THE STElREOSEZlECTIVE SYNTHESIS OF 13crLABELLE~ LYSINES

BY

Ross D. WithereII

Submitted in Partiai Fulnllment of the Requirements for the degree of Master of Science

Dalhousie University Haiifax, Nova Scotia

August 1999

@ CopyriDght by Ross D. WÏthereir, 1999

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For Mom and Dad

Table of Contents

Chapter 1 - Introduction

Distinct Pathways for D- and L-Lysine Catabolisn in Clostridia

Lysine Cataboiism in F. nucIeatum

Isotopicdy Labeiled Substrates Required for Lysine CataboiÏsm Studies

Synthesis of Carbon-Labefled Lysines

Lysine Labeiled at Carbon 1

Lysine Labelled at Carbon 6

Lysines Labeiied at Carbons 2,3,4, andor 5

Synthesis of Amino Acids by the Stereoselective Derivatization of Glycine

Bis-Lactim Ether Methods

Enolates of SchB Base Derivatives

Enolates of unidazolidinones, Oxazoiidinones, and Oxazoiine

Methods Based on the EnoIate Derivatization of Transition Metai Complexes

Preparation of 44odo butyronitdes Carbon-LabeiIed at Positions 2,3, and 4

A Synthetic Plan for the Repmtion D- and L - [ Z $ - ' ~ C C L ~ ~ ~ ~ ~ and D and ~- [4 ,5 - '~~~1~ys ine

Chapter 2 - DeveIopment of a Synthetic Route to 4-Iodobutyrontrie

2.1 Aikylation of Cyanoacetate Esters

2.2 AIkyIation of CyanornaIonate Esters

Preparation of Cyanodonate Esters

Tautornerkation of DiethyI Cyanomalonate

Preparation of Diethyl and DimethyI Cyanoacetate Saits

NucIeophilicity of Carbanions Derived fiom Cyanornahate Esters

Development of AkyIation Reaction Conditions

Modification of Bromoacetic Acid

Preparation of Methyl Bromoacetate

Preparation of Ethyi Cyanoacetate

Functionai Group Modincations

Decarboetholrylation of 2-Carboethoxy-2-cyanosuccinate Esters

Reduction of 3-Cyanopropionate Esters

-1 Iodide Preparation

2.43.1 Attempted Tosyiation of 4-HyditoxybutyronitriIe

2.43.2 Direct Preparation of Iodides h m AIcohols

2.5 Conclusions

Chapter 3 - StereoseIective Synthesis of I3c2-~abeLIed Lysines

3 .I Preparation of [4-13~]4~odobatyronitrile fiom [~-~~~]~romoacet ic Acid

3.2 Preparation of [2,3-t3~~410dobutyronitriie fiom [2-'3~~romoacetic Acid

3.3 Synthesis oMi-BPB-GIycme Complexes

3.4 Alkylation ofNi-BPB-Gly Complexes

3.5 Isolation of LabeUed Lysines

3 -6 Conclusion

Chapter 4 - Experimentd

General

Synthesis of Cyauosuccinate Esters

Synthesis of Cyanomaionate Esters

Preparation of SaIts fiom Cyanomaionate Esters

Experiments to Probe the NucfeopfiiIicity of Dialkyl CyanornaIonate Salts

Allcylation of Cyanomaionate Esters

Esterifkation of Bromoacetic Acid

Conversion of Bromoacetic Acid into EthyI Cyanoacetate

Decarboalkoxylation of Ethyl Methyl2-Carboethoxy-2-cyanosucchate

Reduction of Methyi 3-Cyanopropionate

Conversion of 4-Hydroxybutyronitde to 4IodobutyroaitriIe

Preparation of D- and L-Ni-BPB-Gly Cornpiexes

AUcyIation of Ni-BPB-GIy Complexes

Chiral OPA HPLC Assesment of AUcyIation Stereosefectivity

Isolation of LabeHed Lysines

List of Figures

Figure 1: The Pathway of Lysine Catabolism by Closûidia

Figure 2: Conversion of 3-Keto-5-aminohexanoic Acid to Acetate and Butyrate by Clostridia

Figure 3: Spootaneous Cyciization of 2-Amino-5-keto-hexanoic Acid to h'-~~rrohe-2-meth~~-5-c~boxylic acid

Figure 4: Borsook's Synthesis of DL-[l -'4~]~ysine

Figure 5: Maimind's Synthesis of D L - [ I - ' ~ C ] L ~ S ~ ~ ~

Figure 6: Rothstein's Syntiiesis of ~t-[l-~~~]~~sine

Figue 7: Arnstein's Synthesis of ~~-[l-'~~]l~sine

Figure 8: Bolster's Synthesis of DL-[I sine Figine 9: OIyny k' s S ynthesis of D L - [ ~ - ' ~ C ] L ~ S ~ ~ ~

Figure 10: Lindstedt's S ynthesis of ~ ~ - [ 6 - " ~ ] ~ ~ s i n e

Figure 1 1 : Rothstein's Synthesis of D L - [ ~ - ' ~ C ] L ~ S ~ ~ ~

Figure 12: W illis' s S ynthesis of L-16-' k ] ~ ~ s i n e

Figure 13 : Pichat's Synthesis of D L - [ ~ - ' ~ C ] L ~ S ~ ~

Figure 14: Field's Synthems of D L - [ ~ - " C ] L ~ S ~ ~ ~

Figure 16: Gouid's S ynthesis of ~~-[3-'~~,2-'SN]~~sine

Figure 17: Leete's Synthesk of DL-[~,,S-'~C~]L~S~~~

Figure 18: Preparation of a Bis-Lactim Ether

Figure 19: AUcyIation of a Bis-Lactirn Ether, and Subsequent Hydroiysis

Figure 2 1 : Raap's Stereoselective Synthesis of ~ - [ 2 - ~ ~ ~ ] ~ ~ s i n e

Figure 22: LabeUed 44odobutyronitriIesüsed by Raap to Stereoselectivefy Prepare LabeIled Lysines

Figure 23 : An Asymmetric Enolate AUcyIation of a Schiff Base Derived f?om GLycine

Figure 24: An Impmved Asymmetric Enolate Alkylation of a Schiff Base Derived fkom Glycine

Figure 25: a-Amino Acids are Derivatized with Retention of Configuration

Figure 26: Preparation of a Complementary Pair of Glycine Templates

Figure 27: AlkyIation of an Imidazolidinone Glycine Template

Figure 28 : Fasth' s S tereoselective S ynthesis of L-[6-' sine Figure 29: Synthesis of Belokon's Chiral Complex

Figure 30: Stereoselective Synthesis of Amino Acids Using Belokon's Method

Figure 3 I : Davis's Stereoselective Synthesis of Amino Acids using Belokon's Method

Figure 32: Raap's Synthesis of [2-L3CJ4~odobutyr~nit~e

Figure 33: Raap's Synthesis of [3y4-13~2]4~odo butyronitriIe

Figure 34: Retrosynthetic Auaiysis for the Synthesis of 44odobutyronitriIe h m Bromoacetic Acid

Figure 35: Mechanism for the Formation of Dhethyl Cyanosuccinate and Dimethyl3-Carbomethoxy-3-cyanopentanedioate

Figure 36: aallcyIation of Ethyl Cyanoacetate with Methyl Bromoacetate

Figure 37: A Modined Route to 4-Iodobutyronitrile Proceedmg via AUryIation of Diethyl CyanornaIonate

Figure 38: Rapoport's Stnitegy for the ControlIed MonoaUcyIation of Mdonate Esters

Figure 39: Tautomerizatl011 of DiethyI CyanornaIonate

Figure 40: 'H NMR Spectrum ofDiethy1 Cyanomalo~l~lfe in ChIoroform-d before Addition of TriethyIarnine

Figure 41: 'H NMR Spectrum of Diethyl Cyanomalonate in Chloroform-d after Addition of Triethylamine

Figure 42: DiethyI Cyanodonate and its Tautorner Yield the same Anion Upon Deprotonation

Figure 43: DiaUryL Cyanomalonate Saits as Synthetic Equivdents for the Carbanions of Acetonitde and Cyanoacetate Esters

Figure 44: Dimethyl BenyIcyanomalonate

Figure 45: Ethyl Methyl2-Cyano-2-carboetfioxysuccinate and Diethyl2-Cyano-2-carboethoxysuccinate

Figure 46: 'H NMR S p e m of Ethyl Methyl2Cyano-2-carboethoxysuccinate

Figure 47: 13c NMR Spectnun of Ethyl Methyl2-Cyano-2-carboethoxysuccinate

Figure 48: Possible Mechanism for the Formation of Diethyl3-Carboethoxy-3-Cyanogiutarate

Figure 49: Synthesis of Cyanoacetic Acid fkom Chloroacetic Acid

F i p 50: Decarboethoxylation of Ethyi Cyanoacetate

Figure 51 : DecarboethoxyIation of Ethyl Methyl2-Carboethoxy-2-Cyanosuccinate

Figure 52: Methmoi-Assisted Borohydride Reduction of Methyl3-Cyanopropionate

Figure 53: Tosyiation of 4-HydroxybutyronitriIe and Subsequent Conversion to the Chioride

Figure 54: An Example o f a Direct Conversion of an AlcohoI into an Iodide

Figure 55: Conversion of 4-Hydroxybutyronitrile into 4Iodobutyronitrile

Figure 56: Spthetic Route for the Prepdon of [ 4 - 1 3 ~ ] 4 ~ o d o b u t y r o ~ e

Figure 57: Synthetic Route for the Preparation of [2,3-'3~J-4-rodobiityronitnIe]4~odob~&e

List of Tables

Table 1 : Distribution of ' 4 ~ in B ~ t y ~ ~ l t e fomied h m Lysine and Acetate in Media Supplemented with [~-'~c]~cetate, DL+ -'4~]~ysine, and D L - [ ~ - ~ ~ c sine

Table 2: Prepmtions ofDimethy1 CyanosuccÏnate fiom Methyi Cyanoacetate and Methyl Bromoacetate

Table 3: Preparations of Diethyi Cyanomalonate fiom Ethyl Cyanoacetate 44

Table 4: SaIts Prepared fiom DirnethyI and DiethyI Cyanomdonate 49

Table 5: Summary of Results from Aikylations of Some Cyanomalonate Saits 51

Table 6: Results of Triai Alkyiations of DiethyI Cyanomdonate with Bromoacetate Esters

Table 7: Components Required for the Synthesis of the Desired Amino Acids 74

Table 8: Yields of Chromatographed Ni-BPB-Gly Complexes 75

TabIe 9: Alkylations of Ni-BPB-GIy Complexes with LabeUed 4Iodobutyronitriles 77

Table 10: Yields of Isolated LabeiIed Lysines 78

TabIe L 1 : ReIative Amounts of "c2-~abelled and Udabelled Lysine Present in the Recovered SarnpIes

A method for the asymmetrk synthesis of the amino acid Lysine h m glycine and

44odobutyronitriIe was developed 44odobutyronitrile was prepared fiom bromoacetic

acid in 15% yield via an eight-siep route in which the four carbons of iodobutyronitrile

can be specScally LabelIed h m [*~]bromoacetic acid. To avoid the multiple alkylation

problem encountered during the reaction of ethyI cyanoacetate with esters of bromoacetic

acid under basic conditions, the alkylation of the tertiary carbanion derived fkom diethyl

cyanomalonate was investigated. The cyanomalonate carbanion, which couid be formed

in the reaction mixture or supplied as a prefomed sdt, reacted with s e v d allcyl

broddes and iodides in polar aprotic solvents. This unprecedented carbon-carbon

bond-forrning reaction is the pivotai step in the synthetic sequence that was empIoyed to

prepare two novel, isotopicaiiy Iabelled compounds, [4-13~]4iodobutyronitrile and [2,3-

13~2]-4-iodobutyroni~et in good yield The "c-labelled iodobutyronitriIes were

subsequentiy employed in the asymmetric synthesis of the D- and L- isomen of [2,3-

13~z]lysine and [4,5-'3~z]lyshe via Belokon's method for the stereoselective

derivatkation of glycine. This represents the first hown synthesis of these '3~-1abe~ed

amino acids, which are required to elucidate the pathway of lysine catabolism in a

number of anaerobic bacteria

BPB

CoA

DMF

DMSO

DW

HPLC

KHP

NMR

OPA

TLC

TMS

2-[(N-Be~lzyIproIyl)amino]benzop henone

Coenyme A

N'-Dimethyffomamide

Dimethy1 S u I f i d e

Distilied Water

High Performance Liquid Chmmatography

Potassium Hydrogen Phthalate

Nuclear Magnetic Resonance

O-P hthaidehyde

Thin Layer Chromatography

Tetramethyldane

Acknowledgements

I am indebted to Dr. Robert White, my supe~sor, for bis patience, and

leadership over the past three years. In addition, 1 wish to thank Drs. Bruce %dey and

Stuart Grossert for reading my thesis and providing valuable suggestions. I also thank Dr.

Jim Pincock for his contri'butions as a member of my committee.

1 am etemaily gratefbi to my Iabmates Lizpe, Kamani, Feh , Cormac, Sat,

She- Nicole, and KaPm for making Me in the Iab a pleasure. Also, for their constant

support and companionship, 1 thank rny fiends in and outside of the department

(includhg the 'îv6lfpac9"): you know who you are!

I am unable to express just how appreciative 1 am of my parents. Their love,

support, and guidance have been instrumentai in the completion of this thesis, and I

humbly dedicate this work to them.

NSERC is thanked for financiai support of this project in the form of a research

grant, as weii as funding for myself in the foun of a PGS-A schoiarship. Cambridge

Isotopes Labontory generously provîded 5 g of each of the IabeDed bromoacetic acids

recpired for the IabeiIed work described herein,

Chapter 1

Introduction

Anaerobic bactena ofthe genus Fusubacterium cataboiize a number of D- and L-

amino acids as a source of energy and carbon a t o m ~ . ' ~ " D-Lysine is the only D-amino

acid that is &ed both by the penodontai pathogen F. nucleatzm and the

gastrointestid organism F. vanCum! It has been suggested that the D and L isomers of

lysine are caatabiized via different piitbways in F. nudear~m~ but the evidence presented

for the D pathway is inconcIttsive, and it may be that a lysine racemase converts D-lysine

into the L-isomer prior to catabolism.

There Îs a coderab1e amount of evidence for the inûiguing suggestion that D-

and L-lysine are cataboiized by anaerobic bacten0a of the genus Closhidm via different

pathwaysP The existence of distinct pathways in clostridia provides precedence for the

claim that fbsobacteria aiso utilize separate pathways for the cataboiism of the different

stereoisomers of lysine.

To addrrss whether or not lysine is catabolized via a distinct pathway in F.

mcleatzm and F. vdm, hciividUa1 '3~-~abeffed Lysine enantiomers are needed as

substrates for feeding e>tperiments. To this end, the stereoseiective chemicai synthesis of

13~-labeLted Lysines is the god of this thesis.

1.1 Distinct Pathways for D- and L-Lysine Citabolism in Clostridia

The anaaobic catabolism of lysine by clostridia fb t received attention in the

1950's when it was observed that this amho acid was decomposed into equimolar

amoimts of acetate, butyrate, and amrnonia?' This fermentation was interesthg because

none of the h w n metabolic pathways involving Iysine couId account for the distn'bution

of 14c Ï n the acetate and butyrate derived from 14c-1abeLled lysine.

Since the initial publications conceming lysine catabolism, much work has been

done to elucidate the pathway in clostn*dia, and in 1973, Stadtman outlined the details of

two pathways in a landmark review (Figure 1). Although the pathway as presented by

Stadtman is a quarter-century old, and much work has been published on the subject

since, the missing steps in Figure 1 have not yet been elucidated. It is clear, though, that

carbon 6 of L-lysine becomes the methyi carbon in butyrate, and carbons 1 and 2 of L-

lysine end up in the acetate unit, with carbon 1 remaining as the carboxylate

hctiondity. h the case of D-lysine an entirely different pathway Ïs exercised, in which

carbon 6 of the amino acid becomes the methyl carbon of acetate, while carbons 1 and 2

of lysine are incorporated into butyrate, wîth carbon 1 remaining oxidized. Although a

thorough revÎew of the work nported on the elucidation of this pathway is beyond the

scope of this thesis, a brief description of what has been gleaned about the biochemistry

at each step is instructive.

in addition to IysÏne, a number ofrdated compoimds were tested as possible

substrates for conversion by clostridia into acetate and butyrate, hcIudmg a-wacetyI-~-

lysine (I), a-sacetyI-~-lyshe, and ~- .acety~-~~-~ys ine? Of these, ody a-.acetyI-L-

Iysine behaved a s a substrate, and mass balance and Iabehg e x m e n t ? showed that

Figure 1: The Pathway of Lysme CataboIism by ~lostridiib

Chapter I - introduction

this acyIated amino acid was merely deacylated to ~4ysine (Figure 1, Step 1) before

entering the metaboiic pathway. It has been demonstrated that acylated amino acid 1 is

not a substrate for reactions II, V, or VI in Figure 1 P In addition, it has been shown6 that

the enzyme responsible for the acylation and deacyiation, a-N-acetyI-L-Iysine deacylase,

is not a generd deacylase, as it is inactive towards substrates such as wa~etylgiycine, N-

acetyivaiiue, IîLacetyl-a-aminocaproic acid, and IV-acetyI-01-sminobutyric acid, among

others. Stadtman suggests6 that in clostridia, some L-lysine is acetylated in order to

reserve a certain amount of that amino acid for protein synthesis.

LP-Lysine (2) was the nrst metabolite identified in the L-Lysine fermentation

pathway. The enzyme that catalyses the rearrangement of L-Lysine to 2, ~-lysine-2,3-

aminomutase (Figure 1, Step II), has been isofated fiom cZosn7'dium SB~,"*" and is

yeiIow in colour due to bond pyridoxaf phosphate. The nature and activity of this

enzyme has been the subject of the majority of the literature published with respect to the

pathway in Figure 1 since 1973. Cofactors required for the activity of L-lysinee2$-

aminomutase include pyridoxd phosphate, ~ e ~ + , and Sadenosyhethionine.

The enzyme responsible for the rearrangement (F4gure 1, Step m) of 2 into 3 3 -

diaminohexanoic acid (3) is refmd to as fLIysine mutase.'* Cofactors necessary for

optimal activity of B-lysine mutase incIude pyridoxd phosphate,13v14 as weii as B12

coenzyme, a thiol, ATP, a magnesium or rnanganese ion, a rnonovdent cation, and

pyruvatee ""

Diamino acid 3 is W o n n e d into 3-keto-5-aminohe~oic acid (4) by 33-

diaminohexanoate dehydrogenase (Figure 1, Step IV), which is dependent upon NAD+ or

NAW 16J?,lB *

Chapter I - Introduction 5

Although many of the steps in the fermentation of Iysing by cIostrÏdÏa had been

elucidated before Stadtmanys review: a route by which 3-keto-5-aminohexanoic acid

became butyrate, acetate, and ammonia (Figure 2) was not proposed tmtil several years

later, by Barker's group.

In the £kt step in Figure 2,3-keto-5-aminohexanoic acid fiom the tlysine

pathway is cleaved between carbons 2 and 3 by an acetyi-CoA-assisted enyme. The

mechanism for this cIeavage was reported by ~ a r k e r ' ~ to be novel, and to probably

proceed via the intermediate illustrated in Figure 2. Barker has isolated and p d e d ' g the

3 -keto-5-amino hexanoate cleavage enzyme.

Barker has also isolated, purifie& and studied the L-3-aminobu~ryl coenzyme A

deaminase which is responsible for the conversion of L-3-aminobutyryl-CoA (5) to

crotonyl-CoA (6) in step II of Figure 2.20 in addition, a transferase which cataLyzes the

W e r of CoA between butyryi CoA and acetoacetate has been isolated and prrrined by

the same research group?'

One other noteworthy intermediate in Figure 2 is 3-hydroqbutyry1-CoA (7). This

species is centrai in the mechanism which f o m butyrate by condensing two acetate

units. This mechanism has been suggested by ~arker: and has aiso been detected in other

anaerobic bacteria (see Section 1-12).

WhiIe the idea of separate pathways for the cataboiisrn of D- and L-lysine is

estabfished in the Iiterature,"w much less infornation is avdable in support of the

pathway for the o-isomer.

The presence ofa Iysine racemase (Fi- I, Step V) was reported" in 1970 by

Barker's group when it was detected as an interfiefence in experiments to characterize

Chapter 1 - Introduction

Figure 2: Conversion of 3-Keto-St--iminohe'l[~~oic Acid to Acetate and Butyrate by Clostridia

Iysine 2,3-aminomutase, the active enyme in step II of Figure 1. Surprisingîy W e is

known about the nature of thÏs racemase-

O-lysine is remranged to 2,Sdiaminohexanoic acid (8) by ~a-lysine mutase

(Figure 1, Step VI). The diamino acid 8 that is fonned Ïs very u n d in nature, and had

not been previous1y reportedP D-a-lysine mutase is dependant upon Bu coenzyme and

pyridoxd phosphate:4fs and is dso activated by ATP? ~-a-Lysine mutase is cIoseIy

related to 8-Lysine mutase, which catalyses step III of Figure 1, and it has been suggested

that the two mutases may in fact be interconverhale forms of a sinde proteid

2,S-Diaminohexanoic acid (8) is oxidatively deamimted by a 2,s-

diaminohexmate dehydrogenase (Figure 1, Step VII). This dehydrogenase, which is

linked to a pyn0dine nucleotide, shows an even p a t e r activity towards 2,4

diaminopentanoate, and it has been speculated that the enzyme may exist priinariiy to

faciütate fermentation of ornithine? another metabolic pathway that has been detected in

clostridia? 2-Amino-5-keto-hexhc acid (9) has not been isolated fiom clostridia, but

the cyciic ~~-~~o[ine-2-meth~i-5i:arboxy1ic acid (IO) has. The pynoiine carbxylic

acid 10 can be seen as arising fiom the spontaneous cychzation of ketoamino acid 9,

Figure 3.

Figure 3: Spontaneous Cyciikation of 2-Amin~-S-keto-hex4notc Acid to ~ ~ - ~ o i i n ~ 2 r n e t a ~ 1 - ~ r b o ~ l i c acid

Stadtman has postuIated6 that acetate is derived h m carbons 5 and 6 of Zamino-

5-keto-hexanoic acid (9) by an eventuai thiolytic cleavage. However, the nature of these

subsequent cataboiïc reactions is unknownC

13 Lysine Cataboiism in F. nucieatum

In 1982, Barker et al. published an a c c o d of lysine degradation by F.

nudeatm. Based on isotopic feeding experiments, and the detection in ceU-fke extracts

of the enymes responsible for the fermentation of L-lysine into butyrate, as in Figure 1,

they suggested that the F. r m c l e u ~ pathway is likely to be the same as the one found in

cIostndia Interestingiy, none of the enzymes responsible from the D-lysine pathway in

clostridia were detected, and the rationale for the D pathway is based soIely on the

interpretation of isotopic incorporations.

In separate experiments, F. mcleatmn was incubated with [LL4~]acetate, DL+ - L4CJlysine, and ~ ~ - [ 6 - ' ~ ~ ] 1 ~ s i n e . The Iabelled acetate yielded radioactive butyrate,

iIIusttating that acetate is converted to butyrate. Further, the specinc radioactivity of

butyrate, which is a measure of radiodvity per mole, was nearly twice that of acetate,

showing that butyrate was generated fiom two uni& of acetate. When racemic 11-

14~lysine was fed, the radioactivity was mainly recovered in the acetate product,

whereas with [6-14~]~ysine, the radioactivity ended up rnostly in butyrate. This suggests

that, at Ieast under the experimentaL conditions emphyed, the formation of acetate and

butyrate by the cleavage of the bond between cvbons 2 and 3 of lysine was the dominant

The 14c e~chmen t at each butynrte carbon was detefmined, and the d t s are

sumnarized in Table 1. Radioactivity h m [Lt4~]acetate was mostly incorporated into

carbons 1 and 3 of butyrate, as expected for the condensation of two acetate units (Figure

2); the greater radioactivity at carbon 3 compared with carbon 1 was consistent with the

role ofacetyI-CoA in the (2423 cleavage step.

Table 1: Distribution of I4c h Butyrate formed from Lysine and Acetate m Media Sup plemented with [1-"~1~cetate, ~ ~ - [ l - ' ~ q ~ ~ s i n e , and ~ ~ - [ 6 - ' ~ q ~ ~ s i n e

Carbon Atom Expt 1: Expt. 2: Expt 3: [I -14c]~cetate Feeding (%) [I -14~]~ysine [6-'4~]~ysine

Feeding (%) Feeding (%) 1 42.0 61.7 O S

The butyrae derived nom [ ~ - ' ~ ~ ] l ~ s i n e containeci more Iabel at carbon 1 than at

carbon 3 (1.6 times as much). Both of these carbons are labelied by the couphg of two

acetate which are fonned with label at carbon 1 via the L-lysine degradation

pathway. However, the higher enrichment at carbon 1 of butyrate is com*stent with two

possibIe routes. Carbon 1 of butyrate can be &&y IabeUed via D-lysine degradation

(Figure 1) or by the conversion of the [Li4~]acetoacetate derived h m L-[I -14~]~ysine

into butyrate (Figure 2). The relatively large amornt of radioactivity at carbon 3 of

butyrate indicates that cfeavage ofthe acetoacetate intermediate to acetate miits is

sipnincant, but it is dso conceivabie that a podon is converted to bntyrate *out

cieavage.

In the third experiment in Table 1, catabolism of [6-L4~]lysÏne resuited in

signincant isotopic enrichment at carbons 2 and 4 of butyrate, with carbon 4 exhibiting

4.4 h e s more radioactfvty. This resuIt can also be expiained by the pathways in Figures

1 and 2. The presence of Iabd at two positions cm again be attnauted to the couphg of

two acetate units, derived fiom either ~-[6-'~c]l~sine (Figure 1) or by cleavage of

crotonyl-CoA formed from ~-[6-"c]l~sine (Figm 2). The high proportion of label at

carbon 4 of butyrate is consistent with the conversion of crotonyl-CoA to butyrate as the

major route.

That the distribiition of label in experiment 3 is more asymmetricaiiy distniuted

than that fotmd in experiment 2 can be rationalized by considerhg the formation of

acetate fkom four-carbon intemediates derived h m L-lysine. Crotonyl-CoA is labeiied

more dirrctiy fiom ~ - [ 6 - ~ ~ ~ ~ ~ s i n e and more label h m t[Lt4~]lysine is located in

acetoacetate (Figure 2). Because fewer steps are needed, acetate is obtained more

efficientiy fiom acetoacetate, and this accounts for the more even distribution of

radioactivity in carbons 1 and 3 of the butyrate derived fkom D L - [ I - ' ~ C ] I ~ S ~ ~ ~ .

The different distri%utions of label over two carbons in butyrate in the two

[r4q1ysine experiments does not provide strong support for the D pathway. Ifcatabolism

of O-lysine by the D pathway in Figure 1 had been signincant, the dimiution of Iabel

over carbons 1 and 3 in butyrate wouid have been more unequai in the DL-[I-'~C]I~S~I~

experiment, since the D pathway suggests that carbon 1 ofbutyrirte Ïs deriveci more

directiy fiom carbon 1 of Lysine, and more equd in carbons 2 and 4 of bntyrate in the DL-

[6-L4CJ1ysine expCnment, since carbon 2 ofacefate is derived direct[y h m catbon 6 of D-

lysine.

Chapter 1 - Introduction

13 lsotopicaiiy Labelid Substrates Required for Lysine Catabolùm Stadies

The two pathways for the catabolism of D- and L- Lysine lead to different

distniutions of the six carbon atoms of lysine within the metaboiic end-products acetate

and butyrate (Figure l), and in principle, can be distinguished by determining the

distriibution of isotopic label in the acetate and butyrate products derived fiom

specincdy IabeiIed lysine substrates. However, as d e s d e d above, the isotopic

experiments conducted by Barker et d? did not provide resuits that concIusively support

two distinct pathways for Lysine catabolism in F. mccleuhmr. The 14~-labeIIed lysines

employed by Barker et al? were racemic, and stereochemically pure substrates are

needed to provide definitive redts for the cataboiism of each Lysine enantiomer.

The use of stereochemicaiiy pure lysines labefled with 13c instead of 14c wodd

d o w NMR anaiysis of the product acids to determine not only the amount of

incorporation of Iabel, but also the exact position of e~chment. A second advantage of

this approach is that the enrichent of adjacent carbons with I3c aiiows carbon-carbon

bonds to be labeiied. The adjacent 13c nuclei couple to each other and their "C NMR

signais appear as doublets. Thus the NMR spectni of metabolic products derived fkom

13~z-1abefJed substrates wouId contain two doublets in their 13c NMEL spectnim, ifthe

labelied bond remained intact during the conversion of substrate to product Ifthe bond

13 13 was cleaved by a metaboiic transfomation, then no C- C couphg wouid be observed

in the product NMR spectnria Upon cIeavage the ' 3 ~ ~ ~ c h e d fragments mDr with

dabeiied materiai, diluting the U~ enrichment and mhhizhg the chances for the

recombhation of two n~ atoms.

Chapter 1 - Iatroduction 12

In each of the pathways (Figrne I), one carbon-carbon bond in Iysim is broken to

generate C2 and C4 products f?om the Cs lysine backbone, In the L-pathway the C2-C3

bond is cleaved, whereas a C M 5 cIeavage is proposed for the fiagmentaton of lysine.

Rather than focussing on the bond cleavage, the bond Iabebg approach wodd provide

definitive information on the bond that remains mtact. Therefore, according to the two

routes show in Figure 1, ~-[4,5-'~~~]lysine and ~-[2 ,3-~~~~]1ysine wouid be degraded to

butyrate sampIes containing intact 13ct units.

In the complementary experiments, no 13c coupihg shouid be observed in

butyrate derived fkom ~ - [ 4 ? 5 - ' ~ ~ $ ~ ~ î n e and ~ - [ 2 , 3 - ' ~ ~ ~ 1 ~ s i n e , ZD- and ~4ysine were

13 13 not interconverted by a lysine racemase. Ifa racemase was present, C- C couphg

wouid aiso be observed in these experiments. Moreover, ifonly one pathway and a

13 13 racemase hction, then C- C coupling wodd be retained with either [2,3-'3~$ysine

or [ 4 , ~ - ' ~ ~ ~ ~ 1 ~ s i n e , not both.

Before this approach to the investigation of lysine cataboiism cm be executed,

mfncient quantities of the required IabeIIed substrates must be availabIe. The rernainder

of this thesis wiU be devoted to the design and execution of the synthesis of D- and L-[2J-

L3~z]~ysine as weil as D- and ~ - [ 4 , 5 - ~ ~ ~ ~ ] l ~ s i n e .

1.4 Synthesis of Carbon-LabeUed Lysines

Became carbon-labeIled lysines are requîred to conduct feedmg experiments to

estabtish the nature of Iysine catabolisn in anaerobic bacteria, a srwey of Iiterattne

preparafions of IabeIIed f o m of this amino acid k presented here L y b 1abelIed at

carbons I and 6 are by far the most common f o m of the Iabelled amino acid reporteci m

Chapter 1 - Introduction 13

the fiterahne, doubtlessly because ofthe ease of introduction of label at these positions

via labelled cyanide and carbon dioxide. In addition tu the non-stereoselective syntheses

of carbon-Iabekd lysines desm'bed in the foUowÏng sections, severd stereoseIective

syntheses have been reported and are recounted in sections 13.1 and 13.3.

1.4.1 Lysine Labelied at Carbon 1

Borsook and coIIeagues2' reported one of the eariïest syntheses of lysine labelIed

at carbon 1 in 1950 (Figure 4). based upon au dry's^^ syathesis of unlabeiled lysine

which empIoyed the cIassicaI ~trecke? reaction to introduce the a-amino and a-

carboxyl groups. In the Bonook procedure, tetrahydropyran was hydrated and converted

to the bisulnte addition compound 11, which was then reacted with aqueous sodium

[i4~]cyanide. The resulting cyanohydrh was convexted to the hydroxybutylhydantoin 12,

which was then transfonned into the aminobutyfhydantoin 13 via the corresponding

bromide Barium hydroxide hydrolysis of aminobutyhydantoin 13 aorded DL-[2-

14~]lysine, which was isoIated as the dihydrochlonde in IO-15% yieId fiom the IabeIIed

cyanide.

A similar route descnîed by Mallnind and CO-workedO (Figure 5), improved

Borsook's procedure by incIuding the damino hctionality in a protected form before

the IabeI was introduced, thus reducing the number of reaction steps invoIving labelled

compounds. Mer preparafion of the bisulnte adduct ofthe aldehyde, conversion to lysine

proceeded via the route used by Borsook (Figure 4).

Figure 4: Borsook's Synthesis of ~ ~ - [ l - ~ ' q ~ ~ s i n e

Figure 5: M.imind9s Synthesis of ~t[l-'~C&~siiic

Chapter t - Introduction 15

In anothez variation ofBonook's method, ~ o t h s t e i n ~ ' ~ proceeded from ethyl5-

formylvalerate (14), via the hydantoin 15, to [l-'4~]a-arninop~e~c acid (Figure 6). This

IabeIIed amino acid was converted to D L - [ I - ' ~ C ] I ~ S ' ~ ~ ~ via a Schmidt reaction, using

hydrazoic acid in 100% sulfiüic acid The yield of IabelIed lysine was 66.5% with respect

to labeikd cyanide. In uniabeiled nuis, potassium cyanide produced a slightly higher

yield than sodium cyanide.

Figure 6: Rothstein7s Synthesis of ~ ~ - [ l - ' ~ q ~ ~ s u i e

In 1952, Arnstein and c o ~ e a ~ e s ~ ~ reported a preparation of ~~-[l-'~~]l~sine

fiom cyclohexanone (Figure 7) that was based on ~darnson'? syathesk of unIabeiIed

lysine. In this syn&esis, the sodium enoIate of cycIohexanone was carboxylated with ' 4 ~ -

carbon dioxide generated fmm IabelIed b ~ u m carbonate. nie redtant carboxylic acid

16 was esterified wvith diazomethane, and the methyl esta 17 was mted with hydrazoic

Md and dry hydrochionc acid in chloroform, generating the lactam ester 18. HydroLysis

of thk Iactam ester followed by a Schmidt miction afEorded D L - [ I - ' ~ ~ I ~ s ~ ~ in 12%

yidd fiom barhm [14qcarbonate.

Chapter 1 - introduction

Figure 7: Arnsteh's Synthesis of D L - [ I - ' * ~ L ~ S ~ ~

More recentiy, Bolster and co-worker~~~ have reported an interesthg synthesis of

D t [ l -' 'cllysine from 1 ,~-~entyIenedüsoc~anide~~ (19, Figure 8). The düsocyanÏde 19

was Iithiated with butyl lithium and the redtant anion was carboxylated with

carbon bon dioxide. The lithium carboxylate 20 was hydrolyzed to field DL+ - I I Cllysine with a radiochernicd yieid of up to 14%, without correction for decay. The

advantage of this procedure is that the total preparation thne is ody 50 minutes, which is

important when L 1 ~ , with a haif-Iife of 20.3 min, is present

1 . 4 Lysme Labeiieà at Cubon 6

The fkst synthesis O ~ D L - [ ~ - " C ] I ~ S ~ ~ ~ was reported by Olynyk and CO-workers3'

(Figure 9). The label was introduced in the fïrst step, produchg 4-chIoro-[l-

14~]butyronitriIe (21), which was used to aUcyIate the sodium saIt of diethyi donate.

The redtant cyanodiester 22 was converted to lysine via the hydroxyunine 23 and the

protected amho acid ester 24.

A few years Iata, ~indsted?~ reported a slightly different approach which, iike

some of the methods for the preparation of lysines IabelIed at carbon 1, invoIved

formation of a cyanohydrin fiom an aidehyde-bisulnte adduct The cyanohydrin was

catdytically reduced and deprotected m one step, affording ~~-[6-'~~]1ysine (Figure 10).

Figure IO: LindstedVs Synthesis of ~ ~ - [ 6 - * ~ ~ ~ S r n e

Cbapter 1 - introduction

At about the same the, Rothstein and CI&' descriied an hast identical

synthesis in whkh the alcohol26 corresponding to the aldehyde 25 (but prepared fiom

other pre~urs~rs4~ was transformed into the bromide 27 for subsequent attack by labekd

cyanide (Figure 1 1). As with Lindstedt's synthesis, the desired IabeiIed lysine was then

generated via reduction and hydro1ysis.

NHAc 26

NHAc NH Ac

2. HCI

Figure Il: Rothsteh9s Synthesh of ~ ~ - [ 6 - * ~ q ~ ~ s i n e

GoIebiewski and spenser4' have made use of a nearIy identicai synthetic route fur

the preparation of D L - [ ~ - ' ~ c ] I ~ s ~ ~ ~ ~ in which the aicohol26 was synthesized and then

converted to the tosylate for subsequent reaction with potassium ['3~]cyanide,

Additionally, Reed and co-worker~~~ have prepared D L - [ ~ - ~ ~ C , ~ - ' % J J I in this

fahion, activating the dcohoI 26 via mesyIation, and protecting the a-nitrogen as the

phthalunide. As weII, Sutherland and ~ i n i s ~ ~ have generated ~-[6-'~C)1~sine fiom the

commercidy avaiIabIe methyi ester of~benzyIoxycarbony1-L-giutamic acid (28, Figrne

12). The starting amino acid ester 28 was reducd to the chiraI aIcohoL29, which was

then tosyIated and subjected to cyanide dispIacement, Subsequent reduction and

hydrolysis afliorded ~-[6'~C&she. AItEiough thk IySme synthesis resufted Ïu an

Chapter 1 - introduction 19

opticdy active product, it can not be desmkd as stereoselective, as the stereochemistcy

was aiready defïned in the stamng materiai.

Figure 12: Willis's Synthesis of ~-[6-' 3 ~ ] ~ y s i n e

A somewhat Werent stnitegy was employed by Pichat and c o ~ e a ~ u e s ~ ~ (Figure

13), whereby label was introduced by reaction of ['4~]cyanide with benzamidoIactone 30

in DMF. Reduction and hydroIytis of the product afforded ~ ~ - [ 6 - ' ~ ~ ] 1 ~ s i n e in 61% yieid

with respect to IabeUed cyanide.

- DMF

30 w

Figure 13: Pichat9s synthesis of ~ ~ - [ 6 - ' ~ q ~ ~ ~ i n e

Chapter t - introduction

1.43 Lysines Labelled at Carbons 2,3,4, andlor 5

The fkst description of a preparation of lysine IabeUed at a n o m t e r . carbon

was provided by Fields, Wah, and ~othchÏId~~ (Figure 14). In this pathway, ethyl[2-

L4~]acetamidocyanoacetate (32) was prepared nom [2-L4~]acetic acid via ethyl[2-

'4~]isonitrosocyauoacetate (31), and then alkylated with 4iodobtxtylphthalimide. Upon

hydrolysis of the protected amino acid 33. ~ ~ - [ 2 - ' ~ ~ ] l ~ s i n e was obtained.

Figure 14: Fields% Synthesis of o ~ - [ 2 - ' ~ ~ ] ~ ~ s i n e

Amstein and c o ~ e a ~ u e s ~ ~ related another synthesis of ~ t [ 2 - ' ~ ~ ] l ~ s i n e (Figure

15). The 14c Cabel was introduced f h n Iabeiied diethyi maionate, which was converted

to the phthalimide 35 via the bromomalonate 34. m a t i o n of IabeiIed phthahide 35

with 4iodobutyIphthahnide and subsequent hyddysis afforded D L - [ ~ - ' ~ C P ~ S ~ ~ .

GO& employed a simiIar strategy to make ~~-[3-'~~,2-'SN]1ysine (Figure 16).

In this case, the 4iodobutyIphuiaIimide was Iabeiied at carbon 4, and the iodide was

dispIaced by 'ZN-~abefled phthalimide.

Figure 15: Arnstein's synthesis of ~L- [2 - '~q~~s ine

Figure 16: Godd9s Synthesir of D L - [ ~ - ' ~ C , % ~ L ~ S ~ ~

~ e e t e ~ ~ has deveIoped a synthesis of DL-[~,S-'~C~]I~S~~~ fiom [13c ~acetylene

(Figure 17). The dilithium sait of the IabelIed acetylene was carboxylated and in

subsequent steps the ciiacid 36 was reduced to the saturated di01 37. This di01 was

converted to the corresponding iodobutyIphthalimide 38, which was then transformed

into lysine by coupling with ethyl acetamidocyanoacetate, in a fashion s h d a r to Field's

synthetis4s of ~~-[2-'~~]1~sine (Figure 14).

Figure 17: L

1 Synthesis of Amino Acids by the Stereoseiectbe Derivatization of Gkycine

A cornpiete review ofthe many methods used to stereospecincaiIy synthesize a-

amino acids is presented in the cuaent scientinc herz~ture,*~~ and wodd be weiE beyond

the scope ofthis thesis. instead, an ovemiew ofsome of the most commonly empioyed

methods that are based upon the asymmetnc derivatiiration ofa nudeophile derived fiom

glycine. Glycine, which is readily availabIe in seved isotopic fomis, is a suitable starting

materid for the synthesis of many amino acids, incIuding Iysine? by these routes.

1.5.1 Bis-Lactim Ether Methods

One of the most commoniy employed methods for the synthetis of optically

active a-amino acids is based upon the aUryIatÏon of bis-lactim ethers, via the

corresponding enolate. 50*519 The generai procedure involves generating a bis-lactim ether

by couphg glycine via peptide bonds to a stereochemicaiiy pure arnino acid, such as L-

valine, and then treatuig the cyclic product with trimethyioxonium tetxafIuoroborate

(Figure 18).

Figure 18: PrepuPtion of a Bis-La* Ether

The bis-Iactim ether 39 in Figure 18 is the most WideIy used glycine tempIate, and

is commerciaiiy available in both isomeric forms. Deprotonation of39 by butyilithium,

folIowed by treatment with an eleztrophiie favors substitution at the site truns to the

isopropyf group. Thus, hydrolysis of the aikyIated bis-lactim ether afZords an optically

active product (Figure 19). In most cases, the new amino acid ester is furnished in excess

of 75% yield, with an enantiomeric excess usually betier than 80%. A major drawback of

this methodology is that the separation of the product amino acid ester fiom the L-valine

methyl ester is sometllnes problematic.

025 N HCI

Figure 19: Alkylation of a Bis-Lactim Ether, and Subsequent Hydroîysis

A StereoseIective preparati~n of ~-[6-'~~]i~sine has been accomplished by Raap,

van der WieIen and ~u~tenburg~ via aUryIation of the bis-Iactim ether h m gIycine and

~vaiu ie (Figure 20). The Iakiied iodobutyronitrile was prepared by =acting

['3~]cyanide with 1-bromo-3-chloropmpane, and then treating the intermediate 4-chloro-

[~-'~CIbutyronitriIe with sodium iodide Ïn acaone. ~ [ 6 - ~ ~ ] ~ ~ s i n e with an enantiomeric

excess of dose to 100% was isoIated in an ove& yieid of 35-40%, based on I&iIed

cyanide.

Figure 20: Raap9s Stereoselective Synthesis of ~-[6-~C&~sine

Raap and co-~orkers~**~ have aiso descnied stereoselective syntheses for L-[2-

13~] - , L-[~-~~c]-, ~ 4 3 ,4-L3~+, and ~-[5,6-"~~]1~sine. t - ~ - ' ~ ~ ] ~ ~ s i n e was synthesized

by preparing the bis-lactim ether 40 fiom D-vahe and [2-"~]gI~cine. Aikylation of 40

with 4iodob~tyronitriIe~ foiIowed by reduction ofthe cyan0 groq and hydrolysis,

afForded the target amino acid with an enantiomenc excess of better than 97% (Figure

21). The yield fiom the IabelIed bis-lactim ether was 49%.

OEt OEt

_Il_t

Figure 21: Rup9s Stereoselecüve Synthesis of t [~-~~CJIqsine

Chape I - Introduction 26

The other isotoporners of L-lysine were prepared by alkyIation ofunlabeffed bis- @

Iactim ether 39 with the appropriately Iabeiied 44odobutyronitriles (Figure 22), followed

by reduction and hydrdysis, as in Figure 21 above. The preparation ofthese IabeUed

butyronitrile by Raap and colleagues, as weii as by other groups, wiil be reviewed in

section 1.6 of thk thesis.

Figure 22: Labeiled 44odobutyronitües ased by Raap to Stereoselectively Prepare Labeiled Lysines

1 Enolates of Schiff Base Derivatives

A second general methodology involves fominig a Schiff base nom glycine and a

chual non-racemic catbonyI cornpourd Deprotonation of the a-carbon followed by

aUcyI5ttion may Sord a diastereomeric prectusor to the desired opticdy active amho

acid, as üIustnited by the exampie, reported by Yarnada et al., " in Figure 23. The

reaction sequence in Figure 23 was among the ikst published srampIes ofan asymmetric

aikyIation of a glycine M e t Depending on the eIecb~)phile useci, yieIds of amino acid

ester ranged 50 to 79%, with optical p d e s ratlging h m 66 to 83%.

Ot-Bu

Figure 23: An Asymmetric Enolate AUIyIation of a Schiff Base Derived from Glycine

Many different systems have been reported since that of Yamada and associates,

often providing higher overd yields and even greater stereoselectivity. A particularly

interesthg application of this method Ïs provided by Katsuki and c ~ - w o r k e r s ~ ~ * ~ ~ (Figure

24). Yields of amho acid isolated fiom Katsuki's system range fiom 41 to 8 1%, but the

enantiomeric excesses are all better than 95%.

1 . 5 Enolates of Imiciazoüdinones, Oxazolidinones, and Oxazoüne

This family ofmethods is most often used to alkyIate chval a-amui0 acids, while

retahhg the orîginaI configuration about the a-carbon. In general, carbons 1 and 2 ofthe

chirai amino acid to be modifieci are incorporated into a five-membered heterocyciic ring

that bears a second chna[ center (Figure 25). The stereochemistry of the second chiral

center is controIIed dnring cyciization by the configuration ofthe a-cah011, When the a-

carbon is deprotonated and &yIated, it is the stereo~he~stry of this second c W center

that ensures that the configuration of the a-carbon is retahed, A system reported by

seebach5' is illustrative Figure 25).

OMOM

1 IM HCI

Figure 24: An Improved Asymmehic Enolate AiIqlation of P Schiff Base Derivod from Glycine

17545 Oc M d Tube

Figure 2 5 cc-Amino Ad& are Derivabd w î h Retention of Configaratr'on

Cbapter I - Introduction

Seebach has applicd this system to a number ofamino acids and

e l e c t r ~ ~ h i l e s ~ ~ * ~ ~ * ~ ~ For example, ~+valine was methylated with methyl iodide in 67%

yieId and the product had an enantiomeric excess of 95%.

Seebach has ais0 explored the preparation and use of the correspondhg glycine

de ri vat ive^^^^^ (Figures 26 and 27). YieIds of allcylated imidazo tidinone often exceeded

80%, d y with diastereomeric excesses of greater than 95%.

Figure 26: Preparation of a CompIementary Pau of Giycine Templates

The imidazoiidinone route has been empIoyed by Fastfi and co-worker~~~ for the

generation of both D- and ~-[6-''~]l~sine (Figure 28, only the synthesis ofthe L-isomer is

shown). The "c-1abeLIed iodobutyronitde was prepared nom hydmgen [L'qcyanide and

1,3aüodopropane. UnfortunateIy, no yieIds or measures of stemchernical printy were

reported for the product Iysines.

Chapter I - Introduction

Figure 27: Aikylation of an Imidazoüdinone Glycine Temphte

Figure 28: Fwth's Stereoselecthe Syntbesis of ~ - [6I 'm~s ine

1.5.4 Methods Based on the Enolate Derhaîkation of Transition Metai Complexes

~elokon~' and CO-workers have deveIoped a methodoIogy based on the formation

and use of ~ i ~ + complexes and, to a Lesser extent, cu2+ complexes that d o w asymmetric

substitution at the a-carbon ofgiycme (Figures 29 and 30). The ligand 41 is

commerciaiiy avdable in the t f o r - and is ofken referred to as L-BPB. The most

commody ased ofthe complexes prepared in Figure 29 is the one derived fiom L-probe

where R = Ph, and M = Ni. The Ni cornplex (42), also h o w n as Ni-L-BPB-Gly, is

commercidy avaiiable.

Figure 29: Synthesis of Belokon's Chiral Compler

The stereoselectMty achieved by using these transition metaI complexes Ïs as a

resuit of the position of the phenyI ring nom the N-beiiylprohe moiety, which

effectively blocks substitution at one fae of the enolate ddved h m deprotonation at

the a-carbon of the glycine hgment (Figure 30). The position of this aromatic ring is in

trmi determked by the stereochemistry of the proihe moiety, so that complexes

contauiing L-prohe alkyIate to preferentidy generate L-arnino acids. Similady,

complexes formed fkom ~ p r o h e preferestiaily affiotd D-amino acids.

Chapter I - htroduction

Figure 30: Stereoselective Synthesis of Amko Acids Using Belokon's Method

This method was origiaally appiied towards the stereoselective synthesis of B-

hydroxy-a-amino acids6* by using aidehydes and ketones as electrophiles, but more

recentiy has been appiied to the synthesis of a host of amino acids by miction of the

deprotonated complex with -1 halides.

avis^^ has applied this methodology towards the stereoselective syntheses of D-

and L-Lysine and other related amino acids (Figrne 3 I, only L preparations are shown).

AUcylations of complex proceeded in yields ranging fkom 75.94% with respect to

complex. The diastereoselectivity of the aUcy1ation step generaliy aorded product of

better than 90% enantiomeric pur@.

1.6 Preparation of 4-Iodobatyronitriles Carbon-Labellcd at Positions 2,3, and 4

Ifa method based on the asymmetric derivatization of glycine is to be empIoyed

in the stereoselective synthesis of L3~-labe~ed D- and L-lysine, elecftophiies of the

appropriate structure and Iabehg pattern must be synthesized, The stereoseiective

preparation of Iabelled Lysnies by Raap (Section 1.5.1) and by Fasth (Section 1.5.3), and

the synthesis of non-racemic dabelled lysines by Davis (Section 1.5.4) aü ilInstrate the

Chapter 1 - Introduction

1. NaOH =WQI2m _Il__t

DMF

Lysine, d e n n = 3

Figure 3 1: Davis's S tereoselective SynthesU of Amino Acids using Belokon's Method

usefulness of 4-halobutyroaitdes for the introduction of the side-chah nitrogen atom. In

the BelokodDavis approach, the cyaao group does not interfere with the alkylation of the

Ni cornplex by intmducing a second nucleophiie or electrophile, and it is stable under the

conditions used for the hydrofysis of the akyIated cornplex. Reduction of the cyano

group af301:ds lysine. Thus ody three steps are required in order to introduce fabel into

lysine nom a 4halobutyronitde.

There are a vast number of occurrences in the Iiterature of 4hdobutyroni.triles, 4-

@-toluenesulfonate)butyr~nitriIe~ and other s idar eIectrophiIes carbon-Iabeiled in the

cyano group, and a review of these compormds is beyond the scope of this thesis. By

contrast, the= are ody a few instances of4-iodobutyronitdes carbon-Iabeiled at

positions 2,3, andlor 4, and none of the corresponding chlorides and bromides have been

reported As the work of Raap and othersnga illustrates, these labelleci iodobutyronitdes

are useful as premrsors of lysines that are labelled at carbons 3 through 5.

In 1990, Raap and othersn descrîbed the synthesls of [2-"~]4iodobutyroaibrile

fkum [2-13~]acetoaitrile in 74% yield (Figrrre 32). More re~entl~:~ this approach has

been used to generate [1 ,2-2-'3~~]4iodobutyronitrïIe fkom [L3~z]acetonitde. These

iodobutyroni~es have fomd utility in the synthesis of IabeUed lysine, as desmied in

section 1 .5. 1. It is noteworthy bt, in 1984, Brocker and E3end7 used this method to

prepare [2-'4~]4@-toluend~nate)buty~ni.tri~e, an intermediate in the synthesis of [2-

14c]-3 ,4-epithio butyronitdey but did not generate the iodide.

The synthesis of [3y413~214iodobutyr~nitrile54 (Figure 33) involved more steps

than that of the other isotopomers, and the labelled carbons were derived fiom ethyl [I ,2-

13~2]bromoacetate.

1.6.1 A Synthetic Plan for the Preparation II- and 142 $ '~~j~ys ine

and D- and ~ - [ 4 $ - ' ~ ~ ~ 1 ~ y s i . . e

Since the fea~rcbility ofBelokonrs method (Section 13.4) has been demo-ed

in this Iab by ~avls,6~ and the chhi reagents re+d for the synthesis by this route of D-

and L-lysine are available, BeIokon's method (Figure 3 I) is an athactive candidate for the

stereose1ectÎve synthesis of D- and L-[2 J - ' ~ c ~ ] I ~ s ~ ~ as weU as D= and ~-[4,5-~~~]1~sine.

C h a p e t - introduction 35

Figure 33: Raap9s Synthesis of [3,4U~214~odobutyronitnre

In the case of the [2,3-'3~z]lysines, the label at position 2 cm be suppiïed by [2-

13~]g~ycine. The labelled glycine can be incorporated into the chiral complex 42 by

methods established for uniabeUed glycine,""66 as o&ed in Figure 29 of Section 1.5.4.

In order to label carbon 3 of [2,3-"~$~sine and carbons 4 and 5 of [4,5-

13~$ysine, the syntheas of appropriately IabeiIed electrophiies must be accomplished.

As outilied in the previous section, a 4haIobutyronitiIe has ken niccessfuIIy used to

prepare D- and L-lysine by the Belokon method.

A retrosynthetic anaiysis for the synthesis of 44odobutyronitrile h m

bromoacetic acid is given in Figure 34. The choice of bromoacetic acid as the startulg

either position, and it was one of the a s m d nimiber of IabefIed compounds avdabIe as

an Isotope Research Grant fkom Cambridge Isotope Labonitories (CIL). As the resdt of a

successfiil grant application, 5g of each of [I-'~cI- and [2-13~bromoacetic acid were

supplied by C L for the synthesis of lysine.

The retr~synthe~c Figure (Figure 34) is anaïogous to that nsed by Raap et ai. to

synthesize the four-cabon chah of4-iodobutyronitriIe by forming the centd carbon-

! Carbon-Carbon Bond Formation

Figure 34: Retrosyntheüc Airdysis for the Synthesis of 4-Iodobutyronitriie fkom Bromoacetic Acid

carbon bond. In thk approach, the eIectrophiIe must be asymmetncaf in order to place

Iabel specincally at carbons 3 or 4 of 4-iodobntyronitdee An estet of bromoacetic acid

meets this requirement, but introduces the r e m m e n t for subsecpent reduction and

iodination steps.

Whïle the cyano group is useful for the generation of the side-chah amino group

in lysine (Section lrl), it also stabilues the carbanion nucleophile used in carbon-carbon

bond formation. The cyanoacetate ester, rather than acetonitde, is easiIy denved h m

bromoacetic acid by cyanide dispIacement and estefication. The use of a cyanoacetate

ester, however, makes necessary the subsequent decarboalkoxylation step.

In the sequence proposed (Figure 34), carbon 4 of 4iodobutyrontrile is derived

ftom C-1 of bromoacetic acid, and carbons 2 and 3 of Ciodobutyronitrile are derived

Eom C-2 of bromoacetic acid. Thus, the appropriate electmphiles cm be prepared h m

bromoacetic acid, and coupling of [4-[3~]4iodobutyronitde with [2-'3~]glYcine would

provide [2,3-"~$~sine. [4,5-'3~Ct]~ysine couid be generated by couphg [ ~ , 3 - ' ~ ~ 2 ] 4

iodobutyronitrile with unlabeiIed glycine.

Chapter 2

Development of a Synthetic Route to 4-Iodobutyrontriie

The key step in the retrosynthetic sequence proposed in Figure 34 is the carbon-

carbon bond-forming step to generate the contiguous 4-carbon chah in 4-

iodobutyronitriie. The commercial availability of bromoacetate and cyanoacetate esters

dowed this step to be investigated initidy.

2.1 Aikyiation of Cyanoacetate Esten

Raap and coworked4 have prepared diethyl cyanosuccinate by adding one

equivaient of ethyl bromoacetate to a stirred solution of ethyl cyanoacetate in dry ethano1

containing 2 equivaients of sodium ethoxide. The resulting solution was stirred at O°C for

2 hours, and at room temperature for an additiond hour. Mer a standard work-up

procedine, a 98% yield of diethyi cyanosuccinate was reported.

An atternpt was made in the present study to repiicate this procedine in methmol,

with the correspondhg methyl esters, using varying amomts of base, as summarized in

Table 2. In the first experiment (Run A), a dry solution of methano1 containing 2

equivaents of sodium methoxide was charged in seqwnce with one ecpbaient ofeach of

methyI cyanoacetate and methyl bromoacetate, as desCnbed in the experimentai section

of this thesrCs. The d e product isolated fiom this reaction had a mass correspondkg to a

67% yield, but it was not pure. The 03 obtained consisted maidy of a mixtitre of de&

dmie~yl qmosuccinate 43 and dimethyI 3~bornet60xy-3-cyanopentanedioate 44, the

Chapter 2 - Development of a Synthetic Route to 4-1odobutyronia.ies 39

dicondensation product, in an approximately 3 to I ratio. As weii, some tmeacted methyt

cyanoacetate was detected by 'H NMR, dong with some minor midentined impurities.

Table 2: Preparatiom of Dimethyl Cyanosucciiute fkom Methyl Cyanoacetate and Methyl Bromoacetate

Run Equivdents of Methoxide Recovered Ratio of 43,44, and unreacted Methyl

Cyanoacetate

In R n B, the addition ofa single equivdent of methoxide Ied to a product

mixture consisisting of dimethyl cyanosuccinate, dimethyI3-carbomethoxy-3-

cyaaopentanedioate, and unreacted methyl cyanoacetate in relative ratios of 3:8:7. When

an aimost six-fold excess of methoxide was used (Run C), the dative amormts were

1355, a remit similar to that obtained with ody a two-fold excess of base.

The Merent amounts of dialkylation product can be rationaked by considering

the likeIy mechanisrns for the 6rst and second aikyIation steps (Figure 3 5).

The monoalkyIated pmduct dimethyi cyanomccinate dso contains an acidic

hydmgen. The electton-withdrawing effect of the new substituent wtlI stabilue the

carbanion fkom 43- As a resuit, this carbanion wÏiI be a weaker base than the

deprotonated methyi cyanoacetate. Men two or more equivaients of methoXide are use&

bot6 carbanions wiü be present, and the major product 43 is obtained by alkylation of the

methyl cyanoacetate carbanion, Le., the stronger base is the better nucleophüe. When

Ciiapter 2 - Devebpment ofa Synthetic Route to 4-Iodobutpnitriles

Figure 35: Mechanism for the Formation of Dimethyl Cyanosuccinate and Dimethyi 3-Carbomethoxy-3-cylaopentanedioate

oniy one equivalent of methoxide is employed, the carbanion of cyanoacetate wiïl be

protonated as the more acidic cyanosuccinate product is formed. As a remit, the

carbanion of methyl cyanoacetate is quniched, Ieaving oniy the carbanion of

cyanosuccinate to react with the remainnig methyl bromoacetate to fom 44.

An attempt was made to generate the diaikyl cyanosuccinate fiom ethyl

cyanoacetate and methyi bromoacetate m dry acetone, using potassium carbonate as the

base. Both at room tempeme and at -1 B0C, a product mixture was obtained which

contained an equimolar amount ofunreacted ethyl cyanoacetate and the comsponduig 2-

carboethoxy-2-~y~~1opentanedioate (45, Figrire 36). Based on the amount of starthg

eiectrophiIe, the yieId of diaIkyIated product was 98%. Becrmse ofthe fairy low

solubility of potassiam carbonate in acetone, oniy a very s a d amount ofbase was

present in soIution at any time. The carbanion of the monoaIlry1a;ted species was the

weakest base in the system, and the recovery ofonly the dialkyiated product 45 was

Chapter 2 - DeveIopment ofa Synthetic Route to 4-lodobatyrodd1e~

consistent with this base king present in the bighest concentration thughout the

reaction. The ease with which the dialkyIated product 45 was obtained in acetone

suggested the fmiility of using a tertiary carbanion in the aikyIatim reaction to avoid

diaikylated product.

Figure 36: Didqiation of Ethyl Cyanoacetate with Methyl Bromoacetate

2 3 Alkylation of Cyanomdonate Esten

Since the desired cyanosuccinate ester was to be decarboethoxylated in the next

step (Figure 34), the possibility of reactuig ethyi cyanoacetate with ethyl chloroformate to

genenite diethyl cyanodonate (46) was considered. If the cyanomalonate couid be

generated and alkyiated with methyl bromoacetate, the resuItant species wodd dso

provide the desired 3cyanopropionate 47 upon decarboethoxylation (Figure 37).

This strategy is andogous to that used by Rapoport's groUp6' to achieve

monoallryIation of malonic acid esters. The undesired second alkylation was avoided by

usîng an alkoxycarbnyl moiety as a bIocking group (Figme 38). In this sequence, the

diaikyI maIonate was converted to the comsponding methane trÏcar60xylate 48 and then

reacted with an electrophüe. CarefbUy controiIed decarbodkoxyIation ofthe product

fiom this eIectrophiIic substitution yieIded either the monoester 49 or the

Chapter 2 - Development of a Synthetic Route to 4-Iodobutyroniirües 42

monosubstituted diaUryI donate 50, as desired. SimiIar decarboaIkoxy1ations are

descriid in greater detail in secfion 24.1.

Figure 37: A Modined Route to 44odobutyronitriIe Proceeding via Alkylation of Diethyl Cyanomaionate

Figure 38: Rapoport's Strategy for the Controiied M o n o ~ I . t î o n of Milouate Esters

Chapter 2 - Devefopment ofa Synthdc Route to 4-lodobutpnitriles 43

2.2.1 Preparation of Cyanomaionate Esten

A üterature preparation of diethyl cyanomalonate achieved ody a 42% yie~d)

ethyI cyanoacetate was deprotonated by an equivaent of sodium ethoxide in ethanol, and

then refluxed with ethyI chloroformate for 3 hours. The product diethyi cyanomalonate is

quite acidic, with a pK, of -1.3", and wodd protonate the anion ofethy1 cyanoacetate,

quenching the reaction. Thus, the use of a single equivaient of base could account for the

low reported yield.

Since ethyl cyanoacetate reacted smoothly with potassium carbonate and ethyl

bromoacetate in acetone (Section XI), a s i d a r system was adopted to prepare diethyl

cyanomalonate. Excess base was employed, so thnit the acidic product wodd not quench

the anion of unreacted cyanoacetate. InterestingIy, this reaction was non-stoichiometnc;

optimum yieIds were obtained using an excess of ethyl chloroformate (Table 3). Using

too much electrophiie, however, redted in diminished yield due to the formation of the

dicondensation product diethyL2-cai:boethoxy-2-cyanorndonate, which was identined by

NMR. Whether too iittie or too much eIectrophiIe was being used was easily probed by

adding s e v d volumes of chlorofomi to the acetone reaction mixture. The desired

product precipitated as its potassium saIt, and fltration and evaporation of the organic

solvent, foliowed by NMFt d y s i s of the residue, reveaied the presence of either

unreacted ethyi cyanoacetate or the dicondensation produçt

Because the product was ÏsoIated fkom the reaction mixture as a sak, the organic

carbon acid was obtaked by extradon fkom an acidÎfÏed aqueous solution (pH < 1) of

the s d t Dietayl cyanomalonate was thus obtained m high yields (up to 95%) and in high

punty-

CIiapter 2 - Development of a Synthtic Route to éEadobutyroaih.iles 44

Table 3: Preparatioiw of DiethyI Cyanomdonate h m Ethyt Cyanoacetate

Run Ratio of Electrophile Yield of Diethyl Other Species Isolated

B 1 k 1 20 Ethyl Cyanoacetate

cyanomalonate

Dimethyl cyanomalonate and ethyi methyl cyanomalonate were ais0 prepared in

high yield (86% and 8 1%, respectiveIy) by the procedure developed for generathg the

diethyl ester. The mixed ester was synthesized fkom methyl cyanoacetate and ethyl

chiorofonnate, but in p~c ip le , the combination of ethyl cyanoacetate and methyl

chloroformate could also have been employed. The potassium saits of dimethyl

cyanomalonate and ethyl methyl cyanomaionate were not soluble in boiIing acetone, and

as a result, the reaction mixtures in the preparation of these cornpounds codd not be

mixed by magnetic stirring as the miction proceeded. This probIem was not encountered

in the synthesis of the diethyI ester 46, as the corresponding potassium sait was much

more solubie in refluxing acetone.

2.23 Tautomerization of Diethyl Cyanomdonate

The diethyl and dimethyl esters of cyanomalonate appear onfy a few times in the

chemicai literatme and, surptisingiy, complete NMR data have not been reported. When

chIorofom-d soInti011~ of diethy1 cyanomalonate (46) were anaIyzed by NMR

spectroscopy, minor signds w m observed in the 'H and 13c ~pectfa which were

tentatively atûiiuted to the presence of a smalI amount of tautorner SI (Figure 39).

Chapter 2 - Devefoprnent of a Syntbetic Route to Q-Todobutyronitriles

Very Rapid L

Et0 OEt - OEt

46 51

Figure 39: Tautorneriution of Diethyl Cyrnomalonate

The muItiplets arising fiom the ethyi groups of the tautomer 51 were located in

the chiorofomxi 'H NMR s p e m Figure 40) as shoulders on the downfield side of the

corresponding signals fkom the diethyl cyanornalonate. The signal at 3.50 ppm likely

arose fiom the hydroxylic proton of the minor tautomer. Interestingly, the ethyI groups of

the en01 tautomer gave rise to only one trÎplet and one quartet. This reveded that the

acidic proton was being transferred between the two oxygen atoms at a rate which was

mpid on the NMR timescaie (Figure 39), causing the two ethyl groups to appear

equivaient The transition state for this equilr%ration would contain a six-rnembered ring,

so it is not surprishg that this process was rapid The en01 tautomer was aIso apparentiy

symmetric in the 13c NMR spectrum. The carbon bearing the cyano functionality

appeared surprisingiy fat upfieId in the "C NMR (60.54 ppm), but the assignment of this

resonance was c o b e d by ' 3 ~ NEilR spectroscopy of diethyI cyanornaIonate IabelIed in

thÏs position (77, Section 3.2)- This musuai shft was aIso keIy due to the rapid

equiIicbration process desmiid above.

The presence of this tautorner in acetonitde has been previody sugg~d,7t but

no NMR data have ever been presented to support this clamL hdecd, ody a 'H chernid

Chapter 2 - Development ofa Synthetic Route to 44odobutyroni~es

shift of 4.79 ppm, correspondi. to the acidic proton ofthe major tautorner 46 in

acetonitde, has been reported To co&m that the minor signais in chIoroformd were

due to tautorner 51, and not an impurity, a slight excess of ûiethylamine was added

direcdy to the NMR sample, and the 'H spectcum was recorded again (Figure 41).

Initially, the 'H NMR spectnim contained a minor quartet at 4.42 pprn in addition

to the more intense quartet at 4.35 ppm. Upon addition of base, however, only one quartet

was observed, at 3.84 pprn (the quartet appearing at 2.95 pprn was due to the tnethyl

ammonium present). The triplets origïnaüy appearing between 1.3 and 1.4 pprn have

ken replaced by a singie triplet, which is obscured by the triplet arising fiom the

triethylammonium ion. The hydroxytic proton resomce has, as expected, disappeared.

Figure 40: 'H N M R Spectrum of Diethyl Qmnomdonate in Chioroform-d before Addifion of Triethylimme

Chapter 2 - DeveIopment of a Synthetic Route to Clodobutyronitriles

Figure 41: 'H NMR Spectnun of Diethyl Cyanomaionate in ChIoroforrn-d after Addition of Triethylamine

These r e d t s are consistent with the presence of a srnail amount of the tautorner

51, as both tautomers wouid yield the same anion upon deprotonation (Figure 42).

Figare 42: DiethyI Cyanomaionate and its Tautorner YieId the same Anion Upon Deprotonation

Chapter 2 - Development of a Synthdc Route to 4-IadobutyroaitriIes

2.23 Preparrtion of Diethyl and DimethyI Cyraoacetate Sdts

A aumber of saits were prepared h m diethyl and dimethyi cyanomdonate, and

are iIIustnited in Table 4. AU of the saits were quite soluble in water and DMSO, and ail

but the dimethy1 saits 57 and 58 had appreciable solubility Ïn boiling acetone. The

tetrabutylammonium sdt 56 was aIso very solubIe in chloroform and dichioromethane.

AU of the saits were stored at room temperature for extended perïods (severai months)

and showed no evidence of hygroscopicity.

The sodium and potassium saits 54,57, and 58 were prepared by treating the

corresponding carbon acid with either sodium or potassium akoxide. In hindsight, the

use of aqueous sodium or potassium bicarbonate, foiiowed by evaporation or

lyophüuation, wouid have iikely provided the desired saits without the use of such strong

bases.

2.2.4 Nucleophüicity of Carbanions Derived h m Cyanomilonate Esten

A series of experiments was conducted to demonstrate the nucleophilicity of some

of the saits (Table 4) fomed h m diethyl and dimethyl cyanornaIonate. These

experiments were performed within NMR tubes in perdeuterated solvents, to facilitate

monitoring reaction progress. The nucleophilicity of the diaQI cyanornaIonate saits was

ofsome interest because these saits may be of generai use as synthetic equivaients for the

anions of acetonitde and of cyanoacetate esters (Figure 43). These cyanomaîonate saits

may be employed when mttitiple aIky1ations are to be avoided (as in the present study) in

a manmr ariaIogous to ~ a p ~ p ~ a ~ s ~ monoalkyIations of maionate esters (Figure 38). As

w d , the reIatively Iow basicity ofthese saItn permits the5 use iu the presence base-

-ter 2 - Devetopment ofa Synthetk Route to 44odobutyronitriles

sensitive substrates, and alIows their use in cases when a cornpethg E2 elimination

reaction might otherwise be problematic.

Table 4: S a h Prepared nom DimethyI and Diethyl Cyanomaionate

# Anion Cation Metting Point (OC)

52 C@CH2CH3 a 2 a 3 Not Measured

NCB( CEI~CH~-N C@CH2CH3

HJ \ CH2CH3

Not Measured

Not Measund

305 - 309 (dec.)

Chapter 2 - DeveIopment ofa Syntiietic Route to CIodobutymaitriIes

Figure 43: Dirlkyl Cyrnomaionate Sdts as Synthetic Equhlenb for the Carbanions of Acetonitriïe and Cyanoacetate Esters

The resuits of the triai experiments are summarized in Table 5. AIthough an effort

was made to use an equimolar amount of nucleophile and electrophile in each trial, one of

the reagents was aiways in slight excess. The Iimiting reagent was detemiined by NMR

spectroscopy. The extent of reaction reported in Table 5 was caicdated by dividing the

amount of product by the total amount of product formed plus unreacted lùniting

reactant, and muitiplying by 100%. The reIative amounts were determined by integration

of NMR spectra.

Not surprisingiy, the reactions involving aiIyL and benyl bromides

@uns A - E) proceeded most rapicüy, as these were the most reactive eIectrophÏIes used.

The potassium sait 58 reacted neariy comp1eteiy with b-1 bromide at room

temperatme ui DMSO. The product 59 (Figure 44) was cIeanLy ÏsoIated in a 74% yield,

and was characterized by NMR and MS, in order to demonstrate that the &on was

reasonabIy high-yieIding, and to c o d h the identity of the alkylation p r o d ~ n None of

Table 5: Summary of Results from Alkylations of Some Cyanomalonate Salta

. Ru Nucleophile Electrophile Limi ting Reagent Solvent Time Temperature Extent of Reaction

("C) (W DMSO 40min RT 14 58 Benzyl Bromide Electrophile

(0.38 mmol) (0.37 mmol)

52 Allyl Bmmide Electrophile (0.44 mmol) (0.43 mmol)

56 Allyl Bromide Nucleophile (0.53 m o l ) (0.53 mrnol)

52 Allyl Bromide Electrophile (0.40 m o l ) (0.40 mmol)

56 Allyl Bromide Electrophile (0.53 mmol) (0.53 m o l )

56 Iodomethane Nucleophile (0.38 mrnol) (0.38 mmol)

56 Ethyl pToluenesu1 fonste Electrophile (0.36 mmol) (0.37 mmol)

DMSO 20min RT 50 min

4 h

DMSO 2.5 h 50

Acetone 22 h 50

Acetone 2.5 h 50

Acetone 5 min RT 50 min 2.5 h 6 h

Acetone 23 h 50

Chapter 2 - DeveIopment of a Synthetic Route to 4-lodobutyronitriles 53

the other akyIation products were isolated, or chacterized beyond 'H and I3c NMR

spectroscopy.

Figure 44: Dimethyl Benzylcyanomrionate

TetrabutyIamrnonium s d t 56 was also fkirly reactive with methyI iodide (Run F)

in acetone, methylating rapidy at room temperature. This salt was much more slowly

dqIated with ethyl tosylate (Run G) in acetone, even when subjected to d d heating.

Simple aUcyI bromides were, in generd, unreactive towards the diethyl saits 52

and 53 in acetone and acetonitde, although ethyI bromide did aikyIate the

triethyiammonium sait 53 upon heating in DMSO. An attempt (Run L) was made to

promote alkyIation of 53 in acetone with 2-bromopropane using potassium iodide

cataiysis, but no reaction was observed. No experiments were pcrformed to determine

whether secondary or tertiary aUcyI bromides would react in DMSO. Care must be taken

when usuig this solvent because ofthe potentiai that exim for decarboalkoxyIation upon

Chapter 2 - Development of a SyntfietiCc Route to 44odobutyronitriles 54

The nucIeophüicÏty of diaUcyl cyanornaIonate saIts towards bromoacetate esters

was of particuiar importance to the synthesis of 4-iodobutyronitciIes (Figure 3 9 , and was

the subject of a more rigorous study, which is outIined in the foIIowing sections.

23.5 Development of Akylatîon Reaction Conditions

The important carbon-carbon bond foLIIii]Zg reaction codd now be attempted

without any possiiiity of dialkylation. Because the diethyl cyanomalonate had been

suecessfuly prepared in acetone, îhis solvent was chosen for the akylation of

cyanomalonate esters. With potassium carbonate or bicarbonate as base, and with iodide

cataiysis, diethyl cyanomaionate was alkyIated with methyI bromoacetate to W s h the

desired ethyi methyl2-cyano-2-carboetEioxysuccinate (60) in better than 80% yields

(Table 6, Run A).

Table 6: Resdb of TNl AlkyIations of DiethyI CyanornaIonite with Bromoacetate Esters

Run Reaction Conditions Resuit A Diethyl cyanomaionate (19.8 mmol), M C O S (21 .O mmol), 97% YieId

Methyl Bromoacetate (19.8 mmol), KI (2.0 m o l ) of 60

B Diethyi cyanomaionate (38.6 mol ) , K2C03 (1 9.5 rnmol), 9 1% Yieid Ethyl Bromoacetate (38-1 mmoi), KI (1 -9 mrnol) of 61

C Diethyi cyanomalonate (19.7 m o l ) , KHC03 (205 rnmoi), 8 1% YieId MethyL Bromoacetate (19.6 mmol) of 60.

D Diethyl cyanornatomte (2-7 mmoI), &CO3 (5.5 mmoi), 77% YieId EthyL Bromoacetate (2-7 mol), KI (0.4 mmoI) of 61, with

some 62

Chapter 2 - DeveIopment of a Synthetic Route to Clodobutyro~es

In a similar fashion, diethyI cyanornaIonate couid aiso be akylated with ethyl

bromoacetate (Table 6, Rtm B), forming die- 2-cyano-2-~arboethoxry~~c~ (61).

A slight excess of nucleophile couid be empIoyed ui the alkylation reaction, as the

unreacted potassium sait was easily removed either by filtration h m chlorofonn, or by

washing with aqueous base. To the best of this author's knowledge, the succhte

products 60 and 61 (Figure 45) have not ken previously reported in the literature, and

the 'H and "C NMR spectra of 60 are given in Figures 46 and 47.

Figure 45: Ethyl Methyl Z-Cyino-2-carboethoxysuccinate and DiethyI 2~~0-2 -~boe thoxysucc ina te

The importance of the potassium iodide catalysis became apparent when an

attempt was made to perform the aikyIation of diethyI cyanomaionate with methyl

bromoacetate in the absence of thîs sait (Table 6, Run C). Afier 28 h m of reflux, and

the d work-q, a m d e product was isoIated that, if pure, wodd have conesponded to

an 88% yield. However, NMR adysis reveded that the product oiI was an

approxmiately 7: 1 m i x e of ethyi methyi 2-carboethoxy-2-cyanosuccinate and

unreacted methy1 bromoacetate, so that, in actuality, ody an 8 1% yield of product was

fomied.

Chapter 2 - Developmmt of a Synthetic Route to 4-Iodobutyronitdes 58

AIso of importance was the amount of base present. In a trial experiment (Run D)

where a two-fold excess of diasic potassium carbonate was employed, the product oil

was composed of a 2.4: 1 mixture ofdiethyi 2-carboethoxy-2..cyano~~cCinate (61) and

diethyl3-carboethoxy-3 -cyanogiutarate (62. Figure 48).

Although it is not knom with any certainty how 62 is formed in the presence of

excess carbonate, it is possible that the mechanism involves a decarboethoxy1ation step,

as des&ed in greater detail in Section 2.4.1. The ethyl iodoacetate s h o w in Figure 48 is

formed in situ h m potassium iodide and ethyl bromoacetate. The tmspecified

nucleophile codd be carbonate ion, or possibly the enolate ion denved fiom acetone. I f

the enolate fiom acetone was to participate as the nucleophile in Figure 48, ethyl

acetoacetate wouid be produced, but none of this P-keto ester was observed by NMR in

the product momÛe.

Figare 48: Possible MechaniSm for the Formation of DiethyI 3-CPrboethoxy-3-C yanogIatarate (62)

Chapter 2 - DeveIopment of a Synt&etiCc Route to 41odobutyronitriles

2.3 Modification of Bromoacetic Acid

While the key carbon-carbon bond forming step has been established i?i the previous

section, p r e h h r y steps were needed to convert the availabie I3Glabefled bromoacetic

acid into the substrates for the aiky1ation d o n in the synthesis of [2 J - ' ~ c c ~

iodobutyronitriie. In order to accornpltsh this, two d o r m a t i o n s needed to be

performed. The acid needed to be esterifieci, and the bromide substituent had to be

replaced with cyanide.

2.3.1 Preparation of Methyl Bromoacetate

In tria1 reactions, methyl bromoacetate was generated quickIy and cleanly fiom

bromoacetic acid by following BIackts general procedure for the preparation and use of

diammahane." However, after rotary evaporation of the ether solvent, the yield of

methyi bromoacetate (- 80%) was Iower than exmpeaed, suggesting that a signincant

amount of the rnethyl ester was Iost on evaporation. This was remedied by simply

distiliing off the buik of the ether at atmospheric pressure, and using a mixture of methyl

bromoacetate aud a smaU amotmt of residuai ether Ï n the subsequent reaction. The

residual ether was not probIematic; the yieId fÏom the subsequent reaction ushg methyl

bromoacetate prepared in this manner was essentially the same as yields fFom reactions

using cornmerciai methyL bromoacetate, demonstrating both that the contauünating ether

did not interfixe, and that the esterification proceeded quantitativeIy.

Cbapter 2 - Development of a Synthetic Route to 4-IodobutyronitriIes

23.2 Preparaüon of Ethyl Cyanoacetate

Since the feasfiility of preparing diethyl cyanomalonate fkom ethyI cyanoacetate

in high yield has been demonstnited (Section 2.24, a i i that rcmained was to convert

bromoacetic acid into ethyi cyanoacetate in good yield,

An aqueous preparation of cyanoacetic acid nom chloroacetic acid has been

described in the literaturen (Figure 49) in which the starting acid is deprotonated with

bicarbonate and then treated with a slight excess of cyanide ion at 55OC.

Figure 49: Synthesis of Cyanoacetic Acid fkom Chioroacetic Acid

This method was successfuiiy empIoyed to convert bromoacetic acid into

potassium cyanoacetate. The cyanoacetate product was not isoiated fiom the potassium

bromide produced in this reaction, nor fiom the excess potassium cyanide and potassium

bicarbonate that remained. mead, the entire mixture was stixred in DMF at -55OC with

excess iodoethane. Ethyl cyanoacetate was formed in an approximately 80% yield with

respect to bromoacetic acid, and it was more easily isolated fiom the reaction mixture

tban the cyanoacetate saIt produced in the first step. The ethyl cyanoacetate product was

contamhated wit6 a very smaü amourit ofunidentitied impunty which was detected by

NMR This contamiuant was not removed before the ethyI cyauoacetate was comrerted

mto dÎethyI cyanornaIonate, but the diethyl cyanomaionate was dl isoiated ni hi& yieId,

with no apparent imprrrities. The overaII *Id of diethyl cyanornaIonate was 68% with

CIiapter 2 - Dwebpment of a Syntbetic Rom to 44odobutyronitriles 61

respect to bromoacetic acid, which conesponds to an average yield of 88% for each of

the t h e transformations.

2.4 Functional Group Modifications

The conversion ofthe product of the aUcyIation step, ethyl methyl2-carboethoxy-

2-cyanosuccinate (60) into the desired 4iodobutytonitriie, required t h e M e r

hctionai group modincations: decarboethoxyIation, mluction of an ester, and

iodination (Figure 34).

2.4.1 DecarboethoxyIation of 2-Carboethoxy-2-eyuiosuccinate Esters

The decarboethoxyIation of a-cyano esters, maionate esters, P-keto esters and

related compoimds has been reviewed thoroughiy by ~ r a ~ c h o ? ~ * ' ~ hi polar aprotic me&

such as DMSO and DMF containing a small amotmt of water and a cataiytic amount of

chloride or cyanide ion, these compounds undergo decarboethoxyIaîion via the

mechanism outlined in Figure 50 for ethyI cyanoacetate. As shown, the dtimate products

are the decarboethoxyIated substrate, alcohol, and carbon dioxide, and the cataIytic anion

is regenerated.

Two such de~boethoxyIation steps are required for the generation of methyl3-

cyanopropionate (63) fiom ethyL methyi 2-carboeth0xy-2-cym0~~~cinate (Figrire 51).

The rate of the decarbethoxyIation is govemed by the rate of formation of the carbanion

by chIoride dispIacement As a dt, the ntst decarboethoxyIation ofethyi meihyl2-

carboethoxy-2-cyanosuccinate ta &y1 methyi cyanosuccinate was expected to proceed

Cbapter 2 - Development of a Synthetic Route to 4Iodobufymnitriles

Figure 50: Decarboethoxylation of Ethyl Cyanoacetate

more quickiy than the decarboethoxyIation of ethyi methyf cyanosuccinate to the desired

cyanopropionate 63 as the anion formed in the fkst step receives inductive and resonance

stabiIisration fkom two strongiy withdrawing substituents, whenas the anion formed fiom

the second step is stabilized ody by the cyan0 group.

Methyl3-cyanopropionate was successfully generated fiom ethyI methyf 3-

carboetholry-3-cyanosuccinate 60 by heating a mixturr of this oitrile ester, DMSO,

distiIied water, and sodium chhide at 440°C for approxhatefy 24 hours. These

conditions yiefded methyI3-cyanopropionate in about 80% yield. The buik of the DMSO

was removed by takmg up the cooled reaction mixture in concentrated brine and

Via Tetrahedraî interniediate _L OEt v-.

CH2C02Me e:&

mo

Va Teînhedral iuterrriediate

+H+

N OMe N OMe

Figure 51: Deearboethoxylaüon of Ethyi Methyl2-Carboethory-2-Cymosuccinate

extrachg the desired product with ether. The residual DMSO codd be removed by

chromatographing the crude product on silica gel, with ether as efuant Aiternatively, the

rernaining DMSO codd be eliminated by taking up the m d e product in concentrated

b ~ e and repeating the extraction process a second (or third) tirne.

2.43 Reduction of 3-Cyanopropionate Esters

The selective reduction of methyl esters in the presence of a cyano gmup may be

accomplished with sodium borohydnde m t-butyf aicohoI in the presence of a srnaII

amount of methanof6, or with Iithium borohydride in an ethered soIvent The sodium

borohydride method successfiilly M s h e d the desired nitde aIcohol64 in good yieids

(-70%); Raap et al. used the identical reaction with s i d a . success."

The rnechanism for this methmol-assisfed reduction is not known for strre, but the

active reduchg species Ïs thought to be the trimethoxy borohydnde that is formed in

Chapter 2 - DeveIopment of a Synthetic Route to 4-rodobatyronitriles 64

situn (Figure 52). The aidehyde intermediate couid dso be reduced by this he thoxy

species, or by any u~veacted sodium borohydride.

H/ equiv. MeOH \ QocH - ?--ocd -

Figure 52: Methmol-Assisted Borohydride Reduction of MethyI3-Cynnopropionate

2.43 -1 Iodide Preparation

The nnal reqirired fiinctionai grottp tranformation was the conversion of the

hydroxy group into an iodide (Figure 33). There were a number of estabfished methods

for accomplishuig t6is ttansfonnation based on the a b i . to convert the hydroxy group

hto a much better Ieawig grotp, so that nucIeophiIic dispIacement with iodide codd

occur*

Chapter 2 - DeveIopment of a Synthetic Route to 41odobrityronitriles

2.4.3.1 Attempted Tosylation of 4-Hydroxybutgroaibile

Raap and CO-workd obtained 4-iodobutpnitrde fiom 4hydroxybutyronitrile

via theptoluendonate ester 65, which was prepared in pyridine fiom the aicohol64

and tosyl chionde. An attempt was made to replicate this remit, but in this author's han&

a mixture of the desired tosylate and the correspondhg 4chlorobutyronitrile 66 was

obtained, with ody 54% totai recovery of the carbon backbone. The formation of

chloride 66 cm be explained by nucleophiüc displacement oftosylate (Figure 53), either

in the reaction mixture, or in the work-up, when hydrochioric acid was introduced.

Figure 53: TosyIation of 4Hydrosybutyronitrile and Sttbsequent Conversion to the Chioride

Kabalka has reported7' that in a ninnber of cases, tosylatiom done in pyridine

have affiorded Iow yieIds due to the formation of pyrÎdinium salts, as a remit of pyridine

displacement of tosylate. Ih response to this probfem, he has reported the successful

tosyIation of dcohofs in chIorofomi, after the removaI ofany stabtlisTmg ethanol, ushg

1.5 equivdents oftosyi chlonde and 2 equivaients ofpyriCdine. As an dteniative to

Chapter 2 - DeveIopment of a Synthetic Route to 4lodobutyroniaires 66

preparuig the desüed tosylate teing Kabalka's procedure, the possibfity of preparing 4-

iodobutyronitrile nom 4-hydroxybutyronitde was expIored, and uItmiateIy utiiised-

2.433 Direct Preparation of Iodides fiom AicoholP

A farnily of methods based upon the use of tnphenylphosphine exists for the

conversion of aicohoIs to the conesponding iodides. One such system has been

successfully employed by Smith and co~eagues'~ to quantitativeIy convert aIcoho167

înto iodide 68 (Figure 54).

Figure 54: An Example of a Direct Conversion of an Aicohol into in Iodide

In the present study, the teaction conditions utilized by Smith and CO-workers

were used to prepare &iodobntyn>nitrile in approxhately 70% yield h m the aicohoi 64.

This yield represents chromatographed product, as silica geI chromatography was

necessary to remove the tripheny1phosphine oxide, which was formed as an unwanted by-

product of the reaction. The iodmation IikeIy proceeded as show11 m Figure 55,

Figure 55: Conversion of 4-Hydroxybutyronitrüe into 4-Iodobutyronitrile

2 Conclusion

Based on the work descn'bed within this chapter, synthetic methodology was in

place for the synthesis of Ciodobutyronitrile fiom bromoacetic acid The substrates in the

pivotal carbon-carbon bond forrning step, diethyi cyanomaionate and methyI

brornoacetate, were both readily prepared fiom this common precursor, and the

hctionai group transformations that foliowed this step ail proceeded with acceptable

yields (70 - 80%). The application of this synthetic methodology towards Iabeiled

substrates is the focus of the fïrst two sections of Chapter 3.

Chapter 3

Stereoselective Synthesis of '3~2-~abelled Lysines

The synthesis of 4iodobutyronitrile fiom bromoacetic acid desmbed in the

previous chapter was appiied toward the preparation of 14-13c]- and [2,3-'3~z]4

iodobutyronitde fkom [Luc]- and [2-13~Jbromoacetic acid respectiveiy. Chapter 3 will

focus on the successful synthesis of these Iabeiied electrophiles, dong with their use in

the stereoselective preparation of D- and ~-[2,3-'~~2]1~sine as welI a s D- and ~ 4 4 3 -

L3~z]~ysine via the method of BeIokon (introduced in Section 1.5.4).

3.1 Preparation of [ 4 - 1 3 ~ ~ ~ o d o b u o e from [l-'3~~romoacetic Acid

Based conservatively on yieIds obtained from triai experiments on udabeHed

substrates, the pathway outlined in Figure 56 was expected to affiord [4-13c]4

iodobutyronitde (73) in approximately 30% yield with respect to [I-'3~bromoacetic

acid. This corresponded to about 2.1 g (1 1 m o l ) of nitrile iodide 73 pet 5.0 g of

bromoacetic acid. These ~uantities of IabeiIed iodobutyronitrile were considered adequate

for the preparation of at least I mm01 each of D- and ~-[2,3-"~2]1~siae, so the

preparation of [4-'3~]4iod~butyronitrile was undertaken.

[l -'3~~romoacetic acid in ether was converted to methyI. [1 -'3~romoacetate

using diammethane by the procedure desmied for UnlabeiIed substrate (Section 23.1).

The exact amount of [1~3~~omoacetic acid esmified was 5.78 g, coClSiderabIy more

than t6e mass of 5 g reported on the bottie sappiied by CIL. The IabeUed methyI

Chapter 3 - Stemseleetive SyntF&s ofUC&abelled ~ysines 69

bromoacetate contained a sma1.I amount of ether after the bulk solvent was W e d off at

atmospheric pressure,

UnIabeIIed diediyl cyanomdonate was aikylated with methyl [I -"~]brornoacetate

in acetone with potassium iodide catalysis, and with potassium bicarbonate as base. The

Figure 56: Synthetic Route rot the Preparation of [d'3~4~odobutyronitrile

amount of diethyl cyanomaionate was based on the quantitative esterScation of

[~-~~~Jbromoacetic acid, and the recovered yieId of meuiyl ethy1 [4-13~]-2~boethoxy-

2-cyanosuccinate (70) was 78% with respect to the initiai [~-'~~]bromoacetic acid. This

yield was jwt slightiy Iowet thaa typical yieIds fiom the same allrjfation reaction

(Section 22.5) using dabelled substrats.

The methyI ethyl [ 4 1 3 ~ ~ 2 ~ b o e t h ~ r r y - 2 - c y ~ ~ ~ ~ c i n a t e was

decarMoxyIated in two separate, eqtxai portions. This was forpracticd reasons, as the

Chapta 3 - Stereoselective SynttiesEs of UC&abeiicd Lysines 70

size of the oiI bath and the volumes of solvent used in the work up wouId have become

unwieldy. Both nms attonied methyl [1-'3~-3-cyanopropionate (71) in approximately

80% yieId afta the chromatographie removal ofany traces ofDMS0, aithough an

unidentified con taminant was present, as detected by NMR spectroscopy.

One of the two portions of the [1-'~~]-3-c~ano~ro~~omte was reduced to the

aIcohol72 in the same marner as uniabeiIed W e ester (Section 2.4.2), using sodium

borohydride and methanol in refluxing t-butyl akohol. A crude yield of nearly 80% was

obtained, aithough NMR anaiysis reveaied that this crude product contained a signincant

amount of an unidentifîed impurity which was removed by dica gel chmmatography.

The nnal yield of chromatographed [4-'3~]4hydroxybutyr~nitde (72) was 52% with

respect to labelled nitde ester 71. This slightIy Iower-than-anticipated yieId was lürely

due to the unknown impurity in the labelled nitrile ester 71, which amse in the

dewboethoxyiation step.

The iodination of IabeiIed nitrile aIcohoI72 was performed as with uniabelled

substrate (Section 2.43.2), by treatment of 72 with triphenylphosphine, hnidazole, and

iodhe, to &ord 4.25 m o l of chromatographed [4-'3C)4iodobutyr~ni~e (73). This

comsponded to a 67% yield in the finai step, and the product, which was characterized

by NMR spectroscopy, was of Sufflcient purity for use towards the stereoselective

synthesis of D- and ~-[2,3-'~c&~k Accouuting for the fkt that oniy one haif of the

labeiied nitrile ester 71 was converted mto the correspondhg iodide, [4'3q4

iodobutyronitde was obtained in a 2 1% yieId with respect to [~-~~~Ibromoacetic acid.

33 Preparation of [~,3-~~~l41odobutyronitriIe fkom [2-'3~~rornoacetic Acid

In the case of [ 2 , 3 - ' 3 ~ z ] 4 i o d o b e (81), two equivaIents of IabeIIed

brornoacetic acid were incorporateci into each equivaient ofproduct nitrile iodide, and the

conversion of sorne [2-'3~]bromoacetic acid (74) into diethy1 [2-'3~]cYan~rnaI~nate (77)

was required (Fi- 57). Based on these considerations, 5.0 g of [2-t3~]bromoacetic acid

was expected to yield appmximately 0.75 g (3.9 m o l ) of the doubly labeiied nitrile

iodide 81.

r. KHCQ, KCN/ ~ 2 0 2, a 3 a Z I / DMF -

OH N ~ , E 7 ~ 4 E t Acetone .qo4 2

76 77

N$o" OMe NaCI, H20 - DMSO 140 O c

Figure 57: Synthetic Route for the Preparation of [2~ - '~~~~41odobutyn ,n i t~ l e

Chapter 3 - Stemselecthe Synthesis of LIC&he~ed Lysines 72

The actuai mass of [2-"~]bromoacetic acid provided by CIL was 4.903 g (-35.04

-01). This sampIe was spIit into unequai portions, one of which was converteci hto the

methyI ester 75, and the other was transformeci into diethyI [ 2 - ' 3 ~ ] c y a n o m a l o n a t e . o o ~ t e Based

on a quantitative esterification reaction, and a cornervative expectation of a 60% yield of

labeued cyanomaionate 77,37.5% of the labelled bromoacetic acid 74 was es tded , and

the remainder was converted to diethy1 [2-13~]cyaaomaIonate.

Methyi [2-"c]bromoacetate 75 was prepared with diazomethane in the usuai

manner (Section 2.3.1) and was aiso used without complete removai of the ether solvent.

FoUowing the procedure developed for unlabeL1ed bromoacetic acid (Section

23.2) involving cyanide displacernent of bromide, and esterification with iodoethme,

ethyl [2-t3~]cyan~maIonate (77) was obtained in a 64% yield with respect to [2-

'3~]bromoacetic acid. The intermediate ethyi [2-"c]cyanoacetate (76) was obtained in an

approximately 87% yield fiom the acid, but as expected fiom triai experiments, this

IabeIied nitrile ester contained a srnail amount of unidentified contaminant wbich was not

removed before the m e ester 76 was used, The minor en01 tautorner that was observed

for uniabelIed substrate in chioroformd was not detected in the 'H NMR spectnnn of

IabelIed matenal (76), which was recorded in acetone-d6. However, a resonance appeared

at 61.12 ppm m the 13c NMR spectrum of 76 which corresponded to the ' 3 ~ - ~ a b e ~ e d

quaternafy carbon of the en01 tautorner. No other 13c NMR resonances due to IabelIed

en01 tautorner were detected, and the height ofthe peak at 61 ppm was appmrrimateiy 2%

ofthe height of the peak arim h m the labened carbon of the major tautorner.

The I3c-l3c bond was formed foIiowing the procedme developed nSmg

dabeiled snbstraîes (Section 21.5). An 8% excess of IabeiIed cyanomaIonate 77 was

Chapter 3 - Stereosektive Synthesis ofUcr~iibe~ed ~ysines 73

use& and the recovered methyi ethyI [2,3-'3~z]-2-carboetbov-2-cyano~ccinate (78)

corresponded to a 79% yield with respect to electmphiie. A very smaii peak at -5 ppm in

the '.'c NMR spectnna of the product oiI is Iürely due to a very smalI amount of methyt

[2-'3~]iodoacetate, fomed as the intemediate in the dcylation reaction.

Methyl ethyl [2,3-'3~2]-2carboeth~xy-2-cyan~~cckiate (78) was

decarboethoxylated in one portion with sodium chioride in wet DMSO at 144OC to afTord

methyI 2,3-13~~3-cyanopropionate (79) in 70% yield after chromatography. NMR

analysis of the chromatographed product reveaied a d amount of impunty, but unlike

methyl[1-'~~]-3-c~ano~ro~ionate (71, Section 3.1), methyI [2~-'~~~]-3-c~ano~m~ionate

was suitable for use without M e r purifîcation.

The reduction of the methyl [2,3-13~z]-3cyanopropionate to 4hydroxy-[2,3-

13~2]propionitrile (80) also proceeded smoothiy, affiording clean product in 71% yield

after coiumn chromatography. The labeiled nitde aicohol80 was then converted to [2,3-

'3~2]4iodobutyronitr~e (81) in the normal manneTt and upon column chromatography,

the desired labelled electrophiie was recovered as 0.546 g of a faintly yellow oii. NMR

anaIysis revealed that iodobutyronitrile 81 was quite pure, except for a srnali amount of

residual ether. Because this ether was not expected to be problematic in the foIIowing

alkylation steps, it was not removed Eom the IabeUed material. Mer correcting for the

presence of this residud solvent by NMR integrtition, the a d yield of otherwtse clean

[2,3-"~~]4iodobutyronitrile was 0.504 g (2.56 mmd, 49% yidd h m [2 f -*c2]4

hydroxybutyronitrile)). The overd precentage yield of [2 ~ - ~ ~ ~ ~ ] 4 i o d o b n t y r o n i t d e fiom

[2-13CJbromoacetic acid was I4.6%* accotmtnig for the fact that two equivdents ofthe

Iabeiled acid were dtimately incorporated into the labelled iodobutyn,~e 81-

With the required IabeIied 4-iodobutyronitdes 73 and 81 in hami, it remïned to

prepare the appropriate Belokon-type complexes for the asymetric derivatkation of

giycine (Figure 29, Section 1.5.4). The uniabefled Ni-L-BPB-GIy (42) requKed for the

preparation of [4,5-L3~2]~~Ysine was commercially available, but the complexes

recpired for the other target amino acids (Table 7) had to be synthesized according to

estabfished procedines65 that had aIready been successfuly empioyed by a ~olleague!~

N~-L-BPB-[~-'~C]G~~ (82) was prepared fiom commercial L-BPB ligand (41) and

[2-'3~]g~yche, which was purchased fiom Cambridge Isotope Laboratorïes (Figure

Table 7: Components Requbed for the SynthesY of the Desired Amino Acids

T q e t Requked Complex Required Aikylating Agent [2,3-"~z]-~-~~sine N~-L-BPB-[~-'~CC]GI~ (82) [4"~]41odobut~ronitrile (73) [2,3 - " C Z ] - D - L ~ S ~ ~ N~-D-BPB-[~-'~C]G~~ (83) [4-"~]41odobutyronitrile (73) [4y5 - ' 3~~-~ -~ys ine Ni+BPB-Gly (42) [ z J - ' ~ c ~ ] ~ I o ~ o butyronitri~e (81) [ & 5 - " ~ 2 ] - ~ - ~ ~ ~ i n e N~D-BPB-Gly (84) [2,3-t3~~~4~odobutyronitrile (81)

58). It had previously been e s t ab l i~hed~~*~~ that a fie-fold excess ofgiycine was required

to obtain an optimum yieId of Ni-L-BPB-GIy. This would normaiiy be of no

consequence, as glycine is a readiIy available and inexpensive reagent However, since

'3~-labelled glycine was otiIized, it was necessary to recover the unreacted excess amino

acid fbm the d o n mixture. This was accompIished SMpIy by taking up the rea&on

mixture in distilled water and extracthg the N~-L-BPB-[~-'~C]G(~ cornpiex (a), as welI

as any trace amormts ofunconsumed iigand, with chforofom. The Iabelîed glycine was

then separated h m other watex-sduble species by ion exchange chromatography. The

efficiency ofgiycine recovery, based on the assumption that 20% of the giycine was

consurned in the formation O~N~-L-BPB-[~-'~C]GI~ complex, was 87%.

The o complexes 83 and 84 wete prepared in the same mamer as the Nk-BPB-

[2-'3~]~1y complex, using D-BPB Ligand that had previously been prepared in this

Iaboratory by ~avisP6 In each case, the complex was prnined by column chromatography

and isolated in good yield (TabIe 8). NMR spectroscopy reveded that the synthetic

complexes were of high punty.

41 82

Figure 58: Synthesis of N~-L-BPB-[~-'~c]GI~

Table 8: Yields of Chromatographed Ni-BPB-G$ Complexes

3.4 Aikyiations of Ni-BPB-C;!(Jr Compiaes

The aD@ation of the Ni-BPB-GIy compIexes were perfonned in DMF with

NaOEE, as per the procedure developed by DavLp for 4bromobutyronitri1e.~~ The product

of the alkyIation O~N~-L-BPB-[Z-'~C]GI~ (TabIe 9, Run A) was chromatographed to

remove trace amounts of DMF, and to impmve the dtimate enantiomeric excess of the

[ ~ J - ' ~ C ~ ] - L - L ~ S ~ ~ ~ by separating out the smaii amount of e p h that wodd Iead to the D

isomer. Unfortunately, the recovered yield of alkylated complex was signincandy

reduced,

The stereoselecfivity ofeach aUcyIation was measmeci by hydrolysuig a s m d

amount of the aUcyIated complex, and determining the reiative amounts of each isomer

of the redting cyano amino acid (Figure 3 1) by chiral OPA HPLC. The

stereoselectivities reported in Table 9 are expressed as a percentage arrived at by dividuig

the amount of major isomer by the total amount of both isomers. The relative arnounts of

each isomer were detemiined by integration of the two peaks Li the chromatognun. For

Runs B - D, crude aikylated compIex was hydrolyzed and chiral OPA HPLC analysis of

the cyano amino acid indicated that the stereoseiectivity of the ais,Iation step was

approximately 90% (Le., a 90:IO mixtrire of enantiomers was obtained). The chiral

andysis of the product fiom Run A was performed after the aIkylated complex had been

chromatographed. No unchrornatographed product from Run A was avaiiable; it is Iikely

that the chromatographie step increased the enantiomeric punty nom the approximateIy

90% oberved in the other nms to the hi& Ievel fotmd for Rtm A. No chromatography

was performed on the products fiom runs B - D owing to the Iarge Ioss of materiai

suffered in Run A.

Table 9: Akylatioas of Ni-BPB-GIy Complexes wïth LabetIed 4-Iodobutyronitrlles

Rua CompIex EIectrophile Y ieId StereoseIectivity (%) ofAiIqIation

(%) A N~-L-BPB-[~-'~C]G~~ [413C]4~odobutyronitriIe 45 98.8

(a) (73) B N~-D-BPB-[~-"C]G~~ [4-"~]41odobutyronitrile 81 92.1

(83) (73) C Ni+BPB-GIy [2 f -"~~]41odobutyronitde 93 93 -4

(42) QU D Ni-D-BPB-O$ [2,3-13~2]4~odobutyronitrilee 98 87.1

(84) (81)

3.5 Isolation of Labeiied Lysines

The nnal chemicai steps required for the isolation of the IabeIied Lysines were the

hydrolysis of the aikylated compIexes and the cobaIt@') chloride-mediated sodium

borohydride reduction of the cyano amino acids thus produced (Figure 3 1). In pruiciple,

the intemediate cyano amino acids codd have been isolated via ion exchange

chromatography, but it was much more convenient to perfonn the hydrolysis and

subsequent reduction in one pot. Mer acid hydrolysis of the aUcy1ated compIex was

complete, water was added to the reaction pot, and iiberated BPB Ligand was recovered

by chloroform extraction. The aqueous Iayer was made basic, and the reduction was then

pediormed. The four Iabeiied lysines were then isolated by ion exchange chromatography

(Table IO).

The identities of the four product amino acids were confïrmed by 'H and 13c

NMR auatysis. In each case, the 'H NMR spectrom was similar to that ofunIabeiIed

lysine, ciiffiering ody in that many of the signafs were @te compficated owing to the

'.JCaH and '.JCCH couphgs that were present in the spectra ofthe IabeiIed sampIes, in

Chapter 3 - StereoseIectÏve Syntüesis of '3C+-Labe~ Lysines

Table 10: YieIàs of Isoiated LabeUed Lysines

Run h d u c t Yield (g) Yield (mol ) A [2*3 -"C2]-~-~ysine 0.1 15 0.773 B [2,3-13c2 sine 0.064 0.43 C [4,5-' 3~2]-~-~ys ine 0.135 0,913 D [4,5-13~2]-~~ysine 0.084 0.56

addition to the 2~H-H and 3 ~ ~ . a splitthgs n o d y present As a result of this extensive

couphg, none of the multiplets was nrst order, and due to extensive signai ovalap most

could not be reacüiy analyzed. The a-proton of each of the [2,3-"~21-labe~ed lysines was

an exception; this proton appeared as an apparent triplet of multiplets, with the three

mdtiplets separated by approxUnately 70 H z

Ody the labelled carbons were detected in the 13c NMR spectra and in each case

13 13 the C- C bond was readily observed as a pair of emfched doublets. AU four lysines

were found to be contaminated with s m d amounts of the precursory '3~-labelIed qano

amino acids, identined by the resonances of the IabeiIed carbons in the "C NMR spectra.

These impurities were present likely as a resuit of incomplete reduction of the nitde

hctionaIities in each case. Because the rnethyIenic protons were essentidy coincident

in the 'H NMR spectra, it was not possible to accurately mess the acaial amont of

impunty in each sampIe. Aithough I3c NMR integmtion is not n o d y usefuI for

accurate quantitative wodr, the relative amotmts of 5-cyanonorvaline in each lysine

sampie couid be at Ieast qdhtheLy estimated based on relative peak heights. The 123-

'3~~]-~-lysine contained the most contamniaat, with S-cyanonorvaüne m a h g up

appmximateIy 12% of the whole. In the case of [2,3-*~z~-~-l~sine and [4 ,5- '3~&~

lysine, contamhnt made up about 5% of the totai amino acid. [4,5J3~&~-~ysine

appeared to be fke of con taminant No attempt was made to remove the Unpurïty b m

each sample, as it remains to be determined whether 5-cyanonorvaiine wodd have any

bearing on the planned feeding experiments. As an dtemative to a purification based on

chromatographie separation of the cyano amino acid fiom the lysine, the contaminating

cyano amino acid in each sample may simply be reduced to Lysine by a second treatment

with cobalt @) c W d e and sodium borohydnde. Additionaliy, the Iysine samples might

be successfully purined by a crystalization method; this wouid also WceIy improve the

stereochemicai purity of each sampie, but wouid IikeIy resuit in a signincant loss of

material,

The 'H NMR spectra of the labelled lysine samples contained signais consistent

with significant quantities of unlabelled rnatenaI (Table 1 1), dthough no mono-labeiied

amino acid (which wouid give rise to an enriched shgiet in the NMR spectnim) was

present. If incompletely IabeiIed substrates had been employed in the 13c-13c bond

forming steps, a statisticd distribution of di-IabelIed, mono-labeIIed, and lmlabeiIed

mated wouid have been present, Suice the Lysine sampies were composed of only di-

and dabelled amino acid, the contaminating imlabelled material must have been

introduced after the labelIed Iysine was synthesized. Since each lysine was contaminated

with approximately the same amount of UnlabeIied materiai, it is likeiy that the

con taminant was introduced dirring the ion-exchange chrornatography of the product

amino acids. It is possible that the resin thus empIoyed was incompIeteIy regenerated

before use, and stül contained a smaii amount of dabeIIed Iysiae h m a previous

chromatography.

Table 11: Rehtive Amonnts of *cr~abeffed and Uniabeiled Lysine Present in the Recovered Samplea

Amino Acid % "~~-~abelIed % Unlabeiied [ 2 ~ - ' ~ ~ ~ ] - ~ - ~ y s i n e A 67 33

3.6 Conclusion

The D- and L- isomers of [2y3-13~iJlysine and [4,5-"~$~sine have been

successfùiiy synthesized in quantities SUfficient for the proposed feeding experiments.

The optical purity of each sample has not yet been detemhed, as reliable polarimetry

cannot be performed on impure samples, particdarily when the contaminant is opticaily

active. ln addition, the opticaf purifl of the contaminant lysine is not yet known for

certain; this must also be determined before an accmte assessrnent of the opticai puity

of the IabeiIed amino acids cm be made. avis^^ has shown that the stereochemistry at

the a-carbon of Iysine is not afEected by the hydrolysis, reduction, and ion-exhange

chromatography employed to generate this amino acid fiom the aikylated Ni-BPB-GIy

complex. It is therefore reasonable to assume that the stereochemicai pmity of each 13cr

Iabeiied Iysine is as determined by the chiraI OPA HPLC experiments perfomed to

measure the opticai purity ofthe aUryIated BPB-Ni-G1y compIexes (Table 9). 123-"~~1-

L-Lysine was, on the basis ofthis asmption, prepared with an exmtiomaic punty

approachg 99%, while the other IabeIIed lysines have optical prnities ofclose to 90%.

These stemchernicd pdes shouid be adepte for the proposed feeding experiments9

dthough they might be improved upon by a recrystaiüzation, which couId aiso remove

the smalI amotmt of 5-cyanonorvaline present in the samples.

The percentage of 13c enrichment also remains to be accurately determined.

Aithough 'H NMR gives a rough estimate of the amount of unlabelled lysine present in

the IabelIed sampIes, electrospmy mass spectrometry experiments will be performed in

order to determine with a much higher degree of accuracy the amount of I3c present in

the amino acids. These mass spectrometric experiments would have been necessary even

if the accidental dilution of "C label had not ocurred.

Chapter 4

1.1 Gened

AU NMR spectra were recorded on a Bniker Spectrospin AC250F spectrometer.

'H spectra were acquired at 250.13 MHz, and I3c spectra at 62.9 MHz, in the solvents

indicated AU chemicai shifts are reported with respect to TMS, although this standard

was ody used to directiy calibrate those spectra recorded in chlorofom-d. mer 'H

NMR spectra were caliirated against the residual protons in the corresponding

perdeuteraîed solvent, which incIuded acet011e-d~ (2.04 ppm), a~et0n.itriIe-d~ (1.93 ppm),

DMSO-d6 (2.49 pprn), methmol-& (3.30 ppm). "C NMR spectra were calibrated against

the central îine ofthe signal produced by the methyl carbon of acetowd6 (29.8 ppm),

acetonitrile-d3 (1.3 pprn), D M S O d (39.5 ppm), and methanol-& (49.0 ppm), or the

centrai line of chioroform-d (77.0 ppm). 13c NMR spectra recordeci in DzO were

c a l i t e d against a srnd amount of added methanol, at 49.5 ppm. AU couphg constants

are reported in EIz, and spiitting patterns are designated as fofiows: singiet (s), doublet

(d), tripiet (t), m e t (q), and multipiet (rn). In many of the '3~auiched compounds,

tiniabeiIed quateniary carbons were mt detected in the I3c NMR experhents, as they

were below the dynamic mge ofthe instrument Men assessmg the pMty ofprodwts

using NMR d . * s , a "minor" impinity was deked as one f i c h gbes rise to signais in

the 'H NMR spectnmi that integrate for Iess th IO% of the major peaks present, When

a component was desmied as a percentage ofa mixture, this percentage was caiculated

based on the number of moles present.

Electron impact m a s spectra were obtained using a CE mode1 21-1 10 mass

spectrometer at 70 eV.

Meltmg points were rneasured using a Gdenkamp m e b g point apparatus, and

values were read to the nearest 0.5 degrees and are reported unconected.

Freeze-dryhg was pafomied using an Edwards Mode1 E2M8 high vacuum pump

comected to an Edwards Moddyo fkeze dryer.

[1-13c]- and [2-L3~]bromoacetic acid were supplied by Cambridge Isotope

Laboratories in 99% I3c enrichment at the specined carbons. The lot numbers were

P-7564 and P-7074 respedvely.

4.2 Preparatr'on of Cyanosuccinate Esters

Dimethyl Cyanosuccinate (43): Raap's proced~s4 for the synthesis of the

correspondhg diethyl ester was foilowed to synthesize dimethyl cyanosuccinate (Table 2,

Run A). Sodium metai (0.61 g, 27 mmol) was dissolved in distîiIed methano1 (40 mL),

and the resuIting solution was cooled to O°C in an ice water bath. Methyl cyanoacetate

(1.30 1 g, 13.13 m o l ) was added to this solution, and stirred at O°C for 1 h. Methyl

brornoacetate (2.002 g* 13.09 mmoi) was added; the reaction was stked for 4 h at O°C,

and then for a htther 2 h at room temperature. The reaction was quenched by adding an

agneoos soIution ofpotassium bisiunte, untiI pH 7. The solvent was removed in v m o to

&ord a white residue, which was washed with d e r (4 x 100 mL). The combkd

&es were dned (anhydmus magnesium d a t e ) ancl, upon removd of solvent by

rotary evaporation, a paie yeiiow oii (1 -49 g) was obtained. NMR analysis reveaied that

the crude product was composed of three major components: the desired dimethyl

cyanosuccinate, dimethy13-carbornethoxy-3-cyanopentanedioate, and unreacted methyl

cyanoacetate. The relative ratio of these components was approxmiateiy 3 : 1 : 1. Dimethyl

cyanosuccinate: 'H NMR (chioroform-d) 6: 3.97 (t, J = 6.41 tlz, lHy -CH-), 3.86 (s, 3H, -

0CH3), 3.77 (s, 3H, -OCH3), 3.01 (app. t, J = 6.71 & îH, -CH2-) ; 13c NMR

(chforofomi-d) 6: 169.50 (Ça-), 165.47 (-Cor), 115.69 (-CN), 53.90 (-0CH3), 52.58 (-

0CH3), 33.34 (-CH2-), 32.66 (-CH-). DimethyI 3-carbomethoxy-3-cyanopentanedioate:

'H NMR (chforoform-d) 6: 3.89 (s, 3H7 -oc&), 3 -75 (s, 6H, -OCH3), 3 .O8 (A2Bt J =

16.78 Hz, 4H7 -CH2-); 13c NMR (chlorofomi-d) 6: 168.62 (-CO2-), 167.66 (-COz-),

1 173 3 (-CN), 5424 (wOCH3), 52-50 (-0CH3), 42.15 (-C-CN), 3 9.47 (-CH2-).

Dimethyl cyanosuccinate aiso formed when a single equivaIent of base was

empfoyed (TabIe 2, Run B). The reaaion mixture containhg methyI cyanoacetate (130

g, 13.1 mmol) and methyI bromoacetate (2.00 g, 13. I mmol) was stirred for 3 h at O°C

and then for 2 h at room temperature. The product (1.848 g) was a yeiLow oil which, by

NMR d y s i s , was composed of a 3 :8:7 ratio of dimethyl cyanosuccinate, dimethyl3-

carbomethoxy-3-~yanopentanedioate, and me&yl cyanoacetate.

In a thkd expriment (Table 2, Run C), a six-fold excess of sodium (1 -68 g, 73.1

m o i ) was employeci, and a Iarger vofume of methan01 (125 mL) was thus necessary.

MethyI cyanoacetate (1 2 9 g, 13 .O m o l ) and methy1 bromoacetate (2.0 I g, 1 3.1 mm09

were used, and the maciion was stined at O°C for 3 h, and at room temperature for 2 h.

The yeIIow product oii (1.09 g) contained dimethyI cyanosuccinate, dÏmethyl 3-

carbornethoxy-3-cyanopentanedioate, and methyl cyanoacetate in an approximateIy

1 3 5 5 ratio.

An attempt was aIso made to synthesize a mixed cyanosuccinate ester in acetone.

A mixture of ethyl cyanoacetate (0.74 g, 6.5 mmol), rnethyl brornoacetate (1 .O0 g, 6.53

mmol), and anhydrous potassium carbonate (1.8 1 g, 13.1 mmoI) in acetone (12 mL) was

s tkd in a dark freezer at -M°C for 19 k The reactioa was then taken up in ether (100

mL) and dried (anhydrous magnesium sulfate)). Removd of solvent by rotary evaporation

afForded a coIourless oii (1.198 g) which, by NMR andysis, was composed of an

equimoIar mixture of ethyl cyanoacetate and dimethyl3-carboethoxy-3-

cyanopenatanedioate (45), the diaikyIation produn This yield corresponded to a 98%

conversion of methyl bromoacetate to dimethyl3-cmboeuioxy-3-cyanopeatanedioate.

Dimethy 13-carboethoxy-3 -cyanopentanedioate: ' H NMR (chloro form-d) 6: 4.33 (q, 6.99

Hz, 2H, -OCH2-), 3 -75 (s, 6H. -OCH3), 3 -08 (A2&, J = 16.95 HZ), 1.36 (t, J = 6.99 HZ,

3H, -CH3); 13c NMR (chioroform-d) 6: 168.66 (-CO2-), 166.96 (-CO2-), 1 17-42 (-CN),

63 -6 1 (-0CH2), 52.43 (-0CH3), 42.24 (-c-CN), 39.33 (-CH2-), 13 -92 (-CH3).

4 3 Preparatioa of Cyanomaionate Esters

Diethyl Cyanomdonate (46): In an optimized procedure (Table 3, Run A),

anhydrous potassium carbonate (19.8% g, 14395 mmol) and ethyl chioroformate (16.50

mL, 173 mmoi) were added to a stirred solution of ethyl cyanoacetate (5.023 g, 44.40

mmol) in acetone (50 EL). The & h g suspension was sthed at reflux for 6 h, and

then dowed to COOL The d o n mumire was then srrspended in chioroform (300 mL)

to Induce precipitation of any dissolveci potassmm sait The yebw O ~ C Iiqmd was

removed via suction filtration, and the off-whte soiid that remained was washed with

chIomfomi (3 x 150 mL) and dissoIved in water (350 d). The acpeous solution was

washed with chlorofom (2 x LOO mL), acidSed to pH -0.6 (c. HCI), and extracted with

ether (4 x 200 mL). The etlhereai soIution was dried with anhydmus caicium donde and

the solvent was removed in vacuo to a o r d 46 (7.7û2 g, 94%) as a pale pink ooil. Major

tautornec 'H NMR (chloroform-d) 6: 4.53 (s, IH, CH), 435 (q, J = 7.07 Hz, 4H, -CH2-),

135 (t, J = 7.07 H z , 6H, -CH3); ' 3 ~ IWR (chiorofonm-d) 6: 160.58 (-Ca-), 1 1 1.49 (-

CN), 63.98 (-0CH2-), 44.65 (CH), 13.75 (-CH3); MS (70 eV): MO. 1 (13). 85.1 (53), 68.0

(47), 40.1 (I4), 39.1 (1 1), 29.4 (IOO), 28.7 (17). 27.9 (44). 27.2 (12). High resolution MS

(70eV): calculated for CaiiNO4 = 185.0688 amu, found = 185.0696 t 0.0008 mu).

Minor tautorner: 'H NMR (chioroform-d) 6: 4.42 (e J = 7.1 1 Hz, 4H, -OC&-), 3.49 (s,

rH, -OH), I .4O (t, 1 = 7.1 1 Hz, 6H, -CH3); 13c NMR (chioroform-d) 6: 176.1 8 (-CO2-),

1 13 -87 (-CN), 64.3 1 (-0CH2-), 60.54 (-GCN), 14-15 (-CH3),

in Rtm B (Table 3) the amount of each chemicai used was as foilows: ethyl

cyanoacetate (5.016 g, 44.35 mmol), acetone (60 mL), anhydrous potassiimi carbonate

(18.78 g, 136 mmol), and ethyi chioroformate (5.235 g, 4824 mm01). Reflux was

maintained for 19 h, and work-up was performed as above, except that onIy three

extractions with ether (200 mL each) were perfonned. D i a cyanomaionate (1.644 g,

20%) was isoIated as a pink oü.

In Rtm C (TabIe 3) ethyI cyanoacetate (1.047 g, 926 mmol), acetone (13 mL),

anhydrous potas& carbonate (3.80 g, 27.5 mmol), and etayI chlorofonnate (5.00 mL,

52 mmol, 5.6 equivaients) wem use& and diethy1 cyanornaIonate (0.528 g, 3 1%) was

obtained as a purp1e oil. The fÏItered chIoroform, together ~ Î ~ E L the chIorofomi used to

wash the basic aqueous solution, was dried with anhydrous caIcium chioride and

evaporated to aord a yeiiow oü (2.377 g), which was subjected to NMR anaiysis and

fond to be composed mainiy of diethyl carboethoxycyanomalonate. Diethyl

carboethoxycyanodonate: 'H NMR (chlorofom-d, ) 6: 4.40 (q, J = 7.07 Hz, 6H, -

0CH2-), 1.37 (t, J = 7.07 Hz, 9H, -CH3); 13c NMR (cidoroform-d) 6: 159.46 (-COr-),

1 1 1.1 1 (-CN), 64.73 (-0CH2-), 63 -61 (-GCN), 13.57 (-CH3).

An experiment (Section 2.22) was conducted to CO& that the minor species

observed in the chloroform-d NMR spectra of synthetic diethyl cyanornaIonate was

indeed the en01 tautorner. A solution of 46 (0.106 g, 0.57 m o l ) in chloroform-d (1 mi,)

was prepared, and the 'H and 13c NMR spectra (descn'bed above) were obtained An

excess amount of triethytamine (120 pL, 0.86 mmoi) was added to the sarnple and, after

severai inversions of the NMR tube, the NMR spectra were again obtained, A correction

factor of 0.66 was appüed in the integration of the 'H resonances arising fiom

~ethylamineltriethylarnmonium in order to account for the fact that a 1.5-fold exceu of

base was used. 'H NMR (chloroforma) 6: 3.86 (q, I = 7.22 Hz, 4H, -OC&), 2.76 (q, J =

6.78 Hz, 6H, -NCH2-), 1 .O2 (t, J = 722 Hz, 6H, -OCH2C&), 1.00 (t, l= 6.78 9H, -

NCH2C&); I3c N1MR (~hlotoform-d) 6: 169.90 (-CO2-), 124.14 (-CN), 57.95 (-0CH2),

55.80 (-Ca, 46.07 (-NCH2-), 14.77 (-0CHQ13), 9.49 (-NCH&H3).

Dimethy t Cyanomdonate: Methy1 cyanoacetate (6.1 5 g, 62- 1 mmol), aahydrous

potassium carbonate (28.15 g, 203 -7 mmol), acetone (100 mL), and methy1 chIoroformate

(18.8 mL, 243 mm09 were mixed and refluxed for 6 h, and the work-up proceeded as for

the diethyI ester 46 except that d e r amounts of water (250 mL) and ether (4 x LOO mL

extractions) were wed The product dimethyl cyanomalonate (8.44 g, 87%) was isolated

as a yeiiow oil. Major tautomer: 'HNMR (chloroforma) 6: 4.67 (s, lH, -CH), 3.90 (s,

6H, -ocH3); "C NMR (chl0r0fom-d) 6: 161.1 5 (-Ca-), 1 I 1.57 (-CN), 54.48 (-oc&),

44.24 (CH). Minor tautomer: 'H NMR (chloroformd) 8: 3 .!JO (s, 6H, -OC&), 3.45 (s,

1H, -OH); I3c MAR (cbloroform-d) 6: 176.40 (-COr), 60.44 (-c-CN), 54.67 (-oc&).

Ethyl Methyl CyanornaIonate: Methyi cyanoacetate (6.1 5 g, 62.1 rnmol),

anhydrous potassium carbonate (28.36 g, 2051 mmoi), and ethyl chloroformate (23.0

mL, 241 mrnol) in acetone (100 mi,) were stirred at reflux for 3 h, when the amount of

soIid that had fonned rendered the mixture unstirrable, The entire miction mixture was

taken up in water (600 mL), which was then washed with chioroform (2 x 200 d)

before acidincation (c. HCL) to pH O. The aqueous layer was exûacted with ether (3 x

200 mL). The combined extracts were dried (anhydrous caicium chloride) and

concentrated in vamo to &ord ethyi methyi cyanomalonate (8 S6 g, 8 1 %) as a paie

yeiIow 03. Major tautomer: 'H NMR (chioroform-d) 6: 458 (s, IH, -CH), 434 (q, J =

7.74 Hz, 2H, -OCH2-), 3.89 (s, 3K, -OC&), I .35 (t, J = 7-74 Hz, 3H, -CH3); I3c NMR

(chlorof0mi-d) 6: 16 1.1 8 (-C*), 160.53 (-CO2-), 1 1 1 -48 (-CN), 64.1 1 (-0CH2-), 54.42

(-0CH3), 44.46 (s-CN), I3 -75 (-CH3). Minor tautomer: 'H NMR (chlorofom-d) 6: 4.43

(q, J = 7-98 Hic, 2H, -OCH2-), 3.96 (s, 3H, -OCH3), 3.51 (s, lH, -OH), 1-41 (t, J = 7.98

EIz, 3H, -CH3); L 3 ~ NMR (chiorofom-d) S: 64.79 (-OCH& 60.53 (-c-CN), 5427 (-

0CH3), 14-16 (-a).

Chapter 4 - Experimentzu

Diethyl[2-*~~~rnomaIonate (77): The IabeiIed cyanomaionate ester 77

required for the synthesis O ~ D - and ~-[4,5-~~2]1~sine was prepared fiom ethyI [2-

'3~]cyanoacetate (Section 4-7,2.183 g, 19.13 mmol), anhydrous potassium carbonate

(8.63 g, 62.4 mmol), and ethyi chioroformate (5.74 g, 52.9 m o l ) according to the

procedure deveioped for ualabeiIed material. Clean diethyl [2-'3~]cya.omaionate (2.63 8

g, 74%) was thus isolated as a paie yeilow oü. Diethyl[2-"~]cyanomalonate: 'H NMR

(acetonedi) 6: 5.14 (ci, L~c-H = 141 Hz, IH, -CH), 430 (q, J = 722 tf5 4H, -OCHT), 129

(t, 1 = 7.22 Hz, 6H, -CH3); 13c NMR (acet~ne-d~) 6: 162.13 (d, 'JCc = 60.08 Hz, -COz-),

113.09 (d, 'JcC = 65.80 - 0 , 6 4 2 3 (-OC&), 45.59 ("c-e~ched, s, -CH), 14.12 (-

CH3).

4.4 Pnparntion of Saib fkom Cyanomaionate Esters

Triethyiammonium sait (52) of Diethyl Cyanomaionate: Triethylamine (1 -441

g, 14.24 mmoi) was added to a solution of diethyi cyanomaio~te (1.437 g, 7.76 m o l ) in

ether (25 mL), Rotary evaporation of the solvent e d e d an off-white solid, which was

rirised with copious amounts of ether in a Smtered gIass h e I . The solid was dried in

varno, and 52 (1 -915 g, 86%) was thus obtained as an off white powder.

Tnethylâmmonium sait ofdiethyi cyanomaiomte: 'H NMR (acetoned6) 6: 3 -99 (q, 5 =

7-15 Hk, 4H, -0CHz-), 3.26 (m, 6H, -NCH2-), 1.27 (t, 7.16 H.& 9H, -NCH2CE&), 1.15 (t,

7.1 5 EIz, 6H, - 0 C H m ) ; 13c NMR (acetone6) 8: 170.72 (-C*), 58.19 (-OCHT),

46.74 (-NCH2-), 1 S M (-OCHs3), 8 8.6 (-NCHQ!13).

Pyridiniuxn Sait (53) of Diethyl Cyanomaionate: Diethyl cyanomaionate was

prepared as above (Section 4.3) iÏom ethyI cyanoacetate (7.01 & 62.0 mmol), rahydrous

potassium carbonate (28.37 g, 205.3 mmol) and ethyI chioroformate (23.0 mL, 241

mmol), and worked up in the d marner to &fiord a yelIow oiI which was taken up in

pyridine (25 mL). White crystds formed, and crystallization was aided with the addition

of hexane (50 d). The resultant suspension was suction-filtered, and the white powder

that remained was iinsed with copious amomts of hexane. The white crystals were dried

in v a m to Bfford 53 (14.420 g, 88%). Pyridinium sait of diethyi cyanornaIonate: 'H

NMR (acetonitriIe-d3) 6: 8.94 (m, W, ortho H), 8.52 (m, 1 H, para H), 8.0 1 (m, 2H, meta

H), 4.24 (q, J = 7.10 Hz, 4H, -OCH2-), 1.32 (t, J = 7. IO Hz, 6H, -CH3); "C NMR

(acetonitrile-d3) 6: 1 70.3 9 (-CO2-), 145.88 (-CH-), 144.08 (-CH-), 127.79 (-CH- ), 1 1 8.33

(-CN), 59.79 (-0CH2-), 1 5.1 8 (-CH3).

Sodium Sait (54) of Diethyl Cyanomaionate: An ethanolie solution of sodium

ethoxide (33.19 mL, 0.5046 M, standardized against KHP) was added to diethyl

cyanornaIonate (3.183 g, 17.19 mol) , and then the buik of the ethanol was removed in

vanro. Upon addition of ether (80 mL), white crystais formed, whic6 were separated via

suction filtration. Thorough rinsuig of the powder, foiIowed by removai of the ether

under vacuum finnished a soiid (3.463 gF mp = 180.0 - 207.0°C) which, by NMR

analysis, stiII contained some sodium ethoxÎde. The rnajonty of this powder (3.234 g)

was redissoIved in eh1101 (40 mL), and an additional portion ofdiethyI cyanomaionate

(0376 g, 2.03 m o i ) was added. As before, the ethan01 was removed and repIaced with

ether (80 mL), to mitiate CrystaIliZation, The soEd was separated by sucfion nitration

with ether rinsing, and upon drying uuder vacuum, 54 (2.975 g, 75% with respect to totai

diethyI cyanodonate) was obtained. Sodium sait of diethyI cyanodonate: mp: 204.5 -

207.0°C; 'H NMR (acetonitrile-d3) 6: 3 9 8 (q, J = 8.44 Hz, 4H, -OCH2-), 1.15 (t, J = 8.44

Ek, 6H, -CH3); 13c NMR (acet011itriie-d~) 6: 171.57 (-COr-), 124.06 (-CN), 59.12 (-

0CH2-), 57.02 (-c-CN), 15.15 (-CH3).

Triethylammonium Salt (55) of Dimethyl Cyanomalonate: Triethylamine

(0 -695 g, 6.8 8 mmol) was stirred into a solution of dimethyi cyanornaionate (1 .O7 1 g,

6.82 mmol in ether (20 mL). Precipitation of a white soiid was induced by addition of a

mixture of hexanes (40 mL), and the resuitùig suspension was cooled in an ice water bath

for 15 min before suction filtration. Rinsing of the crystais (mixed hexanes) foUowed by

drying under vacuum aEorded 55 (1 -458 g, 83%). Triethyiammonium sait of dimethyi

cyanomalonate: 'H NMR (chioroformii) 6: 3.59 (s, 6H, -OCH3), 3.2 1 (q, .f = 722 Hz,

6H, -NCHr), 1 .3O (t, J = 7.22 H i , 9H, -NCH2C&); 13c NMR (chiorofom-d) 6: 170.34

(-CO2-), 124.16 (-CN), 55.68 (-c-CN), 49.95 (-0CH3), 46.16 (-WH2-), 8.48 (-CH3).

Tehgbutyiammonium Salt (56) of Dimethyl Cyanomalonate: A methanoiic

solution of tetrabutyIammonium hydroxide (7.15 mL, 25%) was added to a portion of

dimethyl cyanomalonate (1.01 1 g9 6.44 mmol). Precipitation of the deshed salt was

induced by adding ether (300 mL), and the paie yeUow powder (2.412 g) was isolated by

suaion filtration, foiIowed by riPsiog (ether and petroIemn ether) and drying under

vacuum. The powder was dissolved in dichioromethane (15 EL), and then cyta lhed by

the addition of ether (80 mL). An off white solid was sgain recovered by suction

filtration. One more cryskdhation h m dichlofornethane with etha was paformed, and

56 (1 -799 g, 70%) was isolated as an off-whte powder. TetrabutyIammonium sait of

dimethy1 cyanomdonate: mp: LM.0 - 1 15.SaC; 'H NMR (chloroform-d) 6: 3.58 (s, 6H, -

oc&), 3.26 (m, 8H, -NCH2-), 1.64 (quinte& J = 733 Hz, 8H, -NCH2C&-), 1-43 (sextet,

J = 7.33 HZ, 8H, -NCH2CH2C&), 1 .O0 ((5 J = 7.0 1 HZ, 1 W, -CH3).

Sodium Salt (57) of Dimethyl Cyanomaionate: Sodium meta1 (0.143 g, 623

m o l ) was added to a stimng solution of dimethyi cyanomalonate (1.028 g, 6.54 mmol)

in methanol(30 mL). Upon dissolution of the sodium, a mixture of hexanes (50 mL) and

ether (50 mL) was added, and a white precipitate formed The precipitate was isolated by

suction filtration, washed with hexanes, and dried under vacuum to &rd 57 (1 .O03 g,

86%). mp: 3 18 - 325OC (dec.); Sodium sa i t of dimethyl cyanomalonate: 'H NMR

(DMSOd6) 6: 3.44 (s, -OC&); 13c NMR (DMSO-ds) 6: 169.00 (-COz-), 122.87 (-CN),

55.04 (-C-CN), 49.40 (-0CH3).

Potassium SaIt (58) of Dimethyl Cyanomaïonate: Potassium metai (0273 g,

6.99 mmol) was carefiilly dissolved m Z-butanol (100 mL), and this solution was added

to dimethyl cyanomaionate (1.161 g, 7-39 mmoi). A white precipitate formed instantiy,

and a mixtrne of hexanes (600 mL) was added. The white soiid was cokcted by suction

mtration, washed with petroleurn ether? and dried under vacuum to yield 58 (1 23 231 g,

85%)). Potassium sait of dim&yI cyanomaionate: mp: 305 - 30g°C (dec.); 'H NMR

@MSO-<ls) 6: 3.44 (s, -OCH3); ' 3 ~ NMR (DMSOd;) 6: 172.37 (-Ca-), 127.02 (-LN),

56.92 (-CCN) 5 I -84 (am3).

4.5 Erperiments to Probe the NucIeophiliety of D-1 C@nomilonate Sdts

ui a typicd experiment, the salt was weighed mto a smd vid, and dissolved in a

s m d (- 0.5 mL) amount of deuterated solvent. Then, a solution of electrophile in a

similar volume of solvent was prepared in a second vid, and this mixture was transfemd

via pipette into the fust vial. The combined mixtrne was transferred between the two

viais several &es, and then into an NMR tube, fitted with a vented cap. The reaction

was monitored by NMR, and was heated, ifnecessary, in a thermostated (f - SOC) water

bath. The actuai amounts used in the individuai experiments are specined in Table 5. The

amounts of nudeophile and electrophüe were caicuiated based on the m a s

(nucIeophiles) and volumes (electrophiles) employed. The limiting reagent in each

experiment, however, was determined by 'H N M R integrations, to compensate for

experimental inaccuracies in weighing and pipetting. The extent of reaction was

monitored by meamhg by 'H NMR integration the relative amounts of s t m g

materials and product. The extent of &on was then cddated (TabIe 5) by dividing

the amount of product by the total amount of product formed ptus ~~lfeacted limituig

reagent,

DimethyI benyicyanomalonate (59,0.069 g, 74%), the product fiom the

aikylation of 58 with benyl brodde, was isolated fiom the reaction mixture in order to

determine a reactïon yield, as weiI as to demonstrate that the pmduct of the allcyIatioas

couId be easily isolated. Mer the 48h reaction p e d , the d o n medium was taken up

in EtzO (75 d), and washed with water (2 x 50 d). Atter dzying (dydrous

magnesium sulfate), HnItmng, and evaporation of solvent, 59 was obtained as a cIear

coiou~1ess oü, DhethyC benzylcyanornaIonaîe: 'H NMR (chIomforma) 6: 7.34 (s, SE&

H-Ar), 3.85 (s, 6H, -0CH3), 3.54 (s, W, -CH2-); 13c NMR (chloroform-d) 6: 164.54 (-

~OZ-), 134.33 (aryi C a , 130.8 1 (aryl -CH-), lî9.3 9 (aryl -CH-), 128.86 (aryl -CH-),

1 15.32 (-CN), 54.85 (-0CH3), 40.10 (-CHr).

4.6 mlat ion of Cyimomdonate Esters

Ethyl Methyl2-Carboethoxy-2-cyanosuccinate (60): in an optimized reaction

(Table 6, Run A) potassium bicarbonate (2.104 g, 2 1 .O 1 mmol) was added to a solution

of diethyi cyanornaIonate (3.658 g, 19.75 mmol) in acetone (50 mL). The stined mixture

was refluxed for 15 min, foiiowing which the heat was removed, and methyl

bromoacetate (3.022 g, 19.75 mmol) and potassilmi iodide (0.324 g, 1.95 mmol) were

added Mer a further 18 h of heating, the reaction was alIowed to cool, and moted with

chiotofomi (300 d), which was dried over anhydrous calcium chloride, and the soiid

residue was removed via suction Htratioa The chloroform was removed in vacuo, to

fumish a pink oil contauüng some white soiid. This mixture was taken up in ether (130

mL) and washed with aqueous sodium bisuifite (2 x 100 mL, 10 g in 200 mL). The

organic Iayer was dried (dydrous calcium chforide), decolourized (neutrai carbon), and

suction-filtered thmugh a Celite pad. The ether was removed on the rotary evaporator to

&ord 60 (4.935 g, 97%) as a faintIy yeIiow oiI. EthyI methyi 2-carboethoxy-2-

cyanosuccinate: 'H NMR (chloroforma) 6: 4.36 (q, J = 6.96 Hz, 4H, -OC&), 3.76 (s,

3H, -om3), 3 2 9 (s, 2H, -CH2C02-), 1 35 (t, J = 6.96 Hi, 6H, -CH3); 13c NMR

(chioroforn-d) 6: 168.47 (-CC)& 162.59 (-COz-), 114.46 (-CN), 6424 (-OC&=), 52.44

(-0CH3). 5 I.64 (s-CN), 38.05 (-m2C&-), 13-60 (-CH3); M S (70 eV): I X . 2 (56),

153. I (79,139. I (22), 126.1 (37), 125.0 (M), 98 (97), 29.4 (1 00), 28.7 (Z), 27.9 (44).

Egh resolution MS (70eV): calculated for Ci rHr = 257.0899 amu, fomd =

257.0910 f 0.0008 mu).

The synthesis of ethyl meth. 2-carboethoxy-2-cyanomccinate fiom diethyl

cyanornaIonate (3.641 g, 19.66 mmol) was repeated without potassium iodide catalysis

(Table 6, RF C). The reflux was maintained for 24 h an& d e r a work-up as descriied

above, a paie yeiiow liquid (4.450 g, 88%) was obtained. NMR anaiysis revealed that the

mde product was actually a mktme of desired ethyl methyi 2carboethoxy-2-

cyanosucciiiate (- 87% by number of moles present) and unreacted methyl bromoacetate

(- 13%), dong with a negligible amotmt of ether.

Diethyl2-Carboethoxy-2-cyanosuccinate (61): The above procedure for ethyl

rnethyl2-carboethoxy-2-~ymo~~ccinate (60) was adapted for the preparation of diethyl

2-carboethoxy-2-qanosuccinate (Table 6, Rtm B). A mixtlrre of diethyl cyanornaIonate

(7.147 g, 38.60 mmol) and anhydrous potassium carbonate (2.694 g, 19.49 mrnol) in

acetone (50 mL) was s h e d at reflux for 20 min, before the addition of ethyl

bromoacetate (6.457 g, 3 8.66 mmol) and potassium iodide (0.320 g, 1 9 3 mmol). After 14

h at reflux, the reaction was cooled and worked up in the same manner as the mixed ester

60, except for the amount of aqueons sodium bisulnte (5 x 100 EL, 7.5 g in 500 mL)

used for washing. Cornpouad 61 (9.5616,9 1%) was isolated as a pale yellow oil A smaii

m t (- 4% by number, as detammed by NhilR) of dÏethy13-catboethoxy-3-

cyaiaogiutarate was aIso present Die&yI 2~ethoxy-2~anosucCinate: 'H NMR

(chIor0fom-d) 6: 4.35 (q, I = 7.22 Eh, 4H, €&CH2-), 422 (q, I = 7-06 H i , W, -

CO&S2-)), 3-27 (s, W, -CH2C&-), 135 (t, I = 7.22 OC&), 1 3 (t, I = 7.06 &

3H, OC&); 13c NMR (chIoroformoform-d) 6: 168.02 (-Ca-), 162.82 (-Ca-), 1 14.65 (-CN),

64.37 (-0CH2-), 61 -65 (-0CH2-), 5 1.8 1 (S-CN), 3829 (-CH2C&), 14.04 (-CH3).

Diethyl3-catboethoxy-3-cyano@utarate: 'H NMR (chloroforma) 6: 4.32 (q, .J = 7.22 Hz,

2H, -C02CH2-), 4.20 (e J = 7.3 3 H i , 4H, -C&CH2-), 3 -07 (m, 4H, -CH2C@-), 1 3 2 (t, J

= 7.22 H i , 3H, -CH3), 1.29 (t, J = 7.33 Hz, 6H, -CH3); "C NMR (chloroforxn-d) 6:

167.62 (-CO2-), 164.64 (-CO2-), 1 17.1 1 (-CN), 63 .O9 (-0CH2-), 6 1.43 (-0CH2-), .

in a preliminary experiment (Table 6, Run D), an attempt was made to synthesize

diethyl2-carboethoxy-2-cyanosuccinate 61 in the presence of a two-fold excess of

anhydrous potassium carbonate- This nui also difTered fkom the successful synthesis of

diethyl 2-carboettioxyL2~yano~ccinate Ïn that alI of the reagents were mixed together at

once, so that no preliminary reflux to d o w the formation of the potassium sait of diethyl

cyanomaioiÿite was pdonned. A mixture of diethyf cyanomaionate (0.492 g, 2.66

mmol), anhydrous potassium carbonate (0.758 g, 5.49 mmof), potassium iodide (0.068 g,

0.41 mmol), and ethyl bromoacetate (0.445 g, 2.66 m o l ) in acetone (13 mL) was

prepared and stked at reflux for 18 h. The reaction mixture was then cooled, taken up in

chioroform (IO0 mL), and suction nItaed to separate a yebw liquid fiom an off-white

solid The chioroform was dried over anhydrous cdcirmi chloride and then evaporated in

vucuo to Sord a pale yeiiow oii(1.32I9g). NMR anaiysis reveaied that the oil was

composed of diethyi 2-carboethoxy-2-cyanosuccinate (0252 g, 35% yieId, 18% of

mixîure), diethyl3 -carboethoxy-3-cyano@utarate (O. 1 1 1 g, 15% yield, 7% of mixhire),

and 4hydroxy4methyI-2-penainone (0959 & 75% of mixture). The latter compound

was Iikely fomed fkom the =Ivent acetone dukg the s<penment 4Hydto~y4m&.l-

2-pentanone: 'H NMR (ch1oroform-d) 6: 3.90 (s, fH, -OH), 2.65 (s, W, -CH&), 2.19 (s,

3% -(CO)CHz), 125 (s, 6H, -CH& 13c NMR (chloroform-d) 6: 21 0.48 (-CO-), 69-14 (s,

OCOH), 53 -60 (-CH2-), 3 1.39 (-(CO)cH3), 28.87 (-CH3).

Ethyl Methyl [4-'3~~-2-~arboeth~xy-2-cyan~~u~~inate (70): The labelled

nitrile triester 70 required for the synthesis of [4- '3~]4iodobutyr~ni~e was prepared in

acetone (IO0 mL) Eom diethyl cyanomaionate (7.674 g, 41.44 mmol), potassium

bicarbonate (4215 g, 42.10 mmol), potassium iodide (0.551 g, 3.32 mmol), and methyl

[I-"~]bromoacetate (- 41.38 m o l , Secfion 4.6). The reaction was maintained at reflux

for 1 1.5 h and after work up, ethyi methyl [ 4 - L 3 ~ ] - 2 ~ b ~ e t h ~ x y - 2 ~ y a n ~ ~ c c i n a t e

(8.3 59 g, 78%) was obtained as a faintly yellow oil. Ethyl methyl [4-*3~]-2-carboethoxy-

2-cyanosuccinate: 'H NMR (acetone-d6) 6: 4.33 (q, J = 7.08 Hz, 4H,

-OCH2-), 3.72 (d, 3~C-H = 4.228 HZ, 3H, -OCH3), 3.36 (d, 'JC-H = 733 H g W, -CH2COr),

129 (t, J = 7-08 tIz, 6H, -CH3); 13c NMR (acetone-ds) 6: 169.62 ('3~-enrïched, -

C02CH3), 64.90 (-OC&), 52.77 (d, 'JCC = 2.86 & -oc&), 38.64 (d, '.JCc = 6020 HZ, -

-CH2C02-), - 14-06 (-CH3).

Ethyl Methyl[2~-'~~~~-2-~arboetho~-2~~~~0mccinate (78): The established

procedure was aiso employed in the synthesis of ethyL methyl [2,3-'3~z]-2-carboeth~xy-

2-cyanosuccinateY an intermediate in the pahway towards [ 2 , 3 - " ~ ~ ] 4 i o d o b ~ ~ e .

78 (2.695 g, 79%) was prepared in acetone (50 mL) Eom potassium bicarbonate (1.495 g,

14.94 mmoi), methy1 [2-u~]brom~acetate (1 3.12 mmol, Section 4.6), diethyl[2-

13CJcyan~md~nate(2.638 g* 14-17 mmoI, Section 43 , and potassinm iodide (0.248 g,

1.49 mmol), and wodred up in the us& mamer. EthyI methyl [2,3-3~2~-2-carboeth~xy-

2-cyanosucCinate: 'H NMR (acetone46) 6: 4.33 (q, J = 7.02 Hz, 4Hy -OC&-), 3.72 (s,

3H. -oc&), 3.36 (dd, = 136.10 El& 2~c.H = 4.28 Hz, 2H, -CH2C&-), 1-30 (t, J =

7.02 Hz, 6H, -a); 13c NMEt (acetone-d) 6: 169.53 (d, IJCc = 60.08 Hz, <&CH3),

163 5 0 (d, 'JCC = 58.18 EIz, -nCH$X3) , 64.88 (S. -OCH2-), 52.90 (13c-enricheci, d,

'JCC = 37.67 Hz, -CN), 38.63 (13ce~ched, d, 'Jcç = 37.67 Hz, <HzC&), 14.06 (s,

-CH3).

4.7 Esterification of Bromoacetic Acid

The method for the methyl estuincation of the bromoacetic acid was h s t

developed and optimued using unlabeIied substrate, before being applied to the Iabeiied

subûtrates. Because of the high vapour pressure of the product, best redts were obtained

when rotary evaporation was avoided in the work-up. As such, the IabelIed methyl

bromoacetates contained a smaii amount of residuai ether.

Methyl Bromoacetate: The method of ~1ackR was used to prepare the

diazomethane using a diazomethane kit (Aldrich) that consisted of giassware having no

sharp edges or ground glas joints. A soIution containing potassium hydroxide (4 g),

water (6 mL), and ethanol(I8 mL) was heated to 60°C in a round-bottom fia& Mted

with a double-necked adapter. This adapter was, in nim, fitted with a dropphg addition

finine1 and a sW-head. The stilI head was aîtached, via a condenser and a receiving

adapter, to au ice/HZO-cooled soIution ofbmmoacetic acid (497 g, 35.8 mm09 in ether

(20 mL). The soIution of bromoacetic acid was contindy cooled and magnetically

stirred with a micro stirriag bar thtoughout the reactioil An ethered soIution of Diazaid

(15 g in 100 mL ether) was slowïy dnpped via the addition funne1 into the heated

aqueous solution, at a rate of about one drop per second. The diazomethane, diluted with

ether, was didieci through the condenser into the acid solution. The rate of Diazaid

addition was adjusted to approximately match the rate of diazomethane distiliation, aml

the distillation of diazomethane continued in this fashion untii a faint yellow colour

persïsted in the receiving flask. At this point, the addition of Diazald was stopped, and

ether (40 d) was added to the fl ask containhg the basic aqueous solution and then

distilled into the receiving flask to ensure that ail of the diazomethane that had been

formed was transferred. The glassware was disassernbled, and the excess diazomethane,

dong with the bulk of the ether, was distüied into a solution of acetic acid in ether in

order to destroy unreacted diezomethane. The total volume of the product liquid was

reduced in this manner to - 5 mL of a clear coIourless mixture of methyi bromoacetate

with ether, and was used in subsequent reactions without removai of the residuai solvent.

Methyt [l-'3CJ~romoacetate (69): Methyl [Lt3c] bromoacetate was p r e p d

fiom [~-~~~]brornoacetic acid (5.79 g, 41.4 m o l ) as above and the product was used

without any M e r puTification or chmcterization.

Methyl [2-13q~romoacetate (75): The above method was aiso used to convert

[2-13~~romoacetÏc acid (1.84 g, 13.1 mmol) into methyi [2-U~]bmmoimcetate.

4.8 Conversion of Bromoacetic Acid into Ethyl Cyanoacetate

Aithough ethyl cyanoacetate was commercially avaiIabIe and inexpensive, a

method for its preparation in high yieId fiom bromoacetic acid was developed in order to

synthesize the ethyi [2-'3~]cyanoacetae required for the synthesis of [2,3-13c2]-4-

iodobutyronitde.

Ethyl Cyanoacetate: A stirred solution of bromoacetic acid (2.74 g, L 9.7 mmol)

in water (10 mL) was heated to W C , and potassium bicarbonate (2.28 g, 22.8 mmol)

was slowiy and carefuly added The heat source was removed, and potassium cyanide

(1.48 g, 22.8 mmol) was added in portions. The reaction mixture, which became

noticeably warmer upon potassium cyanide addition, was stirred for 40 min. The buik of

the water was removed on the rotary evaporator, and the remainder was fiozen with

Iiquid nitrogen and lyophilwd ovemÏght DMF (20 mL), and iodoethane (4.0 mL, 50

mmol) were added to the white soüd that remained, and the resultant mixture was stirred

at 57OC for 8 h. The mction was taken up m a mÏxture of sodium bisulnte (5 g in 10 mL

water) and saturated brine (90 mL), and this aqueous mi- was extnicted with ether (4

x 100 mL). The ether was dned (magnesium suIfate), dedourized with netitrai carbon,

and concentrated by distilIation at atmospheric pressure to a yelIow residue that

was chromatographed on silica gel (60 g) with ether as eluant (IO0 mL M o n s ) . Those

fiactions containhg ethyI cyanoacetate were combined, and the bulk ofthe ether was

ciistiiled off at atmospheric pressure- The remainmg soIvent was removed with a Stream

ofnitrogeri, &orcikg eth.1 cyanoacetate as a yeIIow oïl (199 g, 89%), which contained a

d amount of tmidesfified impudy. EthyI cyanoacetate: 'H NMR (chioroform-d) 6:

4.27 (e J = 7-07 HZ, W. -OCH2-), 3.50 (s, 2EI, -CH2CN), 133 (t, J = 7.07 HZ, 3H, -CH3);

NMR (chlorofom-d) 6: 163.15 (-C*), 1 13 36 'CN), 62.96 (-OC&-), 24.79 (-

C H m , 14.1 5 (-CH3). UnidentifÎed inpinity: 'LH NMR (cblorofom-d) 6: 2.07 - 1-80 -

(m), 1.23 - 1-04 (III)*

Ethyl [z-"~lCyanoacetate (76): The procedure described above for the

preparation o f unlabeIIed ethyl cyanoacetate was repeated with [2-13~]bromoacetic acid

on a 3 .O7 g (21 -9 mmol) scale, to synthesize 76 in 87% yield after column

chromatography. Ethyi [2-'3~]cyanoacetate: 'H NMR (acetone-ds) S: 4.21 (q, J = 7.06

Eh, m, -OCHr), 3.80 (d, ' J ~ . ~ = 137.25 Hz, 2H, -CH2CN), 126 (t, J = 7.06 Hz, 3H, -

CH3); 1 3 ~ NMR (acetone-de) 6: 62.95 (s, -OCH2-), 24.97 (13~-enriched, <HzCN), 1432

(-CH3)3).

4.9 Decarboalkoxylation of Ethyl Methyl2-Carboethoxy-2-cyan0~~~~inate

MethyL3-Cyaaopropionate (63): A stined mixture of ethyl rnethyl2-

carboethoxy-2-cyanosuchte (3.96 g9 15.4 mmol), water (12 mL), sodium chioride

(039 g, 6.6 mmol), and DMSO (45 mL) was heated in an oil bath at 132°C for 14 h The

brown reaction mixture was cooIed to RT and taken up in saturated brine (100 mL),

ôefore extraction with ether (4 x 100 mL). The combined organic extracts were drÏed

with anhydmus magnesium sulfate, treated with neutrai decoiouaPng carbon, and

reduced to about 100 mL by didation at atmosphenc pressure. The remahhg organic

mixhire was shaken with satucated brine (IO0 mL) and separated. The aqueous Iayer was

extracted with d e r (3 x IO0 mL) and the comhed organic layers (- 400 EL) were

again dried with anhydrous magtlesium sulfate and treaîed with neutraI decoIourizing

carbon. The organic solution was nItered through Celite at the aspirator, and the b& of

the solvent was removed by atmospheric distillation. The remaining ether was blown off

with nitrogen for 15 min, aEording the product as a yellow oil(1 A2 g9 82%), wfüch

contained a very smaü amount of unidentified imputity. Methyl3-cyanopropionate: 'H

NMR (acetone-d6) 6: 3.68 (s, 3IE, -()CH3), 2.74 (m, 4H, -CH2CHÎ-); 13c NMR (acetone-

66) 6: 169.72 (-C*), 1 19-86 (-CN), 52.24 (-0CH3), 3025 (-cH2C&-), 13 -22 (-cH2CN).

Methyt [l-13~-3-Cyanopropionate (71): The above procedure was employed

to decarboethoxylate ethyl rnethyi [1-~~~]-3-c~ano-3-carboetho~~~ccinate (3.952 g,

15.25 xmnol). The &on was maintained at 132OC for 21 h. Ody one senes of

extractions was performed in the work-up, and the crude product was chromatographed

on silica gel with ether (50 mL htions) as eluant. Fractions containhg methyi [Li3c]-

3-cyanopropionate were identined by spotting a TLC plate with a ad amount of each

fiaction, and immersing the plate in Iz vapour. These M o n s were combined, and the

bufk of the ether was distiIIed off at atmospheric pressure. The remaining solvent was

blown off with a stnam of nitmgen to a o r d methyI [ 1 - ' ~ ~ - 3 ~ a n o ~ r o ~ i o n a t e (1 -390 g,

79%) as a yeiiow oil. The product contahed a very s m d amotmt of contaminant that was

detected by NMR but not identifie& and was used without fkther pudication. Methyl

[1-~q-3c~ano~ro~ionate: 'H NMR (a~et0ne-d~) 6: 3.69 (d, = 3.97 Hz, 3H, am3),

2-72 (m, QEE, -CH2CH2-); 13c NMR (acetone-t&) 6: 171.68 ('3~-enrÏched, -Ca-), 52 16

(4 = 1-91 Hz, -O-), 30.14 (d, 'JCC = 59-13 Eiz, D2C&-), 13-13 (d, cc = 190

II55 -m2w-

Methyl[2~-'~~~~-3-~y.mo~ro~ionate (79): The estabIished procedure was &O

used to eEect the decarboethoxyIation of ethyi methyl [2,3-'3~t]-2-carboeth~xy-22

cyanosuccinate (2.695 g, 10.40 mmol). The reaction was heated at 144°C for 18 h, and

the product was worked up and chromatographed in the same manner as methyl [LL3c]-

3-cyanopropionate. The yield of 79, isolated as a yeflow oil, was 0.844 g (7 1 %). Methyi

[2,3-L3~2~-3-cyanopropionate: 'H NMR (acetone-4,) 6: 3.69 (s, 3H, -OCH3), 2.72 (dm,

JdouMcl = 130.55 Hz, 4H, -CH2CH2-); I3c NMR (acetone-ds) 6: 171 -72 (4 ' J ~ ~ = 59.13

Hz, -CO2-), 1 19.84 (dd, ' J ~ ~ = 57.22 HZ, 'J= = 2.86 Hz), 52.22 (s, -ocH3), 30.24 (I3c-

enriched, ci, '&-- = 35.76 Hz, <H2C02-), 1321 ("c-enricheci, d, '.JCC = 35.76 H z , -

CH2CN). -

4.10 Reduction of MethyI3-Cyanopropionate

4EIydroxybrityronittiIe: Methyl3-cyanopropionate (1.35 g, 1 1.9 mmol, Section

4.8) was s h d into t-buty1 aIcohoI (52 mL) dong with sodium borohydnde (1.04 g, 275

mmol). The mixture was brought to reflux, and methanol (1 1 mL) was added in smaü

portions over a 40 min period. When the addition of methanol was complete, the mixture

was stined at reflux for a fûrther 90 min. The bulk ofthe solvent was removed by

distillation at atmospheric pressure, and the residue was taken up in saturated brine (50

mL). The aqueous layer was extracted with chlorofom (4 x 50 mL) and ethyl acetate (3

x 50 II&), and the combined orgarilarilc extracts were dried wÏth anhydrous magnesiun

SUIfate, treated with neutral decoIomizhg carbon., and fütered thtough CeIite at the

aspirator- The bulk ofthe organic solvent was rernoved by rotary evaporation, and the

remainder was blown off with a stream of nitmgen, to &ord 4-hydroxybutyronitde

(0.63 g, 62%) as a faintly yellow oii that recyired no fiirther prirification, 4-

Hydroxybutyronitrile: 'H NMR (acetoned6) 6: 3.64 (t, J = 5.79 Hz, 2H, -CH$l-), 2.59 (s,

1H, -OH), 2.52 (t, J = 7.33 Hz. W, -CH2CN), 1.81 (apparent p, J = 6.56 Hz, W, - CH2aCHr); "C NMR (acetone*) 6: 120.73 (-CN), 60.37 (-CH20H), 293 1 (-

CH&H2CH2-), 13 -9 1 (-C32CN).

[4-13~4~ydroxyb~tyr~nitr i te (72): The above procedure was used to prepare

[413~4hydroxybutyr~nitrile h m methyl [I -'3~]-3-cyanopropionate (1 -390 g, 12.1 8

mol) . After the normal work-up, the cmde product was chromatographed on silica gel

(150 g) with diethyl ether as eluant, and 72 (0.547 g) was recovered in 52% yield [4-

13~]4hydr~xybutyl.onitrile: 'H NMR (acetone-de) 6: 3 -89 (s, IH, -OH), 3.65 (dm, 'JC.~

= 139.48 Hz, W, -CH+), 2.52 (m, W, -CH2CN), 1-89 (m, W, -CH2C&CH2-);

NMR (a~et0ne-d~) 6: 60.27 ( " ~ e ~ c h e d , S. -CH20-), 29.24 (d, 'JCC = 38.1 5 & -

CHsH2C&), 13.79 (s, 4H2CN).

[2,3-'3~+4-~Ydr~xy btttyronitriIe (80): [2,3-'3~~j4~ydr~vbutyr~nitrile was

aiso synthesized according to the estabIished procedure h m [2,3-"~~1-3-

cyanopropionate (0.844 g, 7.33 mmoI). Mer chromatographing with diethyl ether on

&ca gel (100 g), 80 (0.451 g) was recovered, in 71% field. [2,3-13c2]4

Hydroxybutyronitrile: 'H NMR (acetoned6) 6: 3 -79 (m, W, -CW-), 2.5 1 (dm, ' =

1 15.96 H& 2H, -C&CN), 1.8 I (dm, 'IC.H = 13032 Ek, 2EE, -CH2CHHC&), 1.59 (s, IH,

-OH); "C NMR (acetone4) 6: 60.29 (ci, ' J C ~ = 38.15 EIz, -CH20A), 2926 ('b

emiched, d, 'JCC = 33.86 H q <Ha, 13-77 ('3~-enriched, d, = 33.86 Hz, - CHa2CH2-).

4.11 Conversion of 4-Kydrorybutytonitrile to 4-Iodobtatyronltrüe

4-Iodobutyronitriie: A stirred solution of 4-hydroxybutyroni~e (0.63 g, 7.5

m o l ) in ether (10 mL) and acetonitriie (6 mL) was cooIed to O°C with an ice bath and

charged with triphenylphosphine (2.04 g, 7.76 m o l ) and imidazole (0.53 g, 7.9 mmoi).

Upon dissolution of these reagents, I2 (2.71 g? 10.7 mrnol) was sIowly added to the

cooled solution. As the I2 dissolve& it formed a dark orange colour which rapidly

disappeared, as the I2 was consumed. Near the end of the addition, the orange colour

persisted, as an excess of 12 was used. Aiso, about half-way through the I2 addition, white

triphenyiphosphine oxide rapidly precipitated. When the addition of I2 was cornplete, the

ice bath was removed, and the reaction was aiiowed to stir for 20 min. The dark orange

reaction mixture was taken up in dichioromethane (75 mL), and washed successively

with aqueous soIutions of sodium bisulnte (2.5 g in 25 mL), CuS045H20 (3 g in 25 rnL

saturated brine), HCl(0.5 M, 25 d), and sodium bisulfite (2.5 g in 25 mL). The

combined aqueous washes were back-emcted with dichIoromethane (100 mL) and the

organic Iayers were combined and dned with anhydrous magnesiun d a t e . RemovaI of

the organic solvent, f'irst by distülation at atmospheric pressure, and then by stmmhg

with nitrogen afEorded a yeiiow Ii@d (4.18 g), in which crystais formed upon standing

ovemight The entire mDaure was dissolved in acetone (25 mL), and a mudl portion (- I

mL) ofthe resuiting soIution was transfmd imo a mal[ round bottom. The acetone was

remuved by rotary evaporation, and the entire residue was dissdved in a~et0nltriIe-d~ for

NMR d y s i s . The dative and absoIute amounts of 4-iodobntyronitde (Il8 g, 88%)

and triphenyIphosphine oxide (2.90 g), which were the only major species present, were

thus detennined. 4-IodobutytoniMe: 'H NMR (acetoait12e-d~) 6: 3.27 (t, J = 6.71 Hk,

2H, -CH& 2.50 (t, J = 7.01 Hz, W, -CH2CN), 2.07 (quintet, J = 6.86 Eh, W, -

CH2C&CH2-); l3c NMR (acetonitde-d') 6: I 19.83 (-CN), 30.87 (-CH&H2CH2-), 18-75

(-cH2C19, 4.64 (-cH21).

[4-13~4~odobutyronitriIe (73): The above method was employed to prepare

[ 4 - ' 3 ~ ] 4 i o d o b o e (0.83 2 g, 67%) fiom [4-'3~]4hydroxybutyroni~1e (0.547 g,

629 mmol). Labeiied product was separated from the triphenyIphosphine oxide by

chromatography on süica gel (25 g) with ether (20 mL fractions) as eluant The [4-I3c]4

iodo butyronitrile was isolated as a yellow oii. [4-"~]-4-10dobutyronitrile: 'H NMR

(a~et0ne-d~) 6: 3.3 8 (dt, ' J C . ~ = 151 Eh, IH-H = 7.0 1 Hz, 2Ht -CH21), 2.62 (app. q, I = 6.50

H z , 2H, -CH2CN), 2.16 (m, W, -CH2CHtCH2-); NMR (acetoned) 6: 30.02 (d, JCC

= 36.24 Hz, -CHa2CH2-), 18.54 (s, -Ha, 4.1 8 ('3~-auiched, s, -CH21).

[2 ~-'~~~141odobatyronitriIe (81): [2,3-'3~~~4~odobutyronitde (0.504 g,

49%) was dso prepared by this method h m [2~-'~~~]4h~drox~butyronitriIe (0,451 g,

5.18 mmol). The nnal chtomatographed product had an actuai mass of0546 g, but

contained a smaif amount ofether that was quantÏfied using NMR spectroscopy.

smaiI amount of ether was not removed, but was comcted for in the subsequent

aUryIation step. [2~- '2~2]4~odobutyr~ni~e: 'H NMR (a~et0ne-d~) 6: 3.30 (m, W, -

CHa, 2.60 (dm, ' J C ~ = 150.08 H& 2H, -CH2CN), 2.14 (dm, 'J=* = 120.06 W, -

Chapter 4 - Experimentai 107

CHzCBCHr); 13c NMR (acetone-da) 6: 30.00 ( ' 3 ~ - e ~ c h e d , d, 'J== = 33.86 Hz, -

CHcHzCH2-), 18.48 (13Genriched, d, '.JCC = 33.86 Hz, -cH2CN), 4. I3 (d, 'J=< = 3624

EIz, -CH21)-

4.12 Preparation of D- and bNi-BPB-Gb Complexes

UnlabeiIed L-N~-BPB-G~~ cornpiex 42 was pmchased fiom Acros Organics and

used as received. UnlabeUed D - N ~ ~ P B - G I ~ complex 84 as weU as the labeued

complexes 82 and 83 were prepared according to the method of avis:^ which was based

upon Belokon's approach? L-BPB ligand 41 required for the synthesis of labelled

complex was acqukd fiom Amos Organics, and a suficient quantity of D-BPB ligand

had been previously preprved by Davis, and was used for the synthesis of the D

complexes 83 and 84.

D-NbBPB-Gly Complu (84): A mixture of PBPB ligand (0.782 g, 2.03 moi) ,

giycine (0.880 g, 117 mol) , NÏ(N03k6H20 (1.132 g, 3.89 mmoi), and sodium

rnethoxide solution in methano1 (0.9 1 M, 18 mL) was prepared. An additional portion of

methano1 (7 mL) was added, and the resulting mixture was stirred at 5S°C. The greenish

blue suspension rapidly changed into a da& orange-brown solution, and the progress of

d o n was monitored by TLC (silica gel with chioroform eluant). When the BPB Iigand

was completely consumed (4 h), the d o n mUmne was taken up in water (100 mL) and

extracted with chioroform (3 x IO0 mL). The cblorofonn was dned with anhydrous

magnesiun Matey and then removed by rotary evaporation to fiord a datk red oiL This

03 was chromatograqhed on f i c a geI, and opon evaporation of the el- &4 was

afKorded as a red oil. Crystallization of the product was induced by adding ether (- 1

mLJ. The yieid of clean ~-Ni-BPl3-Gly upon rotary evaporation of the ether and drying

on high vacuum was 0.681 g (67 %). The 'H NMR spectrum of the product was quite

complicated, but matched that obtained fiom the commercial L-Ni-BPB-Gly exactly.

Assignments reported below were made on the basis of values pubiished by Belokon et

alBo The spectrum contained a pair of AB systems, corresponding to the methylene

portions of the ben@ group and the glycine fragment, and one proton from each of these

methylene groups absorbed at 3.65 ppm. D-Ni-BPB-Gly: 'H NMR (chloroform-d) 6: 8.38

- 6.63 (m, 14H, H-Ar), 4.50 (d, J= 13 Hz, 1H, one methylenic proton fiom the benyl

group), 3.78 (ci, J = 20 Hz, 1 H, one methyIenic proton from the glycine kgment), 3.65

(ci, J = 20 Hz, 1H, one methylenic proton from the glycine firagment), 3.65 (4 J = 13 Hz,

IH, one methyIenic proton fiom the ben@ group), 3.50 - 3.23 (m. 3H, -CH- and -CH2-

of proiine moiety), 2.50 (m, 2H, -CHr of proline moeity), 2.23 (rn, W, -CH2- of proIine

moiety). 13c NMR (chioroform-d): 18 1.39 (s, -C-), 133.29 (s, aryi -CH-), 13 3.2 1 (s,

~1 -CH-), 132.25 (S. aryl -CH-), 13 I .76 (s, q l -CH-), 129.77 (s, VI -CH-), 129.53 (s,

aryf -CH-), 129.36 (s, aryI -CH-), 129.15 (s, aryl -CH-), 128.95 (s, aryl -CH-), 12628 (s,

aryi -CH-), I25.69 (s, aryi -CH-), 124.28 (s, aryi -CH-), 120.88 (s, ql -CH-), 71 .O4 (s, - CH- of probe moiety), 63.12 (s, -CHT ofbenyl group), 61.3 1 (s, -CH2- of glycine),

57.47 (s, -CH2N- of prohe moiety), 30.74 (s, -- of prohe moiety), 23.72 (s, -CHr

of pmline moiety).

L - N ~ - B P B - [ ~ - ' ~ ~ G I ~ Cornples (82): The above procedure was used to prepare

L-N~-BPB-[~-'~C]GI~ cornplex k m L-BPB ligand (0962 g, 250 mmol) and [2-

'3~]glycine (1 -014 g, 13.33 mmol). The reaction was stopped prernaturely (afler 2.5 h)

because an unexpected blue-green precipitate had formeci, and it appeaced as ifwater

fiom the heating bath may have entered the d o n mixture. Although TLC andysis

reveaied that some L-BPB Ligand remained when the work up commenced, the final yield

of chromatographed 82 (1 .O49 g, 84%) was acceptable. The unreacted [2-"~]~l~cine was

recovered as described below. The L~ NMR spectnrm of 82 was ahost identical to that

of rmlabelied complex, diBering only in that the AB multiplet of the giycine methylene

group appeared as an ABX multiplet owing to the presence of Iabeiied carbon. Each of

these protons coupled to the attached carbon with a coupling constant ofapproximately

90.0 H i Ody the labelled carbon was apparent in the 13c NMR spectnim. L-N~-BPB-[~-

1 3 ~ ] ~ l y : 'H NMR (chiorofom-d) 6: 8.38 - 6.63 (m, 14H, Ha), 4.50 (cf, J= 13 & lH,

one methylenic proton fkom the benyl group), 3.78 (d, ' J C ~ = 90.0 Hk, 'JH.H = 20 HZ,

1 H, one methiyenic proton fiom the giycine hgrnent), 3.65 = 90.0 HZ, 2 ~ H - H =

20.0 Hz. rH, one rnethiyenic proton fiom the giycine hgment), 3.65 (d, J = 13 Hz, lH,

one methyIenic proton fiom the bepyl group), 3.50 - 323 (m, 3H, -CH- and -CH2N- of

prohe moiety), 2.50 (m, 2H, -CH2- of proiine moiety), 2.23 (m, W, of proiine

moiety); 13c NMR (chloroform-d) 6: 6I .3 (13c-e~ched, s, -CHt- of glycine moiety).

D - N ~ - B P B - [ ~ ' ~ ~ G ~ ~ Cornplex (83): D-Ni-BPB-GIy complex contaidg IabeI at

the a-carbn ofthe glycine moiety was prepared in a sin3ar fashon as dabeiied D-

compIex, as desmiid above. The d o n was performed &g the ~-isomer of BPB

ligand (0.678 g, 1.76 mmoI), and Iabei was introduced via [2-'3~]gIYcine (0.666 g, 8.76

mmol). The yiefd of clean, chromatographed 83 was 0.514 g (58%), and the 'H and I3c

NMR spectra were identical to the spectra generated h m 82, above.

Recovery of Excess ~ - ' ~ ~ ] ~ l y c i n e fkom P r e p d o n of Cornplex: The

procedure developed by avis^ was employed to recover the excess [2-'3~]glycine that

was used in the formation of N~-L-BPB-[~-'~C]G~~. The aqueous layer (- 100 mL) fiom

the synthesis of tabeiied complex was adjwted to pH 4.77 with HCl(0.5 M), and appiied

to an Amberfite RI20 (m column (2.5 x 30 cm). The coIumn was rinsed with water (1

L), and the [2-13~]glycine was eluted with aqueous NH3 (0.5 M). Those fiactions

containhg glycine, as determined by testing with ninhydrin reagent, were combined and

the NH3, dong with the buik of the water, was removed by rotary evaporatioa The h k

yellow aqueous solution that remained was brought up to a volume of 60 rnL with

nanopure H20, and was decolourized by refi uxing with neutcal carbon. The aqueous

solution was then fïitered through Cefite at the aspirator, reduced on the rotary

evaporator, and then lyophiked, [ 2 - ' 3 ~ ] ~ ~ Y ~ i n e (0.707 g, 83% recovery) was obtained

as ff uffy white crystais. [2-13~]~~ycine: 'H NMR @z0) 6: 3 -52 (d, L~C.H = 145.1 &); 'Ic

NMR ( ' O ) 6: 4428 ('3~-enriched, s, -CH2-).

4.13 AlkyIation of Ni-BPB-Gly Complexes

W i the reqnKed compIexes and eIectrophiIes in hanci, the crucial

enantioseIective alkyIations were undertaken. The procedure for these reactions was used

by Davis for 4-bromobntyronitti1e.~~

Alkylation O ~ N ~ - L - B P B [ ~ / ~ G ~ ~ with [4-*C'j41odobutgronitrile: A stmed

solution of N~-L-BPB-[~-'~c]G (82,0.972 g, 1.95 mmol) in DMF (6 mC) was charged

with fiesh finely ground NaOH (0.3 14 g, 7.86 mmol). The mixture changed fkom orange

to green-brown, and [4-'3~]4iodobutyr~nitrile (73,0.415 g, 2.12 mmol) was added. The

reaction became noticeably warm to the touch, and within 30 min was cornpiete, as

determined by TLC anaiysis (silica gel, chIorofonn eluaat). The reaction was poured onto

0.5 M acetic acid (50 ML) and extnicted with dichioromethane (3 x 50 d). The organic

extract was dned (anhydrous magneshm sulfate), filtered, and rotary evaporated to

e o r d a red 02. This oü was chromatographed on silica gel (200 g), eluting first with

chloroform, then with a mixture ofchiorofom and acetone with a gradua1 increase in the

proportion of the latter solvent up to 100%. Those hctÎons (125 mL each) containhg

ailqfated complex (as determined by NMR) were combined and concentrated to af5ord

the desired product (0.503 g, 45%) as orange crystais. NMR spectroscopy codbmed the

identity of the product, akhough due to the midi amount of sample that was analyzed,

oniy those 13c resonances corresponding to IabelIed carbon were detected. It is

noteworthy that the appearance of the pair of doublets in the I3c NMR spectrum c o ~ s

13 13 the presenct of the mtact C- C bond formed in this step. 'H NMR (chioroform-d) 6:

8.38 - 6-61 (m, 14 H, H-Ar), 4.61 (d, IH, one methylenic proton fkom the benzyl group),

439 - 3.58 (m, 5 EE, includes one methylenic proton fkom the benyl group, a-proton of

prohe moiety, a-proton of the cyan0 amino acid moiety, and -CH2N- of prohe moiety),

2.92 - 1.81 (m, I OH, contains remaining -CHr units ofprohe and cyan0 amho acid

moieties); 13c NMR (c6lorofonn-d) 6: 70.83 ( 1 3 ~ ~ c h e d , d, cc = 32.4 Hz, a-carbon

of giycine moiety), 35.3 t (13~-enric6ed, d, 'jCc = 32.4 EIz, ~2CHZCE12CN).

AikyIation of N ~ - D - B P B - [ ~ - ' ~ ~ G ~ with [~41odobatyronitriIe: The

remahhg 3 cornpiexes were akyIated in the same maMer as N~-L-BPB-[~-'~C]GI~, but

since no chromatography was pafomied, a slightly modined work-up was performed to

remove the DMF, as weii as the acetic acid empioyed to quench the reaction. Ni-D-BPB-

[2- '3~]~ ly (83,0.508 g, 1 .O2 mrnol) was aUcy Iated with 73 (0222 g, 1.13 mmol) in DMF

(4 mL) after treatment wÏth powdered NaOH (0.168 g, 4.2 1 mmol). The reaction was

quenched &et 35 min with chiiied 0.5 M acetic acid (15 mL). The aqueous mixture was

thai satlnated with NaCl in order to sait out the desired product, and the resulting solid

was separated by sudon fltration and washed with saturated brine. Product was

sepamted fiom the excess salt in the soiid by extraction into chloroform (125 mL). Upon

drying (anbydrous magnesiun sulfate) and rotary evaporation of the chlorofonn, the

desired product was isoIated as a dark red soiid (0.467 g, 8 1%)). The 'H and 13c NMR

spectra of the product were the same as for the chromatographed product fiom the

aUcyIation of 82, except that a negIigibIe amount of DMF was present No second pair of

doublets due to minor epimer fomed in the alkylation was observed in the 13c NMR

spectnrm.

AUryIation of Ni-L-BPB-Gly with [Z ~-'~~~141odobatyronitrile: The

established procedure was used to aIkyiate 42 (0.736 g, 1.48 mmol) with [2,3-'3~z14

iodobutyronitde (0.329 g, 1.67 mmol) and powdered NaOH (0244 g, 6.09 mmol) m

DMF (6 mL). After the same work-up as was empioyed in the alkyIation o fNbBPB-

[2-I3~Cily, aIkylated product (0.783 g, 93%) was obtained as a a d soüd The 'H NMR

spectrum ofthis product was M a r to those obtanied k m the aikyiated IabeHed

Chapter 4 - Experimental

complexes above, except that the many multiplets upfieId fiom 4.5 ppm appeared

differently. This was due to the fact the labeiied carbons were in different positions in this

molecde, and this changed the splitting patterns of many of the upfield protons in the

spectnim. As before, ody the Iabelled carbons were observed in the 13c NMR spectrum,

although a second pair of doublets was also detected. This less intense (9 % of major

signal, estimated by peak heights) pair was iikely due to the presence of a minor epimer

formed in the allqlation. A much more reiiable anaiysis of the relative amounts of each

epimer was accomplished by chiral OPA HPLC as d e s d e d beIow. The minor epimer

codd not be distinguished fiom the major one in the very complicated 'H NMR

spectnmi. Major epimet: 'H NMR (chloroform-d) 6: 8.38 - 6.6 I (m, 14H, K-Ar), 4.6 1

(m, IH, one methylenic proton fiom the benyl p u p ) 4.21 - 3.25 (m, SH, inchdes one

methylenic proton fiom the benzyl ~ O U P , a-proton of the prolule moiety, a-proton of the

cyano amino acid rnoiety, and -CHZN- of the proline moiety), 2.80 - 1 -40 (m. I OH,

contains remaining -CHr units of prohe and cyano amino acid moieties); 13c NMR

(chioroform-d) 6: 22.94 (13~-enriched, d, 'JCC = 33.4 Eh, -CH&HZCHZ-), 16-89 (13c-

e ~ c h e d , d, l ~ c c = 33.4 Hz , -Ha. Mkor epimec "C NMR (chlorofonn-d) 6: 22.79

("c-emiched, d, 'J== = 3 3.4 Hz, -CHZH2C&-), 16.80 (13~-ennched, d, 'J== = 3 3 -4 Hz,

-cH2CN)* -

Aikylation OTN~-WBPB-GI~ wÎth [2,3-'3~+440dobatyronitriIe: N~D-BPB-

GIy (84,0.423 g, 0.86 m o i ) was aIkyIated with 81 (0.1 88 g, 095 m o l ) and powdered

NaOH (0.147 g, 3.67 mmoi) in DMF (3 m.) to fûmish, a f k tIie u s d work-op, the

desired product (0.475 g, 98%) as a da& red soIrLdC The 'H and U~ NMR spectnt were the

same as for the product the aIkyIation of 42 above; a negligible amomt of DMF was

again present The relative amount of minor epimer present was estimated at less than

10% of the major epimer, based on I3c NMR peak heights, although a much more

meanin@ determination was made by chiral OPA HPLC, as desded below.

4.14 Chirai OPA HPLC Assessrnent of Alkylation Stereoselecüvity

The method used to determine the stereseIectivity of the akyfation reactions, and

thus the dtimate optical pun*ty of the product amino acids, was the chiral OPA HPLC

procedure developed by avis^^ for the enantiomerk anaiysis of 5-cyanonorvaline, the

cyano amino acid precursor to lysine.

For each alkyIated cornplex, a sample solution of 5-cyanonorvaiine was prepared

by hydrolyzhg a srnail portion of the complex (1 - 4 mg) with HCI (1 mL, l .S M) in

methanol(1 mL) at 60°C for 5 min, and then washing with a smaii volume ofchlorofom

in order to remove the h i e d BPB Iigand.

Derivatization reagent (80 a), prepared by dissolving N-acetyl-L-cysteine (1 .O

mg) in commerd, incornpiete O-phthaidehyde reagent (1 -0 mL, Sigma Chernicd Co.,

St Louis, MO, USA), was mked with sampIe solution (20 pL, as prepared above), and

the reaction was dowed to proceed for 3 min at ambient temperature with occasional

agitation. Mobile phase used for the separation (100 @) was added, and a 20 pL portion

was injected. The fluorescent isoindoIe derivatives were separaieci by @ent elution at a

total fIow rate of2.O d . on a NucIeosiI 5 Cl8 coIumn (4.6 x 250 mm, Phenomenex,

Tomce, CA, U S A ) B ~ * ~ D- and t-5-Cyamnorvaüae had retention tmies of 9.0 and 8.1

min, respectiveIy, when a gradient of composition (min, 96 acetonitrile) 0.0,O; 3.0,8;

I0.0,8; 11.0,12; 20.0, 12; 22.0, O was created by tuking acetonitrile with an aqueous

s01ution of acetonitrile (5%). copper @) acetate (2.5 mM), L-prohe (5 mM), ammonium

acetate (26 mM), adjusted to pH 7.0 with 5 M NaOH.

The dative amonnts of L 3 ~ IabelIed D- and L-5-cyawnorvalioe were determined

by eiectronic integration of the chrornatograms, and the results, expressed as

stereoselectivity of aUcytation, are given in Table 9.

4.1 5 Isoïation of LabeiIed Lysines

The Iabeiied Lysines were obtained fiom the alkylated complexes via the 5-

cyanonorvalines in a one-pot procedure reported by ~avis.6~

[ 2 ~ - ' ~ ~ t ] - t - ~ ~ s i n e : Alkylated complex (0.476 g, 0.84 rnrnol) prepared fiom Ni-

L-BPB-[~-'~c]GI~ (82) and [4-"~]4iodobutyronitrile was dissolved in a mixture of

methanol(6 mL) and HCI(1.5 M, 6 mL), and the resuiting solution was swirled at 5S°C

for 25 mui, diiring wbich time the dark red coiour of dissolved complex was rapidIy

replaced by a goIden yeiIow coiour. The mixture was taken up m nanopure water (100

mL), and neutralized with potassium bicarbonate (2.3 g). A yelIow precipitate formed,

and was extracted with chloroform (2 x 100 mL). The faintly green aqwous solution that

remained was charged with cobalt (ït) chfonde hexahydrate (0.444 g, 1.87 mmoI), and

upon dissoLution of this compotmd, sodium borohydnde (0.342 g, 9.05 m o i ) was

carefcully added EvoIution of gas was observed, and the mixture rapicüy tiimed bIack

Mer stinuig for 6 h at r o m temperature, the murture was brought to pH 3 with HCL (15

M), and acetone (IO d) was addeé Mer a m e r 3 6 stirring the e t I y pinbpurpIe

solution was brought to pH 6 with potassium bicarbonate, and appIied to an ionexchange

c o h (Amberfite IR-120, H' form, 2.5 x 40 cm). The column was washed with

nanopure water (1 L), and labeiled amino acid was eIuted with aqueous ammonium

hydroxide (0.5 M, 200 mL hctïons). Fractions containhg amino acid (detected with

ninhydrin reagent) were combined and concentrated at reduced pressure to af5ord a

yeilow oa. This 02 was taken up in nanopure water (50 mL) and was reflinred in the

presence of deco1ou .g carbon. Mer suctiou-fiItration through Ceiite, the buIk of the

water was evaporatcd in vacuo, and the remainder was removed via Lyophibtion. [2,3-

1 3 ~ 2 ] - ~ - ~ y s i n e (0.1 15 g, 93% ficm allrylated cornplex) was thus obtained as a v q

faintiy yeilow powder. A s m d (- 6 % of the whole, estimated by 13c peak heights)

amount of unreduced [2,3-13~t]-~-5-cyanono~aline was also detected Ul the 13c NMR

spectnim. No udabelled carbons fiom either amino acid were detected. Signals arising

h m this contaminant couid not be disthguished h m signais due to IabeiIed lysine in

the 'H NMR spectnun. The 'H NMR spe- aiso contained signais arising fiom

unlabeued Lysine con taminant. The protons attached to carbons 2 and 3 of the rmlabeiied

lysine were readiiy differentiated nom the corresponding protons on "~~labelled

material as a resuit of the L~c-H coupiings that occurred in the latter amino acid. The other

protons, however, were completely coincident Based on 'H NMR integration, the Iysine

was composed of approxîmately 67% 'Vr labelled materid, and 33% dabeiied

material. No monoIabeiled material, which wodd give rise to an enriched singiet m the

13 C NMR spectnmi, was detecteâ. [2~-~~c~]- t -~ysine: 'H NMR n o ) 6: 3.65 (dm,

= 142.6 E i i IH, a-proton), 3.00 (t, J =733 Hz, 2H, -a2M%2)9 1-82 (dm, I J C . ~ = 133 Hk,

2K, -C&CH2CH2CHZNH2), 1-70 (III, 2EI, -CH2CH2CH2CH2NH2), 1-46 (a W, -

CH2C&CH2CW2NH2); "C NMR (DzO) 6: 5537 ( L 3 ~ d ~ h e d , ci, 'I== = 34.8 C-2),

3 1-32 ('3~-enriched, d, ' = 34.8 Eh, C-3). [2,3-"~~]-~-5-~yanono~valine: 13c NMR

(40) 5: 55.03 (''~-emiched, d, ' J== = 34.8 Hz, G2), 3 1 .O5 (U~-enriched, d, 'J== =

34.8 Hz, C-3). UnIaWed lysine: 'H NMR (&O) 6: 3.65 (apparent t, J = 6.1 Hz, lH, a-

proton). 1.84 (apparent q, J = 4 . 5 Hz, -C&CH2CH2CW2NH2).

[ ~ J - ' ~ C + D - L ~ S ~ ~ ~ : In a nmilar rnanner, [2,3-'3~2]-o-lysine (0.064 g, 52%) was

generated fiom the corresponding alkylated D complex (0.467 g, 0.83 m o l ) , using

sodium borohydride (0.3 18 g, 8.40 mmol) aud cobalt 0 chioride hexahydrate (0.410 g,

I.72 m o l ) to effect the reduction. The 'H and 13c NMR spectra obtained fiom the

isolated [2,3- '3~t~-~-~ysine were similar to those generated h m the L isomer. The only

notable merence was that the contaminating 5-cyanonorvaline was present in a slightly

higher (12% of the whoIe) relative amount, As for the other IabeiIed lysine samples, a

considerable amon\unt of rmlabeffed material was present Based on 'H NMR

integrations, 62% of the Lysine was 13~2-1abelIed, and the remahder was unlabeiled.

[4$3~z]-~-~ysine: [4,5-13~z]-~-~ysine (0.135 g, 66%) was prepared h m the

precursory IabeUed aUryIated complex (formed fiom unlabeIIed L-complex and

[23-"~2~4iodobutyronitrile, 0.783 g, 1 3 8 mmol) using sodium borohydride (0.50 1 g,

13.25 mmof) and cobdt (II) chioride (0.6943,2.92 m o l ) as descrÏbed above. The white

powder recoveicd der lyophilisation was subjected to NMR analysis. The 'H NMR

spectnmi was simüar to those obtauied fiom [2,3-U~+lysines, mering ody in the

appearatlce of the muitipIets absorbmg between 1 and 2 ppm, as a d t of the différent

dis t r i ion of Iabel in this mateneai, As expected, the a-proton resonance appeared as a

13 13 narrow multiplet, as it wodd in dabeiled samples. The presence of the intact C- C

unit again manifested itseifas a pair of emiched doublets in the 13c NMR spectnim,

which exhiibited a siight =type "Ieaning". No rmlabeiled carbons were detecteà, and if

any [4,5-13~z]-~-5-cyanonowaline was present, it was beyond the detection limits of the

instrument Unlabeiied lysine was also present in this sample, and those 'H NMR

absorptions arising fiom udabelled material that codd be disthguished from resonances

due to 1abelIed materiai are Listed below. The lysine was composed of approximately 70%

'3~rlabeIIed materid, and 30% tmiabeIled amino acid [ ~ , S - ' ~ C ~ ] - L - L ~ S ~ ~ ~ : 'H NMR

(D20) 6: 3.64 (m, 1& a-proton), 3.0 1 (apparent t, J = - 6.7 Hz, W, - C m 2 ) , 1-83 (m,

2H, -C&CH2CH2CH2NH2), 1-71 (dm, l~c.H = 122.1 Hz, W, -CH2CH2C&CHs),

i .45 (dm, ' J ~ . ~ = 1 18.5 HZ, W, - C H ~ C H z C H ~ ) ; NMR (D20) 6: 27.21 (13c-

enricheci, 6, ' I ~ ~ = 34.3 Ek, C-5), 22-16 (13c-e~ched, d, 'J=< = 34.3 tIz, C-4).

UnIabelIed lysine: IH NMR (D20) 6: 1 .?l (apparent qa te t , J = 7.02 Hz, 2H, -

CH2CH2C&CH2NH2), 1.45 (apparent sextet, J = 6.92 Hz, 2H, -CH2C&CH2CH2B&).

[4,5-13~+~-~ysine FoHowing the estabfished procedures, [4,5-'3~z]-o-lysine

(0.084 g, 67%) was ppreared nom the aUcyIation prodttct (0.475 g, 0.84 mmoI) of

dabellecl N~-D-BPB-GI~ and [2,3-u~~]4iodobutyr~~e, sodium borohydride (0328

g, 8.68 mmoI), and cobalt (IL) chioride hexahydnte (0.782 g, 3.29 mol). [ 4 , 5 - ' 3 ~ & ~

Lysine prepared in this marner had the same NMR spcctraI propeaies as the SimiIariIy

IabelIed L isomer. A smalI amotmt (- 5% of the whoIe, estimated by ' 3 ~ peak height) of

[ 4 , 5 - ' 3 ~ ~ - ~ 5 - c y a n o n o ~ a ~ e was detected by 13c NMR Also, there was a considerable

amount of unlabeUed Lysine present By 'H NMR integration, approximately 61% of the

Lysine present was '3~t-~abelled, with the remainder composed of unlabelied materid.

[4,~~~~~~o-~-~~anonofvaline: "C NMR 0) 6: 21 -43 ("c-eariched, ci, ' J ~ = 33 -4

Hz, C-4), 16.89 (13~-emiche4 d, 'J== = 33-4 Hz, C-5).

References

1) Jackuis, KC.; Barker, H A J. BacterioI. 1951,bI. 10 1.

2) Loesche, W.J.; Gibbons, M. Arck Oral Bol. 13,13,19 1.

3) Rogers, AH.; Zilm, P.S.; Gdy, N.J.; Pfennig, AL.; Marsh, PD. Oral Microbid h m o l . 1991,6,250.

4) RamezaxU, M.; MacIntosh, SB.; White, RL. Amino Acidr 1999, in press.

5) Barker, HA.; Kahn, HA.; Hednck, L. J. Bacteriol. 1982,152,20 1.

6) Stadtman, T.C. A&. E'oI., 1973,38,413.

7) Stadtman, T.C. J. Bacterid, 1954,67,3 14.

8) Stadman, TC.; White, F.H., Jr. J. Bucteriol., 1%4,67,651.

9) Stadtman, T.C. J. Biol. Chem., 1963,238,2766.

10) Chirpich, Tl'.; Zappia, V.; Costilow, RN.; Barker, HA. J. Biol. Chem, 1970,245, 1778.

i 1) Zappia, V.; Barker, HA. Biochem Biophys. Acta, 1970,20 7,505.

12) Stadtman, T.C.; Renz, P. Arch. Biocliem Biophys., 1970,125,226.

13) Baker, I J.; van der Drift, C.; Stadtman, T.C. Biochemi~ny, 1973,12.

14) Baker, JJ.; van der Drift, C.; Stadtman, T C Fed Proc., 1972,31,494.

15) Stadtman, TC; Grant, M A . Metho& Nt E~ymology, 1971,178,206.

16) Rimeman, EA.; Barker, HA. J. Biol. Chen, 1968,243,615 1.

17) Baker, JJ., P m . Thesis, University of Caiifornia., Berkeley, Caüfomia, 1970.

18) Baker, I.J.; Barker, ELA. J. Biol. Chenr. 1972,247,7724.

19) Yorifitji, T.; Jeng, LM.; Barker, H A J. B i d C k 1977, 252,20.

21) Barker, HA.; Jeng, LM.; NeE, N.; Robertson, J.M.; Tam, F K ; Hosaka, S. J Biol. Chem 1978,253,12 19.

22) Barker, HA. Ana Rw. Biochem- 1981,50.23.

23) Mchemey, M.J. in Chapter 8 of Biolog~ ofAnaerobic Microorgmim, a publication of Wiley-Interscience, New York, NY, 1988.

24) Morley, C.G.D.; Stadtman, TC. Biochemistry 1970,9,4890.

25) Morley, G.C.D.; Stadtmatl, T.C. Biochemistry 1972,11,600.

26) Stadtman, T.C. Unpublished experirnents cited in Ref. 6.

27) Borsook, H.; Deasy, CL.; Haagen-Smit, A.J.; Keighiey, G.; Lowy, P.H. J. Biol. Chem 1950,184,529.

28) Gaudry, R Canad J. Res., Sect. B 1948,26,387.

29) Sîrecker, A Liebigs Ana Chem 1850,75,27.

30) Maiminci, V1; Tokarev, B.V.; Shemyakin, MM. Doua& Abd N d SSSR 1953, 92,8 1.

32) Rothstein, M. Biochem. Prepurations 1961,8,80.

33) Anistein, H.R.V.; Hunter. GD.; Muir, HM.; Neuberger, A. J. Gem Soc. 1952, 1329.

34) Adamson, D.W. J. Chem. Soc. 1939,1564.

35) Bolster, LM.; Vaaiburg, W.; Van Dijk, TH.; Zijlstra, JB.; Paans, AM.; Wynberg, H.; WoIdring, MG. Int. J. Appl. Radfat- Isot. 1985,36,263.

36) Stafforst, D.; Schi3IKopff, U. Liebigs A m Chem 1980,28.

37) Oiyny4 P.; Camp, DB.; Griffith, M.; WoisIowski, S.; Helmkamp, RJI. J. Org. Chem 1948,13,465.

38) Lindstedt, S. Acta Chem S c d 1953,7,340.

39) Rothstein, U; CIaus, CJ. J. Am Chem Soc. 1953,75,2981.

40) Moe, OA.; Warner, D.T.J. Am Chem. Soc. 1948,70,2763.

4 1) Golebiewski, W.M.; Spenser, ID. C m J Chem. 1988,66,1734.

42) Reed, LW.; Purvis, M.B.; Kingston, D.GJ.; Biot, A.; Gossele, F. J. Org. Chem 1989, 54,1161.

43) Sutherland, A.; W U s , C.L. J. Lobelled Compd Rudiophm 1996,38,95.

44) Pichat, L.; Tostain, J.; Baret, C. Bull Soc. Chim. Fr. 1970,5,1837.

45) Fields, M.; Waiz, DE.; Rothchild, S. J. Am Chem. Soc. 1951, 73,1000.

46) Gould, S.J.; Thinrvengadam, TK. J. Am. Chem Soc. 1981,103,6752.

47) Leete, E. J. Nat. P d 1982, 45, 197.

48) Williams, RM. S)nthesis of Optically Active a-llniino Acidr, Pergamon Press, Oxford, UK & New York, NY, 1989.

49) Duthder, RO. Tetrahedron 1994,50,1539.

51) SchoiIkopf, U. Pure & Appl. Chem. 1983,55,1799.

53) Raap, J.; Van der Wielen, CM.; Lugtenburg, J. Red Trav. Chim Pays-Bas 1990, 109,277,

54) h p , J. Wolthuis, W.N.E.; Hehenkamp, JJ.J.; Lugtenburg, J. Amino Acidr, 19%,8, 171,

55) Yamada, S-1.; Oguri, T.; Shioiri, T. J. C. S. Chem Comm. 1976,136.

56) Ikegami, S.; Uchiyama, H.; Hayama, T.; Karsuki, T.; Yamaguchi, M. Tetrahedron 1988,445333,

57) Ikegami, S.; Hayama, T.; Katsuk, T.; Yamaguchi, M. Tetrahe&on Len. 1986,27, 3403.

58) Na& EL; Seebach, D. Helv. Chim Acta 1985,68,135.

59) Seebach, D.; Aebi, JB.; Na& R; Weber, T. Helv. C h Acta 1985,68,144.

60) Cdderadi, G; Seebach, D. Hefv- Chim Acta 1985,68,1592.

6 1) Aebi, JD.; Seebach, D. HeIv. Chmi, Acta 1985,68,1507.

62) Fitzi, EL; Seebach, D. Angew. Chen I i Ed EngL 1986,25,345.

63) Seebach, D.; Juaristi, E.; MilIer, DD.; Scbickli, C.; Weber, T. Hdv. Chim. Acta 1987,76237.

64) Fasth, J.; HodeIdt, IL; Laangstrom, B. Acta Chem S c d 1995,49,30 1.

65) Belokon, YN.; Bdychev, AG.; Vitt, S.V.; Stmchkov, Y.T.; Batsanov, A.S.; Timfeeva, T.V.; Tsryapkin, V A ; Ryzhov, MG.; Lysova, L A ; Bakmutov, VI.; Beiikov, VM. J, Am. Chemt Soc, 1985,107,4252.

66) Davis, D.T., PhD. Thesis, Dalhousie University, Halifax, Nova Scotia, 1997.

67) Brocker, EX.; Benn, M.H. Food Chem. Tmcol. 1984,22,227.

68) Padgett, HE.; Csendes, LG.; Rapoport, H. J. Org. Chem 1979,44,3492.

69) Cadogan, J.I.G.; Hey, DH.; Sharp, J.T. J. Chem. Soc. (C) 1966,1743.

70) Mignonac, G.; Miquel, k; Bonnemaison, C. Bull. Soc. Chim. Frmce 1958,1958, 1323,

71) BnepxlSki, B.; Schroeder, G.; OIejnik, J.; Jarczewski, A.; Grech, E.; Milart, P. J Mol. Struct. 1997, Nd, 99.

72) Black, T.H. AIdrchimiica Acta 1983,16,3.

73) Gilman, H.; Blatt, AH. Orgm'c Sjmthesis Cd. VOL 1,1941,254.

74) Krapcho, A.P. S)mthesis 1982,805.

75) Krapcho, A.F. Synthesis 1982,893.

77) SudiMi Borohycaide, A Pubfication of ThiokoWeatron Division, Danvers, MA, 1979,23.

78) Kabalka, G.W.; Vanna, M.; Varnia, RS. J. Org. Chem. 1986, 51, 2386.

79) Smith, AB. m, Rano, TA.; Chida, N.; SulikoWSki, GA.; Wood, IL. J . An. Chem Soc- 1992,114,8008.

80) Lam, S. J. Chromatogr. 1986,355,157.

81) White, RL.; Smith, K.C. Cm J. C h 1994,72,1645.