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Seton Hall University Dissertations and Theses
(ETDs)
Seton Hall University Dissertations and Theses
Spring 5-14-2016
Synthesis and Evaluation of Biological Activity of a
Potential Immunomodulatory Zwitterionic
Polysaccharide
Vikram Basava
[email protected]
Follow this and additional works at: http://scholarship.shu.edu/dissertations
Part of the Biochemistry Commons, Immunology of Infectious Disease Commons, and the
Organic Chemistry Commons
Recommended Citation
Basava, Vikram, "Synthesis and Evaluation of Biological Activity of a Potential Immunomodulatory Zwitterionic Polysaccharide"
(2016). Seton Hall University Dissertations and Theses (ETDs). Paper 2170.
Synthesis and Evaluation of Biological Activity of a Potential
Immunomodulatory Zwitterionic Polysaccharide
A Dissertation
In the Department of Chemistry and Biochemistry submitted to the
faculty of the Graduate School of Arts and Sciences in partial
fulfillment of the requirements for the
degree of
Doctor of Philosophy
In Chemistry
at
Seton Hall University
by
Vikram Basava
May 2016
1
© 2014 (Vikram Basava)
2
3
ACKNOWLEDGEMENTS
I am greatly thankful to the support and encouragement that I have received from Dr.
Cecilia H. Marzabadi. Her ideas and guidance have made this dissertation possible.
She always had the time to answer all my questions and let me try reactions with the
ideas which I came up with.
I would like to thank the Department of Chemistry and Biochemistry for giving me the
opportunity to pursue my PhD at Seton Hall University. I am extremely thankful for Dr.
Sergiu Gorun for helping me in getting the x-ray crystal structure of the anhydro sugar
which I synthesized. I would like to acknowledge Dr. Constantine Bitsaktsis for
performing biological tests on the PSA 1 analogue which was synthesized in my project.
I would again like to thank Dr. Bitsaktsis along with Dr. James E. Hanson for taking their
time to review my dissertation write-up.
I am thankful for the support of Dr. Robert L. Augustine and Dr. Setrak K. Tanielyan at
the CAC. I would even like to thank Dr. Mahesh K. Lakshman for his support at the
CCNY, CUNY.
I could have not completed this dissertation without the unlimited support from my family
and friends. I particularly would like to thank my mom, Dr. Barnana Vijaya Kumari, as
well as my dad, Basava Vijaya Bhaskara Rao, and my sister, Shilpa Basava, for
supporting me morally throughout my PhD programme. I would even like to thank Irene,
Kyle, Nada, Sumiea and Emi for their endless support.
4
Table of Contents
Acknowledgements
4
Abstract
7
Chapter 1 Introduction
8
1.1 General Information
8
1.2 Antigen presenting cells (APCs)
10
1.3 Major Histocompatibility Complex
11
1.4 Stimulation of T-cell activation by dendritic cells
13
1.5 Zwitterionic polysaccharides as T-cell dependent antigens
13
1.6 T-cell dependent immune responses: Peritonitis and abscess formation
15
1.7 Bacteroides fragilis
16
1.8 PSA structure
18
1.9 ZPS-mediated CD4+ T-cell activation
18
1.10
Cytokines involved in T-cell-dependent immune responses
19
1.11
Toll-like receptor (TLR) signaling
20
1.12
Aim of the present study
22
Chapter 2 Synthesis and Biological Evaluation of a Potential Immunomodulatory
23
Zwitterionic Polysaccharide
2.1 Introduction
23
2.2 Results and Discussion
37
2.2.1 Synthesis of the pyruvate containing galactose moiety
37
2.2.2 Synthesis of the phthalimido aduct
44
2.2.3 Synthesis of the mono-silylated 1,4-cylcohexanediol
48
2.2.4 Synthesis of the title compound
49
2.2.5 Biological evaluation
51
Chapter 3 Synthesis of Chemical Reactivity of 3,6-Anhydro-D-glucal
56
3.1 General Information
56
3.2 Background
68
3.3 Results
71
3.4 Summary
75
Chapter 4
4.1 Experimental
76
5
4.2 NMR Spectra
4.3 References
93
164
6
ABSTRACT
Synthesis and Evaluation of Biological Activity of a Potential
Immunomodulatory Zwitterionic Polysaccharide
Vikram Basava
Doctor of Philosophy in Chemistry
Seton Hall University
Dr. Cecilia H. Marzabadi
The gram-negative anaerobic bacterium Bacteroides fragilis is an integral component of
the normal gastrointestinal flora. The bacterium colonizes the intestinal tract of human
beings as it has no reservoir other than mammals. An unprecedented proportion of the
genomic DNA of B. fragilus is involved in the production of capsular polysaccharides.
These capsular polysaccharides are important virulence factors in most extracellular
bacterial pathogens. Eight of these polysaccharides have been identified thus far, out of
which two were found to be zwitterionic polymers, PSA1 and PSA2. PSA1 was found to
stimulate T-cell lineage of the immune system because of the dual charge structural
feature of the molecule.
This dissertation discusses about the synthesis of a PSA1 analogue and evaluating its
immunomodulatory activity. Also discussed are the synthesis and the chemical reactivity
of 3,6-anhydro-D-glucal which was formed unexpectedly in the synthesis of L-rhamnal.
7
Chapter 1
Introduction
1.1 General Information
The human immune system utilizes a variety of defense mechanisms to maintain the
integrity of the human body. Recognizing, as well as differentiating between self and
foreign antigens and defending the body from diverse infectious invaders are the major
tasks of the immune system. The defense mechanisms consist of several protective
measures such as natural barriers, non-specific (innate) as well as specific (adaptive)
immune responses. Natural barriers are the first line of defense and are comprised of
both physical, as well as, chemical barriers. Skin, being the toughest layer for a
pathogen to overcome, is a physical barrier. Chemical barriers come into action when
the integrity of the skin is compromised, often due to injury. Each region of the body has
its own strategy and contributes to host defense against pathogens. Mucous
membranes are protected by the secretion of anti-microbial immunoglobulins that can
be found in saliva or tears. Mucous as well as cilia located in lower respiratory tract
prevent colonization from pathogens. Similarly, the acidic pH in the gastrointestinal tract
is an inhospitable environment for most microorganisms.1
The second line of defense is the innate immune system, which acts on the antigens
that penetrated the first line of defense, i.e., the skin and mucous membranes. Antigens
are toxins or other foreign substances that induce an immune response in a body by
producing antibodies. Innate immune responses which are found in all living organisms
8
are antigen independent and act immediately on the clearance of pathogens with a
maximal response. The response is cell mediated and humoral, but, does not generate
a lasting protective immunity because of the lack of immunologic memory.
Microorganisms are detected and destroyed within minutes or hours by the various
mechanisms of the innate immunity and occasionally causing perceptible disease in a
normal, healthy individual.1
The cells of the immune system originate from the stem cell. The stem cells produce
myeloid progenitor cells as well as lymphoid progenitor cells. Myeloid progenitor cells in
the bone marrow give rise to neutrophils, eosinophils, basophils, monocytes and
dendritic cells. The lymphoid progenitor cells give rise to T-cells and B-cells.
Macrophages and dendritic cells, which play a key role in innate and adaptive
immunity, are derived from monocytes. Mast cells, which are fixed in tissues, develop
from the same precursor cell as the circulating basophils. B-cells are produced in the
bone marrow and are released into the blood the lymphatic systems. B-cells can further
develop into plasma cells that secrete antibodies. Precursor T-cells undergo
differentiation in the thymus into two distinct types of T-cells, the CD4+ T-helper cells
and the CD8+ cytotoxic T-cells. Macrophages and dendritic cells function as bridges
between innate immunity and adaptive immunity, since they present antigens to the
immunocompetent T-cells which initiate an immunological response. Phagocytes
involved in the innate immune system are macrophages, mast cells, dendritic cells and
granulocytes. They act upon the antigens by breaking them down with the digestive
enzymes in their lysosomes. Only when an infectious organism breaches these early
lines of defense will an adaptive immune response ensue.1
9
The third line of defense is the adaptive immune system which comprises of the
antigen-specific B-cell and T-cell lymphocytes. T-cells get involved with the B-cells,
which have the ability to rearrange genes of the immunoglobulin family, as soon as the
innate immune system signals the presence of a danger to the T-cells of the adaptive
immune system. This results in the creation of diverse antigen-specific clones and
immunological memory.2 The adaptive immune system then activates antigen-specific
lymphocytes to proliferate and to differentiate into effector and memory cells that
eliminate pathogens. Memory cells can prevent recurrence of disease caused by the
same microbe. This highly sophisticated and potent adaptive immune system needs to
be conducted, instructed and regulated by antigen presenting cells (APCs).
1.2 Antigen Presenting Cells (APCs)
Antigen Presenting Cells (APCs) include dendritic cells (DCs), macrophages as well as
the B-cells. Bone-marrow derived APCs are present on peripheral blood and lymphatic
tissues as well as in other organs to guard the host. 1 DCs carry the antigens of the
pathogen to the secondary lymphatic organs, the lymph nodes and the spleen. They
also help in intracellular engulfing and degrading the antigens.
The activity of the DCs depends upon the extent of their maturation. Immature DCs
have a tremendous phagocytic activity to ingest antigens via pinocytosis or receptormediated endocytosis. They have a number of receptors that enhance the uptake of
antigens,
and
they
are
specialized
to
convert
these
antigens
into
Major
Histocompatibility Complex (MHC)-peptide complexes that can be recognized by
lymphocytes. Mature DCs lose their phagocytic activity and activate the adaptive
immune system response by transforming into highly-effective APCs which have a very
10
short life span with no proliferation capacity. 3 Upon antigen-mediated activation, DCs
influence both the innate and the adaptive immune responses, thereby determining how
the immune system responds to the presence of infectious agents. 1
1.3 Antigen processing and Major Histocompatibility Complex (MHC)-dependent
antigen presentation in APCs
Activation of the adaptive immune system by DCs is mediated by cytokine secretion and
by direct presentation of antigens to T-cells. T-cells, which circulate between blood and
peripheral lymphoid tissues, consist of T-helper, T-cytotoxic and T-regulatory cells. The
CD4+ T-helper cells depend on the major histocompatibility complex (MHC) class II
peptide-contact to initiate and modulate receptor- and cytokine-mediated functions of
the immune system.4 CD8+ T-cytotoxic cells depend on the MHC class I by recognizing
the endogenous antigens and mediating the cytotoxic immune system. 5 MHC is a
membrane glycoprotein encoded in a cluster of genes. It displays peptide antigen to Tcells. T-regulatory cells suppress the immune response and protect the host from
aggressive effector mechanisms of the immune system.
Extracellular pathogens and proteins captured by APCs are either recycled to the
plasma membrane or transported to the lysosome for degradation. They are then
presented to the CD4+ T-cells through MHC class II proteins to induce an immune
response.1,
6
Proteases, glycosidases and lipases present in endolysosomes are
activated by low pH giving rise to protein degradation into smaller peptide fragments.
The peptide needs to get loaded onto the MHCII molecule for it to present the antigen to
a CD4+ T-cell.7 The MHCII molecules are present only on the professional APCs, unlike
11
the MHCI molecules. The MHCII molecule complex consists of two transmembrane
glycoproteins, the α and the β, which form a non-covalently bound complex. The open
ends on the peptide-binding groove of the MHCII molecules get filled and blocked when
the invariant chain prevents uncontrolled early binding thereby targeting it to an acidic
endosomal compartment. The activated proteases cleave the MHCII:invariant chain
complex when it enters the acidic endosomal pathway leaving a short fragment called
class II-associated invariant-chain peptide (CLIP). Intracellular antigen loading of MHCII
is dependent on HLA-DM, a MHCII-like molecule in humans (H-2M in mice) which is
found predominantly in the MHCII compartment.8 The antigen first binds to the MHCII
molecule, catalyzes the release of the CLIP fragment from the MHCII binding groove,
stabilizes the MHCII molecule to prevent aggregation and aids in rapid and tight binding
of peptides to the empty MHCII molecule. The MHCII:peptide complex is then
transported from the lysosome to the cell surface and the peptide presented to CD4 + Tcells.9 Once recognized, the CD4+ T- cells activate other effector cells of the immune
system, like macrophages, which kill the intravesicular pathogens they harbor, or B-cells
to secrete immunoglobulins against the foreign bodies.
The B-cell antigen receptors are a membrane-bound form of antibodies which will be
released upon activation, plasma cell differentiation and proliferation of the B-cell with a
specific antigen. The MHCII:peptide complex must be stable at the cell surface. If it isn’t
stable, the pathogen in the infected cell could escape detection or be picked by MHC
molecules of an infected cell and be wrongfully destructed by cytotoxic T-cells.1
There are about 30,000 antigen-receptor molecules, called T-cell receptors (TCRs) on
the T-cells which consist of TCRα and TCRβ polypeptide chains linked by a disulfide
12
bond. The TCR on the cell surface recognizes processed and presented peptide
antigens. The α:β heterodimers have a similar structure to the Fab fragment of an
immunoglobulin molecule which account for antigen recognition by most T-cells.1
1.4 Stimulation of T-cell activation by Dendritic cells (DCs)
An immunological synapse made up of MHCII:peptide complexes on the DC and the
TCR on the T-cells is formed when the mature DCs interact with the T-cells. This
interaction leads to the activation of the T-cells by an intracellular signaling pathway
which leads to the induction of differentiation and proliferation. The naïve T-cells
proliferate and their progeny adhering to the APCs develop into armed effector T-cells.1
The secretion of cytokines leads to the DCs further directing T-cell development
towards Th1 and Th2 phenotypes via cytokine production.10 Interaction of immature or
tolerogenic DCs with T-cells can result in T-cell deletion, anergy, or the generation of
Treg cells.11
1.5 Zwitterionic polysaccharides as T-cell-dependent antigens
MHCII proteins were previously only known to present proteins, peptides or
superantigens. Most of the known bacterial polysaccharides do not get processed or
presented on the APC surface to T-cells, as they fall apart in the lysosomes of the
APCs.6 But, recent in vivo and in vitro studies showed that some polysaccharides of
certain symbiotic bacteria get transported from lysosomes to the cell surface to get
presented by MHCII protein. This results in the activation of CD4+ T-cells in vitro and the
induction of CD4+ T-cell-dependent abscesses in vivo.12,13,14,15,16 Hence, this discovery
of the MHCII pathway being responsible for carbohydrate antigen presentation and
13
eventual T-cell recognition alters the previous fundamental model of MHCII-antigen
binding.
The CD4+ T-cell activation by bacterial polysaccharides is dependent on the transport of
bacterial polysaccharides via MHCII. The functional role of HLA-DM is to help the
bacterial polysaccharide to compete with peptides for MHCII binding.13,14
The few polysaccharides which induce the CD4+ T-cell-dependent formation of
abscesses in vivo are characterized by the presence of opposite charged motifs on
each repeating unit. They are therefore termed as zwitterionic polysaccharides (ZPS).
They include polysaccharides like PS A1 and PS A2, Sp1, and CP5 from the
commensal
bacteria
Bacteroides
fragilis,
Streptococcus
pneumonia
and
Staphylococcus aureus, respectively (Fig. 1.1).17 The presence of zwitterionic charges
is critical for the antigenic function of the ZPSs. Removal of either of the charges on the
ZPS resulted in compounds that failed to stimulate CD4+ T-cells.18
14
Fig. 1.1: Subunits of PSA1, PSA2, Sp1 and CP5
1.6 T-cell-dependent immune responses: Peritonitis and abscess formation
Secondary peritonitis, the most common one, is observed because of the loss of
integrity in the gastrointestinal tract. This is due to the contamination of the peritoneal
space by symbiotic intestinal bacteria.19 Development of intraperitoneal abscesses, a
bacterial infection, is followed by colonic leakage of commensal intestinal bacteria. It is
seen in patients with inflammatory bowel diseases (IBD) like Crohn’s disease or
ulcerative colitis. Almost a third of patients infected with general peritonitis die in spite of
improved diagnostic modalities, potent antibiotics, modern intensive care and
aggressive surgical treatment.20 The surgical treatment for generalized peritonitis is
through peritoneal lavage, drainage of localized abscesses, removal of the
contaminating source by repairing, and drainage of the peritoneal cavity. However, even
in the presence of improvised therapy, residual abscesses form in patients resulting in
substantial morbidity and mortality.21
Abscess formation is known to be a CD4+ T-cell-dependent immune response induced
by ZPS.18 The zwitterionic charge promotes CD4+ T-cell-dependent abscess induction
15
in experimental murine and rat models.22 Chemical abrogation of the zwitterionic charge
demonstrated that the charged nature of ZPS is critical for its ability to induce abscess
formation. No CD4+ T-cell-dependent immune responses are observed for ZPS
derivatives that have been made neutral or only possess a single charge (cation or
anion).18 In contrast, uncharged polysaccharides induce T-cell-dependent immune
responses after they are modified to possess a zwitterionic charge.18,23,24
1.7 Bacteroides fragilis
Bacteroides fragilis is a Gram-negative, anaerobic, bile-resistant, non-spore forming,
rod-shaped bacterium whose primary known environmental reservoir is the human
lower gastrointestinal tract where it lives as a commensal organism. They are significant
clinical pathogens and are found in most anaerobic infections, with an associated
mortality of more than 19%. The bacteria maintain a complex and generally beneficial
relationship with the host when retained in the gut. They fermentation of carbohydrates
in the human intestine results in the formation of a pool of volatile fatty acids which are
reabsorbed through the intestine and utilized by the host as an energy source. 25 But,
when they escape from the gut, they can cause significant pathology including
bacteremia and abscess formation in multiple body sites. Though B. fragilis accounts for
only 0.5% of the human colonic flora, it is the most commonly isolated anaerobic
pathogen because of its potent virulence factors. They have the most antibiotic
resistance mechanisms and the highest resistance rates of all anaerobic pathogens.
Clinically, Bacteroides sp. have exhibited increasing resistance to many antibiotics
including cefoxitin, clindamycin, metronidazole, carbapenems and fluoroquinolines (e.g.,
gatifloxacin, levofloxacin and moxifloxacin).
16
A distinctive feature of B. fragilis is the large proportion of the genome devoted to
carbohydrate metabolism, including the degradation of dietary polysaccharides and the
production of surface polysaccharides.26,27,28 The ability to express multiple capsular
polysaccharides has been shown to enhance intestinal colonization by B. fragilis.29 Thus
far, eight different capsular polysaccharides, each synthesized from different genomic
loci, have been identified. Two of the capsular polysaccharides of B-fragilis,
polysaccharide A (PSA) and polysaccharide B (PSB) belong to a class of carbohydrates
known as zwitterionic polysaccharides (ZPSs). Previously, carbohydrates had been
characterized as classic T-cell-independent antigens that induce a specific IgM
response but fail to evoke an IgG or memory response.30,31 However, it is now known
that ZPSs are capable of inducing CD4+ T-cell-dependent immune responses. All of the
known ZPSs have immunomodulatory properties that depend on the presence of both
positively and negatively charged groups within the repeating units. 18,22,32 Of all the
known ZPSs, PSA is the best characterized.
PSA is a unique and potent antigen. Among carbohydrates, this polysaccharide stands
out as a T-cell dependent rather than a T-cell-independent antigen. Although it is
processed and presented along the same pathways as conventional peptide antigens,
PSA modulates the host immune system more extensively than many other known Tcell-dependent antigens.
17
1.8 PSA Structure
Fig 1.2: Structure of Polysaccharide A (PSA)
PSA is a zwitterionic polysaccharide, with an average molecular mass of 100 kDa,
comprising of repeating tetrasaccharide units. The positive charge is situated at the free
amino group of the rare 2-acetamido-4-aminofucose moiety and the negative charge
resides on the carboxylate of the pyruvate ketal spanning C4 and C6 in the
galactopyranosyl residue.
1.9 ZPS-mediated CD4+ T-cell activation
Activation of CD4+ T-cells requires the specific interaction of the α-β T-cell receptor
(TCR) on the T-cell with the antigen presented in the context of MHCII on a professional
APC.33 To induce an immune response, PSA must be processed in an endocytic
pathway and presented on an MHCII molecule in a manner analogous to that by which
peptide antigens are presented. The notable difference is that, in the endosome, the
degradation of PSA to a molecule size small enough for presentation by the MHCII
18
molecule is a non-enzymatic chemical process. Whereas, protein degradation occurs
enzymatically.33,34 Once taken up into an APC, PSA follows the same processing
pathway as peptide antigens. Upon entry into the endosome of an APC, PSA is directed
to a MHCII compartment. The up-regulation of inducible nitric oxide synthetase, which is
associated with the oxidative burst, results in increased nitric oxide (NO) production. NO
breaks down PSA through deamination, a chemical reaction in which PSA is oxidized
and then depolymerized to a much smaller molecular size while retaining the
zwitterionic motif and the overall structure of the repeating unit. Finally, co-localization of
PSA, MHCII, and α-β TCRs can be detected on the cell surface of murine splenocytes
by confocal microscopy. However, no PSA is found on the cells lacking MHCII. 34 In the
absence of direct contact between APCs and T-cells, ZPSs cannot stimulate T-cell
proliferation or induce cytokine production in vitro.16
1.10
Cytokines involved in T-cell-dependent immune responses
Cytokines are of great importance in CD4+ T-cell-induced abscess formation. It has
been suggested that cytokines may be responsible for triggering the migration of
immune cells into the peritoneal cavity following contamination with B. fragilis.35
Cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, IL15 and IL-18 are involved in T-cell activation as well as proliferation.36 It was
demonstrated that ZPS PSA induces the production of TNF-α and IL-1α on peritoneal
cells which act as significant immune mediators for abscess formation.37 When TNF-α is
blocked, expression of the ICAM-1 (intercellular adhesion molecule-1) is significantly
reduced leading to the accumulation of polymorphonuclear leukocytes (PMNLs) within
the abdominal cavity. This then inhibits the formation of a peritoneal sepsis.
19
Furthermore, PSA stimulates the secretion of chemokines such as IL-8 which stimulate
T-cells.
IL-10, one of the most potent anti-inflammatory cytokines in the immune system, is
required for PSA suppression of the inflammatory immune response. It plays a critical
role in protection against inflammation in several animal models.
During sepsis and peritonitis, IL-6 is considered as an import mediator of immunologic
alterations in the host, along with TNF-α and IL-1.38 From the results observed, IL-6 is
the most potent proinflammatory cytokines because it stimulates the expression of the
broadest spectrum of acute phase proteins in inflammation and infection.39
IL-6 is a pleiotropic inflammatory cytokine which is produced by a variety of cells. It acts
on a wide range of tissues by stimulating the activation, survival and proliferation of
CD4+ T-cells and acts on T-cells as an anti-apoptotic factor.40 IL-6 prevents apoptosis of
normal and resting T-cells in the absence of additional cytokines. It has also been
identified as a migration factor for human primary T-cells.41 In vitro experiments showed
chemotactic activity of IL-6 on peripheral blood lymphocytes, lymphokine-activated killer
cells, and CD4+ and CD8+ T-lymphocytes.42 IL-6 was also found to be linked to T-celldependent immune diseases and autoimmune diseases such as the inflammatory
bowel diseases, ulcerative colitis and Crohn’s disease.43
1.11
Toll-like receptor (TLR) signaling
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate
immune system. They are single, membrane spanning, non-catalytic receptors usually
expressed in cells such as macrophages and dendritic cells. TLRs play a critical role in
20
host defense by sensing the presence of microbes. They mediate inflammatory
responses, coordinate innate and adaptive immunity, prime naïve T-cells, and induce
memory to facilitate the elimination of pathogens, upon activation.44,45 The TLRs include
TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12,
and TLR13. TLR12 and TLR13 are not found in humans. TLRs recognize certain highly
conserved microbial components known as pathogen-associated molecular patterns
(PAMPs). They even recognize other molecules like bacterial lipopolysaccharides,
flagellin, bacterial DNA, and viral double-stranded RNA.46 Nevertheless, both
pathogenic and commensal microbes produce the microbial ligands which are
recognized by TLRs. Although TLR signaling was traditionally associated with the
sensing of the pathogens, it is now appreciated that commensal bacteria are also
recognized by TLRs, and that this interaction is critical for protection against epithelial
injury and intestinal homeostasis.47
The triggering of TLRs on DCs leads to the induction of co-stimulatory signals (e.g.
MHCII) and to the production of cytokines that orchestrate T-cell recruitment, activation,
differentiation and survival and are essential for the induction of adaptive immune
responses.48 Likewise, the decrease in processing and presentation of PSA combined
with the decreased production of IL-12 by dendritic cells activated by PSA in the
absence of TLR2 signaling demonstrates that in addition to the stimulation of a specific
CD4+ T-cell immune response, innate immune pathways are required for PSA to
establish a Th1/Th2 balance.
21
1.12
Aim of the present study
From what was discussed above, it is quite evident that ZPSs like PSA have the
potential to induce CD4+ T-cell-dependent immune responses in vitro and are also
capable of inducing abscess formation in vivo in a CD4+ T-cell-dependent manner. But,
not much work has been done to date to study the potential to induce CD4 + T-celldependent immune responses of simplified, zwitterionic polysaccharides which are
analogous to PSA.
We propose to study the synthesis of an immunomodulatory zwitterionic polysaccharide
as well as its interactions with APCs and T-cells. The molecule synthesized will be
evaluated for its ability to produce IL-6 and TNF-α in APCs. IFN-γ (T-cell response) and
IgM (B-cell response) production will be studied using mouse spleen cells in co-culture
with APCs. Finally, a cell proliferation assay will be done on the spleen cells of mouse.
The initiation of both innate and adaptive responses by the ligands could prove to be a
major step forward in the development of a carbohydrate-based vaccine.49
22
Chapter 2
Synthesis and Biological Evaluation of a Potential Immunomodulatory
Zwitterionic Polysaccharide
2.1 General Information
A considerable amount of work has been done for the synthesis of various zwitterionic
molecules. The compounds synthesized were found to be of incredible importance in
the field of science. Kowalczky50 reported the synthesis of alkylammonium zwitterionic
amino acid derivatives (Fig 2.1). The molecules synthesized were characterized by
FTIR, NMR spectroscopy and DFT calculations. The pharmacotherapeutic potential of
the molecules synthesized was estimated by Prediction of Activity Spectra for
Substances (PASS) which showed that the compounds could be used for as creatinase
inhibitors,
polyamine-transporting
ATPase
inhibitors,
hydrogen
dehydrogenase
inhibitors and alkylacetylglycero-phosphatase inhibitors. They were even predicted to be
useful as antiviral, antitoxic and antimutagenic agents.
23
Fig. 2.1 Synthesis of 3-(3-dimethylamonio)propylammonio propanoate bromide (a), 4(3-dimethylamonio)propylammonio
butanoate
bromide
(b),
5-(3dimethylamonio)propylammonio pentanoate bromide (c)
Similarly, Szoka, et al.,51 reported and synthesized a novel class of zwitterionic lipids
with head groups containing a 3o or 4o amine and a carboxylate group. These lipids
were found to form stable liposomes that exhibit head group dependent, pH-responsive
biophysical characteristics which could make them suitable for drug delivery
applications.
Zwitterionic structure monomers such as phoshorylcholine52, sulfobetaine53 and
carboxybetaine54 have been found to reduce protein absorption and platelet adhesion.
Xu, et al.,55
incorporated these charged groups into polyurethanes (Fig 2.2) by
synthesizing them from a polycarbonatediol with alkyne groups and 3-((azidoethyl)
24
dimethylammonio)propane-1-sulfonate
using
the
copper
catalyzed
1,3-dipolar
cycloaddition (click) reaction.
Fig. 2.2 Polyurethane
Likewise, Bergstrom56 and others described the synthesis and the structure–activity
relationships of zwitterionic spirocyclic compounds which are used as potent cysteinecysteine chemokine receptor-1 (CCR1) antagonists (Fig 2.3). A spirocyclic amine was
treated with 3-bromo-2,2-dimethyl-propan-1-ol in the presence of NaI and Et3N in DMF
to give the desired adduct which upon coupling with 4-fluoro-2-hydroxybenzoate in a
Mitsunobu type reaction gave the ester adduct. The ester adduct was then treated with
the racemate of methyl pyrollidine-3-carboxylate in the presence of carbonyl diimidazole (CDI) in N, N-dimethylformamide (DMF) to give a mixture of the
diastereomers.
25
Fig. 2.3 (a) NaI, Et3N, DMF, 78 oC 60 h; (b) methyl 4-fluoro-2-hydroxybenzoate, Ph3P, DEAD, THF, rt
20 h; (c) (R)-methyl pyrrolidine-3-carboxylate/(S)-methyl pyrrolidine-3-carboxylate, CDI, DMF, rt, 20 h
Of the few known zwitterionic capsular polysaccharides, the structures of only a handful
of them (PS A1 and PS A2, Sp1, and CP5 from the commensal bacteria Bacteroides
fragilis, Streptococcus pneumonia and Staphylococcus aureus, respectively) have been
characterized thus far (Fig 2.4).
26
Fig. 2.4 Zwitterionic polysaccharides (Subunits)
Bundle, et al.,57 first synthesized the hexasaccharide representing two repeating units of
the zwitterionic capsular polysaccharide from Streptococcus pneumoniae type 1 (Sp1).
The synthesis involved the stereoselective construction of 1,4-cis-α-galactose linkages
based on a reactive trichloroacetimidate donor that incorporates a 6-O-acetyl group
which has been proposed to account for the α-selectivity in glycosylation.
Later, Codee, et al.,58 came up with a modular approach towards the synthesis of all
possible trimer repeating units of the Sp1 polysaccharide. For efficiently introducing αgalacturonic acid bonds stereoselectively, galacturonic-[3,6]-lactone building blocks
27
were used. The building blocks not only acted as donor galactosides, but they were also
functional as reactive acceptor glycosides when equipped with a free hydroxyl group
(Fig 2.5).
Fig. 2.5 Three frame-shifted trimer repeats of Sp1 polysaccharide
Similarly, Bundle, et al., were successful in synthesizing a trisaccharide repeat of the
zwitterionic
Sp1
polysaccharide.
2-Azido-4-benzylamino-4N,3-O-carbonyl-2,4,6-
trideoxy-D-galactopyranosyl trichloroacetimidate (B), was synthesized in six steps from
6-deoxy-D-glucal. The glucal was glycosylated with selectively protected α-1,3-linked
methyl galabioside (C, D) to produce the trisaccharide skeleton of Sp1 (Fig 2.6).
28
Fig. 2.6 Trisaccharide repeat of zwitterionic Sp1 (A) and the monosaccharide synthons (B, C, D)
PS A1, one of the zwitterionic capsular polysaccharides of Bacteroides fragilis, was first
synthesized successfully by van der Marel, et al.,59 in its fully protected form (Path I, Fig
2.7). One of the drawbacks of this synthesis is that the overall yield was low because of
very low nucleophilicity of molecule B.
Seeberger, et al.,60 later synthesized the same molecule by the [3+1] glycosylation
technique, in its unprotected form (Path II, Fig 2.7). Glycosylation in this process took
place between the highly electro deficient trisaccharide glycosylating agent C, and the
pyruvated galactose nucleophile D. The –OH group on C3 of the galactose residue
acted as a very good nucleophile.
29
Fig. 2.7 Retrosynthesis of PS A1
For the de novo synthesis of the 2-acetamido-2-amino-2,4,6-trideoxy-D-galactose
(AAT), N-Cbz-L-threonine (i) was used as the starting material (Fig 2.8). It was first
converted to a methyl ester followed by acetylation to give (ii). Treatment of (ii) with
lithium bis(trimethylsilylamide) (LHMDS) resulted in a Dieckmann cyclization 61,
62
to give
enoate (iii). The treatment of (iii) with 1,2-diisobutylaluminium hydride (DIBAL)63 gave
(iv) which upon reduction under Luche64 conditions gave allylic alcohol (v). Acetylation of
(v) followed by azidonitration65 gave (vii). Cleavage of the anomeric nitrate in (v) with pTolSH/DIPEA66 followed by reaction with 2,2,2-triflouroacetamidoyl chloride resulted in
the formation of the N-phenyl trifluoroacetimidate sugar (A).
30
Fig. 2.8 Synthesis of the AAT building block (A)
Reagents and conditions (a) i., FmocCl, py., 0 °C, 79%; ii., BzCl, py., 0 °C, 82%; iii., TsOH, MeOH, 40
°C; iv., CH3C(O)CO2Me, BF3·OEt2, CH3CN, 23 °C, 39%, two steps; (b) i., i-PrOH, NIS, AgOTf, CH2Cl2, 0
°C; ii., NEt3, CH2Cl2, 23 °C, 49%, two steps; (c) LevOH, DIPC, DMAP, CH2Cl2, 0 to 23 °C, 90%; (d) i.,
NIS, THF, H2O, 23 °C; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 0 °C, 83%, two steps; (e) i., NBS, EtOAc, H 2O,
23 °C, 91%; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23 °C, 87%
The galactosamine derivative was earlier synthesized (Fig 2.9) by the initial treatment of
(viii) with naphthyl bromide and NaH followed by cleavage of the benzylidene ring with
TES and TFA in CH2Cl2 to give the 6-O-benzylated galactosamine derivative, (ix).67
Fig. 2.9 Synthesis of the galactosamine derivative (ix)
Reagents and conditions (a) NapBr, NaH, THF, 0 oC to 23 oC, 84%; (b) TES, TFA, CH2Cl2, 0 oC to 23
oC, 81%
31
The galactosamine derivative obtained, (ix), was then glycosylated (Fig 2.10) with the
AAT, (A), in the presence of TMSOTf in CH2Cl2 at 0 oC to give the disaccharide (x).
Fig. 2.10 Glycosylation of the AAT building block (x)
Reagents and conditions (a) TMSOTf, CH2Cl2, 0 °C, 74%
Similarly, the galactofuranose building block (xii), was synthesized (Fig 2.11) by
reacting the known thiol68 (xi) with N-bromosuccinimide (NBS) to form the lactol,
followed by the formation of the N-phenyl trifluoroacetimidate derivative (xii).
Fig. 2.11 Synthesis of the galactofuranose building block (xii)
Reagents and conditions (a) i., NBS, EtOAc, H2O, 23 °C, 91%; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23
°C, 87%
32
The last building block, the pyruvated galactose sugar (D), was synthesized from the
benzylidene galactoside (xiii).69 The C3 and C2 hydroxyl groups were initially protected
with Fmoc70 and Bz groups respectively (Fig 2.12). Cleavage of the benzylidene acetal
was followed by the formation of the pyruvate ketal71 (xiv) using BF3. OEt2. Finally,
glycosylation of (xiv) with isopropanol in the presence of NIS/AgOTf followed by removal
of the Fmoc protecting group gave the pyruvated galactose nucleophile (D).
Fig. 2.12 Synthesis of the pyruvated galactose nucleophile (D)
Reagents and conditions (a) i., FmocCl, py., 0 °C, 79%; ii., BzCl, py., 0 °C, 82%; iii., TsOH, MeOH, 40
°C; iv., CH3C(O)CO2Me, BF3·OEt2, CH3CN, 23 °C, 39%, two steps; (b) i., i-PrOH, NIS, AgOTf, CH2Cl2, 0
°C; ii., NEt3, CH2Cl2, 23 °C, 49%, two steps
In the synthesis of the trisaccharide (C), the first step involved the removal of the
naphthyl ether group using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (Fig
2.13). The resulting alcohol (xvi) was glycosylated with galactofuranose N-phenyl
trifluoroacetimidate (xii) to give the trisaccharide adduct (xvii) as the β-anomer. The
anomeric tert-butyl-dimethylsilyl (TBDMS) group was removed in the presence of
tetrabutylammonium fluoride (TBAF) to give a lactol that upon treatment with 2,2,2triflouroacetamidoyl chloride in the presence of Cs2CO3 resulted in the formation of the
N-phenyl trifluoroacetimidate adduct (xviii). Thioethyl glycoside (C) was later synthesized
33
from glycosyl imidate (xviii) by the reaction of the latter with ethanethiol (EtSH) in the
presence of TMSOTf at 0 oC in the presence of 4 Ao molecular sieves.
Fig. 2.13 Synthesis of the trisaccharide, C
Reagents and conditions (a) DDQ, MeOH, CH2Cl2, 23 °C, 86%; (b) TMSOTf, CH2Cl2, −30 °C, 90%; (c)
i., TBAF, AcOH, THF, 0 °C; ii., F3CC(NPh)Cl, Cs2CO3, CH2Cl2, 23 °C, 82%, two steps; (d) EtSH, TMSOTf,
4 Ao MS, CH2Cl2, 0 °C, 96%
34
The resulting trisaccharide (C) formed was made to react with pyruvated galactose
moiety (D) in the presence of dimethyl (methylthio) sulfonium triflate (DMTST)72 and
2,4,6-tri-tert-butylpyrimidine (TTBP) at 0 oC to give the totally protected PS A1 adduct
(xix) in 58% yield (Fig 2.14).
Fig. 2.14 Formation of the tetrasaccharide, (xix)
Reagents and conditions (a) dimethyl (methylthio) sulfonium triflate (DMTST), 2,4,6-tri-tertbutylpyrimidine (TTBP), 4 Ao MS, 0 oC, CH2Cl2
Finally, the azide groups of fully-protected PS A1 (xix) were converted to acetamides
using ethanethioic acid (AcSH) and pyridine (Fig 2.15). Global deprotection of the
acetamide adduct of PS A1 was performed in two different steps. Initially, the compound
was hydrogenated in the presence of Pearlman’s catalyst 73 which removed the benzyl
as well as the CBz groups. Next, NaOMe in MeOH/H2O was added dropwise to form the
final PS A1 tetrasaccharide repeating unit in 46% yield.
35
Fig. 2.15 Deprotection and formation of PS A1 tetrasaccharide repeating unit
Reagents and conditions (a) AcSH, py., 23 °C, 67%; (d) i., H2, Pd(OH)2/C, MeOH, 23 °C; ii., THF, then
0.5 M NaOMe in 1:1 MeOH/H2O, 23 °C, 46%, two steps
Earlier studies revealed that peptides synthesized to mimic the zwitterionic charge motif
associated with zwitterionic polysaccharides exhibited biologic properties. Lysineaspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T-cells
in vitro and gave protection against abscesses induced by bacteria such as Bacteroides
fragilis and Staphylococcus aureus.74
Alterations made in the charge motifs of the zwitterionic polysaccharides were shown to
have either partially or totally removed the stimulatory properties of the sugars.75 It was
36
also proved that the non-charged portions of the saccharide chain are not essential for
the stimulation by PS A1. Modifications made to the non-charged sugars failed to
diminish the production of cytokines.
Discussed hereafter is the synthesis of an analogue of the PS A1 zwitterionic
polysaccharide that could be of utmost importance in investigating its biological activity.
Since only the charged sugar residues have been implicated in binding and processing
of PS A1, the synthesis has been carried out using a commercially-available
cycloalkane diol as the bridging moiety. As the galactofuranose moiety has zero charge
on it, and as it doesn’t contribute to the size of the PS A1 moiety in increasing the
distance between the charges (Calculated: 13 Ao; Observed: 15 Ao), we decided to not
include it in the synthesis of the analogue.
2.2 Results and Discussions
2.2.1 Synthesis of the pyruvate-containing galactose moiety
The synthesis of the pyruvated sugar moiety started with the commercially available βD-galactose pentaacetate (1). It was first made to react with 1.2 equivalents of
thiophenol (PhSH) in the presence of BF3.OEt2 to give predominantly the anomeric βsubstituted thiophenol adduct (2) in 97% yield (Scheme 2.1).76
Scheme 2.1 Synthesis of β-thiophenyl-2,3,4,6-tetra-O-acetyl-D-galactopyranose
37
The thiophenyl adduct obtained was then de-acetylated using Zemplen’s conditions to
yield 98% of the galactopyranose sugar (3) in 98% yield (Scheme 2.2).77
Scheme 2.2 De-acetylation using Zemplen’s conditions
A series of reactions were then set up to selectively protect the C4 and C6 hydroxyl
functional groups. The best possible route chosen involved first forming of the 4, 6-Obenzylidene protecting group. As the benzylidene ring is preferentially six membered,
there is a greater chance of ring formation between the C4 and the C6 hydroxyl groups.
The de-acetylated thiophenyl adduct (3) was treated with benzaldehyde dimethylacetal
in the presence of camphor sulfonic acid in DMF at 65 oC (Scheme 2.3).78 However,
TLC analysis of the reaction mixture showed that under these conditions esentially no
reaction took place. A similar reaction was also carried out using the dimethyl acetal in
the presence of p-toluenesulfonic acid. But, again there was no reactivity observed
when monitoring by TLC.79 Reactions were set up separately with (3) using
benzaldehyde and benzaldehyde dimethyacetal in the presence of pyridinium ptoluenesulfonic acid. These reactions also did not show the formation of product.
38
X
Scheme 2.3 Various attempts in synthesizing the benzylidene sugar moiety
When the reaction when set up in the presence of ZnI2 and benzaldehyde, none of the
desired product was obtained (Scheme 2.3). But, the same reaction (Scheme 2.4)
when repeated in the presence of ZnCl2 did give the desired benzylidene sugar (4) in
65% yield.80 The formation of product was supported by the 1H NMR spectrum that
showed a singlet at
5.53 ppm which represents the axial hydrogen on the
benzylidene ring.
39
Scheme 2.4 Synthesis of the benzylidene sugar moiety
Compound (4) was subsequently reacted with dibutyltin oxide and benzoyl chloride in
the presence of 4 Ao molecular sieves in toluene to give a mixture of 2-O-benzoyl and 3O-benzoyl sugar derivatives (Scheme 2.5). The latter, being the major product, was
easily separated by flash column chromatography yielding 50% of the 3-O-benzoyl
adduct (5a).81
Scheme 2.5 Selective benzoylation using organo tin chemistry
Various attempts were made to protect the hydroxyl group at the C-2 position. A
benzylation reaction was initially carried out by reacting compound (5a) with benzyl
40
chloride in the presence of NaH and 15-crown-5 in DMF (Scheme 2.6).82 As the
reaction failed to occur, the benzylation reaction was attempted by using
benzyltrichloroacetimidate and trifluoromethane sulfonic acid.83 But, again there was no
product formation. Reaction of (5a) with p-nitro-benzyl bromide in the presence of NaH
and DMF did not show the addition of the benzyl group at the C-2 position. Reaction of
p-methoxylbenzyl chloride with (5a) in the presence of NaH and 15-crown-5-ether did
not show the benzylation reaction happening, but instead resulted in debenzoylation
giving (4) as the final product.84 This offside reaction could have occurred because of
the formation of NaOH when NaH reacted with trace amounts of water in the solution.
41
Scheme 2.6 Attempted benzylation reactions at the C-2 hydroxyl group
The reason for the lack of reactivity of the C-2 was likely due to the bulky nature of the
protecting group used and the sterically crowded environment at that position. Hence, a
smaller protecting group was used. A reaction was carried out between (5a) and acetyl
chloride in the presence of triethylamine and N, N-dimethylaminopyridine which resulted
in the formation of the 2-O-acetyl adduct (6) in 69% yield (Scheme 2.7).
42
Scheme 2.7 Successful acetylation at the C-2 position
The next step was to introduce a pyruvate moiety into the sugar. This could be done by
cleaving the benzylidene ring and subsequently introducing the pyruvate functional
group into the sugar. The benzylidene ring can be cleaved in different ways. Depending
on the reagents used, there may form a 4-O-benzyl-6-hydroxy sugar or a 4-hydroxy-6O-benzyl sugar. But, for introduction of the pyruvate ring into the sugar, formation of the
4,6-diol is imperative. Therefore, compound (6) was treated with 20% trifluoroacetic acid
in CH2Cl2 to give a 4,6-diol sugar (7a, 45% yield).85 The diol sugar upon treatment with
methyl pyruvate in the presence of BF3-Et2O gave the desired pyruvate sugar (7) in
80% yield (Scheme 2.8).86
43
Scheme 2.8 Addition of the pyruvate group to the sugar moiety
2.2.2 Synthesis of the phthalimido adduct
Various attempts were made in synthesizing the positive charged moiety having the
amino group on it. First, glucosamine was made to react with acetyl chloride which gave
the per-acetylated-2-acetamido α-glucopyranosyl chloride with a very good yield.87 The
product was then reacted with thiophenol in the presence of Et3N in acetonitrile which
gave the desired β-thiophenyl adduct.88 The obtained product showed no reactivity any
further probably because of the formation of the stable oxazoline ring with the
acetamido group at the C-2 position. Attempts in reacting the glucopyranosyl chloride
with 1, 4-cyclohexanediol in the presence of N-iodosuccinimide and Yb(Otf)3 to yield the
desired O-glycoside were not fruitful (Scheme 2.9).89
44
Scheme 2.9 Earlier attempts in synthesizing the thioglycoside
Subsequently, glucosamine first was per-acetylated, and the product was treated with
BF3. Et2O to give the oxazoline derivative. The oxazoline derivative when treated with
thiophenol and camphor sulfonic acid (CSA) showed no reactivity.90 This can be
attributed to the stability of the oxazoline ring. One-pot reaction for the addition of the
thiophenyl moiety to the per-acetylated glucosamine in the presence of BF3. Et2O upon
sonication did give the desired thiophenyl adduct.91 The reaction was found to have not
reached completion (Scheme 2.9).
45
Having observed problems with the 2-acetamido group because of its formation of a
stable oxazoline ring, plans were made to incorporate a phthalimido group as it doesn’t
take part in the neighbouring group participation. Glucosamine HCl was first reacted
with NaOMe in methanol to get glucosamine which was further reacted with phthalic
anhydride in the presence of Et3N.92 The product (8) was then acetylated by reacting it
with acetic anhydride and di-azo-bicyclooctane (DABCO) to give the desired peracetylated 2-phthalimido adduct (9) in 30% overall yield (Scheme 2.10).93
Scheme 2.10 Synthesis of the 2-phthalimido adduct
46
The 2-phthalimido adduct (9) was then made to react with thiophenol in the presence of
a mild base, Et3N. TLC of the reaction did not show the formation of the desired
product. This was observed because of the bulkiness of the thiophenol group (Scheme
2.11).
Scheme 2.11 Substitution of thiophenol at the anomeric carbon atom
Therefore, a smaller thiol, like ethanethiol, was chosen as the leaving group. Reaction
of (9) with ethanethiol and BF3. Et2O in anh. CH2Cl2 gave the desired ethanethiol adduct
(10) in 79% yield (Scheme 2.12).94
Scheme 2.12 Substitution of ethanethiol at the anomeric carbon atom
47
2.2.3 Synthesis of the monosilylated 1, 4-cyclohexanediol
Synthesis of the of one mono-protected hydroxyl group of 1, 4-cyclohexanediol
selectively was not as challenging as the synthesis of the other subunits. First, the
cis/trans mixture of 1, 4-cyclohexanediol was reacted with 1.2 equivalents of benzyl
chloride and NaH in anh. DMF which gave a separable cis/trans mixture of monobenzylated product (11a, 11b) with an overall yield of 88% (Scheme 2.13).
Scheme 2.13 Synthesis of the mono-benzylated adduct of 1, 4-cyclohexanediol
But, from the earlier experiences, it was quite evident that debenzylation occurs when
the reactions were carried out at elevated temperatures. Hence, attempts were made in
substituting the benzyl group with a more robust silyl moiety. Reaction of 1, 4cyclohexanediol with 1.2 equivalents of tert-butyldimethylsilyl chloride and imidazole in
anh. DMF gave the desired mono-silylated product (12a, 12b) in 50% yield as a white
fluffy solid (Scheme 2.14).95
48
Scheme 2.14 Synthesis of the mono-silylated adduct of 1, 4-cyclohexanediol
2.2.4 Synthesis of the final product
Addition of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (10) to the cismono-silylated adduct of 1,4-cyclohexanediol (12a) in the presence of trimethylsilyl
trifluoromethanesulfonate (TMSOTf) and NIS in CH2Cl2 at -78 oC gave the desired
product (13) in 1.5 h. The same reaction when allowed to warm up to room temperature
gave the desilylated product (14) in 7 h (Scheme 2.15).96
49
Scheme 2.16 Reaction of compound (10) with (12) in the presence of TMSOTf and NIS
Further reaction (Scheme 2.17) of the product (14) with the earlier obtained pyruvate
adduct (7) in the presence of TMSOTf and NIS at -40 oC gave the final desired
polysaccharide subunit (15). As the yields were incredibly low, no further steps were
taken in deprotecting the final compound, and the obtained product was sent for
biological evaluation.
50
Scheme 2.17 Synthesis of the PSA1 analogue
2.3 Biological evaluation
The protected PSA1 analogue (15) was evaluated using ELISA assays for the
production of proinflammatory cytokines TNF-α and IL-6 (innate immunity). The cells
used were the RAW 264.7 and the mouse peritoneal macrophages (C57, BL/6, Jackson
Laboratories, Maine, US). Initial results showed (Fig. 16) that the cytokines produced by
15 were negligible in comparison to those produced by the lipopolysaccharides, when
analyzed on the RAW264.7 and the mouse peritoneal macrophages.
51
Fig. 16 Innate Immunity: Production of TNFα and IL-6 by RAW264.7 and the mouse peritoneal
macrophages
The compound (15) was further evaluated for adaptive immune responses on the
spleen cells. The responses which were analyzed were for the T- and B-cell
proliferation, levels of INF-γ in the supernatants which indicates a T-cell response, and
polyclonal IgM and IgG levels in the supernatants which signifies a B-cell response. It
was found that neither the T-cell nor the B-cell responses were observed with (15).
52
To elucidate the toll like receptor responsible for cytokine production, the peritoneal
macrophages from TLR2 and TLR4 knockout mice (female, 4-6 weeks old, C57BL/6)
were obtained. The cells were cultured with variable concentrations of the compound
(15) and, the TNF-α and IL-6 levels in the supernatants were measured at different time
points (Fig. 17).
Fig. 17 Innate Immunity: Production of TNFα and IL-6 by the peritoneal macrophages from TLR2 and
TLR4 knockout mice
53
It was observed that the production of both TNF-α and IL-6 was reduced in only the
TLR4 knockout mice suggesting that signaling of (15) occurs only through TLR4.
Furthermore, the graphical representation showed that the magnitude of TLR4 signaling
with (15) was greater than the known TLR4 agonist, E. coli LPS (lipopolysaccharide).
Similarly, the extent of binding of compound (15) to the TLR4 receptors was illustrated
by treating the peritoneal exudate cells (PECs) with different concentrations of the
compound. The cells were pre-incubated with the compound, after which they were
washed. To the washed cells were then added the antibody followed by the
phytoerythrin dye (PE) and, the number of the PE positive cells was observed by flow
cytometry (Fig. 18).
54
***
80
N.S.
60
40
20
ro
l
1
g/
m
l
5
g/
m
l
10
g
/m
l
0
co
nt
% TLR-4 expression
PECs
Fig. 18 Results from flow cytometry
The graphs in the figure above show that the increase in the concentration of the
compound (15) results in an increased binding of the TLR-4 receptors to the compound.
The graphs with TLR-2 show that the presence of the compound (15) doesn’t interfere
with the cell count. This clearly depicts that the PSA1 analogue doesn’t bind to the TLR2 receptors.
55
Chapter 3
Synthesis and Chemical Reactivity of 3,6-Anhydro-D-glucal
3.1 General Information
Anhydrosugars (or intramolecular anhydrides) are the derivatives of monosaccharides
obtained by the intramolecular elimination of a water molecule resulting in the formation
of a new three-, four-, five-, six- or seven-membered heterocyclic rings. Their advantage
of easy removal has led to the development of numerous derivatives, the synthesis and
properties of which are described in the literature.97, 98, 99, 100, 101 These anhydrosugars
are an important class of sugars which are used for the preparation of modified
carbohydrates
including
the
C-glycosides102,
nucleosides103
and
heterocyclic
compounds104. They are also used in the synthesis of complex enantiomerically pure
products where in the chirality of the parent sugar is transferred to the target via the
chiron approach.105 They constitute very versatile starting materials not only in
carbohydrate chemistry but also for the synthesis of non-carbohydrate and non-natural
products. Anhydrosugars are the suitable monomers for preparing stereoregular
polysaccharides and their specifically substituted derivatives.1
For the synthesis of the anhydro skeleton in a normal sugar molecule, one of the
hydroxyl groups of the diol which is taking part in the anhydro ring formation is
converted into a leaving group such as a halogen, tosylate or triflate. The other hydroxyl
group acts as a nucleophile resulting in the formation of the anhydro ring by an
intramolecular SN2 approach (Fig. 3.1).
56
Formation of the anhydrosugar by elimination of the water molecule
Mechanism for the formation of the anhydrosugar
Fig. 3.1 Synthesis of anhydrosugar
The reaction protocols proposed by Mitsunobu106 and Castro107 for the synthesis of the
anhydrosugars involve activation of the hydroxyl groups under milder conditions (Fig.
3.2).
Fig. 3.2 Proposed protocols of Mitsunobu and Castro
57
The formation of anhydrosugars, may involve various carbon atoms of the sugar, thus
leading to their categorization into different classes. They are divided into two main
categories: 1. the anhydrosugars which involve the participation of the anomeric carbon
atom, called glycosanes, which resemble the intramolecular glycosides; 2. the
anhydrosugars which do not involve the anomeric carbon atom, called simply as
anhydrosugars.1, 108, 109 The latter group comprises of purely synthetic materials which
are exclusively used in synthetic carbohydrate chemistry.
Classification of the anhydrosugars is also based on the heterocyclic ring formed upon
dehydration.
Oxirane
(sugar
epoxide),
oxetane
(oxacyclobutane),
oxolane
(tetrahydrofuran) and oxane (tetrahydropyran) are the various heterocyclic anhydrides
known. Oxirane and oxetane rings show higher reactivity, whereas oxane and oxolane
rings show comparatively low reactivity. The position of the anhydro ring, the steric
arrangement as well as the conformation of the molecule also play a major role in
determining the reactivity of the heterocyclic anhydrides.
3.1.1 Glycosanes
Compounds which are formed by the dehydration of the anomeric hydroxyl group and
the terminal alcohol of a sugar molecule (1,6-anhydropyranoses, 1,6-anhydrofuranoses)
or with any other alcohol on the sugar ring (1,2-, 1,3-, 1,4-anhydrosugars, etc) come
under the class of anomeric anhydrosugars or glycosanes. The most common of these
are the 1,6- and 1,2-anhydrosugars, while the others are less commonly seen in
carbohydrate chemistry. They resemble conventional glycosides in their sensitivity to
58
acids by undergoing acid hydrolysis. But, highly strained anhydrosugars tend to
undergo hydrolysis even in alkaline solutions.
The formation of the carbocation at the anomeric carbon atom controls the
regioselectivity of the opening of the ring.110 Nucleophilic attack occurs preferentially at
the anomeric carbon atom resulting in the formation of various glycosyl derivatives or
glycosides. The anhydrosugars act as monomers in polymerization reactions to form
stereoregular polysaccharides, as the configuration of the corresponding anomeric, as
well as the nonanomeric carbon atoms, remain the same.
3.1.1.1 1,2-Anhydroaldoses
1,2-Anhydroaldopyranoses have the 2,7-dioxabicyclo[4.1.0]heptanes skeleton which
preferentially adopts 4H5 and 5H4 conformations.111,112 1,2-anhydrohexofuranoses have
the more flexible 2,6-dioxabicyclo[3.1.0]hexane skeleton which adopts an
E
conformation. (Fig. 3.3).
Fig. 3.3 1,2-Anhydroaldoses
Brigl was the first to synthesize the 1,2-anhydrosugar (Fig. 3.4).113 1,2-anhydro-3,4,6-triO-acetyl-α-D-glucopyranose or ‘Brigl’s anhydride’ was prepared from β-D-glucopyranose
pentaacetate by sequential treatment with PCl5 and NH3.114 The product was used in
the synthesis of glycosides115 and α-D-linked disaccharides, like sucrose.116
59
Fig. 3.4 Synthesis of Brigl’s anhydride
Subsequently, Danishefsky’s research group synthesized the 1,2-anhydrosugar by
treating a glycal with dimethyldioxirane (Fig. 3.5).117 The product obtained upon
treatment with a nucleophile gave the 2-hydroxy-β-D-glycoside.
Fig. 3.5 Synthesis of 1,2-anhydride using dimethyldioxirane
Similarly, Marzabadi, et al. synthesized the 1,2-anhydro glucal by making a halohydrin
undergo a base induced cyclization (Fig. 3.6).118 Many other 1,2-anhydrides were
synthesized later on.
Fig. 3.6 Synthesis of 1,2-anhydride from a halohydrin
60
3.1.1.2 1,3-Anhydroaldoses
1,3-Anhydrosugars have the 2,6-dioxabicyclo[3.1.1]heptane skeleton and may adopt the
5C ,
2
B2,5 or E2 conformation of the pyranose ring. The presence of axial substituents at
C-2, C-4 and C-5 positions destabilize the 5C2 conformation (Fig. 3.7). The oxetane ring
of 1,3-anhydrosugars is less reactive towards acids in comparison to the sensitivity of
the oxirane ring in both acidic, as well as, in alkaline aqueous solutions.
Fig. 3.7 1,3-Anhydroaldoses
Many of the 1,3-anhydrohexopyranoses of gluco-,119, 120 manno-,121, 122 galacto-,123 talo,124 and 6-deoxy derivatives125,
126,
127
were earlier synthesized via analogous
procedures. They were obtained by reacting partially-protected glycosyl halides with
strong bases like NaH and potassium tert-butoxide (Fig. 3.8).
Fig. 3.8 Synthesis of a 1,3-anhydrohexose derivative
61
3.1.1.3 1,4-Anhydroaldoses
The 1,4-anhydroaldoses have the 2,7-dioxabicyclo[2.2.1]heptane skeleton and adopt
two non-interconvertible conformations, B1,4 and
1,4B
(Fig. 3.9). They tend to be very
stable in alkaline conditions, but get readily hydrolyzed in the presence of acids. The
synthesis is similar to that of 1,3-anhydrosugars, and many compounds in the Dgluco,128,
129, 130
D-galacto,131, 132
6-deoxy-L-manno, L-talo,133 L-arabino, D-ribo, D-xylo
and D-lyxo configurations have been synthesized.134, 135, 136, 137, 138
Fig. 3.9 1,4-Anhydroaldoses
3.1.1.4 1,6-Anhydroaldoses
These compounds have the 6,8-dioxabicyclo[3.2.1]octane skeleton and have limited
steric flexibility. Most of them adopt themselves to 1C4 conformation, while some of them
are more flattened in B3,0 conformation. An inversion in the orientation at all the
stereocenters is observed in comparison to the 4C1(D) and 1C4(L) conformations of the
parent compounds. These anhydrosugars resemble methyl β-D-hexopyranosides in
their chemical activity. They are very stable in alkaline conditions, but are very sensitive
to acids (Fig. 3.10).
62
Fig. 3.10 1,6-Anhydroaldoses
Their synthesis includes cyclization of the hexopyranosyl derivatives having a good
leaving group like halides, azides,139,
140, 141
tosylates142 or 2,4,6-trimethylbenzoates143
at the C-1 position and alkali-sensitive glycosides, like phenyl glycosides,144,
145
by the
action of strong bases.
3.1.2 Anhydrosugars
Compounds which are formed by the dehydration of any two hydroxyl groups on the
sugar molecule, other than the hydroxyl group on the anomeric carbon atom, are the
anhydrosugars. These heterocyclic anhydrides include the oxirane (sugar epoxides, 3membered), oxetane (oxacyclobutane, 4-membered), oxolane (tetrahydrofuran, 5membered) and oxane (tetrahydropyran, 6-membered) derivatives.
3.1.2.1 Oxirane
Oxiranes are the anhydro sugars having a fused oxirane ring to a pyranose or a
furanose
moiety.
These
have
the
3,7-dioxabicyclo[4.1.0]heptane
and
3,6-
dioxabicyclo[3.1.0]hexane basic skeletons that adopt either 5H0 or 1H0 conformations in
63
the ground state (Fig. 3.11). Generally the 2,3- and 3,4-anhydrohexopyranoses adopt
the flexible half-chair conformation (H) depending on the presence or the absence of
additional fused rings.
Fig. 3.11 Oxiranes
Though there exist many synthetic procedures for oxiranes, the most common
procedure followed is the intramolecular SN2 nucleophilic displacement reaction.
Usually, a tosyloxy or a mesyloxy group gets displaced by a vicinyl hydroxyl group that
has been deprotonated by a base such as sodium methoxide.146, 147, 148
These epoxy sugars are generally used in the synthesis of various derivatives of sugars
like halo-, amino-, thio-, azido-, deoxy- and branched-chain derivatives. The oxirane
ring, which is more reactive than oxetane and oxolane rings, opens in the presence of
nucleophiles both in acidic and in basic conditions. But, it tends to be stable to catalytic
debenzylation reactions (only palladium), in acylations and alkylations in basic media,
and in detosylation reactions in the presence of nucleophiles.
64
3.1.2.2 Oxetane
Anhydrosugars containing a fused oxetane ring in the 2,6-dioxabicyclo[3.2.0]heptane or
the 2,7-dioxabicyclo[4.2.0]octane skeleton are called oxetanes (Fig. 3.12).
Fig. 3.12 Oxetanes
3.1.2.3 Oxolane
These include the bicyclic 2,5-anhydro- as well as the 3,6-anhydro-aldose derivatives.
Their
general
skeleton
is
either
2,5-dioxabicyclo[2.2.1]heptane,
2,6-
dioxabicylco[3.2.1]octane or 2,6-dioxabicyclo[3.3.0]octane (Fig. 3.13). They exist even
as monocyclic oxolane derivatives possessing a free aldehyde group. As they are quite
stable in acidic and basic conditions, reactivity of these anhydrosugars is dependent on
the presence or the absence of a free aldehyde group.
65
Fig. 3.13 Oxolanes
From the conformational analysis, it can be inferred that the relatively strained 2,6dioxabicylco[3.2.1]octane skeletons of 3,6-anhydro-D-gluco- and –D-manno-pyranose
tend to rapidly recyclize into the less strained 2,6-dioxabicyclo[3.3.0]octane skeletons of
the 3,6-anhydrohexofuranose in the presence of methanolic HCl. Such a rearrangement
was not observed in the galacto- and talo- configurations.149, 150
Many 2,5-anhydropentoses have been described so far, of L-arabino,151 D-lyxo, D-ribo
and D-xylo sugars.152 Chitose or 2,5-anhydro-D-mannose is the most common 2,5anhydrohexose derivative.153, 154,
155
It can be prepared by the deamination of 2-amino-
2-deoxy-D-glucose. Other isomers include the derivatives of D-allose,156, 157 D-altrose, Lidose,158, 159 D-glucose160, 161 and L-talose162.
Among oxolanes, 3,6-anhydroaldohexose derivatives form the major class. As per the
configuration and conformation of all the eight possible 3,6-anhydroaldohexoses
described, some of them exist as bicyclic pyranose, while others exist as bicyclic
66
furanose forms.163,
164
Hence, 3,6-anhydro derivatives of glucose and mannose adopt
both the pyranose and the cis-fused forms, while those of galactose and talose adopt
the pyranose form only. Gulose and idose can adopt only the cis-fused form. Likewise,
free 3,6-anhydro-D-allose and –D-altrose can only exist in the acyclic form.165, 166
3.1.2.4 Oxane
Pyranose
and
furanose
derivatives
adopting
either
dioxabicyclo[2.2.2]octane skeleton with the pyranose ring in
2,5B
the less flexible 2,6-dioxabicyclo[3.2.1]octane skeleton in
the
rigid
2,5-
or B2,5 conformation or
4C
0
conformation are
categorized as oxanes (Fig. 3.14). They comprise of 2,6-anhydrohexoses and 1,5anhydroketoses. Among the 2,6-anhydrohexoses, pyranoid derivatives in configurations
of altro,167 ido,168 manno169 and talo170 configurations have been reported. Of the
furanose derivatives, 2,6-anhydro-D-mannofuranose and its derivatives are known.171
Fig. 3.14 Oxanes
They can be synthesized by the reaction of sodium methoxide on the corresponding 6tosylate sugar (Fig. 3.15).172 1,5-anhydro-D-fructose is one of the 1,5-anhydroketose
67
sugars which is of particular interest.173 It is the central intermediate formed in the
alternative glycogen catabolic pathway, also called as the Anhydrofructose (AF)
pathway.174
Fig. 3.15 Synthesis of a 2,6-anhydropyranose derivative
3.2 Background
The most widely known compounds from the tetrahedral class of anhydrosugars are
3,6-anhydrofuranoses.175 Furanodictine A and B [Fig. 3.16] are good examples of this
class that show neuronal differentiation activity. 176 3,6-Anhydro-D- and L-galactoses and
their partially methylated derivatives are typical constituents of polysaccharides of red
algae.177 3,6-Anhydro sugars forming bicyclic nucleosides have shown antiviral
activity.178 The puckering in the bicyclic carbohydrate moiety is believed to cause the
molecule to be locked in the proper conformation for biological activity to be
observed.179, 180
68
Fig. 3.16 3,6-Anhydrofuranoses
3,6-anhydrohexoses are considered to be of more importance as they are used to
synthesize new nucleoside analogues.181 Reinhold et al. (1974) first synthesized 3,6anhydrohexose sugars by selective tosylation at the C-6 carbon atom followed by
treatment of the intermediate tosylates with a strong base (sodium methoxide) (Fig.
3.17).
182
Kreimeier et al. and Gross et al. substituted the hydroxyl group at C-6 by
(Me2N)3P and halogens, upon which the tosylated sugars were treated with a strong
base, as mentioned earlier.183,
184
Liptak et al. later confirmed that the presence of
benzyloxy and methoxy groups at the C-3 position would help in the formation of the
3,6-anhydride bond as they act as good internal nucleophiles. 185
Fig. 3.17 Synthesis of 3,6-anhydropyranose
Similarly, Shibayama, et al. and Santoyo-Gonzales et al. described the synthesis of 3,6anhydrohexofuranose derivatives by the basic solvolysis of 5,6-thionocarbonates,
sulfates and sulfites.186, 187 A cyclic 3,5,6-orthoester was shown to have reacted likewise
by basic solvolysis.188 Ishido et al. described the photochemical cyclization of a
69
substituted oxolane ring (Fig. 3.18).189 Yamaguchi et al. showed that the mercaptolysis
of agar as a convenient method for the synthesis of 3,6-anhydro-L-galactose diethyl
dithioacetal.190
Fig. 3.18 Solvolysis
Very little research has been done on the preparation of anhydro-D-glycal sugars, which
are of great importance in the synthesis of complex new derivatives. 3,6-Anhydro-Dglucal was earlier synthesized by Tucker et al. from 3,4-di-O-acetyl-6-O-tosyl-D-glucal
using Deacidite FF IP (OH-) resin in 66% yield (Fig. 3.19).191 4,6-O-anhydro-D-glucal
was previously reported as a byproduct (40% yield) when an equimolar amount of
lithium aluminum hydride in THF was reacted with 6-O-tosyl-D-glucal, though the
structure of this molecule was not fully characterized.192
Fig. 3.19 Synthesis of 3,6-anhydro-D-glucal
70
The results presented describe the synthesis of 3,6-anhydro-D-glucal from 6-O-tosyl-Dglucal using excess LiAlH4 in THF as the reagent. With this combination of reagents, an
82% yield of anhydro-D-glycal was realized.193 The unusual chemical reactivity of the
resulting anhydro glycal was also investigated. Also described is the synthesis of an
unusual 1,4:3,6-dianhydrosugar via N-iodosuccinimide (NIS) addition to the 3,6anhydro-D-glucal.
3.3 Results: Synthesis of 3,6-anhydro-D-glucal and a study of its chemical
reactivity
3,4,6-Tri-O-acetyl-D-glucal was deacetylated using Zemplen conditions to give D-glucal
16 in 98% yield.194 The resulting D-glucal 16 was selectively tosylated at the primary
hydroxyl using p-toluenesulfonyl chloride in a mixture of pyridine and dichloromethane.
The 6-O-tosylated-D-glucal 17 was obtained in 56% isolated yield. Treatment of 17 with
three equivalents of LiAlH4 in THF did not produce the anticipated 6-deoxy-D-glycal, as
previously reported, but instead gave the 3,6-anhydro sugar 18 in 82% yield (Scheme
3.1).195
Scheme 3.1 Synthesis of 3,6-anhydro-D-glucal
Structural assignments for 18 were based on 1D and 2D 1H and
13C-NMR
experiments.
The proposed bridged structure was supported by the observation of long range
71
coupling between the vinylic proton at C2 and the bridgehead proton at C4 (W-coupling)
in the proton NMR. In the 1H-1H-COSY (Appendix), coupling between H4 and H6 and
H6’ was also seen, while in the 1H-NMR the H6 and H6’ peaks only appeared as broad
doublets of doublets. X-ray quality crystals were obtained and the structure of the
molecule established unambiguously (Fig. 3.18), detailed information of which is given
in the Appendix.196
Fig. 3.18 ORTEP representation of compound 18
Compound 18 was subjected to a series of different glycal addition reactions. Treatment
of compound 18 with NIS in the presence of methanol led to the formation of a mixture
of β-gluco-, -manno- and -manno-2-deoxy-2-iodo-O-methylglycosides (19a, 19b, 19c)
in a ratio of 5.2: 1.5: 1 with an overall yield of 93% (Scheme 3.2).197 None of the -gluco
diastereomer was detected in the mixture.
72
Scheme 3.2 Synthesis of 2-deoxy-2-iodo-O-methylglycosides
Treatment of 18 with thiophenol in the presence of a catalytic amount of ceric (IV)
ammonium nitrate (CAN) gave a mixture of α- and β-2-deoxy sugars (20a, 20b) in a
ratio 1.85:1 with a 65% yield. Also formed in the reaction was an α-, β-mixture of the
Ferrier rearranged adducts (21a, 21b) with a 35% yield in a ratio of 2.5:1, formed upon
the breakdown of the anhydride ring (Scheme 3.3).198
Scheme 3.3 Addition of thiophenol to 18 in the presence and absence of CAN
73
When the anhydro sugar 18 was reacted with thiophenol and a catalytic amount of ceric
(IV) ammonium nitrate in the presence of sodium iodide, a reagent known to enhance selectivity, a mixture of α- and β-2-deoxy sugars (20a, 20b) was again obtained in this
reaction (34%), but only the α-adduct 21a from the Ferrier rearrangement was isolated
(65%) (Scheme 3.3).199
Because of the success of the NIS addition with methanol, attempts were made to
synthesize disaccharides from the anhydro sugar donor. Compound 18 was treated with
an equimolar amount of 1,2:3,4-di-O-isopropylidine-D-galactopyranose (DIPG) in the
presence of N-iodosuccinimide to give 2-deoxy-2-iodo-1,4:3,6-dianhydride 22 as the
major product (48%). An inseparable mixture of 2-deoxy-2-iodo sugars (23a, 23b, 23c)
was also formed in low yield, and residual starting materials were isolated (~20%)
(Scheme 3.4). When the same reaction was repeated in the absence of 1,2:3,4-di-Oisopropylidine-D-galactopyranose and in the presence of 4 Ǻ molecular sieves, 22 was
obtained in a much higher yield of 76% (Scheme 3.5).
Scheme 3.4 Synthesis of 1,4:3,6-dianhydride 22 from 18 in the presence of DIPG
74
Scheme 3.5 Synthesis of 1,4:3,6-dianhydride 22 from 18 in the absence of DIPG
3.4 Summary
In summary, good yields of 3,6-anhydro-D-glucal 18 have been obtained from 6-Otosyl-D-glucal 17 via reaction with excess lithium aluminum hydride. The resulting
anhydro glycal was studied under a variety of reaction conditions. Iodoglycosylation of
18 with methanol gave good yields of a mixture of 2-deoxy-2-iodomethylglycosides 19ac, but attempts to use the primary hydroxyl sugar donor 1,2:3,4-diisopropylidine-Dgalactopyranose gave the dianhydro sugar 22 and iodohydrins 23a-c. These observed
products may result due to the steric hindrance present in the anhydro glycal reactant or
in the transition state for the reaction. Alternatively, unfavorable dipole-dipole
interactions in the transition state for the sugar nucleophile approaching the glycal
double bond may be responsible for the observed products. β-Anomers were favored
over α-anomers by a ratio of ~ 4:1. Furthermore, the formation of 22 is favored because
the intramolecular attack by the bridgehead hydroxyl group is anticipated to be faster
entropically than is the attack by the solvated nucleophiles. The small methanol
nucleophile and adventitious water can more easily access the double bond and
mixtures of product resulted from their attack. In those cases where the DIPG was
excluded and freshly powdered and activated 4 Ao molecular sieves were added, only
the dianhydro sugar 22 was obtained. Further reactions with 18 are ongoing.
75
4.1 Experimental
General Methods.
Melting points (mp) were recorded on a Mel-Temp melting point apparatus (Laboratory Devices,
Inc., USA) and are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Varian Inova (500 MHz) spectrometer. NMR samples were dissolved in CDCl3,
and chemical shifts were reported in ppm relative to the residual non-deuterated solvent (CHCl3
as δ = 7.26 ppm). 1H NMR data are reported as follows: chemical shift, integration, multiplicity
(s = singlet; d = doublet; d, d = doublet of a doublet; d, d, d = doublet of a doublet of a doublet;
m = multiplet), coupling constant, and assignment. 13C NMR spectra were recorded on a Varian
Inova (125 MHz) spectrometer. The samples were dissolved in CDCl3, and chemical shifts are
reported in ppm relative to the solvent (CDCl3 as δ = 77.0 ppm). 1H and 13C NMR spectra were
measured at room temperature and the chemical shifts are reported in parts per million, unless
otherwise noted. 1H NMR assignments were done using gCOSY and 13C NMR assignments were
done using gHMQC. Elemental analysis was performed at Robertson Microlit Laboratories Inc.,
Ledgewood, NJ. High resolution and low resolution mass spectra (HRMS and LRMS) were
obtained at the Mass Spectrometry Lab, University of Illinois, Champaign-Urbana, IL and are
reported in m/z (electrospray ionization). X-ray crystallographic data for compound 2 was
deposited at the Cambridge Crystallographic Data Center (CCDC); deposition number CCDC
988697.20 Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel
coated aluminum sheet 60 F254 (Analtech, # 157017). Silica gel (particle size 40-63 µm, 230-400
mesh Silicycle Siliflash P60) was used for flash column chromatography. Preparative thin-layer
chromatographic separations were carried out on 2000 µm silica gel coated glass plate 60 F254
(Analtech). Rotary evaporation was performed using a Buchi R-114 rotary evaporator. Unless
76
otherwise noted, non-aqueous reactions were carried out in oven-dried (125 oC) glassware under
nitrogen. Dry CH3CN, THF and methanol were purchased from Pharmco-Aaper products, Inc.
All other commercially available reagents were used as received.
Thiophenyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (2): To a solution of penta-O-acetylβ-D-galactopyranose (9 g, 23.056 mmol) in 30 mL of anhydrous CHCl3 in a 100 mL r. b. f. was
added 2.84 mL of thiophenol (3.048 g, 1.2 eq, 27.667 mmol) under an atmosphere of N2. To the
mixture was then added 7.3 mL of BF3. Et2O and stirred at room temperature for 3 days. The
reaction was then terminated by diluting with excess CHCl3, followed by washings with
saturated NaHCO3 soln. (2 x 50 mL) and 10% w/v NaOH soln. (2 x 50 mL). The organic extract
was dried over anhydrous Na2SO4 and concentrated in vacuo to yield the crude product. The
product was purified by flash column chromatography using 30%, 40% and 50% EtOAc in
hexanes to give 9.809 g (97%) of the desired compound as a white solid. Rf (1:1 EtOAc/
hexanes): 0.66; 1H NMR (500 MHz, CDCl3) δ 7.49-7.51 (m, 2H, Ar), 7.26-7.31 (m, 3H, Ar),
5.41 (dd, 1H, J3,4 = 3.4 Hz, J4,5 = 1 Hz, H-4), 5.23 (dd, 1H, J1,2 = 10 Hz, J2,3 = 9.8 Hz, H-2), 5.04
(dd, J2,3 = 10.2 Hz, J3,4 = 3.4 Hz, H-3), 4.71 (d, 1H, J1,2 = 10.3, H-1), 4.18 (dd, 1H, J6,6’ = 11.45,
J5,6’ = 6.8, H-6’), 4.1 (dd, 1H, J5,6 = 5.8, J6,6’ = 12.1, H-6), 3.93-3.95 (m, 1H, H-5); 13C NMR (125
MHz, CDCl3) δ 170.36, 170.19, 170.04, 169.42 (C=O, OAc), 132.57, 132.47, 128.89, 128.16
(CH, Ar), 86.6 (C-1), 74.42 (C-5), 72 (C-2), 67.26, 67.22 (C-3, C-4), 61.63 (C-6), 20.85, 20.66,
20.63, 20.58 (CH, OAc).
Thiophenyl-β-D-galactopyranose (3): To a solution of the above obtained thiophenyl adduct
(8.33 g, 18.93 mmol) in 30 mL of anhydrous CH3OH was added 0.1 eq of 5% NaOCH3 in
CH3OH solution. The mixture was stirred at room temperature for 4 h under an atmosphere of
N2. The reaction upon reaching completion was rotary evaporated to get rid of CH3OH. The
77
resulting syrupy product was then dried under high vacuum to give 5.1589 g (100%) of the
desired deacetylated product as a yellowish-white powder. Rf (5% CH3OH/CHCl3): 0.03; 1H
NMR (500 MHz, CDCl3) δ 7.43-7.45 (d, 2H, JAr,Ar = 6.3 Hz, Ar), 7.27-7.31 (m, 2H, Ar), 7.187.21 (m, 2H, Ar), 4.72 (s, 4H, -OH), 4.57 (d, 1H, J1,2 = 9.8 Hz, H-1), 3.71 (d, 1H, J3,4 = 3.4 Hz,
H-3), 3.46-3.53 (m, 3H, H-5, 6, 6’), 3.42 (d, 1H, J1,2 = 9.3 Hz, H-2), 3.35 (dd, 1H, J3,4 = 3.4 Hz,
J4,5 = 9 Hz, H-4); 13C NMR (125 MHz, CDCl3) δ 140.76, 134.49, 134, 131.3 (CH, Ar), 92.95 (C1), 84.37 (C-5), 79.9 (C-2), 74.46 (C-6), 73.54 (C-3), 65.77 (C-4).
Thiophenyl-4,6-O-benzylidene-β-D-galactopyranose (4): Into an oven-dried 250 mL r. b. f.
was taken ZnCl2 (14.27 g, 5 eq) and flame fused under an atmosphere of N2 to make it totally
anhydrous. To the cooled r. b. f. containing fused ZnCl2 was added benzaldehyde (12.21 g, 5.5
eq) and thiophenyl-β-D-galactopyranose (5.69 g, 20.945 mmol) and the mixture was stirred
under an atmosphere of N2 overnight. Additionally, 15 mL of benzaldehyde was added to the
mixture, for it being the only solvent used in this reaction. When the reaction was found to have
almost reached completion, it was diluted with excess CHCl3 and washed with saturated
NaHCO3 solution (2 x 50 mL). The organic layer was separated and dried over anhydrous
Na2SO4, and evaporated in vacuo to get a crude viscous oily mass. The crude product was
purified by flash column chromatography using gradient dilution technique (40%, 50%, 60%,
70%, 80% and 90% EtOAc in hexanes) to result in 5.2 g (70%) of the desired benzylidene
adduct as a solid white powder. Rf (80% EtOAc/hexanes): 0.245; 1H NMR (500 MHz, CDCl3) δ
8.12-8.14 (m, 2H, Ar), 7.69-7.71 (m, 2H, Ar), 7.61-7.65 (m, 1H, Ar), 7.48-7.51 (m, 1H, Ar),
7.41-7.43 (m, 2H, Ar), 7.37-7.4 (m, 2H, Ar), 5.53 (s, 1H, Bn-Ar), 4.53 (dd, 1H, J1,2 = 4.4 Hz, J1,3
= 2.2 Hz, H-1), 4.4 (dd, 1H, J6,6’ = 12.7 Hz, J5,6’ = 1.5 Hz, H-6), 4.23 (d, 1H, J3,4 = 1.5 Hz, H-4),
4.04 (dd, 1H, J6,6’ = 12.45 Hz, J5,6’ = 2 Hz, H-6’), 3.71-3.75 (m, 2H, H-2, 5), 3.55 (d, 1H, J3,4 = 1
78
Hz);
13
C NMR (125 MHz, CDCl3) δ 137.6, 133.7, 133.69, 130.18, 129.34, 128.93, 128.48,
128.23, 128.18, 126.54 (CH, Ar), 101.4 (Bn-Ar), 87 (C-1), 75.4 (C-4), 73.8 (C-5), 70.06 (C-3),
69.28 (C-6), 68.8 (C-2).
Thiophenyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose (5a): 4.834 g (13.4 eq) of
thiophenyl-4,6-O-benzylidene-β-D-galactopyranose was taken into an oven-dried 250 mL screwcap tube and to it was added 60 mL of anhydrous toluene. The sealed tube was heated to 117 oC
upon thorough stirring for three days in the presence of 4 Ao molecular sieves. After three days,
the tube was cooled to r.t., filtered and evaporated in vacuo to yield a syrupy liquid. To the
syrupy liquid was added 15 mL of anhydrous CHCl3 and an ice-cooled solution of benzoyl
chloride (1.977 g, 1.05 eq) in 15 mL of anhydrous CHCl3. The mixture was stirred for 2 h at r. t.
after which it was evaporated in vacuo to yield 7.838 g of the crude product as a yellowishorange solid. Gradient dilution technique (30%, 40%, 50% and 60% EtOAc in hexanes) was
used to purify the crude by flash column chromatography to yield 1.928 g (31%) of the desired
product as an oily colourless liquid. 0.71 g of a mixture of the 2-O-benzyl- and the 3-O-benzyladducts was also obtained. Rf (30% EtOAc/hexanes): 0.327 (Rf of the 2-O-benzyl adduct: 0.39);
1
H NMR (500 MHz, CDCl3) δ 8.03-8.05 (m. 2H, Ar), 7.72-7.7 (m, 2H, Ar), 7.53-7.56 (m, 1H,
Ar), 7.38-7.42 (m, 5H, Ar), 7.31-7.38 (m, 4H, Ar), 7.25-7.28 (m, 1H, Ar), 5.51 (s, 1H, Bn-Ar),
5.19 (dd, 1H, J2,3 = 11.3 Hz, J3,4 = 3.1 Hz, H-3), 4.68 (d, 1H, J1,2 = 9.5 Hz, H-1), 4.51 (s, 1H, H4), 4.42 (d, 1H, J6,6’ = 12.3 Hz, H-6), 4.06-4.18 (m, 2H, H-2, 6’), 3.7 (s, 1H, H-5); 13C NMR (125
MHz, CDCl3) δ 165.9 (-CO-Ar), 137.3, 133, 130, 129.9, 128, 127.8, 126.3 (CH, Ar), 108.9 (BnAr), 93.1 (C-1), 79.3 (C-4), 79.2 (C-5), 74.4 (C-3), 71.2 (C-2), 67.8 (C-6); LRMS (ESI): Calcd
C26H24O6S: 464.1. Found for (M+Na)+: 487.1.
79
Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose
(6):
To
a
solution of the above obtained thiophenyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose
(2 g, 4.31 mmol) in 10 mL of N, N, N-triethylamine was added N, N-dimethylaminopyridine
(38.67 mg, 0.08 eq) and stirred at 0 oC for 30 min. To the ice-cold solution was added 507.54 mg
(1.5 eq) of acetyl chloride drop-wise and the reaction was vigorously stirred at 0 oC initially and
at r.t. for 2 days. The reaction was terminated by diluting with CH2Cl2 (50 mL) and, by washing
with 2 N HCl (2 x 25 mL) and saturated NaHCO3 solution (2 x 25 mL). The organic layer was
dried over anhydrous MgSO4 and evaporate in vacuo to give 2.44 g of the crude product as an
orange fluffy solid. Purification was carried out by flash column chromatography using 30%
EtOAc in hexanes as the eluent. 1.3 g (60%) of the desired product was obtained as a yellow oily
liquid. Rf (30% EtOAc/hexanes): 0.44; 1H NMR (500 MHz, CDCl3) δ 7.99-7.97 (m, 2H, Ar),
7.63-7.64 (m, 2H, Ar), 7.52-7.55 (m, 1H, Ar), 7.31-7.41 (m, 8H, Ar), 5,56 (d, 1H, J2,3 = 9.9 Hz,
H-2), 5.48 (s, 1H, Bn-Ar), 5.2 (dd, 1H, J1,2 = 9.9 Hz, J2,3 = 9.8 Hz, H-3), 4.79 (d, 1H, J1,2 = 9.8
Hz, H-1), 4.54 (s, 1H, H-4), 4.42 (d, 1H, J6,6’ = 12.4 Hz, H-6), 4.06 (d, 1H, J6,6’ = 12.4 Hz, H-6’),
3.69 (s, 1H, H-5), 2.01 (s, 3H, -CO-CH3);
13
C NMR (125 MHz, CDCl3) δ 170.1 (-CO-CH3),
165.9 (-CO-Ar), 137.3, 133.9, 133, 130, 129.3, 128.6, 127.8, 125 (CH, Ar), 108.9 (Bn-Ar), 89.8
(C-1), 79.6 (C-4), 78.9 (C-5), 73.8 (C-3), 70.4 (C-2), 67.8 (C-6), 21.0 (CH3); LRMS (ESI): Calcd
C28H26O7S: 506.1. Found for (M+NH4)+: 524.2.
Thiophenyl-2-O-acetyl-3-O-benzoyl-β-D-galactopyranose (7a): To a solution of thiophenyl-2O-acetyl-3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranose in anhydrous dichloromethane
(20 mL) at -15 oC was added drop-wise a mixture of 20% v/v trifluoroacetic acid (880 mg, 6 eq)
in CH2Cl2. The mixture was brought to r.t. and stirred overnight in an atmosphere of N2. The
reaction was terminated by the addition of excess CH2Cl2 (30 mL), and washed with saturated
80
NaHCO3 solution (2 x 25 mL) and water (2 x 25 mL). The organic layer was separated and dried
over anhydrous Na2SO4, and evaporated in vacuo to yield 820 mg of the crude 4,6-diol sugar.
The compound was purified by flash column chromatography by gradient dilution method (30%,
40%, 50% and 60% EtOAc in hexanes) to yield 244 mg (45%) of the pure product. Rf (30%
EtOAc/hexanes): 0.137; 1H NMR (500 MHz, CDCl3) δ 7.98-8.0 (m, 3H, Ar), 7.93-7.95 (m, 2H,
Ar), 7.52-7.58 (m, 3H, Ar), 7.44-7.45 (m, 2H, Ar), 5.58 (d, 1H, J1,2 = 9.9 Hz, H-2), 5.22 (dd, 1H,
J2,3 = 9.9 Hz, J3,4 = 3.1 Hz, H-3), 5.07 (d, 1H, J1,2 = 10.1 Hz, H-1), 4.42 (d, 1H, J3,4 = 3.1 Hz, H4), 4.06 (m, 1H, H-5), 3.84 (m, 2H, H-6, 6’), 1.97 (s, 3H, -CO-CH3);
13
C NMR (125 MHz,
CDCl3) δ 170.1 (-CO-CH3), 165.9 (-CO-Ar),133.9, 133, 129.3, 129.3, 129.9, 128, 125.1 (CH,
Ar), 89.8 (C-1), 86.3 (C-5), 73.3 (C-3), 70.1 (C-2), 67.7 (C-4), 61.2 (C-6), 21.0 (CH3); HRMS
(ESI): Calcd C21H23O7S for (M+H)+: 419.1165. Found: 419.1173.
Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-pyruvate-β-D-galactopyranose
(7):
To
a
suspension of thiophenyl-2-O-acetyl-3-O-benzoyl-β-D-galactopyranose (10 mg, 0.024 mmol) in
methyl pyruvate (57.5 mg, 23.5 eq) was added BF3-Etherate drop-wise. The mixture was allowed
to stir at r.t. overnight in an atmosphere of N2. The reaction was terminated by the addition of 1
mL of N, N, N-triethylamine to neutralize the acid. The mixture was poured into 25 mL of
saturate NaHCO3 solution and extracted with CH2Cl2 (2 x 50 mL). The organic layer was dried
over anhydrous Na2SO4 and evaporated in vivo to give the desired pyruvate adduct in its crude
form. The crude product was purified by preparatory TLC using 50% EtOAc in hexanes mixture
as the eluent (with trace amounts of N, N, N-triethylamine) to give 10 mg (80%) of the pyruvate
adduct. Rf (50% EtOAc/hexanes): 0.77; 1H NMR (500 MHz, CDCl3) δ 7.97-8.01 (m, 3H, Ar),
7.92-7.95 (m, 2H, Ar), 7.5-7.54 (m, 2H, Ar), 7.44-7.49 (m, 3H, Ar), 5.45-5.47 (m, 1H, H-2),
5.29-5.34 (m, 1H, H-3), 5.08 (dd, 1H, J1,2 = 10 Hz, J1,3 = 2.8 Hz, H-1), 4.63 (dd, 1H, J3,4 = 7.8 Hz,
81
J4,5 = 3.4, H-4), 4.09-4.1 (m, 1H, H-6), 4.06-4.07 (m, 1H, H-6’), 3.89-3.9 (m, 1H, H-5), 3.27 (s,
3H, -OCH3), 2.01 (s, 3H, -CO-CH3), 1.63 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3) δ 170.2 (CO-CH3), 168.9 (-CO-OCH3), 165.9 (-CO-Ar) 133.9, 133, 130.1, 129.3, 128.6, 125.1 (Ar),
114.9 (Pyr), 89.8 (C-1), 79.1 (C-4), 78.9 (C-5), 73.8 (C-3), 70.4 (C-2), 65.1 (C-6), 52.5 (-COOCH3), 25.5 (CH3), 21.0 (-CO-CH3); HRMS (ESI): Calcd C25H26O9NaS for (M+Na)+: 525.1195.
Found: 525.1205.
4-O-tert-butyldimethylsilyl-cyclohexanol (12a): To a solution of cis/trans mixture of 1, 4cyclohexanediol (200 mg, 1.72 mmol) in N, N-dimethylformamide (5 mL) was added 2.5 eq of
imidazole (292. 7 mg) and the mixture was stirred at r.t. 30 min. To the solution was added tertbutyldimethylsilyl chloride (1.25 eq, 292.7 mg) and the mixture was stirred for 3 days in an
atmosphere of N2. At the end of the third day, the mixture was diluted with excess CH2Cl2 (30
mL), washed with water (5 x 25 mL) and saturated NaCl (aq) solution (1 x 25 mL). The organic
layer was dried over anhydrous Na2SO4 and then evaporated in vacuo to give 276 mg of the
crude product. Purification was done by flash column chromatography using 50% EtOAc in
hexanes as the eluent which gave 196 mg (50%) of the product. Rf (50% EtOAc/hexanes): 0.87;
1
H NMR (500 MHz, CDCl3) δ 3.79-3.83 (m, 1H, H-1), 3.65-3.70 (m, 1H, H-4), 1.6-1.77 (m, 6H,
H-2a, 2e, 3e, 5e, 6a, 6e), 1.46-1.5 (m, 1H, H-3a), 1.28-1.32 (m, 1H, H-5a), 0.89 (s, 9H, t-butyl),
0.04 (s, 6H, CH3); 13C NMR (125 MHz, CDCl3) δ 78.1 (C-4), 69.7 (C-1), 32.1 (C-2, 6), 31.3 (C3, 5), 30.9 (-CCH3, t-butyl), 25.9 (-CCH3, t-butyl), -2.0 (CH3).
2-Phthalimido-per-O-acetyl-β-D-glucopyranose (9): To a solution of NaOCH3 (2 g, 37.037
mmol) in CH3OH (60 mL) in an oven dried 250 mL r. b. f. was added glucosamine HCl (10 g,
46.3 mmol) and the mixture was stirred at r. t. in an atmosphere of N2 for 30 min. 6.852 g of
phthalic anhydride (1 eq) was added to the mixture and was stirred at r. t. for 20 h. The reaction
82
was terminated by evaporating the solvent in vacuo. To the solid residue obtained was added 60
mL of acetic anhydride and 50 mL of pyridine and the mixture was stirred at r. t. for 42 h for peracetylating the sugar. The reaction was terminated by adding 100 g of ice and stirring the
mixture for 30 min. The mixture was extracted with CH2Cl2 (2 x 150 mL). The combined
organic extracts were washed with 3M H2SO4 solution (4 x 100 mL), water (2 x 150 mL),
saturated NaHCO3 and water (2 x 150 mL) again sequentially. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated to dryness in vacuo to give 18.42 g of a yellow
fluffy solid. Flash column chromatography of the crude product by gradient dilution method
(40%, 50% and 60% EtOAc/hexanes) gave 6.54 g (30%) of the desired pure product. R f (40%
EtOAc/hexanes): 0.32; 1H NMR (500 MHz, CDCl3) δ 7.85-7.87 (m, 2H, Ar), 7.73-7.76 (m, 2H,
Ar), 6.51 (d, 1H, J1,2 = 8.9 Hz, H-1), 5.88 (dd, 1H, J2,3 = 10.6 Hz, J3,4 = 9.1 Hz, H-3), 5.21 (d, 1H,
J3,4 = 10 Hz, H-4), 4.46 (dd, 1H, J2,3 = 10.6 Hz, J1,2 = 8.9 Hz, H-2), 4.36 (dd, 1H, J6,6’ = 12.4 Hz,
J5,6 = 4.4 Hz, H-6), 4.11-4.14 (m, 1H, H-6’), 4.01-4.04 (m, 1H, H-5), 2.11 (s, 3H, -CO-CH3),
2.04 (s, 3H, -CO-CH3), 2.00 (s, 3H, -CO-CH3), 1.86 (s, 3H, -CO-CH3);
13
C NMR (125 MHz,
CDCl3) δ 172.9, 170.2 (-CO-CH3), 167.6 (-CO-N-), 132.2, 123.7 (Ar), 93.4 (C-1), 74.7 (C-5),
70.8 (C-3), 69.9 (C-4), 61.9 (C-2), 62.4 (C-6), 21.0 (-CO-CH3).
Thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (10): To a solution of 2phthalimido-per-O-acetyl-β-D-glucopyranose (1.5 g, 3.14 mmol) in 30 mL of CHCl3 in a clean
oven dried 100 mL r. b. f. was added ethanethiol (1.2 eq, 234 mg) and the mixture was stirred at
r. t. for 15 min in an atmosphere of N2. To the mixture was added BF3. Et2O drop-wise and the
mixture was stirred at r. t. for 4 days. When TLC showed that the reaction reached completion,
the mixture was diluted with 100 mL of CH2Cl2, washed with saturated NaHCO3 solution (2 x
150 mL) and 10% w/v NaOH (aq) solution (1 x 100 mL). The combined organic layers were
83
dried over anhydrous Na2SO4 and concentrated to dryness in vacuo to give 1.35 g of a red fluffy
solid. The crude product was purified by flash column chromatography, using 50% EtOAc in
hexanes as the eluent, to yield 1.188 g (79%) of the pure product. Rf (50% EtOAc/hexanes):
0.49; 1H NMR (500 MHz, CDCl3) δ 7.85-7.86 (m, 2H, Ar), 7.73-7.76 (m, 2H, Ar), 5.82 (dd, 1H,
J2,3 = 10.3 Hz, J3,4 = 9.2 Hz, H-3), 5.48 (d, 1H, J1,2 = 10.7 Hz, H-1), 5.17 (dd, 1H, J4,5 = 10.1 Hz,
J3,4 = 9.2 Hz, H-4), 4.39 (d, 1H, J1,2 = 10.4 Hz, H-2), 4.3 (dd, 1H, J6,6’ = 12.4 Hz, J5,6 = 4.9 Hz, H6), 4.17 (dd, 1H, J6,6’ = 12.3 Hz, J5,6’ = 2.3 Hz, H-6’), 3.89 (ddd, 1H, J4,5 = 10.25 Hz, J5,6 = 4.85
Hz, J5,6’ = 2.4 Hz, H-5), 2.62-2.71 (m, 2H, -S-CH2-CH3), 2.10 (s, 3H, -CO-CH3), 2.03 (s, 3H, CO-CH3), 1.86 (s, 3H, -CO-CH3), 1.21 (t, 3H, JCH2,CH3 = 7.3 Hz, -S-CH2-CH3); 13C NMR (125
MHz, CDCl3) δ 170.2 (-CO-CH3), 169.6 (-CO-N-), 132.2, 123.8 (Ar), 81.3 (C-1), 76.12 (C-5),
71.7 (C-3), 69.1 (C-4), 62.48 (C-6), 53.86 (C-2), 24.52 (-S-CH2-CH3), 20.91, 20.77, 20.6 (-COCH3), 15.04 (-S-CH2-CH3).
(4-tert-butyldimethylsilyl-cyclohexyl)-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose
(13): Into a clean oven dried 25 mL r. b. f. was taken 575 mg (1.2 mmol) of thioethyl-2phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose, To it was added 15 mL anhydrous CH2Cl2
and the mixture was cooled to -78 oC (acetone/dry ice) upon stirring in an inert atmosphere. To
the mixture was added N-iodosuccinimide (2.5 eq, 675 mg) and stirred at -78 oC for 30 min.
Keeping the temperature constant, 10 µL (0.05 eq) of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) was added drop-wise to the mixture upon vigorous stirring. To the mixture was added
drop-wise a solution of 4-O-tert-butyldimethylsilyl-cyclohexanol (1.2 eq, 331.2 mg) in CH2Cl2
(2 mL) and the reaction was stirred at -78 oC for 90 min. The reaction was terminated and the
mixture was diluted with excess CH2Cl2 (100 mL) and washed with saturated NaHCO3 solution
(1 x 50 mL). The organic layer was dried over anhydrous MgSO4, filtered and concentrated to
84
dryness in vacuo to yield a gram of the crude product as a dark brown oily syrup. Purification
was done by flash column chromatography using gradient dilution (30%, 40%, 50%, 60%, 70%,
80%, 90% EtOAc in hexanes) which yielded 503 mg (65%) of the product as a pale yellow oily
syrup. Rf (60% EtOAc/hexanes): 0.83; 1H NMR (500 MHz, CDCl3) δ 7.84-7.87 (m, 2H, Ar),
7.71-7.75 (m, 2H, Ar), 5.76-5.83 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.75 Hz, J1,3 = 2.4 Hz, H-1),
5.13-5.18 (m, 1H, H-4), 4.28-4.35 (m, 2H, H-2, 6), 4.16 (d, 1H, J6,6’ = 12.2 Hz, H-6’) 3.84-3.87
(m, 1H, H-5), 3.59-3.68 (m, 1H, Cyclohexyl-H-1), 3.49-3.53 (m, 1H, Cyclohexyl-H-4), 2.11 (s,
3H, -CO-CH3), 2.03 (s, 3H, -CO-CH3), 1.86 (s, 3H, -CO-CH3), 1.68-1.8 (m, 2H, Cyclohexyl-H2e, 6e), 1.24-1.40 (m, 4H, Cyclohexyl-H-3e, 2a, 5e, 6a), 1.19-1.40 (m, 1H, Cyclohexyl-H-3a), 0.83
(s, 5H, H-t-butyl), 0.74 (s, 4H, H-t-butyl), -0.04 (s, 3H, CH3), -0.08 (s, 3H, CH3), -0.12 (s, 3H,
CH3);
13
C NMR (125 MHz, CDCl3) δ 172.96, 171.06 (-CO-CH3), 169.84 (-CO-N-), 134.57,
123.92 (Ar), 97.05 (C-1), 72.11 (C-5), 71.21 (C-3), 69.59 (C-4), 62.60 (C-6), 49.66 (C-2), 31.13
(-CCH3, t-butyl), 26.15, 26.02 (-CCH3, t-butyl), 21.13, 21, 20.82 (-CO-CH3), -4.43 (CH3);
LRMS (ESI): Calcd C32H45NO11Si: 647.3. Found for (M+H)+: 648.3.
Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (14): Into a clean oven dried
10 mL r. b. f. was taken 25 mg (0.052 mmol) of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-Dglucopyranose, To it was added 5 mL anhydrous CH2Cl2 and the mixture was cooled to -78 oC
upon stirring in an inert atmosphere. To the mixture was added N-iodosuccinimide (2.5 eq, 29.34
mg) and stirred at -78 oC (acetone/dry ice) for 30 min. Keeping the temperature constant, 0.46
µL (0.05 eq) of trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added drop-wise to the
mixture upon vigorous stirring. To the mixture was added drop-wise a solution of 4-O-tertbutyldimethylsilyl-cyclohexanol (1.2 eq, 14.35 mg) in CH2Cl2 (1 mL) and the reaction was
stirred at -78 oC for 7 h. The reaction was terminated and the mixture was diluted with CH 2Cl2
85
(25 mL) and washed with saturated NaHCO3 solution (1 x 25 mL). The organic layer was dried
over anhydrous MgSO4, filtered and concentrated to dryness in vacuo to yield the crude product
as a orange-brown oily syrup. Purification was done by preparatory TLC technique using 80%
EtOAc in hexanes as the solvent which yielded 32 mg (95%) of the product as a yellowish oily
syrup. Rf (60% EtOAc/hexanes): 0.157; 1H NMR (500 MHz, CDCl3) δ 7.84-7.86 (m, 2H, Ar),
7.72-7.76 (m, 2H, Ar), 5.75-5.82 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.4 Hz, J1,3 = 6.4 Hz, H-1),
5.15-5.19 (m, 1H, H-4), 4.28-4.36 (m, 2H, H-2, 6), 4.16 (m, 1H, J6,6’ = 10.8 Hz, H-6’), 3.85-3.88
(m, 1H, H-5), 3.73-3.77 (m, 1H, Cyclohexyl-H-1), 3.56-3.65 (m, 1H, Cyclohexyl-H-4), 2.11 (s,
3H, -CO-CH3), 2.03 (s, 3H, -CO-CH3), 1.95-1.98 (m, 1H, Cyclohexyl-H-2e), 1.86 (s, 3H, -COCH3), 1.69-1.76 (m, 1H, Cyclohexyl-H-6e), 1.33-1.43 (m, 2H, Cyclohexyl-H-3e, 5e), 1.17-1.31
(m, 2H, Cyclohexyl-H-3a, 5a);
13
C NMR (125 MHz, CDCl3) δ 170.73, 170.22 (-CO-CH3),
169.51, 165.52 (-CO-N-), 134.35, 131.38, 123.6 (Ar), 96.85 (C-1), 76.86 (Cyclohexyl-C-1),
71.78 (C-3, 5), 70.88 (C-4), 69.19 (Cyclohexyl-C-4), 62.23 (C-6), 54.86 (C-2), 30.47, 30.06
(Cyclohexyl-C-2, 3, 5, 6), 20.78, 20.65, 20.46 (-CO-CH3). LRMS (ESI): Calcd C26H31NO11:
533.2. Found for (M+H)+: 534.2.
(Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-2-O-acetyl-3-O-benzoyl4,6-O-pyruvate-β-D-galactopyranose (15): 61.279 mg (1.3 eq) of thiophenyl-2-O-acetyl-3-Obenzoyl-4,6-O-pyruvate-β-D-galactopyranose and 5 mL of anhydrous CH2Cl2 were taken in a
clean oven tried 10 mL r. b. f. and the mixture was stirred at -42 oC (CH3CN/dry ice) in an inert
atmosphere for 15 min. 68.625 mg (2.5 eq) of N-iodosuccinimide was added to the mixture and
stirred for 30 min at -42 oC. Keeping the temperature constant, 1.3 µL of TMSOTf (0.05 eq) was
added to the mixture drop-wise upon vigorous stirring. To the mixture was added a solution of
cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose (50 mg, 0.0939 mmol) in
86
anhydrous CH2Cl2 (1 mL) and the reaction was run at -42 oC for 3 h upon continuous stirring.
The reaction was terminated by adding excess CH2Cl2 (40 mL), washed with saturated NaHCO3
solution (1 x 50 mL) and 10% w/v Na2S2O3 solution (1 x 50 mL). The organic layer was
separated, dried over anhydrous MgSO4, filtered and concentrated to dryness in vacuo to yield
135 mg of the crude product as an orange oily syrup. Purification was done by preparatory TLC
technique using 60 % EtOAc in hexanes as the solvent, which gave 50 mg (57.6%) of the
product as an off white coloured solid. 1H NMR (500 MHz, CDCl3) δ 7.99-8.04 (m, 2H, Ar),
7.91-7.96 (m, 1H, Ar), 7.83-7.88 (m, 1H, Ar), 7.73-7.77 (m, 1H, Ar), 7.49-7.54 (m, 1H, Ar),
7.36-7.41 (m, 1H, Ar), 7.35-7.41 (m, 2H, Ar), 5.73-5.80 (m, 1H, Hglu-1), 5.62-5.66 (m, 1H,
Hgal-1), 5.36-5.46 (m, 2H, Hglu-3, Hgal-2), 5.13-5.19 (m, 1H, Hgal-3), 4.48-4.60 (m, 1H, Hglu-4),
4.26-4.34 (m, 2H, Hgal-4, Hglu-2), 4.10-4.27 (m, 2H, Hglu-5, 6), 3.93-4.08 (m, 2H, Hglu-6’, Hgal-6),
3.80-3.90 (m, 2H, Hgal-6’, Cyclohexyl-H-1), 3.73-3.78 (m, 1H, Hgal-5), 3.61-3.7 (m, 3H, -OCH3),
3.41-3.53 (m, 1H, Cyclohexyl-H-4), 2.11 (s, 1H, -CO-CH3), 2.10 (s, 1H, -CO-CH3), 2.06 (s, 1H,
-CO-CH3), 1.87 (s, 1H, -CO-CH3), 1.86 (s, 1H, -CO-CH3), 1.85 (s, 1H, -CO-CH3), 1.65-1.83 (m,
4H, Cyclohexyl-H-2a, 2e, 6a, 6e), 1.57 (s, 3H, -CO-CH3), 1.37-1.42 (m, 1H, Cyclohexyl-H3e),1.27-1.31 (m, 1H, Cyclohexyl-H-5e) 1.25 (s, 3H, CH3), 0.86-0.92 (m, 1H, Cyclohexyl-H-3a);
C NMR (125 MHz, CDCl3) δ 170.70, 170.20 (-CO-CH3), 169.49 (-CO-OCH3), 165.89 (-CO-
13
N-), 134.37, 133.16, 131.31, 129.88, 129.77, 129.67, 128.42, 128.36, 127.47, 123.61 (Ar),
100.36 (pyr), 98.69 (Cgal-1), 96.65 (Cglu-1), 96.12 (Cyclohexyl-C-1), 95.91 (Cyclohexyl-C-4),
72.04 (Cgal-4), 71.93 (Cgal-5), 71.79 (Cglu-5), 70.82 (Cgal-3), 69.22 (Cglu-3), 69.13 (Cgal-4), 66.7
(Cgal-2), 65.34 (Cgal-6), 64.01 (Cglu-6), 62.17 (Cglu-2), 54.8 (CO-OCH3), 31.93, 29.70, 29.36,
28.98 (Cyclohexyl-C-2, 3, 5, 6), 25.72 (CH3), 20.78, 20.65, 20.47 (-CO-CH3); HRMS (ESI):
Calcd C45H51NO20Na for (M+Na)+: 948.2902. Found: 948.2899.
87
4.2. 6-O-p-toluenesulfonyl-D-arabino-hex-1-enitol (17).
To a solution of D-glucal (7.6 g, 52.5 mmol) in anhydrous pyridine was added dropwise a
solution of p-toluenesulfonyl chloride (15.02 g, 78.8 mmol) in anhydrous CH2Cl2 at 0 oC upon
vigorous stirring. The reaction was terminated after 3 h by the addition of water, followed by
washing with saturated CuSO4 (3 x 30 mL), water (5 x 30 mL) and brine (1 x 30 mL). The
organic extracts were dried under anhydrous MgSO4 and concentrated in vacuo to yield crude
17. Although the compound was synthesized earlier,13 no spectroscopic data was reported. The
product was purified by flash chromatography using 3%, 4% and 5% CH3OH in CHCl3 mixtures
to give 8.18 g (56% yield) of 17 as a colorless oily mass which crystallized upon freezing: Rf
(1:20 CH3OH/CHCl3): 0.46; mp 50–51 oC; 1H NMR (500 MHz, CDCl3) δ 7.81 (d, 2H, J = 6.3
Hz, Ar), 7.35 (d, 2H, J = 7.9 Hz, Ar), 6.24 (d, 1H, J1,2 = 6.1, J1,3 = 1.8 Hz, H-1), 4.74 (dd, 1H, J2,1
= 6.3, J2,3 = 2.2 Hz, H-2), 4.47 (dd, 1H, J6,6’ = 11.25, J6,5 = 4Hz, H-6), 4.28 (dd, 1H, J6,6’ = 11.45,
J6’,5 = 2.5 Hz, H-6’), 4.26 (d, 1H, J3,4 = 6.8 Hz, H-3), 3.91 (ddd, 1H, J5,4 = 9.75, J5,6 = 3.9, J5,6’ = 2
Hz, H-5), 3.76 (dd, 1H, J4,3 = 8.3, J4,5 =8.3 Hz, H-4), 3.09 (s, 1H, 4-OH), 2.45 (s, 3H, PhCH3),
2.23 (s, 1H, 3-OH);
13
C NMR (125 MHz, CDCl3) δ 145.19 (CH, Ar), 143.99 (C-1), 132.61,
129.93 , 128.02 (CH, Ar), 103.04 (C-2), 75.7 (C-5), 69.57 (C-3), 69.35 (C-4), 67.92 (C-6), 21.66
(PhCH3).
4.3. 3,6-Anhydro-2-deoxy-D-arabino-hex-1-enitol (18).
To a solution of 17 (200 mg, 0.33 mmol) in 20 mL of tetrahydrofuran (THF) was added
dropwise 0.83 mL of 2.4 M LiAlH4 in THF (2 mmol) at 0 oC. The mixture was refluxed at 73 oC
for two days. It was then cooled to 0 oC and neutralized with 15% w/v NaOH and water, and
filtered through a Celite@ bed upon dilution with ethyl acetate (EtOAc). The mixture was
concentrated in vacuo and the product was purified by flash chromatography using 1:1 mixture
88
of EtOAc/Hexanes to give 73 mg (84%) of 18 as a white solid: Rf (1:20 CH3OH/CHCl3): 0.42;
22
D]
-24.17 (c 0.58, CHCl3); 1H NMR (500 MHz, CDCl3) δ 6.54 (d, 1H, J1,2 = 5.0 Hz, H-1),
5.02 (ddd, 1H, J2,1 = 5.25, J2,3 = 5.5, J2,4 = 1.5 Hz, H-2), 4.36 (br dd, 1H, J5,6 =3.65, J5,6’ =3.65
Hz, H-5), 4.27 (d, 1H, J6’,6 =11.2 Hz, H-6’), 4.23-4.2 (m, 1H, H-4), 4.18 (dd, 1H, J6,6’ = 11.3, J5,6
= 4.5 Hz, H-6), 4.03 (br dd, 1H, J3,4 =4.5, J3,2= 5.5 Hz, H-3), 2.09 (d, 1H, J4,-OH = 10.7 Hz, 4-OH);
C NMR (125 MHz, CDCl3) δ 146.53 (C-1), 100.41 (C-2), 76.56 (C-5), 74.71 (C-6), 68.76 (C-
13
3), 66.26 (C-4); Anal. Calcd. for C6H8O3: C, 56.24; H, 6.29. Found: C, 56.15; H, 6.42.
4.4 . General procedure for the synthesis of 3,6-anhydro-O-glycosides.
To a solution of 50 mg (0.39 mmol) of 18 in 5 mL of anhydrous CH3CN at 0 oC was added
105.5 mg (0.47mmol) of N-iodosuccinimide. To the solution was then added 1.2 equivalents of
the desired nucleophile and the reaction mixture was stirred at r. t. overnight. The solvent was
evaporated in vacuo and the crude product was purified by preparatory thin-layer
chromatography (TLC) using 1:50 CH3OH/CHCl3 as the solvent.
4.4.1. Methyl-3,6-anhydro-2-deoxy-2-iodo-gluco- and manno-pyranosides (19a, 19b, 19c).
Data for the mixture of diastereomers: 1H NMR (500 MHz, CDCl3) δ 5.31 (s, 1H), 5.03 (d,
1H, J = 7.8 Hz), 4.72 (s, 1H), 4.47 (dd, 1H, J = 4.4 Hz, J = 3.4 Hz), 4.42-4.44 (m, 1H), 4.39-4.36
(m, 2H), 4.28-4.32 (m, 2H), 4.24 (d, 2H, J = 10.7 Hz), 4.15-4.12 (m, 1H), 4.09-4.06 (m, 3H),
3.96 (dd, 1H, J = 10 Hz, J = 5 Hz), 3.91 (dd, 1H, J = 5 Hz, J = 10 Hz), 3.54 (s, 1H), 3.48 (s, 1H),
3.46 (s, 1H), 2.64 (d, 1H, J = 8.8 Hz), 2.44 (d, 1H, J = 5 Hz), 2.41 (d, 1H, J = 8.8 Hz), 2.04 (d,
1H, J = 5.3 Hz); 13C NMR (125 MHz, CDCl3) δ 80.43, 72.81, 69.06, 29.57, 26.60, 20.28; HRMS
(ESI): Calcd C7H11O4NaI for (M+Na)+: 308.9611. Found: 308.9600.
4.5. General procedure for the synthesis of 2-deoxythioglycosides.
89
To a stirred solution of 50 mg (0.39 mmol) of 18 and 21 mg (0.039 mmol) of Ce(NH4)2(NO3)6
in anhydrous CH3CN (5 mL) was added a solution of 0.2 mL (1.95 mmol) of PhSH in anhydrous
CH3CN (5 mL) at 0 oC and allowed to stir overnight at r.t.. The solvent was evaporated in vacuo
and the residue was diluted with excess CH2Cl2, washed with 10% NaOH, saturated NaHCO3
and saturated NaCl and, dried over anhydrous Na2SO4. The organic solvent was evaporated in
vacuo and the crude (150 mg) obtained was purified by preparatory TLC using 1:50
CH3OH/CHCl3 as the solvent to give mixtures of compounds 20a, 20b (60 mg) and 21a, 21b (33
mg).
4.5.1. Thiophenyl-3,6-anhydro-2-deoxy-D-glucopyranoside (20a, 20b).
Data for α-anomer (20b): Rf (1:50 CH3OH/CHCl3): 0.84; 1H NMR (500 MHz, CDCl3) δ 7.547.50 (m, 2H, Ar), 7.33-7.24 (m, 3H, Ar), 5.73 (dd, 1H, J1,2 = 8.0, J1,2’ = 3.2 Hz, H-1), 4.69-4.67
(m, 1H, H-5), 4.59 (dd, 1H, J2,3 = 5.4, J3,4 = 1.2 Hz, H-3), 4.29-4.26 (m, 1H, H-4), 3.99 (dd, 1H,
J6,6’ = 9.7, J5,6 = 4.4 Hz, H-6), 3.84 (dd, 1H, J6,6’ = 9, J5,6’ = 5.5 Hz, H-6’), 3.06 (d, 1H, J4,-OH =
10.7 Hz, 4-OH), 2.63 (ddd, 1H, J2,2’ = 14.15, J1,2 = 8.55, J2,3 = 6.3 Hz, H-2), 2.35 (ddd, 1H, J2,2’ =
14.1, J1,2’ = 3.15, J2’,3 = 1.3 Hz, H-2’); 13C NMR (125 MHz, CDCl3) δ 132.3, 130.8, 129.1 (CH,
Ar), 89.8 (C-1), 85.17 (C-5), 82.9 (C-3), 74.86 (C-6), 71.83 (C-4), 40.6 (C-2); HRMS (ESI):
Calcd C12H14O3NaS for (M+Na)+: 261.0570. Found: 261.0561. Data for β-anomer (20a): Rf
(1:50 CH3OH/CHCl3): 0.76; 1H NMR (500 MHz, CDCl3) δ 7.54-7.50 (m, 2H, Ar), 7.33-7.24 (m,
3H, Ar), 5.66 (dd, 1H, J1,2 = 6.8, J1,2’ = 1.3 Hz, H-1), 4.69-4.67 (m, 1H, H-5), 4.64 (dd, 1H, J3,4 =
5.4, J2,3 = 1.2 Hz, H-3), 4.26-4.25 (m, 1H, H-4), 3.82 (dd, 1H, J6,6’ = 9.55, J5,6’ = 5.7 Hz, H-6’),
3.63 (dd, 1H, J6,6’ = 9.5, J5,6 = 6.2 Hz, H-6), 2.58 (d, 1H, J4,-OH = 7.4 Hz, 4-OH), 2.5 (ddd, 1H,
J2,2’ = 14.1, J1,2 = 6.35, J2,3 = 2 Hz, H-2), 2.17-2.22 (m, 1H, H-2’); 13C NMR (125 MHz, CDCl3) δ
90
129, 127.78, 127.35 (CH, Ar), 88.41 (C-1), 82.52 (C-5), 82.39 (C-3), 73.16 (C-6), 72.12 (C-4),
40.77 (C-2); HRMS (ESI): Calcd C12H14O3NaS for (M+Na)+: 261.0570. Found: 261.0561.
4.5.2. Thiophenyl-2,3-dideoxy-D-glucopyranoside (21a, 21b).
Data for α-anomer (21a): Rf (1:50 CH3OH/CHCl3): 0.19; 1H NMR (500 MHz, CDCl3) δ
7.51-7.53 (m, 2H, Ar), 7.26-7.33 (m, 3H, Ar), 6.0 (dd, 1H, J1,2 = 10.2 Hz, J2,3 = 2 Hz, H-2), 5.98
(d, 1H, J1,2 = 10.2 Hz, H-1), 5.74 (dd, 1H, J3,4 = 1.2 Hz, J2,3 = 2.7 Hz, H-3), 4.32 (dd, 1H, J3,4 = 2
Hz, J4,5 = 8.75 Hz, H-4), 4.03-4.07 (m, 1H, H-5), 3.86-3.92 (m, 2H, H-6, 6’), 1.85 (d, 1H, J4,-OH =
7.3 Hz, 4-OH), 1.82 (dd, 1H, J6,-OH = 6.35 Hz, J6’,-OH = 6.4 Hz, 6-OH);
13
C NMR (125 MHz,
CDCl3) δ 131.77, 129.03, 127.32 (CH, Ar), 131.72 (C-1), 127.12 (C-2), 83.61 (C-3), 71.88 (C-5),
64.29 (C-4), 62.91 (C-6); HRMS (ESI): Calcd C12H14O3NaS for (M+Na)+: 261.0561. Found:
261.0561. Data for β-anomer (21b): Rf (1:50 CH3OH/CHCl3): 0.19; 1H NMR (500 MHz,
CDCl3) δ 7.51-7.53 (m, 2H, Ar), 7.26-7.33 (m, 3H, Ar), 5.88 (dd, 1H, J1,2 = 2 Hz, J2,3 = 1.9 Hz,
H-2), 5.86 (dd, 1H, J1,2 = 1.5 Hz, J2,3 = 1.4 Hz, H-1), 4.72-4.78 (m, 1H, H-4), 4.54-4.60 (m, 1H,
H-5), 4.11-4.16 (m, 2H, H-6, 6’), 2.18-2.24 (m, 4-OH, 6-OH);
13
C NMR (125 MHz, CDCl3) δ
135.10, 132.5, 131.69 (CH, Ar), 131.62 (C-1), 128.84 (C-2), 81.74 (C-3), 79.74 (C-5), 63.40 (C4), 62.75 (C-6); HRMS (ESI): Calcd C12H14O3NaS for (M+Na)+: 261.0561. Found: 261.0561.
4.6. General procedure for the preparation of the dianhydrosugar.
To 100 mg (0.78 mmol) of 18 in 10 mL of CH3CN was added 210 mg (0.94 mmol) of Niodosuccinimide in the presence of 4 Ǻ molecular sieves, and allowed to stir overnight at r.t. The
solvent was removed in vacuo and the residue was diluted with excess CH2Cl2, washed with 10%
Na2S2O3, saturated NaCl and dried over anhydrous Na2SO4. The organic filtrate was evaporated
in vacuo and the crude product (170 mg) was purified by flash chromatography using 2:3
91
EtOAc/Hexanes to give 150 mg (0.61 mmol, 76 %) of 22. In the absence of activated sieves, a
mixture of 2-deoxy-2-iodo-hexopyranoses (23a-c), were also obtained (30%).
4.6.1. 1,4:3,6-Dianhydro-2-deoxy-2-iodo-D-glucopyranose (22).
Data for the dianhydrosugar: Rf (1:50 CH3OH/CHCl3): 0.9; 1H NMR (500 MHz, CDCl3) δ
5.51 (br s, 1H, H-1), 5.09 (dd, 1H, J4,3= 4.25, J4,5 = 4.5 Hz, H-4), 4.27 (dd, 1H, J5,4 = 3.5, J5,6 =
3.25 Hz, H-5), 4.18 (dd, 1H, J3,2 = 7.0, J3,4 = 5.0 Hz, H-3), 4.15 (d, 1H, J6,6’ = 10.8 Hz, H-6’), 4.0
(dd, 1H, J2,3 = 7.0, J2,1 = 2.0 Hz, H-2), 3.94 (dd, 1H, J6,6’ =10.7, J5,6 = 3.0 Hz, H-6);
13
C NMR
(125 MHz, CDCl3) δ 100.20 (C-1), 79.87 (C-4), 77.02 (C-5), 73.28 (C-3), 71.30 (C-6), 32.85 (C2); LRMS (ESI) m/Z (rel. intensity) 254.9 (M+H)+ (79).
4.6.2. 3,6-anhydro-2-deoxy-2-iodo-D-hexopyranose (23a, 23b, 23c).
Data for mixture of diastereomers: Rf 1:50 CH3OH/CHCl3): 0.12; 1H NMR (500 MHz,
CDCl3) δ 5.76 (s, 1H), 5.68 (d, 1H, J = 5 Hz), 5.38 (s, 1H), 5.02 (dd, 1H, J = 5.4 Hz, J = 4.9 Hz),
4.89 (d, 1H, J =), 4.83 (ddd, 1H, J = 15 Hz, J = 5 Hz, J = 5 Hz), 4.73-4.70 (m, 2H), 4.48 (d, 1H, J
= 1.4 Hz), 4.44 (d, 1H, J = 4.9 Hz), 4.40-4.37 (m, 2H), 4.19 (dd, 1H, J = 4.9 Hz, J = 4.4 Hz), 4.14
(dd, 1H, J =), 4.09-4.06 (m, 2H), 4.04-3.98 (m, 2H), 3.79-3.76 (m, 2H), 3.62 (dd, 1H, J = 9.5 Hz,
J = 6.6 Hz);
C NMR (125 MHz, CDCl3) δ 107.69, 99.48, 96.06, 90.68, 84.04, 83.32, 82.26,
13
76.37, 75.55, 71.75, 70.55, 46.57, 46.56, 26.66, 25.12, 23.61, 8.72, 8.71; LRMS (ESI): Calcd
C6H9IO4: 271.9. Found for (M+Na)+: 294.8.
92
4.2 NMR Spectra
93
0.3
0.2
0.1
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
4.0
3.5
2.11
2.09
2.03
1.96
1.0
3.0
2.5
1.96
2.13
5.41
5.41
5.41
5.23
5.21
5.05
5.05
4.72
4.19
4.70
4.18
4.16
4.11
4.10
4.10
4.09
3.95
3.93
7.51
7.51
7.50
7.31
7.30
7.32
7.30
7.26
Normalized Intensity
vb_276_pure_1H.esp
0.9
0.8
0.7
0.6
0.5
0.4
0
2.0
1.5
94
0.3
0.2
170.35
170.18
170.03
169.41
0.4
0.6
200
180
160
140
0.7
120
100
Chemical Shift (ppm)
80
61.63
74.42
72.00
67.22
86.60
128.16
77.31
77.06
76.80
0.8
0.5
60
20.63
20.58
20.85
Normalized Intensity
132.56
128.89
vb_276_pure_13C.esp
1.0
0.9
0.1
0
40
20
95
4
6
7.5
7.0
6.5
6.0
5.5
5.0 4.5 4.0 3.5
F2 Chemical Shift (ppm)
3.0
2.5
2.0
1.5
1.0
96
F1 Chemical Shift (ppm)
2
0
40
60
80
100
120
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
0
97
F1 Chemical Shift ( ppm)
20
8.5
8.0
7.5
7.0
0.4
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
4.0
3.5
0.2
3.0
2.50
2.50
2.50
3.41
3.36
3.36
3.52
3.49
3.43
7.44
4.72
4.58
4.57
3.47
0.8
3.34
3.34
3.53
0.6
3.72
3.71
7.28
7.45
0.9
2.51
2.49
3.54
0.3
7.18
0.5
7.20
0.7
7.46
Normalized Intensity
7.29
vb_277_pure_1H.esp
1.0
0.1
0
2.5
2.0
1.5
98
1.0 vb_277_pure_13C.esp
0.9
129.21
128.74
0.8
0.6
0.5
60.44
0.2
69.14
68.20
74.58
0.3
79.10
126.03
87.68
0.4
135.51
Normalized Intensity
0.7
0.1
150
140
130
120
110
100
90
80
70
Chemical Shift (ppm)
60
50
40
30
20
10
99
2
4
6
8
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
0
100
F1 Chemical Shift ( ppm)
0
40
60
80
100
120
9
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
0
101
F1 Chemical Shift (ppm)
20
0.2
3.75
3.71
0.3
0.5
8.5
8.0
7.70
7.5
0.4
7.0
6.5
6.0
5.5
Chemical Shift (ppm)
5.0
4.5
4.0
3.56
3.55
0.8
3.72
3.73
7.50
0.7
7.32
7.30
7.29
7.72
7.70
7.39
7.39
5.53
1.0
3.74
0.6
8.14
8.14
8.12
8.12
0.9
4.54
4.52
4.41
4.41
4.39
4.23
4.06
4.23
4.05
7.63
Normalized Intensity
vb_285_B_1H.esp
0.1
0
3.5
3.0
102
2.5
77.29
77.04
76.79
vb_285_B_13C.esp
1.0
0.8
0.4
75.39
73.82
70.06
69.28
68.80
101.39
0.5
87.00
0.6
0.3
137.62
Normalized Intensity
0.7
128.47
128.23
126.53
133.71
130.18
0.9
0.2
0.1
170
160
150
140
130
120
110
100
90
Chemical Shift (ppm)
80
70
60
50
40
103
30
4
6
8
9
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
104
F1 Chemical Shift (ppm)
2
60
80
100
120
140
9
8
7
6
5
F2 Chemic al Shif t (ppm)
4
3
2
105
F1 Chemical Shift ( ppm)
40
0.3
0.2
8.5
8.0
7.5
0.4
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
1.27
1.26
0.8
3.5
3.0
2.5
2.0
0.96
0.95
0.94
1.28
1.25
3.70
5.51
0.6
1.81
4.69
4.67
4.51
4.51
4.15 4.41
4.13
4.08 4.12
4.12
4.06
5.21
5.20
5.19
5.18
0.5
8.05
8.03
7.72
7.70
7.42
7.26
7.41
0.9
7.25
7.25
7.54
Normalized Intensity
7.27
1.0 vb_153_B.esp
0.7
0.1
0
1.5
1.0
106
4
6
8
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
107
F1 Chemical Shift (ppm)
2
2.01
1.0
vb_162_A.esp
0.9
7.26
0.8
7.35
0.6
0.1
3.69
4.07
4.05
4.80
4.78
4.54
4.43
4.40
5.21
5.19
5.58
7.52
0.2
5.56
7.41
0.3
5.48
0.4
7.63
7.39
7.37
0.5
7.99
7.97
Normalized Intensity
0.7
0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
108
1.0
4
6
8.0
7.5
7.0
6.5
6.0
5.5 5.0 4.5 4.0
F2 Chemic al Shif t (ppm)
3.5
3.0
2.5
2.0
1.5
109
F1 Chemical Shift (ppm)
2
1.97
vb_169_B
0.25
1.97
0.15
M02(dd)
M07(m)
1.96
M04(d)
4.42
4.42
5.23
5.08
5.06
M01(t)
5.60
5.58
5.56
0.05
M06(m)
M03(d)
4.07
4.05
4.04 3.84
3.84
3.83
0.10
8.00
8.00
7.98
7.66 7.95 7.98
7.64 7.93
7.47
7.45
7.42
7.44
7.42
Normalized Intensity
0.20
0
1.37
8.0
7.5
7.0
6.5
6.0
1.81 1.85
1.73
5.5
5.0
4.5
Chemical Shift (ppm)
1.23 4.00
4.0
3.5
3.0
2.5
2.0
110
4
6
8
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
111
F1 Chemical Shift ( ppm)
2
0.1
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
1.99
1.63
1.62
1.62
1.59
5.47
5.47
5.45
5.32
5.31
5.30
5.10
5.09
5.08
5.07
4.63
4.09
4.07
4.07
4.07
3.90
3.27
3.26
3.25
7.99
7.99
7.98
7.98
7.94
7.93
7.52
7.52
7.50
7.47
7.45
Normalized Intensity
0.5
2.00
1.63
3.27
vb_171_pure
0.9
0.8
0.7
0.6
0.4
0.3
0.2
0
2.5
2.0
1.5
1.0
112
0.5
4
6
8
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
113
F1 Chemical Shift ( ppm)
2
0.2
0.1
9.0
8.5
8.0
7.5
7.0
6.5
4.49
4.47
4.47
4.37
4.36
4.35
4.16
4.14
4.13
4.11
4.01
5.23
5.21
5.91
5.89
5.88
5.87
7.26
0.6
6.52
6.50
2.12
0.3
7.86
7.86
7.76
7.74 7.75
7.75
7.73
7.74
7.87
Normalized Intensity
1.0
2.11
2.04
2.00
1.86
vb_259_pure_1H.esp
0.9
0.8
0.7
0.5
0.4
0
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
114
4
6
8
8
7
6
5
F2 Chemic al Shif t (ppm)
4
3
2
115
F1 Chemical Shift ( ppm)
2
0.2
9
0.1
8
7
6
5
4
Chemical Shift (ppm)
3
1.26
2.13 2.11
4.42
4.40
4.32
4.30
4.29
3.91 4.19
3.90 4.19
3.89
2.72
2.71
2.69
2.67
2.65
5.85
5.83
5.81 5.50
5.48
5.20
5.18
5.16
7.75
7.75
7.74
7.74
7.86
Normalized Intensity
0.3
1.23
1.20
1.22
2.11
2.03
1.86
7.26
vb_266_pure_1H.esp
0.7
0.6
0.5
0.4
0
2
1
116
0.05
180
160
140
120
100
80
Chemical Shift (ppm)
60
24.37
20.77
20.63
20.45
14.90
53.71
75.97
71.59
68.95
62.33
81.22
123.72
170.70
169.47
Normalized Intensity
77.26
77.01
76.76
vb_266_pure_13C.esp
0.30
0.25
0.20
0.15
0.10
0
40
20
0
117
4
6
8
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
118
F1 Chemical Shift ( ppm)
2
100
8
7
6
5
4
F2 Chemic al Shif t (ppm)
3
2
1
119
F1 Chemical Shift ( ppm)
50
vb_238_A_1H
TMS
0.89
0.9
0.8
0.04
0.6
0.5
0.4
0.3
1.73
1.71
1.70
1.66
1.65
1.50
1.48
1.46
1.30
1.30
0.2
3.81
3.67
3.67
Normalized Intensity
0.7
0.1
0
5.0
4.5
4.0
3.5
3.0
2.5
2.0
Chemical Shift (ppm)
1.5
1.0
0.5
0
120
0.05
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Chemical Shift (ppm)
0.15
2.5
2.0
-0.03
-0.04
1.52
0.25
1.5
1.0
0.5
0.05
1.86
0.30
2.11
2.03
0.74
0.40
0.73
1.54
1.26
1.25
1.25
0.88
5.78
5.78
5.76
5.45
5.44
5.43
5.43
5.17
5.17
5.15
4.34
4.33
4.32
4.31
4.31
4.30
4.17
3.87
3.86
3.66
3.63
7.86
7.85
7.85
7.84
7.75
7.25
7.25
Normalized Intensity
0.83
7.26
vb_268_pure_1H
0.35
0.20
0.10
0
0
121
0.04
0.03
180
160
140
120
100
80
Chemical Shift (ppm)
60
49.66
0.07
26.15
26.07
0.08
40
-4.43
21.13
21.00
20.82
72.11
0.05
71.21
69.59
62.60
97.05
134.57
172.96
169.84
0.06
123.92
Normalized Intensity
77.61
77.35
77.10
vb_268_pure_13C
0.02
0.01
0
-0.01
20
0
122
2
4
6
8
8
7
6
5
4
3
F2 Chemical Shift (ppm)
2
1
0
123
F1 Chemical Shift (ppm)
0
50
100
8
7
6
5
4
3
F2 Chemical Shift (ppm)
2
1
0
124
F1 Chemical Shift (ppm)
0
0.2
0.1
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
0.4
1.87
1.86 1.87
1.86
7.26
2.11
2.03
0.7
2.5
1.95
1.74
1.72
1.39
1.38
1.37
1.26
1.25
2.12
5.81
5.80
5.79
5.78
5.46
5.45
5.45
5.44
5.43
5.17
5.16
4.34
4.34
4.32
4.32
4.30
4.18
4.17
4.15
3.87
3.85
3.75
3.60
7.87
7.86
7.86
7.85
7.75
7.27
7.75
7.27 7.75
7.26
7.26
Normalized Intensity
vb_269_B_1H
0.6
0.5
0.3
0
2.0
1.5
1.0
125
0.010
180
160
140
0.015
120
100
80
Chemical Shift (ppm)
54.81
70.83
60
40
28.78
27.07
20.79
20.47
20.66
62.20
71.77
0.025
30.45
69.22
68.87
96.84
96.46
123.62
134.37
0.020
98.66
131.36
170.24
169.51
Normalized Intensity
77.26
77.01
76.76
vb_269_B_13C
0.030
0.005
0
20
0
126
4
6
8
8
7
6
5
4
F2 Chemical Shift (ppm)
3
2
1
127
F1 Chemical Shift (ppm)
2
40
60
80
100
120
8
7
6
5
4
F2 Chemical Shift (ppm)
3
2
1
128
F1 Chemical Shift (ppm)
20
0.2
0.1
8
7
6
5
4
Chemical Shift (ppm)
3
2
1.23
1.21
0.87
0.86
1.57
2.11
1.85
0.6
1.41 1.54
1.61
2.11
3.64
0.5
1.75
2.12
3.84
3.68
3.67
0.4
3.62
3.48
5.41
5.39
5.16
5.15
5.13
4.32
4.31
4.31
4.30
4.28
3.85 4.06
5.77
5.75
5.64
1.25
0.7
0
1
129
0.06
0.3
8.02
8.00
7.85
7.75
7.75
7.74
7.52 7.38
8.04
Normalized Intensity
2.03
7.26
vb_272_A_1H.esp
0.8
0.04
0.03
0.01
180
160
140
120
100
80
Chemical Shift (ppm)
60
62.17
40
14.12
22.69
29.36
31.93
25.72
55.83
54.80
54.72
52.40
69.22
66.70
69.13
0.05
71.93
0.06
20.79
20.47
29.70
123.61
20.65
134.37
129.88 129.67 128.42
0.07
71.79
70.82
133.16
0.08
100.36
98.69
96.65
96.12
95.91
95.35
95.03
91.37
127.47
147.56
170.20
165.89 169.49
163.34
0.02
170.70
Normalized Intensity
77.28
77.03
76.78
128.36
vb_272_pure_13C
0.09
0
20
130
4
6
8
8
7
6
5
4
F2 Chemical Shift (ppm)
3
2
1
131
F1 Chemical Shift (ppm)
2
0.2
0.1
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
2.23
0.3
7.37
7.26
7.82
7.81
0.4
4.75
4.75
4.74
4.73
4.47
4.46
4.30
4.26
4.30
3.91
4.28
3.90
4.27
3.90
3.89
3.76
3.10
3.10
6.25
6.24
6.23
Normalized Intensity
2.45
vb_196_A_1H
1.0
0.9
0.8
0.7
0.6
0.5
0
2.5
2.0
1.5
1.0
132
0.5
0.2
0.1
180
160
140
21.66
0.3
77.00
76.75
77.26
1.0
120
100
80
Chemical Shift (ppm)
75.70
69.57
69.35
67.92
103.03
129.93
128.01
0.4
132.60
145.18
143.99
Normalized Intensity
vb_196_A_13C
0.9
0.8
0.7
0.6
0.5
0
60
40
20
133
4
5
6
7
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
F2 Chemical Shift (ppm)
4.0
3.5
3.0
2.5
134
F1 Chemical Shift (ppm)
3
20
60
80
100
120
140
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
F2 Chemical Shift (ppm)
4.0
3.5
3.0
2.5
2.0
135
F1 Chemical Shift (ppm)
40
1.0 VB_188_A_1H.esp
0.9
0.8
Normalized Intensity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
2.5
2.0
136
1.5
77.27
77.02
76.76
vb_188_A_13C
1.0
0.9
0.8
0.6
0.5
68.76
66.26
100.41
0.3
74.71
0.4
146.52
Normalized Intensity
0.7
0.2
0.1
0
152
144
136
128
120
112
104
Chemical Shift (ppm)
96
88
80
72
64
137
3
4
5
6
6.5
6.0
5.5
5.0
4.5
4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
2.0
138
F1 Chemical Shift (ppm)
2
80
100
120
140
6.5
6.0
5.5
5.0
4.5
4.0
3.5
F2 Chemical Shift (ppm)
3.0
2.5
2.0
1.5
139
F1 Chemical Shift (ppm)
60
5.04
5.03
0.05
5.5
5.0
4.5
4.0
3.5
3.0
Chemical Shift (ppm)
2.5
2.0
1.27
2.04
2.03
2.41
2.40
2.65
2.64
4.38
4.29 4.37
4.25
4.09 4.23
4.09
4.07
4.06
3.93
3.92
4.72
4.48
4.47
5.31
1.25
3.46
Normalized Intensity
3.55
vb_190_pure_1H
0.15
0.10
0
1.5
140
77.25
77.00
76.75
vb_190_13C
29.57
20.28
72
26.60
80
69.06
72.81
0.05
80.43
Normalized Intensity
0.10
0
96
88
64
56
48
40
Chemical Shift (ppm)
32
24
16
8
141
2
3
4
5
5.5
5.0
4.5
4.0
3.5
3.0
F2 Chemical Shift (ppm)
2.5
2.0
1.5
1.0
142
F1 Chemical Shift (ppm)
1
16
32
40
48
56
64
72
80
5.5
5.0
4.5
4.0
3.5
3.0
F2 Chemical Shift (ppm)
2.5
2.0
1.5
143
F1 Chemical Shift (ppm)
24
0.1
7.5
7.0
6.5
6.0
5.5
5.0
4.5
Chemical Shift (ppm)
4.59
4.69
4.69
4.28
4.27
4.26
4.00
3.98
3.85 3.84
3.81 3.82
3.65
3.80
3.64
3.63
3.62
4.0
3.5
2.67
2.66
2.65
2.64
2.63
2.49
2.62
2.49
2.37
2.22
2.37
2.18
2.34
2.04
4.25
4.25
4.31
0.2
4.58
4.58
0.3
4.69
3.08
3.06
3.83
4.68
7.26
0.6
5.64
0.4
5.74
5.74
5.73
5.72
5.66
7.33
0.5
7.30
7.53
0.8
7.50
7.52
7.32
0.9
7.25
7.24
7.49
Normalized Intensity
7.32
vb_195_B_1H
1.0
0.7
3.0
2.5
2.0
144
0.2
135.35
0.3
144
136
128
0.4
120
112
104
96
88
80
Chemical Shift (ppm)
72
40.77
40.60
71.83
74.85
85.17
82.90
89.80
127.35
0.7
73.19
0.6
132.32
130.80
129.15
1.0
82.52
82.38
88.41
Normalized Intensity
vb_195_B_13C
0.9
0.8
0.5
0.1
0
64
56
48
40
145
3
4
5
6
7
8
8.0
7.5
7.0
6.5
6.0
5.5 5.0 4.5 4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
2.0
1.5
146
F1 Chemical Shift (ppm)
2
60
80
100
120
140
8.0
7.5
7.0
6.5
6.0
5.5 5.0 4.5 4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
2.0
1.5
147
F1 Chemical Shift (ppm)
40
0.05
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
1.81
1.83
4.33
4.32
4.06
4.06
3.92
3.91
4.04 3.89
3.88
3.86
3.87
3.86
3.85
5.93
5.95
5.75
5.74
5.74
5.99
5.98
7.33
1.86
1.84
5.95
7.53
7.53
7.51 7.51
7.32
0.10
6.01
6.00
7.51 7.34
Normalized Intensity
7.26
vb_199_B_1H
0
3.0
2.5
2.0
148
0.05
136
128
131.77
120
112
104
96
88
Chemical Shift (ppm)
64.29
62.91
80
71.88
83.61
129.03
127.56
127.12
Normalized Intensity
77.26
77.00
76.75
vb_199_B_13C
0.15
0.10
72
64
56
149
2
4
5
6
7
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
2.0
150
F1 Chemical Shift (ppm)
3
64
72
88
96
104
112
120
128
136
7.5
7.0
6.5
6.0
5.5
F2 Chemical Shift (ppm)
5.0
4.5
4.0
3.5
151
F1 Chemical Shift (ppm)
80
0.05
7.5
0.10
0.25
5.98
5.97
5.97
7.0
6.5
6.0
5.5
5.0
4.5
4.0
Chemical Shift (ppm)
3.5
3.0
2.5
2.0
152
1.38
1.30 1.27
1.64
1.71
5.94
0.30
5.94
5.82 5.89 5.93
5.74
5.73
5.74
5.62 5.63
5.43
5.62
5.42
5.42
5.30
4.64
4.63
4.58
4.57 4.32
4.56 4.33
4.05
4.24
4.05
4.06
3.90
4.04
3.89
3.88
3.86
3.85 3.85
3.87
3.83 3.84
3.59
3.57
3.57
3.56
3.00
2.98
2.33
2.31
2.30
2.22
2.23 2.20 2.19
2.04 2.18
2.02 2.03
6.00
5.99
5.99
7.26
7.33
0.20
7.25
7.25
7.24
7.24
0.15
7.50
7.53
0.35
7.53
1.26
7.31
7.53
0.50
7.49
Normalized Intensity
7.26
7.51
vb_195_C_1H
0.45
0.40
0
1.5
0.05
0.10
135
130
125
120
115
110
105
100
95
90
Chemical Shift (ppm)
85
80
75
71.87
64.18
62.82
83.65
127.53
127.00
0.20
70
65
153
62.74
64.56
81.74
79.73
0.15
132.48
131.68
129.02
131.74
0.25
131.52
135.09
Normalized Intensity
77.27
77.01
76.76
vb_195_C_13C
0.35
0.30
0
60
1
3
4
5
6
7
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
F2 Chemical Shift (ppm)
3.0
2.5
2.0
1.5
154
1.0
F1 Chemical Shift (ppm)
2
64
80
88
96
104
112
120
128
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
2.0
1.5
1.0
155
F1 Chemical Shift (ppm)
72
0.5
0.4
5.5
4.18
4.20 4.17
0.6
5.0
4.5
4.02
4.01 4.02
3.96
3.95
4.27
4.17
0.9
3.94
3.93
0.7
4.28
4.27
5.11
5.09
5.52
0.8
5.10
Normalized Intensity
4.15
vb_197_A_1H
1.0
0.3
0.2
0.1
Chemical Shift (ppm)
4.0
156
77.02
vb_197_A_13C
1.0
0.9
77.28
76.77
0.8
0.6
32.85
79.87
0.4
73.28
71.30
0.5
100.20
Normalized Intensity
0.7
0.3
0.2
0.1
0
105
100
95
90
85
80
75
70
65
60
Chemical Shift (ppm)
55
50
45
40
35
30
157
25
3.0
4.0
4.5
5.0
5.5
5.5
5.0
4.5
F2 Chemical Shift (ppm)
4.0
3.5
3.0
158
F1 Chemical Shift (ppm)
3.5
32
40
56
64
72
80
88
96
104
5.5
5.0
4.5
F2 Chemical Shift (ppm)
4.0
3.5
3.0
159
F1 Chemical Shift (ppm)
48
0.05
5.5
5.0
5.02
5.02
5.01
5.01
5.03
5.76
4.5
Chemical Shift (ppm)
4.39
4.74
4.73
4.20
4.19
4.18 4.09
4.14
4.09
4.07
4.06 4.07
4.06
4.04
4.02
4.01
3.79
3.78
3.76
3.77
3.76
3.63
3.62
3.61
4.38
0.10
4.37
4.48
4.48
4.47
4.45
4.44
4.83
4.79 4.75
4.74
4.89
4.89
5.41
5.69
5.68
Normalized Intensity
4.30
vb_197_B_1H
0.25
0.20
0.15
0
4.0
160
77.26
77.00
76.75
vb_197_B_13C
0.13
0.12
0.11
0.09
46.58
8.73
0.08
0.07
0.02
26.66
25.12
99.47
0.03
84.04
83.32
82.26
0.04
90.68
0.05
76.36
76.26
75.55
71.76
70.55
0.06
107.69
Normalized Intensity
0.10
0.01
0
104
96
88
80
72
64
56
48
Chemical Shift (ppm)
40
32
24
16
8
161
3.5
4.0
4.5
5.0
F1 Chemical Shift (ppm)
3.0
5.5
5.5
5.0
4.5
F2 Chemical Shift (ppm)
4.0
3.5
3.0
162
30
50
60
70
80
90
100
5.5
5.0
4.5
4.0
F2 Chemical Shift (ppm)
3.5
3.0
2.5
163
F1 Chemical Shift (ppm)
40
4.3 REFERENCES
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