A Thesis Entitled Thio-Arylglycosides with Various Aglycon Para-Substituents, a Useful Tool for Mechanistic Investigation Of

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A Thesis

entitled

Thio-arylglycosides with Various Aglycon Para-Substituents, a Useful Tool for

Mechanistic Investigation of Chemical Glycosylations

Xiaoning Li

Submitted as partial fulfillment of the requirements for

the Master of Science Degree in Chemistry

______Advisor: Dr. Xuefei Huang

______College of Graduate Studies

The University of Toledo

August 2007

An Abstract of

Thio-arylglycosides with Various Aglycon Para-Substituents, a Useful Tool for

Mechanistic Investigation of Chemical Glycosylations

Xiaoning Li

Submitted as partial fulfillment of the requirements for

the Master of Science Degree in Chemistry

The University of Toledo

August 2007

Oligosaccharides are usually found as protein or lipid conjugates in cellular

systems. They play crucial roles in many biological processes. Among many approaches, organic synthesis is a very important way to obtain the desired oligosaccharides for biological studies. To date, no general synthetic procedures are available for oligosaccharide synthesis. Laborious synthetic transformations are generally required in order to obtain the desired regio- and/or stereo-selective control in oligosaccharide synthesis, due to their diverse and complex structures and many chemical equivalent

hydroxyl functional groups. To achieve a rapid synthetic routine with high yields, a key

step - glycosylation in oligosaccharide synthesis needs to be well understood. Thus an

insight into the mechanism of glycosylation will provide valuable information potentially

leading to the development of generalized glycosylation method.

In this work, kinetic properties of glycosylation were evaluated by model

reactions between three different series of glycosyl donors and three different glycosyl

acceptors. The glycosylation mechanism was analyzed in the context of a linear-free

energy relationship. In order to do that, three series of glycosyl donors and one glycosyl acceptor were synthesized and the Relative Reactivity Values (RRVs) of these donors

were then determined by HPLC competitive assay. Hammett plots were plotted using

obtained RRVs. A linear correlation was found that allows an accurate prediction of

glycosylation reactivities. The negative slopes of the Hammett plots (ρ-values) show that

electron donating substituents increase the rate of the reactions and the magnitudes of

slopes can be rationalized by neighboring group participation and electronic properties of

the glycon protective groups. RRVmethanol values (using methanol as acceptor under

standardized reaction conditions) have been employed as a quantitative measure to guide the design of building blocks with requisite anomeric reactivities for reactivity based chemoselective glycosylation. We extensively studied glycosylation reactivity by varying the reaction conditions such as by using different carbohydrate acceptors, glycosylation promoters, solvent or by quenching reaction at different time. Our results indicate that the reactivity differential was found to be dependent upon the identity of the acceptor with the less reactive carbohydrate acceptors resulting in lower reactivity differential. This result points out that the rate determining step of glycosylation reaction should involve

iii

the nucleophilic attack of the acceptor onto oxacarbenium ions or similar reactive

intermediates as deduced from Hammett plots. The solvent could also play a role in

varying the relative reactivity between two glycosyl donors. However, the RRVs were

consistent under different promoter activation or by quenching at different reaction time.

The electron density of sulphur atom on glycosyl donor and the bonding energy between

glycosyl donor and promoter were found as qualitative indicators for RRVs. The

knowledge gained from our results can provide valuable insights into further improvement of the powerful chemoselective armed-disarmed glycosylation methodology and understanding of chemical glycosylation in general.

ACKOWNLEDGEMENTS

First of all, I would like to thank Dr. Xuefei Huang, my research advisor for his

instructions on my graduate study and encouragements to my research activities, as well

as his understanding of my careless omissions sometimes.

I would like to thank all my lab members: Adeline Miermone, Balasubramanian

Srinivasan, Bin Sun, Bo Yang, Gilbert Wasonga, Kheireddine El-boubbou, Lijun Huang,

Luyuan Zhou, Nardos Teumelsan, Xiaowei Lu, Youlin Zeng, Yuqing Jing, Zhen Wang. I

have learnt a lot from discussions with them either on group meetings or at casual time.

There are many other people I would like to thank:

To my committee members, Dr. Xiche Hu and Dr. Steven Sucheck for their discussions and help.

To Dr. Viranga Tillekeratne, Dr. Richard Hudson, Dr. James T. Slama for their inspiring discussions on Organic Journal Clubs.

To Dr. Yong Wah Kim for his NMR training.

To Charlene Hansen-Morlock and Pam Samples for their day-to-day help.

To the University of Toledo Department of Chemistry for the financial support.

Finally but not least significantly to my family: my parents and brothers; as well as to many friends of mine either in America or in China

TABLE OF CONTENTS

Abstract...... ii

Acknowledgements...... v

Table of Contents...... vi

List of Figures...... viii

List of Tables...... x

List of Schemes………………………………………………………………...... xi

List of Abbreviations...... xiii

1. Background ………………………………………………………...... 1

1.1 Oligosaccharide Synthesis…………...... 1

1.2 Glycosylation Method and Thioglycosides...... 1

1.3 Reactivity-based Chemoselective Glycosylation...... 2

1.4 Reactivity Tuning of Thio-arylglycosides with Para-substituted Aglycons...... 3

1.5 Using Varyingly Substituted Thio-arylglycosides System as a Tool for

Mechanistic Investigation of Glycosylation...... 5

2. Results and Discussion ...... 7

2.1 Preparation of Glycosyl Donors...... 7

2.2 Determination of the Rate of Glycosylation by NMR...... 11

2.3 Determination of Relative Reactivity Values (RRVs)...... ……...... 13

2.4 Results of the Relative Reactivity Studies...... 16

2.5 Prediction of RRVs…………………………………………………...... 18

2.5.1 Prediction of Donor RRVs by Hammett Equation and Correlation of

1 H-NMR Anomeric Proton Chemical Shifts with σp

Values………………………………………...... 18

2.5.2 A Computational Approach to Prediction of RRVs……...... 20

2.6 Effects of Acceptor and Other Conditions on RRVs………………...... 27

2.6.1 Reaction Time and Quantity of Promoter Used…………...... 27

2.6.2 Effect of Different Promoters on RRVs…………………...... 28

2.6.3 Effect of Saccharide Acceptors on RRVs………………...... 29

2.6.4 Solvent Effect on RRVs…………………………………...... 32

2.7 An Approach to Mechanistic Investigation on Glycosylation with RRVs Using

Linear Free Energy Relationship...... 36

3. Conclusion …………………………………………………………...... 39

Experimental Section ...……….……………………………………...... 40

References ………………………………………………………………...... 56

Appendix: Spectral Data……………………………………………...... 66

vii

LIST OF FIGURES

1. Background

Figure 1. Blood Group O Antigen...... 1

2. Results and Discussion

Figure 2. Changes of a) donor concentration, b) reaction rate vs time; and c)

reaction rate vs donor fraction conversion as measured by 1H-NMR...... 12

Figure 3. HPLC Competitive Assay...... 14

Figure 4. HPLC chromatograms a) before and b) after competitive glycosylation

between donors 3d and 3e...... 15

Figure 5. Hammett plots of log(RRV) of the three donor series vs σp...... 17

1 Figure 6. Linear correlation of σp value and H chemical shift of anomeric

proton...... 19

Figure 7. Correlation of log(RRV) of GlcN donor series vs calculated Mulliken

charges...... 22

Figure 8. Correlation of log(RRV) of GlcN donor series vs calculated bonding

energies (simplified model)...... 24

Figure 9. Correlation of log(RRV) of GlcN donor series vs calculated bonding

energies (with glycon part involved)...... 26

viii

Figure 10. Different acceptors’ effect on Hammett plot of Gal donor series...... 30

Figure 11. HPLC chromatograms a) before and b) after competitive glycosylation

between donors 1e and 1d by using A2 acceptor...... 31

Figure 12. Solvent effect on Hammett plot of Gal donor series...... 33,34

Figure 13. Neighboring group participation leads to smaller absolute values for GlcN

donor series...... 38

LIST OF TABLES

2. Results and Discussion

Table 1. Measured relative reactivities of GlcN donor series with methanol as the

acceptor...... 16

Table 2. Relative Reactivity Values (RRVs) of three series of donors measured

with methanol as the acceptor...... 17

Table 3. Comparison between calculated and experimental log(RRV) values...... 19

Table 4. Chemical shifts of anomeric protons of four series of donors in CDCl3...20

Table 5. Mulliken charges on sulfur atoms of GlcN donor series...... 22

Table 6. S-E bonding energies of GlcN donor series...... 26

Table 7. Relative reactivities of donors 1a-1e using A1 and A2 as acceptors...... 29

Table 8. Relative reactivities of donors 1a-1e using A1 and A2 as acceptors in

Et2O...... 33

LIST OF SCHEMES

1. Background

Scheme 1. Glycosylation between two monosaccharide units...... 2

Scheme 2. Glycosylation of thioglycoside activated by NIS...... 2

Scheme 3. Reactivity-based chemoselective glycosylation...... 3

Scheme 4. Reactivity-based one-pot synthesis by varying thioarylglycosyl donors.....4

2. Results and Discussion

Scheme 5. Post-synthetic aglycon modification of substituted aryl-thioglycosides.....7

Scheme 6. Synthesis of thio-galactose (Gal) donor 1a-1e...... 8

Scheme 7. Synthesis of thio-glucosamine (GlcN) donor 2a-2e...... 9

Scheme 8. Synthesis of aminophenyl glucoside 3f...... 9

Scheme 9. Synthesis of thio-glucose (GlcBn) donors 3a-3e...... 10

Scheme 10. Product and by-product from benzylation of compound 5...... 10

Scheme 11. Saccharide acceptors...... 11

Scheme 12. The observed induction period can be explained by the generation of

iodonium ion for donor activation...... 13

Scheme 13. Calculation scheme and model molecules...... 21

Scheme 14. Calculation scheme and model molecules (simplified model)...... 23

Scheme 15. Bonding energy calculation within the simplified model series...... 24

Scheme 16. A simplified optimization scheme by using Z-matrix...... 25

Scheme 17. Evaluation of reaction time and quantity of promoter used...... 27

Scheme 18. Similar activation process induce by different promoters...... 28

Scheme 19. High yield glycosylation with methanol under different conditions...... 31

Scheme 20. An example of reactivity-based disaccharide synthesis...... 32

Scheme 21. An example of solvent effect on reactivity-based chemoselective

glycosylation...... 34

Scheme 22. General mechanism for glycosylation of thioglycosides...... 37

xii

LIST OF ABBREVIATIONS

Ac Acetate

Bn Benzyl

Bz Benzoyl

DMTST Dimethyl(MethylThio)Sulfonium Triflate

HPLC High Performance Liquid Chromatography

HF Hartree Fock

LG Leaving Group

Me Methyl

NIS N-Iodosuccinimide

NMR Nuclear Magnetic Resonance

NPhth N-Phthalimido

PES Potential Energy Surface

PG Protecting Group

RRV Relative Reactivity Value

RDS Rate Determining Step

TBDPS tert-butyl-diphenyl-silyl

p-TsOH para-Toluene sulfonic acid

xiii

1. Background

1.1 Oligosaccharide Synthesis

An oligosaccharide is referred to as a saccharide polymer containing a small

number (2 ~ 8) of monosaccharide units. An example is human blood group O-Antigen

(Figure 1). In oligosaccharide synthesis, usually the desired monosaccharide building blocks are prepared first and then linked together through a process called glycosylation

(Scheme 1).

Figure1.Blood Group O Antigen.

Glucosamine unit Fucose unit

OH HO OH OH OH OHO O O HO O O OR O NHAc OH

O OH OH HO Galactose unit

1.2 Glycosylation Method and Thioglycosides

Glycosylation is meant to have two separated saccharide units linked together via a glycosidic bond. This process is performed by a nucleophilic displacement of a leaving group (X) attached on the anomeric carbon of one saccharide moiety (donor) by an –OH group on the other saccharide unit (acceptor) or some other kind of acceptors (see

Scheme 1). Glycosylation can be triggered by adding a kind of chemical reagent

(promoter) to activate the donor. The new bond formed through glycosylation is called

glycosidic bond. There are many methodologies available for glycosylation. The main

difference among them is from the choices of different glycosyl building block’s leaving

groups and/or promoters. 1-11

Scheme 1. Glycosylation between two monosaccharide units. Disaccharide O Promoter O E O X O HO Y O O PGO PGO X PGO + Y + PGO O Donor Acceptor

Glycosidic Bond Activation Step Coupling Step

Thioglycoside donors have thioalkyl or thioaryl leaving groups attached at the

anomeric carbon. Thioglycosides are popular glycosyl donors with organic chemists,

because they can be easily prepared, are tolerate various reaction conditions, and are

stable for long period of time. Two normally used promoters with thioglycosides are N- iodosuccinimide/H+ (NIS/H+) and Dimethyl(methylthio)-sulfonium triflate (DMTST).50

The proposed mechanism of glycosylation using NIS is proposed in Scheme 2.

Scheme 2. Glycosylation of thioglycoside activated by NIS.

I H H O N O N O O + I

I O O O O O PGO S + L PGO HO Y PGO O Y Donor Oxacarbenium ion Acceptor

1.3 Reactivity-Based Chemoselective Glycosylation

Reactivity-based chemoselective glycosylation refers to the chemoselective

activation of two glycosyl donors with sufficiently different anomeric activities.12-21 The

more reactive donor will be activated preferably and glycosylate the less reactive donor

which has a built in acceptor site. The leaving group on the less reactive donor will

remain within the product and can be utilized for further glycosylation. For example, in

Scheme 3, both building blocks A and B with the thio-leaving groups undergo the same

promoter activation. If anomeric reactivities of A and B can be tuned properly through

protective group manipulation and/or algycon adjustment, which leads to a highly

reactive A compared to B, the desired disaacharide product can be produced (Scheme 3a).

In order to achieve high yield in this process, the side reactions like in Scheme 3b should be avoided and that requires the reactivity of donor B much lower than that of donor A.

Scheme 3. Reactivity-based chemoselective glycosylation a) Major reaction pathway:

O O A: S A: LA Promoter O O O S Further Synthesis LB B: O Solvent B: O HO S HO S Non-reducing End LB LB

b) One competitive reaction pathway:

B: O B: O HO S HO LB Promoter O O HO O S L B: O B: O B S Solvent HO HO S major by-product from self-coupling reaction LB LB

note: L represents aryl or arkyl groups

The concept of reactivity-based chemoselective glycosylation is not aimed at the

regio-selective control of just a single step glycosylation but at the automated design of a

rapid, multi-step, one-pot synthetic protocol for oligosaccharide synthesis.19

1.4 Reactivity Tuning of Thio-arylglycosides with Para-substituted Aglycons

Leaving group has an important effect on anomeric reactivities, either through electronic effect21-23 or through steric hinderance24. A reactivity based one-pot method utilizing thioaryl glycosyl building blocks differing only in para-substituents of aglycon has been reported.25 Different anomeric reactivities of glycosyl donors are easily achieved by simple post-synthetic modification of the aglycon substituents. This method allows preparing building blocks with different anomeric reactivities from a common precursor through simple transformations. It has been demonstrated that the anomeric reactivities are sufficiently differentiated with this sole structural distinction, which allows us to perform multi-step one-pot synthesis as the example shown in Scheme 4.

The aglycon modification was able to result in similarity of structures but variety of anomeric reactivities between building blocks, and thus it provides us a useful tool to probe glycosylation reactions.

Scheme 4. Reactivity-based one-pot synthesis by varying thioarylglycosyl donors.

BzO OH O BzO S BzO OTBDPS OH BzO 1a' OBz NO NIS, TfOH in CH2Cl2 2 O + O BzO S BzO S ο ο ο ο -70 C to -50 C NIS, TfOH in CH2Cl2 -70 C to -50 C OBz OMe OBz Br 1e 1b'

BzO OTBDPS OH O BzO O BzO O 1 BzO BzO OBz BzO O 1g OMe BzO O BzO OBz p-TolSCl, AgOTf, -50 οC O 39% overall BzO O OBz yield in one-pot BzO O BzO BzO OMe

1.5 Using Varyingly Substituted Thio-arylglycosides System as a Tool for

Mechanistic Investigation of Glycosylation

Most of glycosylation reactions were believed to proceed through the generation

of oxacarbenium ion intermediates followed by nucleophilic attack of the acceptor as

shown in Scheme 1.26-29 The glycosyl oxacarbenium ions have been obtained in the gas

phase,30 and they have not been isolated in solutions30-35 due to their highly reactive

nature. The lifetime of these reactive species in solution are estimated to be around 10-12 seconds.36-38 However, the existence of oxacarbenium ions has been inferred from either glycoside product distribution or kinetic isotope effect.34,36,39,40 We report here the

behavior of oxacarbenium ions can be evaluated through the kinetic study of

glycosylations of series of para-substituted thio-arylglycosides.

The transient nature of oxacarbenium ions make people assume the generation of

these species to be the Rate Determining Step (RDS) of the overall glycosylation

reaction.19,36,39,41 It has been found that more electron withdrawing protective groups on

the glycon ring were able to lead to lower rate of glycosylation,3,42-44 which could be

rationalized as the positively charged oxacarbenium ion becomes more difficult to form.

To make another approach to studying of RDS in glycosylation, we investigate here on

the electronegativity effect of aglycon using our para-substituted thio-arylglycosides

system.

The understanding of RDS in glycosylation will aid in the design of building

blocks with suitable reactivities. In practice, reactivities of various donors can be

quantitatively measured by their Relative Reactivity Values (RRVs). A database is

established with RRVs of thiotolyl building blocks containing various protective groups

tabulated.3,19 The effect of acceptor on RRVs was not taken into consideration in

constructing the database, since excess of methanol was used as the model acceptor. The

idea behind this data base is the reactivity-based chemoselective glycosylation, which has

achieved great success, as witnessed by the assembly of many complex

oligosaccharides.3,13,15,19,45-47 However, the yields for glycosylation are often lower than those predicted based on RRV differences. That inspires us to evaluate on those effects, such as reaction time, usage of different acceptors, promoters and solvents, etc, which could change the RRVs from those measured under standard conditions. We also want to point out that the RRVs can be predicted by using σp values of the substitutes in the

Hammett plots.

2. Results and Discussion

2.1 Preparation of Glycosyl Donors

As mentioned before, an effective synthetic procedure was designed by aglycon

adjustment through modification of substituted aryl-thioglycosides.25 The donors were

prepared from p-nitrophenylthioglycosides in this method. The nitro group on the aglycon can then be converted to different “Y” substituents (Scheme 5) through an

aminophenyl glycoside intermediate. The modification has been proved to give great

effect on tuning donor’s reactivity.25

Scheme 5. Post-synthetic aglycon modification of substituted aryl-thioglycosides.

O O O PGO S PGO S PGO S NO2 NH2 Y

Aminophenyl Glycoside Intermediate Y=OMe, NHAc, Br, N3, NPhth, etc

BzO OTBDPS OBn OBn O HO O BnO O BzO S BnO S BnO S OBz Y NPhth Y OBn Y

1a: Y = NO2 2a: Y = NO2 3a: Y = NO2 1b: Y = Br 2b: Y = Br 3b: Y = Br 1c: Y = N3 2c: Y = N3 3c: Y = N3 1d: Y = NHAc 2d: Y = NHAc 3d: Y = NHAc 1e: Y = OMe 2e: Y = OMe 3e: Y = OMe 1f: Y = NH2 2f: Y = NH2 3f: Y = NH2

We have picked up 1a-1e, 2a-2e and 3a-3e for our investigation (Scheme 5).

These three series of donors are selected to represent thioglycoside donors encountered in typical glycosylation reactions. Thiogalactosides 1a-1e (Gal series) contain multiple

electron withdrawing protective groups (OBz) on the glycon ring with a participating

neighboring group (OBz) on C2. Thioglucosamine series 2a-2e (GlcN series) bear a

participating neighboring group (NPhth) on C2 and several electron donating groups

(OBn), while glucosides 3a-3e (GlcBn series) only have electron donating protective

groups (OBn) but no participating neighboring groups on C2. 1a-1e and 2a-2e are

prepared as described previously through post-synthetic modifications of aglycons

(Scheme 6 and Scheme 7).25 Instead of spending five synthetic steps on average to synthesize each donor starting from individual aryl thiol, it only took one to two steps to acquire building blocks 1b-1e and 2b-2e divergently from common synthetic intermediates 1f and 2f.

Scheme 6. Synthesis of thio-galactose (Gal) donors 1a - 1e.

OTBDMS AcO OAc AcO OAc HO O2NSH O O 1) NaOMe/MeOH O AcO OAc AcO S HO S 2) TBDMSCl in Pyridine OH OAc OAc NO2 MS AW-300 NO2 1-1 1-3 100% SnCl4/DCM 1-2 80~90%

BzO OH BzO OTBDPS 1) BzCl in Pyridine O TBDPSCl in DMF O BzO S BzO S 2) HCl in MeOH OBz OBz NO2 NO2 1-4 80% 1a 95%

SnCl2 in EtOH

BzO OTBDPS BzO OTBDPS BzO OTBDPS t-Butyl nitrite 1) HBF4, NaNO2 in THF-H2O O then CuBr O O BzO S BzO S BzO S 2) O2, p-TsOH in MeOH OBz OBz ο Br NH2 then sealed at 75 C OBz OMe 1b 66% 1f 65~72% 1e 74%

CH3COOH/THF, NaNO2 Ac2O in MeOH then NaN3

BzO OTBDPS BzO OTBDPS O O BzO S BzO S OBz OBz NHAc N3 1c 87% 1d 96%

Scheme 7. Synthesis of thio-glucosamine (GlcN) donors 2a - 2e.

OH 1) NaOMe in MeOH; OAc O2NSH OAc HO O then Phthalic anhydride AcO O AcO O HO OH AcO OAc AcO S + - 2) Ac2O in Pyridine MS AW-300 NPhth NH3 Cl NPhth NO2 2-1 2-2 60~80% SnCl4/CH2Cl2 2-3 71~87%

OBn 1) NaOMe/MeOH Ph O 1) NaH in DMF then BnBr O O HO O 2) Benzaldehyde HO S 2) NaCNBH /THF BnO S 3 NPhth -dimethyl acetal NPhth NO NO2 2 then 2M HCl in Et2O CSA/Toluene 2-4 65~89% 2a 65~88%

OBn OBn SnCl in EtOH 1) HBF , NaNO in THF-H O 2 HO O 4 2 2 HO O BnO S BnO S 2) O2, p-TsOH in MeOH NPhth NH NPhth OMe 2 ο 2f 60~71% then sealed at 75 C 2e 70~78%

Ac O in MeOH t-butyl nitrite CH3COOH/THF, NaNO2 2 then CuBr then NaN3 OBn OBn OBn HO O HO O HO O BnO S BnO S BnO S NPhth NPhth NPhth NHAc Br N3 2b 66~68% 2c 80~87% 2e 100%

3a-3e (GlcBn series) were initially prepared by the same synthetic routine as the

Gal and GlcN series, but the aminophenyl glucoside 3f was found to be very prone to air oxidation (Scheme 8), which could not be practically used for further syntheses. The four electron-donating benzyl groups on glycon ring are responsible for the highly reactive nature of compound 3f.

Scheme 8. Synthesis of aminophenyl glucoside 3f.

OAc OAc O2NSH AcO O AcO O 1) NaOMe/MeOH AcO OAc AcO S OAc OAc MS AW-300 NO2 2) NaH, DMF; 4 then BnBr SnCl4/DCM 4a 87%

OBn OBn 1) NaOMe/MeOH BnO O BnO O BnO S BnO S 2) NaH, DMF; OBn OBn NO NH2 2 then BnBr 3a 45% 3f: prone to oxidation (color change to purple)

Due to the simpler protective group patterns on glucose series donor 3a-3e, we

performed the synthesis from glucose penta-acetate 4 and the corresponding thiophenols

for 3a,48 3b and 3e (Scheme 9). Tin (IV) chloride mediated glycosylation produced

thioglycosides 4a, 4b and 4e30,49 in excellent yields. Replacement of the four acetates on

the glycon ring with benzyl groups was accomplished by treatment of 4a, 4b and 4e with

NaOMe followed by benzylation. The yield for formation of 3a was lower due to competitive reduction of the nitro moiety by NaH (Scheme 10).

Scheme 9. Synthesis of thio-glucose (Glu) donors 3a - 3e.

a) OAc YSH OAc OBn 1) NaOMe/MeOH AcO O AcO O BnO O AcO OAc AcO S BnO S OAc 2) NaH, DMF; MS AW-300 OAc Y OBn Y 4 SnCl /DCM then BnBr 4 4a: Y = NO 87% 2 3a: Y = NO2 45% 4b: Y = Br 90% 3b: Y = Br 92% 4e: Y = OMe 87% 3e: Y = OMe 97% b) OBn SnCl2/EtOH; then 3a BnO O Ac2O BnO S 84% OBn NHAc 3d

c) 1) NaOMe/MeOH OBn 1) SnCl2/EtOH OAc 4a 2) NaNO2,NaN3 AcO O BnO O AcO S 2) NaH, DMF; BnO S CH3COOH/THF OAc then BnBr OBn N N3 3 59% for 2 steps 4c 78% for 2 steps 3c

Scheme 10. Product and by-product from benzylation of compound 5.

OH NaH/DMF OBn HO O then BnBr BnO O HO S BnO S + A complex by-product through OH OBn reduction of the nitro group NO2 NO2 5 3a desired product

Initial attempts to synthesize acetamido substituted donor 3d from 4-acetamido

thiophenol and glucoside 4 failed due to complexation of tin (IV) chloride by the

acetamido moiety. Instead, a one-pot procedure of stannous chloride reduction of 3a

followed by immediate acetylation in the same reaction flask led to donor 3d in 84%

yield (Scheme 9b). Stannous chloride reduction of 4-nitrophenyl glucoside 4a with

subsequent diazonium salt formation and in situ displacement by sodium azide produced

the azido bearing glucoside 4c in 59% yield for the two steps. Removal of acetates

followed by benzylation gave donor 3c in 78% yield (Scheme 9c).

Two saccharide acceptors: A1 and A2 are used to evaluate the effect of different acceptors on measurement of RRVs. A2 is synthesized directly from 2a via glycosylation with 1-bromo-3-propanol as shown in Scheme 11. The primary hydroxyl group in 1- bromo-3-propanol is much reactive than the secondary hydroxyl group on 2a. When excess of 1-bromo-3-propanol was used, glycosylation of 2a with it was achieved in good yield with little self couplings of donor.

Scheme 11. Saccharide acceptors

O OH OBn O HO O O BnO O Br O NPhth O A1 A2

OBn OBn HO Br HO O HO O BnO O Br BnO S NPhth NPhth NIS, TfOH in Et2O/CH2Cl2 NO2 2a A2 78%

2.2 Determination of the Rate of Glycosylation by NMR

We first investigated the possibility of real time monitoring of glycosylation

reaction by NMR. The reaction of 1b and methanol with N-iodosuccinimide (NIS) and

catalytic amount of trifluoromethane sulfonic acid (TfOH) as the promoter system50 was performed at room temperature in the NMR tube. After the reaction was initiated, the

NMR tube was put in a 600 MHz NMR spectrometer immediately and then scanned every 8 seconds. The signal within each 8 seconds was just averaged to give a spectrum representing of the reaction mixture during that reaction period. As the reaction proceeded, the amount of donor decreased as determined from integration of the anomeric proton (Figure 2a).

Figure 2. Changes of a) donor concentration, b) reaction rate vs time; and c) reaction rate vs donor fraction 1 conversion0 003 500630089 00as500 measured by H-NMR. a) b) 0.04 8 0.03 6

4 0.02

2 0.01 Reaction Rate (mM/s) Rate Reaction

Donor Concentration (mM) 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (seconds) Time (seconds) 000050 0 000300005 5000600008 5003003500 0.04 BzO OTBDPS O BzO S 0.03 OBz Br Donor: 1b [8.54 mM] 0.02 Acceptor: CH3OH [12.31 mM] Promoter: NIS/Acetonitrile-D3 [10.89 mM] 0.01

Reaction Rate (mM/s) Rate Reaction TfOH [1.068 mM]

0 Solvent: CDCl3 0 0.2 0.4 0.6 0.8 Fraction conversion Reaction Conditions

Interestingly, the rate of reaction as calculated from the speed of donor

consumption rose during the first few minutes of the reaction before decreasing with time

in the manner expected for a reaction exhibiting positive-order kinetics in substrate

concentration (Figure 2b). The presence of such an induction period at the onset of the

reaction51 could be attributed to the reaction of NIS with TfOH to generate iodonium ion,

which is the active form of promoter (Scheme 12). As the glycosylation progressed

passing the induction period, the observed pseudo-linear decrease of the reaction rate

indicated that glycosylation is apparent first order to donor under this condition (Figure

2b, c).

Scheme 12. The observed induction period can be explained by the generation of iodonium ion for donor activation.

O I H N O F C S OH N O O 3 O O F3C S O I O O

I thioglycoside O + O SR I S activation R

However, it was difficult to accurately determine the absolute rate via this method

for the following reasons: (1) It is laborious to make a series of measurements on NMR,

which needs calibration of internal reference and integration of each NMR spectrum

(usually during reaction the baseline noises become bigger and thus make the accurate

integration of such spectra very difficult); (2) Subtle variation of the reaction condition could cause substantial rate fluctuation and thus gives inconsistency among individual measurement of reaction.

2.3 Determination of Relative Reactivity Values (RRVs)

Due to the difficulties in measuring the absolute reaction rate by NMR, we

adapted a competitive glycosylation condition to measure the relative glycosylation rates: two different glycosyl donors are added in the same reaction flask with methanol as the acceptor.19,20,23,52 Upon addition of a limiting amount of the promoter, the two donors

compete for promoter activation. The consumption of these two donors can be

quantitatively measured on HPLC chromatogram (see Figure 3).

Figure 3. HPLC Competitive Assay.

Signal (mAU) OAc AcO O Donor 2 AcO OAc Donor 1 NPhth 80 Internal reference 60 Internal O Reference Donor 1: O PG O S OCH3 1 L1 PG1O 40 Promoter/Solvent Before Donor 2: O CH3OH O 20 S OCH3 Reaction PG O PG2O 2 L2 0 After note: L represents the aryl- or arkyl groups Reaction

0 51015 Retention Time (s)

From the result of NMR monitoring glycosylation, the glycosylation process is

apparent first order to donor concentration under the experimental condition (see Figure

2). The rate of glycosylation can be described by kobs[D], where kobs is the apparent first

order rate constant and [D] is the donor concentration. Thus, the relative reactivity

between these two donors in the competitive assay can be calculated according to

equation (1) ([R] is the concentration of an internal reference; 0 is designated as the

starting time and t is designated as the finishing time). Donors’ concentration in the

equation was quantified from chromatogram integration following HPLC separation (a

HPLC spectrum is shown in Figure 4).19,23 An internal reference R inert to glycosylation

is added to the reaction mixture in order to normalize the amounts of compounds between

different experiments and to minimize the errors caused by injected sample volume

changes.

[D1]t [R]0 k log( ) − log([D1]0 ) obs1 = [R]t (1) [D2]t [R]0 kobs2 log( ) − log([D2] ) [R]t 0

Figure 4. HPLC chromatograms a) before and b) after competitive glycosylation between donors 3d and 3e (1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-α- D-glucopyranoside was used as the reference for this experiment).

Before Glycosylation After Glycosylation

Donor 3d Donor 3d Reference Donor 3e Donor 3e Reference

3e 3d Reference Before reaction 6078.54 49870.2 5544.6 log([3e]t [R]0 ) − log([3e] ) k3e [R]t 0 After reaction = = 1.23 3804.96 35515 6887.77 [3d ]t [R]0 k3d log( ) − log([3d]0 ) Note: Peak areas from HPLC spectra (unit: mAU*s) [R]t

In all cases, baseline separation of desired peaks in HPLC chromatogram is achieved. The reactivity differentials between the two competing donors are kept below

20 fold to ensure accuracy of the experiment. When greater than 20 folds of donor reactivity differential was observed or baseline separation fails, a third donor with intermediate anomeric reactivity was used to bridge the two donors of interest. For each pair of donors, the reaction was repeated at least three times. Usually, less than 10% differential is obtained among all measurements, with the average value taken as the

relative reactivity value between the two donors, an example of which is shown in Table

Table 1. Measured relative reactivities of GlcN Donor Series 2a-2e with methanol as the acceptor Entry Donor pair (more Experiment Experiment Experiment Average number reactive/less reactive) #1 #2 #3 value 1 4g/2a 4.49 4.58 4.37 4.49 2 2b/4g 7.06 7.04 7.51 7.21 3 2d/2b 5.27 5.32 4.95 5.18 4 2d/2c 3.97 3.35 3.98 3.74 5 2e/2d 1.88 1.89 1.93 1.90

AcO AcO OAc AcO O AcO O AcO S AcO 4g is used to bridge the donors 2a and 2b; OAc S reactivity of donor 6 was arbitrarily set as 1 4g 6

2.4 The Results of the Relative Reactivity Studies

With the experimental conditions established (see experimental section), the

Relative Reactivity Values (RRVs) for three series of donors (Gal 1a-1e, GlcN 2a-2e,

GlcBn 3a-3e) were obtained using MeOH as the acceptor, with the RRV of thiomannoside 6 set as 119 (Table 2). Although donors 2a-2e contain a free hydroxyl group, due to the much higher reactivity of methanol and excess methanol used,53 glycosylation of 2a-2e with methanol was achieved with little self couplings of donors. It is evident that the para-substituents on the aglycon of thioaryl glycoside donors significantly impact anomeric reactivities, with over three orders of magnitude difference obtained by a single substitution (3e vs 3a). This effect is much larger than that resulting from changing one protective group on the glycon ring, which is generally less than 10 fold.3 The much larger influence of aglycon on anomeric reactivity is due to the direct conjugation of para-substituents with the anomeric sulfur atom.

Table 2. Relative Reactivity Values (RRVs) of three series of donors (Gal 1a-1e, GlcN

2a-2e, GlcBn 3a-3e) measured with methanol as the acceptor (Reactivity of thiomannoside 6 was set as 1).

Y NO2 Br N3 NHAc OMe BzO OTBDPS 1a - 1e 0.042 (1a) 0.89 (1b) 3.12 (1c) 18.1 (1d) 21.7 (1e) O BzO S OBz Y OBn 2a - 2e 0.601 (2a) 19.2 (2b) 27.1 (2c) 101 (2d) 191 (2e) HO O BnO S NPhth Y OBn 3a - 3e 3.19 (3a) 161 (3b) 657 (3c) 2482 (3d) 3772 (3e) BnO O BnO S OBn Y

Hammett analyses have been very useful to provide insights into glycosylase mechanisms.54-60 For our glycosylations, Hammett plots were constructed by plotting log(RRV) values of the three series of donors against σp values of the para- substituents61,62 (Figure 5).

Figure 5. Hammett plots of log(RRV) vs σp using methanol as the acceptor

4 GlcBn 3a-3e ρ = - 3.4, R2 = 0.99 GlcN 2a-2e ρ = - 2.7, R2 = 0.98

2 logRRV 0 Gal 1a-1e ρ = - 3.0, R2 = 0.96

- 2

- 0.3 0.1 0.5 0.9 σp

Excellent linear correlations were observed within each donor series, suggesting

that the same glycosylation mechanism is operative over a large range of reactivities.58,63

Furthermore, the slopes of the Hammett plots (ρ values) were all negative indicating that the reactive intermediates are cationic in nature and electron donating substituents such as methoxy expedite the reaction compared with electron withdrawing substituents such as nitro.

2.5 Prediction of RRVs

A database is established with RRVs of thiotolyl building blocks containing various protective groups tabulated.3,19 In order to do that, a large number of

thioglycoside building blocks should be synthesized and tested for their reactivities.

Building block preparation is a time-consuming process and it requires much experience

to design and fine tune the protective groups in order to achieve exact anomeric reactivities.3,14,19 It will be highly desirable if reactivities of the building blocks can be

predicted prior to the actual synthesis.

2.5.1 Prediction of Donor RRVs by Hammett Equation and Correlation of 1H-

NMR Anomeric Proton Chemical Shifts with σp Values

With the excellent linear correlations between log(RRV) and σp and large number

61 of σp values available, σp can be employed as a predictor of RRV. For example,

log(RRV) of galactoside 1g is calculated to be 1.38 from the Hammett plot (Figure 5),

while the experimentally determined value is 1.28 (σp = -0.14 for methyl group). The

same is applicable to thioglycosides 2g and 3g (Table 3).

Table 3. Comparison between calculated and experimental log(RRV) values

Donor Calculated Experimental

log(RRV) value log(RRV) value

BzO OTBDPS 1g O 1.38 1.28 BzO S OBz

OBn 2g HO O 2.28 2.41 BnO S NPhth

OBn 3g BnO O 3.68 3.42 BnO S OBn

Since the only structural differences among our donor series are in their aglycon

substituents, we envision that the chemical shifts of their anomeric protons should only

be affected by the electronic properties of the para-substituents on aglycons. Excellent

linear correlations between the chemical shifts of the anomeric protons (Table 4) and σp values were observed in all four donor series (Figure 6), which corroborates with previous observations that chemical shifts can be used to predict relative reactivities.19,20,25

Figure 6. Linear correlation of σp value and 1H chemical shift of anomeric proton

R2 = 0.99 5.6 GlcN 2a - 2f

5.2 (ppm) 2 δ R = 0.95 Gal 1a - 1f R2 = 0.98 2 4.8 GlcAc R = 0.99 5 - 9, 15 GlcBn 3a - 3f

4.4 - 0.4 0 0.4 0.8 σp

Table 4. Chemical shifts of anomeric protons of three series of donors (Gal 1a-1f, GlcN

2a-2f, GlcBn 3a-3e) in CDCl3.

Y NO2 Br N3 NHAc OMe NH2

BzO OTBDPS 1a - 1f 5.09 4.90 4.88 4.90 4.82 4.78 O BzO S OBz Y (1a) (1b) (1c) (1d) (1e) (1f)

OBn 2a - 2f 5.69 5.50 5.47 5.46 5.40 5.35 HO O BnO S NPhth Y (2a) (2b) (2c) (2d) (2e) (2f)

OBn 3a - 3e 4.82 4.62 4.56 4.57 4.53 4.47 BnO O BnO S OBn Y (3a) (3b) (3c) (3d) (3e) (3f)

OAc 4.88 4.65 4.62 4.59 4.54 4.51 AcO O AcO S OAc Y (4a) (4b) (4c) (4d) (4e) (4f)

2.5.2 A Computational Approach to Prediction of RRVs

Anomeric reactivities have been estimated by calculating the energy difference between the ground state and the oxacarbenium ion.41 With the large number of atoms in typical fully protected glycosyl donors, global minima of the structures can be difficult to obtain.64,65 Furthermore, the need to include solvation for accurate determination of oxacarbenium ion energy renders this approaches challenging.41 We plan to explore a calculation method which can indicate the RRVs without laborious synthetic work.

With the intuition that the electron density on sulfur atom may affect the efficiency of electrophilic attack by promoter, we first attempt to calculate the Mulliken charge on the sulfur atom of the donor. The calculation process is shown in Scheme 13.

Scheme 13. Calculation scheme and model molecules

MacroModel Gaussian 03 input Monte Carlo Multiple Minimum (MCMM) HF 6-31G* Optimization Force Field: MM2, MM3 and OPLS_2005 HF 6-31+G* Single Point

Y OBn NO2 HO O BnO S Br NPhth Y N3 NHAc Glc-series OMe

We first optimized the molecular conformation using the software package

MacroModel® with Monte Carlo Multiple Minimum (MCMM) method (in Scheme 13).

The molecular conformation was then further optimized using Gaussian03® with HF 6-

31G* basis set. After optimization, the Mulliken charge was given under a single point

calculation with HF 6-31+G* basis set.

The optimization was performed on two Linux desktop computers (P4

3.6GHz/2GB). For each donor in GlcN-series, the calculation in Scheme 13 took roughly

2~4 weeks. Due to the large degree of freedom for the optimization of GlcN molecules,

the optimization routine could only survey a limited portion of the Potential Energy

Surface (PES) and the global minimum thus was difficult to obtain. We took the criteria

that the final optimized conformations within the same series should be roughly the same

except at the substituent site. Thus we tuned the input structures iteratively for Gaussian

03, until the output structures presented certain consistency within this series. The

calculation results are shown in Table 5.

Table 5. Mulliken charges on sulfur atoms of GlcN donor series 2a-2e.

Y NO2 Br N3 NHAc OMe

OBn 2a - 2e HO O 0.1933 0.1801 0.1740 0.1785 0.1664 BnO S NPhth Y (2a) (2b) (2c) (2d) (2e)

A good linear correlation between the Mulliken charges of (2a, 2b, 2c, 2e) and their log(RRVs) was observed within this donor series (Figure 7), but donor 2d was

found an exception from the linear relationship. The difference of the Mulliken charge

between donor 2c and 2d is about 1 fold, which is inaccurate to represent the relative

reactivity between them (about 4 fold).

Figure 7. Correlation of log(RRV) of GlcN donor series vs calculated Mulliken charges

3 2d R2=0.84

0 0.165 0.17 0.18 0.19 Mulliken Charge on "S"

The Mulliken charge can qualitatively predict the RRVs within a donor series but is not good enough for quantitative prediction. We also found the Mulliken charge could

vary dramatically from different input structures or calculating schemes (e.g. changing

the basis set). On the other hand, the energies calculated by Gaussian were relatively

consistent within different calculation trials under the calculation Scheme 13. We thus

want to find other prediction variables derived from energy calculation. From our kinetic

study (Scheme 13), glycosylation was initiated by an electrophilic attack of promoter (E+) on the sulfur atom of donor to generate the reactive intermediate. With more reactive donor used, the electrophilic attack should be easier to take place. It hints to us that we can use the S-E (sulfur-promoter) bonding energy to evaluate the easiness of forming the sulfonium intermediate, thus can further use it to predict the RRVs.

For a quick reveal of the method, we calculated the bonding energy based on a simplified model (M1) which used the methyl group to simulate the saccharide glycon part (see Scheme 14).

Scheme 14. Calculation scheme and model molecules (simplified model) Y NO S 2 H3C Br MCMM N Force Field: MM3 HF 6-31G* Opt--->single point Y 3 M1 NHAc OMe

The simplified molecules were optimized easily by Gaussian03 with consistent

output structures. The S-E bonding energies within these model molecules were calculated based on the optimized conformations. Br+ was chosen as promoter instead of

I+, which is unavailable from Gaussian03 package. The energies of Br+ (E0) and the

intermediates (E2) were calculated as shown in Scheme 15. The bonding energy of S-E

was calculated as the energy difference between E0+E1 and E2.

Scheme 15.Bonding energy calculation within the simplified model series

Bonding Energy = E0 +E1 -E2 E0 Y Br Br NO2 S E2 H3C Br S N3 H3C Y E1 NHAc Y OMe

To our delight, the bonding energy had a good linear correlation with the experimental log(RRV) (see Figure 8). Further investigation has to be performed in order to prove our hypothesis.

Figure 8. Correlation of log(RRV) of GlcN donor series vs calculated bonding energies (simplified model)

ρ =0.03, R2=0.99

OMe 2 Br NHAc

logRRV (GlcN) N3 1

NO2

0 20 40 60 80 S-E Bonding Energy Shift (kJ/mol) note: the RRV of 2a is set as 1 arbitrarily

The good correlation between the S-E bonding energy and log(RRV) encouraged us to include the saccharide glycon in the bonding energy calculation. If a good linear correlation can still hold after the saccharide glycon is involved, the glycon effect can thus be extensively evaluated by examining the magnitude of slope as in Figure 8. In

order to do that, we need to develop an efficient optimization process for relative large

molecules. A new optimization scheme thus is proposed in Scheme 16.

In the new optimization scheme, the protecting groups (PG) on the sugar ring and

latent leaving group (LG) on the aglycon will be optimized separately within their

simplified models. The output structure of each group will be saved in Z-matrix form by

using molecular internal coordinates. We suppose that the conformations of these functional groups (PG and LG) will not change too much after “connect” them to the whole donor molecules. Then their conformations will be frozen and only their relative positions will be optimized in the whole molecules. The degree of freedom for optimization thus can be reduced to a limited number, which makes the optimization faster and more consistent.

Scheme 16. A simplified optimization scheme by using Z-matrix

O LG: Leaving Group on aglycon PGO LG PG: Protecting Group on sugar ring donor to be optimized

save the struture of the sugar fragment optimize leaving group optimize protecting group with molecular internal coordinate - Zmatrix with simplified model O with simplified model PGO CH3 O file 1 H3CLG

save the optimized struture of PG save the optimized structure of LG in Zmatrix form in Zmatrix form file 2 file 3 freeze freeze structure structure

combine file 1, 2 and 3 to reform the struture of donor with PG and LG frozen Optimize O PGO LG

With the procedure in Scheme 16, the optimization went smoothly for the 2a-2e

molecules. For each donor, the optimization was achieved within one week and the conformations within the same series followed exactly the same pattern. The bonding

energies were then calculated in the same way as for the simplified model in Scheme 15,

and the result is shown in Table 6.

Table 6. S-E bonding energies of GlcN donor series 2a-2e. (unit: kJ/mol)

Y NO2 Br N3 NHAc OMe

OBn 2a - 2e Br 648.39 689.97 706.06 699.53 705.48 HO O BnO S NPhth Y (2a) (2b) (2c) (2d) (2e)

The bonding energies of 2a, 2b, 2d and 2e have good linear correlation with the donors’ RRVs (see Figure 9). The exception is donor 2c, which is aberrant from the linear relationship about 10kJ/mol.

Figure 9. Correlation of log(RRVs) of GlcN donor series vs calculated bonding energies (with glycon part involved)

3 2e 2d

log(RRVs) 2b 2

1 2a Donor 2c

0 640 660 680 700 S-E bonding energy (kJ/mol) note: the RRV of 2a is set as 1 arbitrarily

2.6 Effects of Acceptor and Other Conditions on RRVs

Wong and co-workers built up a reactivity data base for many thioglycosyl

building blocks.3,19 The standardized reaction condition mentioned in section 2.3 was

used to quantitatively assign a Relative Reactivity Value (RRV) to each assay building block. In practice, the reaction conditions are usually different from this standardized condition. The most significant change is that the saccharide acceptors will be used instead of methanol in most cases. Some other effects, such as reaction time, promoter and solvent, could also play a role in varying the magnitude of RRVs. Here we report our studies on these effects, which could change the reactivity of donors from the value scaled in the RRVsmethanol system.

2.6.1 Reaction Time and Quantity of Promoter Used

The reaction time did not affect the relative reactivity much. For example, when

donor 2a competed with donor 4g, k2a/k4g values of 4.80, 4.28 and 4.49 were obtained

when the reactions were quenched at 3, 5 and 120 minutes respectively. The quantity of

promoter used seemed to have some effect on the RRVs. When less promoter was used in reaction, the RRV between 2a and 4g became bigger (6.09). In conclusion, the reaction time dose not affect the RRV too much, and less promoter may lead to large RRV (see

Scheme 17).

Scheme 17. Evaluation of reaction time and quantity of promoter used.

OBn OAc HO O AcO O NIS, TfOH/CH Cl BnO S + AcO S 2 2 NPhth HPLC Competitive Assay NO2 OAc CH OH 2a 4g 3

1. stop reaction at different time: 2. use less promoter (0.6 e.q. NIS + 0.06 e.q. TfOH)

4g(left) 2a(left) reaction time RRV 4g(left) 2a(left) reaction time RRV >95% 100% 1min ~ 78% 96% 2hrs 6.09 73% 93% 3min 4.80 51% 84% 5min 4.28 34% 78% 2hrs 4.42

2.6.2 Effect of Different Promoters on RRVs

Next we examined the effect of promoter. The relative reactivities between donor

pair of 1b and 4g were measured with two promoter systems, NIS/TfOH and p-

TolSCl/AgOTf. The observed relative reactivities were similar using both systems (3.02

for NIS/TfOH and 2.86 for p-TolSCl/AgOTf). These results suggest that the relative

reactivities are independent of promoter identity, which is consistent with the

observations by Wong and coworkers.19 The consistency of RRVs when different

promoters are used can be rationalized by their similar mechanistic functionality to

activate the donor for glycosylation. The mechanisms of glycosylations under three kinds

of promoters are shown as Scheme 18a-c, their common functionality is as Scheme 18d.

Due to the same effect of promoters on RRVs, we can use the stronger promoter

p-TolSCl/AgOTf when glycosylation using NIS/TfOH was sluggish.

Scheme 18. Similar activation process induced by different promoters a) N-Iodosuccinimide (NIS), TfOH

O I H N O F C S OH N O O 3 O O F3C S O I O O I O O O SR I+ S PGO R b) (dimethylthio)-methylsulfonium triflate (DMTST)

S O O + S O SR + SS S PGO R OTf OTf c) p-TolSCl/AgOTf

SCl +Ag OTf S OTf + AgCl

S O O O SR S OTf S + OTf R PGO

d) A general mechanism

+ E O promoter E O O PGO S PGO S PGO Y Y

2.6.3 Effect of Saccharide Acceptors on RRVs

Prior competitive glycosylation studies have been mainly focused on systematic

investigations of the effect of donor structural change on glycosylation.3,20,23 In order to

examine the effect of acceptor on our glycosylation, instead of using methanol as the

acceptor as described above, we performed competitive glycosylation reactions of Gal

series donors 1a-1e using galactoside A1 and glucosamine A2 respectively as

representatives of primary and secondary carbohydrate acceptors. The reactions of donor

1a with A1 or A2 were slow with NIS/TfOH as the promoter system. Instead, the usage of a stronger promoter p-TolSOTf, formed in situ through reaction of p-TolSCl and

AgOTf,66 expedited the reactions allowing us to determine the relative reactivities of

these donors (Table 5). Comparable reactivity range has been reported by Oscarson and

coworkers, when relative reactivities of a series of perbenzoylated thioglycosides containing various alkyl and aryl thiol aglycons were measured in glycosylation of a carbohydrate acceptor.23

Table 7. Relative reactivities of donors 1a-1e using A1 and A2 as acceptors (reactivity of donor 1a was set as 1)

Donor Y=NO2 (1a)Y=Br (1b) Y=N3 (1c)Y=NHAc (1d) Y=OMe (1e)

Methanol 1 21 74 428 515

Acceptor A1 1 12 72 177 234

Acceptor A2 1 6 12 51 62

Excellent linear correlation was obtained in the Hammett plot for reaction of

donors 1a-1e with acceptor A1 (ρ = – 2.66) (Figure 10). Interestingly, compared with

methanol acceptor for the same donor series, the range of relative donor reactivities is

much smaller (k1e/k1a 234 with acceptor A1 and 515 with methanol acceptor) and the

reaction rate is less sensitive to changes in para-substituents of the aglycon as reflected by

the smaller absolute ρ value (2.66 vs 3.01). Although acceptors are known to affect glycosylation regio-selectivity67 and yield,68,69 this is the first report that they drastically

impact relative reactivities of donors. This suggests RDS of the glycosylation reaction

cannot be prior to engagement of the acceptor.

Figure 10. Different acceptors' effect on Hammett plot of Gal donor series

Methanol: ρ =-3.01, R2=0.96 acceptor A1: ρ =-2.66, R2=0.96 log RRV 2.5 acceptor A2: ρ =-1.95, R2=0.93

1.5

0.5

0 0.2 0.4 0.6 0.8 σp

For glycosylations of a very unreactive acceptor A2 by donors 1a-1e, the same

general trend was observed that electron donating aglycon substituents facilitate the reaction but with smaller range of reactivity compared with that obtained using methanol acceptor (Table 7 and Figure 10).

However, after reaction, apparent amount of acceptor A2 left in HPLC chromatogram also indicates a lower yield could be expected by using this acceptor (see

Figure 11). The methanol acceptor, on the other hand, always gives excellent yield toward glycosylation based on our numerous tentative experiments (see Scheme 19 for some examples). When using saccharide acceptors in glycosylation, the decreased relative reactivity between two building blocks is not the solo factor accounting for lower yield. The nature of glycosylation also becomes less productive towards less efficient saccharide acceptors and glycosylation yields are thus expected to become less than those glycosylations with methanol acceptor.

Figure 11. HPLC chromatograms a) beforeandb)after competitive glycosylation between donors 1e and 1d with A2 acceptor

Donor 1e Donor 1d Donor 1d Donor 1e

Reference Reference Acceptor A2

Acceptor A2 usually has 10%~40% left

Scheme 19. High yield glycosylation with methanol under different conditions. a) OBn OBn DMTST/CH2Cl2 HO O HO O 93% BnO S CH OH BnO OCH3 NPhth 3 NO2 NPhth b) BzO OBz BzO OBz NIS, TfOH/CH2Cl2 O O 97% BzO S CH3OH BzO OCH3 OBz OBz c) BzO OTBDPS BzO OTBDPS p-TolSOTf/CH2Cl2 O O 94% BzO S BzO OCH CH3OH 3 OBz Br OBz

In the programmable reactivity based chemoselective glycosylation, RRVsmethanol are utilized as a quantitative measure to guide building block selection.3,13,15,19,45-47

However, glycosylation yields can be much lower than those indicated by RRVmethanol differentials.24,14 For example, although galactoside 10 is 13.7 fold more reactive than

glucosamine 11 based on RRVmethanol, the yield for disaccharide 12 is only 40% (Scheme

20).14 This is presumably because the relative reactivity differential between 10 and 11

in competitive glycosylation of the secondary acceptor 11 is much smaller than that

predicted based on RRVmethanol, leading to reduced chemoselectivity and low glycosylation yield. Therefore, in design of complex oligosaccharide synthesis, the

RRVmethanol differential of two selected building blocks need to be large enough to

accommodate decrease of selectivity, which can be more than one order of magnitude in glycosylating an unreactive carbohydrate acceptor.

Scheme 20. An example of reactivity-based disaccharide synthesis

BnO OBn OBn BnO OBn NIS, TfOH OBn O HO O O PMBO STol AcO STol 40% PMBO O O LevO TrocHN LevO AcO STol TrocHN 10 11 12 RRVmethanol = 4150 RRVmethanol = 302

2.6.4 Solvent Effect on RRVs

In order to evaluate the effect of different solvent on the relative reactivity values,

we performed the competitive glycosylation reactions of Gal series donors 1a-1e using galactoside A1 and glucosamine A2 as acceptors in diethyl ether and compared the results with those reactions performed in methylene chloride. (in Table 8)

Table 8. Relative reactivities of donors 1a-1e using A1 and A2 as acceptors in Et2O

(reactivity of donor 1a was set as 1)

Donor Y=NO2 (1a)Y=Br (1b) Y=N3 (1c)Y=NHAc (1d) Y=OMe (1e)

Acceptor A1 1 7.63 20.0 34.3 47.1

Acceptor A2 1 1.26 3.98 14.3 17.5

Excellent linear correlation was obtained in the Hammett plot for reaction of

donors 1a-1e with acceptor A1 in Et2O (ρ = – 1.85) (Figure 12). The linear correlation

failed to some extend with acceptor A2 in Et2O, which may be due to donor decomposition competing with glycosylation resulting from low acceptor nucleophilicity in Et2O as evident from multiple unidentified side products in HPLC chromatogram.

Compared with reaction in CH2Cl2 for the same donor series, the range of relative donor

reactivities is much smaller with both acceptors.

Figure 12. Solvent effect on Hammett plot of Gal donor series

acceptor A1 2.5 2

log RRV log in CH2Cl2: ρ =2.66, R =0.96 2 in Et2O: ρ =1.85, R =0.99

1.5

0.5

0 0.20.40.60.8 σp

Figure 12.(continued) Solvent effect on Hammett plot of Gal donor series

acceptor A2 2 in CH2Cl2: ρ =1.95, R =0.93

log RRV log 2 1.5 in Et2O: ρ =1.35, R =0.73

0.5

0 0.2 0.4 0.6 0.8 σp

The solvent effects have influence on both reactivity of donors70 and stereoselectivity of glycosylation outcomes.71-73 This study either supplements or supports the previous studies through a kinetic approach.

Scheme 21. An example of solvent effect in reactivity based chemoselective glycosylation

SEt

BnO O NIS/AgOTf OBn NIS/AgOTf BnO OBn CH2Cl2 Et2O O O A complex product 13 BzO SPh mixture O BnO OBz OBn BnO OBn HO O BzO SPh 14 OBz

A very interesting experimental result about the solvent effect on glycosylation between donor 13 and acceptor 14 is shown in Scheme 21.74 In Scheme 21, when the glycosylation was performed in diethyl ether, only donor 13 was activated by the promoter and reaction gave the desired disaccharide. If the reaction was performed in methylene chloride, a complex product mixture was obtained.74 This could be rationalized as that in diethyl ether the reactivity difference between these two building

34 blocks was greater than that in methylene chloride, and thus seems contradictory to our result that the Gal-series donors have smaller reactivity range in diethyl ether than in methylene chloride. The contradiction can be explained by the fact that only limited amount of NIS can be dissolved when diethyl ether is used as the solvent. Thus the reaction in diethyl ether would proceed with a very low concentration of promoter. Based on our previous study, a less promoter concentration could lead to greater reactivity difference between donors (see Scheme 17). Another explanation could be made from the real-time NMR study. The glycosylation process with NIS needs an induction period to generate the activated form of promoter- iodonium ion (see Figure 2). Due to the low solubility of NIS in diethyl ether, the non-linear behavior in induction period may be dominating throughout the whole reaction time as NIS gradually dissolves. The result in

Scheme 21 thus can not question our results, which follow the pseudo-first-order kinetics

(in our reactions, promoters are prepared as solution either in acetonitrile or in any other suitable solvents).

It is apparently that glycosylation is affected by those factors as efficiency of acceptors, solvent effect and promoter used, etc. All those factors work together to change the outcome of glycosylation. Thus the design of oligosaccharide synthesis is a complicated work due to these concerns and our work is expected to give some insight to it by focusing on the key step-glycosylation.

2.7 An Approach to Mechanistic Investigation on Glycosylation with RRVs

Using Linear Free Energy Relationship

It has been generally accepted that glycosylation of thioglycosyl donors is a three-

step process:19,41 step A: generation of a glycosyl sulfonium ion by electrophilic addition

of promoter onto the anomeric sulfur atom; step B: conversion of the sulfonium ion to a

reactive intermediate (oxacarbenium ion or others); step C: attack of the nucleophilic

glycosyl acceptor on the reactive intermediate to form the glycoside product (Scheme

22a). Although formation of oxacarbenium ion (step B) has been commonly postulated to be the RDS of glycosylation,19,20,52 the fact that the acceptor has a significant effect on

glycosylation rate suggests RDS of the glycosylation reaction cannot be prior to

engagement of the acceptor. Experimentally, in reaction of donor 1a-1e with acceptor

A1, β disaccharide 17 is exclusively formed implying that the dioxonium ion 16 is the kinetically competent reactive intermediate (Scheme 22b). Acceptor A1 cannot directly react with the glycosyl sulfonium ion 15 to form glycoside products since such a reaction pathway will generate significant amount of α glycoside. It is thus most likely step C is the RDS of the overall reaction.

As A1 is a worse nucleophile than methanol, it reacts slower with the reactive

intermediate 16. Therefore, rate differences established in pre-RDS steps resulting from

differential aglycons become less pronounced leading to a smaller absolute ρ value (2.7

vs 3.0, Figure 5). Secondary hydroxyl group in glucosamine A2 is less reactive69 than

the primary alcohol in A1, causing further decrease in the absolute value of ρ (2.0 vs 2.7).

Scheme 22. General mechanism for glycosylation of thioglycosides a) + E O promoter E O O PGO S PGO S PGO Y Step A Y Step B or glycosyl sulfonium ion other reactive O intermediates HO O O PGO O Step C b) BzO OTBDPS BzO OTBDPS p-TolSOTf BzO OTBDPS O O BzO S O BzO Step A BzO S Y Step B BzO Y O BzO STol O 1a-1e - OTf 16 Ph 15 A1 BzO OTBDPS O BzO O Step C BzO O RDS O 17 O O O

In all Hammett plots, ρ values were negative demonstrating building blocks with

electron donating groups react faster. This seems to be counter-intuitive as p-nitro

thiophenol is a better leaving group than p-methoxy thiophenol. With our building

blocks differing only in their aglycon substituents, the oxacarbenium ion intermediates

generated within the same donor series should have the same structure as aglycons have already been cleaved. Thus the rate constants for step C for the same donor series should be the same (Scheme 22a). The fact that building blocks with electron withdrawing aglycons react slower can be explained by assuming step A is a fast equilibrium. As the sulfur atoms in p-nitrophenyl glycosides are less electron rich, their activation by an electrophilic promoter will be slower than the corresponding p-methoxyphenyl glycosides. This will generate lower concentrations of the glycosyl sulfonium ions and oxacarbenium ions from p-nitrophenyl glycosides, leading to lower overall glycosylation rates.

The absolute ρ value of Gal donor series 1a-1e is larger than that of the GlcN series 2a-2e (3.0 vs 2.7, Figure 5), suggesting that Gal series of donors are more sensitive to para-substituents on their aglycons. This can be rationalized by the presence of more electron withdrawing protective groups on the glycon ring in the Gal donor series, resulting in more electron deficient cationic intermediates upon activation.

Since OH is an equally potent electron donating moiety as benzyl group,19 GlcN series 2a-2e contain one more electron withdrawing protective group on the glycon ring as compared to GlcBn series 3a-3e donors. Yet, the GlcN series are less sensitive to aglycon changes as shown by a smaller absolute ρ value in its Hammett plot (2.7 vs 3.4,

Figure 5). This presumably results from the participating neighboring group (NPhth) on

C-2 of GlcN donors. With participation of NPhth, the positive charge of the oxacarbenium ion of GlcN donors becomes more dispersed; hence the electron donating para-substituent on aglycon has lesser stabilizing effect leading to a smaller absolute ρ value (Figure 13). Similar phenomena of smaller absolute ρ value due to neighboring group participation have been observed in several nucleophilic displacement reactions.75,76

Figure 13. Neighboring group participation leads to smaller absolute ρ values for GlcN donor series.

OBn + BnO I δ δ+ OBn I HO O S Y + HO O HO O BnO + δ BnO S + δ BnO δ O N Oδ+ N Y + + O O δ N O δ O + S Y δ I

transition state with more dispersed charge less sensitive to Y

OBn I BnO + OBn δ BnO O I S + BnO O BnO BnO O δ + S BnO δ OBn Y BnO BnO δ+ Y S Y BnO I

transition state with more concentrated charge more sensitive to Y

3. Conclusion

We have demonstrated that simple modification of the para-substituents of

thioaryl glycosides can render over three orders of magnitude difference in anomeric

reactivities of glycosyl donors. The reactivity differential was found to be dependent

upon the identity of the acceptor with the more reactive methanol resulting in a larger

range of donor anomeric reactivities. Except for acceptor’s influence, the other reaction

conditions, such as reaction time, identity of promoter and solvent were also evaluated on

their potential effect on glycosylations. Hammett analyses demonstrate that electron

donating substituents greatly expedite glycosylation. Both anomeric reactivities and

chemical shifts of the anomeric protons linearly correlate with σp values of the

substituents, which allows prediction of anomeric reactivities even prior to syntheses of

the building blocks.

Donors different only in their aglycons provide a unique mechanistic probe for

glycosylation, because they generate comparable reactive intermediates after cleavage of aglycons. Our results suggest that the RDS of glycosylation reaction is nucleophilic

attack of the acceptor on oxocarbenium ion or other reactive intermediates generated

following donor activation. Although additional factors such as choice of solvent and

reaction temperature need to be taken into account, this study presents valuable insights

into designing efficient armed-disarmed chemoselective glycosylation and thioglycoside

glycosylation in general.

4. Experimental Section

General Synthesis Procedures: All reactions were carried out under nitrogen with

anhydrous solvents in flamed-dried glassware, unless otherwise noted. Glycosylation reactions with acceptors other than methanol were performed in the presence of molecular sieves, which were flame-dried right before the reaction under high vacuum.

When methanol was used as acceptor in glycosylation, it is pre-dried by molecular sieve

3A and the donors which would be used were also azeotropically evaporated with toluene to remove residue moisture. Chemicals used were reagent grade as supplied except where noted. HPLC solvents were all HPLC grade and used as bought. Analytical thin-layer chromatography was performed using silica gel 60 F254 glass plates (EM Science);

Compound spots were visualized by UV light (254 nm) and/or by staining with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O244H2O (24.0 g) in 6%

H2SO4 (500 ml). Flash column chromatography was performed on silica gel 60 (230-400

Mesh, EM Science) or Al2O3 for any air sensitive compound.

Standard condition for the competitive glycosylation19: the two donors (0.05 mmol

each) were dried together with ~50mg reference compound using high vacuum pump

overnight. They were then dissolved in 5mL dry DCM. This solution mixture was

transferred to three pre-dried HPLC vials (1mL each). The mixture contents in each vial

was analyzed in ~10 μL each time (3 times for every vial) by HP 1050 series HPLC

system, and the constancy of the three spectrums given from the same vial was required

to confirm that the HPLC system was working in good conditions (sometimes long time

working could cause low illuminating of the UV detector). After that, 2.04 μL of dry

MeOH was added to each vial; 20 μL of 0.5 M NIS solution (dissolved in pre-dried

acetonitrile) and 10μL of 0.1 M TfOH (in Et2O) solution were added to each vial to

initiate the reaction. The reaction was then left at room temperature for 2 hours and worked up by diluting with methylene chloride (2 mL), washing with saturated aqueous sodium thiosulfate containing 10% sodium hydrogen bicarbonate, drying with Na2SO4 and evaporating to dryness. The dryness was then dissolved in 1 mL dry methylene chloride. The contents of the mixture in each vial were analyzed by HPLC by the same procedure (~10 μL each time; 3 times for every vial; averaging the results to reduce experimental uncertainties).

HPLC system: all the measurements were performed under a HP 1050 series

HPLC system with a SUPELCO normal phase analytic HPLC column (25 cm * 4.6 mm

ID). The HPLC condition: gradient system (Hexanes : Ethyl Acetate 10 : 90 ~ 70 : 30); flow rate = 1.0 mL/min; UV detector: λ=254nm. HP 1050 series includes: pumping unit; low pressure gradient unit; mixer; auto injector; UV-Vis detector.

Measurement of RRVs:

Measurement of RRVs: Gal-Series with Methanol as Acceptor in CH2Cl2

BzO OTBDPS trial 1: 22.37 trial 1: 1.02 BzO OTBDPS RRV=0.042 trial 2: 21.43 OBn RRV=0.89 O trial 2: 1.11 O BzO S trial 3: 25.45 HO O trial 3: 1.12 BzO S BnO S OBz ave.: 23.08 ave.: 1.08 OBz NO2 NPhth Br 1a 2a NO2 1b

trial 1: 2.84 trial 2: 3.38 ave.: 3.02 trial 3: 2.84 *note 1) The RRV of 4g was set as 2.7 arbitrarily base on the data base built (1 by Wong group (see Ref. 19); OAc RRV=2.7* 2) Competitive glycosylation between 1e and 1d used isopropanol as AcO O acceptor due to the overlay of product with 1e when usingmethanol AcO S as acceptor. 4g OAc

trial 1: 1.12 trial 2: 1.13 ave.: 1.16 trial 3: 1.22

OTBDPS (2 trial 1: 1.16 OTBDPS trial 1: 6.26 OTBDPS BzO RRV=21.7* BzO RRV=18.1 BzO RRV=3.12 O trial 2: 1.24 O trial 2: 5.80 O BzO S trial 3: 1.22 BzO S trial 3: 5.20 BzO S OBz OMe ave.: 1.20 OBz NHAc ave.: 5.75 OBz N 1e 1d 1c 3

Measurement of RRVs: GlcN-Series with Methanol as Acceptor in CH2Cl2

trial 1: 4.49 trial 1: 7.06 OBn RRV=2.7*(1 OBn RRV=19.2 RRV=0.601 trial 2: 4.58 OAc trial 2: 7.04 HO O HO O AcO O BnO S trial 3: 4.37 trial 3: 7.51 BnO S ave.: 4.49 AcO S ave.: 7.21 NPhth NO OAc NPhth Br 2a 2 4g 2b

trial 1: 5.27 trial 2: 5.32 ave.: 5.18 trial 3: 5.49

*note 1) The RRV of 4g was set as 2.7 arbitrarily base on the data base built OBn RRV=101 by Wong group (see Ref. 19); HO O 2) Competitive glycosylation between 2e and 2d used isopropanol as BnO S acceptor due to the overlay of product with 2e when using methanol NPhth NHAc as acceptor. 2d

trial 1: 3.97 trial 2: 3.35 ave.: 3.74 trial 3: 3.98

trial 1: 1.88 OBn (2 OBn OBn RRV=191* trial 2: 1.89 RRV=101 RRV=27.1 HO O HO O 3.74 HO O BnO S trial 3: 1.93 BnO S BnO S ave.: 1.90 NPhth OMe NPhth NHAc NPhth 2e 2d 2c N3

Measurement of RRVs: GlcBn-Series with Methanol as Acceptor in CH2Cl2

trial 1: 1.20 (1 OBn RRV=3.19 OAc RRV=2.7* BzO OTBDPS RRV=18.1 trial 2: 1.16 (2 BnO O AcO O 6.67* O BnO S trial 3: 1.17 BzO S ave.: 1.18 AcO S OBn NO OAc OBz NHAc 3a 2 4g 1d

trial 1: 8.90 trial 2: 9.60 ave.: 8.89 trial 3: 8.15

OBn RRV=3772(3 *note 1) The RRV of 4g was set as 2.7 arbitrarily base on the OBn RRV=161 BnO O data base built by Wong group (see Ref. 19); BnO O BnO S 2) Previous result; BnO S OBn 3) Competitive glycosylation between 3e and 3d used 3e OMe 3b OBn Br isopropanol as acceptor due to the overlay of product with 3e when using methanol as acceptor. trial 1: 1.34 trial 1: 1.46 trial 2: 1.28 ave.: 1.52 trial 2: 1.37 ave.: 1.39 trial 3: 1.94 trial 3: 1.33

trial 1: 3.64 trial 1: 5.16 OBn OBn OBn RRV=2482 trial 2: 3.76 RRV=657 trial 2: 6.15 RRV=116 BnO O BnO O BnO O BnO S trial 3: 3.94 BnO S trial 3: 5.73 BnO S OBn ave.: 3.78 OBn ave.: 5.68 OBn 3d NHAc 3c N3 3g NBnAc

Measurement of RRVs: Gal-Series with A1 as Acceptor in CH2Cl2 (reaction in 50~100 mg scale)

OTBDPS BzO OTBDPS RRV=1* OAc BzO RRV=11.7 O O 17.21 AcO O 1.48 BzO S AcO S BzO S OBz OBz NO2 OAc NHAc Br 1a 4d 1b

*note: The RRV of 1a was set as 1. 2.02

OAc AcO O AcO S 4g OAc

3.08

BzO OTBDPS BzO OTBDPS BzO OTBDPS RRV=234 RRV=177 RRV=72.3 O O O BzO S 1.32 BzO S 2.46 BzO S OBz OMe OBz NHAc OBz N 1e 1d 1c 3

Measurement of RRVs: Gal-Series with A2 as Acceptor in CH2Cl2 (reaction in 50~100 mg scale)

OTBDPS OTBDPS BzO RRV=1* OAc BzO RRV=5.95 O O 14.91 AcO O 2.51 BzO S AcO S BzO S OBz OBz NO2 OAc NHAc Br 1a 4d 1b

*note: The RRV of 1a was set as 1. 2.29

OAc AcO O AcO S 4g OAc

1.15

BzO OTBDPS BzO OTBDPS BzO OTBDPS RRV=62.49 RRV=51.41 RRV=11.80 O O O BzO S 1.22 BzO S 4.36 BzO S OBz OMe OBz NHAc OBz N 1e 1d 1c 3

Measurement of RRVs: Gal-Series with A1 as Acceptor in Et2O (reactionin50~100mgscale)

OTBDPS BzO OTBDPS RRV=1* OAc BzO RRV=7.63 O O 5.92 AcO O 1.29 BzO S AcO S BzO S OBz OBz NO2 OAc NHAc Br 1a 4d 1b

*note: The RRV of 1a was set as 1. 3.39

OAc AcO O AcO S 4g OAc

1.29

BzO OTBDPS BzO OTBDPS BzO OTBDPS RRV=47.1 RRV=34.3 RRV=20.0 O O O BzO S 1.38 BzO S 1.71 BzO S OBz OMe OBz NHAc OBz N 1e 1d 1c 3

Measurement of RRVs: Gal-Series with A2 as Acceptor in Et2O (reactionin50~100mgscale)

OTBDPS BzO OTBDPS RRV=1* OAc BzO RRV=1.26 O O 1.29 AcO O 1.02 BzO S AcO S BzO S OBz OBz NO2 OAc NHAc Br 1a 4d 1b

*note: The RRV of 1a was set as 1. 2.14

OAc AcO O AcO S 4g OAc

1.48

BzO OTBDPS BzO OTBDPS BzO OTBDPS RRV=17.5 RRV=14.3 RRV=3.98 O O O BzO S 1.22 BzO S 3.60 BzO S OBz OMe OBz NHAc OBz N 1e 1d 1c 3

For the preparation of compounds 1a, 1b, 1c, 1d, 1e, 1f and 2a, 2b, 2e, 2f see ref25

p-Azidophenyl-3,6-di-O-benzyl-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranosi-

de (2c). Compound 2f (597 mg, 1 mmol) was dissolved in a mixture solvent consisted of

° 8 N acetic acid (20 mL) and THF (20 mL) at 0 C. A 5% aqueous solution of NaNO2 (2.6

mL) was added to this solution and the mixture was stirred for 10 minutes. A 5% aqueous

solution of NaN3 (6.5 ml) was then added and the reaction mixture was allowed to warm

up to room temperature in 10 minutes. The reaction was again cooled to 0 °C, neutralized

with 10 % aqueous NaOH, extracted with EtOAc, washed with water and brine, and dried

over anhydrous Na2SO4. After filtration, removal of all solvents under reduced pressure,

compound 2c was obtained by flash chromatography in 87% yield (542 mg). 1H NMR

(400 MHz, CDCl3) δ 2.95 (s, 1H, 4-OH), 3.66-3.71 (m, 1H, H5), 3.76-3.88 (m, 3H, H2,

H6, H6’), 4.18 (t, 1H, J = 10.2 Hz, H3), 4.23 (t, 1H, J = 10.2 Hz, H4), 4.52 (d, 1H, J=12.4

Hz, PhCH2O), 4.57 (d, 1H, J=12.4 Hz, PhCH2O), 4.62 (d, 1H, J=12.4 Hz, PhCH2O), 4.72

13 (d, 1H, J=12.4 Hz, PhCH2O), 5.47 (d, 1H, J = 10.2 Hz, H1), 6.82-7.84 (m, 18H); C

NMR (100 MHz, CDCl3) δ 54.6, 70.7, 74.0, 74.1, 74.8, 78.2, 79.9, 83.6 (C1), 119.6,

127.8-128.8, 135.1, 137.9, 138.1, 140.4, 167.8, 168.4; MS (ESI) m/z calcd. For

+ C34H30N4NaO6S [M + Na] 645.2; found 645.3.

p-N-Acetamidophenyl 2-deoxy-2-N-phthalimido-3,6-di-O-benzyl-1-thio-β-D-glucopy

-ranoside (2d). Compound 2f (59.7 mg, 0.1 mmol) was dissolved in MeOH (10 mL) at 0

°C. A solution of acetic anhydride (47 μL, 0.5 mmol) was added to this solution and the mixture was stirred for 10 minutes at 0 °C, then the reaction mixture was neutralized with

solid NaHCO3, extracted with EtOAc, washed with water and brine, and dried over

anhydrous Na2SO4. After filtration and removal of all solvents under reduced pressure,

compound 2d was obtained by flash chromatography in 97% yield (62.0 mg). 1H NMR

(400 MHz, CDCl3) δ 2.12 (s, 3H, NHCOCH3), 3.19 (s, 1H, 4-OH), 3.64-3.68 (m, 1H, H5),

3.77-3.86 (m, 3H, H2, H6, H6’), 4.18 (t, 1H, J = 9.6 Hz, H3), 4.22 (t, 1H, J = 9.6 Hz, H4),

4.51 (d, 1H, J=12.4 Hz, PhCH2O), 4.54 (d, 1H, J=12.4 Hz, PhCH2O), 4.59 (d, 1H, J=12.4

Hz, PhCH2O), 4.72 (d, 1H, J=12.4 Hz, PhCH2O), 5.46 (d, 1H, J = 9.6 Hz, H1), 6.91-7.83

13 (m, 18H), 7.57 (bs, 1H, NHAc); C NMR (100 MHz, CDCl3) δ 29.8 (NHCOCH3), 54.6,

70.7, 74.0 (2 X OCH2Ph, C6), 74.2, 74.8, 78.1, 79.8, 84.0 (C1), 120.2, 123.6, 123.7,

128.0-128.8, 134.2-134.4, 137.9, 138.1, 138.4, 167.7, 168.4, 168.8; MS (ESI) m/z calcd.

+ for C36H34N2NaO7S [M + Na] 661.2; found 661.4.

4a: p-Nitrophenyl-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside The nitrophenyl

-thio-leaving group was introduce using the same method as described in ref22. 1H NMR

(600 MHz, CDCl3) δ 2.01 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 2.09 (s, 3H, COCH3),

2.12 (s, 3H, COCH3), 3.82-3.85 (m, 1H, H5), 4.21 (dd, 1H, J=2.4, 12.6 Hz, H6), 4.26 (dd,

1H, J=5.4, 12.6 Hz, H6), 4.88 (d, 1H, J1,2=10.2 Hz, H1), 5.06 (t, 1H, J=10.2 Hz, H2), 5.09

(t, 1H, J=10.2 Hz, H3), 5.28 (t, 1H, J=10.2 Hz, H4), 7.58-7.61 (m, 2H, phenyl), 8.16-8.18

13 (m, 2H, phenyl); C NMR (150 MHz, CDCl3) δ 20.0-21.0 (4 X COCH3), 62.3 (C6), 68.2,

69.7, 73.8, 76.3 (C2, C3, C4, C5), 84.5 (C1), 124.1, 131.2, 142.0, 147.2 (phenyl), 169.4-

+ 170.7 (4 X COCH3); MS (ESI) m/z 508.2 [M + Na] . calcd. for C20H23NNaO11S 508.09 p-Nitrophenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside (3a). To a 50 mL

round-bottom flask were added the tetraacetate 513 (1.11 g, 2.28 mmol) and a 2:3 mixture

° of MeOH-CH2Cl2 (25 mL). The mixture was cooled to 0 C, treated with a 0.5 M NaOMe

solution in MeOH (1.50 mL, 0.75 mmol) and stirred for 10 minutes followed by warming

up to room temperature and further stirring for 2 hours. The reaction was neutralized

with acetic acid to pH around 7. The suspension was concentrated under reduced pressure,

diluted with CH2Cl2 and filtered to afford the white solid tetraol (0.84 g). The crude tetraol was dried under vacuum in a 25 ml round-bottom flask overnight and azeotropically distilled in toluene to remove any residue water. It was then suspended in anhydrous DMF (15 mL) with freshly activated MS 4 Å (1 g). The suspension was stirred for 30 minutes at room temperature, then cooled to 0 °C in an ice-bath followed by slow

addition of 95% NaH (0.35 g, 13.8 mmol). The resulting suspension was stirred for 30

minutes at 0 °C then treated with BnBr (1.42 mL, 11.9 mmol). The mixture was warmed

to room temperature and stirred for another 4 hours. Then the mixture was cooled back

to 0 °C and quenched with acetic acid to pH=7 and diluted with EtOAc. The organic

phase was collected and washed with saturated NaHCO3 and dried over Na2SO4. The residue was purified by flash chromatography to give the compound 3a in 45% yield for

1 two steps (0.65 g). H NMR (600 MHz, CDCl3) δ 3.57 (t, 1H, J1,2 = 9.6 Hz, H2), 3.58-

3.61 (m, 1H, H5), 3.65-3.70 (m, 2H, 2 X H6), 3.75 (t, 1H, J = 9.6 Hz, H3), 3.78 (t, 1H, J =

9.6 Hz, H4), 4.52 (d, 1H, J=11.4 Hz, PhCH2O), 4.57 (d, 1H, J=11.4 Hz, PhCH2O), 4.60

(d, 1H, J=11.4 Hz, PhCH2O), 4.76 (d, 1H, J=11.4 Hz, PhCH2O), 4.82 (q, 2H, J=11.4 Hz,

PhCH2O), 4.82 (d, 1H, J1,2 = 9.6 Hz, H1), 4.87 (d, 1H, J=11.4 Hz, PhCH2O), 4.90 (d, 1H,

13 J=11.4 Hz, PhCH2O), 7.19-7.36 (m, 20H), 7.59-7.61 (m, 2H), 7.99-8.01 (m, 2H); C

NMR (150 MHz, CDCl3) δ 69.1, 73.7, 75.4, 75.9, 76.1, 77.9, 79.4, 80.9, 86.0, 86.8 (C1),

124.1, 128.0-129.6, 137.7, 138.0, 138.1, 138.4, 144.6, 146.4; MS (ESI) m/z calcd. for

+ C40H39NNaO7S [M + Na] 700.2; found 700.4.

p-Bromophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (4b). Glucose

pentaacetate 4 (5 g, 12.9 mmol) and p-bromo thiophenol (2.9 g, 15.5 mmol) were

dissolved in CH2Cl2 under nitrogen at 0 °C. Borontrifluoride etherate (2.4 mL, 19.4

mmol) was added and the reaction mixture was stirred for 18 hours at room temperature.

After dilution with CH2Cl2, the organic layer was washed with saturated NaHCO3 and

brine. The organic layer was dried over Na2SO4, the solvent was removed and the residue was purified by column chromatography on silica gel to afford 6 (6.2 g) in 90% yield. 1H

NMR (600 MHz, CDCl3) δ 2.00 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.09 (s, 3H,

COCH3), 2.10 (s, 3H, COCH3), 3.70-3.73 (m, 1H, H5), 4.18 (dd, 1H, J=2.4, 12.0 Hz, H6),

4.21 (dd, 1H, J=5.4, 12.0 Hz, H6), 4.65 (d, 1H, J= 10.2 Hz, H1), 4.94 (t, 1H, J = 10.2 Hz,

H2), 5.02 (t, 1H, J = 10.2 Hz, H3), 5.22 (t, 1H, J = 10.2 Hz, H4), 7.37-7.38 (m, 2H), 7.44-

13 7.46 (m, 2H); C NMR (150 MHz, CDCl3) δ 20.8-21.0 (4 X COCH3), 31.2 (p-OCH3),

62.2 (C6), 68.2, 69.9, 74.1, 76.0, 85.5 (C1), 119.6, 126.7, 136.0, 141.1, 169.5-170.1 (4 X

+ COCH3); MS (ESI) m/z calcd. for C20H23BrNaO9S [M + Na] 541.0; found 541.0.

p-Bromophenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside (3b). Compound

3b was prepared as described in the synthesis of compound 3a starting from compound 6

1 in 92% yield. H NMR (600 MHz, CDCl3) δ 3.48 (t, 1H, J1,2 = 9.6 Hz, H2), 3.50-3.52 (m,

1H, H5), 3.63 (t, 1H, J = 9.6 Hz, H3), 3.70 (t, 1H, J = 9.6 Hz, H4), 3.69-3.71 (m, 1H, H6),

3.77 (dd, 1H, J = 1.8, 10.8 Hz, H6’), 4.53 (d, 1H, J=12.0 Hz, PhCH2O), 4.58 (d, 2H,

J=12.0 Hz, PhCH2O), 4.62 (d, 1H, J1,2 = 9.6 Hz, H1), 4.73 (d, 1H, J = 10.8 Hz, PhCH2O),

4.81 (d, 1H, J = 10.8 Hz, PhCH2O), 4.83 (d, 1H, J = 10.8 Hz, PhCH2O), 4.84 (d, 1H, J =

12.0 Hz, PhCH2O), 4.88 (d, 1H, J = 10.8 Hz, PhCH2O), 7.18-7.20 (m, 2H), 7.27-7.37 (m,

13 20H), 7.43-7.44 (m, 2H); C NMR (150 MHz, CDCl3) δ 69.2, 73.6, 75.3, 75.7, 76.1 (4 X

OCH2Ph, C6), 78.0, 79.2, 80.9, 86.9, 87.6 (C1), 122.0, 127.9-128.7, 132.2, 133.0, 133.7,

+ 138.1, 138.4, 138.5; MS (ESI) m/z calcd. for C40H39BrNaO5S [M + Na] 733.2; found

733.4.

p-Azidophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (4c). The mixture

of 4a (181 mg, 0.37 mmol) and SnCl2.H2O (336 mg, 1.49 mmol) in EtOH (6 mL) and

CH2Cl2 (2 mL) was refluxed at 78 °C for 6 hours, and then cooled down to room

temperature. EtOH was removed under vacuum followed by addition of EtOAc (20 mL).

The resulting mixture was filtered through Celite, extracted with saturated aqueous

NaHCO3 (20 mL) and the organic phase was collected. Purification by flash

chromatography gave p-aminophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside

1 4f in 100% yield (170 mg). H NMR (400 MHz, CDCl3) δ 2.01 (s, 3H, COCH3), 2.05 (s,

3H, COCH3), 2.09 (s, 3H, COCH3), 2.12 (s, 3H, COCH3), 3.64-3.69 (m, 1H, H5), 3.82 (bs,

2H, NH2), 4.18-4.22 (m, 2H, H6, H6’), 4.51 (d, 1H, J1,2=10.0 Hz, H1), 4.88 (dd, 1H, J=9.6,

10.0 Hz, H2), 5.00 (t, 1H, J=9.6 Hz, H3), 5.19 (t, 1H, J=9.6 Hz, H4), 6.60-6.62 (m, 2H),

7.27-7.31 (m, 2H). Upon isolation, compound 4f (455 mg, 1 mmol) was immediately

dissolved in a mixture solvent consisted of 8 N acetic acid (20 mL) and THF (20 mL) at 0

° C. A 5% aqueous solution of NaNO2 (2.6 mL) was added to this solution and the mixture

was stirred for 10 minutes. A 5% aqueous solution of NaN3 (6.5 mL) was then added and

the reaction mixture was allowed to warm up to room temperature in 10 minutes. The

reaction was again cooled to 0 °C, neutralized with 10 % aqueous NaOH, extracted with

EtOAc, washed with water and brine, and dried over anhydrous Na2SO4. After filtration,

removal of all solvents under reduced pressure, compound 4c was obtained by flash

1 chromatography in 59% yield (280 mg). H NMR (600 MHz, CDCl3) δ 1.99 (s, 3H,

COCH3), 2.02 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 3.70-3.73 (m,

1H, H5), 4.18 (dd, 1H, J=1.8, 12.0 Hz, H6), 4.21 (dd, 1H, J=4.8, 12.0 Hz, H6’), 4.62 (d,

1H, J1,2=9.6 Hz, H1), 4.91 (t, 1H, J=9.6 Hz, H2), 5.01 (t, 1H, J=9.6 Hz, H3), 5.21 (t, 1H,

13 J=9.6 Hz, H4), 6.98-6.99 (m, 2H), 7.49-7.51 (m, 2H); C NMR (150 MHz, CDCl3) δ

20.8-21.0, 31.2 (4 X COCH3), 62.2 (C6), 68.2, 69.9, 74.1, 76.0, 85.5 (C1), 119.6, 126.7,

136.0, 141.1, 169.5-170.1 (4 X COCH3); MS (ESI) m/z calcd. for C20H23N3NaO9S [M +

Na]+ 504.1; found 504.1. p-Azidophenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside (3c). Compound

3c was prepared as described in the synthesis of compound 3a starting from compound 9

1 in 78% yield. H NMR (600 MHz, CDCl3) δ 3.46 (t, 1H, J1,2 = 9.6 Hz, H2), 3.47-3.50 (m,

1H, H5), 3.63 (t, 1H, J = 9.6 Hz, H3), 3.70 (t, 1H, J = 9.6 Hz, H4), 3.71-3.73 (m, 1H, H6),

3.78 (d, 1H, J = 10.2 Hz, H6’), 4.53 (d, 1H, J = 10.2 Hz, PhCH2O), 4.58 (d, 1H, J1,2 = 9.6

Hz, H1), 4.58-4.60 (m, 2H, PhCH2O), 4.73 (d, 1H, J = 10.2 Hz, PhCH2O), 4.81 (d, 1H, J

= 10.8 Hz, PhCH2O), 4.84 (d, 1H, J = 10.2 Hz, PhCH2O), 4.85 (d, 1H, J = 10.8 Hz,

PhCH2O), 4.88 (d, 1H, J = 10.2 Hz, PhCH2O), 6.82-6.83 (m, 2H), 7.19-7.39 (m, 20H),

13 7.56-7.57 (m, 2H); C NMR (150 MHz, CDCl3) δ 69.2, 73.6, 75.3, 75.7, 76.1 (4 X

OCH2Ph, C6), 78.0, 79.2, 80.9, 86.9, 87.6 (C1), 119.7, 127.9-128.7, 129.7, 134.3, 138.1,

+ 138.2, 138.4, 138.5, 139.9; MS (ESI) m/z calcd. for C40H39N3NaO5S [M + Na] 696.3; found 696.4. p-N-Acetamidophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (4d). The

mixture of 4a (1.46 g, 3.00 mmol) and SnCl2.H2O (1.38 g, 6.12 mmol) in EtOH (30 mL)

and CH2Cl2 (10 mL) was refluxed at 78 °C for 6 hours, and then cooled down to room

temperature. A solution of acetic anhydride (0.94 mL, 10.0 mmol) was added to this

solution and the mixture was stirred for 2 hours at room temperature, then the reaction

mixture was neutralized with solid NaHCO3, extracted with EtOAc, washed with water

and brine, and dried over anhydrous Na2SO4. After filtration and removal of all solvents

under reduced pressure, compound 4d was obtained by flash chromatography in 63%

1 yield (0.94 g). H NMR (400 MHz, CDCl3) δ 1.98 (s, 3H, COCH3), 2.01 (s, 3H, COCH3),

2.08 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.18 (s, 3H, NHCH3), 3.67-3.71 (m, 1H, H5),

4.11-4.18 (m, 2H, 2 X H6), 4.59 (d, 1H, J=9.6 Hz, H1), 4.91 (t, 1H, J=9.6 Hz, H2), 5.01 (t,

1H, J=9.6 Hz, H3), 5.20 (t, 1H, J=9.6 Hz, H4), 7.38 (m, 1H, NHAc), 7.44-7.49 (m, 4H);

13 C NMR (100 MHz, CDCl3) δ 20.8-21.0 (4 X COCH3), 24.9 (NHCOCH3), 62.3, 68.3,

70.1, 74.2, 76.0, 86.0 (C1), 120.1, 126.0, 133.7, 135.1, 138.8, 169.5, 169.6, 170.4 170.9 (4

+ X COCH3); MS (ESI) m/z calcd. for C22H27NNaO10S [M + Na] 520.1; found 520.2. p-N-benzylatedacetamidephenyl-2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside

(3g) Compound 3g was prepared as described in the synthesis of compound 3a using

1 compound 4d as starting material. H NMR (600 MHz, CDCl3) δ 1.78 (s, 3H,

NBnCOCH3), 3.41-3.47 (m, 2H, H2, H5), 3.57 (t, 1H, J6’,6=9.6 Hz, H6’), 3.61-3.65 (m, 2H,

H3, H4), 3.69-3.71 (m, 1H, H6 ), 4.45 (d, 1H, J = 12.0 Hz), 4.49-4.52 (m, 2H), 4.60 (d, 1H,

J = 10.2 Hz), 4.66 (d, 1H, J = 10.2 Hz), 4.72-4.83 (m, 6H), 6.70-6.71 (m, 2H, phenyl),

13 7.08-7.28 (m, 25H, phenyl), 7.39-7.41 (m, 2H, phenyl); C NMR (100 MHz, CDCl3) δ

23.0 (COCH3), 52.9, 69.2, 73.6, 75.4, 75.8, 76.1 (5 X OCH2Ph, C6), 78.0, 79.2, 81.0, 86.9

(C2, C3, C4 and C5), 87.3 (C1), 127.6 (phenyl), 127.9-128.7 (aromatic), 128.8, 129.0

(phenyl), 132.0, 137.5, 138.0, 138.1, 138.4, 138.5 (aromatic), 141.9 (COCH3); MS (ESI)

m/z 802.6.

p-N-Acetamidophenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside (3d).

Compound 3d was prepared as described in the synthesis of compound 15 starting from

1 compound 3a in 84% yield. H NMR (600 MHz, CDCl3) δ 2.17 (s, 3H, NHAc), 3.46 (t,

1H, J1,2 = 9.6 Hz, H2), 3.47-3.49 (m, 1H, H5), 3.62 (t, 1H, J = 9.6 Hz, H3), 3.69 (t, 1H, J =

9.6 Hz, H4), 3.70 (dd, 1H, J = 4.2, 10.8 Hz, H6), 3.75 (dd, 1H, J = 1.2, 10.8 Hz, H6), 4.53

(d, 1H, J = 10.2 Hz, PhCH2O), 4.57 (d, 1H, J1,2 = 9.6 Hz, H1), 4.56-4.60 (m, 2H,

PhCH2O), 4.73 (d, 1H, J = 10.2 Hz, PhCH2O), 4.81 (d, 1H, J = 10.2 Hz, PhCH2O), 4.83

(d, 1H, J = 10.2 Hz, PhCH2O), 4.87 (d, 1H, J = 10.2 Hz, PhCH2O), 4.88 (d, 1H, J = 10.2

Hz, PhCH2O), 7.07 (bs, 1H, NHAc), 7.17-7.19 (m, 2H), 7.26-7.40 (m, 20H), 7.54-7.55

13 (m, 2H); C NMR (150 MHz, CDCl3) δ 24.9 (NHCOCH3), 73.6, 75.3, 75.6, 76.0, 78.0,

79.3, 81.0, 87.0, 87.9, 89.5 (C1), 120.2, 127.9-128.7, 133.9, 137.8, 138.2, 169.0; MS (ESI)

+ m/z calcd. for C42H43NNaO6S [M + Na] 712.3; found 712.6.

p-Methoxyphenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-glucopyranoside (3e).

Compound 3e was prepared as described in the synthesis of compound 3a starting from

49 1 compound 7 in 97% yield. H NMR (600 MHz, CDCl3) δ 3.44 (t, 1H, J1,2 = 9.6 Hz, H2),

3.46-3.47 (m, 1H, H5), 3.62 (t, 1H, J = 9.6 Hz, H3), 3.69 (t, 1H, J = 9.6 Hz, H4), 3.72-3.76

(m, 1H, 1 X H6), 3.75 (s, 3H, OCH3), 3.78 (dd, 1H, J = 1.2, 9.6 Hz, 1 X H6), 4.53 (d, 1H,

J1,2 = 9.6 Hz, H1), 4.54 (d, 1H, J = 10.2 Hz, PhCH2O), 4.59 (d, 1H, J = 10.2 Hz,

PhCH2O), 4.60 (d, 1H, J = 10.2 Hz, PhCH2O), 4.74 (d, 1H, J = 10.2 Hz, PhCH2O), 4.82

(d, 1H, J = 10.2 Hz, PhCH2O), 4.84 (d, 1H, J = 10.2 Hz, PhCH2O), 4.89 (d, 1H, J = 10.2

Hz, PhCH2O), 4.90 (d, 1H, J = 10.2 Hz, PhCH2O), 6.72-6.76 (m, 2H), 7.19-7.42 (m,

13 20H), 7.53-7.55 (m, 2H); C NMR (150 MHz, CDCl3) δ 55.5 (OCH3), 69.3, 73.6, 75.3,

75.6, 76.1, 78.0, 79.2, 80.9, 87.0, 88.1(C1), 114.6, 123.6, 127.8-128.7, 135.4, 138.3-138.6,

+ 139.9; MS (ESI) m/z calcd. for C41H42NaO6S [M + Na] 685.3; found 685.4.

3-Bromopropanyl-2-N-phthalimido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranoside

(A2). To a mixture of 2a (626 mg, 1 mmol), 3-bromopropanol (0.26 mL, 3 mmol), and

MS 4Å (800 mg) in CH2Cl2 (20 mL) was added NIS (587 mg, 3.3 mmol) under nitrogen.

The mixture was stirred for 30 minutes at -40 °C. TfOH (0.4 mL, 0.1 M in Et2O) was added. The reaction mixture was stirred for 1 hour, during which the temperature was raised to room temperature. The reaction mixture was then diluted with CH2Cl2 and filtered through celite. The solution was washed with saturated aqueous

Na2S2O3/NaHCO3, brine, and dried over Na2SO4. The residue was purified by flash chromatography to give the compound 17 in 74% yield (451 mg). 1H NMR (400 MHz,

CDCl3) δ 1.81-1.89, 1.96-2.04 (m, 2H, BrCH2CH2CH2O), 3.03 (d, 1H, J = 2.4 Hz, 4-OH),

3.14-3.26 (m, 2H, BrCH2CH2CH2O), 3.50-3.56, 3.63-3.68 (m, 2H, BrCH2CH2CH2O),

3.79 (dd, 1H, J=4.8, 10.4 Hz, H6), 3.80-3.84 (m, 1H, H5), 3.87 (t, 1H, J=9.6 Hz, H2),

3.89 (dd, 1H, J=5.2, 10.4 Hz, H6), 4.14 (t, 1H, J = 9.6 Hz), 4.25 (t, 1H, J = 9.6 Hz), 4.54

(d, 1H, J=12.0 Hz, PhCH2O), 4.59 (d, 1H, J=12.0 Hz, PhCH2O), 4.66 (d, 1H, J=12.0 Hz,

PhCH2O), 4.75 (d, 1H, J=12.0 Hz, PhCH2O), 5.14 (d, 1H, J = 9.6 Hz, H1), 6.94-7.81 (m,

13 14H); C NMR (100 MHz, CDCl3) δ 30.4, 32.4, 55.6 (BrCH2CH2CH2), 67.1, 70.7, 74.0,

74.1, 74.3, 74.6, 78.9, 98.7 (C1), 123.6, 127.7-128.8, 131.8, 134.2, 138.0, 138.4; MS (ESI)

+ m/z calcd. for C31H32BrNNaO7 [M + Na] 632.1; found 632.2.

Procedure for large scale competitive glycosylation for yield determination:

Methanol was pre-dried by molecular sieves 3Å overnight. Donors 10 and 1b were dried

under high vacuum overnight and azeotropically evaporated with toluene to remove any

residue moisture. To a solution of donors 10 (80.5 mg, 0.177 mmol) and 1b (160 mg,

0.177 mmol), methanol (36 μL, 0.885 mmol) in CH2Cl2 (17 mL), a solution of AgOTf

(91 mg, 0.354 mmol) in acetonitrile (0.05 mL) was added under N2. The mixture was

stirred for 15 minutes and cooled down to -40oC followed by addition of p-TolSCl (28

mg, 25 μL, 0.177 mmol). The reaction was stirred for 30 minutes from -40oC to room

temperature and quenched with several drops of triethylamine. CH2Cl2 (20 mL) was

added and all insoluble material was filtered off. The filtrate was washed with saturated

aqueous NaHCO3 and dried over Na2SO4. Purification by flash chromatography gave

unreacted donor 10 (23.1 mg), unreacted donor 1b (103.4 mg), methyl 2,3,4,6-tetra-O- acetyl-β-D-glucopyranoside (41.6 mg, 0.115 mmol, 92% yield based on the amount of

donor 10 consumed) and methyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-

galactopyranoside (45.4 mg, 0.060 mmol, 94% yield based on the amount of donor 1b

consumed). Methyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 1H NMR (400 MHz,

CDCl3) δ 2.00-2.10 (m, 12H, 12 X COCH3), 3.40 (s, 3H, OCH3), 3.95-3.98 (m, 1H, H5),

4.19-4.26 (m, 2H, H6), 4.89 (t, 1H, J = 9.6 Hz, H2), 4.95 (d, 1H, J = 9.6 Hz, H1), 5.06 (t,

13 H, J= 9.6 Hz, H4), 5.47 (t, 1H, J= 9.6 Hz, H3); C NMR (100 MHz, CDCl3) δ 20.8-21.0

(4 X COCH3), 55.7 (OCH3), 62.1 (C6), 67.3, 68.7, 70.3, 71.0, 97.0 (C1), 169.4-171.0

+ (COCH3); MS (ESI) m/z. calcd. for C15H22NaO10 [M + Na] 385.1; found 385.2. Methyl

2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-1-β-D-galactopyranoside 1H NMR

(600 MHz, CDCl3) δ 0.99 (s, 9H, (CH3)3), 3.52 (s, 3H, OCH3), 3.82-3.84 (m, 2H, 2 X H6),

4.06 (t, 1H, J = 7.0Hz, H5), 4.66 (d, 1H, J = 7.0 Hz, H1), 5.60-5.62 (dd, 1H, J = 10.8 Hz,

3.6 Hz, H3), 5.67-5.70 (dd, 1H, J = 10.8, 7.0 Hz, H2), 6.02 (d, 1H, J = 3.6 Hz, H4), 7.09-

7.11 (m, 2H), 7.22-7.25 (m, 2H), 7.35-7.49 (m, 12H), 7.64-7.66 (m, 2H), 7.77-7.79 (m,

13 2H), 7.93-7.95 (m, 2H), 8.00-8.01 (m, 2H); C NMR (150 MHz, CDCl3) δ 19.2, 26.8,

29.9, 57.4, 61.5, 68.1, 70.2, 72.1, 74.0, 102.6, 127.8-130.2, 132.8-133.5, 135.7-135.8,

+ 165.6-165.9; MS (ESI) m/z calcd. for C44H44O9SiNa [M + Na] 767.3; found 767.5.

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SPECTRAL DATA

1 H-NMR of compound 2c (600MHz, CDCl3)...... 69

13 C-NMR of compound 2c (150 MHz, CDCl3)...... 70

1 H-NMR of compound 2d (600 MHz, CDCl3)...... 71

13 C-NMR of compound 2d (150 MHz, CDCl3)...... 72

1 H-NMR of compound 3a (600 MHz, CDCl3)...... 73

13 C-NMR of compound 3a (150 MHz, CDCl3)...... 74

COSY NMR of compound 3a (600 MHz, CDCl3)...... 75

1 H-NMR of compound 3b (600 MHz, CDCl3)...... 76

13 C-NMR of compound 3b (150 MHz, CDCl3)...... 77

COSY NMR of compound 3b (600 MHz, CDCl3)...... 78

1 H-NMR of compound 3c (600 MHz, CDCl3)...... 79

13 C-NMR of compound 3c (150 MHz, CDCl3)...... 80

COSY NMR of compound 3c (600 MHz, CDCl3)...... 81

1 H-NMR of compound 3d (600 MHz, CDCl3) ...... 82

13 C-NMR of compound 3d (150 MHz, CDCl3)...... 83

COSY NMR of compound 3d (600 MHz, CDCl3)...... 84

1 H-NMR of compound 3e (600 MHz, CDCl3) ...... 85

13 C-NMR of compound 3e (150 MHz, CDCl3) ...... 86

COSY NMR of compound 3e (600 MHz, CDCl3) ...... 87

1 H-NMR of compound 3g (600 MHz, CDCl3) ...... 88

13 C-NMR of compound 3g (150 MHz, CDCl3) ...... 89

1 H-NMR of compound 4a (600 MHz, CDCl3) ...... 90

13 C-NMR of compound 4a (150 MHz, CDCl3) ...... 91

1 H-NMR of compound 4b (600 MHz, CDCl3)...... 92

13 C-NMR of compound 4b (150 MHz, CDCl3)...... 93

1 H-NMR of compound 4c (600 MHz, CDCl3)...... 94

13 C-NMR of compound 4c (150 MHz, CDCl3)...... 95

1 H-NMR of compound 4d (600 MHz, CDCl3)...... 96

13 C-NMR of compound 4d (150 MHz, CDCl3) ...... 97

1 H-NMR of compound 4f (600 MHz, CDCl3)...... 98

1 H-NMR of Product of Large Scale Glycosylation of A2 (600 MHz, CDCl3) ...... 99

13 C-NMR of Product of Large Scale Glycosylation of A2 (150 MHz, CDCl3) ...... 100

1 H-NMR of Product of Large Scale Glycosylation of P1 (600 MHz, CDCl3...... 101

13 C-NMR of Product of Large Scale Glycosylation of P1 (150 MHz, CDCl3) ...... 102

1 H-NMR of Product of Large Scale Glycosylation of P2 (600 MHz, CDCl3) ...... 103

13 C-NMR of Product of Large Scale Glycosylation of P2 (150 MHz, CDCl3) ...... 104

Selected HPLC Spectra for 1a~1e Series Measurement...... 105

Selected HPLC Spectra for 2a~2e Series Measurement...... 108

Selected HPLC Spectra for 3a~3e Series Measurement...... 110

Selected HPLC Spectra for 1a~1e Series Measurement (Acceptor A1) ...... 113

Selected HPLC Spectra for 1a~1e Series Measurement (Acceptor A2) ...... 116

1 H-NMR (600 MHz, CDCl3) of 2c

OBn HO O BnO S NPhth 2c N3

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 2c

OBn HO O BnO S NPhth N 2c 3

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 2d

OBn HO O BnO S 2d NPhth NHAc

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 2d

OBn HO O BnO S 2d NPhth NHAc

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 3a

OBn BnO O BnO S OBn NO 3a 2

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3a

OBn BnO O BnO S OBn 3a NO2

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

COSY-NMR (600 MHz, CDCl3) of 3a

OBn BnO O BnO S OBn 3a NO2

-1

g1,2 4

5 ppm

11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

1 H-NMR (600 MHz, CDCl3) of 3b

OBn BnO O BnO S OBn 3b Br

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3b

OBn BnO O BnO S OBn Br

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

COSY-NMR (600 MHz, CDCl3) of 3b

OBn BnO O BnO S OBn Br

-1

3 g1,2

5 ppm

11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

1 H-NMR (600 MHz, CDCl3) of 3c

OBn BnO O BnO S OBn 3c N3

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3c

OBn BnO O BnO S OBn 3c N3

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

COSY-NMR (600 MHz, CDCl3) of 3c

OBn BnO O BnO S OBn 3c N3

3.5

4.0

4.5 g1,2

5.0

5.5 ppm

6.0

6.5

7.0

7.5

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 ppm

1 H-NMR (600 MHz, CDCl3) of 3d

OBn BnO O BnO S OBn NHAc

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3d

OBn BnO O BnO S OBn NHAc

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

COSY-NMR (600 MHz, CDCl3) of 3d

OBn BnO O BnO S OBn NHAc

3.3

3.4 g1,2 3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2 ppm

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5.0

5.0 4.5 4.0 3.5 ppm

1 H-NMR (600 MHz, CDCl3) of 3e

OBn BnO O BnO S OBn 3e OMe

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3e

OBn BnO O BnO S OBn 3e OMe

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

COSY-NMR (600 MHz, CDCl3) of 3e

OBn BnO O BnO S OBn 3e OMe

-1

3 g1,2

5 ppm

11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm

1 H-NMR (600 MHz, CDCl3) of 3g

OBn BnO O BnO S 3g OBn NBnAc

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 3g

OBn BnO O BnO S 3g OBn NBnAc

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 4a

OAc AcO O AcO S OAc 4a NO2

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 4a

OAc AcO O AcO S OAc 4a NO2

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 4b

OAc AcO O AcO S 4b OAc Br

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 4b

OAc AcO O AcO S OAc Br 4b

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 4c

OAc AcO O AcO S OAc 4c N3

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 4c

OAc AcO O AcO S OAc 4c N3

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 4d

OAc AcO O AcO S 4d OAc NHAc

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of 4d

OAc AcO O AcO S OAc NHAc 4d

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of 4f

OAc AcO O AcO S OAc NH 4f 2

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

1 H-NMR (600 MHz, CDCl3) of A2

OBn HO O BnO O Br NPhth A2

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

13 C-NMR (150 MHz, CDCl3) of A2

OBn AcO O BnO O Br A2 NPhth

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

100

1 H-NMR (600 MHz, CDCl3) of P1

OAc AcO O AcO OMe OAc P1

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

101

13 C-NMR (150 MHz, CDCl3) of P1

OAc AcO O AcO OMe OAc P1

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

102

1 H-NMR (600 MHz, CDCl3) of P2

BzO OTBDPS O BzO OMe OBz P2

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)

103

13 C-NMR (150 MHz, CDCl3) of P2

BzO OTBDPS O BzO OMe OBz P2

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Chemical Shift (ppm)

104

Acceptor:MeOH

1a 2a 1a 2a reference reference