A De Novo Approach Towards Glycosylation using Iterative Pd-/B-Dual Catalysis: Applications in Natural Products Synthesis, Digitoxin analogues, Oligomannose

motifs and their SAR Studies

by Sumit O. Bajaj

B.Sc. in Chemistry, Physics, Maths, Amravati University M.Sc. in Chemistry, Sant Gadge Baba Amravati University

A dissertation submitted to

The Faculty of the College of Science of

Northeastern University in partial fulfillment of the requirements

for the Doctor of Philosophy in the

July 10, 2015

Dissertation directed by

George A. ODoherty Professor of Chemistry and Chemical Biology

ii

Dedication

To the loving memory of my beloved Grandfather Late Seth Shri Mansaramji Hariramji Bajaj, Lohi, Maharashtra, India, the person behind my success.

ToMy Grandmother Shrimati Saraswatibai Mansaramji Bajaj, the inspiration behind my success and my positive attitude.

iii

Acknowledgements

First, I would like to express my sincere gratitude towards my advisor Prof. George A.

ODoherty for his excellent mentorship, guidance, encouraging conversation during the course of

my studies at Northeastern University. I am grateful that he was always there to support me

when circumstances became difficult in my graduate studies. I am very thankful to him for

providing an excellent opportunity to work on research projects and learn new techniques.

Whenever I encountered problem, he was always been very helpful to resolve these through

critical thinking, discussion and positive attitude. Whenever I told him that the reaction is not

working, his favorite quote Try it again. I want to express my gratitude to Prof. James Aggen,

Prof. Roman Manetsch, Prof. Ke Zhang, Prof. Graham Jones and Prof. Michael Pollastri, to be

on/part of my dissertation committee, discussing chemistry and their valuable suggestions on my

thesis. Everyone was friendly and supportive.

I feel that I am fortunate to work in the ODoherty group where all the group members

both past and present, were most helpful during my tenure. Specially, I would like to thank the

ODoherty group members- Dr. Miaosheng Li, Dr. Mingde Shan, Dr. Xiaomei Yu, Dr. Sang-

woo Kang, Dr. Qian Chen, Dr. Rajender Vemula, Dr. Yalan Xing, Dr. Bulan Wu, Dr. Mingzong

Li, Dr. Hongyan Li, Dr. Leo Wang, Dr. Ehesan Sharif, Dr. Qi Zhang, Dr. Yanping Wang, Dr.

Michael Cuccarese, Melvin Rajaratnam, Dr. Yashan Zhong, Dr. Pei Shi, Debarpita Ray, Yu Li,

Jiamin Zheng, Yuzi Ma, Chao Liang, Xiaofan Liu, Alhanouf Aljahdali and all the other graduate

students, postdocs and undergraduates for their friendship and assistance in the department of

chemistry and chemical biology at Northeastern University, Boston MA.

I would like to thank my grandfather late Shri. Mansaramji Hariramji Bajaj, to whom this

dissertation is dedicated for the enormous love, care and strong blessings in the form of

iv

Ashirwad. His positive attitude, guidance to work hard and honestly made me a completely

different personality than what I was before. Most importantly I would like to thank my

Grandmother Shrimati Saraswatibai Mansaramji Bajaj for her enormous love, care and advice

she gave me for my successful career. I would like to thank my maternal grandparents Shri.

Narayandasji Lahoti and Sau. Shantabai N. Lahoti. I would like to thank my grandfather Shri.

Laxminarayanji H. Bajaj.

I would like to bow my head towards my parents Shri. Omprakash M. Bajaj, Sau. Pushpa

O. Bajaj for their enormous love, education, guidance they showered on me. I would like to

thank my elder brother Amit O. Bajaj and younger brother Satish O. Bajaj for their guidance and

affection they showered on me. I would like to especially thank my sister-in-law Sau. Jyoti A.

Bajaj and Sau. Kirti S. Bajaj for their enormous care, very delicious food and affection they

showed towards me during my PhD career. The most important personality which reminds me of

my childhood days, my loving niece, my sweetheart Palak Amit Bajaj, I would like to thank her

about this, its totally a different feeling when I hear Sumit Chachu from her.

Then comes the turn of all my uncles Shri Manaklalji M. Bajaj, Kamalkishorji M. Bajaj,

Madhusudanji L. Bajaj, Chandraprakashji M. Bajaj and Sanjay N. Lahoti, all aunts for their love,

inspiration and guidance.

I am so happy that I met my beloved wife, Ms. Deepa R. Atal during my visit to India in

2015. I would like to thank her for enormous care, love and being a very strong positive

motivator, supporter during my final Ph.D dissertation writing, even words are shorter to express

my feelings towards her. I would also like to thank my Father-in-law, Advocate Ratanlalji B.

Atal and mother-in-law Prabhadevi R. Atal for their enormous support and love. Special thanks

v

to Dr. Mohanji Biyani, Sandeepji Rathi, Advocate Riteshji G. Boob, Mr. Manishji Daga, Dr.

Jyoti M. Biyani, Mrs. Kanchan M. Daga, Ms. Vrunda R. Atal (CA), Girdhar M. Biyani, Krishna

Daga, Soham Daga, Nandini Biyani, Naman Biyani for their love, guidance and support in my

decisions.

I would like to thank my brothers Deepak M. Bajaj, Harshal K. Bajaj, Swapnil Bajaj,

Niket C. Bajaj, to all my sister-in-laws Sau. Aarti D. Bajaj, Neha H. Bajaj, to all my sisters

Hemal M. Bajaj, Suhati K. Bajaj, Shital K. Bajaj, and Mangla M. Bajaj and Monali C. Bajaj, also

to all other nieces Urvi D. Bajaj, Rucha D. Bajaj, Devanshi R. Rathi, Harshita R. Harkanth,

nephew Manan N. Gandhi, Rishab Rathi and Vidhan N. Gandhi. I would also like to thanks my

brother in laws for their encouragement throughout my studies.

I would like to thank all my friends/roommates Dr. Ehesan Sharif, Dr. Ashish Bambal, Javeeria

Ghani, Dr. Somu Chatterjee, Dr. Subodh Kumar, Dr. Manoj Gupta, Mrs Nikita Manoj Gupta, Dr.

Yamini Sharma, Ritika Tulshan, Manjari Tulshan, Amol bhai, Sandeep Lahiri, Chander Prasad

Padamalai, Sanjiv Kaushik, Valmik Doshi, Nimit Shah, Harsha Shekhar, Tathagat Pathak, Pratik

Patel, Kunal Sharma, Ankith Gupta, Arun, Sanjog Rathi, Rakesh Dand, Nimish Somaiya, Alok

Vishwakarma, Nilesh Dhurve, Swati Gupta, Subhash for their help, friendship and guidance in

daily chores. I would like to thank Dr. Novruz G. Akhmedov and Dr. Roger Kautz to help me to

crack down some oligosaccharide structures through NMR confirmation and their friendship. I

would like to thank the Northeastern University for the financial support. I would also like to

thank Cara Shockley, Andy Bean for their help and constant support in the administrative work.

I would also like to thanks Dr. Stewart Campbell, Dr. Jeff Arnold, Mr. Richard Laura, Dr. Greg

Mercer and Dr. Tesmol George for their mentorship, useful suggestions/discussions during my

vi

internship at Corden Pharma, MA. Finally, I would like to thanks every single individual whom I

interacted. I would like to thanks people from my village for the care, love and blessings they

showered on me. I would like to Pranam and thanks Ashutoshji Swami and Avdhoot Swamiji for

their spiritual guidance.

Finally, I bow my head to the ALMIGHTY GOD who gave me enough patience, strength,

courage, positive attitude and good people in the form of parents, brothers, sisters, wife, in-laws,

niece, nephews, friends and mentors to handle and solve the problems during my PhD career.

Without the above mentioned people and gods grace, I would not have been here defending my

thesis.

vii

Abstract of Dissertation

Carbohydrates containing natural products regularly play an impressive role in the

biological activities, like target binding, tissue targeting, cell-cell interaction, cell-cell

recognition and membrane transportation. Despite of the important biological properties, these

highly complex oligosaccharide natural products are still challenging synthetic targets to make.

The ODoherty group has been using a de novo asymmetric approach to build on the desired

functionality and chirality within the molecule starting from achiral moiety. The importance of

this approach comes from the fact that all the stereocenter(s)/chirality in the molecule are built

using an achiral starting material (2-acetyl furan), in contrast to the traditional approaches which

starts from the known carbohydrate starting materials. This approach relies on a highly dia-

stereoselective Palladium (0)-catalyzed glycosylation reaction to control the anomeric

stereochemistry, followed by post-glycosylation transformations (Luche reduction, Upjohn

dihydroxylation) to install the desired functionality and stereochemistry in the sugar moiety. In

this regard, we have developed Pd-/B-dual catalysis for the regio- and stereo-selective synthesis

of complex oligosaccharide class of natural products which in turn help us to expand the SAR

network towards the anti-cancer studies.

Oligo-mannose related motifs got most attention as they are ubiquitously present on the

surface of the mammalian cells and has been looked as an emerging field towards the studies

related to cancer/HIV vaccine development. Building oligosaccharide motifs in a regioselective

manner was always seen as a challenge, to address this issue we have developed and utilized the

Pd-/B-dual catalysis for the regioselective glycosylation of mannose related oligosaccharides

based on protecting group free approach. Working towards this goal, we successfully bis-

glycosylated -D-mannose to form trisaccharides and pentasaccharide oligomannose motifs

viii

using the Pd-/B-catalysis.

Mezzettiasides, a class of partially acetylated highly complex oligosaccharide natural

products possess anti-cancer activities in the M range and were isolated from the fruit and stem

bark of Mezzettia leptopoda. They are divided into 3 subclass i.e., disaccharides (Mezzettiaside-

9-11), trisaccharides (Mezzettiaside 2-3 and 8), tetrasaccharides (Mezzettiaside 5-7) attached to

each other in 1,3-linked fashion. Inspired from the synthesis and biological importance of a

related class of natural products, the cleistriosides and cleistetrosides, we synthesized and

evaluated the SAR studies of mezzettiasides by exploiting a de novo asymmetric approach (i.e.,

building chirality in the molecules from achiral starting material). This highly divergent first total

synthesis features the iterative use of Pd-/B-dual catalyst using nucleophilic boron/electrophilic

palladium for the regio-/stereo-selective glycosylation. It is worth noting that syntheses of all this

10 divergent members were completed in a range of 13-22 linear steps, yet only 42 total steps

were required. Only 1 protecting group (AcCl) was used throughout the synthesis of 9 final

products whereas, for the synthesis of 10th member, the same protecting group (AcCl) was used

two times. Along the line, four new disaccharide unnatural analogues were synthesized for

studying the different acetylation pattern (Ac/n-Pr/i-Pr/t-Bu) and concluded that smaller the

group, better the activity and less sterically hindered acetylating agent are more potent than the

sterically hindered ones. Chapter 2 describes this in details.

Based on the developed Pd-/B-dual catalyzed glycosylation methodology, we planned to

utilize this concept towards the synthesis of digitoxin analogues using protecting group free

strategy, planning towards the synthesis of digitoxin 1,3-linked analogues. Digitoxin is a

naturally occurring cardiac glycoside which has been used in the treatment of congestive heart

failure but due to toxicity, its applications in the clinics are limited. Recent study focuses

ix

digitoxins potential application in cancer and viral (HCMV) infections. In this regards, we and

several other groups have recently reported that digitoxin can be studied towards its potential as

an anti-cancer agent. Our final goal is to synthesize the novel molecule(s) which will selectively

target the tumor cells while maintaining the efficacy and high potency with low toxicity. In this

regard, we have made a library of digitoxin oligosaccharide (mono- to tetra-saccharides) with -

L/D and performed SAR studies. From the SAR studies it was concluded that sugar

stereochemistry plays an important role in the anticancer studies. Chapter 3 describes this in

detail.

With the great success of 1,3-linked digitoxin oligosaccharide analogues, we planned to

shift our focus towards the amino version of the digitoxin. As amines are the most interesting

pharmaceuticals and are present as a backbone in the most active molecules, may be because

amine containing compound are much more soluble, can form H-bonding, interact with the

proteins and can pass through the membrane faster and has a tremendous effect on the potency.

Working towards this aim of synthesizing digitoxin amino analogues, we invested our Pd-/B-

methodology for the synthesis of mono and di-saccharide amino analogues and finally compared

the importance of NH2 vs OH group towards the potency by varying the installation of amine

group on different carbohydrate chain. Along this path, we have also successfully glycosylated

the natural digitoxin molecule on the C-4 position of the last digitoxose sugar so that we can

make the tetrasaccharide amino compounds with -L/D stereochemistry and check its

applications towards the cancer cell lines.

x

Table of Contents

Dedication....ii

Acknowledgements........iii

Abstract of dissertation..vii

Table of contents..x

List of figures....xiii

List of schemes.xiv

List of tables..xvi

List of abbreviations....xvii

Chapter 1: Introduction to carbohydrates and development of Pd-/B-mediated dual catalyzed

regio/stereo-selective glycosylation methodology...1

1.1 Background of carbohydrates..1

1.2 Classical methods for glycosylation1

1.3 De Novo approach to carbohydrates and synthesis of Boc-pyranones3

1.4 De Novo approach using Pd-catalysis towards glycosylation..5

1.5 The aims...7

1.6 Development of Pd-/B-mediated dual catalysis towards regio/stereo-selective

glycosylations8

1.7 Effect of C-4 substitution on the regioselectivity..13

1.8 Iterative use of Pd-/B-dual catalysis towards Mannose oligosaccharide structures..15

1.9 Conclusion.20

1.10 References21

xi

Chapter 2: First Total Synthesis of Mezzettiaside natural products via the iterative use of a Pd-

/B-mediated dual catalysis and SAR study22

2.1 Background of Mezzettiasides...22

2.2 De Novo synthesis of Boc-pyranone (2.1.1 or 1.2.3).24

2.3 Retrosynthetic analysis of Mezzettiaside family members24

2.4 Synthesis of the key intermediate 2.4.2 using a dual Pd-/B-catalysis for the regioselective

glycosylation26

2.5 Synthetic approach to mezzettiaside 2-11..28

2.6 Synthesis of four new disaccharide analogues...35

2.7 Bioactivity of all 10 mezzettiasides and four new disaccharide analogues towards anti-

cancer H460 cancer cell line/anti-bacterial strain....37

2.8 Conclusion.38

2.9 References..39

Chapter 3: Synthesis of cardiac glycosides 1,3-linked oligosaccharides via enzymatic like

regio/stereo-selective glycosylation and SAR study.....41

3.1 Introduction to Na+/K+-ATPase pump and Cardiac Glycosides (CGs)42

3.2 Synthesis of monosaccharide digitoxin analogues46

3.3 Synthesis of 1,3-linked digitoxin analogues (di-/tri-/tetra-saccharide)..48

3.4 Biological evaluations of 1,3-linked oligosaccharides..52

3.5 Studies directed towards the synthesis of monosaccharide C4 amino-digitoxin

analogues....53

3.6 Carbohydrate as mimic to hydrated ions...55

3.7 Synthesis of Boc-azide as a glycosyl donor...56

xii

3.8 Regioselective synthesis of disaccharide amino-digitoxin analogues...57

3.9 Conclusion.59

3.10 References60

Chapter 4: Experimental Section..62

4.1 Chapter 1 Experimental Procedure62

4.2 Chapter 2 Experimental Procedure73

4.3 Chapter 3 Experimental Procedure..128

Appendix: 1H and 13C NMR Spectrum

xiii

List of Figures

Figure 2.1: Mezzettiaside family of natural products.23

Figure 3.1: General representation of cardiac Glycosides ..45

Figure 3.2: Crystal structure of Ouabain, a cardiac glycoside.....46

Figure 3.3: General representation of digitoxin...........46

Figure 3.4: Carbohydrate as a mimic to hydrated ions....55

xiv

List of Schemes

Scheme 1.1: Classical method for O-glycosylation...2

Scheme 1.2: De Novo synthesis of Boc-pyranones ( / -L/D-Boc-pyranones).4

Scheme 1.3: Diastereoselective Palladium catalyzed glycosylation through Pd- -allyl

Complex6

Scheme 1.4: Post-glycosylation transformations...7

Scheme 1.5: Tin-oxide mediated Benzylation...8

Scheme 1.6: General representation of Cleistrio-/Cleistetro-side natural products..9

Scheme 1.7: Regioselective glycosylation using Taylors catalyst.10

Scheme 1.8: Regio-/Stereo-selective glycosylation11

Scheme 1.9: Regio-/Stereo-selective synthesis of disaccharide using Pd-/B-dual catalysis...13

Scheme 1.10: General representation of effect of C-4 substitution on regioselectivity......14

Scheme 1.11: Plausible mechanistic representation of bis-glycosylation on Mannose...17

Scheme 1.12: Bis-glycosylation on methyl- -D-mannose..18

Scheme 1.13: Iterative Bis-glycosylation on trisaccharide..19

Scheme 1.14: Utilization of Pd-/B-methodology towards bis-glycosylation..20

Scheme 2.1: Synthesis of -L-Boc-pyranone..24

Scheme 2.2: Retrosynthetic analysis of mezzettiaside 2-11....25

Scheme 2.3: Synthesis of key intermediate 2.3.426

Scheme 2.4: Synthesis of key intermediate 2.4.227

Scheme 2.5: Synthesis of mezzettiaside-9 (9).28

Scheme 2.6: Synthesis of mezzettiaside-10 (10) 29

Scheme 2.7: Synthesis of mezzettiaside-11 (11).29

xv

Scheme 2.8: Synthetic approach to mezzettiaside-8 (8)..30

Scheme 2.9: Approach to mezzettiaside-4 (4) ....31

Scheme 2.10: Approach to mezzettiaside-2 (2)...31

Scheme 2.11: Synthesis of mezzettiaside-3 (3)...32

Scheme 2.12: Approach towards mezzettiaside-5 (5) .33

Scheme 2.13: Synthesis of mezzettiaside-6 (6) and mezzettiaside-7 (7).34

Scheme 2.14: Approach towards unnatural disaccharide analogues (2.14.1-2.14.2)..36

Scheme 2.15: Approach towards unnatural disaccharide analogues (2.15.1-2.15.2)..36

Scheme 3.1: De Novo synthesis of glycosyl donors and -L-/D-Digitoxin

monosaccharides.47

Scheme 3.2: Regioselective glycosylation using Pd-/B-catalysis.......49

Scheme 3.3: Synthesis of trisaccharide-digitoxin analogues...50

Scheme 3.4: Synthesis of tetrasaccharide-digitoxin analogues...51

Scheme 3.5: Synthesis of C-4-amino-digitoxin monosaccharide analogues..54

Scheme 3.6: Synthesis of -L-Boc-azide56

Scheme 3.7: Synthesis of disaccharide amino-digitoxin analogue..57

Scheme 3.8: Synthesis of digitoxin disaccharide amino analogue..58

Scheme 3.9: Synthesis of diamino-digitoxin disaccharide analogue...59

xvi

List of Tables

Table 1.1: Optimization of regio-/stereo-selective glycosylation....12

Table 1.2: Effect of C-4 substitution on regioselectivity ....14

Table 2.1: Optimization of the regio-selectivity of glycosylation...27

Table 2.2: Anticancer activity ( M) for the isolated mezzettiasides...35

Table 2.3: Anticancer/antibacterial data for the 10 natural and 4 synthetic disaccharide

analogues37

Table 3.1: Anticancer data of digitoxin analogues using H460 cell line.52

Table 3.2: Anticancer data of monosaccharide C-4 amino-digitoxin analogues.55

xvii

List of Abbreviations

Ac Acetyl

Anal. Analysis

Bn Benzyl

Boc t-Butoxycarbonyl

bp Boiling point

Bu Butyl

BuLi n-Butyllithium

Calcd Calculated

CI Chemical Ionization

ClAc Chloroacetyl

CTAB Cetyltrimethylammonium bromide

d Doublet

DBA trans, trans-dibenzylideneacetone

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

de Diastereomeric excess

DEAD Diethyl azodicarboxylate

Chemical shift (ppm)

DMAP 4-Dimethylaminopyridine

DMSO Dimethyl sulfoxide

ee Enantiomeric excess

EI Electron ionization

ent Enantiomer

xviii

equiv Equivalent(s)

ESI Electron spray ionization

Et Ethyl

EtOAc Ethyl acetate

g Gram(s)

h Hour(s)

HRMS High resolution mass spectrum

Hz Hertz (cycles per second)

IR Infrared

J Spin-spin coupling constant

mol Mole(s)

m Multiplet

Me Methyl

mg Milligram(s)

MHz Megahertz

MIC Minimum inhibitory concentration

min Minute(s)

mmol Millimole(s)

mp Melting point

MS Mass spectrum

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NBS N-Bromosuccinimide

NBSH o-Nitrobenzenesulfonylhydrazide

xix

NMO N-Methylmorpholine N-oxide

NMR Nuclear magnetic resonance

Ph Phenyl

PMB p-Methoxybenzyl

Ppm Parts per million

Py Pyridine

q Quartet

Rf Ratio to front

rt Room temperature

t Triplet

THF Tetrahydrofuran

TLC Thin layer chromatography

p-TsOH p-toluene sulfonic acid

TBAI Tetrabutylammonium iodide

1

Chapter 1

Introduction to carbohydrates and development of Pd-/B-mediated dual

catalyzed regio-/stereo-selective glycosylation methodology

1.1 Background of Carbohydrates

Carbohydrates containing natural products play an impressive role in the biological processes

(like cell death, cell-cell recognition, target binding, cell-cell interaction, tissue targeting and

membrane transportation).1,2 These attracted the synthetic community to study the role of

carbohydrates in biologically active natural/unnatural products. Although, nature provides

biologically active carbohydrate motifs but chemists are hampered by the limited number of

sugar isomers when they try to perform carbohydrate based structure activity relationship (SAR)

studies. Often times aglycon core of natural products are devoid of activities when the

carbohydrate motif is removed (e.g., Jadomycin A vs Jadomycin B3 and Digitoxigenin vs

Digitoxin). Hence the need for alternative carbohydrate synthetic methodologies which

systematically installs rare natural and unnatural sugars are required to enable drug discovery

efforts. These highly complex carbohydrate natural products are challenging synthetic targets. A

practical total synthetic approach toward these natural/unnatural products is one that enables the

supply of abundant material for SAR studies.

1.2 Classical methods for glycosylation

Glycosylation is one of the most meticulously studied reactions in chemistry. The reaction

is a process in which a nucleophile acts as a glycosyl acceptor and attacks the electrophile to

form a glycosidic bond in presence of an activator or Lewis acid catalyst.3 The new glycosidic

bond is formed by the displacement of the leaving group (e.g., -OTf, -Cl, -Br, -I, -OC=(NH)-R, t-

2

Boc) at the anomeric position of the sugar by a nucleophile, often in the presence of an activator.

In this process, the nucleophile is referred as glycosyl acceptor, whereas, the incoming group

acts as a glycosyl donor (electrophile).

The most typically used glycosylation methods are the Koenigs-Knorr glycosylation and

Schmidt glycosylation. The Koenigs-Knorr glycosylation4,5 involves the nucleophilic

displacement of halide (chloride/bromide as leaving group) at the anomeric position in presence

of promoter. Whereas, the Schmidt glycosylation6 displaces a trichloroacetamidate. The

generally accepted mechanism involves the ionization of the leaving group at the anomeric

center followed by a nucleophilic addition.

Scheme 1.1: Classical method for O-glycosylation

Using traditional glycosylation methodologies, the nucleophile can attack from two

possible faces: top ( ) or bottom ( ) face (Scheme 1.1). If the nucleophile attacks from the top

face, a -isomer is formed, whereas, if the nucleophile attacks from the bottom face, the -

isomer is obtained. The traditional glycosylation is a powerful methodology but does suffer from

the following drawbacks:

a) It often produces a mixture of anomers ( / ) in varying ratios thus affecting the yield (Scheme

1.1).

b) It often requires stoichiometric amount of promoter/catalyst (e.g., TMSOTf, TfOH, AgOTf).

3

c) It requires protection/deprotection manipulation which increases total number of steps.

d) It is sensitive to moisture, temperature, solvent dependent and highly relies upon the structure

of donor and acceptor.

In order to overcome these problems, there is a continuing need to develop new

glycosylation methodology which will address the concerns and rely on catalytic amount of

activator. In this regards, the ODoherty lab has developed a Pd-catalyzed glycosylation

methodology under mild reaction conditions thus maintaining the good anomeric selectivity

(Section 1.4).

1.3 De novo Achmatowicz approach to carbohydrates and synthesis of Boc-pyranones

In contrast to the classical glycosylation methods where carbohydrates containing starting

materials being used for the synthesis of carbohydrate structures, the ODoherty group used a de

novo asymmetric approach to build carbohydrate motifs. This de novo approach to sugars installs

the desired functionality and chirality from achiral moieties (e.g., 2-acetylfuran 1). The

uniqueness of this approach comes from the fact that all the stereocenters in the molecule are

built onto the achiral framework of 2-acetylfuran 1. We call this concept a de novo asymmetric

approach towards sugars. This approach relies on a highly diastereoselective palladium(0)-

catalyzed glycosylation in combination with asymmetric catalysis to control the anomeric

stereochemistry.7

The de novo asymmetric approach begins with the synthesis of the key glycosyl donors

(Boc-pyranones) using Noyori reduction and Achmatowicz oxidative rearrangement. The

synthesis of Boc-pyranone (1.2.3, 1.2.4, (ent)-1.2.3 and (ent)-1.2.4) starts with achiral 2-

acetylfuran 1, which underwent an enantioselective Noyori asymmetric hydrogenation using the

4

Noyori catalyst (Noyori (S,S) or Noyori (R,R)) and sodium formate (hydrogen source). This

highly scalable procedure provided the corresponding chiral furanols (1.2.1 and ent-1.2.1) with

excellent enantiocontrol. The furan alcohols (1.2.1/ent-1.2.1) underwent Achmatowicz oxidative

rearrangement8 upon exposure to aqueous NBS conditions (NBS/NaOAc/H2O) to provide an

equilibrating mixture of hemiacetals 1.2.2/ent-1.2.2. Hemiacetal 1.2.2 with the C-5 L-

configuration was protected with Boc2O/DMAP ( 78 C) to provide a separable mixture of -L-

Boc-pyranone 1.2.3 and -L-Boc-pyranone 1.2.4 in ~3:1 ratio. Alternatively, when hemiacetal

ent-1.2.2 was heated to 80 C with Boc2O/DMAP in benzene, -D-/ -D- oc-pyranone (ent-

1.2.3/ent-1.2.4) were obtained in 1:1 ratio (Scheme 1.2).7

Scheme 1.2: De Novo synthesis of Boc-pyranones ( / -L/D-Boc-pyranones)

Thus in just a three step sequence, achiral starting material 1 was converted into any possible

stereoisomers of the Boc-pyranones. This practical procedure provides the four stereoisomers on

multigram scale. Once the / are separated, the stereochemistry can be transformed with the

complete control using our Pd-chemistry. In turn, this stereochemistry is used to install the

5

remaining stereocenters with the post glycosylation reaction (Luche reduction (NaBH4/CeCl3)

and Upjohn dihydroxylation (OsO4/NMO)) to provide an acceptor (i.e., a glycosyl donor was

converted to glycosyl acceptor using post-glycosylation transformation).

1.4 De Novo approach using Pd-catalysis towards glycosylation

The ODohertys Pd-catalyzed steroselective glycosylation has been used for the synthesis

of natural/unnatural carbohydrates motif(s) with interesting biological activity. This Pd-catalyzed

reaction is highly stereospecific (i.e., -pyranones results in -anomeric products) and occurs

under mild reaction conditions (i.e., 0 C) in the presence of catalytic amount (1-5 mol%) of

Pd2(dba)3CHCl3/4PPh3 as a catalyst. The advantage of this Pd-catalyzed methodology is: a) its

compatibility with wide variety of solvents (e.g., CH2Cl2, THF, DMF, CH3CN); b) its reliance on

a catalytic amount of Pd-catalyst and c) its predictable stereochemistry.

The reaction proceeds through a Pd- -allyl intermediate complex which serves as the

glycosyl donor and reacts with an alcohol nucleophile (glycosyl acceptor) with net retention of

stereochemistry. Thus the incoming nucleophile retains the anomeric stereochemistry of the

starting material, (i.e., double inversion of stereochemistry, Scheme 1.3). This is seen as

advantageous over traditional glycosylation reactions which often provide mixture of products at

the anomeric position.7

6

Scheme 1.3: Diastereoselective Palladium catalyzed glycosylation through Pd- -allyl complex This Pd-catalyzed glycosylation can be applied towards a wide range of alcohol nucleophiles

under mild reaction condition [Pd2(dba)3CHCl3/4PPh3 (2.5 mol%)] with decent yields. The

enone portion of the Boc-pyranone not only serves as an anomeric directing group during Pd(0)

catalyzed glycosylation, it also serves as a triol protecting group (i.e., masked enone as an atom-

less protecting group). That is to suggest that the post-glycosylation transformations function as

triol deprotection steps, for example, reduction of the enone 1.4.1 under Luche conditions

(NaBH4/CeCl3) gave C-4 allylic alcohol 1.4.2. Then the resulting allylic alcohol 1.4.2 was

subsequently dihydroxylated using the Upjohn condition (OsO4/NMO) to form a manno-triol

1.4.3a (X = -OH). Similarly, when a diimide (NBSH/Et3N) reduction was performed on the

double bond, amicetose sugar 1.4.3b (X = -H) was obtained. The allylic alcohol 1.4.2 can act as

an acceptor and can be expanded to prepare series of 1,4-linked motifs using Pd-catalyzed

glycosylation with Boc-pyranone 1.2.3, thus providing disaccharide 1.4.5 after post

glycosylation transformations (Scheme 1.4).

7

Scheme 1.4: Post-glycosylation transformations

1.5 The aims

The initial aim of my PhD studies was to develop a general methodology for regio-/stereo-

selective Pd-catalyzed glycosylation of manno-1,2 diols at the equatorial C-3 position. In this

regards, we have developed a Pd-/B-dual catalyzed glycosylation methodology for the regio-

/stereo-selective glycosylations of 1,2 and 1,3 diols. An important feature of this methodology is

how it can enables the minimal use of protecting groups and in some applications protecting

group free synthesis of natural/unnatural oligosaccharide structures. The applications of this Pd-

/B-dual catalyzed methodology have been successfully applied towards the following synthetic

applications:

One pot regio/stereo-selective bis-glycosylation of mannose related oligosaccharides.

First total synthesis of mezzettiaside 2-11, anti-cancer natural products using divergent

approach (see Chapter 2).

8

Protecting group free synthesis of digitoxin oligosaccharide and amino analogues

towards cancer studies (see Chapter 3).

1.6 Development of Pd-/B-mediated dual catalysis towards regio-/stereo-selective

glycosylations

The regio-/stereo-selective functionalization of a single hydroxyl group in presence of other

unmasked hydroxyl group(s) in the carbohydrate motif is seen as a challenge as it is the key for

building oligosaccharide with the minimal use of protecting groups. Even when enzymatic

approaches are available, there is no assurance that the enzymatic glycosylation will be general

(e.g., able to transfer a / -D-/L-sugar to its corresponding / -D-/L products). Previous

regioselective studies on carbohydrate mainly focused on organotin mediated regioselective

glycosylation using stoichiometric dibutyltin oxide (Bu2SnO), tributyltin oxide (Bu3Sn)2O and

dibutyltin dimethoxide (Bu2Sn(OMe)2) (Scheme 1.5).

Scheme 1.5: Tin-oxide mediated Benzylation

The problems associated with the tin chemistry were its toxicity, requirement for stoichiometric

amount, absolute need for anhydrous conditions (Dean-Stark) and long reaction time. Most

9

problematically, in our hands these reactions suffered from poor reproducibility in terms of

regio-selectivity and yields. Other problems associated with tin chemistry are the requirement for

a two steps procedure, in which the sugar (glycosyl acceptor 1.6.1) is first reacted with tin oxide

in methanol or benzene/toluene to form an intermediate tin-acetal 1.6.2. Then intermediate 1.6.2

reacts with the corresponding electrophile Boc-pyranone 1.2.3 to give the glycosylated product

in presence of palladium catalyst. For instance, our Pd-catalyzed regioselective glycosylation

between 1.6.2 and 1.2.3 yielded a mixture of C-3:C-2 (7:1) products. This particular reaction was

the representative of the issue of poor reproducibility (Scheme 1.6).9

Scheme 1.6: General representation of Cleitrio/Cleistetro-side natural product

While progress was made towards the regioselective acylation/tosylation/alkylation (Prof.

Taylors work) of the carbohydrate motifs10, the regioselective glycosylation of the unprotected

carbohydrates to form a specific linkage by using tin-chemistry has been still an unsolved

problem. In this regards, the scope for the development of new methodology for stereo- and

10

regio-selective glycosylation that does not rely on tin chemistry was seen as a potential major

improvement.

To address the issues related to regio-/stereo-selectivity, reproducibility and yield in the

glycosylation reaction, there was a strong desire from the synthetic carbohydrate community to

develop alternative methodology which could use a catalytic method for the regio-/stereo-

selective glycosylation of polyol sugars. The most advantageous part of this endeavor would be

the avoidance of unnecessary protection/deprotection steps.

In this regard, Prof. Taylor recently developed a borinate ester (Ph2BOCH2CH2NH2)

catalyst that promoted the regioselective acylation/tosylation/alkylation/glycosylation. The

Taylors catalyst (Ph2BOCH2CH2NH2) via intermediate of a cyclic boronate can direct the

incoming group to the equatorial C-3 of an unprotected rhamnose, mannose and pyranose sugars,

using 1.1 equiv. of Ag2O for glycosylation (Scheme 1.7).10

Scheme 1.7: Regioselective glycosylation using Taylors catalyst

11

Inspired by this approach, we were interested in developing a general methodology which not

only provides the desired regioselectivity, but also imparts / -stereoselectivity. This in turn

allows libraries of biologically active carbohydrates structures to be synthesized in fewer steps

with complete control of regio/stereo-selectivity. Working towards this aim, we have developed

a new methodology which utilizes a combination of nucleophilic boron and electrophilic

palladium(0) catalysis to install the incoming glycosyl donor selectively to get an equatorial C-3

and/or C-6 hydroxy of a pyranose with complete control of -stereochemistry. Discussed in this

Chapter is the development of Pd-/B-mediated dual catalyzed glycosylation methodology, in

which the nucleophilic boron imparts the regioselectivity whereas, the electrophilic Pd controls

the -selectivity.

With the above strategy in mind, we started exploring our initial attempts for the

regioselective glycosylation on the C-4 substituted rhamnose motif 1.8.1 (glycosyl acceptor)

with Boc-pyranone (glycosyl donor 1.2.3). Initially, rhamnose 1.8.1 underwent regio/stereo-

selective Pd-catalyzed glycosylation in presence of tin oxide (Bu2SnO, 1.1 eqvi.) to give an

inseparable mixture of C-3:C-2 (5:1) glycosylated product (1.8.2 and 1.8.3).

Scheme 1.8: Regio/Stereo-selective glycosylation

12

Inspiring by this problem, we investigated the use of a Pd-/B-dual catalyzed glycosylation. The

Taylor catalyst ultimately led to the regio-/stereo-selective C-3 glycosylation in good yields of

C-4 substituted sugars. We began our studies with the C-4 substituted rhamnose 1.8.1. When diol

1.8.1 was reacted with 15 mol% borinate catalyst and glycosyl donor 1.2.3 (see Chapter 2, for

application to the mezzettiasides) using CH2Cl2 as a reaction solvent, a regiomeric ratio for

1.8.2:1.8.3 of ~2.5:1 was observed (Table 1.1).

Table 1.1: Optimization of regio/stereo-selective glycosylation

Reducing the amount of borinate catalyst to 10 mol% in CH3CN/CH2Cl2 had no significant effect

on the regiomeric ratio. Whereas, when 15 mol% catalyst was used in CH3CN/THF (1:0.1), the

regiomeric ratio improved two fold (i.e., 6:1). Finally, the glycosylation condition was optimized

to 30 mol% borinate catalyst in CH3CN/THF (1:0.1) which provided the best regioselectivity

ratio (7.5:1) in decent yield (77%) (Table 1.1).11

The plausible mechanism for the Pd-/B-mediated dual catalyzed glycosylation is shown in

Scheme 1.9. Initially, the diol forms an anionic tetra-coordinated borinate intermediate 1.8.1a

13

whereas, the Boc-pyranone forms a cationic palladium -allyl complex. From the observations,

we can conclude that the reaction solvent plays a critical role in regioselectivity, as shown in

Table 1.1.

Scheme 1.9: Regio/Stereo-selective synthesis of disaccharide using Pd-/B-dual catalysis

1.7 Effect of C-4 substitution on the regio-selectivity

With the successful development of the iterative Pd-/B-mediated dual catalysis

methodology, we explored a range of C-4 substituted sugars to gauge its effect on the

regioselectivity. When rhamnose 1.10.4 was used as starting material (i.e., C-4 = -OH), the

pyranone donor was regio/stereo-selectively delivered exclusively at the C-3 position of

rhamnose (Entry 1, Table 1.2) to provide 1.10.5. Whereas, when C-4 was changed to an acetoxy

group 1.10.6, the ratio of the incoming group being transferred to the C-3 vs C-2 was 15:1 (Entry

2). In addition, when the steric at C-4 was further increased to a hexanoate (i.e., C-4 = -

OC(O)(CH2)4CH3, 1.8.1), the ratio dropped to 7.5:1 (Entry 3). Finally, when C-4 was changed to

an azide (i.e, C-4 = -N3), the C-3/C-2 ratio was found to be 16:1 (Entry 4). From these

observations we concluded that both steric and electron withdrawing group at C-4 affects the

14

regioselectivity, electron withdrawing groups at C-4 reduces the selectivity. Smaller the

substituent at C-4 being better for the regioselectivity (Table 1.2)

Scheme 1.10: General representation of effect of C-4 substitution on regioselectivity

Table 1.2: Effect of C-4 substitution on regioselectivity

With the successful development of an iterative Pd-/B-mediated dual catalysis methodology

towards regio- and stereo-selective glycosylation, we wanted to expand its application towards

the synthesis of biologically active natural/unnatural compounds having range of sugar units

15

(mono- to oligo-saccharides).

We were also fascinated at the possibility of using this methodology to exploit an SAR

study of a biologically active natural products. In this regard, we have successfully used this

methodology for the first total synthesis of the mezzettiasides.11,12 The mezzettiasides are a

family of anti-cancer natural products (details will be discussed in Chapter 2). Finally Chapter 3

talks about the use of Pd-/B-dual catalysis towards the protecting group free synthesis of

digitoxin oligosaccharide analogues and its amino sugar analogues.

Presented in this chapter, is the use of this Pd-/B-dual catalyzed regio/stereo-selective

glycosylation in the iterative bis-glycosylation of mannose to form oligosaccharide structures. In

this regards, we initially started to explore the glycosylation reactions between methyl- -D-

mannose 1.11.1 and Boc-pyranone (ent)-1.2.3. The glycosyl acceptor (methyl- -D-mannose

1.11.1) underwent bis-glycosylation using the Pd-/B-dual catalysis to form trisaccharide enone

1.11.3 (Scheme 1.12). The most interesting feature of this methodology is that, in just 6 steps

starting material methyl- -D-mannose was efficiently converted to a fully functionalized

pentasaccharide motif.

1.8 Iterative use of Pd-/B-dual catalysis towards Mannose oligosaccharide structures

Carbohydrates not only act as energy storage but also play key roles in number of

biological processes (e.g., viral/bacterial infections, angiogenesis, tumor cell metastasis,

inflammation, immune response).13 Due to this vast scope of carbohydrates in biology, it has

become an emerging subject in drug discovery.

Working towards this goal, we planned to synthesize mannose related oligosaccharide

motifs to uncover the potential applications of our above mentioned Pd-/B-catalyzed

16

glycosylation methodology towards the synthesis of oligosaccharide motifs. Of the

oligosaccharides, the oligomannose containing motifs has gotten most attention as oligomannose

related structures are ubiquitously present on the surface of the mammalian cells and are

involved in processes such as protein quality control, cell-cell communication, cell-cell

differentiation, viral infection and tumor migration.13

Herein, we report regioselective and stereoselective Pd-/B-dual catalysis strategy for the

protecting group free synthesis of 3,6-linked mannose trisaccharides. The approach started with

the bis-glycosylation of methyl- -D-mannopyranoside 1.11.1 and -pyranose (glycosyl donor,

ent-1.2.3). Initially it forms a six membered cyclic intermediate 1.11.1a (Scheme 1.11) which

reacts with the palladium -allyl complex at C-6 position to give a C-6 glycosylated product

1.11.2. Subsequently it then forms a five membered ring tetra-coordinated boron intermediate

1.11.2a which then react regioselectively to install the -D-Boc-pyranone (glycosyl donor)

equatorially at the C-3 position 1.11.3. Thus in a net one pot reaction 1.11.1 is converted into

trisaccharide 1.11.3 cleanly in a 68% yield (Scheme 1.11). The most important feature of this

iterative bis-glycosylation is that no protecting groups were required for the synthesis of

oligosaccharides. A schematic representation of bis-glycosylated oligosaccharide is shown in

Scheme 1.11.

17

Scheme 1.11: Plausible mechanistic representation of bis-glycosylation on Mannose

The synthesis of oligosaccharide motifs starts from commercially available methyl- -D-

mannopyranoside 1.11.1 which upon treatment with borinate catalyst (35 mol%) and

Pd2(dba)3CHCl3/4PPh3 catalyst (5 mol%) underwent iterative regio/stereo-selective bis-

glycosylation using -D-Boc-pyranone ent-1.2.3 to provide trisaccharide dienone 1.11.3 (68%).

Dienone 1.11.3 was reduced (NaBH4/CeCl3) to corresponding bis-allylic alcohol 1.12.1 in 90%

yield. Alcohol 1.12.1 was dihydroxylated using OsO4/NMO to provide fully functionalized

trisaccharide compound 1.12.2 (79%) (Scheme 1.12).

18

Scheme 1.12: Bis-glycosylation of methyl- -D-mannose

Building upon this success we were intrigued to see if we could explore Pd-/B-dual catalysis on

the trisaccharide moiety 1.12.2 at the C-3/C-3 hydroxyl positions respectively to form a

pentasaccharide motif without using any protecting group. This was accomplished by bis-

glycosylating trisaccharide motif 1.12.2 with -D-Boc-pyranone ent-1.2.3 regoiselectively at C-

3/C-3 positions respectively with full control of -selectivity to give pentasaccharide dienone

1.13.1. Dienone 1.13.1 underwent a Luche reduction (NaBH4/CeCl3) followed by Upjohn

dihyroxylation (OsO4/NMO)) to form pentasaccharide 1.13.2 (Scheme 1.13).

19

Scheme 1.13: Iterative Bis-glycosylation on trisaccharide

Finally, we have shown that we can successfully regio/stereo-selectively glycosylate in

one pot using the Pd-/B-dual catalysis. The importance of this bis-glycosylation comes from the

fact that pentasaccharide motif 1.13.2 can be prepared in just 6 steps, without the use of any

protecting group(s). In order to demonstrate the stereochemical variability, we glycosylated

methyl- -D-mannose 1.11.1 with -L-Boc-pyranone 1.2.3 to obtain trisaccharide enone 1.14.1,

which further underwent post-glycosylation transformations (NaBH4/CeCl3 and OsO4/NMO)

providing fully functionalized trisaccharide moiety 1.14.2 (Scheme 1.14).

Scheme 1.14: Utilization of Pd-/B-methodology towards bis-glycosylation

20

1.9 Conclusion

In summary, we have successfully developed a de novo methodology using nucleophilic

boron and electrophilic palladium which can be used towards regioselective installation of sugars

with complete retention of anomeric stereochemistry. Utilization of this method was shown in

the iterative bis-glycosylation of manno-pyranose motif to form mannose related oligosaccharide

structures. The most important feature of the Pd-/B-dual catalyzed reaction is the lack of

protecting groups. For instance, the synthesis of the pentasaccharide motif only required 6 steps.

Thus the synthesis is highly efficient and affordable toward carbohydrate motifs for medicinal

chemistry studies.

Future studies focusing on utilizing galactose (as an acceptor) and cyclitols/carbasugar

(as donors) are under investigation in the ODoherty group. Applications of this developed

methodology towards different carbohydrate structures can be seen in the total synthesis of

mezzettiaside natural products using minimal protecting group and in the protecting group free

synthesis of digitoxin oligosaccharide analogues as discussed in Chapter 2 and 3 respectively.

21

1.10 References

1. For useful reviews see: (a) P. R. Crocker, T. Feizi, Curr. Opin. Struct. Biol., 1996, 6, 679; (b) E. Herbert, Biosci. Rep., 2000, 20, 213.

2. For useful reviews see: (a) H. Lis, N. Sharon, Chem. Rev., 1998, 98, 637; (b) S. Houlton,

Chem. Br., 2002, 38, 46. 3. (a) L. Wang, R. L. White, L. C. Vining, Microbiology, 2002, 148, 1091; (b) M. Shimizu, H.

Togo, M. Yokoyama, Synthesis, 1998, 799. 4. W. Koenig, E. Knorr, Ber., 1901, 34, 957.

5. H. Paulsen, Angew. Chem. Int. Ed. Engl., 1982, 21, 155; (b) G-J. Boons, Carbohydrate Chemistry; Blackie Publishers; London, 1998; (c) S. H. Khan, R. A. ONeill, Modern Methods in Carbohydrates Synthesis; Harwood Academic: Amsterdam, 1996; (d) K. C. Nicolaou, H. J. Mitchell, Angew. Chem. Int. Ed., 2001, 40, 1576; (e) D. P. Galoni, D. Y. Gin, Nature, 2007, 446, 1000.

6. R. R. Schmidt, J. Michel, Angew. Chem. Int. Ed. Engl., 1980, 19, 731; (b) R. R. Schmidt,

Angew. Chem. Int. Ed. Engl., 1986, 25, 212.

7. (a) R. S. Babu, G. A. ODoherty, J. Am. Chem. Soc. 2003, 125, 12406; (b) Concurrent with these studies was the similar discovery by Feringa, Lee; A. C. Comely, R. Eelkema, A. J. Minnaard, B. L. Feringa, J. Am. Chem. Soc., 2003, 125, 8714; (c) H. Kim, H. Men, C. Lee, J. Am. Chem. Soc., 2004, 126, 1336; (d) The poor reactivity in Pd-catalyzed allylation reaction of alcohols as well as a nice solution to this problem was reported, see: H. Kim, C. Lee, Org. Lett., 2002, 4, 4369; (e) For a related Rh system, see: P. A. Evans, L. J. Kennedy, Org. Lett., 2000, 2, 2213; (f) S. O. Bajaj, J. R. Farnsworth, G. A. ODoherty, Org. Synth., 2014, 91, 338.

8. O. Achmatowicz, R. Bielski, Carbohydr. Res., 1977, 55, 165.

9. B. Wu; M. Li, G. A. ODoherty, Org. Lett., 2010, 12(23), 5466.

10. (a) D. Lee, M. S Taylor, J. Am. Chem. Soc., 2011, 133, 3724; (b) C. Gouliaras, D. Lee, L. Chan; M. S. Taylor, J. Am. Chem. Soc., 2011, 133, 13926; (c) C. A. McClary, M. S. Taylor, Carbohydr. Res., 2013, 381, 112; (d) E. Dimitrijevic, M. S. Taylor, Chem. Sci., 2013, 4, 3298; (e) T. M. Beale, M. S. Taylor, Org. Lett., 2013, 15, 1358.

11. S. O. Bajaj, E. U. Sharif, N. G. Akhmedov, G. A. ODoherty, Chem. Sci., 2014, 5, 2230. 12. S. O. Bajaj, P. Shi, P. J. Beuning and G. A. ODoherty, Med. Chem. Comm., 2014, 5, 1138.

22

Chapter 2

First total synthesis of Mezzettiaside natural products via the iterative use of a

Pd-/B-mediated dual Catalysis and SAR study

2.1 Background of Mezzettiasides

The zeal for natural products with interesting biological properties, led to the discovery of

Mezzettiasides, a class of partially acetylated oligosaccharide natural products which are known

to be active against cancer cell lines, particularly towards Lu1, Col2 and KB (5 to > 20 M).1,2

This family of oligorhamnoside was isolated from the fruit and stem bark of Mezzettia leptopoda

in Malaysia in 1986.1 The important feature of this class of anti-cancer natural products is that,

all the carbohydrate units have unique acylation pattern and are 1,3-linked oligorhamnose motifs.

This unique acylation pattern makes them a challenging target for synthesis. Mezzettiaside

family members are divided into three subclasses: disaccharide (Mezzettiaside 9-11),

trisaccharide (Mezzettiaside 2-4 & 8) and tetrasaccharide (Mezzettiaside 5-7) (Fig. 2.1).

As part of a program aimed at the synthesis and biological investigation of

anticancer/antibiotic oligosaccharide natural products,3 we became intrigued in the synthesis of

mezzettiasides. The interest came out from our synthesis and study of a related class of natural

products, the cleistriosides and cleistetrosides3, where we found that the pattern of acylation had

a significant effect on the biological activity. As a continuation of these studies, we desired

access to all the ten members of the mezzettiaside natural products.3,4 While re-isolation of this

material from its various sources was considered, we rather planned to synthesize all the

mezzettiasides using our de novo asymmetric approach.

Thus, we envisioned the synthesis of all the mezzettiasides using asymmetric divergent

approach utilizing Pd-catalyzed glycosylation in combination with the nucleophilic boron (Pd-

23

/B-) dual catalysis methodology as discussed in Chapter 1.5 The divergent approach allowed us

the use of minimal protecting group (i.e., only one protecting group (AcCl) was used in the

synthesis of 9 natural products, whereas for the 10th member uses AcCl twice). Herein, we

disclose our successful de novo asymmetric approach to all the ten members of the mezzettiaside

where all the stereocenters were derived from achiral starting material 2-acetyl furan 1. Figure

2.1 shows all the 10 mezzettiasides natural products.

Figure 2.1: Mezzettiasides family of natural products

(Contributions: Sumit O. Bajaj performed all the synthetic work presented here under the

guidance of Prof. ODoherty and Dr. Sharif. Dr. Akhmedov helped with the structure analysis of

some final molecules using detailed NMR studies. The biological evaluation was performed by

Dr. Shi under the guidance of Prof. ODoherty and Prof. Beuning).

24

Along with the first total synthesis of this family of natural products, we have also

planned to synthesize four new disaccharides analogues (2.14.1-2.14.2 and 2.15.1-2.15.2) with

different (R1 = n-Pr, i-Pr, t-Bu, i-Bu) acylation pattern. These products enabled structure activity

relationship (SAR) studies6 where the importance of the sugar chain length and acylation pattern

at various positions of the sugar moiety on the biological activity was investigated.

2.2 De Novo synthesis of Boc-pyranone (2.1.1 or 1.2.3)

The synthesis of Boc-pyranone ( -L) 2.1.1 from inexpensive achiral starting material 2-

acetyl furan 1 is outlined in Scheme 2.1. Pyranone 2.1.1 is a key building block for all the

mezzettiaside family of natural products. The uniqueness of this de novo route is that we can

install the stereochemistry in the sugar products from achiral material 2-acetyl furan 1, using

asymmetric catalysis. This concept is referred to as a de novo approach to carbohydrates.7

Boc-pyranone 2.1.1 could be easily prepared in three steps (Noyori (S,S) enantioselective

asymmetric hydrogenation9, Achmatowicz oxidative rearrangement (NBS/NaOAc/H2O) and

Boc-protection (Boc2)O) from achiral 2-acetyl furan 1 (Chapter 1). In the Pd-catalyzed

glycosylation reaction, the Boc-pyranone 2.1.1 or 1.2.3 acts as a glycosyl donor and the reacting

alcohol is the glycosyl acceptor.7,8

Scheme 2.1: Synthesis of -L-Boc-pyranone

2.3 Retrosynthetic analysis of Mezzettiaside family members

The retrosynthetic route we devised for the mezzettiaside natural products is outlined in

25

Scheme 2.2. We envisioned that the three tetrasaccharides (mezzettiasdie 5-7) and the four

trisaccharides (mezzettiaside 2-4 & 8) could be obtained from a common pyranone trisaccharide

intermediate 2.8.1. In turn, intermediate 2.8.1 as well as the final three disaccharides

(mezzettiaside-9-11) could be prepared from a disaccharide allylic alcohol. Finally, allylic

alcohol 2.4.2 could be obtained from ester diol 2.3.4, whereas, diol 2.3.4 can be made from Boc-

protected pyranone 2.1.1 in 4 steps (Scheme 2.2).

Scheme 2.2: Retrosynthetic analysis of mezzettiaside 2-11

We planned to synthesize all the mezzettiaside using divergent approach. Key to the

success of this approach is the strategic use of enone atomless/minimal protecting groups (enone

of a pyranone as a masked triol and the minimal use of chloroacetate as the only other protecting

group) in combination with the iterative use of a highly regio- and stereo-selective B-/Pd-

catalyzed glycosylation makes this route more interesting.

26

2.4 Synthesis of the key intermediate 2.4.2 using a dual Pd-/B-catalysis for the regio-

selective glycosylation

The synthesis of key intermediate 2.4.2 began with 2-acetyl furan 1. In a 3 steps sequence

acetyl furan 1 (Noyori reduction, Achmatowicz reaction9 and Boc-protection) was converted to

Boc-pyranone 2.1.1. Pyranone 2.1.1 in a palladium catalyzed glycosylation with octan-1-ol

provided enone 2.3.1. Enone 2.3.1 was reduced under Luche reduction condition (NaBH4/CeCl3)

followed by esterification (hexanoic acid, DCC/DMAP) provided C-4 hexanoate motif 2.3.3.

Finally Upjohn syn-dihydroxylation (OsO4/NMO)10 provided diol 2.3.4 (Scheme 2.3).

Scheme 2.3: Synthesis of key intermediate 2.3.4

The C-4 hexanoate diol 2.3.4 was studied in the regioselective glycosylation reaction at O-

3 hydroxy group with stoichiometric amount of tin catalyst (Bu2SnO).3 The main drawback of

the tin chemistry was the requirement for stoichiometric amount. Also, the reproducibility in

regio-isomeric ratio and yield was the hardest part to optimize. As discussed in Chapter 1, we

have developed a new methodology based on catalytic borinate ester to control the

regiochemistry in the glycosylation process. Our optimized condition for the glycosylation

(Entry 2, Table 2.1) uses 30 mol% borinate catalyst (Ph2BOCH2CH2NH2)11,12 and 2.5%

palladium catalyst to provide a mixture of C3:C2 (2.3.5/2.3.6) in 7.5:1 ratio.

27

.

Table 2.1: Optimization of the regio-selectivity of glycosylation

The glycosylated mixture 2.3.5/2.3.6 was chloroacetylated ((ClAc)2O/Py) to provide 2.4.1 and its

C-2 regiomer followed by Luche reduction (NaBH4/CeCl3) to provide desired pure allylic

alcohol 2.4.2 in excellent yield (77%, 2 steps) (Scheme 2.4).

Scheme 2.4: Synthesis of key interemediate 2.4.2

28

2.5 Synthetic approach to mezzettiaside 2-11

The key intermediate 2.4.2 was transformed into disaccharides mezzettiaside 9-11 using a

divergent route. In order to obtain mezzettiaside-9, disaccharide allylic alcohol 2.4.2 was

chloroacetylated ((ClAc)2O/Py) at the C-4 position, followed by dihydroxylation to give diol

2.5.1 (77%, 2 steps). Peracetylation of 2.5.1 (Ac2O/DMAP) then removal of both chloroacetates

(thiourea/NaHCO3/n-Bu4NI)13 provided mezzettiaside-9 (9) (83%, 2 steps) (Scheme 2.5).

Scheme 2.5: Synthesis of mezzettiaside-9 (9)

In a similar fashion, disaccharide allylic alcohol 2.4.2 was acetylated (Ac2O/DMAP), followed

by dihydroxylation (OsO4/NMO) to give C-4 acetate diol 2.6.1 (80%, 2 steps). Diol 2.6.1

underwent treatment with (CH3C(OEt)3/p-TsOH/AcOH) to regioselectively install an axial C-2

acetate in alcohol 2.6.2 (85%). Alcohol 2.6.2 upon chloroacetate deprotection

(thiourea/NaHCO3/n-Bu4NI) furnished mezzettiaside-10 (10) (86%) (Scheme 2.6).

29

Scheme 2.6: Synthesis of mezzettiaside-10 (10)

Allylic alcohol 2.4.2 was dihydroxylated under Upjohn condition (OsO4/NMO), to provide

a triol which upon peracetylation (Ac2O/DMAP) gave triacetate 2.7.1 (81%, 2 steps). Finally

deprotection of the chloroacetate group (thiourea/NaHCO3/n-Bu4NI) gave the desired

mezzettiaside-11 (11) (82%) (Scheme 2.7).

Scheme 2.7: Synthesis of mezzettiaside 11(11)

As mentioned earlier, this route is divergent so the disaccharide intermediate 2.6.2 was

utilized towards the synthesis of trisaccharides mezzettiaside 2-4 and 8 (Scheme 2.8-2.11).

Alcohol 2.6.2 upon treatment with Boc-pyranone 2.1.1 underwent a Pd-catalyzed glycosylation

to give trisaccharide enone 2.8.1 (68%) in presence of Pd2(dba)3CHCl3/4PPh3. Enone 2.8.1 upon

30

post-glycosylation transformations provided trisaccharide triol 2.8.2 (79%, 2 steps). Triol 2.8.2

was then regioselectively C-2 acetylated ((CH3C(OEt)3/p-TsOH/AcOH) to give trisaccharide

diol 2.8.3 (79%). Diol 2.8.3 upon subsequent chloroacetate deprotection provided mezzettiaside-

8 (8) (85%) (Scheme 2.8).

Scheme 2.8: Synthetic approach to mezzettiaside-8 (8)

In order to obtain mezzettiaside-4, trisaccharide enone 2.8.1 was reduced (NaBH4/CeCl3)

and acetylated (Ac2O/Py) to give allylic acetate 2.9.1 (71%, 2 steps). Allylic acetate 2.9.1 was

dihydroxylated (OsO4/NMO) and deprotected (CH3C(OEt)3/p-TsOH/AcOH) to give

mezzettiaside-4 (4) (70%, 2 steps) (Scheme 2.9).

31

Scheme 2.9: Approach to mezzettiaside-4 (4)

Allylic acetate 2.9.1 upon syn-dihydroxylation (OsO4/NMO) gave diol 2.10.1 (85%).

Exposure of diol 2.10.1 to the orthoester mediated C-2 acylation ((CH3C(OEt)3/p-TsOH/AcOH))

gave alcohol 2.10.2 (84%). Removal of chloroacetate group (thiourea/NaHCO3/TBAI)13 from

alcohol 2.10.2 gave mezzettiaside-2 (2) (77%) (Scheme 2.10).

Scheme 2.10: Approach to mezzettiaside-2 (2)

To regioselectively install a C-3 acetate on diol 2.10.1, the Taylor type boron mediated

acylation (Ph2BOCH2CH2NH2/AcCl) was utilized providing intermediate C-2 alcohol 2.11.1

(71%).11 Chloroacetate deprotection (thiourea/NaHCO3/TBAI) of alcohol 2.11.1 provided

mezzettiaside-3 (3) (82%) (Scheme 2.11).

32

Scheme 2.11: Synthesis of mezzettiaside-3 (3)

Similarly, tetrasaccharides mezzettiaside 5-7 were prepared from trisaccharide intermediate

2.10.1. Intermediate diol 2.10.1 underwent a Pd-/B-dual catalyzed C-3 regioselective

glycosylation (Ph2BOCH2CH2NH2/Pd(PPh3)2) with Boc-pyranone 2.1.1 to provide

tetrasaccharide enone 2.12.1 (76%) with exclusively -stereochemistry. Enone 2.12.1 upon

subsequent post-glycosylation transformations (NaBH4/CeCl3 then OsO4/NMO) gave triol 2.12.2

(73%, 2 steps). Triol 2.12.2 upon C-3 regioselective acetylation (Ph2BOCH2CH2NH2,

AcCl/DIPEA)11 and chloroacetate deprotection (thiourea/NaHCO3/TBAI) provided

mezzettiaside-5 (5) in good yield (58%, 2 steps) (Scheme 2.12).

33

Scheme 2.12: Approach towards mezzettiaside-5 (5)

Finally, the synthesis of the last two tetrasaccharides mezzettiaside 6-7 began with intermediate

2.12.2, which upon chloroacetate deprotection gave mezzettiaside-7 (7) (83%). Whereas,

exposure of intermediate 2.12.2 to the regioselective C-2 acylation (CH3C(OEt)3/p-TsOH) gave

mezzettiaside-6 (6) after deprotection (thiourea/NaHCO3/TBAI) of the chloroacetate group

(61%, 2 steps) (Scheme 2.13).

34

Scheme 2.13: Synthesis of mezzettiaside-6 (6) and mezzettiaside-7 (7)

With all the mezzettiasides 2-11 in hand, we next wanted to explore the effect of the various

acylation patterns on anticancer and antibacterial activities. In addition we also wanted to explore

analogues of the simplest disaccharide mezzettiasides 9-11, as these offered the greatest

opportunity for structural modification. The reported activities for Mezzettiaside 9-11 showed a

great dependence on the degree of acylation. To address this question we decided to make C-3

ester analogues of mezzettiaside-10. The anticancer data reported for the isolated material

[disaccharide (mezzettiaside 9-11) and trisaccharide (mezzettiaside 2-4 & 8)] were found to have

the broadest range of activity against the lung/colon (Lu1, Col2, Table 2.2) cancer cell lines. We

decided to use the NCI H460 non-small cell lung cancer cell line for our evaluation. For

cleistetrosides/cleistriosides, we found the greatest antibacterial activity was seen with the

B.subtilis strain, so we decided to use this strain for MIC screening of antibacterial activity.

Cell Line M-2 M-3 M-4 M-8 M-9 M-10 M-11

Lu1 8.6 11.8 19.4 19.7 5.4 >20 6.1

Col2 4.3 4.9 6.2 8.2 >20 >20 9.0

KB 6.2 14.3 15.4 >20 11.3 >20 12.7

aResults are expressed as ED50 values ( g/mL) bLu1 = human lung cancer; Co12 = human colon cancer; KB = human oral epidermoid carcinoma

35

Table 2.2: Anticancer activity ( M) for the isolated mezzettiasides

We performed an MTT anti-cancer assay on NCI-H460 cancer cell line, which confirmed that

among the disaccharides/trisaccharides/tetrasaccharides, trisaccharides mezzettiaside 2-4 & 8

were more active than disaccharides 9-11 and tetrasaccharides 5-7. Initially, we were concerned

about the stability of the acetate groups towards ester hydrolysis or 1,2 migration and no

evidence of this has been observed.

Among the three types of mezzettiasides (di-, tri- and tetra-saccharides) a strong

dependency upon the degree and location of acylation on activity can be seen. To further explore

this effect, we decided to install the bulkier group (n-butyrate, i-butyrate, i-valerate and pivalate)

on the disaccharide motif at C-3 position of the intermediate 2.6.2. These more hindered esters

which might be stable towards hydrolysis and then compared the effect of acylation vs

glycosylation on the disaccharide 2.6.2 at C-3 position. Working towards this, we planned to

synthesize four new disaccharide unnatural analogues and investigated their SAR studies.

2.6 Synthesis of four new disaccharide analogues

The synthesis of new analogues started with disaccharide alcohol intermediate 2.6.2.

Disaccharide 2.6.2 upon esterification with n-butanoic acid/DCC/DMAP provided C-3 butanoate

which upon chloroacetate group deprotection (thiourea/NaHCO3/Bu4NI) gave disaccharide

2.14.1 (70%, 2 steps). In a similar fashion, intermediate 2.6.2 upon treatment with pivaloyl

chloride/Py. underwent acylation followed by deprotection (thiourea/NaHCO3/Bu4NI) of the

chloroacetate to provide disaccharide analogue 2.14.2 (58%, 2 steps) (Scheme 2.14).

36

Scheme 2.14: Approach towards unnatural disaccharide analogues 2.14.1-2.14.2

Along these lines, intermediate 2.6.2 was esterified with iso-butyryl chloride/pyridine and the

chloroacetate was deprotected (thiourea/NaHCO3/Bu4NI) yielding disaccharide 2.15.1 (68%, 2

steps). Similarly when 2.6.2 was treated with iso-valeric acid in presence of DCC/DMAP, it

underwent esterification to provide disaccharide analogue 2.15.2 (58%, 2 steps) (Scheme 2.15).6

Scheme 2.15: Approach towards unnatural disaccharide analogues 2.15.1-2.15.2

2.7 Bioactivity of all 10 mezzettiasides and four new disaccharide analogues towards anti-

cancer H460 cancer cell line/anti-bacterial strain

All 10 synthetically prepared natural compounds and 4 new disaccharide analogues were

tested for cytotoxicity against cancer cell lines and bacterial strains. These results from these

studies are outlined in Table 2.3. Disaccharide mezzettiaside-11 and trisaccharides mezzettiaside

37

2-4 and 8 were found to be the most cytotoxic towards H460 cell. Whereas, mezzettiaside-4, 8

and 9 were most cytotoxic against the B.Subtilis strain.6 The synthetic unnatural disaccharide

analogues 2.14.1-2.14.2 and 2.15.1-2.15.2 were synthesized to compare the effect of

glycosylation (mezzettiaside 2-4 and 8) to acylation. These acetylated products had reduced

activities which led us to conclude that installing sugar at C-3 of the disaccharide was more

effective than acylation (2.14.1-2.14.2 and 2.15.1-2.15.2).

a All the MIC ( M) are an average of at least three independent experiments bAll the IC50 ( M) are an average of at least three independent experiments

Table 2.3: Anti-cancer/anti-bacterial data for the 10 natural and 4 synthetic disaccharide (2.14.1-2.14.2 and 2.15.1-2.15.2) analogues

R R1 R2 MICa ( M)

IC50b( M)

B.subtilis H460 Mezze-2 Ac H Ac >128 17.0 Mezze-3 Ac Ac H >128 10.0 Mezze-4 Ac H H 8 9.0 Mezze-8 H H Ac 16 24.4 Mezze-5 H Ac H >128 40.0 Mezze-6 H H Ac 64 259.6 Mezze-7 H H H 32 17.7

R R1 MICa ( M)

IC50b ( M)

B.subtilis H460 Mezze-9 H Ac 16 151.4

Mezze-10 Ac H >128 >500 Mezze-11 Ac Ac >128 15.2

2.14.1 Ac n-PrCO >128 46.6 2.14.2 Ac t-BuCO >128 141.1 2.15.1 Ac i-PrCO >128 32.0 2.15.2 Ac i-BuCO >128 79.3

38

2.8 Conclusion

In conclusion, we have shown the utility of our highly divergent de novo Pd-/B-dual

catalyzed approach towards the first total synthesis of mezzettiaside family of natural products.

In addition, this synthetic approach allowed the synthesis of the entire family of mezzettiaside

(10 members). The synthesis concluded the regio/stereo-selectively installation of the sugars at

the C-3 position to form 1,3-linked oligo-rhamnoside natural products. The key feature of this

synthesis is the use of a divergent approach to synthesize all 10 natural products. The synthesis

occured in the range of 13-22 longest linear steps and yet only 42 total steps were required for

the synthesis of all members. In combination with the natural products mezzettiaside 2-11, four

new disaccharide analogues 2.14.1-2.14.2 and 2.15.1-2.15.2 were successfully synthesized with

different pattern of acylation (Ac/n-Pr/i-Pr/t-Bu) to compare the effect of glycosylation vs

acylation.

All fourteen molecules (Mezze-2-11, 2.14.1-2.14.2 and 2.15.1-2.15.2) were tested against

NCI H460 cancer cell lines and B. Subtilis bacterial strain to confirm that sugar chain length,

substitution at various positions varies the biological activity. Finally, this SAR study uncovered

the anti-cancer data for the tetrasaccharide mezzettiaside 5-7. The synthesis makes use of

inexpensive, commercially available achiral starting material (2-acetyl furan 1) and builds on

chirality using a de novo approach. Overall 10-20 chiral centers were built and masked enone

was used as an atom-less protecting group. In order to build all the 10 members of the natural

product library, only one protecting group (AcCl) was used. The synthesis was amenable for the

preparation of enough quantities for the biological testing against cancer-cell line and bacterial

strains.

39

2.9. References

1. (a) J. T Etse, A. I. Gray, C. Lavaud, G. Massiot, J-M. Nuzillard and P. G. Waterman, J. Chem. Soc., Perkin Trans. 1, 1991, 861; (b) D. A. Powell, W. S. York, H. V. Halbeek, J. T. Etse, A. I. Gray and P. G. Waterman, Can. J. Chem., 1990, 68, 1044. 2. B. Cui, H. Chai, T. Santisuk, V. Reutrakul, N. R. Farnsworth, G. A. Cordell, J. M. Pezzuto, A. D. Kinghorn, J. Nat. Prod., 1998, 61, 1535. 3. B. Wu, M. Li, G. A. ODoherty, Org. Lett., 2010, 12 (23), 5466; (b) V. Seidel, F. Bailleul, P. G. Waterman, J. Nat. Prod., 2000, 63, 6. 4. P. Shi, M. C. Silva, H. L. Wang, N. G. Akhmedov, M. Li, P. J. Beuning, G. A. ODoherty, ACS Med. Chem. Lett., 2012, 3, 1086. 5. (a) S. O. Bajaj, E. U. Sharif, N. G. Akhmedov, G. A. ODoherty, Chem. Sci., 2014, 5, 2230; (b) S. Y. Ko, A. W. Lee, S. Masamune, L. A. Reed, K. B. Sharpless, Science, 1983, 20, 949; (c) A. B. Northrup, D. W. C. MacMillan, Science, 2004, 305, 1752; (d) Md. M. Ahmed, B. P. Berry, T. J. Hunter, D. J. Tomcik, G. A. O'Doherty, Org. Lett., 2005, 7, 745; (e) J. M. Harris, M. D. Keranen, H. Nguyen, V. G. Young, G. A. O'Doherty, Carbohydr. Res., 2000, 328, 17; (f) J. M. Harris, M. D. Keranen, G. A. O'Doherty, J. Org. Chem., 1999, 64, 2982. 6. S. O. Bajaj, P. Shi, P. J. Beuning, G. A. ODoherty, Med. Chem. Comm., 2014, 5, 1138.

7. (a) M. Li, J. G. Scott, G. A. O'Doherty, Tetrahedron Lett., 2004, 45, 1005; (b) H. Guo, G. A. O'Doherty, Org. Lett., 2005, 7, 3921. 8. S. O. Bajaj, J. R. Farnsworth, G. A. ODoherty, Org. Synth., 2014, 91, 338.

9. (a) O. Achmatowicz, P. Bukowski, B. Szechner, Z. Zwierzchowska, A. Zamojski, Tetrahedron, 1971, 27, 1973; (b) A. R. Noyori, T. Ohkuma, M. Kitamura, J. Am. Chem. Soc., 1987, 109, 5856.

10. In general, we have found the NaBH4 (with and without CeCl3) reduction of -pyranones and the OsO4 catalyzed dihydroxylation of the resulting allylic alcohol to proceed with virtually complete diastereocontrol, see ref. 4, 6, 7 and 10. 11. (a) D. Lee, M. S Taylor, J. Am. Chem. Soc., 2011, 133, 3724; (b) C. Gouliaras, D. Lee, L. Chan, M. S. Taylor, J. Am. Chem. Soc., 2011, 133, 13926; (c) C. A. McClary, M. S. Taylor, Carbohydr. Res., 2013, 381, 112; (d) E. Dimitrijevic, M. S. Taylor, Chem. Sci., 2013, 4, 3298; (e) T. M. Beale, M. S. Taylor, Org. Lett., 2013, 15, 1358. 12. While the Taylor catalyst has been used in regioselective glycosylations (glycosylbromide with excess Ag2O (>1.0 equiv.), ref. 12), this is the first example of its use to catalytically induce regiocontrol in a catalyzed glycosylation.

40

13. (a) M. H. Clausen, R. Madsen, Chem.-Eur. J., 2003, 9, 3821; (b) M. Naruto, K. Ohno, N. Takeuchi, Tetrahedron Lett., 1979, 251. S. O. Bajaj, E. U. Sharif, N. G. Akhmedov, G. A. ODoherty, Chem. Sci., 2014, 5, 2230. Reproduced by permission of The Royal Society of Chemistry (www.rsc.org/follow). S. O. Bajaj, P. Shi, P. J. Beuning, G. A. ODoherty, Med. Chem. Comm., 2014, 5, 1138. Reproduced by permission of The Royal Society of Chemistry (www.rsc.org/follow).

41

Chapter 3

Synthesis of cardiac glycosides 1,3-linked oligosaccharides via enzymatic like

regio/-stereo-selective glycosylation and SAR study

Synthetic chemistry has always been challenged by natures ability to assemble natural

products. This challenge comes from natures seemingly limitless ability to make complex

structures with specific biological properties. Nature accomplishes this synthetic feat by

designing enzymes that selectively zip these structures together without the formation of

byproducts or the use of protecting groups. Nowhere is this synthetic efficiency more pronounces

than in oligosaccharide biosynthesis, where glycosyltranferase enzymes transfer the desired

sugar regio- and stereo-selectively onto a polyol containing carbohydrate without the need for

protecting groups. To do this the glycosyltransferase utilizes two distinct binding sites, one to

selectively recognize and activate the nucleophilic component (glycosyl acceptor) and the other

to selectively recognize and activate the electrophilic component (glycosyl donor). Both these

enzyme binding sites require great structural complexity in order to ensure substrate selectivity,

which is compensated by the catalysts large turnover number. It is worth noting that this

substrate selectivity perforces an inherant substrate inflexibility when trying to use a given

glycosyltransferase for the transfer of unnatural substrates.

In an effort to match natures prowess at using enzymes to assemble oligosaccharide motifs

without the use of protecting groups, we have been exploring the use of asymmetric catalysis for

the protecting group free synthesis of oligosaccharide structures. This alternative approach offers

several advantages over biosynthetic approaches, in terms of the range of substrate

42

stereochemistry that can be added and the divergent synthetic direction the glycosylated product

can be taken. In this regard, we have shown that our de novo approach to carbohydrates could be

used to construct stereochemically diverse libraries of oligosaccharides containing D- and L-

sugar.1,2 For example, in only seven steps all D-sugar, all L-sugar or mixed D-/L-sugar

trisaccharide with rhamno- or amiceto-substitution/stereochemistry can be assembled without the

use of protecting groups.1,2 This approach, which solely focuses on the use of Pd- -allyl catalysis

to activate the glycosyl donor, relys on the glycosyl acceptor to contain a single or singlely

reactive alcohol. Thus to more truly mimic glycosyltranferase like catalysis, an additional

component, which regioselectively activates the glycosyl acceptor, needs to be added to this

process. To do this we turned to borinate chemistry, which has been shown to regioselectively

activate 1,2- and 1,3-diols (Chapter 1). More specifically, the selective functionalization of the 3-

and 6-positions of manno- and galacto-pyranose sugars. Herein, we describe the use of this

approach for the synthesis and SAR-study of the oligosaccharide portion of the cardiac

glycosides as anticancer agents.

3.1 Introduction to Na+/K+-ATPase pump and Cardiac Glycosides (CGs)

Na+/K+-ATPase are known to regulate several vital cellular functions, like homeostasis,

cell volume (ions inside and outside the cells), signal transducer and membrane potential.3,4,5 The

pump is important for the movement of ions across cell membranes (e.g., muscle contraction)

and also for creating charge imbalances across the cell membranes (e.g., electrical impulses).3,4,5

The pumping process begins with the binding of two extracellular K+, three intracellular Na+ ions

and a molecule of ATP. Once all the ions are bound ATP is converted into ADP and inorganic

phosphate. The ion exchange occurs across the cell membrane, with the energy for the process

43

coming from ATP hydrolysis.6,7 The Na+/K+-ATPase is divided into two subunits and .8 The

and subunits are known to exist in various isoforms (i.e., 1, 2, 3, 4 and 1, 2, 3),

the expression of which is associated with various tissue types.9 For instance, the 1 isoform is

present ubiquitously,whereas, the 2 is expressed mostly in the heart muscle and plays a key role

in the maintenance of blood pressure and cardiac function.10

Cardiac glycosides are known for inhibiting Na+/K+-ATPase and increase intracellular

sodium ions. The Na+/K+-ATPase is a P-type pump that actively transports two K+ inside and

three Na+ outside, because of this the intracellular sodium levels are low, which helps to sustain

an adequate electrochemical gradient across the plasma membrane. Electrochemical gradients

are necessary for proper cellular function.3,4,5 In addition to the Na+/K+-pump, the Na+/Ca2+

exchange pump extrudes Na+ ions out of the cell and introduces Ca2+ ions into the cell.11 In heart

muscle cells, the inhibition of the Na+/K+-ATPase leads to modulation of heart contractions,

whereas, in cancer cells this leads to apoptotic induced cell death.12,13,14

Recent reports suggest that some cancers have highly expressed 115,16,17,18,19 and/or

320,21,22 isoforms and the selective binding to these isoforms may lead to a better cancer

therapy.23,24,25 By targeting the 1 and 3 isoform selectively, one can inhibit cancer cell growth

and reduce the off target toxicity. More recently, we and others have shown that cardiac

glycosides with improved anticancer activity can be found by modifying the carbohydrate

portion of the molecule.12 Specifically, we prepared 1,4-linked mono-, di- and tri-saccharide

cardiac glycosides with varying substituion and stereochemistry and screened for anticancer

cytotoxicity (Scheme 3.1).26 In general, these studies have found that improved anti-cancer

activity can be found by changing the substitution and stereochemistry of the sugars (e.g., -L-

44

rhamno- and -L-amiceto-). In addition, we found that reducing the length of the trisaccharide to

a monosaccharide significantly increases the anticancer activity. In fact, a similar dependance of

chain length can be found when one looks at the cardiotonic activity of the cardiac glycosides.

Taken as a whole, one can conclude from the evidence that natures choice of a trisaccharide is

not to increase activity but rather increase the selectivity for the heart muscle tissue.

On the basis of crystal structure of the Na+/K+-ATPase bound to ouabain (Fig. 3.2), a

related cardiac glycoside, it could be seen that cardiac glycosides binds to the -subunit of

Na+/K+-ATPase. The crystal structure of ouabain also shows that there is little or no opportunity

for SAR-substituion of the aglycon region whereas, the solvent exposed cone shaped region

where the carbohydrate resides is an ideal space for SAR-type exploration. Following natures

lead, we chose to modify the carbohydrate subunits of digitoxin (a component of digitalis) to

explore this space and to find new -1 and/or -3-selective Na+/K+-ATPase inhibitor as

anticancer agents. We chose to use a synthesis led SAR-study to map out this extracellular

carbohydrate binding site, which is circumscribed by the structurally ill-defined-extracellular

loops27 of the Na+/K+-ATPase. This large solvent exposed pocket that allows for the exchange of

Na+ and K+ ions is flexible enough to fit a wide range of polar structures, like sugars. As this

space is still chiral, it should response differently to the addition of a D- vs L-sugar to a given

ligand untill the growing oligosaccharide chain is free from the protein environment.

Cardiac glycosides structures (digitoxin, digoxin and ouabain) are divided into two

portions: the aglycon (steroid core) having an unsaturated lactone at C17 position and the

carbohydrate motif (sugar) attached to the core at the C3 position (Fig. 3.1).28 Of particular

interest to us was digitoxin, a cardiac glycoside used in the treatment of congestive heart failure,

arrthymia by enhancing the cardiac contraction.29 Digitoxin was isolated from the flower of

45

digitalis purpurea, (aka, foxglove) which has long been known for its inotropic effect (increases

strength of muscular contraction). Despite the narrow therapeutic window, digitoxin is still in

clinical use around the world.30

Figure 3.1. General representation of Cardiac Glycosides

Our previous efforts to explore this space with oligosaccharides led to the synthesis and

evaluation of several 1,4-linked di- and tri-saccharide structures, which all had significantly

reduced anti-cancer activities. Having found no success with 1,4-linked oligosaccharide, we

decided to explore 1,3-linked oligosaccharide. Herein, we describe the use of our dual Pd-/B-

catalyzed glycosylation for the exploration of the carbohydrate binding region of the Na+/K+-

ATPase. The ultimate aim of this project is to identify novel carbohydrate motifs that can

selectively target Na+/K+-ATPase, which in turn can be used as anticancer agents. The ideal

anticancer drug will not only be effective but also selective against tumor cells.

46

Figure 3.2: Crystal Structure of Ouabain, a cardiac glycoside

The general representation of digitoxin, a cardiac glycoside is shown in Fig. 3.3, where the

steroid core (digitoxigenin) is a pharmacophore and the carbohydrate portion controls the

isoform selectivity.

Figure 3.3. General representation of digitoxin

3.2 Synthesis of monosaccharide digitoxin analogues

From the initial studies by Dr. Hua-Yu Leo Wang on the cardiac glycosides, he

concluded that L-sugars with rhamno-3.1.3a and amiceto-3.1.3b substitution gave the most

47

active analogues.13 The synthesis of these monosaccharide began with the preparation of the D-

/L-Boc-pyranones (glycosyl-donors) from achiral furan. Specifically, 2-acetyl furan 1 underwent

a three step procedure (Noyori reduction, Achmatowicz rearrangement and Boc-protection) to

form the D- and L-Boc-pyranones 3.1.4 and 3.1.1 respectively.

The Boc-pyranones 3.1.1/3.1.4 can be further treated with digitoxigenin under Pd-

catalyzed glycosylation to provide enones, which were further reduced to allylic alcohols

3.1.2/3.1.5 using Luche reduction condition (NaBH4/CeCl3). Allylic alcohols 3.1.2/3.1.5 were

dihydroxylated (OsO4/NMO) to provide 3.1.3a/3.1.6a or reduced using o-nitrobenzenesulfonyl

hydrazine (NBSH) reduction to give 3.1.3b/3.1.6b (Scheme 3.1). The four monosaccharides

were screened for their cyctotoxicity against H460 cancer cell lines (Table 1). This data indicated

that the -L-monosaccharides were better than its corresponding -D analogues.26

IC50 (nM)X =

Mono--OH H

-L-Sugar 35 39-D-Sugar 706 387

Scheme 3.1: De Novo synthesis of glycosyl donors and -L-/D-Digitoxin monosaccharides

48

We next decided to explore the effects of further carbohydrate substitution at the C-3

position of the -L-rhamnose sugar on the most active monosaccharide 3.1.3a. To do this, we

envisioned the addition of either -L- or D-sugar with either rhamno- or amiceto-substitution

(3.2.3a/3.2.6a or 3.2.3b/3.2.6b). This stereodivergent substituion served as stereochemical

control element to ensure the space being occupied is still under the influence of the extracellular

loops of the protein.

3.3 Synthesis of 1,3-linked digitoxin analogues (di-/tri-/tetra-saccharide)

To install the D- and L- sugars we turned our attention towards the use of the dual Pd-/B-

catalyzed glycosylation (Scheme 3.2). The synthesis of disaccharides starts with the

monosaccharide rhamno-digitoxin 3.1.3a. Monosaccharide 3.1.3a upon exposure to our

regioselective Pd-/B-dual catalysis glycosylation methodology reacted with the incoming donor

(Boc-pyranones 3.1.1/3.1.4) selectively at C-3 equatorial alcohol position with -selectivity to

obtain corresponding enones 3.2.1/3.2.4. The enones 3.2.1/3.2.4 were further reduced under

NaBH4/CeCl3 condition to give disaccharide allylic alcohols 3.2.2/3.2.5 respectively. An Upjohn

dihydroxylation (OsO4/NMO) provided 3.2.3a/3.2.6a. Alternatively, a diimide reduction using

NBSH was used to give 3.2.3b/3.2.6b (Scheme 3.2).

49

Scheme 3.2: Regioselective glycosylation using Pd-/B-catalysis

The four di-saccharides were screened for their cyctotoxicity against the human non-

small cell lung cancer cell line, H460 (Table 3.1). These results showed that only the bis-L-

rhamno-disaccharide 3.2.3a retained its potency (3.2.3a: IC50 = 39 nM). The chiral nature of the

space was indicated by the ne