SYNTHESIS OF MULTIPLE SIALIC ACID-TERMINATED DENDRIMERS WITH A
POLY(AMIDOAMINE) CORE FOR THE STUDY OF THE EFFECT OF
GENERATION AND DISTANCE OF SUGAR TO CORE ON BINDING AFFINITY
Russell G. Clayton Jr.
B.A., University of California, Davis 2004
THESIS
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
CHEMISTRY
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
FALL
2010
SYNTHESIS OF MULTIPLE SIALIC ACID-TERMINATED DENDRIMERS WITH A
POLY(AMIDOAMINE) CORE FOR THE STUDY OF THE EFFECT OF
GENERATION AND DISTANCE OF SUGAR TO CORE ON BINDING AFFINITY
A Thesis
by
Russell G. Clayton Jr.
Approved by:
__________________________________, Committee Chair
Katherine McReynolds, Ph.D.
__________________________________, Committee Member
Cynthia Kellen-Yuen, Ph.D.
__________________________________, Committee Member
Claudia Lucero, Ph.D.
____________________________
Date
ii
Student: Russell G. Clayton Jr.
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 credit is to be awarded for
the thesis.
__________________________, Department Chair
Susan Crawford, Ph.D.
Department of Chemistry
iii
___________________
Date
Abstract
of
SYNTHESIS OF MULTIPLE SIALIC ACID-TERMINATED DENDRIMERS WITH A
POLY(AMIDOAMINE) CORE FOR THE STUDY OF THE EFFECT OF
GENERATION AND DISTANCE OF SUGAR TO CORE ON BINDING AFFINITY
by
Russell G. Clayton Jr.
This thesis discusses the synthesis and characterization of four sialic acidconjugated poly(amidoamine) glycodendrimers as potential inhibitors of the V3 loop of
the HIV virus. Known to inhibit the HIV virus by binding to the V3 loop of gp120 are the
carbohydrates heparin sulfate (HS) and dextrin sulfate (DS). Unfortunately, HS and DS
are also commonly used as anticoagulants and blood thinning agents. Sulfated colominc
acid, an α-2, 8 linked homopolymer of sialic acid, is also known to have anti HIV
properties without the negative side effects of HS and DS. The drawback to using
carbohydrates is that the body will quickly break them down. This leaves little time for
the carbohydrates to reach their intended destination. Therefore, a vehicle is necessary to
transport the carbohydrates to their site of action before they can be degraded by the
body. Dendrimers can meet this need. Dendrimers are large, spherical, macromolecules,
whose surface can be functionalized with any sort of molecule including carbohydrates.
iv
They have been used for a variety of applications, including medical uses, and are known
to be of low toxicity. In addition, dendrimers are multivalent, which means that there are
multiple functional groups available to bind either concurrently, or in tandem to a ligand,
increasing the overall binding strength of the parent molecule.
The work presented in this thesis looks at numerous aspects of dendrimers and
their synthesis. In building a dendrimer, there are two possible synthetic routes,
convergent and divergent. A convergent synthesis involves building from the outside
inward, while a divergent synthesis builds from the inside outward. This work utilized
both routes to test which method provided better yields and simplified purifications. In
addition to testing the synthetic strategies, two sets of glycodendrimers were made that
differed by the addition of a linker between the poly(amidoamine) and the sialic acid.
This will allow future work to use these two sets of glycodendrimers to test the effect of
dendridic radii on binding strengths. Finally, the synthetic strategy involving all
carbohydrates eschewed protecting group chemistry to increase yields and reduce steps.
Each additional step takes time and materials, and every reaction risks losing yield. This
led to increased yields and decrease synthesis times.
_______________________, Committee Chair
Katherine McReynolds, Ph.D.
_______________________
Date
v
ACKNOWLEDGMENTS
Dr. McReynolds
Dr. Kellen-Yuen and Dr. Lucero
McReynolds research group
Especially:
Michelle Waterson
Rachel Blackeye
Family
John and Janice Webb
Russ and Susan Clayton
Beverly and August Bencivengo
Friends
All those who supported me and listened to me vent. Then prayed for me, that I
could make it through the storm.
vi
TABLE OF CONTENTS
Page
Abstract ...................................................................................................................................... v
Acknowledgments.................................................................................................................... vi
List of Tables ........................................................................................................................ viii
List of Figures .......................................................................................................................... ix
List of Schemes ........................................................................................................................ xi
Chapter
1. INTRODUCTION …………….………………………………………………………… 1
HIV/AIDS Chemotherapy Background ....................................................................... 2
Polyanions as Inhibitors. ............................................................................................ 10
Dendrimers for Drug Delivery ................................................................................... 15
Research Goals .......................................................................................................... 20
2. RESULTS & DISCUSSION............................................................................................. 28
Synthesis .................................................................................................................... 29
Glycodendrimer Purification ..................................................................................... 32
3. CONCLUSIONS & FUTURE WORK ............................................................................. 47
Future Directions ....................................................................................................... 50
4. EXPERIMENTAL ............................................................................................................ 52
Appendix A.
1
Appendix B.
13
H Nuclear Magnetic Resonance Spectra ....................................................... 62
C Nuclear Magnetic Resonance Spectra ....................................................... 70
Appendix C. Matrix Assisted Laser Desorption Ionization Mass Spectroscopy .................. 77
Bibliography ........................................................................................................................... 81
vii
LIST OF TABLES
Page
1.
Table 1 Monomer and oligomer sialic acid and their inhibition of
A/Memphis/102/72 adsorbtion to erthrocytes….…………………………….. 20
viii
LIST OF FIGURES
Page
1.
Figure 1 A mature HIV virion……………………….…………………………. 3
2.
Figure 2 HIV fusion to a host cell...……...……….……………………………. 4
3.
Figure 3 HIV replication within a host cell…….………………………………. 5
4.
Figure 4 The fusion inhibitor Fuzeon™.……….………………………………. 8
5.
Figure 5 The entry inhibitor Selzentry™…………….…………………………. 8
6.
Figure 6 Polyanionic oligosaccaharides………….……………………………. 10
7.
Figure 7 Model of gp120, V3 loop, and gp120 with the V3 loop overlaid……. 12
8.
Figure 8 Colominic acid is a α-2,8 linked polymer of sialic acid..……………. 13
9.
Figure 9 A generation 2 PAMAM dendrimer……….…………………………. 15
10.
Figure 10 Divergent and convergent synthesis strategies for dendrimers...……. 17
11.
Figure 11 Tetrameric glycol-DATEG-PAMAM dendrimers (1)….……………. 23
12.
Figure 12 Tetrameric glycoPAMAM dendrimers (2)...…………...……………. 23
13.
Figure 13 Octameric glycol-DATEG-PAMAM dendrimers (3)..……...………. 24
14.
Figure 14 Octameric glycoPAMAM dendrimers (4)…...…….…...……………. 24
15.
Figure 15 16-mer glycol-DATEG-PAMAM dendrimers (5)....……...…………. 25
16.
Figure 16 16-mer glycoPAMAM dendrimers (6).…………………...…………. 26
17.
Figure 17 1H NMR of sialic acid (10)……………………………………......…. 33
18.
Figure 18 1H NMR of sugar-linker complex (11)……………….…………...…. 34
19.
Figure 19 1H NMR of Tetrameric glyco-DATEG-PAMAM dendrimer (1)....…. 35
20.
Figure 20 1H NMR of Tetrameric glycoPAMAM dendrimer (2).…………...…. 36
ix
21.
Figure 21 1H NMR of Octameric glyco-DATE-PAMAM dendrimers (3)..……. 38
22.
Figure 22 MALDI MS of Octameric glyco-DATE-PAMAM dendrimers (3)…. 39
23.
Figure 23 An overlay of Octameric glycol-DATEG-PAMAM dendrimer..….... 42
24.
Figure 24 Relaxation time effect on 1H NMR.…….…………...………………. 44
25.
Figure 25 Representations of T1 and T2 in NMR relaxation.……...……………. 46
26.
Figure 26 Negative controls to be used in future biological assays.……………. 51
x
LIST OF SCHEMES
Page
1.
Scheme 1 Monoprotection of diaminotriethylene glycol………………………. 30
2.
Scheme 2 Amide coupling of sialic acid to linker followed by deprotection..…. 30
3.
Scheme 3 Amide coupling of sugar-linker to PAMAM G= -0.5, 0.5, and 1.5…. 31
4.
Scheme 4 Amide coupling of sialic acid to PAMAM G= 0, 1, and 2….………. 31
5.
Scheme 5 Sulfation of sialic acid residues on compounds 1-6.…………...……. 50
xi
1
Chapter 1
INTRODUCTION
By the end of 2008, UNAIDS (Joint United Nations Programme on HIV/AIDS)
and the WHO (World Health Organization) estimated that 33.4 million people were
living with HIV. Of these 33.4 million people, 2.7 million were newly infected and 2
million people died of AIDS in 2008.1 In 1996, the treatment regimen HAART (highly
active antiretroviral therapy) emerged and has prolonged the lifespan of HIV infected
individuals by delaying the onset of AIDS.2 HAART therapy typically consists of a
mixture of drugs that inhibit reverse transcriptase and protease functions. Since the virus
can readily mutate, new drugs are needed to add to the HAART regimen in order for it to
retain its effectiveness.
Recently, two new classes of drugs have emerged that warrant attention. They are
fusion inhibitors (FIs) and entry inhibitors (EIs). The fusion inhibitor, Fuzeon™, inhibits
the ability of HIV to create a fusion pore, and thus infect a cell.3 Fuzeon™ also shows
activity against drug resistant strains of HIV, which gives it the potential of being a
potent inhibitor. The entry inhibitor Selzentry™ works as an antagonist to the CCR5 coreceptor on gp120.4 Thus far, these are the only drugs in both of these classes and
therefore it is important to develop new fusion and entry inhibitors that can add greater
diversity to the HAART therapy.
2
HIV/AIDS Chemotherapy Background:
Of the estimated 33.4 million people worldwide infected with HIV, 1.1 million of
those people reside in the United States.5 Because of HAART, the quality of life for HIV
patients has been dramatically improved in the last decade. HAART is a form of therapy
that involves mixing three or more drugs chosen from six different mechanistic classes to
tailor a treatment to an individual. Unfortunately, as the virus spreads, individuals begin
to get infected with strains of the virus that have already been exposed to HAART. This
means that a particular virus has likely developed a resistance to some of the HAART
drugs. As a result, HAART therapy needs to be ever-evolving, like HIV, to provide
continuing success in improving a patient’s quality of life after infection. The current
understanding of the biology of HIV has lead to the development of numerous drugs and
significant advances in controlling HIV. Once the virus’ biology is more thoroughly
understood, more effective drugs to block viral processes and inhibit the virus can be
developed.
While a great deal is already known about the HIV virus, more knowledge about
the structure and function will allow for the development of additional ways to combat it.
The HIV virus is spherical, with a diameter between 100-120 nm. The outermost shell is
a phospholipid bilayer that is unevenly clustered with 14 ± 7 spikes of the trimer protein
complex gp41/gp120 which are mobile within the bilayer. Gp41 and gp120 are two
separate proteins held together by noncovalent interactions.6 In the center of the virion is
a cone-shaped capsid which contains the viral RNA, viral protease, reverse transcriptase,
3
and integrase proteins required for
viral replication. Figure 1 shows
a schematic of a mature HIV
virion.7
Once HIV infection has
occurred, the virus will seek out
cells it can use for replication.
The primary receptor for gp120 is
CD4, an extracellular
glycoprotein found on various
immune cells. Thus, the most
common cells infected are T-
Figure 1. A mature HIV virion. gp120 (glycoprotein
120), gp41 (glycoprotein 41), RNA (Riboneucleic
acid), MA (matrix protein, p17), NC (nucleocapsid
protein, p7), CA (Capsid layer, p24), RT (Reverse
Transcriptase).7
cells and macrophages. Initially, gp120 binds with CD4, which causes conformational
changes in gp120. These changes in gp120 reveal a previously hidden pocket, which
induces further binding between gp120 and a coreceptor. The coreceptor type involved is
dependent on the host cell type. Because of the ability of HIV to recognize multiple
coreceptors, the virion is infectious towards numerous targets. This allows the virus more
opportunity for infection and replication, although in general only one of two coreceptors
is used: CXCR4 is the coreceptor on T-cells,8 while CCR5 is the coreceptor on
macrophages.9 Once the coreceptor has bound to gp120, further conformational changes
occur allowing the N-terminal region of gp41 to extend and insert itself into the bilayer of
the host cell, as seen in Figure 2.7
4
Figure 2. HIV fusion to a host cell. CD4 binds to gp120 causing structural changes and
allowing for the binding of either coreceptor CCR5 or CXCR4. This causes further
structural changes and allows the fusion domain of gp41 to fuse with the host cells
membrane.7
Once the virus has anchored itself to the host cell via the gp41 trimer, the gp41s
form a hairpin turn to bring their C-terminal and N-terminal regions in contact with one
another to form a six-helix bundle. The folding of gp41 allows the opening of a fusion
pore between HIV and the host cell. The fusion pore then allows the virion core to be
released into the host cell.10 This begins the replication process as seen in Figure 3.
5
Figure 3. HIV replication within a host cell.7 1) The virus attaches to and fuses with the
cellular membrane releasing the core into the host. 2) The core is unpackaged, the viral
RNA is reverse transcribed into DNA via reverse transcriptase (RT) and moved into the
nucleus. 3) Viral DNA is integrated into host DNA via viral integrase (IN). 4) The viral
DNA is transcribed into viral mRNA 5) mRNA is translated into viral proteins. 6) Viral
proteins and genetic material are moved to the edge of the host cell membrane, packaged
and budded off. Protease (PR) cleaves the proteins into functional forms producing a
mature virion.
6
Once the virion core is released into the host cytoplasm, it is opened and its
contents released. The viral RNA is then reverse transcribed by the viral enzyme reverse
transcriptase to produce viral DNA. The viral DNA is next moved into the nucleus, where
it is inserted into the host genome by the enzyme viral integrase. The viral DNA is
transcribed into viral mRNA and moved out of the nucleus and into the ribosomes. The
viral proteins, including gp160 (a precursor of gp41/gp120 complex), are then created. As
translation progresses, the proteins and genetic material required for a new virion are
collected near the outer membrane of the cell. When all required components are present,
a portion of the cell membrane pinches off and produces an immature virion. The
protease protein then cleaves the p55 into structural and functional proteins thus maturing
the virus.7
As there are a variety of intricate steps occurring in the viral replication cycle,
there are many opportunities for disruption by anti-viral drugs. Currently, there are six
mechanistic classes of drugs available for use in HAART treatment: NRTIs (nucleoside
reverse transcriptase inhibitors), NNRTIs (non-nucleoside reverse transcriptase
inhibitors), INIs (integrase inhibitors), PIs (protease inhibitors), FIs (fusion inhibitors),
and EIs (entry inhibitors). NRTIs work by competitively binding to reverse transcriptase
(RT) and thus blocking the substrate binding site from viral RNA. NNRTIs, on the other
hand, bind to a pocket that, while not a part of the actual substrate binding site, is closely
associated with it. Once the NNRTIs binds to this pocket, a conformational change
occurs to the RT substrate binding site which reduces the catalytic properties of RT. INIs
work by preventing the covalent bonding of the 3’ end of viral DNA to the host cell
7
DNA. Without insertion of the viral DNA, transcription cannot occur and viral mRNA
will not be produced. PIs function by preventing the maturation of HIV virions after they
have budded off from the host cell. They accomplish this by preventing gag (p55) from
being cleaved into structural proteins (p17, p24, p7, p6, p2, and p1) and functional
proteins (protease, RT/RNaseH, and integrase).3 The final two mechanistic classes of
drugs (EIs and FIs) function by preventing HIV from fusing to or transferring the viral
core to the host cell.
Currently, only one EI and one FI are available for HIV therapy. The FI Fuzeon™
(Figure 4) is a peptide sequence of 36 amino acids, derived from gp41. It works by
binding to gp41 once the gp41 has anchored itself into the host cell membrane. This
prevents the formation of the six-helix bundle, and thus, the creation of the fusion pore.
The only EI currently approved by the FDA is called Selzentry™ (Figure 5).4 Viral
inhibition is accomplished through binding of the drug to a hydrophobic pocket in the
transmembrane helices of CCR5 and alteration in the conformation of the loops where
HIV binds.11
8
Figure 4. The fusion inhibitor Fuzeon™. A schematic of gp160 before it is cleaved into
gp41/gp120. Fuzeon™, also known as T-20, is a 36 amino acid sequence derived from
the gp41 region.3
O
N
N
H
N
N
N
F
F
Maraviroc
SelzentryTM
Figure 5. The entry inhibitor Selzentry™. also known generically as Maraviroc.
As there is only one FI and one EI each that are FDA approved, there is ample
room to develop more drugs within these classes. There are a variety of ways in which
the infection of a host cell by HIV can be inhibited. There are still alternate approaches
9
available for disrupting the interactions between gp120 and its coreceptors. Blocking the
V3 loop of gp120 will yield drugs with a different function from Fuzeon™ and
Selzentry™, creating a seventh mechanistic drug class and allow for even greater
flexibility with HAART therapy.
The usefulness in developing a seventh mechanistic drug class can also been seen
when considering side effects of current drugs. NNRTIs, NRTIs, and PIs have been in
use long enough that long-term side effects have cropped up. These effects can include
cardiovascular disease (heart attacks), hepatotoxicity (acute liver disease), renal
dysfunction (kidney tubular dysfunction), lipodystrophy (redistribution of fat within the
body), and distal sensory peripheral neuropathy (numbness of the extremities). The side
effects observed are generally dependant on the class of drug being used. For example,
HAART-related incidence of cardiovascular disease is more commonly seen from the use
of PIs, whereas there is no increased risk associated with NNRTIs.12 Also of concern
when considering new and current drugs is patient compliance with taking the
medications. When patients stop taking medications, this can lead to a resurgence of viral
loads and the emergence of drug resistance.13 This makes it imperative that new drugs be
explored to help fight HIV/AIDS. By developing new drugs, the quality of life for
patients can be improved by tailoring a HAART regimen to an individual who may
already be at risk for another adverse health condition. Thus the newer classes of drugs,
FIs and EIs, provide a starting point for the development of additional drugs for the use in
HAART to which HIV has not yet developed resistance.14
10
Polyanions as inhibitors:
In terms of abundance, no other biological molecule surpasses carbohydrates.
They can be found everywhere, including on proteins and cells.15 In addition to their
prevalence, carbohydrates come in many different shapes, and it is these shapes that help
other biological molecules distinguish one carbohydrate from another. This allows
carbohydrates to serve a myriad of purposes. Numerous carbohydrates have been found
to be involved in, or to disrupt, the process of HIV fusion. Among the polysaccharides
-
O
O
O3SHN
products dextran sulfate
HO
O
O
HO
O
CO 2-
studied are the natural
OSO3-
and heparin sulfate (Figure
6).16 Both dextran sulfate
1)
OSO3-
and heparin sulfate are
polymeric carbohydrates
with varying molecular
weights that can exceed
O
RO
R=H,SO3-
RO
OR
2)
RO
O
500,000 Da.
Ample research has
O
been conducted on these
RO
OR
two polysaccharides. Both
O
have been found to be good
Figure 6. Polyanionic oligosaccaharides. 1) Heparin
Sulfate 2) Dextran Sulfate
inhibitors of HIV. Dextran
11
sulfate (MW 5000 Da) was found to have a 50% inhibitory concentration (IC50) of 9.1
μg/mL for the protein gp120, while heparin sulfate had an IC50 of 7.0 μg/mL. When
dextran sulfate was tested with molecular weights of 5000 Da, 8000 Da, and 500,000 Da.
all oligosaccharides gave similar IC50s, suggesting that it may not be necessary to use the
maximum length of an oligosaccharide to achieve inhibition, but rather a smaller piece
may be sufficient.17 The drawback to using these oligosaccharides is that their more
traditional medical use is as anticoagulants (blood thinners). With heparin sulfate, both
the antiviral and anticoagulant properties decreased with a decrease in molecular weight.
For dextran sulfate (MW 8000 Da), the IC50 for HIV was 10.7 μg/mL while the dosage
required to double the APTT (activated partial thromboplastin time) was 50 μg/mL.17
APTT is a simple test that looks at the time it takes for a sample of blood to clot. The
normal range is between 25 and 35 seconds for clotting to occur. Because antiviral
activity decreases with molecular weight, heparin sulfate would not be able to function in
vivo as a drug. Unfortunately, free carbohydrates do not last long in the body and the
dosage of dextran sulfate required to maintain the IC50 value would be more than 50
μg/mL (vida infra).
The mechanism by which dextran sulfate and heparin sulfate were found to work
is through their binding of the V3 loop within gp120,3 which is a very basic region. Some
mutants can have a V3 loop with a net charge as high as +9, with a sequence like
12
X-B-B-B-X-B-X-X-B-X where X is a
hydropathic residue and B is a basic
residue. This motif is similar to others
that have been found to bind heparin.18
Since gp120 is a trimeric protein, each
monomer has one V3 loop. Figure 7
depicts the orientation of the V3 loop
within the trimer of gp120.
Through binding assays, heparin
sulfate and dextran sulfate were found
to compete with antibodies that bind to
the V3 loop while not interfering with
the binding of CD4 to its receptor on
gp120.19 This indicates that polyanionic
carbohydrates are selective
inhibitors of the V3 region of gp120.
Figure 7. Model of gp120, V3 loop, and gp120
with the V3 loop overlaid.18
Another polysaccharide that has
proven to be a viable inhibitor of HIV in vitro is sulfated colominic acid. Colominic acid
is a homopolymer of N-acetylneuraminic acid (sialic acid). In colominic acid, sialic acid
units are connected by α-2,8 ketosidic linkages (Figure 8). Like dextran sulfate and
heparin sulfate, colominic acid, when sulfated, is able to block HIV entry into host
cells.14 Yang et al. looked at sulfated colominic acid for anti-HIV activity and APTT.14
13
Sullfated colominic acid has been shown to have an affinity for the V3 loop of gp120
yielding an IC50 value as low as 0.07 μg/mL. At a concentration of 10 μg/mL, sulfated
colominic acid was found to have no appreciable effect on APTT. Sulfated colominic
acid at a concentration of 10 μg/mL produced an APTT of 53.8 seconds. This makes
sulfated colominic acid a strong candidate for use as an HIV entry inhibitor. Additionally,
at concentrations of 100 μg/mL, there was no observed toxicity to host cells. A more indepth look at sulfated colominic acid is warranted due to its affect on HIV, minimal
effect on APTT and low cellular toxicity.
OR
OR
OR
CO 2-
CO 2-
OR
RO
O
AcHN
RO
CO 2-
OR
OR
O
O AcHN
RO
O
O AcHN
OR
RO
Figure 8. Colominic acid is a α-2,8 linked polymer of sialic acid. (R= H or SO3-)
Sulfated colominic acid is a polysaccharide like dextran sulfate and heparin
sulfate. These carbohydrates can be easily degraded in both the mouth and stomach when
ingested orally. During digestion, the carbohydrate polymers are broken down into single
carbohydrates, whose binding is known to be weaker than in polymeric form. When the
polysaccharides are digested, they lose their multivalent properties. Multivalency, which
occurs when there are multiple ligands available to bind simultaneously or in tandem to a
receptor and increases the overall binding strength , is what is responsible for their antiHIV abilities. In a study by Hiebert et al.,20 patients were given either a single four gram
14
dose of dextran sulfate (MW 8000 Da) orally once a day for four days, or four one gram
doses per day for up to a year. For the short term dosing, 2.2 μg/mL was the peak
concentration of dextran sulfate achieved in the plasma, and for the long-term study, 2.4
μg/mL was the peak concentration. This indicates that orally administered
polysaccharides would be poorly absorbed into the bloodstream by themselves. With an
effective concentration required for a 50% response (EC50 concentration) of about 10
μg/mL, the required dose of dextran sulfate would be huge (at least five times the dosages
in the test). Since sulfated colominic acid is also a polysaccharide, it is likely that it will
suffer the same problems with degradation as dextran sulfate. It is therefore important to
find a method of delivering the sulfated colominic acid without it being degraded, while
retaining its bioavailability after transport. Dendrimers provide an excellent solution to
this dilemma.
Dendrimers are highly branched polymeric macromolecules. Higher generation
dendrimers can potentially hold tens to hundreds of sialic acid residues. This will allow
sialic acid to mimic the multivalency of sulfated colominic acid. Multivalency is key to
how sulfated colominic acid binds to gp120. A single sialic acid residue will bind to
gp120, but weakly. With multiple sialic acid residues, as in sulfated colominic acid, there
can be numerous binding events occurring concurrently, or in tandem. This increases the
overall binding strength of the entire molecule. As the number of sialic residues
increases, the binding strength will increase as well.
15
Dendrimers for drug delivery:
Dendrimers are spherical, highly branched macromolecular polymers with ends
that can be functionalized (Figure 9). The synthesis of dendrimers was first reported in
1986 by Donald Tomalia, who published the synthesis of a poly(amido(amine)) starburst
polymer.21
Figure 9. A generation 2 PAMAM dendrimer. R can be either H or some other
functional group.22
There are two methods of synthesizing dendrimers, the divergent and the
convergent approach. The divergent synthetic method follows a fairly straightforward
16
route (Figure 10). Starting with the core, the endpoints are branched, thus doubling the
number of endpoints. Each branching step constitutes a new generation of the dendrimer.
The core is generation 0, the first branching is generation 1, and so on. Once the
dendrimer has reached the desired generation, the ends can be functionalized as desired.
Alternatively, convergent synthesis starts from the outside and builds inward. The
terminal functional group is used as the starting point and added to a branch. These
branches are added to another branch, and so on, until the branches are added to the core.
17
Figure 10. Divergent and convergent synthesis strategies for dendrimers.23
18
The divergent and convergent synthetic methods each have their pros and cons.
While the divergent method is the most straightforward synthetically, it has numerous
drawbacks. As the generation increases, so do the number of reactive points. This means
that each subsequent generation will have that many more reactions required per
generational increase. This can lend itself to long reaction times and can also lead to
incomplete reactions. In the case of dendrimers, a complete reaction actually requires
several reactions to occur on the same molecule. For example, on the PAMAM
dendrimer, going from generation zero to generation one requires four separate reactions
to happen. If less than four reactions occur, then there could be up to four different
products from the overall reaction. This can include branching at only three points, not
four, or even branching at only two, one, or even none of the possible branch points.
Additional synthetic problems arise as the chemical properties and masses of the various
products become more similar as the generations increase, which causes difficulty in
separating the incomplete from the complete dendrimers.
On the other hand, even though the convergent synthesis is not as straightforward,
it does eliminate long reaction times and difficult purifications. Starting from the outside
and working in, the number of reactive points (regardless of which generation is being
built), are minimized. Using this method, the size of incompletely reacted pieces varies
greatly from that of completely reacted pieces, making separation easier. Unfortunately,
as the dendrimer pieces become sufficiently large they can suffer from steric issues, thus
reducing the ability to branch or functionalize their reactive points.24 Overall, a
19
convergent synthesis method should allow more consistency between dendrimers which
will be necessary in medical applications.
Dendrimers have a variety of applications in medicine. They have been used in
gene therapy, drug delivery, as magnetic resonance imaging (MRI) contrast reagents, in
prion research, burn treatment, and in electron paramagnetic resonance (EPR) imaging.25
There are two properties that some dendrimers, like PAMAM (poly(amido amine)),
possess that make them important for medical use. The first is their low toxicity in vivo
and the second is the ability to increase the affinities of bound carbohydrates to a ligand
through multivalency. The low toxicity of PAMAM was demonstrated by a study using
rats that were injected with PAMAM generations 3, 5, and 7.26 Over periods of seven or
30 days, the animals’ body weights were measured. There was no statistical difference in
the body weights among test subjects, and only three of the 20 rats died. Of the three that
died, two died from causes unrelated to the experiment. In regards to the multivalent
effect, it has been shown that the binding of a single saccharide to a ligand may be weak,
but a polysaccharide can have a stronger association with its target ligand. Table 1 shows
an example of the affect multivalency has in binding strengths in terms of the
concentrations required to block influenza binding to an erythrocyte.27 Alone, 4-O-acetylNeuAc binds to influenza surface hemagglutinin (IC50 of 56,000 µM) (1:1), but when
there are multiple 4-O-acetyl-NeuAc residues available for binding, as in Equine α2M,
the potency of binding, and thus inhibition, is driven up by many orders of magnitude
(IC50 of 0.00052 µM). Also shown are the IC50s for various other macroglobulins and
20
NeuAc (sialic acid). As can be clearly seen, the macroglobulins inhibit at least one order
of magnitude better than NeuAc or 4-O-acetyl-NeuAc alone due to multivalency.
Table 1: Monomer and oligomer sialic acid and their inhibition of A/Memphis/102/72
adsorption to erthrocytes.27
Inhibitor
α-Methyl-NeuAc
Human α2M,
oligosaccharides
Equine α2M,
oligosaccharides
NeuAc (sialic acid)
4-O-Acetyl-NeuAc
Human α2M
Concentration for
50% inhibitiona
Relative inhibitory
potencyb
μM
2,000
1.0
1,100
1.8
2,300
43,000
56,000
0.9
0.047
0.036
0.26
7,700
Equine α2M
0.00052
3,800,000
aEach glycoprotein, oligosaccharide, or free sialic acid was examined for its
ability to inhibit A/Memphis/102/72 adsorption to resialyated erythrocytes
modifoed to contain 18 nmol/mL packed cells NAc-Neuraminic Acid (NeuAc)
in the NeuAcα2,6Gal linkage.
bInhibitory potency is expressed relative to α-Methyl-NeuAc.
Research Goals:
Carbohydrates, like heparin sulfate, dextran sulfate, and sulfated colominic acid
are known to block the V3 loop region of gp120. Unfortunately, heparin sulfate and
dextran sulfate can cause thinning of the blood and an increased time for coagulation.
Fortunately, sulfated colominic acid has no appreciable effect on coagulation at
theraputic concentrations. An additional drawback to the use of carbohydrates as drugs is
that the body readily and quickly metabolizes them. This leads to the requirement of large
21
doses to maintain even low concentrations of the carbohydrate within the body. To utilize
carbohydrates more effectively as drugs, a system will be necessary to transport them to
the desired site of action, while minimizing degradation. Dendrimers have been used in
numerous biological applications, and are quite capable of fulfilling this function.25
Glycodendrimers can achieve strong binding due to the multivalent effect, which increase
with increasing dendritic generations. What sets these glycodendrimers apart from the
other drugs that inhibit HIV entering into a host cell is that they target the V3 loop of
gp120. All of the other drugs currently on the market function by targeting the enzymes
required for viral replication and are only effective once a cell has been infected.
In this work, a series of poly(amidoamine) (PAMAM) dendrimers functionalized
with sialic acid were built. The purpose for synthesizing the different PAMAM
dendrimers was two-fold. The first goal was to explore the ease of synthesis using both
convergent and divergent synthesis methods. The PAMAM generation 0, 1, and 2
dendrimers represented divergent syntheses as the full dendrimers had already been
synthesized and the sialic acid residues could be simply appended to the surface.
PAMAM generations –0.5, 0.5, and 1.5 represented a more convergent-like synthesis
since sialic acid was first reacted with a diaminotriethylene glycol linker, then the linker
was attached to the PAMAM dendrimers. Thus the synthesis of these molecules followed
a quasi-convergent path, going from the outside inward.
The second goal for this project was to evaluate the effect of the dendrimer radii
on their ability to bind to gp120. With the diaminotriethylene glycol linker inserted
between the sugar and the PAMAM, the radius of the glycodendrimer was increased.
22
This produced three pairs of glycodendrimers with the same number of sialic acid
residues, but differing molecular radii. Thus, it could be determined how the molecular
radius affected the binding strength for glycodendrimers terminating in 4, 8, or 16 sialic
acid residues.
For this project, the synthesis of a series of six sialic acid-PAMAM
glycodendrimers (Figures 11-16) were attempted, but only four were successfully
synthesized. Three of the glycodendrimers were made from PAMAM generations 0, 1,
and 2. These full generations of PAMAM contain amine-terminated branches. Sialic acid
was easily coupled to the amine via an amide formation reaction using the C-1 carboxylic
acid of sialic acid. The fourth glycodendrimer was part of the group of three dendrimers
whose syntheses were attempted via the convergent approach, i.e. PAMAM generations
-0.5, 0.5, and 1.5. Unlike the full generations (0, 1, and 2), the branches of the half
generations were all terminated in carboxylic acid residues. Half generations exist when
the terminal 1,2-diaminoethane from all branches, is not present, which leaves a
carboxylic acid behind. To couple sialic acid to the PAMAM half generations, a
diaminotriethylene glycol linker was employed, along with amide coupling chemistry.
Unfortunately, the only glycodendrimer that was successfully synthesized through this
approach was the glycodendrimer based on PAMAM generation –0.5 (1).
It should be noted here that the glycodendrimers were named as follows: the
number of end points (either tetramer, octamer, or 16-mer), followed by glyco. If
diaminotriethylene glycol was present between PAMAM and sialic acid, the abbreviation
23
DATEG (diaminotriethylene glycol) appears. The end of the name always terminates in
PAMAM dendrimer.
O
HO
OH OH
OH
O
AcHN
N
H
HO
HO
OH OH
OH
HO
H
N
O
N
H
O
O
AcHN
O
O
O
N
H
H
N
O
O
HN
O
OH
OH
NHAc
O
O
O
OH OH
OH
H
N
O
NHAc
O
O
1
OH
H
N
N
N
O
O
OH OH
OH
OH
O
Figure 11. Tetrameric glyco-DATEG-PAMAM dendrimer (1).
O
OH
OH
OH
NHAc
OH
HO
O
O
HN
H
N
O
AcHN
OH
O
HO
OH
OH
OH
NH
O
HN
N
N
2
NH
O
HN
OH
OH
NHAc
O
OH
HO
OH
O
NH
O
AcHN
O
N
H
OH
HO
O
Figure 12. Tetrameric glycoPAMAM dendrimer (2).
OH
OH
OH
24
HO
OH
OH
OH
O
NHAc
O
OH
OH
AcHN
O
HO
HN
OH
O
N
H
O
O
O
NH
O
O
NH
O
O
O
OH
N
H
H
N
O
N
H
N
H
N
O
O
AcHN
OH
O
OH
OH
HO
OH
OH
NHAc
O
O
N
O
OH
O
OH
OH
OH
O
HO
O
NH
HN
N
N
NH
HN
OH
OH
HO
O
3
O
OH
OH
O
H
N
O
O
N
H
AcHN
H
N
O
N
N
H
N
O
OH
OH
O
HO
NHAc
O
O
OH
OH
O
O
O
HN
O
O
HN
O
O
O
H
N
O
HO
NH
OH
O
NHAc
HO
HO
HO
HO
O
O
HO
HO
HO
AcHN
OH
Figure 13. Octameric glyco-DATEG-PAMAM dendrimer (3).
OH
NHAc
HO
OH
OH
OH
HO
OH
O
O
OH
AcHN
O
OH
HO
HN
H
N
O
NH
O
NH
O
O
OH
HO
N
H
N
O
HN
OH
O
NH
O
OH
O
OH
AcHN
N
HO
OH
NHAc
OH
OH
N
H
HN
O
O
OH
HN
O
N
O
N
4
NH
NHAc
O
OH
NH
O
HN
O
O
N
H
N
OH
OH
OH
AcHN
HO
OH
N
OH
HO
O
O
NH
H
N
OH
OH
O
O
HN
O
HN
NH
O
N
H
OH
HO
O
O
O
NHAc
HO
HO
HO
HO
HO
OH
AcHN
HO
Figure 14. Octameric glycoPAMAM dendrimer (4).
OH
25
OH
OH
N H Ac O H
N H Ac O H
OH
OH
HO
HO
O
O
OH
OH
O
OH
N H Ac
O
OH
NH
OH OH
Ac H N
O
OH
O
OH
O
NH
NH
O
O
OH
O
O
HN
Ac H N
OH
O
OH
O
O
HN
O
HO
O
O
O
OH
OH
OH
O
O
HO
OH
OH
NH
HN
O
NH
O
N
O
N H Ac
OH OH
N
H
O
OH
OH
NH
N
O
O
NH
O
O
HN
O
O
HN
O
HN
O
O
OH
O
OH
OH
N
O
OH
O
O
N
O
O
H
N
O
Ac H N
O
N
N
H
HO
OH
N
H
O
N
H
NH
N
NH
O
N H Ac
O
N
H
HN
O
O
HO
HO
HO
N
OH
OH
O
H
N
O
Ac H N
HO
N
OH
OH
O
O
HN
5
HN
H
N
N
HO
NH
H
N
O
OH
H
N
N
O
O
N
H
O
O
N
O
O
O
NH
O
O
O
NH
O
HN
O
O
N
HN
HO
HO
Ac H N
O
O
N
HN
O
H
N
O
HO HO
NH
O
HO
O
OH
O
NH
O
HO
N H Ac
NH
O
O
HO
O
HN
HO
O
O
HN
O
HO
HO
O
HO
O
OH
N H Ac
O
HO HO
HN
HO
HN
OH
O
O
Ac H N
HO
HO
O
O
OH
HO
HO
Ac H N
HO
HO
O
O
O
HO
OH
H O Ac H N
HO
HO
Figure 15. 16-mer glyco-DATEG-PAMAM dendrimer (5).
HO
HO
O
O
NH
N H Ac
O
HO
O
N
HO
HO
26
OH
OH
NHAc
HO
OH
NHAc
OH
OH
HO
OH
OH
O
OH
NHAc
O
HO
OH
OH
OH
OH
OH
O
O
OH
AcHN
O
HO
O
HN
NH
OH
O
OH
NH
OH
NH
OH
O
NH
HN
OH
HN
O
AcHN
HN
O
O
HO
OH
O
O
NHAc
OH
N
N
OH
O
OH
O
OH
O
HN
NH
NH
HN
O
O
HN
O
O
OH
H
N
O
N
OH
H
N
O
O
N
H
N
N
H
O
N
OH
OH
NH
O
O
O
OH
OH
N
H
N
H
N
HO
AcHN
HO
NHAc
O
NH
HN
HO
HO
O
O
HO
N
N
OH
O
O
OH
OH
OH
NHAc
OH
HN
6
H
N
N
H
N
O
N
H
AcHN
O
HO
H
N
OH
OH
OH
OH
O
O
O
NH
O
O
NH
HN
HO
O
N
H
O
O
HN
N
N
N
O
H
N
NH
O
HN
NH
O
HO
O
HO
O
HO
N
N
HO
AcHN
O
O
HO
OH
O
O
NH
NH
NHAc
O
HO
NH
HN
O
HO
HN
HO
HN
HO
HO
HN
NH
O
O
OH
NHAC
O
O
HO
O
HO
HO
HO
HO
OH
O
O
HO
HO
HO
AcHN
OH
HO
OH
HO
HO
HO
AcHN
AcHN
HO
HO
Figure 16. 16-mer glycoPAMAM dendrimer (6).
It is important to build these molecules because they have the potential to create
an additional inhibitory mechanism for HAART treatment. These dendrimers are
intended to be inhibitors of the HIV entry process through blocking the interactions
between the host cell coreceptors and the V3 loop of gp120. There are currently only two
drugs with FDA approval that are designed to inhibit viral infection at the earliest stage,
Selzentry™ and Fuzeon™.4 Selzentry™ works by binding to CCR5 and altering the
27
binding site for gp120,11 while Fuzeon™ functions by blocking the ability of gp41 to fold
onto itself and bringing the virion into contact with the host cell. The novelty of
Selzentry™ and Fuzeon™ leave room for the further development of new drugs.
Glycodendrimers therefore have the potential to be used in tandem with all of the other
drugs currently available since their mode of operation is unique. This will increase their
usefulness within HAART therapy and open the door for future research on dendrimers
as potential HIV drugs.
28
Chapter 2
RESULTS & DISCUSSION
The purpose of this work was to examine the ease of synthesizing
glycodendrimers of various generations using both convergent and divergent synthetic
strategies. In addition, the synthesis of these glycodendrimers will allow the effect that
both generation and dendritic radii have on the binding to gp120 to be determined. An
important part of the synthetic strategy revolves around a nearly complete lack of
protecting group chemistry. This will have a significant impact on the overall project, the
most obvious effect being the reduction in the number of steps in the synthesis and a
general increase in yield. Because protecting group strategies require the introduction and
removal of a protecting group, by eliminating these steps not only is time saved, but also
numerous purifications are avoided and the yield of the synthesis is increased. What may
be less obvious is how the chemical properties of a carbohydrate change once it has been
protected. In carbohydrate chemistry, it is common practice to use acetates and acetals to
protect the free hydroxyls. This has the effect of masking numerous potential reaction
points and changes the solubility by allowing a carbohydrate to dissolve in a less polar
organic solvent. With the sole exception of the preparation of compound 8, no protecting
group chemistry was used in this study. To achieve this, a synthetic route was devised
where the reactions were conducted in a polar solvent, and the reagents would not target
the unprotected hydroxyls.
29
To test the convergent strategy for the synthesis of dendrimers, sialic acid was
coupled to a hydrophilic linker, which was then coupled to PAMAM. Amide formation
was used to connect the sialic acid to the linker, and subsequently the sugar-linker
complex to PAMAM. Since a full generation of PAMAM is amine terminated and the
linker used was also amine terminated, the half generations of PAMAM ( –0.5, 0.5, and
1.5) were employed in these cases. These three PAMAM dendrimers are all carboxyl
terminated, thus allowing amide formation as a method to couple the sialic acid-linker
complex directly to the PAMAM core. For the divergent strategy of dendrimer formation,
the carboxy group of sialic acid was coupled directly to the terminal amine of PAMAM
generations 0, 1 and 2 via an amide formation reaction.
Synthesis:
Generations –0.5, 0.5, and 1.5 were coupled to the sugar-linker complex through a
convergent synthetic method. To achieve this, the linker (diaminotriethylene glycol, 7)
was singly protected with di-tert-butyldicarbonate ((Boc)2O). Since both ends of the
linker are chemically equivalent, this reaction affords three products: unreacted starting
material (7), the mono-protected linker (8) and the di-protected linker (9). Monoprotected linker (8) was isolated by normal phase flash chromatography using 1:1
chloroform and methanol, giving the desired product (8) in 51% yield (Scheme 1).
30
H2N
O
O
NH 2
7
O
(Boc)2O, TEA
O
H2N
NH 2
O
H2N
O
O
MeOH, 35oC, 24 hrs., 51%
N
H
8
O
7
O
H
N
O
O
O
O
N
H
O
9
Scheme 1. Monoprotection of diaminotriethylene glycol.
The mono protected linker (8) was next coupled to sialic acid (10) using
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), and
N,N-diisopropylethyl amine (Hunig’s Base), in N,N-dimethylformamide (DMF) under
nitrogen gas. The reaction was heated to 35oC and stirred overnight. The solvents and
volatile compounds were then evaporated under reduced pressure. The Boc protecting
group was next removed by dissolving the crude material in 1:2 dichloromethane and
trifluroacetic acid and stirring for two hours (Scheme 2).
O
H2N
O
O
N
H
8
OH
HO
OH
OH
+
O
1)BOP, Hunig's Base, DMF, N2, 35oC, 24 hrs. HO
2)TFA, CH2Cl2, 2 hrs, 88% (2 steps)
O
AcHN
CO 2-
OH OH
OH
H
N
O
AcHN
HO
O
O
11
HO
10
Scheme 2. Amide coupling of sialic acid to linker followed by deprotection.
O
NH 2
31
Compound 11 was isolated by reverse phase high pressure liquid chromatography
(RP-HPLC), using a linear gradient between water with 0.1% trifluoroacetic acid (TFA)
and acetonitrile (ACN) with 0.1% TFA. Next, the linker (11) was attached to PAMAM
via an amide coupling using BOP and Hunig’s base in DMF at 35oC and under nitrogen
for 24 hours, 7 days, or 14 days, producing glycodendrimers 1 and 3 (Scheme 3).28
Glycodendrimer 5, unfortunately, was not isolated.
PAMAM
(CO2 H)X
X= 4, 8, 16
OH
O
BOP, Hunig's Base
OH
OH
HO
O
Linker
AcHN
HO
NH2
OH
DMF, N2, 35oC
24 hrs., 7 days, 14 days
O
OH
OH
HO
O
Linker
AcHN
HO
PAMAM
O
X
11
NH
1, 3, 5
X= 4, 8, 16
Scheme 3. Amide coupling of sugar-linker PAMAM generations -0.5, 0.5, and 1.5
To make compounds 2, 4, and 6, sialic acid (10) was mixed with PAMAM and
was allowed to react with BOP and Hunig’s base while dissolved in DMF and heated to
35oC under nitrogen gas for 24 hours, 7 days or 14 days. This produced glycodendrimers
2, 4, and 6 via an amide formation reaction (Scheme 4). This synthesis route followed a
divergent methodology.
OH
OH
OH
HO
+
O
AcHN
HO
10
CO2 -
X(H 2 N)
PAMAM
X= 4, 8, 16
OH
BOP, Hunig's Base
DMF, N2, 35oC
24 hrs., 7 days, 14 days
OH
OH
HO
H
N
O
AcHN
X
X= 4, 8, 16
HO
2, 4, 6
Scheme 4. Amide coupling of sialic acid to PAMAM generations 0, 1, and 2
O
PAMAM
32
Glycodendrimer Purification:
Due to their size and polarity of the reagents and products of the glycodendrimer
reactions, none of the reactions could be monitored by thin layer chromatography. As a
result, all glycodendrimer reactions were allowed to react for long periods of time.
Compounds 1 and 2 were allowed to react for 24 hours since they were not very sterically
hindered. Compounds 3, 4, 5, and 6 were allowed to react for a full week per generation
of core used. As was shown in the cases of compounds 1, 2, 4, and 6, these times proved
to be sufficient.
Tetrameric glycol-DATEG-PAMAM dendrimer (1): After 24 hours, all volatile
compounds and solvents were evaporated under reduced pressure. The crude material
was then dialyzed with 500 molecular weight cutoff (MWCO) tubing in deionized (D.I.)
water. The pore size of this tubing was such that all starting materials, including
PAMAM and sugar-linker could easily pass through, but the glycodendrimers could not.
The water was changed every hour for four hours, then allowed to stir overnight at 4oC.
The crude material was removed from the tubing and lyophilized. The dry material was
then purified with a C18 column using RP-HPLC. Using a linear gradient between water
with 0.1% trifluoroacetic acid (TFA) and acetonitrile (ACN) with 0.1% TFA, pure
compound 1 was collected with a yield of 6.4%. Matrix-assisted laser desorption
ionization (MALDI) mass spectrometry (MS) confirmed the presence of compound 1. 1H
NMR data for compound 1 was not easy to interpret. While most of the individual peaks
from sialic acid (Figure 17) and from PAMAM could be clearly seen, there was an
33
overlap of peaks from sialic acid and the linker. The defining feature in the 1H NMR that
identifies this as being compound 1 and not just unreacted sugar linker (11) and
PAMAM, is a shift in one of the linker peaks. In the 1H NMR of sugar linker (11)
(Figure 18), there is a peak at δ 3.19. This peak corresponds to the CH2 next to the
primary amine. When sugar linker (11) is coupled to PAMAM, Hp (See Appendix A for
peak assignments) shifts as the amine becomes an amide. In the 1H NMR of compound 1,
the peak that was at δ 3.19 (Figure 18) can now be seen at δ 3.43 (Figure 19).
SpinWorks 3:
1
.8
7
9
PPM
0
.9
1
1
0
.9
6
0
4.0
0
.9
3
5
1
.0
2
2
3.8
0
.9
5
8
3.6
file: C:\Research\NMR\Sialic Acid\1\fid
1
.0
0
0
3.4
expt: <zg30>
3.2
3.0
2.8
2.6
2.4
freq. of 0 ppm: 500.129953 MHz
transmitter freq.: 500.133089 MHz
processed size: 32768 complex points
time domain size: 65536 points
LB: 0.300
width: 10330.58 Hz = 20.6557 ppm = 0.157632 Hz/pt
Hz/cm: 52.012
number of scans: 64
GF: 0.0000
ppm/cm: 0.10400
2
.6
1
1
2.2
0
.9
5
6
2.0
1.8
34
Figure 17. 1H NMR of sialic acid (10).
Figure 18. 1H NMR of sugar-linker complex (11).
35
Figure 19. 1H NMR of Tetrameric glycol-DATEG-PAMAM dendrimer (1)
Tetrameric glycoPAMAM dendrimer (2): After 24 hours, the solvents and volatile
compounds were evaporated off under reduced pressure. 500 MWCO dialysis tubing was
filled with the crude product and immersed in DI water. The water was stirred and
changed once an hour for four hours, then left to stir overnight at 4oC. The crude material
from the tubing was then lyophilized and purified with a C18 column using RP-HPLC. A
linear gradient was run using water with 0.1% TFA and ACN with 0.1% TFA providing
compound 2 in an 11.4% yield. MALDI MS confirmed compound 2. The 1H NMR of
compound 2 (Figure 20) clearly showed all sialic acid-derived peaks (Figure 17). The
36
peaks from the PAMAM could be easily integrated to give a total of 9H; 8H for the 4
CH2s of the dendrimers, and 1H for ¼ of the C2H4 center of the dendrimer (All 1H NMR
were integrated to show ¼ of the protons expected).
SpinWorks 3:
1
.9
9
5
PPM
0
.9
9
1
1
.1
7
4
4.0
1
.0
5
6
1
.1
1
9
3.8
1
.1
6
6
3.6
file: ...esearch\NMR\RC_I_157_FinProd\1\fid
6
.9
1
2
3.4
2
.1
5
0
3.2
expt: <zg30>
3.0
1
.0
0
0
2.8
2.6
2.4
3
.1
0
6
2.2
1
.0
1
0
2.0
1.8
freq. of 0 ppm: 500.129953 MHz
transmitter freq.: 500.133089 MHz
processed size: 32768 complex points
time domain size: 65536 points
LB: 0.300
width: 10330.58 Hz = 20.6557 ppm = 0.157632 Hz/pt
Hz/cm: 52.046
GF: 0.0000
ppm/cm: 0.10406
number of scans: 64
Figure 20. 1H NMR of Tetrameric glycoPAMAM dendrimer (2)
Octameric glyco-DATEG-PAMAM dendrimer (3): After seven days, all solvents
and volatile compounds were evaporated under reduced pressure. The crude material was
37
dialyzed against pure water in 500 MWCO tubing. The tubing was stirred for four hours,
with water changes every hour. The tubing was then allowed to stir overnight at 4oC. The
material was collected and lyophilized. The dry product was further purified with a C18
column using RP-HPLC, but the product collected appeared to still be impure. Because
the fully and partially substituted glycodendrimers are sufficiently similar chemically and
structurally, they could not be easily purified with RP-HPLC alone. The product from
RP-HPLC was then further purified with size exclusion chromatography (SEC) using fast
paced liquid chromatography (FPLC), using a buffer containing 0.03 M ammonium
bicarbonate (NH4HCO3) as the mobile phase. The 1H NMR of what should have been
compound 3 appeared to be very similar to the 1H NMR of compound 1, which isn’t
surprising since these glycodendrimers are polymers. However, the 1H NMR showed that
the major product present was not the completely substituted glycodendrimer (Figure
21). Where the two differ is in a single peak at δ 3.29, which is not present in the 1H
NMR of any of the other glycodendrimers.
38
Figure 21. 1H NMR of Octameric glyco-DATEG-PAMAM dendrimer (3)
For compound 3, the calculated molecular weight is 4462 Da., however, MALDI
MS (Figure 22) showed small peaks for (M+H) m/z=4467, (M-H2O) m/z=4448, and (M2H2O) m/z=4428, along with impurity peaks (M-cmpd 11-H2O) m/z=4027 and (M-2
cmpd 11) m/z=3619. Compound 3 has more than 180 carbon atoms, so it is likely there
are at least two 13C atoms adding weight beyond the calculated. The impurity seen in
MALDI MS corresponds to octameric glyco-DATEG-PAMAM dendrimer (3) that is
missing either one or two sugar-linker complexes (11) and water molecules. While
39
MALDI MS shows evidence that 3 was synthesized, it was not formed in a large enough
quantity to get confirmatory 1H or 13C data.
Figure 22. MALDI MS of Octameric glyco-DATEG-PAMAM dendrimer (3). S= sugar
linker complex (11)
Octameric glycoPAMAM dendrimer (4): After seven days, all solvents and
volatile compounds were evaporated under reduced pressure. The crude material was
dialyzed in 500 MWCO dialysis tubing and stirred for four hours with water changes
every hour. The tubing was then allowed to stir overnight at 4oC. The dialyzed crude
product was next further purified on a C18 column with RP-HPLC and a linear gradient
40
of water with 0.1% TFA and ACN with 0.1% TFA. The collected product was impure,
and further purified with SEC-FPLC using 0.03 M NH4HCO3.
The 1H NMR of the product collected from the SEC-FPLC looked very different
from the initial 1H NMRs collected for compounds 1 and 2 and the partially pure
compound 4 from RP-HPLC. The difference included the shifting of three major peaks
that were previously obscured in other spectra. The unusual difference in the 1H NMR
data proved to be surprising in that glycodendrimers are all polymeric in nature and as
their generation increases, the additional size comes simply from repeating units.
Therefore, there is no reason to expect the 1H NMRs for compounds 1 and 2 to be
significantly different from the 1H NMR of compound 4. Since the 1H NMR appeared
clean based on integration, the sample was analyzed via MALDI MS, which showed the
product to be compound 4 with a 14% yield.
The question that remained was why there was such a difference between spectra
when there should not have been. The answer lies in the methods of purification used.
Compounds 1 and 2 had only been purified by RP-HPLC, which uses acidified solvents.
Compound 4 had been purified by RP-HPLC as well, but was then subjected to SECFPLC, using basic solvents. To test the hypothesis that the pH was the cause for the
dramatic difference in 1H NMR data, a crude sample of compound 4 was taken directly
from the HPLC and analyzed by 1H NMR. The sample was then lyophilized, re-dissolved
in 0.3 M NH4HCO3 (ten times the FPLC concentration) and stirred for an hour. After
stirring, the sample was lyophilized, dissolved in DI water and lyophilized again to
remove excess NH4HCO3. Figure 23 shows an overlay of the 1H NMR from the acidified
41
glycodendrimer (bottom) and the base treated glycodendrimer (top). The top 1H NMR
illustrates what was common for compounds 1, 4, and 6. Compound 1 was purified by
SEC-FPLC at a later point in time, but initial purification was done solely through RPHPLC. If compound 2 had been purified by SEC-FPLC, it too would have looked like the
top spectra. When the pH was made more acidic, the proton peaks that correspond to the
PAMAM dendrimers were shifted downfield. This shifting is consistent with what is
known about the effect that protonated amines have on their neighboring protons. The
protonation of the amine can easily cause a 0.8 to 1.0 ppm downfield shift in the CH2
signal.29
42
Figure 23. An overlay of octameric glycoPAMAM dendrimer (4). Acidic (bottom) and
basic (top) show a pH-dependant shifting of dendrimer peaks.
16-mer glyco-DATEG-PAMAM dendrimer (5): After 14 days, all solvents and
volatile compounds were evaporated under reduced pressure. The crude material was
placed into 500 MWCO dialysis tubing. The tubing was suspended in DI water and
stirred for four hours, with water changes every hour. The tubing was then allowed to stir
overnight at 4oC. The crude material from the dialysis tubing was collected and
lyophilized. The dry material was then purified with a C18 column using RP-HPLC. The
still crude product from RP-HPLC was further purified with SEC-FPLC. Even with
multiple passes through both RP-HPLC and SEC-FPLC, compound 5 was not isolated.
43
Since no 1H NMR data was collected that indicated pure or even near-pure compound 5
was present, no samples were sent in for MALDI-MS analysis.
16-mer glycoPAMAM dendrimer (6): After reacting for 14 days, all solvents and
volatile compounds were evaporated under reduced pressure. The crude material was
loaded into 500 MWCO dialysis tubing and suspended in DI water, and stirred for four
hours with water changes every hour. The tubing was then allowed to stir overnight at
4oC. The dialyzed crude was next passed through a C18 column with RP-HPLC using a
linear gradient of water with 0.1% TFA and ACN with 0.1% TFA. The collected product
was impure so it was further purified with SEC-FPLC using 0.03 M NH4HCO3,
providing compound 6 in a 28% yield. MALDI MS confirmed the presence of compound
6. 1H NMR of compound 6 displayed peaks that were nearly identical to compound 4. As
with compounds 2 and 4, there was no overlap between peaks from sialic acid and
PAMAM-based protons.
One difficulty that arose and is most evident in compound 6, was the broadening
of the peaks associated with the PAMAM dendrimers in the 1H NMR. In the 1H NMRs of
sialic acid-containing compounds, the visible peaks of sialic acid were always well
defined. The only peaks that were difficult to identify were those with more complex
splitting patterns. On the other hand, it was seen that as the dendrimers grew larger, the
peaks associated with the core protons became more broadened. As a result, it was
difficult to integrate all the peaks. In addition, the peaks associated with PAMAM
integrated to more protons than what should have been seen. Figure 24 shows a
comparison of 1H NMR peaks from compounds 1, 4, and 6 which correspond to
44
glycodendrimer generations -0.5, 0, and 2. On the left side are signals produced by sialic
acid, while on the right side are signals produced by protons from the PAMAM core. As
can be easily seen, the signals due to sialic acid are always sharp and well defined,
whereas the signals displayed from the PAMAM core became more broad and intense as
the glycodendrimer generation increased.
Figure 24. Relaxation time effect on 1H NMR. Side by side comparison of the peaks
from sialic acid (left) and PAMAM (right) for three generations of glycodendrimers. As
the generation of the glycodendrimer increased so do the relaxation times associated with
the protons buried deep within the glycodendrimer.
The cause of the broadening is an increase in the relaxation times of the nuclei
buried within the PAMAM core. When these times increase, the peaks become broader
45
and less defined. As a molecule tumbles in a solvent, it can transfer energy to
neighboring nuclei or to the solvent, thus quickly relaxing. In the rigid center of a
glycodendrimer, there is less tumbling, and so those protons take longer to relax. There
are actually two types of nuclear relaxation times T1 and T2. While a molecule is sitting
within the magnetic field, a radio frequency pulse is applied to the nuclei. This causes
two things to happen (Figure 25). First, some of the nuclei that are in the lower energy
spin state (+½) are excited into the higher energy spin state (-½). When the radio pulse is
stopped, those energized nuclei can return to their original spin state. The time it takes for
this to occur is known as T1. The second type of relaxation time occurs when the axis of
spin for the nuclei line up with the magnetic field. Once they are allowed to relax, they
will return to their original orientation within the magnetic field. The time a nucleus takes
to return the axis of rotation to the original direction is known as T2. Both T1 and T2
affect the appearance of the NMR signals. A change in T1 will lead to a change in the
intensity of the associated peak, which also affects integration of the peak. When T2
increases, the associated peaks becomes more broad and less defined. Since the sialic
acid-based protons sit on the outermost perimeter, they will always display sharp peaks
since they are free to tumble about and interact with other nuclei and the solvent. The
internal protons on the other hand, cannot, causing an increase in relaxation times.30
46
Figure 25. Representations of T1 and T2 in NMR relaxation. T1 shows the energy
difference between +½ and -½ spin states. T2 shows the natural (a) and the pulse induced
(b) orientations for nuclei.
47
Chapter 3
CONCLUSIONS & FUTURE WORK
The primary focus of this research was to develop an efficient synthetic strategy
to evaluate convergent and divergent synthetic methods to determine which method was
simpler and produced higher yields in the synthesis of sialic aicd-coupled
glycodendrimers. In evaluating the convergent versus divergent methodology, it was a
surprise to find that the divergent synthetic method produced the desired products in
higher yields. The addition of the diaminetriethylene glycol linker was expected to aid the
reactions by reducing steric hinderance. However, what likely happened was that the
linker was too floppy such that the primary amine was unable to come into contact and
react effectively with the carboxylic acid on PAMAM. In the case of the convergent
synthetic method, two of the three attempted glycodendrimers were not isolated in large
enough quantities to fully characterize. While compound 3 was identified via MALDI
MS, the quantities present were too small for clear 1H and 13C NMR data to be obtained.
In the case of compound 5, the 1H NMRs were not clear enough to warrant MALDI MS
analysis. Compound 1, while it was isolated and characterized, was only present in a
6.4% yield. The reverse of this was the divergent synthetic method. Compounds 2, 4, and
6 were all isolated and characterized via 1H NMR, 13C NMR and MALDI MS in yields of
11.4%, 14%, and 28%, respectively. These results clearly indicate that for our system,
divergent synthesis was the superior method.
In addition to testing the divergent and convergent synthetic methods, the
avoidance of protecting group chemistry presented a hurdle of its own. The final
48
glycodendrimers were synthesized without using protecting group chemistry. Typically in
carbohydrate chemistry, protecting groups are used to mask various functional groups
and to change the solubility of the protected carbohydrate. In this work, since the
carbohydrates were never protected, the solvents used needed to be capable of dissolving
polar molecules and the reagents needed to be specific for amide chemistry. The reagent
BOP was chosen due to its prevalence in amino acid synthesis. BOP was effective in
selecting the carboxylic acid over all other functional groups. This allowed for the use of
amide coupling chemistry in the absence of protecting groups. The end result of this was
a reduction in total number of steps required for the synthesis, and an increase in yield
due to the loss of additional steps.
In doing this work, there were two NMR-related observations made that will
impact future work in this area. The first observation came in the form of protonated
amines within the PAMAM core. Early purifications on the two smallest
glycodendrimers, compounds 1 and 2, were accomplished on a C18 column using RPHPLC. For both of these compounds, the early 1H NMR spectra were taken while they
were in their protonated form. On the other hand, compounds 4 and 6 required SECFPLC for further purification. This resulted in both of these glycodendrimers being
exposed to basic ammonium bicarbonate for three or more days. This led to all of the
internal amines being converted back to their neutral form, allowing the peaks from
neighboring CH2s to shift back to their original positions between δ 2.4 and 3.0. This had
a significant impact on the appearance of the 1H NMR of these glycodendrimers.
Ultimately, what occurred was that in an area where three peaks would normally be
49
expected (in the basic spectra), only one was observed (in the acidic spectra). In general,
this would only be a small problem since integration on a 1H NMR spectrum can be used
to indicate how many protons make up a peak. Unfortunately, this involves a second
observation, peak broadening in higher generation dendrimers, which can have a
significant impact for future researchers in this area.
As the generation of the glycodendrimer increased there was broadening that was
associated with the peaks caused by the dendrimers. Coincidentally, the broadened peaks
include the peaks that shift in acidic solutions. The reason the peaks are broadened is due
to the relaxation time of the nuclei within the dendrimer core. As a dendrimer grows in
size, the internal bonds become more rigid and restricted in their movement. This
restricted movement prevents tumbling and interaction with the solvent, which causes an
increase in the relaxation times. When both T1 and T2 increase, as is seen in the
increasing generation of dendrimers, the peaks associated with them become less well
defined and their intensities change. This results in a loss of the ability to identify peaks
by J-values, splitting patterns and integration. When peaks that should be well-defined or
isolated from other peaks shift and begin to overlap with other peaks, it can make 1H
NMR identification of a compound very difficult. Therefore it is important to understand
the NMR phenomena presented here to aid in the identification of future glycodendrimer
products rather than discarding them from what could appear as impure NMR spectra.
50
Future Directions:
The attempted synthesis of glycodendrimers 1-6 was only the first part of the
ultimate goal for this project. Once all six glycodendrimers are fully synthesized and
characterized, they will be sulfated to allow them to bind to the V3 loop of gp120
(Scheme 5). Elemental analysis, 1H and 13C NMR will be used to determine the degree
and location of sulfation.
HO
OH OH
OH
R'
O
AcHN
HO
SO3-Pyr
RO
DMF, 50oC, 2 hr
O
OR OR
OR
RO
R= H or
R'
O
AcHN
O
SO3-
R'= PAMAM or Linker-PAMAM
Scheme 5. Sulfation of sialic acid residues on compounds 1-6.
Along with these six glycodendrimers, some negative controls will also be
synthesized or utilized. The purpose of these negative controls will be to show that any
positive results on a binding assay between the glycodendrimers and gp120 are coming
from the glycodendrimers, and not just one of the pieces that make up the
glycodendrimer. These may include, but are not limited to, acetylated PAMAM (12),
sulfated PAMAM (13), sialic acid (10), sialic acid with linker (14), sulfated monomeric
sialic acid(15), and sulfated sialic acid with linker (16) (Figure 26). All of these
molecules, including the six glycodendrimers, will be evaluated by an enzyme-linked
immunosorbent assay (ELISA) to determine the strength of binding to gp120. For the
ELISA assay, gp120 will be immobilized on a solid support. The gp120 will then be
51
incubated with an antibody specific for the V3 loop on gp120, plus the molecules to be
assayed. The antibody bound to gp120 will have a enzyme tag associated with it,
therefore the strength of the competitors will be measured by the depth of color
developed when the substrate is added after a final wash step of the well. Once binding
strength has been determined for all glycodendrimers, they will be compared to one
another. From this comparison, trends pertaining to binding strength versus generation
and binding strength verses dendritic radii can be determined and ultimately used to
deduce how the dendridic radii affects binding strength.
NHSO3-
HN
-O 3SHN
NH
O
O
O
O
O
O
N
N
N
N
N
N
N
N
13
12
N
N
N
N
O
O
O
O
O
O
NHSO3-
HN
-O 3SHN
NH
OH
OH
CO 2-
OH
HO
OH
O
H
N
O
HO
HO
O
10
OR
O
OR
RO
O
14
O
OR
OR
AcHN
H
N
O
15
N
H
CO 2-
OR
RO
RO
O
O
AcHN
AcHN
OR
O
OH
OH
HO
O
O
AcHN
R= H or SO3-
R= H or
SO3-
RO
O
16
Figure 26: Negative controls to be used in future biological assays.
N
H
O
52
Chapter 4
EXPERIMENTAL
All nuclear magnetic resonance (NMR) spectroscopy was performed on a Bruker
Avance III 500 or an Avance 300 NMR spectrometer with either D2O or CDCl3 solvents
purchased from Acros. For 13C analysis, 3-(trimethylsilyl) tetradutero sodium propionate
(TSP) from Wilmad was used as a zero point reference. Flash chromatography was
performed using flash silica gel (32-63 μM) from Dynamic Adsorbents Inc. Dialysis
purification was done using Spectra/por® Biotech Cellulose Ester dialysis membrane
from Spectrum Laboratories Inc. Reverse phase high pressure liquid chromatography
(RP-HPLC) was conducted on a Hewlett Packard TI-series 1050 using a Grace Prevail C18 5µM 10 x 250 mm column (semi-prep scale). Fast pace liquid chromatography
(FPLC) was performed with a Pharmacia pump P-500 with a L.C. Controller LCC-500
Plus. The column used in conjunction with the FPLC was a Bio-Rad 2.5 cm x 120 cm
Econo column filled with Bio-Rad Bio-Gel® P-10.
The reagents used came from a variety of sources: All poly amido(amine)
(PAMAM) dendrimers were purchased from Aldrich. Benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) was purchased from
Nova Biochem. N,N-diethylisopropyl amine (Hunig’s base) was purchased from Alfa
Aesar. Trifluroacetic acid (TFA), triethylamine (TEA), Ammonium bicarbonate, and
N,N-dimethylformamide (DMF) were purchased from EMD. Di-tert-butyl dicarbonate
((Boc)2O) was purchased from Acros. Diaminotriethylene glycol was obtained from
Huntsman. N-Acetylneuraminic acid (sialic acid) was purchased from Nacalai Tesque.
53
Copies of all 1H NMR spectra are located in Appendix A, 13C NMR data are
contained in Appendix B, and MALDI MS data can be found in Appendix C. Integrations
on the 1H NMRs for compounds 1-6 was set for ¼ of total protons, the equivalent of 1
branch of the full glycodendrimer.
All glycodendrimers are named as follows: the number of end points, either
tetramer, octamer, or 16-mer, followed by glycol. If there is a linker present between
PAMAM and sialic acid, the abbreviation DATEG (diamino triethylene glycol) will
appear. The end is always PAMAM dendrimer.
{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester (8):
Diamino triethyleneglycol 7 (5.0 g, 33.8 mmol) was weighed into a flame-dried 500 mL
round-bottomed flask. Methanol (135 mL) was added, followed by TEA (340 mg, 3.4
mmol), then (Boc)2O (7.38 g, 33.8 mmol). The reaction was heated to 35oC and stirred
overnight. The solvents were evaporated under reduced pressure and the crude residue
was purified by flash chromatography using 1:1 methanol:chloroform, giving 8 as a
viscous golden oil (4.25 g, 17.1 mmol, 51% yield). 1H NMR (D2O): δ 1.41 (s, 9H, HT),
2.79 (t, 2H, J=5.4 Hz, HS), 3.25 (t, 2H, J=5.4 Hz, HP), 3.57 (q, 4H, J= 5.0 Hz, 9.7 Hz,
HQ), 3.66 (s, 4H, HR). 13C NMR (D2O, TSP internal std): δ 30.4, 42.6, 72.1, 72.2, 74.9,
161.
54
N-Acetylneuraminic acid {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-amide (11):
Sialic acid 10 (100 mg, 0.32 mmol) was weighed into a flame-dried 25 mL roundbottomed flask, dissolved into DMF (2 mL) and placed under nitrogen. BOP (210 mg,
0.48 mmol) was added to the solution as a solid. In a separate flask, the linker 8 (87 mg,
0.35 mmol) was dissolved in DMF (1 mL). Hunig’s base (170 mg, 1.28 mmol) was then
added. This mixture was next added to the sialic acid solution. The reaction was heated to
35oC and stirred for 24 hours. The solvent was evaporated under reduced pressure. The
crude was dissolved in 1:2 dichloromethane:TFA (12 mL) and stirred for 2 hours before
the solvents were evaporated under reduced pressure. The crude material was purified
with RP-HPLC using a gradient of deionized water/0.1% trifluoroacetic acid and
acetonitrile/0.1% trifluoroacetic acid giving 11, as a gummy white solid (155 mg, 0.35
mmol, 88% yeild). 1H NMR (D2O): δ 1.67 (t, 1H, J=12.2 Hz, HB), 2.03 (s, 3H, HC), 2.31
(dd, 1H, J=4.7 Hz, 13 Hz, HA), 3.19 (t, 3H, J= 4.8 Hz, HP), 3.43 (m, 2H, HS), 3.55-3.73
(M, 11H, HG,H,J,Q,R) 3.82 (dd, 1H, J=2.6 Hz, 11.9 Hz, HE), 3.89 (t, 1H, J=10.3 Hz, HI),
4.05 (m, 2H, HD,F) 13C NMR (D2O, TSP internal std): δ 22.3, 39.0, 39.3, 39.8, 52.4, 63.4,
66.6, 67.0, 68.4, 68.9, 69.7, 69.8, 70.4, 70.7, 96.0, 173, 175. MALDI-TOF:[M + H]+
(C17H34N3O10) calcd m/z = 439.2, Found m/z = 440.2.
Tetrameric glycol-DATEG-PAMAM dendrimer (1):
PAMAM G = -0.5 (24 mg, 0.055 mmol) was weighed into a flame-dried 10 mL roundbottomed flask and dissolved in DMF (1 mL), and flushed with nitrogen. Hunig’s base
(71 mg, 0.55 mmol) was added, followed by BOP (120 mg, 0.27 mmol). The sugar linker
55
11 (100 mg, 0.23 mmol) was placed into a second 10 mL round-bottomed flask and
dissolved in DMF (1 mL). The two solutions were mixed together, heated to 35oC, and
stirred under nitrogen for 24 hours. The solvents were evaporated under reduced pressure
and the crude product was dialyzed with 500 MWCO tubing in a 1L flask against
nanopure D.I. water. The water was changed once every hour for four hours, and allowed
to stir overnight at 4oC. The remaining crude material was then lyophilized. The dialyzed
crude material was then purified using RP-HPLC with a linear gradient between
water/0.1% trifluoroacetic acid and acetonitrile/0.1% trifluoroacetic acid. Fractions
containing the product were collected, grouped, and purified through size exclusion
chromatography with 0.03 M ammonium bicarbonate as the mobile phase. This provided
compound 1, as a fluffy white solid (3 mg, 0.0017 mmol, 6.4% yield). 1H NMR (D2O): δ
1.73 (m, 1H, HB), 2.09 (s, 3H, HC), 2.36 (dd, 1H, J= 5.0Hz, 16.4Hz, HA), 2.49 (t, 2H,
J=7.2Hz, HM), 2.68 (s, 1H, HK), 2.87 (t, 2H, J=7.1Hz, HL), 3.43 (t, 3H, J=5.4Hz, HP),
3.49 (m, 3H, HS), 3.67 (m, 17H, HG,J,Q,R), 3.78 (m, 1H, HH), 3.88 (dd, 1H, J=2.6Hz,
11.8Hz, HE), 3.95 (t, 1H, J=9.7Hz, HI), 4.11 (m, 2H, HD,F). 13C NMR (D2O, TSP
internal): δ 24.9, 35.2, 41.6, 41.7, 42.4, 51.90, 51.92, 54.9, 66.0, 69.5, 71.0, 71.4, 71.6,
71.6, 72.2, 72.3, 72.9, 73.2, 98.3, 98.3, 98.4, 175.6, 177.6, 177.7. MALDI-TOF:[M +
Na]+ (C82H148N14O44Na) calcd m/z = 2057, Found m/z = 2060.4.
Tetrameric glycoPAMAM dendrimer (2):
Sialic acid 10 (200 mg, 0.66 mmol) was weighed into a flame-dried 10 mL roundbottomed flask, flushed with nitrogen, then dissolved into DMF (4 mL). BOP (400 mg,
56
0.9 mmol) was then added as a solid. PAMAM G=0 (20% wt. in methanol) (75 mg, 0.15
mmol) was next weighed into a separate 10 mL round-bottomed flask and the methanol
was evaporated under reduced pressure. DMF (1.5 mL) and Hunig’s base (190 mg, 1.5
mmol) were added to the flask with PAMAM. The PAMAM solution was then added to
the sialic acid solution, heated to 35oC, and the reaction was stirred for four days under
nitrogen. The crude reaction product was dialyzed with 500 MWCO tubing against
nanopure D.I. water. The water was changed once an hour for three hours, then left
stirring overnight at 4oC. The crude material was then purified using RP-HPLC with a
linear gradient between water/0.1% trifluoroacetic acid and acetonitrile/0.1%
trifluoroacetic acid providing compound 2, as a fluffy white solid (29.4 mg, 0.017 mmol,
11.4% yield). 1H NMR (D2O): δ 1.69 (t, 1H, J=12.3Hz, HB), 2.08 (s, 3H, HC), 2.34 (dd,
1H, J=4.9Hz,13Hz, HA) , 2.76 (m, 2H, HM), 3.40 (m, 7H, HK,L,N,O), 3.61 (d, 1H, J=9.3Hz,
HG), 3.66 (dd, 1H, J=6.3Hz, 11.8Hz, HJ), 3.77 (m, 1H, HH), 3.87 (dd, 1H, J= 2.4Hz,
11.8Hz, HE), 3.94 (t, 1H, J=10.3Hz, HI), 4.09 (m, 2H, HD,F). 13C NMR (D2O, TSP
internal std): δ 25.1, 32.7, 41.56, 41.6, 42.7, 51.6, 52.5, 55.2, 66.2, 69.7, 71.3, 73.2, 73.5,
98.6, 118.2, 120.5, 165.8, 166.1, 175.6, 175.9, 177.9. MALDI-TOF:[M + Na]+
(C66H116N14O36Na) calcd m/z =1703, Found m/z =1702.7
Octameric glyco-DATEG-PAMAM dendrimer (3):
Sugar linker 11 (53 mg, 0.12 mmol) was weighed into a flame-dried 10 mL roundbottomed flask, flushed with nitrogen, then dissolved into DMF (1 mL). BOP (58 mg,
0.13 mmol) was added as a solid. PAMAM G=0.5 (20% wt. in methanol) (16 mg, 0.013
57
mmol) was weighed into a separate 10 mL round-bottomed flask and the methanol was
removed in vacuo. Next, DMF (1 mL), then Hunig’s base (34 mg, 0.26 mmol), were
added to the PAMAM. The PAMAM mixture was added to the sugar linker. The reaction
was heated to 35oC for seven days. The solvents were evaporated in vacuo and the crude
was freeze-dried. The crude was dialyzed with 500 MWCO tubing against nanopure
D.I.Water. The water was changed every hour for four hours, then stirred overnight at
4oC. The crude material was next purified using RP-HPLC with a linear gradient between
water/0.1% trifluoroacetic acid and acetonitrile/0.1% trifluoroacetic acid. This was
followed by size exclusion chromatography in 0.03 M ammonium bicarbonate buffer. 1H
and 13C NMR showed the PAMAM was not fully substituted. For compound 3, the
calculated molecular weight was 4462 Da. MALDI MS showed small peaks for (M+H)
m/z=4467, (M-H2O) m/z=4448, and (M-2H2O) m/z=4428, but also found were (M-cmpd
11-H2O) m/z=4027 and (M-2 cmpd 11) m/z=3619. While MALDI MS shows evidence
that 3 was synthesized, it was not present in a large enough quantity to get clear 1H or 13C
data.
Octameric glycoPAMAM dendrimer (4):
Sialic acid 10 (190 mg, 0.62 mmol) was weighed into a flame-dried 10 mL roundbottomed flask, flushed with nitrogen, then dissolved in DMF (4 mL). BOP (310 mg, 0.7
mmol) was added as a solid. PAMAM G=1 (29% wt. in methanol) (100 mg, 0.07 mmol)
was weighed into a separate 10 mL round-bottomed flask and the methanol was
58
evaporated off under reduced pressure. DMF (2 mL) and Hunig’s base (180 mg, 1.4
mmol) were added to the PAMAM. The two solutions were mixed and the reaction was
stirred under nitrogen for seven days. The solvents were then evaporated in vacuo and the
crude was freeze-dried. The crude solid was next dialyzed with 500 MWCO tubing
against nanopure D.I. water. The water was changed every hour for four hours then
stirred overnight at 4oC. The crude material was next purified using RP-HPLC with a
linear gradient between water/0.1% trifluoroacetic acid and acetonitrile/0.1%
trifluoroacetic acid. This was followed by size exclusion chromatography using Biogel P10 gel in 0.03 M ammonium bicarbonate buffer producing compound 4, as a fluffy white
solid (28 mg, 0.0101 mmol, 14% yield). 1H NMR (D2O): δ 1.68 (t, 2H, J=12.3Hz, HB),
2.07 (s, 6H, HC), 2.33 (dd, 2H, J= 5.0Hz, 13.1Hz, HA), 2.43 (m, 6H, HM), 2.63 (m, 1H,
HK), 2.83 (m, 6H, HL), 3.33 (m, 12H, HN,O), 3.60 (d, 2H, J=9.6Hz, HG), 3.66 (dd, 2H,
J=6.2Hz, 11.8Hz, HJ), 3.77 (m, 2H, HH), 3.86 (dd, 2H, J=2.4Hz, 11.8Hz, HE), 3.93 (t, 2H,
J=10.2Hz, HI), 4.07 (m, 4H, HD,F). 13C NMR (D2O, TSP internal std.): δ 25.0, 35.3, 35.5,
39.6, 41.3, 41.7, 42.5, 51.8, 52.8, 54.0, 55.0, 66.0, 69.5, 69.54, 71.1, 72.9, 73.2, 98.4,
175.7, 177.6, 177.9.
MALDI-TOF:[M + H]+ (C150H264N34O76) calcd m/z =3759, Found m/z =3762.5.
16-mer glyco-DATEG-PAMAM dendrimer (5):
Sugar linker 11 (75 mg, 0.17 mmol) was weighed into a flame-dried 10 mL roundbottomed flask, flushed with nitrogen, then dissolved into DMF (1 mL). BOP (93 mg,
0.21 mmol) was added as a solid. PAMAM G=1.5 (20% wt. in methanol) (25 mg, 0.0085
59
mmol) was weighed into a separate round-bottomed flask and the methanol was
evaporated off in vacuo. Next, DMF (1 mL), then Hunig’s base (44 mg, 0.34 mmol) were
added to PAMAM. The PAMAM mixture was added to the sugar-linker. The reaction
was heated to 35oC and run for 14 days. The solvents were then evaporated in vacuo, and
the crude was freeze-dried. The crude solid was next dialyzed with 500 MWCO tubing
against nanopure D.I.water. The water was changed every hour for four hours, then
stirred overnight at 4oC. The crude material was then purified using RP-HPLC with a
linear gradient between water/0.1% trifluoroacetic acid and acetonitrile/0.1%
trifluoroacetic acid. This was followed by size exclusion chromatography using Biogel P10 gel in 0.03 M ammonium bicarbonate buffer. 1H NMR showed that no fractions
contained the fully substituted product. Compound 5 was not isolated. No clear 1H NMR
was collected that indicated the presence of compound 5, therefore, no samples were sent
out for MALDI-MS analysis.
16-mer glycoPAMAM dendrimer (6):
Sialic acid 10 (46 mg, 0.15 mmol) was weighed into a flame-dried 10 mL roundbottomed flask, flushed with nitrogen, then dissolved in DMF (1 mL). BOP (84 mg, 0.19
mmol) was added as a solid. PAMAM G=2 (20% wt. in methanol) (25 mg, 0.0077 mmol)
was weighed into a separate round-bottomed flask and the methanol was evaporated
under reduced pressure. DMF (1 mL) and Hunig’s base (40 mg, 0.31 mmol) were added
to the PAMAM. The two solutions were mixed, heated to 35oC, and reaction was stirred
under nitrogen for 14 days. The solvents were then evaporated under reduced pressure
60
and the crude sample was freeze-dried. The crude solid was then dialyzed with 500
MWCO tubing. The water was changed once an hour for four hours, then stirred
overnight at 4oC. Initial purification was conducted using RP-HPLC with a linear
gradient between water/0.1% trifluoroacetic acid and acetonitrile/0.1% trifluoroacetic
acid. This was followed by size exclusion chromatography using Biogel P-10 gel in 0.03
M ammonium bicarbonate buffer providing compound 6, as a fluffy white solid (17 mg,
0.00215 mmol, 28% yield). 1H NMR (D2O): δ 1.70 (m, 4H, HB), 2.10 (s, 12H, HC), 2.35
(dd, 4H, J=4.9Hz, 13.1Hz, HA), 2.46 (m, 14H, HM), 2.68 (m, 1H, HK), 2.86 (m, 14H, HL),
3.36 (m, 24H, HN,O), 3.63 (d, 4H, J=9.5 Hz, HG), 3.69 (dd, 4H, J= 6.2 Hz, 11.8Hz, HJ),
3.79 (m, 4H, HH), 3.89 (dd, 4H, J= 2.4 Hz, 11.8Hz, HE), 3.96 (t, 4H, J=10.2 Hz, HI), 4.10
(d, 8H, J=10.8 Hz, HD,F). 13C NMR (D2O, TSP internal std): δ 25.0, 35.5, 39.6, 41.3,
41.7, 51.9, 54.1, 55.0, 66.0, 69.45, 69.5, 71.1, 73.0, 73.3, 98.32, 98.34, 98.36, 98.38,
175.7, 177.7, 177.9, 166.6 MALDI-TOF:[M + Na]+ (C318H560N74O156Na) calcd m/z
=7935, Found m/z =7937.
61
APPENDICES
62
APPENDIX A
1
H Nuclear Magnetic Resonance Spectra
Diaminotriethylene glycol (compound 7), 1H NMR at 300 MHz in CDCl3.
63
Sialic acid (compound 10) 1H NMR at 500 MHz in D2O31
64
{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester (compound 8) 1H
NMR at 500 MHz in D2O
65
N-Acetylneuraminic acid {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-amide (compound 11)
1
H NMR at 500 MHz in D2O
66
Tetrameric glyco-DATEG-PAMAM dendrimer (compound 1) 1H NMR at 500 MHz in
D2O
67
Tetrameric glycoPAMAM dendrimer (compound 2) 1H NMR at 500 MHz in D2O
68
Octomeric glycoPAMAM dendrimer (compound 4) 1H NMR at 500 MHz in D2O
69
16-mer glycoPAMAM dendrimer (compound 6) 1H NMR at 500 MHz in D2O
70
APPENDIX B
13
C Nuclear Magnetic Resonance Spectra
Sialic acid (compound 10) 13C NMR at 500 MHz in D2O
71
{2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester (compound 8) 13C
NMR at 500 MHz in D2O
72
N-Acetylneuraminic acid {2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-amide (compound 11)
13
C NMR at 500 MHz in D2O
73
Tetrameric glyco-DATEG-PAMAM dendrimer (compound 1) 13C NMR at 500 MHz in
D2O
74
Tetrameric glycoPAMAM dendrimer (compound 2) 13C NMR at 500 MHz in D2O
75
Octomeric glycoPAMAM dendrimer (compound 4) 13C NMR at 500 MHz in D2O
76
16-mer glycoPAMAM dendrimer (compound 6) 13C NMR at 500 MHz in D2O
77
APPENDIX C
Matrix Assisted Laser Desorption Ionization Mass Spectroscpoy
Tetrameric glyco-DATEG-PAMAM dendrimer (compound 1) MALDI MS
78
Tetrameric glycoPAMAM dendrimer (compound 2) MALDI MS
79
Octomeric glycoPAMAM dendrimer (compound 4) MALDI MS
80
16-mer glycoPAMAM dendrimer (compound 6) MALDI MS
81
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