Studies Towards the Total Synthesis of the Naturally-Occurring Anticancer Agent Halichomycin

Home , Diethylaluminium cyanide, Endiandric acid c, Hexamethylphosphoramide, Lithium diisopropylamide

STUDIES TOWARDS THE TOTAL SYNTHESIS OF THE NATURALLY-OCCURRING ANTICANCER AGENT HALICHOMYCIN

b y

Maxine Lai-Fun Cheung

Me,

OMe

Me 'NH Me Me

Me Me

A thesis presented to the University of London in partial fulfilment of the requirements for the degree of Doctor of Philosophy June 2003

The Christopher Ingold Laboratories Department of Chemistry University College London ProQuest Number: 10015856

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 LIST OF ABBREVIATIONS

Ac acetyl

AIBN 2 ,2 -azoblsisobutyronjtrlle

9-BBN 9-borablcyclo[3.3.1]nonane

Bn benzyl

Bu^ f-butyl

Cy cyclohexyl

DBU 1,8-diazabicyclo[5.4.0]undec-

DDQ 2,3-dlchloro-5,6-dicyano-1,4-

DEAD diethyl azodicarboxylate

DEIPS dlethylisopropylsllyl

DET diethyl tartrate

DIBAL diisobutylaluminium hydride

DIPT diisopropyl tartrate

DMAP 4-dimethylaminopyridine

DMF A/,A/-dimethylformamide

DMSO dimethyl sulfoxide

EDCI 1 -(3-dimethylaminopropyl)-3-

Et ethyl

HMPA hexamethylphosphoramide

LDA lithium diisopropylamide mCPBA 3-chloroperoxybenzoic acid

Me methyl

NMO 4-methylmorpholine A/-oxide

PCC pyridinium chlorochromate

PDC pyridinium dichromate

Ph phenyl PMB 4-methoxybenzyl

PPTS pyridinium 4-toluenesulfonate

TBAF tetra-A7-butylammonium fluoride

TBS fe/t-butyldimethylsilyl

TES triethylsilyl

Tf trifiuoromethanesulfonyl

TFA trifluoroacetic acid

TFAA trifluoroacetic anhydride

THF tetrahydrofuran

THP tetrahydropyranyl

TMS trimethylsilyl

TPAP tetra-n-propylammonium perruthenate

TRIS tris(hydroxymethyl)aminomethane

Ts 4-toluenesulfonyl Dedicated with love to Dean ACKNOWLEDGEMENTS

I would like to thank Professor Karl J. Hale for his devoted and dedicated supervision over the last 3 years. I am grateful to him for giving me the opportunity to study such a remarkable molecule.

I would especially like to thank all the people who helped me during my PhD, in particular Pascal for all his advice and guidance in the lab. To Soraya - Thanks for making me feel welcome and training me ‘from scratch’ when I arrived! Special thanks to all my friends in the lab who helped make my time at UCL enjoyable, especially Ying, Audrey, Sven, Marcus,

Linos and Shahid - You will not be forgotten!

Many thanks to all the technical staff for their excellent services, particularly Dr. Jorge

Gonzalez Outereirino, Dr. Abil Aliev and Charles Willoughby for all their help and advice. I am thankful to Mike Cocksedge and staff at the London School of Pharmacy for HRMS, and to

Jonathan Steed at King’s College London for performing the X-ray crystallography analysis. I am also grateful to the EPSRC and Pfizer for financial support.

Finally, I am indebted to my parents for their love, support and help throughout my life, and especially to my boyfriend Dean for his never-ending patience, encouragement, devotion and invaluable advice, and it is to him that I dedicate this thesis. ABSTRACT

This thesis is split into 4 sections, and is concerned with the development of a

biomimetic total synthesis of the naturally-occurring antitumour agent, halichomycin. The first

section describes a range of biomimetic natural product syntheses that have been devised since

1917, and shows the levels of complexity that have been transcended by various groups in

these efforts. The second part of the thesis discusses the discovery and structure elucidation of

halichomycin and Wood’s synthetic approach to a region of this compound.

In addition to earlier ventures, our eventual biosynthetic proposal for tricyclic assembly

in halichomycin is described in this thesis. Synthetic efforts towards halichomycin have so far

resulted in the synthesis of an advanced AB-carbon backbone intermediate 252.

In Chapter 3, our group’s original retrosynthetic analysis of halichomycin is described

along with our attempts to implement this approach. A biosynthetic proposal for my assembly is

then presented, and it will be shown how this served as a source of inspiration for a new

synthetic strategy to halichomycin. Our progress towards this objective will then be reviewed.

Specifically, the synthesis of an advanced precursor 252 of pre-halichomycin will be discussed.

Noteworthy features of our synthesis include the Roush asymmetric crotylboration

reaction between 210 and 211, en route to the anti-anti alcohol 212, and the face-selective

enolate alkylation of 236 to give 237. Aldehyde 256 was converted to 254 via an Evans aldol

reaction, followed by protection, then reduction. A Wittig-Stille sequence on 254 then completed the synthesis of 252.

Evans Aldol, Wittig-Stille TE90 OTBS Protection Me sequence and Reduction

0PM B 254 OPMB

Me C 02tt OPMB OMe 252 HO CHO B ''NH Me ,NH 2 Me . 24 i^e' 19 7 steps ^ Me

Me Halichomycin Pre-Halichomycin 0 OTBOPS 250

210 Roush E n —/ Face-selective Asymmetric_ 3 steps^ i6\^y\Me Enolate

Crotylboration Me Me Alkylation

IPr02C ",/'''B Me

iPiOgC

IV CONTENTS

List of Abbreviations I

Acknowledgement ...... Ill

Abstract ...... IV

Contents ...... V

1.0 INTRODUCTION

1.1 Biomimetic Syntheses

1 .1 . 1 Robinson’s Tropinone Synthesis ...... 2

1.1.2 O.L Chapman’s Biomimetic Synthesis of Carpanone ...... 6

1.1.3 W.S. Johnson’s Steroid Polyene Cyclisations ...... 7

1.1.4 K.C. Nicolaou’s Biomimetic Approach to Endiandric Acids A-G ...... 14

1.1.5 Heathcock’s Biomimetic Total Synthesis of Methyl Homosecodaphniphyllate .... 17

1 .1 . 6 W.S. Johnson’s Total Synthesis of d/-/?-Amyrin ...... 28

1.1.7 Paterson’s Studies in Biomimetic Polyether Synthesis ...... 32

1.1.8 Baldwin’s Studies on the Biomimetic Synthesis of the Manzamine Alkaloids ...... 36

1.1.9 Sulikowski’s Progress toward a Biomimetic Synthesis of Phomoidride B ...... 44

1.1.10 Epilogue ...... 48

2.0 BACKGROUND

2.1 The Discovery of the Antitumour Agent, Halichomycin ...... 49

2.2 The Yale Approach to the Synthesis of the C(1 )-C(7) Segment of Halichomycin: 52

The Wood Synthesis

3.0 RESULTS AND DISCUSSION

3.1 Original Retrosynthetic Analysis of Halichomycin and Preliminary Efforts to

Implement a Conventional Synthetic Approach

3.1.1 Original Retrosynthetic Analysis of Halichomycin ...... 54

3.1.2 Attempted Implementation of the Original Retrosynthetic Strategy for Halichomycin 57

3.1.3 A Biogenetically-Modeiled Total Synthesis of Halichomycin ...... 69

4.0 EXPERIMENTAL ...... 76

References

V Chapter 1: Introduction

1.0 INTRODUCTION

Numerous molecules have now been created by biomimetic approaches. A biomimetic synthesis is one that attempts to mimic the way Nature would biosynthesise the molecule.

Given that much of this thesis will be spent discussing our attempts at implementing a biogenetically-modelled synthesis of the molecule halichomycin, we thought that it would be of interest to the reader to see what other types of biomimetic syntheses have been carried out during the 20*" Century.

Below is a table outlining the biomimetic syntheses that will be discussed in this thesis by way of introduction to our own work in this field.

Year Organic Chemist Work

1901 R. Willstatter First Synthesis of Tropinone via a long sequence of reactions 1917 R. Robinson Synthesis of Tropinone in one step

1923 R. Willstatter Synthesis of Cocaine

1971 O.L. Chapman Biomimetic Synthesis of Carpanone

1976 W.S.Johnson Biomimetic Approaches to Polyene Cyclisations

1982 K.C. Nicolaou Biomimetic Approaches to Endiandric Acids A-C: The Endiandric Acid Cascade 1992 C.H. Heathcock Biomimetic Studies on the Daphniphyllum Alkaloids: Total Synthesis of Homo secodaphniphyllate 1993 W.S.Johnson Total Synthesis of p-Amyrin

1993 1. Paterson Studies in Biomimetic Polyether Synthesis

1999 J.E. Baldwin Studies on the Biomimetic Synthesis of the Manzamine Alkaloids

2 0 0 2 C.A. Sulikowski Studies on the Biomimetic Synthesis of Phomoidride B

Table 1: Overview of Biomimetic Synthetic Studies Covered in this Introduction Chapter 1: Introduction

1.1 BIOMIMETIC SYNTHESES

1.1.1 Robinson’s Tropinone Synthesis^ (1917)

Me N COgMe

OCOPh

Fig. 1 : Structures of atropine 1, tropinone 2, and cocaine 3

Atropine (the racemate of structure 1) is an abundant alkaloid with remarkable

physiological properties, its most weli known being its capacity to cause the pupil of the eye to dilate. During the determination of its structure, a key degradation product, tropinone 2, was obtained by Willstatter.

In 1901, Willstatter synthesised tropinone by a long sequence of reactions^ (Scheme 1), which involved the elimination of amino groups by exhaustive Hofmann méthylation, the addition of bromine to the two new double bonds, substitution of bromide by dimethylamine, and eiimination of hydrogen bromide by dimethylamine.

Na/EtOH 1. Mel 2. AgOH

1. HBr 1. Mel NMe; quinoline 2. AgOH

Na/EtOH MeoN

waim 1. NaOH

NMe) = 0 NMe\— OH ^ — NMe\— Br

Scheme 1 : Willstatter's first route to tropinone Chapter 1: Introduction

In 1917, Robinson synthesised tropinone In one step\ following a very simple

disconnection of the molecule (Scheme 2).

CHO HsC dislocation + HaNMe

CHO HgC

Scheme 2; Robinson's simple dislocation of tropinone

Robinson retrosynthetlcally fractured the molecule to produce succlndlaldehyde, methylamlne and acetone, and subsequently proved that tropinone could be obtained In low yield by the condensation of these three fragments In aqueous solution. Replacement of acetone by the calcium salt of acetone dicarboxyllc acid, Improved this yield. A salt of tropinone dicarboxyllc acid was found to be the Initial product, which lost two molecules of carbon dioxide with the formation of tropinone when the solution was acidified and heated.^

COgH CHO aqueous solution + H2NMe + NMe)=0 = 1

CHO COgH

Scheme 3: Robinson's One-Step Synthesis of Tropinone

Essentially this Is a double Mannlch reaction, but at that time, this kind of reaction was not known as such, as It Is today. The methylamlne and aldehyde combine reverslbly to produce a protonated Imlne (I) (Scheme 4).

c o P c o P CHO MeNH2 3 -—

CHO CHO b o P c o p (1) (II)

C 0 2 ®

[HNMe V = o

CHO^

\) 0 ^ (III) Scheme 4: Synthesis of tropinone via a double Mannich reaction Chapter 1: Introduction

The enol of acetone dicarboxylate (II) is also present In solution. The combination of this enol, nucleophlllc on carbon, with the electrophlllc protonated Imlne, results In carbon-carbon bond formation to Initially give (III). Cyclisation then ensues to give (IV), which subsequently undergoes dehydration to give the active Internal electrophlle (the Imlnlum Ion), which finally e cycllses, so delivering (VI) (Scheme 5).

CO? HNMe HNMe K:?rNMeF=0 ^ o

(IV) (V) (VI)

Scheme 5

Robinson drew attention^ to the significance of firstly, the near-physlologlcal conditions used In the reaction, and secondly, to how closely related the starting materials are to compounds known to occur In nature.

The problem of 0 - 0 bond formation was avoided In Willstatter’s synthesis, since he chose a starting material which already possessed all the carbon-carbon bonds of tropinone and, hence, his synthesis only required a modification of the starting functional groups, and the

Introduction and removal of heteroatoms. In extensive researches,'^ Schopf and his co-workers raised the yield of tropinone to more than 90%, principally by a careful choice of buffer solution.

Willstatter also found^ a very simple route to tropinone (Scheme 6 ).

.COgEt .COgEt

Kolbe MeNh(2 NMe NMe electrolysis 2. Na

9 'COaEt 8

Scheme 6: Willstatter's much later route to tropinone Chapter 1: Introduction

In this pathway, one carbon-carbon bond (6->7) was set up in an electrochemical (and possibly,

homolytic) step, and the other (tetrahydro - 8 = V l^ 9 ) by the cyclic version of the Claisen condensation, a Dieckman condensation (Scheme 7).

EtO EtO

N M e > = 0 GEt

(VI) (VII) (VIII)

Scheme 7

Another advance on Robinson’s tropinone-forming strategy can be found in Willstatter's

synthesis® of cocaine 3 (Scheme 8 ). Reaction of the monoester of acetone dicarboxylic acid 10 with methylamine and succindialdehyde produced the monoester of tropinone dicarboxylic acid, which was therefore capable of losing only one carboxylic group upon acidification and heating.

The ketoester 11, so produced, was reduced to the hydroxyester 12 with sodium amalgam, and it was at this point that, for the first time, stereochemical issues emerged. Fortunately for

Willstatter, the methoxycarbonyl group had taken up the natural configuration of being positioned cis to the NMe bridge in 11, and this was retained in the reduction that led to 12.

CO2M6 C02Me COaMe CQpMe

. PhCOCl + HpNMe + NMe)"OCOPh 2. Separate diastereomers

10 11 12 a mixture of diastereomers

Scheme 8 : Willstatter's synthesis of cocaine 3

Benzoylation of the mixture of diastereomers in 12 and fractional crystallisation to remove the unwanted isomer thereafter produced racemic cocaine, which was then resolved to afford the natural material 3. Chapter 1: Introduction

1.1.2 O.L. Chapman’s Biomimetic Synthesis of Carpanone.^ (1971)

In 1969, Australian scientists® elucidated the structure of the hexacyclic lignan carpanone 13 (Scheme 9), having isolated it from the bark of the Carpano tree. Carpanone possesses five contiguous stereogenic centres that span four of its ring systems; together, these structural features conspired to make this a target molecule of great complexity for 1971.

Oxidative phenolic Me

Intramolecular^^'/,. Diels-Alder , H | O' < O'

13; carpanone 14 15

Scheme 9: Presumed biosynthesis and retrosynthetic analysis of carpanone 13

Even though there is no element of symmetry present in carpanone’s complex structure, the original isolation group suggested® that carpanone could potentially form in Nature through the intramolecular cycloaddition of a Cg-symmetric bis(quinodimethide) (Scheme 10).

Me Me Me Me Oxidative C-C ,0 dimérisation bond rotation

OH O ; T U- 0

14 (Cg- symmetric)15 14 13: carpanone

Scheme 10: Biogenesis of Carpanone proposed by Brophy.

Such an intramolecular cycloaddition would be capable of simultaneously producing two rings and setting three contiguous stereocentres. An oxidative dimérisation (yO^phenolic coupling) was thought to produce the Cg-symmetric bis(quinodimethide) 14.

In 1971, Chapman and coworkers disclosed a remarkable two-step chemical synthesis of carpanone^ based upon the aforementioned biosynthetic proposal. Chapman and coworkers’ elegant biomimetic synthesis of carpanone commenced with the base-induced isomérisation of

2-allyl-4,5-methylenedioxyphenol 16® to 2-[(E)-prop-1-enyl]-4,5-methylenedioxyphenol 15

(Scheme 1 1 ). Chapter 1: Introduction

NaOAc, f-BuOK MeOH-HgO. DMSO PdClz, 38°C O -P d -0 ' ooC (46% yield) 16 15 17

phenolic coupling -Pd(0)

Me Me ■X*- DIels-Alder U - 0 14 (±)-Carpanone 13

Scheme 11 : Chapman's Synthesis of Carpanone 13

Compound 15 was the key intermediate in this synthesis: it was oxidatively dimerised with palladium dichloride to give carpanone 13 presumably through the intermediacy of the Cg- sym metric and highly reactive bis(quinodimethide) 14.

At that time, phenolic couplings had traditionally been accomplished with one-electron oxidants but Chapman’s group anticipated that PdClg, containing as it does a divalent metal, would facilitate the crucial oxidative coupling step by bringing two phenolic units together through complexation (intermediate 17). They envisioned that the stereochemical outcome of the key intramolecular phenolic coupling would also govern the stereochemical course of the critical carbon-carbon bond forming event (17^14). Carpanone 13 was formed in 46% yield by treating a rapidly stirred solution of 15 and sodium acetate in MeOH-HgO at 38°C with PdClg.

Once the putative bis(quinodimethide) 14 was formed, it was suggested to undergo an intramolecular Diels-Alder reaction to furnish carpanone 13, and it is this spectacular sequential transformation that is responsible for the creation of two new rings and all five contiguous stereocentres.

1.1.3 W.S. Johnson’s Steroid Polyene Cycllzatlons^° (1976)

For many years the synthesis of polycyclic natural products such as steroids involved, for the most part, step-by-step annélations, i.e. each new ring was added independently to the Chapter 1: Introduction

next. The biomimetic approach to steroids differs in that the plan envisages the production of a number of rings stereospecifically in a single-pot reaction by the ring closure of an acyclic chain having appropriately-placed trans- olefinic bonds; a process analogous to the known biogenetic conversion of squalene into polycyclic triterpenoids, e.g., lanosterol^\ the precursor of cholesterol. (Scheme 12).

In 1955, Stork^^ and Eschenmoser^^ independently pointed out that the stereochemical course of the biological cyclisation of squalene could be rationalised on stereoelectronic grounds.

Squalene Lanosterol

Scheme 12: Conversion of squalene to lanosterol

Their important hypothesis, which immediately stimulated serious biomimetic studies, can be illustrated by considering the conversion of squalene oxide 18, which is a known biogenetic intermediate^"*, into the plant triterpenoid dammaradienol 19 (Scheme 13).

H-i-A

trans trans tranS' transj trans HO

H Scheme 13: Conversion of squalene oxide 18, into dammaradienol 19. Chapter 1: Introduction

The process may be regarded as a sequence of trans-anti-parallel electrophilic additions to the oiefinic bonds, in the same stereochemicai sense that bromine adds stereospecifically to alkenes. Thus protonation of the oxygen atom of squalene oxide 18 generates an incipient cationic centre at C-2, which is attacked by the 6,7-olefinic bond, thereby initiating formation of

the a bond between C-2 and 0-7. Concomitantly, the cationic centre developing at C - 6 invites

an attack by the 1 0 ,1 1 -olefinic bond which generates the a bond between C - 6 and C- 1 1 , and so on. This series of attacks occurs in a “trans” manner, yieiding the trans-tused ring system found in the product 19. It is the aW-trans geometry of the olefinic bonds in squalene that results in all frans-fusion of the four rings in product 19.

Because the five-membered D-ring widely occurs in naturai products, particularly in the

D ring of steroids, W.S. Johnson thought that it would be of special interest to search for systems that would give five-membered ring closure in biomimetic processes. Since CC triple bonds located in the 5,6-position relative to a developing cationic centre have a tendency to cyciize so as to give five-membered rings^®, it seemed reasonable to examine the behaviour of such bonds as participants in polyene cyclisations. The dieneaikynol 20 was examined as a model system and it was indeed, found to undergo facile acid-catalysed cyclisation to give exclusively the 6/5 trans-fused ring system in high yield.Thus upon treatment of 20 with formic acid, the enol formate 22 was produced in 90% yield. (Scheme 14).

c©

.0 0 HO

HCO2H in pentane 25°C

OH 20 22 23

Scheme 14: Formic acid treatment of 20, followed by hydrolysis Chapter 1: Introduction

Hydrolysis yielded the ketone 23 which has all of the structural features of the C/D ring system of progesterone. The cyclization of substance 20 involves either an intermediate or a transition state having the properties of a vinyl cation 21. Johnson found it especially interesting that such

cations could be generated efficiently under extremely mild conditions (e.g., with 1 % trifluoroacetic acid in a solvent at -78°C) from a relatively stable tertiary allylic alcohol. The energy for this transformation probably is provided by the conversion of ti to a bonds.

Having scored this success, W.S Johnson’s attention soon turned to the total synthesis of steroids via the application of acetylenic participation in biomimetic polyene cyclisations. The trienynol 24 appeared to be a promising substrate for cyclisation directly to the steroid nucleus.

It was converted (65% yield) into the pregnone 26 which, in turn, was readily transformed into progesterone 27^^. Ethylene carbonate was added to the cyclisation mixture to serve as a nucleophile for capturing the vinyl cation, possibly in the form of the stabilised cation 25.

(Scheme 15).

F3CCO2H

F2 CHCH3 HO n 0 0 ,-30°C

24 Ï

H2O

l.t-BuOCrOaH

2. DDQ (-H2) 3 .H2 , Rh(PPh3)3l o H Progesterone 27 26

Scheme 15: Johnson's formation of progesterone 27 from the trienyol 24

10 Chapter 1: Introduction

In other cyclisation studies the trienynoi 24 was treated with trichioroacetic acid in 2- nitropropane giving the oxyimino compound 28 ° This product was converted by hydrogenolysis with lithium tetrahydridoaluminate into the dioi 29 which was transformed with periodate into the 17-keto compound and thence into testosterone benzoate

.0—N=C(CH3)2

OH OCOCeHe OH

8 steps

o H Testosterone benzoate 29

Scheme 16: Formation of testosterone benzoate from the trienyol 24

Another substrate that proved useful for synthesising steroids is the cyclopentenol 31 which, on cyclisation, produced the crystalline ketone 32 in 71% yieid.^° Conversion of this product, via ozonoiysis and intramolecuiar aldoi condensation, into (±)-progesterone was effected in 80% yield (Scheme 17).

The question of the mechanism of stereospecific cationic polyene cyclisations, whether enzymic or biomimetic, has been open to debate. On one hand, the intermediacy of partially cyclised cations has been shown to be consistent with the observed stereochemical course of such cyclisations^^ On the other hand, a concerted process in which all of the new 0 0 bonds

11 Chapter 1: Introduction

are formed synchronously represents an equally satisfactory rationalisation of the facts. During this time, W.S. Johnson felt that, whilst very little direct evidence had been obtained for deciding between the “stepwise” and the “synchronous” mechanism; the balance was in favour of the latter.

1. 03

2. 5% KOH + 1 7 a - epimer o

(±)-progesterone 27

Scheme 17: Conversion of cyclopentenol 31 to (±)-progesterone 27

Kinetic studies of a series of substituted dienic p-nitrobenzenesulfonates in both acetic acid^ and in trifluoroethanol^® have shown small, but incremental, rate enhancements by substituting the vinyl hydrogens of the terminal olefinic bond by methyl groups. These effects represent a necessary, but not sufficient, condition for the synchronous mechanism.

In connection with a total synthesis of estrone 35 from 33^"^, a thorough study was made of the key step, namely the Lewis acid-catalysed biomimetic cyclisation 33-^34 (Scheme 18) which proceeds in high yield. In addition to the product 34, there was always obtained, some of the isomer resulting from cyclisation ortho, instead of para, to the OR group. The para/ortho ratio varied depending on the nature of the allylic leaving group in the substrate^®. For example.

12 Chapter 1: Introduction

in the case of 33 having a free hydroxyl group, the ratio was 8.4:1 ; on the other hand, when the leaving group was trimethylsilyloxy the ratio was 2.6:1. Thus the effect of the leaving group is

“felt” in the bonding of the aromatic nucleus to the internal olefinic bond, suggestive of a synchronous process. Moreover, kinetic studies of the cyclisation of the substrate 33 having other groups {i.e., CHg, H, CFg) in place of OH in the aromatic nucleus was found to give decreasing rates as the groups became more electronegative^®. These findings are also consistent with a synchronous process. Although the evidence points strongly toward a synchronous process in the case of the cyclisation 33^34, it should be emphasized that this result does not constitute proof that all cationic polyene cyclisations are synchronous.

OH RO RO 34 33

HO RO 35

Scheme 18: Biomimetic cyclisation of 33->34

W.S. Johnson concluded that acid-catalysed biomimetic polyene cyclisation of acyclic chains is a viable synthetic tool for the stereospecific formation of polycyclic systems. Acetal as well as allylic alcohol functions are useful initiators for these cyclisations, and methylacetylenic end groups have been found to be particularly useful terminators which have shown to give exclusively five-membered ring formation, and thus make possible the total synthesis of the steroid nucleus in a single step starting from a one-ring substrate.

Whilst this discussion has focussed entirely on the state of biomimetic steroid synthesis to the end of 1976, further developments will be discussed in section 1.1.6 in connection with the biomimetic synthesis of p-amyrin by Johnson.

13 Chapter 1: Introduction

1.1.4 K.C. Nicolaou’s Biomimetic Approach to Endiandric Acids A-G: The Endiandric

Acid Cascade.^^ (1982)

The endiandric acids are a notable class of secondary metabolites, that were isolated from the Australian plant Endiandra introrsa {Lauraceae) by David St. Black’s group^^ in the early

1980’s. They possess a striking novel molecular architecture, that is based upon some truly intricate structural inter-relationships. Endiandric acids A, B, and 0 consist of four fused carbocyclic rings, a phenyl substituent and a carboxyl group (Scheme 19). Although they

•Ph ■Ph HOgC.

B C

Scheme 19: The Endiandric Acids A-D contain eight stereogenic centres, the endiandric acids are found as racemates in nature, which is very unusual for natural products. In order to explain this observation, St. Black proposed that the “biosynthesis” of these molecules involved achiral polyunsaturated precursors, and proceeded through a series of nonenzymatic electrocyclisations (Schemes 20 and 23).

St. Black postulated a cascade of reactions as the pathway by which endiandric acids A-

D are formed in nature. Thus, endiandric acids E, F, and G were proposed as immediate precursors of the tetracyclic endiandric acids A, B and C; the final skeletal assembly being effected by an intramolecular Diels-Alder reaction.^® Endiandric acid D does not form a tetracycle, as it cannot undergo an intramolecular Diels-Alder reaction. Another feature of the

St. Black hypothesis was the contention that endiandric acids D-G might arise from the sequential electrocyclisations of achiral polyenes IIV through the intermediacy of 1,2-trans- disubstituted cyclooctatrienes.

At the time of their discovery, the molecular frameworks of the endiandric acids were unprecedented, and in 1982, intrigued by these unique structures and St. Black’s hypothesis,

K.C. Nicolaou’s group at the University of Pennsylvania began a program directed towards their total synthesis.^®

14 Chapter 1: Introduction

-Ph

O f Q c

HO2C

endiandric acid D endiandric acid E endiandric acid F endiandric acid G

Diels-Alder Diels-Alder Diels-Alder

a: conrotatory 8 n electron electrocycllsatlon b: disrotatory 6 tc electron Ph electrocycllsatlon

endiandric acid A acid B endiandric acid Cendiandric

Scheme 20: The endianidric acid cascade (R=H=Me): The Black Hypothesis

By assembling compound II (Scheme 20) from compound 36, K.C. Nicoiaou’s group were able to demonstrate that a “biomimetic” one-step synthesis of the endiandric acids could be achieved involving the cascade of reactions proposed by St. Black. (Scheme 21)^®

COOMe

Hg, Undlar

Heat R=Me PhMe

DAE

Scheme 21 : One-Step Generation of Endiandric Acids A, E, and D Methyl Esters from Acetylenic Precursor 36

15 Chapter 1: Introduction

Mild hydrogenation (Hg, Undlar catalyst, quinoline, CHgCI, 25°C) of the acetylenic precursor 36 followed by brief heating of II at 100°C (toluene), produced endiandric acid A methyl ester. In this reaction, the endiandric acid A complex polycyclic framework was essentially formed in a single step, by creating stereospecifically four new rings and eight asymmetric centres from an achiral open-chain precursor. Endiandric acid D methyl ester was not isolated under these conditions, although, when the hydrogenation mixture was carefully examined prior to

thermolysis, endiandric acids D and E methyl esters were found to be present in 1 2% and 10% yields, respectively.^®

Similarly, mild hydrogenation of the diacetylene 37 (Scheme 22) as described for 36, followed by brief heating of the resulting mixture at 100°C, led to the isolation of endiandric acid

B methyl ester and endiandric acid C methyl ester in ca. 28% yield. (B:C ca. 4.5:1). Now, in this reaction, the complex polycyclic structures of endiandric acids B and C (which appear to be unrelated), were also formed in a one-pot process.

COaMe Ph

MeOaC' ■Ph

B C

.COaMe MeOaC

Scheme 22: One-Step Generation of Endiandric Acids B, C, F, and G Methyl Esters from Acetylenic Precursors?

By operating exclusively at 25°C, K.C. Nicolaou’s group were again, able to detect and isolate endiandric acids F and G methyl esters in ca. 15% and 12% yields, respectively. It was at this stage, that it became very apparent that the genesis of endiandric acids in nature from

16 Chapter 1: Introduction

polyunsaturated achiral precursors was feasible without enzyme participation thereby confirming

David St. Black’s hypothesis.

In conclusion, K.C. Nicolaou’s group has demonstrated in an elegant series of papers^®,

that Woodward and Hoffmann thermally-allowed 8 n and 6 ti electrocyclic cyclisations can be used very effectively for the stereospecific construction of these complex natural products

(Scheme 23).

conrotatory 8 n disrotatory 6 electron electron 1 Qr." electrocycllsatlon Y y electrocycllsatlon

conformational switch

disrotatory 6 n electron

^ electrocycllsatlon

Scheme 23: Thermally allowed 8 n electron and 6 t i electron electrocyclisations (Woodward-Hoffmann rules)

Nicolaou’s “biomimetic” approach to the endiandric acids provided a final verification of

Black’s non-enzymatic hypothesis for the creation of the endiandric acids. Nicolaou also deduced that endiandric acids E-G, which have not as yet been found in Nature should be stable enough to be isolated from Endiandra introrsa {Lauraceae)}^

1.1.5 Heathcock’s Biomimetic Totai Synthesis of Methyi Homosecodaphniphyiiate-The

Daphniphyllum Aikaioids.^ (1992)

Daphniphylline (38) and secodaphniphylline (39) represent two of the three basic

classes of C-30 Daphniphyllum alkaloids. They are accompanied in nature by their C - 2 2 counterparts, methyl homodaphniphyllate (40) and methyl homosecodaphniphyllate (41).

(Scheme 24). Daphniphylline is more common than secodaphniphylline, e.g., 1000 kg of D. macropodum leaves yielded 100 g of 38 and only 1.1 g of 39.®°

17 Chapter 1: Introduction

AcO

HN j

38 40

Scheme 24: C-30 Daphniphyllum Alkaloids (38 & 39), and their C-22 counterparts (40 & 41)

Examination of the skeleton of secodaphniphylline reveals that the unbroken squalene molecule can be traced through the pentacyclic domain. In order to convert squalene into

secodaphniphylline, four C-C bonds need to be formed: Cio to C 1 4 : Ce to C15 : C3 to the C 15

methyl group: C 7 to the C 10 methyl group. In addition, a nitrogen must be inserted between the

Cio and C 15 methyl groups. For daphniphylline, the nitrogen must be positioned between Cio and its methyl group. Thus, it is likely that secodaphniphylline precedes daphniphylline biosynthetically. A plausible biosynthetic link between the two skeletons is the unsaturated amine 42 (Scheme 25).

squalene secodaphniphylline skeleton

daphniphylline 42 skeleton

Scheme 25: Conversion of squalene into both the secodaphniphylline and daphniphylline skeletons

18 Chapter 1: Introduction

With the intention of imitating this hypothetical biosynthetic scheme in the laboratory,

Heathcock focused his attention to the methyl homosecodaphniphyllate as a synthetic target.

The key manoeuvre in his retrosynthetic plan (Scheme 26) was an intramolecular Diels-Alder reaction followed by an intramolecular ene reaction. Although thermal Diels-Alder reactions of 2- aza dienes usually require temperatures of over 200°C,®^ Heathcock hoped that the suggested conversion of 45 to 44 might occur under acid catalysis under milder conditions. It was thought that the required aza diene for this process might arise from the monocyclic dialdehyde (46), which could potentially be derived from a three-component condensation of an enolate, an a,p- unsaturated carbonyl compound, and the known homogeranyl iodide (49).^^

Whenever there is an alkyl substituent at the 2-position of the unsaturated ester, such as in the enoate 48, the 1,4-addition of ester enolates is not favourable. Heathcock found the

.OR .OR OR COaMe

R' OROR

47 OHO 48

OHO

Scheme 26: Retrosynthetic analysis of secodaphniphylline preliminary experiments with ester 50^^ and enoates 51 or 52 (Scheme 27) discouraging.

Formation of the enolate of ester 50 with LDA in THF at -78°C followed by the addition of enoate

51 did not produce any of the desired Michael adduct. Instead, a low yield of the 1,2-adduct was

19 Chapter 1: Introduction

isolated. Use of HMPA as a cosolvent did not alter this result. Formation of the enolate of ester

50 with LDA in THF or THF/HMPA and addition of enoate 52 at -78°C gave no reaction; warming slowly to 0°C did not induce the desired Michael addition. Thus, the undesired 1,2- addition was prevented using tert-butyl enoate 52 but 1,4-addition was still not observed.

Addition of enoates 51 or 52 to the dianion®'* of the acid corresponding to ester 50 also resulted in no 1,4-adduct.

,OBn Me02C‘ MeOgC fBu02C 50 51 52

Scheme 27; Ester 50 and enoates 51 & 52

In connection with his study of the stereochemistry of the Michael reactions of the lithium enolates of amides, Yamaguchi found that the lithium enolate of A/-propionylpyrrolidine adds smoothly to the ethyl ester corresponding to enoate 51 to give two diastereomeric products in a ratio of 1:1.®® Since the stereoisomers result from stereorandom protonation of the ester enolate, it appears that the Michael addition itself occurs with high stereoselectivity. On this basis, Heathcock prepared amide 53. Treatment of the lithium enolate of 53 successively with ester 51 at -78°C, followed by homoprenyl iodide (54)®® at room temperature gave three products in a total yield of 87% (Scheme 28).

•OBn .OBn . LDA, THF i. ester 5 1 ,-78°C

N .OBn

MeOgC'

55 56a,b

Scheme 28

The major isomer 55, was obtained in 78% yield. The two minor isomers (56a,b) were obtained as an inseparable mixture in 9% yield.

20 Chapter 1: Introduction

In the alkylation step, the halide would approach the enolate trans to the bulky side chain, and therefore lead to 55 (Scheme 29). The eventual production of methyl homosecodaphniphyllate by this approach validated the assignment of the relative stereochemistry of the two cyclopentyl stereocentres.

•OBn OBn

N 55

,OMe LiO OMe

Scheme 29

The foregoing tandem Michael addition-alkylation was repeated using homogeranyl iodide as the alkylating reagent to give three diastereomeric adducts in a total yield of 99%

(Scheme 30). The major isomer (87% yield) was 57, and the two minor isomers, 58 and 59, were isolated as a 2:1 mixture in a combined yield of 12%.

i. LDA. THF OBn OBn OBn ii. ester 51,-78 °C iii. I I

OBn -78^25 °C, 16 h (99%)

Scheme 30: The tandem Michael addition-alkylation using homogeranyl iodide

Thus, the tandem Michael addition-alkylation process allowed the assembly of amide

57, which contained all of the carbons present in the skeleton of methyl homosecodaphniphyllate, in one step from relatively simple starting materials.

21 Chapter 1: Introduction

Their next task was transformation of the ester and amide groups of 57 into aidehyde groups. A good deal of experimentation with the model ester-amide 55 led to a three-stage synthesis. (Scheme 31). Thus, treatment of 57 with excess DIBAL gave hydroxy amide 60,

OBn OBn OBn OBn

i. KOH, EtOH, DIBAL, H2 0 , 95 °C HOCH2 toluene, -78 °C ii. HCI, H2O HOCH2' (83%) (100%) (96%)

61a,b 62a,b

Scheme 31 : Summary of three-stage protocol to 62a, b

which was treated with 5 M KOH at 95 °C for 2 h. Acidification of the saponification mixture gave lactone 61 as a 1:1 mixture of diastereomers in quantitative yield. Lithium aluminium

hydride reduction of the lactone mixture gave a 1 : 1 mixture of epimeric diols 62a and 62b in nearly quantitative yield.

Swern oxidation®^ under normal conditions (oxalyl chloride, DMSG, EtgN, CHgClg) was performed on diols 62a,b. Gaseous ammonia was then passed over the surface of the stirring solution, followed by evaporation of the solvent and volatile by-products to give a residue that was taken up in acetic acid containing NH^OAc. The resulting solution was heated at 70 °C and worked up to obtain pentacyclic amine 63 in 82% yield. (Scheme 32).

•OBn

.OBn

1. Swern, CH 2 CI2 2. NH3 HOCH; 3. HOAc, NH4OAC, 70 °C, 1.5 h HOCH; >- (82%)

62a, b

Scheme 32: Formation of pentacyclic amine 63

22 Chapter 1: Introduction

Whilst optimising the conditions for the tetracyciisation process (62^63), Heathcock found that a significant amount of by-product was formed when 4 equivalents of the Swern

oxidant was used. This by-product, which was obtained in about 10% yield when excess

oxidant was added, was assigned structure 64. It was presumed that compound 64 formed from

amine 63 via the immonium ion 65, which could result from the presence of some residual

methylenating species that is left over from the Swern oxidation. (Scheme 33).

OBn OBn OBn

OAc

63 65 64

Scheme 33: Formation of byproduct 64 from amine 63 via immonium ion 65

Several reaction intermediates during the conversion of 62a,b into 63 were isolated and

characterized. Swern oxidation gave the dialdehydes 6 6 , in 55% yield. This reaction was repeated without isolation to the dialdehydes and ammonia was added to afford aza diene 67, in

44% yield. This reaction was repeated, and 67 was dissolved in acetic acid for 10 mins,

affording imine 6 8 in 90% yield. Tic revealed that the Diels-Alder reaction was complete in less

than 5 mins. When the acetic acid solution was heated at 70°C for 1 .5 h, pentacyclic amine 63 was obtained. (Scheme 34).

The mechanism of the tetracyclization process may be completely stepwise (Scheme

35), or it could involve a concerted Diels-Alder reaction to give 68-H+, followed by a concerted ene reaction®® to give 63-H+. Since subsequent mechanistic investigations®® showed that the E stereochemistry of the homogeranyl double bond was preserved throughout the process,

Heathcock suggested that 6 8 -H+ was formed by a concerted, highly asynchronous, Diels-Alder reaction. Although unnecessary, when NH^OAc is used in the tetracyciisation reaction, the yield

23 Chapter 1: Introduction

.OBn .OBn OBn

HOCH; Swern OHO NH;

HOCH; OHC

62a,b OBn OBn

NH4OAC. NH4OAC, HOAc, 2 5 ° C HOAc, 70°C

0 0 03

Scheme 34: Reaction intermediates in the conversion of 62a,b into 63 appears to be slightly improved. The excess acetate ion may stabilize the cationic intermediates in the reaction. An ancillary experiment also showed that the reaction works well without

EtgN HCI which results from the Swern reaction. Thus, NH 3 was added to purified dialdehydes

6 6 at 0°C, and once the solvent was removed, acetic acid and NH^OAc was added to the

residue and heated for 1 h at 70°C to afford amine 63 in 90% yield.

.OBn OBn OBn

OBn OBn 67-H+ 6 8 -H+

63-H+ Scheme 35: Mechanism of the tetracyciisation process via a concerted Diels-Alder reaction

24 Chapter 1: Introduction

Further insights into the tetracyciisation mechanism also came from performing the cyclisation of

62a,b with methylamine instead of ammonia. Now the pentacyclic saturated amine 69 was obtained in 75% yield (Scheme 36). This was also produced when benzylamine was used in the second stage of the tetracyciisation protocol.

OBn

1. Swern, GHzGIz 2. GHaNHz or PhGHzNHz HO GHz 3. HOAc, NH4 OAC, 85 °C, 10 h HO GHz (65-80%)

62a, b 69

Scheme 36

The mechanism first suggested to Heathcock by Professor Steven Pedersen, that accounts for the formation of 69 is shown in Scheme 37. After reaction of dialdehyde 66 with the primary amine to give 70, treatment with acetic acid at room temperature is performed, which immediately transforms 70 into the tetracyclic immonium salt 72. The postulated intermediate N- methyldihydropyridinium salt 71 was never observed, which suggests that it undergoes a very rapid intramolecular Diels-Alder reaction. Further reaction of 72 occurred when the acetic acid solution was heated at 80°C for 10 h or kept at room temperature for several days. Cyclisation of 72 could be expected to provide the amino cation 73, which should undergo intramolecular hydride transfer to give 74/° Compound 69 then arises via hydrolysis of 74.

25 Chapter 1: Introduction

OBn OBn OBn

OHC

OHC RCHg' NH

.OBn OBn OBn

H2O 69

72 73 74

Scheme 37: Proposed mechanism for the formation of 69

With an efficient synthesis of amine 63 in hand, Heathcock investigated its conversion to

methyl homosecodaphniphyllate. (Scheme 38) On large scale ( 2 g of amine 63) a large catalyst

ratio (100 mass percent Pd/C), and a long reaction time (47 h) are required to accomplish the

hydrogenation step. Addition of 5 equivalents of HCI and further reaction for 60 h completes the

hydrogenolysis to give 75-H \ which is the HCI salt of amino alcohol 75. in 96% yield from amine

63. Neutralisation of the HCI salt gave the amino alcohol 75 Oxidation of salt 75-H+ with excess Jones reagent*^ directly afforded the amino acid 76 Excess Jones reagent was added at 0°C, and the excess oxidant was quenched with isopropyl alcohol after 30 mins. The solvent was removed to give the crude amino acid salt (76-H+), which was taken up in methanol, then stirred overnight at roomtemperature. This reaction mixture was then worked up to obtain (±)-

methyl homosecodaphniphyllate (41) in 8 6 % yield from 75-H+.

OBn i- H2. Pd/C OH ii. Hz, Pd/C. HCI Jones MeOH, H+ (96%) (86%)

HN- HN-

76-H+ (±)-41

Scheme 38: Conversion of 63 to (±)-methyl homosecodaphniphyllate 41

26 Chapter 1: Introduction

The total synthesis of (+)-methyl homosecodaphniphyllate (41) is summarised in

Scheme 39. This synthesis required nine steps and proceeded in 48% overall yield starting from the simple materials 49, 51, and 53. The key steps in this synthesis were the tandem Michael

addition-alkylation process, which assembled all of the carbons required for the skeleton, and the tetracyciisation reaction, which gave the complete pentacyclic skeleton in one operation.

OBn OBn

O' ■Ph

HOCH2' 3 steps CO2 M6 HOCH2 ' < y (87%) (81%) 51

62a,b 49 OBn COaMe

2 steps 3 steps

(82%) (83%) HN- HN-

63 (±)-41

Scheme 39: Summary of total synthesis of (+/-)- methyl homosecodaphniphyllate (41 )

All of the steps in this synthesis can be carried out efficiently on gram scale. More than

3.5 g of (±)-methyl homosecodaphniphyllate has been synthesised using this route. Since the tetracyciisation process is so efficient, and proceeds under such mild conditions with common reagents, Heathcock believes that it is probably biomimetic.

27 Chapter 1: Introduction

1.1.6 W.S. Johnson’s Total Synthesis of d/-^Amyrin: The Fluorine Atom as a Catlon-

Stablllzlng Auxiliary In Biomimetic Polyene Cyclisations.^^ (1993)

HO 77

Scheme 40

y^Amyrin 77 is Isolated from the latex of rubber trees and from Erythroxylum coca, and

is the parent compound of the oleanane family of pentacyclic triterpenoids. Possessing five

fused rings and eight chiral centres, the prospect of assembling this compound has captivated

synthetic chemists for several decades, and has resulted in two formal total syntheses,'^®® *’ and a

number of reports on the conversion of other triterpenes to

In 1993, Johnson disclosed his total synthesis of racemic yg-amyrin 77 in ca. 0.2%

overall yield, based on a biomimetic polyene cyclisation that generates in one step (78-^79), six of the eight chiral centres of the pentacyclic triterpene in 65-70% yield.'^^

pro-C-17

pro-C-13

SiMes

Scheme 41

28 Chapter 1: Introduction

The route evolved out of Johnson’s earlier studies,'^ which found that a suitably

positioned fluorine atom on the polyolefin could effectively stabilise incipient cationic centres

during acid-catalysed cyclisation, leading to much higher yields of tetracyclic and pentacyclic

compounds. In this study Johnson also discovered that the fluorine atom, acting as a cation-

stabilising (C-S) auxiliary at the pro-C-13 position in substrate 80, controlled the regiochemistry

of the cyclisation, producing exclusively the 6 -membered ring C in pentacycle 81. In order to

apply these findings to the synthesis of yg-amyrin, Johnson modified the substrate 8 0 ^ so that

the olefinic bond at pro-C-17 possessed the cis configuration for producing the cis D/E ring

fusion found in 77.

A linear sequence was used to access cyclopentenol 78 from mesityl oxide, with fluoro

dienynol 82 featuring as a key intermediate. The latter was prepared"^ in nine steps, with an

overall yield of 20%. Transposition of the trans trisubstituted alkene in 84 to the cis isomer 85 was achieved by the epoxidation/phosphine elimination route of Vedejs and Fuchs.’*® Thus compound 84 was treated with lithium diphenylphosphide, followed by iodomethane, to obtain the cis trisubstituted alkene 85 in 81% overall yield from 82 (99:1, cis.trans). Tetra-n-

butylammonium fluoride removed the silyl protecting group’*® to give alcohol 8 6 , which provided the acetate 87 in 83% yield upon acétylation.

Fortunately, when acetate 87 was treated with the sodium enolate of keto ester 88 in the

presence of palladium tetrakis(triphenylphosphine),’*^ an 8 8 : 1 2 mixture of the pro-C-13 trans keto ester 90 was produced, along with its c/s isomer, which was separated to give 90 in 73% yield.

Decarbethoxylation of keto ester 90, followed by reduction of the ketone, gave cyclopropylcarbinol 92 in 95% yield, therefore improving the efficiency of the conversion of

86-»92, which is now achievable in four steps and in 61% overall yield.

29 Chapter 1: Introduction

\ NaH (60%) 1 . 2 ,6 -lutidine, 1 . Ph2PLi A (Ph3P)4Pd ©uMe2SiOTf : 2. Mel F ■' ^ PhsP Et0 2 C 2. mCPBA Y l 1 3. TBAF 98% 4. DMAP R0 -" o 8 8 o 82: R=H 84: R=Si(Me) 2^-butyl 85: R=Si(Me) 2f-butyl 83: R=Si(Me) 2^-butyl 8 6 : R=H 87: R=C0CH3 cJy Y < Aq. NaOH (5%) in THF/MeOH ONa O , (1:1) 97% 89

t-BuLi (1.7M) LiAIHa 98% SM 63 HO'

ZhBig (Anhydrous) 82% 2,6-lutidine 1 • NaH (60%)/THF ifPBr3

aM8 3 , NaOH (ac^ % aM6 3 — -— ► 2. Nal/Acetonitrile's%'l\/ 92% 83% oO o 96 97 PPTSPPTS 97% Acetone/Water (10:1)

(10%) MeULiBr Aq. NaOH aM0 3 aM6 3 aM0 3

Scheme 42: Synthesis of cyclopentenol 78

Incorporation of the propargylsilane moiety was accomplished by treatment of 92 with

fert-butyllithium and trimethylchlorosilane, and gave alcohol 93 in 79% yield. The Brady-Julia

rearrangement^® of 93 entailed bromination with phosphorous tribromide, followed by zinc

bromide-catalysed rearrangement to give bromide 94 in 82% yield {trans.cis > 97:3). Keto ester

95 was then alkylated with bromide 94, to give keto ester 96, which could then be decarboxylated by heating with aqueous base giving ketone 97. Conversion to diketone 98 by treatment with pyridinium p-toluenesulfonate followed by cyclodehydration with aqueous base at ambient temperature afforded the cyclopentenone 99 in 61% overall yield from bromide 94 without the base-sensitive propargylsilane moiety being isomerised. Addition of methyllithium to cyclopentenone 99, completed the synthesis of cyclopentenol 78 in 97% yield. Thus,

30 Chapter 1: Introduction

cyclopentenol 78, bearing the propargylsilane terminating group and having the 3-trans, 7-trans,

1 1 -c/s alkene stereochemistry, was synthesised in over 18% yield for the 8 6 steps from alcohol

82. Optimal conditions for the cyclisation, added a solution of cyclopentenol 78 in dichloromethane to trifluoroacetic acid in dichloromethane at -70°C, and produced the pentacycle 79 in 65-70% yield. Johnson’s preparation of yg-amyrin from pentacycle 79, was accomplished using a straight-forward series of transformations, which entailed: (a) removal of the allene moiety from ring E, (b) elimination of the fluorine atom to produce the C-12 alkene, and (c) construction of the correctly functionalised six-membered ring A with the trans A/B ring fusion (Scheme 43).

\ TFA/CH2CI2 Aq. NaOH W -70°C/70% 93% O ^3M63 I 75%

OH 100 101

80 % CH2 CI2 -10° C 2 ,2 -dimethyl OH UAIH4 O -prop^ane-1,3-dioi

PPTS/CeHe 100°C/3 h 76% o 1 0 2 104 103 1. n-BuLi (in hexane) 2 . CS2 3. Mel

1, BusSnH/Toluene OCSSCHg 2 , azobis(isobutyronitriie) Hydrolysis

PhSH formaldehyde triethanolamine 120-125°C 8 h 73% NaBHj MeOH/THF(1:1) 1. L1/NH3

26% overall yield from 108 2. Mel O ' ^ 108 CHgSPh Scheme 43: Synthesis of p-amyrin 77 from pentacycle 79

The Sharpless method'^® was employed to oxidise pentacycle 79, using ruthenium trichloride and sodium metaperiodate, which produced the triketone 100 in 75% yield. When this was treated

with aqueous base, the enone 1 0 1 was produced, which upon standing in a solution of tin

31 Chapter 1: Introduction

tetrachloride in dichloromethane at -10°C, underwent regioselective elimination of hydrogen

fluoride, producing alkene 102 in 70% overall yield from triketone 100.

Selective removal of the more sterically hindered C-22 ketone, involved the use of a

lithium aluminum hydride reduction on 103, down to the alcohols 104 (1:1 mixture) followed by

the formation of the methyl xanthate 105 by reaction with carbon disulfide and iodomethane,

which produced an overall yield of 77% from 103. Xanthate 105 was then reduced using

tributyltin hydride and azobis(isobutyronitrile) to afford the C-22 deoxygenated pentacycle 106.

Hydrolysis of 106, produced the ketone 107 in 87% overall yield from 105.

With rings B, C, D, and E assembled, only the modification of ring A was required by

the introduction of the 4,4-dimethyl groups and reduction of the C-3 ketone to the Sy^alcohol.

Thus, the 4-phenylthiomethylated enone 108 was produced in 73% yield by the treatment of 107

with benzenethiol and formaldehyde in the presence of triethanolamine. Enone 108 was then

converted to the 4,4-dimethyl ketone 109 by reductive méthylation, using lithium in ammonia,

followed by quenching of the C-4 enolate with iodomethane. Finally, the crude ketone 109 was

reduced using sodium borohydride, then careful purification using chromatography afforded d l-^

amyrin 77, in an overall yield of 19% from ketone 107.

1.1.7 Paterson’s Studies In Biomimetic Poiyether Synthesis: Construction of an ABCD-

Ring Subunit of Etheromycin Using Poiyepoxide Cascade Cyclisations. (1993)

The polyether antibiotic etheromycin 110 is characterised structurally by a complex

array of six ether rings (A-F) with a multitude of associated stereocentres®^ The Cane-Celmer-

Westley hypothesis®^ suggested that it might be derived biosynthetically from the cyclisation of

polyepoxide 111 (Scheme 44). This would require the 0(7) hydroxyl of 111 to form the A-ring

hemiacetal, and the 0(9) hydroxyl to trigger the epoxide ring-opening cascade to give 110 by its

internal attack on the 0(13)-keto group. It is worth noting that for this to be synthetically viable, the alternative cyclisation mode for polyepoxide 111, where the 0(7) and 0(9) hydroxyls become

involved in bicyclic acetal formation (blue arrow), has to be less favourable.

32 Chapter 1: Introduction

OH OH /O H 111 vOH OMe 27

OH OMe

etheromycin 1 1 0 ">h

OMe

Scheme 44: Cyclisation of polyepoxide 111

Through a series of studies, Paterson has shown that the aforementioned polyepoxide ring- opening strategy is feasible for the construction of the fragment 113 and the similar

fragment 115. (Scheme 45).

CSA

113

112 OH HO OH 27

HF >■

.0©

114 115

P1 = P2 = TBS OH OPz OH

Scheme 45: Acid-mediated cascade cyclisations of polyepoxide precursors to the ODE and BCD rings

33 Chapter 1: Introduction

By analogy with Paterson’s earlier work“ ^, the enones 117 and 118 (Scheme 46) were

chosen as potential acyclic precursors for the A BCD rings of etheromycin, where the C 13 ketone group is constrained by the trans double bond from premature internal opening of the neighbouring epoxide. The reductive removal of this double bond and oxidation at 0 ^ should give a p-diketone^ to trigger the cyclisation cascade.

OBz OH OBz HO

OMe H CEI PS BnO' OPMB BnO' OPMB 117 118

Scheme 46

The synthesis of the more complex enones 117 and 118 is shown below. (Scheme 47).

E^O 4 steps TBDPSO. 4e.c^ OHC ref 53a ^ 9 (63%)

OPMB OPMB

OBz OPMBOBz OPMB OBz CHO 3— C (84%) (74%) ''OH (38%) OMe H 125 CEI PS BnO' BnO' BnO' OPMB 126 119 1 2 0

OBz HO b,c (86%)

OMe H CEI PS BnO' OPMB

OBz OBz HO OBz OH CHO

O +123 (51%) (80%)

BnO' 128 BnO'117 OPMB

Scheme 47: a) EtgiPrSiCI, imidazole, DMAP, DMF, 25°C, 16h; b) DDQ, HgO, CHgClg, 25°C, 3 min; c) Dess-Martin periodinane, CHgClg, 25°C, 0.5 h; d) PMB 0 C(CCl3 )=NH, caf TfOH, EtgO, 25°C, 40 min; e) TBAF, THF, 25°C, 0.5 h; f)

Ba(0 H)2 .8 H2 0 (0.7eq), aq. THF, 25°C, 40 min; g) LDA, THF, -78°C, ZnCl 2 , 120, 1 h; h) 0.5 M HCI, THF, 25°C, 10 h; I)

PPTS, MeOH, (MeO) 3 CH, 25°C, 10 h; j) 124, n-BuLi, THF, 0°C; 127, -78°C, 1 min.

The previously reported^^^ acetal 126 was converted into the aldehyde 120, in five steps by protecting group adjustment followed by Dess-Martin oxidation. The synthesis of the diepoxide

34 Chapter 1: Introduction

1 2 1 from 1 2 2 relies on sequential Sharpless asymmetric epoxidation^® for control of the epoxide stereochemistry. Subsequent conversion into the aldehyde 123 was followed, in turn, by a

Ba( 0 H)2 -mediated^^ Horner-Emmons reaction with 124 to give the enone 125, in 59% overall yield. Using the Zn enolate^^ derived from 125, the aldol coupling with the aldehyde 120 gave an 84% yield of the enone adducts 118. The alternative cyclisation precursor 117 has Cg as the free ketone, and both the 7-OH and 9 -0 H are protected as the acetonide. This was prepared from 126 (precursor of 119)^^ starting with the DDQ deprotection of the PMB ether, followed by

oxidation to give the aldehyde 127 in 8 6 % yield. Addition of a p-ketophosphonate dianion 124, to 126, then produced 128, and a Horner-Emmons coupling of 128 with the aldehyde 123, again

mediated by Ba( 0 H)2 ,®® gave the enone 117 as a mixture of epimers at and 0 ^ 2

Paterson had predicted that the diethylisopropylsilyl (DEIPS) protecting groupé® would come off more rapidly than the acetonide would be hydrolysed in 117, which would allow the

0(11 )-0H to be oxidised to the corresponding ketone 129. Installation of a ketone at 0(11) could be expected to favour the subsequent cyclisation cascade since it would reduce the degrees of freedom in the cyclisation precursor and give “turn-like” structure to this molecule.

The free 9-OH in 129 was now unmasked to participate in the cyclisation cascade, to obtain 118.

OBz no OBz

OMeH OMe H 129 CEI PS CEI PS BnO' OPMB BnO' OPMB

(30%)

OBz OBz O 130a Ri=Me;R 2 =H 130b Ri=H; R2=Me

OMe H (51%) BnO' BnO' OPMB OPMB OH OH

OBz OH O OBz O

BnO' OPMB BnO' OPMB 131

Scheme 48: a) Hg, Rh/AlgOg, THF, 25°C, 0.5 h; b) Dess-Martin periodinane, CHjClj, 25°C, 0.5 h; c) 0.5 M HCI, THF,

25°C, 2.5 h; d) PPTS, (MeO) 3 CH, MeOH, 25°C, 8 h; e) ( 0 0 0 1 )2 , DM80, OHgOlg, -78°0, 45 min, EtgN, -78°0->-20°0; f) nBUgSnH, (Ph3 P)4 Pd (0.5 mol%), THF, 25°0, 45 min; g) 0.5 M HOI, THF, 25°0, 16 h.

35 Chapter 1: Introduction

Thus, 118 was primed for cyclisation by careful reduction of the enone double bond by Hg and

Rh/AlgOg in THF (Scheme 48), followed by oxidation using Dess-Martin periodinane. The resulting p-diketone 129 was treated directly with 0.5 M HCI in THF to generate the polyether sequence in 130a and the 12 epi compound 130b in 30% overall yield (70:30 ratio). The major ketone 130a was then subjected to acetal formation (PPTS, (MeOjgCH, MeOH), leading to the isolation of the fully cyclised compound 116, with all the ABCD rings in place. The stereochemistry in 116 was determined by the assignment of the ^H and NMR spectra, followed by extensive NOE experiments.^® Less satisfactory results were obtained when starting with the acetonide-containing enone 117. A lower overall yield was obtained, presumably due to the inefficiency of acetonide removal relative to cleavage of the DEIPS ether in 129.

Paterson anticipates that future work will achieve a biomimetic synthesis of the complete

ABGDEF ring system.

1.1.8 Baldwin’s Studies on the Biomimetic Synthesis of the Manzamlne Alkalolds.^^

(1999)

The manzamines are a structurally unique class of alkaloids, which were first isolated by

Higa from a collection of the marine sponge Haliclona species gathered off Manzamo, Okinawa, in Japan.Manzamine A (132) was found to have in vitro activity against P388 mouse leukaemia cells (ICso=0.07 ^g mL'^ or 2.4 ^ig mL"" ^®) and X-ray crystallography established its structure (Scheme 49).

,0 H

132 133 134

Scheme 49: Manzamine A (132), B (133) and C (134)

36 Chapter 1: Introduction

Higa’s group®° later reported the isolation of manzamines B (133) and C (134) from the same sponge. Since biosynthetic investigations on marine sponges are notoriously difficult,®^ no biosynthetic work, to date, has been published on the manzamines. However, in 1992, Baldwin and Whitehead put forward, a now famous proposal, in which they postulated that the manzamine alkaloids are formed from just four building blocks: tryptophan, ammonia, a Cio unit

(a symmetrical dialdehyde) and a C 3 unit (an acrolein equivalent).®^ Their careful analysis of the manzamine B (133) structure revealed that it potentially derived from a highly symmetric set of precursors (Scheme 50). Their retrosynthetic analysis indicated that by the removal of the epoxide and the tryptophan unit from 133 an amino aldehyde 135 could be obtained. Aldehyde

135 could itself be derived by hydrolysis of the iminium ion 136. A redox equilibrium might relate the manzamine precursors 136 and 137. Disconnection of 137 by an intramolecular endo Diels-

Alder reaction gives macrocycle 138, which is the conjugate base of 139. It was thought that the

other three building blocks (two equivalents of ammonia, and the C 3 and C 10 units) could combine to give the symmetrical macrocycle 139. Thus, Baldwin provided a simple but extremely elegant explanation for the biosynthesis of a very complex molecule.

©N tryptophan >

N©

133 135 136 137

139 138

Scheme 50: The Baldwin-Whitehead hypothesis for the biosynthesis of the manzamine alkaloids

Since their biosynthetic proposal was first published a host of other manzamine-related alkaloids have been discovered. Baldwin’s hypothesis has since been adapted and modified on a number

37 Chapter 1: Introduction

of occasions to explain the possible biosynthetic origin of these new alkaloids. Many of these natural products bear a striking resemblance to intermediates from the hypothesis, such as ircinal (140, the aldehyde precursor to 132) and keramaphidin (141, the reduced form of

136 and 137) (Scheme 51).

O ^ H

,\'OH

140 141

Scheme 51 : Ircinal A (140) and keramaphidin B (141)

Concurrent with the publication of the manzamine biosynthetic hypothesis, an investigation into the validity of the hypothesis was initiated, which has so far led to the biomimetic synthesis of keramaphidin B (141) by Baldwin’s group.

The pentacyclic alkaloid, keramaphidin B (141) was isolated independently by

Kobayashi at a/.^ The structure of 141 was established through a combination of NMR spectroscopy and X-ray crystallography. Even though there was approximately 97% of the (+)- form in the sponge, 141 crystallised as a racemate. This observation strengthens the hypothesis of a Diels-Alder cycloaddition with the achiral precursor 138 proposed in the manzamine biosynthesis, since both enantiomers of the reduced cycloadduct 141 occur naturally. Baldwin’s group and others confirmed the feasibility of the proposed key Diels-Alder reaction by previous model studies which they had conducted.Formation of 143, the core structure of keramaphidin B (Scheme 52), was achieved by treating 142 with a pH 8.3 TRIS/HCI buffer solution followed by quenching with sodium borohydride.®®

i) pH 8.3 TRIS/HCI

ii) NaBH4 , HgO, MeOH

142 25% 143, 25-35%

Scheme 52: Formation of tricyclic core structure 143

38 Chapter 1: Introduction

With this knowledge in hand, Baldwin’s group set out to verify whether the same Diels-Alder

reaction could be performed Intramolecularly. Since this investigation required an efficient synthesis of the macrocycle 139, tetrahyd ropy ran was treated with acetyl bromide to give 5-

bromopentylacetate,®® which was heated with triphenylphosphine followed by hydrolysis to afford the phosphonium salt 144 (Scheme 53).®®® Purification by chromatography was not needed for all three steps, and 100 grams of 144 were routinely prepared. The commercially available 3-

Pyridin-3-yl-1-propanol, was converted into aldehyde 145 by Swern oxidation.®^ Wittig olefination of 145 occurred with the ylide generated from 144. f-BuOK gave a disappointing result. The product yield was a moderate 43-66% and the cis/trans selectivity was around

85:15. Also, if there was any moisture present in the reactions, a substantial amount of diphenylphosphine oxide was isolated (10-39%).®®

a. b ^ Phap-" Û 144 X=OH 146 X=OTHP 147 X=OTHP g r l 4 8 X=OH OH 150 X=l 151 X=OTs N 145

NC. CN

153 149

139 152

OMe keramaphidin B (141)

154 OMe

Scheme 53: Synthesis of bis-dihydropyridine 139: a) AcBr, cat. Zn. heat, 95%; b) PhaP, 100C; then K 2 CO3 ,

H2 O, MeOH. 76%; c) 3.4-dihydro-2H-pyran. PPTS. CH 2 CI2 , 93%; d) (0 0 0 1 )2 , DMSO. EI3 N. -65 to 250, 90%; e) KHMDS, IM F. -780 to RT; then 147, -780 to RT; then 145, -780 to RT. 83% (Z:E~99:1); f) 3M HCI. MeOH.

94%; g) PhaP, I2 , imidazole. MeCN, 56-90%; h) TsCI, EtaN. CH 2 CI2 , OC. 95%; i) Nal, butan-2-one, heat. 192 h;

j) NaBH4 , MeOH. -780 to RT. 56% over two steps; k) m CPBA, CH2 CI2 , 00. 98%; I) TFAA, CH 2 CI2 , 100%; m)

KCN, H2 O. pH 3-4. 92%; n) CFaC0 2 Ag. quant.; o) MeONa, MeOH. 8 6 %; p) camphorsulfonic acid (CSA). quant.; q) MeOH/IM aq. buffer (1:1 pH 7.3); then NaBH^, MeOH. -780 to RT. 0.2-0.3%.

39 Chapter 1: Introduction

In order to eliminate the effect of the alkoxide group on the Z/E ratio, protection of the alcohol group in 144 was sought. Hydroxyphosphonium salt 144 was masked as its tetrahydropyranyl (THP) derivative 146 in 93% yield.®® When the ylide derived from 146 was used in the Wittig reaction, with potassium hexamethyldisilazide (KHMDS) as base, a far superior Z/E ratio of 99:1 was obtained and the yield of isolated alkene 147 was 83% contaminated with 5% diphenylphosphine oxide. Dilute hydrochloric acid in methanol removed the THP protecting group to give 148 (94%).^° The significant improvement in yield and quality of material, counteracted the incorporation of the additional protection and deprotection step in the synthesis. Armed with alcohol 148, Baldwin turned his attention to its activation and cyclodimérisation. Investigation into the cyclodimérisation revealed that an iodide leaving group was optimum, thus, providing the desired bis-pyridinium salt 149 in 40-44% yield.

Triphenylphosphine/iodine/imidazole was used to convert alcohol 148 into iodide 150, but it was discovered that the yield of iodination varied from 56-90%, since 150 was unstable and difficult to handle (it polymerises at room temperature over a period of hours). Given the instability of

150, together with the moderate yield of the cyclodimérisation, an alternative protocol was sought. TsCi and NEtg was used to prepare the tosylate 151 (95%) from alcohol 148, and in the condensed state, it decomposed only slowly over a week of standing at room temperature.

Next, Baldwin focussed on optimising the cyclodimérisation reaction, which consists of two separate 8^2 reactions. The first is the intermoiecuiar reaction, favoured by a high concentration. However, high concentration reaction conditions also favour non-productive polymerisation. The second 8^2 process is the ring-closing reaction, which one would expect to be favoured by low concentration. The assumption that operating at a low concentration should be beneficial on balance was tested in practice. A solution of 151 in butan-2-one was added slowly to a refluxing solution of Nal in the same solvent; this effected a one-pot

Finkelstein/dimerisation/macrocyclisation reaction. The crude product was reduced with NaBH^

in MeOH to give bis-tetrahydropyridine 152 in 56% yield over two steps. 8 ince partial oxidation of 152 was required to obtain the precursor for the study of the Dieis-Alder reaction, the modified

Poionovski reaction (also known as the Polonovski-Potier reaction)^^ was used. Treatment of

40 Chapter 1: Introduction

152 with m-CPBA (m-chloroperbenzoic acid) gave two separable diastereomeric A/-oxides.

Trifluoroacetic anhydride (TFAA) was then used on either diastereomer to give the proposed manzamine biosynthetic precursor 139. Compound 139 was stored either as its bis-cyano derivative 153 (by treatment of 139 with KCN) or as its bis-methoxy derivative 154 (by treatment of 139 with NaOMe).

Armed with a reliable route to 139, the proposed Diels-Alder reaction was investigated.

In the hope of optimising the biomimetic Diels-Alder reaction, extensive studies were conducted; the reaction time was varied prior to quenching; a variety of solvents were tested; the buffer was varied and the effect of pH changes on the reaction was also examined.^^ Dissolution of 139 in

a 1 : 1 mixture of 1 M aq. TRIS/HCI (pH 7.3) and MeOH, followed by NaBH^ reduction of the reaction after one hour at low temperature, gave the best result. A small amount of keramaphidin B (141) was observed in the ’ H NMR spectrum of the crude product when the reaction was performed on a preparative scale. After extensive purification by flash chromatography and repeated HPLC, 0.2-0.3% of 141 was obtained.^^ The major product from the reduction was the recyclable bis-tetrahydropyridine 152, which arose from the disproportionation of 139, giving a mixture of tetrahydropyridine and pyridinium salt,^^ which got reduced by the sodium borohydride quench giving 152. While the result clearly demonstrated the validity of Baldwin’s proposal, the low yield of keramaphidin B (141) was not satisfactory. In order to find out more on the kinetic barrier of this reaction, a molecular modelling study was carried out on the Diels-Alder precursor 138 (Scheme 54).

155

Scheme 54: Transition states of 138 and 155

41 Chapter 1: Introduction

This investigation revealed that there are conformations available to the macrocycle which are

close to the required transition state.®^ This result suggested that the low yield obtained in the

Diels-Alder reaction is due to the kinetic preference of 138 to disproportionate, and not the

inaccessibility of the two reactive moieties. It is reasonable to envisage an in vivo Diels-

Alderase mediating the conversion of bis-iminium salt 139 to keramaphidin B (141) in Nature.

The putative enzyme could catalyse the reaction by limiting the conformational mobility of the

substrate, and thus minimising the loss in entropy in forming the transition state.^'^ Moreover, an

enzymatically mediated reaction would avoid the problem of disproportionation, which is the

preferred reaction of 138.

In the light of the unfavourable disproportionation in the bis-dihydropyridine route,

Baldwin’s group wondered whether they were using the correct oxidation state of pyridine in their

hypothesis. For example, if a pyridinium salt was used as the diene and a tetrahydropyridine was used as the dienophile in 156, the [4+2] cycloaddition would directly form pentacyclic 136

(Scheme 55).

® N Diels-Alder

156 156 136

HO' MG’

HN Diels-Alder

157 135 135

Scheme 55: Alternative biosynthetic hypotheses

This new alternative biosynthetic hypotheses exhibits two advantages over the original bis- dihydropyridine route: i) the pyridine moieties are at the tetrahydropyridine and pyridine

42 Chapter 1: Introduction

oxidation states, therefore avoiding the disproportionation problem; and II) the postulated redox equilibrium between the cycloadduct 137 and the ircinal precursor 136 (Scheme 50) is no longer needed.

Marazano and co-workers recently proposed that the biomimetic Diels-Alder reaction might involve substituted 5-amino-2,4-pentadienals as the diene.^® This route would not only avoid the problems with disproportionation, but would have the added advantage of less ring strain during the Diels-Alder cycloaddition. Ring-opening of pyridinium salt 156, affords amino- aldehyde 157. This could then undergo a Diels-Alder reaction to directly afford the ircinal 135

(Scheme 55).^®

Because of the low yields of keramaphidin B (141) obtained from the intramolecular

Diels-Alder cycloaddition, Baldwin investigated a two-step synthesis by an intermolecular cycloaddition followed by two ring-closing metathesis reactions (Scheme 56). The keramaphidin

B core (158) was achieved in a reproducible 22% yield via a biomimetic cycloaddition.

,Flu=" i) pH 8.3 160 TRIS/HCI + DCM (1 mM) 40°C

158 159 141

Scheme 56: Synthesis of Keramaphidin B (141) by ring-closing metathesis

The ring-closing metathesis of 158 could theoretically give six “mono-cyclised” products, like

159, and three “bi-cyclised” products (one of which is keramaphidin B) together with oligomers and E-double bond isomers. However, Baldwin’s group hoped that there would be a preference for the naturally occurring product 141. They investigated both the Schrock molybdenum

43 Chapter 1: Introduction

catalyst and the Grubbs ruthenium catalyst 160, the latter giving a better profile. When this reaction was performed on a preparative scale, mono-cyclised 159 (10-20%) and keramaphidin

B (1-2%) were obtained. So even though there did seem to be a bias towards the natural product 141, it was still evident that there was difficulty in forming 11- and 13-membered rings by ring-closing metathesis.

1.1.9 Sulikowski’s Progress toward a Biomimetic Synthesis of Phomoidride B.^ (2002)

In 1997, phomoidrides A and B (161)^® (Scheme 57) were isolated by a group of workers at Pfizer.

HOOC" 161

Scheme 57: Structure of phomoidride B (161)

These fungal secondary metabolites belong to the nonadride group of natural products, and NMR analysis was used to assign their structures. Phomoidride A and phomoidride B are modest inhibitors of ras farnesyl transferase and squalene synthase. Their unique structures and their potentially useful biological profiles have generated considerable interest in their total synthesis.

Relative to previously identified nonadrides, the unusual core structure of the phomoidrides inspired Sulikowski and coworkers to study their biosynthesis,^® and they have now concluded that the core structure common to phomoidride B (161) is assembled through the decarboxylative homodimerization of a 16-carbon unsaturated anhydride (Scheme 58). This dimerization induces the loss of carbon dioxide, together with the stereoselective formation of three carbon-carbon bonds (a, b and c). Sulikowski®® and others®^ have worked towards biomimetic approaches to these compounds based on the homodimerization hypothesis outlined in Scheme 58.

44 Chapter 1: Introduction

-CO H 163 (phomoidride core)

26 HO

Scheme 58: Homodimerization hypothesis of a 16-carbon unsaturated anhydride

Previously, Sulikowski hypothesized that the intramoiecular cyclisation (Scheme 59) may give the desired regio- and stereocontrol by the pathway shown. It was expected that

deprotonation at C 1 3 wouid induce an intramolecuiar Michael addition at C 14 with an exo approach of the two anhydride units resulting in the production of 5 lactone intermediate 166.

base ""H

164 X = 0 166 165 X = N-alkyl

O OH

167 168

Scheme 59: Hypothesized intramolecular cyclisation

It was further hypothesized that the second intramolecuiar Michael addition (166^167) would create good stereocontrol due to conformational restrictions from the first formed carbon-carbon

45 Chapter 1: Introduction

bond (C 1 3 -C1 4 ). Unfortunately, although deprotonation at C 1 3 occurred as predicted,

fragmentation to give anhydride 169 was the only thing observed (Scheme 60).

o DBU

MeCN

164 169

Scheme 60: Fragmentation to give anhydride 169

The reluctance of ester 164 to adopt the required s-cis conformation was thought to be

responsible for the observed absence of any cyclized products. Sulikowski then focused attention toward examining the cyclisation of tertiary amide 165.

The synthesis of tertiary amide 180, started with a malonate anion addition to the crystalline mucobromic acid derivative 170 to produce fe/t-butyl malonate 171 (Scheme 61).

O, ^^nC3H7 Br 1 ) (Na)CH(C0 2 tBu)2 2) TFA (neat) 174

3) BH3THF Pd(Ph3P)2Cl2 (31-49%) KOAc, PhH, heat OH TBSO -X-TBSO (54-61%) 170

171 X=Y=C 0 2 t-Bu

172 X=CÜ 2 H: Y=H 173 X=CH20H; Y=H

DEDCI, DMAP 176 2) HF.pyridine NHC Hq Dess-Martin periodinane TBSO ® (48-71%) OTBS 178X=TBSO, H 179 X=HO, H 177 180 X=0 HOOC

Scheme 61: Synthesis of tertiary amide 180

Acid-catalysed removal of the ferf-butyl groups induced spontaneous decarboxylation and

isolation of monocarboxylic acid 172, which was reduced with BH 3 THF to give alcohol 173 in 31-

49% overall yield from 170. Diene 175 was produced in 54-61% yield, via a Suzuki-Miyaura

46 Chapter 1: Introduction

cross-coupling®^ of 173 and vinyl boronate 174. The primary hydroxyl group of 175 was

transformed into secondary amine 176 by n-butylamine displacement of the in situ derived

triflate. Amide 178 was produced via the coupling of amine 176 and carboxylic acid 177 using

EDCI-DMAP. HF-pyridine efficiently removed the TBS group, and oxidation of the resultant

bisacetal using Dess-Martin periodinane®® afforded the bisanhydride 180 (48-71%, three steps).

The base-catalyzed cyclisation of tertiary amide 180 was examined next. Amide 180 was added

to an acetonitrile solution containing 0.3 equivalents of triethylamine. A deep red colour

appeared immediately, indicative of enolate formation as in the case of ester 164. But in

contrast to 164, the red colour immediately dispersed to return to a colourless solution. TLC of

the reaction mixture, indicated that the starting materiai was consumed within 10 mins. NMR

analysis revealed that two polycyclised products 181 (49%) and 182 (31%) were produced.

(Scheme 62).

181 182

183 184

Scheme 62: Polycyclisation products

Crystallisation of compound 181 from acetonitrile, followed by single-crystal X-ray analysis allowed a full stereochemical assignment to be made for 181. ID NOES Y spectra suggested that 182 differs from 181 in stereochemistry at the spirocyclic carbon. A much slower cyclisation occurred (48 h) when the reaction solvent was changed from acetonitrile to dichloromethane.

47 Chapter 1: Introduction

giving cycioheptane 183 (18%) and monocyciized product 184 (54%). (Stereochemistry of these two have not been assigned).

1.1.10 Epilogue

Cleariy, the syntheses we have described show both the successes and the pitfalls often encountered when attempting to implement biogenetically-modelled syntheses of complex natural products. Although, when successful, such biomimetic approaches can often allow breathtakingly complex structures to be assembled in a comparatively short number of steps, and so allow multi-gram quantities of the target natural product to be readily obtained (eg Methyl homosecodaphniphyllate), but they are often inter-married with very high risks and possible failure (eg Baldwin’s approach to Manzamine, and Sulikowski’s phomoidride synthesis).

We hope that this brief overview has given the reader a taste of the potential highs and lows of biomimetic synthesis. We will now discuss the target molecule of this thesis, halichomycin.

48 Chapter 2: Background

2.0 BACKGROUND

2.1 The Discovery of the Antitumour Agent, Halichomycin.

Halichomycin is a structurally unprecedented macrolide of marine origin which was first encountered and characterised in 1994 by Numata and coworkers^ at Osaka University in

Japan. They discovered this unusual tricyclic hemimacrolactam in culture filtrates of

Streptomyces hygroscopicus, an actinomycete found in the gastrointestinal tract of Halichoeres bleekeri, a well-known marine fish. (Scheme 63). 85

Scheme 63: Photo of Halichoeres bleekeri

Hence, they named this molecule halichomycin. (Scheme 64).

'Me,

'O Me

Me NH 22 29.

Me Me

Halichomycin

Scheme 64: Structure of Halichomycin

49 Chapter 2: Background

Halichoeres bleekeri has several synonyms, the most widely used being halichoeres chloropterus] it is also commonly known as the Pastel-green wrasse. They belong to the

Wrasse Family, and are of the Ray-finned fish Class. These harmless fish have been known to grow to a maximum size of 19 cm long (males/unsexed), and are marine reef-associated, typically living in a depth range of 0-10 m. Halichoeres bleekeri are tropical fish that are generally confined to Indo-Australian waters ranging from the Philippines to the Great Barrier

Reef, Australia.

Recognition of this species is based upon the head of the males having intricate reticulate patterns of bands that vary from one individual to another, and the anus having small blackish spots upon it. Juveniles and females usually contain dark dots on the dorsal and posterior areas; these are, however, lost in males.

Ecological studies of halichoeres bleekeri have shown that they prefer to inhabit shallow protected coral reefs and nearby silty sand and rubble bottoms. They usually feed on hard- shelled prey, including molluscs, crustaceans and sea urchins.®® (Scheme 65).

Scheme 65: Other variations of halichoeres bleekeri

The halichomycin-producing microorganism cultured by Numata and coworkers®^ was maintained at 27°C for a week in a medium (20 I) containing 0.1% casein and 1% starch in artificial seawater which had been adjusted to pH 7.5. The AcOEt extract of the culture filtrate

50 Chapter 2: Background

was then purified by bioassay-directed fractionation employing a combination of Sephadex LH-

20 and silica gel column chromatographies and reverse-phase HPLC to afford halichomycin (4

mg) as an oily material that exhibited potent cytotoxicity (EDgo 0.13 pg/ml) in the P-388

lymphocytic leukaemia test system in cell culture.®®

Halichomycin evokes interest not only because of its antitumour profile but also because

of its unprecedented structure which poses important questions for and challenges current

theories of polyketide biosynthesis. The powerful anticancer properties could in the future prove

to be of enormous clinical value for the treatment of human cancers.

The molecular formula of halichomycin was established to be C 3 3 H4 9 NO5 by HREIMS.

Its unique structure was derived after extensive NMR and mass spectral analysis. A detailed

examination of the and spectral data®^ by DEPT and COSY experiments revealed

the presence of an amide, a conjugated ketone, five double bonds, a methoxy, an ethyl, seven

methyls, three of which are allylic methyls, a methylene, and ten sp® methines, including five

methines which are linked to an oxygen atom, one of which is further bonded to a nitrogen atom.

Numata and coworkers®'* also used HMBO correlations, and NOESY crosspeaks to help

elucidate the structure of halichomycin and establish its relative stereochemistry. The highly

ornate skeleton of halichomycin consists of an unusual combination of two heavily adorned

medium-sized ether rings intertwined and annulated onto one another and interwoven within an

extensively functionalised 13-membered hemimacrolactam. The dense array of functionality

present within these ring systems is linked in a very novel way. The extended core of

stereodefined double bonds and multiple asymmetric centres, provides a synthetic challenge of

the highest quality.

Apart from ourselves, J.L. Wood and coworkers®^ at Yale University in the USA are the only group to have reported a synthetic approach to a segment of halichomycin. They have devised a synthesis of the C(1)-C(7) segment of halichomycin, which will now be discussed.

51 Chapter 2: Background

2.2 The Yale Approach to the Synthesis of the C(1)-C(7) Segment of Halichomycin:

The Wood Synthesis.

As part of an ongoing program directed toward the synthesis of halichomycin, J.L. Wood and coworkers®^ began investigating known methods for the stereocontrolled formation of tri substituted olefins which possessed allylic stereogenic centres®®. Although they were initially attracted to the commonly used chiral starting material methyl-3-hydroxy-2-methylpropionate,®®® ‘^ the number of oxidation state and protecting group changes required to manipulate this material led them to consider alternatives. This ultimately resulted in the development of an efficient two- step preparation of 186, which is a synthetic intermediate equating to the C(1)-C(7) segment of

185. A stereoselective aldol coupling between an acylated oxazolidinone 187 and a masked (3-

dicarbonyl 188 served as the key step (Scheme 6 6 ).

Me Me, OEt Me ,0 Me N Me Me """NH Me Me Me Bn 186

185 Me Me Me

OEt

PhaP.

-I Me Me Me Bn 188 189 187

Scheme 6 6 : The Wood Retrosynthesis

In the Wood synthesis, 3-ethoxy-2-methylpropenal 190 was employed as a masked p- dicarbonyl equivalent.®® Even though many precedents exist for this electrophilic precursor being converted to the required type of tri-substituted olefin,®® its reactivity in a stereoselective

52 Chapter 2: Background

aldol reaction was previously unknown.®^ It was found that under typical Evans’ aldol conditions,®^ the coupling of 187 and 190 gave a labile aldol product 191 which underwent clean conversion to 192 when warmed to room temperature and in situ treatment with 1N HCI/MeOH

(1 :1 ) (Scheme 67, 87-95% yield). Consistently high yields of the desired olefin were produced after repeated experimentation, but it was found that if the triflate was of poor quality, or close attention was not paid to the imide/triflate stoichiometry, a significant amount of a minor byproduct was observed (ca. 5-10%).

A, 1. Et2B0Tf. EI3 N 3 . 1N HOI 2. Me (87-95% yield) Me OEt Me Me Bn 187 190

LIA]H4 EtgO Me Me (-)-193 CHO O N

Me Me COaEt 192 PhH, heat Me Me Me Me ‘PPh 3 OEt 189 (94% yield)

Scheme 67: The Wood Synthesis to the 0(1 )-C(7) segment of halichomycin

Since the reaction was able to produce E or Z isomers of either syn- or anti-a\do\ products,®^ the reaction was evaluated through chemical correlation. Comparison of the spectral and chlroptlcal data of the 1,5-diol [(-)-193], derived from the reduction of 192, with (+)-193, concluded that the coupling of 187 and 190 had proceeded as anticipated to furnish 192. Once the structure of 192 was confirmed, they then completed the C(1)-C(7) synthon and found that the homologation of 192 furnishes 186 in 94% yield.®®

53 Chapter 3: Discussion

3.0 RESULTS AND DISCUSSION: Towards a Bioqenetically-Modelled Asymmetric

Total Synthesis of Halichomycin.

3.1 Original Retrosynthetic Analysis of Halichomycin and Preliminary Efforts to

implement a Conventional Synthetic Approach.

3.1.1 Original Retrosynthetic Analysis of Halichomycin.

Halichomycin abounds within all the structural features one would traditionally associate with molecular complexity. It possesses an unusual combination of medium- and large-sized ether rings intertwined and annulated onto one another; it has a dense array of functionality linked in a very novel way; Me it also bears reactive functional groups. Its diverse \ I Me n is^ omb spread of substituents, contiguously domiciled over three adjoining ring systems leaves

/ 2 1 a sole ring-carbon atom unadorned, and this is ® Halichomycin positioned at 0(9). Taking all of these issues into consideration, this extended core of stereodefined double bonds and multiple asymmetric centres conspires to make the halichomycin problem an unusually forbidding and awesome synthetic challenge of the highest quality.

A particularly demanding task is viable retrosynthetic disassembly of the multiple intersecting ring systems, whose ring sizes and functionality arrays offer limited possibilities for strategic ring closure. After careful evaluation of several retrosynthetic plans, a strategy for the

synthesis of halichomycin was eventually identified; it is outlined in Scheme 6 8 .

Our originally proposed pathway for halichomycin had as its centrepiece, a base- mediated intramolecular Michael addition reaction on 194 for closure of the 11-membered 0 ring.

Our selection of this ring closure strategy was guided by molecular models of 194, which clearly indicated that this transformation could potentially be facile, due to the extended dienone 5-

54 Chapter 3: Discussion

carbon being positioned close to the C(7)-hydroxyl, as a result of steric and geometric constraints imparted by the pre-existing A and B rings. These same constraints also appear to rule oult the possibility of other Michael addition processes competing with the one desired. It was allso thought that the predefined dieneone geometry would help ensure that the stereochemistry at C(22) was controlled. The correct stereochemistry at C(21) should follow from the intermediary enolate protonating from its less-hindered face, exo- to the newly- generated tricyclic array.

A OMe OMe L/; Me 0-~~y Intramolecular Me 0 - ^ W 1..C Kishi-NozakI Me „ O \ TBDPSO ^ B '" n H ------^ ^ NPMB 7 NPMB ' Michael Addition Coupling Me H -oT o Me Me 196 Me Halichomycin

Me. Me, OMe O O Enolate TBCPSO' displacement I BLKSO» Me Br Me 7 "'Me Bromination with inversion 199 0PM B OPMB OTBDPS OTBDPS Me' CHO OfBDPS 200 PMBHN' 197 Keto-phosphonate 198 OMEM Ring closure

O II P(0Et)2 C H o ( OTBS Alkynyl-ester Q ^ y ^ O Elaboration of ÇTES generation and ~ = r ^ TBDPSO, TBDPSO, OTBDPS OTBDPS keto-phosphonate selective Me )"'M e .. Me Me carbocupration Me Me Me Me OPMB OTBDPS OPMB 202 OPMB 203 201

Reduction

0 ^ 0 (Y CHO Ph-V^ T OBn Evans Syn OBn Me""^ HO, OTBDPS OTBDPS 205 OPMB Aldol Me Me Me Me Me''' 206 OPMB 204 Scheme 68: Original Retrosynthetic Analysis of Halichomycin

55 Chapter 3: Discussion

Somewhat surprisingly, there are no reports of the alkoxy-Michael cyclisation being used for the construction of medium sized ether rings, although it has been used successfully on many occasions for the formation of tetrahydropyran and 1,3-dioxane ring systems.^"* To the best of

our knowledge, only one report®^ exists on the closure of an 1 1 -membered ring using a heteronucleophile Michael addition tactic, and this involves the monoaddition of an internally- tethered thiolate anion onto a multiply-extended trienone. As already mentioned, according to molecular models of the tethered electrophile in compound 194 suggest that it is well positioned to be able to engage in the desired alkoxy-Michael addition process. Clearly, a Michael cyclisation tactic would not only simplify construction of this ornate medium-sized ether ring, it would also greatly simplify the protecting group strategy that would be needed for the overall synthesis. We envisioned introducing the dienone array of 194 via a Kishi-Nozaki NiClg/CrClg mediated coupling®® between dienyl iodide 195 and aldehyde 196. Kishi has previously shown that such couplings are fully compatible with sensitive a,p-unsaturated ester functionality, during his synthetic work on halichondrin and palytoxin.®® We were confident therefore that this methodology would be suitable here.

The next bond which was selected for disconnection was the a,p-bond in the un saturated lactam B-ring of 196; this choice allowed amide 197 to be selected as a sub-target.

Scenarios that we envisioned for forming the a,ô-trisubstituted dienone in 196, included a Ti(0)- mediated dicarbonyl coupling of 197 according to the protocol of McMurry®^ and Dauben.®®

Alternatively, a Horner-Emmons style macrocyclisation®® might prove useful for closing the 13- membered ring. Our plan for stereoselectively constructing the glycosyl-amide motif, associated with the A-ring of 197, would attempt an 8^2 type displacement on bromide 199 with the amide anion derived from 198. In turn, we envisioned compound 199 being accessible by electrophilic bromination of the enolate obtained from ketone 200 with NBS. Molecular models of this enolate indicated that its top-face would be considerably less hindered and more accessible than its underside. As a result, we predicted that bromination would proceed from the desired direction to give a-bromoketone 199. Further analysis of 200 revealed that its (E)-disubstitued alkene

56 Chapter 3: Discussion

was strategic for disassembly, so again, we thought that a Horner-Emmons cyclisation on 201 looked a good bet for ring-closure. Potentially, the trisubstituted olefin geometry in compound

202, could have been derived from a Corey carbocupration^°° reaction on a methyl alkynoate precursor, which in turn, might have been accessible from iodide 203. The fact that a syn- relationship exists between the 0(7) and 0(8) stereocentres in 203, and that one of these centres is a masked secondary alcohol, suggested that the 0(7)-0(8) bond in 203 might be constructed through an Evans asymmetric aldol reaction,which led to the selection of 204,

205 and 206 as sub-targets.

3.1.2 Attempted Implementation of the Original Retrosynthetic Strategy for

Halichomycin.

Since a key intermediate in our intended route to halichomycin was the bromide 199

(Scheme 6 8 ), an early aim of our synthetic programme was to create the intermediate 204.

OH TBDPSCI (1 eq) OTBDPS DIBAL (2.2 eq) OH OTBDPS SOg.Py (4eq) Imidazole (2 eg) CH2CI2 (0.5M) EtaN(IOeq), DMSO MeO MeO V DMF(1.4M) V -78°C /3h rs CH2CI2, 0 °C /3 h rs Me Me Me 0°C to r.t., 24 hrs 98% 88% 207 89% 208 209

CCfelPr 211 (2 eq) OH O OTBDPS •B-0 ‘^'"CCfelPr OH

OTBDPS Toluene, -78°C / 24 hrs Me Me Me Me Me 210 212 213

Scheme 69: Synthesis of the anti-anti alcohol 212

For this we needed first to create the anti-anti alcohol 212 (Scheme 69). The commercially available Roche methyl ester 207 was protected as a TBDPS (terf-butyldiphenylsilyl) ether in

8 8 % yield. The NMR spectrum of 208 contained a 9 proton singlet at 8 1.01, and a 10 proton

phenyl multiplet centred around 8 7.60 which confirmed its identity. Reduction of the methyl

57 Chapter 3: Discussion

ester in 208 with DIBAL (dllsobutyialuminium hydride) at-78°C in dichloromethane furnished the primary alcohol 209 in 98% yield, whose IR spectrum now displayed a broad peak at 3385 cm \ indicative of the newly installed OH group. Oxidation of this alcohol with sulfur trioxide pyridine complex, DMSO (dimethylsulfoxide) and triethylamine in dichloromethane was next effected to

obtain the aldehyde 210 in 8 8 % yield. A doublet at ô 9.75 in the 500 MHz ^H NMR spectrum in

CDCI3 , along with an intense aldehyde carbonyl absorption at 1735cm’^ (and the disappearance of the broad OH peak) in the IR spectrum confirmed the formation of aldehyde 210. Other

methods evaluated included the Swern oxidation in DMSO and ( 0 0 0 1 ) 2 (oxalyl chloride), (yields of between 61-82%), and the TRAP oxidation.The TRAP and Swern oxidation were found to give many more impurities (as judged by tic), and were therefore deemed less satisfactory.

The 0(17), 0(16) and 0(25) stereocentres in 212 were set via a Roush asymmetric crotylboration^°® reaction between aldehyde 210 and the (R,R)-diisopropyl tartrate (E)- crotylboronate^*^ 211. The latter reagent is prepared by metalation of trans-2-butene with n- butyllithium and KO^Bu (potassium fert-butoxide) in tetrahydrofuran, and treatment of the

resulting (£)-crotylpotassium with (/-RrO) 3 B (triisopropylborate); after aqueous hydrolysis, and trans estérification with DIRT (diisopropyl tartrate) 211 is obtained in good yield. 500 MHz ^H

NMR analysis of alcohol 212 in ODOI3 confirmed the presence of the terminal alkene as there

was a doublet of doublet of doublets at 6 5.98 and a multiplet at 5 5.05, and the aldehyde singlet was now missing. The broad absorption at 3498 cm'^ in the IR spectrum confirmed the presence of a free hydroxyl group, and the (M+H)+ peak at miz 383.2406 in the high resolution

mass spectrum confirmed that 2 1 2 had an empirical formula of C 2 4 H3 4 0 2 Si.

According to Roush^°^, the asymmetric crotylboration reaction between aldehyde 210 and (£)-crotylboronate 211 should be carried out in toluene at -78°C, for the desired anti-anti alcohol 212 to be produced as the major reaction product with a 9:1 level of diastereoselectivity.

In our hands, however, this reaction has so far consistently yielded a 2:1 ratio, which has been confirmed by three independent workers!!

58 Chapter 3: Discussion

OTBDPS OTBDPS .Me Me OH via chair transition state 0 ^ 8 rx Me Me /P0 2 C"'< ^ 212 /P1O2C

OTBDPS

Me [Favoured Transition State A | B'

iPrO

Disfavoured Transition State B iPrO Me

OTBDPS

Scheme 70: Synthesis of anti-anti alcohol via favoured chair transition state A

It has been suggested^°^ that crotylboronate 211 preferentially reacts with aldehyde 210 by way of transition state A (Scheme 70), in which:- i) the aldehyde and the two tartrate ester units occupy axial positions with respect to the

dioxaborolane unit, and ii) the tartrate esters are syn-coplanar to the adjacent dioxaborolane C -0 bonds.

According to Roush, the stereochemically favoured transition state A is stabilised by a

favourable dipole-dipole interaction between the aldehyde carbonyl ( 8 +) and the proximate ester

carbonyl oxygen ( 8 -), while the alternative transition state B is destabilised by unfavourable interactions between the non-bonding electrons on the ester carbonyl and the boron-complexed aldehyde.

59 Chapter 3: Discussion

An alternative method for the formation of 212 has also been investigated, involving the use of indium and crotylbromide^°® in tetrahydrofuran and water. However, this reaction proved to be unfavourable since the undesired diastereomer 213 was now the major product in a 2:1 ratio as revealed by NMR analysis on the crude material.

After removal of the minor undesired diastereomer of our 2:1 mixture, by flash column

chromatography, the desired alcohol 2 1 2 was 0-benzylated^°® (Scheme 71) under mildly acidic conditions using catalytic amounts of TfOH (trifluoromethanesulfonic acid) and benzyl-2,2,2- trichloroacetimidate as the benzylating agent; the benzyl ether 214 was isolated in 80% yield.

500 MHz ^H NMR analysis in CDCI 3 now revealed a total of 15 protons between 5 7.71 (4H) and

Ô 7.46 (1 1 H) which indicated an extra phenyl group was present in 214. Moreover, the lack of an OH absorption in the IR spectrum further corroborated that the alcohol had been protected as its benzyl ether. Numerous attempts were made to benzylate the alcohol. Other less successful efforts at obtaining 214, involved the use of PPTS (pyridinium para-toluene sulfonate) at room temperature, and alkylation under basic conditions using n-butyllithium and benzyl bromide. The former proceeded very slowly to give 214, while the latter reaction did not lead to any of 214 being produced.

1 o'^ccig BHa-THFOM. 1.1 eq) OH OBn ______(1.5 eq) 2 " THF (0.2M)

Caf. TfOH (0.765 eq) OTBDPS o°C to r.t., 3 hrs then, OTBDPS Me Me CH2CI2 (0.3IVI), r.t., Me Me H2O2 / MeOH, 6 8 % Me Me 2 hrs, 80% 212 214 215

RUCI3 (0.1 eq), Nal 0 4 (2.1 eq)

CCI4/CH3 CN/H2 O (2:2:3) (0.3M) r.t., 24 hrs, 61% n-BuaBOTf (1.1 eq) CH2CI2 (0.3M) 0 - ^ 0 EtaN, 0°C, then cool ^ I EtaN (1 eq), PvCI (1 eq) to -78°C, then add OBn TH F/0°C , n-BuLi (1 eq) OBn men add, OTBDPS OTBDPS o .. Me Me Me CHD Me Me O Me Me 218 OPMB 204 219 (1 eq) 216 OPMB

Scheme 71 : Our synthesis of carboxylic acid 216, and the attempted Evans Asymmetric Aldol Reaction

60 Chapter 3: Discussion

Hydroboration^°^ of the terminal alkene in 214 using a 1M solution of borane in

tetrahydrofuran at 0 °C, followed by oxidation, procured the primary alcohol 215 in 6 8 % yield.

The appearance of a broad absorption band at 3389 cm"' on the IR spectrum, and the absence of the olefinic proton signals in the 500 MHz NMR spectrum confirmed that 215 had been formed. This reaction was initially attempted using 9-BBN (9-borabicyclo[3.3.1 jnonane) in THF at 0°C, and later with catecholborane.'°® The former reaction was unsuccessful, with starting material remaining unchanged as judged by tic analysis. The latter process was found to produce variable yields of 215, and was therefore deemed unreliable. We believe that this is probably due to inconsistent quality catecholborane being sold commercially.

Further oxidation of alcohol 215 to acid 216 was accomplished with RuO^ generated in situ from NalO^ (sodium periodate) and RuClg in carbon tetrachloride, acetonitrile and water at room temperature.''® The appearance of an intense absorption at 1707 cm"* was consistent with the newly formed carbonyl group in 216 PDC in DM F at room temperature was also evaluated for this oxidation, but the reaction took longer and not much of the product was formed according to tic analysis. Acylation of acid 216 with the lithiated oxazolidinone 217 was then carried out to

obtain 219 in 58% yield. The 500 MHz ^H NMR spectrum in CDCI3 now displayed multiplets at 5

7.61 (4H) and 5 7.38 (16H) which indicated an extra phenyl group was present. The lack of a broad OH stretch, and the presence of two strong absorptions at 1787 cm"' and 1695 cm '\ further corroborated the production of 219. Also, the presence of an (M+H)+ peak at m/z

664.3450 in the high resolution mass spectrum indicated that 219 had an empirical formula of

C4 iH 4 9 0 5 NSi. An Evans asymmetric aldol reactionwas now attempted between the (Z)-di-n- butylboron enolate (obtained from 219), and aldehyde 218 This reaction failed to deliver any of the syn-aldol adduct 204. The starting material appeared to have degraded, as shown by tic.

The problem was thought to lie in the benzylated oxygen behaving as a nucleophile towards the

Lewis-acid activated carbonyl group, thus forming a lactone ring. It was reasoned that use of a para-bromobenzyl group instead of the benzyl group (Scheme 72) would cause this oxygen to be less nucleophilic, but again, the aldol reaction was unsuccessful.

61 Chapter 3: Discussion

NH A. /— Br BH3 THF (1M, 1.1 eq) Br OH B r ^ (1 2eq) ? THF (0.2M) Cat. TfOH (0.765 eqT 0°C to rt. 3 h then, OTBDPS Me Me CH2CI2 (0.3M), r.t.. Me Me ■H2O2 / MeOH. 60% Me Me 2 hrs, 60% 212 220 221

RuClg (0.1 eq). NalO^ (2.1 eq)

CCI4/CH3CN/H2 O (2:2:3) (0.3M) r.t., 24 hrs. 61%

n-Bu2B0 Tf (1.1 eq) 0 ^ 0 CH2CI2 (0.3M) .0 ^ 0 O EtaN, 0°C. then cool ( Et3N (leq), PvCI (1 eq) N^^O Br r ^ QBnBr to-780C, then add, OBnBr THF/0°C, n-BuLI (1 eq) . then add. 'OTBDPS Ph'"^ OTBDPS o ' OTBDPS Me Me CHO Me Me 217 O Me Me OPMB 224 M.-S 223 ( (1 eq) 222 ORVIB Ph

Scheme 72: Attempted Evans Asymmetric Aldol Reaction using the para-bromobenzyl molecule

Concerns about the PMB group on aldehyde 218 being somehow cleaved further encouraged us to investigate different protecting groups, such as the benzyl group for this aldol. Again, these new aldol processes still failed to proceed satisfactorily. In view of this, we decided to investigate an alternative approach for creating 204.

o A r ' o \ ^

Diol Protection, Evans Syn-Aldol then OTBDPS Protection T T Nitnie Reductloi ^ Me Me Me Me Me Me

° 225

DIBAL Reduction. Sharpless Epoxidation, then Regioseiective opening of epoxide

Et0 2 C OBn OBn OBn , Diol Generation WIttIg Reaction / OHC OTBDPS and Cleavage OTBDPS Me Me Me Me Me Me 214 229 228

Scheme 73: Retrosynthetic analysis of a new route to the syn-aldol adduct 225

We envisaged creating 225 from aldehyde 226 via an Evans asymmetric aldol reaction.

We thought that 226 could be derived from nitrile 227 by protection of the diol as its acetonide

ring, followed by DIBAL reduction on the nitrile group. A Wittig reaction using Ph 3 P=CHC0 2 Et

62 Chapter 3: Discussion

would give the (£)-olefin 228 from aldehyde 229, which could be derived from the benzyl ether

214 by OSO4 induced diol generation, followed by diol cleavage.

?" ....

n MO (1.5eq) OTBDPS 0°C. 20 min. OTBDPS Me Me r.t., 5.5 hrs Me Me 93% Me Me 76% 214 230 229

Ph3P=CHC0 2 E t(2 eq) EtOsC. DIBAL (2.2 eq) HO' OBn CH2CI2 (0.3M) E CH2CI2 (0.5M) r.t., 48 h, 76% I I OTBDPS -7 8 °C /2 h , 92% | | OTBDPS Me Me Me Me 228 231

Scheme 74: Synthesis of the allylic alcohol 231

The terminal alkene in 214 was reacted with a catalytic amount of OsO^ (osmium tetroxide) and NMO (4-methylmorpholine-N-oxide), in acetone, f-BuGH, and water at room temperature to access the diol 230 in 76% yield. The very broad absorption at 3411 cm'^ in the

IR spectrum of 230, and the absence of the olefinic protons in the NMR spectrum confirmed

230 had been made. This diol was now cleaved using Pb(OAc ) 4 in tetrahydrofuran at 0°C, the aldehyde 229 being isolated in 93% yield. Initially, a one-step conversion of alkene 214 to

aldehyde 229 was attempted using OSO 4 and Nal 0 4 , and although a clean reaction, this route only gave 229 in 45-50% yield. The overall yield of aldehyde 229 via the two-step route was calculated to be 72%. Hence this route was favoured, since it led to a yield improvement of greater than 20%. Aldehyde 229 was next reacted with carbethoxymethylenetriphenylphosphorane in CHgClg at room temperature; the desired (E)-olefin 228 was isolated in

76% yield. Its 500 MHz ^H NMR spectrum was now devoid of any aldehyde signal, and the presence of an (M+Na^ peak at m/z 567.2907 in the high resolution mass spectrum indicated

that 228 had an empirical formula of C 3 4 H4 4 0 4 SiNa. DIBAL reduction of the ester in 228

furnished the allylic alcohol 231 in 92% yield. The olefinic peaks at 5 5.77 ( 1 H) and 5 5.63 (1H)

were confirmed by the 500 MHz ^H NMR spectrum of 231 in CDCI 3 . The IR spectrum also had a

63 Chapter 3: Discussion

broad OH band at 3389 cm \ but now lacked the Intense carbonyl absorption at 1718 cm‘\ The

catalytic Sharpless epoxidation of allylic alcohol 231 using (+)-DET (diethyl tartrate), Ti( 0 -'Pr) 4

(titanium (IV) isopropoxide) and TBHP (tert-butylhydroperoxide) in dichloromethane at -35°C

provided the epoxy alcohol 232 (Scheme 75) in 87% yield. The loss of the NMR olefinic

signals and the presence of an (M+Na)+ peak at m/z 541.2750 in the high resolution mass

spectrum indicated that 232 had an empirical formula of CagH^gO^SINa.

(+)-DET (1 eq), Ti( 0 iPr)4 (1 eq) HO OBn 4A molecular sieves Et2AICN (5 eq). Toluene (0.3M)

OTBCPS TBHP (10 eq), CH 2CI2 OTBDPS 0°C to rt / 2.5 h, 48% Me Me -4 5°C /24h , 87% Me Me 231

Me 2C(OMe )2 / Acetone PPTS (0.25 eq)

NC Y Y OTBDPS 40 °C / 6 h, 78% NO T T OTBDPS Me Me Me Me Me Me 227 234

A. ° ^ ^ 2 3 5 , n-BugBOTf (1.1 eq),

\ CH2CI2 (0.5M) DIBAL (1 eq), CH 2CI2 (0.26M) X ------' -78°C /1 h, 60% OHC' Y ^ OTBDPS ^ \ OTBDPS Me Me EtsN (1.2 eq), 0°C, then cool to Me Me -78°C and add 226 (1.1 eq, 0.16M) 226 225 Ph

Scheme 75: Sharpless epoxidation and another attempted Evans asymmetric aidoi reaction

Regioseiective opening of the epoxide 232 using a 1 M solution of EtgAICN (diethylaluminum

cyanide) in toluene rendered the 1,2-dlol 227 as the major product in 48% yield. The minor

regioisomer 233 was also formed in 28% yield. In an attempt to try and increase the yield of the

major product, the solvent was changed from toluene to dichloromethane, although this made no

real difference to the ratio according to tic. Other efforts to improve the situation included the

use of a 1M solution of (CHajgAICI (dimethylaluminium chloride) in hexane with KCN (potassium

cyanide), and also Ti( 0 -'Pr) 4 with lithium acetylide in THF, but both these systems failed to open the epoxide. Protection of diol 227 in the form of an acetonide ring, using 2,2-dimethoxypropane

64 Chapter 3: Discussion

and catalytic PPTS in acetone at 40°C, successfully afforded 234 in 78% yield. The two new singlets in the 500 MHz NMR spectrum at S 1.38 (3H) and ô 1.26 (3H) unveiled the presence of the two new methyl groups in 234, and a very distinctive characteristic peak at 109.5 in the

NMR gave further confirmation of 234. DIBAL reduction of the nitrile in 234 at -78°C in dichloromethane then gave the aldehyde 226, which was used in an Evans asymmetric aldol reactionwith the acylated oxazolidinone 235. However, this failed to generate any of the syn- aldol adduct 225 even though this reaction was repeated by another member of the group.

In view of these unforeseen events, we elected to abandon this route and try a different approach to forming the A-ring of halichomycin.

OBn LDA (1.3 eq), THF-HMPA (10:1, 0.2M). Hz / Pd(OH)2 (20%, 0.2 eq), ^ HO. MeOH (0.09M), 2d, r.t., 8 8 % Y " ^ 'OTBDPS then add (1.2 eq) O Me Me Me Me p N Br 216 236 In THF at -78°C dropwise and stir at -78°C for 2 h, 82% Me,

LIBH4 (1 0 eq), THF/M eO H HO- Scheme 76 ' OTBCPS (100:1, 0.2M). reflux, 3 hrs, 79% Me Me Me Me OTBCPS 237 238

We decided to create a lactone ring from our previously formed carboxylic acid 216 by

hydrogenolysis of the 0 -benzyl group, using 2 0 % Pd(OH ) 2 (palladium hydroxide) on carbon catalyst in methanol; this effected a clean, but rather slow, deprotection to permit in situ butyrolactonisation. The benzyl signals in the ^H NMR spectrum of 236 were now absent, and replacement of the broad OH absorption with a strong carbonyl stretch at 1778 cm'^

(characteristic in 5-ring lactones in the IR), confirmed that 236 had been formed. A stereoselective C-alkylation of butyrolactone 236 was achieved by low-temperature énolisation with LDA and addition of the allylic bromide (Scheme 76).^°® Total stereocontrol was observed In this reaction, and was attributable to the stereodirecting influence of the C(25)-Me group (which hinders syn-approach of the bulky electrophile to the enolate). Preservation of the reaction

65 Chapter 3: Discussion

temperature at -78°C throughout also contributed to the high selectivity attained. In this regard, premature warming markedly lowered the selectivity levels that were observed. The configuration of the newly induced stereocenter in 237 was verified by NOE analysis.

Having fulfilled its role in stereoselective attachment of the C( 8 )-methallyl unit, the butyrolactone ring of 237 was reductively ring-opened with lithium borohydride to give the diol

238. Initial attempts to open the lactone 237, with DIBAL at -78°C in dichloromethane were unsuccessful, the hemi-acetal being the only product recovered. Identical results were obtained

when LiEtgBH (super-hydride) also at -78°C in THF was used. UAIH 4 (lithium aluminium hydride) in diethyl ether at 0°C was also problematicalWith diol 238 in hand, we now proposed a new route to the A-ring of halichomycin (Scheme 77), which would feature an intramolecular Stille process as the key ring-forming step. We believed that by protecting the

Me' OTBCPS ^ OTBCPS

Me. BugSn^

SnBua TBSO

Me Me OTBCPS Me OTBCPS

Scheme 77: Proposed route to the A-ring

primary alcohol in 238, using TESCI (triethylsilylchloride) and EtgN, in dichloromethane at 0°C, the secondary alcohol would be left open for attack.

Unfortunately, reaction of the secondary alcohol in 238 with KgCOg and t- butylbromoacetate in dioxane at reflux, did not lead to any of the desired product. The alcohol remained un reacted (Scheme 78).

66 Chapter 3: Discussion

Imidazole (2.2 eq), DMF (0.1 M) 0°C.

add TESCI (1.2 eq) t e s q - lE S O over 5 mins. then stir OTBDPS at 0°C for 1.5 h, OTBDPS OTBDPS 70%

Scheme 78: Attempted attack of secondary alcohol

An OTBS for OTES protecting group interchange was now effected, and a similar reaction was then attempted using n-BuLi, HMPA (hexamethylphosphoramide), and f-butylbromoacetate in

THF at -78°C. But this reaction was also unsuccessful.

Our attentions then focused on trying to attach a different group onto the secondary alcohol.

TBSO TBSO

Me OTBDPS IVIe OTBDPS Me' OTBDPS 240

Me,Me,

KH (1.2eq). THF (0.3M), TBSO- TBSO- TBSO -H Me t — \ Bu4NI (4 eq). 0°C to rt MeMe Me OTBDPS then stir for 40 mins. Me Me 240 241 242

Scheme 79: Other attempted attacks on the secondary alcohol

Reaction of the secondary alcohol in 240 was attempted using KH (potassium hydride), n-Bu^NI

(tetrabutylammonium iodide) and 4-bromo-1-butene in THF at 0°C, but to our disappointment, this was unsuccessful, as was the reaction using allyltrichloroacetimidate. The former gave no reaction at all, whereas the latter produced a suspected desilylated product. However, reaction of 240 with KH, n-Bu^NI and allyl bromide in THF was found to desilylate the OTBDPS group, replacing it with the allyl group, forming the allyl ether 241. But the secondary alcohol in 240 was also found to react slightly, producing a small (20%) amount of 242 (Scheme 79).

Although these approaches were largely unsuccessful, this new evidence nevertheless proved that it was indeed possible to attack the secondary alcohol. This led us to consider the

67 Chapter 3: Discussion

possibility of changing the terminal OTBDPS protecting group to a smaller OPMB (para- methoxybenzyl) group. We thought that such a tactic might potentially side-step the steric over crowding problems, associated with the attacking of the secondary alcohol, which we reasoned was the more likely origin of this lack of reactivity.

An OPMB for OTBDPS protecting group interchange was now effected much earlier in the sequence, which successfully delivered the PMB-ether 244 (Scheme 80).

XNH 40% aq. HF/THF/MeCN P M B 'O C Q 3 (1:2:1) PRIS (0.5 eg), OTBDPS (0 .09M), r.t., I OH CH2Cl2(0.1M), Me Me 24 \ h, 85% Me Me r.t.. 7 h, 97% Me Me 237 243 244

TBSCI (1.1 eg) UBH4 (10 eg), NaH (10 eg), DMF (0.1 M), Imidazole (2.1 eg) THF / MeOH HO TBSO 0°C, 30 mln, then add.

(100:1, 0.2M) DMF (0.1 M) (1 0 eg) OPMB 0°C to ft, 2 h, 98% reflux, 3 h, 79% Me OPMB o°C to rt, 24 h, 75% 246

^^„#K,^SnBi^(1 2 eq) Cl'' I (0.1 eg) (Ph3P)4Pd (0.1 eg) jbsq. 3 TBSO PCy TBSO DMF (0.06M) Toluene (0.002M) OPMB Heat (100-110°C) OPMB reflux 1 h Me OPMB 63% 249

Scheme 80: Route towards the formation of the A-ring of halichomycin

The 500 MHz NMR spectrum of 244 in CDCI3 was devoid of the 9 proton singlet (expected for the OTBDPS group), but now had present, the aryl protons of the OPMB group as apparent doublets at 5 7.21 (2H) and 5 6.85 (2H), and the OMe group resonating as a singlet at 5 3.78

(3H). The butyrolactone unit in 244 was reductively ring-opened with lithium borohydride, as before, producing the new did 245 in 79% yield. Present in the IR spectrum was a very broad absorption at 3276 cm \ which corresponded to the OH groups, and the absence of the strong carbonyl stretch at 1778 cm^ gave further confirmation of the successful ring opening reaction.

Selective protection of the primary alcohol in 245 as a TBS-ether was achieved, as before, compound 246 being isolated in excellent yield (98%). 0-Allylation of the secondary alcohol in

68 Chapter 3: Discussion

246 was then accomplished in 75% yield using sodium hydride in DMF, and allylbromide at 0®C.

The IR spectrum of 247 was now devoid of any broad OH absorption, and the 500 MHz ^H NMR

spectrum in CDCI 3 had olefinic signals at 5 5.83 (2H), 5 5.18 ( 1 H), and 5 5.06 ( 1 H). A Stille

reaction between vinyl iodide 247 and allyl tributyltin using (Ph 3 P)4 Pd as catalyst in DMF at reflux, furnished compound 248 in 63% yield. A ring-closing metathesis reaction was now attempted using Grubbs catalyst (1®* generation) in toluene at reflux. Although compound 249 was actually formed in yields of between 4-20%, the reaction was very unreliable indeed. It also had to be carried out at very high dilution in toluene {of. 0.002M), as polymerisation occurred faster than ring closure!

3.1.3 A Blogenetically-Modelied Total Synthesis of Halichomycin

At this point, we decided that we had to identify a much better synthetic pathway to halichomycin. At about this time, we came across an article by Kobayashi and Ishibashi in which they commented that the biosynthetic provenance “appeared to be strange”^” . This caused us to think about how Nature might be biosynthetically assembling halichomycin’s tricyclic ring system.

W-Acylcarbinol -amine |_| formation = 0 Dehydration CHO Me,NH2 N-^

Me Me Me Me Me Me Me g Putative intemai Pre-Halichomycin nucieophiiic addition, ketoreduction, and 0-methylation

OMe^^ OMe OMe Stereospecific enoi intramoiecuiar 16 "nh ------— O >.11 M e/ protonation ^ Me^NH Michaei addition s '"M e NH at 0(21)

c-' 21 M o Me Me / Me Me Me Halichomycin C H+

Scheme 81 : Biosynthetic proposal for ring formation in halichomycin

69 Chapter 3: Discussion

Our biosynthetic proposal for ring assembly in halichomycin invokes the branched

precursor (putative pre-halichomycin) as an intermediate and postulates internal macrocyclic N-

acylcarbinolamine formation^as the first step in ring formation (Scheme 81). Intermediate A is

then thought to undergo dehydration to the a-keto-N-acylimine B Molecular models of B show

that it can adopt a conformation appropriate for stereoselective internal attack of the C(16)-

hydroxyl upon the A/-acylimine carbon,^which would lead to the hemimacrolactam C after

C(14)-ketone reduction and 0-methylation with S-adenosylmethionine. Models further show that

the C(7)-hydroxyl of C can readily approach the C(22)-position of the dienone by a trajectory

suitable for acid-catalysed Michael ring-closure, a process that could later be followed by

stereospecific enol protonation at C(21). As for our open-chain putative halichomycin precursor,

much of its skeleton looks as though it is propionate- and acetate- derived, although it is not at

all clear how the bonds adjacent to the C(8)-C(9) bond are being constructed by a conventional

polyketide biosynthesis pathway. The attractive feature about our proposal for ring assembly is that all the rings are formed sequentially yet in tandem fashion. From a total synthesis

perspective, such an approach would be particularly appealing since it would massively simplify the synthetic problem at hand. It would also accomplish ring formation in tandem fashion rather than by one ring at a time.

To experimentally test this simple biogenetic hypothesis, by both chemical and biological

means, we embarked on an asymmetric total synthesis of the putative halichomycin precursor

250, and so far we have put in place a synthetic strategy for creating the key AB-carbon backbone intermediate 252, needed for this venture.

Our new synthetic plan would also allow us to channel into our original retrosynthetic analysis. As before, we envisaged constructing the C(18)-C(24) dienone sector of 250 through a

Kishi-Nozaki-Hiyama-Takai coupling^^® between 251 and 195, followed by oxidation (Scheme

82). We thought that aldehyde 251 could be derived from 252 by ammonolysis, O- debenzylation, and primary alcohol oxidation. A double Wittig sequence involving aldehyde 254

70 Chapter 3: Discussion

and ylide 255 was envisioned for stereospecific elaboration of the dienoate array in 252, while a

Stille reaction^with p-stannylenone 253 was planned for fashioning the dienone perimeter.

Kishi-Nozaki Coupling, ...V. "OMe Selective 0-Desilylation t e s o „ ,^ 4 v OTBS/ Me O B "NH \n,Y C \ "Me Me Me 19' ^ and Double Oxidation 'M e 'O Me Me Me “X- O 2512 + II Halichomycin Pre-Hallchomycin Me ‘ CONHg 250 19

256 BuaSn 253 O OTBS Evans Aidol, Protection TE90^7 > 1^ ^OTBS ^ s o '"Me 4: OTBS and Reduction Me 254 Me''e 8 Me'"' 6^ OPMB Y Me N - ^ 0 PPha OPMB OPMB ' X 257 X Me COgEt Me COgEt Ph 255 252

Me,

Scheme 82: Present Retrosynthetic Pathway of Halichomycin HO-

Me Me OPMB 245

We reasoned that the syn-relationship between the C( 6 ) and C(7) stereocentres of 254 could potentially be controlled through an Evans asymmetric aldol reactionbetween 256 and 257.

The former would be obtainable from our previously prepared diol 245. Differential 0-silylation of diol 245 procured compound 259 in 94% over 2 steps. Absence of the broad OH absorption

at 3494 cm"' in its IR spectrum, and a 9 proton triplet at 8 0.92, a 6 proton quartet at 8 0.55, a 9

proton singlet at 8 0.85 and signals at 8 0.05 (3H) and 8 0.02 (3H), all confirmed that 259 had been made (Scheme 83).

71 Chapter 3: Discussion

Imidazole (2.2 eq), 2,6-Lutidine (20 eq), DMF (0.1 M). QOC, CHgClg, -50°C, add TESCI (1.2 eq) add TBSOTf (3 eq) JESO over 5 mins, then over 5 mins, then OPMB stir at 0°C for IV,/ 'oPMB stir for 0.5 h, OPMB 1.5 h. 70% 89% 258

TPAP (0.05 eq), NMO (2 eq), 2% aq. HF. THF / MeCN (1:1), CHgClg (0.01M), 4A MS,

r.t., 1.5 h. 8 6 % r.t., 40 mins, 90% OPMB OPMB

M e—V ) = 0 M e ^ N (4 eq)

Ph O , (n-Bu)2B0 Tf (4 eq) EtgN (4.2 eq). CHgClg, 0°C TESOTf (5 eq) over 5 mins, 0.5 h, then cool to -78°C, add in CH2CI2 (0.02M), OPMB 2,6-Lutidine (20 eq) at -50°C OPMB Me Me then warn to r.t., for 45 min OTBS (1 eq) in CHgCiz, Me Me Q (52%, 2 steps) OFMB Me*^N H Me Me y ^ stir for 35 min, then warm p h * * ^ o to r.t., for 1 h. 261

Scheme 83: Latest route towards halichomycin

Selective cleavage of the primary OTES group with 2% aqueous HF, in a 1:1 mixture of THF and

acetonitrile, furnished the primary alcohol 260 in 8 6 % yield. The IR spectrum now had a very

broad absorption at 3441 cm \ and was also devoid of the triplet and quartet at 6 0.92 and Ô 0.55 in the 500 MHz ^H NMR spectrum in CDCI3. This now permitted oxidation to the aldehyde 256 with TPAP/NMO^^^ in 90% yield. The 500 MHz ^H NMR spectrum in CDCI3 now had an aldehyde signal at 5 9.60 and the IR spectrum now displayed the absence of a broad OH absorption. The Evans asymmetric aldol addition between 256 and 257 required the use of a significant excess of the propionimide enolate (4 eq.) to drive the reaction to completion, which made the purification of 261 exceedingly difficult. The IR spectrum of 261 now contained a broad OH absorption at 3560 cm \ two very intense carbonyl absorptions at 1785 cm'^ and 1695 cm'^ The 500 MHz ^H NMR spectrum was now missing the aldehyde signal at 5 9.60, but had

new phenyl signals present at ô 7.32 (3H) and 5 7.27 (2 H). The difficulty in purifying aldol

adduct 261 was overcome by the subsequent 0 -triethylsilylation reaction, which allowed the protected aldol adduct 262 to be isolated pure by simple flash column chromatography.

72 Chapter 3: Discussion

Significantly, no other aldol adducts were observed in this addition. The structure of 262 was verified by X-ray crystallography (Scheme 84).^''®

Scheme 84: X-ray Crystal Structure of Compound 262

Attention now shifted toward the stereospecific elaboration of the two diene arrays present within 252 (Scheme 85).

Reductive removal'’"'® of the oxazolidinone unit from 262 with lithium borohydride (10 eq.) in

Et2 0 /H2 0 (160:1, 0.02 M) furnished the primary alcohol 263 in excellent yield (82%). Previous experience in the group revealed that these unusual conditions were optimal. The IR spectrum now displayed a broad OH absorption at 3465 cm'^ but was devoid of any carbonyl absorptions.

Also, the presence of an (M+Cs)"' peak at miz 881.2470 in the high resolution mass spectrum

indicated that 263 had an empirical formula of C 3 5 H6 5 0 5 Si2 lCs. A TPAP oxidation'’^^ in dichloromethane converted 263 into the aldehyde 254 in 81% yield. The absence of the broad

OH absorption and the appearance of an intense carbonyl stretch at 1723 cm'^ in the IR spectrum suggested that oxidation had been successful. The aldehyde signal at 9.76 in the 500

73 Chapter 3: Discussion

MHz NMR spectrum of 254 further confirmed our assertion. Aldehyde 254 reacted readily

with 255 in toluene at reflux to give 264 with complete stereocontrol.

UBH4 (10 eq) in EtgO/HgO TPAP (0.1 eq). NMO (2 eq). (160:1. 0.02M). at 0°C CH2CI2 (0.02M). 4A MS OPMB then warm to r.t., and OPMB r.t.. 1 h 10 min. 81 % Me Me stir for 1.5 h, 82% Me Me O M e^-^N 263

Me Me JPh3 255 OTBS OTBS Me C(%Et ( 1 0 eq), PhMe (0 .0 1 M) /-BU2AIH (2.2 eq). PhMe (0.048M) TESO, OPMB 5 h. 90% OPMB -78°C, 0.5 h, 87% Me Me Me Me Me'''

254 264 Sole isomer

Me JPhg 255 OTBS Mn0 2 (20 eq). CHCI 3 (0.02M) Me CQEt (15 eq), PhMe (0 .0 1 M) TE90, OPMB Heat. 6 h. 98% OPMB Heat. 16 h. 97% Me Me Me Me

Me' Me CHO

265

Me, ° 253 snBus(2 eq). (CHgCN) 2PdCl2 Me OTBS. TESO TESO OTBS OPMB (0.5 eq). /-Pr 2NEt (10 eq). DMF (0.01 M). 5 h. 40% Me OEt Me Me Me PMBO- 267 Me Me 252 Me COaEt Sole Isomer Sole Isomer

Scheme 85: Route towards the AB-carbon backbone intermediate 252

The aldehyde signal was now absent from the 500 MHz ^H NMR spectrum in CDCI3, and a new olefinic signal was present at 5 6.73. Also, the intense carbonyl stretch in the IR spectrum had now shifted to 1712 cm \ which would be characteristic of an a,p-unsaturated ester. DIBAL reduction of the newly formed ester in 264 afforded the primary alcohol 265 in 87% yield. Mild

74 Chapter 3: Discussion

allylic alcohol oxidation of 265 with MnOg (manganese dioxide) generated the aldehyde 266 in

98% yield. Previous experience in the group revealed that 20 equivalents of MnOg was required

to give optimal yield. An aldehyde signal at 6 9.54 now appeared in the NMR spectrum. The absence of a broad OH absorption and a new intense carbonyl stretch at 1692 cm'^ indicated we were dealing with an enal. Aldehyde 266 then willingly engaged in a second Wittig reaction with

255, to form the (E,E)-dienoate 267, as a single geometrical isomer in 97% yield. The dienone unit was fashioned by a Stille coupling^between the vinyl iodide 267 and the (3-stannylenone

253 (compound 253 was best prepared according to the route shown in Scheme 8 6 ).

BuaSnH (1.2 eq). AIBN (0.05 eg) PPTS (1.5 ^),^MeOH (0.3M), ^ OH

PhMe (0.3M), heat, 3 h, 72% (50%. plus 30% recovered starting material) 268 269 270

TBSCI (1 eq) (0.03M in CHgClg) added dropwise to the dioi, Qy TPAP (0.05 eq), NMO (2.2 eq) and Imidazoie (2 eq) i 4A MS, CH 2Ci2 (0.26M)

in CH2CI2 (0.26M) at 0°C, ^ ^ SnBua r.t.. 1 h, 75% stir 10 min, 70%

Scheme 8 6 : Synthesis of stannylenone 253

To our delight, the desired tetraene 252 was isolated as a single geometrical isomer in 33-40% yield. However, it was formed alongside a significant quantity of the stannane homocoupling product. We are hoping to improve the yield of 252 in the near future, and to reduce the amount of dimérisation that is occurring with stannylenone 253. Future hopes are for isotopically labelled 250 to be prepared from 252, and for 250 to be converted into halichomycin itself by both chemical and biochemical means.

In conclusion, we have presented a biosynthetic proposal for ring formation in the antitumour agent halichomycin, in which macrocyclisation of the putative prehalichomycin is the first step. A dehydration to the a-keto A/-acylamine is then invoked, followed by a tandem nucieophiiic addition of the C(16)-hydroxyl to form the hemimacrolactam. A stereospecific

Michael ring closure and enol protonation complete C-ring assembly. So far, synthetic efforts towards obtaining 250 have resulted in the formation of compound 252.

75 Chapter 4: Experimental

4.0 EXPERIMENTAL

Materials and Methods.

Reactions were carried out under a nitrogen atmosphere with freshly distilled dry solvents unless

otherwise noted. Hexanes refers to the distillate fraction of petroleum spirit that is collected at

40-60°C and are distilled prior to use. All solvents were of reagent grade. Dichloromethane, toluene, benzene and acetonitrile were distilled from calcium hydride under nitrogen. Diethyl

ether and THF were distilled from sodium under nitrogen. All other reagents were used ‘as

supplied’ from the manufacturer unless otherwise stated. Flash column chromatography was carried out according to Still et with Kieselgel 60 40/60Â (220-240 mesh) silica gel.

Precoated silica gel plates (250pm) with a fluorescent indicator (E. Merck) were used for

analytical thin layer chromatography. The plates were initially examined under UV light and then developed with either a sulfuric acid stain [EtOHiHgSO^ip-MeOCgH^CHO (95:4:1)] or iodine

unless otherwise stated. Evaporation refers to the removal of solvents at < 40°C on a Büchi

rotary evaporator. ^H NMR spectra were acquired at 500 MHz with a Brucker DRX 500 and

NMR spectra were acquired at 125 MHz with a Brucker DRX 500. 2-D NMR spectra were also

recorded on a Brucker DRX 500. Chemical shifts are reported in 5-values relative to the residual

CHCI3 peak in CDCI 3 at 6 7.24 and the central peak at 5 77ppm for All NMR spectra were

recorded in deuterated solvent solutions. All infrared spectra were recorded on a Perkin-Elmer

1605 FT-IR spectrophotometer. Optical rotations were measured on an Optical Activity, Polaar

2000 automatic polarimeter. High resolution mass spectra were measured at the London School of Pharmacy on a V.G. 7070H or VG-ZAB instrument with a Finnigan Incos II data system.

76 Chapter 4: Experimental

(/?)-3-(terf-Butyl-diphenyl-silanyloxy)-2-methyl-propionic acid methyl ester 208

O OTBDPS

l-mlfc-24

(Spectral data agreed with those previously reported by Roush, ref. 103)

To a cooled (0°C), stirred, solution of primary alcohol 207 (5 g, 4.6 ml, 42.3 mmol) in dry DMF

(40 ml, 1.05 M) was added imidazole (5.7 g, 84.7 mmol, 2 eq) in a single portion. TBDPSCI

(11.6 g, 11.0 ml, 42.3 mmol, 1 eq.) was then added via syringe over 4 min., and the reaction mixture was allowed to warm up to and stir at RT overnight. (TLC mobile phase: hexane/ethyl

acetate, 1 2 :1 ). The reaction mixture was quenched by the careful addition of NaHCOg (10 g) and HgO (50 ml). The aqueous phase was extracted with diethyl ether (3 X 50 ml). The

combined organic phases were washed with brine ( 1 0 0 ml), dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg)

with hexanes/ethyl acetate (20:1) as eluent provided the silyl methyl ester 208 (13.3 g, 8 8 %) as a clear oil.

'H-NMR (500 MHz, in CDCIg): Ô 7.60 (d, J = 6.5 Hz, 4M), 7.4-7.35 (m, 6 H), 3.85-3.78 (dd, J =

6.9, 9.8 Hz, 1H), 3.70-3.6 (dd, J = 5.8, 9.8 Hz, 1H), 3.68 (s, 3H), 2.80-2.65 (m, 1H), 1.13 (d, J =

7.1 Hz, 3H), 1.01 (s, 9H) ppm.

IR (neat film ): 3070 (m), 3049 (m), 2935 (s), 2859 (s), 1963 (w), 1895 (w), 1741 (s), 1467 (s),

1429 (s), 1199 (s), 1110 (s), 823 (m), 740 (m), 704 (s) cm \

HRMS: (FAB, MNOBA matrix) for CgiHgaOg^^Si (M +H)\ calcd: 357.1874, found 357.1886.

77 Chapter 4: Experimental

'"C-NMR (125 MHz, in CDCI3 ): Ô 175.4, 135.5, 133.5, 133.4, 129.6, 127.6, 65.9, 51.5, 42.4,

26.7, 19.2, 13.5 ppm.

78 Chapter 4: Experimental

(S)-3-(tert-Butyl-diphenyl-silanyloxy)-2-methyl-propan-1-ol 209

OH OTBDPS

l-mlfc-70

(Spectral data agreed with those previousiy reported by Roush, ref. 103)

To a cooled (-78°C), stirred, solution of the methyl ester 208 ( 1 g, 2.8 mmol) in CHgClg (6.7 ml,

0.42 M), was added DIBAL (5.1 ml, 6.17 mmol, 2.2 eq) via syringe over 5 min. The reaction mixture stirred for a further 1.5 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was diluted with CHgClg (20 ml), and then quenched with 10% Rochelle’s salt (15 ml) at

0 °C. The resulting mixture was left to stir at RT for 45 min. The aqueous phase was extracted with CHgClg (3 X 20 ml). The combined organic phases were dried (IVIgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg), with hexanes/ethyl acetate (10:1) as eluent, provided the primary alcohol 209 (0.9 g, 98%) as a clear oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.67-7.65 (dd, J = 1 .2 , 7.7 Hz, 4H), 7.45-7.38 (m, 6 H), 3.76-

3.71 (dd, J = 4.5, 10.0 Hz, 1H), 3.66 (d, J = 6.1 Hz, 2H), 3.60-3.57 (dd, J = 7.6, 10.1 Hz, 1H),

2.42 (s, 1H), 2.00 (m, 1 H), 1.03 (s, 9H), 0.80 (d, 3H) ppm.

IR (neat film): 3385 (br), 2959 (s), 2932 (s), 2859 (s), 1961 (w), 1892 (w), 1469 (m), 1427 (m),

1110 (s), 1039 (s), 704 (s) cm \

HRMS: (FAB, MNOBA matrix) for CgoHggOz^^i (M+H)+, calcd: 329.1948, found 329.1937.

79 Chapter 4: Experimental

'=’C-NMR (125 MHz, in CDCI3 ): Ô 135.6, 135.5, 133.2, 129.8, 127.8, 6 8 .6 , 67.6, 37.4, 26.8, 19.1,

13.2 ppm.

80 Chapter 4: Experimental

(/?)-3-(fe/t-Butyl-diphenyl-silanyloxy)-2-methyl-propionaldehyde 210

O OTBDPS

l-mlfc-53

(Spectral data agreed with those previously reported by Roush, ref. 103)

To a stirred solution of primary alcohol 209 (10.1 g, 30.8 mmol) in CHgClg (35 ml, 0.88 M) was added dry DMSG (50 ml) via syringe at RT The reaction mixture was then cooled to 0 °C, where upon dry EtgN (42.9 ml, 307.7 mmol, 10 eq) was added to the reaction flask in one portion. The sulfur trioxide-pyridine complex (19.6 g, 123.1 mmol, 4 eq) was then added in one portion, and the resulting mixture left to stir for 3 h. (TLC mobile phase: hexane/ethyl acetate,

12:1). The reaction mixture was quenched by the addition of water (100 ml). The aqueous phase was extracted with CHgClg (3 X 50 ml). The combined organic phases were washed twice with brine (100 ml), dried (IVIgSO^), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (50:1) as eluent

provided aldehyde 210 (8 . 8 g, 8 8 %) as a colourless amorphous solid.

’H-NMR (500 MHz, in CDCI3): Ô 9.75 (d, J = 1 . 8 Hz, 1 H), 7.60 (d, J = 6.5 Hz, 4H), 7.49-7.32 (m,

6 H), 3.95-3.82 (m, 2H), 2.59-2.50 (m, 1 H), 1.08 (d, J = 7.0 Hz, 3H), 1.02 (s, 9H) ppm.

IR (neat film ): 2932 (m), 2859 (m), 2715 (w), 1895 (w), 1735 (s), 1469 (m), 1428 (m), 1110 (s),

1033 (m), 741 (m) cm \

HRMS: (FAB, MNOBA matrix) for CgoHgeOa^^Si (M +Na)\ calcd: 349.1586, found 349.1600.

81 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 204.4, 135.6, 133.2, 129.8, 127.8, 76.8, 64.1, 48.8, 26.8, 19.2,

10.3 ppm.

82 Chapter 4: Experimental

(£)-crotylboronate 2 1 1

COpiPr

(Spectra data agreed with those previously reported by Roush, ref. 103)

An oven dried three-neck round bottom flask equipped with a magnetic stir bar and an internal thermometer, was charged with THF (17.5 ml) and KO'Bu which was dried overnight on a high vacuum pump (2.4 g, 21.3 mmol). The mixture was cooled to -78 ®C, whereupon trans-2- butene (2.1 ml, 22.5 mmol), condensed into a two-neck graduated cylinder, immersed in a -78

°C dry ice bath was added via cannula. n-BuLi (2.5 M solution in hexanes, 8.5 ml, 21.3 mmol)

was then added dropwise over 1 0 min. via syringe at such a rate that the internal temperature did not rise above -65 °C. The cooling bath was then removed, and the reaction mixture was allowed to warm to -50 °C, and was maintained at this temperature for 15 mins. The reaction mixture was then recooled to -78 °C. Triisopropylborate (4.0 g, 4.9 ml, 21.3 mmol) was then

added dropwise over 6 min. and the reaction mixture was maintained at -78 °C for 10 min. and

then rapidly poured into a separating funnel containing 1 M aq. MCI (40 ml) saturated with NaCI

(5 g). The aqueous layer was adjusted to pH 1 with 1 M HCI, whereupon DIPT-(L) (5.0 g, 21.3 mmol) in ether (7.5 ml) was quickly added. The aqueous layer was extracted with ether (4 X 100 ml). (TLC mobile phase: hexane/ethyl acetate, 3:1). The combined organic phases were dried

(MgSOJ for approx. 4 h. stirring at r.t. and under an Ng atmosphere. This was then quickly

filtered and concentrated in vacuo to provide the (£)-crotylboronate 2 1 1 as a clear oil, which was used crude for the next step.

83 Chapter 4: Experimental

{2R, 3S, 4S)-1-(te/t-Butyl-diphenyl>silanyloxy)-2,4-dimethyl-hex-5-en-3-ol 212

OTBDPS Me Me

l-mlfc-39 top spot

(Spectra data agreed with those previously reported by Roush, ref. 103)

To a stirred solution of powdered 4Â molecular sieves (10 g) in toluene (54 ml), was added a

solution of crotylboronate (35.2 g, 122.5 mmol, 2 eq) in toluene (122.5ml, 1 M). The reaction

mixture was stirred at RT for 1 0 min. and then cooled to -78 °C. A solution of the aldehyde 210

(20 g, 61.3 mmol, 1 eq) in toluene (204 ml, 0.3 M) was then added via a dropping funnel over a

period of 1 h. After the addition was complete, the reaction mixture was maintained at -7 8 °C overnight, until all the starting material had completely reacted as monitored by (TLC mobile phase: hexane/ethyl acetate, 10:1). Excess ethanollc NaBH^ (406 mg in 5.4 ml absolute ethanol) was then added dropwise via pipette, the cooling bath was then removed, and the reaction mixture was allowed to warm to 0 °C, whereupon it was diluted with an aqueous

solution of NaOH (6 . 6 g, 1 M) and stirred vigorously for 2 h. The aqueous layer was extracted with ether (3 X 200 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification, and separation of diastereomers by multiple flash column

chromatography (SiOg), with hexanes/diethyl ether ( 1 0 0 :1 ) as eluent provided the secondary

alcohol 212 (15.4 g, 6 6 %) as a thick slightly cloudy yellow oil.

'H-NMR (500 MHz, in CDCI3): 6 7.65 (m, 4H), 7.42-7.31 (m, 6 H), 5.98 (ddd, J = 8.3, 10.4, 17.0

Hz, 1H), 5.05-5.01 (m, 2H), 3.71 (dd, J = 4.4, 14.5 Hz, 1H), 3.65 (dd, J = 4.3, 7.5 Hz, 1H), 3.50

(s, 1H), 3.42-3.40 (dd, V=1.9 Hz, 1H), 2.39-2.35 (m, 1H), 1.82-1.79 (m, 1H), 1.09 (d, J = 6 . 8 Hz,

3H). 1 .02 (s, 9H), 0.75 (d, J = 6.9 Hz, 3H) ppm.

84 Chapter 4: Experimental

IR (neat film ): 3498 (br), 3070 (w), 2960 (m), 2932 (m), 2861 (w), 1466 (m), 1427 (m), 1110 (s),

1003 (m), 913 (w), 703 (s) cm \

HRMS: (FAB, MNOBA matrix) for (M+H)\ calcd: 383.2395, found 383.2406.

'^C-NMR (125 MHz, In CDCI3): Ô 139.8, 135.6, 132.9, 132.8, 129.8, 127.8, 115.1, 79.8, 69.0,

41.2, 37.8, 26.8, 19.1, 17.8, 13.5 ppm.

[a]% :-17.4 (c 0.43, CHCI3).

85 Chapter 4: Experimental

{2R, 3R, 4/7)-1-(fert-Butyl-diphenyl-silanyloxy)-2,4-dinriethyl-hex-5-en-3-ol 213

OTBDPS Me Me

l-mlfc-39

(Spectral data agreed with those previously reported by Roush, ref. 103)

'H-NMR (500 MHz, In CDCI3): Ô 7.67 (m, 4H), 7.40 (m, 6 H), 5.82 (ddd, J = 8.3, 10.4, 17.0 Hz,

1H), 5.09 (m, 2H), 3.71 (d, J = 5.14 Hz, 2H), 3.58 (dd, J = 2.6, 8.5 Hz, 1H), 2.44 (s, 1H), 2.20

(sextet, J = 7.26 Hz, 1H), 1.83 (m, 1H), 1.06 (s, 9H), 0.95 (d, J = 1.0 Hz, 3H), 0.93 (d, J = 0.9 Hz,

3H) ppm.

IR (neat film): 3499 (br), 3070 (w), 2961 (s), 1961 (w), 1893 (w), 1826 (w), 1739 (w), 1465 (m),

1426 (m), 1388 (w), 1231 (w), 1109 (s), 1000 (m), 914 (w) cm \

HRMS: (FAB, MNOBA matrix) for Cg^Ha^Og^^i (M+H)\ calcd: 383.2395, found 383.2406.

'®C-NMR (125 MHz, in CDCis): Ô 141.8, 135.6, 135.5, 133.4, 133.2, 129.7, 127.7, 115.4, 76.1,

68.4, 41.7, 36.6, 26.9, 19.2, 16.7, 9.5 ppm.

[a ]% : -4.4 (c 0.09, CHCI 3 ).

86 Chapter 4: Experimental

((2/?,3S,4S)-3-Benzyloxy-2,4-dimethyl-hex-5-enyloxy)-fe/f-butyl-diphenyl-silane 214

OBn

OTBDPS Me Me

l-mlfc-78

To a stirred solution of the secondary alcohol 212 (9.75 g, 25.5 mmol) and benzyl-2,2,2- trichloroacetimidate (7.1 ml, 38.2 mmol, 1.5 eq) in CHgClg (100 ml, 0.25 M), was added trifluoromethanesulfonic acid (0.17 ml, 1.95 mmol, 0.077 eq) in CHgClg (20 ml, 0.1 M), dropwise over 3 min. at RT The reaction mixture was stirred for approximately 3 h. whereupon a light orange slurry formed. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then diluted with CHgClg (50 ml) and solid NaHCOg (3 g) was added, and the reaction mixture stirred for a further 10 min. whereupon the colour of the slurry turned a light yellow. The organic layer was washed, first with a saturated solution of NaHCOg (1 X 100 ml), and then with

HgO (1 X 100 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo to yield an orange/brown sludge. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/diethyl ether (50:1) as eluent provided the benzyl ether 214

(9.6 g, 80%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.71-7.66 (m, 4H), 7.46-7.21 (m, 1 1 H), 5.94 (ddd, J = 8.3, 10.4,

17.0 Hz, 1H), 5.05-4.99 (m, 2H), 4.50 (dd, J = 11.1, 21.5 Hz, 2H), 3.84 (dd, J=5.4, 9.8 Hz, 1H),

3.76 (dd, J=3.8, 9.8 Hz, 1H), 3.35 (dd, J=3.4, 8.2 Hz, 1H), 2.56-2.48 (m, 1H), 1.94-1.85 (m, 1H),

1.13 (d, J=6.9 Hz, 3H), 1.12 (s, 9H), 1.03 (d, J=6.89 Hz, 3H) ppm.

IR (neat film): 3582 (w), 3342 (w), 3068 (m), 3033 (m), 2961 (m), 2932 (m), 2861 (m), 1767 (s),

1665 (m), 1458 (s), 1227 (s), 1109 (s), 1003 (m), 827 (s), 701 (s) cm \

87 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for (M+H)\ calcd: 473.2887, found 473.2876.

'^C-NMR (125 MHz, in CDCI3 ): Ô 140.7, 138.9, 135.7, 133.7, 129.5, 128.3, 127.6, 127.5, 127.2,

1 1 4 .4 , 84.6, 74.6, 65.5, 40.6, 38.9, 26.9, 19.3, 18.3, 14.5 ppm.

[ a f \ : +4.8 (c 0.4, CH^Cy.

88 Chapter 4: Experimental

[(2R,3S,4S)-3-(4-Bromo-benzyloxy)]-2,4-dimethyl-hex-5-enyloxy]-fe#t-butyl-diphenyl-silane

220

OBnBr

OTBDPS Me Me

l-mlfc-64

To a stirred solution of the secondary alcohol 212 (1.0 g, 2.6 mmol) and 4-bromobenzyl

trichloroacetimidate^^^ (1.04 g, 3.1 mmol, 1 . 2 eq.) in CHgClg (5.0 ml, 0.52 M), was added trifluoromethanesulfonic acid (0.02 ml, 0.26 mmol, 0.1 eq.) in CHgClg (20 ml, 0.1 M), dropwise over 3 min. at RT. The reaction mixture was stirred for approximately 3 h. whereupon a light orange slurry formed. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then diluted with CHgClg (50 ml) and solid NaHCOg (3 g) was added, and the reaction mixture stirred for a further 10 min. whereupon the colour of the slurry turned a light yellow. The organic layer was washed, first with a saturated solution of NaHCOg (1 X 100 ml), and then with

HgO ( 1 X 100 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo to yield an orange/brown sludge. Purification of the crude residue by flash column chromatography (SiOg), with hexanes/diethyl ether (50:1) as eluent, provided the bromobenzyl ether 220 (1.0 g, 72%) as a yellow oil.

'H-NMR (500 MHz, in CDCIg): 5 7.62 (m, 3H), 7.36 (m, 9H), 6.99 (m, 2H), 5.85 (ddd, J = 8.4,

10.4, 18.7 Hz, 1H), 5.00 (m, 2H), 4.50 (dd, J = 11.3, 21.5 Hz, 1H), 3.75 (dd, J = 5.3, 9.8 Hz, 1H),

3.69 (dd, J = 3.8, 9.8 Hz, 1H), 3.27 (dd, J = 3.4, 8.2 Hz, 1 H), 2.45 (m, 1 H), 1.85 (m, 1 H), 1.08 (d,

J = 6.9 Hz, 3H), 1.07 (s, 9H), 0.97 (d, J = 6.9 Hz, 3H) ppm.

89 Chapter 4: Experimental

IR (neat film): 2962 (s), 2858 (s), 2354 (w), 1898 (w), 1821 (w), 1768 (m), 1728 (w), 1712 (w),

1696 (w), 1666 (w), 1644 (w), 1592 (w), 1552 (w), 1536 (w), 1489 (s), 1468 (m), 1428 (m), 1389

(m). 1306 (w), 1226 (w), 1111 (s), 1073 (s), 1010 (s), 914 (w), 824 (m) cm \

HRMS: (FAB, MNOBA matrix) for CaiHagO/^Br^sSi (M+H)\ calcd: 551.19806, found 551.19714.

'^C-NMR (125 MHz, In CDCI3): Ô 140.5, 137.9, 135.7, 135.6, 135.5, 133.8, 133.6, 131.2, 129.8,

129.6, 129.5, 127.7, 127.6, 121.0, 114.6, 84.9, 73.9, 65.5, 40.5, 40.5, 38.9, 26.9, 26.7, 19.3,

18.3, 14.5 ppm.

[a Y \:+ 7 A (cO.38, CHgCy.

90 Chapter 4: Experimental

(3S,4S,5R)-4-Benzyloxy-6-(feft-butyl-diphenyl-silanyloxy)-3,5-dimethyl-hexan - 1 -ol 215

OBn

OTBDPS Me Me

l-mlfc-134

To a cooled (0°C), stirred solution of the benzyl ether 214 (1.0 g, 2.12 mmol), in THF (7 ml, 0.3

M) was added BH 3 THF (1.0 M in THF, 2.12 ml, 2.12 mmol, 1 eq) dropwise over 3 min., via syringe. The reaction mixture was allowed to warm up to and stir at RT for 3 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was quenched at 0 °C, by the careful addition of aqueous NaHCOg (3.0 M, 7.0 ml, 21.2 mmol, 10 eq.), followed by HgOg (27.5 wt % in

HgO, 4.0 ml, 31.7 mmol, 15 eq.) and methanol ( 1 ml). The resulting mixture was then left to stir

at RT for a further 1 h. Solid KgCOg was added to the reaction mixture at 0 °C until completely saturated, and the aqueous phase was extracted from ether (3 X 20 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg), with hexanes/ethyl acetate (50:1->10:1) as eluent

provided the primary alcohol 215 (0.7 g, 6 8 %) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.65 (m, 4H), 7.43-7.05 (m, 11H), 4.53 (d, J = 3.4 Hz, 2H), 3.77

(m, 2H), 3.70 (m, 1 H), 3.52 (m, 1H), 3.32 (dd, J = 3.6, 8.3 Hz, 1 H), 1.99-1.92 (m, 2H), 1.64-1.57

(m, 2H), 1 .02 (s, 9H), 1.04 (d, J = 7.0 Hz, 3H), 1.00 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 3584 (w), 3389 (br), 3070 (w), 2960 (s), 2931 (s), 2857 (m), 2349 (w), 1428 (m),

1390 (w), 1361 (w), 1112 (s), 701 (s) cm \

HRMS: (FAB, MNOBA matrix) for CaiH^gOa^^Si (M-hH)+, calcd: 491.2986, found 491.2981.

91 Chapter 4: Experimental

'"C-NMR (125 MHz, in CDCI3 ): Ô 138.4, 135.7, 135.6, 133.8, 133.7, 129.5, 128.3, 127.5, 127.4,

85.4, 75.2, 65.7, 59.8, 38.7, 32.2, 26.9, 19.3, 17.4, 14.8 ppm.

92 Chapter 4: Experimental

(3S,4S,5/?)-4-(4-Bromo-benzyloxy)-6-(fe/t-butyl-diphenyl-silanyloxy)-3,5-dimethyl-hexan-1- ol 2 2 1

OTBDPS Me Me

l-mlfc-91

To a cooled (0 °C) stirred solution of the bromo benzyl ether 220 (1.0 g, 1.81 mmol), in THF (5

ml, 0.36 M) was added BH 3 THF (1.0 M solution in THF, 1.81 ml, 1.81 mmol, 1 eq) dropwise over

3 min. via syringe. The reaction mixture was allowed to warm up to and stir at RT for 3 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was quenched at 0 °C, by the careful addition of aqueous NaHCOg (3.0 M, 6.0 ml, 18.1 mmol, 10 eq.), followed by HgOg (27.5 wt % in HgO, 3.3 ml, 27.2 mmol, 15 eq) and methanol (1 ml). The resulting mixture was then left

to stir at RT for a further 1 h. Solid K 2 CO3 was added to the reaction mixture at 0 °C until completely saturated, and the aqueous phase was extracted from ether (3 X 20 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate

(50:1^10:1) as eluent provided the primary alcohol 221 (0.6 g, 62%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.59-7.54 (m, 4H), 7.33-7.28 (m, 2H), 7.28-7.19 (m, 6 H), 6.87-

6.83 (m, 2H), 4.39 (d, J = 11.2 Hz, 1 H), 4.33 (d, J = 11.2 Hz, 1 H), 3.68-3.56 (m, 3H), 3.46-3.40

(m. 1H), 3.22-3.20 (dd, J = 3.38, 8.63 Hz, 1 H), 1.91-1.84 (m, 2H), 1.56-1.44 (m, 2H), 0.98 (s,

9H), 0.94 (d, J = 6.97 Hz, 3H), 0.91 (d, J = 6.90 Hz, 3H) ppm.

IR (neat film): 3364 (br), 3071 (w), 2959 (s), 2930 (s), 2858 (s), 1591 (m), 1488 (m), 1428 (m),

1112 (s), 1071 (s), 1012 (s), 740 (m), 702 (s) cm '.

93 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for CsiH^iOg^^Si^^BrNa (IVI+Na)\ calcd: 591.1924, found

591.1906.

'®C-NMR (125 MHz, in CDCI3 ): Ô 137.3, 135.7, 133.5, 131.5, 131.2, 129.5, 129.0, 128.5, 127.5,

127.4, 121.3, 85.4, 74.4, 65.5, 64.4, 59.7, 38.5, 33.3, 32.1, 26.9, 19.3, 17.4, 14.7 ppm.

94 Chapter 4: Experimental

(3S,4S,5R)-4-Benzyloxy-6-(ferf-butyl-dlphenyl-silanyloxy)-3,5-dimethyl-hexanoic acid 216

OBn

O Me Me

lll-mlfc- 1

Carbon tetrachloride ( 2 . 0 ml), acetonitrile (2 . 0 ml) and HgO (3.0 ml) were added to the flask

containing the alcohol 215 (420 mg, 0 . 8 6 mmol). Sodium periodate (380 mg, 1.80 mmol, 2.1 eq) was then added to this diphasic solution followed by ruthenium (III) chloride (17.8 mg, 0.086 mmol, 0.1 eq), and the resulting mixture was stirred vigorously at RT overnight. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was then diluted with CHgClg (5 ml), and the aqueous phase was extracted with CHgClg (3 X 20 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash

column chromatography (SiOg) with hexanes/ethyl acetate ( 2 0 :1 ^ 1 0 :1 ) as eluent provided the acid 216 (273 mg, 63%) as a yellowish oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.64-7.57 (m, 4H), 7.42-7.10 (m, 1 1 H), 4.50 (s, 2H), 3.74-3.70

(m, 2H), 3.31 (dd, J = 4.1, 7.2 Hz, 1 H), 2.50 (dd, J = 3.7, 15.0 Hz, 1H), 2.29-2.16 (m, 2H), 2.20

(dd, J = 9.2, 15.0 Hz, 1H), 1.90-1.83 (m, 1H), 1.06 (s, 9H), 1.04 (d, J = 6.9 Hz, 3H), 1.00 (d, J =

6.9 Hz, 3H) ppm.

IR (neat film): 3070 (m), 2858 (m), 1959 (w), 1888 (w), 1707 (s), 1428 (s), 1295 (m), 1075 (br),

824 (s), 739 (s), 701 (s) cm'L

HRMS: (FAB, MNOBA matrix) for CaiH^^O/^Si (M+H)\ calcd: 505.2793, found 505.2774.

95 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 178.2, 138.3, 135.7, 133.7, 133.6, 129.6, 128.7, 127.6, 127.4,

84.8, 74.9, 65.4, 38.6, 36.3, 32.4, 26.9, 19.3, 18.1, 14.6 ppm.

[a]% :-4 .2 (c 0.7, CHgCy.

96 Chapter 4: Experimental

(3S,4S,5R)-4-(4-Bromo-benzyloxy)-6-(te/t-butyl-diphenyl-silanyloxy)-3,5-dimethyl- hexanoic acid 2 2 2

o '

O Me Me

l-mlfc-92

Carbon tetrachloride (2.0 ml), acetonitrile ( 2 . 0 ml) and HgO (3.0 ml) were added to the flask containing the alcohol 221 (580 mg, 1.02 mmol). Sodium periodate (460 mg, 2.14 mmol, 2.1 eq) was then added to this biphasic solution, followed by ruthenium (III) chloride (21.1 mg, 0.102 mmol, 0.1 eq), and the resulting mixture was stirred vigorously at RT overnight. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was then diluted with CHgClg (5 ml), and the aqueous phase was extracted with CHgClg (3 X 20 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg), with hexanes/ethyl acetate (20:1->10:1) as eluent provided the acid 222 (362 mg, 61%) as a yellowish oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.65-7.60 (m, 4H), 7.43-7.38 (m, 2H), 7.38-7.31 (m, 6 H), 6.98

(d, J = 8.3 Hz, 2H), 4.45 (dd, J = 1 1.3, 21.5 Hz, 2H), 3.75-3.68 (m, 2H), 3.28 (dd, J = 4.2, 7.2 Hz,

1 H), 2.47 (dd, J = 4.1, 15.3 Hz, 1H), 2.35-2.27 (m, 1 H), 2.19 (dd, J = 9.1, 15.2 Hz, 1 H), 1.92-1.84

(m, 1H), 1.07 (s, 9H), 1.04 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 3584 (w), 3071 (w), 3049 (w), 2961 (m), 2931 (m), 2858 (m), 1707 (s), 1428 (s),

1112 (s), 1072 (s), 1012 (s), 740 (m), 703 (s) cm \

HRMS: (FAB, MNOBA matrix) for CaiHggG/^Si^^BrNa (M+Na)\ calcd: 605.1685, found

605.1685.

97 Chapter 4: Experimental

^^C-NMR (125 MHz, in CDCI3 ): S 179.5, 137.5, 135.7, 135.6, 133.6, 133.5, 131.2, 130.5, 129.6,

129.0, 127.6, 121.2, 84.9, 74.0, 65.4, 38.6, 36.4, 32.5, 26.9, 19.3, 18.2, 14.6 ppm.

[a]%:-10.8 (cO.3, CHgCy.

98 Chapter 4: Experimental

(S)-4-Benzyl-3-[(3S,4S,5/?)-4-benzyloxy-6-(teit-butyl-diphenyl-silanyloxy)-3,5-dimethyl-

hexanoyl]-oxazolidin- 2 -one 219

OBn

^Y^^^Y^OTBDPS Me Me

l-mlfc-51(b)

To a stirred solution of the acid 216 (250 mg, 0.495 mmol) in THF (5 ml, 0.1 M), was added Et^N

(0.07 ml, 0.495 mmol, 1 eq) at RT PvCI (0.06 ml, 0.495 mmol, 1 eq) was then syringed into the

reaction mixture at 0 °C, and stirred for 20 min. To the oxazolidinone (82 mg, 0.495 mmol, 1

eq), dissolved in THF was then added n-BuLi (2.5 M in hexanes, 0.2 ml, 0.495 mmol, 1 eq) at -

78 °C and stirred for 20 min. The mixed anhydride solution was then added to the now lithiated

oxazolidinone via syringe at -78 °C. The resulting mixture continued to stir at this temperature for a further 4 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was then

diluted with ether (5 ml) and water (10 ml) was added. The aqueous phase was extracted with

ether (3X10 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated

in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with

hexanes/ethyl acetate (50:1->10:1) as eluent provided 219 (177 mg, 54%) as a colourless oil.

'H-NMR (300 MHz, in CDCI3): Ô 7.61-7.52 (m, 5H), 7.38-7.03 (m, 14H), 4.50 (dd, J = 11.0, 17.8

Hz, 2H), 4.50-4.45 (m, 1H), 3.95-3.88 (dd, J = 2.6 Hz, 1 H), 3.78-3.58 (m, 3H), 3.26-3.08 (m, 3H),

2.72-2.52 (m, 2H), 2.51-2.39 (m, 1H), 1.98-1.85 (m, 1H), 1.09 (d, J = 6.9 Hz, 3H), 1.02 (s, 9H),

0.89 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 2962 (s), 1960 (w), 1787 (s), 1695 (s), 1604 (m), 1589 (m), 1454 (s), 1427 (s),

1382 (s), 1294 (s), 1209 (s), 1088 (s), 824 (m), 745 (m) cm '.

99 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for C^iH^gOsN'^Si (M+H)+, calcd: 664.3457, found 664.3450.

'^C-NMR (125 MHz, in CDCI3 ): Ô 172.9, 153.4, 138.9, 135.7, 135.6, 133.8, 129.7, 129.5, 128.8,

128.3, 127.7, 127.1, 85.6, 74.3, 65.8, 65*5, 55.2, 38.7, 38.0, 37.9, 32.2, 26.9, 19.3, 18.4, 14.7 ppm.

100 Chapter 4: Experimental

(S)-4-Benzyl-3-[(3S,4S,5/?)-4-(4-bromo-benzyIoxy)-6-(feft-butyl-diphenyl-silanyloxy)-3,5- dimethyl-hexanoyl]-oxazolidin-2-one 223

N. .O OBnBr

OTBDPS Me Me

l-mlfc-68

To a stirred solution of the acid 222 (360 mg, 0.62 mmol) in THF (5 ml, 0.12 M), was added EtgN

(0.09 ml, 0.62 mmol, 1 eq) at RT PvCI (0.08 ml, 0.62 mmol, 1 eq) was then syringed into the

reaction mixture at 0 °C, and stirred for 20 min. To the oxazolidinone ( 1 0 0 mg, 0.62 mmol, 1 eq), dissolved in THF was then added n-BuLi (2.5 M solution in hexanes, 0.25 ml, 0.62 mmol, 1 eq) at -78 °C and stirred for 20 min. The chilled mixed anhydride solution (-78°C) was then added dropwise to the now lithiated oxazolidinone via syringe. The resulting mixture continued to stir at this temperature for a further 4 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was then diluted with ether (5 ml) and water (10 ml) was added. The aqueous phase was extracted with ether (3X10 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (50:1^10:1) as eluent provided 223 (266 mg,

58%) as a colourless oil.

'H-NMR (300 MHz, in CDCI3): Ô 7.62 (m, 5H), 7.34 (m, 10H), 7.08 (d, J = 8.3 Hz, 2H), 7.03 (d, J

= 8.3 Hz, 2H), 4.53 (m, 1H), 4.46 (s, 2H), 4.04 (dd, J = 2.7, 9.0 Hz, 1H), 3.91 (t, J = 8.4 Hz, 1 H),

3.75 (m, 1 H), 3.68 (m, 1 H), 3.27 (t, J = 5.7 Hz, 1 H), 3.22 (dd, J = 3.3, 13.3 Hz, 1H), 3.14 (dd, J =

5.0, 16.6 Hz, 1 H), 2.80 (dd, J = 8.4, 16.6 Hz, 1H), 2.66 (dd, J = 9.5, 12.7 Hz, 1 H), 2.48 (m, 1 H),

1.95 (m, 1 H), 1.06 (s, 9H), 1.02 (d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H) ppm.

101 Chapter 4: Experimental

IR (neat film): 3070 (m), 2962 (s), 1784 (s), 1698 (s), 1590 (w). 1487 (m), 1427 (m), 1386 (s),

1297 (m), 1206 (m), 1109 (s), 824 (m), 742 (m), 705 (s), 613 (w) cm \

HRMS: (FAB, MNOBA matrix) for (M+H)\ calcd: 742.25631, found

742.25589.

'"C-NMR (125 MHz, In CDCI3): Ô 176.4, 172.9, 153.4, 137.9, 135.7, 135.6, 135.3, 133.7, 131.2,

129.7, 129.3, 128.8, 121.1, 88.3, 85.6, 73.8, 65.9, 65.4, 55.2, 38.6, 37.9, 32.1, 26.9, 19.2, 18.4,

14.7 ppm.

[aFD:+20 (c0.1,CH2CW.

102 Chapter 4: Experimental

(3/?,4/?,5R)-3-Benzyloxy-5-(te/t-butyl-diphenyl-silanyloxy)-2,4-dimethyl-pentanal 229

OBn OHC

Me Me

l-mlfc-81

To a cooled (0°C), stirred solution of the alkene 214 (820 mg, 1.74 mmol) in THF (5 ml, 0.34 M), was added osmium tetroxide solution (0.04 M in HgO/MegCO, 0.86 ml, 0.035 mmol, 0.02 eq).

The resulting light brown solution was stirred at 0 °C for 10 min before NalO^ (2.22 g, 10.41 mmol, 6 eq.) was added. The reaction mixture stirred at 0 °C for 35 min and then allowed to warm up to RT where it was stirred overnight. (TLC mobile phase: hexane/ethyl acetate, 10:1).

The reaction mixture was quenched by the careful addition of solid sodium sulfite (3 g), followed by HgO (10 ml). The aqueous phase was extracted with ethyl acetate (3 X 15 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (50:1) as eluent provided the aldehyde 229 (413 mg, 50%) as a colourless oil.

'H-NMR (300 MHz, in CDCI3 ): Ô 9.80 (d,1H), 7.71 (d, 5H), 7.51-7.21 (m, 10H), 4.58 (d, 2H),

4.04-3.94 (m, 1H), 3.89-3.71 (m, 2H), 2.86-2.72 (m, 1H), 2.15-2.02 (m, 1H), 1.19 (d, 3H), 1.11

(s, 9H), 0.99 (d, 3H) ppm.

103 Chapter 4: Experimental

(3S,4/?,5/?)-4-Benzyloxy-6-(te/t-butyl-diphenyl-silanyloxy)-3,5-dimethyl-hexane-1,2-diol 230

OH OBn HO OTBDPS Me Me

l-mlfc-109

To a stirred solution of the alkene 214 (1.0 g, 2.12 mmol, 1 eq) in a 1:1 acetone/water mixture, was added OsO^ (0.04 M solution in acetone/water, 2.6 ml, 0.106 mmol, 0.05 eq), followed by t-

butanol (3.7 ml). 4-Methylmorpholine-A/-oxide (0.372 g, 3.18 mmol, 1.5 eq) was then added in one portion, and the resulting mixture was stirred for 5.5 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was quenched by the addition of a saturated aqueous solution of sodium sulfite, and left to stir for a further 1 h. The aqueous phase was then extracted with ethyl acetate (3 X 20 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (4:1->2:1) as eluent provided the diol 230

(0.82 g, 76%) as a colourless oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.67-7.61 (m, 4M), 7.42-7.06 (m, 11H), 4.59-4.52 (dd, J =

10.92, 21.56 Hz, 2H), 3.74 (dd, J = 4.9, 10.0 Hz, 2H), 3.70-3.64 (m, 3.67 Hz, 2H), 3.50-3.43 (m,

2H), 2.09-2.01 (m, 1H), 2.00-1.95 (m, 1H), 1.89-1.82 (m, 1H), 1.06 (s, 9H), 1.03 (d, J = 6.9 Hz,

3H), 0.87 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 3411 (br), 3070 (w), 2930 (m), 2859 (m), 2359 (w), 1954 (w), 1892 (w), 1821 (w),

1591 (w), 1425 (s), 1260 (w), 1109 (s), 824 (m), 734 (m), 704 (s) cm \

HRMS: (FAB, MNOBA matrix) for CgiH^gO/^SiNa (M+Na)\ calcd: 529.2742, found 529.2750.

104 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3): Ô 137.9, 137.8, 135.8, 135.7, 133.7, 133.5, 129.7, 129.6, 128.5,

127.8, 127.7, 127.6, 86.7, 75.6, 74.8, 74.3, 71.3, 65.5, 65.4, 64.5, 39.5, 38.7, 37.9, 35.4, 27.0,

26.9, 19.3, 19.2, 14.8, 14.6, 12.7 ppm.

[a ro :-5 7 (c 0.1, CH 2CI2 ).

105 Chapter 4: Experimental

(£)-(4S,5S,6f?)-5-Benzyloxy-7-(terf-butyl-diphenyl-silanyloxy)-4,6-dimethyl-hept-2-enoic acid ethyl ester 228

OBn

OTBDPS Me Me

l-mlfc-98

To a stirred solution of aldehyde 229 (4.0 g, 8.43 mmol, 1 eq) in CHgClg (41.3 ml, 0.2 M), was added carbethoxymethylenetriphenylphosphorane (5.87 g, 16.9 mmol, 2 eq) in one portion, and the reaction mixture was left to stir for 48 h. (TLC mobile phase: hexane/ethyl acetate, 10:1).

The solvent was then removed under reduced pressure, and the residue was subjected to flash column chromatography (SiOg) with hexanes/ethyl acetate (100:1->50:1->30:1) as eluent.

Alkene 228 (3.5 g, 76%) was obtained as a colourless oil.

H-NMR (500 MHz, in CDCia): Ô 7.65-7.60 (m, 4H), 7.45-7.15 (m, 11H), 7.03 (dd, J = 8.6, 15.8

Hz, 1H), 5.75 (d, J = 15.3 Hz, 1H), 4.51 (s, 2H), 4.18-4.14 (m, 2H), 3.76 (dd, J = 5.3, 9.9 Hz,

1H), 3.69-3.66 (dd, J = 4.1, 9.9 Hz, 1H), 3.39-3.37 (dd, J = 3.6, 7.8 Hz, 1H), 2.66-2.65 (m, 1H),

1.89-1.80 (m, 1H), 1.28-1.25 (t, 3H), 1.12 (d, J = 6.9 Hz, 3H), 1.06 (s, 9H), 0.94 (d, J = 6.9 Hz,

3H) ppm.

IR (neat film): 3584 (w), 3070 (w), 2960 (m), 2931 (m), 2858 (m), 1718 (s), 1651 (m), 1266 (m),

1183 (m), 1112 (s), 701 (s) cm \

HRMS: (FAB, MNOBA matrix) for Cg^H^^O/^SiNa (M+Na)\ calcd: 567.2890, found 567.2907.

106 Chapter 4: Experimental

'"C-NMR (125 MHz, in CDCI3 ): Ô 166.6, 151.1, 138.6, 135.7, 133.7, 133.6, 129.6, 128.8, 128.3,

128.2, 127.7, 127.6, 127.4, 121.2, 84.2, 74.7, 65.4, 60.1, 39.4, 38.9, 26.9, 19.3, 17.5, 14.4, 14.3 ppm.

[a]%: +20.7 (cO.42, CHgCy.

107 Chapter 4: Experimental

(E)-(4S,5S,6R)-5-Benzyloxy-7-(fe/t-butyl-diphenyl-silanyloxy)-4,6-dimethyl-hept-2-en-1-ol 231

OTBDPS Me Me

l-mlfc-99

To a cooled (-78 ®C), stirred solution of the ester 228 (2.92 g, 5.36 mmol, 1 eq) in CHgClg (11.2 ml, 0.48 M), was added DIBAL (8.4 ml 1.5 M in PhMe, 11.8 mmol, 2.2 eq) in a slow stream via syringe over 5 min and the reaction mixture was left to stir for 3.5 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then diluted with CHgClg (40 ml) and slowly quenched at 0 °C with a solution of 10% aq. Rochelle’s salt (28 ml). The resulting mixture was left to stir vigorously at RT for 1.5 h. The aqueous phase was extracted with CHgClg (1 X 40 ml) then with diethyl ether (2 X 40 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (15:1^10:1) as eluent afforded the primary alcohol 231 (2.5 g, 92%) as a thick clear oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.66-7.62 (m, 4H), 7.43-7.15 (m, 11H), 5.77-5.70 (dd, J = 8.5,

15.3 Hz, 1H), 5.63-5.57 (m, 1H), 4.51 (s, 2H), 4.03 (d, J = 5.8 Hz, 2H), 3.77 (dd, J = 5.5, 10.2

Hz, 1H), 3.71 (dd, J = 3.8, 9.8 Hz, 1H), 3.30 (dd, J = 3.4, 8.1 Hz, 1H), 2.50-2.47 (m, 1H), 1.84-

1.82 (m, 1H), 1.40 (s, 1H), 1.11 (d, J = 6.9 Hz, 3H), 1.08 (s, 9H), 0.97 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 3585 (w), 3389 (br), 3069 (w), 2961 (m), 2930 (m), 2857 (m), 2359 (w), 1427

(m), 1112 (s), 1092 (m), 824 (m), 700 (s) cm \

HRMS: (FAB, MNOBA matrix) for CggH^gOg^^SiNa (M +Na)\ calcd: 525.2803, found 525.2801.

108 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): 5 138.8, 135.8, 135.7, 135.6,134.8, 133.8, 129.5, 129.1, 128.8,

128.3, 128.2, 127.6, 127.5, 127.4, 84.8, 65.6, 63.8, 39.1, 38.7, 26.9, 19.3, 18.7, 14.6 ppm.

[a ]% :-4 .5 (c 0.4, CH^Cy.

109 Chapter 4: Experimental

{(2S,3S)-3-[(1/?,2/?,3/?)-2-Benzyloxy-4-(fe/t-butyl-diphenyl-silanyloxy)-1,3-dimethyl-butyl]- oxiranyl}-methanol 232

OTBDPS Me Me

l-mlfc- 1 0 0

To a cooled (-35 °C), stirred solution of flame dried 4Â molecular sieves (1.14 g) in CHgClg (0.3

M solution in CHgClg), was added (+)-diethyltartrate (0.11 ml, 0.64 mmol, 1 eq) via syringe followed by titanium (IV) isopropoxide (0.19 ml, 0.64 mmol, 1 eq). f-Butyl hydrogen peroxide

(0.72 ml, 3.18 mmol, 1 0 eq) was then added, and the reaction mixture stirred at -35 °C for a

further 55 min. Alkene 231 (320 mg, 0.64 mmol, 1 eq) was then added dropwise over 5 min as a solution in CHgClg (2.1 ml, 0.3 M), and the resulting mixture stirred at -30 °C for 15 min, before being transferred to a freezer (-33°C) and left to stand overnight. (TLC mobile phase: hexane/ethyl acetate, 4:1). The reaction mixture was allowed to warm to 0 °C and quenched by carefully pouring into a solution of FeSO^THgO (10 g) and citric acid (15 g) in water (20 ml). The resulting mixture was stirred vigorously at 0 °C for 0.5 h. The aqueous phase was extracted with diethyl ether (3 X 20 ml). The combined organic phases were then stirred vigorously with 15%

NaOH in brine (20 ml) at 0 °C for 1 h. The aqueous phase was then extracted with diethyl ether

(3 X 20 ml). The combined organic phases were dried (IVIgSO^), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (10:1->7:1) as eluent afforded the epoxide 232 (288 mg, 87%) as a thick clear oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.65-7.62 (m, 5H), 7.41-7.39 (m, 2H), 7.35-7.34 (m, 4H), 7.26-

7.25 (m, 3H), 7.18 (m, 2H), 4.55 (dd, J = 12.5, 21.3 Hz, 2H), 3.78-3.70 (m, 2H), 3.50-3.43 (ddd, J

110 Chapter 4: Experimental

= 4.3, 7.2, 11.7, 1H), 3.39-3.35 (t, J = 5.9 Hz, 1H), 2.99-2.93 (m, 2H), 2.04-1.97 (m, 1 H), 1.72-

1.65 (m, 2H), 1.08 (d, J = 6.9 Hz, 3H), 1.06 (s, 9H), 0.99 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 3581 (w), 3439 (br), 3074 (w), 2965 (m), 2931 (m), 2857 (m), 2363 (w), 1455

(m), 1110 (s), 704 (s) cm \

HRMS: (FAB, MNOBA matrix) for CagH^gO/^SiNa (M+Na)+, calcd: 541.2756, found 541.2750.

'^C-NMR (125 MHz, in CDCij): Ô 138.6, 135.7, 133.7, 129.6, 128.3, 127.6, 127.3, 84.2, 74.7,

65.2, 61.7, 59.3, 58.1, 38.4, 26.9, 19.3, 15.1, 14.9 ppm.

[a]% :-13.3 (c 0.1, CHgCy.

I l l Chapter 4: Experimental

(2S,3S,4S,5R)-4-Benzyloxy-6-(te/t-butyl-diphenyl-silanyloxy)-2-((R)-1,2-dihydroxy-ethyl)- 3,5-dimethyl-hexanenitrile 227

(2R,3R,4S,5R,6R)-5-Benzyloxy-7-(ferf-butyl-diphenyl-silanyloxy)-3-hydroxy-2-hydroxy methyl-4, 6 -dimethyl-heptanenitrile 233

OH OH OK OBn ONOBn

NO OTBDPS HO''' Me Me Me Me

227 233 l-mifc-120 Bottom spot (227)

To a cooled (0 °C), stirred, solution of the epoxide 232 (90 mg, 0.17 mmol, 1 eq) in toluene (1.7

ml, 0.1 M), was added a solution of diethylaluminium cyanide (0.9 ml ( 1 M sol" in PhMe), 0.87

mmol, 5 eq) dropwise via syringe over 1 min. The resulting mixture was left to stir for 2.5 h. at

RT (TLC mobile phase: hexane/ethyl acetate, 1 :1 ). The reaction mixture was then diluted with water (5 ml), and the aqueous phase was extracted with ethyl acetate (3 X 10 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate

(5:1 ^3 :1 ) as eluent to provide the undesired isomer 233 (26.8 mg, 29%) as a colourless oil, followed by 1,2-diol 227 (44.5 mg, 48%) as an oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.68-7.62 (m, 4M), 7.44-7.25 (m, 9H), 7.10-7.05 (m, 2H), 4.62-

4.54 (dd, J = 13.1, 23.5 Hz, 2H). 3.78 (dd, J = 4.7, 10.2 Hz, 3H), 3.72 (dd, J = 4.14, 10.13 Hz,

1H), 3.63 (dd, J = 5.02, 7.695 Hz, 2H), 2.94 (dd, J = 2.14, 9.155 Hz, 1 H), 2.50-2.42 (m, 1H),

2.04-1.97 (m, 2H), 1.23 (d, J = 7.13 Hz, 3H), 1.07 (s, 9H), 1.01 (d, J = 7.03 Hz, 3H) ppm.

IR (neat film): 3412 (br), 3066 (w), 2960 (m), 2934 (m), 2861 (m), 2363 (w), 2237 (w), 1965

(w), 1892 (w), 1825 (w), 1586 (w), 1109 (s) cm \

112 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for (M+H)\ calcd: 546.3023, found 546.3040.

^®C-NMR (125 MHz, in CDCI3): Ô 136.6, 135.7, 133.3, 133.2, 129.8, 128.6, 127.8, 127.7, 120.0

83.3, 75.8, 67.5, 64.9, 64.8, 38.4, 36.2, 35.9, 26.9, 19.2, 15.0, 14.6 ppm.

l-mlfc-120 Top spot (233)

'H-NMR (500 MHz, In CDCI3): Ô 7.62 (m, 4H), 7.42 (m, 2H), 7.35 (m, 4H), 7.26 (m, 3H), 7.03 (m,

2 H), 4.51 (s, 2H), 4.23 (d, J = 10.4 Hz, IN), 4.11 (s, 1H), 3.93 (m, 2H), 3.85 (dd, J = 4.6, 9.9 Hz,

1 H), 3.75 (dd, J = 3.2, 9.9 Hz, 1 H), 3.62 (dd, J = 1.6, 9.7 Hz, 1 H), 2.83 (ddd, J = 4.3, 5.9, 10.3

Hz, 1H), 1.91 (m, 1H), 1.82 (m, 1H), 0.99 (d, J = 8.9 Hz, 3H), 0.91 (s, 9H), 0.84 (d, J = 8.5 Hz,

3H)ppm.

IR (neat film): 3454 (br), 3067 (m), 2954 (s), 2848 (m), 2355 (w), 2243 (w), 1961 (w), 1890 (w),

1820 (w), 1580 (w), 1426 (m), 1 1 0 2 (s) cm \

HRMS: (FAB, MNOBA matrix) for CagH^gNO^SiNa (M+Na)\ calcd: 568.2865, found 568.2859.

'^C-NMR (125 MHz, In CDCI3): Ô 137.3, 135.8, 135.7, 133.5, 133.3, 129.8, 129.7, 128.6, 128.1,

127.8, 127.7, 118.2, 8 6 .6 , 76.2, 70.7, 65.3, 62.1,38.7, 37.6, 35.6, 27.0, 19.3, 14.4, 11.9 ppm.

113 Chapter 4: Experimental

(2S,3S,4S,5/?)-4-Benzyloxy-6-(feff-butyl-dlphenyl-silanyloxy)-2-((A?)-2,2-dimethyl-[1,3] dioxolan-4-yl)-3,5-dimethyl-hexanenitrile 234

O-

OBn

NC OTBDPS Me Me

l-mlfc-126

To a stirred solution of diol 227 (69 mg, 0.13 mmol) in acetone (0.27 ml, 0.4 M), was added 2,2-

dimethoxypropane (0.27 ml), followed by a catalytic amount of pyridinium para-toluene sulfonate

( 8 mg, 0.032 mmol, 0.25 eq). The reaction mixture was then warmed to 40 °C and stirred

vigorously for 8 h. (TLC mobile phase: hexane/ethyl acetate, 4:1). The solvent was then

removed under reduced pressure and the resulting residue purified by flash column

chromatography (SiOg), eluting with hexanes/ethyl acetate ( 2 0 :1 ) to provide the protected diol

234 (72.3 mg, 95%) as a clear oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.68-7.62 (m, 4H), 7.43-7.19 (m, 11H), 4.56 (s, 2H), 4.35-4.31

(q, J = 6 . 6 Hz, 1 H), 3.98 (dd, J = 5.9, 8 . 6 Hz, 1H), 3.79-3.74 (m, 2H), 3.65 (dd, J = 5.9, 10.2 Hz,

1 H), 3.55 (dd, J = 4.7, 7.3 Hz, 1H), 3.05 (dd, J = 3.7, 6.7 Hz, 1 H), 2.38-2.33 (m, 1H), 2.11-2.03

(m, 1 H), 1.38 (s, 3H), 1.26 (s, 3H), 1.06 (s, 9H), 1.05 (d, J = 6.01 Hz, 3H), 1.02 (d, J = 6 . 8 Hz,

3H)ppm.

IR (neat film): 3402 (br), 3071 (w), 2961 (m), 2930 (m), 2860 (m), 2358 (w), 2238 (w), 1958

(w), 1893 (w), 1823 (w), 1732 (w), 1632 (m), 1426 (s), 1220 (m), 1109 (s), 1069 (s), 824 (m),

731 (m), 703 (s) cm \

114 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 138.3, 135.7, 135.6, 133.6, 129.6, 128.7, 127.8, 127.6, 119.4,

109.5, 83.8, 74.2, 73.8, 68.0, 65.3, 39.3, 38.4, 35.7, 26.9, 26.7, 25.5, 19.2, 16.3, 15.1, 13.8 ppm.

[aF o:-1 4 .3 (c 0.3, CHgCy.

115 Chapter 4: Experimental

(2/?,3S,4S,5/?)-4-Benzyloxy-6-(fe/t-butyl-diphenyl-silanyloxy)-2-((/?)-2,2-dimethyl-[1,3 ]dioxolan-4-yl)-3,5-dimethyl-hexanal 226

OBn

O H C ^ ' Y " "Y "OTBDPS Me Me

l-mlfc-127

To a cooled (-78°C), stirred, solution of nitrile 234 (70 mg, 0.12 mmol) in CHgClg (1.0 ml, 0.1 M), was added DIBAL (1.5 M in PhMe, 0.085 ml, 0.12 mmol, 1 eq) via syringe over 1 min, and the reaction mixture stirred for 1 h. (TLC mobile phase: hexane/ethyl acetate, 5:1). The reaction mixture was then diluted with CHgClg (2.3ml) and then slowly quenched at 0 °C with 10% aq.

Rochelle’s salt (1.7 ml). The resulting mixture was left to stir for a further 0.5 h. The aqueous phase was extracted with CHgClg (1X5 ml) and then with diethyl ether (2X3 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (10:1) as eluent furnished aldehyde 226 (21.2 mg, 30%) as a colourless oil.

116 Chapter 4: Experimental

(4S,5S)-5-[(/?)-2-(fe/t-Butyl-dlphenyl-silanyloxy)-1-methyl-ethyl]-4-methyl-dihydro-furan-2- one 236

OTBDPS Me Me

l-mlfc-138

To a stirred solution of the benzyl ether 216 (44.5 g, 8 8 . 2 mmol) in methanol (220 ml, 0.4 M), was added 20% palladium hydroxide on carbon (21.3 g, 17.7 mmol, 0.2 eq) at RT under Ng. The flask was evacuated and purged several times with Hg gas, and the black slurry stirred

vigorously for 6 d. (TLC mobile phase: hexane/ethyl acetate, 2:1). The reaction mixture was then filtered through a CELITE™ pad and the filtered catalyst washed thoroughly with copious amounts of methanol. The filtrate was then concentrated In vacuo. Purification of the residue by

flash column chromatography (SiOg) with hexanes/ethyl acetate ( 2 0 :1 ) as eluent provided lactone 236 (29.0 g, 83%) as a thick yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.63 (m, 4H), 7.37 (m, 6 H), 4.11 (t, J = 6.5 Hz, 1 H), 3.68 (m,

2H), 2.64 (dd, J = 8 .8 , 17.6 Hz, 1H), 2.46 (m, 1 H), 2.12 (dd, J = 8.9, 16.8 Hz, 1 H), 1.96 (sept, J =

6 . 8 Hz, 1H), 1.14 (d, J = 6.7 Hz, 3H), 1.04 (s, 9H), 0.97 (d, J = 6.9 Hz, 3H) ppm.

IR (neat film): 2962 (m), 2361 (w), 1778 (s), 1589 (w), 1467 (m), 1426 (m), 1386 (m), 1324 (w),

1263 (w), 1212 (m), 1172 (m), 1110 (s), 1003 (m), 702 (s) cm \

HRMS: (FAB, MNOBA matrix) for Cg^HagOa'^iNa (M+Na)+, calcd: 419.2000, found 419.2018.

'^C-NMR (125 MHz, in CDCia): Ô 176.8, 135.9, 133.9, 133.8, 130.0, 128.1, 8 8 .8 , 65.4, 39.9,

37.6, 32.7, 27.3, 19.8, 19.7, 13.5 ppm.

117 Chapter 4: Experimental

[aro: +15.5 (d.ll.CHgCy.

118 Chapter 4: Experimental

(3S,4S,5S)-5-[(/?)-2-(ferf-Butyl-diphenyl-silanyloxy)-1-methyl-ethyl]-3-((Z)-3-lodo-2-methyl- allyl)-4-methyl-dihydro-furan-2-one 237

OTBDPS Me Me

ll-mlfc-20

To a cooled (-78°C) solution of diisopropylamine (4.8 ml, 34.1 mmol, 2 eq) in THF (15 ml, 2.2 M),

was added n-butyllithium ( 1 . 6 M in hexanes, 21 ml, 34.1 mmol, 2 eq) dropwise over 15 min. The milky white solution was left to stir for 0.5 h, whereupon a solution of the lactone 236 (6.77 g,

17.1 mmol, 1 eq) in THF (80 ml, 0.2 M) was added dropwise via syringe over 12 min. To the resulting transparent red solution was then added HMPA (14.8 ml, 85.4 mmol, 5 eq). The

resulting deep orange/red solution was then left to stir for 1 h, whereafter the bromide ( 6 . 6 8 g,

25.6 mmol, 1.5 eq) in THF (25 ml, 1.0 M) was added dropwise via syringe over 25 min. The resulting dark brown mixture was then stirred at -78 °C for a further 3.5 h. (TLC mobile phase: hexane/ethyl acetate, 5:1 ). The reaction mixture was then quenched with ether and water at -78

°C. The aqueous phase was extracted with diethyl ether (3 X 100 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. The crude residue was subjected to flash column chromatography (SiOg) with hexanes/ethyl acetate (30:1) as eluent to obtain the alkylated lactone 237 (6.9 g, 71%) as a thick yellow oil.

’H-NMR (500 MHz, in CDCI3): Ô 7.62 (m, 4H), 7.37 (m, 6 H), 5.81 (s, 1 H), 4.02 (dd, J = 6.0, 8 . 8

Hz, 1H), 3.67 (m, 2H), 2.62 (m, 2H), 2.44 (m, 1H), 2.21 (m, 1H), 2.01 (m, 1H), 1.89 (s, 3H), 1.14

(d, J = 6.5 Hz, 3H), 1.04 (s, 9H), 1 .00 (d, J = 7.0 Hz, 3H) ppm.

IR (neat film): 3069 (m), 2930 (s), 1770 (s), 1588 (w), 1470 (m), 1427 (s), 1384 (m), 1265 (m),

1187 (m), 1111 (s), 1006 (m) cm \

119 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for CgsHgylOg^^SINa (M+Na)"", calcd: 599.1432, found 599.1454.

'®C-NMR (125 MHz, in CDCy: Ô 177.8, 144.4, 135.6, 133.5, 133.4, 129.7, 127.7, 8 6 .1 , 64.8,

46.3, 39.4, 38.4, 38.2, 26.9, 23.7, 19.3, 18.2, 13.3 ppm.

[a ]% : +5.0 (c 0.4, CHgCy.

120 Chapter 4: Experimental

(3S,4S,5S)-5-((R)-2-Hydroxy-1-methyl-ethyI)-3-((Z)-3-iodo-2-methyl-allyl)-4-methyl-dihydro- furan-2-one 243

Me Me

lll-mlfc-27

To a stirred solution of the alkylated lactone 237 (6.46 g, 11.2 mmol) In a 2:1 mixture of THF

(62.7 ml) and CH 3 CN (30 ml), was added 40% HP (30 ml) dropwise over 5 min, using a plastic syringe. The resulting mixture was then left to stir vigorously for 24 h at RT. (TLC mobile phase: hexane/ethyl acetate, 2:1). Solid NaHCOg (XS) was then added to the reaction mixture in small

portions over 10 min followed by a saturated solution of aq. NaHCOg (190 ml) slowly over 2 0 min. After stirring at room temperature for 20 min, the aqueous phase was extracted with ethyl acetate (3 X 150 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude reaction mixture by flash column

chromatography (SiOg) with hexanes/ethyl acetate (5:1-> 2 :1 ) as eluent furnished the alcohol 243

(3.2 g, 85%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 6.07 (s, IN), 3.93 (dd, J = 7.1, 8.3 Hz, 1 H), 3.66 (m, 2H), 2.63

(d, J = 7.0 Hz, 2H), 2.44 (m, 1H), 2.22 (m, 1 H), 1.95 (m, 1 H), 1.91 (s, 3H), 1.16 (d, J = 6.5 Hz,

3H), 1.03 (d, J = 7.0 Hz, 3H) ppm.

IR (neat film): 3437 (br), 3057 (w), 2966 (m), 2935 (m), 1767 (s), 1613 (w), 1442 (m), 1384 (m),

1327 (m), 1267 (m), 1190 (m), 1038 (m), 1002 (m), 950 (w), 829 (w), 771 (w), 728 (w) cm \

HRMS: (FAB, MNOBA matrix) for C 1 2 H 2 0 IO 3 (M + H )\ calcd: 339.0458, found 339.0457.

121 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): 5 177.6, 144.4, 144.2, 87.3, 86.9, 77.8, 64.6, 64.5, 46.3, 39.8,

39.4, 38.4, 23.5, 18.4, 13.5 ppm.

[a ]% :- 1 1 . 2 (c 0 .1 ,CH 2C y.

122 Chapter 4: Experimental

(3S,4S,5S)-3-((Z)-3-lodo-2-methyl-allyl)-5-[(/?)-2-(4-methoxy-benzyloxy)-1-methyl-ethyl]-4- methyl-dihydro-furan-2-one 244

OPMB Me Me

lll-mlfc-28

To a stirred solution of the alcohol 243 (2.91 g, 8 . 6 mmol) in CHgClg (50 ml, 0.17 M), was added a solution of PMB-trichloroacetimidate (2.67 g, 9.5 mmol, 1.1 eq) via syringe at RT. Pyridinium para-toluene sulfonate (1.08 g, 4.3 mmol, 0.5 eq) was then added in a single portion, and the resulting mixture was allowed to stir vigorously overnight. (TLC mobile phase: hexane/ethyl acetate, 2:1). The reaction mixture was then quenched by the addition of aqueous NaHCOg, and the aqueous phase was extracted with CHgClg (3 X 50 ml). The combined organic extracts were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude residue by

flash column chromatography (SiOg) with hexanes/ethyl acetate ( 2 0 :1 ) as eluent afforded the protected alcohol 244 (3.8 g, 97%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.21 (d, J = 8 . 6 Hz, 2H), 6.85 (d, J = 8 . 6 Hz, 2H), 5.80 (s, 1H),

4.37 (s, 2H), 3.95 (dd, J = 5.5, 8 . 8 Hz, 1 H), 3.78 (s, 3H), 3.49 (dd, J = 5.9, 9.3 Hz, 1 H), 3.39 (dd,

J = 5.7, 9.3 Hz, 1 H), 2.62 (m, 2H), 2.42 ( m, 1H), 2.27 (m, 1 H), 2.09 (m, 1 H), 1.89 (s, 3H), 1.12

(d, J = 6.5 Hz, 3H), 1.03 (d, J = 7.0 Hz, 3H) ppm.

IR (neat film): 3061 (w), 2964 (m), 1768 (s), 1612 (m), 1586 (w), 1513 (s), 1459 (m), 1382 (m),

1359 (m), 1322 (m), 1302 (m), 1247 (s), 1177 (s), 1093 (m), 1034 (m), 1006 (m), 980 (m), 953

(w), 821 (m), 770 (w), 678 (w), 651 (w) cm \

HRMS: (FAB, MNOBA matrix) for CgoHgylO^Na (M+Na)\ calcd: 481.0841, found 481.0852.

123 Chapter 4: Experimental

'"C-NMR (125 MHz, in CDCI3): Ô 177.8, 159.2, 144.4, 130.3, 129.2, 113.8, 86.5, 72.8, 71.1,

55.3, 46.2, 38.6, 38.3, 37.1, 23.7, 18.1, 14.0 ppm.

[a ro :+ 5 .9 (c 0.4, CHgCy.

124 Chapter 4: Experimental

(Z)-(2/?,3S,4S,5S)-5-Hydroxymethyl-8-lodo-1-(4-methoxy-benzyIoxy)-2,4,7-trimethyl-oct-7- en-3-ol 245

OPMB

ll-mlfc-25

To a stirred solution of lactone 244 (3.4 g, 7.4 mmol) in a 100:1 mixture of THF (70 ml) and methanol (0.7 ml) at RT was added lithium borohydride (1.62 g, 74.2 mmol, 10 eq) in a single

portion. The resulting mixture was heated at reflux for 1 .5 h. (TLC mobile phase: hexane/ethyl acetate, 2:1). The reaction mixture was then diluted with ethyl acetate (30 ml) and cooled to 0

°C, whereupon HgO (100 ml) was then added with caution. The aqueous phase was extracted

with ethyl acetate (3 X 1 0 0 ml). The combined organic extracts were dried (IVIgSOJ, filtered and concentrated in vacuo. Purification of the residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (5:1 ^2 :1 ) as eluent provided the diol 245 (2.8 g, 82%) as a colourless amorphous solid.

H-NMR (500 MHz, In CDCI3 ): 6 7.18 (m, 2H), 6.84 (m, 2H), 5.86 (s, 1H), 4.38 (s, 2H), 3.78 (s,

3H), 3.69 (dd, J = 3.7, 9.2 Hz, 1H), 3.64 (m, 1H), 3.50 (m, 1H), 3.42 (dd, J = 4.7, 9.2 Hz, 1H),

3.37 (m, 2H), 2.30 (m, 1 H), 2.23 (m, 2H), 1.95 (m, 1 H), 1.89 (s, 3H), 1.07 (d, J = 7.1 Hz, 3H),

0.87 (d, J = 7.0 Hz, 3H) ppm.

IR (neat film): 3276 (br), 2931 (m), 2878 (m), 2373 (w), 2343 (w), 1613 (m), 1584 (w), 1513 (s),

1458 (m), 1365 (m), 1338 (m), 1301 (m), 1252 (s), 1172 (m), 1090 (s) cm \

HRMS: (FAB, MNOBA matrix) for C 2 0 H3 2 IO4 (M+H)\ calcd: 463.1331, found 463.1345.

125 Chapter 4: Experimental

’^C-NMR (125 MHz, in CDCI3): Ô 159.3, 147.4, 129.7, 129.3, 113.9, 79.1, 75.3, 73.4, 73.0, 63.8,

65.2, 40.4, 36.8, 36.6, 34.7, 23.5, 15.7, 12.9 ppm.

[ a f o: +15.2 (c 0.5, CHgCy.

M.pt: 94-96°C

126 Chapter 4: Experimental

(Z)-(2/?,3S,4S,5S)-5-(fe/t-Butyl-dimethyl-silanyloxymethyl)-8-iodo-1-(4-methoxy-benzyl oxy)-2,4,7-trimethyl-oct-7-en-3-ol 246

OPMB

ll-mlfc-26

To a cooled (0 °C), stirred, solution of the diol 245 (2.52 g, 5.6 mmol) in dry DMF (50 ml, 0.1 M),

was added imidazole ( 0 . 8 g, 1 1 . 8 mmol, 2 . 1 eq) in a single portion. TBSCI (0.932 g, 6.2 mmol,

1 . 1 eq) was then added in a single portion, and the reaction mixture continued to stir for a further

2 h. (TLC mobile phase: hexane/ethyl acetate, 2 :1 ). The reaction mixture was then quenched with the careful addition of a saturated solution of NaHCOg. The aqueous phase was extracted with diethyl ether (3 X 50 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (20:1) gave the protected alcohol 246 (3.2 g, 98%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3 ): 6 7.20 (m, 2H), 6 . 8 6 (m, 2H), 5.85 (s, 1H), 4.35 (dd, J = 11.5,

46.5 Hz, 2H), 3.78 (s, 3H), 3.64 (dd, J = 3.9, 9.1 Hz, 1H), 3.51 (dd, J = 4.4, 10.2 Hz, 1H), 3.45

(dd, J = 3.9, 9.2 Hz, 1H), 3.37 (m, 1H), 3.24 (m, 1H), 2.28 (m, 1H), 2.15 (dd, J = 11.2, 13.9 Hz,

1H), 2.08 (td, J = 1. 8 , 7.2 Hz, 1 H), 1.97 (m, 1 H), 1.90 (d, J = 5.8 Hz, 3H), 1.03 (d, J = 7.0 Hz,

3H), 0.86 (s, 9H), 0.85 (d, J = 6.9 Hz, 3H), 0.021 (s, 3H), 0.014 (s, 3H) ppm.

IR (neat film ): 3499 (m), 2856 (s), 1613 (m), 1514 (s), 1464 (m), 1360 (w), 1302 (w), 1251 (s),

1174 (m), 1092 (s), 834 (s), 777 (m) cm'L

HRMS: (FAB, MNOBA matrix) for CgeH^glO/^Si (M+H)+, calcd: 577.2218, found 577.2210.

127 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3): 5 159.2, 147.4, 129.9, 129.3, 113.9, 79.3, 75.1, 73.8, 73.2, 63.6,

55.2, 38.1,35.8, 35.1,34.9, 26.1,23.8, 18.3, 15.3, 11.6, -5.6, -5.7 ppm.

[a fo : +16.9 (c 0.4, CHgCy.

128 Chapter 4: Experimental

[(2S,3S,4S,5/?)-4-Allyloxy-2-((Z)-3-iodo-2-methyl-allyl)-6-(4-methoxy-benzyloxy)-3,5- dimethyl-hexyloxy]-fe/t-butyl-dimethyl-silane 247

OPMB

ll-mlfc-27

To a cooled (0 °C), stirred solution of the secondary alcohol 246 (1.0 g, 1.7 mmol) in dry DMF

(17 ml, 0.1 M), was added sodium hydride (60% dispersion in oil, 0.41 g, 17.3 mmol, 10 eq) in small portions over 5 min. The grey suspension was left to stir for 0.5 h whereupon allylbromide

(2 . 1 ml, 17.3 mmol, 10 eq) was added dropwise over 5 min. The resulting mixture was stirred for a further 5 min at 0 °C, then allowed to warm to RT whereafter stirring was continued overnight.

(TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then re-cooled to 0

°G, and diluted with diethyl ether (10 ml), followed by the slow addition of methanol (20 ml) to destroy the surplus active sodium hydride. A saturated solution of NaHCOg was then added, and the aqueous phase was extracted with diethyl ether (3 X 30 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (50:1) as eluent gave the allyl ether 247 (0.79 g, 75%) as a yellow oil.

’H-NMR (500 MHz, in CDCI3 ): Ô 7.24 (m, 2H), 6.85 (m, 2H), 5.83 (m, 2H), 5.18 (ddd, J = 1.8,

3.7, 17.2 Hz, 1 H), 5.06 (ddd, J = 1.5, 3.4, 10.5 Hz, 1H), 4.51 (s, 2H), 4.00 (dt, 1.6, 3.5 Hz,

2H), 3.78 (s, 3H), 3.52 (dd, J = 3.7, 8.9 Hz, 1 H), 3.44 (m, 2H), 3.37 (t, J = 10.1 Hz, 1H), 3.18 (dd,

J = 4.9, 7.2 Hz, 1H), 2.33 (dd, J = 2.6, 13.3 Hz, 1 H), 2.16 (m, 2H), 2.10 (m, 1 H), 2.05 (m, 1 H),

1.87 (s, 3H), 1.01 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 7.1 Hz, 3H), 0.85 (s, 9H), 0.009 (s, 3H), 0.006

(s, 3H) ppm.

129 Chapter 4: Experimental

IR (neat film): 2856 (s), 1613 (m), 1513 (s), 1463 (m), 1360 (w), 1302 (w), 1250 (s). 1173 (w),

1088 (s), 1005 (w), 921 (w), 834 (s), 776 (m), 675 (w) cm '.

HRMS: (FAB, MNOBA matrix) for CggHgolO/^Si (M+H)+, calcd: 617.2524, found 617.2523.

'^C-NMR (125 MHz, In CDCI3): 5 159.1, 147.3, 135.4, 130.9, 129.1, 115.3, 113.7, 86.0, 75.3,

74.4, 72.7, 72.4, 63.6, 55.3, 37.6, 37.1,36.0, 33.1, 26.0, 23.6, 18.3, 15.1, 12.1, -5.4, -5.6 ppm.

[a]%:+33 (cO.4, CHgCy.

130 Chapter 4: Experimental

{(Z)-(S)-2-[(1S,2S,3R)-2-Allyloxy-4-(4-methoxy-benzyloxy)-1,3-dimethyl-butyl]-4-methyl octa-4,7-dienyloxy}-tert-butyl-dimethyl-silane 248

Me^ / tbso ^ A ,

Me^ Me OPMB

ll-mlfc-38

To a stirred solution of the vinyl iodide 247 (0.8 g, 1.3 mmol) in dry DMF (20 ml, 0.06 M), was

added (Ph 3 P)4 Pd (0.15 g, 0.13 mmol, 0.1 eq) in a single portion, followed by allyl tri-n-butyltin

(0.48 ml, 1 . 6 mmol, 1. 2 eq) via syringe over 3 min, at RT. The resulting mixture was then heated

(100 °C - 1 10 °C) for 5 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then quenched with NaHCOg, and the aqueous phase was extracted with diethyl ether (3 X

25 ml). The combined organic phases were dried (MgSO^), filtered and concentrated in vacuo.

Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (100:1) as eluent revealed the product 248 (0.43 g, 63%) as a clear oil.

'H-NMR (500 MHz, in C D C y: Ô 7.24 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.88-5.82 (m,

1H), 5.82-5.75 (m, 1H), 5.20 (dd, J = 1.9, 17.2 Hz, 1H), 5.16 (t, J = 8.7 Hz, 1H), 5.04 (dd, J = 1. 8 ,

10.5 Hz, 1H), 4.98 (dd, J = 1.9, 18.9 Hz, 1H), 4.93 (dd, J = 1.5, 11.6 Hz, 1H), 4.40 (s, 2H), 4.01

(dd, J = 1.5, 5.1 Hz, 2H), 3.78 (s, 3H), 3.52 (dd, J = 3.6, 8 . 8 Hz, 1H), 3.44 (d, J = 8.7 Hz, 1H),

3.42 (d, J = 3.5 Hz, 1 H), 3.27 (t, J = 8.9 Hz, 1 H), 3.17 (q, J = 5.4 Hz, 1H), 2.56 (m, 2H), 1.98 (m,

2H), 2.00 (m, 3H), 1.69 (s, 3H), 1 .01 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 7.1 Hz, 3H), 0.84 (s, 9H), -

0.01 (s, 6 H) ppm.

131 Chapter 4: Experimental

^®C-NMR (125 MHz, in CDCI3 ): 5 159.0, 137.6, 135.8, 135.6, 130.9, 129.2, 123.3, 115.1, 114.2,

113.7, 86.0, 74.1, 72.7, 72.4, 63.6, 55.3, 37.2, 36.8, 33.4, 32.4, 28.5, 26.0, 23.8, 18.3, 15.3, 12.1,

-5.4, -5.5 ppm.

132 Chapter 4: Experimental

fe/t-Butyl-{(6Z,9Z)-(2S,3S,4S)-2-[(/?)-2-(4-methoxy-benzyloxy)-1-methyl-ethyl]-3,6- dimethyl-oxacycloundeca-6,9-dien-4-ylmethoxy}-dimethyl-silane 249

TBSO

Me Me' OPMB

ll-mlfc-51

To a stirred solution of the aikene 248 (50 mg, 0.09 mmol) in toluene (50 ml, 0.002 M), was added bis(tricyclohexylphosphine)benzylidene ruthenium(IV)dichloride (Grubbs catalyst) (7.7 mg, 0.009 mmol, 0.1 eq) in a single portion at RT. The resulting mixture was heated at reflux for

1 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The solvent was removed under reduced pressure, and the crude product was purified using flash column chromatography (SiOg) with hexanes/ethyl acetate (100:1) as eiuent to provide 249 (1.81 mg, 4%) as a clear oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.24 (d, J = 8.2 Hz, 2H), 6.84 (d, J = 11.4 Hz, 2H), 5.60 (ddd, J

= 4.2, 10.8, 21.7 Hz, 1H), 5.56-5.51 (m, 1H), 5.32 (t, J = 8.4 Hz, 1H), 4.40 (dd, J = 11.5, 21.5 Hz,

2H), 4.20 (dd, J = 5.8, 11 . 6 Hz, 1H), 4.06 (t, J = 9.5 Hz, 1H), 3.78 (s, 3H), 3.57 (m, 2H), 3.48 (dd,

J = 7.7, 10.1 Hz, 1 H), 3.28 (t, J = 8.9 Hz, 1H), 3.22 (dd, J = 2.4, 9.5 Hz, 1 H), 3.12 (q, J= 10.5

Hz, 1 H), 2.39 (m, 2H), 2.10 (m, 2H), 1.89 (m, 1H), 1.74 (s, 3H), 1.41 (t, J = 12.7 Hz, 1H), 1.11 (d,

J = 6.9 Hz, 3H), 0.85 (s, 9H), 0.77 (d, J = 7.0 Hz, 3H), -0.002 (s, 6 H) ppm.

'^C-NMR (125 MHz, In CDCI3): Ô 159.0, 138.5, 133.9, 131.0, 129.1, 124.0, 122.0, 113.7, 90.2,

77.6, 72.8, 71.4, 69.6, 65.6, 55.2, 40.2, 36.4, 36.3, 27.7, 27.0, 26.8, 26.0, 18.3, 16.8, 11.8, -5.3, -

5.5 ppm.

133 Chapter 4: Experimental

Tributyl-[(E)-2-((S)-2,2-dimethyl-[1,3]dioxolan-4-yl)-vinyl]-stannane 269

ll-mlfc-101

To a stirred solution of the alkyne^^^ 268 (800 mg, 6.3 mmol) In toluene (21 ml, 0.3 M), was added tributyltin hydride (2.1 ml, 7.6 mmol, 1.2 eq) In a slow stream over 2 min. The reaction mixture was brought to reflux whereupon AIBN was added In small portions over 10 min. The reactants were heated at reflux for a further 40 min. (TLC mobile phase: hexane/ethyl acetate,

10:1). The reaction mixture was allowed to cool to RT and the solvent was removed under reduced pressure. Purification of the crude product by flash column chromatography (SlOg) with pure hexanes->hexanes/dlethyl ether (100:1) as eluent gave the vinyl stannane 269 (1.8 g,

72%) as a colourless oil.

'H-NMR (500 MHz, in CDCI3): Ô 6.28 (dd, J = 0.9, 18.9 Hz, 1H), 5.92 (dd, J = 6.7, 18.9 Hz, 1H),

4.45 (m, 1 H), 4.07 (dd, J = 6.2, 8.1 Hz, 1H), 3.58 (t, J = 7.9 Hz, 1 H), 1 .49-1.43 (m, 6 H), 1.42 (s,

3H), 1.37 (s, 3H), 1.32-1.24 (m, 6 H), 0.87-0.84 (m, 15H) ppm.

IR (neat film): 2925 (s), 1459 (m), 1374 (m), 1219 (m), 1155 (w), 1062 (s), 990 (w), 863 (m) cm'

'"C-NMR (125 MHz, in CDCig): Ô 145.2, 133.3, 109.3, 80.0, 69.3, 29.0, 27.2, 26.7, 25.9, 13.7,

9.4 ppm.

134 Chapter 4: Experimental

(£)-(S)-4>tributylstannanyl-but-3-ene-1,2-diol 270

ll-mlfc-113

To a stirred solution of vinyi stannane 269 (3.0 g, 7.2 mmoi) in methanol (24 ml, 0.3 M) was added pyridinium para-toluenesulfonate (2.7 g, 10.8 mmol, 1.5 eq) in a single portion at RT. The resulting mixture was stirred vigorously overnight. (TLC mobile phase: hexane/ethyl acetate,

2 :1 ). The solvent was removed under reduced pressure, and the crude product purified by flash

column chromatography (SiOg) with hexanes/ethyi acetate (5:1 ^ 2 :1 ) as eluent to provide 269

(0.79 g, 30%) and dioi 270 (1.3 g, 50%) as a yeiiow oil. (Percentage yield: 50%).

'H-NMR (500 MHz, in CDCI3): Ô 6.29 (dd, v/= 1.4, 19.2 Hz, IN), 5.95 (dd, J = 5.1, 19.2 Hz, 1H),

4.21 (m, 1 H), 3.68-3.64 (m, 1H), 3.50-3.47 (m, 1 H), 2.07 (d, J = 4.4 Hz, 1 H), 1.84 (q, J = 4.8 Hz,

1 H), 1.49-1.45 (m, 6 H), 1.31-1.27 (m, 6 H), 0.89-0.86 (m, 15H) ppm.

IR (neat film): 3379 (br), 2924 (s), 1460 (m), 1074 (m), 1027 (m), 874 (m), 663 (w) cm L

HRMS: (FAB, MNOBA matrix) for CisHs^OgSnNa (M+Na)\ caicd: 401.1490, found 401.1478.

'^C-NMR (125 MHz, In CDCI3): Ô 146.1, 131.1, 75.3, 66.1, 29.2, 26.9, 13.8, 9.3 ppm.

135 Chapter 4: Experimental

(E)-(S)-1-(ferf-Butyl-dimethyl-sllanyloxy)-4-tributylstannanyl-but-3-en-2-ol 271

ll-mlfc-115

To a cooled (0 °C) solution of the diol 270 (600 mg, 1 . 6 mmol) in CHgClg ( 6 ml, 0.2 M), was added imidazole (217 mg, 3.2 mmol, 2 eq) in a single portion. A solution of TBSCI (0.24 g, 1.6 mmol, 1 eq) in CHgClg (53 ml, 0.03 M) was then added dropwise via syringe over 4 min, and the resulting mixture stirred for 10 min at 0 °C. (TLC mobile phase: hexane/ethyl acetate, 3:1). The

reaction mixture was then quenched with a saturated solution of aq. NaHCOg ( 1 0 ml), and the aqueous phase was extracted with CHgClg (3X10 ml). The combined organic phases were dried (IVIgSOJ, filtered, and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/diethyl ether (50:1) as eluent procured the protected alcohol 271 (0.5 g, 70%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 6.27 (dd, J = 1.4, 19.2 Hz, 1H), 5.95 (dd, J = 5.0, 19.1 Hz, 1 H),

4.13-4.11 (m, 1 H), 3.65 (dd, J = 3.7, 9.9 Hz, 1H), 3.43 (dd, J = 7.4, 9.9 Hz, 1 H), 2.51 (d, J = 3.7

Hz, 1 H), 1.51 -1.45 (m, 6 H), 1.32-1.25 (m, 6 H), 0.86-0.84 (m, 21H), 0.059 (s, 9H) ppm.

IR (neat film): 3420 (br), 2928 (s), 1604 (w), 1461 (m), 1379 (w), 1255 (m), 1105 (s) cm \

'®C-NMR (125 MHz, in CDCig): Ô 146.3, 130.3, 75.2, 67.0, 29.0, 27.3, 25.9, 13.7, 9.4, 1.0, -5.4 ppm.

136 Chapter 4: Experimental

(E)-1-(terf-Butyl-dimethyl-silanyloxy)-4-tributylstannanyl-but-3-en-2-one253

ll-mlfc-116

To a stirred solution of the secondary alcohol 271 (200 mg, 0.4 mmol) and 4Â molecular sieves

(200 mg) in CHgClg (1.4 ml, 0.3 M), was added NMO (110 mg, 0.9 mmol, 2 . 2 eq) in a single portion followed by TRAP (7.2 mg, 0.02 mmol, 0.05 eq) also in a single portion. The resulting

mixture was stirred at RT for 1 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was then filtered and the filtrate and washings concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/diethyl ether (50:1) as eluent gave the ketone 253 (147 mg, 75%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.73 (d, J = 19.6 Hz, 1 H), 6.84 (d, J = 19.6 Hz, 1H), 4.35 (s,

2H), 1.51-1.41 (m, 6 H), 1.30-1.21 (m, 6 H), 0.96-0.85 (m, 21H), 0.07 (s, 9H) ppm.

IR (neat film): 2933 (s), 1696 (m), 1565 (m), 1464 (m), 1436 (m), 1255 (m), 1109 (s), 839 (s) c m \

HRMS: (FAB, MNOBA matrix) for CggH^yOg^'SiSn (M+H)+, calcd: 491.2380, found 491.2367.

'"C-NMR (125 MHz, in CDCig): Ô 195.9, 152.3, 140.0, 67.8, 28.4, 26.7, 25.2, 17.8, 13.1,9.1, -5.4 ppm.

137 Chapter 4: Experimental

(Z)-(2/?,3S,4S,5S)-8-lodo-1-(4-methoxy-benzyloxy)-2,4,7-trimethyl-5-triethylsilanyloxy methyl-oct-7-en-3-ol 258

Me^ /

T E S O . ^

Me^ Me OPMB

lll-mlfc-30

To a cooled (0°C) solution of diol 245 (12.1 g, 26.2 mmol), in dry DMF (130 ml, 0.2 M), was added imidazole (3.9 g, 57.6 mmol, 2.2 eq) in a single portion. A solution of TESCI (5.25 ml,

31.4 mmol, 1.2 eq) was then added dropwise via syringe over 20 min, and the resulting mixture was stirred at 0 °C for 1.5 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was quenched by the careful addition of a saturated solution of aq. NaHCOg (150 ml).

The aqueous phase was extracted with diethyl ether (3 X 50 ml). The combined organic phases were dried (MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (30:1->20:1) provided alcohol

258 (12.4 g, 82%) as a clear oil.

'H-NMR (500 MHz, in CDCI3): Ô 7.20 (m, 2H), 6 . 8 6 (m, 2H), 5.83 (s, 1 H), 4.43 (t, J = 11.4 Hz,

1H), 4.36 (d, J = 11.4 Hz, 1H), 3.78 (s, 3H), 3.65 (dd, J = 3.9, 9.2 Hz, 1 H), 3.52 (dd, J = 4.3, 10.3

Hz, 1H), 3.47 (m, 1H), 3.36 (m, 1H), 3.28 (m, 1H), 2.26 (m, 2H), 2.14 (dd, J = 11.3, 13.9 Hz, 1H),

2.10 (dt, J = 1.7, 7.2, 8.9 Hz, 1H), 1.99 (m, 1H), 1.90 (s, 3H), 1.03 (d, J = 7.0 Hz, 3H), 0.92 (t, J =

7.9 Hz, 9H), 0.86 (d, J = 7.0 Hz, 3H), 0.55 (q, J = 7.9 Hz, 6 H) ppm.

IR (neat film ): 3494 (br), 2956 (s), 2362 (m), 1613 (m), 1514 (s), 1460 (m), 1417 (m), 1380 (m),

1301 (w), 1249 (s), 1173 (m), 1092 (s) cm \

138 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for (M+H)\ calcd: 577.2196, found 577.2210.

'"C-NMR (125 MHz, in CDCij): 5 159.2, 147.4, 130.0, 129.2, 113.8, 79.4, 75.1, 73.9, 73.2, 55.3,

38.2, 36.6, 35.1, 34.8, 23.5, 15.2, 11.6, 6 .8 , 4.4 ppm.

i2 5 . [a ]% :+ 2 1 . 8 (c 0.4, CHgOy.

139 Chapter 4: Experimental

1-[(Z)-(2/?,3S,4S,5S)-3-(fert-Butyl-dimethyl-silanyloxy)-8-iodo-2,4,7-trimethyl-5-triethyl silanyioxymethyl-oct-7-enyloxymethyl]-4-methoxy-benzene 259

Me^ /

T E S O ^ OTBS

Me^ Me^ OPMB

lll-mlfc-33

To a stirred solution of the secondary alcohol 258 (12.5 g, 21.7 mmol) in CHgClg (216 ml, 0.1 M), was added 2,6-lutidine (50.4 ml, 216mmol, 20eq.) via syringe over 12 mins. The reaction mixture was then cooled to -50 °C, whereupon a dropwise solution of TBSOTf (14.9 ml, 65 mmol, 3 eq) was added over 20 min. The resulting mixture was stirred at -50 ®C for a further 0.5 h (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was quenched by the

careful addition of a saturated solution of aq. NaHCOa ( 2 0 0 ml), and the resulting mixture was stirred for 5 min. The aqueous phase was extracted with CHgClg (3 X 100 ml). The combined organic phases were dried (IVIgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (100:1) gave the disilyl ether 259 (13.4 g, 90%) as a yellow oil.

H-NIVIR (500 MHz, in CDCI3): Ô 7.22 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 5.84 (s, 1H),

4.39 (dd, J = 1 1 . 1 Hz, 2H), 3.78 (s, 3H), 3.62 (dd, J = 3.1, 6.2 Hz, 1H), 3.57 (dd, J = 4.2, 8.9 Hz,

1H), 3.43 (dd, J = 3.9, 10.1 Hz, 1 H), 3.34 (t, J = 7.8 Hz, 1 H), 3.26 (t, J = 8.0 Hz, 1H), 2.37 (d, J =

12.4 Hz, 1H), 2.17 (t, J = 11.8 Hz, 1H), 2.05 (m, 3H), 1.87 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.91

(t, J = 7.9 Hz, 9H), 0.90 (d, J = 7.0 Hz, 3H), 0.85 (s, 9H), 0.54 (q, J = 7.9 Hz, 6 H), 0.05 (s. 3H),

0.02 (s, 3H) ppm.

140 Chapter 4: Experimental

IR (neat film): 2955 (s), 2361 (w), 1727 (w), 1613 (m), 1587 (w), 1513 (m), 1463 (m), 1362 (w),

1301 (w), 1250 (s), 1172 (w), 1085 (s), 1037 (m), 1006 (m), 835 (m) cm '.

HRMS: (FAB, MNOBA matrix) for (M+Na)\ calcd: 713.28940, found

713.28834.

'"C-NMR (125 MHz, In CDCI3 ): Ô 159.1, 147.1, 130.9, 129.1, 113.7, 79.0, 75.5, 73.0, 72.7, 63.0,

55.3, 38.3, 38.2, 36.7, 34.4, 26.3, 23.6, 18.4, 14.9, 12.7, 6.9, 4.4, -3.3, -4.4 ppm.

[a]%:+28.5 (cO.2, CHgCy.

141 Chapter 4: Experimental

(2S,3S,4S,5/?)-4-(ferf-Butyl-dimethyl-silanyloxy)-2-((Z)-3-iodo-2-methyl-allyl)-6-(4-methoxy- benzyloxy)-3,5-dimethyl-hexan-1 -ol 260

Me^

/ \ | OTBS

Me^ Me ^OPMB

lll-mlfc-35

To a stirred solution of the protected diol 259 (12.3 g, 17.8 mmol) in a 1:1 mixture of THF (100 ml) and acetonitrile (100 ml), was added 2% aq. HP (48 ml, 2.7 eq) dropwise over 15 min. The reaction mixture was stirred vigorously at RT for 1.5 h. (TLC mobile phase: hexane/ethyl

acetate, 10:1), The reaction mixture was quenched by the addition of solid NaHCOg ( 2 g), followed by careful addition of a saturated solution of aq. NaHCOg (100 ml). The aqueous phase

was extracted with ethyl acetate (3 X 1 0 0 ml). The combined organic phases were dried

(MgSOJ, filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (30:1->10:1) gave the primary alcohol 260

(9.6 g, 94%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.22 (m, 2H), 6.84 (m, 2H), 5.90 (s, 1H), 4.39 (dd, J = 11.5,

23.5 Hz, 2H), 3.78 (s, 3H), 3.67 (dd, J = 3.1, 5.7 Hz, 1H), 3.58 (dd, J = 4.3, 9.1 Hz, 1H), 3.52

(dd, J = 3.5, 11.2 Hz, 1H), 3.41 (dd, J = 5.9, 11.2 Hz, 1 H), 3.26 (dd, J = 7.8, 9.0 Hz, 1H), 2.31

(m, 2H), 1.96 (m, 3H), 1.87 (d, J = 1.1 Hz, 3H), 1.03 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H),

0.87 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H) ppm.

IR (neat film): 3441 (br), 2955 (s), 2361 (w), 1739 (w), 1612 (m), 1586 (w), 1513 (s), 1464 (m),

1363 (m), 1301 (m), 1249 (s), 1174 (m), 1091 (m), 1038 (s), 834 (s), 773 (s), 677 (m) cm \

HRMS: (FAB, MNOBA matrix) for CgeH^gO/^ilNa (M+Na)\ calcd: 599.2041, found 599.2030.

142 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 159.1, 147.1, 130.8, 129.3, 113.7, 78.2, 75.7, 72.7, 63.3, 55.3,

38.8, 37.8, 37.5, 36.6, 26.2, 23.5, 18.4, 15.8, 13.6, -3.6, -4.4 ppm.

[a f\:+ 2 4 A (cO.2, CH^Cy.

143 Chapter 4: Experimental

(2S,3S,4S,5/?)-4-(te/t-Butyl-dinnethyl-silanyloxy)-2-((Z)-3-iodo-2-methyl-allyl)-6-(4-methoxy- benzyloxy)-3,5-dimethyl-hexanal 256

Me OPMB

lll-mlfc-37

To a stirred solution of the primary alcohol 260 (8 . 6 g, 14.9 mmol) and flame-dried 4Â powdered

molecular sieves (10 g) in CHgClg (700 ml, 0 . 0 2 M), was added NMO (3.51 g, 29.9 mmol, 2 eq) in a single portion, followed by TRAP (0.26 g, 0.7 mmol, 0.05 eq), and the resulting black slurry was stirred at RT for 40 min. (TLC mobile phase: hexane/ethyl acetate, 9:1). The reaction mixture was then filtered through a small pad of CELITE™, and the pad washed thoroughly with copious amounts of CHgClg. The filtrate and washings were concentrated in vacuo. Purification

of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate ( 1 0 :1 ) yielded the pure aldehyde 256 (7.8 g, 91%) as a yellow oil.

H-NMR (500 MHz, in CDCI3 ): Ô 9.60 (d, J = 2.7 Hz, 1 H), 7.23 (d, J = 8.3 Hz, 2H), 6.85 (d, J =

8.3 Hz, 2H), 4.38 (m, 2H), 3.78 (s, 3H), 3.56 (t, J = 4.7 Hz, 1 H), 3.52 (dd, J = 5.0, 9.2 Hz, 1 H),

3.23 (t, J = 8 . 8 Hz, 1 H), 2.64 (m, 2H), 2.42 (dd, J = 3.0, 13.2 Hz, 1 H), 2.18 (m, 1 H), 1.98 (m, 1 H),

1.83 (s, 3H), 0.98 (d, J = 7.0 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.86 (s, 9H), 0.048 (s, 3H), 0.031

(s, 3H) ppm.

IR (neat film): 2955 (s), 2708 (w), 1723 (s), 1612 (m), 1586 (w), 1513 (s), 1464 (m), 1383 (m),

1301 (m), 1249 (s), 1174 (m), 1091 (s), 1038 (s), 1006 (m), 836 (s), 774 (m) cm \

HRMS: (FAB, MNOBA matrix) for Cg^H^O/^SilNa (M+Na)+, calcd: 597.1845, found 597.1873.

144 Chapter 4: Experimental

'®C-NMR (125 MHz, in CDCI3 ): Ô 203.8, 159.1, 144.8, 130.7, 129.2, 113.7, 72.7, 72.2, 55.3,

50.9, 37.7, 37.0, 35.9, 26.1, 23.8, 18.3, 15.2, 13.7, -3.7, -4.3 ppm.

[ a f o : +19.0 (c 0.3, CH 2 CI2 ).

145 Chapter 4: Experimental

(4R,5S)-3-[(2/?,3S,4S,5S,6S,7R)-6-(fe/t-Butyl-dimethyl-silanyloxy)-3-hydroxy-4-((Z)-3-iodo- 2-methyl-allyl)-8-(4-methoxy-benzyloxy)-2,5,7-trimethyl-octanoyl]-4-methyl-5“phenyl-

oxazolidin- 2 -one 261

K \ OTBS HO. OPMB \ Me Me Me"'"' > 0 N

“TP h * ^ 0

lll-mlfc-45

To a cooled (0 °C) solution of the imide 257 (1.99 g, 8 . 6 mmol, 4 eq) in CHgClg (55 ml, 0.15 M),

was added n-BugBOTf (1.0 M in CHgClg, 8 . 6 ml, 8 . 6 mmol) dropwise via syringe over 10 min.

The resulting mixture was stirred at 0 °C for 10 min, whereafter the reaction mixture turned yellow. Dropwise addition of EtgN (1.25 ml, 9 mmol, 4.2 M) over 3 min turned the reaction

mixture clear again, and the reactants were maintained at 0 °C for 30 min. After cooling to -78

°C, whereupon the aldehyde 256 (1.23 g, 2 . 1 mmol, 1 eq) in CHgClg (50 ml, 0.04 M) was added dropwise over 40 min. The resulting mixture stirred at -78 °C for 35 min, and was then allowed to warm up to RT for a further 1 h. (TLC mobile phase: hexane/ethyl acetate, 5:1). It was then quenched by the addition of a pH 7 phosphate buffer solution (40 ml) and MeOH (40 ml). The

reaction mixture was then cooled to 0 °C, whereupon aq. HgOg (27.5 wt %, 25 ml) was added, and the resulting solution stirred at 0°C for 0.5 h. The aqueous phase was extracted with CHgClg

(3 X 50 ml). The combined organic phases were dried (IVIgSOJ, filtered and concentrated in

vacuo. Purification of the residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (20:1) gave an inseparable 3:1 mixture of the imide and the aldol adduct 261.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.32 (m, 3H), 7.27 (m, 2 H), 7.05 (m, 2H), 6.80 (m, 2H), 5.92 (s,

1 H), 5.58 (d, J = 7.2 Hz, 1 H), 4.67 (dq, J = 6 .6 , 6 . 8 Hz, 1 H), 4.39 (d, J = 2.1 Hz, 2 H), 3.87 (m.

146 Chapter 4: Experimental

1 H), 3.81 (dd, J = 7.1, 13.9 Hz, 1H), 3.73 (s, 3H), 3.66 (dd, J = 3.7, 8.7 Hz, 1 H), 3.47 (dd, J =

2.7, 6.5 Hz, 1 H), 3.13 (t, J = 9.1 Hz, 1H), 2.63 (dd, J = 11. 6 , 13.5 Hz, 1H), 2.52 (m, 1 H), 2.09 (m,

2H), 1.95 (s, 3H), 1.83 (m, 1H), 1.30 (d, J = 6 . 6 Hz, 3H), 1.06 (d, J = 7.2 Hz, 3H), 1 .01 (d, J = 6 . 8

Hz, 3H), 0.86 (s, 9H), 0.76 (d, J = 6 . 6 Hz, 3H), 0.06 (s, 3H), 0.05 (s, 3H) ppm.

IR (neat film ): 3560 (br), 2932 (s), 2857 (m), 2356 (w), 1785 (s), 1695 (s), 1612 (m), 1514 (s),

1456 (m), 1375 (m), 1347 (s), 1302 (w), 1250 (s), 1196 (s) cm \

HRMS: (FAB, MNOBA matrix) for CagHsgOyNF^SI (M+H)\ calcd: 808.31053, found 808.31033.

'^C-NMR (125 MHz, In CDCI3 ): Ô 177.0, 159.6, 152.7, 148.0, 133.8, 131.1, 129.8, 128.9, 125.9,

114.2, 81.9, 79.2, 77.0, 73.8, 73.6, 73.5, 55.6, 54.9, 41.1,39.3, 38.5, 35.6, 34.7, 26.8, 23.4, 18.9,

16.7, 16.0, 14.8, 14.6, -2.8, -3.7 ppm.

+26.7 (cO.1, CHgCy.

147 Chapter 4: Experimental

(4/?,5S)-3-[(2/?,3S,4S,5S,6S,7R)-6-(ferf-Butyl-dimethyl-silanyloxy)-4-((Z)-3-iodo-2-methyl- allyl)-8-(4-methoxy-benzyloxy)-2,5,7-trimethyl-3-triethylsilanyloxy-octanoyl]-4-methyl-5- phenyl-oxazolidin-2-one 262

OPMB Me Me

lll-mlfc-46

To a cooled (-50 °C) solution of the alcohol 261 (2.1 g, 2.6 mmol) in CHgClg (120 ml, 0.02 M) was added a solution of 2,6-lutidine (3.0 ml, 25.9 mmol, 10 eq) dropwise via syringe. This was

followed by the dropwise addition of TESOTf (1.76 ml, 7.8 mmol, 3 eq) over 8 mins. The reaction mixture was stirred at 0 °C for 5 min, then at RT for 45 min. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was quenched by the careful addition of saturated aq. NaHCOg (100 ml), and the resulting solution was stirred for 5 min. The aqueous phase was extracted with CHgClg (3 X 50 ml). The combined organic phases were dried

(IVIgSO^), filtered and concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (30:1) gave the protected aldol adduct 262

(4.5 g, 56% over 2 steps) as a colourless amorphous solid.

'H-NMR (500 MHz, in CDCIg): Ô 7.32 (m, 3H), 7.23 (m, 2H), 7.13 (m, 2H), 6.81 (m, 2H), 5.86 (s,

1 H), 5.62 (d, J = 7.2 Hz, 1 H), 4.72 (dq, J = 6 .6 , 6 . 8 Hz, 1 H), 4.41 (d, J = 1 1 .5 Hz, 1 H), 4.38 (d, J

= 11.5 Hz, 1H), 4.05 (t, J = 5.3 Hz, 1H), 3.75 (s, 3H), 3.67 (dd, J = 5.2, 6.9 Hz, 1 H), 3.61 (dd, J =

3.8, 8.9 Hz, 1H), 3.49 (dd, J = 3.3, 6.1 Hz, 1H), 3.16 (t, J = 8.9 Hz, 1 H), 2.52 (d, J = 15.3 Hz,

1H), 2.34 (dd, J = 9.8, 15.3 Hz, 1 H), 2.06 (m, 2H), 1.95 (s, 3H), 1.89 (m, 1 H), 1.23 (d, J = 6.9 Hz,

148 Chapter 4: Experimental

3H), 1.01 (d, J = 4.6 Hz, 3H), 0.98 (m, 9H), 0.94 (s, 3H), 0.84 (s, 9H), 0.79 (d, J = 6 . 6 Hz, 3H),

0.74 (m, 6 H), 0.06 (s, 3H), 0.008 (s, 3H) ppm.

IR (neat film ): 2879 (m), 1783 (s), 1694 (s), 1612 (m), 1514 (m), 1460 (m), 1345 (m), 1307 (w),

1250 (s), 1201 (m), 1119 (m), 1015 (s), 830 (m), 769 (m), 741 (m) cm \

HRMS: (FAB, MNOBA matrix) for C^sHygO/^iglNNa (M+Na)^, calcd: 944.3810, found

944.3790.

'^C-NMR (125 MHz, in CDCI3 ): Ô 175.9, 159.1, 152.3, 147.5, 133.6, 130.8, 129.2, 128.6, 125.5,

113.7, 80.8, 78.7, 75.5, 75.0, 73.1, 72.9, 55.2, 54.6, 42.1, 38.4, 38.2, 37.8, 34.8, 26.3, 23.6, 18.4,

16.2, 14.5, 14.4, 14.0, 7.3, 5.6, -3.2, -4.2 ppm.

W o : +3.27 (c 0.2, CH2 CI2 ).

M.pt: 109-1 i r e .

149 Chapter 4: Experimental

(2S,3S,4S,5S,6S,7/?)-6-(tert-Butyl-dimethyl-sllanyloxy)-4-((Z)-3-iodo-2-methyl-allyl)-8-(4- methoxy-benzyloxy)-2,5,7-trimethyl-3-triethyIsilanyloxy-octan-1-ol 263

OPMB Me Me

lll-mlfc-57

To a cooled (0 °C) solution of the protected aldol adduct 262 (160 mg, 0.17 mmol) in a 172:1

mixture of diethyl ether ( 8 . 6 ml) and HgO (0.05 ml) was added ÜBH 4 (37 mg, 1.7 mmol, 10 eq) in a single portion. The reactants were stirred at 0 °C for 10 min, and then warmed to RT; stirring

was continued for a further 2 h. (TLC mobile phase: hexane/ethyl acetate, 1 0 :1 ). The reaction mixture was then quenched by the careful addition of ethyl acetate (5 ml) and HgO (5 ml). After a further 5 mins the aqueous phase was extracted with ethyl acetate (3X10 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the

crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate ( 2 0 :1 ) yielded the primary alcohol 263 (104 mg, 82%) as a colourless oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.22 (m, 2H), 6.85 (m, 2H), 5.83 (s, 1 H), 4.42 (dd, J = 11.6,

23.5 Hz, 2H), 3.81 (s, 1H), 3.80 (s, 3H), 3.58 (dd, J = 3.9, 6 . 8 Hz), 3.56 (dd, J = 3.8, 8.9 Hz, 1H),

3.29 (m, 2H), 3.24 (t, J = 8.7 Hz, 1H), 2.55 (d, J = 14.1 Hz, 1H), 2.05 (m, 4H), 1.91 (s, 3H), 1.65

(m, 1H), 1 .00 (d, J = 6.9 Hz, 3H), 0.94 (t, J = 8.2 Hz, 9H), 0.90 (d, J = 6.9 Hz, 3H), 0.83 (s, 9H),

0.73 (d, J = 6 . 8 Hz, 3H), 0.62 (q, J = 8.2 Hz, 6 H), 0.021 (s, 3H), 0.018 (s, 3H) ppm.

IR (neat film): 3465 (br), 2878 (s), 1741 (w), 1613 (m), 1587 (w), 1513 (m), 1463 (m), 1381 (m),

1302 (w), 1250 (s), 1174 (w), 1085 (s), 1040 (s), 838 (m), 775 (m), 740 (m) cm \

HRMS: (FAB, MNOBA matrix) for CasHesOs^^SiglCs (M+Cs)+, calcd: 881.2483, found 881.2470.

150 Chapter 4: Experimental

"C-NMR (125 MHz, in CDCI3 ): Ô 159.1, 148.6, 130.8, 129.2, 113.7, 79.4, 75.2, 74.2, 72.9, 72.6,

66.0, 55.3, 39.3, 38.3, 38.0, 37.4, 34.5, 26.3, 23.7, 18.5, 15.0, 13.2, 10.6, 7.2, 6 .6 , 5.8, 5.5, -2.9,

-4.3 ppm.

[a]% :+18.4 (c 0.3, CHgCy.

151 Chapter 4: Experimental

(2/?,3S,4S,5S,6S,7/?)-6-(fe/t-Butyl-dimethyl-silanyloxy)-4-((Z)-3-iodo-2-methyl-allyl)-8-(4- methoxy-benzyloxy)-2,5,7-trimethyl-3-triethylsilanyloxy-octanol 254

OPMB Me Me

lll-mlfc-58

To a stirred solution of the primary alcohol 263 (3.03 g, 4.1 mmol) and flame-dried 4Â powdered

molecular sieves (10 g) in CHgClg (200 ml, 0.02 M) was added NMO (1.04 g, 8.9 mmol, 2.2 eq)

in a single portion, followed by TRAP (140 mg, 0.4 mmol, 0.1 eq). The resulting black slurry was

stirred at RT for 1 h. (TLC mobile phase: hexane/ethyl acetate, 9:1). The reaction mixture was then filtered through a small pad of CELITE™, and washed through with copious amounts of

CHgClg. The solvent was concentrated in vacuo. Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl acetate (30:1) as eluent yielded the pure aldehyde 254 (2.47 g, 81%) as a pale yellow oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 9.76 (s, 1H), 7.22 (m, 2H), 6.84 (m, 2H), 5.83 (s, 1H), 4.36 (d, J

= 11.6 Hz, 1 H), 4.43 (d, J = 11 . 6 Hz, 1 H), 4.23 (dd, J = 1 .3, 9.7 Hz, 1 H), 3.78 (s, 3H), 3.58 (dd, J

= 3.6, 7.0 Hz, 1H), 3.52 (dd, J = 3.9, 8 . 8 Hz, 1H), 3.25 (dd, J = 7.7, 8 . 8 Hz, 1H), 2.63 (d, J = 14.6

Hz, 1H), 2.40 (qd, J = 1.4, 6 . 8 Hz, 1H), 2.21 (dd, J = 9.2, 14.6 Hz, 1H), 2.12 (m, 2H), 2.02 (m,

1H), 1.91 (s, 3H), 1.00 (d, J = 6.9 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.94 (d, J = 8.0 Hz, 3H), 0.90

(t, J = 8.2 Hz, 9H), 0.83 (s, 9H), 0.54 (q, J = 8.2 Hz, 6 H), 0.054 (s, 3H), 0.0263 (s, 3H) ppm.

IR (neat film): 2956 (s), 2706 (w), 2362 (w), 1723 (s), 1613 (m), 1513 (s), 1463 (m), 1381 (w),

1301 (w), 1250 (s), 1173 (w), 1082 (s), 1036 (s), 932 (w), 835 (s), 776 (m) cm \

152 Chapter 4: Experimental

HRMS: (FAB, MNOBA matrix) for CgsHeaOs^^SlglCs (M+Cs)\ calcd: 879.2320, found 879.2313.

'®C-NMR (125 MHz, In CDCI3 ): Ô 159.1, 148.6, 130.8, 129.2, 113.7, 79.4, 75.2, 74.2, 72.9, 72.6,

65.9, 55.3, 39.3, 38.3, 38.0, 37.4, 34.5, 26.3, 23.7, 18.5, 15.0, 13.2, 10.6, 7.2, 6 .6 , 5.8, 5.5, -2.9,

-4.3 ppm.

[a]% :+ 8 .3 (cO.6 , CHgCy.

153 Chapter 4: Experimental

(£)-(4S,5R,6S,7S,8S,9/?)-8-(terf-Butyl-dimethyl-silanyloxy)-6-((Z)-3-iodo-2-methyl-allyl)-10- (4-methoxy-benzyloxy)-2,4,7,9-tetramethyl-5-triethylsilanyloxy-dec-2-enoic acid ethyl ester 264

OTBS TESO, OPMB Me Me Me

lll-mlfc-59

To a stirred solution of the aldehyde 254 (340 mg, 0.46 mmol) in dry toluene (45 ml, 0.01 M), was added (carbethoxyethylidene) triphenylphosphorane (1.65 g, 4.6 mmol, 10 eq) in a single portion, and the resulting mixture was heated to reflux for 5 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The solvent was then removed under reduced pressure, and the crude residue was purified by flash column chromatography (SiOg) with hexanes/ethyl acetate (50:1) as eluent to give the ester 264 (343 mg, 90%) as a colourless oil.

'H-NMR (500 MHz, In CDCI3): 5 7.21 (d, J = 8 . 6 Hz, 2H), 6.83 (d, J = 8 . 6 Hz, 2H), 6.73 (dd, J =

1.4, 9.6 Hz, 1H), 5.85 (s, 1H), 4.37 (dd, J = 11.6, 23.5 Hz, 2H), 4.20-4.10 (m, 2H), 3.78 (s, 3H),

3.52 (m, 3H), 3.20 (t, J = 8.1 Hz, 1 H), 2.50 (m, 2H), 2.21 (dd, J = 9.4, 15.1 Hz, 1H), 2.13 (m, 1H),

2.05 (m, 1 H), 1.98 (m, 1H), 1.91 (s, 3H), 1.83 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 0.98 (d, J = 6.9

Hz, 3H), 0.95 (t, J = 8.1 Hz, 9H), 0.88 (dd, J = 2.7, 6 . 8 Hz, 6 H), 0.82 (s, 9H), 0.65 (q, J = 8.1 Hz,

6 H), 0.05 (s, 3H), 0.019 (s, 3H) ppm.

IR (neat film): 2879 (s), 1712 (s), 1650 (w), 1613 (m), 1587 (w), 1513 (s), 1463 (m), 1366 (m),

1250 (s), 1173 (m), 1083 (s), 1032 (s), 836 (m), 774 (m), 742 (m) cm \

HRMS: (FAB, MNOBA matrix) for C^oHyiOg^^SigCs (M-kCs)+, calcd: 963.2846, found 963.2888.

154 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 168.3, 159.1, 147.5, 146.1, 130.8, 129.1, 126.6, 113.7, 79.9,

77.6, 75.3, 72.7, 72.6, 60.4, 55.3, 38.1, 37.9, 37.6, 36.6, 34.2, 26.2, 23.7, 18.4, 14.3, 13.8, 13.3,

7.2, 5.6, -3.1, -4.3 ppm.

[ay% : +3Q.5 (c 0.2, CH^CIg).

155 Chapter 4: Experimental

(£)-(4S,5R,6S,7S,8S,9R)“8-(fe/t-Butyl-dîmethyl-silanyloxy)-6-((Z)—iodo-2-methyl-allyl)-10- (4-methoxy-benzyloxy)-2,4,7,9-tetramethyl-5-triethylsilanyloxy-dec-2-en-1-ol 265

OPMB Me Me

lll-mlfc-62

To a cooled (-78°C) solution of the ester 264 (100 mg, 0.12 mmol, 1 eq) in CHgClg (2.5 ml, 0.048

M) was added DIBAL (1.5 M in PhMe, 0.18 ml, 0.27 mmol, 2.2 eq) in a slow stream over 1 min,

and the reaction mixture was allowed to stir for 0.5 h. (TLC mobile phase: hexane/ethyl acetate,

1 0 :1 ). The reaction mixture was then diluted with ethyl acetate (4 ml) and warmed to 0 °C, whereafter 10% aq. Rochelle’s salt (10 ml) solution was added. The resulting mixture was stirred vigorously at RT for 20 min. The aqueous phase was then extracted with ethyl acetate (5

X 5 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo.

Purification of the crude residue by flash column chromatography (SiOg), with hexanes/ethyl

acetate (20:1->8:1), provided the allylic alcohol 265 (82 mg, 87%) as a thick clear oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 7.21 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.86 (s, 1H),

5.27 (dd, J = 1.1, 9.2 Hz, 1 H), 4.38 (dd, J = 11.8, 23.5 Hz, 2H), 3.94 (s, 2H), 3.78 (s, 6 H), 3.57

(dd, J = 3.5, 9.1 Hz, 1 H), 3.47 (dd, J = 3.4, 5.4 Hz, 1H), 3.41 (t, J = 5.1 Hz, 1H), 3.12 (t, J = 8.9

Hz, 1H), 2.42 (m, 2H), 2.27 (dd, J = 9.8, 15.5 Hz, 1H), 2.01 (m, 3H), 1.85 (s, 3H), 1.67 (s, 3H),

0.97 (d, J = 6.9 Hz, 3H), 0.95 (t, J = 8.1 Hz, 9H), 0.88 (d, J = 6.7 Hz, 3H), 0.82 (s, 9H), 0.65 (q, J

= 8.1 Hz, 6 H), 0.05 (s, 3H), 0.021 (s, 3H) ppm.

156 Chapter 4: Experimental

IR (neat film ): 3434 (br), 2956 (s). 1613 (m), 1513 (s), 1465 (m), 1381 (m), 1250 (s), 1174 (m),

1084 (s), 1037 (s) cm \

HRMS: (FAB, MNOBA matrix) for CagHegOs^sSiglCs (M+Cs)\ calcd: 921.2804, found 921.2783.

^®C-NMR (125 MHz, In CDCI3 ): Ô 159.1, 147.2, 134.2, 130.5, 130.2, 129.6, 113.8, 81.1, 79.2,

75.2, 72.7, 69.0, 55.3, 37.5, 36.1, 35.4, 26.2, 23.8, 18.3, 16.9, 16.5, 14.3, 14.0, 7.3, 6 .6 , 5.8, -

3.4, -4.2 ppm.

[a ]% : +21.9 (c 0.1, CHgOy.

157 Chapter 4: Experimental

(£)-(4S,5/?,6S,7S,8S,9/?)-8-(te/t-Butyl-dimethyl-silanyloxy)-6-((Z)—iodo-2-methyl-allyl)-10- (4-methoxy-benzyloxy)-2,4,7,9-tetramethyl-5-triethylsiianyloxy-dec-2-enal 266

OTBS TESO, OPMB Me Me Me

lll-mlfc-63

To a stirred solution of the allylic alcohol 265 (100 mg, 0.13 mmol) in CHCI 3 (6.4 ml, 0.02 M), was added MnOg (332 mg, 3.8 mmol, 30 eq) in a single portion, and the resulting black suspension heated at reflux for 28 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was filtered through a small pad of CELITE™, and the pad washed thoroughly with copious amounts of ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue purified by flash column chromatography (SiOg) with hexanes/ethyl acetate

(20:1 ) to give the aldehyde 266 (100 mg, 98%) as a yellow oil.

'H-NMR (500 MHz, in CDCI3 ): Ô 9.54 (s, 1H), 7.23 (m, 2H), 6.85 (m, 2H), 6.45 (dd, J = 1.2, 9.5

Hz, 1H), 5.87 (s, IN), 4.39 (dd, J = 11.6, 23.5 Hz, 2H), 3.78 (s, 3H), 3.60 (dd, J = 2.9, 7.0 Hz,

1H), 3.55 (m, 2H), 3.22 (t, J = 8.2 Hz, 1 H), 2.71 (m, 1 H), 2.52 (d, J = 14.9 Hz, 1H), 2.22 (dd, J =

9.2, 15.0 Hz, 1H), 2.10 (m, 1H), 2.01 (m, 1H), 1.91 (s, 3H), 1.76 (s, 3H), 0.99 (d, J = 6.9 Hz, 3H),

0.93 (m, 9H), 0.90 (d, J = 7.2 Hz, 3H), 0.83 (s, 9H), 0.64 (m, 6 H), 0.05 (s, 3H), 0.02 (s, 3H) ppm.

IR (neat film ): 2957 (s), 2706 (w), 1692 (s), 1641 (w), 1613 (w), 1513 (m), 1464 (m), 1381 (w),

1300 (w), 1250 (s), 1174 (w), 1084 (s), 1008 (s), 835 (m), 775 (m), 740 (m) cm '.

HRMS: (FAB, MNOBA matrix) for CagHeyOg^^iglCs (M+Cs)+, calcd: 919.2650, found 919.2626.

158 Chapter 4: Experimental

'®C-NMR (125 MHz, In CDCI3 ): Ô 195.4, 159.1, 158.6, 147.3, 138.0, 130.8, 129.2, 113.7, 79.7,

77.6, 75.5, 72.7, 55.3, 38.2, 37.8, 37.2, 34.4, 26.2, 23.9, 18.5, 15.3, 13.9, 13.3, 10.1, 7.2, 5.7, -

3.1, -4.3 ppm.

[a ]% : +36.2 (cO.2, CH^Cy.

159 Chapter 4: Experimental

(2E,4£)-(6S,7R,8S,9S,10S,11 R)-10-(fert-Butyl-dimethyl-silanyloxy)-8-((Z)-3-iodo-2-methyl- allyl)-12-(4-methoxy-benzyloxy)-2,4,6,9,11 -pentamethyl-7-triethylsilanyloxy-dodeca-2,4- dienoic acid ethyl ester 267

OTBS TESO OPMB Me Me Me

M e ^ COaEt

III- PD-157

To a stirred solution of aldehyde 266 (81 mg, 0.103 mmol) in dry toluene (5 ml, 0.02 M), was added (carbethoxyethylidene)triphenylphosphorane (370 mg, 1.03 mmol, 10 eq) in a single portion, and the resulting mixture was heated at reflux for 16 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The solvent was then removed under reduced pressure.

Purification of the crude residue by flash column chromatography (SiOg) with hexanes/ethyl

acetate (50:1->40:1) provided the (E,E)-dienoate 267 ( 8 6 mg, 97%) as a colourless oil.

'H-NMR (500 MHz, In CDCI3 ): Ô 7.21 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 6.84 (d, J = 8.5 Hz, 2H),

5.84 (s, 1H), 5.53 (d, J = 9.3 Hz, 1H), 4.39 (dd, J = 11.6, 23.5 Hz, 2H), 4.18 (q, J = 7.1 Hz, 2H),

3.77 (s, 3H), 3.56 (dd, J = 3.7, 8.9 Hz, 1 H), 3.52-3.48 (m, 2H), 3.20 (t, J = 8.7 Hz, 1H), 2.48 (d, J

= 13.4 Hz, 2 H), 2.25 (dd, J = 9.0, 15.0 Hz, 1 H), 2.10 (m, 1 H), 2.00 (m, 2H), 1.97 (s, 3H), 1.88 (s,

3H), 1.85 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H), 0.99 (d, J = 6 . 8 Hz, 3H), 0.94 (s, 3H), 0.92 (t, J = 7.9

Hz, 9H), 0.89 (d, J = 7.5 Hz, 3H), 0.82 (s, 9H), 0.62 (q, J = 7.9 Hz, 6 H), 0.03 (s, 3H), 0.0001 (s,

3H) ppm.

HRMS: (FAB, MNOBA matrix) for C^gHysOg^^SiglCs (M+Cs)\ calcd: 1003.3236, found

1003.3201.

160 Chapter 4: Experimental

'^C-NMR (125 MHz, in CDCI3 ): Ô 169.1, 159.0, 147.5, 143.0, 141.1, 131.2, 130.8, 129.1, 125.3,

113.6, 79.8, 78.2, 75.1, 72.7, 72.6, 60.5, 55.2, 38.2, 37.8, 37.7, 36.4, 34.9, 26.1, 23.8, 18.4, 17.1,

15.8, 15.1, 14.2, 14.1, 13.9, 13.2, 7.2, 5.6, -3.2, -4.4 ppm.

161 Chapter 4: Experimental

(2E,4E,10Z,12E)-(6S,7R,8S)-15-(fe/t-Butyl-dimethyl-sllanyloxy)-8-[(1 S,2S,3R)-2-(fe/t-butyl- dimethyl-silanyloxy)-4-(4-methoxy-benzyloxy)-1,3-dimethyl-butyl]-2,4,6,10-tetramethyl-14- oxo-7-triethylsilanyloxy-pentadeca-2,4,10,12-tetraenoic acid ethyl ester 252

TBSP TESO OTBS Me Me OPMB Me

III- PD-158

To a stirred solution of the vinyl iodide 267 (44 mg, 0.05 mmol, 1 eq) and the stannane 253 (51 mg, 0.1 mmol, 2 eq) in dry DMF (2.5 ml, 0.01 M), was added Hunig’s base (0.087 ml, 0.5 mmol,

10 eq) dropwise over 1 min followed by (CH 3 CN)2 PdCl 2 (6.4 mg, 0.025 mmol, 0.5 eq) in a single portion. The resulting mixture was stirred at room temperature for 5 h. (TLC mobile phase: hexane/ethyl acetate, 10:1). The reaction mixture was quenched by the addition of water (2.5 ml). The aqueous phase was extracted with ethyl acetate (3X5 ml). The combined organic phases were dried (MgSOJ, filtered, and concentrated in vacuo. Purification of the crude

residue by flash column chromatography (SI 0 2 ) with hexanes/ethyl acetate (40:1^30:1) provided the desired tetraene 252 (18 mg, 40%) as a yellow oil.

'H-NMR (500 MHz, In CDCI3): Ô 7.67 (dd, J = 11.9, 15.0 Hz, 1 H), 7.23 (d, J = 8.7 Hz, 2H), 7.05

(s, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.43 (d, J = 15.1 Hz, 1H), 6.05 (d, J = 11 . 8 Hz, 1H), 5.46 (d, J =

9.3 Hz, 1 H), 4.39 (dd, J = 1 1 .7, 23.5 Hz, 2H), 4.24 (d, J = 4.2 Hz, 2H), 4.16 (m, 2H), 3.77 (s, 3H),

3.56 (dd, J = 3.8, 8.9 Hz, 1 H), 3.51 (dd, J = 3.7, 6.5 Hz, 1 H), 3.42 (dd, J = 3.6, 6.5 Hz, 1H), 3.22

(t, J = 8.5 Hz, 1 H), 2.57 (m, 1H), 2.46-2.41 (m, 2H), 2.13 (m, 1H), 2.05 (m, 1 H), 2.00 (m, 1H),

1 .93 (d, 6 H), 1 .73 (s, 3H), 1.27 (t, J = 7.^ Hz, 3H), 0.99 (d, J = 6 . 8 Hz, 3H), 0.93 (s, 3H), 0.92 (t.

162 Chapter 4: Experimental

J = 4.3 Hz, 9H), 0.90 (s, 9H), 0.87 (d. J = 6.7 Hz, 3H), 0.59 (q, J = 7.8 Hz, 6H), 0.08 (d, J = 3.1

Hz, 6H), 0.04 (s, 3H), 0.007 (s, 3H) ppm.

HRMS: (FAB, MNOBA matrix) for CsgHg^Og^^isNa (M+Na)+, calcd: 965.6110, found 965.6154.

'^C-NMR (125 MHz, in CDCIg): Ô 199.3, 169.0, 159.1, 153.2, 142.8, 141.2, 139.1, 130.8, 129.1,

125.8, 125.3, 122.2, 113.7, 80.3, 78.3, 77.5, 76.7, 72.7, 72.6, 69.1, 60.5, 55.2, 37.8, 37.7, 36.2,

32.0, 26.2, 26.1, 25.8, 25.6, 25.4, 24.6, 18.4, 16.7, 14.8, 14.3, 13.9, 13.3, 7.2, 5.7, 5.5, -3.0, -4.4,

-5.4 ppm.

163 References

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of such additions usually lies toward the A/-acylcarbinolamine to the extent of 5 kcal/mol.

See: J. Auerbach, M. Zamore, S.M. Weinreb, J. Org. Chem., 1976, 41, 725. For reviews

on this topic, see: (a) B.C. Challis, J.A. Challis, Comprehensive Organic Chemistry, D.

Barton, W.D. Ollis, Eds., Pergamon: Oxford, 1979; Vol. 2, Chapter 9.9, p 957. (b) H E.

Zaugg, W.B. Martin, Org. React, 1965, 14, 52.

113. The addition of alcohols to A/-acylimines derived from /V-acylcarbinolamines is well

precedented in the chemical literature. See: H.E. Zaugg, W.B. Martin, Org. React,

1965, 14, 52.

114. K.J. Hale, P. Dimopoulos, M.L.-F. Cheung, M. Frigerio, J.W. Steed, P.C. Levett, Org.

Lett., 2002, 4(6), 897.

115. Review: P. Cintas, Sy/7f/?es/s, 1992, 248.

116. (a) J.K. Stille, B.L Groh, J. Am. Chem. Soc., 1987, 109, 813. (b) V. Farina, V.

Krishnamurthy, W. Scott, Org. React, 1998, 50, 1.

117. S.V. Ley, J. Norman, W.P. Griffith, S.P. Marsden, Synthesis, 1994, 639.

118. J.W. Steed, The Department of Chemistry, King’s College London.

171 References

119. K.C. Nicolaou, T.K. Chakraborty, A.D. Piscopio, N. Minowa, P. Bertinato, J. Am. Chem.

Soc., 1993, 115,44^9.

120. W.C. Still, M. Kahn, A. Mitra, J. Org. Chem., 1978, 43, 2923.

121. The 4-Bromo-benzyltrichloroacetimidate was synthesised in 98% yield from 4-

Bromobenzyl alcohol and trichloroacetonitrile (prepared according to M. Alterman, H.O.

Andersson, J. Med. Chem., 1999, 42, 3835).

122. B. Jiang, P. Ma, Synthetic Communications, 1995, 3641.

172 STUDIES TOWARDS THE TOTAL SYNTHESIS OF THE NATURALLY-OCCURRING ANTICANCER AGENT HALICHOMYCIN

Maxine Lai-Fun Cheung

Appendix

A thesis presented to the University of London in partial fulfilment of the requirements for the degree of Doctor of Philosophy June 2003

The Christopher Ingold Laboratories Department of Chemistry University College London I - m l f c - 2 4 PROTON CDC13

OTBDPS

MeO Me

"x-T-i" r-'i—i—r "I f -i I } i I t -f " I" j I I I I r' I "t T I I r I ' I I I I ' I I I I I I "I ‘f I I 'I I 3 2 1 0 ppm CARBON CDC13

OTBDPS

MeO Me

200 180 160 140 120 100 80 60 40 20 0 p p m 79.70 %T f I 1 V ext f ile : ÜÜSE1846 Ident :'b _8 Wiri'"'lüüüPPM Acq;^'/-JUL-2UÜ0 15 : 6U : 49 +U:45 Cal : FABMM27 07 0 Ü_1 ZAB-SE4F FAB+ Magnet BpM:135 Bpl:45666304 TIC : 1051850880 Flags : HALL F ile T ext:I-mlfc-24 FABMS MATRIX TGT 4.6E7 1 0 0 % 13S

95J L4.3E7

90J L4.1E7 279 85 j 213 L3.9E7

80J OTBDPS L3.7E7 151 L3.4E7 101 ISl L3.2E7 299 651 L3.0E7

sol L2.7E7 55i 18S L2.5E7 121 50J L2.3E7 197 45J L2.1E7

40J L1.8E7 3 Si L1.6E7

3 0 i L1.4E7 2 Si L1.1E7 16S 2 0 i L9.1E6 ISi 357 L6.8E6 239 loi 221 L4.6E6 Si 289 L2.3E6 313 ^ 401 418 434 450 469 487 0. 11111 . 1 11 111 lljllll[l 1 l lu l|ilIjlHljii 1 il![Itlljl 1 11 ill jllIijlHI llijililjlllljllll|[iliilililililili lliljlJi|iiilililiJiliy.iill l|lilîiiylili|iliulilljiliîlilyiiâUiiîiiiîiiyiiifaiiiyi*.yiiiyiiifai*iy.M^*LiyiiiLtiiYiiilîiiîiiipiiy.irfîiiyiiii — ULO.OEO • U Ù U lÔO 1^0 140 160 180 260 ' 2^6 ' 246 ' 2^6 ' 266 ' 366 ' 3^6 340 360 380 400 420 440 460 4Ô0 5(J0 m/ I-mlfc-70 PROTON CDC13

O H O TBDPS %

f r

UU -A A. / w llV ^ A

"1“ I "("'“r I" I"'I I " I "I "I "I"" I' "I" r "I i | i T ' n I ' I 1 I ! ' T " r " 'I t ' T ■ 'I T ' ' ' I " I' I I I I " I r I I 9 8 1 0 ppm CARBON CDC13

OH OTBDPS

200 180 160 140 120 100 80 60 40 20 0 ppm PERKIN ELNER

to O

01 o

1.83 -'I— 4000 3500 3000 2500 2000 1500 1000

OH O TBDPS

Me File: ÜÜSE828A Ident : IÜL3 6 Win 100 Ü PPM AcqT%4 -MAR- 2 0 0 0 lO : 50 ;'3'Ü""+1: 28 Cal : FABLM2 3Ü3ÜÜ_1 ZAB-SE4F FAB+ Magnet BpM:199 Bpl:7501285 TIC:83685176 Flags : HALL F ile Text:I-mlfc-70COL FABMS MATRIX MNOBA + NÀ 100% 199 7.5E6 95^ L7.1E6

90J l6 .8E6 85: L6.4E6 so: OH OTBDPS L6.0E6

v s J L5.6E6 70J Me L5.3E6 65J L4.9E6 60j L4.5E6 55J L4.1E6 50 j L3.8E6

4 5 ^ L3.4E6 40J L3.0E6 35: L2.6E6 30: L2.3E6 L1.9E6 25: 193 20: L1.5E6 271 329 15J L1.1E6 183 l Û i ' 239 L7.5E5 251 211. . . 229 L3.8E5 223 243 ,259 275 289 303 311 319 342 0 iL i.tJ.i LO.OEO jW'liW4 i 4 iH^^W V V Y Y V V v ‘i‘"i“i‘ ' ur,v 170 1Ô0 190 200 210 220 230 240 250 260 270 200 2^0 3Ô0 310 3^0 330 340 350 m/z I-mlfc-53col protonpp.ser CDC13 v servSOO A

Current Data Parameters NAME FeblB-2000kjh EXPNO 10 PROCNO 1

F2 - Acquisition Parameters D a te . 20000219 Time 10.21 INSTRUM drxSOO PROBHD 5 mm Multinu PULPROG zg30 TO 65536 SOLVENT C0C13 NS 16 OS 2 SWH 10330.578 Hz FIORES 0.157632 Hz AO 3.1719923 sec RG 2 03 .2 DW 48.400 usee OE 6 .0 0 usee TE 3 00 .0 K 01 1,00000000 sec

------CHANNEL f l ------NUCl IH PI 11.50 usee PLl 0 .0 0 dB SFOl 500.1330885 MHz

F2 - Processing param eters SI 32760 SF 500.1300237 MHz WOW EM SSB 0 LB 0 .3 0 Hz 60 0 PC 1.00

ID NMR plot parameters CX 30 .0 0 cm F IP 11.000 ppm FI 5501.43 Hz F2P -1 .0 0 0 ppm F2 -5 0 0 .1 3 Hz PPMCM 0.40000 ppm/cm HZCM 200.05202 Hz/cm

OTBDPS

Me P-T ppm I-mlfc-53col cl3 cp d .ser CDC13 v servSOO 4

Current Data Parameters NAME Febl8-2000k)h EXPNO 11 ^ S o S S 2 o in PROCNO 1 mtn m g>f\j oj F2 - A c q u is itio n Parameters Date_ 20000219 \ll/ 'W Time 12.58 INSTRUM drxSOO PROBHD 5 mm M ultinu PULPROG zgpgao TO 65536 SOLVENT C0C13 NS 3072 OS 4 SWH 31446.541 Hz FIORES 0.479836 Hz AO 1.0420724sec RG 2048 DW 15.900 usee OE 6.0 0 usee OTBDPS TE 300.0 K 01 . 2.00000000 sec dll 0.03000000 sec d l2 0.00002000 sec

Me NUCl 13C PI 5.00 usee PLl 0.00 dB SFOl 125.7715719 MHz

mmmmmm■■ CHANNEL 12 mmaagmmj CP0PRG2 w altzlG NUC2 IH PCP02 95.00 usee PL2 0.0 0 dB PL12 19.00 dB PL13 19.00 dB SF02 500.1320005 MHz

F2 - Processing parameters SI 32768 SF 125.7577907 MHz NOW EM SSB 0 LB 1.00 Hz 68 0 PC 1.40

10 NMR p lo t parameters CX 30.00 cm F IP 235.024 ppm Fl 29556.15 Hz F2P -1 5.032 ppm F2 -1890.41 Hz PPMCM 8.33522 ppm/cm ...... I HZCM 1048.21851 Hz/cm ppm 220 200 180 160 1-40 120 100 40 20 F*EHKIM BLJ^H

77 .16 %T

CO CD cn -vf I i

o o

hn o (3 O h* o 03 'Î^ IS o

Ci .. CD CO

• v -l

3500 3000 2500 1500 1 0 0 0 cm"*

OTBDPS

Me P ile: ÜÜSE541 ïdéritT6’3 Win 1000PPM Acq: ^^-FEB-2ÜÛÜ 14:01:58 +0:48 Cai :FABMM2 2ü^UU_l ZAB-SE4F FAB+ Magnet BpM:269 Bpl:52767404 TIC :1065750464 Flags :HALL F ile Text :I-mlfc-53COL FABMS MATRIX MNOBA + NA 100%, 269 5 3E7 L5 0E7

90J L4 7E7 L4 5E7 80i 14 2E7 OTBDPS 75J L4 0E7

l 3 7E7 70j Me 65: 239 i.3 4E7 60j L3 2E7

5 s j L2 9E7 50Î 12 6E7 4sj 12 4E7 40J 12 1E7 35 j L i 8E7 249 3 0 j Ll 6E7 25 J L i 3E7 20Î Ll 1E7 211 15^ 11 9E6 10 j 227 15 3E6 327 5j J _ 223 259 309 319 12 6E6 15 43 73 2?J-,289 297 305 .1339 , 357 365 379 /392 OJ iW m li l'l't444H44 Ti'i'i'rr i o OEO 2Ô0 210 220 230 240 250 260Î 270 280 Ao 3Ô0 31 0 320I 330 340 350 350 370 6 0 3 ^ 0 4Ô0 m/z I —m l f c — 3 9 to p > sp >o t PROTON CDC13

OTBDPS Me Me

r r i - i T - r i r ppm I - m l f c -3 91 o p s p o t CARBON CDC13

OTBDPS Me Me

0 ppm 220 200 180 160 140 120 100 80 60 40 20 I-mlfc-39topspot DEPT CDC13

Me Me

“ n 1 ------r 1 ' I ' I ' r 220 200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-39topspot COSY CDC13

ppm

-1

-2 OH < OTBDPS Me Me

- 4

-5

- 7

-8

■nr T - r y r ppm I—mlfc—3 9top spot HMQC CDC13

ppm

-100

OH -120

OTBDPS Me Me -140

-160

8 7 6 5 4 3 2 1 ppm o

CVI o o

'H 24.46 3500 2500 2000 15003000 1000

ÇH

Me Me |£^± JLe : o o SSX~3X. ic ie n t : 1 Ü X6 W in 1 O O Ü P PM A-Cq : XV - JAJST-ii O U U 1 X : X J : 4 6 -t- Ü : 44 CaX : FABLMX V U X U U—X ZAB-SE4F FAB+ Magnet BpM:123 Bpl:18100224 TIC :1120851200 FlagsiHALLi File Text :I-mlfc-39-COL FABMS MATRIX MNOBA + NA 100% 239 1 5E7 95Î Ll 4E7 90i Ll 3E7 85j 229 Ll 2E7 OH 80J Ll 2E7 75J 'OTBDPS Ll 1E7 Me Me 70i Ll 0E7 L9 5E6 65 269 60i L8 8E6 L8 1E6 5 5 J

50J L7 3E6 213 383 45^ L6 6E6 40i L5 9E6 20' L5 1E6 35J 325 3oi u 4E6 25J 24 9 303 L3 7E6 257 2 0 : L2 9E6

L2 2E6 I 5 J 289 309 405 loJ Ll 5E6 75 31 337 365 448 5J 352 395 L7 3E5 411422 439 460 478 490 01 mini Lo OEO 2Ô0 2É0 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m/z I-m lfc-39 bottom spot PROTON CDC13

OTBDPS Me Me

,dU UL IL

■T' r-'i—I—I—I- I I- 1 "I" ", "I—I I I —r-j—I—r-T—r—i—i—i i \ j i i i" i r r-r-i—r"I " r r I 1 I I I I I ' I I I p I" "I" I ' I f I r "I "f " T '"! " I'“T'"|

8 6 5 4 3 2 1 0 ppm I-mlfc-39 bottom spot CARBON CDC13

OTBDPS Me Me

78.70 XT

ai i "V • io

MVl O o

o V*

3500 3000 2500 2000 1500 1000

0 0 /0 1 /1 4 17: 40 f t OH SCAN: 16 scans, 16.0cm-1 OTBDPS Me Me File:Ü0ÔEl32' Ident :li_lb Win lUUUPPM Acq: 1V-JAN-2ÜÜÜ“ 11 :17 : 58 +Ü:49 Cal : FABLM17 OTTODZr ZAB-SE4F FAB+ Magnet BpM:197 BpI:17379328 TIC : 467104064 Flags:HALL F ile Text :I-mlfc-39(UNDESIRED) FABMS MATRIX MNOBA + NA 100% 239 1E6 9 s i 7E6 9 o i 3E6

85J 9E6 80 j OH SE6

OTBDPS 1E6 229 70z 269 M e M e 7E6 6 Si 3E6 6o i 9E6 SSi SE6

s o i 1E6 4 Si 7E6 40i 383 3E6 35, 8E6 3o i 4E6 249 32S 2 si 0E6

2 0 i 6E6 15i 2S7 2E6 30S lOi 297 lES lES 335 395 =: 3» 35235. 419 '437 449 461 478 490 fi4 OEO 2Ô0 220 240 260 280 300 320 340 36060 36036 400 42o 440 460 460 500 m/z I —m l f c —7 8 PROTON CDC13

OBn

OTBDPS Me Me

I I-I »"<* "H" - r - p r - r - 7 4 ppm I —m l f c —7 8 CARBON CDC13

OBn

OTBDPS Me Me

200 180 160 140 120 100 80 60 40 20 0 ppm DEPT CDC13

OBn

OTBDPS Me Me

I I I I I r I'l l I j'r I'l r I n -ri-j'i i i i i i i 'i i "|"r r i'i'i i i n prn-rT'r rTr|-n "i'i ri i-rr-j'r i i ri T ? in j' r i i i r i i-i 'i | r i i i i < r i r j' r i rr i i i i'f |' I'n n t i i i j' r i l 'i-i-i-n l y r r'f T vt r-n -j~n-iTi i i i i | i i i i i in i j i i -j r'lT I'l-rn t p i i r t i i i i'j i r i rri-n 'iy i'iTviTrTi'j vi i i i i r i i j i i i rr i i i i | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm I —ml f c —7 8 COSY CDC13

J l i __I il L _____ A. J ppm

J - 1.0 -1.5

< — 2.0

< -2.5 OBn

OTBDPS m m Me Me -3.5 -4.0

-4.5

-5.5

m - 6.0

-7.5

ppm I —m l f G —7 8 HMQC CDC13

ppm

OBn OTBDPS 70 Me Me 80

100

^110

i-120

i-130

rl40

fe-150

1 ppm F 'E riK lN ELMEPi

76 .69 %T

CVJ CD in cn CO cu

'«J

o CO CD

6 .5 2 3500 3000 2500 2000 1500 1000 cm“^ 500

OBn 00/01/14 17:21 ft SCAN: 16 scans, 16.0cm-1 OTBDPS Me Me F iLT'eTD" 0 S El'ü B 8 Id.ent:b_l.u Win lüUUPPM Acq: 2-MAY-2Ü0Ü 11:'2Ü:Ü1 +ü:27 Cal : FABMM0 2 ObUOl,! ZAB-SE4F FAB+ Magnet BpM:135 Bpl:5095766 TIC:69232328 Flags:HALL F ile Text:I-mlfC-78 PURE FABMS MATRIX TGT 100%, 211 6.4E5

90. 6.1E5 85, 5.8E5 239 OBn 5.4E5

75. 5.1E5 Me Me 70. 4.7E5 4.4E5 4.1E5

55. 3.7E5 247 3.4E5 21 289 3.1E5 45. 259 269 2.7E5 40. 227 35. 2.4E5

30. 2.0E5

25. 1.7E5 1.4E5 20. 31 1.0E5 15. 277 303 473 10. 329 6.8E4 337 365 379 15 407 437 457 503 519 541E

200 m/z I-m lfc-64 PROTON CDC13

OBnBr

OTBDPS Me Me

5 ppm I-mlfc-64 CARBON CDC13

OBnBr

OTBDPS Me Me

#wmWmiWww#W#W'

200 180 160 140 120 100 80 60 40 20 ppm I-mlfc-64 DEPT CDC13

OBnBr

OTBDPS Me Me

M#W*WWw

pr i"i I i"i 11 f j' I 'I iT'i i' M I I'i'i Ï r 111"f f ^TTV r'l 1T i I I I I' I'l 'rriT i j'l' i n i x i rTj* vi rr r i 11 t | ^ t c n 'i 11 i i ^ i i r i"i r r 'i r j i r i ' 1 1 I Ï I'j'i I Ml I I I I I I I I I' 11 I I T| I I 11 f'rri ? j 1 T'l 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 3020 10 ppm I-mlfc-64 COSY CDC13

Ml I JL li ppm hO

-1

■ i -2

-f -4

OBnBr

OTBDPS — 6 Me Me

J L -7

T l-n 'IT ’I-l-r I T I'T-T-TT 0 ppm I—mlfc—64 HMQC CDC13

ppm

100

120

OBnBr -140

OTBDPS Me Me -160

-180

8 7 6 5 4 3 21 0 ppm mSMKfN ELMGr?

283.04 XT

o o CIO O) cu i l to

275.90 c u r so r I CD 7 7 8 .9 cm“*

o o s

cu o i • v l

209.90 jdL.. 3000 2500 2000 1500 1000 cm

OBnBr

OTBDPS Me Me Base: Positive Ion FAB 210000 171

OBnBr Sample: I-MLFC-64 OTBDPS 150000 Instrument Resolution: 7,500 Me Me Theoretical Mass (C31H3902BrSi): 551.19806 (M+H) Measured Mass: 551.19714 Error: 1.7ppm

100000

247

553 199 50000 -

269 391 495 323 II I 417 „ , 646 J 04 10Ô 150 200 250 30Ô 350 % 450 5Ô0 550 600 650 700 750 m/z I - m l f c - 1 3 4 PROTON CDC13

OBn HO OTBDPS Me Me

I 'I I IT I I I I I I' r I I I I ' I ' I ' I "'I''r I'l I I'l' I j i i i "t't '»■ i "i~i i -1 i | i"i 't ' i" vi t r | r i i i i -r-i i i "j ' i' r i I I I I I 1 I...... I "I- I "I

8 7 6 5 4 3 2 0 ppm I-mlfc-134 CARBON GDC 13

OBn HO OTBDPS Me Me

n r 2 00 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-134 DEPT CDC13

OBn HO OTBDPS Me Me

, [T I 1 I ■ 1 1 I'H I'll n I. I [ ■ I I I I IT M [ I 11 ■ ■ I r r , 11 I I i r r r r r |...... j r r i ,,,,,, | T i vri i < ii r. i [ i i ■ [ ri ■ n l i T r [ i I T | ...... | ...... I"' ...... I " " '^^^1 I " " ...... <...... <...... 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 .40 30 20 10 0 ppm I-m l£c-13 4 COSY CDC13

ppm

hl.O

# e [-1.5

H2.0

OBn HO OTBDPS h3.5 Me Me

h7.5

ppm I-ml£c-134 HMQC CDC13

^__ L ppm Me Me

HlOO

H llO

■rl20

• 1-130

i-1 4 0

É-150

2 6 5 4 3 1 ppm r’SHKlN EUMEFT

88.10 %r

;

n UK. go

29.75- 3500 3000

OBn 00/04/07 08: 17 f t HO X: 16 scans, 2.0cm -l, apod none OTBDPS Me Me F^ile : üD 5 e 1Ü ^6 ïdent: 5 5 ^ 5 7 "Win TÜ'Ü'U PPM A cq : 2-M AY-2üUU ll:U/:bj +3:ÜU Cal:FABMMU%UbUU_l ZAB-SE4F FAB+ Magnet BpM:137 B pl:17762988 TIC : 487312288 F lag s:HALL F ile Text:I-m lfc-75 FABMS MATRIX MNOBA + NA 100% 305 r3 .0 E 6 95J 239 2.9E6 90i 2.7E6 21 325 85J 2.6E6

80j 2.4E6 OBn 289 2.3E6 227 HO OTBDPS 70: 2.1E6 Me Me 2.0E6 1.8E6

55. 269 1.7E6 1.5E6

45. 1.4E6 40. 249 1.2E6 491 35. 1.1E6

30. 9.0E5 25. 7.5E5

20. 336 6.0E5 15. 381 4.5E5 349 421 3.0E5 10. 391 433 401 623 581 1.5E5 443 473 529 550 LO.OEO

2Ô0 250 3Ô0 4Ô0350 700 m/z I-mlfc-91 PROTON CDC13

HO OTBDPS Me Me

I'll I I I ' I I I I I I I “I I "I I I I t I I I ! I I" I" r I ' I f I I—I " I I I I I—I—I—I—I—r —i ""7 -'i ""T- I I ■'I I ' I r r

4 2 1 0 ppm I-mlfc-91 CARBON CDC13

Me , Me

turn M i Jm iI m

200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-91 DEPT CDC13

HO OTBDPS Me Me

I I i-TTi r |-ri'Ti-i ri i i 111 rirrn lyt 'i i I'ri'i Ti-j'rrr i i r i i i j I i i n i ri'r j' i in rrn'Vj'viTTi'n i i j i i ii i i 'i i i j i r i n ii i i | i i i i i t t >"i~j i i ri-i ri Ti-pi in i i i t i |'l i"t ri'r r i i ji fi i i' I'l i i j it i i t i n'r|Tn"ri'n "i'i"|"r t i i i"i i i'i j'l'ifr'i i i ri | i J | , i ri i | , u i i i i | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm I - m l f c - 9 1 COSY CDC13

LA. ppm

-f -4 -t h 4- 0

-1

-2

-3

- 4

-5

HO OTBDPS - 6 Me Me

-7

0 ppm I-mlfc-91 HMQC CDC13

À j À j L ppm "1 ' ■ ' r— ' 1 1 ...... T - 1 1 1 1 1 1 1 1 1 1 1 1 1 - 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I f 1 - 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 t 1 1 1 1 1 1 1 1 - 40 1 1 1 1 1 1 r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 f 1 - 60 1 1 1 t * 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 , 1 1 1 1 1 1 1 1 T 1 - 80 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J. J J L _ 1 _L J. -100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ------1------■1------I------t------1 - -120 1 1 1 1 1 1 1 1 1 1 1 1 1 . f ' } » ' 1 1 1 1 1 1 1 1 1 1 1 1 1 -140 1 1 1 1 1 V 1 1 1 1 1 1 1 1 ,/ 1 1 1 1 1 1 1 1 1 1 1 HO 1 1 1 1 1 1 1 1 OTBDPS 4------1------1 _ -1------1------4- _ - 4 ------160 1 1 1 1 1 1 1 1 Me Me 1 1 1 1 1 1 1 1 1 1 1 1 _J__ 1 , „ 1., I I' r I 'I I T f i ■!' I I 1 I I I I I I I-I I f I' I T ‘1 I'I I I I r T I I I I I I I I I I I I I I I I ■!■ r ~i i i i t i" i i—i i i i i i i i i r v i -r i ' i- r- i-r r r • ■ I ' 7 2 0 ppm 64.61 %r

o o

o O)

32.98 3500 3000 2500 2000 1500 1000

HO OTBDPS Me Me l^ile: Ü05E132Ü Ident';~7_21 Win 1ÜÜÜPPM Acg:22-MAY-2UUU 14:19:^1 +U:4b Cal:FABMM2^UbUU_l ZAB-SE4F FAB+ Magnet BpM:169 BpI:7287877 TIC :115566808 FlagszHALL F ile Text:I-m lfc-91 FABMS MATRIX MNOBA + NA 100È 305 1.1E6 L1.1E6 L1.0E6 L9.7E5 L9.1E5 L8.5E5 OTBDPS L8.0E5 Me Me L7.4E5 L6.8E5 L6.3E5 L5.7E5 L5.1E5 L4.6E5 L4.0E5 L3.4E5 L2.8E5 L2.3E5 L1.7E5

493 513 L1.1E5 L5.7E4 607 / 648 6|66 689: i Iw

2Ô0 650 700 m/z Ill-mlfc-1 PROTON CDC13

OBn

O Me Me

I I I I I I >'j rv I I I rt-i I I I ■III» I 1'I I I I I I I 1""1" I I f 1 I I I *11 T"r*TT r I ' l"|' I I I T’l" I 1 I

12 11 10 8 6 2 1 0 ppm Ill-mlfc-1 CARBON CDC13

OBn HO

O Me Me

200 1 8 0 160 140 120 100 80 60 40 20 0 ppm Ill-mlfc-1 DEPT CDC13

OBn HO

O Me Me

U.,.,,|u , I I I I I...... I...... ""!'...... "I"",,,,,I IT', I...... T'" " " " l ' ...... I...... 'I...... 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm COSY CDC13

ppm

- 1.0

- 1 .5

- 2.0 4 m - 2 .5 OBn

0 Me Me -3.5

-4.0

-4.5 4

-5.5

ppm III-m lfc-1 HMQC CDC13

ppm

OBn HO O Me Me

-100

-120

-140

-160

I I I I j "r !■ I I I I I I I I I I I I 1 ppm I Mk . i

“ Î I o 05 tJl m' I— CO O g o to o Q) CO ro tn g 3070.0 o o n o B !

IM o o o 1959.1 1088.2*

1707.1

1589.0 Hk g o 1427.7

1295.0

1205.1

o 1027.6 o o 939.0

823.7

739.2 701.1

614.3 F ile :"Ü'USE122 b ïdent: ; b_13 Win lÜÜUPPM Acq:12-MAY-2UUU 12:UÜ:jU +Ü:3U Ca± : FABMMi:^ U b U U_U. ZAB-SE4F FAB+ Magnet BpM:201 BpI:13895908 TIC : 315195584 Flags:HALL File Text:I-m lfc-84 FABMS MATRIX TGT 100% 201 1.4E7 L1.3E7

90J L1.3E7 85i L1.2E7

80J L1.1E7 OBn L1.0E7 75i HO OTBDPS 70i L9.7E6 185 Me Me 65J L9.0E6 60i L8.3E6

55J L7.6E6 50 j L6.9E6 45J L6.3E6 289 40 j 1.5 . 6E6 L4.9E6 3SJ 171 30J L4.2E6 25J L3.5E6 505 20J L2.8E6 213 15i L2.1E6 229249 L1.4E6 10 j 319 309 5i L6.9E5 34^1 I'l,^397 469 527 565 597 639 0 I |i l|i.liLij|.iij.ipu«Yj.Lid.LLtlJ|L»»YlilLU, LO.OEO 150 2Ô0 250 3Ô0 3 ^ 0 ' ' 4 6 0 450 500 iso 600 700 m/z I-m lfc-92 PROTON CDC13

HO OTBDPS Me Me

I !■ I r - I 1 I ■! I I I I I > l-T ' < "I r I I I—I—I r r I 'I—i—i—r ' i * i" i—i i‘ f \—r ' I"' ‘ ' r"i“"i : " I I' I ‘ I “ I" r i‘t--i i - t j 9 8 4 2 1 ppm I - m l f c - 9 2 CARBON CDC13

Br o HO

O Me Me

J v J U iiMiinHftl KM

2 0 0 180 160 140 12 0 100 80 60 40 20 0 ppm I-mlfc-92 DEPT CDC13

O Me Me

1 ri Ï Tt*TT j 1 T'l'f r I I' I I I I |"1‘ I TT'I r I'I'I f u n n I I y IT 11 I I I I vt'vi ry r r i i i |'t n n n i i jt t i rv i i-mpTTT-n i m | i i i i I'l i n i i i i i i i i i i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm

-H H I- 4- -0

-1

-2 J4

- 3

- 4

- 5

O Me Me

- 7

7 6 4 32 1 0 ppm5 I-mlfc-92 HMQC CDC13

ppm

j _ -100

Br 120 o HO 140 O Me Me

-160

8 7 6 5 43 2 1 0 ppm PERKIN ELMER

71.09

Oi 33.72

PEAKS 00 613.3 I cm"'

7.90 4000 3500 3000 2500 2000 1500 1000

0 Me Me File:ÜÜ5E1319'Ident;11_15 Win'lOOOPPM Acq;22-MAY-2UÜ0 14:U/:j7 +U:4l Cal;FABMM22U5ÜÜ_1 ZAB-SE4F FAB+ Magnet BpM:135 BpI:4843111 TIC:92807840 Flags:HALL F ile Text:I-m lfc-92 FABMS MATRIX MNOBA + NA 100% 1.6E6 1.5E6 1.4E6 1.3E6 1.3E6 1.2E6

OTBDPS 1.1E6 0 Me Me 1.0E6 9.5E5 8.7E5 7.9E5 7.1E5 6.3E5 5.5E5 4.7E5 3.9E5 3.2E5 2.4E5 1.6E5 7.9E4 469 507 527 I 643 663 685 'iJilMUkU 0 .OEO 200 m/z I-mlfc-51(b) PROTON CDC13

'Y " OBn

Me Me

n ’ T 1 0 ppm I-mlfc-51(b) CARBON CDC13

OBn Ph OTBDPS Me Me

W# ####

200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-51(b) DEPT CDC13

OTBDPS Me Me

■tli. <_ ifc- tu -A— «iéJ ppm -0

-1

-2

-3 i

A -4

-5 OBn Ph OTBDPS Me Me

-7

7 6 5 4 3 2 1 0 ppm I-mlfc-51(b) HMQC CDC13

i _ j 4 ilL ^ - k Mill ^ -■ ■«— _A J al ppm

100

110

120

1 — - h - 130

rl40

rl50

OBn rl60 Ph OTBDPS Me Me i-170

8 6 5 4 32 17 ppm 4 5 .5 2

4 5 .0 2 CURSOR

•>r1

7 .3 4 30003500 2500 2000 1500 1000 cm"

02/11/02 11:04 X: 4 scans. 16.0cm-l, apod none, fla t OBn Ph OTBDPS Me Me Bæe: VG70-SE Positive Ion FAB 125000 Sample: l-MLFC-51b Instrument Resolution: 7,500 110000 - Theoretical Mass (C41H4905NSI): 664.34578 (M+H) 100000 Measured Mass: 664.34504 Error: 1.2ppm

70000 OTBDPS 60000- Me Me 50000-

40000

30000 -

10000 - I-mlfc-68 PROTON CDC13

Y OBnBr

Y''% ^OTBDPS Me Me

JULA—J iuL-JU u v

I I l'I T '1'1 !■ 1 I I - I - I—f v r I I I - I- I - I" I ' I T - r - p r I I I ' T"T' I" I r t t i | I I I" I "I "I ' Ï I' ' I ' 1 I " r I I "I I' I I 1 ppm I-m lfc-68 CARBON CDC13

^ OBnBr Ph OTBDPS Me Me

200 180 160 140 120 100 80 60 40 20 0 ppm I - m l f c - 6 8 DEPT CDC13

OBnBr Ph OTBDPS Me Me

MbJ I»

^ ‘ ‘ ‘ ^ I ^ ^ ^ ' I ‘ ‘ ‘ ‘ ' ' ' ' ' I ' ^ I ■ I ■ ■ J...... I I ...... I ■ ■ ■ ■ j I . I 1 I . f i I I I I I r I I' I I I I n n 'l I I 'I I II I I I j rr I I'l I I i"i j I'l i i i r r i i j i r i ii i i i i j i i j r i i i ii i i i j i i i i i i i ■ri-|' i' n r r i i i i j' r i i i i i i i i | i I'l i i i i i i j i i I'l' i i i n j'...... j'l i i i i i i i i | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm

4 4 H I- -0

i -2

- 4

OBnBr Ph OTBDPS Me Me

- 7

7 6 5 4 3 2 1 0 ppm I-mlfc-68 HMQC CDC13

ppm

- I -

100

-120

-140

O BnBr -160 Ph OTBDPS -180 Me M e

8 7 6 5 4 3 2 1 0 ppm FERKIN ELMER

37.96

30.56 CURSOR 560.3 ctn*"‘

to cu o

18.92 25003000 2000 1500 1000

02/08/19 15:11 OBnBr X: 16 scans, 16.0cm -l, apod none, f l a t Ph OTBDPS Me Me Base: Positive ion FAB 70000 171 65000 300 Sample: I-MLFC-68 OBnBr 60000 -1 Instrument Resolution: 7,500 Theoretical Mass (C41H4805BrNSi): 742.25631 (M+H) 55000 OTBDPS Measured Mass: 742.25589 Me Me 50000 Error: 0.6ppm 199 45000 556 40000 -I 744

35000 339 438 30000 -

25000

20000 -I 261 239 478 15000

10000 141 684 5000 376

ilLlIliAjjylili. iil.illlliilll|iillllLl 100 200 300 400 500 600 700 800 m/z I-mlfC“109 PROTON GDC 13

O H OBn HO OTBDPS Me Me

—j—I -I I—I ■ V I I 1—I j" I—I—I—I I I I I—r“j—T—r—I—TT—I—1—1—I j I I I—r -1 I" r I i ' |' i i r t i i i i t ' j " i—r i ' i—r I I !■ r I r i- i ' r-r- -1—T T I T“ *“ p 6 5 4 3 2 1 0 ppm I-mlfc-109 CARBON CDC13

O H OBn HO OTBDPS Me Me

At 1

I I I "i"i"i' I' I'll I I 11" I Ill'll Y IT ri"r r I r i‘ I' I i i i i i r r Vj i r'l i i i | i i i i i i i i r | i i i i i i i i i | - r r-i-1 i i i i i | i i i' i r r i-vr-j i r i i i i- r i r | i 'i i r i i r i i | i i i-i-i' i i "i i j i i r i "i i i i ‘i |-i-r-i-< i r 'i i -r j ri"i 'lit iT i"|-r i i r tT T~ri‘y i-1 i i 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm I - m l f c - 1 0 9 DEPT CDC13

OH OBn HO OTBDPS Me Me

'I 11 j I'l ri I'l II I'l 11 II i I I'll I I rr rr I I I I j I I I I I I it 'i j it i i f r r i i 11 i i i i i I'ti'prri r r i i i I j i i rnT ii r | I'l i i i i r t r j ri i i i i i 'i i j i i i i i i i i r j I'l in i i i i | i i i i I'ti I'l | r r i-rr i"ii r j i i i i i r rri j iT i-ri ri i i j i i i i i i i i i | i i i rriTi cj r i i i n i I't ji i r i i i i i i j i i-rm i i r j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm 0

-2

- 3

-4

-5

OH OBn

HO — 6 OTBDPS Me Me

-7

0 ppm I-mlfc-109 HMQC CDC13

ÜL ppm

-100

-120 OH OBn HO OTBDPS -140 Me Me

-160

■i-r nr-j—I-1 i i i i i i i j i r 0 ppm 61.78

17.20 ICUHSOR 56 3.5 cm ' -i

19.47 3000

00/07/26 16:43 OH OBn SCAN: 16 scans. 4.0cm-l, flat, smooth HO OTBDPS Me Me F ile;'üü5ElB2b Ident :l_b Win lÜÜ'üPPM Acq:'27-JÜL-2ÜÜÜ~'ll : Ü9 :4V +ü ;^4 cal :FABMM^7UVUU_l ZAB-SE4F FAB+ Magnet BpM:529 BpI:5880423 TIC:86413384 Flags:HALL F ile Text :I-mlfc-109 FABMS MATRIX MNOBA + NA

1 0 0 % 529 5.9E6

95J L5.6E6 90i L5.3E6

85J L5.0E6

80J L4.7E6 L4.4E6 75i OH OBn 70J HO L4.1E6 OTBDPS L3.8E6 65J Me Me 60 L3.5E6

5 5 j L3.2E6 50J L2.9E6 45J L2.6E6 40J L2.4E6

3 s j L2.1E6 30J L1.8E6 25J L1.5E6 20; L1.2E6 15; L8.8E5 L5.9E5 loJ 239 211 5; 227 289 L2.9E5 307 329 349 365 382 422 440 470 489 507 ^'f 545 587 0 LO.OEO 2Ô0 220 240 260 280 3Ô0 320 340 360 380 4Ô0 420 440 460 400 5(J0 5^0 540 560 580 6Ô0 m/z I-mlfc-98 PROTON CDC13

OBn

OTBDPS Me Me

I I I I ■ I I I I I I < I i » ' r"T- r I I I ■'!■ T-7 —I IT I l l —I I ■ I'nr—I’ I "I ■ r-r i—i- - I - I - I —I— I— I— I— I— I— I— I— 1— 1— I— I— I— I— I I I— I— r —I— I— I— r— 1—1— I— I ' l 'I ...... I— r —T—1— r—r—r - j — r —t— r— r — i— i— i— i— i— j— r —i— i— i— i— i— i— i— i— | 9 8 7 6 5 4 3 2 1 0 ppm I-m lf c - 9 8 CARBON CDC13

Et0 2 C OBn

OTBDPS Me Me

lüLi blL .ill * U. iLwl lint.

200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-98 DEPT CDC13

OBn

OTBDPS Me Me

1U.J JL wiAiwXU

'rrr^ i tttj i "rri p* r rri n Ti'j'i ft r i i i r'l j i i i 'i 11 im j ii i i m r m-11 r m-r r rr | i iT i i n r» j i i i i i 111 i j i r i i tt i I'l | fin r rx iTj vn"r r rr i i j'l i i n i r ii | i i i i i i i i r^Tn'TT rrr ^ jin r ri ri I'j I'l i -i rri' i Tj"i"rTV i"i i i ij i i* rn r r i r j rni i i i i i | 'i i i i i i i i i j i x i i ti "i 'i"i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm I —m lf C “ 98 COSY CDC13

L l I I AA 11 11 ^ iik. ppm

I @ h i

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OBn F-5

^y^Y^OTBDPS Me Me

h v

65 4 3 2 1 0 ppm7 I-mlfc-98 HMQC CDC13

L l II 11 A ^ M — M ppm

H H 20

h l O O

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hl40

OBn hl60 OTBDPS Me Me

8 7 65 4 3 2 1 0 ppm t/Sf a_ M E r?

€8.10 XT

26 .64 3500 3000 2500 2000 1500 1000

OBn

OTBDPS Me Me F ile:0 'ü'5Ël496 ïdent : 6 3 " 'Win ' 100Ü£>PM Acq i l'9-JUN-2‘0~0'T) ' 0'8ï2'gT59 +0:27 "Cal ; FABLM19ü6üÜ_r ZAB-SE4F FAB+ Magnet BpM:183 B pl:19367936 TIC:911397184 F lags:HALL F ile T ext:I-m lfc-98 FABMS MATRIX MNOBA + NA 100% 239 1.3E7

9sJ Î.1.3E7 90J L1.2E7 85J 269 L1.1E7

80J L1.1E7 319 75: OBn L1.0E7

OTBDPS L9.3E6 7 0 : 211 Me Me L8 . 6E6 6 5 : 677 60J L8.0E6 227 5 5 j L7.3E6 50: L6. 6E6 45: 299 L6.0E6 40: L5.3E6 35: 289 L4.6E6 30: L4.0E6 L3.3E6 2 5 : 259 359

2 0 : L2.7E6 467 i5 _ [ 392 487 i_2 .0E6: 392 567 1 0 : 329 L1.3E6 379 437 5: 409 L6.6E5 49 545 457 499 523 1555 635 657 ji 1577 599 LO.OEO 0 lu ... T r "n' 200 250 3Ô0 350 4Ô0 450 500 550 650 700 m/z I - m l f c - 9 9 PROTON CDC13

HO' OBn

Y ^ j ^ O T B D P S Me Me

JX. 1»JL ______/V

t- I ' I - r I -I—!■ I- r I -I—I—r T~i—I—r—r—I—1 1 —1—I I I—I—I—I—[—I—I—I—r—r—I—r—r—i—|—i—i—i—i—i—r—r—i—r—|—r—r—i—r- i—r i—i—r—p-i—i—i—i—i—i—r—i—i—|—r—i—i i~'i t—i i ■ i " i i i 0 ppm I-mlfc-99 CARBON CDC13

HO' OBn

^I^^N^^OTBDPS Me Me

«W « «dMtil11 ulLw* iwk«nL

200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-99 DEPT CDC13

HO OBn

OTBDPS Me Me

■ I mi WHii U I

I I 1 I i‘i I'l I I r i i"i n n -r r-i i i I I I I I I I I I I I 'l iI I I In I T I TT I n I "|“m | V j I I 1 T 1 1 I 'r i r i * «' # j i i i i r i i i v j i * i ■ : # i i i | i i i r n y r r v i 'i t t t r j 'i 'T r i t i 'I'l i 11 i n i 11t 11 t' i i i i i i t i '| n i n “fT i"i"i'I'lv ri"i"j i' t“n rr'ci'i"|T*'n'ri“riT T T " f t'l'i"i'r| t t 'i 'i''I i"i"i i '| i"i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm

4- -f H i - 0

-1

-2

-3

- 4

- 5

HO OBn 4 1 OTBDPS - 6 Me Me

- 7

7 6 4 35 2 1 0 ppm I - m l f c - 9 9 HMQC CDC13

ppm

H - 20

-j - 60

H O ^ QBn -100

Me Me -120

-140

-160

8 7 6 5 4 3 2 1 0 ppm PErUClN ELM ER

77.07-1 XT

CXI ot>

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2 0 ,8 0 * “^— cm 3500 3000 2500 2000 1500 1000

00/06/14 15:24 ft HO OBn X: 16 scans, 2.0cm -l, apod none OTBDPS Me Me File; 0'ügEl497"'ldent ;4 3 Win l'ÜOÜPPM Acq:l^-JUN-2UÜU U8:i4:49 +U : Cal : FABLMI^UbUO! ZAB-SE4F FAB+ Magnet BpM:121 BpI:19959808 TIC : 514926720 Flags:HALL F ile Text :I-mlfC“99 FABMS MATRIX MNOBA + NA 100%. 239 269 5 9E6 95i L5 6E6

90J L5 3E6 85J L5 0E6

80J L4 7E6 209 HO OBn 635 l4 4E6 75J 249 70i OTBDPS L4 2E6 Me Me 65J L3 9E6 60J L3 6E6 55i L3 3E6 50 j L3 0E6 227 45J L2 7E6

40J L2 4E6

L2 1E6 289 30 j L i 8E6 259 525 2 5 : L l 5E6 L l 2E6 20J 377

l 8 9E5 i d 299 319 359 503 30S loJ 329 391 L5 9E5 339 41 677 L3 0E5 5Î 49 615 444 485 541 567 593 651 0. liuuUiUll lJ)l|lLlln^lilllliyuil Uiki l o OEO

2 Ô0 250 300 350 4Ô0 450 5Ô0 550 6 Ô0 650t 7Ô0 m/z I-mlfc-100 PROTON CDC13

j " l'"l I' l' I'" !' r " f f “I ■ r"“t " J I' ' r “'i t t ' c 0 ppm I-m lfc-100 CARBON CDC13

OBnHO

OTBDPS Me Me

Miu

200 180 160 140 120 100 80 60 40 20 0 ppm I “inlfC“100 DEPT CDC13

H O ^ OBn o r Y ^ N ^ o t b d p s Me Me

I'l iT'rri't'iTTT r-rn | t ii n i i i i |i n r f n rr r'l I 'lTi'i i i i p i i n i i ri'i i r r i r i"i r i | i r i rn i i i i i n i‘i irii i i i rf i i i i i-j i i i "i r n-rr j-i r r ri'i' r i'i"i i r 11 n "ii r |f i 11111111 r'ln I i I rI i rir i I i r'rri'i'Ir r r n -| i i 'i-n i vr r n ri -ii i r|i-|"r r i m -n i TTri'i'i'i rn i i ri r \ t i i r i ryi-i i i n -ivi'i i-n Ti-n-n -|-n nI'I'l 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm

4 44 K f- 0

-1

-2

- 3

- 4

- 5

- 7

76 5 4 3 2 1 0 ppm I-mlfc-100 COSY CDC13

ppm

4 44 +- 0

-1

-2

-3

-4

-5

-7

76 5 4 3 2 1 0 ppm ppm T

1 - 80

-100

120

OBn -140 Me Me 160

8 7 6 5 43 2 1 0 ppm FERKtN ELMER

66.22

IIP 3 3 .37

704.0

2 7 .3 9 3500 2500

00/06/21 10:04 ft X: 16 scans. 2.0cm -l, apod none OTBDPS Me Me File:00'SEl717 Ident :'2Z6 Win' TÜÜÜPPM Acq:13-JUL-2ÜUÜ 09:37:44 +ü:lb Cal:FABLM13U /UU_l ZAB-SE4F FAB+ Magnet BpM;136 BpI:22062694 TIC : 769738496 Flags:HALL F ile Text :I-mlfc-100 FABMS MATRIX MNOBA + NA 100& 239 _1.0E7 9.5E6 L9.0E6

85, L8.5E6 8.0E6 80 HO OBn

75. OTBDPS 21 7.0E6 70 Me Me 541 65. 6.5E6 6.0E6

55, 5.5E6 269 50, 5.0E6 227 45, 289 4.5E6 40, 4.0E6

35. 3.5E6 651 249 3.0E6 25, 2.5E6

2 0 , 2.0E6 15, 1.5E6 309 329 631 1.0E6 461 409 L5.0E5 379 519 47 0 531 557 577 609 1 0 OEO 200 250 v i o m/z I-m lfc-120 bottom spot PROTON CDC13

OH OH, O Bn

NC OTBDPS Me Me

'' T 9 8 4 ppm I-mlfc-120 bottom spot CARBON CDC13

NC OTBDPS Me Me

#WW#wwMmww * * i*wJL

1 ------'— 200 180 160 140 120 100 80 60 40 20 0 ppm I-mlfc-120 bottom spot DEPT CDC13

OH .OK 'OBn

NO OTBDPS Me Me

JnUl, Jww

I'l' I I'l rn I r'l' 11 I 11 I I'l I 'I i rn i i i i i | i i i rri riTpTrn i I i i i j i i i i i i i i i | i i i i i i i i i j i ri i iii rr | i i rn'i r t'i'jti i i i i m j' i' i'i i ri 11 i |'i i ri' rTi'i i j i i i i i i r ri j' it i"i i rn I'pnTnri rr r j i i i rry i n j i i i i i r ii i j'rri"i i i i i i i r i i"i"i i i i j' i i ■ i ■ ■ i ■ t t i ■ i ■ j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm I-mlfc-120 bottom spot COSY CDC13

_ A _ m 1La JL JUA. JU L1 1 ppm

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2 -4

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7 6 3 2 1 0 ppm54 I-mlfc-120 bottom spot HMQC CDC13

ppm

-100

OH -120 OH O Bn

NC OTBDPS -140 Me Me

-160

7 6 5 4 3 2 1 0 ppm r^Ef^KtN ECMEr?

57.95 (O (O

CVI

CVI (O o • too ow 00 ) CM

27.76 3500 3000 2500 2000 1500 1000

NC OTBDPS Me Me Fiie:ÜÜgËia23A ''ïd 'en 't';'31 T "V? i n l'ÜÜÜPPM Acq;27-JUL~2ÜÜ"Ü 11:U1:Ü2 +0:36 Cal ;FABMM^VUVuO ZAB-SE4F FAB+ Magnet BpM:154 B pl:10172007 TIC :150876800 F lag s:HALL F ile Text :I-mlfc-120 BOTTOM SPOT FABMS MATRIX MNOBA + NA

1 0 0 % 5 4 6 3.3E6 95J L3.1E6

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-yiM,. ,,, ...f- y ,M.n , , I'M—r-i'-r I I i" i" ' I'"I !■" I "I I I -I 'l" I" I I t I T"» r -1— r -1— |-'T"i— I— r-T— n — r nr i—i—> r 'i i i i—i—r-|—i—i—r-i—r-r-r ppm I-mlfc-120 top spot CARBON CDC13

k ^ C N OBn

HO' Y^^Y^OTBDPS Me Me

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1 I I I'f i I I I" I ' 'I I I I I I I I i I I I r I T TT-r r i-i'r in r r i I'l'! r 1- 1 [ r II'I T-rv-rT p r i-n - i i "i i i ' |-T-i-r-i"i t i r r | r i i"i i i I'l'iyi i-i i T : : i i j i ii i ' i ■ i r r r j t 'r r'r t-i-i-r r^ -rri- i i i i i'r-f t t " f i t t t t 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm I-mlfc-120 top spot DEPT CDC13

HO"" y '^ Y ^ O T B D P S Me Me

'I I I I I I I I I I I I I I'l'i'i I I I I in I I I I I I I T n rrrn-i-rrn-ii-i-r T|"n-| rr r-r i i j i i-M n rn | ri 11 i i i' 11 j tTr r iT i'vr j-r i' rn n I'l | i ri i i rr i m -j t r 'i i -r i r i-i |"i rn > i i i i j i i i i i t ri i | i i i i r i i i i j I'l i i i i 'I'l r j rm -n-rri j i i i i ri i i i | i i i i i i i ■ i [ i M J ■ i i . i | ■ i i ■ j i i . j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm I-mlfc-120 top spot COSY CDC13

_ w L JL.JL_XA.AWl. j l l ppm

-1

-2

i -4

-5 OH

k ^ C N ,OBn

HO"" y ' % ^ otbdps Me Me -7

TT 0 ppm HMQC CDC13

ppm

: 80

100

-120 OH

k ^ C N DBn -140

Me Me -160

8 7 6 5 4 3 2 1 ppm r^ERKlN E L N E f?

5 6 .5 9 -

-r

25.43 3500 3000 2500 2000 1500 1000 cm“‘

OH 00/07/20 10:31 ft X: 16 scans, 16.0cm-l, apod none, flat HO OTBDPS Me Me pilë':ÜÜSÉ1824 Idëht:4_5 Win lÜO'ÜPPM Acq:27-JUL-2'ü'ÜÜ " 11 : Ü5 :3 3 '+ü :33 Cal :FABMM^vu /uu__i ZAB-SE4F FAB+ Magnet BpM:136 Bpl:2492160 TIC:48940080 F lags:HALL F ile Text:I-m lfc-120 TOP SPOT FABMS MATRIX MNOBA + NA 1 0 0 % 568 7.9E5 95_ L7.5E5 90_ L7.1E5

8 5 - L6.7E5 80i L6.3E5 OH 75J L5.9E5 70J 'OBn L5.6E5

65J HO' OTBDPS L5.2E5 60 j Me Me L4.8E5

55J L4.4E5

50J L4.0E5 45: 13.SES 40: L3.2E5 307 ■ 35: L2.8E5 30: L2.4E5 25: L2.0E5 546 20: L1.6E5 15: L1.2E5

10- L7.9E4 349 3gQ 584 L4.0E4 5_ B36 I 360 -20/1 /11 o 4.-SW zihsf 489 ^20 528 0: Liililub |Ayih^LO . OEO 2Ô0 2^0 24o 2è0 2È0 3Ô0 3^0 340 3è0 300 4Ô0 4^0 44o 4èO 4È0 5Ô0 520 5^40 56 0 5Ô06Ô0 m/z I - m l f c - 1 2 6 PROTON CDC13

OBn

NC''Y'^|^O TeDPS; Me Me

ppm I-mlfc-126 CARBON CDC13

OBn

NC OTBDPS Me Me

\ F I V I I ' I» ^ I / * i'Ti'~vI ' v“i * f' “t'T I I * T I * ]r I H I ^ I f I 1I Ir'lT r" n 'T r't T I | I i i i i r i i w nT"T" p r r"^nr"T i i i i i i T"| ' rT 1 7" I i i i i ' j 'i'"i "i f i ' r i 7 i j i"TT^ i'"r 1 1" 1 "T | i t r“i "ri" i 1 i"|"i t 7 ■?■!* 1 1 i 1 |“r“ 1 T'i ' i i n'T 'j" t 1 I I ['I I'l t r r ( I i -j'T'T'V'7 TT'f 'f I j T't i'l' i 'i r \ “ t |‘ 1 ■> 1 1 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm I - m l f c - 1 2 6 DEPT CDC13

OBn

NO OTBDPS Me Me

ppm

^ 0

h i

TT-r^ 0 ppm I-mlfc-126 HMQC CDC13

ppm

-100

-120 OBn

NO OTBDPS -140 Me Me

-160

V 0 ppm O O 5 s O) tn n tji § m 81 3402.3

O O 3071.0 — "3g ? 2 ^ . % & = 2060.1

Q) n-

2 3 5 8 .1 - 2237.6

1950.2 1092.9 1022.6 1732.3

1631.0

1426r&-: 1300.0

1255.3 1220.2 - 1159.9 1109.7 1069.6-

023.6 730.3 703.1 I-mlfc-138 PROTON CDC13

OTBDPS Me Me

2 1 ppm I-mlfc-138 CARBON CDC13

OTBDPS Me Me

# « w # W

n n 2 0 0 180 160 140 1 2 0 1 00 80 60 40 20 ppm I-ml£c-138 DEPT CDC13

OTBDPS Me Me

"n'Tflp "" T

IIIII III 11II11 rt'i I n I "I n I [ I 111 I ir n -j i r I I'TT ITI'I'I'» I I I I I ITT T iy i t r i'l I II I j f t I I I'M I I'j II I II I H I I'l I I I I I II I'l I I I'l II I n |T1'I I I I I IT | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm ppm

- 1.0 J # 2 -1.5

- 2.0

-2.5

-3.0

-3.5

OTBDPS -4.0 Me Me -4.5

-5.0

-5.5

-6.5

ppm I-ml£c-138 HMQC CDC13

Jii ppm

OTBDPS 40 Me Me

hioo

hl20

hl40

hl60

ppm ■V'}

r. 2 9 .5 7 ^ 3500 3000 2500 2000 1500 1000 c r - ‘ 500

00/09/23 18: 18 A 1 a f SCA;;: 16 scans. .Ocm« 4 Mt 3 û« s m o o th OTBDPS Me Me file"'(T2^E555 Ident ; 31_32" Win 1ÜÜÜPPM AcqTl8-FEB-2Ü02 1U:U8:04 +1:49 Cal:FABLMlüU2U^_l ZAB-SE4F FAB+ Kârgnet BpM:135 Bpl: 16668672 TIC : 308963456 Flags :HALL F i l e Text :II-ml f c - 15 FABMS MATRIX MNOBA + NA 100% 135 1.7E7 95i L1.6E7 289 90J L1.5E7 L1.4E7

80i L1.3E7 L1.3E7 199 309 OTBDPS 70J Me Me L1.2E7

6 5 Î L1.1E7 60: L1.0E7

5 5 j L9.2E6 50i L8.3E6 L7.5E6 45Î 339 319 40J L6.7E6 35J L5.8E6 3 0i L5.0E6 121 25i L4.2E6 20J 183 L3.3E6 154 15 : 165 419 L2.5E6 239 L1.7E6 loJ 145 261 211 227 249 269 L8.3E5 5J 279 297 . . 3 95 351 365 379 I 409 429 oJ ilWi LO.OEO 1Ô0 1^0 140 160 180 2Ô0 2^0 240 '''66 260 ' 2Ô026' 3Ô0 320 340 3 60 380 40 0 420 440 m/z I I - m l f c - 2 0 PROTON CDC13

f OTBDPS Me Me

I ■ ■ 9 4 0 ppm II-m lfc-20 CARBON CDC13

OTBDPS Me Me

2 0 0 180 1 60 140 1 2 0 1 0 0 80 60 40 20 ppm

i II-m lfc-20 DEPT CDC13

OTBDPS Me Me

11 11111 I'l rm ■1...... F 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-m lfc-20 COSY CDC13

jUL J lX -A —A. ppm

J - 1.0 2 ; -1.5

- 2.0 o # 0# o -2.5 *#

-3.5 OTBDPS Me Me -4.0 -4.5

-5.0

-5.5

- 6.0

-7,0

-7.5 1 ppm II-mlfc-20 HMQC CDC13

ppm

OTBDPS Me Me

-100

-120

-140

-160

-180

ppm P S riK lN ELMS?

45.10

o o o

29.37 1500 3000 2500 2000

02/03/21 11:39 X: 16 scans, 4.0cm-l, flat OTBDPS Me Me F i l é : Ü25E56Ü "ïdent ; 13_14+riZ13 Win"'lüUUPPM Acq:ia-FEB-^ü02 10:35:04 +U:46 Cal ; FABLMlbUl^ Olj. ZAB-SE4F FAB+ Magnet BpM:199 Bpl:14280295 TIC : 4440887 68 FlagstHALL F ile T ext:II-m lfc-20 FABMS MATRIX MNOBA + NA 100% 470 7.5E6 7.1E6 6.8E6 6.4E6 6.0E6 5.6E6 5.3E6 4.9E6 4.5E6 4.1E6 3.8E6 3.4E6 3.0E6 2.6E6 2.3E6 1.9E6 1.5E6 1.1E6 7.5E5

371392410 3.8E5 429 450 615 G55 flilàllliifiiiilliitfi.ili.iHjii. lilnlulilllii^i^uuiiüiitaiLiAlltililii..llllll O.OEO 200 250 6 ^ 0 m/z III-mlfc-27 PROTON CDC13

OH M© M©

,j...,—r r i I r I ■! i | i T i i i--i- 1 i i' j ' ' i I'v r i I'l i' [ i i i i i i i i-r-|— I I I I I I' 'I I r' I" I I I I ppm 9 8 6 4 3 2 1 0 III-m lfc-27 CARBON CDC13

OH Me Me

WWW##!* ‘ ' ''"""I""""...... «luAidlOÉiiÉilÉMlUMt.lllilÉ^MUMÉ^Éj

“T“ - f - "“ I 60 40 20 ppm 200 1 80 160 140 120 100 80 III-mlfc-27 DEPT CDC13

OH Me Me

- 1.0

-1.5

- 2.0

-2.5

-3.0

-3.5

-4.0

OH -4.5 Me Me -5.0

-5.5

-6.5

-7.0

1-7.5

ppm III-mlfc-27 HMQC CDC13

ppm

# 20 OH Me Me

-100

-120

-140

-160 fîB rV C /N ELMER

5 9 .9 8 -

(V 1 \ 1 m \ "V

8 § s

o r i

T 3500 3000 2500 2000 1500 1000 cm1-1

02/07/13 15:35 X: 16 scans. 16.0cm-l, apod none, fla t OH Me Me F ile :023E55Tldent:14_37-14Zï5 win' lOOOPPM Àcq:lS-FEB-2002 1Ü:Q1:Ü8 +2:03 Cal ;FABLM18U2U2_l ZAB- SE4F FAB+ Magnet BpM:107 Bpl:5553664 TIC : 129780576 F lag s:HALL F ile Text:II-m lfc-22 FABMS MATRIX MNOBA + NA 5.6E6 1 0 0 %107 5.3E6

90J 5.0E6 85J .4.7E6 soi OH 339 .4.4E6 123 Me Me .4.2E6

7 0 : .3 .9E6

65^ 3.6E6 181 211 .3 .3E6 6 0 : 361 137 5 5 j .3 .1E6 309 5 0 : .2 *8E6

4 5 : .2 .5E6

4 0 : .2 .2E6

3 5 : .1.9E6

3 0 : .1.7E6 147 2 5 : 193 .1.4E6 .1.1E6 2 0 : 169 321 1 5 : .8.3E5

lo J .5.6E5 .57 223 293 5: ")39 263 .2 .8E5 2,41' 79 377 391 417 0: lll|llil|liii|llll|ll ijii.iji ii iiljiiiLliii|..-^.- ijii.àji, Wj ‘rn - t I...... I "ï "'rI '"f 'T I. -f-f-f'-T -1* .O.OEO lÔO 120 140 150 180 200 220 240Î 0 260 2ào 30300 320 340 3é0 3éo 400 420 440 m/z III-m lfc-28 PROTON CDC13

OPMB Me Me

i l

-T" -n - ■r I' I I | r rr i ■ p* i i" i i |' r r 1 ' r 'i'" i ■ > ' I I I I ' r I ' I I I I I 9 8 3 2 1 0 ppm III-mlfc-28 CARBON CDC13

OPMB Me Me

JUL A hiM

“I —r “T“ 40 20 0 ppm 200 180 160 140 120 100 80 60 III-mlfc-28 DEPT CDC13

OPMB Me Me

,.».Tyrrr.n-,^ ,| ...... | ■ ■. | ,,,,,, , ■ ■. . r r r , . p ...... | ...... ,,,,,, ...... | | ...... | " ,...... | ...... | ...... |...... | ...... | ...... 190 ISO 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm III-mlfc-28 COSY CDC13

ppm

** -1.5

— 2.0 oo ## -2.5 * 2 OPMB Me Me -3.5

-4.0

-4.5

ppm III-m lfc-28 HMQC CDC13

ppm

40 OPMB Me Me

-100

-120

-140

-160

-180

T T I ■ ' ' ' T 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 ppm f^EREKfiSr ELMEr7

5 8 .6 0

•n

5 7 .2 1 CQ cursor I O 6 3 1 .0 cm“*

1 3 .4 0 3 0 00 2500 2000 1500 1000

O 02/07/13 15:51 X: 16 scans, 16.0cm-l. apod none, fla t OPMB Me Me Fiie:ü2'gE'bb7 Ideri"t':B_19 Win'lüüüPPM A c q ; 1B-FEB-2ÜÜ2 lü : 20 ; Ü1 +U:"49 CâTTFÂBLMiwu2U^_i ZAB-SE4F FAB+ Magnet BpM:121 Bpl:9259691 TIC : 142327712 Flags : HALL File Text:II-mlfc-23 FABMS MATRIX MNOBA + NA 1.1E6 100% 241 1.1E6 331 1.0E6 481 9.7E5 457 9.1E5

75, .5E5 70 . 0E5 7.4E5 181 OPMB Me Me 6.8E5 6.2E5

256 5.7E5 45, 5.1E5 4.5E5

35, 272 4 . 0E5 227 3.4E5 195 289 211 2.8E5 25. 307

2 0 , 2.3E5 1.7E5 591 321 1.1E5 471 497 ^ ^ ^ ^ 3 9 5 4 1 1427 5.7E4 511 533 579 O.OEO 250 3Ô0 m/z I I - m l f c - 2 5 PROTON CDC13

HO OH

Me' Me' OPMB

ppm II-mlfc-25 CARBON CDC13

HO OH

Me^ Me OPMB

iw*W inruJfitiii fnTi'TlAT" "T"...... Hun tiiitiWiinfîT^VS ' I'l" T— ... WWW#*##, JUwws##*" /

”T" —f— 20 ppm 200 180 160 140 120 100 80 60 40 II-m lfc-2 5 DEPT CDC13 .

HO OH

Me' Me' OPMB

i II-mlfc-25 COSY CDC13

ppm

o o - 1.0

HO OH

Me' - 2.0 Me' OPMB 1 -4.0

1-7.5 ppm II-mlfc-25 HMQC CDC13

ppm

HO p H 20 Me' Me' OPMB

e m

-100

-120

-140

-160 PERKIN ELMER

as. 09

nri ii

o o

11,29-L T 3000 2500 2000 1500

02/03/21 12: 14 X: 16 scans, 4.0cm-l, flat, smooth HO OH

Me' OPMB FiléVÜ'2^E556 'Idéiït :22 ÂcqTlB-FEB-2"ÜÜ2 +1:17 Cal :FABLMlbü2 ZAB-SE4F FAB+ Magnet B pl:9404423 TIC : 121775688 F lags:HALL F ile T ext:II-m lfc-25 FABMS MATRIX MNOBA + NA 100%. 176

463 307

HO OH

289 Me' Me' OPMB

256273

422 443 45

lilllllljlillllilllil lillliliiLlitlililillj|i|ilillllil|ü ltM IJ 2 0 0 250 600 m/z II-mlfc-26 PROTON GDC 13

TBSO OH

Me' Me' 'OPMB

r —i— I— I— I— I— I— I— |'- 'i - I — I— I— I— I— I—' ' I— 1—1—' I '' r— 1—1—' I— I— I— r— I— i— i— i— i— i— i— i— i— i— i— i— i— r —i— r —i— i— i— i— i— r— i— t— i— i— r— r I'l I -1 I I I I "I 'I'l' I I 8 4 0 ppm I I - m l f c - 2 6 CARBON CDC13

TBSOOH

Me Me' OPMB

JVJ

2 0 0 180 160 140 1 2 0 1 0 0 80 60 40 2 0 0 ppm II-m lfc-26 DEPT CDC13

TBSO OH

Me Me’ OPMB

nir

I I I i n I I I'l I VI rTTTTTTTTTTTTTTI "I i"i 'n"iT I 'lTITI ini'r I I 'I I'i"r 11 I I I I I I 1 11 I M I V M I r I I I r t t ï t I T-rrt r n-r |-r n-r-i i m i | i i i i r i i n | i ii i ri i n | ii i i i i i i i | m i t r iT i r j'Ti i r r r i - r r p T 'i'»ii m i |'f v i i m i i r r i'T i'-rn I 't i i i i i i i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 2 0 10 ppm .II-mlfc-26 COSY CDC13

ppm

3 - 1

-2

TBSO OH

Me‘ Me 'OPMB -7

7 6 5 4 3 2 1 0 ppm II-m lfc -2 6 HMQC CDC13

ppm

-100

-120 TBSO OH

Me' -140 Me OPMB

------1 -160

7 6 54 3 2 1 0 ppm f^EHKlN E L M S ?

5 4 .9 6 XT

3 2 5 5 .9 6

6 1 8 .7 cm"*

CVI CO * CM CD O)

1 .9 8 iWSe 3500 3000 2500 2000 1500 1000

TBSO OH FTTë :ü2gEbb8 "Ident :b_l'ü Win lüüÜPPM Acq: 18-£^EB-2 0Ü'2 1Ü:24:T9 +0:29 Câl:FABLMlWU^UZ_l ZAB-SE4F FAB+ Magnet BpM:79 Bpl:14286166 TIC : 158431040 Flags:HALL Text:II-mlfc-26 FABMS MATRIX MNOBA + NA 100% 577 _1.3E6 L1.2E6 L1.1E6

l1.1E6

l1.0E6

l 9.4E5

L8.8 E 5

OPMB l8.2E5

l7.5E5

l6.9E5 L6.3 E 5 L5.7E5 L5.0E5

l4.4E5 L3.8E5

l3.1E5 L2.5E5 -1.9E5

l1.3E5

l 6.3E4

lO.OEO 6É0 m/z II-m lfc -2 7 PROTON CDC13

TBSO

Me' Me' OPMB

.i|.. y .1 >1 III. I '- 'i'- I I' I •■f I ' 1 r ' T‘T i-.T.'-T'-v—I i>-"r I r" I I i-TT r 1—r-nr"r T’I . “P" I ■ ■ ■ ■ T"

' ' ' 9 8 7 5 3 1 0 ppm I I - m l f c - 2 7 CARBON CDC13

TBSO

Me OPMBMe'

200 180 160 140 120 100 80 60 40 20 0 ppm II-m lfc -2 7 DEPT CDC13

TBSO Me Me' OPMB

■i"i II I T'l 11 I' I I I'l I I I'l'i'i f r ITT n I I I I 11 I I I I I I j I II "I ir i i I'l i | I'l 11 rt-f i i | i i i i i i i 11 |"i r i n rn i | I'l i i ir i i rp i i ii i n i j i i i i i i i i' ij i i i I'l n i i I'l'rn i ■ i ■ j i i j ■ ■ i i i i ■ ■ ■ j ■ i i . ^ ■ i i i j i ■ ■ i i i ■ i ■ [ ■ i > ■ ■ ■ ^ | ' ■ ' > i ' ' ■ ■ [ ■ i ‘ ^ ' p * ' ' ' ^ | 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-m lfc -2 7 COSY CDC13

UL jyuL JlAJl É ppm

-1

- 5 -2

C»

TBSO tD 8 Me Me' OPMB -7

7 6 5 4 2 1 0 ppm I I - m l f c - 2 7 HMQC CDC13

JUL juU. J ppm

-100

-120

TBSO -140

Me Me' OPMB -160

7 6 5 4 3 2 1 0 ppm i=*erWC//S» ELMS?

54# 48 - %T

oî «

56.21 1

65 0.9 cm"^ -

I Î to to to 4 to m to OJ 0 ) Oi

8 .9 6 - ’*—j 3500

02/08/03 10:48 X: 16 scans, 16.0cm~l, apod none, f l a t TBSO |F ile:ü 2 'gE559 Ident : 36_38 Wiii' lüÜÜPPM Acq : 1B-FEB-2ÜÜ2 1Ü:29:Ü9 +2 : ü'8' Cal : FAÈLM1BU2 U^_l ZAB-SE4F FAB+ Magnet BpM:89 B p l:19180204TIC : 593664576 F lags:HALL F ile T ex t:II-m lfc-27 FABMS MATRIX MNOBA + NA 100% 214

OPMB

.2E5 .3E5 .4E5

395 421

653 679 II- m lf c - 3 8 PROTON CDC13

TBSO

Me OPMBMe'

^ * I'll I I" i~I” I I ' I ' r I 'r I""I "I I ' Î "r '1 ' 1 i i * |" i t i i r i i"i 'r" ^ " i i i i ~t -y-y i —i" i i i "i~ i • i r i i r i' r 'i I r- i i r*'i" I—i—t i T i -i"'!' -j- T—I I T—r i 1 - 1 I I'l I r 'r I 'l r | T"'r i r-r i r i r ’ • ‘ I 6 5 4 3 2 1 0 ppm II- m lf c - 3 8 CARBON CDC13

TBSO

Me Me' OPMB

n r 200 180 160 140 120 100 80 60 40 20 0 ppm II-mlfc-38 DEPT Me CDC13

TBSO

Me" Me" OPMB

piTiTTim jTiTrTri'iTj'Tn fi"i~i i i | ri i n~rm'p'i' r i rri i i pi i i i T'l n i j ri i n i i I'l j i r n i' i i i i j r rri i i i i' i | i I'l in i r r | i i r"i i t t i i | i r i i i i i i i j i r 11 r i i i i -j fTi ri i i"i » 1 1 i v i i i r i i j i i i i rrm -j n i n i"i' i i j i r t rr i i ri'j' i i'i'i"i~n i i'|Trri'r"i i n~jT rnTTTi'f jTTiT n r i i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-mlfc-38 COSY CDC13

JUL jyiiJJJLiLa ppm

■0

1 /

_ 1 , L L L . I t I I I I Me T T«T*- ■ r r r

TBSO k Me I Me b P M B 0 I " I I I •II» I l_ _ A .IÇ . h 7 I $ Id

1 T H 'I' 1 "I" I T r I I' i"i I f I I I 'I ■ I I I I "I y I i r i r i m I I I I !■ I I ...... I I I I 7 0 ppm II-mlfc-51 PROTON CDC13

TBSO

Me' Me' OPMB

T -r-ry I J I I » I I « I f I i' I I T ^ \ I r ' 1" "T ™T' I ' “ I ■ I '■'! ' 1 I I I i- i-T r r I I I ‘I—I I ' I 'l r r i i r-|' nr 6 2 1 0 ppm II-mlfc-51 CARBON CDC13

TBSO

Me' Me' 'OPMB

200 180 160 140 120 1 0 0 80 60 40 20 ppm II-mlfc-51 DEPT CDC13 Me

TBSO

Me Me’ OPMB

' ‘ ' ' ' ^ [ ‘ ...... I ■ ■ ■ I I M I I I ■ 11 ; ■ I I r 11 I I I t ■ ■ ^ ; . I J I . I I M I I j I ■ I < ^ I I ■ < j T n t 11 m j i i i t i i 11 i | t t i i i n i i'j i t i r i i i'i r y n r i i i i n | i r ii i i 'i i 11 I 'l i' i n~i i r j I'l i m i i r i j I'l i r~i r i r r | n i i i i n i j i i i i i i i r i'pr r'f i i m i j i i r i'i i "i i t j i i t i i i i"i i 1 1 n i i i 11 i j 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-mlfc-51 COSY CDC13

-Xs__A. J u U L ppm J

J -1

I------2

a- - 4------I------3

« 4 TBSO

Me' Me OPMB

-7

7 4 3 2 1 0 ppm II-m lfc-101 PROTON CDCL3

I ' I-' I 'I " I » " I I j I ' I " I ' t ' I I I'l I p-"»...... I ' I ...... I' I" j I I T - I ' j " I I' ' I" j ■ > ' I I I I ■ I ' r'■ I- I I I V r ' r" I f ' i i - r- [—1 - i" ■i -i " j ■ 1 1 r | 1 11 1 | 1 1 1 1 " 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 ppm II-m lfc-101 CARBON 13 CDCL3

#wU#L

' I ' I ...... I., . 111 . . JI 111 . . 1...... I J------.. 111. r-rprn "T" 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-mlfc-101 DEPT CDCL3

-rp- m "|" I f I I I I I' I' I I I I rp i I'll i-r I 'I I' I II '11' “T ^ 20 10 ppm 150 140 130 120 110 90 80 70 60 50 40 II-m lfc-101 HMQC CDCL3

Il Ll > L ppm

-100

-120

-140

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 ppm eu 00 en (O CD 00

53.48

659.6 cm“ ^ o eu o

en eu euO)

18.54 3000 2500 2000 1500 1000 II-mlfc-113 PROTON CDCL3

OH HO

Ï I"" I f t I "" T'""1 I I I Ï 1 I 1 I 1 I I I I I " I I rI "I j...... I I I I I 1------1------1------1------1------1------1------1------1------1------1------1------1------1------1 j 1------1------■ I1------"' 1 i------" 1I------1 I------1-----T------I"1------I1- } 1 I ' I 'M I I 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 ppm II-m lfc-113 CARBON 13 CDCL3

A W||#wWW w w & A iiL #

■rrr‘>*‘i^vi I iTi'i'fi I I f 'li 'i' i I I I I I I » 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm II-m lfc-113 DEPT CDCL3

OH HO

I I I I r'i' i I I I I I I I'l I "I m I I I'lT i , , ^ , y-j I J I I I I j ...... I.■'■■■"■ . I...... ■ "I"""' ■■■'I'l I I ...... ' j"...... J I I I I f I I I I J I » I I I I I I I j I I I I I I I I I I > » » » * ' » ■ ^,,,,,,,,, I j , 150 140 130 120 110 100 90 80 70 60 50 40 30 2 0 1 0 ppm ppm

o to J - 1.0

- 2.0

-2.5

-3.0

-3.5 OH *4 HO -4.0 4

-7.5

7 ppm II-m lfc-113 HMQC CDCL3

ppm 10

20 OH HO 40

hlOO hiio

f- 1 2 0

H130

hl40

150

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 ppm ( f s i

54.98-

445.2 cm“ ‘ CO cu O) o

o o X-#

25.11 3500 3000 2500 2000 1500 1000

01/11/05 10:41 X: 16 scans. IB.Ocra-l. apod none, flat, smooth Pile:UiaB3316 Id.ent:Ul_lV Win 1ÜÜU&DÜ Xcq:13-N0V-%ÜUl U:bV:ia +u:4ü Cal:FAamxjxx

OH TBSO

• r~ • ' • I ' • ' ' I ' ■ ■ ‘ I ^ ‘ ‘ I ■ ^ ■ I ■ ■ ‘ ■ I ■ ■ ■ ' I ■ • ■ ■ j ■ ■ ■ ■ I'T 'f- r I...... I ■■ I I |- I . r-. 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 0.0 ppm II-mlfc-115 CARBON CDCL3

OH TBSO

m iN*<*wwwwiirw^^

n r p n [ I I I ■ I ii 'i 'r'i I I I I 'p r i^ t'i'T'i'iT ^'rTTi T i'i i i j i i I't < i i > i [ i i r i'i "i | r-t i ■!' i j i i fi i i r i i | i i i i i"i i i i j t i "T" 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-mlfc-115 DEPT CDCL3

TBSO

I I I I I I iT'i'i iTTi 'iT i I I I I I'l I I I r i't'i I I ri"iTTi-r'| r 'l i it t i n i i'i i i m i i i | m 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-mlfc-115 COSY CDCL3

ppir

-0

-1

-2

-3

-4 OH TBSO

-7

0 ppm 53.994

o cu '3-

v-l

3500 3000 2500 2000 1500

01/11/05 14:24

X: 16 scans, I6.0cm-1. apod none, flat = ^ S n B u II-mlfc-116 PROTON CDCL3

TBSO

-r-t-i-y-T- 6 4 2 0 ppm II-mlfc-116 CARBON 13 CDCL3

TBSO

I '1111 T^^^nn^^M TT^*nrm n"TT^^T^ri"r^^^^TfTn^^T^T^TTr^nT“r ^ ^ ^ r F i “n ^ ^ T ^ T r T T T ^ ^ 7 7 t'ritTj rm^nn^n^T^TT^rtT^rT^^nrrf^nH I r 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm II-mlfc-116 DEPT CDCL3

TBSO

I I [ M 1 1 r i I I n I I I I I I I I I 'p 'i I I I I I I I I'l I I I I I I I t I I I I I I I I I I J1-I ■n I j rrri 111 11 j 111 i i 11 i i j i » ■ » » » > » » | « » « » * » » ■ « | » ,...... 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm ppm

o ri 9

-2

-3

-4

-7

-8

ppm II-mlfc-116 HMQC CDCL3

ppm

40 TBSO

-100

-120

-140

ppm f=BFeCW tajMGr?

51.10

48.33

620.5 cm***

- Oj

28 3500 3000 2500 2000 1500 1000

01/10/27 14:43 X: 16 scans, 16.0cm -l, apod none, f l a t , smooth TBSO ^ ^ S n B Us Pile;ül^Ë4317 I 3ent::2_5 Win' lOOOPPM Acq:13-kOV-2001 15 :10:013 +0:14 Cal fPÆ5mi31T01'Zl ZAB-SE4F FAB-f Magnet BpM:184 Bpl: 1096320 TIC:39226884 Flags :HALL File Text:II-mlfc-116 FABMS MATRIX MNOBA + NA 433 -7.4E5 I.7.1E5 L6.7E5 16.3E5 L5.9E5 5.6E5 5.2E5 4.8E5 .4.5E5 4.1E5 3.7E5 3.3E5 3.0E5 2.6E5 2.2E5 1.9E5 1.5E5 1.1E5 919 7.4E4 586 732 3.7E4 liL l 723 il 807 870 36 L O.OEO eio 650 7Ô0 750 800 8^0 90 950 m/z III-mlfc-30 PROTON CDC13

TESO OH

'OPMB

I I ' l ' ...... I I I I I r " i I I ' T I " j I I I I I I I I I I -I I I I I I I ' l"T ' I ' ' ■ ‘ .. 8 7 5 4 3 2 ppm III-mlfc-30 CARBON CDC13

TESO OH Me' Me 'OPMB

AUL JuJl

ppm 200 180 160 140 120 100 80 60 40 20 III-mlfc-30 DEPT CDC13

TESO OH

M e ' Me' OPMB

I 111,1 |I I'l l I n I I 11 rriTMi n 11111 111 H'n i 11 11 11 [ II 11 111 rij 11 11 i-jTrin t [ i 111 1111 i [ i 111 11 u i [ 111 11 i i rrpi'rti n i i [ i i i i i i J J i | ■ J ■ , i ■ ■ , ■ | . i . ■ . i i ■ j ' ' | ' 1 ‘ ' ...... | ...... I " I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm III-m lfc-30 COSY CDC13

ppm -0.5

- 1.0

-1.5 Me - 2.0

1 -2.5 TESO p H

Me -3.0 Me' b P M B -3.5 m 1 -4.0

-4.5

-5.0

-5.5

-7.0

*-7.5 ppm III-mlfc-30 HMQC CDC13

^ ^ — ppm

- 20

TESO OH

Me' Me' OPMB

-100

-120

-140

-160

ppm ffE R K Z J V

37.62

37.38 CURSOR 897.2 c m -* - o

3500 3000 2500 2000 1500 c n r * 1 0 0 0

TESO OH

Me' Me OPMB lase: 8 + 1 fe: VG70-SE Positive Ion FAB 600000Q 121

550000C Sample: III-MLFC-30 Instrument Resolution: 9,000 500000G Theoretical Mass (C26H4504ISi): 577.22097 (M+H) 450000C Measured Mass: 577.22086 Error: 0.2ppm 400000G Me 350000C

300000G 577 307 TESO PH 250000C Me' 200000G OPMBMe'

150000C

100000G 220 500000

m/z Ill-mlfc-33 PROTON CDC13

TESO GIBS

M e ' Me' OPMB

uu /

' ' I ...... ' r '1 t " nr" I I ' !■ I i " I I ' [■ 1- r I I I r I I I' I I i' 9 7 4 3 ppm III-mlfc-33 CARBON CDC13

TESO OTBS

M e ' Me ‘OPMB

WWW* w ww#wW wwww w ##ww m #J « A WWW wmiwNmm «AwwAJw »h J "Lw

“T“ ppm 200 180 160 140 120 100 80 60 40 20 III-mlfc-33 DEPT CDC13

TESO OTBS

Me' OPMBMe'

I I'lj i i r n ri»rp i->TiT»ii^Triri11 1 1 ^ r m - r r i 1 1 1 11 r r i'r r ix p -ryi i r n 11 i i 1 1 1 1'l 11 y i i i 1 1 1 1 1 1 [ 1 1 i i i i r iTj i T ii-niT ri p r i ' i r n t i ['n "n"rTTTT| 1 1 i i | 'i"i't'n T i"i r[ 1 1 1 111 r i "I |"i ri rt r i 111111111 n 11 ■ m ' • ■ • ■ j t ■ ■.. | ■ 1 1 1 1 1 1 1 [ i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm III-mlfc-33 COSY CDC13

ppm

r “ 1 r O

-I rl

J r 2

I -3

h 4

Me ------1 y h5 in OTBS ♦ J Me r6 Ms OPMB

i t h i ♦

I" I i~ i'I ■! I I I T-i "r~i-r'T I ~i■ I [VI iT T -i i-i t I I I I I i'~[-i "I-1 [-I-I-1 i-'r-i~i T ~i-| I I I I I'I' I !■ I I r i-i T - i - r ' i ' - f | - T i-r I" I-I i i r | i "i i i i 2 1 0 ppm III-mlfc-33 HMQC CDC13

ppm TESO OTBS

M e' Me OPMB

••

-100

-120

-140

-160 r 0 ppm 45.92 %T

o (U o

33.64

732.4 cm~% s

5.00 3000 2500 2000 1500 1000 cm'

Me 02/07/20 15: 04 X: 16 scans, 16.0cm -l, apod none, f l a t TESO OTBS

Me’ Me OPMB B U e H ^ ? :^ 1 n t:^ 4 § 3 ® 0 Î)6 Sample: VG70-SE Positive Ion FAB 100% 121

90%

80% Sample: lll-MLFC-33 70% Instrument Resolution: 6,000 Theoretical Mass (C32H5904ISI2): 713.28940 (M+Na) 60% Measured Mass: 713.28834 Error: 1.5ppm 50% Me 40%

30% TESO OTBS

20%] Me Me OPMB 360 10% 165 430 71.3 0% 251 J__jL J A_ 51.0 50 200 300 400 600 700 800 m/z III-mlfc-35 PROTON CDC13

HO OTBS

Me' Me' OPMB

9 8 7 6 5 4 3 21 0 ppm III-mlfc-35 CARBON CDC13

HO OTBS

'OPMB

^ ---- JUU

ppm 200 180 160 140 120 100 80 60 40 20 III-mlfc-35 DEPT CDC13

HO OTBS

Me Me* OPMB

111111 rrrr'TjT riT 'n 'i

Me ppm

HO OTBS -0.5

Me' - 1.0 Me' OPMB -1.5

- 2.0

-4.0

-5.0

-6.5

-7.0

0 ppm III-mlfc-35 HMQC CDC13

ppm HO OTBS

Me' Me' OPMB

-100

-120

-140

-160 T' 0 ppm 6 2 .4 8

«n

O 6 5 .9 9

3 g 4 9 6 .4 cm“ *

fO CD 'O f

- 6.11 3500 3000 2500 2000 1500 1000

HO OTBS

Me' Me OPMB F‘iIë";'02SE2959 "laent Win lüÜÜPPM Acq:22-JUL-20Û2 14 :34 +0:17 Cal : FABLM22Ü7Û2_1 ZAB-SE4F FAB+ Magnet BpM:121 Bpl:15169127 TIC:32870864 Flags:HALL File Text:III-mlfc-35 FABMS MATRIX MNOBA + NA 100%, 2 .3E5 L2.2E5

90J L2.1E5 85 j L2.0E5 80i Me L1.9E5

f 5 J / “ ^ 1 L1.7E5 H O ^ J OTBS 70i L1.6E5 M e ^ 6 5 : L1.5E5 Me^ OPMB 60J L1.4E5

5 5 : L1.3E5 50Î L1.2E5 c/- 45J 709 L1.0E5 40i L9.3E4 180 286 35: L8.1E4 30 j L7.0E4 599 25: L5.8E4

2 0 : L4.6E4 323 15J 256 577 L3.5E4 269 197 227 lO j 438 L2.3E4 307 337 5i 392 L1.2E4 380 418 473 0 •I486 531 ^, 620 661 6941,725 765 i liil J I,A.I| iJHi |||»*1||| n |I 4-1 '“f.“V ' O.OEO i 200 250 3Ô0 50 400 450 5Ô0 550 600 650 700 750 800 m/z III-m lfc-37 PROTON CDC13

OHC OTBS Me' O PM BMe'

I ' ' ~ r - 8 6 ppm III-m lfc-37 CARBON CDC13

OTBS OHC Me' Me OPMB

Jv.

■"I 20 ppm 200 180 160 140 120 100 80 60 40 III-m lfc-37 DEPT CDC13

OTBS OHC Me Me OPMB

"T” 20 ppm 200 180 160 140 120 100 80 60 40 III-m lfc-37 COSY CDC13

ppm

- 0.0

-0.5 mo Me i - 1.0

OTBS OHC Me Me 'OPMB ## -2.5 -3.0

—m

1-7.5

0 ppm III-m lfc-37 HMQC CDC13

Me JIA ppm

PTBS OHC Me 20 Me OPMB 40

-100

-120

-140

-160

-180

-200

0 ppm FBFOC/M

4 8 .5 9 XT

5 0 .8 0 o

6 4 7 .3 CUT*

1 2 .7 5 3000 2500 2000 1500 1000 cm'

02/07/19 10:46 X: 16 scans. 16.0cm-l. apod none, fla t OHC OTBS Me' OPMBMe File:"0'2^E2958 Ident : Win TÜ'üüPPM Acq: 22-JUL-;^üü2 14:3ü:bV +0:42Cal :FABLM2^UV02_1 ZAB-SE4F FAB+ Magnet BpM:121 Bpl:10899456 TIC:47482044 Flags:HALL F i l e Text: III-m lfc-37 FABMS MATRIX MNOBA + NA

1 0 0 % 707 3 .2E5

95J L3.1E5

90J L2.9E5 85J Me L2.8E5 80: L2.6E5 OTBS 7 5 : OHC L2.4E5 70: Me' L2.3E5 Me' OPMB 65: 184 L2.1E5 60: L1.9E5

5 5 : L1.8E5 50: 436 L1.6E5 45: L1.5E5 40: L1.3E5 286 597 35: L1.1E5 392 30: L9.7E4 25: L8.1E4 241 20: L6.5E4

IS J 323 L4.9E4 418 581 lo J L3.2E4 L1.6E4 5: 452 723 P,?^,361 iT 471 499 524 , L 613 659 J73^ 780 0 >h^Li. f .III |ll|Jl<^l«i^jlll^Jll ■/ r .O.OEO 150 2 5 0 3 50 400t :50 5Ô0 551 600 650 700 750 800 m/z III-m lfc-45 PROTON CDC13

OTBS HO, OPMB Me Me Me Me

1 1 I I I" f ' I"' r"i |“i“i I I I v i-'i'-i I I I r "I r i "i r i i‘ i I'l'ii r r "x" i— r i -i r r i" \"'\ | i" i ■ t "i ' i* r t “ i ■ i' ‘T"t i i i "r i i "i" i i i ■ i ■ t - ,- r-r"!—Tr " ’p I— I r I r I i ' I—i "i I— ‘i" r - x —r I— i j— i I— i I— i I— i I— i ■ I—i' I— i "r-i I— I— I—i I— i ■ I—i" I—i— I—i- I—r I— I—' I—r I—i^'i I— I 3 2 1 0 ppm III-m lfc-45 CARBON CDC13

OPMB Me Me

M e ^ N

200 1 80 1 6 0 140 120 100 80 60 40 20 0 ppm III-m lfc-45 DEPT CDC13

OPMB Me Me

Me" " ^ 0 M e . N

P h ^ O

Tin I 11 Tl'l ! 1 f"lf"| T I I' l"l l"I t'PITt' rr t t‘l"I 'T'TTT'TTI' l"T"f TTf"l“IT’l' I Ï l'1 I TTI I I"! Ill I'I I 1 I'C II* ri-rTTi-riii-n-rn-r-ri-r-riTn-r-rTrn-T t I'l i i in » n I "'I'' n-in-n ri i i r r i i n i r i r i i i i » i 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm ppm

-0

q I------1

I------o* -2

Me 1 -4 r \ OTBS

OPMB r 5 Me

Me,. N

P hX>=° ^ O

— — -4 -I— — t I— ~ - 9 -7

• ^ 8

7 6 5 4 3 2 18 0 ppm III-mlfc-45 HMQC CDC13

ppm

r \ OTBS

OPMB 20 ^ Me

M e . N 40

, . 1 . ^ 60

-100

-120

-1 4 0

-1 6 0

•5 0 ppm FEFVC/A/ BJMER

2 4 6 .4 4

1 9 0 .0 8

1146.1 OPMB cm~* - Me Me

1 16.6 5

02/11/02 12:23 X: 4 scans, 16.0cm~l, apod none, fla t (MP è+006' Sampfe: VG70-SE Positive Ion FAB 1100000 136

-j 1000000 Sample: lll-IVILFC-45 Instrument Resolution: 14,000 900000 Theoretical Mass (C39H5807NISi): 808.31053 (M+H) 800000 - Measured Mass: 808.31033 .Me Error: 0.25ppm 700000 - OTBS

600000 - HO OPMB Me Me 500000 307 Me Me 400000

300000 Ph

808 200000

100000 220 554 460 676 496 0 L__U 63.0 j . 750 0 00 200 300 400 O T 600 700 800 900 1000 m/z Ill-mlfc-46 PROTON CDC13

OTBS TESO, OPMB Me Me Me Me

Ph'

A A aJ\. 1 _ J L

"™ n 2 1 ppm III-m lfc-46 CARBON CDC13 Me

K \ OTBS T E S O ^

L Me Me Me'"" / = 0 M e . N \ — n

P h ^ 0

I” “T“ 200 180 160 140 120 100 80 60 40 20 ppm III-m lfc-46 DEPT CDC13

OPMB Me Me

I M 11111111111 n ryWTTTrmrp r .... I'" 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm III-m lfc-46 COSY C D C 1 3

jy ppm “0

Me O o -1

OTBS TESO, OPMB -2 Me Me ■m Me # o Me -3

Ph’

T y 0 ppm III-mlfc-46 HMQC CDC13

JU ppm

OPMB Me Me

M e ^ N

•100

110

120

130

140

150 ’T FERKIN ELMER

3 3 .0 3 - %T

CO cu o

3 2 .0 2 CURSOR 55 4.6 cm~‘ (O I o o o o r^ cu

o o

1.15 Me 3000 20002500 1500 1000 1^ \ OTBS

OPMB 02/08/13 17:26 X: 256 scans, 16.0cm~l. apod none, f l a t M% e ^ N P h ^ O ZT IPile-0r5E4ÜVÿ Idenb;lü_'lt) Win lÜUUPfM Acq: 1-NUV-2ÜÜ1 +U!4H cax:FAt)bMmXim ZAB-SE4F FAB+ Magnet BpM:121 Bpl:9357312 TIC:124271016 Flags:HALL File TextIII-mlf c-46 FABMS MATRIX MNOBA + NA -X20.00- ^8 .5E6 100% 199 L8 .0E6 95 11 .6E6 90- Me 11 .2E6 85_ le .8E6 SO K \ ÇTBS TESO. le ,3E6 TS. Ls .9E6 TO Me Me ) = 0 Ls .SES SS. M e . h \ — o Ls .1E6 6 0 . P h ^ C ) U .TES S5- u .2ES SO. i-3 .8E6 4S- -3 .4ES 4 0 - -3 .OES 3S- -2 .SES 3 0 . -2 .lE S 2 5 - 432 892 -1 .TES 2 0 - 2T4 610 -1 .3ES 1S- 944 -8 .SES 1 0 - 332 920 L4 .2ES S- WjAWLO.OEO 0 900 1000 m/z III-m lfc-57 PROTON CDC13

OTBS TESO, OPMB Me Me Me OH

ppm III-m lfc-57 CARBON CDC13

OTBS TESO, OPMB Me Me Me OH

20 ppm 200 180 160 140 120 100 80 60 40 III-m lfc-57 DEPT CDC13

OTBS TESO, OPMB Me Me Me

...... | n r | i ■ 11 ■ 1111| i I'l 11 m n m n i i ■ n 11J m . 11 rj| m ill p i I I 11 n n'l 1III III H'l 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm III-mlfc-57 COSY CDC13

-0.5

- 1.0

-1.5

OTBS - 2.0 TESO, OPMB -2.5 Me Me Me -3.0

OH -3.5

-4.0 =s -4.5

-5.5

- 6.0

-6.5

L 7 . 5 r I I- I' I r 0 ppm III-mlfc-57 HMQC CDC13

ppm

OTBS TESO, OPMB 60 Me Me Me eo OH

-100

-120

-140

-160 r 0 ppm PERKIN ELMER

53.18

55.32 CURSOR 663.6

o m

15.82 3500 3000 2500 2000

OTBS 02/08/22 10:50 TESO X: 64 scans, 16.0cm-1, apod none, flat OPMB Me Me Me n a Pile :Ü1SE4UÜÜ Ident :10__23 Win lOOOPPM Acq: 1-NOV-2ÜÜ1 12:17:25 +0:53 Cal;FABLM0111U1_1 ZAB-SE4F FAB+ Magnet BpM:121 BpI:9334492 TIC:98530368 Flags :HALL File TextIII-mlfc-57 FABMS MATRIX MNOBA + NA 203 M------— ------X 2 0 . 0 0 ------

436 OTBS TESO 365 OPMB Me Me Me OH

539 581

O.OEO lO'OO m / z Ill-mlfc-58 PROTON CDC13

OTBS TESO. OPMB Me

JL A j ____ _

I I >-T“l ...... I' I I" ' ' ' I 7 4 3 2 1 ppm III-m lfc-58 CARBON CDC13

OPMB Me Me

“T “ —r 200 180 160 140 120 100 80 60 40 20 ppm III-m lfc-58 DEPT GDC 13

OPMB Me Me

r|> 1111 n I I'j I iTi'i 11 r i I II iiTt I I I p 1 11II 11 n 1111 11 n I p iit-i i i iTpn r i i 1111 j 11111'rr r r j n - p -rr r n n r p x i ^n^^nrTTTTTTTyn^ r""""I"' I I I I 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 p p m III-m lfc-58 COSY CDC13

JL. ê I ■ â I IÂ AA A l i ppm

OTBS TESO OPMB

• # 0 • cm

r 10 8 7 6 59 4 3 2 1 III-m lfc-58 HMQC CDC13

OTBS ppm TESO. OPMB

M e

-100

-120

-140

-160

-180

-200

10 0 ppm oi o ^ CD CD ro n CD D) 3 !-»• o œ o o o 2956.1 ro 2079.0 CD o o 2705.6 !

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1725.8

1612.8

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1381.0

1301.4 1249.9

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1081.9 —

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835.3 775.6

CD IPile'ül'gË4üül ïdenb:4_lü Win lüOüP^M Acq: i-NOV-:^uui 12:22:150 +U;Jb ual;FABbMÜlllUl_r ZAB-SE4F FAB+ Magnet BpM:121 Bpl;9275802 TIC:106816880 Flags:HALL File TextIII-mlfc-58 FABMS MATRIX MNOBA + NA -1 185 -X20.00- L9

3 56 i 9 Ls Me La 393 \ OTBS L7 T E S O ^ L7 539 ,v\'\ Me Me .6 Me' ^ > = 0 H III-m lfc-59 PROTON CDC13

OTBS TESO OPMB Me Me Me'

JL JI A .1 i — i

r " 1 ppm III-mlfc-59 CARBON CDC13

OTBS TESO, OPMB Me Me Me

20 ppm 200 180 160 140 120 100 80 60 40 III-mlfc-59 DEPT CDC13

OTBS TESO, OPMB Me Me Me

Me COaEt

I IIII I-TJ>'TT I I I i I I'j 11 11 [1IITI 1111 [ I 111 T| 111 |TT| n Ml I |in I'l >1 F11 r 111 r r t Ti 11III m i I [ 11 ii 11 i n j 1111 i n i i | r iit i i ri i [ 111111 > 111 11 11 n 11 i 111 11111 > rprn i 11111 | i r r r j 11, rnTTiyriTrn 111 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm ppm 0.0

-0.5

- 1.0

-1.5

- 2.0

-2.5

-4.0

OTBS TESO, OPMB Me Me Me

P-7.5 III-mlfc~59 HMQC CDC13

— I ! > 1 i 1 ppm

-100 Me

OTBS 1------120 TESO, OPMB Me Me Me -140

-160

7 6 5 4 3 2 1 0 ppm CO sPi“ ^ 8 X œ iO | til k CD O) to I-- CO o o ro CD 3 >-*► CO w

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1711.7- 1649.6- 1612.7 — 1506.0

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APPENDIX

Table 1 . Crystal data and structure refinement for lll-mlfc-46. Identification code lll-mlfc-46

Empirical formula (^4 5 ^ 72(^7^ 12! N Formula weight 944.3790 Temperature 110(2) K Wavelength 0.71073 Â Crystal system Monoclinic Space group P21 Unit cell dimensions a = 14.6699(5) Â a= b = 7.7774(3) Â p= C = 21.9785(8) Â y = Volume 2396.37(15) Â3 Z 4 Density (calculated) 1.278 Mg/m3 Absorption coefficient 0.764 mm'"' F(OOO) 972 Crystal size 0.50 X 0.05 X 0.05 mm^ Theta range for data collection 2.78 to 25.00°. Index ranges -15<=h<=17, -9<=k<=9, -26<=l<:

Reflections collected 1 2 2 0 1 Independent reflections 8240 [R(int) = 0.0804] Completeness to theta = 25.00° 99.7 % Absorption correction Scalepack Max. and min. transmission 0.9628 and 0.7011 Refinement method Full-matrix least-squares on F^ Data / restraints / parameters 8240/13/520 Goodness-of-fit on F^ 1.154 Final R indices [l>2sigma(l)] R1 =0.0921, wR2 = 0.1143

R indices (all data) R 1 =0.1218, wR2 = 0.1227 Absolute structure parameter -0.06(2) Extinction coefficient 0.0011(4) Largest diff. peak and hole 1.992 and -1.000 e.Â'^ Appendix

Table 2 . Atomic coordinates ( x 1 0 ^i and equivalent isotropic displacement parameters (Â^x 103) for lll-mlfc-46. U(eq) is defined as one third of the trace of the orthogonalized U'i tensor.

U(eq)

1(1 ) 8136(1) 228(1) 375(1) 30(1

Si(1 ) 4472(1) -2366(3) 1261(1) 16(1

Si(2) 9687(1) -4374(2) 1551(1) 1 2 ( 1

0 ( 1 ) 6570(3) 645(7) 6500(2) 25(2

0 (2 ) 5790(3) -2355(7) 3716(2) 18(1

0(3) 5523(3) -2530(6) 1814(2) 1 2 ( 1 0(4) 8898(2) -4616(9) 1957(2) 13(1 0(5) 8739(4) -1776(7) 3553(2) 23(1

0 (6 ) 8638(4) -7088(7) 3900(2) 26(1 0(7) 8110(4) -6126(8) 4686(3) 23(2

N(1) 8450(4) -4175(8) 4057(3) 1 0 ( 2

0 ( 1 ) 7448(6) 1501(12) 6767(4) 43(3

0 (2 ) 6318(4) 312(15) 5864(3) 2 0 ( 2 0(3) 6859(5) 763(9) 5457(3) 18(2

0(4) 6511(4) 341(14) 4814(3) 2 2 ( 2

0(5) 5664(5) -532(10) 4566(3) 2 1 ( 2

0 (6 ) 5139(5) -973(9) 4982(3) 19(2 0(7) 5461(5) -556(9) 5622(3) 17(2

0 (8 ) 5290(6) -940(11) 3864(3) 30(2 0(9) 5477(5) -2661(11) 3040(3) 23(2

0 ( 1 0 ) 6113(5) -3949(10) 2865(3) 13(2

0 ( 1 1 ) 6028(6) -5719(11) 3133(4) 26(2

0 ( 1 2 ) 5945(5) -4074(9) 2135(3) 13(2 0(13) 4571(5) -2950(12) 461(3) 24(2

0(14) 3584(5) -3849(10) 1442(3) 2 0 ( 2 0(15) 4107(4) -38(12) 1267(3) 18(2 0(16) 3849(5) 422(16) 1874(3) 36(2 0(17) 3222(5) 298(19) 690(3) 42(2 0(18) 4911(6) 1134(11) 1219(4) 38(2 Appendix

C(19) 6885(4) -4465(9) 1973(3) 6 (2 )

C(20) 6697(4) -5193(8) 1292(3) 1 1 (2 )

C(2 1 ) 7592(5) -2933(9) 2130(3) 1 1 (2 ) C(22) 7455(4) -1702(8) 1561(3) 9(2) C(23) 7898(4) 27(12) 1716(3) 13(2) C(24) 7918(6) 893(10) 2341(3) 28(2)

C(25) 8197(5) 968(10) 1307(3) 2 0 (2 ) C(27) 8982(4) -4659(14) 698(3) 17(2) C(28) 9471(5) -4188(10) 188(3) 26(2) C(29) 10560(5) -6166(10) 1814(3) 19(2) C(30) 11146(7) -6682(12) 1378(4) 41(3) C(31) 10295(5) -2239(10) 1737(3) 18(2) C(32) 11131(6) -1924(12) 1477(4) 26(2)

C(33) 8649(5) -3490(9) 2397(3) 1 0 (2 ) C(34) 8894(4) -4448(11) 3045(3) 14(2) C(35) 9969(4) -4911(16) 3279(3) 26(2) C(36) 8692(5) -3327(11) 3551(3) 14(2) C(37) 8424(6) -5915(11) 4178(4) 15(2) C(38) 8205(6) -3126(11) 4553(4) 18(2) C(39) 9082(5) -2192(11) 4982(3) 30(2) C(40) 7756(5) -4493(12) 4870(3) 17(2) C(41) 7973(4) -4433(10) 5581(3) 14(2)

C(42) 8844(5) -4993(13) 5985(3) 2 1 (2 ) C(43) 9035(5) -4806(16) 6641(3) 31(2) C(44) 8367(6) -4097(10) 6892(4) 28(2) C(45) 7503(6) -3556(10) 6493(4) 30(2)

C(46) 7306(6) -3746(10) 5842(3) 2 1 (2 )

111 Appendix

Table 3. Bond lengths [A] and angles [°] for lll-mlfc-46.

l(1)-C(25) 2.105(7) Si(1)-0(3) 1.664(4) Si(1)-C(13) 1.862(7) Si(1)-C(14) 1.868(7) Si(1)-C(15) 1.889(9) Si(2)-0(4) 1.666(4) Si(2)-C(29) 1.866(7) Si(2)-C(27) 1.869(6) Si(2)-C(31) 1.872(8) 0(1)-C(2) 1.360(7) 0(1)-C(1) 1.415(9) 0(2)-C(8) 1.413(8) 0(2)-C(9) 1.440(8) 0(3)-C(12) 1.437(8) 0(4)-C(33) 1.430(8) 0(5)-C(36) 1.208(9) 0(6)-C(37) 1.191(10) 0(7)-C(37) 1.338(10) O(7)-C(40) 1.472(10) N(1)-C(37) 1.382(10) N(1)-C(36) 1.425(9) N(1)-C(38) 1.487(10) C(2)-C(7) 1.388(10) C(2)-C(3) 1.404(10) C(3)-C(4) 1.393(9) C(4)-C(5) 1.379(10) C(5)-C(6) 1.399(10) C(5)-C(8) 1.511(10) C(6)-C(7) 1.386(9) C(9)-C(10) 1.495(10)

C(1 0 )-C(1 1 ) 1.517(10) C(10)-C(12) 1.551(9) C(12)-C(19) 1.553(8) C(15)-C(18) 1.519(10) C(15)-C(16) 1.533(10)

IV Appendix

C(15)-C(17) 1.546(8) C(19)-C(20) 1.547(8) C(19)-C(21) 1.550(9) C(21)-C(22) 1.541(9) C(21)-C(33) 1.549(9) C(22)-C(23) 1.488(11) C(23)-C(25) 1.331(10) C(23)-C(24) 1.522(9) C(27)-C(28) 1.542(9) C(29)-C(30) 1.518(11) C(31)-C(32) 1.517(10) C(33)-C(34) 1.554(9) C(34)-C(36) 1.510(10) C(34)-C(35) 1.550(9) C(38)-C(40) 1.524(12) C(38)-C(39) 1.535(10) C(40)-C(41) 1.502(8) C(41)-C(46) 1.379(10) C(41)-C(42) 1.393(9) C(42)-C(43) 1.392(9) C(43)-C(44) 1.375(11) C(44)-C(45) 1.377(11) C(45)-C(46) 1.383(10)

0(3)-Si(1)-C(13) 111.0(3) 0(3)-Si(1)-C(14) 110.5(3) C(13)-Si(1)-C(14) 107.7(4) 0(3)-Si(1)-C(15) 105.8(3) C(13)-Si(1)-C(15) 110.0(4) C(14)-Si(1)-C(15) 112.0(3) 0(4)-Si(2)-C(29) 105.8(3) 0(4)-Si(2)-C(27) 104.9(2) C(29)-Si(2)-C(27) 110.4(4) 0(4)-Si(2)-C(31) 110.2(3) C(29)-Si(2)-C(31) 110.8(4) C(27)-Si(2)-C(31) 114.3(4) C(2)-0(1)-C(1) 117.0(6) Appendix

C(8)-0(2)-C(9) 109.8(5) C(12)-0(3)-Si(1) 126.5(4) C(33)-0(4)-Si(2) 130.5(5)

C(37)-O(7)-C(40) 1 1 1 .0 (6 ) C(37)-N(1)-C(36) 129.1(7)

C(37)-N(1)-C(38) 1 1 1 .8 (8 ) C(36)-N(1)-C(38) 119.2(6) 0(1)-C(2)-C(7) 115.3(6) 0(1)-C(2)-C(3) 125.0(7) C(7)-C(2)-C(3) 119.7(6) C(4)-C(3)-C(2) 118.8(6) C(5)-C(4)-C(3) 122.3(7) C(4)-C(5)-C(6) 117.9(6) C(4)-C(5)-C(8) 121.3(7) C(6)-C(5)-C(8) 120.7(7) C(7)-C(6)-C(5) 121.2(7) C(6)-C(7)-C(2) 120.1(7)

0(2)-C(8)-C(5) 1 1 0 .0 (6 )

O(2)-C(9)-C(10) 1 1 0 .1 (6 ) C(9)-C(10)-C(11) 112.3(7) C(9)-C(10)-C(12) 112.7(6) C(11)-C(10)-C(12) 109.8(6) O(3)-C(12)-C(10) 111.7(6) 0(3)-C(12)-C(19) 109.5(5) C(10)-C(12)-C(19) 111.6(5) C(18)-C(15)-C(16) 108.4(7) C(18)-C(15)-C(17) 108.9(7) C(16)-C(15)-C(17) 108.1(6) C(18)-C(15)-Si(1) 110.3(5) C(16)-C(15)-Si(1) 111.9(6) C(17)-C(15)-Si(1) 109.1(7) C(20)-C(19)-C(21) 114.5(5) C(20)-C(19)-C(12) 112.1(5) C(21)-C(19)-C(12) 112.3(6) C(22)-C(21)-C(33) 110.9(5) C(22)-C(21)-C(19) 112.2(5) C(33)-C(21)-C(19) 113.5(6)

VI Appendix

C(23)-C(22)-C(21) 115.5(5) C(25)-C(23)-C(22) 123.4(6) C(25)-C(23)-C(24) 116.4(8) C(22)-C(23)-C(24) 119.9(6) C(23)-C(25)-l(1) 125.4(6) C(28)-C(27)-Si(2) 117.6(5) C(30)-C(29)-Si(2) 117.8(6) C(32)-C(31)-Si(2) 116.5(6) 0(4)-C(33)-C(21) 110.0(5) 0(4)-C(33)-C(34) 106.9(5) C(21)-C(33)-C(34) 114.6(5) C(36)-C(34)-C(35) 107.0(5) C(36)-C(34)-C(33) 110.9(6) C(35)-C(34)-C(33) 110.7(5) 0(5)-C(36)-N(1) 119.1(7) 0(5)-C(36)-C(34) 123.7(7) N(1)-C(36)-C(34) 117.2(7) 0(6)-C(37)-0(7) 122.9(8) 0(6)-C(37)-N(1) 128.7(9) 0(7)-C(37)-N(1) 108.4(8) N(1)-C(38)-C(40) 100.5(6) N(1)-C(38)-C(39) 111.7(7) C(40)-C(38)-C(39) 116.5(7) O(7)-C(40)-C(41) 109.2(6) O(7)-C(40)-C(38) 104.0(5) C(41)-C(40)-C(38) 117.8(7) C(46)-C(41)-C(42) 119.1(6) C(46)-C(41)-C(40) 119.0(6)

C(42)-C(41)-C(40) 1 2 1 .8 (6 ) C(43)-C(42)-C(41) 119.7(7) C(44)-C(43)-C(42) 120.5(7) C(43)-C(44)-C(45) 119.9(7) C(44)-C(45)-C(46) 119.9(8) C(41)-C(46)-C(45) 120.9(7)

Symmetry transformations used to generate equivalent atoms:

Vll Appendix

Table 4. Anisotropic displacement parameters (Â^x 1 0 ^) for lll-mifc-46. The anisotropic displacement factor exponent takes the form: -2n^[ h^ + ... + 2 h k a* b* j

U" \J22 JJ33 U23 y l 3 \J^2

1(1 ) 42(1) 23(1) 31(1) 1 2 (1 ) 2 1 (1 ) 6 ( 1 )

Si(1 ) 1 2 ( 1 ) 2 0 ( 1 ) 14(1) -2 (1 ) 2 (1 ) -1 (1 )

Si(2) 1 1 ( 1 ) 1 2 (2 ) 14(1) 1 ( 1 ) 6 ( 1 ) 2 ( 1 )

0 ( 1 ) 24(3) 32(5) 16(2) ■7(2) 1 (2 ) 1(3)

0 (2 ) 19(3) 24(3) 9(2) - 1 (2 ) 3(2) 8 (2 )

0(3) 7(2) 13(3) 14(2) 2 (2 ) 1 (2 ) 0 (2 )

0(4) 13(2) 16(3) 1 2 (2 ) 0(3) 5(2) 0(3)

0(5) 42(3) 12(4) 16(3) 1 (2 ) 10(3) -3(3)

0 (6 ) 42(3) 14(3) 23(3) -1(3) 11(3) 0(3) 0(7) 38(4) 19(4) 15(3) 2(3) 15(3) -4(3) N(1) 10(3) 10(4) 7(3) -4(2) -3(2) -1(3)

0 (1 ) 41(6) 51(7) 28(5) -19(4) -4(4) -18(5)

0 (2 ) 23(4) 17(4) 18(3) -7(5) 1(3) 14(5) 0(3) 11(3) 15(5) 29(4) -1(3) 7(3) 5(3) 0(4) 25(4) 19(5) 24(3) 3(5) 10(3) 13(5) 0(5) 26(4) 21(5) 16(4) -4(3) 9(3) 12(4)

0 (6 ) 12(4) 14(5) 27(4) -8(4) 2(3) 6(4) 0(7) 21(4) 17(4) 16(4) -2(3) 11(3) -4(3)

0 (8 ) 36(5) 27(5) 26(5) -6(4) 10(4) 21(4) 0(9) 25(4) 25(5) 18(4) -9(4) 5(3) 3(4)

0 (1 0 ) 10(4) 14(4) 14(4) -2(3) 1(3) -9(3)

0 ( 1 1 ) 34(5) 20(5) 24(5) 2(4) 11(4) -3(4)

0 (1 2 ) 7(3) 17(4) 13(3) 4(3) 2(3) -3(3) 0(13) 10(4) 45(6) 17(4) -1(4) 2(3) 2(4) 0(14) 11(4) 25(5) 24(4) -1(4) 4(3) -1(3) 0(15) 9(3) 12(5) 31(4) 4(4) 2(3) 1(4) 0(16) 35(4) 34(6) 39(4) -2(5) 11(3) 14(6) 0(17) 33(4) 34(5) 49(5) -8(7) -4(4) 6(7) 0(18) 40(5) 21(5) 52(6) 6(4) 12(5) 5(4)

0(19) 8(3) 3(5) 8(3) 1(3) 2 (2 ) 5(3)

Vlll Appendix

C(2 0 ) 8(3) 8(4) 16(3) -3(2) 5(2) -5(3)

C(2 1 ) 19(4) 7(4) 7(3) -1(3) 2(3) -5(3)

C(2 2 ) 7(3) 7(4) 12(4) 0(3) 4(3) 1(3) C(23) 12(3) 2(5) 20(3) -3(4) -1(3) 4(4) C(24) 35(5) 16(5) 28(4) -1(3) 3(4) -3(4) C(25) 16(4) 14(4) 29(4) 3(3) 4(3) 2(3) C(27) 20(3) 14(4) 17(3) 8(5) 4(3) 2(5) C(28) 33(4) 29(6) 17(4) -5(3) 8(3) -5(4) C(29) 22(4) 12(5) 22(4) -4(3) 6(3) 9(4) C(30) 54(6) 31(7) 44(6) 5(5) 25(5) 22(5) C(31) 18(4) 17(5) 17(4) 5(4) 4(3) 1(4) C(32) 21(5) 25(6) 33(5) -1(4) 9(4) -9(4) C(33) 13(4) 10(4) 10(4) -3(3) 8(3) 1(3) C(34) 17(3) 17(6) 9(3) 2(3) 5(3) -3(4)

C(35) 20(3) 44(6) 12(3) -1(5) 1(3) -2 (6 ) C(36) 14(4) 24(6) 3(4) 4(3) 3(3) 0(4) C(37) 24(5) 2(5) 14(4) 3(3) -2(4) 0(4) C(38) 20(5) 19(5) 16(4) -10(4) 6(4) 6(4) C(39) 41(5) 34(6) 18(4) -10(4) 14(4) -21(5)

C(40) 16(3) 2 1 (6 ) 16(3) -3(4) 7(3) 2(4)

C(41) 19(3) 7(4) 14(3) 2(3) 2 (2 ) -4(3) C(42) 29(4) 15(5) 19(3) 3(4) 9(3) 1(5) C(43) 30(4) 30(5) 23(4) 18(6) -6(3) -4(6) C(44) 48(5) 18(5) 15(4) -7(3) 3(4) -11(4) C(45) 47(6) 26(6) 22(5) 0(4) 17(4) 4(4) C(46) 22(4) 23(5) 17(4) 1(3) 1(3) 2(4)

IX Appendix

Table 5. Hydrogen coordinates ( x 10^) and isotropic displacement parameters (Â^x 10 for lll-mlfc-46.

U(eq)

H(1A) 7971 785 6718 65 H(1B) 7537 1712 7221 65

H(1 C) 7444 2600 6548 65

H(3) 7450 1345 5616 2 2

H(4) 6869 6 6 6 4536 27

H(6 ) 4552 -1569 4821 2 2

H(7) 5093 -864 5897 2 1

H(8 A) 4601 -1214 3752 35

H(8 B) 5369 73 3612 35 H(9A) 5490 -1570 2810 27 H(9B) 4813 -3094 2912 27 H(10) 6785 -3555 3058 16 H(11A) 6250 -5673 3599 39

H(1 1 B) 6420 -6538 2981 39 H(11C) 5360 -6090 2992 39 H(12) 5492 -5044 1970 15 H(13A) 5091 -2295 377 37 H(13B) 3970 -2680 135 37 H(13C) 4703 -4183 450 37 H(14A) 3750 -5041 1375 30 H(14B) 2946 -3589 1160 30 H(14C) 3591 -3696 1886 30 H(16A) 4424 378 2240 54 H(16B) 3379 -402 1936 54 H(16C) 3579 1583 1833 54 H(17A) 2718 -522 696 63 H(17B) 3396 158 295 63 H(17C) 2992 1472 713 63 H(18A) 4698 2333 1194 57 Appendix

H(18B) 5093 844 836 57 H(18C) 5463 982 1596 57 H(19) 7201 -5414 2266 7 H(20A) 6345 -4344 982 16 H(20B) 6320 -6251 1250 16 H(20C) 7306 -5446 1213 16 H(21) 7437 -2260 2475 13

H(22A) 7724 -2254 1246 1 0

H(22B) 6763 -1549 1355 1 0 H(24A) 8295 194 2698 42 H(24B) 7266 1007 2368 42 H(24C) 8208 2035 2361 42 H(25) 8460 2066 1447 24

H(27A) 8783 -5878 635 2 1

H(27B) 8395 -3959 618 2 1 H(28A) 9639 -2965 223 39 H(28B) 9034 -4420 -236 39 H(28C) 10050 -4879 253 39

H(29A) 11009 -5845 2231 2 2

H(29B) 10206 -7193 1885 2 2 H(30A) 10725 -7180 986 61 H(30B) 11625 -7534 1593 61 H(30C) 11465 -5666 1273 61

H(31A) 9815 -1330 1570 2 1

H(31B) 10524 -2109 2206 2 1 H(32A) 11628 -2785 1651 39 H(32B) 11390 -772 1601 39

H(32C) 10915 -2013 1 0 1 2 39

H(33) 9058 -2440 2450 1 2 H(34) 8506 -5524 2997 17 H(35A) 10350 -3868 3295 39 H(35B) 10116 -5738 2985 39

H(35C) 1 0 1 2 1 -5420 3705 39

H(38) 7710 -2259 4342 2 2 H(39A) 9559 -3039 5204 45 H(39B) 8892 -1491 5294 45 H(39C) 9356 -1448 4721 45

XI Appendix

H(40) 7048 -4451 4676 2 1 H(42) 9304 -5499 5814 25 H(43) 9632 -5171 6917 37 H(44) 8500 -3980 7340 34

H(45) 7043 -3053 6 6 6 6 36 H(46) 6703 -3397 5570 26