Next, the viral RNA
(yellow) is copied into double stranded DNA
(red) using the viral enzyme reverse
transcriptase. After this, the viral DNA enters
the nucleus and is integrated into the host
chromosomes (green and yellow) by the viral
enzyme integrase. In the nucleus, viral RNA
(yellow) is created through transcription. The
viral RNA is then transferred out of the
nucleus and polypetides are formed during
translation. The polypeptides are cleaved by
both viral and host proteases into active viral
components. The viral proteins and RNA
come together and bud from the host cell,
forming new virions.9,14,17
Available Treatments for HIV
In 2009, the treatment for HIV consisted of 25 drug/drug combinations. These
drugs are still being used today and fall into 6 different categories.17 Each class of drugs
inhibits a different viral course of action, from viral attachment, to viral protein
9
replication within the host cell. There are five groups of drugs involved in enzymatic
inhibition and one group that targets coreceptor/cell entry.17 Starting with enzyme-based
inhibitors, there are three groups that target viral reverse transcriptase (RT): nucleoside
RT inhibitors (NRTIs), nucleotide RT inhibitors (NtRTIs), and non-nucleoside RT
inhibitors (NNRTIs). The NRTIs (zidovudine (AZT), didanosine (ddI), zalcitabine
(ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), and emtricitabine ((-)FTC))
inhibit the normal nucleotide substrate from binding to the RT active site, and incorporate
into viral DNA as chain terminators to stop DNA production. Two nucleoside RT
inhibitors, AZT and ddI, can be seen in Figure 6.17 The next RT inhibitor class is the
NtRTI group. This group inhibits by incorporating into the 3’ terminus of the viral DNA,
terminating the production of viral DNA. There is one widely prescribed NtRTI on the
market today, called tenofovir disoproxil fumarate (TDF, Viread®). The last class of RT
inhibitors is the NNRTI group. NNRTI (nevirapine, delavirdine, efavirenz, and
etravirine, Figure 7) inhibitors bind to an allosteric site on RT, causing enzyme
inactivitation.17
Figure 6. Nucleoside RT inhibitors: zidovudine (left), didanosine (right).17
10
Figure 7. Two non-nucleoside RT inhibitors: efavirenz (left), etravirine (right).17
Another class of HIV inhibitors targets viral integrase to prevent the insertion of
viral DNA into the host genome.17 Integrase has two important functions, which are 3′processing followed by the strand transfer of viral DNA into the host genome. Before
strand transfer takes place, integrase first cleaves viral DNA at the 3′ conserved
dinucleotide CA (cytosine adenine). Following this, the complex composed of integrase
and the cleaved DNA translocates to the nucleus, where it binds to the host genes in a
sequence-dependent manner. There is one integrase inhibitor (INI) that blocks, the strand
transfer process called raltegravir.17
The last targeted enzyme within the host cell is viral protease. There are 10 viral
protease inhibitors (PIs) available (saquinavir, ritonavir, indinavir, nelfinavir, amprenavir,
lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir), all of which compete with
the viral polyproteins to bind within the protease active site (Figure 8). Since these PIs
11
cannot be cleaved like the viral polyproteins, they remain in the active site causing
inactivity of the enzyme.17
Figure 8. Examples of two protease inhibitors: ritonavir (left), amprenavir (right).17
In addition to the enzymatic inhibitors which work inside the host cell to prevent
viral replication, there are two types of inhibitors that work externally to the host cell at
the earliest stages of infection. The first class is comprised of fusion inhibitors (FIs).
Enfuvirtide (DP-178, pentafuside, T-20, Fuzeon®, Figure 9) is the only fusion inhibitor
currently available. It is a polypeptide that blocks the virus from entering the host cell by
engaging in a coil-coil interaction with the heptad repeats (HR) of viral gp41, as shown in
Figure 10. Unlike the previously discussed drugs, which can be taken orally, enfuvirtide
is injected subcutaneously twice a day. This makes enfuvirtide more difficult to use
compared to other inhibitors. The second type of inhibitor that works outside the cell are
the co-receptor inhibitors (CRIs). Maraviroc, is currently the only one drug that falls into
this group. It works on the host cell surface by interacting with the CCR5 or CXCR4 coreceptors, altering their conformation. When this process occurs, there is no molecular
12
recognition between viral gp120 and these co-receptors, so ultimately, viral fusion is
prevented.17
Figure 9. The polypeptide enfuvirtide containing 36 amino acids.17
Figure 10. Enfuvirtide (yellow) interacts with viral gp41’s HR1(heptad repeat region 1,
red) and HR2 (heptad repeat region 2, blue). This interaction blocks gp41 from folding
back on itself, stopping the virion membrane (green) and the host cell membrane (gray)
from fusing.17
13
Because the groups of drugs described above are used to inhibit HIV replication
via different mechanisms, they are used in combinations as a stronger line of defense
against viral invasion.17 Since 1996, drug combinations have been used to fight against
HIV. As previously mentioned, this treatment is known as HAART.2,17 This is a
regimen that combines at least three anti-HIV drugs from the different classes of
inhibitors.17 In the past, some patients were taking more than 20 pills daily. Recently,
some HIV inhibitors were combined into one pill, making patient compliance easier.
Atripla®, for example, was created in 2006 and contains three different RT inhibitors,
tenofovir (NtRTI), efavirenz (NRTI), and emtricitabine (NNRTI).17 Although there are
different drugs/drug combinations to combat HIV replication, there are also drawbacks
associated with them.2
The Effects of HIV Treatment
Since the introduction of HAART, the survival rate for infected patients has risen,
but not without some complications. The level of complications encountered depends on
how well the patients adhere to treatment, and how far along the disease has progressed
before treatment is initiated.2,17-19 When patients follow the recommended regimens, they
usually take several drugs a day from at least two different groups of inhibitors. For some
patients, the side effects of the drugs are problematic, and they end up not taking the
recommended dosage. Other patients are advised to discontinue use of the drugs because
of new health conditions arising from the treatment. Various studies have been
performed to evaluate the different side effects associated with these antiviral drugs.
14
One study involving 109 patients revealed that the most common side effects were
fatigue, stiff and painful joints, aching muscles, diarrhea, depression, and neuropathy.20
The worst problems experienced were changes in physical appearance, through weight
gain and lipodystrophy, sleeplessness, joint stiffness and pain, fatigue, and neuropathy.
The less severe problems patients experienced were fever and cold-like symptoms.20
Another problem that occurs with the HAART therapy is viral resistance. Viral
mutations have been reported with each class of drugs, which leads to inefficient drug
therapy for patients.2,21 In the end, when HAART therapy fails, patients have no alternate
methods to turn to, therefore new methods are needed to combat HIV.
Developing Research and Treatment for HIV
Since the discovery of HIV, developing a vaccine has been a very active area of
research. Today, the search for a vaccine is still underway; however, some think there is
little hope that one will ever be created.22 However, if a vaccine can be created and
implemented, some issues involving efficacy, safety, and ethics must first be addressed.
The biggest problem is that viral particles may hide in the body undetected in HIV
reservoirs, such as the brain, bone marrow, and the lymph nodes.23 Therefore, if a vaccine
is administered, its efficiency at preventing infection may not be completely realized.22
The latest vaccine (MRKAd5 HIV-1 gag/pol/nef) created by Merck, was a failure. Their
overall results indicated that there was an increase in HIV infection among patients
receiving the vaccine compared to the control group in their study.24 While the search for
a viable vaccine is still underway, some new anti-viral agents are under development.
15
Inhibition of virion maturation is a new area of research for HIV drug therapy.25
Viral maturation happens after the virus buds from the infected cell, when it matures into
a viable particle capable of infection. Scientists are seeking target sites to stop this late
stage in viral replication.25 One target is the viral Gag polyprotein, which forms the viral
membrane (Figure 11).18 After budding occurs, Gag processing mediated by viral
protease continues, which involves the cleavage of the capsid and spacer peptides to form
a mature capsid. Recently, it has been found that dimethylsuccinyl betulinic acid (PA457 or bevirimat) may inhibit this processing event in virion maturation.18 While
bevirimat (Figure 12) is in phase II clinical trials, some scientists anticipate viral
resistance. Because viral resistance has always been a issue with drug therapy
researchers are always seeking different avenues to treat HIV.
16
Figure 11. Gag assembly that leads to virion maturation. (A) Composition of Gag: the Nterminal myristate (Myr), matrix (MA) and +++ denotes a highly basic domain, capsid
(CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2) and the Ctemirmal p6 and Pr55Gag (polyprotein precursor) domains. (B) Illustration of viral
assembly, release, and maturation.18
Figure 12. The structure of bevirimat.25
17
Gene therapy is another active area of research that is being investigated to
combat HIV. It is known that some individuals are highly resistant to HIV infection, due
to a mutation in the CCR5 gene in their CD4 cells. The CCR5 coreceptor works with the
CD4 receptor to initiate viral infection (vide supra). Some people do not express the
CCR5 coreceptor, due to a 32-base pair deletion in the CCR5 gene.27 If CCR5 is not
present on the host cell to aid the CD4 receptor, then the host cell is resistant to HIV
infectivity. There is one anecdotal report of apparently successful “gene therapy” based
on this. Recently, an HIV patient with acute myeloid leukemia was treated with
chemotherapy; the leukemia went into remission but eventually returned. The next line
of defense against leukemia for this HIV patient was a bone marrow transplant. The HIV
patient was given two bone marrow transplants from a donor who had two copies of this
mutated gene. Four years later, his leukemia and HIV have not returned. The successful
results of this case has led to more research with the mutated CCR5 gene.26
A small trial was recently performed to mimic the success of this HIV/leukemia
patient.26 In the treatment process, the patients undergo leukapheresis to remove their
CD4 cells. The CD4 cells are then modified with a zinc-finger nuclease to change the
DNA in the CCR5 gene. The mutated CD4 cells are then infused back into the patients.
In this study, it was estimated that about 25% of the cells put back into the patients were
modified by the enzyme. In some patients these mutated cells persisted for 3 months, but
eventually dropped from 25% to just above 5%. As of now, it is not clear if these
remaining mutated cells will evolve, but this study has given hope that gene therapy has
18
the potential to cure HIV one day.26 When or if this day will arrive remains unclear. In
the meanwhile, researchers are seeking other ways to fight this virus.
Anionic Sulfated Compounds
It has been established that sulfated polysaccharides have the ability to inhibit
HIV.28,29 Two naturally occurring molecules, dextran sulfate and heparin sulfate (Figure
13), have demonstrated the inhibition of HIV-1 infection among CD4 positive cells in
vitro, at low (µg/mL) concentrations.28 Inhibition is due to the binding between the
sulfated polysaccharides and multiple basic amino acids found in 3rd variable loop region
within gp120. Although these compounds displayed inhibitory properties against HIV,
there are some significant drawbacks to their use, such as cytotoxicity and increased
blood clotting time (anticoagulant effect). One sulfated polysaccharide found to work
without these detrimental effects is known as sulfated colominic acid (SCA, Figure 14),
an α-28 linked homopolymer of sialic acid. In one study, when compared to dextran
sulfate, SCA demonstrated no anti-coagulant activity even at 10 µg/mL, while dextran
showed decreased blood clotting at 1 µg/mL.30 It was also found that SCA (6 - 12%
sulfur by weight) had more potent anti-viral activity compared to unsulfated colominic
acid, and that T-cell growth was not impeded by concentrations up to 100μg/mL.25
Based on these results, some scientists have evaluated SCA further, specifically by
combining it with the multivalent effect (vide infra).
19
Figure 13. Structure of two sulfated polysaccharides.5
Figure 14. Sulfated colominic acid.5
The Multivalent Effect and Dendrimers
The multivalent effect is a process that improves binding interactions through the
attachment of multiple molecules to many receptors at one time.31 Molecules that have
this effect are multivalent and have been studied from both natural and synthetic sources.
In nature, this process can be illustrated with the influenza virus as it enters the host cell.
It has been estimated that between 300 and 600 hemagglutinin spikes on the viral surface
20
allow it to develop a strong interaction with sialyl moieties on the host cell prior to
infection.32 This same interaction has been accomplished using a multivalent synthetic
compound known as a dendrimer (vide infra).33
Dendrimers are branched macromolecules that are globular in shape and
structurally well defined. The main components are the core, the branching structures,
and the terminal groups.34 They are assembled either convergently or divergently (Figure
15).35 When a dendrimer is synthesized convergently, the outermost portions are created
first then added to a core molecule. The divergent approach consists of building from the
core outward. Each method develops a branched system within the compound. Each
point of branching leads to a different generation of molecules with an increasing number
of reactive ends (Figure 16).35 The multiple termini give it the property of being
multivalent. The first class of dendrimers that were created are known as
poly(amidoamine), or PAMAM (Figure 17). Since the formation of PAMAM in 1984,
the study of dendrimers has become an active research area.34 Dendrimer research is
increasing because of the potential these compounds have for molecular recognition and
improved binding affinity. Dendrimers could lead to prospective anti-viral compounds,
in addition to numerous other applications.34-38
21
Figure 15. Divergent dendrimer synthesis is a method that builds from the core outward.
The convergent method combines the branching units and surface groups of the
dendrimer together before adding them to the core.35
Figure 16. A generation (G) 7 dendrimer is developed from a poly(amidoamine)-NH3
core.36
22
Figure 17. A generation 2 PAMAM dendrimer.37
Dendrimers are versatile molecules, making them perfect for numerous
applications. Because dendrimers can take on different sizes and shapes, they have been
synthesized to mimic various molecules.29 Dendrimers have been used as DNA and drug
carriers.30,33 They have been created and functionalized to mimic carbohydrate-protein
interactions and enzyme-like catalysis as well.29 One example of a dendrimer mimicking
enzyme activity was demonstrated by Zhang and co-workers.34 They created a
generation 3 dendrimer (Figure 18) that had the catalytic activity resembling glutathione
23
peroxidase, a mammalian enzyme that catalyzes the reduction of hydroperoxides which
build up inside the body.
Figure 18. Generation 3 dendrimer mimic of glutathione peroxidase.34
Dendrimers also have the potential to inhibit viral infection.33 Since it has been
established that the influenza virus binds to a host cell before infection via a multivalent
interaction (vide supra), a study was performed to mimic this interaction utilizing a
dendrimer.33 The host cell mimic employed in this research was a PAMAM-based
dendrimer functionalized with sialic acid residues (Figure 19). This study demonstrated
the inhibition of the influenza virus in both in vitro and in vivo. When tested in vitro, the
dendrimer blocked viral infection at concentrations of 0.058 - 0.195 mM. When this
24
dendrimer was evaluated in vivo, it displayed no toxic side-effects in the test subjects (14
mice), yet still resulted in the potent inhibition of the influenza virus. The mice were
exposed to a lethal dose of the virus mixed with the G4 sialic dendrimer (9 µg per g of
body weight). After 14 days there was a 100% survival rate, versus 6% in the control
group.33 Through this study, it has been verified that dendrimers have the potential to
inhibit viral infection through molecular recognition and the multivalent effect. Because
of this, more research has been performed utilizing dendrimers as anti-viral agents.39
OH
HO
O
H
N
H3C
CO2-
OH
HO
H
N
S
H
N
C
G4 PAMAM dendrimer
S
O
Figure 19. Sialic acid conjugated with a generation 4 (G4) PAMAM dendrimer.33
Another study that utilized dendrimer constructs for viral inhibition was
performed in 2003.39 This study developed different generation dendrimers
functionalized with sulfated galactose residues to inhibit HIV infection. The research
demonstrated that the sulfated 3-(β-D-galacto pyranosylthio)-propionic acid derivatives
(Figure 20) had the ability to inhibit HIV-1 in CD4 negative cells. Generation 3 - 5
dendrimers had EC50 (effective concentration for 50 % inhibition) values of 90 μM, 70
μM, and 20 μM, respectively. These glycodendrimers also demonstrated no cytotoxicity
25
up to 3 mg/mL. Thus, the dendrimers in this study showed potential to inhibit HIV.
After further analysis, the dendrimers described here may become a novel class of antiHIV agents.39
OR
OR
O
H
N
S
RO
OR
R = H or SO3-
O
G3-G5 DAB-Am dendrimers
Figure 20. 3-(β-D-galactopyranosylthio)-propionic acid with generations 3 thru 5 (G3G5) polypropylenimine (DAB-Am) dendrimers.39
One dendrimer that has been analyzed extensively for its anti-viral properties is
SPL7013 (Vivagel®, Figure 21).6,7 This dendrimer has been found to inhibit both HSV
(herpes simplex virus)-2 and HIV-1.6 It is now in clinical trials as a microbicide agent.
Microbicides are molecules that are used topically, to prevent sexually transmitted
diseases. The most recent clinical study performed with the SPL7013 dendrimer involved
ex vivo assays. The cervicovaginal fluid (CVF) from 11 women were collected after the
vaginal application of one dose of SPL7013 gel.6 The samples were tested with and
without seminal fluid over 24 hours for antiviral activity, with both HIV-1 and HSV-2.
26
After a 3 hour period post dose of SPL7013, without the addition of seminal fluid, there
was still antiviral activity among samples from all women, with averages of 96% (HIV-1)
and 94% (HSV-2) inhibition. After a 24 hour period, 6/11 subjects still demonstrated
high antiviral activity with >90% inhibition of both viruses. The assays tested with the
seminal fluid were conducted with samples from 3 women. Results revealed similar
inhibition values at 3 hours, but the results were mixed at 24 hours, resulting in an overall
decrease in inhibition.6 While this dendrimer displayed antiviral activity, more testing is
required before it can be used as an HIV inhibitor to prevent HIV infection.
Microbicides such as SPL7013 are important in the prevention of the HIV
infection. This area of research is just as important as the more traditional treatments for
HIV patients. Hopefully, in the near future there will be topical medications available for
people to protect themselves from this disease, as well as treatments for people already
infected.
27
Figure 21. The dendrimer SPL7013. The divalent benzhydrylamine (BHA) core is in
shown in red. The lysine residues are in green, purple, brown, and black, each
representing a new branching point, forming a generation 4 dendrimer. The terminal
groups in blue are naphthalene disulfonic acid (DNAA).7
Overview of the Current Project
There is overwhelming evidence that new HIV inhibitors are needed. Many drugs
have been created to combat viral replication, but they are not without drawbacks. Viral
resistance and harmful side effects are problems that have led to new research directed
towards fighting this virus. Most of the drugs created thus far attack the virus from
28
within an infected host cell. A new area of research to fight against the virus involves
attacking it before it enters the host cell. Molecules that have shown the potential to
inhibit the virus from entering the host cell are sulfated glycodendrimers. Various studies
have demonstrated that there is high binding affinity between sulfated glycodendrimers
and viral gp120. Because of these results, more research is necessary in this area.
In this study, two glycodendrimers have been created utilizing maltose and tris
(2-aminoethyl)amine (tris). Although maltose is not known to have any anti-HIV
properties, it is hypothesized that once it is attached to a dendrimer and sulfated, it will
exhibit a strong binding affinity to viral gp120 and will inhibit viral infection.
Tris(2-aminoethyl)amine was selected to build the cores because of its synthetic
versatility and simple structure. Cores of various sizes may be created from the terminal
amines, from small and to very large. To create the two glycodendrimers (Figures 22
and 23) utilizing maltose and tris(2-aminoethyl)amine, the following compounds were
synthesized: a carboxy-terminated hexavalent core, a trivalent amino core, an oxime
sugar-linker, and maltonic acid. An amino hexavalent core (Figure 24) was also created,
but was not utilized in this study. The amino hexavalent core will employed at a later
date to lend further versatility for future dendrimer construction.
Two different methods were employed to create the two glycodendrimers. The
convergent process was used to synthesize the hexavalent dendrimer containing six
sugars, while the divergent process was employed to create the smaller trivalent
dendrimer. Once these glycodendrimers are sulfated, they will be tested in a competitive
gp120 binding assay for binding affinity. If significant gp120-glycodendrimer binding
29
affinity is observed, these glycodendrimers will be further evaluated in a viral inhibition
assay. In the end, this research could lead to new therapeutic treatments for HIV patients.
HO
HO
HO
HO
OH
O
OHO
HO
O
HO
HO
OH
OH
HO
HO
O
O
O
NH
HN
O
O
O
O
HO
HO
OH
OH
OH
O HO
OH
HO
N
N
N
HO HO
N
N
O
OH
O
O
O
O
O
O
N
N
H
O
O
O
N
H
N
O
HN
O
O
N
HOHO
HO O
OH
HO
HO
O HO
OH
O
O
O
NH
O
O
N
HO
HO
HO
OH
O
HO
O
HO
O
HO
OH
OH
Figure 22. Hexavalent maltose amino-oxime glycodendrimer.
N
HO
HO
OH O
HO
O
HO
OH
OH
30
OH
HO
OH HO
O
HO
HO
OH
O
OH
O HO
O
OH
HO
O
HO
HO
HO
NH
O
O
O
N
H
O
HN
HN
O
N
NH
O
O
HN
O
HO
HO
HO
HO
O
HO
O
HO
HO
HO
Figure 23. Trivalent maltose amino-amide glycodendrimer.
HO
HO
31
NH2
H2N
O
O
N
H2N
O
O
N
N
N
O
H2N
Figure 24. Hexavalent amino core.
O
H2N
NH2
32
Chapter 2
RESULTS AND DISCUSSION
The main goal of this research was to create two glycodendrimers that were
constructed with the disaccharide maltose and a commercially available amino tris-core.
Once created, the glycodendrimers were to be utilized in another study in our lab, by
another group member. This included sulfating the glycodendrimers and testing their
inhibition properties on a competitive gp120 binding assay. If the sulfated
glycodendrimers demonstrate activity, they will then be sent to Duke University, where
our collaborator, Dr. Celia LaBranche, would evaluate them for their ability to inhibit
HIV infection in human cells. As stated previously, new anti-viral therapies are needed to
fight against HIV. Scientists have long known that sulfated molecules have the ability to
inhibit HIV infection. The sulfated glycodendrimers created herein have the potential to
stop viral infection as well. The compounds created in this study will add to future
knowledge directed towards the fight against HIV.
Another aim in this study was to employ two different methodologies to
synthesize the glycodendrimers. The larger dendrimer (Figure 22) containing six sugars
was synthesized using a convergent approach. This method involved building the outer
portions of the dendrimer first before attaching them to the core. The smaller dendrimer
(Figure 23), containing three sugars was created via the divergent method. In this
method, the glycodendrimer was synthesized from the core outwards.
33
The final objective of this research was to add to the library of dendrimer core
molecules with an amino hexavalent core (Figure 24).
It is important to possess
molecules of all shapes and sizes, so a variety glycodendrimers may be eventually
created. The amino terminated core is therefore a nice addition. The primary amines on
this molecule could be used to add either aldonic acid sugars directly to it, or to create
dendrimers of different generations, whereby the primary amines could serve as
branching points. All the compounds synthesized to create the glycodendrimers in this
study, can be added to our library of molecules and eventually be used in future research
to synthesize other glycodendrimers.
3-(3-Hydroxypropoxy)propanenitrile (1)
Compounds 1 (the target molecule) and 2 were synthesized via a Michael
addition reaction, which is presented in Scheme 1. In a traditional Michael reaction, a
nucleophile adds across an olefin or alkyne to produce one major product. An example
of this can be seen in a study by Krishna and Jayaraman, in which an alcohol with
acryonitrile are combined resulting in a nearly quantitative yield of a single product
(Scheme 2).40 The reaction reported here stems from their study, but has been adjusted
to fit a diol versus a mono-alcohol.
34
O
OH
NC
HO
OH
Compound 1
74% Yield
+
CN
40% NaOH
+
Acrylonitrile
1, 3 - propanediol
NC
CN
O
O
Compound 2
7% Yield
Scheme 1: Ethereal nitrile linkers (1 and 2) prepared via a Michael reaction, using
1,3-propanediol and acrylonitrile.
OH
+
benzyl alcohol
CN
acrylonitirle
40% NaOH
BnO
CN
O-benzyl-2-cyano ethanol
99% Yield
Scheme 2: Benzyl alcohol and acrylonitrile employed in a Michael reaction to create Obenzyl-2-cyano ethanol.40
In Scheme 1, two nucleophilic sites exist in 1,3-propanediol, creating two
different products (Compounds 1 and 2) in the reaction with acrylonitrile. To reduce the
formation of product 2, an excess of the 1,3-propanediol (2.23 equivalents) was used. In
addition, the NaOH catalyst (40% w/v) and limiting reagent (acryl
onitrile) were added dropwise, while the mixture was stirred vigorously.
35
The reaction was monitored by TLC and stopped after 3 days. Usually TLC is
used to monitor the disappearance of the limiting reagent and the formation of new
products. In Scheme 1, the limiting reagent is acrylonitrile, which has a low boiling
point, so it is difficult to visualize by TLC. It was apparent through TLC that two new
products were being formed within the first day, but since it was unknown if the
acrylonitrile was completely consumed or not, the reaction time was based on a previous
reaction, which utilized similar starting materials.41 In that previous reaction, the yields
were greatest during the third day, while the yields for the desired product decreased for
reactions run for 2 or 4 days. Therefore, the reaction in Scheme 1 was allowed to run for
3 days also.
After the reaction was complete, it was quenched with DI water, and then
neutralized with 1 M HCl, followed by lyophilization. The residue was then purified by
flash chromatography where Compound 2 eluted first, followed by Compound 1.
Compound 1 was isolated with a 74% yield. Compound 1 was characterized by both 1H
and 13C NMR spectroscopy.
The 1H NMR for Compound 1 can be seen in Figure 25. The chemical shifts of
each set of protons in Compound 1 are found in the appropriate range, starting with the
apparent pentet at δ 1.7.
Looking at Compound 1, this signal corresponds to the
methylene protons (2), which is the result of two overlapping triplets from CH2 groups
(1) and (3). The next chemical shift at δ 2.5 is CH2 group (5) adjacent the nitrile. Further
downfield, the hydroxyl proton can be seen at δ 2.9. Since the most electronegative
group within Compound 1 is the hydroxyl group, it stands to reason that the group
36
adjacent to it be the most deshielded and will be downfield of the other signals.
Therefore the triplet at δ 3.58 is assigned as the methylene group (1). The triplets at δ
3.53 and δ 3.50 almost overlap; they correspond to CH2 groups (3) and (4). Notice these
protons are in very similar chemical environments, causing almost identical chemical
shifts.
In Figure 25, none of the coupling constants have identical coupling partners
with the assigned CH2 groups, but the values are close. The reason why the J-value 6.10
Hz for CH2 group (2) is not identical to the coupling constants (both were J = 6.05 Hz)
for CH2 groups (1) and (3) is because it is not a true pentet. The pentet is the result of two
overlapping triplets that are not well resolved. The reason CH2 groups (4, J = 6.25 Hz)
and (5, J = 6.20 Hz), do not have exact J-values, is mostly likely due to the merging
triplets of CH2 groups (3) and (4), distorting the splitting patterns. Further confirmation
of Compound 1 was conducted via 13C NMR (See Appendix B).
37
2
5
HO
2, J = 6.10Hz
O
CN
1
3
4
1, J = 6.05Hz
4, J = 6.25 Hz
3, J = 6.05Hz
5, J = 6.20Hz
Figure 25. 1H NMR (500 MHz, CDCl3) of Compound 1. A larger version of this
spectrum may be viewed in Appendix A.
3-(2-Cyanoethoxy)propyl p-toluenesulfonate (3)
Compound 3 was created under biphasic conditions, as seen in Scheme 3. This
reaction was performed because the alcoholic OH on Compound 1 needed to be
converted into a better leaving group, for the subsequent reaction. Since tosylates are
good leaving groups, tosyl chloride was used in this procedure to synthesize Compound
3, a tosyl ester.
38
CN
HO
O
TsCl, CH2Cl2
50% NaOH, 50°C
Compound 1
CN
TsO
O
Compound 3
30.4% Yield
Scheme 3. Compound 1 was tosylated under basic conditions to produce Compound 3.
In this reaction, vigorous stirring and heating were needed to mix the biphasic
solution. First, Compound 1 was dissolved in dichloromethane and heated to 50 degrees
Celsius. Next, 2 equivalents of tosyl chloride were added, followed by aqueous NaOH,
which was added dropwise. The reaction was stirred and allowed to reflux for 6 days
before stopping.
The reaction was monitored by TLC, and after the third day there were still
visible signs of starting material, Compound 1. Because of this, more NaOH,
dichloromethane, and tosyl chloride were added. The temperature was also increased to
54ºC. After Compound 1 was no longer seen by TLC, the reaction was stopped on the
sixth day.
Once the reaction was removed from heat, the organic phase was extracted and
concentrated by rotary evaporation. Next, the residue was purified via flash
chromatography. The desired product 3, eluted as the third band, followed by a mixture
of Compounds 1 and 3. The yield for pure Compound 3 was 30%. Compound 3 was
characterized by 1H and 13C NMR spectroscopy.
39
The 1H NMR for Compound 3 can be seen in Figure 26. The doublets at δ 7.7
and δ 7.3, which arise from the tosyl group, display a para-substitution pattern for an
aromatic ring. The doublet at δ 7.7 corresponds to the CH groups at position (4, 5) since
they are near the tosyl SO3 electron withdrawing group. The doublet at δ 7.3 relates to the
two protons at position (2, 3), which are adjacent a methyl group, moving them slightly
upfield. As seen in Figure 26, groups that couple with identical J-values are CH2 groups
(7) and (8) with J-value 5.95 Hz and CH2 groups (9) and (10) with the coupling constant
6.35 Hz. The last set of CH2 groups (6) and (7) couple with J-values 6.00 Hz and 5.95
Hz, respectively. The remaining singlet at 2.4 ppm, corresponds to the methyl group at
position (1), which has no neighboring protons, therefore appears as a singlet.
Compound 3 was also characterized via a 13C NMR (Appendix B).
40
2
4,5 J = 8.25Hz
5
O
1
2,3 J = 7.95Hz
S
O
3
4
7
O
10
O
CN
6
8
9
1
9, J = 6.35Hz
8, J = 5.95Hz
10, J = 6.35Hz
7, J = 5.95Hz
6, J = 6.00Hz
Figure 26. 1H NMR (500 MHz, CDCl3) of Compound 3. This spectrum may also be
seen expanded in Appendix A.
It should be noted that the reaction in Scheme 3 was performed numerous times
and yields were low in each trial. At first it was thought that yields were low due to the
biphasic system. Because of this pyridine was employed, replacing the aqueous NaOH,
in order to create a more homogenous mixture. Through 1H NMR it was apparent that
product formation was actually worse using pyridine, so the residue was not completely
purified and no yields were calculated.
To increase yields in the future, a few variables could be changed to create
Compound 3. In a previous reaction similar to the procedure in Scheme 3, the heat was
increased to 90ºC by mistake for a short period and the yield reached 51.5%. This is one
41
variable that could be investigated to increase yields. Due to time constraints, this was
not performed in this study.
Hexavalent nitrile core (4)
The hexavalent nitrile core 4, which can be seen in Scheme 4, was created for
multiple reasons. The terminal nitrile groups on the hexavalent core 4 make it very
versatile, since it could be hydrolyzed to create a carboxy core or reduced to form an
amine-terminated core.
CN
O
CN
NC
O
O
H2N
TsO
O
CN
+
N
NH2
N
K2CO3, CH3CN
N
N
O
90°C
Compound 3
CN
N
NH2
Tris(2-aminoethyl)amine
O
NC
O
Compound 4
91% Yield
CN
Scheme 4: Six equivalents of tosyl linker 3 were added to the commercially available
tris(2-aminoethyl)amine to form the hexavalent core 4.
To perform the reaction seen in Scheme 4, a very dry environment was required.
All glassware used was flame-dried and placed under nitrogen gas before adding any
starting materials or reagents. Once the acetonitrile and potassium carbonate were added
together, the reaction was stirred and heated to 89ºC. Following this, tris(2-aminoethly)
42
amine was added to the mix. Then a combination of the tosyl nitrile linker 3 (7.67
equivalents) in acetonitrile was added in portions, over a four hour period. Over the next
24 hours, the heat was lowered to 82ºC and allowed to stir at this temperature until
stopped.
The reaction was halted after two days, since a similar reaction done by DeCampo
and co-workers was found to be optimize in that time frame.42 TLC was performed to
analyze reaction progress. Due to the polarity of the limiting reagent (tris 2-(aminoethyl)
amine) and the desired product (Compound 4), it was difficult to verify the completeness
of the reaction. It was apparent that there was product formation after the first day, and
after the second day it appeared that the tris(2-aminoethyl)amine was consumed.
Because both the tris-core and hexavalent core 4 have similar Rf values with any solvent
system, the reaction was stopped based on a color change in the reaction spot on the TLC
plate. When the color for the tris-core, which was yellowish, had disappeared from view
on the TLC plate, the reaction was thought to be complete. This happened after the
second day, which coincides with the reaction done by DeCampo and co-workers, which
was also complete in two days.42
Once the reaction was stopped, it was filtered and condensed in vacuo. Next, an
extraction was performed on the residue and the resulting solution was concentrated
through evaporation. Since the hexavalent core 4 was found to be amphiphilic, the
excess tosyl-linker 3 was removed using a water rinse. The tosic acid formed in this
reaction was removed via a batch rinse with cationic resin. A 91.1% yield was obtained
43
for Compound 4, which was characterized by, 1H and 13C NMR, as well as mass
spectrometry.
The 1H NMR for Compound 4 is given in Figure 27. Looking at this spectrum,
the three peaks furthest downfield at δ 3.59, δ 3.47, and δ 2.56 are sharp and well-defined
triplets, whereas the chemical shifts at δ 2.46 and δ 1.66 are less clear. The reason for
both the poor integration and broad and an unclear splitting patterns has to do with the
size of the molecule and the relaxation times in the NMR experiment.43 Because
Compound 4 is a large molecule, it tumbles slowly in solution. The inner atoms have
limited motion and are not able to interact with neighboring nuclei very well. This causes
an increase in the time it takes for the nuclei to relax back to their original orientation
after having been pulsed with energy during an NMR experiment.44 Nuclei in a magnetic
field precess randomly about the magnet in different spin states until a radio frequency
pulse is applied. When energy is absorbed, nuclei change from a low energy spin state to
a high energy spin state and the nuclei become more organized as they precess in their
spin states about the magnetic field (Figure 28).45 When the nuclei release this energy,
they return (relax) back to their original orientation. The time it takes for the nucleus to
relax (i.e. emit energy) can be broken up into two components, longitudinal relaxation
time (T1) and transverse relaxation time (T2). T1 corresponds to the time it takes for
nuclei to return to their original spin state as they release energy to neighboring nuclei. T2
corresponds to the amount of time it takes for organized precessing nuclei to return to
their original axis of rotation about the magnetic field.43,44 T1 and T2 relaxation times
affect the NMR signal as energy is released. T1 affects the intensity of the observed
44
NMR signal and thus the integration of the peaks, while T2 affects the width of the peaks.
In large molecules both relaxation times may be affected due to the increased time it
takes to relax, resulting in line broadening.43
CN
6
5
3
7
O
1
4
N
N
2
O
3
7
5
6
CN
3
5,6
J = 6.27Hz
7, J = 6.21Hz
J = 6.27Hz
1,2,3
4
Figure 27. 1H NMR (300 MHz, CDCl3) of Compound 4. An extended spectrum of this
NMR may be viewed in Appendix A.
45
T1
T2
Figure 28. An illustration of T1 and T2 in a NMR experiment. T1 (left) represents the
different energy spin states. T2 (right) demonstrates nuclei precessing about the magnetic
field (a) before being pulsed with energy and (b) after irradiation.45
In Figure 27, it is apparent which chemical shifts have been affected by the
relaxation time. Looking at Compound 4, the CH2 groups that are more susceptible to
line broadening are (1-4), which are buried within this molecule. The broad apparent
pentet at δ 1.66 is assigned to CH2 group (4) since it is the most shielded CH2 group due
to the adjacent methylene groups. This leaves CH2 groups (1-3) which gives the broad
signal at δ 2.46. The triplet at δ 2.56 was assigned to CH2 group (7) because it is adjacent
a nitrile. The last two triplets between δ 3.47 and δ 3.59 were assigned to CH2 groups
(5-6) since they are adjacent the electronegative oxygen atom. Since CH2 groups (5) and
(6) have identical coupling constants, it is unclear which couples to CH2 group (7).
Further confirmation of Compound 4 can be viewed in the 13C NMR (Appendix B) and
the IR (Figure 29, Appendix C) spectra. The IR shows a nitrile peak at 2251 cm-1
(Figure 29). This aspect will be important later when discussing the hydrolysis of
Compound 4. A matrix assisted laser desorption ionization-time of flight mass
46
spectrometry (MALDI-TOF MS) spectrum was also collected for Compound 4
(Appendix D).
87.2
80
70
1667.75
60
1328.21
1414.44
50
844.40
951.82
Nitrile
665.83
%T 40
3017.84
2251.04
30
CN
1467.07
1224.85
O
20
N
1367.96
N
O
10
CN
2873.23
3
755.07
1118.35
-2.0
4000.0
3000
2000
1500
1000
600.0
cm-1
Figure 29. IR of Compound 4, with a nitrile peak at 2251 cm-1. This spectrum can be
seen in Appendix C in an expanded view.
Methyl ester core (5)
As can be seen in Scheme 5, Compound 5 is an intermediate, which was
synthesized in order to reduce the overall salt formation that results from the direct
hydrolysis of Compound 4 to a carboxylic acid. In prior reactions that were basecatalyzed, the salt formation was extensive and it was time consuming to remove, as the
47
target Compound 6 was also completely water soluble. Therefore, to minimize salt
formation, an acid-catalyzed reaction was employed as the first step in Scheme 5, in
which hydrochloric acid is created in situ utilizing acetyl chloride and methanol.
O
CN
O
O
CN
O
O
O
O
O
Acetyl chloride, MeOH
N
N
O
CN
0°C
O
25°C
N
O
N
O
N
N
O
N
O
CN
O
O
N
O
NC
O
O
Compound 4
O
Compound 5
O
33% Yield
NC
O
O
O
LiOH,
H2O,
mw
O
OH
O
OH
O
O
HO
O
N
O
N
N
HO
O
N
O
O
O
Compound 6
67% Yield
O
OH
O
OH
Scheme 5: The hexavalent core 4 undergoes esterification in the first step to form the
hexa-methoxy Compound 5. Hydrolysis takes place in the second step to form
carboxylic acid Compound 6.
48
This esterification reaction commenced with the combination of the hexavalent
nitrile core 4 and dry methanol. The mixture was cooled in an ice bath before adding the
acetyl chloride to keep the hydrochloric acid in the solution upon formation.46 The
mixture was stirred overnight at 25ºC.
Due to the similar polarities of both Compound 4 and Compound 5, the reaction
was analyzed with colorimetry via TLC. Since these compounds appeared different when
stained with ninhydrin, it was used to differentiate between both products. Starting
material 4 had appeared brown by TLC using ninhydrin stain, while Compound 5 looked
yellowish. When Compound 4 was no longer visible, the reaction was stopped.
After 24 hours was concentrated in vacuo and then freeze dried overnight. Next,
to remove any unreacted starting material or partially formed products, dialysis was
performed. It had been established previously that the starting material, Compound 4, is
not retained while in dialysis tubing. During one trial, when Compound 4 was purified
by dialysis, using 100 MWCO (molecular weight cut-off) tubing, some product was
found to have gone through the tubing.41 It should be noted that the methyl ester 5 may
also have the ability to escape the dialysis tubing. This may explain the low yields. After
the sample remaining in the 100 MWCO tubing was freeze dried, it was analyzed by
NMR spectroscopy.
The 1H and 13C NMRs performed on Compound 5 were inconclusive. Multiple
samples were analyzed by NMR spectroscopy, using both CD3OD and D2O solvents.
During some NMR experiments, the NMR would not even lock and at other times when a
signal was obtained, there was extreme peak broadening (vide supra) and integrations
49
could not be attained. Because of these difficulties Compound 5 was analyzed by IR
(Figure 30) instead. In this spectrum, a strong ester carbonyl stretch was observed at
1733 cm-1, and the sharp nitrile peak at 2254 cm-1 (Figure 29) was no longer visible,
indicating the successful production of Compound 5. This product was carried forward
without any further purification.
83.6
80
75
70
2924.15
%T 65
O
O
O
60
N
1732.95
Ester
carbonyl
1197.27
1112.88
N
O
55
O
O
3
50.0
4000.0
3000
2000
1500
1000
cm-1
Figure 30. IR of the Compound 5 displays a strong ester carbonyl peak at 1733 cm-1.
An expanded version of this spectrum may be viewed in Appendix C.
Hexavalent carboxy core (6)
The hexavalent core was next functionalized with terminal carboxylic acids to
assist in amide coupling which are used to create the large glycodendrimers in this
research. Compound 6 was created from the second reaction shown in Scheme 5. This
400.0
50
ester hydrolysis reaction was performed in a microwave to reduce the reaction times from
days to minutes.41,47
To synthesize Compound 6, the methyl ester 5 and nanopure water were
combined, followed by the addition of lithium hydroxide. The mixture was placed in a
microwave and heated for 25 minutes at a power setting of 400W. The first 2 minutes in
this process were used to ramp the temperature slowly up to 60ºC, where the reaction was
held for the remainder of the reaction time. After the mixture was heated for 23 more
minutes, it was cooled to room temperature.
When the reaction had cooled to room temperature, it was concentrated in vacuo
to remove the water. The sample was then reconstituted in methanol, stirred, then
centrifuged to remove the leftover lithium hydroxide. This was followed by size
exclusion FPLC (fast paced liquid chromatography), which was performed to remove any
side products and any remaining salt. A strong signal at 214 nm was observed on the
chromatogram, indicating the formation of the desired product, Compound 6. The peaks
that demonstrated an absorbance at 214 nm were collected and resulted in 66% yield
(68.1 mg) of Compound 6. The identification of Compound 6 was verified through first
by NMR spectroscopy and then mass spectrometry.
The 1H NMR spectrum for Compound 6 is given in Figure 31. The spectrum
demonstrates peak broadening, due to the slow relaxation times (vide supra) for
Compound 6. The peaks at δ 1.80-1.90 and δ 2.75-2.90 are very broad and indistinct
splitting patterns. The signal at δ 3.58 displays some peak broadening as well. The two
well-defined peaks at δ 2.45 and δ 3.71 were assigned to CH2 groups (7) and (6),
51
respectively, since they have the same coupling constant (J = 6.60 Hz) and are positioned
in the appropriate area. The other CH2 groups were assigned by their expected chemical
shifts. Methylene (4) would be the most shielded and upfield, and while CH2 groups (13) are expected to have similar polarities and therefore should appear at similar chemical
shifts. Assignments were further confirmed by integration of the assigned peaks, which
added up to the correct proton count needed for Compound 6, as can be seen in Figure
31. Characteristics of Compound 6 can also be seen in the 13C NMR in Appendix B.
Compound 6 was additionally analyzed by time of flight negative mode electrospray
mass spectrometry (TOF ES- MS) available for view in Appendix D.
O
6
5
3
OH
O
7
1
4
N
N
2
5
6, J = 6.60Hz
7
O
3
OH
6
O
3
7, J = 6.60Hz
5, J = 6.10Hz
1,2,3
4
Figure 31. 1H NMR (500 MHz, D2O) of Compound 6. An extended view of this
spectrum may be found in Appendix A.
52
Boc-protected oxime sugar-linker (8)
Compound 8 was synthesized under acidic conditions (Scheme 6). Since this was
a condensation reaction in which water was formed through the coupling of maltose and
Compound 7 (made previously by a group member), keeping free acidic protons
available in the reaction was important. The buffer used here was 0.1M ammonium
acetate (pH 4.5). The Boc-protected linker 7 used in this reaction was employed for two
reasons. The first was for purification purposes. Creating a less polar molecule than
maltose would make separation easier via flash chromatography. The second reason was
to make characterization obvious. The Boc-protecting group creates a unique singlet
around δ 1.40 that integrates for 9H in 1H NMR spectroscopy.
53
OH
O
HO
HO
OH
HO
+
O
O
HO
Maltose
H2N
H
N
O
O
Boc
Compound 7
HO
OH
Buffer NH4OAc (4.5)
OH
O
HO
HO
OH
HO
O
OH
O
N
HO
O
Boc
HO
Compound 8
76% Yield
TFA
CH2Cl2
OH
HO
HO
H
N
O
OH
HO
O
OH
HO
O
N
NH3+
O
HO
Compound 9
Quantitative Yield
Scheme 6: The coupling of maltose and a Boc-protected amino Compound 7 under
acidic conditions to form Compound 8, followed by a deprotection step creating an
amino oxime sugar-linker 9.
Maltose, Compound 7, and an ammonium acetate buffer were combined, and the
pH was monitored via pH paper. The pH remained at ~4.5 during the entire reaction.
The reaction was also monitored by TLC. When Compound 7 disappeared from view,
the reaction was stopped and the sample was lyophilized. The sample was purified
further through flash chromatography. Unreacted Compound 7 eluted first, then the
54
desired product (Compound 8), followed by maltose. Compound 8 was collected and
concentrated by rotary evaporation. As expected, the Boc-protecting group did promote
separation from maltose, yielding 76% of Compound 8. Identification of Compound 8
with the Boc was also apparent as seen through 1H and 13C NMR spectroscopy.
Before the analysis of Compound 8 by 1H NMR spectroscopy can be examined,
the NMR properties of maltose should be discussed. Because maltose (Figure 32) comes
in two forms, there are distinctive chemical shifts for each structure. In α-maltose, there
are two anomeric protons in the equatorial positions that form two doublets at δ 5.24 and
δ5.42. In β-maltose, there is one anomeric proton in the axial and one in the equatorial
position at δ 4.67 and δ 5.42, respectively. When maltose is coupled to another molecule,
the doublet corresponding to the non-reducing anomeric proton should always be visible
downfield, left of the D2O solvent peak. The other sugar hydrogens overlap, creating a
complex splitting pattern which will always be apparent between 3.20 and 4.20 ppm.
55
OH
OH
O
O
HO
HO
HO
HO
H'
OH
HO
O
O
OH
HO
H
Maltose
OH
HO
O
O
HO
H'
HO
Maltose
OH
HO
H
D2O
H'
H
J = 3.81Hz
H
J = 7.95Hz
J = 3.78Hz
Figure 32. 1H NMR (300 MHz, D2O) of Maltose. α-Maltose with two equatorial
positioned protons in red (H' = unreactive proton) and β-maltose with one axial proton
(green) and one equatorial proton (red) emphasized.
Looking at the 1H NMR spectrum for Compound 8 (Figure 33), the doublets
that appear furthest downfield at δ 7.60 and δ 7.00 correspond to the vinylic hydrogens of
the E and Z stereoisomers.48 The chemical shift at δ 7.60 arises from the E isomer, since
this isomer (trans) is more stable then the Z (cis) isomer, and therefore should be seen in
larger quantity. As can been seen in Figure 33, the signal corresponding to the E proton
(δ 7.60) is stronger than the Z proton peak (δ 7.00), demonstrating the production of more
of the E isomer Compound 8, in a 6:1 ratio. The next major signal left of the HOD peak
in Figure 33 relates to the nonreducing anomeric proton. As stated previously there
56
should always be a doublet in this area (above 4.80 ppm) representing this proton. The
other peaks ranging from δ 5.40 to δ 3.40 correspond to sugar hydrogens and some CH2
groups (3-5). Since these groups overlap, coupling constants could not be measured.
The peaks that did exhibit clear coupling were seen at δ 3.14 and δ 1.78 which
correspond to CH2 groups (7) and (6), respectively. Since CH2 group (6) is more
shielded versus the other CH2 groups it was assigned to the most upfield of these two
peaks. The peak at δ 1.45 is the signal from the methyl groups on the Boc protecting
group. Since these CH3 groups have no neighbors to create a splitting pattern, one singlet
is formed. Compound 8 was also analyzed by 13C NMR (see Appendix B).
E J = 6.05 Hz
1
Z
J = 5.50 Hz
OH
O
HO
HO
H'
HO
OH
O
7
7
OH
2
O
N
O
HO
HO
3
H
N
5
4
O
7
6
O
HE,Z
7
D2O
1,2-4
1
J = 3.90 Hz
6, J = 6.40Hz
5, J = 6.45Hz
H'
Figure 33. 1H NMR (500 MHz, D2O) of Compound 8. This spectrum may also be
viewed (expanded) in Appendix A.
57
E,Z oxime sugar-linker (9)
The Boc-protecting group was removed from Compound 8 by the addition of
TFA (Scheme 6). The reaction was analyzed by TLC using ninhydrin stain. It was
evident that the reaction was complete after 3 hours by TLC, as indicated by a color
change from the light pink spot of the starting material being replaced by a strong purplepink band. When the reaction was complete, all solvents were removed through
evaporation and lyophilization. After the sample was freeze dried, it was dissolved in
ammonium bicarbonate (0.03M) instead of water. This was to help remove TFA that was
not removed during rotary evaporation. The yield of Compound 9 was quantitative. The
product was identified by 1H NMR (Figure 34), 13C NMR (see Appendix B), and Mass
spectrometry (see Appendix D).
The 1H NMR spectrum for Compound 9 is presented in Figure 34. Notice that
the singlet corresponding to the removal of the Boc group at 1.45 ppm is absent. This is
the only real difference between Figures 33 and 34, except that the coupling constants
for CH2 groups (6) and (7) could no longer be measured.
58
1
J = 5.95 Hz
E
J = 5.55 Hz
Z
OH
O
H'
HO
HO
OH
OH
HO
O
HO
2
5
O
N
O
3
HO
4
NH3+
6
HE,Z
D2O
1,2-4
1
J = 3.85 Hz
6
5
J = 3.90 Hz
H'
J = 5.15 Hz
Figure 34. 1H NMR (500 MHz, D2O) of Compound 9. This spectrum may also be
viewed (expanded) in Appendix A.
Hexavalent maltose amino-oxime glycodendrimer (10)
Compound 6 and the oxime sugar-linker Compound 9 were coupled through
amide bond formation under dry conditions (Scheme 7). Because Compounds 9 and 10
are both hydrophilic, they were dissolved together in nanopure water and lyophilized
overnight. When they were removed from the freeze dryer, they were immediately
placed under nitrogen gas. Six sugar-linkers 9 were needed per one carboxy core 10,
thus 6.3 equivalents of the Compound 9 were used. After the solvent dimethyl sulfoxide
(DMSO) was added, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU) was mixed into the reaction. TBTU was added in excess (14.4 equivalents) in
59
order to neutralize any residual TFA left from the formation of the sugar linker 9. It was
recommended to add at least 1.2 equivalents of TBTU for every carboxylic acid,49 so 7.2
equivalents were added for Compound 10 and 7.2 for any remaining TFA. The base,
N,N-diisopropylethylamine (DIPEA) was added last to a pH of 8.5. Basic conditions
were important in this reaction to activate TBTU. DIPEA was added in portions of 100
μL and allowed to stir for ~ 30 minutes before checking the pH, using wet pH paper.
After 300 μL were added, the desired pH (8-9) was reached. The reaction was allowed to
stir for 24 hours before stopping. This reaction was stopped based on the work done by
Baek and Roy.49 In their work, product formation was complete within 24 hours as
shown by a negative ninhydrin test upon TLC. This negative ninhydrin test would not
work for this reaction, because the limiting reactant here is not the amine Compound 9,
which would always give a positive result. Even so, the reaction in this study was stopped
at 24 hours, since Baek and Roy’s reaction was complete in this amount of time.49
60
O
HO
OH
O
OH
O
O
HO
HO
O
N
HO
O
N
O
HO
O
HO
OH
+
O
N
OH
OH
TFA-
N
NH3+
O
O
HO
Compound 9
O
N
Compound 6
O
O
O
TBTU, DIPEA,
DMSO, N2
O
OH
OH
HO
HO
OH
O
HO
O
HO
OH
OH
N
O
O
H
N
O
HO
O
Compound 10
66% Yield
OH
O
HO
HO
HO
OH
OH
O
HO
HO
N
N
O
N
O
O
N
H
O
3
Scheme 7: Glycodendrimer 10 formed through the coupling of 6 and 9.
When the reaction was stopped, it was first concentrated by rotary evaporation.
Next, a large volume of water was added to the reaction mixture and it was lyophilized
overnight to remove any remaining DMSO. The following day, an extraction was
performed to remove any side-products that were organic soluble. The aqueous phase
was freeze dried again. After lyophilization, a white precipitate (ppt.) formed in the
sample when it was dissolved back into water, so filtration was performed on the sample.
The ppt. collected was not analyzed and discarded because it was thought to be TBTU
byproducts. This conclusion was based on previous trials using a similar reagent BOP
61
(benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate), which
reacted the same way after being freeze dried. Compound 10 was then further purified
by dialysis. Since the desired product 10 had a molecular weight (MW) of 3567 g/mol,
the dialysis membrane used had a 2000 molecular weight cut off (MWCO). After 7.5
hours of dialysis, a fluffy yellow-orange solid was recovered through lyophilization and
analyzed by 1H and 13CNMR spectroscopy.
Given that Compound 10 is a large molecule (exact mass = 3567.73 g/mol) most
peaks in its 1H NMR (Figure 35) were broad due to long relaxation times (vide supra).
To get a more accurate proton count, only one arm of the dendrimer was integrated. The
observed (38) and expected (38) proton count were a match after this spectrum was
integrated. Even though the peaks were not well-defined in Figure 35, all the CHn
groups were assigned by comparing starting materials to the product. A comparison
spectrum can be seen in Figure 36. The two peaks between δ 1.80-2.10 correspond to
CH2 groups (6) and (11), the most shielded protons. The signal at δ 2.50 relates to CH2
group (8), which is adjacent to the carbonyl group. Next, the very broad peak between δ
2.80-3.20 was assigned to CH2 groups (12-14), which were the protons most hidden
within the core. The chemical shift at δ 3.25 was assigned to CH2 group (7), and the
peaks spanning from δ 3.30 to 5.40 were assigned to CH2 groups from the sugar-linker
(1,3, and 5) and groups (9) and (10) from the core. The last two peaks at δ 7.60 and δ
7.00 corresponded to the E and Z oxime protons. Again, the E proton was assigned to the
more intense doublet (vide supra). The intensity for both signals have decreased though,
which was most likely due to the closing of the sugar ring.48
62
J = 5.95 Hz
E
J = 5.40 Hz
Z
1
HO
HO
OH
O
H'
HO
OH
OH
O
HO
HO
H
N
5
2
N
O
O
4
3
7
8
6
O
HE,Z
10
O
9
13
N
11
N
12
3
2
1-4,8,9
1
J = 3.70 Hz
6
11-13
7
5,10
H'
Figure 35. 1H NMR (500 MHz, D2O) of Compound 10. This spectrum may also be
viewed (expanded) in Appendix A.
63
Figure 36. A spectra comparison of: (top), the hexavalent carboxy core 6, (middle), the
hexavalent amino-oxime glycodendrimer 10, (bottom) of the E,Z oxime sugar-linker 9.
Ring closure is a phenomenon that occurs among carbohydrates. Scheme 8
demonstrates a proposed mechanism of the oxime formation and the ring closing of
Compound 8.48 In this scheme, after maltose is placed under mildly acidic conditions
(pH 4 - 5), it is oxidized into an open chain aldehyde (aldehydo). Next, the electrophilic
aldehydo is attacked by the primary amine end of Compound 7 forming the carbinal
oxyamine intermediate. When the intermediate loses water, Compound 8 is formed.
The last step in Scheme 8 demonstrates the two structures of Compound 8 that seem to
appear to be in equilibrium by 1H NMR spectroscopy. It has been found that over time,
64
all of the oxime Compounds 8, 9, and 10 start to undergo ring closure as they are being
analyzed by 1H NMR spectroscopy. This explains the decreased signal intensity for the E
and Z isomer compounds.
OH
O
OH
HO
HO
OH
HO
O
HO
Maltose
Buffer NH4OAc (4.5)
aldehydo
O
HO
HO
O
OH
HO
HO
OH
O
HO
OH
O
HO
H
H2N
H
N
O
O
Boc
Compound 7
OH
O
HO
HO
OH
HO
O
OH
OH
HO
HO
Oxyamine intermediate
H
H
N
O
Boc
O
N
H
- H2O
OH
O
HO
HO
OH
HO
O
OH
N
HO
O
H
N
O
HO
Boc
H
Compound 8
H2O
OH
O
HO
HO
OH
HO
O
O
H
N
HO
O
O
HO
H
Scheme 8: Proposed oxime formation and ring closing mechanism.48
H
N
Boc
65
The 13C NMR of Compound 10 can be viewed in Appendix B. The next
analysis performed on Compound 10 was matrix assisted laser desorption-ionization
time of flight mass spectrometry (MALDI-TOF MS, Figure 37). The peaks seen at
3622.790 m/z and 3640.374 m/z are the mass of Compound 10 with the addition of
water. The mass 3622.790 is [M+H+3H2O] and the mass 3640.374 is [M+H+4H2O].
Compound 10 [M+H] = 3568.735 is a close match to the peak at 3570.280 m/z. Because
this is a large molecule, the mass may vary due to a mixture of isotopes. Since
Compound 10 has 144 carbons, there is most likely some 13C present, causing an
increase in the mass. After Compound 10 was characterized in this study, it carried
forward in another study in our lab.
66
[M+H]
C144H270N16O84
Exact Mass: 3567.73
1
HO
HO
OH
O
HO
O
HO
OH
OH
N
O
H
N
O
N
N
O
HO
O
2
3
[M+H+4H2O]
[M+H+3H2O]
Figure 37. MALDI-TOF of Compound 10. This spectrum may also be viewed
expanded in Appendix D.
The ultimate purpose for the synthesis of Compound 10 was to test it for any
anti-viral properties. Therefore, once Compound 10 was synthesized, it was sulfonated
(Scheme 9) to maximize its inhibition potential.50 This sulfated Compound 10 has been
sent off for an elemental analysis. It has been determined by Columbia Analytical
Services in Tucson Arizona, that Compound 10 has a sulfur percentage of 12.93%. This
measures out between 21 or 22 sulfate groups on Compound 10, translating to nearly 2
sulfate groups per sugar. Soon it will be evaluated for its binding affinity to the HIV
67
protein gp120 in a competitive binding assay. The other glycodendrimer created in this
study will eventually be tested in the same fashion.
HO
HO
OH
O
HO
O
HO
OH
OH
N
O
O
H
N
O
HO
O
Compound 10
N
OH
O
HO
HO
HO
OH
OH
O
HO
N
O
N
O
O
N
H
HO
O
3
SO3-pyridine
DMF, N2, 0°C
1 hour
RO
RO
OR
O
RO
O
RO
OR
OR
N
O
O
H
N
O
RO
O
Sulfated
Compound 10
OR
O
RO
RO
RO
OR
OR
O
RO
N
N
O
N
O
RO
O
N
H
R = H or SO3-
Scheme 9. Conditions to sulfate glycodendrimer 10.50
O
3
68
Synthesis of the Boc-protected trivalent amino amide core (12)
To create the glycodendrimer containing three disaccharides, a trivalent core was
needed. To begin the process, Compound 12 was created from the Boc-protected linker
11 (which was made previously by a group member) and tris(2-aminoethyl)amine
(Scheme 10). Compound 11 was freeze-dried beforehand to remove moisture. After 11
and tris(2-aminoethyl)amine were combined, they were immediately placed under
nitrogen gas. The following reactants were then added sequentially: BOP, DMF, and
DIPEA. BOP promotes the amide coupling of 11 and tris(2-aminoethyl)amine, therefore
an excess was used. After the components were dissolved in DMF, DIPEA was added
and the reaction was allowed to mix for three days at which time the reaction was stopped
based on the results of a previous trial.
69
NH2
N
O
H
N
O
N
H2N
NH2
N
H
BOP, DIPEA,DMF,
O
Tris (2-aminoethyl) amine
N2, 0°C
O
Compound 12
27% Yield
RT
3
+
O
HO2C
TFA
CH2Cl2
Boc
N
H
Compound 11
TFAH
N
O
NH3+
N
O
Compound 13
Quantitative Yield
3
Scheme 10: The formation of Boc-protected core 12 using the commercially available
tris-core and a previously synthesized carboxylic acid 11. In the second step, the trivalent
amino core 13 was created by the removal of the Boc protecting group.
When the coupling reaction was stopped, the reaction was concentrated in vacuo,
followed by three extractions. They were performed in succession to help remove BOP
byproducts and DIPEA. The sample was concentrated by evaporation and lyophilized
overnight. The organic phase was dried with anhydrous sodium sulfate overnight,
followed by purification via flash chromatography. Three flash columns were
necessitated for this sample due to the BOP byproducts and leftover Compound 11. In
the first separation, the solvent system was 8:1 chloroform:methanol. This step removed
leftover linker 11. The second flash column was performed to elute BOP byproduct first
with less polar solvents. After the residue was added to the column it was eluted first
with ethyl acetate, then ethyl acetate:methanol (95:5). Lastly, when methanol alone was
70
added the product eluted. Although this process did remove some BOP byproducts, some
impurities remained, so, for the last flash column, a shorter silica plug was employed.
The same solvents were used as above and a pure sample of Compound 12 (27% yield)
was collected after the BOP byproduct eluted. This was confirmed by NMR
spectroscopy.
When the 1H NMR (Figure 38) for Compound 12 was integrated, there was
almost a perfect match of 63 protons even though some peak broadening (vide supra) was
visible. The peaks relating to the amide hydrogens are seen at δ 5.30 and 7.10. The
broad peak at δ 3.30 was assigned to CH2 groups (1) and (2), since they are protons
within the core structure. The broad peak at δ 2.58 and the triplet at δ 2.43 were assigned
to CH2 groups (4) and (7). CH2 group (4) was assigned to this area (δ 2.43 and δ 2.58)
because protons adjacent a carbonyl group were found in this range. CH2 group (7) was
also assigned in this region, because once Compound 12 was deprotected, a peak in this
area disappeared, which most likely corresponded to CH2 group (7). The remaining
peaks were sharp and well-defined. The sharp singlet at δ 1.40 is indicative of the Boc
group. The triplets between δ 3.45 and δ 3.66 corresponded to the protons adjacent the
electronegative oxygen atom, CH2 groups (5) and (6). Although some J-values were
measured, none were used to assign any sets of proton, since none were identical. A 13C
NMR can be viewed in Appendix B that confirms the formation of Compound 12.
71
3,8
CDCl3
9
3
1
4
H
N
O
7
9
O
N
2
5
6
O
N
H
O
9
8
3
9
5,6
J = 5.90Hz
J = 5.15Hz
1,2
4,7
J = 5.80Hz
Figure 38. 1H NMR (500 MHz, CDCl3) of Compound 12. This spectrum may also be
viewed (expanded) in Appendix A.
Synthesis of the trivalent amino-amide core (13)
To deprotect Compound 12, it was placed in TFA and dichloromethane (Scheme
10). When the disappearance of Compound 12 was confirmed by TLC, the reaction was
quenched with water and evaporated. To further remove TFA, the sample was
lyophilized several times. Compound 13 was recovered in quantitative yields and
verified by NMR and mass spectrometry (Appendix D).
The 1H NMR (Figure 39) for Compound 13 was easy to analyze. Now that the
bulky Boc group was removed, the inner core protons peaks were well-defined and
displayed clear splitting patterns. Through the coupling constants, every CH2 group
72
could be assigned, even though some values were not identical. The signal at δ 2.52 was
assigned to CH2 group (3), because it was the most shielded set of protons. Since the
peak at δ 3.71 was in the appropriate area and it had a J-value (5.95 Hz) close to CH2
group (3, J = 6.00 Hz), it was assigned CH2 group (4). The next set of triplets that were
found to couple were CH2 group (5) at δ 3.12 (J = 5.05 Hz) and CH2 group (6) at δ 3.65
(J = 5.05 Hz). The last set triplets between δ 3.38 and δ 3.55 corresponded to CH2 (1)
and CH2 (2), which have different coupling constants of 6.05 Hz and 6.25 Hz. Since CH2
groups (1) and (2) appear to have similar chemical environments, they were not assigned
to one of these particular signals. Another aspect of Figure 39 that identifies the
formation of Compound 13 is the proton count. Each triplet displayed 6 protons, adding
up to a total of 36, which matches the expected number of protons in Compound 13.
73
1
3
H
N
6
O
NH3+
N
2
O
4
4, J = 5.95 Hz
5
3
5, J = 5.05 Hz
1,2
3, J = 6.00 Hz
J = 6.25 Hz
J = 6.05 Hz
6, J = 5.05 Hz
Figure 39. 1H NMR (500 MHz, D2O) of Compound 13. This spectrum may also be
viewed in Appendix A.
Synthesis of maltonic acid (14)
Scheme 11 outlines the reaction performed to create Compound 14. In this
reaction, a three necked flask was required. Methanol and iodine were combined
together in the flask and an addition funnel was attached. The reaction was heated (40ºC)
before the addition of maltose, which was added to the mixture after being dissolved in a
small amount of boiling water. To this concoction, a mixture of KOH and methanol was
added dropwise from the addition funnel. The formation of Compound 14 could be
visualized as it precipitated out of solution. After about 85 minutes, the reaction was
74
removed from the oil bath. The sample was then vacuum filtered. The precipitate was
rinsed with cold methanol, then cold ethyl ether. The filtrate was subsequently filtered
and washed a second time to capture the fine precipitate product. From the first filtration,
6.74 grams were collected, and from the second, 2.54 grams were obtained. After
lyophilization, this desired product 14 resembled off-white sugar crystals (85% yield).
Compound 14 was characterized via NMR spectroscopy.
OH
OH
O
HO
HO
O
H'
HO
O
HO
I2, MeOH
OH
O
KOH, 40°C
HO
OH
Maltose
HO
HO
H'
HO
Maltonic acid
( Compound 14 )
O
HO
OH
OH
K+
O-
HO
O
85% Yeild
Scheme 11: Maltonic acid (Compound 14) was synthesized from maltose.
In Figure 40, the 1H NMR spectrum for Compound 14 can be viewed. The nonreducing end anomeric hydrogen can be seen at δ 5.16, with a J-value of 3.84 Hz. The
reducing end anomeric proton is absent, demonstrating the oxidation of this anomeric
carbon to a carboxylic acid. The proton count coincides with the expected 13 hydrogens
for this molecule that would be visible in the D2O solvent. Compound 14 was also
confirmed by 13C NMR (Appendix B).
75
OH
O
D2O
HO
HO
H'
HO
O
HO
OH
OH
OH
HO
O
J = 3.81Hz
9.84Hz
H'
J = 3.84 Hz
J = 9.54Hz
Figure 40. 1H NMR (300 MHz, D2O) of Compound 14. This spectrum may also be
found (expanded) in Appendix A.
Synthesis of trivalent amino-amide glycodendrimer (15)
Compound 15 was created utilizing the conditions outlined in Scheme 12 to form
the amide bonds. The trivalent core 13 was placed under nitrogen gas first, followed by
the addition of Compound 14. Four equivalents of Compound 14 were used in this
reaction as there were 3 amine sites on Compound 13. Compounds 13 and 14 were
dried further under nitrogen gas for one hour before the addition of BOP. After the
solvent DMF was added to the mixture, the base DIPEA was added in excess (7
equivalents). This reaction was allowed to stir for 3 days before stopping.
76
OH
H
N
O
O
+
NH3+
N
O
HO
HO
OH
HO
OH
O
Compound 13
OH
HO
Compound 14
3
HO
O
BOP,
DIPEA,
DMF,
N2
OH
O
HO
HO
OH
OH
O
OH
HO
Compound 15
3.4% Yield
OH
O
O
H
N
N
O
N
H
3
Scheme 12: The formation of Compound 15 through the coupling of Compound 13
and Compound 14.
When the reaction was stopped it was concentrated in vacuo. To remove leftover
DMF, the residue was reconstituted in MeOH and then toluene and concentrated using
rotary evaporation. Next, an extraction was performed using water and CHCl3. The
aqueous layer collected was lyophilized overnight before purifying by size exclusion
FPLC while monitoring at 214 nm. Multiple samples were collected at this absorbance
and concentrated by lyophilization. After 1H NMR analysis showed the presence of
impurities, this sample was further purified by RP-HPLC to remove the remaining
partially formed products and BOP byproducts. To do this, different methods were
performed. A linear gradient was applied for the first trial, starting with 100% H2O, 0.0%
77
acetonitrile (ACN), and 0.10% TFA and ending with 100% ACN, 0.0% H2O. In this 60
minute run, the sample collected at 22-28 minutes demonstrated impurities, partially
formed products and BOP byproducts based on 1H NMR analysis. For the next trial, a
more polar method was employed. This trial started with the elution of 100% H2O,
0.10% TFA for the first 30 minutes, then a linear gradient was applied for the next 30
minutes ending with 100% ACN, 0.10% TFA. This time, the sample was collected at 5054 minutes. This sample still had the same impurities as before, so a new method was
performed using a less polar conditions. The last method used started with 90% H2O,
10% ACN and ended with 70% H2O, 30% ACN after 70 minutes. A pure sample of
Compound 15 (white solid) was collected in low yield (3.4%) between 13-14 minutes.
The low yield was most likely due to incomplete product formation when the reaction
was stopped after 3 days. It was also possible that the low yield was due to the many
purification steps performed on Compound 15, causing product loss.
The sample collected by HPLC was first characterized by 1H NMR spectroscopy
(Figure 41). In this spectrum, there is a coupling (J = 2.35 Hz) between the doublet at δ
4.34 and the doublet of doublets at δ 4.22. These protons are part of the sugar moiety.
The non-reducing sugar proton, can be seen in the insert, with a J value of 3.94 Hz. The
signal at δ 2.58 was assigned to the CH2 group (5) located adjacent to the carbonyl, which
shields it, moving it upfield. The other protons in Compound 15 are found overlapping,
between δ 3.40 and 4.00. To integrate this spectrum, the chemical shift at δ 2.58 was
calibrated for 6 protons. Since Compound 15 was evaluated in D2O, the overall proton
count (75) was the same as the expected protons (75). Compound 15 was also analyzed
78
by 13C NMR (see Appendix B). The mass spectrum for Compound 15 can be found in
Appendix D, further confirming this product.
J = 3.95Hz
H'
1
OH
O
HO
HO
H'
OH
OH
O
OH
HO
OH
O
O
3
H
N
2
6
4
O
5
N
H
N
7
1-4,6,7
3
J = 2.35Hz
5, J = 5.90Hz
J = 2.35Hz
J = 6.25Hz
Figure 41. 1H NMR (500 MHz, D2O) of Compound 15. This spectrum is also in
Appendix A, in expanded view.
Synthesis of the Boc-protected hexavalent amino core (16)
Compound 16 was created using the conditions seen in Scheme 13. Compound
4 was placed under nitrogen gas then dissolved in dry MeOH. (Boc)2O was added in
excess (12 equivalents) to accommodate the six reactive sites on Compound 4. The
catalyst, NiCl2·6 H2O (Nickel chloride hexahydrate, 0.60 equivalents), was added next,
followed by sodium borohydride (NaBH4). Since NaBH4 was added directly to the
79
reaction, it was added in portions to keep the hydrogen formation in check. After each
addition, more NaBH4 was added, once the formation of bubbles had ceased. The
reaction was monitored by TLC using ninhydrin stain to verify the disappearance of the
starting material (Compound 4) before ending the procedure.
NC
Boc
NH
Boc
O
HN
CN
O
NC
O
O
N
O
N
(Boc)2O, NiCl2 . 6H2O
N
N
H
N
Boc
O
NC
O
Compound 4
N
H
N
Boc
N
MeOH, N2
N
O
O
O
CN
N
Compound 16
O
O
NC
Boc
NH
HN
Boc
TFA,
CH2Cl2
NH2
H2N
O
O
N
H2N
O
N
O
NH2
N
N
Compound 17
O
NH2
O
NH2
Scheme 13: The hexavalent nitrile core 4 was reduced forming Compound 16, followed
by deprotection to form an amine Compound 17.
80
After 24 hours, the reaction was stopped with the addition of tris (2aminoethyl)amine to complex with the nickel chloride hexahydrate. After 30 minutes of
stirring, the reaction mixture was purple in color, confirming the complex formation. The
reaction was concentrated in vacuo and then extracted two times. To analyze Compound
16, a 1H NMR was performed. Due to the long relaxation times (vide supra) of this
molecule, the chemical shifts and integrations were impossible to determine. Because of
this, a sample of Compound 16 was analyzed by IR (Figure 42). After verifying the
disappearance of the nitrile peak (2251 cm-1) from Compound 4 and the appearance of
the carbamate carbonyl peak at 1693 cm-1. Compound 16 was carried forward and used
in the second step in Scheme 13.
90.0
88
86
84
1391.18
82
80
3344.25
Carbamate
NH
1526.51
2865.66
2931.76
%T
1251.06
1365.79
Boc
78
N
H
Carbamate
carbonyl
O
76
N
N
1172.26
O
74
1115.89
1693.84
H
N
Boc
72
3
70.0
3750.0
3000
2000
1500
1000
750.0
cm-1
Figure 42. IR of Compound 16 displaying a carbamate carbonyl peak at 1693.8 cm-1.
81
Synthesis of the hexavalent amino core (17)
To produce Compound 17, Compound 16 was dissolved in CH2Cl2, followed by
the addition of TFA (Scheme 13). The mixture was stirred at room temperature and
monitored by TLC with ninhydrin staining. Compound 16 displayed a pinkish streaky
band with ninhydrin stain, so when this was absent from view via TLC, the reaction was
stopped. This occurred after 85 minutes.
Once the reaction was stopped, it was concentrated in vacuo and then freeze
dried. The sample was then dissolved in water and extracted with CHCl3. The aqueous
layer was lyophilized overnight, and further purified by RP-HPLC. The samples
collected between 20-70 minutes resembled formation of Compound 17 by NMR
analysis, even the waste collected in between each peak. Therefore, all the samples
collected in this time frame were pooled together and lyophilized. Because Compound
17 does not have a chromophore, it was thought that, since it was complexed with TFA, it
would be visible through UV detection. This was not the case though; peaks that were
seen were very weak. Because HPLC did not purify Compound 17, FPLC was used
next. During this process, the flow rate decreased from 0.25 mL/min to ~ 0.10 mL/min
and the elution of Compound 17 took four days. It is unclear at this time why this
occurred. The fractions that demonstrated an absorption at 225 nm, were pooled together
and concentrated by lyophilization. Compound 17 was analyzed by NMR spectroscopy
and mass spectrometry.
Most the peaks in the 1H NMR spectrum (Figure 43) were broad due to long
relaxation times (vide supra). Even though all the chemical shifts were unclear, it was
82
still obvious where some CH2 groups should appear. Starting with the peaks furthest
upfield, these relate to the most shielded protons, CH2 groups (4) and (7), since they are
both adjacent methylene groups. The set of peaks between δ 2.55 and δ 3.10
corresponded to CH2 groups (1, 2, 3, and 8). The last set of peaks between δ 3.55 and δ
3.65 were the most deshielded set of protons, CH2 groups (5) and (6), which are adjacent
an oxygen atom. The expected proton count for Compound 17 in a D2O solvent is 84,
which is what is observed in Figure 43. The formation of Compound 17 was further
confirmed through 13C NMR spectroscopy, which can be found in Appendix B.
7
4
N
N
4
2
5,6
NH2
O
1
7
O
3
8
6
5
3
5
1,2,3,8
6
NH2
8
3
4,7
Figure 43. 1H NMR (500 MHz, D2O) of Compound 17 created from Scheme 13. This
spectrum is also in Appendix A, in an expanded view.
83
The mass spectrum for Compound 17 can be seen in Figure 44. The peak that
most correlated to Compound 17, in Figure 44 was 838.88 m/z [M+2H]. Since
Compound 17 had six primary amines and four tertiary amines, it was likely that, since it
was created under acidic conditions, it was harboring some acidic protons, adding to the
mass. It was also possible that since this is a large molecule, it may have contained some
isotopes that would increase the mass.
C42H96N10O6
Exact Mass: 836.7514
NH2
O
N
N
O
NH2
3
[M+2H]
Figure 44. Time of flight positive mode electrospray mass spectroscopy (TOF ES+ MS)
of Compound 17.
84
Chapter 3
CONCLUSIONS AND FUTURE WORK
The fight against HIV is ongoing. At least 33.3 million people are currently
infected worldwide.1,2 There are treatments for HIV, but they are not without drawbacks,
like viral resistance and harmful side-effects.1 Most drug treatments thus far attack HIV
from within the cell. There are few drugs that prevent HIV from binding to the host cell
and inhibit infectivity at a very early stage in the process. It has been established that
sulfated molecules have an affinity to HIV through ionic interactions, between the viral
surface gp120 and the polyanionic sulfated compounds. Some studies have shown that
sulfated glycodendrimers have an affinity to HIV through these interactions and thus
have the potential to prevent HIV infection.5,6,39
As sulfated glycodendrimers have shown the potential to inhibit HIV infection,
the synthesis of this class of molecules is of profound interest in the fight against HIV. In
this research, two glycodendrimers, 10 and 14, were created. A hexavalent amino core
17 was also created, which will be utilized later to synthesize other glycodendrimers.
Creating these glycodendrimers is only one part of process. To further prepare these
molecules for the testing of their HIV inhibition potential, both glycodendrimers need to
be sulfated. As mentioned before (vide supra), Compound 10 has already been sulfated
(Scheme 7) by another group member.50 Eventually this process will be performed on
Compound 14 as well.
85
After Compounds 10 and 14 are sulfated, they will be tested with an ELISA
(enzyme-linked immunosorbent assay). This is a quick competitive gp120 binding assay
that will be utilized to screen the glycodendrimers for binding affinity.5 If Compounds
10 and 14 demonstrate binding affinity, the samples will be tested on active viral particles
by a collaborator at Duke University. At Duke University, a luciferase reporter gene
assay will be used to determine how well Compounds 10 and/or 14 are able to inhibit
HIV-1 infection.5 If Compounds 10 and 14 demonstrate inhibitory properties, it may
lead to the development of new anti-viral agents to fight HIV infection. These
glycodendrimers may be used as microbicides, preventing the spread of HIV. While it is
important to find treatments for HIV patients, it is also important to help find ways to
protect individuals not infected with the virus. As the old saying goes, “An ounce of
prevention is worth a pound of cure”.51
86
Chapter 4
EXPERIMENTAL
Materials
Dichloromethane (CH2Cl2), tris(2-aminoethyl)amine, potassium carbonate
(K2CO3), acetyl chloride and p-toluenesulfonyl chloride (TsCl), Di-tert-butyl dicarbonate
((Boc)2O), N,N-diisopropylethylamine (DIPEA), and dimethyl sulfoxide (DMSO), were
purchased from Acros Organics, and sodium hydroxide (NaOH) and potassium
hydroxide (KOH) from Spectrum Chemicals. Ethyl acetate (EtOAc), hexane, and silica
gel were purchased from Whatman. Maltose and 1,3-propanediol were obtained from
Sigma-Aldrich, and methanol (MeOH) and lithium hydroxide (LiOH) from Fisher
Scientific. Nickel chloride (NiCl2·6H2O) was purchased from Alfa-Aesar. Anhydrous
MeOH, dichloromethane (CH2Cl2), and acetonitrile (ACN), and sodium borohydride
(NaBH4) were obtained from EMSci. Trifluroacetic acid (TFA), triethylamine (TEA),
ammonium bicarbonate (NH4HCO3), dimethylformamide (DMF) were purchased from
EMD and O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)
from Chem-Impex International. Benzotriazol-1-yloxy-tris(dimethylamino) phosphonium
hexafluorophosphate (BOP) was obtained from Advanced Chemical Technology. The
100, 500, and 2000 MW cellulose ester membrane were purchased from Spectra/Por®,
and the P-10 Bio-Gel used was from Bio-Rad. The materials used in this research were
not purified prior to use, and only when reactions conditions involved the use of nitrogen
gas were all glassware, syringes, stir-bars, etc., flame or oven-dried.
87
Instrumentation
A Bruker Avance 300 or Avance III 500 NMR (NSF, CHE MRI-0922676) were
used for all NMR measurements. All lyophilization was performed using the Freeze
Dry/Shell Freeze System (LABCONCO 7522800). A 2032 DryFast Ultra® was used for
all rotary evaporation. All centrifugation was performed using Centrific Model 228
(Fisher Scientific). A Hewlett Packard TI-series 1050 with a Grace Prevail C-18 5µM
column (10 x 250 mm) were used for the reverse phase high pressure liquid
chromatography (RP-HPLC). Fast pace liquid chromatography (FPLC) was performed
on two different pump systems; one was a Pharmacia pump P-500 with a L.C. Controller
LCC-500 Plus, and the other was a Bio-Rad BioLogic DuoFlow F10 Pumphead with a
Dell Controller. A Bio-Rad Econo-Column (2.5 x 120 cm) was used with both FPLC
systems. The microwave synthesis was performed with a microwave accelerated reaction
system (MARS®). A Perkin Elmer System 2000 FT-IR Spectrometer was used for all IR
measurements.
Characterization
The 1H and 13C spectra were analyzed using Spinworks or Topspin. Solvents
used were either deuterium oxide (D2O) or chloroform-d (CDCl3) and purchased from
Acros. Expanded versions of all spectra can be located in the Appendices; 1H NMR in
Appendix A, 13C NMR in Appendix B, infrared (IR) in Appendix C and mass spectra in
Appendix D. The electrospray time-of-flight (ES-TOF) were collected from Ohio State
88
University, Mass Spectrometry & Proteomics Facility. The MALDI-MS were obtained
from Ohio State University, Mass Spectrometry & Proteomics Facility, and the
University of the Pacific Mass Spectrometry Facility.
Methods
Synthesis of 3-(3-hydroxypropoxy)propanenitrile (1)40
Using a syringe, 62.5 μl of 40% NaOH (W/V) was added dropwise to 1,3propandiol (0.95 mL, 13.1mmol) in a round bottom flask (25 mL). The mixture was
placed in a water bath upon the addition of acrylonitrile (0.39 mL, 5.88 mmol), which
was added dropwise. TLC (90% EtOAc, 10% hexane, with visualization using I2 and
molybdenum stain) was performed to verify the disappearance of the starting material,
before stopping the reaction. The procedure was allowed to run 3 days, and then
quenched with DI H2O. Next it was acidified to ~ pH 7 with 1 M HCl. The residue was
next concentrated by freeze drying and purified by flash silica gel chromatography (90%
EtOAc, 10% hexane) and isolated by rotary evaporation. Compound 1 (clear pale
yellow liquid) was collected at Rf = 0.36. From the reaction, 559 mg (74% yield) of
Compound 1 was obtained. 1H NMR (500 MHz, CDCl3): δ1.70 (app. pentet, 2H, J =
6.10Hz), 2.50 (t, 2H, J = 6.20 Hz), 2.93 (s, 1H), 3.50 (t, 2H, J = 6.05Hz), 3.52 (t, 2H, J =
6.25Hz), 3.58 (t, 2H, J = 6.05Hz).
68.6, and 118.
C NMR (300 MHz, CDCl3): δ 18.5, 31.8, 59.6, 65.1,
13
89
Synthesis of 3-(2-cyanoethoxy)propyl p-toluenesulfonate (3)42
The starting material (1, 1.0 g, 7.9 mmol) was dissolved in dichloromethane (13
mL) and heated to 50-54ºC in a round bottom flask (50 mL). Tosyl chloride (3.00 g, 15.8
mmol) was added to the mixture, followed by the dropwise addition 2.9 mL of 50%
NaOH (W/V) dropwise. The reaction was monitored using TLC (50% EtOAc 50% hexane
and UV, molybdenum stain) and after the disappearance (6 days) of Compound 1, the
reaction was allowed to reach room temperature. An extraction was performed with
water (3x), followed by rotary evaporation of the organic phase. Next, the product was
purified through silica gel flash chromatography (50% EtOAc and 50% hexane) and
collected at the Rf value of 0.39. Compound 3 (679 mg, 30.4% yield, viscous cloudy
white liquid) was characterized by NMR spectroscopy. 1H NMR (500 MHz, CDCl3):
δ1.92 (m, 2H, J = 5.95Hz), 2.43 (s, 3H), 2.50 (t, 2H, J = 6.35Hz), 3.50 (t, 2H, J =
5.95Hz), 3.54 (t, 2H, J = 6.35Hz), 4.12 (t, 2H, J = 6.00Hz), 7.34 (d, 2H, J = 7.95Hz), 7.7
(d, 2H, J = 8.25Hz).
C NMR (300 MHz, CDCl3): δ 18.4, 21.3, 28.8, 65.1, 66.2, 67.2,
13
118, 128, 130, 133, and 145.
Synthesis of hexavalent nitrile core (4)42
In a round bottom flask (100mL) with a condenser attached, anhydrous
acetonitrile (30.0 mL) and K2CO3 (2.83 g, 20.5 mmol) were mixed under N2. Upon the
addition of the tris(2-aminoethyl)amine (0.40 mL, 2.67 mmol) the reaction was heated to
89ºC. Next, a mixture of the Compound 3 (5.80 g, 20.5 mmol), and dry acetonitrile (2
90
mL) were added in portions over a four hour period and the temperature was adjusted to
85ºC. Over the next 20 hours, the temperature was lowered to 82ºC and allowed to reflux
at this temperature until the reaction was stopped. Silica gel TLC (CHCl3 : MeOH :
triethylamine (1:1:0.1%), I2, ninhydrin stain) was performed to verify the disappearance
of the starting material (tris-core). On the second day when the yellowish color verifying
the presence of the tris-core on the TLC plate was missing, the reaction was stopped.
After the reaction cooled to room temperature, it was filtered, through filter paper and
concentrated in vacuo. The residue (bronze colored oil) was then extracted using H2O
(200mL) and CHCl3 (3x 75 mL). Next, the organic layer was concentrated in vacuo and
then washed in water followed by centrifugation (3400 rpm) for 10 minutes. The
supernatant was decanted into a round bottom flask (100 mL) containing an anion
exchange resin (AG 1-X8, 50-500 mesh, chloride form) and stirred overnight, then
filtered and lyophilized. This process was repeated with the pellet collected to obtain any
remnants of the desired product 4 not collected in the first water wash. From the
combined samples, a dark bronze viscous liquid was obtained for Compound 4 (2.03g,
91% yield). 1H NMR (300 MHz, CDCl3): δ 1.66 (m, 12H), 2.46 (m, 24H), 2.56 (t, 12H, J
= 6.21Hz), 3.48 (t, 12H, J = 6.27Hz), 3.59 (t, 12H, J = 6.27Hz); 13C NMR (500 MHz,
CDCl3): δ 18.5, 27.0, 50.7, 52.1, 53.0, 65.0, 69.0, 118. IR (CHCl3): 665.8, 755.1, 844.4,
951.8, 1118, 1225, 1328, 1368, 1414, 1467, 1668, 2251, 2873, and 3018 cm-1. MALDITOF MS [M + H] (C42H72N10O6) Calcd: m/z = 813.6, Found: m/z = 813.8.
91
Synthesis of methyl ester core (5)46,52
Compound 4 (504 mg, .619 mmol) and anhydrous MeOH (2.0 mL) were
combined in a round bottom flask (10 mL) and stirred in an ice bath. After the acetyl
chloride (1.6 mL) was added, the reaction was stirred overnight at room temperature.
TLC (CHCl3 : MeOH : H2O (6:4:0.5), I2, ninhydrin) was performed to verify the
disappearance of the starting material by colorimetry. After 24 hours, the brownish color
representing Compound 4 had disappeared and the reaction was stopped. It was
evaporated, and freeze dried overnight, and then dialyzed for 2 hours in 100 MWCO
tubing against 4L of water. A 217 mg (33% yield) sample (off-white chunky solid) was
recovered after lyophilization. After Compound 5 was characterized, it was carried
forward in the following reaction to create a carboxylic acid terminated core. IR
(acetone): 1113, 1197, 1733, and 2924 cm-1.
Synthesis of the hexavalent carboxy core (5)47
Compound 5 (109 mg, 0.10 mmol) was transferred to a round bottom flask (25
mL) and dissolved into nanopure water (1.5 mL). After LiOH (60 mg, 2.5 mmol) was
added to the mixture, it was heated to 60ºC in a microwave (400 W) for 5 minutes before
the addition of more H2O (1.0 mL). The reaction was reheated to 60ºC for 20 more
minutes in the microwave. When the sample reached room temperature, it was
concentrated in vacuo, washed with MeOH (2 mL) and centrifuged (3400 rpm) for 10
minutes. The supernatant was decanted and concentrated in vacuo and further purified
92
using size exclusion FPLC (2.5cm x 120cm column, Bio-Rad P-10 size exclusion gel).
The column was eluted with 0.03M ammonium bicarbonate at a rate of 0.25mL/min and
monitored at 214 nm. The peaks collected resulted in 66% yield (68.1 mg) of
Compound 6 (tannish colored chunky solid). 1H NMR (500 MHz, D2O): δ 1.85 (m,
12H), 2.45 (t, 12H, J = 6.60Hz), 2.75-2.91 (m, 24H), 3.58 (t, 12H, J = 6.10Hz), 3.71 (t,
12H, J = 6.60 Hz); 13C NMR (500 MHz, D2O, internal MeOH reference): δ 26.0, 37.8,
49.4, 50.2, 50.8, 67.8, 68.5, 180. ES TOF MS [M - H] (C42H78N4O18) Calcd: m/z =
925.5233, Found: m/z = 925.5223.
Synthesis of the Boc-protected oxime sugar-linker (8)53
Compound 7 (125 mg, 0.54 mmol) and maltose (222 mg, 0.62 mmol) were
combined in a round bottom flask (25 mL). Ammonium acetate (0.1M, 5 mL, pH 4.5)
was added next. After the mixture stirred for 30 minutes, the pH was checked. The pH
was found to be 4.5 and the reaction was allowed stir overnight. Before stopping the
reaction after 20 hours, the pH was checked again. Throughout the reaction the pH
remained around 4.5. TLC (CHCl3:MeOH:H2O, 6:4:0.5, with visualization using
molybdenum stain) was performed to verify the disappearance of Compound 7 (Rf =
0.80) before stopping the reaction. After the reaction was complete, it was freeze dried.
The residue was then purified with flash silica gel chromatography using the same TLC
conditions as described above. From the flash column, Compound 8 (Rf = 0.53, 231 mg,
76% yield, white crystalline solid) was recovered and analyzed by NMR spectroscopy.
1
H NMR (500 MHz, D2O): δ 1.45, (s, 9H), 1.78 (t, 2H, J = 6.60Hz), 3.14 (t, 2H, J =
93
6.60Hz) 3.48 (m, 1H), 3.60-4.00 (m, 14H), 4.12 (m, 0.2H), 4.35 (m, 2H), 4.60 (m, 1H),
5.00 (m, 0.2H), 5.12 (d, 1Hα, J = 3.90Hz), 5.40 (m, 0.1H), 6.98 (d, 0.1HZ, J = 5.50Hz)
and 7.60 (d, 0.6HE, J = 6.05Hz); 13C NMR (500 MHz, D2O, internal MeOH reference): δ
16.9, 27.8, 28.7, 28.8, 37.1, 48.9, 57.4, 60.4, 60.5, 60.8, 62.1, 62.3, 65.7, 68.3, 68.4, 68.5,
68.7, 71.3, 71.7, 71.8, 72.2, 72.3, 72.5, 72.7, 72.9, 73.0, 73.1, 73.4, 75.8, 76.9, 77.2, 80.2,
81.5, 90.2, 99.8, 100, 101, 152, 153, 158.
Synthesis of the E,Z oxime sugar-linker (9)54
Dichloromethane (3 mL) and Compound 8 (231 mg) were combined in a round
bottom flask (25 mL). Next, TFA (1.5 mL) was added dropwise over a 16 minute period.
The reaction was stirred at room temperature and monitored by TLC (6:4:1
CHCl3:MeOH:H2O, ninhydrin stain). When the starting material (Rf = 0.64, light pink
color) was absent from view by TLC and replaced by a new product (Rf = 0.42, streaky
pink purplish color), the reaction was stopped (3 hours). The reaction was quenched with
water (0.5 mL) and then concentrated in vacuo. The residue was dissolved in ammonium
bicarbonate and then lyophilized. This was repeated one additional time. The desired
product 9 (white crystalline solid) was produced in quantitative yield. 1H NMR (500
MHz, D2O): δ 1.94 (m, 2H), 3.08 (m, 2H), 3.40 (m, 1H), 3.50-4.00 (m, 15H), 4.10 (m,
0.1H), 4.30 (m, 2H), 4.50 (t, 0.4H, J = 5.15 Hz), 4.95 (m, 0.1H), 5.12 (d, 0.5Hα, J = 3.90
Hz), 5.40 (d, 0.4H, J = 3.95 Hz), 6.98 (d, 0.09HZ, J = 5.55 Hz) and 7.60 (d, 0.4HE, J =
5.95 Hz); 13C NMR (500 MHz, D2O, internal MeOH reference): δ 26.5, 26.6, 37.6, 37.7,
37.7, 37.8, 60.5, 60.6, 60.7, 60.8, 60.9, 62.2, 62.4, 65.7, 68.0, 68.4, 68.5, 68.8, 69.0, 69.3,
94
69.4, 70.0, 71.3, 71.4, 71.7, 71.8, 71.9, 72.1, 72.6, 72.7, 72.9, 73.0, 73.1, 73.2, 73.3, 74.1,
74.6, 75.8, 76.2, 76.9, 77.0, 77.2, 80.2, 81.2, 90.0, 91.9, 95.8, 100, 152, 153. ES TOF
MS [M+Na] (C17H34N2O12) Calcd: m/z = 481.2009, Found: m/z = 481.1995.
Synthesis of hexavalent maltose amino-oxime glycodendrimer (10)49
Compound 9 (331 mg, 0.578 mmol) and Compound 10 (84.8 mg, 0.0916 mmol)
were added together to a round bottom flask (50 mL) and dissolved in nanopure water
(2mL). The mixture was freeze dried overnight. The lyophilized sample was placed
under N2 gas and dissolved in DMSO (2 mL). The addition of TBTU (424 mg, 1.32
mmol) was next, followed by DIPEA (300 μL). DIPEA was added in portions (100 μL),
to a pH of 9. When the reaction was complete after 24 hours, it was lyophilized. Next,
an extraction was performed. The residue was dissolved in water (50 mL) and impurities
were extracted 3x with CHCl3 (20mL). After the aqueous phase was lyophilized, the
residue was dissolved back into water and any insoluble products were filtered through
glass wool. Dialysis was performed next using 2000 MWCO tubing for a total of 7.5
hours at 4ºC, with water changes at least every hour. After the sample was freeze dried
again, it was filtered through glass wool to remove any ppt. The sample recovered (215
mg, 66% yield) had the appearance of light brown sugar. In the 1H NMR only one arm
was integrated (500 MHz, D2O): δ 1.79-2.00 (m, 4H), 2.59 (m, 2H), 2.70-3.20 (m, 4H),
3.30 (m, 2H), 3.45 (m, 2H), 3.50-4.01 (m, 19H), 4.26 (m, 2H), 4.54 (m, 0.4H), 5.00 (m,
0.1H) 5.12 (d, 0.6H, J = 3.70 Hz), 5.40 (m, 0.3H), 7.00 (d, 0.1HZ, J = 5.40 Hz), and 7.60
(d, 0.5HE, J = 5.95 Hz). 13C NMR (500 MHz, D2O, internal MeOH reference): δ 24.5,
95
28.6, 35.9, 36.6, 39.2, 39.6, 47.6, 50.3, 50.4, 51.2, 51.3, 51.4, 60.5, 60.6, 60.7, 60.9, 61.0,
62.2, 62.5, 65.8, 66.8, 67.6, 68.4, 68.5, 68.6, 68.7, 68.9, 69.4, 69.5, 71.4, 71.8, 72.3, 72.4,
72.6, 72.8, 72.9, 73.0, 73.1, 73.2, 73.5, 75.9, 76.9, 77.3, 80.4, 81.6, 90.3, 99.8, 100, 101,
152, 154, 174. MADLI-TOF MS, [M+H] (C144H270N16O84) Calcd: m/z = 3568.735,
Found: m/z = 3570.280.
Synthesis of the Boc-protected trivalent amino-amide core (12)55
Tris(2-aminoethyl)amine (51.0 μL, 0.342 mmol) was added to recently freeze
dried Compound 11 (290 mg, 1.25 mmol) in a round bottom flask (25 mL). The sample
was immediately placed under nitrogen gas. BOP (605 mg, 1.37 mmol) was added next,
followed by anhydrous DMF (1 mL). After the mixture was stirred for 5 minutes,
DIPEA (400 μL, 2.39 mmol) was added. The reaction was allowed to stir for three days
before stopping. After the sample was concentrated by rotary evaporation, an extraction
was performed. The residue was dissolved in nanopure water (40 mL) and extracted 3x
with chloroform (20 mL). The organic layer was then extracted 3x with water (20 mL).
The organic phase was concentrated in vacuo before purifying by flash silica gel
chromatography (3x). With the first flash column CHCl3:MeOH (8:1 and ninhydrin
stain) were employed to elute Compound 12 (Rf = 0.48). On the second flash column,
the product was eluted with ethyl acetate (100%), then ethyl acetate:methanol (95:5),
followed by methanol (100%). Compound 12 was visible eluting during the addition of
100% methanol and collected. For the last column, a silica plug 4 cm in height was
utilized and the same solvent conditions as column two. When Compound 12 was
96
visible eluting during methanol addition, it was collected and concentrated in vacuo. The
sample (72.3 mg, 27% yield) characterized by NMR. 1H NMR (500 MHz, CDCl3): δ
1.42 (s, 27H), δ 2.43 (t, 6H, J = 5.80 Hz), 2.52 (m, 6H), 3.22 (m, 12H), 3.45 (t, 6H, J =
5.15 Hz), 3.66 (t, 6H, J = 5.90 Hz), 5.30 (s, 3H), 7.05 (s, 2H).
13
C NMR (500 MHz,
CDCl3): δ 29.4, 37.6, 41.2, 55.3, 70.8, 80.1, 157, 173.
Synthesis of the trivalent amino-amide core (13)54
Compound 12 (72.3 mg, 0.0913 mmol) and CH2Cl2 were added together in a
round bottom flask (10 mL). TFA (500 μL, 50% (v/v)) was added next dropwise. TLC
(methanol, 100%) was performed to verify the disappearance of Compound 12. The
reaction was stopped and quenched with water (0.5 mL) after 1 hour. The residue was
concentrated in vacuo. The sample was then lyophilized (2x) overnight. Compound 12
(pale rust colored sticky solid) was characterized by 1H NMR (500MHz, D2O): δ 2.52 (t,
6H, J = 6.00 Hz), 3.12 (t, 6H, J = 5.00 Hz), 3.38 (t, 6H, J = 6.25 Hz), 3.55 (t, 6H, J = 6.25
Hz), 3.66 (t, 6H, J = 5.05 Hz), 3.72 (t, 6H, J = 6.00 Hz).
13
C NMR (500 MHz, D2O,
internal MeOH reference): δ 34.5, 35.7, 39.1, 53.4, 66.3, 66.6, 175. ES-TOF MS [M+H]
(C21H45N7O6) Calcd: m/z = 492.3509, Found: m/z = 492.3500.
Synthesis of maltonic acid (14)56
All glassware used in this experiment was rinsed with methanol prior to use.
Methanol (238 mL) and iodine (13.4 g, 52.7 mmol) were combined together in a threeneck round bottom flask (1 L) and heated to 40ºC. Maltose (10.0 g, 27.8 mmol) was
97
dissolved in boiling water (~20 mL) before adding to the mixture of methanol/iodine.
Next, a mixture of KOH (14.3 g, 254 mmol) and MeOH (357 mL) were mixed together
and then added dropwise to the reaction via an addition funnel. After 85 minutes, the
reaction was removed from the oil bath and allowed to stir at room temperature for 95
minutes. The residue was filtered and rinsed with cold MeOH (1400 mL) followed by
cold ethyl ether (1000 mL). To collect product lost through the filtration, the eluent
collected was filtered again, this time by gravity through 4 layers of filter paper, and
rinsed with cold ethyl ether (500 mL). After lyophilization, the residue (off-white
crystals, 9.28 g, 85% yield) was characterized through NMR spectroscopy. 1H NMR
(300 MHz, D2O): 3.14 (t, 1H, J = 9.54Hz), 3.51-3.55 (dd, 1H, J = 3.81Hz, 9.54Hz), 3.643.98 (m, 9H), 4.07-4.12 (m, 2H), 5.14 (d, 1H, J = 3.84Hz).
13
C NMR (300 MHz, D2O,
internal MeOH reference): δ 60.4, 62.2, 69.4, 71.8, 72.4, 72.6, 72.7, 73.0, 82.4, 100, 178.
Synthesis of the trivalent maltose amino-amide glycodendrimer (15)55
Compound 13 (83.7 mg, 0.100 mmol) and Compound 14 (168 mg, 0.424 mmol)
were added together in a round bottom flask (50 mL) and placed under nitrogen gas.
After 1 hour, BOP (199 mg, 0.450 mmol) was added to the mixture followed by DMF (5
mL) and DIPEA (70.0 μL, 0.418 mmol). The reaction was stirred at room temperature
for 3 days before stopping by the addition of MeOH (20 mL). Once the sample was
concentrated in vacuo, toluene (20 mL) was added next, followed by rotary evaporation.
The sample was reconstituted in water (50 mL) and extracted with 20 mL CHCl3 (4x).
98
The aqueous phase was freeze dried overnight and then purified via FPLC (2.5cm x
120cm column, Bio-Rad P-10 size exclusion gel). The column was eluted with 0.03M
ammonium bicarbonate at a rate of 0.25mL/min while monitoring at 214 nm. The peaks
collected were concentrated by lyophilization. The sample was further purified by RPHPLC. This procedure was performed multiple times with different gradients. The only
constant value in each method was the addition of 0.10% TFA (v/v) in each trial. The first
trial started with a linear gradient with 100% H2O, 0.0% ACN and ended with 100%
ACN, 0.0% H2O after 60 minutes. The sample collected between 22 and 28 minutes was
concentrated by lyophilization. For the next trial, in the first 30 minutes 100% H2O was
maintained, then a linear gradient was applied for the next 30 minutes ending with 100%
ACN, 0.0% H2O. This time the sample was collected at 54 minutes. After
lyophilization, the sample was purified again through RP-HPLC. The next method
started with 90% H2O, 10% ACN and ended with 70% H2O, 30% ACN after 70 minutes.
A pure sample of Compound 15 (5.2 mg, 3.4% yield) eluted between 13-14 minutes.
After the sample was freeze dried, the residue (white sticky solid) was characterized by
NMR spectroscopy. 1H NMR (500 MHz, D2O): δ 2.59 (t, 6H, J = 5.90 Hz), 3.45-3.47
(m, 14H), 3.63-4.00 (m, 46H), 4.21-4.23 (dd, 3H, J = 2.35 Hz, 6.25 Hz), 4.34 (d, 3H, J =
2.35 Hz), 5.12 (d, 3H, J = 3.95 Hz). 13C NMR (500 MHz, D2O, internal MeOH
reference): δ 34.7, 35.9, 38.7, 53.8, 60.5, 62.3, 66.4, 66.8, 69.5, 71.8, 71.9, 72.0, 72.4,
72.6, 73.0, 82.1, 101, 174, 175. MALDI-TOF, [M+Na+H] (C57H105N7O39) Calcd: m/z =
1535.645, Found: m/z = 1535.159.
99
Synthesis of the Boc-protected hexavalent amino core (16)57
Compound 4 (65.4 mg, 0.0805 mmol) was placed in a flame dried round bottom
flask (25 mL) and placed under nitrogen gas. Compound 4 was dissolved in MeOH (5
mL) followed by the addition of (Boc)2O ( 211 mg, 0.966 mmol) and NiCl2·6H2O (11.5
mg, 0.0483 mmol). Next, NaBH4 (128 mg, 3.38 mmol) was added to the reaction in
portions over a 25 minute period. Silica gel TLC (CHCl3 : MeOH : H2O (6:4:0.5),
ninhydrin staining) was performed to verify the disappearance of the starting material
(Compound 4) before ending the procedure. After 24 hours, the yellow/brownish
streaky band verifying the presence of Compound 4 had disappeared, and the reaction
was stopped. A new pinkish band was present on the TLC plate, representing new
product formation. Tris (2-aminoethyl)amine (72.3 μL, 0.483 mmol) was added to the
sample and stirred for 30 minutes. An extraction was performed after the sample was
concentrated in vacuo. For this procedure, the sample was dissolved in ethyl acetate (50
mL), and impurities were extracted with 20 mL of sodium bicarbonate (NaHCO3 x3).
The organic layer was concentrated in vacuo, and then reconstituted in CHCl3 (20 mL).
Impurities were extracted with H2O (10 mL x2). After evaporation, a sample of
Compound 16 (vicous light brownish liquid) was dissolved in CHCl3 and analyzed by IR
(CHCl3): 1116, 1172, 1251, 1366, 1391, 1526, 1694, 2866, 2932, and 3344 cm-1. This
sample (60.2 mg, 54% yield) was carried forward without further purification.
100
Synthesis of the hexavalent amino core (17)54
Compound 16 (60.2 mg, 0.0435 mmol) and CH2Cl2 (2 mL) were added together in a
round bottom flask (10 mL). TFA (1 mL, 50% (v/v)) was added next dropwise. TLC
(CHCl3:MeOH:H2O, 6:4:0.5, ninhydrin stain) was performed to verify the disappearance
of Compound 16. The reaction was stopped after 85 minutes and concentrated in vacuo
and then the sample was freeze dried. After this, the sample was dissolved in water (20
mL) and extracted with 10 mL of CHCl3 (3x). The aqueous layer was then lyophilized
overnight and purified further with RP-HPLC. The linear gradient used here started with
90% H2O: 10% ACN: 0.10% TFA and ended with 70% H2O: 30% ACN: 0.10% TFA
after 70 minutes. The sample collected between 20-70 minutes was lyophilized and
purified by FPLC (2.5cm x 120cm column, Bio-Rad P-10 size exclusion gel). The
column was eluted with 0.03M ammonium bicarbonate at a rate of 0.25mL/min and
monitored at 214 nm. The peaks collected were concentrated by lyophilization. When
this product eluted the flow rate decreased from 0.25mL/min to 0.10mL/min. and it took
4 days to elute. To visualize the peaks the y-axis had to be expanded to see the product
eluting, since Compound 17 has no chromophore. The desired product 17 (5.4 mg, 16%
yield) collected was freeze dried and characterized first by NMR. 1H NMR (500MHz,
D2O): δ 1.70-1.95 (m, 24H), 2.55-2.75 (m, 24H), 2.95-3.15 (m, 12H), 3.55-3.65 (m,
24H).
13
C NMR (500 MHz, D2O, internal MeOH reference): δ 25.5, 25.6, 27.9, 28.1,
29.7, 37.6, 38.5, 49.1, 50.2, 50.5, 50.6, 50.7, 51.0, 68.0, 68.1, 68.6, 68.7, 68.9, 69.1, 69.2.
ES TOF MS [M+2H] (C42H96N10O6) Calcd: m/z = 838.77, Found: m/z = 838.88.
101
APPENDICES
102
APPENDIX A
1
H Spectra
103
3-(3-Hydroxypropoxy)propanenitrile (Compound 1), 1H NMR, 500 MHz, CDCl3.
104
3-(2-Cyanoethoxy)propyl p-toluenesulfonate (Compound 3), 1H NMR, 500 MHz,
CDCl3.
105
Hexavalent nitrile core (Compound 4), 1H NMR, 300 MHz, CDCl3.
106
Hexavalent carboxy core (Compound 6), 1H NMR, 500 MHz, D2O.
107
Boc-protected oxime sugar-linker (Compound 8), 1H NMR, 500 MHz, D2O.
108
E,Z oxime sugar-linker (Compound 9), 1H NMR, 500 MHz, D2O.
109
Hexavalent maltose amino-oxime glycodendrimer (Compound 10), 1H NMR, 500 MHz,
D2O.
110
Boc-protected trivalent amino-amide core (Compound 12), 1H NMR, 500 MHz, CDCl3.
111
Trivalent amino-amide core (Compound 13), 1H NMR, 500 MHz, D2O.
112
Maltonic acid (Compound 14), 1H NMR, 300 MHz, D2O.
113
Trivalent maltose amine-amide glycodendrimer (Compound 15), 1H NMR, 500 MHz,
D2O.
114
Hexavalent amino core (Compound 17), 1H NMR, 500 MHz, D2O.
115
APPENDIX B
13
C Spectra
116
3-(3-Hydroxypropoxy)propanenitrile (Compound 1), 13C NMR, 300 MHz, CDCl3.
117
3-(2-Cyanoethoxy)propyl p-toluenesulfonate (Compound 3), 13C NMR, 300 MHz,
CDCl3.
118
Hexavalent nitrile core (Compound 4), 13C NMR, 500 MHz, CDCl3.
119
Hexavalent carboxy core (Compound 6), 13C NMR, 500 MHz, D2O.
120
Boc-protected oxime sugar-linker (Compound 8), 13C NMR, 500 MHz, D2O.
121
E,Z oxime sugar-linker (Compound 9), 13C NMR, 500 MHz, D2O.
122
Hexavalent maltose amino-oxime glycodendrimer (Compound 10), 13C NMR, 500 MHz,
D2O.
123
Boc-protected trivalent amino-amide core (Compound 12), 13C NMR, 500 MHz, CDCl3.
124
Trivalent amino-amide core (Compound 13), 13C NMR, 500 MHz, D2O.
125
Maltonic acid (Compound 14), 13C NMR, 300 MHZ, D2O
126
Trivalent maltose amino-amide glycodendrimer (Compound 14), 13C NMR, 500 MHz,
D2O.
127
Hexavalent amino core (Compound 17), 13C NMR, 500 MHz, D2O.
128
APPENDIX C
Infrared Spectra
129
Hexavalent nitrile core (Compound 4), IR with CHCl3.
130
Hexavalent methyl ester (Compound 5), IR with acetone.
131
Boc-protected hexavalent amino core (Compound 15), IR with CHCl3.
132
APPENDIX D
Mass Spectra
133
Hexavalent nitrile core (Compound 4), MALDI TOF MS.
134
Hexavalent carboxy core (Compound 6), ESI TOF MS.
135
E,Z oxime sugar-linker (Compound 9), ESI TOF MS.
136
Hexavalent maltose oxime-amino glycodendrimer (Compound 10), MALDI TOF.
137
Trivalent amino-amide core (Compound 13), ESI TOF MS.
138
Trivalent maltose amino-amide glycodendrimer (Compound 15), MALDI TOF MS.
139
Hexavalent amino core (Compound 17), ESI TOF MS.
140
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