synthetic studies of heparan derivatives: glycosyl

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Synthetic studies of heparan derivatives: Glycosylcouplings and post-glycosylative modificationsHari Raj KhatriPurdue University

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To the best of my knowledge and as understood by the student in the Thesis/Dissertation Agreement, Publication Delay, and Certification/Disclaimer (Graduate School Form 32), this thesis/dissertation adheres to the provisions of Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.

Hari Raj Khatri

SYNTHETIC STUDIES OF HEPARAN DERIVATIVES: GLYCOSYL COUPLINGSAND POST-GLYCOSYLATIVE MODIFICATIONS

Doctor of Philosophy

Alexander Wei

Chengde Mao

Christopher Uyeda

Alexander Wei

Hilkka I. Kenttämaa

Timothy Zwier 05/26/2016

i

SYNTHETIC STUDIES OF HEPARAN DERIVATIVES: GLYCOSYL

COUPLINGS AND POST-GLYCOSYLATIVE MODIFICATIONS

A Dissertation

Submitted to the Faculty

of

Purdue University

by

Hari Raj Khatri

In Partial Fulfillment of the

Requirements of the Degree

of

Doctor of Philosophy

August 2016

Purdue University

West Lafayette, Indiana

ii

To my parents Ram Prasad Neupane and Yam Maya Neupane

and my wife Luna

iii

ACKNOWLEDGMENTS

Pursuing a PhD degree at Purdue University has been a memorable

journey for me and I want to take a moment to express my sincere thanks to

all the wonderful people who came across during my six years of journey.

Firstly, I would like to express my sincere thanks to my advisor, Prof.

Alexander Wei for his continuous support, guidance, patience and motivation

throughout my graduate studies. Alex’s continuous support, constructive

comments and directions were always pivotal at key moments in my work. He

provided me every opportunity to grow as an independent researcher and

supported both academically and emotionally through some of my rough days

to finish this thesis.

I would also like to acknowledge my thesis committee members, Prof.

Hilkka I. Kenttämaa, Prof. Chengde Mao, Prof. David Colby, and Prof.

Christopher Uyeda for their valuable time and suggestions throughout my

PhD.

I am very thankful to my current and past lab members, Dr. Naveen

Kadasala, Yu Bai, Anna V Morales-de-Echegaray, Lu Lin, Anh Nguyen, Nigam

Arora, Jianxin Wang, Dr. Andrew Evans, Dr. Jonathan Mehtala, Dr. Thora

Moltais and especially to Dr. Panuwat Padungros, Dr. Laura Auberch, Dr.

iv

Matthew Casselman, Dr. Kulbhushan Durugkar and Dr. Oscar Morales – they

were the primary resources from whom I learnt a lot of research techniques.

I would like to thank Dr. James Riederman and soon to be Dr. Damodar

Koirala for running ESI-MS for my samples when necessary.

A special thanks to my parents, Ram Prasad and Yam Maya who are miles

away from here, for their unconditional love, moral support and

encouragement. Today, all their hardships and sacrifices have finally paid off. I

would also like to thank my family, friends and relatives who have always been

there to support me.

Finally, there is one special person in my life who deserves huge thanks,

my wife Luna who has always been an anchor and my support system through

thick and thin in my life.

v

TABLE OF CONTENTS

Page

LIST OF TABLES ............................................................................................ vii

LIST OF FIGURES ........................................................................................ viiii

LIST OF ABBREVIATIONS ............................................................................ viii

ABSTRACT .................................................................................................... xiv

CHAPTER I: HEPARIN-LIKE OLIGOSACCHARIDES FOR FGF-2 BINDING INTRODUCTION .............................................................................................. 1

1.1 Introduction to Heparin (HP) and Heparan Sulfate (HS) ....................... 1 1.2 Heparin-Binding Proteins and Their Biological Importance ................... 2 1.3 Heparin Interaction with Fibroblast Growth Factor-2 ............................. 4 1.3.1 Structure of FGF-2 ........................................................................ 5 1.3.2 FGF-2 Synthesis and Storage ...................................................... 7 1.3.3 Role of FGF-2 in Disease and Therapy ........................................ 7 1.3.4 HSPG Binding Sites of FGF-2 ...................................................... 8 1.3.5 Interaction of FGF-2 and HS Disaccharides ................................. 9 1.4 Synthesis of Heparin-like Oligosaccharides ........................................ 10 1.5 References.......................................................................................... 13

CHAPTER II: SYNTHETIC STUDIES TOWARD L-IDURONSYLDISACCHARIDES BY POST-GLYCOSYLATION MODIFICATION ............... 16

2.1 Introduction ..................................................................................................... 162.1.1 L-Iduronic acid ............................................................................. 17

2.1.2 4-Deoxypentenosides (4-DPs) and Glycals ................................ 17 2.2 Synthesis of 4’-Deoxypentenosyl Donors and Disaccharides ............. 18 2.3 A New Approach to Disaccharides with Terminal L-ido

Configuration ....................................................................................... 21 2.4 Synthesis of Glycosyl Donors ............................................................ 22 2.6 Synthesis of Glycosyl Acceptors ......................................................... 24

vi

Page

2.7 Synthesis of Disaccharides by BSP/Tf2O-Mediated Glycosylation ...... 27 2.8 Post-Glycosylative Modification of Disaccharides ............................... 31

2.8.1 Synthesis of 5’-DTC-Activated Derivatives ................................ 312.8.2 C-Furylation of Glycosyl DTCs .................................................. 34

2.9 Conclusions ........................................................................................ 37 2.10 General Methods and Experiments................................................... 38 2.10.1 Experimental ............................................................................. 38 2.11 References ........................................................................................ 66

CHAPTER III: SYNTHETIC STUDIES ON STEREOSELECTIVE GLYCOSYLATION OF GLUCOSAMINE DONORS .................................... 67

3.1 Stereoselective Glycosidic Bond Formation with D-Glucosamine ....... 67 3.2 Synthesis of Trichloroacetimidate Donor 42 ........................................ 70 3.3 Synthesis of Disaccharide by Coupling of Trichloroacetimidate

Donor 42 .............................................................................................. 72 3.4 Synthesis of C2-N-Diphenylphosphinic Amide Trichloroacetimidate

Donor 50 .............................................................................................. 76 3.5 Coupling Reactions of Trichloroacetimidate Donor 50

by Ni(II) Catalyst ................................................................................. 78 3.6 Conclusion and Future Directions ....................................................... 80 3.7 General Methods and Experiments ..................................................... 80 3.7.1Experimental ................................................................................ 80 3.8 References.......................................................................................... 93

VITA ............................................................................................................... 95

PUBLICATION ............................................................................................... 96

vii

LIST OF TABLES

Table Page

Table 1.1 Select heparin-binding proteins and their characteristics ...................... 3

Table 1.2 Dissociation constants of FGF-2 binding HS-disaccharides ............... 10

Table 2.1 BSP/Tf2O- mediated glycosyl coupling for disaccharides synthesis ... 30

Table 2.2 Copper(I/II) triflate-mediated furylation of �-glucosyl DTC 30 ............. 35

Table 3.1 Preparation of glycosyl thioimidates ................................................... 75

Table 3.2 Glycosylation with trichloroacetimidate donor 51 using [Ni(4-F-PhCN)4(OTf)2] catalyst ........................................................................................ 79

viii

LIST OF FIGURES

Figure Page

Figure 1.1 Structure of heparin and heparan sulfate repeat unit........................... 1

Figure 1.2 Pentasaccharide HP sequence with anticoagulant properties ............. 4 Figure 1.3 (A) 18-kDa FGF-2, a 146-amino acid protein (B) X-ray crystal structure of the dimerized FGF-2 dimer/FGFR ectodomain complex, with the HS-binding groove revealed by sulfate anions. (C) Cartoon of 5-molecule complex on the cell surface comprised of two FGF-2s, two FGFRs and HSPG. .. 6

Figure 1.4 (A) FGF-2/FGFR binding interactions, based on x-ray structural data. (B) X-ray crystal structure of Site 2’ binding to unsulfated trisaccharide. (C) Unsulfated trisaccharide L-IdoA(�1�4)GlcNAc(�1�4)GlcA(�1�4)OMe ...... 9 Figure 1.5 Diversity-oriented approaches for the synthesis of disaccharide sulfoforms ........................................................................................................... 12

Figure 2.1 Epimerization of D-glucuronic acid (D-GlcA) ...................................... 16

Figure 2.2 Structure of glycal and 4-DPs derived from �-glucopyranoside ......... 18

Figure 2.3 Synthesis of 5’-furyl-substituted disaccharide L-ido configuration ...... 19

Figure 2.4 Attempted addition of 2-furyl unit to a 4’-epoxypentenosyl disaccharide ....................................................................................................... 20

Figue 2.5 Attempted addition of furan to a 5’-DTC-substituted pentenoside ...... 21

Figure 2.6 A revised approach toward disaccharide with terminal L- ido configuration(R = Bn).......................................................................................... 22

Figure 2.7 Synthesis of glycosyl donor 4 ............................................................ 23

Figure 2.8 Synthesis of 4-DP thioglycoside donor 6 ........................................... 23

Figure 2.9 Synthesis of acceptor 12 ................................................................... 25

ix

Figure Page

Figure 2.10 Synthesis of acceptors 14, 16 and 18 .............................................. 27

Figure 2.11 Mechanism of BSP/Tf2O-mediated glycosyl coupling ...................... 28

Figure 2.12 Synthesis of fully protected C5’-DTC disaccharide 28 ..................... 33

Figure 2.13 Synthesis of �-glucosyl DTC 30 and �-C5’-DTC disaccharide 32 ... 34

Figure 2.14 CuOTf-mediated C-furylation of 5’-DTC disaccharide 32 ................ 36

Figure 2.15 CuOTf-mediated C-furylation of 5’-DTC disaccharide 28 ................ 36

Figure 3.1 Heparan trisaccharide ligand with high affinity for secondary binding site on FGF-2 ......................................................................................... 69 Figure 3.2 Retrosynthetic analysis of target trisaccharide .................................. 70

Figure 3.3 Synthesis of trichloroacetimidate donor 42 ........................................ 72

Figure 3.4 Glycosyl coupling of donor 42 with acceptor 43 ................................ 73

Figure 3.5 Synthesis of diazo transfer reagent 45 and azidoglucose 46 ............ 74

Figure 3.6 Working hypothesis to S- or O-glycosides from lactol 48 and phenyl diphenylphosphinite ................................................................................ 76 Figure 3.7 Synthesis of trichloroacetimidate glucosamine donor 50 ................... 77

x

LIST OF ABBREVIATIONS

[�]D specific optical rotation; deg mL/(g dm)

Ac Acetyl

aq Aqueous

AT-III Antithrombin III

BAIB Bisacetoxyiodobenzene

Bn Benzyl

br Broad peak

BDMA Benzylidene dimethyl acetal

BSP Benzenesulfinyl piperidine

Bu Butyl

Bz Benzoyl

Calcd Calculated

CAN Ceric ammonium nitrate

Cbz Carbonylbenzyloxy

CSA Camphorsulfonic acid

� Chemical shift

d Doublet

DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene

xi

DCE 1,2-Dichloroethane

DCM Dichloromethane

DDQ 2,3-Dichloro-5,6-dicyano-p-benzoquinone

DIPEA N,N-Diisopropylethylamine

DMF N,N-Dimethylformamide

DMFDNpA N,N-Dimethylformamide dineopentyl acetal

DMDO Dimethyldioxirane

DTC Dithiocarbamate

ESI Electrospray ionization

Et Ethyl

Equiv Equivalent

FGF Fibroblast growth factor

FGFR Fibroblast growth factor receptor

FT Fourier transform

Glc Glucose

GlcA Glucuronic acid

GlcN Glucosamine

h Hour

HBP Heparin-binding protein

HIV Human immunodeficiency virus

HMW High molecular weight

HMDS Hexamethyldisilazane

HRESI High resolution electrospray ionization

xii

HS Heparan sulfate

HP Heparin

HSPG Heparan sulfate proteoglycan

HSV Herpes simplex virus

IdoA Iduronic acid

Im Imidazole

iPr Isopropyl

IR Infrared (spectroscopy)

LMW Low molecular weight

MS Molecular sieves

MAPK Mitogen-activated protein kinases

Me Methyl

Min Minute

MS Mass spectrometry

NIS N-Iodosuccinimide

NMR Nuclear magnetic resonance (spectroscopy)

o/n Overnight

Ph Phenyl

Phth Phthaloyl

PMB p-Methoxybenzyl

PMBDMA p-Methoxybenzaldehyde dimethyl acetal

PMP p-Methoxyphenyl

Py Pyridine

xiii

q Quartet

rt Room temperature

rxn Reaction

s Singlet

SEM Trimethylsilylethoxymethyl

t Triplet

TBAI Tetrabutylammonium iodide

TCA Trichloroacetimidate

TBDPS Tert-butyldiphenylsilyl

TEMPO 2,2,6,6-Tetramethylpiperidine 1-oxyl

Tf2O Trifluoromethanesulfonic anhydride

TfOH Trifluoromethanesulfonic acid

THF Tetrahydrofuran

TLC Thin layer chromatography

TMS Trimethyl silyl

TsOH p-Toluenesulfonic acid

TTBP 2,4,6-Tri-tert-butylpyrimidine

xiv

ABSTRACT

Khatri, Hari Raj. Ph. D., Purdue University, August 2016. Synthetic Studies of Heparan Derivatives: Glycosyl Couplings and Post-Glycosylative Modifications. Major Professor: Alexander Wei.

Heparan sulfate (HS) and closely related heparin are comprised of

alternating units of D-glucosamine and either D-glucuronic acid (D-GluA) or L-

iduronic acid (L-IdoA), and support variable degrees of sulfation which can

interact with a large number of proteins with diverse biological functions. HS

oligosaccharides can be constructed from readily accessible D-GlcN and D-GlcA

derivatives, but the inclusion of L-IdoA is less straightforward. To address this,

our laboratory has developed alternative synthetic strategies for HS-like

oligosaccharides to incorporate either D-GlcA or L-IdoA in a synthetically efficient

manner by nucleophilic ring opening of 4-epoxypyranosides, which can be made

from readily available D-hexoses in few steps. These are derived from 4-

deoxypentenosides (4-DPs), unsaturated pyranosides that can be linked with

other sugars, enabling us to investigate synthetic strategies involving post-

glycosylative modifications. Terminal 4-DPs have been generated at a late stage

from �-1,4-linked disaccharides, and also by stereoselective coupling of 4-DP

thioglycoside donors with various acceptors. The 4’-enol ether can be modified

by stereoselective epoxidation and ring opening by a dithiocarbamate auxiliary,

xv

which can be activated by copper(I) salts for carbon nucleophile addition with

terminal L-ido configuration. We also explored stereoselective glycosylation of a

novel glucosamine donor with a N-diphenylphosphinamido group.

1

CHAPTER I: HEPARIN-LIKE OLIGOSACCHARIDES FOR FGF-2 BINDING

1.1 Introduction to Heparin (HP) and Heparan Sulfate (HS)

Heparin (HP) and heparan sulfate (HS) are linear, highly sulfated

polysaccharides with disaccharide repeating units containing (1 4) linked �-D-

glucosamine (GlcN) and uronic acid, either �-D-glucuronic acid (�-GlcA) or �-L-

iduronic acid (�-L-IdoA).1 Chemically, the distribution of uronic acids in heparin is

approximately 90% L-IdoA and 10% D-GlcA. HP is a more highly sulfated

glycosaminoglycan (2.3–2.8 sulfo per disaccharide)2 with molecular weight

ranging from 5–40 kDa. In HP, the amines may be functionalized with acetyl or

sulfo groups, and the C2 position of the uronic acid moiety may also be sulfated

(Figure 1).

Figure 1.1 Structure of heparin and heparan sulfate repeat unit

2

HS is structurally similar to HP, but with molecular weights ranging from 5–50

kDa and with a lower percentage of L-IdoA. HS has a lower sulfate density (0.6–

1.5 sulfo/disaccharide) and is heterogeneous in its sulfation patterns.2 HP is often

referred to as the more completely modified version of HS and also possesses

the highest negative charge density of any known biological macromolecule.

However, heparin is found exclusively inside storage vesicles of mast cells of

some animal species, whereas heparan sulfates are ubiquitous to cell surfaces of

vertebrates and invertebrate species.3, 4

The structural microheterogeneity within HP and HS arises during its

biosynthesis. The sequential biosynthetic processes: a) N-deacetylation and N-

sulfation of GlcN; b) C5 epimerization of D-GlcA into L-IdoA; c) 2-O-sulfonation of

L-IdoA and D-GlcA, and d) 6-O-sulfonation and 3-O-sulfonation of GlcN all do not

go to completion.5 While heparan sulfate contains all of the structural variations

found in heparin, the frequency of occurrence of the later post-glycosylative

modifications is higher in HS, and the extent of structural heterogeneity is also

greater.

1.2 Heparin-Binding Proteins and Their Biological Importance

Glycosaminoglycans (GAGs) are a class of complex polysaccharides and

primary constituents on the surface of every eukaryotic cell and in many

extracellular matrices. The heterogeneity of HP/HS GAGs arising from their

variable sulfation pattern and backbone structure enables them to interact with a

variety of basic proteins such as growth factors, enzymes and morphogens, as

3

well as proteins presented by microbial pathogens.6 Among the hundreds of

proteins known to bind HP/HS in the literature, some representative ones are

listed in Table 1.1.

Table 1.1 Select heparin-binding proteins and their characteristics 1

HP-binding protein, with example

Physiological role(s) Kd minimumsize (DP*)

HP ligand function

Proteases/esterases

Antithrombin III (AT-III)

Coagulation cascade (serpin) ~20 nM

5 Enhances AT-III-mediated inhibition of blood coagulation factor1,7

Growth factors

FGF-1 and FGF-2

cell proliferation, differentiation, morphogenesis, angiogenesis

nM 4–6 Activates signal transduction8-11

Chemokines

PF-4

Inflammation, wound healing 16 nM 12 Inactivates HP and HSPG interaction12,13

Pathogen coat proteinsHIV-1 gp 120

Viral entry 0.3 �M

10 Inhibits the binding of V3 loop antibodies to HIV-1gp 12014,15

Adhesion proteins

L-selectin

adhesion, inflammation, metastasis

46 �M �4 Blocks interaction between SLex and L-selectin16,17

*DP = degree of polymerization

One well-studied example of a HP–protein interaction involves a specific

pentasaccharide sequence (Figure 1.2) and the serine protease inhibitor

antithrombin III (AT-III), which in its activated form inhibits the blood clotting

cascade.18 Antithrombin-III in its native form is a weak protease inhibitor but

upon binding with pentasaccharide undergoes a conformational change, resulting

in a 300-fold increase in the inhibition of the critical serine protease Factor Xa

4

(but not of thrombin). This rare sequence contains a highly charged GlcNS,3S,6S

unit, which is critical for AT III-binding and subsequent anticoagulant properties18.

A synthetic analog of this pentasaccharide is now marketed as the anticoagulant

drug Arixtra.

Figure 1.2 Pentasaccharide HP sequence with anticoagulant properties

1.3 Heparin Interaction with Fibroblast Growth Factor -2

Cell-surface heparan sulfate proteoglycans (HSPGs) in mammalian cells

frequently act as ligand for extracellular signaling proteins. FGFs are large family

of monomeric, globular heparin binding growth factors having separate binding

regions for heparin or HS and fibroblast growth factor receptors (FGFRs). FGF-2

is one of the most studied proteins that have affinity for HS, and is implicated in

many physiological and pathological processes, such as embryonic

development, wound healing, angiogenesis, and tumorigenesis.19

5

1.3.1 Structure of FGF-2

FGF-2 (a.k.a. basic FGF) is an 18–34 kDa protein with no signaling sequence

for secretion. The main portion of FGF-2 consists of 12 anti-parallel �-sheets

organized into trigonal pyramidal structure.20 There are five isoforms of FGF-2

(18, 22, 22.5, 24, and 34 kDa) which can be generated by alternate CUG-

translation of start sites and distributed in different subcellular locations.21 The

smallest of these, FGF-218k, has strong affinity for extracellular HSPGs (Figure

1.3). The importance of HS for mediating FGF-2 signaling was demonstrated

when FGF-2 failed to activate HSPG-deficient Chinese hamster ovary (CHO)

cells transfected with FGFR1, but succeeded when exogenous heparin was

added.22 HSPGs are known to stabilize the dimerization of FGF-2, especially

when binding to FGFR dimers at the cell surface (Figure 1.3). Studies suggest

that this 5-molecule complex activates the mitogen-activated protein kinase

(MAPK) signaling pathway, leading to cell proliferation.23,24

6

Figure 1.3 (A) 18-kDa FGF-2, a 146-amino acid protein.25 (B) X-ray crystal structure of the dimerized FGF-2 dimer/FGFR ectodomain complex, with the HS-binding groove revealed by sulfate anions.26 (C) Cartoon of 5-molecule complex on the cell surface comprised of two FGF-2s, two FGFRs, and HSPG.

Side view

Front view

FGFR

Top view FGFB

A

HSPG

growthfactors

cell-surfacereceptors

C

7

1.3.2 FGF-2 Synthesis and Storage

FGF-2 is produced by a wide variety of cell types and is localized within the

basement membrane (BM), a specialized part of the extracellular matrix

underneath the endothelial layer, and also on the surfaces of endothelial cells.27

On the cell surface, FGF-2 binds to high-affinity transmembrane receptors and

low-affinity HSPGs. Such interactions permit the sequestering of FGF-2s on the

cell surface and extracellular matrix (ECM), facilitating intracellular transduction

and stabilization or storage of FGF-2 within the basement membrane.22 This has

been shown to stimulate cell proliferation and differentiation, implicating the BM

as a reservoir for FGF-2. It has been suggested that FGF-2 is released from the

BM in response to cell damage or non-lethal tissue membrane disruptions.18 The

storage of FGF-2 within the ECM is diffusion controlled, thanks to its rapid and

reversible binding with HSPGs.28 Disruption of interactions between FGF-2 and

HSPG by proteolytic degradation (e.g. heparanases), or by competitive binding

with soluble heparin can also enhance FGF-2 release.19,20,29

1.3.3 Role of FGF-2 in Disease and Therapy

FGF-2 proteins are involved in the development and differentiations of various

range of organs and transport-systems: brain, blood vessel, bone, lung, liver,

skin, muscle, eye, reproductive system, and hematopoiesis.19 Elevated

expression of FGF-2 has been demonstrated in a variety of tumor types both in

vivo and vitro.30 FGF-2 expression can also correlate with the advanced stages

of tumor growth: For example: FGF-2 is overexpressed by numerous human

8

cancer cell lines including those derived from leukemic, and bladder, gastric,

overian, and squamous cell carcinoma.31 FGF-2 stored within the extracellular

matrix can be mobilized by heparanase, which plays a key role in many

cancers.32 Similarly, the triggered release of stored FGF-2 is also involved in

wound healing and the repair or replacement of injured tissues.33,34 Given their

importance in various cellular growth and differentiation processes, therapeutics

that target FGF-2 activity have been a topic of active investigation.30,34

1.3.4 HSPG Binding Sites of FGF-2

FGF-2 has three high-affinity binding sites that are recognized by HSPGs,

which function as modulators of FGF-2 activity. One of these (Site 1, Figure 1.4)

has been extensively characterized because of its direct role between FGF-2 and

FGFR, which is lined with multiple basic residues.35,36 Two other carbohydrate-

binding domains (Sites 2 and 2') have been identified, and are reported to

associate with unsulfated heparan derivatives at submicromolar concentrations.37

Site 2 features polar residues, whereas Site 2' is lined with hydrophobic ones

(Figure 1.4 A). When compared with the x-ray crystal structure of the FGF�FGFR

complex, it becomes clear that Site 2 is associated directly with FGFR, and thus

not specific for heparan. However, Site 2' is remote and thus available as a

binding domain for heparan. This has been validated by an x-ray crystal structure

of FGF-2 complexed with an unsulfated heparin trisaccharide, L-

IdoA(�1�4)GlcNAc(�1�4)-D-GlcA(�1�4)OMe (Figure 1.4B,C). This

9

trisaccharide and its C5" epimer, D-GlcA(�1�4)GlcNAc(�1�4)-D-

GlcA(�1�4)OMe, were both found to have submicromolar binding with FGF-2.

Figure 1.4 (A) FGF-2/FGFR binding interactions, based on x-ray structural data.26 (B) X-ray crystal structure of Site 2’ binding to unsulfated trisaccharide.37 (C) unsulfated trisaccharide L-IdoA(�1�4)-D-GlcNAc(�1�4)-D-GlcA(�1�4)OMe.38

1.3.5 Interaction of FGF-2 and HS Disaccharides

Huang and co-workers have synthesized a library of 48 HS-based

disaccharides, and studied their interactions with FGF-2.39,40 Three highly

sulfated epitopes were observed to bind FGF-2 with moderate affinity; based on

this, the authors inferred that the 3’-O-sulfate increased binding affinity, despite

its absence on the native HS ligands for FGF-2 (Table 1.2).40

B

C

10

Table 1.2 Dissociation constants of FGF-2 binding HS-disaccharides40

Disaccharide structures Sulfonation sites KD FGF-2 (�M)

OHO-O3SO

O-O3SHN

OH

OOH MeO

OSO3-

-OOC

N-sulfate(GlcN)

3’-O-sulfate (GlcN)

2-O-sulfate (IdoA)

1.7 ± 0.3

N-sulfate(GlcN)


2-O-sulfate (IdoA)

17.5 ± 1.8

N-sulfonate(GlcN)



2-O-sulfate (IdoA)

9.0 ± 0.2

1.4 Synthesis of Heparin-like Oligosaccharides

Chemical synthesis is a powerful tool for providing access to well-defined

heparin-like oligosaccharides. However, the synthesis of heparin-like

oligosaccharides presents several challenges: a) easy access to L-iduronic acid

and L-idose; b) differences in the chemical reactivity of L-idoA and D-GlcA as

building blocks; c) stereoselective formation of the 1,2-cis linkage on GlcN; d)

optimization of suitable protecting groups for the facile installation of sulfate

esters at desired positions; and e) reliable methods for backbone homologation.41

Synthetic strategies of heparin-like oligosaccharides can be either target-oriented

11

or diversity-oriented: the latter can support an iterative approach to a variety of

structures, while the former is often convergent with a focus on step

economy.42,43 However, with respect to the construction of oligosaccharide

libraries, the target-oriented approach tends to be tedious and labor-intensive.

Chemo-enzymatic synthesis44 offers another approach that utilizes specific

enzymes to carry out key transformations, often cutting out several processing

steps. However, the chemo-enzymatic approach is limited by enzyme specificity,

the challenges of purifying polar intermediates, and reaction scale.

In contrast, diversity-oriented synthesis is based on the design of a unified

synthetic route based on common intermediates, one that can be diversified

simply by changing the order of chemical transformations. This approach can be

used to generate a library of oligosaccharides with the same base structure, for

screening structure–binding relationships of target receptors. For example, our

group45 has synthesized a set of monosulfated disaccharide isomers (sulfoforms)

from a single precursor, using an orthogonal protecting group scheme for five

different hydroxyls and one amine (Figure 1.5).45 Diversity-oriented synthesis is

thus an appealing approach for synthesizing all possible epitopes of

oligosaccharides for evaluating structure–activity relationships.

12

Figure 1.5 Diversity-oriented approaches for the synthesis of disaccharide sulfoforms.45

13

1.5 References (1) Capila, I.; Linhardt, R. J. Angew. Chem. Int. Ed. 2002, 41, 390.

(2) Xu, D.; Esko, J. D. Annu. Rev. Biochem. 2014, 83, 129.

(3) Nader, H.; Ferreira, T.; Paiva, J. F.; Medeiros, M.; Jeronimo, S.; Paiva, V.; Dietrich, C. P. J. Biol. Chem. 1984, 259, 1431.

(4) Nader, H. B.; Takahashi, H. K.; Straus, A. H.; Dietrich, C. P. Biochim. Biophys. Acta 1980, 627, 40.

(5) Shriver, Z. H.; Capila, I.; Venkataraman, G.; Sasisekharan, R. Handb. Exp. Pharmacol. 2012, 159.

(6) Raman, R.; Sasisekharan, V.; Sasisekharan, R. Chem. Biol. 2005, 12, 267.

(7) Jin, L.; Abrahams, J. P.; Skinner, R.; Petitou, M.; Pike, R. N.; Carrell, R. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 14683.

(8) DiGabriele, A. D.; Lax, I.; Chen, D. I.; Svahn, C. M.; Jaye, M.; Schlessinger, J.; Hendrickson, W. A. Nature 1998, 393, 812.

(9) Mach, H.; Volkin, D. B.; Burke, C. J.; Middaugh, C. R.; Linhardt, R. J.; Fromm, J. R.; Loganathan, D.; Mattsson, L. Biochemistry 1993, 32, 5480.

(10) Faham, S.; Hileman, R.; Fromm, J.; Linhardt, R.; Rees, D. Science 1996, 271, 1116.

(11) Thompson, L. D.; Pantoliano, M. W.; Springer, B. A. Biochemistry 1994, 33, 3831.

(12) Stringer, S. E.; Gallagher, J. T. J. Biol. Chem. 1997, 272, 20508.

(13) Mikhailov, D.; Young, H. C.; Linhardt, R. J.; Mayo, K. H. J. Biol. Chem. 1999, 274, 25317.

(14) Rider, C. C.; Coombe, D. R.; Harrop, H. A.; Hounsell, E. F.; Bauer, C.; Feeney, J.; Mulloy, B.; Mahmood, N.; Hay, A.; Parish, C. R. Biochemistry 1994, 6974.

(15) Batini�, D.; Robey, F. J. Biol. Chem. 1992, 267, 6664.

(16) Koenig, A.; Norgard-Sumnicht, K.; Linhardt, R.; Varki, A. J. Clin. Invest. 1998, 101, 877.

14

(17) Nelson, R. M.; Cecconi, O.; Roberts, W. G.; Aruffo, A.; Linhardt, R. J.; Bevilacqua, M. P. Blood 1993, 82, 3253.

(18) Laremore, T. N.; Zhang, F.; Dordick, J. S.; Liu, J.; Linhardt, R. J. Curr.Opin. Chem. Biol. 2009, 13, 633.

(19) Bikfalvi, A.; Klein, S.; Pintucci, G.; Rifkin, D. B. Endocr. Rev. 1997, 18, 26.

(20) Nugent, M. A.; Iozzo, R. V. Int. J. Biochem. Cell Biol. 2000, 32, 115.

(21) Sorensen, V.; Nilsen, T.; Wiedlocha, A. Bioessays 2006, 28, 504.

(22) Yayon, A.; Klagsbrun, M.; Esko, J. D.; Leder, P.; Ornitz, D. M. Cell 1991, 64, 841.

(23) Bernfield, M.; Götte, M.; Park, P. W.; Reizes, O.; Fitzgerald, M. L.; Lincecum, J.; Zako, M. Annu. Rev. Biochem. 1999, 68, 729.

(24) Guimond, S.; Maccarana, M.; Olwin, B.; Lindahl, U.; Rapraeger, A. J. Biol. Chem. 1993, 268, 23906.

(25) Zhang, J.; Cousens, L. S.; Barr, P. J.; Sprang, S. R. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 3446.

(26) Plotnikov, A. N.; Schlessinger, J.; Hubbard, S. R.; Mohammadi, M. Cell 1999, 98, 641.

(27) Reisig, K.; Clyne, A. M. Matrix Biol. 2010, 29, 586.

(28) Folkman, J.; Klagsbrun, M.; Sasse, J.; Wadzinski, M.; Ingber, D.; Vlodavsky, I. Am. J. Pathol. 1988, 130, 393.

(29) Dowd, C. J.; Cooney, C. L.; Nugent, M. A. J. Biol. Chem. 1999, 274, 5236.

(30) Goetz, R.; Mohammadi, M. Nature Rev. Mol. Cell Biol. 2013, 14, 166.

(31) Teicher, B. A.; Ellis, L. M. Antiangiogenic agents in cancer therapy; Springer Science & Business Media, 2008.

(32) Arvatz, G.; Shafat, I.; Levy-Adam, F.; Ilan, N.; Vlodavsky, I. Cancer Metast. Rev. 2011, 30, 253.

(33) van Wijk, X. M.; van Kuppevelt, T. H. Angiogenesis 2014, 17, 443.

(34) Carter, E. P.; Fearon, A. E.; Grose, R. P. Trends Cell Biol. 2015, 25, 221.

(35) Pellegrini, L.; Burke, D. F.; von Delft, F.; Mulloy, B.; Blundell, T. L. Nature 2000, 407, 1029.

15

(36) Stauber, D. J.; DiGabriele, A. D.; Hendrickson, W. A. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 49.

(37) Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman, J.; Svahn, C.-M.; Waksman, G. Science 1995, 268, 432.

(38) Westman, J.; Nilsson, M.; Ornitz, D. M.; Svahn, C.-M. J. Carbohydr. Chem. 1995, 14, 95.

(39) Hu, Y.-P.; Zhong, Y.-Q.; Chen, Z.-G.; Chen, C.-Y.; Shi, Z.; Zulueta, M. M. L.; Ku, C.-C.; Lee, P.-Y.; Wang, C.-C.; Hung, S.-C. J. Am. Chem. Soc. 2012, 134, 20722.

(40) Li, Y.-C.; Ho, I.-H.; Ku, C.-C.; Zhong, Y.-Q.; Hu, Y.-P.; Chen, Z.-G.; Chen, C.-Y.; Lin, W.-C.; Zulueta, M. M. L.; Hung, S.-C. ACS Chem. Biol. 2014, 9, 1712.

(41) Dulaney, S. B.; Huang, X. Adv. Carbohydr. Chem. Biochem. 2012, 67, 95.

(42) Hu, Y.-P.; Lin, S.-Y.; Huang, C.-Y.; Zulueta, M. M. L.; Liu, J.-Y.; Chang, W.; Hung, S.-C. Nature Chem. 2011, 3, 557.

(43) Hansen, S. U.; Miller, G. J.; Cliff, M. J.; Jayson, G. C.; Gardiner, J. M. Chem. Sci. 2015, 6, 6158.

(44) Xu, Y.; Masuko, S.; Takieddin, M.; Xu, H.; Liu, R.; Jing, J.; Mousa, S. A.; Linhardt, R. J.; Liu, J. Science 2011, 334, 498.

(45) Fan, R.-H.; Achkar, J.; Hernández-Torres, J. M.; Wei, A. Org. Lett. 2005, 7, 5095.

16

CHAPTER II: SYNTHETIC STUDIES TOWARD L-IDURONSYL

DISACCHARIDES BY POST-GLYCOSYLATION MODIFICATION

2.1 Introduction

2.1.1 L-Iduronic acid

L-Iduronic acid (IdoA) is an important hexuronic monosaccharide component in

glycosaminoglycans (GAGs) that enhances binding affinity with various proteins

such as growth factors, enzymes, morphogens, cell-adhesion molecules, and

cytokines.1 In nature, epimerization of D-glucuronic acid at the C-5 position leads

to L-iduronic acids (Figure 2.1)1,2 however, this transformation is challenging to

perform under synthetic conditions.

Figure 2.1 Epimerization of D-glucuronic acid (D-GlcA)1,2

17

Most of the synthetic approaches to heparin-like oligosaccharides incorporate

L-iduronic acid derivatives using a multistep sequence, and are further

challenged by poor reactivity as a glycosyl donor.3,4

2.1.2 4-Deoxypentenosides (4-DPs) and Glycals

4-Deoxypentenosides (4-DPs) and glycals are cyclic enol ethers derived from

pyranosides. Glycals are unsaturated at C1 and C2, whereas 4-DPs are

unsaturated at C4 and C5 (Figure 2.2). The reactive 1,2-enol ether in glycals can

undergo various of transformations making them important intermediates in

carbohydrate synthesis. 4-DPs have been shown to have similar reactivity

profiles.5 Despite this, they have not been commonly used in oligosaccharide

synthesis. Our group has reported that 4-DPs can be stereoselectively

epoxidized using dimethyldioxirane (DMDO) into �- or �-epoxypyranosides,

which are amenable to anti- or syn-selective ring opening to generate novel

pyranosides with rare or unnatural substituents at the C5 position.6 Inspired by

these studies, we aimed to further investigate the synthetic utility of 4’-

deoxypentenoside (4’-DPs) as advanced intermediates, for late-stage conversion

into terminal L-iduronsyl disaccharides.

18

Figure 2.2 Structure of glycal and 4-DPs derived from �-glucopyranoside

Efficient glycosyl coupling with high stereoselectivity at an advanced stage of a

multi-step synthesis can be challenging, with few alternative strategies. This

challenge could be addressed by developing post-glycosylative modifications

method that allows the stereoselective installation of functional groups at the C5

position of terminal glycosides with a predetermined anomeric configuration. In

this chapter we explore the chemistry of 4’-DP-disaccharides as intermediates for

post-glycosylative modifications en route to L-iduronosyl disaccharides.

2.2 Synthesis of 4’-Deoxypentenosyl Donors and Disaccharides

4’-DP-disaccharide can be synthesized in two ways: a) glycosyl couplings with

4-DP thiophenyl glycosides as donors, and b) the decarboxylative elimination of

disaccharides with terminal GlcA units.7

Our work was inspired by some of the previous results from our laboratory.

Notably, Padungros et al successfully synthesized a 5’-furyl-substituted

disaccharide with terminal L-ido configuration, starting from peracetylated

cellobiose (Figure 2.3).5,6

19

O

BnOOBn

O

OOBn

OBnOBn

OBnHO

O

O

BnOOBn

O

OOBn

OBnOBn

OBnO

O ZnBr

ZnBr2, THF 0°C

O

OBnOOBn

O

OBn

OBn

OBn

OBn1. AcOH:THF:H2O

2. TEMPO, BAIB3. DMFDNPA

O

O

AcO

AcOOAc

AcO

O

OAc

OAc

OAc

OAc

1. HBr-AcOH

2.Ag2CO3, BnOH

O

O

AcO

AcOOAc

AcO

O

OAc

OBn

OAc

OAc

O

O

O

BnOOBn

O

O

OBn

OBn

OBn

OBnPh

DMDO-DCM

1. NaOMe2. PhCH(OMe)2

3. NaH, BnBr

Figure 2.3 Synthesis of 5’-furyl-substituted disaccharide with L-ido configuration8

Unfortunately, this approach failed when using an orthogonally protected

disaccharide intermediate in which the 4’-epoxypentenoside was treated with (2-

furyl)AlEt2 or (2-furyl)ZnBr in THF (Figure 2.4).8 This attempt apparently suffered

from the low reactivity of 4’-epoxypentenosyl disaccharide, as well as the lability

of the 3-O-SEM protecting group.

20

Figure 2.4 Attempted addition of 2-furyl unit to a 4’-epoxypentenosyl disaccharide8 The above hurdles were partially cleared when the 4’-epoxypentenosyl

disaccharide was subjected to epoxide ring opening by diethyl dithiocarbamate

(DTC), generated in situ from CS2 and diethylamine in methanol (Figure 2.5).

Further, the corresponding 5’-DTC could react with furan as a nucleophile when

activated with CuOTf(C6H6)0.5 at low temperature (–30 to –15 °C). Product

formation was confirmed by comparison with the 1H NMR spectrum of the same

compound made previously by Padungros.8 Unfortunately, orthogonally

protected 5’-DTC disaccharides were again unreactive to CuOTf(C6H6)0.5

mediated addition of furan.

21

Figure 2.5 Attempted addition of furan to 5’-DTC-substituted pentenoside

2.3 A New Approach to Disaccharides with Terminal L-Ido Configuration

To circumvent the issue of poor reactivity, we redesigned the 4’-DP-

disaccharide so that (a) the 3-O-SEM protecting group would be replaced with an

acetyl (Ac) or benzyl (Bn) group, and (b) a benzyl group would be installed on the

anomeric position of glucosamine (acceptor) before coupling (Figure 2.6).

22

Figure 2.6 A revised approach toward heparan disaccharides with terminal L-ido configuration (R = Bn).

2.4 Synthesis of Glycosyl Donors

Orthogonally protected donor 4 was synthesized following the adapted

protocol (Figure 2.7).8 Commercially available glucose pentaacetate was

subjected to BF3.Et2O-mediated glycosylation with thiophenol (PhSH) to give 1 in

90% yield, which was converted into 4,6-p-methoxybenzylidene acetal 2 in 80%

yield over two steps. Regioselective 3-O-benzylation of 2 gave compound 3 (90%

yield) followed by 2-O-benzoylation to afford glycosyl donor 4 in 90% yield as a

white crystalline solid, recrystallized from 95% ethanol.

23

Figure 2.7 Synthesis of glycosyl donor 4. Reagents and conditions: 1) BF3.Et2O

(10 equiv), PhSH (2.0 equiv), CH2Cl2, rt (90%); 2) (i) NaOMe (0.4 equiv), 2:3 CH2Cl2:MeOH, (ii) PMBDA (3.0 equiv), d-CSA (0.2 equiv), THF, reflux (73% over two steps); 3) (i) Bu2SnO (1.6 equiv), toluene, reflux, (ii) BnBr (1.6 equiv), CsF (1.6 equiv), DMF, rt (90%); 4) C6H6COCl (2.0 equiv), py, rt (90%).

Orthogonally protected 4-DP monosaccharide donor 6 was synthesized from 4

by hydrolysis to a 4,6-diol, chemoselective oxidation of the primary alcohol to an

acid, then decarboxylative elimination under microwave conditions to afford 4-DP

donor 6 as a white solid (44% yield over three steps, Figure 2.8).

Figure 2.8 Synthesis of 4-DP thioglycoside donor 6. Reagents and conditions: 1) 4:4:1 AcOH:THF:H2O, 70 °C (92%); 2) (i) BAIB (3.0 equiv), TEMPO (0.4 equiv), 2:1 CH2Cl2:H2O, rt, (ii) DMFDNpA (5.0 equiv), DMF, 150 °C, �-wave (48% over two steps).

24

2.6 Synthesis of Glycosyl Acceptors

An orthogonally protected glucosamine acceptor 12 was synthesized from D-

glucosamine (Figure 2.9). Intermediate 7 was synthesized in three steps and

70% yield using an adapted procedure,9 then converted into �-thiotolyl glycoside

8 in 72% yield as a white solid. Intermediate 8 was deacetylated with catalytic

sodium methoxide (NaOMe), and protection of the 4,6-diol with p-anisaldehyde

dimethyl acetal gave 9 in 77% yield over two steps. The phthalimide group of 9

was converted into an azide to give 10 in 80% yield over two steps.

Glucosamine acceptor 12 was obtained from 10 by protecting the C3

hydroxyl as 2-(trimethylsilyl)ethoxymethyl (SEM) ether 11 in 90% yield, followed

by regioselective reductive cleavage of the anisylidene ring with dibutylboron

triflate (Bu2BOTf) and borane–tetrahydrofuran complex (BH3.THF) to afford 4-O-

p-methoxybenzyl (PMB) ether in 90% yield. This was treated with tert-

butyldiphenylsilyl chloride (TBDPS-Cl) to afford 6-O-TBDPS ether in 96% yield.

Last, oxidative cleavage of the 4-O-PMB ether with 2,3-dichloro-5,6-

dicyanobenzoquinone (DDQ) afforded 12 in 78% yield.

25

Figure 2.9 Synthesis of acceptor 12. Reagents and conditions: 1) (i) NaOMe (1.0 equiv), MeOH, rt; (ii) Phth2O (1.1 equiv), Et3N (1.1 equiv), MeOH, rt; (iii) Ac2O, py, rt (70%, in 3 steps); 2) TolSH (2.0 equiv), BF3.Et2O (10 equiv), CH2Cl2, rt (78%); 3) (i) NaOMe (0.4 equiv), 1:1 CH2Cl2:MeOH, 0 °C; (ii) PMBDA (3.0 equiv), d-CSA (0.5 equiv), THF, reflux, (77%, 2 steps); 4) (i) NH2C2H4NH2 (20 equiv), n-BuOH, 100 °C, (ii) Tf2O (5.0 equiv), NaN3 (6.0 equiv), Et3N (2.0 equiv), CuSO4 (0.5 equiv), py, (92%); 5) SEM-Cl (2.0 equiv), i-Pr2NEt (3.0 equiv), TBAI (0.5 equiv), CH2Cl2, 40 °C (90%); 6) (i) Bu2BOTf (1.1 equiv), BH3.THF (10 equiv), 0 °C (90%); (ii) TBDPS-Cl (2.0 equiv), imidazole (3.0 equiv), CH2Cl2, rt (96%); (iii) DDQ (1.7 equiv), 10:1 CH2Cl2:H2O, rt (78%).

Glucosamine acceptors 14 and 16, with acetyl (Ac) and benzyl (Bn) protecting

groups at the C3 position respectively, and glycosyl acceptor 18 were also

prepared (Figure 2.10). Starting from intermediate 10, the C3 hydroxyl was

acetylated, the 4,6-anisylidene acetal was regioselectively cleaved with Bu2BOTf

and BH3.THF to yield a C8 alcohol, followed by 6-O-TBDPS protection to afford

intermediate 13 in 65% yield over three steps. This was converted to an �-benzyl

glycoside by activating the thiophenyl donor with N-iodosuccinimide (NIS) and

26

silver trifluoromethanesulfonate (AgOTf) in the presence of distilled benzyl

alcohol (BnOH), followed by deprotection of the PMB group by treatment with

BF3.Et2O in wet chloroform to afford acceptor 14.

Similarly, glycosyl acceptor 16 was synthesized from intermediate 10 in five

steps via 3-O-benzylation, 4,6-anisylidene acetal reductive cleavage, 6-O-

TBDPS protection, NIS/TfOH-mediated glycosylation, and deprotection of the 4-

O-PMB ether in 59% overall yield. Glycosyl acceptor 18 was synthesized by

regioselective cleavage of the 4,6-O-benzylidene acetal in glucoside 17 using

sodium cyanoborohydride and HCl in ether in 77% yield.

27

10

O

OAcN3

OTBDPS

PMBO

STol O

OAcN3

OTBDPS

HO

OBn

3) i. NaH, BnBr, im, TBAI1:4 DMF:THF

iii. TBDPS-Cl, im.CH2Cl2, rt

2) i. NIS, AgOTf, BnOH1:3 DCE:Et2O

ii. BF3.Et2O, CHCl3

13

10

O

OBnN3

OTBDPS

PMBO

STol O

OBnN3

OTBDPS

HO

OBn

ii. BH3.THF, TMSOTf, THF iii. TBDPS-Cl, im, CH2Cl2

4) i. NIS,TfOH, BnOH1:3 DCE:Et2O

ii. BF3.Et2O, CHCl3

14

15 16

1) i. Ac2O, Pyii. BH3.THF, TMSOTf

THF

O

OBnOBnO

OMeO

Ph

O

OBnOBnHO

OMeOBn

17 18

5) 4Å MS, NaBH3CN

HCl.Et2O, THF, rt

Figure 2.10 Synthesis of acceptors 14, 16 and 18. Reagents and conditions: 1) (i) Ac2O, py, rt (90%); (ii) BH3.THF, TMSOTf, –20 °C, THF (82%); (iii) TBDPS-Cl (2.0 equiv), imidazole (3.0 equiv), CH2Cl2, rt (96%); 2) (i) 4Å MS, NIS (2.0 equiv), AgOTf (0.6 equiv), BnOH (3.0 equiv), 3:1 Et2O:DCE, –20 °C (86% yield, 3:1 �:�); (ii) BF3.Et2O (4.0 equiv), wet CHCl3, –20 °C, (56%); 3) (i) NaH (5.0 equiv), TBAI (0.3 equiv), BnBr (3.0 equiv), imidazole (0.4 equiv), 4:1 THF:DMF, rt (90%); (ii) BH3.THF, TMSOTf, –20 °C, THF (85%); (iii) TBDPS-Cl (2.0 equiv), imidazole (3.0 equiv), CH2Cl2, rt (96%); 4) (i) 4Å MS, NIS (2.0 equiv), TfOH (0.4 equiv), BnOH (3.0 equiv), 3:1 Et2O:DCE, –20 °C (92%, 4:1 �:�); (ii) BF3.Et2O (4.0 equiv), wet CHCl3, –20 °C (87%); 5) 4Å MS, NaBH3CN (10 equiv), HCl.Et2O (excess),THF, rt (77%).

2.7 Synthesis of Disaccharides by BSP/Tf2O-Mediated Glycosylation

Thioglycosides were activated using 1-benzenesulfinyl piperidine and

trifluoromethanesulfonic anhydride (BSP/Tf2O) conditions, developed by Crich

and co-workers,10 as this method has been found to be compatible with a wide

variety of protecting groups in previous work by our research group.11

28

Mechanistically, BSP/Tf2O activation of thioglycosyl donor involves the

generation of N-sulfinyl triflate intermediate I at –60 °C. The highly thiophilic

intermediate activates the thioglycosyl donor to generate disulfonium

intermediate II, followed by concomitant formation of glycosyl triflate and a

cationic disulfide byproduct III. Finally, the glycosyl triflate reacts with the

acceptor (alcohol) in SN2-like fashion to yield �- or �-linked disaccharides,

depending on the configuration of the donor (Figure 2.10).

Figure 2.11 Mechanism of BSP/Tf2O-mediated glycosyl coupling10

A variation of the BSP/Tf2O-mediated coupling protocol was developed in our

lab and used to synthesize heparan disaccharide derivatives (19–22).8 In a

29

typical coupling, a mixture of glycosyl donor, BSP, tri-tert-butylpyrimidine (TTBP),

and activated 4Å molecular sieves were stirred in dichloromethane at –60 °C for

30 minutes, then treated with a dilute solution of Tf2O (precooled to –60 °C) for

30 minutes, followed by dropwise addition of glycosyl acceptor in CH2Cl2 solution

(again precooled to –60 °C) with additional stirring for one hour. The reaction

mixture was slowly warmed to –20 °C, then quenched with cold P(OEt)3 and Et3N

to afford heparan-like disaccharides with high �-stereoselectivity (Table 2.1).

30

Table 2.1 BSP/Tf2O-mediated coupling for heparan disaccharides synthesis

OO

O OBz

SPh

OBnPMP

Entry Donor Acceptor (ROH) Disaccharide

O

HO N3

STol

OSEM

OTBDPS

O

O N3

STol

OSEM

OO

OBzBnO

OPMPOTBDPS

O

HO N3

OBn

OAc

OTBDPS

O

HO N3

OBn

OBn

OTBDPS

O

OBz

SPh

OBn

1

2

3

4

O

HO OBn

OMe

OBn

OBn

O

O N3

OBn

OAc

OO

OBzBnO

OPMPOTBDPS

O

O N3

OBn

OBn

OO

OBzBnO

OPMPOTBDPS

O

O OBn

OMe

OBn

O

OBzBnO

OBn

4

4

4

12

14

16

19

20

21

22

O

OBn

SPh

OBz

BSP, TTBPTf2O, –60 °C

ii) P(OEt)3

i) Acceptor (ROH)–60 to –20 °CSPh

donor

6 18

73%

34%

66%

52%

Yield

30 min

O

OBn

OTf

OBz

O

OBn

OR

OBz

Reagents and conditions: BSP (1.2 equiv), TTBP (2.0 equiv), Tf2O (1.08 equiv), P(OEt)3 (2.0 equiv), Et3N (5.0 equiv).

31

2.8 Post-Glycosylative Modification of Disaccharides

To study the copper triflate-mediated stereoselective addition of furan at the

C5’ position, 4’-DP disaccharide 25 was synthesized from 21 in three steps by

hydrolysis of the 4’,6’-acetal, chemoselective TEMPO/BAIB oxidation, and

decarboxylative elimination following our standard procedure for the synthesis of

4-deoxypentenosides.12 Anisylidene-protected disaccharide 21 was hydrolyzed in

a 8:1:1 mixture of CH3COOH:THF:H2O and stirred at 70 °C for 5 hours to obtain

diol 23 in 86% yield (Figure 2.12). The same reaction could also be carried out

using microwave heating at 60 °C for 20 minutes in 70% yield, however we

preferred thermal conditions for large-scale synthesis.

The C6’ hydroxyl of 23 was selectively oxidized to carboxylic acid 24 by

treatment with BAIB and catalytic TEMPO in 1:2 CH2Cl2:H2O mixture at room

temperature for 45 minutes. This was subjected to decarboxylative elimination in

DMF using DMFDNpA at 150 °C for 45 minutes under microwave conditions, to

afford 4’-DP disaccharide 25 in 61% yield over two steps.

2.8.1 Synthesis of 5’-DTC-Activated Derivatives

4’-DP disaccharide 25 was converted to 4’-O-benzyl-5’-DTC derivative 28 in

three steps (Figure 2.12). 4’-DP disaccharide 25 was epoxidized with

dimethyldioxirane (DMDO) in dichloromethane at –70 °C, which proved to be

highly chemoselective and traceless after workup.13 The reaction mixture was

allowed to stir at 0 °C for 24 hours to afford 4’-epoxypyranosyl disaccharide 26.

The 1H NMR peak at 2.81 ppm (C6D6) indicated �-facioselectivity, which was

32

subsequently confirmed by epoxide ring opening with diethyl DTC yielding the 5’-

� adduct 27 in 72% yield over two steps (H5’: � 5.85 ppm, d, J = 10.0 Hz).8 It is

worth noting that some unreacted 4’-DP carried over from the epoxidation step,

and a slightly more polar byproduct was isolated but not fully characterized. We

speculate this to be the 5’� adduct, derived from the corresponding 4’�-

epoxypentenoside. Lastly, 5’�-DTC disaccharide 27 was treated with sodium

hexamethyldisilazane (NaHMDS) in DMF at –55 °C, followed by benzyl bromide

addition to afford the corresponding 4’-O-benzyl disaccharide 28 in 88% yield.

33

O

O

O

BnOOBn

OBn

N3

OBz

OTBDPS

5) Et2NH, CS2

MeOH-THF0 °C to rt

O

O

O

O

BnOOBn

OBn

N3

OBz

OTBDPSSHO 6) TBAI, BnBr

NaHMDS

DMF, –55 °C

O

O

O

BnOOBn

OBn

N3

OBz

OTBDPSSBnO

Et2N

S

O

O

O

BnO O

O

OBnN3

OBnOTBDPS

OBz

1) AcOH:THF:H2O(4:4:1)

70 °C

2) BAIB, TEMPO

3) DMFDNpA

DMF, 150 °C- wave

OHO

BnO O

O

OBnN3

OBnOTBDPS

OBz

O

HO

HO

BnO O

O

OBnN3

OBnOTBDPS

OBz

O

BnO O

O

OBnN3

OBnOTBDPS

OBz

O

21 23

2425

PMP HO

2:1 DCM:H2Ort

26 27

28

4) DMDO-DCM

–70 °C to 0 °C

NEt2

S

Figure 2.12 Synthesis of fully protected C5’-DTC disaccharide 28. Reagents and conditions: 1) 4:4:1 AcOH:THF:H2O, 70 °C, 6 h (86%); 2) BAIB (3.0 equiv), TEMPO (0.4 equiv), 2:1 CH2Cl2:H2O, rt, 35 min; 3) DMFDNpA (5.0 equiv), DMF, 150 °C, �-wave, 50 min (61% over two steps); 5) DMDO (3.0 equiv), CH2Cl2, –70 °C to 0 °C, 24 h; 5) CS2 (1.2 equiv), Et2NH (2.2 equiv), 1:2 THF:MeOH, 0 °C to rt, 3h (80% over two steps); 6) TBAI (0.4 equiv), BnBr (3.0 equiv), NaHMDS (3.0 equiv), DMF, –55 °C, 2 h (88%). Two additional DTC-activated carbohydrates (30 and 32) were also prepared

as model compounds. Tri-O-benzyl glucal 29 was converted to �-glucosyl DTC

30 in three steps: (i) epoxidation with DMDO in DCM to afford an �-epoxide, (ii)

epoxide ring opening using diethyl-DTC prepared in situ by the reaction of CS2

34

and diethylamine in methanol (80% yield over two steps), and (iii) 2-O-

benzylation to yield protected �-DTC glucoside 30 in 85% yield (Figure 2.13). �-

C5’-DTC disaccharide 32 was synthesized from the 4’-DP derivative of

cellobioside (31) in a similar fashion, in 53% yield over three steps (Figure 2.13).

O

OBnBnO

BnOS

OBnS

NEt2O

OBnBnO

BnO1) DMDO-acetone, 0°C2) CS2, Et2NH, MeOH-THF

3) NaHMDS, BnBrDMF, –55 °C

O

OBn

OBn

OBn

OBn4) DMDO-DCM, –60°C5) CS2, Et2NH, MeOH-THF

6) NaHMDS, BnBrDMF, –55 °C

2930

31 32

O

O

BnOOBn

O

OBn

OBn

OBn

OBn

O

O

BnOOBn

BnOS

NEt2

S

Figure 2.13 Synthesis of �-glucosyl DTC 30 and �-C5’-DTC disaccharide 32. Reagents and conditions: 1) 0.1 M DMDO in acetone (3.0 equiv), CH2Cl2, –30 to –20 °C, 2 h; 2) CS2 (1.2 equiv), Et2NH (2.2 equiv), 1:2 THF:MeOH, 0 °C to rt, 3 h (80% over two steps); 3) TBAI (0.4 equiv), BnBr (3.0 equiv), NaHMDS (3.0 equiv), DMF, –55 °C, 2 h (78%); 4) DMDO in DCM (3.0 equiv), CH2Cl2, –60 to –30 °C, 48 h; 5) CS2 (1.2 equiv), Et2NH (2.2 equiv), 1:2 THF:MeOH, 0 °C to rt, 2 h (61% over two steps); 6) TBAI (0.4 equiv), BnBr (3.0 equiv), NaHMDS (3.0 equiv), DMF, –55 °C, 2 h (88%).

2.8.2 C-Furylation of Glycosyl DTCs Given our laboratory’s earlier success in using DTC glycosides as donors,14

we anticipated DTC glycosides to be amenable toward C-furylation, which could

ultimately be converted to carboxyl group by ozonolysis.5 We thus initiated this

study using �-DTC glucoside 30 and �-5’-DTC disaccharide 32 as model

35

compounds for C-furylation. �-DTC glucoside 30 was treated with furan in the

presence of copper triflate, which had been used previously to activate DTCs for

O-glycosylation.14 C-furyl adduct 33 was formed in 72% isolated yield (entry 5,

Table 2.2).

Table 2.2 Copper(I/II) triflate-mediated furylation of �-glucosyl DTC 30

O

OBnBnO

OBnS

OBn

Cu(OTf)x, furan, 4Å MS O

OBnBnO

OBn

OBn

O

Entry Product/Yield

1 Cu(OTf)2 furan (neat) 5 h No reaction

2 Cu(OTf)2furan-DCM

(1:1)

Solvent Time

16 h Incomplete [a]

3 Cu(OTf)2 furan-DCE(1:3)

furan-DCE(1:1)

–30 to 0 °C

8 h

5 h4

5

Cu(OTf)2

CuOTf(C6H6)0.5

Lewis acid(2.2 equiv)

DCE(30 eq furan)

45 min 72 %

33

NEt2

S

XXX

XXX

Note: CuOTf.(C6H6)0.5 was prepared using literature procedure;15 furan was freshly distilled over CaH2. In entries 1 and 2, the reaction was warmed to 10 °C. [a] 60% conversion by 1H NMR of the crude reaction mixture. The optimized reaction condition was then applied toward �-5’-DTC

disaccharide 32 (Figure 2.14). This did not result in a clean addition; however,

ESI-MS of the crude product yielded a significant signal at m/z 1031,

corresponding to the [M+Na]+ peak of expected disaccharide 34.

36

Figure 2.14 CuOTf-mediated C-furylation of 5’-DTC disaccharide 32. Reagents and conditions: CuOTf(C6H6)0.5 (2.1 equiv), furan (32 equiv), 4Å molecular sieves, CH2Cl2, –30 to –5 °C over 1 h; c = 0.036 M. �-5’-DTC disaccharide 28 was also subjected to furan addition using the

optimized conditions for the synthesis of 33. This produced 5’-furyl adduct 35 in

32% isolated yield, along with recovered starting material (Figure 2.15).

O

O

O

BnOOBn

OBn

N3

OBz

OTBDPSBnO

O

O

O

O

BnOOBn

OBn

N3OBz

OTBDPSBnO

S

NEt2

S

4Å MS, CuOTf·(C6H6)0.5

35

furan, ClCH2CH2Cl

28

Figure 2.15 CuOTf-mediated C-furylation of 5’-DTC disaccharide 28. Reagents and conditions: CuOTf.(C6H6)0.5 (2.5 equiv), furan (30 equiv), 4Å molecular sieves, ClCH2CH2Cl, –30 to –5 °C over 1 h; c = 0.05 M (32%).

37

2.9 Conclusions We have found 4-deoxypentenosyl thioglycosides to be versatile donors using

the BSP/Tf2O-mediated glycosylation protocol. Post-glycosylational C5� additions

were investigated using DMDO oxidation and epoxide ring opening by

nucleophilic DTCs, prepared in situ from diethylamine and CS2, and CuOTf-

mediated furan additions, however C-furylation was not efficient.

38

2.10 General Methods and Experiments

2.10.1 Experimental

General methods: All starting materials and reagents were obtained from

commercial sources and used as received unless otherwise noted. All solvents

used were freshly distilled prior to use. 1H and 13C NMR spectra were recorded

on a Bruker ARX400 operating at 400 MHz and 100 MHz or a Bruker AV500HD

operating at 500 MHz and 125 MHz or Bruker Avance-III-800 operating at 201

MHz respectively, and referenced to the solvent used (7.16 and 128.06 ppm for

C6D6, 7.26 and 77.00 ppm for CDCl3) unless stated otherwise. IR spectra were

acquired from Nicolet- Nexus 670 FTIR by thin film preparation with attenuated

total reflectance (ATR) technique. Optical rotations were measured at room

temperature with a Rudolph Research AUTOPOL® III polarimeter. Mass spectra

were acquired using either a Hewlett-Packard 5989B or a Finnigan 4000 mass

spectrometer. Column chromatrography was performed with silica gel (Sorbent

Tech, 32–63 D). Preparative TLC separation was performed with silica gel Prep

TLC (Sorbent Tech, 500 �m). TLC analysis was monitored with silica gel G60F254

(Sorbent Tech, 250 �m) and detected by UV absorption at 256 nm or dipping in

p-anisaldehyde-sulfuric acid stain solution then charring at 150 °C. 4Å molecular

sieves were flame-dried using a Bunsen burner under reduced pressure prior to

use. Tf2O was freshly distilled from P2O5 prior to use. N-iodosuccinimide (38 mg,

0.168 mmol) was recrystallized from 1:4 acetone:hexanes. BSP was purchased

39

from Sigma–Aldrich and used without further purification. All experiments were

performed under the argon atmosphere with standard Schlenk line techniques.

General method for BSP/Tf2O glycosylation: In a typical procedure, all

glasswares were flame-dried. Thioglycoside donor (234 mg) and acceptor (396

mg, 0.58 mmol) were dried separately by azeotropic distillation with toluene (3 x

5 mL) and dried in high vacuum for 1 h. The glycosyl donor was combined with

the BSP (101 mg, 0.480 mmol), TTBP (198 mg, 0.80 mmol), and freshly

activated 4Å molecular sieves (300 mg) under an inert atmosphere and dissolved

in freshly distilled CH2Cl2 (3 mL), then stirred at rt for 1 h. The solution was

cooled to –60 °C and stirred for an additional 30 min, after which freshly distilled

Tf2O (73 �L, 0.43 mmol) was added dropwise over 5 minutes. The resulting

bright orange solution of glycosyl triflate solution was stirred at –60 °C for 15–30

min and monitored by TLC until the consumption of glycosyl donor was complete,

then treated with a solution of pre-cooled (–60 °C) acceptor (0.63 mmol) in

CH2Cl2 (1.5 mL). Stirring was continued at –60 °C for 1 h, and then slowly

warmed to –20 °C over a period of 4 h. The resulting pale orange solution was

quenched with P(OEt)3 (138 �L, 0.79 mmol) and Et3N (280 �L, 2.02 mmol) pre-

cooled to –20 °C, then stirred for an aadditional 15 min. The yellow solution was

removed from the cooler and slowly warmed to rt over 30 min. The reaction

mixture was then quenched at 0 °C with saturated NaHCO3 (5 mL), and stirred

vigorously for 30 min at rt. The suspension was filtered through Celite, extracted

with CH2Cl2 (3 × 10 mL), washed with brine (3 x 5 mL), dried over Na2SO4,

40

concentrated under reduced pressure, then purified by silica column

chromatography.

General method for NIS glycosylation: Thioglycoside (70 mg, 0.092 mmol)

was dried by azeotropic distillation with toluene (3 x 1 mL) and dissolved in a 1:3

mixture of DCE:Et2O (1.2 mL) under inert atmosphere, then treated with activated

4Å molecular sieves (250 mg), freshly distilled benzyl alcohol (25 �L, 0.24 mmol),

and N-iodosuccinimide (38 mg, 0.168 mmol). The reaction mixture was cooled to

–30 °C for 15 minutes and treated with triflic acid (3.5 �L, 0.039 mmol) or AgOTf

(14 mg, 0.054 mmol), then slowly warmed to –20 °C over one hour. The reaction

mixture was quenched with Et3N (40 �L, 0.29 mmol) at –20 °C, then warmed to

room temperature and passed through Cellite, washed with saturated sodium

thiosulfate solution (3 x 2 mL), dried over anhydrous sodium sulfate,

concentrated to dryness, then purified by silica column chromatography.

General methods for epoxidation of 4’-DP-disaccharides and epoxide

ring opening by DTC: In a typical protocol, the glycal 4’-DP (0.03 mmol) was

dried by azeotropic distillation with toluene (3 × 1 mL) and dissolved in CH2Cl2

(0.8 mL). This solution was cooled to –70 °C for 15 min, treated with freshly

prepared 0.1 M DMDO-DCM solution (1 mL, 0.1 mmol) with stirring for 1 h, then

warmed to –30 °C and stirred for 12–24 h. The mixture was then concentrated

(at 0 °C for the best results), azeotroped with toluene (3 x 1 mL), then dissolved

in dry and degassed THF (0.3 mL) for epoxide ring opening by using freshly

prepared diethyl dithiocarbamate; prepared in situ in degassed MeOH (4.9 mL)

from freshly distilled diethylamine (560 �L, 5.2 mmol) and freshly distilled CS2

41

(160 �L, 2.6 mmol). The ice bath was removed and stirred at rt for 30 minutes to

obtain a colorless to slightly pale-yellow solution of diethyl-DTC

diethylammonium salt (2.6 mmol, 0.53 M in MeOH), which was used

immediately. The calculated volume of freshly prepared DTC solution was added

to the epoxide solution in THF at 0 °C under argon atmosphere with stirring for 3

h. After the completion of reaction, the mixture was concentrated, and purified by

silica column chromatography to obtain the DTC products.

General method for 4’-O-benzylation of DTC disaccharides: 4’-O-Benzyl-

5’-DTC disaccharide was prepared by the adapted protocol: 5’-DTC-disaccharide

(0.1 mmol) was dried by azeotropic distillation with toluene (3 x 1 mL) and

dissolved in dry DMF (2 mL) at room temperature, then treated with TBAI (8 mg,

0.02 mmol), cooled to –50 °C for 15 minutes under argon, and treated with

freshly distilled benzyl bromide (34 �L, 0.28 mmol) and 0.6 M NaHMDS solution

in toluene (500 �L, 0.3 mmol). The reaction mixture was stirred for 2–3 hours

then quenched at –50 °C with saturated ammonium chloride solution (2 mL),

extracted with diethyl ether (3 x 5 mL), washed with brine (3 x 1 mL), dried over

anhydrous sodium sulfate, and purified by column chromatography using a 5–

20% ethyl acetate in hexanes gradient.

General procedure for the CuOTf(C6H6)0.5-mediated furan addition to

glycosyl dithiocarbamate: Glycosyl DTC donor (0.052 mmol) was dried by

azeotropic distillation with toluene (3 × 1 mL), dissolved in degassed DCE (0.65

mL), then introduced into a two-necked flask charged with freshly activated 4Å

molecular sieves (100 mg) and distilled furan (75 �L, 1.04 mmol). The mixture

42

was cooled to –30 °C for 15 minutes and treated with CuOTf.(C6H6)0.5 (27 mg,

0.104 mmol) which was reserved in a solid addition tube. The reaction mixture

was slowly warmed to –5 °C over 45–60 minutes, and turned yellow near

completion. The mixture was quenched with Et3N (35 �L, 0.25 mmol) at –5 °C,

then warmed to room temperature and passed through a pad of Celite and

diluted with water, extracted with ethyl acetate (3 x 5 mL), washed with brine (3 x

2 mL), dried over sodium sulfate, then purified by silica column chromatography.

Thiophenyl 2-O-benzoyl-3-O-benzyl-4,6-O-p-methoxybenzylidene-�-

glucopyranoside (4). Glucose pentaacetate was subjected to Lewis-acid

(BF3.Et2O)-mediated glycosylation with thiophenol (PhSH) to give 1 in 90% yield.

Glucoside 1 was deacetylated and converted to a 4,6-p-methoxybenzylidene

acetal 2 in 80% yield over two steps. Regioselective 3-O-benzylation of 2 gave

compound 3 in 90% yield, and 2-O-benzoylation of compound 3 afforded glycosyl

donor 4 as a white crystalline solid in 90% yield. 1H and 13C NMR were compared

and matched with literature:8 1H NMR (300 MHz, CDCl3): � 8.02 (d, J = 7.3 Hz,

2H), 7.61 (t, J = 7.4 Hz, 1H), 7.53–7.37 (m, 6H), 7.36–7.21 (m, 4H), 7.19–7.01

(m, 4H), 6.92 (d, J = 8.7 Hz, 2 H), 5.57 (s, 1H), 5.29 (dd, J = 8.5, 10.0 Hz, 1H),

4.85 (d, J = 10.1 Hz, 1H), 4.80 (d, J = 11.9 Hz, 1H), 4.66 (d, J = 11.9 Hz, 1H),

4.40 (dd, J = 5.0, 10.5 Hz, 1H), 3.96–3.71 (m, 5H), 3.56 (m, 1H), 1.55 (s, 1H).

Thiophenyl 2-O-benzoyl-3-O-benzyl-4-deoxy-�-pent-4-enopyranoside (6).

Thioglycoside 4 (85 mg, 0.14 mmol) was hydrolyzed by 8:1:1 AcOH:THF:H2O (5

mL) by heating to 70 °C for 3 h. Upon completion of the reaction, the mixture was

cooled to room temperature, concentrated and azeotroped with toluene (3 x 5

43

mL) to remove residual AcOH, redissolved in ethyl acetate (20 mL), washed with

brine (3 x 2 mL), dried over Na2SO4, concentrated to dryness, and purified by

silica gel chromatography using a 20–100% EtOAc in hexanes gradient to afford

diol 5 (66 mg, 96%) as a white crystalline solid.

Diol 5 was subjected to TEMPO/BAIB oxidation, quenched with saturated

sodium thiosulfate solution, extracted with chloroform, dried over sodium

anhydrous Na2SO4, and concentrated to afford crude glucuronic acid (65 mg,

0.13 mmol). This was dried by azeotropic distillation with toluene (3 x 2 mL),

dissolved in distilled DMF (4 mL) in a sealed microwave tube, and treated with

DMFDNpA (185 �L, 0.66 mmol). The reaction mixture was heated at 150 °C for

20 minutes under microwave conditions, then concentrated to dryness and

purified by silica gel chromatography using a 5–10% EtOAc in hexanes gradient

with 1% Et3N to yield thiophenyl 4-DP glycoside 6 as a white solid (36 mg, 48%

over two steps). 1H NMR (400 MHz, C6D6): � 8.09 (d, J = 8.0 Hz, 2H), 7.53 (d, J =

7.5 Hz, 2H), 7.25–6.90 (m, 12H), 6.32 (d, J = 6.4 Hz, 1H), 5.90 (d, J = 1.6 Hz,

1H), 5.76 (d, J = 1.2 Hz, 1H), 4.98 (t, J = 12 Hz, 1H), 4.67 (d, J = 12.4 Hz, 1H),

4.58 (d, J = 12 Hz, 1H), 3.87 (m, 1H). 13C NMR (100 MHz, C6D6): � 164.98,

142.91, 138.59, 138.58, 136.33, 133.13, , 131.53, 129.93, , 129.00, 128.39,

128.06, 127.82, 127.75, 127.58, 127.27, 100.87, 83.42, 70.17, 68.29. IR: 3072,

2886, 1722, 1649, 1265, 1070, 711, 677 cm-1. [�]25D = –201° (c= 0.46, CH2Cl2).

HRESI-MS: m/z calcd. for C25H22O4S [M+Na]+ 441.1136; found 441.1131.

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-phthalimido-�-glucopyranoside (7). The

synthesis of glucosamine 7 was adapted from the literature previously reported

44

from our group.9 Glucosamine hydrochloride (50 g, 232 mmol) was mechanically

agitated with 1 M NaOMe in MeOH (232 mL) for 2 h at 25 °C, followed by

addition of finely powdered phthalic anhydride (19 g,128 mmol) and Et3N (35.5

mL, 255 mmol). The mixture was stirred for 45 min, then treated with a second

portion of phthalic anhydride (19 g, 128 mmol) and stirred for 24 h at 25 °C. The

mixture was cooled to –20 °C for 4 h, then concentrated under reduced pressure

to afford a white solid which was redispersed in pyridine (500 mL) followed by

slow addition of Ac2O (330 mL) at 0 °C. The reaction mixture was mechanically

stirred at 25 °C for 48 h, then concentrated to dryness and purified by

recrystallization in 95% EtOH to afford 7 as an amorphous white solid (77.6 g,

70% over 3 steps). The product was confirmed by comparison with literature

NMR data:9 1H NMR (300 MHz, CDCl3): � 7.84 (dd, J = 3, 6 Hz, 2H), 7.73 (dd, J

= 3.0, 5.7 Hz, 2H), 6.49 (d, J = 8.7 Hz, 1H), 5.86 (dd, J = 9.3, 10.5 Hz, 1H), 5.19

(t, J = 9.3 Hz, 1H), 4.44 (dd, J = 8.7, 10.5 Hz, 1H), 4.34 (dd, J = 3.9, 12.3 Hz,

1H), 4.11 (dd, J = 2.4, 12.3 Hz, 1H), 4.00 (ddd, J = 2.4, 4.8, 10.8 Hz, 1H), 2.08 (s,

3H), 1.97 (s, 3H), 1.84 (s, 3H); 13C NMR (75 MHz, CDCl3): � 170.70, 170.08,

169.55, 168.71, 167.44, 134.57, 131.26, 123.87, 89.82, 72.67, 70.56, 68.32,

61.58, 53.55, 20.82, 20.68, 20.48.

Thiotolyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-�-glucopyranoside (8).

A solution of glucosamine acetate 7 (10 g, 20.9 mmol) and p-thiocresol (3.4 g,

27.25 mmol) in CH2Cl2 (200 mL) cooled to 0 °C was treated with BF3·Et2O by

dropwise addition under argon atmosphere, then stirred at room temperature for

24 h. The reaction mixture was quenched with saturated NaHCO3 solution (50

45

mL), extracted with CH2Cl2 (3 x 100 mL), washed with brine solution (3 x 25 mL),

dried over anhydrous Na2SO4, filtered, then concentrated to dryness and the

crude product was purified by recrystallization in 95% EtOH to afford

thioglycoside 8 as an amorphous white solid (8.5 g, 75%). 1H NMR (400 MHz,

CDCl3): �7.87 (dd, J = 3.1, 5.4 Hz, 2H), 7.76 (dd, J = 3.0, 5.6 Hz, 2H), 7.3 (d, J

=7.1 Hz 2H), (d, J = 7.9 Hz, 2H), 5.77 (t, J = 9.2 Hz, 1H), 5.65 (d, J = 10.6 Hz,

1H), 5.12 (t, J = 10.2, Hz, 1H), 4.27 (m, 2H), 4.20 (dd, J = 2.4, 12.2 Hz, 1H), 3.88

(ddd, J = 2.4, 4.9, 10.4 Hz, 1H), 2.33 (s, 3H), 2.10 (s, 3H), 2.02 (s, 3H), 1.83 (s,

3H); 13C NMR lit.16 (75 MHz, CDCl3): � 170.76, 170.22, 169.58, 167.95, 167.08,

138.89, 134.57, 134.43, 134.04, 131.71, 131.29, 129.77, 127.05, 123.82, 83.22,

75.95, 71.77, 68.79, 62.31, 53.69, 21.29, 20.90, 20.73, 20.54.

Thiotolyl 2-deoxy-4,6-O-p-methoxybenzylidene-2-phthalimido-�-

glucopyranoside (9). A solution of glucosamine triacetate 8 (15.4 g, 28.46

mmol) in 2:3 CH2Cl2:MeOH (300 mL) was cooled to �10 °C and treated with 1 M

NaOMe in MeOH (11.5 mL, 11.5 mmol) with stirring at �10 °C for 2 h. When all

starting material was consumed, the reaction was quenched with activated

Dowex 50X-W-H+ ion-exchange resin, filtered, dried over sodium sulfate, then

concentrated to dryness to obtain the crude 3,4,6-triol. This was dried by

azeotropic distillation with toluene (3 x 15 mL), dissolved in THF (300 mL),

treated with anisylidene dimethyl acetal (11.78 mL, 69.4 mmol) and d-CSA (1.28

g, 5.531 mmol), then heated to reflux for 10 h. When all starting material was

consumed, the reaction was quenched with saturated NaHCO3 solution (50 mL)

at room temperature, extracted with ethyl acetate (3 x 150 mL), dried over

46

Na2SO4, and concentrated to dryness. The crude product was purified by

recrystallization in 95% EtOH to obtain 9 as an amorphous white solid (11.7 g,

77% over 2 steps). 1H NMR (400 MHz, CDCl3): � 7.88 (d, J = 4.3 Hz, 1H), 7.83

(d, J = 2.3 Hz, 1H), 7.77–7.70 (m, 2H), 7.39 (d, J = 9.8 Hz, 2H), 7.27 (d, J = 8.5

Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.61 (d, J = 10.7 Hz,

1H), 5.50 (s, 1H), 4.58 (t, J = 9.7 Hz, 1H), 4.36 (dd, J = 4.8, 10.4 Hz, 1H), 4.29 (t,

J = 10.3 Hz, 1H), 3.80 (m, 4H), 3.64 (td, J = 4.9, 9.6 Hz, 1H), 3.54 (t, J = 9.1 Hz,

1H), 2.65 (s, 1H), 2.30 (s, 3H). 13C NMR (100 MHz, CDCl3): � 160.22, 138.32,

134.10, 133.21, 131.58, 129.61, 129.32, 127.78, 127.55, 123.74, 123.24, 113.65,

101.78, 84.37, 81.76, 77.26, 76.94, 76.62, 70.20, 69.66, 68.46, 60.30, 55.54,

55.22, 21.04, 14.09.

Thiotolyl 2-azido-2-deoxy-4,6-O-p-methoxylbenzylidene-�-gluco-

pyranoside (10). Preparation of TfN3: A suspension of NaN3 (2.8 g, 43.1 mmol)

in distilled pyridine (25 mL) was treated with distilledTf2O (3.6 mL, 21.3 mmol) at

0 °C with stirring for 2 h, and used immediately. A solution of phthalimide 9 (3.8

g, 7.1 mmol) and ethylenediamine (12 mL, 177.8 mmol) in n-butanol (12 mL) in a

pressure tube was heated at 100 °C with stirring for 10 h. The mixture was

cooled down to room temperature, concentrated to dryness, azeotroped with

toluene (3x 15 mL), , then re-dispersed in distilled pyridine (25 mL) and treated

with with Et3N (2.0 mL, 14.4 mmol), CuSO4·5H2O (890 mg, 3.56 mmol) and

freshly prepared TfN3 (25 mL) at 0 °C. The mixture was slowly warmed to 25 °C

and stirred for 3 h. Upon the completion of reaction, the mixture was

concentrated, azeotroped with toluene (3 x 10 mL), then the crude azide was

47

dispersed into ethyl acetate (30 mL) and water (5 mL), extracted with ethyl

acetate (3 x 150 mL), washed with brine (3 x 20 mL), dried over Na2SO4,

concentrated to dryness and purified by silica column chromatography using a 0–

30% ethyl acetate in hexanes gradient to afford glucosamine 10 as an

amorphous white solid (3.0 g, 92%). 1H NMR (400 MHz, CDCl3): � 7.46 (d, J =

8.8 Hz, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 8.7 Hz,

2H), 5.48 (s, 1H), 4.48 (d, J = 10.1 Hz, 1H), 4.35 (dd, J = 3.9, 10.7 Hz, 1H), 3.80

(s, 3H), 3.75 (t, J = 8.8 Hz, 1H), 3.43 (m, 2H), 3.31 (dd, J = 9.0, 10.1 Hz, 1H),

2.90 (s, 1H), 2.37 (s, 3H).

Thiotolyl 2-azido-2-deoxy-4,6-O-p-methoxybenzylidene-3-O-[2-

(trimethylsilyl)ethoxymethyl]-�-glucopyranoside (11). A solution of

glucosamine 10 (0.48 g, 1.1 mmol), TBAI (206 mg, 0.55 mmol) and DIPEA (0.9

mL, 5.6 mmol) in dichloromethane (10 mL) was treated with SEM-Cl (0.6 mL, 3.3

mmol) at room temperature, then refluxed at 40 °C for 12 h. The reaction was

quenched with saturated sodium bicarbonate solution (5 mL) and stirred at room

temperature for 4 h, then extracted with dichloromethane (3 x 50 mL), washed

with brine (3 x 5 mL), dried over anhydrous sodium sulfate, concentrated under

reduced pressure, and purified by silica gel chromatography using a 0–20%

EtOAc in hexanes gradient to yield 3-O-SEM ether 11 as colorless syrup (563

mg, 90%). 1H NMR (400 MHz, CDCl3): � 7.46 (d, J = 7.3 Hz, 2H), 7.35 (d, J =

8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 9.8 Hz, 2H), 5.46 (s, 1H), 4.94

(d, J = 6.7 Hz, 1H), 4.80 (d, J = 6.7 Hz, 1H), 4.47 (d, J = 10.1 Hz, 1H), 4.33 (dd, J

48

= 4.9, 10.5 Hz, 1H), 3.8 (s, 3H), 3.7 (m, 3H), 3.49 (m, 3H), 3.28 (t, J = 9.8 Hz,

1H), 2.37 (s, 3H), 0.88 (t, J = 7.5 Hz, 2H), 0.01 (s, 9H).

Thiotolyl 2-azido-6-O-(tert-butyldiphenylsilyl)-2-deoxy-3-O-[(2-trimethyl-

silylethoxymethyl)]-�-glucopyranoside (12). All glasswares were flame-dried

before use. Glycoside 11 (1.16 g, 2.1 mmol) was azeotroped with toluene (3 x 5

mL), dissolved in a 1 M BH3 solution in THF (20.6 mL, 20.6 mmol), and treated

with a 1 M Bu2BOTf solution in THF (2.3 mL, 2.3 mmol) at –10 °C, then warmed

to 0 °C with stirring for 1 h. The reaction was quenched with Et3N (0.58 mL, 2.4

mmol) followed by the slow addition of cold MeOH (15 mL) at �10 °C until the

effervescence ceased. The reaction mixture was slowly warmed to room

temperature, concentrated, azeotroped with methanol (3 x 10 mL), then purified

by silica gel chromatography using a 0–20% EtOAc in hexanes gradient to afford

the intermediate C6 alcohol as a syrupy solid (1.02 g, 90%).

The C6 alcohol was azeotroped with toluene (3 x 5 mL), dissolved in distilled

dichloromethane (18 mL) at room temperature, treated with imidazole (367 mg,

3.06 mmol) and TBDPS-Cl (530 �L, 2.04 mmol), and stirred for 4 h at room

temperature under argon. The reaction was quenched with saturated ammonium

chloride solution (20 mL), extracted with dichloromethane (3 x 100 mL), dried

over sodium sulfate, concentrated, then purified by silica column chromatography

using a 0–20% ethyl acetate in hexanes gradient to obtain the intermediate 6-O-

TBDPS ether as colorless syrup (1.45 g, 96% yield).

A portion of the orthogonally protected intermediate (1 g, 1.25 mmol) was

dissolved in 1:10 H2O:CH2Cl2 (27.5 mL) and treated with DDQ (480 mg, 2.11

49

mmol) at room temperature for 1 h. The reaction was quenched with saturated

sodium thiosulfate solution (25 mL), washed with sodium bicarbonate solution

(30 mL), extracted with dichloromethane, dried over sodium sulfate, concentrated

to dryness, then purified by silica gel chromatography using a 0–5% acetone in

hexanes gradient to afford C4 alcohol 12 as a colorless syrup (660 mg, 78%

yield). The product was confirmed by comparison with literature NMR data.8 1H

NMR (400 MHz, CDCl3): � 7.76 (dt, J = 7.9, 1.6 Hz, 4H), 7.52 (d, J = 8.0 Hz, 2H),

7.40 (m, 6H), 7.05 (d, J = 8.0 Hz, 2H), 4.93 (d, J = 7.2 Hz, 1H), 4.76 (t, J = 7.5

Hz, 1H), 4.48 (d, J = 1.2 Hz, 1H), 4.37 (d, J = 11.4 Hz, 1H), 4.03 (dd, J = 2.2,

11.1 Hz, 1H), 3.94 (dd, J = 4.5, 11.1 Hz, 1H), 3.82 (m, 1H), 3.6 (m, 1H), 3.32 (m,

2H), 2.32 (s, 3H), 1.07 (s, 9H), 0.97 (m, 2H), 0.01 (s, 9H).

Thiotolyl 3-O-acetyl-2-azido-2-deoxy-4-O-p-methoxybenzyl-6-O-(tert-

butyldiphenylsilyl)-�-glucopyranoside (13). Intermediate 10 (1.4 g, 3.26 mmol)

was acetylated to yield the 3-O-acetate (1.38 g, 90%). A portion of this (1.14 g,

2.42 mmol) was azeotroped with toluene (3 x 5 mL) and treated with 1 M BH3

solution in THF (12.1 mL) and TMSOTf (90 �L, 0.5 mmol) at –30 °C, then slowly

warmed to 0 °C and stirred at this temperature for 6 h. The reaction was

quenched with triethylamine (3.3 mL, 23.86 mmol) followed by slow addition of

methanol (10 mL), then warmed to room temperature and concentrated with

azeotropic distillation using additional methanol (3 x 10 mL), then purified by

silica gel column chromatography using a 10–40% ethyl acetate in hexanes

gradient to afford the C6 alcohol as a white solid (938 mg, 82%).

50

A portion of this C6 alcohol (680 mg, 1.43 mmol) was dissolved in

dichloromethane (11 mL), treated with TBDPS-Cl (740 �L, 2.88 mmol) and

imidazole (290 mg, 4.26 mmol) at 0 °C, then warmed to room temperature and

stirred for an additional 2 h. The reaction was quenched with saturated

ammonium chloride solution (10 mL), extracted with dichloromethane (3 x 50

mL), washed with brine (3 x 5 mL), dried over sodium sulfate, and purified by

silica column chromatography using a 0–10% ethyl acetate in hexanes gradient

to afford 6-O-TBDPS ether 13 as a white solid (920 mg, 90% yield). 1H NMR (400

MHz, CDCl3): � 7.99 (m, 2H), 7.89 (m, 2H), 7.71 (d, J = 7.9 Hz, 2H), 7.34 (m,

5H), 7.20 (m, 3H), 6.92 (d, J = 7.9 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H), 5.41 (t, J =

9.6 Hz, 1H), 4.62 (t, J = 6.6 Hz, 1H), 4.22 (d, J = 10.2 Hz, 1H), 3.94 (m, 2H), 3.76

(t, J = 9.5 Hz, 1H), 3.36 (s, 3H), 3.31 (t, J = 9.8 Hz, 1H), 2.99 (d, J = 9.7 Hz, 1H),

2.04 (s, 3H), 1.73 (s, 3H), 1.27 (s, 9H); 13C NMR (100 MHz, CDCl3): � 169.70,

159.26, 138.55, 135.77, 135.53, 135.11, 134.71, 133.98, 133.21, 132.68, 129.73,

129.55, 129.32, 127.74, 127.64, 127.24, 113.78, 85.96, 79.90, 77.26, 77.14,

76.94, 76.62, 76.22, 74.92, 74.28, 63.03, 62.17, 55.20, 26.84, 26.47, 21.11,

20.84, 19.25, 18.93.

Benzyl 2-azido-3-O-acetyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-�-

glucopyranoside (14). A flame-dried two-necked round-bottom flask was

charged with activated 4Å molecular sieves (650 mg) and equipped with a solid

addition tube containing AgOTf (73 mg, 0.28 mmol). A solution of thioglycoside

13 (312 mg, 0.44 mmol), distilled benzyl alcohol (175 �L, 1.69 mmol), and

recrystallized NIS (217 mg, 0.96 mmol) in 1:3 DCE:ether (5 mL) was added,

51

cooled to –30 °C for 15 minutes, then treated with AgOTf from the solid addition

tube. The reaction mixture was warmed to –20 °C with stirring for 2 h, then

quenched with triethylamine (200 �L, 1.44 mmol) and warmed to room

temperature. The mixture was passed through Celite, then washed with sodium

thiosulfate solution (3 x 5 mL) and brine (3 x 5 mL), dried over sodium sulfate,

then concentrated and purified by column chromatography using 0–10% ethyl

acetate in hexanes gradient to yield �-benzyl glycoside as the major product

(63%, colorless syrup) and �-benzyl glycoside as the minor product (20%).

A portion of �-benzyl glycoside (160 mg, 0.23 mmol) was dissolved in wet

chloroform (4 mL) and treated with BF3.Et2O (145 �L, 1.15 mmol) at –25 °C with

stirring for 30 minutes. The reaction was quenched with saturated sodium

bicarbonate solution (5 mL) at –20 °C, warmed to room temperature, extracted

with dichloromethane (3 x 50 mL), washed with brine (3 x 5 mL), dried over

sodium sulfate, filtered and concentrated, then purified by silica column

chromatography using a 10–30% ethyl acetate in hexanes gradient to give

glucosamine acceptor 14 as a colorless syrup (315 mg, 56%). Note: acetyl

transfer between C3 and C4 of product was observed. 1H NMR (400 MHz,

CDCl3): � 7.71 (ddd, J =1.9, 3.7, 8.0 Hz, 4H), 7.34 (m, 11H), 5.39 (dd, J = 8.6,

10.6 Hz, 1H), 5.00 (d, J = 3.6 Hz, 1H), 4.65 (m, 2H), 3.90 (d, J = 3.8 Hz, 2H),

3.75 (m, 2H), 3.25 (dd, J = 3.5, 10.6 Hz, 1H), 2.77 (s, 1H), 2.19 (s, 3H), 1.09 (s,

9H).

Thiotolyl -2-azido-3-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-4-O-p-

methoxybenzyl-�-glucopyranoside (15). C3 alcohol 10 (1 g, 2.33 mmol) was

52

dissolved in 1:4 DMF:THF (12 mL) along with imidazole (63 mg, 0.93 mmol), and

TBAI (344 mg, 0.93 mmol) and cooled to 0 °C for 30 minutes. The reaction

mixture was treated with 60% sodium hydride (450 mg, 18.75 mmol) in small

portions and stirred for 1 h, then treated with benzyl bromide (830 �L, 6.98

mmol), warmed to room temperature, and stirred for an additional 4 h. The

reaction was quenched with saturated ammonium chloride (10 mL) and stirred for

1 h, then extracted with ethyl acetate (3 x 100 mL), washed with brine (3 x 5 mL),

dried over sodium sulfate, and concentrated to dryness. The crude product was

purified by silica gel chromatography using a 0–20% ethyl acetate in hexanes

gradient to yield the 3-O-benzyl ether as a white solid (1.08 g, 90%).

A portion of this product (273 mg, 0.52 mmol) was treated with BH3.THF (2.6

mL, 2.6 mmol) and TMSOTf (20 �L, 0.21 mmol) at –20 °C, followed by additional

workup and purification steps as described for compound 13 to yield a C6 alcohol

as a colorless syrup (232 mg, 85%). A portion this intermediate (180 mg, 0.34

mmol) was dissolved in in dichloromethane (3.5 mL) and treated with TBDPS-Cl

(177 �L, 0.68 mmol) and imidazole (70 mg, 1.03 mmol) at room temperature for

1 h, followed by additional workup and purification steps as previously described

to afford 6-O-TBDPS ether 15 as a colorless syrup (236 mg, 90%). 1H NMR (400

MHz, CDCl3): � 7.87 (dd, J = 1.4, 7.6 Hz, 2H), 7.79 (dd, J = 1.7, 8.2 Hz, 2H),

7.60 (d, J = 8.1 Hz, 2H), 7.43 (m, 9H), 7.09 (t, J = 9.6 Hz, 2H), 6.85 (d, J = 8.6

Hz, 2H), 4.92 (s, 2H), 4.84 (d, J = 10.3 Hz, 1H), 4.67 (d, J = 10.3 Hz, 1H), 4.42

(d, J = 10.1 Hz, 1H), 4.02 (m, 2H), 3.82 (m, 4H), 3.57 (t, J = 9.3 Hz, 1H), 3.39 (m,

2H), 2.36 (s, 3H), 1.16 (s, 9H).

53

Benzyl 2-azido-3-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-�-

glucopyranoside (16). Glucosamine intermediate 15 (447 mg, 0.58 mmol) was

azeotroped with toluene (3 x 5 mL), then dissolved in 1:3 DCE:ether (4.5 mL) and

treated with NIS (265 mg, 1.17 mmol), distilled benzyl alcohol (125 �L, 1.2

mmol), and 4Å activated molecular sieves (800 mg) under argon atmosphere.

The reaction mixture was cooled to –30 °C for 15 minutes, then treated with triflic

acid (22 �L, 0.25 mmol) and warmed to –10 °C for 1.5 h, followed by standard

aqueous workup as previously described (cf. 14). The crude product was purified

by silica gel chromatography using a 0–10% ethyl acetate in hexanes gradient to

afford the �-benzyl glycoside as the major product and �-benzyl glycoside as the

minor product (410 mg, �:� 3:1 ratio, 93% combined yield).

The �-benzyl glycoside (1.33 g, 1.79 mmol) was subjected to deprotection of

4-O-PMB as previously described (cf. 14). The crude product was purified by

silica gel chromatography using a 10–30% ethyl acetate in hexanes gradient to

afford C4 alcohol 16 as a colorless syrup (970 mg, 87%). 1H NMR (400 MHz,

CDCl3): � 7.53 (m, 20H), 4.94 (d, J = 3.6 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 4.83

(d, J = 11.1 Hz, 1H), 4.70 (d, J = 12.1 Hz, 1H), 4.54 (d, J = 12.1 Hz, 1H), 3.89 (m,

2H), 3.85 (d, J = 3.2 Hz, 2H), 3.72 (d, J = 5.7 Hz, 2H), 3.30 (m, 1H), 2.47 (s, 1H),

1.07 (s, 9H).

Methyl 2,3,6-tri-O-benzyl-�-glucopyranoside (18). 4,6-O-benzylidene acetal

17 (200 mg, 0.433 mmol) was dried by azeotropic distillation with toluene (3 x 5

mL) in a two-necked round-bottomed flask, then dissolved in dry THF (6 mL) and

treated with sodium cyanoborohydride (285 mg, 4.33 mmol) under inert

54

atmosphere. The flask was equipped with an addition funnel charged with 1 M

solution of HCl in ether (5.5 mL, 5.5 mmol). The reaction mixture was cooled to 0

°C for 15 min, treated with dropwise addition of HCl-ether while stirring, then

warmed to room temperature with stirring for another 3 h. The reaction was

diluted with water (5 mL) and ethyl acetate (50 mL), then treated with solid

sodium bicarbonate until a neutral pH was achieved. The mixture was extracted

with ethyl acetate, washed with brine (3 x 5 mL), dried over sodium sulfate, then

concentrated and purified by silica gel chromatography using a 10–40% ethyl

acetate in hexanes gradient to afford C4 alcohol 18 as a colorless syrup (154 mg,

77%). 1H NMR (400 MHz, CDCl3): � 7.55 (m, 15H), 5.05 (d, J = 11.7 Hz, 1H),

4.80 (d, J = 11.7 Hz, 1H), 4.71 (d, J = 3.5 Hz, 1H), 4.4 (m, 4H), 4.09 (t, J = 9.1

Hz, 1H), 3.97 (m, 1H), 3.76 (m, 3H), 3.57 (dd, J =3.5, 9.5 Hz, 1H), 3.20 (s, 3H),

2.39 (s, 1H).

Thiotolyl 2-azido-4-O-(3’-O-benzyl-2’-O-benzoyl-4’,6’-O-p-methoxy-

benzylidene-�-glucopyranosyl)-6-O-tert-butyldiphenylsilyl-3-O-(2-trimethyl-

silylethoxymethyl)-�-glucopyranoside (19). Glycosyl donor 4 (234 mg, 0.40

mmol) and glucosamine acceptor 12 (396 mg, 0.58 mmol) were dried separately

by azeotropic distillation with toluene (3 x 2 mL). Donor 4 was treated with BSP

(101 mg, 0.48 mmol), TTBP (198 mg, 0.8 mmol) and 4Å activated molecular

sieves (500 mg) in distilled CH2Cl2 (4 mL), then cooled to –60 °C for 15 minutes

and treated dropwise with freshly distilled and pre-cooled Tf2O (73 �L, 0.43

mmol), with stirring at –60 °C for another 30 minutes. To this mixture, a pre-

cooled (–60 °C) solution of glucosamine acceptor 12 in CH2Cl2 (3 mL) was added

55

via cannula, stirred for 1 hour at –60 °C, then slowly warmed to –25 °C over a

period of 3 h. The resulting pale orange solution was quenched with pre-cooled

P(OEt)3 (138 �L, 0.79 mmol) and Et3N (280 �L, 2.02 mmol) at –25 °C, then

stirred for another 15 min. This yellow solution was removed from the bath and

warmed to rt over 30 min. The suspension was then filtered through Celite,

diluted with water, extracted with CH2Cl2 (3 × 10 mL), washed with brine (3 x 5

mL), dried over Na2SO4, then concentrated and purified by silica gel

chromatography using a 5–30% EtOAc in hexanes gradient with 1% Et3N to

afford the desired disaccharide 19 as a white foamy solid (340 mg, 73%). 1H

NMR (400 MHz, CDCl3): � 7.78 (dd, J = 7.2, 11.8, Hz, 4H), 7.63 (m, 2H), 7.43 (m,

9H), 7.31 (m, 2H), 7.20 (m, 2H), 7.09 (m, 4H), 6.95 (m, 4H), 5.56 (s, 1H), 5.21 (t,

J = 9.2, Hz, 1H), 5.0 (d, J = 6.9 Hz, 1H), 4.93 (d, J = 8.1 Hz, 1H), 4.87 (d, J = 6.9

Hz, 1H), 4.82 (d, J = 12.2 Hz, 1H), 4.68 (d, J = 12.3 Hz, 1H), 4.40 (dd, J = 5.0,

10.6 Hz, 1H), 4.24 (d, J = 10.1 Hz, 1H), 4.10 (t, J = 9.4 Hz, 1H), 3.84 (s, 3H),

3.76 (m, 4H), 3.65 (m, 3H), 3.55 (t, J = 9.3 Hz, 1H), 3.37 (dt, J = 5.0, 9.7 Hz,

1H), 3.21 (t, J = 9.8 Hz, 1H), 2.96 (d, J = 9.7 Hz, 1H), 2.28 (s, 3H), 1.09 (s, 9H),

0.99 (m, 2H), 0.06 (s, 9H); 13C NMR (101 MHz, CDCl3): � 164.49, 160.04,

138.62, 137.86, 135.76, 135.36, 134.34, 133.41, 133.02, 131.96, 130.10, 129.82,

129.68, 129.58, 129.01, 128.26, 128.12, 127.94, 127.73, 127.42, 127.24, 126.56,

113.59, 101.16, 100.10, 96.60, 85.78, 81.77, 79.72, 78.96, 77.90, 77.25, 77.14,

76.93, 76.61, 74.67, 73.71, 73.43, 68.54, 66.04, 63.74, 60.69, 55.24, 26.75,

21.04, 19.30, 18.05.

56

Benzyl 3-O-acetyl-2-azido-4-O-(2’-O-benzoyl-3’-O-benzyl-4’,6’-O-p-

methoxybenzylidene-�-glucopyranosyl)-6-O-tert-butyldiphenylsilyl-2-deoxy-

�-glucopyranoside (20). Glycosyl donor 4 (50 mg, 0.08 mmol) was subjected to

the BSP glycosylation as described above (cf. 19) using BSP (21 mg, 0.10

mmol), TTBP (42 mg, 0.17 mmol), Tf2O (16 �L, 0.09 mmol), and 4Å mol sieves

(140 mg) in CH2Cl2 (0.6 mL). A solution of pre-cooled (–60 °C) acceptor 14 (68

mg, 0.12 mmol) in CH2Cl2 (0.4 mL) was added to the reaction mixture with

stirring at –60 °C for 1 h, then slowly warmed to –40 °C over 1 h. The reaction

was quenched with pre-cooled P(OEt)3 (27 �L, 0.15 mmol) and Et3N (55 �L, 0.39

mmol) at –40 °C, warmed to room temperature, followed by workup as previously

described. The crude product was purified by silica gel chromatography using a

5–30% EtOAc in hexanes gradient with 1% Et3N to afford disaccharide 20 as an

amorphous solid (30 mg, 34%). 1H NMR (400 MHz, CDCl3): � 7.23 (m, 29H),

5.57 (s, 1H), 5.46 (t, J = 10.0 Hz, 1H), 5.25 (t, J = 8.5 Hz, 1H), 4.92 (d, J = 3.6

Hz, 1H), 4.87 (d, J = 8.0 Hz, 1H), 4.81 (d, J = 12.3 Hz, 1H), 4.68 (d, J = 12.1 Hz,

1H), 4.54 (d, J = 12.4 Hz, 1H), 4.40 (m, 2H), 4.12 (m, 2H), 3.69 (m, 6H), 3.51 (d,

J = 11.5 Hz, 4H), 3.17 (dd, J = 3.6, 10.7 Hz, 1H), 2.12 (s, 3H), 1.06 (s, 9H).

Benzyl 2-azido-4-O-(3’-O-benzyl-2’-O-benzoyl-4’,6’-O-p-

methoxybenzylidene-�-glucopyranosyl)-3-O-benzyl-6-O-tert-butyldiphenyl-

silyl-2-deoxy-�-glucopyranoside (21). Glycosyl donor 4 (100 mg, 0.17 mmol)

and acceptor 16 (92 mg, 0.15 mmol) were subjected to BSP glycosyl coupling

conditions as previously described (cf. 19), using BSP (43 mg, 0.20 mmol), TTBP

(80 mg, 0.32 mmol), 4Å mol sieves (280 mg) in CH2Cl2 (1.2 mL), and Tf2O (31

57

�L, 0.18 mmol). Acceptor 16 was dissolved in CH2Cl2 (0.6 mL) prior to addition.

The reaction mixture was stirred at –60 °C for 2 h, then quenched and worked up

as previously described. The crude product was purified by silica gel

chromatography using a 5–30% EtOAc in hexanes gradient with 1% Et3N to

afford the desired disaccharide 21 as a colorless syrup (124 mg, 66%). 1H NMR

(400 MHz, CDCl3): � 7.73 (ddd, J =1.3, 6.9, 8.1 Hz, 4H), 7.69 (m, J = 2H), 7.54

(dt, J = 1.3, 7.4 Hz, 1H), 7.44 (m, 10H), 7.32 (m, 6H), 7.14 (m, 8H), 6.94 (m, 2H),

5.54 (s, 1H), 5.34 (m, 1H), 5.09 (d, J = 10.2 Hz, 1H), 4.99 (dd, J = 1.3, 8.1 Hz,

1H), 4.89 (d, J = 3.7 Hz, 1H), 4.84 (d, J = 12.2 Hz, 1H), 4.76 (d, J = 10.3 Hz, 1H),

4.70 (d, J = 12.2 Hz, 1H), 4.47 (d, J = 2.4 Hz, 2H), 4.25 (m, 2H), 3.94 (dd, J =

9.0, 10.4 Hz, 1H), 3.84 (d, J = 1.0 Hz, 4H), 3.73 (m, 2H), 3.57 (t, J = 10.4 Hz,

1H), 3.46 (d, J = 11.8 Hz, 1H), 3.36 (m, 2H), 3.27 (d, J = 9.8 Hz, 1H), 1.03 (s, J =

1.2 Hz, 9H); 13C NMR (101 MHz, CDCl3): � 164.91, 160.24, 138.49, 138.05,

136.82, 136.05, 135.54, 133.86, 133.23, 132.55, 130.20, 129.90, 129.47, 128.61,

128.52, 128.41, 128.35, 128.27, 128.06, 127.96, 127.89, 127.76, 127.67, 127.48,

113.79, 101.36, 100.54, 96.71, 82.07, 78.28, 78.16, 75.95, 75.77, 74.03, 73.88,

71.49, 69.88, 68.67, 66.33, 63.12, 60.88, 55.46, 26.88, 19.54.

Methyl 2,3,6-tri-O-benzyl-4-O-(2�-O-benzoyl-3�-O-benzyl-4�-deoxy-�-pent-

4�-enopyranosyl)-�-glucopyranoside (22). Thiophenyl 4-DP donor 6 (55 mg,

0.13 mmol) was treated with BSP (33 mg, 0.16 mmol), TTBP (65 mg, 0.26

mmol), and 4Å molecular sieves (300 mg) in CH2Cl2 (1.0 mL), followed by the

dropwise addition of Tf2O (24 �L, 0.14 mmol) at –60 °C with stirring for 30 min. A

solution of acceptor 18 (80 mg, 0.17 mmol) in CH2Cl2 (0.5 mL) pre-cooled to –60

58

°C was added, with stirring at –60 °C for another 2 h. The reaction was quenched

and worked up as previously described (cf. 19), and the crude product was


with 1% Et3N to yield 4�-DP disaccharide 22 as a white solid (53 mg, 52%). 1H

NMR (400 MHz, C6D6): � 8.06 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.36–

6.96 (m, 21 H), 6.20 (d, J = 6.4 Hz, 1H), 5.84 (t, J = 3.5 Hz, 1H), 5.69 (d, J = 3.8

Hz, 1H), 5.20 (d, J = 11.5 Hz, 1H), 5.02 (d, J = 11.5 Hz, 1H), 4.81 (t, J = 5.6 Hz,

1H), 4.59 (d, J = 3.6 Hz, 1H), 4.55 (m, 4H), 4.33–4.27 (m, 2H), 4.22 (t, J = 9.2

Hz, 1H), 4.00 (t, J = 3.6 Hz, 1H), 3.92 (dd, J = 3.5, 11.0 Hz, 1H), 3.83 (m, 1H),

3.59 (dd, J = 1.8, 10.9 Hz, 1H), 3.51 (dd, J = 3.6, 9.4 Hz, 1H), 3.04 (s, 3H). 13C

NMR (100 MHz, C6D6): � 165.08, 142.80, 140.16, 127.89, 127.65, 127.41,

127.41, 100.17, 98.12, 96.96, 96.95, 80.62, 79.91, 79.91, 77.47, 77.47, 75.02,

73.18, 73.18, 72.82, 70.35, 70.35, 70.17, 69.73, 69.73, 68.65, 54.52, 54.52. IR:

3030, 2924, 1724, 1651, 1601, 1453, 1265, 1107, 1028, 912, 799, 698 cm-1.

[�]25D = +65.14° (c 3.5, CH2Cl2). HRESI-MS: m/z calcd for C47H48NaO10 [M+Na]+

795.3140, found 795.3168.

Benzyl 2-azido-4-O-(2’-O-benzoyl-3’-O-benzyl-�-glucopyranosyl)-3-O-

benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-�-glucopyranoside (23). A

solution of disaccharide 21 (0.45 g, 0.41 mmol) in 4:4:1 AcOH:THF:H2O (9 mL)

was heated to 70 °C and stirred for 5 h, then cooled to rt. and concentrated to

dryness. The crude product was dissolved in ethyl acetate (30 mL), washed with

brine (3 x 2 mL), dried over sodium sulfate, then concentrated and purified by

59


afford 23 as a white crystalline solid (766 mg, 86%). 1H NMR (400 MHz, CDCl3):

� 7.82–7.76 (m, 4H), 7.69 (d, J = 9.2 Hz, 2H), 7.56–7.14 (m, 24H) 5.27 (t, J =

11.0 Hz, 1H), 5.07 (d, J = 10.6 Hz, 1H), 4.91 (m, 2H), 4.81 (d, J = 10.7 Hz, 1H),

4.66 (m, 2H), 4.47 (m, 2H), 4.18 (t, J = 9.5 Hz, 1H), 3.94 (t, J = 9.6 Hz, 1H), 3.72

(m, 3H), 3.52 (m, 3H), 3.37 (dd, J = 3.7 ,10.3 Hz, 1H), 3.27 (m, 2H), 2.36 (s, 1H),

1.08 (s, 9H).

Benzyl 2-azido-4-O-(2’-O-benzoyl-3’-O-benzyl-4-deoxy-�-pent-4’-

enopyranoside)- 3-O-benzyl-6-O-tert-butyldiphenylsilyl-�-D-glucopyranoside

(25). A solution of diol 23 (151 mg, 0.15 mmol) in 2:1 CH2Cl2:H2O (3 mL) was

treated with BAIB (150 mg, 0.46 mmol) and TEMPO (11 mg, 0.07 mmol) with

stirring at room temperature for 50 min. The reaction mixture was quenched with

Na2S2O3 (5 mL) at 0 °C with vigorous stirring for 30 minutes, extracted with

EtOAc (3 × 20 mL), washed with brine (3 x 5 mL), dried over Na2SO4, then

concentrated to dryness to afford crude glucuronic acid 24 (158 mg, 0.16 mmol).

This was azeotroped with toluene (3 x 2 mL), redissolved in degassed DMF (3

mL), treated with DMFDNpA (220 �L, 0.79 mmol), then heated at 150 °C for 50

minutes in a microwave reactor. The reaction mixture was then concentrated to

dryness, azeotroped with toluene (3 x 5 mL), redissolved in ethyl acetate and

was washed with brine (3 x 2 mL), dried over Na2SO4, then concentrated and


with 0.5% Et3N to afford the desired disaccharide 25 as white solid (90 mg, 61%).

1H NMR (400 MHz, C6D6): � 8.05 (d, J = 8.5 Hz, 2H), 7.91–7.88 (m, 3H), 7.85–

60

7.80 (m, 3H), 7.58 (d, J = 8.2 Hz, 2H), 7.35–6.95 (m, 20H), 6.24 (d, J = 6.4 Hz,

1H), 5.92 (t, J = 4.0 Hz, 1H), 5.89 (d, J = 4.4 Hz, 1H), 5.35 (d, J = 11.4 Hz, 1H),

4.86–4.80 (m, 2H), 4.56–4.53 (m, 3H), 4.49 (t, J = 9.5 Hz, 1H), 4.27 (m, 1H), 4.23

(d, J = 4.5 Hz, 1H), 4.12–4.07 (m, 2H), 4.0 (dd, J = 2.3, 12.0 Hz, 1H), 3.72 (dd, J

= 1.6 ,11.9 Hz, 1H), 3.56 (d, J = 11.0 Hz, 1H), 3.17 (dd, J = 3.6, 10.3 Hz, 1H),

1.17 (s, 9H).

Benzyl 2-azido-4-O-(2’-O-benzoyl-3’-O-benzyl-5’-diethyldithiocarbamoyl-

�-glucopyranoside)-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-�-

glucopyranoside (27). Disaccharide 25 (27 mg, 0.3 mmol) was dried by

azeotropic distillation with toluene (3 x 1 mL), dissolved in distilled

dichloromethane (0.3 mL) then cooled to –70 °C and treated with a freshly

prepared solution of 0.1 M DMDO in DCM (0.9 mL, 0.1 mmol). The reaction

mixture was slowly warmed to –30 °C over 4 h and stirred for an additional 20 h

at –30 °C before warming to rt. The reaction was concentrated to dryness and

azeotroped with toluene (3 x 1 mL) to afford epoxide 26, which was used without

further purification. 1H NMR (400 MHz, C6D6): � 8.02 (d, J = 7.8 Hz, 2H), 7.90 (m,

2H), 7.50 (d, J = 7.5 Hz, 2H), 7.30 (d, J = 6.9 Hz, 4H), 7.21 (m, 10H), 7.03 (m,

10H), 5.63 (m, 2H), 5.23 (d, J = 11.5 Hz, 1H), 4.77 (m, 3H), 4.48 (m, 4H), 4.19 (d,

J = 12.3 Hz, 1H), 4.08 (m, 2H), 3.92 (dd, J = 10.3, 15.1 Hz, 1H), 3.65 (d, J = 11.7

Hz, 1H), 3.46 (t, J = 11.8 Hz, 1H), 3.15 (dd, J = 4.2, 10.2 Hz, 1H), 2.81 (d, J = 2.5

Hz, 1H), 1.20 (s, 9H).

61

The crude epoxide 26 (26 mg, 0.03 mmol) was dissolved in degassed THF

(0.35 mL) under argon atmosphere, cooled to 0 °C for 15 minutes, then treated

with freshly prepared diethylammionium diethyldithiocarbamate as a 0.53 M

solution in methanol (170 �L, 0.09 mmol) with stirring for 3 h. The reaction

mixture was concentrated to dryness and purified by silica gel chromatography to

afford disaccharide 27 as colorless syrup (24 mg, 80% over two steps). 1H NMR

(400 MHz, CDCl3): � 7.82 (m, 2H), 7.77 (d, J = 7.2 Hz, 2H), 7.70 (m, 2H), 7.54

1H), 5.30 (t, J = 8.3 Hz, 1H) 5.10 (d, J = 10.3 Hz, 1H), 5.00 (d, J = 8.1 Hz, 1H),

4.87 (m, 2H), 4.76 (d, J = 13.4 Hz, 1H), 4.67 (d, J = 10.3 Hz, 1H), 4.45 (d, J = 5.7

Hz, 2H), 4.22 (t, J = 9.4 Hz, 1H), 4.14 (m, 1H), 4.00 (dt, J = 9.9, 4.8 Hz, 1H), 3.93

(dd, J = 8.8, 10.4 Hz, 1H), 3.63 (t, J = 9.1 Hz, 1H), 3.51–3.44 (m, 2H), 3.35 (d, J

= 4.5 Hz, 1H), 3.33 (d, J = 3.7 Hz, 1H), 3.28 (d, J = 10.0 Hz, 1H), 1.23 (dd, J =

7.8, 14.3 Hz, 6H), 1.09 (s, 9H).

Benzyl 2-azido-4-O-(2’-O-benzoyl-3’,4’-O-dibenzyl-5-diethyldithiocarbam-

oyl-�-D-glucopyranosyl)-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-�-

glucopyranoside (28). Disaccharide 27 (50 mg, 0.045 mmol) was subjected to

low-temperature benzylation following the procedure described below (cf. 32) to

afford 4’-O-benzyl disaccharide 28 as a syrup (47 mg, 88%). 1H NMR (400 MHz,

C6D6): � 8.07 (d, J = 7.4 Hz, 2H), 8.01 (d, J = 7.7 Hz, 2H), 7.97 (d, J = 7.2 Hz,

2H), 7.64 (d, J = 7.5 Hz, 2H), 7.40–7.18 (m, 16H), 7.14–6.90 (m, 11H), 6.46 (d, J

= 10.1 Hz, 1H), 5.92 (t, J = 8.7 Hz, 1H), 5.49 (d, J = 8.1 Hz, 1H), 5.42 (d, J = 11.0

Hz, 1H), 4.83–4.64 (m, 5H), 4.59 (t, J = 9.4 Hz, 1H), 4.46 (d, J = 3.6 Hz, 1H),

4.25 (d, J = 11.5 Hz, 1H), 4.18–3.99 (m, 4H), 3.84 (t, J = 9.2 Hz, 1H), 3.74 (d, J =

62

11.1 Hz, 2H), 3.52 (d, J = 10.0 Hz, 1H), 3.46 (m, 1H), 3.17 (m, 1H), 3.12 (dd, J =

3.6, 10.4 Hz, 1H), 2.87 (m, 1H), 1.39 (s, 9H), 0.93 (t, J = 7.1 Hz, 3H), 0.69 (t, J =

7.1 Hz, 3H).

N,N-Diethyldithiocarbamoyl 2,3,4,6-tetra-O-benzyl-�-glucopyranoside

(30). Glycal 29 (140 mg, 0.34 mmol) was dried by azeotropic distillation with

toluene (3 x 2 mL), dissolved in dichloromethane (0.5 mL), then treated with

freshly prepared DMDO as a 0.1 M solution in acetone (5.6 mL, 0.5 mmol) with

stirring at –30 °C for 2 h. The reaction was concentrated to dryness, azeotroped

with toluene (3 x 1 mL), then treated with freshly prepared diethylammionium

diethyldithiocarbamate as a 0.53 M solution in methanol (550 �L, 0.29 mmol).

This was stirred at rt for 3 h to afford the corresponding �-DTC-glycoside (156

mg, 80% in two steps), which was used without further purification.

A portion of the �-DTC glycoside (140 mg, 0.24 mmol) was subjected to

benzylation as previously described (cf. General Procedures) to afford 2-O-

benzyl ether 30 as a white solid (126 mg, 78%). 1H NMR (400 MHz, C6D6): �

7.55–7.04 (m, 20H), 6.39 (d, J = 8.7 Hz, 1H), 4.92 (m, 6H), 4.78 (d, J = 11.4 Hz,

1H), 4.52 (d, J = 11.9 Hz, 1H), 4.34 (d, J = 11.9 Hz, 1H), 4.10 (ddd, J = 2.4, 6.4,

9.2 Hz, 1H), 3.84 (m, 6H), 3.17 (m, 2H), 1.05 (t, J = 8.4 Hz, 3H), 0.82 (t, J = 7.1

Hz, 3H).

Benzyl 2,3,6-tri-O-benzyl-4-O-(2�,3�,4’-tri-O-benzyl-5�-

diethyldithiocarbamoyl-�-pyranosyl)-�-glucopyranoside (32). 4’-DP

disaccharide 31 (22 mg, 0.026 mmol) was treated with DMDO as a 0.1 M

63

solution in DCM (780 �L, 0.078 mmol) at 0 °C and stirred for 12 h. The reaction

mixture was concentrated to dryness to obtain crude epoxide (25 mg, 0.029

mmol), which was treated with diethylammionium diethyldithiocarbamate as a

0.53 M solution in methanol (245 �L, 0.13 mmol). The reaction mixture was

stirred at 0 °C and warmed to rt for 1.5 h, then concentrated to dryness and

purified by preparative TLC using 20% ethyl acetate in hexanes to afford the 5’�-

DTC disaccharide as colorless syrup (16 mg, 61% over two steps). This was

treated with NaHMDS (80 �L, 0.048 mmol), TBAI (2 mg, 0.0054 mmol) and

benzyl bromide (6 �L, 0.05 mmol) in dry DMF (0.3 mL) at –55 °C for 1 h, followed

by aqueous workup and purification (cf. 30) to afford 4’-O-benzyl disaccharide 32

as a syrup (15 mg, 88%). 1H NMR (400 MHz, C6D6): � 7.72–7.68 (m, 2H), 7.57–

7.53 (m, 2H), 7.38–7.05 (m, 31H), 6.22 (d, J = 10.3 Hz, 1H), 5.29–5.03 (m, 2H),

5.00–4.50 (m, 14H), 4.48–3.97 (m, 3H), 3.98–3.41 (m, 8H), 3.23–2.77 (m, 2H),

0.92 (t, J = 6.5 Hz, 3H), 0.71 (t, J = 8.3 Hz, 3H).

C-Furyl 2,3,4,6-tetra-O-benzyl-�-glucopyranoside (33). DTC glycoside 30

(24 mg, 0.036 mmol) was dried by azeotropic distillation with toluene (3 x 1 mL),

treated with freshly distilled furan (70 �L, 0.97 mmol) and 4Å activated molecular

sieves (50 mg) in distilled dichloroethane (0.4 mL), then cooled to –30 °C for 15

minutes. This mixture was treated with CuOTf(C6H6)0.5 (0.072 mmol) and warmed

to –10 °C in 45 minutes. The reaction was quenched with Et3N (25 �L, 0.18

mmol) at –10 °C, warmed to rt, then passed through Celite and washed with

dichloromethane (20 mL). The organic portion was washed with brine (3 x 1 mL),

dried over sodium sulfate, concentrated to dryness, then purified by preparative

64

TLC using 15% ethyl acetate in hexanes with 1% triethylamine to afford

compound 33 (15 mg, 72% yields).1H NMR (400 MHz, C6D6): � 7.44 (d, J = 1.8

Hz, 1H), 7.29–7.10 (m, 17H), 7.12 (dd, J = 2.6, 7.0 Hz, 2H), 6.44 (d, J = 3.3 Hz,

1H), 6.26 (dd, J =1.8, 3.3, Hz, 1H), 5.03 (d, J = 6.5 Hz, 1H), 4.91 (d, J = 10.8 Hz,

1H), 4.74 (dd, J = 6.9, 10.8 Hz, 2H), 4.55 (d, J = 6.4 Hz, 2H), 4.50 (d, J = 12.1

Hz, 1H), 4.41–4.34 (m 2H), 4.13 (t, J = 9.2 Hz, 1H), 3.86 (dd, J = 6.4, 9.7 Hz,

1H), 3.66–3.56 (m, 2H), 3.53–3.41 (m, 3H).

Benzyl 2,3,6-tri-O-benzyl-4-O-(2’,3’,4’-Tri-O-benzyl-5’-�-furyl-�-pyranosyl)-

�-glucopyranoside (34). 5’�-DTC disaccharide 32 (14 mg, 0.013 mmol) was

treated with freshly distilled furan (30 �L, 0.97 mmol) and 4Å activated molecular

sieves (40 mg) in distilled dichloroethane (0.3 mL), cooled to –30 °C, then treated

with freshly prepared CuOTf(C6H6)0.5 (0.027 mmol) as previously described (cf.

33). The reaction was warmed to –5 °C in 1 h, then quenched with Et3N (10 �L,

0.072 mmol) and worked up as previously described to afford a mixture

containing 5’-furyl adduct 34, which was identified by ESI-MS: m/z calcd. for

C64H64O11Na [M + Na]+, 1031.4346; found, 1031.5700.

Benzyl 2-azido-4-O-(2’-O-benzoyl-3’,4’-O-dibenzyl-5’-�-furyl-�-

glucopyranosyl)-3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-deoxy-�-gluco-

pyranoside (35). 5’�-DTC disaccharide 28 (10 mg, 0.008 mmol) was dried by

azeotropic distillation with toluene (3 x 1mL), treated with freshly distilled furan

(15 �L, 0.21 mmol) and 4Å activated molecular sieves (20 mg) in distilled

ClCH2CH2Cl (0.18 mL) and cooled to –30 °C for 15 minutes, then treated with

65

freshly prepared CuOTf(C6H6)0.5 (0.02 mmol), and warmed to –20 °C over 1 h.

The reaction was quenched with Et3N (6 �L, 0.043 mmol) at –20 °C, and worked

up as previously described (cf. 33) to afford compound 35 (3 mg, 32%). Selected

1H NMR peaks (400 MHz, C6D6): � 8.16 (m, 4H), 7.90 (m, 10H), 7.26 (m, 11H),

7.03 (m, 10H), 6.39 (d, J = 3.3 Hz, 1H), 5.99 (m, 2H), 5.73 (dq, J = 8.3, 3.9 Hz,

2H), 5.52 (d, J = 3.6 Hz, 1H), 5.11 (d, J = 11.2 Hz, 1H), 4.72 (m, 12H), 4.12 (m,

20H), 3.05 (m, 1H), 1.16 (m, 1H), 1.16 (s, 9H). (+)-ESI-MS: m/z calcd. for

C66H67O11N3SiNa [M + Na]+ 1128.4443; found 1128.36.

66

2.11 References

(1) Frihed, T. G.; Bols, M.; Pedersen, C. M. Chem. Rev. 2015, 115, 3615.

(2) Raedts, J.; Kengen, S. W.; Van Der Oost, J. Glycoconj. J. 2011, 28, 57.

(3) Hung, S.-C.; Thopate, S. R.; Puranik, R. Carbohydr. Res. 2001, 331, 369.

(4) Hung, S.-C.; Thopate, S. R.; Chi, F.-C.; Chang, S.-W.; Lee, J.-C.; Wang, C.-C.; Wen, Y.-S. J. Am. Chem. Soc. 2001, 123, 3153.

(5) Cheng, G.; Fan, R.; Hernández-Torres, J. M.; Boulineau, F. P.; Wei, A. Org. Lett. 2007, 9, 4849.

(6) Boulineau, F. P.; Wei, A. Org. Lett. 2002, 4, 2281.

(7) Padungros, P.; Fan, R.-H.; Casselman, M. D.; Cheng, G.; Khatri, H. R.; Wei, A. J. Org. Chem. 2014, 79, 4878.

(8) Padungros, P. Ph. D. Thesis, Purdue University, 2012.

(9) Hernández-Torres, J. M.; Liew, S.-T.; Achkar, J.; Wei, A. Synthesis 2002, 487.

(10) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015.

(11) Fan, R.-H.; Achkar, J.; Hernández-Torres, J. M.; Wei, A. Org. Lett. 2005, 7, 5095.

(12) Boulineau, F. P.; Wei, A. Org. Lett. 2002, 4, 2281.

(13) Mikula, H.; Svatunek, D.; Lumpi, D.; Glöcklhofer, F.; Hametner, C.; Fröhlich, J. Org. Process Res. Dev. 2013, 17, 313.

(14) Padungros, P.; Alberch, L.; Wei, A. Org. Lett. 2012, 14, 3380.

(15) Salomon, R.; Kochi, J. J. Am. Chem. Soc. 1973, 95, 1889.

(16) Morales Collazo, O. Ph. D. Thesis, Purdue University 2014.

67

CHAPTER III: SYNTHETIC STUDIES ON THE STEREOSELECTIVE GLYCOSYLATION OF GLUCOSAMINE DONORS

3.1 Stereoselective Glycosidic Bond Formations with D-Glucosamine

Numerous biomolecules like heparin, heparan sulfate, their derivatives and

natural products such as antibiotics1 (e.g. tunicamycins and neomycin) and

bacterial antioxidants2 (e.g. mycothiol and bacillithiol) contain D-GlcN units as

core components. �-Glucosaminyl bonds can be effectively achieved using

neighboring group participation of acyl groups at C2; however, stereoselective

formation of �-glucosaminyl bonds remains a challenge in carbohydrate

synthesis.3 The presence of non-participating groups masking the C2 amine

(e.g., 2,3-trans-oxazolidinone, p-methoxybenzylidene, or azide) is commonly

employed in the synthesis of 1,2-cis glycosides, but these groups cannot ensure

a highly stereoselective outcome.4,5 While the �-product is often favored by the

stereoelectronic anomeric effect, the stereoselectivity of glycosylation can be

poor, requiring other modes of stereocontrol such as SN2-like reactivity or

directing groups, both of which can be engineered by judicious design of

protecting groups on the donor or acceptor.6

To address �-stereoselectivity in D-glucosaminyl glycosylation, Kerns and co-

workers initially reported a strategy using 2,3-trans-oxazolidinone glycosyl donors

to introduce conformational strain in the activated intermediate; but its application

68

was limited by the high loading of promoter (PhSOTf) as well as side reactions

such as N-glycosylation and -sulfenylation.7,8 Furthermore, �-stereoselectivity

was highly dependent on the choice of acceptor.9 Later modifications such as N-

acetylation10-12 or benzylation13,14 combined with proper tuning of activators and

solvents gave higher stereoselectivity and yields.

Stereoselective synthesis of 1,2-cis-amino glycopeptides via conjugate

addition of serine/threonine to 2-nitrogalactals was reported by Schmidt and co-

workers.15 Gin and co-workers reported the ring opening of aziridine-2-

carboximide with lactol nucleophiles to form �-O-serine glycosides with good

stereoselectivity.16 These methods were of limited substrate scope. Hung and co-

workers reported an optimized set of orthogonal protecting groups for a D-

glucosaminyl donor (with azide group at C2) that afforded glucosamine-linked

oligosaccharides with high �-stereoselectivity, using a 1,6-anhydro-�-L-

idopyranoside as an acceptor and TMSOTf as a promotor.5 However, this

strategy was limited to the axial C4 hydroxyl of L-iduronate acceptors.

A novel method of stereoselective synthesis of 1,2-glucosaminosides via

nickel-catalyzed glycosylation with N-benzylidene glucosamine and

galactosamine trichloroacetimidates was reported by Nguyen and co-

workers.17,18 They found a nickel (II) catalyst that can coordinate with the

approaching acceptor and the C2 benzylideneamino group on the donor,

resulting in stereoselective �-glycosylation.19,20 The ligand on the nickel catalyst

and the substituents on the C2 benzylideneamino moiety were highly influential

in controlling the stereoselectivity of glycosylation. Electron-withdrawing

69

substituents helped to decrease reaction time, which translated into increased �-

selectivity of glycosyl couplings, and was applied toward the synthesis of several

challenging targets.4,21

In Chapter I (Section 1.3.4), we described an intriguing findings by Ornitz et

al. that FGF-2 possesses a secondary binding site for unsulfated heparan

trisaccharides with submicromolar binding affinity.22 Our group has focused on

the synthesis of a heparan trisaccharide ligand (Figure 3.1). Initial synthetic

attempts were based on known glycosyl coupling methods,23 but were hampered

by: (a) low �-stereoselectivity in the coupling of our glucosaminyl donor (unit B)

with glucose-based acceptor (unit A), and (b) difficulties in separating the

anomeric epimers.

Figure 3.1 Heparan trisaccharide ligand with high affinity for secondary binding site on FGF-2.

To address the first bottleneck, we envisioned applying the �-stereoselective

Ni-catalyzed glycosylation methodology by designing an appropriate N-

benzylidene glucosaminyl trichloroacetimidate.20 This can be prepared in a few

steps from a known D-glucosamine derivative (Figure 3.2).

O

OO

O

O

OOH

OH

OH

HO

HO

HO

HO OH

NHAc

OH

NH2

O

O

A

B

C

70

Figure 3.2 Retrosynthetic analysis of target trisaccharide

3.2 Synthesis of Trichloroacetimidate Donor 42

Glucosamine derivative 36 was subjected to reductive cleavage of 4,6-O-

anisylidene acetal using BH3.THF and TMSOTf, followed by 6-O-benzylation to

afford 37 in 70% yield over two steps (Figure 3.3). Compound 37 was then

subjected to azide reduction followed by condensation with p-

flurorobenzaldehyde to produce N-benzylideneamine 38 in 66% yield over two

steps. Normally, oxidation of the anomeric thiotolyl group should produce lactol

39; however, attempts to remove the thiotolyl group after benzylideneamine

O

OP

STolO

O N3PMP

O

OO

O

OOH

OH

OH

HO

HO

HO

HO OH

NHAc

OH

NCbz

O

O

O

OP

OPO

PMBO N

X

NHO O OSPh

OBn OBnOBnHOOBzP'O

PO PO NCbzCCl3

X = CF3, F

O

71

formation with mercuric chloride, NIS/AgOTf, or NIS/NBS oxidation proved

fruitless. On the other hand, the thiotolyl group of 37 was readily oxidized by N-

bromosuccinimide in acetone, which upon hydrolysis and acetylation afforded

compound 40 in 80% yield over two steps. Aza-ylide formation under Staudinger

conditions followed by addition to 4-fluorobenzaldehyde afforded imine 41 in 87%

yield. Hydrolysis of the anomeric acetate with an ammonia–methanol solution,

followed by treatment with DBU and trichloroacetonitrile, produced

trichloroacetimidate (TCA) donor 42 in 54% yield over two steps.

72

Figure 3.3 Synthesis of trichloroacetimidate donor 42. Reagents and conditions: (1) (i) BH3.THF (5.0 equiv), TMSOTf (0.2 equiv), CH2Cl2, –30 to –5 °C, 5 h; (ii) NaH (3.0 equiv), TBAI (0.2 equiv), BnBr (2.0 equiv), imid. (0.5 equiv), 1:4 DMF:THF, 4 h (81%); (2) (i) Bu3P (1.2 equiv), CH2Cl2, 0 °C, 1 h; (ii) p- (CF3)C6H4CHO (1.5 equiv), 0 °C, 2 h (70%); (3) NBS (2.0 equiv), acetone, –30 °C to –20 °C, 1.5 h, (ii) Ac2O (3.0 equiv), Py, rt (80%); (4) (i) Bu3P (1.2 equiv), CH2Cl2, 0 °C, 1 h; (ii) p-FC6H4CHO (1.5 equiv), 0 °C, 2 h (87%); (5) (i) NH3, MeOH, THF; (ii) DBU (0.5 equiv), CCl3CN (3.0 equiv), CH2Cl2, 0 °C, 6 h (54%).

3.3 Synthesis of Disaccharide by Coupling of Trichloroacetimidate Donor 42

The trichloroacetimidate donor was subjected to Ni(4-FC6H4(CN))4(OTf)2

catalyzed coupling with acceptor 43, synthesized previously in our lab. A product

with the appropriate mass was detected by ESI-MS, but could not be isolated

(Figure 3.4).

73

O O

OBnOBn

OTBDPS

O

OBnON

PMBO

BnO

O O

OBnOBn

OTBDPS NHCbz

HOCH2Cl2, rt

Ni(4-F-PhCN)4Cl2, AgOTf

43 44

42 +

F

NHCbz

Figure 3.4 Glycosyl coupling of donor 42 with acceptor 43. Reagents and conditions: Acceptor 43 (1.2 equiv), Ni[4-F-PhCN]4(OTf)2 (10 mol%), CH2Cl2, rt.

With this result, we assessed the pitfalls of the synthetic route: (a) inability to

remove thiotolyl group in the presence of the C2 benzylideneamine, (b)

challenges to efficiently convert the C2 azide lactol into a benzylideneamine; (c)

conversion of the lactol into a trichloroacetimidate donor, which proceeded in

only fair yield.

To address these limitations, we considered to replace the TCA group with a

thioimidate (-S-C(=N)-) moiety: 2-mercaptobenzoxazole (-SBox), 2-mercapto-

benzothiazole (-SBaz), 2-mercaptopyridine (-SPy), and 2-mercatopyrimidine. To

make these donors, we synthesized azidoglucose derivative 46 (Figure 3.5) in

84% yield by a diazo transfer reaction with commercially available glucosamine

hydrochloride and in situ generated sulfonylazide reagent 45.24

74

Figure 3.5 Synthesis of diazo transfer reagent 45 and azidoglucose 46. Reagents and conditions: (1) (i) SO2Cl2 (1.0 equiv), MeCN, (ii) imidazole (2.0 equiv), (iii) 25% HCl in EtOH (excess); (2) (i) imidazole-1-sulfonyl azide hydrochloride (1.2 equiv), CuSO4 (0.2 equiv), K2CO3 (2.4 equiv), MeOH, rt, 10 h; (3) Ac2O (8.0 equiv), py, rt, 4h (84% over two steps).

Glucosamine intermediate 46 was subjected to various Lewis-acid mediated

glycosylation conditions to generate thioimidate donors. Among these, only S-

Box glycoside 47 could be produced in decent yields as an anomeric mixture

(entry 3, Table 3.1). This was subjected to deacetylation using a saturated

solution of NH3 in MeOH or DBU in MeOH followed by 4,6-O-anisylidene

formation. However, saponification proceeded in poor yields with hydrolysis of

the –SBox group as a major side reaction.

75

Table 3.1 Preparation of glycosyl thioimidates

[a] Product could not be differentiated from starting material by TLC, but detected by ESI-MS. [b] Bu3Sn–SBaz was freshly prepared by adapting a literature procedure.25

76

3.4 Synthesis of C2-N-diphenylphosphinic Amide Trichloroacetimidate Donor 50

The preparation of alkyl aryl sulfides from 2-sulfanylbenzothiazole using

phenyl diphenylphosphinite and azide was reported by Mukaiyama and co-

workers by oxidation–reduction condensation, in which chiral secondary and

tertiary alcohols were converted into the corresponding chiral sulfides with

inversion of configuration.26,27 We hypothesized that lactol 48 could form cyclic

intermediate 48a, from which phenoxide acts as a leaving group when treated

with nucleophiles (thiols or alcohols) to afford S- or O-glycosides (Figure 3.6).

O

OAcAcO

OAc

NH

PO Ph

PhOPh

O

OAcAcO

AcO

N3

OH

48 48a

Ph2POPh

Toluene

O

OAcAcO

OAc

NH

P

OPh

Ph

RXHXR

48bExpected

O

OAcAcO

OAc

NH

P

OPh

Ph

OAc

48c

RXH

X = S or O–PhOH

O

OAcAcO

OAc

NH

P

OPh

Ph

OHAc2O

Py

49

Figure 3.6 Working hypothesis to S- or O-glycosides from lactol 48 and phenyl diphenylphosphinite.

We tested our hypothesis toward thioglycosides by treating lactol 48 with

commercially available phenyl diphenylphosphinite (Ph2POPh) in toluene. The

conspicuous absence of phenoxy (PhO) signals was noted in the

77

characterization of lactol 48a by 1H and 13C NMR; (+)ESI-MS also confirmed the

absence of intermediate 48a. Instead, diphenylphosphinic amide 49 was

obtained and confirmed by 1H, 13C NMR, (+)ESI-MS, and acetylation (48c).

Therefore, trichloroacetimidate donor 50 was synthesized from azidoglucose 46

in three steps (Figure 3.7): saponification with saturated NH3 solution in methanol

to afford 48 in 78% yield; heating with phenyl diphenylphosphinite at 60 °C to

afford N-diphenylphosphinamide 49 in 68% yield; treatment with

trichloroacetonitrile and catalytic DBU to afford TCA donor 50 in 60% yield.

Figure 3.7 Synthesis of trichloroacetimidate glucosamine donor 50. Reagents and conditions: (1) 7 M NH3-MeOH (excess), –40 to –10 °C, 1:1 DCM:MeOH, 2 h (78%); (2) Ph2POPh (1.2 equiv), degassed toluene, 60 °C, 2 h (68%); (3) CCl3CN (3.0 equiv), DBU (0.5 equiv), DCM, 0 °C to rt, 6 h (60%).

O OH

OAcN3AcO

OAc

Toluene, 60 C

3) CCl3CN, DBU

DCM, rt

O

OAcAcO

OAc

2) Ph2POPh O OH

OAcNH

AcO

OAc

O

CCl3HN

1) NH3-MeOH

DCM-MeOH 0 °C

O OAc

OAcN3AcO

OAc

46 48 49

50

P

OPh

Ph

NH

P

OPh

Ph49

78

3.5 Coupling Reactions of Trichloroacetimidate Donor 50 by Ni(II) Catalyst

We sought to utilize the method developed by Nguyen and co-workers, in

which trichloroacetimidate donors are coupled with various glycosyl acceptors

under [Ni(4-FPhCN)4(OTf)2]-catalyzed conditions with high �-stereo-

selectivity.19,20 We thus explored the coupling of donor 50 with various acceptors

under similar conditions (Table 3.2).

79

Table 3.2 Glycosylation with trichloroacetimidate donor 51 using [Ni(4-F-PhCN)4(OTf)2] catalyst

[a] Catalyst was prepared in situ at 0 °C, then warmed to rt for 40 min.

80

3.6 Conclusion and Future Directions

In this study, we have investigated [Ni(4-F-PhCN)4(OTf)2]-mediated

glycosylation of trichloroacetimidate donor 50, which produced �-glycosides with

selected acceptors. Trichloroacetimidate donor 50 is a novel glycosyl donor

which can potentially have a broader scope, if issues of reactivity with sterically

encumbered acceptors can be addressed. More studies are needed with variable

substituents on phenyl groups of the phosphinamide moiety.

3.7 General Methods and Experiments

3.7.1 Experimental

General methods of preparation of imidazole-1-sulfonyl azide

hydrochloride 45. The procedure was adapted from the literature.24 In a typical

experiment, NaN3 (4.8 g, 73.85 mmol) was dispersed into dry acetonitrile (70 mL)

and cooled to 0 °C for 15 minutes. SO2Cl2 was added dropwise and stirred for an

additional 30 minutes at 0 °C, then brought to room temperature and stirred for

another 20 h. To the milky white mixture was added recrystallized imidazole (10

g, 147 mmol) at 0 °C in small portions. This was stirred at 0 °C for 30 minutes

and then at room temperature for 3 h. The white precipitate thus obtained was

diluted with ethyl acetate (200 mL) and washed with saturated sodium

bicarbonate solution (2 x 100 mL), water (2 x 100 mL), brine (2 x 50 mL), and

dried over anhydrous MgSO4. The organic portion was concentrated down and

added to 25% HCl in absolute ethanol (8 mL) at 0 °C with vigorous stirring. The

81

white crystals obtained were filtered and washed with hexanes to afford

imidazole-1-sulfonyl azide hydrochloride 45 (10.8 g, 70%). 1H NMR (400 MHz,

D2O): � 9.32 (s, 1H), 8.01 (s, 1H), 7.60 (s, 1H). 13C NMR (101 MHz, D2O) �

138.32, 124.73, 120.53.

General method for the diazo transfer reaction of D-glucosamine

hydrochloride: In a typical experiment, imidazole-1-sulfonyl azide hydrochloride

45 (2.99 mmol, 1.3 equiv) was added to a suspension of D-glucosamine

hydrochloride (2.3 mmol, 1 equiv), potassium carbonate (11.59 mmol, 2.4 equiv),

and copper(II) sulfate pentahydrate (1.2 mmol, 2 mol%) in methanol (22 mL) at

room temperature. The reaction mixture was stirred for at least 10 h and turned

green. Upon completion of the reaction by TLC, the mixture was concentrated,

and azeotroped with toluene (3 x 5 mL), then dispersed into distilled pyridine (20

mL) and acetic anhydride (23 mmol, 10 equiv). Upon completion of the reaction

(TLC), the mixture was concentrated, extracted with ethyl acetate, washed with

brine, and purified by silica column chromatography using 10–50% ethyl acetate

in hexanes to furnish peracetylated azide 46.

General method of preparation 49: In a typical experiment, phenyl

diphenylphosphinite (0.72 mmol, 1.2 equiv) was weighed out in a glove box and

dissolved in distilled and degassed toluene (2 mL). This was added to an

anerobic solution of lactol 48 (0.6 mmol, 1 equiv) in toluene (8 mL) at room

temperature. The reaction mixture was warmed to 60 °C for 2–4 h, then cooled to

room temperature, concentrated to dryness, and purified by silica gel

82

chromatography using a 50–100% ethyl acetate in hexanes gradient to afford 49

as a white solid.

Preparation of D-Glucosamine Trichloroacetimidate Donor 50: To a

solution of compound 49 (0.24 mmol, 1 equiv) in distilled dichloromethane (4 mL)

at 0 °C, freshly distilled trichloroacetonitrile (0.72 mmol, 3 equiv), followed by

freshly distilled DBU (0.12 mmol, 0.5 equiv) were added and stirred at this

temperature for 6–12 h. The reaction did not reach to completion, but was diluted

with dichloromethane and passed through a plug of solid ammonium chloride,

then concentrated and purified by silica gel chromatography using a 50–80%

ethyl acetate in hexanes gradient with 1% triethylamine to obtain isolated product

30–60% yield and recovered starting material.

Preparation of Catalyst precursor Ni(4-FPhCN)4Cl2: This was prepared by

following the reported protocol:19 A flame-dried round-bottom flask was charged

with NiCl2 (87 mg, 0.67 mmol) and 4-fluorobenzonitrile (2.1 g, 16.75 mmol) in

distilled and degassed CH2Cl2 (2 mL), then stirred at room temperature under an

argon atmosphere for 30 h. The reaction mixture was then poured into hexanes

(20 mL) to induce precipitation. After 10 minutes the hexanes layer was

decanted, and the resulting yellow solid was further washed with hexane (2 × 10

mL) then transferred into a pre-tared flask and dried under high vacuum

overnight to afford Ni(4-FPhCN)4Cl2 as a yellowish powder (125 mg, 30%).

General glycosylation with trichloroacetimidate donor using Ni(4-

FPhCN)4(OTf)2: In a typical experiment, Ni(4-FPhCN)4(OTf)2 was generated in

situ from a reaction of Ni(4-FPhCN)4Cl2 (4.81 mg, 0.0078 mmol, 14 mol%),

83

AgOTf (3.82, 0.0148 mmol, 28 mol%), and an activated 4Å molecular sieves (30

mg) in CH2Cl2 (0.3 mL) cooled to 0 °C, then warmed to room temperature with

stirring for 1 hour under an argon atmosphere. The reaction mixture was again

cooled to 0 °C, then treated with a solution of D-glucosamine trichloroacetimidate

donor 50 (0.054 mmol, 1 equiv) and acceptor (0.07 mmol, 1.3 equiv) in degassed

CH2Cl2 (0.3 mL). The reaction mixture was stirred at 0 °C for 1 h, then warmed to

room temperature for 24 h. The reaction was quenched with triethylamine (15 �L,

0.1 mmol), diluted with dichloromethane (10 mL), then passed through Celite and

concentrated. The crude product was purified by silica gel chromatography using

a 30–50% acetone in hexanes gradient with 1% triethylamine to afford desired

disaccharide product.

Thiotolyl 3,6-di-O-benzyl-4,6-O-p-methoxybenzylidene-2-(4-

(trifluoromethyl)benzylideneamino)-�-glucopyranoside (38). Compound 37

(198 mg, 0.32 mmol) was dried with azeotropic distillation with toluene (3 x 2

mL), dissolved in distilled and degassed dichloromethane (5 mL), then cooled to

0 °C under an argon atmosphere. Bu3P (95 �L, 0.38 mmol) was added and

stirred for 1 h at this temperature. When all starting materials were consumed,

the reaction mixture was treated with 4-trifluoromethylbenzaldehyde (60 �L, 0.45

mmol). After stirring 2 h at 0 °C, the reaction was quenched with cold water (2

mL), warmed to room temperature, extracted with dichloromethane (3 x 20 mL),

washed with brine (3 x 5 mL), and dried over sodium sulfate. The crude product

was purified by silica gel chromatography using a 5–20% ethyl acetate in

hexanes gradient with 1% triethylamine to afford C2 benzylideneamine 38 as a

84

yellowish solid (187 mg, 78% over two steps). 1H NMR (400 MHz, CDCl3): �

8.82 (s, 1H), 8.06 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 7.27 (m, 10H),

7.04 (d, J = 7.8 Hz, 4H), 6.83 (m, 4H), 5.22 (d, J = 10.2 Hz, 1H), 4.65 (m, 8H),

4.47 (m, 2H), 4.16 (t, J = 9.0 Hz, 1H), 3.87 (d, J = 24.2 Hz, 2H), 3.80 (m, 10H),

2.31 (s, 3H).

Acetyl 2-azido-3,6-di-O-benzyl-2-deoxy-4,6-p-methoxybenzylidene-

glucopyranoside (40). Compound 37 (212 mg, 0.35 mmol) was dissolved in dry

acetone (9 mL), and cooled to –30 °C, treated with recrystallized N-

bromosuccinimide (123 mg, 0.69 mmol) dissolved in 1 mL of acetone, then

warmed slowly to –20 °C for 1.5 h. The reaction was quenched at –20 °C with

saturated sodium bicarbonate (5 mL), then warmed to room temperature,

extracted with ethyl acetate (3 x 50 mL), washed with brine (3 x 5 mL), dried over

sodium sulfate, then concentrated and purified by silica gel chromatography

using a 20–40% ethyl acetate in hexanes gradient to afford an intermediate

lactol (175 mg, 90%). 1H NMR (400 MHz, CDCl3): � 7.36 (m, 10H), 7.05 (t, J =

8.7 Hz, 2H), 6.81 (m, 2H), 5.32 (m, 1H), 4.89 (q, J = 6.1 Hz, 1H), 4.72 (dd, J =

8.3, 10.5 Hz, 1H), 4.56 (m, 3H), 4.44 (dd, J = 5.5, 10.5 Hz, 1H), 4.00 (dd, J = 9.0,

10.2 Hz, 1H), 3.79 (s, 3H), 3.61 (m, 4H), 3.42 (m, 2H), 3.09 (s, 1H).

The lactol intermediate (175 mg was subjected to acetylation using acetic

anhydride (0.1 mL) and pyridine (3 mL), then concentrated to dryness and

purified by silica gel chromatography to afford glycosyl acetate 40 as a mixture of

anomers (170 mg, �/� =1:1, 90%). 1H NMR (400 MHz, CDCl3): � 7.50–7.28 (m,

28H), 7.13–6.95 (m, 5H), 6.87–6.73 (m, 5H), 4.90 (t, J = 3.5 Hz, 2H), 4.86 (d, J =

85

3.5 Hz, 2H), 4.77–4.68 (m, 2H), 4.63 (dd, J = 5.3, 11.9 Hz, 3H), 4.53–4.37 (m,

6H), 3.93 (m, 1H), 3.87 (d, J = 3.2 Hz, 2H), 3.81 (d, J = 7.1 Hz, 2H), 3.80–3.76

(m, 7H), 3.76–3.68 (m, 6H), 3.67–3.44 (m, 9H), 2.20–2.15 (m, 3H), 2.14 (d, J =

0.9 Hz, 1H), 2.13–2.11 (m, 3H).

Acetyl 3,6-di-O-benzyl-2-deoxy-2-p-fluorobenzylideneamino-4,6-p-

methoxybenzylidene glucopyranoside (41). Compound 40 (70 mg, 0.13 mmol)

dissolved in degassed dichloromethane (2 mL) was treated with tributyl

phosphine (42 �L, 0.17 mmol) at 0 °C for 1 h, followed by addition of p-

fluorobenzaldehyde (40 �L, 0.37 mmol) with stirring at 0 °C for 2 h. Aqueous

workup (cf. 38) afforded benzylideneamine 41 as a yellowish solid and a mixture

of anomers (�/� =1:1, 80 mg, 87% over two steps). 1H NMR (400 MHz, CDCl3):

� 8.83 (s, 1H), 7.94–7.91 (m, 2H), 7.24 (m, 14H), 6.81 (m, 2H), 6.26 (d, J = 3.7

Hz, 1H), 6.18 (d, J = 11.0 Hz, 1H) 4.63 (m, 6H), 3.98 (m, 8H), 2.13 (s, 3H).

3,6-Di-O-benzyl-2-deoxy-2-p-fluorobenzylidenamino-4-O-(4,6-p-methoxy-

benzylidene)-�-glucopyranosyl trichloroacetimidate (42). Glycosyl acetate 41

( 38 mg, 0.06 mmol) was dissolved in THF (0.3 mL) and treated with 7 M

ammonia in methanol (0.3 mL) at –40 °C, then warmed to 0 °C for 2 h. The crude

reaction mixture (30 mg, 0.05 mmol) was concentrated to dryness, redissolved in

distilled dichloromethane (0.8 mL), treated with trichloroacetonitrile (15 �L, 0.15

mmol) and DBU (4 �L, 0.026 mmol), and stirred at 0 °C for 2 h. The reaction

mixture was concentrated and purified by silica gel chromatography using a 10–

30% ethyl acetate in hexanes gradient with 1% triethylamine to afford �-

trichloroacetimidate 42 as an oil (24 mg, 54% over two steps). 1H NMR (500

86

MHz, C6D6): � 8.57 (s, 1H), 8.09 (s, 1H), 7.43 (m, 2H), 7.34 (m, 2H), 7.19 (m,

6H), 7.01 (m, 2H), 7.03 (m, 2H), 6.78 (dd, J = 2.1, 8.7 Hz, 2H), 6.69 (m, 2H), 6.40

(d, J = 8.2 Hz, 1H), 4.86 (d, J = 10.9 Hz, 1H), 4.77 (m, 2H), 4.68 (d, J = 10.9 Hz,

1H), 4.61 (d, J = 11.3 Hz, 1H), 4.56 (d, J = 11.6 Hz, 1H), 4.51 (m, 2H), 4.39 (m,

2H), 4.05 (t, J = 9.2 Hz, 1H), 4.02 (t, J = 9.1 Hz, 1H), 3.74 (m, 5H), 3.31 (s, 4H),

3.29 (m, 2H). 13C NMR (126 MHz, C6D6): � 163.19, 160.88, 159.43, 138.67,

138.60, 132.41, 130.85, 130.25, 130.18, 129.60, 129.31, 128.29, 128.23, 128.20,

128.05, 127.98, 127.88, 127.68, 127.59, 127.49, 127.30, 115.52, 115.35, 113.70,

97.54, 91.17, 83.48, 80.58, 77.02, 76.50, 75.94, 74.72, 74.53, 74.31, 73.97,

73.22, 68.44, 54.41, 36.47, 24.66, 23.18.

1,3,4,6-tetra-O-acetyl-2-azido-2-deoxyglucopyranoside (46). Glucosamine

hydrochloride (0.2 g, 0.93 mmol) was treated with diazo transfer reagent 45 (0.23

g, 1.12 mmol), potassium carbonate (0.335 g, 2.42 mmol), and copper sulfate

pentahydrate (50 mg, 0.2 mmol) in methanol (5.5 mL) with stirring at room

temperature for 12 h. Upon completion for the reaction by TLC, the mixture was

concentrated to dryness, and azeotroped with toluene (3 x 5 mL), then dispersed

into pyridine (5 mL) and treated with acetic anhydride (1 mL) with stirring at room

temperature for 18 h. The reaction mixture was then concentrated and purified by


afford azidoglucose tetraacetate 46 as white solid (�:� = 2:3, 291 mg, 84%). 1H

NMR (400 MHz, CDCl3): � 6.38 (d, J = 3.7 Hz, 1H), 5.65 (d, J = 8.6 Hz, 1H), 5.54

(dd, J = 9.4, 10.5 Hz, 1H), 5.18 (m, 3H), 4.38 (m, 3H), 4.1Hz, 3H), 3.89 (ddd, J =

87

2.2, 4.5, 9.8 Hz, 1H), 3.75 (ddd, J = 2.1, 5.8, 9.9 Hz, 2H), 2.27 (s, 6H), 2.18 (s,

3H), 2.17 (s, 3H), 2.15 (s, 5H), 2.12 (s, 2H), 2.10 (s, 3H).

S-Benzoxazolyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-

glucopyranoside (47). Glycosyl acetate 46 (1.2 g, 3.22 mmol) was dried by

azeotropic distillation with toluene (3 x 2 mL), then treated with a solution of 2-

mecaptobenzoxazole (1.1 g, 7.28 mmol) and activated 4Å molecular sieves (1.5

g) dispersed in dry dichloromethane (30 mL). The reaction mixture was cooled to

0 °C was and treated with BF3.Et2O (2 mL, 15.8 mmol), then warmed to room

temperature and stirred for 48 h. The reaction was quenched with aqueous

sodium bicarbonate solution at 0 °C, then extracted with dichloromethane (3 x

100 mL), washed with brine (3 x 10 mL), dried over sodium sulfate, concentrated

and purified by silica gel chromatography using a 10–50% ethyl acetate in

hexanes gradient to afford S-benzoxazolyl glycoside 47 as a syrup (920 mg, �:�

= 1:1, 62%). Selected major peaks of 1H NMR (400 MHz, CDCl3): � 7.65–7.60

(m), 7.46 (d, J = 8.7 Hz), 7.29 (m), 6.27 (d, J = 3.6 Hz), 5.54 (d, J = 8.3 Hz), 5.42

(m), 5.13–4.98 (m), 4.31–4.18 (m), 4.12–3.99 (m) 3.99–3.85 (m), 3.79 (m), 3.69–

3.60 (m), 2.17 (s), 2.08 (s), 2.07 (s), 2.06 (s), 2.05 (s), 2.02 (s), 2.00 (s).

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-glucopyranose (48). Glycosyl acetate

46 (338 mg, 0.9 mmol) was dissolved in THF (2 mL) and treated dropwise with 7

M ammonia solution in methanol (2 mL) at –40 °C using a cannula, then warmed

to 0 °C and stirred for 2 h. The reaction mixture was concentrated and purified by


88

afford compound 50 as white solid (232 mg, 78%). 1H NMR (400 MHz, CDCl3):

� 5.51 (t, J = 10.5 Hz, 1H), 5.37 (s, 1H), 5.12–4.89 (m, 2H), 4.71 (d, J = 8.0 Hz,

1H), 4.30–4.16 (m, 4H), 4.15–4.02 (m, 4H), 3.69 (m, 1H), 3.39 (dd, J = 3.4, 10.5

Hz, 1H), 2.06 (s, 9H), 2.02 (s, 6H), 1.99 (s, 3H). IR: 3430, 2931, 2113, 1753,

1236 cm-1.

3,4,6-Tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-D-glucopyranose

(49). Azidoglucose 48 (115 mg, 0.35 mmol) was treated with commercially

available phenyl diphenylphosphinite (115 mg, 0.41 mmol) as described

previously (see General Methods) to afford 50 as a white solid (115 mg, 68%).

1H NMR (400 MHz, CDCl3): � 7.77 (m, 4H), 7.42 (m, 6H), 5.39 (s, 1H), 5.33 (t, J

= 9.8 Hz, 1H), 4.86 (t, J = 9.8 Hz, 1H), 4.25 (m, 2H), 4.04 (m, 2H), 3.07 (m, 1H),

2.12 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H). 13C NMR (101 MHz, CDCl3): � 170.77,

169.58, 132.58, 132.48, 132.26, 132.11, 131.60, 131.51, 130.74, 129.42, 128.70,

128.59, 128.47, 92.94, 72.11, 68.62, 66.53, 62.02, 55.06, 20.97, 20.69, 20.58.

31 P NMR (162 MHz, CDCl3): � 26.17.

3,4,6-Tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-�-glucopyranosyl

trichloroacetimidate (50). Compound 49 (200 mg, 0.4 mmol) was treated with

CCl3CN (125 �L, 1.25 mmol) and DBU (30 �L, 0.2 mmol) in dry dichloromethane

(6 mL) at 0 °C for 12 h. The reaction mixture was passed through an ammonium

chloride plug, concentrated, then purified by silica gel chromatography using a

50–90% ethyl acetate in hexanes gradient with 1% triethylamine to afford

trichloroacetimidate 50 as a white crystalline solid (160 mg, 60%). 1H NMR (500

MHz, CDCl3): � 8.88 (s, 1H), 7.80–7.76 (m, 4H), 7.52 (m, 2H), 7.44 (ddt, J = 8.6,

89

10.8, 5.3 Hz, 4H), 6.44 (d, J = 3.7 Hz, 1H), 5.36 (t, J = 10.0 Hz, 1H), 5.11 (t, J =

9.9 Hz, 1H), 4.24 (dd, J = 4.1, 12.5 Hz, 1H), 4.10 (ddd, J = 2.3, 4.2, 10.3 Hz, 1H),

4.05 (dd, J = 2.3, 12.4 Hz, 1H), 3.56 (ddt, J = 9.9, 11.5, 3.7 Hz, 1H), 3.23 (dd, J =

7.0, 11.5 Hz, 1H), 2.16 (s, 3H), 2.03 (s, 6H). 13C NMR (126 MHz, CDCl3): �

171.76, 170.71, 169.41, 160.70, 132.52, 132.36, 132.30, 132.23, 132.00, 131.92,

129.03, 128.93, 128.82, 128.72, 109.83, 96.51, 90.98, 71.68, 70.29, 67.59,

61.55, 53.86, 21.31, 20.80, 20.74. 31P NMR (203 MHz, CDCl3): � 22.88. IR:

3317, 3276, 3185, 2958, 1749, 1677, 1438, 1369, 1224, 1041, 732 cm-1. [�]D25 =

+325° (c 0.02, CH2Cl2).

Isopropyl 3,4,6-tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-�-D-

glucopyranoside (55). Trichloroacetimidate donor 50 (18 mg, 0.027 mmol) was

dried by azeotropic distillation with toluene (3 x 1 mL), then treated with freshly

distilled isopropanol (4 �L, 0.079 mmol) and Ni(4-FPhCN)4(OTf)2 catalyst (17.5

mol%) prepared in situ from AgOTf (2.5 mg, 0.0097 mmol, 35 mol%) and Ni(4-

FPhCN)4Cl2 (3 mg, 0.0048 mmol, 17.5 mol%) in dichloromethane (0.25 mL). The

reaction mixture was stirred at 0 °C for 12 h, at rt for an additional 12 h, then

quenched with triethylamine (10 �L). This was concentrated and purified by

preparative TLC using 45% acetone in hexanes gradient with 1% triethylamine to

afford isopropyl glycoside 55 as a white solid (4 mg, 26%). 1H NMR (500 MHz,

CDCl3): � 7.96 (dd, J = 7.6, 11.8 Hz, 2H), 7.88 (dd, J = 7.7, 11.9 Hz, 2H), 7.45

(m, 6H), 5.07 (t, J = 9.7 Hz, 1H), 4.88 (t, J = 9.6 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H),

4.22 (dd, J = 5.4, 12.2 Hz, 1H), 4.06 (m, 2H), 3.64 (ddd, J = 2.5, 5.3, 9.9 Hz,

1H), 3.15 (sept., J = 8.04 Hz, 1H), 2.82 (dd, J = 4.7, 10.6 Hz, 1H), 2.65 (d, J = 7.4

90

Hz, 1H), 2.18 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.31 (m, 6H). 13C NMR (126

MHz, CDCl3) � 171.66, 170.67, 169.48, 134.05, 133.22, 133.14, 132.07, 131.99,

131.90, 131.85, 131.23, 128.48, 128.44, 128.38, 128.34, 101.18, 74.26, 74.16,

72.14, 71.75, 68.82, 62.38, 56.78, 46.34, 23.45, 21.82, 21.31, 20.76, 20.65. 31P

NMR (203 MHz, CDCl3) : � 21.99.

3,4,6-Tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-�-glucopyranosyl-

(1�3)-4,6-O-benzylidene-D-glucal (56). Trichloroacetimidate donor 50 (25 mg,

0.038 mmol) was treated with glycosyl acceptor 51 (11 mg, 0.047 mmol) and

Ni(4-FPhCN)4(OTf)2 catalyst (16 mol%) prepared in situ from AgOTf (3.3 mg,

0.0128 mmol, 32 mol%) and Ni(4-FPhCN)4Cl2 (3.9 mg, 0.0063 mmol, 16 mol %)

in dichloromethane (1 mL) at 0 °C, following the procedure described above (cf.

55). The crude product was purified by preparative TLC using 45% acetone in

hexanes with 1% triethylamine to afford disaccharide 56 as a white solid (3 mg,

11%). 1H NMR (800 MHz, CDCl3): � 7.88 (dd, J = 7.6, 11.7 Hz, 2H), 7.81 (dd, J =

7.6, 11.6 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.43 (m, 4H), 7.37 (m, 4H), 6.44 (d, J

= 3.1 Hz, 1H), 5.58 (s, 1H), 5.04 (t, J = 9.7 Hz, 1H), 5.00 (dd, J = 2.1, 6.2 Hz,

1H), 4.92 (t, J = 9.6 Hz, 1H), 4.70 (dd, J = 4.4, 7.6 Hz, 2H), 4.37 (dd, J = 5.0, 10.5

Hz, 1H), 2.13 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H). 13C NMR (126 MHz, CDCl3): �

171.52, 170.74, 169.54, 145.25, 137.18, 133.30, 132.79, 132.08, 131.35, 129.49,

128.57, 126.16, 101.99, 101.68, 101.33, 78.94, 74.72, 74.32, 71.72, 68.77,

68.54, 68.42, 61.98, 57.02, 21.23, 20.87, 20.76. 31P NMR (203 MHz, CDCl3): �

22.01.

91

Methyl 3,4,6-tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-�-gluco-

pyranosyl-(1�6)-2,3,4-tri-O-benzyl-�-glucopyranoside (57). Trichloroaceti-

midate donor 50 (20 mg, 0.031 mmol) was treated with glycosyl acceptor 52 (18

mg, 0.038 mmol) and Ni(4-FPhCN)4(OTf)2 catalyst (14 mol%) prepared in situ

from AgOTf (2.36 mg, 0.009 mmol, 28 mol%) and Ni(4-FPhCN)4Cl2 (2.81 mg,

0.0045 mmol, 14 mol%) in dichloromethane (0.5 mL) along with 4Å molecular

sieves (20 mg), as described above (cf. 55). The reaction mixture was stirred for

26 hours at rt followed by workup and purification by preparative TLC using 45%

acetone in hexanes with 1% triethylamine to afford disaccharide 57 as a white

solid (8 mg, 27%). 1H NMR (500 MHz, CDCl3): � 7.97 (dd, J = 8.0, 12 Hz, 2H),

7.85–7.79 (m, 2H), 7.82, 7.31 (m, 19H), 7.05 (m, 2H), 5.06 (d, J = 10.4 Hz, 1H),

4.84 (m, 5H), 4.61 (m, 3H), 4.15 (dd, J = 4.7, 12.3 Hz, 1H), 4.08 (m, 2H), 4.02

(dd, J = 2.5, 12.3 Hz, 1H), 3.81 (dt, J = 10.2, 3.1 Hz, 1H), 3.71 (d, J = 8.0 Hz,

1H), 3.60 (m, 1H), 3.54 (dq, J = 9.8, 3.7 Hz, 2H), 3.47 (ddd, J = 2.5, 4.8, 9.9 Hz,

1H), 3.34 (s, 3H), 3.09 (quint., J = 9.6 Hz, 1H), 2.17 (s, 3H), 1.98 (d, J = 1.5 Hz,

6H). 13C NMR (126 MHz, CDCl3): � 171.80, 170.77, 169.50, 138.63, 138.01,

133.56, 132.14, 131.91, 130.68, 129.64, 128.76, 128.63, 128.59, 128.39, 128.19,

127.99, 102.81, 98.45, 82.29, 80.09, 76.65, 76.13, 74.24, 73.89, 73.55, 72.04,

69.74, 68.70, 67.75, 62.15, 56.54, 55.67, 21.55, 20.84, 20.77. 31P NMR (203

MHz, CDCl3): � 21.52.

92

Methyl 3,4,6-tri-O-acetyl-2-deoxy-2-diphenylphosphinamido-�-gluco-

pyranosyl-(1�6)-2,3,4-tri-O-benzyl-�-glucopyranoside (58). Trichloroacet-

imidate donor 50 (35 mg, 0.054 mmol) was treated with acceptor 53 (45 mg,

0.096 mmol) and Ni(4-FPhCN)4(OTf)2 (14.5 mol%) prepared in situ from AgOTf

(3.3 mg, 0.0148 mmol, 27 mol%) and Ni(4-FPhCN)4Cl2 (4.81 mg, 0.0078 mmol,

14.5 mol%) in dichloromethane (0.55 mL) along with 4Å molecular sieves (30

mg), as described above (cf. 55). The reaction mixture was stirred for 48 h at

room temperature followed by workup and purification by preparative TLC using

45% acetone in hexanes with 1% triethylamine to afford 58 as white solid (8 mg,

16%). 1H NMR (500 MHz, CDCl3): � 8.02 (dd, J = 4.05, 7.5 Hz, 2H), 7.86 (dd, J =

7.2, 11.3 Hz, 2H), 7.49 (m, 4H), 7.31 (m, 15H), 7.13 (m, 2H), 5.00 (d, J = 10.5

Hz, 1H), 4.86 (m, 5H), 4.69 (d, J = 10.8 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H), 4.32

(d, J = 7.2 Hz, 1H), 4.21–4.15 (m, 2H), 4.03 (dd, J = 4.8, 10.0 Hz, 2H), 3.72 (t, J

= 8.9 Hz, 1H), 3.66–3.45 (m, 5H), 3.44–3.39 (m, 4H), 3.11 (quint., J = 9.4 Hz,

1H), 2.19 (s, 3H), 2.0 (s, 3H), 1.97 (s, 3H). 13C NMR (126 MHz, CDCl3): � 171.86,

170.80, 169.56, 138.49, 138.42, 138.31, 133.72, 133.64, 132.22, 132.09, 132.01,

128.76, 128.72, 128.57, 128.54, 128.45, 128.35, 128.26, 127.95, 127.87, 105.07,

103.14, 103.06, 84.95, 82.35, 77.41, 76.02, 74.91, 74.67, 74.61, 74.04, 72.01,

68.66, 68.14, 62.19, 57.67, 56.69, 21.54, 20.87, 20.77.

93

3.8 References

(1) Magnet, S.; Blanchard, J. S. Chem. Rev. 2005, 105, 477.

(2) Helmann, J. D. Antioxid. Redox Signal. 2011, 15, 123.

(3) Bongat, A. F.; Demchenko, A. V. Carbohydr. Res. 2007, 342, 374.

(4) Nigudkar, S. S.; Demchenko, A. V. Chem. Sci. 2015, 6, 2687.

(5) Zulueta, M. M. L.; Lin, S.-Y.; Lin, Y.-T.; Huang, C.-J.; Wang, C.-C.; Ku, C.-C.; Shi, Z.; Chyan, C.-L.; Irene, D.; Lim, L.-H. J. Am. Chem. Soc. 2012, 134, 8988.

(6) Guo, J.; Ye, X.-S. Molecules 2010, 15, 7235.

(7) Benakli, K.; Zha, C.; Kerns, R. J. J. Am. Chem. Soc. 2001, 123, 9461.

(8) Kerns, R. J.; Zha, C.; Benakli, K.; Liang, Y.-Z. Tetrahedron Lett. 2003, 44, 8069.

(9) Wei, P.; Kerns, R. J. J. Org. Chem. 2005, 70, 4195.

(10) Geng, Y.; Zhang, L.-H.; Ye, X.-S. Chem. Commun. 2008, 597.

(11) Geng, Y.; Zhang, L.-H.; Ye, X.-S. Tetrahedron 2008, 64, 4949.

(12) Boysen, M.; Gemma, E.; Lahmann, M.; Oscarson, S. Chem. Commun. 2005, 3044.

(13) Manabe, S.; Ishii, K.; Ito, Y. J. Am. Chem. Soc. 2006, 128, 10666.

(14) Manabe, S.; Ishii, K.; Ito, Y. Eur. J. Org. Chem. 2011, 2011, 497.

(15) Winterfeld, G. A.; Schmidt, R. R. Angew. Chem. Int. Ed. 2001, 40, 2654.

(16) Ryan, D. A.; Gin, D. Y. J. Am. Chem. Soc. 2008, 130, 15228.

(17) Yang, J.; Cooper-Vanosdell, C.; Mensah, E. A.; Nguyen, H. M. J. Org. Chem. 2008, 73, 794.

(18) Mensah, E. A.; Azzarelli, J. M.; Nguyen, H. M. J. Org. Chem. 2009, 74, 1650.

(19) Mensah, E. A.; Yu, F.; Nguyen, H. M. J. Am. Chem. Soc. 2010, 132, 14288.

(20) Mensah, E. A.; Nguyen, H. M. J. Am. Chem. Soc. 2009, 131, 8778.

94

(21) McKay, M. J.; Nguyen, H. M. ACS Catal. 2012, 2, 1563.

(22) Ornitz, D. M.; Herr, A. B.; Nilsson, M.; Westman, J.; Svahn, C.-M.; Waksman, G. Science 1995, 268, 432.

(23) Morales Collazo, O. Ph.D. Thesis, Purdue University 2014.

(24) Goddard-Borger, E. D.; Stick, R. V. Org. Lett. 2007, 9, 3797.

(25) Sabelle, S.; Renard, P.-Y.; Pecorella, K.; de Suzzoni-Dézard, S.; Créminon, C.; Grassi, J.; Mioskowski, C. J. Am. Chem. Soc. 2002, 124, 4874.

(26) Kuroda, K.; Maruyama, Y.; Hayashi, Y.; Mukaiyama, T. Chem. Lett. 2008, 37, 836.

(27) Kuroda, K.; Maruyama, Y.; Hayashi, Y.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 2009, 82, 381.

95

VITA

Hari Raj Khatri hails from Nepal. He completed his Master’s degree in

Chemistry in 2004 from Tribhuvan University, Nepal with a thesis titled “Isolation,

identification and characterization of biologically active compounds from

medicinal plant Bergenia ciliata”. After graduation, he was actively involved in

teaching. He taught for 4 years in various undergraduate colleges in Nepal.

Later, he joined the University of South Dakota, USA for his graduate studies in

2008. In 2010, he earned his second Master’s degree in Chemistry under the

guidance of Dr. Grigoriy A. Sereda. His Master’s thesis was titled “Design and

synthesis of calixarene-templated catalysts”. He joined Professor Alexander

Wei’s research group at Pudue University in 2010 to pursue his PhD. During his

Ph.D., Hari explored the post-glycosylative modification of iduronsyl

disaccharides and also studied the stereoselective glycosylation of C-2-

diphenylphosphinamido trichloroacetimidate donor. He successfully defended his

dissertation in April 2016.

PUBLICATION

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synthetic studies of heparan derivatives: glycosyl

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