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Stable Analogues of Phosphohistidine

Shin Mukai

B. Eng. (Master’s)

Chemistry

School of Biomedical, Biomolecular and Chemical Sciences

This thesis is presented for the degree of Doctor of

Philosophy to The University of Western Australia

iii

Candidate Declaration

The work described in this thesis was carried out by the author during the period of

April 2007 to January 2011 in the School of Biomedical, Biomolecular and Chemical

Sciences at The University of Western Australia under the supervision of Associate

Professor Matthew J. Piggott. Unless duly described, this work is original.

………………………..

Shin Mukai

April 2011

iv

DECLARATION FOR THESES CONTAINING PUBLISHED WORK AND/OR WORK PREPARED FOR

PUBLICATION

The examination of the thesis is an examination of the work of the student. The work must have

been substantially conducted by the student during enrolment in the degree.

Where the thesis includes work to which others have contributed, the thesis must include a

statement that makes the student’s contribution clear to the examiners. This may be in the form

of a description of the precise contribution of the student to the work presented for examination

and/or a statement of the percentage of the work that was done by the student.

In addition, in the case of co-authored publications included in the thesis, each author must give

their signed permission for the work to be included. If signatures from all the authors cannot be

obtained, the statement detailing the student’s contribution to the work must be signed by the

coordinating supervisor.

Please sign one of the statements below.

1. This thesis does not contain work that I have published, nor work under review for publication.

Student Signature

.........................................................................................................................................................

2. This thesis contains only sole-authored work, some of which has been published and/or

prepared for publication under sole authorship. The bibliographical details of the work and where

it appears in the thesis are outlined below.

Student Signature

.........................................................................................................................................................

3. This thesis contains published work and/or work prepared for publication, some of which has

been co-authored. The bibliographical details of the work and where it appears in the thesis are

outlined below.

The student must attach to this declaration a statement for each publication that clarifies the

contribution of the student to the work. This may be in the form of a description of the precise

contributions of the student to the published work and/or a statement of percent contribution by

the student. This statement must be signed by all authors. If signatures from all the authors cannot

be obtained, the statement detailing the student’s contribution to the published work must be

signed by the coordinating supervisor.

Student Signature ………………………………………………………………………………………….

Coordinating Supervisor Signature. ..……………………………………………………………………

v

Summary

Phosphorylation is a crucial post-translational modification of proteins. This chemical

modification acts as an on-off switch for protein function; for example, the catalytic

activity of many enzymes, and most aspects of cell signalling, are controlled by

phosphorylation. Most commonly, the hydroxyl group of serine, threonine and tyrosine

residues is phosphorylated. There is a mass of information about proteins that contain

phosphohydroxyamino acid residues and the kinases that catalyse their phosphorylation.

Phosphorylation of one of the imidazole nitrogens of histidine residues in proteins also

plays a very important role in nature. However, information about phosphohistidine-

containing proteins is limited owing to the chemical lability of the phosphoramidate

functional group and the lack of commercially available phosphohistidine antibodies.

Attempts to raise antibodies using phosphohistidine have failed due to the instability of

the latter towards hydrolysis.

The primary aim of the research described in this thesis was, therefore, to synthesise

stable triazolylphosphonate analogues of N1- and N3-phosphohistidine for the purpose

of generating antibodies, which can recognise phosphohistidine residues in a protein

context.

N

N

HN

NH

O

PO

OO

N

N

HN

NH

O

POO

O3

11

3

or

proteinprotein

protein

protein

N

NH

HN

NH

O

3

1

proteinprotein

histidine kinase

N N

NPO

O

O

HN

O

ORR

N NN

HN

OR

O

PO

OO

R

Figure S1. Histidine phosphorylation and phosphohistidine analogues

Chapter 1 of this thesis provides an introduction to protein phosphorylation and the

history of detection of phosphohistidine in both prokaryotes and eukaryotes. The

chapter also includes a summary of similar work by other groups published during the

completion of the research described in this thesis.

vi

Chapter 2 describes the synthesis of haptens derived from phosphohistidine analogues.

The shape and charge distribution of these analogues are very similar to those of

phosphohistidines, and because the P–N bond has been replaced by a P–C bond, they

are stable to hydrolysis in vivo.

N N

NPO

O

OH3N

O

NH

N NN

H3NNH

O

PO

OO

N NN

H3NNH

O

PO

OO

HN

O

HN

O

O

NN

NP

O

OO

O

N N

NPO

O

OH3N

O

NH

HN

O

HN

O

O

ON

NNP

O

OO

125 129 156

137130126

Figure S2. Structures of haptens designed to mimic phosphohistidine residues, shown

here in their predominant protonation state at physiological pH

As small molecules do not illicit an immune response, haptens must be linked to a

carrier protein before antibody generation can be attempted. Chapter 3 discusses the

bioconjugation strategy, a model amide coupling of a phosphonotriazolylalanine in

water, and attempted protein bioconjugation prior to phosphohistidine antibody

generation.

Chapter 4 describes the synthesis of the „free‟ triazole analogues of N1- and N3-

phopshohistidine 170 and 171, and a brief investigation of their biological activity. The

results suggest that these compounds could potentially act as histidine kinase inhibitors.

N N

NPO

O

OH3N

O

O

N NN

H3NO

O

PO

OO

170 171

Figure S3. „Free‟ phosphohistidine analogues

vii

Chapter 5 details the preparation of Fmoc-protected phosphohistidine analogues 180

and 181 for potential use in (automated) solid-phase peptide synthesis. In addition, this

chapter discusses the potential utility of some of the compounds described in this thesis

for preparing affinity chromatography media for the purpose of isolating histidine

kinases and signalling molecules that recognise the phosphohistidine motif.

N N

NPEtO

EtO

OFmocN

O

O

N NN

FmocNO

O

PO

OEtOEt

H H

180 181

Figure S4. Fmoc derivatives of phosphohistidine analogues

Finally, as the extra nitrogen atom in the triazoles above alters the basicity of the

heterocycle, and therefore might affect phosphohistidine antibody generation, an

imidazole analogue 231 of N1-phosphohistidine and a pyrazole analogue 209 of N3-

phosphohistidine were targeted. Chapter 6 describes the attempted synthesis of these

compounds.

N

NPEtO

EtO

OBocN

O

OR'

N N

BocNOR'

O

PO

OEt

H H

231 209

OEt

Figure S5. Imidazole analogue and pyrazole analogues

The thesis ends with a conclusion, summarising the achievements and outstanding

goals, and possible future work in this area.

viii

Acknowledgements

I would like to extend my sincere appreciation and gratitude to my supervisor, A/Prof.

Matthew Piggott for his advice, support and encouragement. I am also grateful to my

co-supervisor, Prof. Paul Attwood for his guidance and dedication.

I would like to thank Dr Paul Besant for giving me several skills in biochemistry and

encouraging me to challenge a full marathon. It was one of the greatest achievements in

my life to complete the Perth Marathon 2010.

Thanks must go to the Piggott research group members, in particular, Michael Gandy,

Katie Punch and Blake Nguyen for their friendship and assistance.

I would like to thank Dr Lindsay Byrne for his invaluable NMR lessons, Dr Gavin

Flematti for his assistance with HPLC analyses and Dr Anthony Reeder for conducting

mass spectrometry on my samples.

An expression of gratitude is extended to Mr. Kim Foo (First year laboratory), Mr.

Graeme Cuffe (First year laboratory), Mr. Oscar Del Borrello (Second year laboratory),

Dr Sato Juniper (Graduate Research School) and Dr Joanne Edmondston (Student

Services) for their friendly assistance and expertise.

I am grateful to Prof. Allan McKinley for his support throughout the course of my PhD.

I wish to thank the supervisors in my Master‟s research, Prof. Sei-ichi Nishimoto

(Kyoto University) and A/Prof. Kazuhito Tanabe (Kyoto University) for their

encouragement.

ix

The financial assistance of a SIRF scholarship and a UIS scholarship from The

University of Western Australia is gratefully acknowledged.

I would like to extend a big thank-you to my family and friends.

Thankfully, I am alive and healthy, which enables me to pursue my dreams.

One Life, No Regret.

x

Abbreviations

The following abbreviations are used in this thesis.

Abbreviation Full Name

Ac

ADDP

ADP

All

ATP

aq.

B

Bn

Boc

Boc2O

BSA

But

CAN

Cbz

COD

conc.

Cp*

DCC

DCM

DEAD

DIAD

DIPEA

DMF

DMSO

DNA

EDCI

ESI

Fmoc

FmocCl

HATU

acetyl

azodicarboxylic acid dipiperidide

adenosine 5'-diphosphate

allyl

adenosine 5'-triphosphate

aqueous

base

benzyl

tert-butoxycarbonyl

di-tert-butyl dicarbonate

bovine serum albumin

tert-butyl

ceric ammonium nitrate

carbobenzyloxy

1,5-cyclooctadiene

concentrated

pentamethylcyclopentadienylbis(triphenylphosphine)

N,N′-dicyclohexylcarbodiimide

dichloromethane

diethyl azodicarboxylate

diisopropyl azodicarboxylate

N,N-diisopropylethylamine

N,N-dimethylformamide

dimethyl sulfoxide

deoxyribonucleic acid

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

electrospray ionisation

fluorenylmethoxycarbonyl

fluorenylmethoxycarbonyl chloride

O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

xi

HCTU

HMBC

HPLC

HRMS

IR

KLH

MALDI

NBD

Me

MES

Ms

Nu

OVA

Ph

iPr

r.t.

sat.

SDS-PAGE

TFA

TLC

THF

TIPS

TMAD

TMS

TMSBr

Tris

Ts

TsCl

Tza

O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium

hexafluorophosphate

heteronuclear multiple bond coherence

high performance liquid chromatography

high resolution mass spectrometry

infrared (absorption spectrometry)

keyhole limpet hemocyanin

matrix-assisted laser desorption ionisation

norbornadiene

methyl

2-(N-morpholino)ethanesulfonic acid

mesyl (methanesulfonyl)

nucleophile

ovalbumin

phenyl

isopropyl

room tempetature

saturated

sodium dodecyl sulfate polyacrylamide gel electrophoresis

trifluoroacetic acid

thin layer chromatography

tetrahydrofuran

triisopropylsilyl

N,N,N′,N′-tetramethylazodicarboxamide

trimethylsilyl

trimethylsilyl bromide

tris(hydroxymethyl)aminomethane

tosyl (4-toluenesulfonyl)

tosyl (4-toluenesulfonyl) chloride

triazolylalanine

xii

Table of Contents

Candidate declaration iii

Summary v

Acknowledgements viii

Abbreviations x

Chapter 1

Introduction 1

1.1 Protein phosphorylation 2

1.2 Phosphohistidine 3

1.2.1 Synthesis of phosphohistidine 4

1.2.2 The two-component system in prokaryotic signalling 5

1.2.3 Histidine phosphorylation in mammalian cells 7

1.2.4 Instability of phosphohistidine 8

1.3. Detection of phosphohistidine 11

1.3.1 Detection using [32

P]ATP 11

1.3.2 Mass spectrometric detection 12

1.4. Phosphohistidine antibodies 15

1.4.1 Antibodies 17

1.4.2 Polyclonal and monoclonal antibodies 18

1.4.3 Haptens 20

1.4.4 Stable analogues of phosphohistidine 20

1.5 Stable phosphohistidine analogue targets: Aims 24

1.6.1 Similar work published by the Muir group 27

1.6.2 Similar work published by the Webb group 30

xiii

Chapter 2

Synthesis of Triazolylphosphonate Analogues of Phosphohistidine 33

2.1 Strategy 34

2.2 Synthesis of a protected azidoalanine 34

2.2.1 Mitsunobu reaction 35

2.3. Synthesis of diethyl ethynylphosphonate 37

2.4 Azide-Alkyne Cycloadditions 42

2.4.1 Background 42

2.4.2 Copper-catalysed azide-alkyne cycloadditions 43

2.4.3 Ruthenium-catalysed azide-alkyne cycloadditions 45

2.4.4 Synthesis of protected triazolylalaninephosphonates

(NBoc-Tza(POEt2)-OMe) 46

2.5 Synthesis of haptens 51

2.5.1 N-methylamide haptens 52

2.5.2 Dipeptide haptens 54

2.6 Synthesis of haptens ready for bioconjugation through

the carboxyl group 60

2.6.1 Attempts to convert NBoc derivatives to NAc derivatives 60

2.6.2 Synthesis of NAc-Tza(POEt2)-OMe 63

2.6.3 Synthesis of N-acetyl haptens 65

2.6.4 Synthesis of NAc-Tza(POEt2)-OAll 67

2.6.5 Attempted deallylation 70

2.6.6 Synthesis of NAc-Tza(POEt2)-OBn 71

2.6.7 Synthesis of N-acetyl haptens-2 73

Chapter 3

Hapten-Carrier Protein Conjugation Strategies 77

3.1 Introduction to bioconjugation methods 78

xiv

3.2 Trial conjugation-1 80

3.3 Trial conjugation-2 82

Chapter 4

Synthesis of Free Phosphosphohistidine Analogues and Evaluation of

their Ability to Inhibit a Mammalian Histidine Kinase 87

4.1 Possible biological activity of the phosphohistidine analogues 88

4.2 Competitive and non-competitive inhibitors of histidine kinases 88

4.3 Synthesis of free triazolylalaninephosphonates 90

4.4 Inhibition of a mammalian histidine kinase by

the free phosphohistidine analogues 96

Chapter 5

Preparation for Solid-Phase Peptide Synthesis and Affinity Chromatography 101

5.1 Introduction to solid-phase peptide synthesis 102

5.2 SPPS by the Muir and Webb groups 104

5.3 Synthesis of Fmoc derivatives of N1- and

N3-phosphohistidine analogues 104

5.4 Preparation for affinity chromatography 109

Chapter 6

Attempted Synthesis of Pyrazole and Imidazole Analogues of Phosphohistidine 111

6.1 Attempted synthesis of a phosphonopyrazole analogue

of N3-phosphohistidine 112

6.1.1 Synthesis of pyrazolylphosphonates 112

xv

6.1.2 Attempted synthesis of protected pyrazolylalanines 116

6.2 Imidazole analogus of N1-phosphohistidine 121

Conclusion 124

Chapter 7

Experimental 125

7.1 General Details 126

7.2 Experimental for Chapter 2 127





References 184

1

Chapter 1

Introduction

2

1.1 Protein phosphorylation

Protein phosphorylation plays a very important role in the regulation of cellular function

in both prokaryotes and eukaryotes, and has been intensively studied since it was

discovered more than 50 years ago.1 Indeed, investigation of intracellular signalling

almost always involves the study of protein phosphorylation.2 In signalling cascades,

proteins are often phosphorylated, in reactions catalysed by protein kinases, and

dephosphorylated by phosphatases (Scheme 1).1 Protein phosphorylation, in its simplest

form, involves the transfer of the -phosphoryl group from adenosine 5'-triphosphate

(ATP) to a side chain of an amino acid residue, most usually a hydroxyamino acid

residue, in the substrate protein, thereby causing conformational changes or protein-

protein interactions that alter its biological activity.3 For example, phosphorylation of

transcription factors is central to the regulation of gene expression.2 There is increasing

evidence that the activation of nuclear steroid hormone receptors, which are

transcription factors, is also controlled by their phosphorylation status.4

NO

OHOH

OP

OP

OP

N

N

N

NH2

ATP

NO

OHOH

OP

OP

N

N

N

NH2

Protein OH Protein O P

O

O

O

O

O

OOO

OO

ADP

O

OO

O O

Kinase

Phosphatase

Scheme 1. Protein hydroxyphosphorylation

Most commonly, it is the hydroxyl of serine, threonine and tyrosine residues that is

phosphorylated to form a phosphoester.3 There is a mass of information about proteins

that contain phosphoesters, and on the associated kinases.3 The free phosphoamino

acids phosphoserine (2), phosphothreonine (3) and phosphotyrosine (4) are illustrated in

Figure 1.

3

O

H3NO

O

2 3 4

POO

H3NO

O

POO

OP

O

OO

H3NO

O

O

OO

Figure 1. Structures of phosphohydroxyamino acids

There are, however, protein phosphorylation events that occur on other amino acid

residues; perhaps the most important of these is at histidine residues. Although

phosphohydroxyamino acids dominate the protein phosphorylation literature,

phosphohistidine has been reported to occur more abundantly than phosphotyrosine, but

less so than phopshoserine.5

1.2 Phosphohistidine

Histidine kinases catalyse the phosphorylation of either N3 (5) or N1 (6) of the side-

chain imidazole of histidine residues (Scheme 2).5

1,3-Diphosphohistidine can be

chemically synthesised; however, this has never been found to occur naturally.5

N

N

HN

NH

O

PO

OO

N

N

HN

NH

O

POO

O

5 6

3

1 1

3

or

proteinprotein

proteinprotein

N

NH

HN

NH

O

3

1

proteinprotein

histidine kinase

Scheme 2. Protein histidine residue phosphorylation

As mentioned above, two isomers of phosphohistidine exist (Figure 2).5 Isomer 8 is

referred to as N1-phosphohistidine by IUPAC and Chemical Abstracts, whereas 7 is

regarded as N1-phosphohistidine by the majority of the research community.6-8

Both of

these numbering systems have been accepted in the literature; however, to avoid

confusion, the more widely used numbering system has been chosen in this thesis, that

4

is, isomer 7 is referred to as N1-phosphohistidine, and the other, isomer 8, as N3-

phosphohistdine.

H3N

O

O

NN P

O

OO

H3N

O

O

NN

PO

OO

Widely used numbering system

IUPAC numbering system

N1-phosphohistidine

N3-phosphohistidine

N3-phosphohistidine

N1-phosphohistidine

7 8

Figure 2. Two numbering systems for phosphohistidine

1.2.1 Synthesis of phosphohistidine

The first report on synthetic phosphorylation of histidine appeared in 1947, when

phosphorus oxychloride was used as an electrophile (Scheme 3).9 An alternative

employing phosphoramidate was published in 1956 (Scheme 3).10

31P NMR studies on the synthesis using phosphoramidate elucidated that N1-

phosphohistidine (7) is formed more rapidly than N3-phosphohistidine (9), which can

be formed directly from histidine or via hydrolysis of the 1-phosphoramidyl group of

1,3-diphosphohistidine (8).5

5

N

N

H3NO

O

PO

OO

8

3

1

+N

NH

H3NO

O

3

1

P

O

ClCl

Cl

Et3N H2O

or

P

O

HOO

NH3 H2O

(A)

(B)

N

N

H3NO

O

PO

OO

3

1

H2O

P

O

OO

7

N

N

H3NO

O

POO

O

9

1

3

Histidine

Scheme 3. Synthetic phosphorylation of histidine

1.2.2 The two-component system in prokaryotic signalling

Histidine phosphorylation is best understood in prokaryotic signalling, in the context of

the „two-component histidine kinase system‟.11,12

This signal transduction system

frequently occurs to connect external stimuli such as temperature, osmolarity, chemo

attractants and pH to various crucial intracellular events such as gene regulation.5,11

In

general, two component systems consist of a protein histidine kinase and a response-

regulator protein. The histidine kinases are the intracellular parts of membranous sensor

proteins that detect environmental changes. Activation of the sensor usually results in

dimerisation of the sensor proteins and autophosphorylation of a conserved histidine

residue in the kinase domain, that is, a self-catalysed phosphorylation reaction involving

ATP. A phosphoryl transfer reaction then ensues between the phosphohistidine in the

histidine kinase domain and a conserved aspartate residue in the response regulator

6

protein (RRP) (Figure 3).5,11,13,14

This results in the activation of the response-regulator

protein, which most often is a transcription factor that interacts with DNA to control

gene expression.5,11,13,14

The phosphorylated response-regulator protein can be

inactivated by specific phosphatases.15

Thus, the activation level of response-regulator

proteins is controlled by their phosphorylation via histidine kinases and their

dephosphorylation by phosphatases.15

Figure 3. Typical two-component histidine kinase signalling pathway. P = Phosphoryl

group, RRP = Response Regulator Protein

Some two-component systems are associated with the virulence of pathogenic bacteria.

For example, stimulation of the VanS-VanR system by vancomycin, the antibiotic of

last resort, leads to the expression of vancomycin-reisistance genes in some strains of

bacteria.11

The emergence of methicillin-resistant Staphylococcus aureus (MRSA)

bacterial strains resistant to vancomycin is a major concern for humanity.13

7

Consequently, effective methods to deal with those antibiotic-resistant bacterial

infections are being investigated, and inhibitors of bacterial two-component histidine

kinases systems could prove useful as novel antibiotics.13

1.2.3 Histidine phosphorylation in mammalian cells

Protein phosphorylation of histidine residues in mammalian cells, although not as well

studied as two-component systems, is also known, and there is evidence that suggests a

correlation between histidine phosphorylation and various cellular functions and

dysfunctions such as cancer.8,16

While, mechanistically, histidine phosphorylation in

bacterial proteins is reasonably well understood, by comparison there is sparse

information about histidine phosphorylation in mammalian cells. The only well-

characterised mammalian histidine kinase, nucleoside diphosphate kinase (NDPK),

catalyses phosphorylation of ribonucleosides via a phosphohistidyl-enzyme

intermediate, in addition to phosphorylating various proteins such as the beta subunit of

some trimeric G-proteins. 5,11,17

Although there are several reports regarding the

importance of histidine phosphorylation in eukaryotic systems, elucidation of signalling

pathways involving phosphohistidine has been painfully slow.5,11

This is mainly

because phosphohistidine is chemically unstable,5,11

as explained in the following

section.

A mammalian histidine kinase of particular interest is histone H4 histidine kinase

(HHK). Histidine phosphorylation on histone H4 is a vital process in regenerating liver,

and HHK activity is upregulated in liver cancer (Figure 4).18

Although elucidation of

the roles and functions of HHK is not complete, HHK could be a potential therapeutic

target for liver cancer.18

8

Figure 4. Histone H4 kinase (HHK) activity in human foetal liver (Fetal), human liver

cancer (HCCT), normal tissues surrounding the cancer (HCCN) and normal adult liver

(Normal)18

1.2.4 Instability of phosphohistidine

Phosphohistidine has a phosphoramidate bond that differs significantly from the

phosphoester in the phosphohydroxyamino acids. Phosphoramidates are significantly

less hydrolytically stable than phosphoesters.5 The ΔG

o for hydrolysis of the

phosphoester group in the phosphohydroxyamino acids in proteins is in the range 6.5

to 9.5 kcal mol–1

, whereas the ΔGo

for hydrolysis of the phosphoramidate group in

phosphohistidine residues is of the order 12 to 14 kcal mol–1

.19

A detailed review on

phosphohistidine by Attwood et al. discusses the thermodynamic stability of

phosphoramidates in relation to carboxylic amides (Figure 5).5 To briefly summarise,

amides 10 are dramatically more thermodynamically stable (with respect to hydrolysis)

than phosphoramidates as, in the former, the lone pair of electrons on the nitrogen is

delocalised onto the carbonyl, and this interaction makes the hydrolytically susceptible

C–N bond stronger. As a result of the delocalisation, the carbonyl carbon is also less

electrophilic, which hampers the attack of nucleophiles such as water. It follows that the

9

charge-separated canonical form 10a greatly contributes to the stability of amides. The

electron delocalisation characteristic of amides also explains the lack of basicity of the

amide nitrogen, and the restricted rotation around the CN bond.

N

O

N

O

PN

O

O

O

PN

O

O

O

10

11

10a

11a

Figure 5. Resonance structures of amides 10 and phosphoramidate 11

Although phosphoramidates 11 are notionally similar to amides, the nitrogen lone pair

electron in phosphoramidates 11 is not delocalised onto the phosphoryl group and the

canonical form 11a is not a significant resonance contributor. Presumably this is due to

poor overlap between the orbital containing the nitrogen lone pair electrons and the

phosphoryl π-bond, although there is some debate about whether the bond commonly

represented as P=O has any -bond character.20

Phosphohistidine is rapidly hydrolysed under acidic conditions, whereas the

phosphoester amino acids can survive low pH. The half-lives of phosphoserine 2 and

phosphothreonine 4 are approximately 18 h in 1 M HCl at 100 °C, and that of

phosphotyrosine 3 is approximately 5 h (Scheme 4).21

In contrast, the half-lives of N1-

phosphohistidine 7 and N3-phosphohistidine 8 are 18 s and 25 s, respectively, in 1M

HCl at 49 °C (Scheme 4).22

It has been proposed that N3-phosphohistidine is kinetically

10

more stable than N1-phosphohistidine 7, because of anchimeric assistance from α-amino

group in the hydrolysis of 7.5

H3NO

O

NN

Rapid hydrolysis

in 1M HCl at 49 oC

Half life = 18 s

H3NO

O

NNH

PO

OO

H3NO

O

O

Highly stable

in 1M HCl at 100 oC

Half life = 18 h

H3NO

O

OH

P O

OO

H3NO

OStable

in 1M HCl at 100 oC

Half life = 5 h

H3NO

O

O

P

OO O

OH

H3NO

OHighly stable

in 1M HCl at 100 oC

Half life = 18 h

H3NO

O

O

P OO

O

OH

2

3

4

H3NO

O

NN P

O

O

O

Rapid hydrolysis

in 1M HCl at 49 oC

Half life = 25 s

H3NO

O

NNH

8

7

12

13

14

15

16

Acid-stable

Acid-labile

Scheme 4. Acid stability of phopshoamino acids

11

1.3 Detection of phosphohistidine

The acid-lability of phosphohistidine poses some issues when attempting to undertake

biochemical characterisation of phosphorylated proteins. For example, standard

phosphoamino acid analysis involves hydrolysis of the peptide bonds of a protein with 6

M hydrochloric acid.14

The phosphoester amino acids survive this treatment method but

this process results in rapid hydrolysis of the P–N bond of phosphohistidine.14

Additionally, radioactive peptide mapping and Edman degradation are useful for

detection of phosphorylated serine, tyrosine or threonine residues in a protein.23

However, since these methods also require acidic conditions,23

they cannot be utilised to

analyse phosphohistidine. For these reasons, insights into the role of phosphohistidines

have been slow to surface,24

and several alternative techniques have been developed in

order to measure histidine phosphorylation:8

1.3.1 Detection using [32

P]ATP

Using this methodology, a substrate protein subjected to [32

P]ATP, in the presence of

a histidine kinase, is digested to the component amino acids by the protease cocktail,

pronase E.18

Qualitative identification of the resultant phosphoamino acids, is achieved

by comparison with authentic phosphoamino acid standards, by reversed-phase TLC.

The TLC plate is subsequently analysed with a phosphorimager (Figure 6).18

12

Figure 6. Phosphoamino acid analysis of histidine phosphorylation.18

The left panel is a

ninhydrin-stained reversed-phase TLC plate. A digested phosphohistidine-containing

protein was developed on the TLC alongside with free histidine (left lane),

phosphohistidine (PH), phosphotyrosine (PY), phosphoserine (PS), phosphothreonine

(PT) and phosphoarginine (PR) standards. The right panel is a phosphoimage of the

same TLC plate, showing 32

P-labelled phosphohistidine.

1.3.2 Mass spectrometric detection

Mass analysis of phosphohistidine-containing proteins has made detection of

phosphohistidine possible without radioactive materials.5 Solvent conditions and

matrixes for ionisation and duration of exposure to the solvents or matrixes need to be

carefully selected.5 For example, proteolytic digests of phosphohistidine-containing

proteins were performed, and then the purified phosphohistidine was detected by

positive ion electrospray mass spectrometry (Figure 7).25

However, a relatively large

13

amount of phosphoprotein is required so that this detection can be achieved, and

therefore this method has yet to be applied to proteins in cellular extracts.25

Figure 7. Positive ion ESI-MS spectrum of a proteolysed phosphoprotein showing a

peaks corresponding to phosphohistidine (pH), ( [M+H]+, m/z = 236)

25

A combination of enrichment techniques and mass spectrometry analyses enables

researchers to identify phosphorylated proteins, determine the site of phosphorylation

and characterise conditions under which these phosphorylation occurred.26

For example,

enrichment using immobilised metal-ion affinity chromatography (IMAC) has been

developed for phosphoproteins containing phosphohydroxyamino acids i.e.

phosphoserine, phosphotyrosine and phosphothreonine.26

Napper and co-workers

reported a novel combination of IMAC and positive ion matrix-assisted laser desorption

ionisation time of flight mass spectrometry (MALDI-TOF MS).26

Various resin-

immobilised divalent metal cations such as gallium, nickel, iron, zinc, cobalt, copper

have been immobilised on a resin of IMAC to purify phosphoproteins, and copper(II)

ions were adopted to isolate a phosphohistidine-containing protein26

but a significant

14

amount of nonphosphorylated (HPr and HPr-H2O) was observed in the positive ion

MALDI TOF-MS owing to the instability of phosphohistidine (Figure 8).26

Figure 8. Positive ion MALDI TOF-MS of a sample enriched by IMAC. HPr :

histidine-containing protein, P-HPr : phosphorylated histidine-containing protein, HPr-

H2O : histidine-containing protein which lost water.

The negative ionisation mode also caused dephosphorylation of the intact

phosphohistidine-containing protein (Figure 9).26

These results indicate that detection of

histidine phosphorylated proteins utilising a combination of IMAC and mass

spectrometry has proven challenging.

15

Figure 9. Negative ion MALDI TOF-MS of a sample enriched by IMAC.

Abbreviations as in Figure 8

1.4 Phosphohistidine antibodies

As mentioned above, phosphorylation of proteins on hydroxyamino acid residues is

well understood, and most progress in protein phosphorylation research has been made

in this area.3 Phosphohydroxyamino acids are detectable using conventional

methods.3,14

In addition, there are commercially available antibodies that can selectively

recognise phosphotyrosine and both phosphoserine and phosphothreonine residues.5

An example of the convenience that antibodies afford is depicted in Figure 10. If the

task is to identify phosphoproteins and no antibodies are available, a mixture of proteins

in a cell extract has to be separated by denaturing polyacrylamide gel electrophoresis

(SDS-PAGE), and then each protein is hydrolysed to its component amino acids. These

amino acids can be analysed by mass spectrometry, HPLC and so on, as described

above. Where antibodies are available, the detection of phosphoamino acids is

accomplished by a Western blot, which is vastly more concise than the standard amino

acid analysis. In this analytical technique, proteins are transferred to a membrane after

16

their separation by SDS-PAGE, and then the membrane is immersed in a solution of

antibodies. The antibodies that bind to the phosphoamino acid residues can be detected

by commercially available secondary antibodies, which are either tagged with a

fluorophore or an enzyme that catalyses a chromogenic reaction.

HN

NH

proteinprotein

O

H3O+

POO

O

H3NO

O

POO

O

Separation bySDS-PAGE

Hydrolysis of peptidebonds in each protein

Analysis of amino acids

C

C Hydrolysis of peptide bonds isNOT required

Detection by antibodiesfor phosphoamino acids

O O

Figure 10. Conventional phosphoamino acid analysis and detection of phosphoamino

acids using antibodies. Red antibody = anti-phosphohistidine antibody. Black antibody

= secondary antibody. Yellow sphere C = chromophore.

The significant progress in understanding protein phosphorylation and the associated

signalling pathways, in recent times, has been possible due largely to the use of

phosphoester-recognising antibodies. Conversely, progress on phosphohistidine

research has lagged behind due to the lack of such antibodies. Despite significant

investment of time and effort, efforts to generate phosphohistidine antibodies (see

below) have been unsuccessful.

17

1.4.1 Antibodies

Antibodies are glycoproteins referred to as immunoglobulins, produced by specialised B

lymphocytes in response to a large foreign molecule: the antigen. Antibodies comprise

four polypeptides: two identical heavy chains and two identical light chains held

together to form a „Y-shaped‟ protein structure (Figure 11).27,28

The amino acid

sequence of the amino termini of both the the light and heavy chains differs between

individual antibodies, and these parts are thus referred to as „the variable regions‟.27,28

The remaining parts of the polypeptide chains are referred to as „the constant regions‟,

and are common in each class of immunogloblins.27,28

The variable regions of the light

chain and of the heavy chains consist of 110-130 amino acid residues, and form an

antigen binding site that enables each antibody molecule to recognise a specific antigen

(Figure 11).27,28

. As antibodies differ in the binding site, they can function in a variety

of immune responses and at particular stages of the immune response.27,28

Figure 11. Structure of an antibody

18

1.4.2 Polyclonal and monoclonal antibodies

There are two forms in which antibodies for immunoassays are obtained: „polyclonal

antibodies‟ and „monoclonal antibodies‟. Polyclonal antibodies are a mixture of

different antibodies generated by multiple B lymphocytes.27,28

Antigens are generally

complex and possesses numerous surface features, or epitopes, that are specifically

recognised by the antibody. Many different cell lines of B lymphocytes produce

antibodies to the same antigen, and each antibody clone recognises different epitopes on

the same antigen.27,28

A large number of antigen-specific antibody clones are observed

in serum obtained from an immunised animal, and antibodies purified from these

samples are referred to as polyclonal antibodies.27,28

In contrast, monoclonal antibodies are pure antibodies generated by a single clone of B

lymphocytes and recognising a single epitope on an antigen.27,28

The first discovery of

monoclonal antibodies was in the sera of patients suffering from multiple myeloma,

where high levels of an identical antibody were produced by expansion of malignant

plasma cells.29

A hybridoma technology to produce monoclonal antibodies was invented

by Köhler and Milstein in the mid-1970s, and this achievement earnt them the Nobel

Prize in Physiology or Medicine in 1984.30

In this method, isolated splenic B cells are

fused with myeloma cells from the same species, generating immortal hybridomas,

which retain their abilities to produce unique monoclonal antibodies.30

Table 1. Differences between polyclonal antibodies and monoclonal antibodies

Polyclonal antibodies Monoclonal antibodies

from multiple B lymphocytes from a single clone of B lymphocytes

detect multiple epitopes on an antigen detect a single epitope on an antigen

19

Production of polyclonal antibodies does not require complex technology, and

therefore the production time scale is short.29

On the other hand, the production of a

hybridoma, and hence monoclonal antibodies, is complex and time-consuming.29

While

polyclonal antibodies can be expected to be obtained within several months after

commencing immunisation, it can take more than a year to generate hybridomas and

subsequently produce monoclonal antibodies.29

However, they have the considerable

advantage of being a constant and renewable source of antibodies that, more so than

polyclonal antibodies, provide highly reproducible results.29

In addition, monoclonal

antibodies are monospecific, which enables them to efficiently detect target antigens

among various related molecules in tissues or affinity purification.

The high specificity of monoclonal antibodies can, at times, be a disadvantage.29

In the

case where minor structural transformation of an epitope caused by genetic

polymorphism, glycosylation, denaturation and so on occurs, a monoclonal antibody

may not bind to the antigen.29

In contrast, as polyclonal antibodies are heterogeneous

and recognise multiple epitopes on an antigen, they are less likely to be affected even if

a single or small number of epitopes are structurally transformed.29

Polyclonal

antibodies are also less susceptible to changes in pH and salt concentration changes than

monoclonal antibodies.29

Because both polyclonal antibodies and monoclonal

antibodies have advantages and disadvantages, several factors need to be considered in

deciding which antibodies to be used.29

1.4.3 Haptens

Unlike large molecules like proteins, small molecules do not induce an immune

response, that is, although they are antigenic, they are not immunogenic.31

Thus, in the

case where small molecules are utilised as antigens, they are called „haptens‟ and must

20

be conjugated to a suitable carrier molecule (Figure 12).31

The resulting „immunogen‟

causes an immune response and antibodies that recognise the immunogen are generated.

Some of these antibodies specifically recognise the hapten, and thus can be used to

recognise that (or very similar) epitopes in other large molecules.

CarrierMolecule

Small molecule

No immune responseInjected into a rabbit

Smallmolecule

Immune responsegenerated

Injected into a rabbit

Figure 12. Example of a hapten

1.4.4 Stable analogues of phosphohistidine

Attempts to raise selective antibodies to phosphohistidine have been unsuccessful.5 This

is presumably because when phosphohistidine is used as a hapten, it is hydrolysed

before causing a strong immune response.5 In order to overcome this difficulty, other

workers previously designed and synthesised hydrolytically stable phosphohistidine

analogues for the purpose of generating anti-phosphohistidine antibodies (Figure 13).

N

N

H3NO

O

PSO

O

O

H3NO

O

POO

O

NH

H3NO

O

POO

O

N

N

H3NO

O

POO

O

N3-phosphohistidine 17 18 19

Figure 13. Stable analogues of N3-phosphohistidine

21

The Turck group prepared a synthetic peptide containing thiophosphohistidine 17

(Scheme 5).32

In their strategy, the peptide 20 was treated with thiophosphoryl chloride,

followed by RP-HPLC purification to afford the peptide with a thiophosphohistidine

residue, the identity of which was confirmed by ESI-MS.32

LAAAGGGG NH2NH

O

NNH

PSCl3 LAAAGGGG NH2NH

O

NN P

O

SO

Et3N

H2O

20 21

Scheme 5. Synthesis of a thiophosphohistidine-containing peptide by Turck‟s group

The Pirrung group also synthesised thiophosphohistidine using potassium

thiophosphoramidate 22 (Scheme 6).33

H3NO

O

NNH

H3NO

O

NN P

O

OS

P

O

HOS

NH2

H2O

16 23

22

Scheme 6. Synthesis of thiophosphohistidine 23 by Pirrung‟s group

Thiophosphohistidine is hydrolytically more stable than phosphohistidine, possibly

because sulfur has lower electronegativity than oxygen, thereby imparting an increase in

the stability of the thiophosphoramidate P–N bond.5,33

However, attempts to produce

antibodies utilising the thiophosphohistidine analogue as a hapten were unsuccessful.34

The inference for this failure is that thiophosphohistidine is still not sufficiently

hydrolytically stable in vivo to allow an immune response to develop.

The other method of creating a more stable analogue involves replacing the

phosphoramidate P–N bond with a P–C bond (giving a phosphonate), which is

22

hydrolytically inert. Reymond and co-workers made the furylphosphonate analogue 18

of N3-phosphohistidine (8) in five steps from the furan 2435

using a variation of the

amidomalonate synthesis (Scheme 7).36

Radical bromination of the furan 24 using N-

bromosuccinimide gave the bromide 25 in excellent yield.37

α-Alkylation of 26 with 25

provided the diester 27.37

Hydrolysis and decarboxylation of 27 gave the racemic amino

acid derivative 28.37

Enzymatic resolution using papain,38

by which only S-configured

NBoc-protected amino acid esters are hydrolysed, furnished the enantiopure carboxylic

acid 29. The free amino and phosphonic acid groups were revealed by heating the

carboxylic acid in 1M HCl under reflux to afford 30 as a hydrochloride.36

OP OEt

OEt

ONBS

AIBN

CCl4

Reflux

OP OEt

OEt

OBr

BocNH

OEtO

OEt

O

NaH

EtOH

BocNH CO2Et

CO2Et

OP

O

OEt

OEt

1) NaOH, EtOH2) HCl3) Dioxane, reflux

BocNH

OP

O

OEt

OEt

OEt

O

BocNH

OP

O

OEt

OEt

OH

O

Reflux

HClClH3N

OP

O

OH

OH

OH

OPapain

96% 80%

76%

95%

40%

24 25 27

26

30 29 28

Scheme 7. Synthesis of a furylphosphonate analogue of N3-phosphoihistidine by the

Reymond group36

However, successful production of anti-phosphohistidine antibodies using the N3-furan

analogue 30 has not been reported. This is possibly because the furan oxygen is a

weaker hydrogen bond acceptor than the nitrogen of phosphohistidine. Alternatively, as

there is no record in the literature, it may be that it was not attempted.

23

The Reymond group also synthesised the pyrrole analogue 19, and the Attwood

research group attempted to raise antibodies utilising a short peptide containing this

analogue (Scheme 8).39

Although antibodies were raised, they only recognised the

peptide containing 19 and were not cross-reactive with phosphohistidine-containing

peptides.39

H3NNH

HN

NH

O O

O

HN

O

O

O

NHPO

OO

N

O

O

HS

Hapten

H3NNH

HN

NH

O O

O

HN

O

O

O

NHPO

OO

S

N

O

O

KLH

KLH Immunogen

Carrier protein

NH

H3NO

O

PO

O

O

19

Scheme 8. Carrier protein-hapten conjugation strategy used by the Attwood group

There are several possible reasons for this failure:

1. While phosphorylated histidine residues in proteins are generally internal, the

analogue contains a terminal phosphohistidine analogue residue. The negatively charged

carboxylate is very different from the neutral peptide linkage occurring in native

histidine-phosphorylated proteins.

2. The non-phosphorylated ring nitrogen in phosphohistidine residues is a good

hydrogen bond acceptor, whereas the pyrrole nitrogen is a hydrogen bond donor.

Hydrogen bonding to the nitrogen of phosphohistidine is likely to be critical in antibody

recognition.

24

3. The pyrrole analogue only mimics N3-phosphohistidine, and therefore if there are

N1-phosphohistidine residues in a protein of interest, these would probably not be

recognised by the antibodies due to the different spatial arrangement of phosphoryl

group relative to the rest of the hapten.

1.5 Stable phosphohistidine analogue targets: Aims

At the outset, the primary aim of the research described in this thesis was to

1. Synthesise hydrolytically stable phosphohistidine analogues.

2. Produce generic phosphohistidine antibodies (i.e., that recognise the phosphohistidine

epitope in all, or most histidine-phosphorylated proteins).

3. Investigate the intrinsic biological activity of the analogues.

4. Undertake solid-phase peptide synthesis to incorporate the analogues into short

peptides, for the purpose of generating antibodies to specific histidine-phosphorylated

proteins.

Several analogues that addressed all of the deficiencies of the previous phosphohistidine

analogues described above were designed. A subset comprising a triazole (31) and

imidazole (33) analogue of N1-phosphohistidine and a triazole (32) and pyrazole (34)

analogue of N3-phosphohistidine is shown in Figure 14.

25

N

N

H3NO

O

PO

OO

N N

HN

OR'

O

PO

OO

N NN

HN

OR'

O

PO

OO

N

N

POO

OH3N

O

O

RR

N

NPO

O

O

HN

O

OR'

N N

NPO

O

O

HN

O

OR'R R

N3-phosphohistidine

N1-phosphohistidine

32

31

34

33

Figure 14. Primary N1- and N3-phosphohistidine analogue targets

In these compounds, R and R' represent an appropriate protecting group, which can be

removed selectively.

Like the previous phosphohistidine analogues 18 and 19, these compounds are

phosphonates and, instead of the phosphoramidate P–N bond, contain a C–P bond,

which makes them non-hydrolysable. The other reasons why these analogues were

chosen for the potential generation of antibodies are detailed below:

1. The phosphoryl group and the unsubstituted heterocyclic nitrogen in N1- and N3-

phosphohistidines, illustrated in red in Figure 14, would play an important role in the

binding interactions with antibodies. In the analogues, both of these features are

retained.

2. The planar, aromatic nature and sp2-hybridisation of all atoms in the imidazole ring

of phosphohistidine are maintained by the introduction of another one or two

26

heteroatoms, marked in blue, into the heterocycle. The shapes of these analogues are as

similar to the phosphohistidines as possible and any non-covalent antibody-antigen

interactions with the heteroaromatic ring should therefore be transferable.

3. By using the chiral pool, the analogues can be prepared without any need for

enantioselective reactions or resolutions, which simplifies the synthesis and purification

and avoids waste.

With respect to the last statement, the proposed analogues could be obtained from L-

serine, which is an inexpensive and enantiopure starting material (Scheme 9).

Importantly, throughout the synthesis, the reaction conditions have to be chosen

carefully to avoid racemisation.

H3N

O

O

L-serine

HO

N

NPO

O

O

HN

O

ORR

N NN

HN

OR

O

PO

OO

R

N N

NPO

O

O

HN

O

ORR

N N

HN

OR

O

PO

OO

R

Scheme 9. Simplified retrosynthetic analysis for the target phosphohistidine analogues

27

In 2010, two independent groups published work very similar to aspects of the research

described in this thesis. To simplify comparison of our work with theirs, that work is

summarised in the next two sections.

1.6.1 Similar work published by the Muir group

In 2010 the Muir research group, at Rockerfeller University, reported the NBoc-

protected triazolylalaninephosphonate esters 35 and 36 (Figure 15), analogues of N1-

and N3-phosphohistidine, respectively.40

BocNOH

OH

N

N N

P

O

OEt

OEt

BocNOH

OH

N NN

PO

OEtEtO

35 36

Figure 15. Proteted triazolylphosphonate analogues of phosphohistidines synthesised

by the Muir group

The synthesis of 35 and 36 is shown in Scheme 10. A copper(I)-catalysed cycloaddition

of the azide 37 and alkyne 38 gave the 1,4-triazole 36 exclusively.41

In contrast, the

ruthenium(II)-catalysed reaction of 37 and 38 did not proceed as expected, which

required them to protect the carboxylic acid by reaction with benzyl bromide, providing

the benzyl ester 40. The ruthenium(II)-catalysed reaction42

of the azide 40 and alkyne

38 provided the 1,5-triazole 41, and the crude product was subsequently subjected to

hydrogenolysis to afford the free carboxylic acid 35.

28

BocNOH

OH

N

N N

P

O

OEt

OEt

BocNOBn

OH

N NN

PO

OEtEtO

36

BocNOH

OH

N3

37

BocNOBn

OH

N3

BocNOH

OH

N NN

PO

OEtEtO

35

40

41

P H

O

EtO

EtO

38

CuI, DIPEA

DMF, 72%

Br

DIPEA, DMF

63%

Cp*RuCl(COD)

P H

O

EtO

EtO

38

PhMe, r.t.

Pd/C

H2

THF

68% (Two steps)

39

Scheme 10. Synthesis of peptide precursors 35 and 36 by Muir‟s group

Compounds 35 and 36 were used in the solid-phase synthesis of peptides containing the

phosphohistidine analogues, specifically, the histone H4 tail peptides 45 and 46. The

phosphonic acid was revealed during the cleavage of the synthesised peptides from the

resin by hydrofluoric acid (Scheme 11).40

Furthermore, Muir and co-workers extended

the histone H4 tail peptide 46 to full-length histone H4 utilising native chemical

ligation.40

29

H2NHN

RKVLR

O

H2N

Boc SPPS

HATU DIPEA

DMF

HATU DIPEA

DMF

HNRKVLR

O

HN

OBocNH

N

N

N

PO

EtOOEt

NH2

RKVLR

O

HN

ONH

N

N

N

PO

HOOH

CGAKR

O

Ac

HNRKVLR

O

HN

OBocNH

N

N

NPEtO

EtO

NH2

RKVLR

O

HN

ONH

N

N

NPHO

HO

CGAKR

O

Ac

O

O

Boc SPPSCleavage from the resin

Boc SPPSCleavage from the resin

42

36 35

44 43

46 45

Scheme 11. Solid-Phase Peptide Synthesis (SPPS) of histone H4 Tail Peptides

containing stable triazolylphosphonate analogues of phosphohistidine by the Muir

group40

Polyclonal antibodies were raised using peptide 46 as an immunogen, and the antibodies

generated specifically recognised phosphorylated histone H4, but not the non-

phosphorylated protein.40

Although these antibodies do not recognise phosphohistidine

residues in other proteins (i.e. they are not generic), this is the first example of

antibodies that specifically recognise the histidine-phosphorylated form of a protein.

This result showed that the triazolylphosphonate analogues are good mimics of

phosphohistidines and, to some extent, validated the approach to produce generic

phosphohistidine antibodies described in this thesis.

30

1.6.2 Similar work published by the Webb group

Most recently, Webb and co-workers, from the University of Leeds, reported solid-

phase peptide synthesis using an Fmoc-protected triazolylphosphonate analogue 47 of

N3-phosphohistidine (Figure 16).43

FmocNOH

O

N

N N

P

O

OEt

OEt

H

47

Figure 16. SPPS-ready N3-phosphohistidine analogue synthesised by Webb‟s group.43

The Webb group‟s synthesis of 47 is depicted in Scheme 12. A Cu(II)-catalysed diazo-

transfer reaction converted N--Boc-diaminopropionic acid 48 to the azide 37, which

was subjected to a cycloaddition with the alkyne 38 in the presence of a copper(I)

catalyst, affording the triazole 36, reported by the Muir group. The Boc group was

removed by trifluoroacetic acid (TFA) to produce, after neutralisation, the free amino

acid 49. Treatment with FmocCl furnished the Fmoc-protected amino acid 47.

Alternatively, a copper(I)-catalysed cycloaddition of commercially available Fmoc-

azidoalanine 50 and the alkyne 38 afforded 47 in excellent yield.

31

BocNOH

OH

N3

BocNOH

OH

NH2

BocNOH

OH

N

N N

P

O

OEt

OEt

H2NOH

O

N

N N

P

O

OEt

OEt

FmocNOH

O

N

N N

P

O

OEt

OEt

H

FmocNOH

OH

N3

48 37

47

36

4950

TfN3, CuSO4

K2CO3, MeOH

78% CuSO4

sodium ascorbate

P H

O

EtO

EtO

38

tBuOH/H2O

88%

(1) TFA(2) H2O

92%

68%CuSO4

sodium ascorbate

P H

O

EtO

EtO

38

tBuOH/H2O

96%

FmocCl

Dioxane/H2O

Na2CO3

Scheme 12. Synthesis of the SPPS-ready N3-phosphohistidine analogue 47 by Webb‟s

group43

A standard Fmoc SPPS protocol was applied to incorporate 47 into the heptapeptide 51,

based on the phosphocarrier domain of pyruvate, orthophosphate dikinase (Scheme 13).

In contrast to the Muir group‟s approach, the phosphonic acid of the heptapeptide 51

was not revealed during cleavage of 51 from the resin with TFA. However, the

deprotection was achieved using trimethylsilyl bromide (TMSBr), providing the

heptapeptide 52 as a mixture with the mono- and diethyl phosphoesters.43

Purification

of the heptapeptide 52 was accomplished by HPLC.

32

FmocNOH

OH

N

N N

P

O

OEt

OEt

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

NH2

O

S

H

OH

OH

N

NN P

OEt

OEtO

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

NH2

O

S

H

OH

OH

N

NN P

OH

OHO

Standard SPPSconditions, followed byTFA cleavage

1) TMSBr, DCM

2) MeOH/H2O

47

51

52

Scheme 13. Solid-Phase Peptide Synthesis (SPPS) by the Webb group of a stable

phosphohistidine analogue-containing heptapeptide based on the phosphocarrier domain

of pyruvate, phosphate dikinase

33

Chapter 2

Synthesis of Triazolylphosphonate

Analogues of Phosphohistidine

34

2.1 Strategy

The plan to prepare the triazolylalanine (Tza) analogues of phosphohistidine involved

azide-alkyne cycloadditions (Scheme 14). It was envisioned that this reliable reaction

could provide access to both N1- (55) and N3- (56) phosphohistidine analogues.

HN

O

O

N3

R1 R2

P

O

O

O

H

R3

HN

O

O

N

R1 R2HN

O

O

N

R1 R2

N

N

N NP

P

O

O

OO

O

+

R3

R3

R3R3

OR353

54

55 56

Scheme 14. Plan to synthesise the Tza analogues of phosphohistidine. R1–R

3 are

protecting groups that can potentially be removed orthogonally

Thus, a protected azidoalanine 53 and an ethynylphosphonate 54 were initially required.

2.2 Synthesis of a protected azidoalanine

The synthesis of the protected triazole analogues of phosphohistidine began with the

literature preparation of the azide 6044

from the inexpensive, enantiopure starting

material L-serine 57 (Scheme 15). Fischer esterification of 57 gave the methyl ester

hydrochloride 58,45

and the amino group was protected as a t-butyl carbamate, affording

59.45

35

H3N

O

O

OH

BocN

O

OMe

OH

HClH3N

O

OMe

OH

BocN

O

OMe

N3

H

HN3

PPh3

DEAD

THF

SOCl2

MeOH

Boc2O

Et3N

Dioxane

MeCN74%

90%

72%

57 58 59

60

Scheme 15. Synthesis of N-Boc-azidoalanine methyl ester 60

2.2.1 Mitsunobu reaction

The substitution reaction between an alcohol and a weak acid, which was later named

for its inventor, was first reported by Oyo Mitsunobu in 1967. It is utilised to convert an

alcohol to the corresponding ester, nitrile and so on, with inversion of the

configuration46

and has been widely used due to its reliability, effectiveness and

versatility.47

With respect to the mechanism (Scheme 16),46

the reaction commences

with formation of the adduct 63 between triphenylphosphine 61 and an

azodicarboxylate, most commonly diethyl azodicarboxylate (DEAD) 62. The proton of

the acid 64 (the conjugate base of which is the nucleophile) is abstracted by the

generated betaine 63. Due to a strong affinity between oxygen and phosphorus, the

positively charged phosphorus in the phosphonium intermediate 65 is attacked by the

oxygen in the alcohol, (most probably with general base catalysis, as shown in Scheme

16), providing the anion 68 and O-alkylated phosphine oxide 69. The nucleophile 67

undergoes an SN2 reaction with the electrophile 69 to afford the substation product 71

and triphenylphosphine oxide 72.

36

N NEtO OEt

O O

Ph3P

N N OEt

O

EtO

O

PPh3

NuH

N NH

OEt

O

EtO

O

PPh3

R1

R2

O

61

62 63

64

65 66

H

Nu

N NH

OEt

O

EtO

O

R1

R2

OPh3P

NH

NH

OEt

O

EtO

ONu

R1

R2

OPh3P

R1

R2

Nu Ph3P O

68 6970

64

69

67

7271

67

HNu

Scheme 16. Mechanism for the Mitsunobu reaction

In the case where a very weak acid participates in the reaction, the nitrogen anion 68

can attack the electrophile 69, furnishing the undesired side-product 75 (Scheme 17).

N N OEt

O

EtO

O

PPh3

R3

R4

OH

N NH

OEt

O

EtO

O

PPh3

R3

R4

O

N NH

OEt

O

EtO

O

63

65 74

68

R3

R4

OPh3P

R3

R4

NEtO

O

NHEtO

O

Ph3P O69

72

75

73

Scheme 17. Formation of the undesired side-product 75

37

The Mitsunobu reaction of 59 with hydrazoic acid converted the alcohol to the

corresponding azide 6044

in acceptable yield (Scheme 15). The optical rotation of the

azide 60 was close to the published value.48

As mentioned above, DEAD 62 is the most common azo compound used for Mitsunobu

reactions, and the reagent used in the literature synthesis of 60.44

Although it is

commercially available, it is a sea freight item and hard to acquire in Australia.

Accordingly, DEAD was substituted with diisopropyl azodicarboxylate (DIAD), which

is more readily available. However, the azide 60 and the hydrazine by-product derived

from DIAD had similar mobility on silica gel, which hampered the purification of the

azide. It was therefore necessary to synthesise DEAD 62. Fortunately this was achieved

easily, in two steps from hydrazine 76 (Scheme 18). Diethyl hydrazodicarboxylate 7049

was obtained by the reaction of hydrazine 76 and ethyl chloroformate 77. Oxidation of

70 with concentrated nitric acid produced DEAD 62.50

NH2H2N

Cl

O

O

Na2CO3

EtOH/H2O

NH

NH

O

O O

Oconc. HNO3

CHCl3

NN

O

O O

O

60%70%

76 70 62

77

Scheme 18. Synthesis of DEAD

2.3 Synthesis of diethyl ethynylphosphonate

The ethynylphosphonate 8243,51

required for the Huisgen cycloaddition reactions and

their metal-catalysed variants was synthesised as depicted in Scheme 19. TIPS-

acetylene 78 was treated with ethylmagnesium bromide to form the magnesium

acetylide, which was quenched with diethyl chlorophosphate 80,52

affording the TIPS-

protected ethynylphosphonate 81 in good yield (Method A). Although 80 is

commercially available, it is also readily prepared from diethyl phosphite 79 (Scheme

38

19). Deprotection of 81 was achieved with potassium fluoride to furnish the

ethynylphosphonate 82 in acceptable yield.53

TIPS-actylene was used because with

TMS-acetylene 83 the intermediate 84 was not able to be isolated in pure form; the silyl

group was spontaneously cleaved during normal purification techniques. However,

subsequently, it was found that the target alkyne 82 was obtained in the best overall

yield when the deprotection of the crude intermediate 84 was deliberately completed by

treatment with weak aqueous base (Method B).51

Method A

H TIPS

EtMgBr

THF

P

O

EtO

EtOCl

P

O

EtO

EtOH

Et3N

CCl4

P TIPS

O

EtO

EtO

P H

O

EtO

EtO

KF

MeOH80%

61%

85%

78 81

80

79

82

Method B

H TMS

EtMgBr

THF

P

O

EtO

EtOCl

P TMS

O

EtO

EtO

P H

O

EtO

EtO

K2CO3

H2O

75% (overall)80

84 8283

Scheme 19. Synthesis of the ethynylphosphonate 82

Webb and co-workers have recently reported that the ethynylphosphonate 82 can be

synthesised by direct addition of the ethynylmagnesium bromide 85 to diethyl

39

chlorophosphate 80 (Scheme 20).43

Their approach reduces the number of steps but the

alkyne 82 is produced in better yield by the methods described above.

H MgBr

THF

P

O

EtO

EtOCl

P H

O

EtO

EtO

24%

80

85 82

Scheme 20. Synthesis of the alkyne 82 by the Webb group43

Because of the expense of the ethynylsilanes above, alternative syntheses of

ethynylphosphonate 82 were investigated (Scheme 21). The Grignard reagent formed by

treatment of the much cheaper 2-methyl-3-butyn-1-ol 86 with an excess of

ethylmagnesium bromide reacted smoothly with diethyl chlorophosphate 80, affording

the protected ethynylphosphonate 87.

H

EtMgBr

THF

P

O

EtO

EtOCl

P

O

EtO

EtO

OHOH

77%

Cl

P(OEt)3

NiCl2 OH

47%

80

86 87 88

Scheme 21. Synthesis of the protected ethynylphosphonate 87

Compound 87 was also formed by a nickel(II)-catalysed Arbuzov reaction of

triethylphosphite and the chloride 88.54

The Arbuzov reaction is, traditionally, the

reaction of a trialkylphosphite 89 and an alkyl halide 90 to provide the corresponding

phosphonate 93 (Scheme 22).55,56

40

(EtO)3PRX P

O

EtO

OEtR X P

O

EtO

OEtR

+ EtX

8990

91 92 93 94

Scheme 22. Mechanism for the Arbuzov reaction

The Arbuzov reaction can also be applied to aryl or alkynyl halides (Scheme 23), but

requires transition metal catalysis; nickel(II) chloride (NiCl2) 95 is used in many

cases.55-57

In the proposed reaction mechanism,55,56

NiCl2 95 is reduced in situ to a

tetrakis(trialkylphosphite) nickel(0) complex 96. Oxidative addition of 96 to an alkynyl

halide (97 in this case) gives 98, and reductive elimination subsequently furnishes the

desired phosphonate 102, regenerating Ni(0).

41

Ni

PEtO

OEt

OEt

PEtOOEt

OEt

X Ni

PEtO

OEt

OEt

PEtOOEt

OEt

X-

+

Ni

PEtO

OEt

O

PEtO

OEt

OEt

P

OEt

OEtOEt

EtX

P(OEt)3

NiCl2

P(OEt)3

X

P

O

OEt

OEt

[(EtO)3P]2Ni

95

[(EtO)3P]4Ni

96

97

98 99

100

102

101

89

R R R

RR

89

Scheme 23. Mechanism for the Nickel(II)-catalysed Arbuzov reaction

Unfortunately, the deprotection of 87 did not proceed as expected (Scheme 24). Under

each of the conditions attempted,58-60

complete consumption of the starting material 87

was observed, but the desired ethynylphosphonate 82 was not produced. These

alternative routes to the alkyne 82 had to be abandoned at this point, but fortunately

there was sufficient material to proceed with.

42

P

O

EtO

EtO

P H

O

EtO

EtO

OH

(1) KOH, i-PrOH, 70 oC

(2) NaH, toluene, 110 oC

(3) K2CO3, 18-crown-6, toluene, 110 oC

Attempted conditions

87 82

Scheme 24. Attempted deprotection of 82

2.4 Azide-Alkyne Cycloadditions

2.4.1 Background

1,3-Dipolar cycloadditions are pericyclic reactions of a dipolarophile, for example an

alkene or an alkyne, and a 1,3-dipolar compound such as a nitrile oxide or an azide,

producing the corresponding 5-membered heterocyclic compounds.61,62

A particularly

popular example42,63

is the 1,3-dipolar cycloaddition between an azide and an alkyne to

furnish a 1,2,3-triazole, known as a Huisgen cycloaddition after Rolf Huisgen

established the general application of this reaction in the 1960s.61,62

In the thermal

reaction of an azide 103 and an alkyne 104, a mixture of the two regioisomers 105 and

106 is afforded, and electronic and steric factors affect the regioselectivity of the

reaction (Scheme 25).42,63

43

R1 R2

N N N R3

NN

NR3

R2R1

R2 R1

N N N R3

NN

NR3

R1R2

or

NN

NR3

R2R1

NN

NR3

R1R2

+

R1 R2

N N N R3

103 105 106

104

Scheme 25. Mechanism for the Huisgen cycloaddition of azides and alkynes

2.4.2 Copper-catalysed azide-alkyne cycloadditions

In the case of cycloadditions between azides 107 and terminal alkynes 108, the

regioselectivity is able to be controlled by copper(I)-catalysts, which furnish 1,4-

disubstituted 1,2,3-triazoles 109 (Scheme 26).41,63

R1 H

R2 N3N

NN

R2

R1Cu(I)107

108

109

Scheme 26. Cu(I)-catalysed cycloadditions of azides and terminal alkynes

The proposed mechanism for the copper(I)-catalysed reaction is depicted in Scheme 27.

Formation of copper(I) acetylide 110 is the starting point of the reaction, and therefore

no reactions take place in the case of an internal alkyne. The acetylide 110 coordinates

44

to an azide 107 to regiospecifically form a copper(III) metallacycle 112, and subsequent

reductive elimination, followed by hydrolysis of the copper-carbon bond in 113, gives a

1,4-substituted triazole 109.63

NN

N

R1

R2

[LnCu]+

CuLn

NN

N

R1

R2

R1 H

R1 CuLn

N N N R2

R1 CuLn

N N N R2

N N NR2

107

108

110

111

112

113 109

CuLn

R1

C

Acetylideformation

Reductiveelimination

Productrelease

Coordination betweenazide and acetylide

Regiospecific formationof Cu(III) metallacylce

Scheme 27. Proposed mechanism for copper(I)-catalysed cycloadditions of azides and

terminal alkynes63

There are several sources of copper(I),63

including the iodide, bromide and triflate, that

can be used to catalyse these reactions. A nitrogen base, for example, 2,6-lutidine,

triethylamine, diisopropylethylamine or pyridine, is required in these cases in order to

impede the formation of undesired by-products such as diacetylenes, bis-triazoles, and

5-hydroxytriazoles, although this is not always successful.41,63

Accordingly, it is

generally more efficient to generate copper(I) in situ by reducing copper(II) salts.63

The

combination of copper(II) sulfate and sodium ascorbate is particularly popular.63

Under

these conditions, the reaction can be conducted at room temperature and in a wide range

of solvents, such as aqueous tert-butyl alcohol, ethanol, dimethylformamide and water

45

without any organic co-solvent.63

The modular nature, high and reliable yields, atom

economy, wide scope and simple purification of copper(I)-catalysed azide-alkyne

cycloadditions has led them to become the prototypical „click‟ reaction.64,65

In

particular, copper(I)-catalysed cycloadditions are extremely powerful tools for

bioconjugation, both in vitro and in vivo, because of the perfect regioselectivity and

chemoselectivity of the reactants, as well as the high reliability.65

Azide-alkyne

cycloadditions have had a significant impact on a variety of research fields, including

medicinal and materials chemistry.65

2.4.3 Ruthenium-catalysed azide-alkyne cycloadditions

In contrast to copper catalysts, the cycloadditions of azides 114 and terminal alkynes

115 in the presence of ruthenium(II)-catalysts, give 1,5-substituted triazoles 116

predominantly (Scheme 28).42

R1 H

R2 N3N

NN

R2

Ru(II)

114

115

116

R1

Scheme 28. Ruthenium(II)-catalysed cycloadditions of azides and terminal alkynes

A mechanistic proposal for the ruthenium(II)-catalysed reactions is described in Scheme

29.66

Unlike the copper catalysed reaction, the Ru(II)-catalysed variant does not involve

formation of a metal acetylide and can be applied to both terminal and internal

alkynes.42,66

The displacement of the spectator ligands by an azide 114 and alkyne 115

initiates the reaction and produces 118. Subsequent oxidative coupling gives 119,

followed by reductive elimination of the ruthenium and the release of the 1,5-substituted

triazoles 116. Several commercially available ruthenium(II) catalysts, including

46

Cp*RuCl(PPh3)2, Cp*RuCl(COD), Cp*RuCl(NBD) and [Cp*RuCl]4, are effective for

this reaction.42,66

RuCl

L

L

R1

H

N N N R2

RuCl N

N

N

R2

RuCl N

N

NR1

R2

R1 H

RuCl NN

NR2

R1

N NN R2

R1

Displacement ofspectator ligands

Regioselectiveoxidative coupling

Reductiveelimination

Productrelease

116

115

114117

118

119

120

Scheme 29. Proposed mechanism for ruthenium(II)-catalysed azide-alkyne

cycloadditions66

2.4.4 Synthesis of protected triazolylalaninephosphonates (NBoc-Tza(POEt2)-

OMe)

The protected 1,5-subsituted triazole 121 and 1,4-subsituted regioisomer 122 were

prepared by subjecting the azide 60 to thermal cycloaddition with the alkyne 82

(Scheme 30), with 122 predominating (Table 2).48

The identities of the regioisomers,

47

which were easily separated by column chromatography, were confirmed by an HMBC

experiment (Figure 17). Specifically, the 1,4-substituted triazole 122 was identified by a

three-bond correlation between the -protons and the triazolyl carbon. In the 13

C NMR

spectra of both regioisomers the signals arising from the two triazolyl carbons are split

into doublets, due to coupling with the phosphorus nucleus (Figures 18 and 19). In

particular, the C–P coupling constant is very large (220 Hz). In contrast, H–P coupling

was not observed with the triazoles‟ protons in the 1H NMR spectra.

The formation of both isomers was both expected and useful, giving access to both

protected N1-PHis (121) and N3-PHis (122) analogues. However, from the perspective

of preparative efficiency, it was of interest to control the regioselectivity of the

cycloaddition reaction and, accordingly, catalysis was explored. The copper(I)-catalysed

reaction63

furnished the triazole analogue 122 exclusively and in excellent yield (Table

2). The exclusive formation of the isomeric analogue 121 is far more challenging.

Ruthenium(II) catalysis successfully reversed the regioselectivity under these conditions

(Table 2).42

However, the reaction was not regiospecific and it would be preferable to

optimise the reaction conditions to accomplish the exclusive formation of the 1,5-

substituted triazole 121. A possible solution for this matter would be to increase the

amount of the ruthenium catalyst to 10 mol% and conduct the catalysed cycloaddition at

room temperature.40

However, as discussed later in Chapter 4, changing the protecting

groups on the azide substrate also achieved the desired regiospecificity.

48

BocN

O

OMe

N3

HBocN

OMe

OH

N

N N

P

O

OEt

OEt

BocNOMe

OH

N NN

PO

OEtEtO

P H

O

EtO

EtO

+conditions

60

82

121 122

Scheme 30. Azide-alkyne cycloadditions. Conditions: (a) PhMe, 110 ºC (b) CuSO4 (10

mol%), sodium ascorbate, 1:1 t-BuOH-H2O (c) Cp*RuCl(PPh3)2 (1.5 mol%), PhMe, 60

ºC. See table 2 for yields.

Table 2. Yields of azide-alkyne cycloadditions under various conditions.

Product Thermal Cu(I)-catalysed Ru(II)-catalysed

121 21 % – 56 %

122 58 % 95% 21 %

49

BocNOMe

OH

N

C

NN

P

OEt

O

OEt

HH

H

Figure 17. HMBC spectrum of 122 (CDCl3)

50

Figure 18. 1H and

13C NMR spectra of 121 (CDCl3)

51

Figure 19. 1H and

13C NMR spectra of 122 (CDCl3)

52

2.5 Synthesis of haptens

As discussed in Chapter 1, small molecules do not elicit an immune response, and must

be conjugated to a carrier protein before being used to raise antibodies.67

To allow this

conjugation, modification of the phosphohistidine analogues to give a series of haptens

was carried out. It was intended to conjugate through amide linkages so that the haptens

better mimic the naturally occurring phosphohistidine residues in proteins (Figure 20).

To maximise the chances of generating a generic phosphohistidine antibody, for each

regioisomer, three haptens were targeted, with the intention of preparing six different

immunogens for use in antibody generation in the same animal.

H3NNH

O

NN

N

P

O

OO

conjugated to a carrier proteinthrough the amino group

amide mimics peptide bondsin native P-His residues

Figure 20. Example of a target hapten

2.5.1 N-methylamide haptens

The methyl esters 121 and 122 were converted to the amides 123 and 124 simply by

mild treatment with methylamine (Scheme 31).68

Concomitant deprotection69

of the

amine and phosphonic acid, in the presence of the amide, was conducted using

hydrogen bromide in acetic acid, providing the haptens 125 and 126 in good yield.

These haptens can be conjugated through the amino group by coupling with aspartate

and glutamate side chains on the surface of the carrier protein.

53

BocNOMe

OH

N

N N

P

O

OEt

OEt

BocNNH

OH

N

N N

P

O

OEt

OEt

BrH3NNH

O

N

N N

P

O

OH

OH

HBr

AcOH

73 %90%

BocNOMe

OH

N NN

PO

OEtEtO

BocNNH

OH

HBr

AcOH

71 %

MeNH2

MeOH,H2O

85%

121

122

123

124

125

126

NN

N

P

O

EtOOEt

BrH3NNH

O

NN

N

P

O

HOOH

MeNH2

MeOH,H2O

Scheme 31. Synthesis of N-methylamide haptens

Figure 21. 1H NMR spectrum of 125 (D2O)

54


2.5.2 Dipeptide haptens

The dipeptide haptens 129 and 130 were also prepared, as depicted in Scheme 32 and

33. Careful saponification70

of 121 and 122 gave the carboxylic acids 35 and 36,

reported by Muir40

(See section 1.6.1 for an account of very similar work published

during the completion of this research). The NMR spectra of 35 and 36 were compared

with those published.40

The chemical shifts in both the 1H and

13C NMR spectra

acquired in CDCl3 were in accord with those reported, but the signals were considerably

more broadened in the present case, possibly because of some kind of dynamic

phenomenon (Figures 23 and 24). On the other hand, sharp peaks were seen in the NMR

spectra acquired in CD3OD (Figures 23 and 24).

55

BocNOMe

OH

N

N N

P

O

OEt

OEt

BocNOH

OH

N

N N

P

O

OEt

OEt

LiOH

MeOH/H2O

88%122 36

BocNOMe

OH

N NN

PO

OEtEtO

BocNOH

OH

N NN

PO

OEtEtO

LiOH

MeOH/H2O

86%

121 35

0 oC

0oC

Scheme 32. Careful saponification of the methyl esters 121 and 122

(A)

(B)

Figure 23. 1H NMR of 35. (A) CD3OD, (B) CDCl3

56

(A)

(B)

Figure 24. 1H NMR of 36. (A) CD3OD, (B) CDCl3

The specific rotations of the carboxylic acids 35 and 36 were lower in magnitude than

those reported by Muir40

(Table 3), which suggested that the compounds had been

partially racemised in the hydrolysis step, and this was confirmed by enantioselective

HPLC. The enantiomeric excess determined were almost consistent with those

determined from the specific rotations (Table 3). Although this partial racemisation was

unfortunate, it should be noted that even racemic compounds would suffice for antibody

production. Nevertheless, this problem was later overcome (see Chapter 4).

57

Table 3. Specific rotations and enantiomeric excesses for the carboxylic acids 35 and

36.a

[α]D ee

Muir40

This work [α]D HPLC

35 +1.8° (1.0) +1.2° (0.5) 67% 70%

36 +60° (1.0) +40.5° (0.7) 68% 74%

a [α]D values were recorded at 21 C (Muir) and ambient temperature (this work).

Concentrations in g.100 mL–1

in CHCl3 are given in brackets. The ee values were

determined by comparison with the optical rotations reported by Muir and coworkers40

([α]D) and relative integrals of the peaks corresponding to the enantiomers by

enantioselective HPLC.

O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

(HCTU)-mediated peptide coupling reactions of the enantiomerically enriched

carboxylic acids 35 and 36 and N-methyl-L-alaninamide 133, prepared by treatment of

L-serine methyl ester 13271

with methylamine,72

afforded the dipeptides 127 and 128 in

acceptable yields (Scheme 33).73

The 1H and

13C NMR spectra of 127 and 128

contained several minor peaks in addition to those expected for the desired compounds.

These were originally ascribed to the presence of rotamers (a common occurrence with

amides). But, given that the carboxylic acids 35 and 36 were partially racemised, the

additional peaks may actually have been due to diastereomers. Indeed, the

diastereomeric ratio calculated from the 1H NMR spectrum of the dipeptide 127 was in

accord with the enantiomeric ratio of the carboxylic acid 35 (Table 4). On the other

hand, the diastereomeric ratio of the dipeptide 128 did not match the enantiomeric ratio

of the carboxylic acid 35. These data indicate that further epimersation of the Tza

stereocentre may have occurred in the peptide coupling reaction.

58

Table 4. Enantiomeric ratio of 35 and 36 and diastereomeric ratio of 127 and 128

Phosphonoamino acid Enantiomeric ratio Dipeptide Diastereomeric ratioa

35 17:3 127 17:3

36 87:13 128 17:8

a As judged by comparison of relative integrals for the sets of signals in the

1H NMR

spectrum of the column chromatography-purified product.

Removal of the remaining protecting groups was achieved with hydrogen bromide in

acetic acid,69

giving the dipeptide haptens 129 and 130, retaining the diastereomeric

ratio (as judged from the 1H NMR spectra), in good yields (Scheme 33).

BocN

O

NH

H

O

HN

DIPEA

H2NNH

O

HCTU

DMF

60%

BocNOH

OH

N NN

PO

OEtEtO

35

BrH3N

O

NH

O

HN

HBr

AcOH

BocNOH

OH

N

N N

P

O

OEt

OEt

BocNNH

OH

N

N N

O

HN

PO

OEtOEt

BrH3NNH

O

N

N N

O

HN

PO

OHOH

36

DIPEA

H2NNH

O

HCTU

DMF

61%

HBr

AcOH

127

128

129

130

H2NNH

O

ClH3NOEt

O

H3NO

OSOCl2

EtOH MeNH2

131 132 133

133

133N N

N

N

N

N

PPEtOEtO

O O

HOHO

68% over 2 steps

Scheme 33. Synthesis of dipeptide haptens and N-methyl-L-alaninamide

59

Figure 25. 1H NMR spectrum of 127 (CD3OD)


60



61

2.6 Synthesis of haptens ready for bioconjugation through the carboxyl group

Other haptens, which could be linked through the carboxyl group by coupling to lysine

residues on the surface of the carrier protein, were also targeted (Figure 29).

HN

OH

O

NN

N

P

O

OO

conjugated to a carrier proteinthrough the carboxyl group

O

amide mimics peptide bondsin native P-His residues

Figure 29. Example of a hapten

2.6.1 Attempts to convert NBoc derivatives to NAc derivatives

The first strategy to obtain these bioconjugation precursors is shown in Scheme 34. The

Boc-amino protecting group in 122 was removed by TFA, giving the amine 134 in good

yield after neutralisation.74

However, neither acylation of the amine 134 by acetic

anhydride nor peptide coupling with N-acetyl-L-alanine 13875

was successful, judging

by ESI-MS and 1H NMR spectra of the crude products, although the starting materials

seemed to be completely consumed. The reasons for this are unclear, but it is thought

that some inherent property of 134 was responsible.

62

BocNOMe

OH

N

N N

P

O

OEt

OEt

H2NOMe

O

N

N N

P

O

OEt

OEt

TFA

DCM

80%

Ac2O HN

OMe

O

N

N N

P

O

OEt

OEt

O

LiOH

MeOH/H2O

HN

OH

O

N

N N

P

O

OEt

OEt

O

HBr

AcOH

HN

OH

O

N

N N

P

O

OH

OH

O

H2NOMe

O

N

N N

P

O

OEt

OEt

HN

OMe

O

N

N N

P

O

OEt

OEt

O

LiOH

MeOH/H2O

HBr

AcOH

NH

O

HN

OH

O

O

HCTU

DIPEA

DMF

HN

OH

O

N

N N

P

O

OEt

OEt

O

NH

OHN

OH

O

N

N N

P

O

OH

OH

O

NH

O

122 134 135

136137

134 139

140141

138

Et3N

DCM

Scheme 34. Attempted conversion of NBoc derivatives to NAc derivatives

63

Figure 30. 1H NMR of 134 (CDCl3)

2.6.2 Synthesis of NAc-Tza(POEt2)-OMe

Due to the difficulties in replacing the NBoc group with an NAc group, it was decided

to incorporate the NAc group earlier on in the synthesis. The known azide 14376

was

synthesised in two steps from L-serine methyl ester 58 (Scheme 35). An amide coupling

reaction using N,N′-dicyclohexylcarbodiimide (DCC) furnished N-acetyl-L-serine

methyl ester 14277

in excellent yield. The alcohol 142 was converted to the azide 143 by

a Mitsunobu reaction in acceptable yield.

ClH3N

O

OMe

OH

HN

O

OMe

OHO

HN

O

OMe

N3

O

AcOH

DCC

DCM

HN3

DIAD

PPh3

THF90%

60%58 142 143

Scheme 35. Synthesis of the azide 143

Various conditions were applied to azide-alkyne cycloadditions of the azide 143 and the

ethynylphosphonate 82 (Scheme 36), with similar results to those observed with the

NBoc derivative. In the absence of a catalyst, the isomeric triazole analogues 144 and

145 were produced, with the 1,4-substituted triazole 145 predominating (Table 5). The

copper(I)-catalysed reaction furnished the 1,4-substituted triazole 145 exclusively in

64

excellent yield (Table 5). The regioselectivity was successfully reversed in the presence

of the ruthenium(II) catalyst, affording the 1,5-substituted triazole 144 as the major

product (Table 5).

HN

O

OMe

N3

HN

OMe

O

N

N N

P

O

OEt

OEt

HN

OMe

O

N NN

PO

OEtEtO

P H

O

EtO

EtO

+conditions

O O O

143

82

144 145


mol%), sodium ascorbate, 1:1 t-BuOH/H2O (c) Cp*RuCl(PPh3)2 (1.5 mol%), PhMe, 60

ºC

Table 5. Yields of Huisgen cycloadditions under various conditions


144 26% – 57%

145 60% 91% 19%

Figure 31. 1H NMR spectrum of 144 (CDCl3)

65


2.6.3 Synthesis of N-acetyl haptens

The next task was to transform the methyl esters 144 and 145 to the corresponding

carboxylic acids, followed by deprotection of the phosphonic acid to provide the target

haptens. The ester 145 was carefully saponified with lithium hydroxide (Scheme 37).

78

The target carboxylic acid 146 was obtained but the yield was extremely poor. It should

be noted that the saponification using lithium hydroxide needed to be conducted at 0 ºC.

When it was carried out at room temperature, only the elimination product 147 was

observed.

Owing to the poor yield, other conditions for the hydrolysis were investigated. When

potassium carbonate was used as a base,79

the desired carboxylic acid 146 was obtained

in better yield. Under these conditions, the reaction did not proceed at 0 ºC, and

therefore it was performed at room temperature. The optical rotation of the carboxylic

acid 146 obtained under these conditions was greater in magnitude than that using

lithium hydroxide (Table 6), indicating that as expected, enolate formation and

At the time this experiment was conducted, it was not known that these conditions caused partial

racemisation.

66

racemisation accompanied the elimination of the triazole substrate, which presumably

occurred by an E1cB mechanism.

AcNOMe

OH

N

N N

P

O

OEt

OEt

AcNOH

OH

N

N N

P

O

OEt

OEt

+

AcNOH

OH

conditions

145 146 147

Scheme 37. Hydrolysis of the methyl ester 145 under various conditions

Table 6. Results of the hydrolysis of 145.

Ester Conditions 146 (Yield) 147 Specific Rotation

145 LiOH, MeOH, H2O, 0 ºC 31% – +5.1°

145 LiOH, MeOH, H2O, RT – Observed –

145 K2CO3, MeOH, H2O, RT 47% – +8.8°

145 K2CO3, MeOH, H2O, 0 ºC no reaction –


The carboxylic acid 146 synthesised by hydrolysis of the methyl ester 145 using K2CO3

was subjected to hydrogen bromide in acetic acid to reveal the phosphonic acid,69

giving the hapten 137 in good yield (Scheme 38).

67

HN

OH

O

N

N N

P

O

OEt

OEt

O

HBr

AcOH

HN

OH

O

N

N N

P

O

OH

OH

O

70%146 137

Scheme 38. Deprotection of 146

The same strategies were applied to 1,5-substituted triazole 144 (Scheme 39). However,

a mixture of the desired carboxylic acid 148 and elimination product 147, which were

not separable by normal-phase chromatography, was obtained. Accordingly, the poor

yield, difficult purification and assumed racemisation required another approach to be

taken.

AcNOMe

OH

N NN

PO

OEtEtO

Conditions

AcNOH

OH

N NN

PO

OEtEtO

AcNOH

OH

+

144 148

147

Scheme 39. Hydrolysis of the methyl ester 140. Conditions (a) K2CO3, MeOH/H2O, r.t.

(b) LiOH, MeOH/H2O, 0 ºC.

2.6.4 Synthesis of NAc-Tza(POEt2)-OAll

The alternative route was designed to circumvent the problems of racemisation and

elimination during the saponification of methyl esters. An allyl ester was initially

chosen as it can be removed by deprotection using palladium(II)-catalysis.80

Thus, L-

serine 57 was treated with acetic anhydride in acetic acid,75

providing a mixture of N-

acetyl-L-serine 149 and N-acetyl-O-acetyl-L-serine 150 (Scheme 40). The mixture was

esterified with allyl bromide to give the desired ester 151 and undesired ester 152, with

68

the latter dominating.81

The alcohol 151 was converted to the novel azide 153 by a

Mitsunobu reaction in acceptable yield.

AcOH

Ac2OHN

O

OH

OH

H3N

O

O

OHO

HN

O

OH

OO

O

HN

O

O

OHO

HN

O

O

OO

O

DMF

DIPEA

21% (over two steps)


+

Br

57 149 150

151

152

HN

O

O

N3

O

153

HN3

DIAD

PPh3

THF

61%

Scheme 40. Synthesis of N-acetyl azidoalanine allyl ester 153

The azide 153 was subjected to cycloaddition reactions with the ethynylphosphonate 82

under various conditions (Scheme 41). Interestingly, the thermal reaction furnished the

1,5-substituted triazole 154 and 1,4-substituted triazole 155 in the ratio of 1:1 (Table 7),

whereas the 1,4-substituted triazole 145 was a major product in the case of the thermal

reaction of the NAc protected methyl ester 143 (2:1). The reasons for the altered

regioselectivity brought about by the allyl ester are not obvious. As expected, the 1,4-

69

substituted triazole 155 was formed exclusively in the copper(I)-catalysed reaction

(Table 7). The innate tendency of the allyl group to favour more of the 1,5-triazole was

further enhanced with Ru(II) catalysis (Table 7), with the regioselectivity now

significantly better than previously observed.

AcN

O

OAll

N3

HAcN

OAll

OH

N

N N

P

O

OEt

OEt

AcNOAll

OH

N NN

PO

OEtEtO

P H

O

EtO

EtO

+conditions

153

154 155

82



ºC

Table 7. Yields of azide-alkyne cycloadditions under various conditions


154 41 % – 73 %

155 44 % 84% 10 %


70


2.6.5 Attempted deallylation

Despite similar precedents,80

the target carboxylic acids 148 and 146 were not obtained

by palladium(0)-catalysed cleavage of the allylesters 154 or 155 (Scheme 42). Although

what occurred in the reactions are unclear, the starting materials were not completely

consumed and there was no trace of the target compounds, judging by the ESI-MS and

1H NMR spectra of the crude products.

Pd(PPh3)4

Morpholine

THF

HBr

AcOH

AcNOAll

OH

N

N N

P

O

OEt

OEt

Pd(PPh3)4

Morpholine

THF

AcNOH

OH

N

N N

P

O

OEt

OEt

HBr

AcOH

AcNOH

OH

N

N N

P

O

OH

OH

AcNOAll

OH

N NN

PO

OEtEtO

154

AcNOH

OH

N NN

PO

OEtEtO

148

AcNOH

OH

N NN

PO

OHHO

156

155 146 137

Scheme 42. Attempted deallylation

71

In the proposed mechanism for removal of allyl protecting group by a palladium

catalyst,80

palladium(0) species coordinate to the double bond of the allyl protecting

group, followed by oxidative addition to form a -allyl complex (Scheme 43).

R O

O

R OH

OPd+ (II)

L

L

R O

O

Pd (0)

L

L

coordination

Oxidative addition

+

Pd (II)

L

L

B

Pd (0)

L

L +B

Pd (0)

L

L

Scheme 43. Proposed mechanism for removal of the allyl protecting group

As to why this normally reliable deprotection failed in the current case, it is possible

that the Pd formed a stable complex with the substrate, which has many groups capable

of coordinating Pd, shutting down the catalytic cycle.

2.6.6 Synthesis of NAc-Tza(POEt2)-OBn

Due to the difficulties with the deallylation, an alternative route was designed to

circumvent the saponification step, and a benzyl group was chosen as an acid-labile

carboxyl protecting group, which would be cleaved by treatment with hydrogen

bromide in acetic acid, used for deprotection of the phosphonate, thereby also avoiding

one step. The synthesis was similar to that of the allyl esters. The ester 160 was

synthesised in 2 steps75,81

from L-serine 57 with the undesired ester 161 being a by-

product (Scheme 44). The alcohol 160 was transformed to the azide 162 by a Mitsunobu

reaction.

72

AcOH

Ac2OHN

O

OH

OH

H3N

O

O

OHO

HN

O

OH

OO

O

HN

O

O

OHO

HN

O

O

OO

O

DMF

DIPEA



+

Br

HN3

DIAD

PPh3

THF

HN

O

O

N3

O

61%

57

157 158

159

161

160

162


The azide 162 was subjected to azide-alkyne cycloadditions as before (Scheme 45). The

thermal reaction furnished the 1,5-substituted triazole 163 and 1,4-substituted triazole

164 in the ratio of 1:1 (Table 8). As anticipated, the copper(I)-catalysed reaction gave

164 exclusively (Table 8). A remarkable difference observed in the case of the benzyl

esters was the exclusive formation of the 1,5-substituted triazole 163 using the

ruthenium catalyst (Table 8). The findings thus far suggest that bulky carboxyl

protecting groups assist the formation of the 1,5-substituted triazole in the click

reaction, although the exact mechanisms are still being elucidated.

73

HN

O

OBn

N3

HN

OBn

O

N

N N

P

O

OEt

OEt

HN

OBn

O

N NN

PO

OEtEtO

P H

O

EtO

EtO

+conditions

O O O

162

163 164

82



ºC

Table 8. Yields of azide-alkyne cycloadditions under various conditions.


163 43% – 81%

164 45% 87% –

2.6.7 Synthesis of N-acetyl haptens-2

The protected carboxylic acid and phosphonic acid of 163 and 164 were revealed

concomitantly utilising hydrogen bromide in acetic acid,69

giving the haptens 156 and

137 in good yields.. The specific rotation of the hapten 137 obtained in this way was

greater in magnitude than that obtained via saponification, suggesting that the partial

racemisation observed under basic conditions was avoided by using protonolysis (Table

9).

74

HN

OBn

O

N

N N

P

O

OEt

OEt

HN

OBn

O

N NN

PO

OEtEtO

O

O

HBr

AcOH

HN

OH

O

N

N N

P

O

OH

OH

O

67%

HBr

AcOH

69%

HN

OH

O

N NN

PO

OHHO

O

163

164 137

156

Scheme 46. Synthesis of the conjugation precursors

Table 9. Specific rotations of the hapten 137

Hapten 137 1 step from 164 2 steps from 145

[α]D +10.0º (c 1.0, H2O) +6.1º

(c 1.0, H2O)


75




77

Chapter 3

Hapten-Carrier Protein Conjugation Strategies

78

3.1 Introduction to bioconjugation methods

As mentioned in the previous chapter, small molecules must be conjugated to a large

molecule, usually a „carrier‟ protein, in order to cause an immune response.67

Small

molecules can be coupled to carrier proteins by various reactions, which involve

existing functional groups or added reactive groups.82

Among several conjugation

methods, one of the most common procedures is coupling of haptens and carrier

proteins through amide linkages using water-soluble carbodiimides such as N-(3-

dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI) (Scheme 47).83

O

O

aspartate/glutamateresidue

carrierprotein

H2N Hapten

O

NH

carrierprotein

Hapten

EDCI

Scheme 47. An example of a coupling of carrier protein and hapten using EDCI

Reactive esters, for example N-hydroxysuccinimide (NHS) esters, are often used to

allow conjugation through amide linkages (Scheme 48).82

These esters can selectively

react with alkyl amines in the presence of anilines, alcohols, phenols, and histidine.82

The optimum pH for reactions of the alkyl amines and reactive esters in aqueous

systems is 8–9.82

O

O

carrierprotein

N

O

O

H2N Hapten

O

HN

carrierprotein

Hapten

Scheme 48. Coupling of carrier protein and amino-bearing hapten using an activated

ester

79

Commercially available maleimide-activated carrier proteins are also frequently utilised

for the bioconjugation of haptens containing thiol groups, which undergo conjugate

addition with the maleimide to form stable thioethers (Scheme 49).82

The optimum pH

for the reactions is about 7.0, as maleimide can be hydrolysed to a maleamic acid

derivative above pH 8.0.82

Ncarrierprotein

O

O

HS Hapten

Ncarrierprotein

O

O

S Hapten

Scheme 49. Coupling of maleimide-activated carrier protein and a thiol-bearing hapten

Although other carrier proteins such as ovalbumin (OVA) and bovine serum albumin

(BSA) are utilised as carriers, keyhole limpet hemocyanin (KLH) is the most common

immunostimulator.84

KLH is more immunogenic compared with other carrier proteins

because of its high molecular weight.84

KLH also possesses a large number of lysine

and aspartic acid residues to which haptens can be attached.84

The 1,4-triazolylalanine

derivatives 126, 130 and 137 will be conjugated to KLH as depicted in Scheme 50, and

the same strategies will be applied to the 1,5-triazolylalanine derivatives 125, 129 and

156.

The resulting immunogens (i.e., the six different bioconjugates) will be co-administered

to rabbits to maximise the chances of producing phosphohistidine-recognising

antibodies.

80

KLH

O

O


EDCI

BrH3N

O

NH

N

N N

P

O

OO

126 (Hapten 1)

KLH

O

HN

NH

O

N

N N

P

O

OO

carrier protein

Amides mimic peptide bondsin native P-His residues

KLH

O

O


EDCI

BrH3N

O

NH

N

N N

130 (Hapten 2)

KLH

O

carrier protein

HN

O

PO

OO

HN

O

NH

N

N N

HN

O

PO

OO

KLH

carrier protein

H3N

Lysine residue

EDCI

HN

O

OH

N

N N

P

O

OO

137 (Hapten 3)

O

KLHNH

OHN

Immunogen 1

Immunogen 2

N

N N

P

O

O

O

O

Immunogen 3

Scheme 50. Hapten-Carrier protein Conjugation Strategies

3.2 Trial Conjugation-1

A trial conjugation in water was performed using the hapten 126 and azidoacetic acid

166, prepared by the reaction of bromoacetic acid 165 and sodium azide (Scheme 51).85

After purification by Dowex anion exchange chromatography, the slightly impure

desired amide 167 was obtained, as judged by the 1H NMR,

13C NMR and ESI-MS

81

spectra of the product. No trace of undesired phosphoramidates 168 (Scheme 52),

resulting from self-condensation of the phosphonic acid, was observed. This result

indicated that the haptens possessing a phosphonic acid could be linked to a carrier

protein by an EDCI-mediated amide coupling in water.

OHN3

O

EDCI

NaHCO3

H2O

60%

OHBr

O

OHN3

O

NaN3

H2O

61%

BrH3N

O

NH

N

N N

P

O

OHOH

HN

O

NH

N

N N

P

O

OHOH

O

N3

165 166

126

166

167

Scheme 51. Trial conjugation of the hapten 126 and azidoacetic acid 166

R1 NH2

EDCI

P

O

OH

OHR2

P

O

OH

NHR2

R1

168

Scheme 52. Potential, undesired phosphoramidate formation resulting from activation

of a phosphonic acid by EDCI

82

Figure 40. 1H and

13C NMR of 167 (D2O)

3.3 Trial Conjugation-2

Conjugation of the hapten 126 and a protein was then attempted. OVA, which can also

be used as a carrier protein and is more affordable than KLH, was chosen for model

studies. Although several attempts were made (Scheme 53) based on literature

precedents86,87

for the coupling of amino-bearing haptens, none of them were

successful, as judged by MALDI-TOF mass spectra of the products after dialysis, which

was virtually the same as that of unreacted OVA.

83

O

O


EDCI

BrH3N

O

NH

N

N N

P

O

OHOH

126 (Hapten 1)

O

HN

NH

O

N

N N

P

O

OHOH

carrier protein

Amides mimic peptide bondsin native P-His residues

OVA OVA


1) EDCI, NaHCO3, H2O

2) EDCI, H2O

3) EDCI, 0.1M MES buf fer (pH 4.7)

Scheme 53. Attempted conjugation of the hapten 126 and OVA

At this time, we have no explanations for the failure of this coupling, and there was

insufficient time to conduct any further work in this area, but several control

experiments are possible in order to investigate the causes of the failure and search for

suitable bioconjugation conditions.

For example, N-methylalaninamide 133 and N-acetylalanine 138 would be useful for

the control experiments (Scheme 54). Success in these couplings will validate the

bioconjugation procedure and point to an inherent difficulty in amide couplings of the

hapten(s). Given the previously observed inability to acylate 134 with small molecules

(see section 2.6.1), this is a distinct possibility.

84

O

O

carrier protein

OVA

H2NNH

O

EDCI

O

HN

NH

OOVA

OVA

HN

OH

O

EDCI

O

H3N OVANH

OHN

O

133

138

Scheme 54. Possible control experiments for the bioconjugation

In the case where EDCI-mediated peptide coupling reaction is not suitable for the

bioconjugation of the haptens and a carrier protein, other methods without using EDCI

will need to be found. The first candidate is using the activated ester, which allows

conjugation through amide linkages.82

O

O

KLH

N

O

O

BrH3N

O

NH

N

N N

P

O

OO

126 (Hapten 1)

KLH

O

HN

NH

O

N

N N

P

O

OO

Immunogen 4

Scheme 55. Conjugation of the hapten and a carrier protein using an activated ester

Alternatively, now that the azide 167 has been produced in the trial conjugation of the

hapten 126 and azidoacetic acid 166 (Scheme 51), another approach utilising a

copper(I)-catalysed azide-alkyne cycloaddition („click‟) reaction can be proposed

(Scheme 56). KLH possessing alkynes on the surface could be synthesised by the

85

reaction of the lysine residues with the succinimidyl ester 169.88

As the „click‟ reaction

can be conducted in water with no organic co-solvents,63

the cycloaddition of the azide

167 alkyne 170 should give immunogen 5.

KLH NH3

lysine residue

KLH NH

Carrier protein

O

KLH NH

O

N N

N

HN

O

NH

O

N

NN

P

O

O

O

H

N3

HN

O

NH

O

N

NN

P

O

O

O

CuSO4

Sodium ascorbate

Immunogen 5

NO

O

HO

O

H2O

169

167

170

Scheme 56. Proposed conjugation strategy using a copper(I)-catalysed azide-alkyne

cycloaddition (click) reaction

87

Chapter 4

Synthesis of Free Phosphohistidine

Analogues and Evaluation of their Ability to Inhibit a

Mammalian Histidine Kinase

88

4.1 Possible biological activity of phosphohistidine analogues

The possible intrinsic biological activity of the phosphohistidine analogues captured our

interest. For example, they may exhibit antibacterial activity by interfering with protein-

protein recognition in bacterial two-component systems. In addition, as product-mimics,

they could potentially inhibit histidine kinase activity, and therefore antiproliferative

activity in mammalian cells. The roles of histidine kinases in mammalian cells are

poorly understood, but inhibition of their activity could provide some insight into their

functions and lead to the invention of useful medicines, including anticancer drugs.8,18

4.2 Competitive and non-competitive inhibitors of histidine kinases

As with any enzyme, there are two common types of inhibition of histidine kinases:

competitive and non-competitive. In competitive inhibition, an inhibitor prevents a

histidine kinase from binding the substrate protein and/or ATP by blocking the binding

site of the histidine kinase, and thereby, the transfer of a phosphoryl group does not

occur (Figure 41).89

For instance, the Winkler group reported that the novel

thienopyridine TEP inhibited autophosphorylation of a bacterial histidine kinase, His-

HpkA77, competitively with respect to ATP.89

89

Competitive inhibition

Histidine kinase

ATP

Inhibitor

Inhibition by blockingthe binding site

N S

HN Br

N

H2N

TEP

O

NH2

Figure 41. Mechanism of competitive inhibition and the structure of TEP

On the other hand, in non-competitive inhibition, an inhibitor binds to a histidine kinase

on a site different from the active site, which causes structural transformation of the

histidine kinase, thereby preventing binding of the substrates (ATP or the substrate

protein) (Figure 42).15,90

For example, it was found by Hock‟s group that RWJ-49815

and Closantel interfered with autophosphorylation of a bacterial two-component

histidine kinase, Kin A, due to non-specific structural transformation caused by the

inhibitor intercalating in the hydrophobic core of the enzyme.13,15,90

90

Non-competitive inhibition

Histidine kinase

ATP

Inhibitor

Inhibition by causing structuraltranformation of the kinase

O

HN NH2

NH

RWJ-49815

NH

O

CN

Cl

I

OH

I

Cl

Closantel

Figure 42. Mechanism of non-competitive inhibition and the structures of RWJ-49815

and Closantel

4.3 Synthesis of free triazolylalaninephosphonates

In order to evaluate the biological activities, the free triazolylalaninephosphonates 170

and 171 (Figure 43) were synthesised.

H3NO

O

N NN

PO

OO

H3NO

O

N

N N

P

O

O

O

170 171

Figure 43. Structures of ‘free‟ N1-(170) and N3-phosphohistidine (171) analogues

Initial attempts at global deprotection of 121 and 122 using hydrochloric acid or

hydrobromic acid were unsuccessful (Scheme 57).36

Although what occurred was not

completely elucidated, the methyl esters seemed to be stable under these conditions,

judging by 1H NMR spectra of the crude materials.

91

BocNOMe

OH

N NN

PO

OEtEtO

BocNOMe

OH

N

N N

P

O

OEt

OEt

XH3NOH

O

N NN

PO

OHHO

XH3NOH

O

N

N N

P

O

OH

OH

121

122

(X=Cl or Br)

(X=Cl or Br)

H3O Cl or H3O Br

H3O Cl or H3O Br

170

171

Scheme 57. Attempted global deprotections using mineral acids

Therefore, the carboxylic acids 35 and 36, which were synthesised by the hydrolysis of

the methyl esters 121 and 122 as described in the previous section, were utilised for the

synthesis of the free phosphonoamino acids. The amine and phosphonic acid were

concomitantly deprotected by hydrogen bromide in acetic acid to afford the free

analogues 170 and 171 as hydrobromides in acceptable yields (Scheme 58).69

However,

it should be remembered that the saponification to provide the carboxylic acids caused

partial racemisation and, accordingly, the free phosphonoamino acids 170 and 171,

prepared in this way, were not enantiopure but enantiomerically enriched.

BocNOMe

OH

N NN

PO

OEtEtO

BocNOH

OH

N NN

PO

OEtEtO

BocNOMe

OH

N

N N

P

O

OEt

OEt

BocNOH

OH

N

N N

P

O

OEt

OEt

LiOH

MeOH/H2O

88%

LiOH

MeOH/H2O

86%

HBr

AcOH

72%

HBr

AcOH

70%

BrH3NOH

O

N NN

PO

OHHO

BrH3NOH

O

N

N N

P

O

OH

OH

36

35

122

121 170

171

Scheme 58. Synthesis of phosphonoamino acids 170 and 171

92

To avoid racemisation, a strategy similar to that used for the synthesis of the haptens

137 and 156 (section 2.6.7) was used; the methyl ester was replace with a benzyl ester,

which is cleavable by protonolysis or hydrogenolysis (Scheme 59). The amino group of

L-serine 57 was protected to give the t-butyl carbamate 172,91

followed by alkylation

with benzyl bromide 159 to give the benzyl ester 173.81

A Mitsunobu reaction

converted the alcohol 173 to the corresponding azide 17440

in acceptable yield.

H3N

O

O

OH

BocN

O

O

N3

H

BocN

O

OH

OH

HBocN

O

O

OH

HBoc2O

NaOH

Dioxane

H2O

DIPEA

DMF

HN3

DEAD

PPh3

THF

72%

85%

75%

Br

57 172 173

159

174


The azide 174 was subjected to azide-alkyne cycloadditions to furnish the protected

triazolylalanines 175 and 176 (Scheme 60). The thermal reaction gave a 1:1 mixture of

175 and 176, which were separable by flash chromatography (Table 10). In the same

reaction of the analogous methyl ester 60 (Scheme 30, page 48), the 1,5- and 1,4-

triazoles 121 and 122 were produced in the ratio of 1:2. Judging by these results, the

formation of the 1,5-triazole 175 was assisted by the benzyl protecting group. As

anticipated, the copper(I)-catalysed reaction gave the 1,4-triazole 176 exclusively in

good yield (Table 10). As with the N-acetyl analogue 163 (Scheme 45, page 73),

93

exclusive production of the regioisomer 175 was observed in the presence of the

ruthenium(II)-catalyst (Table 10).

BocN

O

O

N

H

NN

P O

OEtEtO

BocN

O

O

N

H

NN

PO

OEtEtO

P H

O

EtO

EtO

BocN

O

O

N3

H

conditions +

174

82

175 176

Scheme 60. Azide-alkyne cycloadditions of benzyl esters. Conditions: (a) PhMe, 110 ºC

(b) CuSO4 (10 mol%), sodium ascorbate, 1:1 t-BuOH-H2O (c) Cp*RuCl(PPh3)2 (1.5

mol%), PhMe, 60 ºC.

Table 10. Yields of Azide-Alkyne cycloadditions under various conditions


175 41% – 80%

176 42% 88% –


94


Gratifyingly, all protecting groups were successfully removed in one step utilising

hydrogen bromide in acetic acid, producing the free phosphonamino acids 170 and 171

as the hydrobromides in acceptable yield (Scheme 61). Their specific rotations were

greater in magnitude than those of the same compounds produced via the methyl esters

(121 and 122). These results suggested that the more enantiopure free analogues were

obtained by employing a benzyl protecting group for the carboxylic acid in place of a

methyl protecting group (Table 11).

BocN

O

OBn

N

H

N

N

PO

EtOOEt

BrH3N

O

OH

NN

N

PO

HOOH

HBr

AcOH

65%

BocN

O

OBn

N

H

N N

P

O

OEt

OEt

BrH3N

O

OH

N

N N

P

O

OH

OH

HBr

AcOH

67%

175

176

170

171

Scheme 61. Improved and simplified methods to synthesise the free phosphonoamino

acids 170 and 171

95

Table 11. Specific rotations of 170 and 171

One step from the benzyl ester Two steps from the methyl ester

[α]D of 170 7.7° (c 0.5 H2O) 5.6° (c 0.8 H2O)

[α]D of 171 25.0° (c 0.5 H2O) 18.8° (c 0.5 H2O)

Figure 46. 1H and


96

Figure 47. 1H and


4.4 Inhibition of a mammalian histidine kinase by the free phosphohistidine

analogues

The roles of histidine kinases and histidine-phosphorylated proteins in mammalian cells

are poorly understood, except for one well characterised mammalian histidine kinase,

NDPK.17

Although the phosphorylation of histone H4 is associated with regeneration in

liver following injury and with hepatocellular carcinoma, it has not been established

whether cause or effect defines this relationship.18

However, inhibition of histidine

kinases‟ activity could provide some insight into their functions, and, in particular,

inhibition of the histone H4 histidine kinase associated with hepatocellular carcinoma18

could be of great clinical importance.8 Accordingly, the inhibitory activity of the free

97

phosphohistidine analogues 170 and 171 against transfer of a phosphoryl group from

[32

P]ATP to histone H4 by a guinea pig liver histidine kinase was measured. The

“kinase” has not been fully characterised but its ability to phosphorylate a histone H4

histidine has been reasonably well investigated.34

Histones are proteins that are

intimately associated with DNA, helping to store it in compact form as chromatin, and

are also involved in the regulation of gene expression.

In these experiments, solutions containing histone as substrate, the guinea pig liver

histidine kinase and [32

P]ATP were incubated with various concentrations of the

analogues. The positive control did not contain the phosphohistidine analogue, and the

phosphorylation rate should be the highest in these samples. The negative controls had

either no histone or no histidine kinase, and no phosphorylation should be observed

(Table 12).

Table 12

Component Assay NaBr control Positive control Negative controls

170 or 171 + – – –

histone + + + + [–]

kinase + + + – [+]

Following incubation, the phosphorylated histone was separated by SDS-PAGE and

quantified with a phosphoimager. As shown in Figures 48 and 49, both of the free

phosphohistidine analogues 170 and 171 inhibited the phosphorylation relative to the

positive control. Unexpectedly, the phosphorylation was also inhibited by sodium

bromide (NaBr), which was included as another control to account for the fact that the

inhibitors were hydrobromides. However, the inhibitory effects of the inhibitors are

greater than that of NaBr.

98

Figure 48. Solutions of varying concentrations of the „free‟ phosphohistidine analogue

170 or sodium bromide (10 mM) were added to a reaction mixture containing histone

(500 g/mL), radiolabeled ATP* (0.2 mM) and a guinea pig liver histidine kinase (200

g/mL) in 0.1 M tris buffer (pH 9.0). *[32

P] ATP 3000 Ci/mmol

99

Figure 49. Solutions of varying concentrations of the „free‟ phosphohistidine analogue

171 or sodium bromide (10 mM) were added to a reaction mixture containing histone

(500 g/mL), radiolabeled ATP* (0.2 mM) and a guinea pig liver histidine kinase (200

g/mL) in 0.1 M tris buffer (pH 9.0). *[32

P] ATP 3000 Ci/mmol

100

These results indicate that the free phosphohistidine analogues 170 and 171 could be

mammalian histidine kinase inhibitors. Owing to time constrains, it remains to repeat

these experiments to determine the IC50 values and to determine what type of inhibition

is occurring, i.e., competitive or non-competitive, with respect to both of the substrates.

Although these results are promising, it may be better to conduct similar experiments on

a well-characterised histidine kinase, such as NDPK or a two-component histidine

kinase like EnvZ or CheA, as the identity of the histone H4 kinase has not yet been

confirmed. Indeed, compounds related to 170 and 171 may assist in the conclusive

confirmation that the guinea pig enzyme used in this study is a histidine kinase. The last

and crucial step in this identification is the purification of this putative histidine kinase.

The commercial sample is not pure, and purification has been challenging.34

As it seems

apparent that 170 and 171 bind to the enzyme, their derivatives may prove useful for

purification by affinity chromatography, as detailed in the next section.

101

Chapter 5

Preparation for Solid-Phase Peptide Synthesis

and Affinity Chromatography

102

5.1 Introduction to solid-phase peptide synthesis

Solid-phase peptide synthesis (SPPS) has been highly useful for producing peptides

with specific sequences since it was first reported a few decades ago.92

The synthetic

peptides can be used for a variety of purposes such as haptens and synthetic vaccines.92

Merrifield and co-workers developed SPPS using NBoc-protected amino acids, as

outlined in Scheme 62.93

An NBoc-protected amino acid is usually attached to a

crosslinked polystyrene resin.92

The Boc protecting group is removed by TFA, followed

by neutralisation of the resultant ammonium group with triethylamine.92

Peptide

coupling of a second Boc-protected amino acid and the resin-bound amino acid is

accomplished using a carbodiimide activating agent such as dicyclohexylcarbodiimide

(DCC).92

The cycle of reactions is repeated as required, and then the synthesised peptide

is usually cleaved from the resin with anhydrous HF.92

ResinO

O

BocN

R1

H 1. TFA

2. Et3N

ResinO

O

H2N

R1

OH

O

BocN

R2

H

CN N

ResinO

OHN

R1O

R2

BocNH

OH

OHN

R1O

R2

FH3N

HF

R3 R4

Scheme 62. Boc SPPS

Although Boc SPPS is fast, and cost of the reagents is relatively low, it does have

several disadvantages.92

The final side-chain functional group deprotection and cleavage

from the resin uses anhydrous HF, requiring special equipment and making the process

103

potentially hazardous.92

In addition, both deprotection and cleavage from the resin can

be achieved using acids, and it follows that the Boc SPPS lacks orthogonality.92

In order to overcome these problems, Fmoc SPPS has been developed (Scheme 63).92

An Fmoc-protected amino acid is usually attached to a resin with 4-

hydroxymethylphenoxy substitution.92

The Fmoc protecting group is base-labile, which

enables the N-terminal α-amino group to be revealed by piperidine.92

Most importantly,

the final cleavage from the resin and deprotection of side-chain functional groups can be

accomplished using TFA, which is vastly safer than hydrofluoric acid.92

Fmoc SPPS

utilises a base for the deprotection and an acid for the final cleavage and side-chain

deprotection, and therefore it possesses orthogonality.92

O ResinO

O

FmocN

R1

H piperidineO Resin

O

O

H2N

R1

OH

O

FmocN

R2

H

O ResinO

OHN

R1O

R2

FmocNH

OH

OHN

R1O

R2

H2N

O ResinO

OHN

R1O

R2

H2N

TFA

piperidine

N

N

N

ON

PF6

Scheme 63. Fmoc SPPS

104

5.2 SPPS by the Muir and Webb groups

As described in the introduction, during the completion of the research described in this

thesis, the Boc-protected N1- and N3-phosphohistidine analogues 35 and 36, and the

Fmoc derivative of the N3-analogue 47 were synthesised and applied the to SPPS by the

Muir40

and Webb43

research groups, respectively (Figure 50).

BocNOH

OH

N

N N

P

O

OEt

OEt

BocNOH

OH

N NN

PO

OEtEtO

FmocNOH

OH

N

N N

P

O

OEt

OEt

35 36 47

Figure 50. Peptide precursors synthesised by the Muir and Webb groups

Comparing these two methods, Muir‟s strategy revealed the phosphonic acid during

cleavage of the synthesised peptide from a resin using anhydrous HF,40

whereas Webb‟s

strategy required an extra step to deprotect the phosphonic acid using trimethylsilyl

bromide after the synthesised peptide had been detached from a resin.43

However, the

latter method is safer and more convenient than the former in that usage of anhydrous

HF can be avoided.

5.3 Synthesis of Fmoc derivatives of N1- and N3-phosphohistidine analogues

The plan in the current project was to synthesise Fmoc derivatives of N1- and N3-

phosphohistidine analogues and incorporate them into peptides using Fmoc SPPS. The

synthesis began with commercially available Fmoc-L-serine monohydrate 177, and the

carboxylic acid was protected as the benzyl ester 17894

in excellent yield (Scheme 64).81

A Mitsunobu reaction converted the alcohol 178 to the novel azide 179 in acceptable

yield.

105

FmocN

O

O

N3

H

FmocN

O

OH

OH

HFmocN

O

O

OH

H

DIPEA

DMF

HN3

DEAD

PPh3

THF

Br

177

159

178 17990%

65%


Cycloadditions of the azide 179 and alkyne 82 were conducted under a variety of

conditions (Scheme 65). The thermal reaction resulted in a 1:1 mixture of the 1,5-(180)

and 1,4-(181) triazoles, and the regioisomers were easily purified by flash

chromatography (Table 13). While the exclusive formation of the 1,4-triazole 181 was

accomplished in excellent yield using copper(I)-catalysis (Table 13), the 1,5-triazole

180 was the sole isomer furnished by the ruthenium(II)-catalysed reaction (Table 13).

FmocN

O

OBn

N3

HFmocN

OBn

OH

N

N N

P

O

OEt

OEt

FmocNOBn

OH

N NN

PO

OEtEtO

P H

O

EtO

EtO

+conditions

179

82

180 181



ºC

Table 13. Yields of Azide-Alkyne cycloadditions under various conditions


180 39% – 78%

181 41% 85% –

106

Figure 51. 1H NMR spectrum of 180

Figure 52. 1H NMR spectrum of 181

Although time did not permit any further synthetic work in this area, the peptide

precursors 182 and 47 for Fmoc SPPS can be obtained by hydrogenolysis of 180 and

181, respectively (Scheme 66). It should be noted that Fmoc chemistry of the N1-

phosphohistidine analogue 182 has not been reported.

107

FmocN

O

OBn

NN

N

PO

OEtEtO

H

H2/Pd

MeOH

FmocN

O

OH

NN

N

PO

OEtEtO

H

180 182

FmocNOBn

OH

N

N N

P

O

OEt

OEt

181

H2/Pd

MeOH

FmocNOH

OH

N

N N

P

O

OEt

OEt

47

Scheme 66. Proposed synthesis of SPPS precursors 182 and 47.

Although Webb and co-workers published the synthesis of a short peptide using the N3-

phosphohistidine analogue 47,43

there is still scope for improvement. The ethyl

phosphonate was not able to be cleanly cleaved under the deprotection conditions,

giving a mixture of free phosphonic acid, ethyl phosphonate and diethyl phosphonate,

which reduced the yield of the target peptide.43

In SPPS of peptides containing

phosphoamino acids, phosphonic and phosphoric acids are usually protected as dibenzyl

or di-tert-butyl phosphonates, because they can be removed easily by treatment with

TFA during cleavage of the synthesised peptides from a resin. In order to apply this

strategy to the current problem, the novel ethynylphosphonate 185 will need to be

synthesised (Scheme 67).

108

H TMS

EtMgBr

THF

P

O

O

OCl

P TMS

O

ButO

ButO

P H

O

ButO

ButO

K2CO3

H2O

184 18583

183

Scheme 67. Proposed synthesis of the novel ethynylphosphonate 185

The Grignard reagent formed by treatment of TMS-acetylene 83 with an excess of

ethylmagnesium bromide should react with di-tert-butyl chlorophosphate 183,95

affording the protected ethynylphosphonate 185 after treatment of the intermediate 184

with a weak base. The phosphonate 185 would be subjected to cycloadditions with the

azide 179 to afford the triazoles 186 and 187, hydrogenolysis of which would provide

the SPPS precursors 188 and 189, respectively.

FmocNOBn

O

N3

HFmocN

OBn

O

N

H

N

N

FmocNOBn

O

N

H

N N

P

O

OBut

OBut

PO

OButButO

+

Conditions

185

186 187

179

H2/Pd

FmocNOH

O

N

H

N

N

FmocNOH

O

N

H

N N

P

O

OBut

OBut

PO

OButButO

+

188 189

Scheme 68. Proposed cycloadditions to furnish SPPS precursors 188 and 189

109

5.4 Preparation for affinity chromatography

There are commercially available affinity chromatography resins carrying immobilised

phosphoserine, phosphotyrosine or phosphothreonine, which can be utilised for

convenient and rapid purification of particular proteins that bind to the phosphoamino

acids. The experiments described in Chapter 4 suggested that there is an affinity

between the free phosphohistidine analogues 170 and 171 and the histidine kinase,

implying that an affinity resin with the covalently bound phosphonotriazololylalanines

could be of great value for purification/isolation of this and other histidine kinases and,

potentially, signaling proteins that recognise phosphohistidine residues.

The Fmoc derivatives 180 and 181 described in this chapter may prove useful in

preparing affinity chromatography resins. It remains to remove the benzyl and ethyl

protecting groups to obtain the precursors 190 and 191 for affinity chromatography

carrying the phosphohistidine analogues (Scheme 69). Deprotection using hydrogen

bromide in acetic acid has been dependable throughout this project, and there is

literature precedent suggesting that fluorenylmethyl carbamates are stable under these

conditions.96

FmocN

O

OBn

N

N N

P

O

OEtOEt

H

FmocN

O

OBn

NN

N

PO

OEtEtO

H

HBr

AcOH

FmocN

O

OH

N

N N

P

O

OHOH

H

FmocN

O

OH

NN

N

PO

OHHO

H

HBr

AcOH

180

181

190

191

Scheme 69. Proposed synthesis of the precursors for affinity chromatography

110

It should be possible to couple the precursors 190 and 191 to a resin through an amide

linkage, followed by removal of the Fmoc protecting group with piperidine to reveal the

amino group (Scheme 70). The haptens described in Chapter 2 may also prove useful

for affinity chromatography. Unfortunately, there was insufficient time to explore these

possibilities.

HN

HNH2N

Agarose

FmocN

O

OH

N

N N

P

O

OHOH

HFmocN

O

OH

NN

N

PO

OHHO

H

FmocN

O

NH

H

NH

NH

FmocN

O

NH

H

NH

NH

N

N NP

O

HOHO

NN

N

PO

OHHO

H2N

O

NH

NH

NH

H2N

O

NH

NH

NH

N

N NP

O

HOHO

NN

N

PO

OHHO

20% piperidine inDMF

190 191

192

194193

196195

Scheme 70. Proposed strategy to prepare affinity chromatography

111

Chapter 6

Attempted Synthesis of Pyrazole and Imidazole Analogues of

Phosphohistidine

112

6.1 Attempted synthesis of a phosphonopyrazole analogue of N3-phosphohistidine

The extra nitrogen atom in the triazoles described in this thesis does lower the basicity

of the heterocycle relative to histidine. Thus, in an attempt to more closely match the

pKa of phosphohistidine, which may be important in antibody recognition, a synthesis of

phosphonopyrazole and phosphonoimidazole analogues has been pursued.

The initial plan to reach the target analogue 199 involved synthesising the

phosphonopyrazole 197 and subjecting it to a displacement reaction with a serine-

derived tosylate 198 (Scheme 71).

N

NHPEtO

EtO

O

RNOR'

O

OTs

H

RNOR'

O

N

H

N

P

O

OEt

OEt

197 199

198

Scheme 71. Plan to synthesise a pyrazole N3-phosphohistidine analogue

6.1.1 Synthesis of pyrazolylphosphonates

The starting point for the synthesis was iodination of pyrazole 200 to give 4-

iodopyrazole 20197

in excellent yield. An initial attempt to install the phosphono group

used a Nickel(II)-catalysed Arbuzov reaction55

of the unprotected pyrazole 201 with

triethyl phosphite (Scheme 72). Unfortunately, no reaction was observed, which

prompted us to protect the pyrazole nitrogen as the p-toluenesulfonamide 203.98

Nevertheless, as judged by the 1H NMR and ESI-MS spectra of the crude product, the

phosphorylation reaction of 203 was unsuccessful, although 203 was completely

consumed (Scheme 72).

113

N

NH

N

NHI

CAN

I2

MeCN

N

NHP

O

EtO

EtO

P(OEt)3

NiCl2

N

NTsI

TsCl

pyridine

DCM

N

NTsP

O

EtO

EtO

P(OEt)3

200 201 202

203 204

93%

63%

150 oC

NiCl2 150 oC

Scheme 72. Attempted Arbuzov reactions to furnish phosphonopyrazoles

The third attempt to synthesise the phosphonate 204 used iodine-magnesium exchange

of the 203 with isopropylmagnesium bromide,99

followed by quenching with diethyl

chlorophosphate 80. 1H NMR and ESI-MS spectra of the crude product indicated the

perfect consumption of 203, but there was no trace of the desired phosphonate 204

(Scheme 73).

N

NTsI

N

NTsP

O

EtO

EtO

iPrMgBr

THF

ClP

O

EtO

EtO

203 204

80

Scheme 73. Attempted iodine-magnesium exchange to give the phosphonopyrazole 204

Because of the difficulties above, another strategy to obtain the phosphonate 204 was

investigated. There is literature precedent in which copper(I)-catalysed coupling of aryl

halides 205 and dibutyl phosphate 206 proceeded in excellent yields (Scheme 74).100

114

I

R CuI

Cs2CO3

H2NNH2

HP

O

BuO

BuO

206 P

R

O

OBu

OBu

PhMe

110C

205 207

R Yield

OMe 85%

NH2 86%

Scheme 74. Precedent for Cu(I)-catalysed C-P bond formation100

According to the procedures,100

Cu(I)-catalysed coupling of the iodides 201 or 203 and

diethyl phosphite 79 was attempted; however, no reaction was observed in the case of

iodopyrazole 201, and only the reduced compound was produced with the N-tosyl

derivative 203 (Scheme 75).

N

NHI

N

NHP

O

EtO

EtO

CuI

HP

O

EtO

EtO

Cs2CO3

H2NNH2

N

NTsI

HP

O

EtO

EtO

CuI

Cs2CO3

H2NNH2

201

N

NTsP

O

EtO

EtO

202

204203

79

79

110 oC

110 oC

Scheme 75. Attempted Cu(I)-catalysed coupling of iodopyrazoles and diethyl phosphite

115

Finally, palladium-catalysed coupling101

of the iodide 203 and diethyl phosphite 79

gave the target phosphonate 204 in acceptable yield (Scheme 76). Historically, N-

detosylation of aromatic heterocycles has been conducted by using a base, such as

potassium hydroxide, in methanol.102

However, the formation of toxic methyl p-

toluenesulfonate is problematic under these conditions.103

The use of aqueous THF with

a phase transfer catalyst in place of methanol can prevent toxic alkyl p-

toluenesulfonates from being generated.102

Detosylation was achieved, in this case, by

heating with potassium hydroxide in aqueous dioxane, providing the target

phosphonopyrazole 202 in good yield, without the need for the phase transfer catalyst.

The unprotected pyrazole 201 also underwent the palladium(II)-catalysed coupling101

with diethyl phosphate 79, furnishing the phosphonopyrazole 202 in acceptable yield

(Scheme 76). Despite more troublesome purification using this strategy, the yield of the

phosphonate was ameliorated compared with the two step procedure.

N

NTsI

N

NTsP

O

EtO

EtO

PPh3

Et3N

HP

O

EtO

EtO

Pd(OAc)2

EtOH

N

NHP

O

EtO

EtO

KOH

Dioxane

H2O

N

NHI

N

NHP

O

EtO

EtO

HP

O

EtO

EtO

Pd(OAc)2

PPh3

Et3N

EtOH

203 204 202

202201

79

79

50%

53%

47%

Scheme 76. Synthesis of the phosphonopyrazole 202

116



6.1.2 Attempted synthesis of protected pyrazolylalanines

Endeavours to synthesise pyrazole analogues of N3-phosphohistidine according to the

plan described in Scheme 71 were made. Displacement reactions104

of the known

tosylate 208,105

with the phosphonopyrazole 202 were initially attempted (Scheme 77).

In the presence of bases, elimination of the leaving group was observed instead of the

117

desired SN2 reaction, and the dehydroalanine derivative 210 was the sole product. In the

absence of bases, no reactions proceeded even at elevated temperatures.

N

NHP

O

EtO

EtO

BocNOMe

OH

N

N

P

O

OEt

OEt


1) NaOH, DMF or MeCN, r.t.

2) K2CO3, DMF or MeCN, r.t.

3) reflux in DMF or MeCN

BocNOMe

OH

BocNOMe

OH

OH

BocNOMe

OH

OTs

TsCl, pyridine, DCM

70%

59 208

209

210

202

Scheme 77. Attempted displacement reactions of phosphonopyrazole 202 and tosylate

208

The failure of these reactions is somewhat surprising given the precedent indicating that

the mesylate 211 was displaced by azide to furnish azidoalanine 212 (Scheme 78).104

BocNOBn

OH

OMs

BocNOBn

OH

212211

NaN3

DMF

40 oC N3

73%

Scheme 78. Precedent for a displacement reaction of mesylate 211 and sodium azide

Owing to the setbacks outlined above, another approach to prepare pyrazole analogues

of N3-phosphohistuidine was investigated. In ring-opening reactions of -lactones such

as 213, the H2C–O bond is cleaved by soft nucleophiles to afford the -functionalised

118

carboxylic acids 214, whereas the carbonyl is attacked by hard nucleophiles to yield the

-hydroxycarbonyl derivatives 215 (Scheme 79).106

O

RNH

O

SoftNucleophiles

HardNucleophiles

RNOH

OH

Nu

RNNu

OH

OH

213

214

215

Scheme 79. Ring opening of the -lactones 213

There is precedent in which the H2C–O bond of the -lactone 216 was cleaved by

pyrazole 200, forming the protected pyrazolylalanine 217 in reasonable yield (Scheme

80).107

The second attempt to synthesise a pyrazole analogue of N3-phosphohistidine

therefore mimicked this approach.

O

CbzNH

O

N

NH

CbzNOH

O

N

H

NMeCN, 50

C

71%200

217

216

Scheme 80. Reported ring opening of a serine-derived -lactone with pyrazole107

The required -lactone 218 was prepared in acceptable yield by exposing NBoc-serine

172 to Mitsunobu conditions (Scheme 81).108

In spite of the promising precedent, no

trace of the desired N-alkylpyrazole 219 was generated under several conditions106-109

119

(Scheme 81). Although the -lactone 218 was completely consumed, what occurred is

still unclear. It is possible that the reduced nucleophilicity of 202 relative to pyrazole

200, owing to the electron-withdrawing phosphoryl group, contributed to the

unexpected result, with decomposition of -lactone 218 occurring more rapidly than the

desired reaction.

BocNOH

OH

OH

DEAD

PPh3

THF

O

OBocNH

BocNOH

OH

N

N

P

O

OEt

OEt

N

NHP

O

EtO

EtO


1) MeCN or DMF, 50 oC

2) K2CO3, MeCN or DMF, 50 oC

3) Cs2CO3, MeCN or DMF, 50 oC

4) NaH, MeCN or DMF, 50 oC

202

45%172

218

219

Scheme 81. Attempted ring opening reactions of the -lactone

Most recently, a Mitsunobu reaction was chosen for the purpose of making the pyrazole

analogue, because of the report of a conventional Mitsunobu reaction of 4-nitropyrazole

220 and the alcohol 221 (Scheme 82).110

Presumably, this reaction is only possible

because of the enhanced acidity of the pyrazole NH due to the electron-withdrawing

nitro group. It was felt that the phosphoryl group in 202 might similarly lower the pKa

sufficiently for a Mitsunobu reaction to be viable.

120

NNH

O O

OH

DEAD

PPh3

THF

69%

O O

NN

NO2

NO2

220

221

222

Scheme 82. An example of a Mitsunobu reaction of a pyrazole.110

When conventional Mitsunobu conditions were applied to the alcohol 59 and

phosphonopyrazole 202, the reaction did not proceed as anticipated, probably because

the pKa of the phosphonopyrazole is not low enough (Scheme 83).

N

NHP

O

EtO

EtO

BocNOMe

OH

OH

BocNOMe

OH

N

N

P

O

OEt

OEtDEAD

PPh3

THF

202

209

59

Scheme 83. Unsuccessful Mitsunobu reaction of diethyl 4-pyrazolylphosphonate

Tsunoda and co-workers proved that a combination of either azodicarboxylic acid

dipiperidide (ADDP, 223)111

or N,N,N′,N′-tetramethylazodicarboxamide (TMAD)112

and

tributylphosphine (PBu3) is a powerful tool for Mitsunobu reactions of alcohols and

weak acids with pKa values higher than 11.111,112

Therefore, in the current work, ADDP

223 was prepared by treating DEAD 62 with piperidine (Scheme 84).113

Due to time

constraints, the Mitsunobu reaction employing ADDP 223 and PBu3 (Scheme 85) was

not attempted, but is planned for future work.111

121

N N O

OO

O N N N

OO

N

62 223

NH

70%

Et2O

Scheme 84. Synthesis of ADDP

N

NHP

O

EtO

EtO

BocNOMe

OH

OH

BocNOMe

OH

N

N

P

O

OEt

OEt

ADDP

PBu3

THF

202

209

59

Scheme 85. Proposed Mitsunobu reaction using ADDP and PBu3

6.2 Imidazole analogues of N1-phosphohistidine

Imidazole analogues of N1-phosphohistidine have also been pursued. The synthesis

began with the preparation of 4(5)-iodoimidazole 226, via the triiodide 225, as reported

previously (Scheme 86).114

Surprisingly, the palladium-catalysed phosphonation101

that

worked well in the synthesis of the phophonopyrazoles 202 and 204 was ineffective for

the preparation of the target phosphonoimidazoles 228 and 230, although ESI-MS

spectra of the crude products indicated the perfect consumption of the starting materials

(Scheme 87).

122

NHN NHN

I

I I

NHN

I

I2

KI

NaOH

H2O

Na2SO3

EtOH

H2O

60% over two steps

NHN

I

224 225 226 227

Scheme 86. Synthesis of 4(5)-iodoimidazole

HP

O

EtO

EtO

Pd(OAc)2

PPh3

Et3N

EtOH

NHN

I

NHN

PO

OEtOEt

226

79

228

NTsNNTsN

229 230

I

TsCl

Et3N

THF

70%

PO

EtOOEt

HP

O

EtO

EtO

Pd(OAc)2

PPh3

Et3N

EtOH

79

Scheme 87. Attempted phosphonation of 4(5)-iodoimidazole

Although time did not permit any further synthetic work in this area, alternative

strategies to synthesise the novel phosphonoimidazole have been considered. Several

articles reported that iodine-magnesium exchange of N-protected iodoimidazoles

proceeded smoothly in DCM despite Grignard reactions normally being performed in

etheral solvents.115,116

It is proposed that 4-iodo-1-p-toluenesulfonylimidazole 229114

may undergo such iodine-magnesium exchange, and quenching the reaction with diethyl

123

chlorophosphate 80 should provide the protected phosphonoimidazole 230 (Scheme

88). The tosyl protecting group could then be removed by potassium hydroxide in

aqueous dioxane to afford the desired phosphonoimidazole 228.

NTsN

I

ClP

O

EtO

EtO

iPrMgBr

DCMNTsN

PO

EtOOEt

NHN

PO

EtOOEt

KOH

Dioxane

H2O229

230 228

80

Scheme 88. Proposed route to the imidazolylphosphonate 228

A Mitsunobu reaction utilising a combination of ADDP and PBu3 could potentially

furnish an imidazole analogue of N1-phosphohistidine 231; however, it is likely to be

complicated by competing formation of the 1,5-substituted imidazole 232 (Scheme 89).

More likely, an alternative approach will be required.

NHN

PO

EtOOEt

BocNOMe

OH

OH

BocNOMe

OH

NN

ADDP

PBu3

THF

BocNOMe

OH

NN

PP

O

EtOOEt

O

OEtEtO

+

228

231 232

55

Scheme 89. Challenging Mitsunobu reaction for the imidazole analogue 231

124

Conclusion

The primary aim of the research described in this thesis was to synthesise a series of

haptens, based on stable N1- and N3-phosphohistidine analogues, to be utilised for

generation of generic phosphohistidine antibodies. The first goal, the synthesis of

haptens incorporating triazolylalaninephosphonates, was achieved. Several strategies to

conjugate the haptens to carrier proteins were attempted, but none have been successful

to date. However, it is likely that with further experimentation, bioconjugation will be

achieved and antibody generation will ensue soon after.

Towards the conclusion of the work described herein, virtually identical research was

published by other groups,40,43

including the generation of polyclonal antibodies that

specifically recognise histidine-phosphorylated histone H4.40

However, the antibodies

do not recognise other histidine-phosphorylated proteins,40

and therefore we are still in

pursuit of generic phosphohistidine antibodies.

The biological activities of the phosphohistidine analogues synthesised in this project

were of particular interest, and accordingly, the free triazolylalaninephosphonate

analogues of N1- and N3-phosphohistidine have been synthesised. In preliminary

experiments, the analogues were found to inhibit a mammalian histidine kinase. This

observation may be the starting point for the discovery of more potent histidine kinase

inhibitors with potential therapeutic use. In addition, these inhibitors may be useful for

affinity chromatography to allow purification/isolation of histidine kinases and,

potentially, other signaling proteins that recognise histidine-phosphorylated proteins.

125

Chapter 7

Experimental

126

General details

All solvents were distilled prior to use; anhydrous solvents and reagents were distilled

under N2. THF for organometallic reactions was distilled from sodium benzophenone

ketyl. All reaction temperatures refer to bath temperatures. Organic extracts were dried

over anhydrous MgSO4 and then filtered. Solvents were evaporated at vacuum or

under a stream of N2.

Flash chromatography was conducted with Merck silica gel 60. Analytical TLC was

performed on Whatman flexible plates (250 m layer, silica gel 60 F254). Spots were

visualised under UV light and by staining with ammonium molybdate or ninhydrin.

Melting points were measured on a Kofler hot stage melting point apparatus and are

uncorrected. IR spectra were acquired using a Perkin-Elmer Spectrum One FTIR

spectrometer. Mass spectra were acquired on an Applied Biosystems QSTAR pulsar I

quadrupole time-of-flight instrument or a Waters LCT Premier XE spectrometer.

Histone phosphorylation was quantified using a Fuji Photo Film BAS-III

phosphoimager. NMR spectra were acquired on Varian Inova 300 (300 MHz, 1H; 75.5

MHz, 13

C; 120 MHz, 31

P), Varian 400 (400 MHz, 1H; 100 MHz,

13C), Bruker Avance

500 (500 MHz, 1H; 125 MHz,

13C) or Bruker AV600 (600 MHz,

1H; 150 MHz,

13C;

240 MHz, 31

P) spectrometers, as indicated. Chemical shifts are expressed in ppm,

relative to CHCl3 (1H, 7.26 ppm), CDCl3 (

13C, 77.0 ppm), CD3SOCD2H (

1H, 2.50

ppm), (CD3)2SO (13

C, 39.5 ppm), CHD2OD (1H, 3.30 ppm), CD3OD (

13C, 49.0 ppm),

and 85% H3PO4 (external capillary, 31

P, 0 ppm), as appropriate. Routine assignments

of 13

C NMR spectra were made with the assistance of DEPT 135 and DEPT 90

experiments.

127

HPLC

HPLC was conducted using a Hewlett Packard 1050 HPLC system equipped with a

multiple wavelength detector (MWD) and a 250 × 10 mm i.d., 5 µm, Apollo C18

reversed-phase column (Grace-Davison), with a 33 mm × 7 mm guard column of the

same material. The samples were eluted at 4 mL/min with 30% (v/v) MeCN-water.

UV absorbance was measured at 220 nm.

Enantioselective chromatography of the carboxylic acids 35 and 36 was carried out

using a 250 × 4.6 mm i.d., 5 µm Chiracel OD-H column (Diacel), eluted at 1.0 mL/min

with 8% isopropanol/hexanes, and detection at 220 nm.

~Chapter 2~

ClH3NOMe

O

OH

(S)-3-Hydroxy-1-methoxy-1-oxopropan-2-aminium chloride (58)

This known compound was synthesised according to the method of Rudd.45

Thionyl chloride (8.70 mL, 121 mmol) was added dropwise to a suspension of L-serine

(12.5 g, 120 mmol) in MeOH (240 mL) at 0 ºC. The resulting mixture was allowed to

warm to room temperature and stirred for 18 h. The MeOH was evaporated, and the

residue was washed with Et2O. The solid was recrystallised from MeOH to afford the

title hydrochloride 58 as a white solid (13.5 g, 74 %). 1H NMR (500 MHz, CD3OD):

4.10-4.15 (m, 1H, -H), 3.90-4.00 (m, 2H, -H), 3.81 (s, 3H, OCH3). 13

C NMR (125

MHz, CDCl3): δ 169.3 (CO), 60.6 (), 56.1 (OCH3), 53.8 (). MS (ESI) m/z: 120

[M+H]+

(where M = free base). []D = +4.1 (c 1.0, MeOH) [lit.45

+3.9 (c 4.05,

MeOH)].

128

BocNOMe

OH

OH

(S)-Methyl-2-(tert-butoxycarbonylamino)-3-hydroxypropanoate (59)

This known compound was synthesised according to the method of Rudd with slight

modifications.45

Boc anhydride (7.00 g, 32.1 mmol) was added to a suspension of the hydrochloride 58

(5.00 g, 32.1 mmol) and triethylamine (13.4 mL, 96.1 mmol) in MeCN (100 mL) at 0

ºC. The resulting mixture was allowed to warm to room temperature and stirred for 6 h,

and then the MeCN was evaporated. The residue was diluted with sat. aq. KHSO4 and

then extracted with DCM (3 × 50 mL). The organic extract was washed with sat. aq.

NaHCO3 (50 mL) and brine (50 mL), dried and concentrated under reduced pressure.

The crude product was subjected to flash chromatography. Elution with 2:3

EtOAc/hexanes gave 59 as a pale yellow oil (6.37 g, 90%). 1H NMR (500 MHz,

CDCl3): 5.62 (br d, J = 7.0 Hz, 1H, NH), 4.28 (m, 1H, -H), 3.88 (m [appearent br d],

1H, -Ha), 3.77 (dd, 1H, J = 11.0, 3.5 Hz, -Hb), 3.69 (s, 3H, OCH3), 1.37 (s, 9H, t-

Bu). 13

C NMR (125 MHz, CDCl3): δ 171.4 (CO) 155.7 (NCO), 80.0 [OCCH3)3], 62.9

(), 55.6 (OCH3), 52.3 (), 28.1 [CCH3)3]. MS (ESI) m/z: 242 [M+Na]+. []D = +10.1

(c 1.0, CH3Cl) [lit.117

+9.7 (c 4.29, CH3Cl)].

Preparation of a 2.5 M hydrazoic acid (HN3) solution.118

Concentrated sulfuric acid (3.60 mL) was added dropwise to a suspension of sodium

azide (8.45 g, 130 mmol) in toluene (51.5 mL) and water (4.20 mL) at 0 ºC. The

resulting mixture was stirred for 1 h at 0 ºC, and then the precipitated Na2SO4 was

filtered off. The obtained HN3 solution was immediately used for a Mitsunobu reaction.

129

NH

NH

O

OO

O

Ethyl hydrazodicarboxylate (70)

This known compound was synthesised according to the methods of Wang.119

Ethyl chloroformate (4.60 mL, 48.4 mmol) was added dropwise to a solution of

hydrazine monohydrate (1.80 mL, 37.2 mmol) at 0 ºC. After the addition, a solution of

Na2CO3 (5.30 g, 50.0 mmol) in water (40 mL) was added to the mixture simultaneously

with ethyl chloroformate (4.60 mL, 48.4 mmol) at 0 ºC. The resulting mixture was stirrd

at 0 ºC for 1 h, and then the precipitate was filtered, washed with cold water (30 mL),

EtOH (30 mL) and Et2O (30 mL) and dried under vacuum, affording 70 as a white solid

(3.93 g, 60%). 1H NMR (300 MHz, d4-DMSO): 9.00 (br s, 2H, NHCH2CH3), 4.06 (q,

J = 6.9 Hz, 4H, NHCH2CH3), 1.20 (t, J = 6.9 Hz, 6H, NHCH2CH3). 13

C NMR (75 MHz,

d4-DMSO): δ 156.9 (NCO), 60.9 (CH2), 14.8 (CH3).

N N O

OO

O

Diethylazodicarboxylate (62)

This known compound was synthesised according to the methods of Steel.120

Concentrated nitric acid (20 mL) was added dropwise to a suspension of 70 (3.00 g,

17.0 mmol) in CHCl3 (20 mL) at 0 ºC. The resulting solution was stirred at 0 ºC for 3 h

and then diluted with water (50 mL). The layers were separated, and the aqueous phase

was extracted with CHCl3 (3 × 60 mL). The organic extract was washed with water (50

mL), sat. aq. NaHCO3 (2 × 50 mL) and brine (50 mL), dried and concentreated under a

stream of N2, affording 62 as an orange liquid (2.07 g, 70%) in a sufficiently pure state.

1H NMR (300 MHz, CDCl3): 4.10 (q, J = 7.2 Hz, 4H, NCH2CH3), 1.01 (t, J = 7.2 Hz,

6H, NCH2CH3) 13

C NMR (75 MHz, CDCl3): δ 159.9 (NCO), 65.0 (CH2), 13.2 (CH3).

130

BocN

O

OMe

N3

H

(S)-Methyl-3-azido-2-(tert-butoxycarbonylamino)propanoate (60)

This compound was synthesised according to the method of Boger44

with slight

modifications.

A stirred solution of the alcohol 59 (3.61 g, 16.5 mmol) and triphenylphosphine (6.49 g,

24.7 mmol) in anhydrous THF (20 mL) at –78 ºC under N2 was treated with a 2.5 M

solution of HN3 in toluene (9.85 mL, 24.7 mmol) and DEAD (3.90 mL, 24.7 mmol).

The resulting solution was allowed to warm to room temperature. After 5 h the solution

was diluted with water (100 mL), the layers were separated and the aqueous phase was

extracted with Et2O (3 × 80 mL). The combined organic phase was washed with brine

(50 mL), dried and concentrated under reduced pressure. The residue was subjected to

flash chromatography. Elution with 1:4 Et2O/hexanes gave 7 as a colourless oil (2.90 g,

72 %). 1H NMR (600 MHz, CDCl3): 5.61 (br d, 1H, NH), 4.28 (m, 1H, -H), 3.55 (s,

3H, COOCH3), 3.49 (m [apparent br d], 2H, -H), 1.23 (s, 9H, t-butyl). 13

C NMR (150

MHz, CDCl3) 169.7, 154.6, 79.5, 77.2, 53.0, 51.9, 27.6. MS (ESI) m/z: 343 [M+Na]+.

[]D = +36.4 (c 2.0, CHCl3) [lit.48

+36.0 (c 0.6, CHCl3)]. The 1H NMR data are

similar to those reported.48

P

O

EtO

EtOCl

Diethyl chlorophosphate (80)

This known compound was synthesised according to the method of Steinberg.52

Triethylamine (1.10 mL, 7.90 mmol) was added to a solution of diethyl phosphite (8.60

mL, 67.0 mmol) in carbon tetrachloride (13 mL) at 0 ºC. The resulting solution was

allowed to warm up to room temperature, stirred for 18 h and then volatile components

131

were removed under reduced pressure. The residue was distilled under vacuum at 50 ºC,

giving 80 as a pale yellow liquid (9.77 g, 85%). 1H NMR (400 MHz, CDCl3): 4.10 (m,

4H, OCH2CH3), 1.33 (t, J = 5.2 Hz, 6H, OCH2CH3) 13

C NMR (100 MHz, CDCl3): δ

65.7 (d, JC,P = 7.4 Hz, CH2), 15.5 (d, JC,P = 8.2 Hz, CH3). 31

P NMR (120 MHz, CDCl3):

δ 5.35.

P TIPS

O

EtO

EtO

Diethyl (triisopropylsilyl)ethynylphosphonate (81)

Ethyl bromide (1.00 mL, 13.7 mmol) was added dropwise to a suspension of stirred Mg

turnings (416 mg, 17.0 mmol) in THF (10 mL) at 0 ºC under N2 and the resulting

mixture was heated at reflux for 30 min. The ethynylmagnesium bromide solution thus

prepared was added dropwise via canula to a solution of TIPS-acetylene (3.00 mL, 13.7

mmol) in THF (10 mL) at 0 ºC under N2. The reaction mixture was allowed to warm to

room temperature and stirring was continued for 1 h before being cooled to 0 ºC and

treated dropwise with diethyl chlorophosphate (2.20 mL, 15.2 mmol). The resulting

solution was allowed to warm to room temperature and after 1 h was diluted with sat.

aq. NH4Cl (30 mL) and extracted with Et2O (3 × 50 mL). The organic extract was

washed with brine (30 mL), dried and concentrated under reduced pressure, and the

residue was subjected to flash chromatography. Elution with 1:4 EtOAc/hexanes gave

81 as a pale yellow oil (3.49 g, 80%). Rf = 0.80 (1:1 EtOAc/hexanes). 1H NMR (600

MHz, CDCl3): 4.11 (m, 4H, OCH2CH3), 1.32 (dt, JH,P = 0.6, J = 7.1 Hz, 6H,

OCH2CH3), 1.03–1.10 (m, 21H, i-Pr). 13

C NMR (150 MHz, CDCl3): δ 106.4 (d, JC,P =

37.6 Hz, C2), 96.3 (d, JC,P = 269.4 Hz, C1), 63.0 (d JC,P = 5.4 Hz), 18.3 (i-Pr CH3), 15.9

(d, JC,P = 6.8 Hz, OCH2CH3), 10.7 (CSi). 31

P NMR (120 MHz, CDCl3): δ –7.38; MS

(ESI) m/z: 319 [M+H]+, 341 [M+Na]

+. This compound has been reported but not

characterised.121

132

P H

O

EtO

EtO

Diethyl ethynylphosphonate (82)

(Method A; Removal of TIPS)

A solution of 81 (1.00 g, 3.14 mmol) and KF (0.38 g, 6.5 mmol) in MeOH (10 mL) was

stirred for 2 h and then concentrated under reduced pressure. The residue was diluted

with water (50 mL) and extracted with Et2O (3 × 40 mL). The organic extract was

washed with brine (30 mL), dried and concentrated under reduced pressure. The residue

was subjected to flash chromatography. Elution with 1:1 Et2O/hexanes gave 82 as a pale

yellow oil (310 mg, 61%). 1H NMR (500 MHz, CDCl3): 4.04 (m, 4H, CH2), 3.00 (d,

JH,P = 13.5 Hz 1H, C), 1.24 (dt, JH,P = 0.5 J = 7.0 Hz, 6H, 2 × CH3). 13

C NMR (125

MHz, CDCl3): δ 88.1 (d, J = 50.6 Hz, CH), 73.8 (d, J = 289 Hz, CP), 63.1 (d, J = 5.5

Hz, OCH2CH3), 15.7 (d, J = 7.0 Hz, OCH2CH3). MS (ESI) m/z: 163 [M+H]+. The

1H

and 13

C NMR data are similar to those reported.43

(Method B; Grignard reaction using TMS acetylene)

The procedure reported by Mignani51

was slightly modified.


turnings (487 mg, 20.0 mmol) in THF (20 mL) at 0 ºC under N2 and the resulting


prepared was added dropwise via canula to a solution of TMS-acetylene (2.30 mL, 16.3






washed with brine (30 mL), dried and concentrated under reduced pressure. The

133

obtained residue was dissolved in 10% aq. Na2CO3 (50 mL), stirred for 18 h, then

diluted with sat. aq. NH4Cl (30 mL) and extracted with Et2O (3 × 50 mL). The organic

extract was washed with brine (30 mL), dried and concentrated under reduced pressure.

The residue was subjected to flash chromatography. Elution with 1:3 EtOAc/hexanes

gave 82 as a pale yellow oil (2.01 g, 77%), identical with the material described above.

P

O

EtO

EtO

OH

Diethyl 3-hydroxy-3-methylbut-1-ynylphosphonate (87)

(Method A: Grignard reaction)


turnings (416 mg, 17.0 mmol) in THF (10 mL) at 0 °C under N2 and the resulting


prepared was added dropwise via canula to a solution of the alkyne 86 (1.13 g, 13.4






washed with brine (30 mL), dried and concentrated under reduced pressure, and the


87 as a pale yellow oil (2.27 g, 77%). Rf = 0.20 (1:1 EtOAc/hexanes). 1H NMR (500

MHz, CDCl3): 3.44 (m, 4H, OCH2CH3), 2.33 (s, 1H, OH), 1.07 (s, 6H, CCH3), 0.69

(dt, JH,P = 1.0, J = 7.0 Hz, 6H, OCH2CH3). 13

C NMR (125 MHz, CDCl3): δ 83.1 (d, JC,P

= 4.5 Hz, C2), 72.7 [C(CH3)2], 72.4 (d, JC,P = 7.0 Hz, C1), 62.0 (d, JC,P = 5.9 Hz,

OCH2CH3), 29.2 (d, JC,P = 4.7 Hz, [C(CH3)2], 14.6 (d, JC,P = 6.8 Hz, OCH2CH3). 31

P

NMR (120 MHz, CDCl3): δ –5.22. MS (ESI) m/z: 243 [M+Na]+.

134

(Method B: Ni(II)-catalysed Arbuzov reaction)

A solution of the chloride 88 (1.00 g, 8.43 mmol) and NiCl2.5H2O (340 mg, 1.55

mmol) in triethylphosphite (5.00 mL, 29.2 mmol) was heated at reflux for 6 h, and

then the excess triethylphosphite was removed under reduced pressure. The residue

was diluted with water (50 mL) and extracted with EtOAc (3 × 50 mL). The organic

extract was washed with brine (50 mL), dried and concentrated under reduced

pressuere. The residue was subjected to flash chromatography. Elution with 2:3

EtOAc/hexanes gave 87 as a pale yellow oil (874 mg, 47%), identical in every respect

with the material described above.

BocNOMe

OH

N

N N

P

O

OEt

OEt

BocNOMe

OH

N NN

PO

OEtEtO

121 122

Cycloadditions of the azide 60 and alkyne 82

Thermal

A solution of the azide 60 (150 mg, 0.614 mmol) and the alkyne 82 (100 mg, 0.614

mmol) in toluene (5 mL) under N2 was heated at reflux for 5 h. The toluene was

evaporated and the residue was subjected to flash chromatography. Elution with 1:1

EtOAc/hexanes gave 121 as a pale yellow oil (53 mg, 21%), identical with the material

described below. Further elution with EtOAc gave 122 as a pale yellow oil (144 mg,

58%), identical with the material described below.

135

Ru(II)-catalysed

(S)-Methyl-2-(tert-butoxycarbonylamino)-3-(5-(diethoxyphosphoryl)-1H-1,2,3-

triazol-1-yl)propanoate (121)

Cp*RuCl(PPh3)2 (5 mg, 1.5 mol%) was added to a solution of the azide 60 (100 mg,

0.41 mmol) and alkyne 82 (67 mg, 4.1 mmol) in toluene (5 mL) under N2. The resulting

solution was stirred at 60 ºC for 24 h, then diluted with water (10 mL) and extracted

with EtOAc (3 × 30 mL). The extract was washed with brine (10 mL), dried and

concentrated under reduced pressure. The residue was subjected to flash

chromatography. Elution with 1:1 EtOAc/hexanes gave 121 as a pale yellow oil (93 mg,

56%). Rf = 0.15 (1:1 EtOAc/hexanes). IR (thin film) cm–1

: 3306 (NH), 1747 (m, C=O),

1715 (s, NC=O). 1H NMR (600 MHz, CDCl3): 7.95 (s, 1H, triazolyl), 5.69 (br d, J =

6.0 Hz, 1H, NH), 4.88–5.00 (m, 3H, -CH & -CH2), 4.17 (m, 4H, 2 × OCH2CH3),

3.76 (s, 3H, OCH3), 1.36 (s, 9H, t-Bu), 1.35 (m, 6H, 2 × OCH2CH3). 13

C NMR (125

MHz, CDCl3): δ 169.5 (CO2), 154.8 (NCO2), 140.0 (d, JC,P = 20.4 Hz, triazolyl CH),

126.7 (d, JC,P = 219.6 Hz, CP), 79.8 [OC(CH3)3], 63.4 (d, JC,P = 5.9 Hz, OCH2CH3a),

63.3 (d, JC,P = 5.8 Hz, OCH2CH3b), 53.2 (), 52.5 (OCH3), 50.4 (), 27.8 [C(CH3)3],

15.9 (d, JC,P = 4.4 Hz, OCH2CH3a), 15.8 (d, JC,P = 4.5 Hz, OCH2CH3b). 31

P NMR (240

MHz, CDCl3): δ 4.03. MS (ESI) m/z: 407 [M+H]+, 429 [M+Na]

+. HRMS (ESI):

observed, 407.1688, [C15H27N4O7P+H]+ requires, 407.1690. []D = –31.0º (c 1.7,

EtOAc). Further elution with EtOAc gave 122 as a pale yellow oil (35 mg, 21%),

identical with the material described below.

136

Cu(I)-catalysed

(S)-Methyl-2-(tert-butoxycarbonylamino)-3-(4-(diethoxyphosphoryl)-1H-1,2,3-


CuSO4.5H2O (30 mg, 0.120 mmol) was added to a solution of the azide 60 (300 mg,

1.23 mmol), the alkyne 82 (200 mg, 1.23 mmol) and sodium ascorbate (24 mg, 0.12

mmol) in 1:1 t-BuOH/water (6 mL). The resulting solution was stirred for 24 h, then

diluted with water (10 mL) and extracted with DCM (3 × 30 mL). The organic extract

was washed with brine (10 mL), dried and concentrated under reduced pressure. The

residue was subjected to flash chromatography. Elution with EtOAc gave 122 as a pale

yellow oil (429 mg, 88%). Rf = 0.05 (1:1 EtOAc/hexanes). IR (thin film) cm–1

: 3293

(NH), 1744 (m, C=O), 1716 (s, NC=O).1H NMR (600 MHz, CDCl3): 8.03 (s, 1H,

triazolyl), 5.78 (br d, J = 7.7 Hz, 1H, NH), 4.76 (dd, J = 13.5, 4.5 Hz, 1H, -Ha), 4.70

(dd, J = 14.0, 6.5 Hz, 1H, -Hb), 4.55 (m, 1H, -H), 3.99 (m, 4H, 2 × OCH2CH3), 3.59

(s, 3H, OCH3), 1.21 (s, 9H, t-Bu), 1.144 (t, J = 7.2 Hz, 3H, OCH2CH3a), 1.140 (t, J =

7.2 Hz, 3H, OCH2CH3b). 13

C NMR (150 MHz, CDCl3): δ 169.1 (CO2), 154.8 (NCO2),

136.7 (d, JC,P = 239 Hz, CP), 131.6 (d, JC,P = 32 Hz, triazolyl CH), 80.0 [OC(CH3)3],

62.59 (d, JC,P = 5.7 Hz, OCH2CH3a), 62.58 (d, JC,P = 5.9 Hz, OCH2CH3b), 53.3 (α),

52.5 (OMe), 50.1 (), 27.7 [C(CH3)3], 15.8 (d, JC,P = 6.5 Hz, OCH2CH3). 31

P NMR (240

MHz, CDCl3): δ 6.97. MS (ESI) m/z: 407 [M+H]+. HRMS (ESI): observed, 407.1696,

[C15H27N4O7P+H]+ requires, 407.1690. [D = –15.9º (c 2.2, EtOAc).

137

BocNNH

OH

NN

N

P

O

EtOOEt

(S)-tert-Butyl-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)-1-(methylamino)-1-

oxopropan-2-ylcarbamate (123)

33% Aqueous methylamine (2.40 mL, 25 mmol) was added to a solution of 121 (240

mg, 0.60 mmol) in MeOH (5 mL). The solution was stirred at room temperature for 4 h,

and then concentrated under reduced pressure. The residue was subjected to flash

chromatography. Elution with 1:20 MeOH/DCM gave 123 as a pale yellow oil (207 mg,

85%). Rf = 0.20 (1:20 MeOH/DCM). IR (thin film) cm–1

: 3307 (NH), 1716 (ester C=O),

1672 (amide C=O). 1H NMR (500 MHz, CD3OD): 8.11 (s, 1H, triazolyl), 5.12 (br d,

1H, J = 11.0 Hz, -H), 4.74 (m, 2H, -H), 4.25 (m, 4H, 2 × OCH2CH3), 2.75 (s, 1H,

NHCH3), 1.38, (t, 6H, J = 7.5 Hz, 2 × OCH2CH3), 1.33 (s, 9H, t-Bu). 13

C NMR

(125MHz, CD3OD): δ 171.5, 157.2, 141.6 (d, JCP = 20.8 Hz, triazolyl CH), 127.9 (d, JCP

= 222 Hz, CP), 80.9, 65.5 (d, JC,P = 5.8 Hz, OCH2CH3a), 65.4 (d, JC,P = 5.9 Hz,

OCH2CH3b), 55.7, 52.3, 28.6, 26.5, 16.54 (d, JC,P = 2.9 Hz, OCH2CH3a), 16.49 (d, JC,P

= 3.0 Hz, OCH2CH3b). 31

P NMR (120 MHz, CD3OD): δ 4.74. MS (ESI) m/z: 406

[M+H]+, 428 [M+Na]

+. HRMS (ESI): observed, 428.1673, [C15H28N5O6P+Na]

+

requires, 428.1669. [D = –33.8o (c 1.0, MeOH).

138

BocNNH

OH

N

N N

P

O

OEt

OEt

(S)-tert-Butyl-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)-1-(methylamino)-1-

oxopropan-2-ylcarbamate (124)

33% aqueous methylamine (3.00 mL, 31 mmol) was added to a solution of triazole 122

(300 mg, 0.74 mmol) in MeOH (5 mL). The solution was stirred at room temperature

for 4 h, and then concentrated under reduced pressure. The residue was subjected to

flash chromatography. Elution with 1:10 MeOH/DCM gave 124 as a pale yellow oil

(270 mg, 90%). Rf = 0.20 (1:9 MeOH/DCM). IR (thin film) cm–1

: 3305 (NH), 1712

(ester C=O), 1666 (amide C=O). 1H NMR (500 MHz, CD3OD): 8.39 (s, 1H, triazolyl),

4.92 (dd, 1H, J = 12.5 Hz, 3.0 Hz, -H), 4.63 (m, 2H, -H), 4.14 (m, 4H, 2 ×

OCH2CH3), 2.70 (s, 1H, CONHCH3), 1.32 (s, 9H, t-Bu), 1.288 (t, 3H, J = 7.0 Hz,

OCH2CH3a ), 1.287 (t, 3H, J = 7.0 Hz, OCH2CH3b). 13

C NMR (125 MHz, CD3OD): δ

171.3, 157.1, 137.4 (d, JC,P = 242.2 Hz,CP), 133.5 (d, JCP = 33.2 Hz, triazolyl CH),

81.0, 64.45 (d, JC,P = 4.2 Hz, OCH2CH3a), 64.50 (d, JC,P = 4.2 Hz, OCH2CH3b), 55.7,

52.3, 28.6, 26.5, 16.6 (d, JC,P = 6.5 Hz, OCH2CH3). 31

P NMR (120 MHz, CD3OD): δ

8.73. MS (ESI) m/z: 406 [M+H]+, 428 [M+Na]

+. HRMS (ESI): observed, 406.1855,

[C15H28N5O6P+H]+ requires, 406.1850. [D = 5.4º (c 1.0, MeOH).

139

BrH3NNH

O

NN

N

P

O

HOOH

(S)-1-(Methylamino)-1-oxo-3-(5-phosphono-1H-1,2,3-triazol-1-yl)propan-2-

aminium bromide (125)

A solution of 123 (100 mg, 0.25 mmol) in 33% HBr in acetic acid (2.00 mL, 8.1 mmol)

was stirred at room temperature for 48 h. The reaction mixture was concentrated under

vacuum and the residue was purified by Dowex cation exchange chromatography.

Elution with water gave the hapten 125 as a white solid (54 mg, 71%), mp 141–143 C.

Rf = 1.0 on reverse-phase TLC (H2O). IR (KBr disk) cm–1

: 300–3500 (br, NH3 + OHs),

1685 (C=O). 1H NMR (600 MHz, D2O): 7.73 (s, 1H, triazolyl), 4.89 (dd, J = 6.6 Hz,

2.4, 2H, -H), 4.37 (t, J = 6.0 Hz, 1H, -H), 2.51 (s, 1H, NHCH3). 13

C NMR (125 MHz,

D2O): δ 166.4, 138.4 (br s, triazolyl CH), 134.6 (d, JC,P = 192.1 Hz, CP), 52.5, 49.0,

25.9. 31

P NMR (120 MHz, D2O): δ 3.48. MS (ESI) m/z: 250 [M+H]+

(where M = free

base). HRMS (ESI): observed, 250.0703, [C6H11N5NaO4P+H]+ requires, 250.0700. [D

= +38.4º (c 1.0, H2O).

BrH3NNH

O

N

N N

P

O

OH

OH

(S)-1-(Methylamino)-1-oxo-3-(4-phosphono-1H-1,2,3-triazol-1-yl)propan-2-

aminium bromide (126)




Elution with water gave 126 as a white solid (56 mg, 73%), mp 145–146 C. Rf = 1.0 on

140

reverse-phase TLC (H2O). IR (KBr disk) cm–1

: 3100–3500 (br, NH3 + OHs), 1646

(C=O). 1H NMR (500 MHz, D2O): 8.14 (s, 1H, triazolyl), 4.97 (d, J = 5.5 Hz, 2H, -

H), 4.54 (t, J = 5.0 Hz, 1H, -H), 2.69 (s, 1H, NHCH3). 13

C NMR (125 MHz, D2O): δ

167.0, 144.5 (d, JC,P = 214.7 Hz, CP), 129.7 (d, JC,P = 28.9 Hz, triazolyl CH), 52.9, 49.8,

26.3. 31

P NMR (120 MHz, D2O): δ 1.55 MS (ESI) m/z: 272 [M+Na]+, 294 [M+2NaH]

+

(where M = free base). HRMS (ESI): observed, 272.0522, [C6H12N5NaO4P+Na]+

requires, 2720519. [D = +27.2º (c 1.0, H2O).

BocNOH

OH

N NN

PO

OEtEtO

(S)-2-(tert-Butoxycarbonylamino)-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoic acid (35)

LiOH.H2O (5 mg, 0.1 mmol) was added to a stirred solution of 121 (51 mg, 0.13 mmol)

in 3:1 MeOH/water (4 mL) at 0 ºC. After 2 h, the reaction mixture was acidified to pH 4

with sat. aq. KHSO4 and then extracted with DCM (3 × 40 mL). The organic extract


residue was subjected to flash chromatography. Elution with 1:9 MeOH/DCM gave 35

as a colourless oil (41 mg, 86 %). Rf = 0.10 (1:9 MeOH/DCM). 1

H NMR (500 MHz,

CD3OD): 8.09 (s, 1H, triazolyl), 5.15 (m, -CH), 4.74 (m, 2H, -CH2), 4.25 (m, 4H, 2

× OCH2CH3), 1.36 (t, J = 7.0 Hz, 6H, 2 × OCH2CH3), 1.30 (s, 9H, t-Bu). 13

C NMR (125

MHz, CD3OD): 175.5 (CO2), 158.0 (NCO), 142.3 (d, JC,P = 20.6 Hz, triazolyl CH),

128.4 (d, JC,P = 222.5 Hz, CP), 81.1 [OC(CH3)3], 66.3 (d, JC,P = 5.5 Hz, OCH2CH3a),

66.2 (d, JC,P = 5.5 Hz, OCH2CH3b), 57.1 (), 54.5 (), 29.5 [C(CH3)3], 17.4 (d, JC,P =

6.3 Hz, OCH2CH3a), 17.3 (d, JC,P = 5.9 Hz, OCH2CH3b). MS (ESI) m/z: 393 [M+H]+,

141

415 [M+Na]+. A sample that had partially degraded was further purified by HPLC in

order to compare to the reported spectra in CDCl3. 1H NMR (500 MHz, CDCl3): 8.00

(br s, 1H, triazolyl), 5.90 (br s, 1H, NH) 5.07 (br m, 1H, -CH), 4.91 (br m, 2H, -

CH2), 4.22 (m, 4H, 2 × OCH2CH3), 1.35–1.40 (m, 6H, 2 × OCH2CH3), 1.36 (s, 9H, t-

Bu). 13

C NMR (125 MHz, CDCl3): δ 171.1 (br, CO2), 155.8 (NCO), 140.5 (br s,

triazolyl CH), 126.7 (br d, JC,P = 231.5 Hz, CP), 80.8 [OC(CH3)3], 64.3 (br s,

OCH2CH3), 53.8 (), 51.1 (), 28.2 [C(CH3)3], 16.1 (br s, OCH2CH3). [D = +1.2o (c

0.5, CDCl3). The NMR data are similar to those reported.40

BocNOH

OH

N

N N

P

O

OEt

OEt

(S)-2-(tert-Butoxycarbonylamino)-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoic acid (36)

LiOH.H2O (5 mg, 0.1 mmol) was added to a stirred solution of 122 (51 mg, 0.13 mmol)

in 3:1 MeOH/water (4 mL) at 0 ºC. After 2 h, the reaction mixture was acidified to pH 4

with sat. aq. KHSO4, and then extracted with DCM (3 × 40 mL). The extract was

washed with brine (10 mL), dried and concentrated under reduced pressure. The residue

was subjected to flash chromatography. Elution with 1:4 MeOH/DCM gave 36 as a

colourless oil (42 mg, 88 %). Rf = 0.10 (1:4 MeOH/DCM). 1H NMR (500 MHz,

CD3OD): 8.39 (s, 1H, triazolyl), 5.01 (br dd, J = 17.5, 10.2 Hz, 1H, -CH2a), 4.71 (m

[apparent dd], 1H, -CH2b), 4.51 (m, 1H, -CH), 4.17 (m, 4H, 2 × OCH2CH3), 1.37 (s,

9H, t-Bu), 1.32 (t, J = 7.1 Hz, 6H, 2 × OCH2CH3). 13


175.2 (CO2), 158.2 (NCO), 138.0 (d, JC,P = 242.2 Hz, CP), 134.2 (d, JC,P = 31.8 Hz,

triazolyl CH), 81.4 [OC(CH3)3], 65.4 (d, JC,P = 3.4 Hz, OCH2CH3a), 65.3 (d, JC,P = 3.3

Hz, OCH2CH3b), 57.5 (), 54.0 (), 29.5 [C(CH3)3], 17.4 (d, JC,P = 6.5 Hz, OCH2CH3).

142

MS (ESI) m/z: 415 [M+Na]+. A sample that had partially degraded was further purified

by HPLC in order to compare to the reported spectra in CDCl3. 1H NMR (600 MHz,

CDCl3): 8.28 (br s, 1H, triazolyl), 6.4 (v br s, OH), 5.55 (br s, 1H, NH), 4.98 (m, 2H,

-CH2), 4.75 (br s, 1H, -H), 4.20 (br m, 4H, 2 × OCH2CH3), 1.43 (s, 9H, t-Bu), 1.34 (2

× overlapping t at 1.34 and 1.33 [apparent q], J = 6.0 Hz, 6H, 2 × OCH2CH3). 13

C NMR

(150 MHz, CDCl3): δ 170.5 (br CO2), 155.4 (NCO), 136.4 (br d, JC,P = 231.6 Hz, CP),

132.5 (br s, triazolyl CH), 80.7 [OC(CH3)3], 63.85 (br s, OCH2CH3a), 63.76 (br s,

OCH2CH3b), 53.7 (br, ), 51.2 (), 28.2 [C(CH3)3], 16.1 (br s, OCH2CH3). [D =

+40.5o (c 0.7, CHCl3). The NMR data are similar to those reported.

40

BocN

O

N

NN

N

HH

O

HN

PO

OEtEtO

tert-Butyl-(S)-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)-1-((S)-1-

(methylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-ylcarbamate (127)

HCTU (635 mg, 1.53 mmol) was added to a solution of 35 (500 mg, 1.27 mmol), N-

methyl-L-alaninamide 133 (196 mg, 1.91 mmol) and diisopropylethylamine (DIPEA)

(450 mL, 2.55 mmol) in DMF (10 mL) at 0 ºC. The resulting solution was stirred at

room temperature for 18 h, then diluted with sat. aq. NH4Cl (20 mL) and extracted with

EtOAc (3 × 40 mL). The organic extract was washed with brine (20 mL), dried and


chromatography. Elution with 1:50 MeOH/DCM gave a 17:3 ratio of 127 and the R,S

diastereomer as a colourless oil (363 mg, 60%). Rf = 0.15 (1:50 MeOH/DCM). IR (thin

film) cm–1

: 3307 (NH), 1600-1750 (broad peak, C=Os). 1H NMR (600 MHz, CD3OD):

8.16 (s, 1H, triazolyl), 5.14 (m [apparent d], 1H, -Ha), 4.80-4.87 (m [under water

signal], 2H, -Hb & -H), 4.39 (q, 1H, J = 7.2 Hz, -Hala), 4.29 (m, 4H, 2 ×

143

OCH2CH3), [2.78, 2.77* (2 s, 3H, NCH3)], 1.36-1.44 (m, 18 H, t-Bu & 2 × OCH2CH3 &

CCH3). 13

C NMR (150 MHz, CD3OD): δ [175.9*, 175.0 (CCO)], 170.7 (CCO), [158.4*,

157.3 (NCO2)], 141.5 (d, JC,P = 21.1 Hz, triazolyl CH), 128.0 (d, JC,P = 221.5 Hz, CP),

[82.1*, 81.0 (OC(CH3)3)], 65.5 (d, JC,P = 5.6 Hz, OCH2CH3a), 65.4 (d, JC,P = 5.4 Hz,

OCH2CH3b), 55.7, 52.4, 50.5, 28.6 [C(CH3)3], 26.4 (NCH3), 18.2 (CCH3) 16.55 (d, JC,P

= 2.9 Hz, OCH2CH3a), 16.51 (d, JC,P = 2.7 Hz, OCH2CH3b). 31

P NMR (120 MHz,

CD3OD): δ 4.64. MS (ESI) m/z: 477 [M+H]+, 499 [M+Na]

+. HRMS (ESI): observed,

477.2227, [C18H33N6O7P+H]+ requires, 477.2221. [D = –19.1º (c 1.0, MeOH).

BocNNH

OH

N

N N

O

HN

PO

OEtOEt

tert-Butyl-(S)-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)-1-((S)-1-

(methylamino)-1-oxopropan-2-ylamino)-1-oxopropan-2-ylcarbamate (128)

HCTU (1.27 g, 3.06 mmol) was added to a solution of 36 (1.00 g, 2.55 mmol), N-

methyl-L-alaninamide 133 (391 mg, 3.82 mmol) and DIPEA (900 mL, 5.10 mmol) in

DMF (20 mL) at 0 ºC. The resulting solution was stirred at room temperature for 18 h,

then diluted with sat. aq. NH4Cl (30 mL) and extracted with EtOAc (3 × 50 mL). The

organic extract was washed with brine (30 mL), dried and concentrated under reduced

pressure. The residue was subjected to flash chromatography. Elution with 1:20

MeOH/DCM gave a 17:8 ratio of 128 and the R,S diastereomer as a colourless oil (741

mg, 61%). Rf = 0.15 (1:20 MeOH/DCM). IR (thin film) cm–1

: 3293 (NH), 1600-1750

(broad peak, C=Os). 1H NMR (500 MHz, CD3OD): [8.46, 8.45* (2 s, 1H, triazolyl)],

[5.00, 4.98* (2 dd, J = 13.5, 4.0 Hz, 1H, -Ha)], 4.72-4.79 (m, 1H, -Hb), 4.58-4.70 (m,

1H, -H), 4.36 (m, 1H, -Hala), 4.21 (m, 4H, 2 × OCH2CH3), [2.78, 2.76* (2 s, 3H,

* Peak corresponding to the minor diastereomer

144

NCH3)], 1.42 (s, 9H, t-Bu), 1.35-1.40 (m, 9H, 2 × OCH2CH3 & CCH3). 13

C NMR (125

MHz, CD3OD): δ [176.05*, 175.96 (2 s, CCO)], [171.7*, 171.4 (2 s, CCO)], [158.4*,

158.2 (2 s, NCO2)], 138.2 (d, JC,P = 242.2 Hz, CP), [134.53, 134.47* (2 d, JC,P = 33.2

Hz, triazolyl CH)], [82.2*, 82.1 (2 s, OC(CH3)3)], 65.44 (d, JC,P = 2.8 Hz, OCH2CH3a),

65.39 (d, JC,P = 2.8 Hz, OCH2CH3b), [56.7*, 56.4 (2 s)], [53.1, 52.7* (2 s)], 51.4,

[29.50*, 29.47 (2 s, C(CH3)3)], 27.3 (NCH3), [19.0, 18.8* (2 s, CCH3)], 17.4 (d, JC,P =

6.4 Hz, OCH2CH3). 31

P NMR (120 MHz, CD3OD): δ 8.73. MS (ESI) m/z: 477 [M+H]+,

499 [M+Na]+. HRMS (ESI): observed, 477.2227, [C18H33N6O7P+H]

+ requires,

477.2221. [D = –5.0° (c 1.0, MeOH).

BrH3N

O

N

NN

N

H

O

HN

PO

OHHO

(S)-1-((S)-1-(Methylamino)-1-oxopropan-2-ylamino)-1-oxo-3-(5-phosphono-1H-

1,2,3-triazol-1-yl)propan-2-aminium bromide (129)

A solution of 127 (100 mg, 0.210 mmol) in 33% HBr in acetic acid (1.8 mL, 7.34

mmol) was stirred at room temperature for 48 h. The reaction mixture was concentrated

under vacuum and the residue was purified by Dowex cation exchange chromatography.

Elution with water gave a 17:3 ratio of the hapten 129 and the R, S diastereomer as a

white solid (55 mg, 66%), mp 137–138 C. Rf = 1.0 on reverse-phase TLC (H2O). IR

(KBr disk) cm–1

: 3000–3500 (br, NH3 + OHs), 1685 (br, C=Os). 1H NMR (500 MHz,

D2O): [7.69*, 7.67 (s, 1H, triazolyl)], [4.86*, 4.75 (2 dd, J = 14.5, 4.5 Hz, 2H, -H)],

4.46 (m, 1H, -H), 4.16 (q, J = 7.0 Hz, 1H, -Hala), [2.62*, 2.60 (2 s, 3H, CH3)], 1.20

(d, 3H, J = 7.5 Hz, CCH3). 13

C NMR (125 MHz, D2O): δ [174.5, 172.8* (2 s, CO)],

162.7 (CO), [138.4, 138.1* (2 d, JC,P = 172.3 Hz, CP), [136.8*, 133.68 (2 d, JC,P = 15.3


145

Hz, triazolyl CH)], 56.1, [49.2*, 50.2 (2 s)], 48.5, [25.23, 25.19* (2 s, CCH3)], [15.8*,

15.6 (2 s, NCH3)]. 31

P NMR (120 MHz, D2O): δ –3.58. MS (ESI) m/z: 321 [M+H]+, 343

[M+Na]+ (where M = free base). HRMS (ESI): observed, 321.1070, [C9H17N6O5P + H]

+

requires, 321.1076. [D = –28.8º (c 1.0, H2O).

BrH3NNH

O

N

N N

O

HN

PO

OHOH

(S)-1-((S)-1-(Methylamino)-1-oxopropan-2-ylamino)-1-oxo-3-(4-phosphono-1H-

1,2,3-triazol-1-yl)propan-2-aminium bromide (130)

128 (100 mg, 0.210 mmol) in 33% HBr in acetic acid (1.8 mL, 7.34 mmol) was stirred

at room temperature for 48 h. The reaction mixture was concentrated under vacuum and

the residue was purified by Dowex cation exchange chromatography. Elution with water

gave a 17:8 ratio of the hapten 130 and the R,S diastereomer as a white solid (53 mg,

63%), mp 145–147 C. Rf = 1.0 on reverse-phase TLC (H2O). IR (KBr disk) cm–1

:

3000–3500 (br, NH3 + OHs), 1685 (C=Os). 1

H NMR (500 MHz, D2O): [8.06, 8.05* (2

s, 1H, triazolyl)], 4.83-4.96 (m, 2H, -H), [4.51, 4.45 (2 m [apparent t], 1H, -H)],

[4.14, 4.04* (2 q, J = 6.0 Hz, 1H, -Hala)], [2.60, 2.54* (2 s, 3H, CH3)], [1.21, 1.12*

(2 d, 3H, J = 6.0 Hz, CCH3)]. 13

C NMR (125 MHz, D2O): δ [174.5*, 174.3 (2 s, CO)],

[166.1*, 166.0 (2 s, CO)], 144.0 (d, JC,P = 220.9 Hz, CP), 130.0 (br s, triazolyl CH),

[52.4*, 52.3 (2 s)], [50.0, 49.8* (2 s)], [49.3, 49.2* (2 s)], [25.9, 25.8* (2 s, CCH3)],

16.4 (NCH3). 31

P NMR (120 MHz, D2O): δ 1.32. MS (ESI) m/z: 321 [M+H]+, 343

[M+Na]+ (where M = free base). HRMS (ESI): observed, 321.1075, [C9H17N6O5P + H]

+

requires, 321.1076. [D = –63.5º (c 1.0, H2O).


146

H2NNH

O

N-Methyl-L-alaninamide (129)

This known compound was synthesised according to the method of Shimizu122

and

Ottenheqm72

with slight modifications.

Thionyl chloride (12.5 mL, 172 mmol) was added dropwise to L-alanine (4.46 g, 50

mmol) in EtOH (50 mL) at 0 ºC. The resulting solution was stirred at room temperature

for 18 h, and then the solvent was removed under reduced pressure. The product was

used for the next step without further purification. The crude hydrochloride was

dissolved in 40% aq. MeNH2 (6.0 mL, 77 mmol) at 0 ºC. The resulting solution was

stirred at room temperatrure for 1 h, and the solvent was removed under reduced


MeOH/DCM gave 129 as a white solid (3.47 g, 68%), mp 210–211 ºC. Rf = 0.10 (1:4

MeOH/DCM). 1H NMR (500 MHz, CD3OD): 3.95 (m, 1H, -H), 2.77 (s, 3H,

NHCH3), 1.50 (d, J = 7.0 Hz, 3H, CHCH3). 13

C NMR (125 MHz, CD3OD): δ 172.6

(CO), 50.4 (), 26.4 (NHCH3), 18.2 (CHCH3). MS (ESI) m/z: 103 [M+H]+, 125

[M+H]+. [D = +8.0 (c 1.0, EtOH) [lit.

123 +7.7 (c 0.7, EtOH)].

H2NOMe

O

N

N N

P

O

OEt

OEt

(S)-Methyl-2-amino-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate

(134)

TFA (1.00 mL, 13.1 mmol) was added to a solution of 122 in DCM (10 mL) at 0 ºC.

The solution was allowed to warm to room temperature and stirred for 5 h, and then

volatile components were removed under reduced pressure. The residue was diluted

with sat. aq. NaHCO3 (30 mL) and extracted with EtOAc (3 × 50 mL). The organic

147


The residue was subjected to flash chromatography. Elution with 1:10 MeOH/DCM

gave 134 as a pale yellow oil (302 mg, 80%). Rf = 0.35 (1:9 MeOH/DCM). IR (thin

film) cm–1

: 3392 (NH), 1741 (C=O). 1H NMR (500 MHz, CDCl3): 8.19 (s, 1H,

triazolyl), 4.75 (dd, J = 13.5, 4.0 Hz, 1H, -Ha), 4.54 (dd, J = 13.5, 7.0 Hz, 1H, -Hb),

4.17 (m, 4H, 2 × OCH2CH3), 3.97 (s, 1H, br s), 3.74 (s, 3H, OCH3), 1.312, (t, J = 7.0

Hz, 3H, OCH2CH3a), 1.311, (t, J = 7.0 Hz, 3H, OCH2CH3b). 13

C NMR (150 MHz,

CDCl3): δ 172.3 (CO2), 136.9 (d, JC,P = 239.6 Hz, CP), 132.0 (d, JC,P = 33.3 Hz,

triazolyl CH), 63.0 (d, JC,P = 5.8 Hz, OCH2CH3), 54.2 (α), 53.3 (OMe), 52.6 (), 16.0

(d, JC,P = 6.5 Hz, OCH2CH3). 31

P NMR (120 MHz, CDCl3): δ 7.73. MS (ESI) m/z: 307

[M+H]+. HRMS (ESI): observed, 307.1160, [C10H19N4O5P+H]

+ requires, 307.1166.

[D = –4.9º (c 1.0, CHCl3).

HN

OH

O

O

N-Acetyl-L-alanine (138)

This known compound was synthesised according to the method of Schmidt with slight

modifications.75

Acetic anhydride (6.30 mL, 67.3 mmol) was added to a suspension of L-alanine (5.00 g,

56.1 mmol) in acetic acid (25 mL). The suspension was stirred at room temperature for

18 h, resulting in a clear solution. The acetic acid and excess acetic anhydride were

removed under vacuum, and the residue was subjected to a C18 reverse-phase

chromatography. Elution with water gave 138 as a colourless oil (5.30 g, 72%). 1H

NMR (500 MHz, CD3OD): 4.33 (m, 1H, -H), 1.93, 1.92 (rotamers, 3H, s, COCH3),

1.33, 1.31 (rotamers, d, J = 7.0 Hz, 3H, CCH3). 13

C NMR (125 MHz, CD3OD): δ 174.7,

148

173.0 52.7, 22.3, 17.4. MS (ESI) m/z: 132 [M+H]+, 154 [M+H]

+. []D = 62.7 (c 1.0,

H2O) [lit.124

62.0 (c 3.0, H2O)].

HN

O

OMe

OHO

(S)-Methyl-2-acetamido-3-hydroxypropanoate (142)

This known compound was synthesised according to the method of Maruyama77

with

slight modifications.

DCC (3.90 g, 18.9 mmol) was added to a suspension of 58 (2.85 g, 18.3 mmol), acetic

acid (1.10 mL, 19.3 mmol) and triethylamine (2.60 mL, 18.7 mmol) in DCM (60 mL) at

0 ºC. The reaction mixture was allowed to warm to room temperature and stirred for 18

h, and then dicyclohexylurea was filtered off. The filtrate was concentrated under

reduced pressure, and the residue was subjected to flash chromatography. Elution with

EtOAc gave 142 as a pale yellow oil (2.66 g, 90%). 1H NMR (500 MHz, CDCl3): 7.28

(br d, 1H, J = 8.0 Hz, NH), 4.34 (m, 1H, -H), 3.71 (dd, 1H, J = 11.0, 4.5 Hz, -Ha),

3.58 (dd, J = 11.5, 3.5 Hz, -Hb), 1.80 (s, 3H, NHCOCH3). 13

C NMR (125 MHz,

CDCl3): δ 171.0, 170.6, 61.7 (COOCH3), 54.2 (-C), 51.8 (-C), 22.0 (NHCOCH3) MS

(ESI) m/z: 162 [M+H]+. [D = +10.5º (c 1.0, MeOH). The

1H NMR data are similar to

those reported.77

HN

O

OMe

N3

O

(S)-Methyl-2-acetamido-3-azidopropanoate (143)

A solution of the alcohol 142 (2.00 g, 12.4 mmol) and triphenylphosphine (3.90 g, 14.9

mmol) in anhydrous THF (50 mL) at 78 ºC under N2 was treated with a 2.5 M HN3

solution in toluene (6.00 mL, 15.1 mmol) and DIAD (3.00 mL, 15.3 mmol). The

resulting solution was allowed to warm to room temperature, stirred for 5 h, and then

149

diluted with water (100 mL). The layers were separated and the aqueous phase was

extracted with Et2O (3 × 100 mL). The organic extract was washed with brine (50 mL),

dried and concentrated under reduced pressure. The residue was subjected to flash

chromatography. Elution with 1:1 Et2O/hexanes gave 143 as a yallow oil (1.39 g, 60

%). 1H NMR (300 MHz, CDCl3): 6.51 (br d, 1H, J = 5.4 Hz, NH), 4.74 (m, 1H, -H),

3.79 (s, 1H, OCH3, 3H), 3.73 (dd, 2H, J = 3.6, 1.2 Hz, -H), 2.05 (s, 3H, NCH3). 13

C

NMR (75 MHz, CDCl3): δ 170.1, 169.5, 53.8, 52.9, 52.2, 22.9. MS (ESI) m/z: 187

[M+H]+, 209 [M+H]

+. [D = +79.3

o (c 1.0, CHCl3). [lit.

125 +74.2 (c 1.4, CHCl3)]. The

1H NMR data are similar to those reported.

125

HN

OMe

O

N

N N

P

O

OEt

OEt

HN

OMe

O

N NN

PO

OEtEtO

O O

144 145


Thermal

A solution of the azide 143 (1.00 g, 5.37 mmol) and the alkyne 82 (871 mg, 5.37 mmol)

in toluene (10 mL) under N2 was heated at reflux for 5 h. The toluene was evaporated,

and the residue was subjected to flash chromatography. Elution with (7:3

EtOAc/hexanes) gave 144 as a pale yellow oil (486 mg, 26%), identical with the

material described below. Further elution with EtOAc gave 145 as a pale yellow oil

(1.12 g, 60%), identical with the material described below.

150

Ru(II)-catalysed

(S)-Methyl-2-acetamido-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoate (144)


1.07 mmol) and the alkyne 82 (173 mg, 1.07 mmol) in toluene (20 mL) under N2. The

resulting solution was stirred at 60 ºC for 24 h, then diluted with water (20 mL) and

extracted with EtOAc (3 × 100 mL). The extract was washed with brine (20 mL), dried

and concentrated under reduced pressure. The residue was subjected to flash

chromatography. Elution with 7:3 EtOAc/hexanes gave 144 as a pale yellow oil (213

mg, 57%). Rf = 0.35 (EtOAc). IR (thin film) cm–1

: 3283 (NH), 1748 (ester C=O), 1671

(amide C=O). 1H NMR (500 MHz, CDCl3): 7.81 (s, 1H, triazolyl), 7.51 (br d, 1H, J =

8.0 Hz, NH), 4.99 (dd, 1H, J = 8.0, 4.0 Hz, -H), 4.88 (dd, 1H, J = 14.0, 4.5 Hz, -Ha),

4.75 (dd, 1H, J = 14.0, 8.0 Hz, -Ha), 4.05 (m, 4H, 2 × OCH2CH3), 3.58 (s, 3H,

OCOCH3) 1.79 (s, 3H, NHCOCH3), 1.20 (m, 6H, 2 × OCH2CH3). 13

C NMR (125 MHz,

CDCl3): δ 170.1, 169.1, 139.9 (d, JC,P = 20.4 Hz, triazolyl CH), 126.7 (JC,P = 220.4 Hz,

CP), 63.6 (d, JC,P = 5.9 Hz, OCH2CH3), 52.5, 51.8, 50.0, 22.2, 15.81 (JC,P = 3.6 Hz,

OCH2CH3a), 15.76 (JC,P = 3.8 Hz, OCH2CH3b). 31

P NMR (120 MHz, CDCl3): δ 4.36.

MS (ESI) m/z: 349 [M+H]+, 371 [M+Na]


[C12H21N4O6P+Na]+ requires, 371.1091. [D = +15.7

o (c 1.0, CH3Cl). Further elution

with EtOAc gave 145 as a pale yellow oil (71 mg, 19%), identical with the material

described below.

151

Cu(I)-catalysed

(S)-Methyl-2-acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoate (145)




diluted with water (10 mL) and extracted with EtOAc (3 × 100 mL). The organic extract


residue was subjected to flash chromatography. Elution with EtOAc gave 145 as a pale

yellow oil (425 mg, 91%). Rf = 0.10 (EtOAc). IR (thin film) cm–1

: 3272 (NH), 1747

(ester C=O), 1673 (amide C=O). 1H NMR (500 MHz, CDCl3): 8.11 (s, 1H, triazolyl),

7.57 (br d, 1H, J = 7.5 Hz, NH), 4.78 (m, 2H, -H), 4.69 (dd, 1H, J = 14.0, 7.0 Hz, -

H), 3.96 (m, 4H, 2 × OCH2CH3), 3.55 (s, 3H, OCOCH3) 1.78 (s, 3H, NHCOCH3), 1.121

(t, 3H, J = 7.0 Hz, OCH2CH3a), 1.118 (t, 3H, J = 7.0 Hz, OCH2CH3b). 13

C NMR (125

MHz, CDCl3): δ 170.5, 168.9, 136.3 (d, JC,P = 240.2 Hz, CP), 131.6 (d, JC,P = 33.1 Hz,

triazolyl CH), 62.7 (d, JC,P = 5.8 Hz, OCH2CH3), 52.4, 52.0, 49.8, 22.0, 15.70 (d, JC,P =

0.6 Hz, OCH2CH3a), 15.65 (JC,P = 0.75 Hz, OCH2CH3b). 31

P NMR (120 MHz, CDCl3):

δ 7.71. MS (ESI) m/z: 349 [M+H]+, 371 [M+Na]


[C12H21N4O6P+Na]+ requires, 371.1091. [D = +62.0

o (c 1.0, CH3Cl).

152

HN

O

OH

NO

N N

P

O

OEtOEt

(S)-2-Acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoic acid

(Method A; saponification using LiOH)

LiOH.H2O (60 mg, 1.44 mmol) was added to a solution of 145 (500 mg, 1.44 mmol) in

3:1 MeOH/water (15 mL) at 0 ºC. After 2 h, the reaction mixture was acidified to pH 4

~ 5 with sat. aq. KHSO4 and then extracted with EtOAc (3 × 50 mL). The organic

extract was washed with brine (10 mL), dried and concentrated under reduced


MeOH/DCM gave 146 as a colourless oil (144 mg, 30 %). [D = +5.1º (c 1.0,

MeOH). Identical in every respect except the optical activity with the material

described below.

(Method B; saponification using K2CO3)

K2CO3 (200 mg, 1.44 mmol) was added to a solution of 145 (500 mg, 1.44 mmol) in 3:1

MeOH/water (15 mL) at 0 ºC. The solution was allowed to warm to room temperature

and stirred for 2 h, and the reaction mixture was acidified to pH 4 with sat. aq. KHSO4

and extracted with EtOAc (3 × 50 mL). The organic extract was washed with brine (10

mL), dried and concentrated under reduced pressure. The residue was subjected to flash

chromatography. Elution with 1:4 MeOH/DCM gave 146 as a colourless oil (226 mg,

47 %). Rf = 0.10 (1:4 MeOH/DCM). IR (thin film) cm–1

: 3293 (NH), 1720 (C=O), 1618

(NC=O).1H NMR (500 MHz, CD3OD): 8.47 (s, 1H, triazolyl), 5.06 (m, 1H, -Ha),

4.78-4.86 (m, 2H, -Hb + -H), 4.20 (m, 4H, 2 × OCH2CH3), 1.96 (s, 3H, NCH3),

1.361 (t, J = 7.0 Hz, 3H, OCH2CH3a), 1.360 (t, J = 7.2 Hz, 3H, OCH2CH3b). 13

C NMR

(125 MHz, CD3OD): δ 175.7 (CO), 173.9 (CO), 138.0 (d, JC,P = 242.3 Hz, CP), 134.3

153

(d, JC,P = 33.3 Hz, triazolyl CH), 65.47 (d, JC,P = 0.9 Hz, OCH2CH3a), 65.43 (d, JC,P =

0.7 Hz, OCH2CH3b), 57.0 (α), 53.8 (), 23.5 (NHCH3), 17.4 (d, JC,P = 6.5 Hz,

OCH2CH3). 31

P NMR (120 MHz, CD3OD): δ 8.90. MS (ESI) m/z: 335 [M+H]+, 357

[M+Na]. HRMS (ESI): observed, 357.1046, [C11H19N4O6P+Na]+ requires, 357.0934.

[D = +8.8º (c 1.0, MeOH).

HN

O

O

OHO

HN

O

O

OAcO

(S)-Allyl-2-acetamido-3-hydroxypropanoate (151)

(S)-Allyl-2-acetamido-3-acetoxypropanoate (152)

Acetic anhydride (6.30 mL, 67.0 mmol) was added to a suspension of L-serine (5.90 g,

56.1 mmol) in acetic acid (25 mL) at room temperature. The mixture was stirred at

room temperature for 18 h, and then concentrated under reduced pressure. The residue

was dissolved in DMF (25 mL), and allyl bromide (4.9 mL 56 mmol) and DIPEA (9.8

mL, 56 mmol) were added to the solution. The resulting solution was stirred at room

temperature for 18 h, then diluted with sat. aq. NH4Cl and extracted with EtOAc (3 × 50

mL). The organic extract was washed with brine, dried and concentrated under reduced


EtOAc/hexanes gave 152 as a white solid (5.79 g, 45%), mp 81–82 C. Rf = 0.40

(EtOAc). IR (thin film) cm–1

: 3294 (NH), 1747 (ester C=O), 1661 (amide C=O). 1H

NMR (500 MHz, CDCl3): 7.27 (d, 1H, J = 8.0 Hz), 5.56 (m, 1H, OCH2CHCH2), 4.98

(dd, 1H, J = 17.0, 1.5 Hz, OCH2CHCH2a), 4.89 (dd, 1H, J = 10.0, 1.0 Hz,

OCH2CHCH2b), 4.53 (1H, m, -H), 4.30 (m, 2H, OCH2CHCH2), 4.04 (dd, 2H, J = 6.5,

5.0 Hz, -H), 1.69 (s, 3H, OCOCH3), 1.68 (s, 3H, NHCOCH3). 13

C NMR (125 MHz,

CDCl3): δ 169.9, 169.6, 168.5, 130.8, 117.7, 65.2, 62.9, 50.9, 21.7, 20.1. MS (ESI) m/z:

230 [M+H]+, 252 [M+Na]

+. HRMS (ESI): observed, 230.1020, [C10H15NO5+H]

+

154

requires, 230.1023. [D = +58.8o (c 1.0, CH3Cl). Further elution with EtOAc gave 151

as a brown oil (2.21 g, 21%). Rf = 0.15 (EtOAc). IR (thin film) cm–1

: 3366 (NH), 1741

(ester C=O), 1655 (amide C=O). 1H NMR (400 MHz, CDCl3): 7.28 (d, 1H, J = 7.6

Hz, NH), 5.77 (m, 1H, OCH2CHCH2), 5.20 (ddd, 1H, J = 17.6, 3.2, 1.6 Hz,

OCH2CHCH2a), 5.11 (ddd, 1H, J = 10.4, 2.4, 1.2 Hz, OCH2CHCH2b), 4.51 (2H, dt, J =

6.0 Hz, 1.2 Hz, OCH2CHCH2), 4.47 (dd, 1H, J = 8.0, 4.0 Hz, -H), 3.83 (dd, 1H, J =

11.2, 4.0 Hz, -Ha), 3.71 (dd, 1H, J = 11.2, 3.2 Hz, -Hb), 1.90 (s, 1H, NHCOCH3). 13

C

NMR (100 MHz, CDCl3): δ 171.2, 170.1, 131.2, 118.3, 65.8, 62.0, 54.5, 22.3. MS (ESI)

m/z: 188 [M+H]+, 210 [M+Na]

+. HRMS (ESI): observed, 188.0914, [C8H13NO4+H]

+

requires, 188.0917. [D = +33.6o (c 1.0, CH3Cl).

HN

O

O

N3

O

(S)-Allyl-2-acetamido-3-azidopropanoate (153)



solution in toluene (5.20 mL, 12.8 mmol) and DEAD (2.00 mL, 12.7 mmol). The

resulting solution was allowed to warm to room temperature, stirred for 5 h and then




chromatography. Elution with 1:1 Et2O/hexanes gave 153 as a brown oil (1.38 g, 61 %).

Rf = 0.40 (Et2O). IR (thin film) cm–1

: 3274 (NH), 2107 (N3) 1743 (ester C=O), 1660

(amide C=O). 1H NMR (500 MHz, CDCl3): 6.84 (d, 1H, J = 7.5 Hz, NH), 5.83 (m,

1H, OCH2CHCH2), 5.27 (dd, 1H, J = 17.0, 1.5 Hz, OCH2CHCH2a), 5.20 (dd, 1H, J =

10.5, 1.0 Hz, CH2CHCH2b), 4.72 (m, 1H, -H), 4.60 (dd, 2H, J = 4.5, 1.5 Hz,

155

OCH2CHCH2), 3.68 (d, 2H, -H), 1.99 (s, 3H, NHCOCH3). 13

C NMR (125 MHz,

CDCl3): δ 170.2, 169.2, 130.9, 119.0, 66.3, 52.1, 52.0, 22.6. MS (ESI) m/z: 235

[M+Na]+. HRMS (ESI): observed, 235.0808, [C8H13NO4+H]

+ requires, 235.0802. [D

= +16.3o (c 1.0, CH3Cl).

HN

O

O

NO

N N

P

O

OEtOEt

HN

O

O

NN

N

PO

EtO

O

OEt

154 155


Thermal



and the residue was subjected to flash chromatography. Elution with 7:3




Ru(II)-catalysed

(S)-Allyl-2-acetamido-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate

(154)

Cp*RuCl(PPh3)2 (15 mg, 19 mmol) was added to a solution of the azide 153 (250 mg,


resulting solution was stirred at 60 ºC for 24 h, then diluted with water (10 mL) and



156



: 3284 (NH), 1747 (ester C=O), 1682


8.0 Hz, NH), 5.78 (m, 1H, OCH2CHCH2), 5.23 (ddd, 1H, J = 17.0, 2.5, 1.0 Hz

OCH2CHCH2a), 5.15 (ddd, 1H, J = 10.5, 2.5, 1.0 Hz, OCH2CHCH2b), 5.11 (m, 1H, -

H) 4.95 (dd, 1H, J = 14.0, 4.5 Hz, -Ha), 4.86 (dd, 1H, J = 14.5, 8.0 Hz, -Hb), 4.56

(m, 2H, OCH2CHCH2), 4.13 (m, 4H, 2 × OCH2CH3), 1.88 (s, 1H, NHCOCH3), 1.29 (m,

6H, 2 × OCH2CH3). 13

C NMR (125 MHz, CDCl3): δ 170.1, 168.5, 140.1 (d, JC,P = 20.4

Hz, triazolyl CH), 131.0, 126.9 (d, JC,P = 220.4 Hz, CP), 118.8, 66.3, 63.73 (JC,P = 4.5

Hz, OCH2CH3a), 63.69 (JC,P = 4.7 Hz, OCH2CH3b), 52.1, 50.2, 22.5, 16.01 (d, JC,P =

3.6 Hz, OCH2CH3a), 15.96 (d, JC,P = 3.8 Hz, OCH2CH3b). 31

P NMR (120 MHz,

CDCl3): δ 4.48. MS (ESI) m/z: 375 [M+H]+, 397 [M+Na]


375.1422, [C14H23N4O6P+H]+ requires, 375.1428. [D = +21.0

o (c 1.0, CH3Cl). Further

elution with EtOAc gave 155 as a pale yellow oil (44 mg, 10%). Identical with the

material described below.

Cu(I)-catalysed

(S)-Allyl-2-acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate

(155)

CuSO4.5H2O (30 mg, 0.12 mmol) was added to a solution of the azide 153 (250 mg, 1.2

mmol), the alkyne 82 (191 mg, 1.2 mmol) and sodium ascorbate (23 mg, 0.12 mmol) in

1:1 t-BuOH/water (6 mL). The resulting solution was stirred at room temperature for 24

h, then diluted with water (10 mL) and extracted with DCM (3 × 30 mL). The organic


The residue was subjected to flash chromatography. Elution with EtOAc gave 155 as a

pale yellow oil (371 mg, 84%). Rf = 0.15 (EtOAc). IR (thin film) cm–1

: 3271 (NH),

157

1745 (ester C=O), 1674 (amide C=O). 1H NMR (500 MHz, CDCl3): 8.10 (s, 1H

triazolyl), 7.21 (br d, 1H, J = 7.5 Hz, NH), 5.80 (m, 1H, OCH2CHCH2), 5.25 (ddd, 1H,

J = 17.5, 3.0, 1.5 Hz, OCH2CHCH2a), 5.19 (ddd, 1H, J = 10.5, 2.5, 1.5 Hz,

OCH2CHCH2b), 4.93 (m, 1H, -H), 4.85 (m, 2H, -H), 4.57 (dt, 2H, J = 5.5 Hz, 1.0

Hz, OCH2CHCH2), 4.09 (m, 4H, 2 × OCH2CH3), 1.92 (s, 1H, NHCOCH3), 1.251 (t, 3H,

J = 7.5 Hz, OCH2CH3a), 1.248 (t, 3H, J = 7.5 Hz, OCH2CH3b). 13

C NMR (125 MHz,

CDCl3): δ 170.6, 168.3, 136.9 (d, JC,P = 239.8 Hz, CH), 131.8 (d, JC,P = 33.1 Hz,

triazolyl CH), 130.9, 119.2, 66.6, 63.0 (d, JC,P = 0.9 Hz, OCH2CH3a), 62.9 (d, JC,P = 0.8

Hz, OCH2CH3b), 52.4, 50.2, 22.5, 16.02 (d, JC,P = 1.3 Hz, OCH2CH3a), 15.96 (d, JC,P =

1.1 Hz, OCH2CH3b). 31

P NMR (120 MHz, CDCl3): δ 7.52. MS (ESI) m/z: 375 [M+H]+.

HRMS (ESI): observed, 375.1433, [C14H23N4O6P+H]+ requires, 375.1428. [D =

+25.3o (c 1.0, CH3Cl).

HN

O

O

OHO

HN

O

O

OO

O

(S)-Benzyl-2-acetamido-3-hydroxypropanoate (160)

(S)-Benzyl-2-acetamido-3-acetoxypropanoate (161)

Acetic anhydride (6.30 mL, 67.0 mmol) was added to a suspension of L-serine (5.90 g,

56.1 mmol) in acetic acid (25 mL) at room temperature. The mixture was stirred at

room temperature for 18 h, and then concentrated under reduced pressure. The residue

was dissolved in DMF (25 mL), and benzyl bromide (6.70 mL 56.4 mmol) and DIPEA

(9.80 mL, 56.3 mmol) were added to the solution. The resulting solution was stirred at

room temperature for 18 h, then diluted with sat. aq. NH4Cl and extracted with EtOAc

(3 × 50 mL). The organic extract was washed with brine (30 mL), dried and


158

chromatography. Elution with 1:1 EtOAc/hexanes gave 161 as a pale yellow oil (7.05 g,

45%). Rf = 0.50 (EtOAc). IR (thin film) cm–1

: 3293 (NH), 1746 (ester C=O), 1661

(amide C=O). 1H NMR (400 MHz, CDCl3): 7.28 (m, 5H, aromatic), 7.00 (br d, 1H, J

= 8.0 Hz), 5.13 (dd, 2H, J = 31.6 Hz, 12.4 Hz, OCH2Ph), 4.87 (m, 1H, -H), 4.42 (dd,

1H, J = 11.2 Hz, 4.0 Hz, -Ha), 4.29 (dd, 1H, J = 11.2 Hz, 3.6 Hz, -Hb), 1.97 (s, 3H,

NHCOCH3), 1.87 (s, 3H, OCOCH3). 13

C NMR (100 MHz, CDCl3): δ 170.1, 169.9,

169.1, 134.7, 128.2, 128.1, 127.9, 67.0, 63.5, 51.3, 22.4, 20.0. MS (ESI) m/z: 280

[M+H]+, 302 [M+Na]

+. HRMS (ESI): observed, 302.1005, [C14H17NO5+Na]

+ requires,

302.0999. [D = +24.5o (c 1.0, CH3Cl). Further elution with EtOAc gave 160 as a white

solid (4.15 g, 31%), mp 60–62 C. Rf = 0.20 (EtOAc). IR (thin film) cm–1

: 3313 (NH),

1742 (ester C=O), 1657 (amide C=O). 1H NMR (400 MHz, CDCl3): ; 7.30 (m, 5H,

aromatic), 7.14 (br d, 1H, J = 7.6 Hz, NH), 5.14 (d, 2H, J = 1.2 Hz, OCH2Ph), 4.63 (m,

1H, -H), 3.95 (dd, 1H, J = 11.6 Hz, 4.0 Hz, -Ha), 3.81 (dd, 1H, J = 11.2 Hz, 3.2 Hz,

-Hb), 1.97 (s, 3H, NHCOCH3). 13

C NMR (100 MHz, CDCl3): δ 171.1, 170.3, 135.0,

128.4, 128.2, 127.8, 67.1, 62.4, 54.7, 22.6. MS (ESI) m/z: 238 [M+H]

+, 260 [M+Na]

+.

HRMS (ESI): observed, 238.1077, [C12H16NO4+H]+ requires, 238.1074. [D = +14.3°

(c 1.0, CH3Cl).

HN

O

O

N3

O

(S)-Benzyl-2-acetamido-3-azidopropanoate (162)


mmol) in anhydrous THF (50 mL) at 78 °C under N2 was treated with a 2.5 M HN3

solution in toluene (4.10 mL, 10.3 mmol) and DIAD (2.00 mL, 10.2 mmol). The


159




chromatography. Elution with 1:1 Et2O/hexanes gave 162 as a yellow oil (1.35 g, 61

%). Rf = 0.45 (Et2O). IR (thin film) cm–1

: 3293 (NH), 2106 (azide), 1743 (ester C=O),

1655 (amide C=O). 1H NMR (500 MHz, CDCl3): 7.26 (m, 5H, aromatic), 7.15 (br d, J

= 7.5 Hz, NH), 5.11 (d, 2H, OCH2Ph), 4.76 (m, 1H, -H), 3.63 (dd, 2H, J = 4.0 Hz, 1.5

Hz), 1.95 (s, 3H, NHCOCH3). 13

C NMR (125 MHz, CDCl3): δ 170.1, 169.2, 134.5,

128.2, 128.1, 127.8, 67.2, 51.9, 51.8, 22.2. MS (ESI) m/z: 263 [M+H]+, 285 [M+Na]

+.

HRMS (ESI): observed, 263.1133, [C12H14N4O3+H]+ requires, 263.1139. [D = +22.8º

(c 1.0, CHCl3).

AcNOBn

OH

N

N N

P

O

OEt

OEt

AcNOBn

OH

N NN

PO

OEtEtO

163 164


Thermal



and the residue was subjected to flash chromatography. Elution with 1:1



45%), identical with the material below.

160

Ru(II)-catalysed

(S)-Benzyl-2-acetamido-3-(5-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoate (163)


0.763 mmol) and the alkyne 82 (124 mg, 0.763 mmol) in toluene (20 mL) under N2.

The resulting solution was stirred at 60 ºC for 24 h, then diluted with water (10 mL) and





: 3290 (NH), 1747 (ester C=O), 1683


8.5 Hz, NH), 7.16 (m, 5H, aromatic), 5.09 (dd, 1H, J = 8.0, 4.5 Hz, -H), 5.02 (d, 2H, J

= 6.5 Hz, OCH2Ph), 4.90 (dd, 1H, J = 14.0, 4.5 Hz, -Ha), 4.81 (dd, 1H, J = 14.0, 8.0

Hz, -Hb), 4.00 (m, 4H, 2 × OCH2CH3), 1.81 (s, 3H, NHCOCH3), 1.18 (m, 6H, 2 ×

OCH2CH3). 13

C NMR (125 MHz, CDCl3): δ 170.1, 168.5, 140.0 (d, JC,P = 20.6 Hz,

triazolyl CH), 134.5, 128.1, 128.0, 127.8, 126.6 (d, JC,P = 220.4 Hz, CP), 67.2, 63.45 (d,

JC,P = 6.2 Hz, OCH2CH3a), 63.40 (d, JC,P = 6.0 Hz, OCH2CH3b), 52.8, 50.0, 22.1, 15.70

(d, JC,P = 4.2 Hz, OCH2CH3a), 15.65 (d, JC,P = 4.2 Hz, OCH2CH3b). 31

P NMR (120

MHz, CDCl3): δ 4.35. MS (ESI) m/z: 425 [M+H]

+, 447 [M+Na]

+. HRMS (ESI):

observed, 425.1590, [C18H25N4O6P+H]+ requires, 425.1584. [D = +14.0° (c 1.0,

CH3Cl).

161

Cu(I)-catalysed

(S)-Benzyl-2-acetamido-3-(4-(diethoxyphosphoryl)-1H-1,2,3-triazol-1-

yl)propanoate (164)


0.763 mmol), the alkyne 82 (124 mg, 0.763 mmol) and sodium ascorbate (16 mg,

0.0808 mmol) in 1:1 t-BuOH/water (10 mL). The resulting solution was stirred at room

temperature for 24 h, then diluted with water (10 mL) and extracted with EtOAc (3 ×

100 mL). The organic extract was washed with brine (10 mL), dried and concentrated

under reduced pressure. The residue was subjected to flash chromatography. Elution

with EtOAc gave 164 as a pale yellow oil (282 mg, 87%). Rf = 0.20 (EtOAc). IR (thin

film) cm–1

: 3271 (NH), 1744 (ester C=O), 1675 (amide C=O). 1H NMR (500 MHz,

CDCl3): 8.10 (s, 1H, triazolyl), 7.67 (br d, 1H, J = 8.0 Hz, NH), 7.15 (m, 5H,

aromatic), 5.00 (s, 2H, OCH2Ph), 4.88 (dd, 1H, J = 7.0, 4.5 Hz, -H), 4.81 (dd, 1H, J =

14.0, 4.5 Hz, -Ha), 4.71 (dd, 1H, J = 14.0, 7.5 Hz, -Hb), 3.98 (m, 4H, 2 × OCH2CH3)

1.79 (s, 3H, NHCOCH3), 1.13 (t, 6H, J = 7.0 Hz, 2 × OCH2CH3). 13

C NMR (125 MHz,

CDCl3): δ 170.3, 168.3, 136.3 (d, JC,P = 239.8 Hz, CP), 134.4, 131.5 (d, JC,P = 33.1 Hz,

triazolyl CH), 128.0, 127.9, 127.7 67.1, 62.5, (d, JC,P = 5.7 Hz, OCH2CH3) 52.1, 49.7,

22.0, 15.6 (d, JC,P = 6.4 Hz, OCH2CH3). 31

P NMR (120 MHz, CDCl3): δ 7.73. MS (ESI)

m/z: 425 [M+H]+, 447 [M+Na]


[C18H25N4O6P+Na]+ requires, 447.1404. [D = +13.9° (c 1.0, CH3Cl).

162

HN

O

OH

NN

N

PO

OHHO

O

(S)-2-Acetamido-3-(5-phosphono-1H-1,2,3-triazol-1-yl)propanoic acid (156)

A solution of 163 (200 mg, 0.47 mmol) in 33% HBr in acetic acid (4.0 mL, 16 mmol)


vacuum and the residue was purified by Dowex anion exchange chromatography.

Elution with water gave 156 as a white solid (90 mg, 69%), mp 146–148 C. IR (KBr

disk) cm–1

: 3000–3500 (br, NH3 + OHs), 1717 (C=O), 1650 (NC=O). 1H NMR (500

MHz, D2O): 7.83 (s, 1H, triazolyl), 4.71 (dd, 1H, J = 14.0, 3.5 Hz, -H), 4.54 (dd, 1H,

J = 9.0, 3.5 Hz, -Ha), 4.46 (dd, 1H, J = 14.0, 9.5 Hz, -Hb), 1.38 (s, 3H, CH3). 13

C

NMR (125 MHz, D2O): δ 173.8, 170.5, 136.1 (d, JC,P = 197.9 Hz, CP), 134.5 (br s,

triazolyl), 51.6, 51.1, 21.5. 31

P NMR (120 MHz, D2O): δ –2.65. MS (ESI) m/z: 279

[M+H]+, 301 [M+Na]

+. HRMS (ESI): observed, 301.0305 [C7H11N4O6P+Na]

+ requires,

301.0308. [D = –23.8° (c 1.0, H2O).

HN

O

OH

N

N N

P

O

OHOH

O

(S)-2-Acetamido-3-(4-phosphono-1H-1,2,3-triazol-1-yl)propanoic acid (137)

(Method A: From the benzylester 164)



vacuum and the residue was subjected to Dowex anion exchange chromatography.

Elution with water gave 137 as a white solid (88 mg, 67%), mp 135–137 C. IR (KBr

disk) cm–1

: 3100–3500 (br, NH3 + OHs), 1732 (C=O), 1637 (NC=O). 1H NMR (500

163

MHz, D2O): 8.10 (s, 1H, triazolyl), 4.71 (dd, 2H, J = 12.5, 5.5 Hz, -H), 4.60 (dd, 1H,

J = 13.5, 7.0 Hz, -H), 1.67 (s, 3H, CH3). 13

C NMR (125 MHz, D2O): δ 173.9, 171.1,

139.9 (d, JC,P = 223.7 Hz, CP), 131.5 (d, JC,P = 30.2 Hz, triazolyl), 52.2, 50.5, 21.5

(CH3). 31

P NMR (120 MHz, D2O): δ 2.68. MS (ESI) m/z: 279 [M+H]+, 301 [M+Na]

+.

HRMS (ESI): observed, 279.0491 [C7H11N4O6P+H]+ requires, 279.0489. [D = +10.0°

(c 1.0, H2O).

(Method B: From the carboxylic acid 146)



vacuum and the residue was subjected to Dowex anion exchange chromatography.

Elution with water gave 137 (117 mg, 70%) as a white solid. [D = +6.1º (c 1.0, H2O).

Identical in every respect except the optical activity with the material described above.

~Chapter 3~

OHN3

O

Azidoacetic acid (166)

This compound was synthesised according to the method of Taylor126

with slight

modifications.

Sodium azide (1.87 g, 28.8 mmol) was added to a solution of bromoacetic acid (1.00 g,

7.20 mmol) in water (10 mL) at 0 ºC. The solution was allowed to warm to room

temperature, stirred for 18 h, then diluted with 1 M HCl (20 mL) and extracted with


concentrated under reduced pressure, affording 166 as a pale yellow oil (371mg, 51%).

1H NMR (500 MHz, CDCl3): 11.55 (br s, 1H, COOH), 3.95 (2H, s, 2H).

13C NMR

164

(125 MHz, CDCl3): δ 173.9 (CO), 49.8 (CH2). MS (ESI) m/z: 102 [M+H]+, 124

[M+Na]+. The


126

HN

O

N

NN

N

P O

OHHO

H

O

N3

(S)-1-(2-(2-Azidoacetamido)-3-(methylamino)-3-oxopropyl)-1H-1,2,3-triazol-4-

ylphosphonic acid (167)

EDCI (37 mg, 0.20 mmol) was added to a solution of 126 (54 mg, 0.16 mmol),

azidoacetic acid 166 (16 mg, 0.16 mmol) and NaHCO3 (19 mg, 0.23 mmol) in water (5

mL). The solution was stirred at room temperature for 18 h and then subjected to

Dowex anion exchange chromatography. Elution with water gave 167 as a white solid

(32 mg, 60%), mp 125–126 C. Rf = 1.0 on reverse-phase TLC (H2O). IR (KBr disk)

cm–1

: 3000–3500 (br, NH3 + OHs), 2119 (N3) 1644 (C=O). 1H NMR (500 MHz, D2O):

8.04 (s, 1H, triazolyl), 4.83-4.90 (m, 2H, -H), 4.72-4.77 (m, 1H, -H), 3.96 (s, 2H,

CH2N3), 2.65 (s, 3H, NCH3). 13

C NMR (125 MHz, D2O): δ 170.6, 169.9, 144.3 (d, JC,P

= 213.6 Hz, CP), 129.3 (d, JC,P = 28.8 Hz, triazolyl CH), 53.2, 51.5, 50.0, 25.9. 31

P

NMR (120 MHz, D2O): δ 1.67. MS (ESI) m/z: 377 [M+2NaH]+. HRMS (ESI):

observed, 377.0464 [C8H12N8O5P+2NaH]+ requires, 377.0458. [D = 1.8

o (c 1.0,

H2O).

165

~Chapter 4~

BocN

O

OH

OH

H

(S)-2-(tert-Butoxycarbonylamino)-3-hydroxypropanoic acid (172)

This known compound was synthesised according to the Method of Sinou with slight

modifications.91

Boc anhydride (7.20 g, 33 mmol) was added to a solution of L-serine (3.00 g, 29 mmol)

in 1:1 dioxane/1 M aq. NaOH (50 mL) at 0 ºC. The resulting solution was stirred at 0 ºC

for 3 h, then acidified to pH 4 ~ 5 with sat. aq. KHSO4 and extracted with EtOAc (3 ×


under reduced pressure, giving 172 as a colourless oil (4.97 g, 85%). 1H NMR (500

MHz, CDCl3): 5.88 (br d, J = 7.5 Hz, 1H, NH), 4.34 (m, 1H, -H), 3.99 (m, 1H, -

Ha), 3.83(m, 1H, -Hb). 13

C NMR (125 MHz, CDCl3): δ 173.9 (CO), 156.1 (NCO),

80.4 [OCCH3)3], 62.8 (), 55.3 (), 27.5 [CCH3)3]. MS (ESI) m/z: 206 [M+H]+, 228

[M+Na]+. [D = 9.1

o (c 1.0, MeOH) [lit.

127 +8.7 (c 2.8, CHCl3)]. The

1H NMR data

are similar to those reported.91

BocN

O

O

OH

H

(S)-Benzyl 2-(tert-butoxycarbonylamino)-3-hydroxypropanoate (173)

Benzyl bromide (1.80 mL, 15.2 mmol) was added to a solution of 172 (3.00 g, 14.6

mmol) and DIPEA (1.30 mL, 15.2 mmol) in DMF (20 mL) under N2 at 0 ºC. The

resulting solution was allowed to warm up to room temperature, stirred for 18 h, then

diluted with sat. aq. NH4Cl (30 mL) and extracted with EtOAc (3 × 50 mL). The


166


EtOAc/hexanes gave 173 as a colourless oil (3.66 g, 85%). 1H NMR (500 MHz,

CDCl3): 7.27-7.27 (m, 5H, aromatic), 5.71 (d, J = 8.0 Hz, 1H, NH), 5.15 (d, J = 2.5

Hz, 2H, OCH2Ph), 4.39 (m, 1H, -H), 3.95 (m [apparent br d], 1H, -Ha), 3.84 (dd, J =

11.0, 3.5 Hz, 1H, -Hb), 1.42 (s, 9H, t-Bu). 13

C NMR (125 MHz, CDCl3): δ 170.8

(CO2), 155.7 (NCO2), 135.1 (ArC), 128.4 (ArH), 128.2 (ArH), 127.9 (ArH), 80.0

[C(CH3)3], 67.1 (OCH2Ph), 62.9 (), 55.7 (), 28.1 [C(CH3)3]. MS (ESI) m/z: 296

[M+H]+, 318 [M+Na]

+. [D = 19.6 (c 1.0, MeOH) [lit.

128 19.0 (c 1.0, CHCl3)]. The

NMR data are similar to those reported.129

BocN

O

O

N3

H

(S)-Benzyl-3-azido-2-(tert-butoxycarbonylamino)propanoate (174)



solution in toluene (2.50 mL, 6.27 mmol) and DEAD (1.00 mL, 6.35 mmol). The





chromatography. Elution with 1:4 Et2O/hexanes gave 174 as a colourless oil (1.18 g, 72

%). Rf = 0.20 (1:4 Et2O/hexanes). 1H NMR (400 MHz, CDCl3): 7.34 (m, 5H,

aromatic), 5.53 (br d, J = 7.6 Hz, 1H, NH), 5.19 (d, J = 2.8 Hz, 2H, OCH2Ph), 4.50 (m,

1H, -H), 3.71 (d, J = 3.6 Hz, 2H, -H), 1.44 (s, 9H, t-Bu). 13

C NMR (100 MHz,

CDCl3): δ 169.3 (CO2), 154.7 (NCO2), 134.6 (ArC), 128.24 (ArH), 128.17 (ArH), 127.9

(ArH), 80.0 [OC(CH3)3], 67.2 (OCH2Ph), 53.3 (α), 52.2 (), 27.8 [C(CH3)3]. MS (ESI)

167

m/z: 267 [M+Na]+. []D = +8.2 (c 1.0, CHCl3) [lit.

40 +8.0 (c 1.0, CHCl3)]. The

1H and

13C NMR data are similar to those reported.

40

BocNOBn

OH

N

N N

P

O

OEt

OEt

BocNOBn

OH

N NN

PO

OEtEtO

175 176


Thermal


mmol) in toluene (10 mL) under N2 was heated at reflux for 5 h. The toluene was

evaporated and the residue was subjected to flash chromatography. Elution with 1:1




Ru(II)-catalysed

(S)-Benzyl-2-(tert-butoxycarbonylamino)-3-(5-(diethoxyphosphoryl)-1H-1,2,3-



0.624 mmol) and the alkyne 82 (101 mg, 0.624 mmol) in toluene (20 mL) under N2.

The resulting solution was stirred at 60 ºC for 24 h, then diluted with water (20 mL) and




mg, 80%). Rf = 0.20 (1:1 EtOAc/hexanes). IR (thin film) cm–1

: 3305 (NH), 1747 (m,

168

C=O), 1716 (s, NC=O). 1H NMR (500 MHz, CDCl3): 7.94 (s, 1H, triazolyl), 7.29 (m,

5H, aromatic), 5.75 (br s, 1H, NH), 5.15 (s, 2H, OCH2Ph), 4.88-5.00 (m, 3H, -H + -

H), 4.14 (m, 4H, 2 × OCH2CH3), 1.34 (s, 9H, t-Bu), 1.27-1.36, (m, 6H, 2 × OCH2CH3).

13C NMR (125 MHz, CDCl3): δ 169.1 (CO2), 155.0 (NCO2), 140.3 (d, JC,P = 20.4 Hz,

triazolyl CH), 134.8 (ArC), 128.5 (ArH), 128.4 (ArH), 128.2 (ArH), 127.0 (d, JC,P =

219.6 Hz, CP), 80.1 [OCCH3)3], 67.6 (OCH2Ph), 63.64 (d, JC,P = 5.7 Hz, OCH2CH3a),

62.58 (d, JC,P = 5.8 Hz, OCH2CH3b), 53.5 (α), 50.6 (), 28.1 [C(CH3)3], 16.09 (d, JC,P =

4.8 Hz, OCH2CH3a), 16.09 (d, JC,P = 4.9 Hz, OCH2CH3b). 31

P NMR (120 MHz,

CDCl3): δ 4.60. MS (ESI) m/z: 483 [M+H]+, 505 [M+Na]


505.1820, [C21H31N4O7P+Na]+ requires, 505.1823. [D = +9.2º (c 1.0, CHCl3).

Cu(I)-catalysed

(S)-Benzyl-2-(tert-butoxycarbonylamino)-3-(4-(diethoxyphosphoryl)-1H-1,2,3-



0.624 mmol) and alkyne 82 (101 mg, 0.624 mmol) and sodium ascorbate (13 mg,

0.0656 mmol) in 1:1 t-BuOH/water (10 mL). The resulting solution was stirred at room

temperature for 24 h, then diluted with water (20 mL) and extracted with EtOAc (3 ×



with EtOAc gave 176 as a pale yellow oil (265 mg, 88%). Rf = 0.10 (1:1

EtOAc/hexanes). IR (thin film) cm–1

: 3293 (NH), 1744 (m, C=O), 1716 (s, NC=O).1H

NMR (500 MHz, CDCl3): 8.03 (s, 1H, triazolyl), 7.27 (5H, m, aromatic) 5.71 (br d, J

= 7.5 Hz, 1H, NH), 5.12 (d, J = 2.0 Hz, 2H, OCH2Ph), 4.84 (m, 2H, -H), 4.68 (m, 1H,

-H), 4.10 (m, 4H, 2 × OCH2CH3), 1.33 (s, 9H, t-Bu), 1.261, (t, J = 7.0 Hz, 3H,

OCH2CH3a), 1.259, (t, J = 7.0 Hz, 3H, OCH2CH3b). 13

C NMR (125 MHz, CDCl3): δ

169

168.6 (CO2), 154.9 (NCO2), 137.0 (d, JC,P = 239.4 Hz, CP), 134.4 (ArC) 131.2 (d, JC,P =

33.1 Hz, triazolyl CH), 128.4 (ArH), 128.4 (ArH), 128.2 (ArH), 80.4 [OC(CH3)3], 67.8

(OCH2Ph), 62.8 (d, JC,P = 5.8 Hz, OCH2CH3), 53.5 (α), 50.5 (), 27.9 [CCH3)3], 15.9 (d,

JC,P = 6.5 Hz, OCH2CH3). 31

P NMR (120 MHz, CDCl3): δ 7.52. MS (ESI) m/z: 483

[M+H]+, 505 [M+Na]

+. HRMS (ESI): observed, 505.1825, [C21H31N4O7P+Na]

+

requires, 505.1823. [D = +11.1º (c 1.0, CHCl3).

BrH3NOH

O

N NN

PO

OHHO

(S)-1-Carboxy-2-(5-phosphono-1H-1,2,3-triazol-1-yl)ethanaminium bromide (170)

(Method A: From the benzyl ester 175)



vacuum and the residue was subjected to Dowex cation exchange chromatography.


reverse-phase TLC (H2O). IR (KBr disk) cm–1

: 3200–3500 (br, NH3 + OHs), 1600–

1750 (br, obscures C=O). 1

H NMR (600 MHz, D2O): 7.86 (s, 1H, triazolyl), 5.11 (dd,

J = 15.0, 3.6 Hz, 1H,-CH) 4.98 (dd, J = 15.6, 7.2 Hz, 1H, -CH), 4.27 (dd, J = 7.8, 3.6

Hz, 1H, -CH). 13

C NMR (125 MHz, D2O): δ 169.6 (CO), 139.9 (d, JC,P = 18.4 Hz,

triazolyl CH), 135.6 (d, JC,P = 191.8 Hz, CP), 53.2 (α), 49.0 (). 31

P NMR (120 MHz,

D2O): δ –3.14. MS (ESI) m/z: 237 [M+H]+ 259 [M+Na]

+, 281 [M+2NaH]

+ (where M =

free base). HRMS (ESI): observed, 259.0201, [C5H10N4O5P+Na]+ requires, 259.0203.

[]D = 7.7° (c 0.5 H2O).

170

Method B (From the carboxylic acid 35)

A solution of 35 (90 mg, 0.23 mmol) in 33% HBr in acetic acid (2.0 mL, 8.1 mmol) was

stirred at room temperature for 48 h. The reaction mixture was concentrated under

vacuum, and the residue was subjected to Dowex cation exchange chromatography.

Elution with water gave 170 as a white solid (51 mg, 70%). [D = –5.6º (c 0.8, H2O).

Identical in every respect except the optical activity with the material described above.

BrH3N

O

OH

N

N N

P

O

OH

OH

(S)-1-Carboxy-2-(4-phosphono-1H-1,2,3-triazol-1-yl)ethanaminium bromide (171)

(Method A: From the benzyl ester 176)





reverse-phase TLC (H2O). IR (KBr disk) cm

–1: 3300–3500 (br, NH3 + OHs), 1716

(C=O). 1H NMR (600 MHz, D2O): 8.12 (s, 1H, triazolyl), 4.98–5.00 (m, 2H, -CH2),

4.47 (dd, J = 5.4, 4.8 Hz, 1H, α-CH). 13

C NMR (125 MHz, D2O): δ 170.7 (CO), 143.7

(d, JC,P = 217.6 Hz, CP), 130.2 (d, JC,P = 31.7 Hz, triazolyl CH), 54.5 (α), 50.0 (). 31

P

NMR (120 MHz, D2O): δ 1.95; MS (ESI) m/z: 237 [M + H]+

(where M = free base).

HRMS (ESI): observed, 237.0385, [C5H10N4O5P]+ requires, 237.0383. []D = 25.0° (c

0.5 H2O).

171

(Method B: From the carboxylic acid 36)

A solution of 36 (90 mg, 0.23 mmol) in 33% HBr in acetic acid (2.0 mL, 8.1 mmol) was

stirred at room temperature for 48 h. The reaction mixture was concentrated under


Elution with water gave 171 as a white solid (52 mg, 72%). [α]D = –18.8° (c 0.5, H2O).

Identical in every respect except the optical activity with the material descrived above.

Inhibition assay

Solutions of varying concentrations of the „free‟ phosphohistidine analogue 170 or 171

(10 mM, 1 mM, 100 M and 10 M) or sodium bromide (10 mM) were added to a

reaction mixture containing histone (500 g/mL), radiolabeled ATP* (0.2 mM) and

transglutaminase from guinea pig liver (200 g/mL) in 0.1 M tris buffer (pH 9.0) at

room temperature. Alongside these samples, separate control reactions were prepared

that contained either no inhibitor (positive control), no kinase or no histone (negative

controls). Each sample was incubated at 37 ºC for 1 h, and the reaction was quenched

by the addition of 2 × SDS sample buffer (12 L). The phophorylated histone was

subsequently separated by polyacrylamide (15%) gel electrophoresis, and the

phosphorylation of the histone was quantified with a phosphoimager.

*[32

P] ATP 3000Ci/mmol

172

~Chapter 5~

FmocN

O

O

OH

H

(S)-Benzyl-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-hydroxypropanoate

(178)

Benzyl bromide (1.40 mL, 11.8 mmol) was added to a solution of Fmoc-L-serine

monohydrate (2.00 g, 5.79 mmol) and DIPEA (1.00 mL, 11.6 mmol) in DMF (20 mL)

at 0 °C. The solution was allowed to warm to room temperature, stirred for 18 h, then

diluted with sat. aq. NH4Cl (30 mL) and extracted with EtOAc (3 × 50 mL). The



EtOAc/hexanes gave 178 as a white solid (2.18 g, 90%). 1H NMR (500 MHz, CDCl3):

7.81 (d, J = 7.5 Hz, 2H), 7.70 (br d, J = 5.5 Hz, 2H), 7.46 (t, J = 7.5 Hz, 4H), 6.47 (br d,

J = 5.0 Hz, 1H, NH), 5.27 (s, 2H, OCH2Ph), 4.66 (m, 1H, -H), 4.53 (m[apparent t],

1H, H-10'a), 4.43 (m [apparent t], 1H, H-10'b), 4.66 (t, J = 7.0 Hz, 1H, H-9'), 4.12

(m[apparent br d], 1H, -Ha), 3.99 (m[apparent br d], 1H, -Hb). 13

C NMR (125 MHz,

CDCl3): δ 170.4, 156.3, 143.5, 143.3, 140.9, 134.8, 128.2, 128.0, 127.7, 127.3, 126.7,

119.6, 67.0, 66.9, 62.4, 56.0, 46.6. MS (ESI) m/z: 440 [M+Na]+. []D = +0.7 (c 1.0,

DCM) [lit.94

+0.6 (c 1.0, DCM)]. The 1H and

13C NMR data are similar to those

reported.94

FmocN

O

OBn

N3

H

(S)-Benzyl-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-azidopropanoate (179)


mmol) in anhydrous THF (40 mL) at –78 ºC under N2 was treated with a 2.5 M HN3

173

solution in toluene (2.30 mL, 5.75 mmol) and DEAD (910 mL, 5.78 mmol). The





chromatography. Elution with 1:4 Et2O/hexanes gave 179 as a white solid (1.38 g, 65

%). mp 70–72 ºC. Rf = 0.15 (1:1 Et2O/hexanes). IR (thin film) cm–1

: 3337 (NH), 2107

(N3) 1650-1750 (br, C=O + NC=O). 1H NMR (500 MHz, CDCl3): 7.80 (d, J = 7.5 Hz,

2H), 7.64 (br d, J = 7.0 Hz, 2H), 7.33-7.46 (m, 9H), 5.85 (br d, J = 7.5 Hz, 1H, NH),

5.26 (d, J = 4.5 Hz, 2H, OCH2Ph), 4.64 (m, 1H, -H), 4.45 (m, 2H, H-10'), 4.26 (t, J =

7.5Hz, 1H, H-9'), 3.79 (br s, 2H, -H). 13

C NMR (125 MHz, CDCl3): δ 169.2 (CO2),

155.6 (NCO2), 143.6, 143.5, 141.2, 134.8 (ArC), 128.6 (ArH), 128.2 (ArH), 127.6

(ArH), 127.0, 125.0, 119.9, 67.8, 67.2, 53.9 (α), 52.4 (), 46.9 (C-9'). MS (ESI) m/z:

443 [M+H]+, 465 [M+Na]

+. HRMS (ESI): observed, 465.1539, [C25H22N4O4+Na]

+

requires 465.1533. [D = +25.7º (c 1.0, CHCl3).

FmocN

O

OBn

N

N N

P

O

OEtOEt

HFmocN

O

OBn

NN

N

PO

EtO

H

180 181

OEt


Thermal


mmol) in toluene (10 mL) was heated at reflux for 5 h. The toluene was evaporated

under vacuum, and the residue was subjected to flash chromatography. Elution with 2:3

174

EtOAc/hexanes gave 180 as a pale yellow oil (53 mg, 39%). Further elution with

EtOAc gave 181 as a pale yellow oil (56 mg, 41%).

Ru(II)-catalysed

(S)-Benzyl-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(5-

(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate (180)



resulting solution was stirred at 60 °C for 24 h, then diluted with water (10 mL) and

extracted with EtOAc (3 × 100 mL). The organic extract was washed with brine (10

mL), dried and concentrated under reduced pressure. The residue was subjected to flash


mg, 78%). Rf = 0.20 (1:1 EtOAc/hexanes). IR (thin film) cm–1

: 3272 (NH), 1700-1750

(br, C=O + NC=O). The NMR spectra showed the presence of the rotamers. 1H NMR

(500 MHz, CDCl3): [7.99, 7.98 (2 s, 1H, triazolyl)], 7.74 (d, J = 7.5 Hz, 2H), 7.54 (d,

J = 7.5 Hz, 2H), 7.23-7.43 (m, 9H), [6.42, 6.26 (2 br d, J = 7.5 Hz, 1H, NH)], 5.25 (d, J

= 5.0 Hz, 2H, OCH2Ph), 4.98–5.21 (m, 3H, -H + -H), 4.07-4.70 (m, 7H, H-10' + H-9'

+ 2 × OCH2CH3), 1.33 (t, J = 7.0 Hz, 3H, OCH2CH3a), 1.31 (t, J = 9.5 Hz, 3H,

OCH2CH3b). 13

C NMR (125 MHz, CDCl3): δ [168.7, 168.4 (2 s, CO2)], [156.9, 155.8,

(2 s, NCO2)], [143.7, 143.5, 141.44, 141.39 (4 s)], [141.15, 141.14, 140.59, 140.56 (4

s)], [140.34, 140.27 (2 d, JC,P = 20.3 Hz, triazolyl CH)], [134.7, 134.6 (2 s, ArC)],

[130.3, 128.3 (2 s)], 128.6 (ArH), 128.53 (ArH), 128.47 (ArH), [127.7, 126.63, 126.62

(3 s)], [127.3, 127.2 (2 d, JC,P = 221.4 Hz, CP)], [127.01, 126.99 (2 s)], [126.5, 126.4 (2

s)], [120.0, 119.9 (2 s)], [67.9, 67.4 (2 s)], [65.0, 64.0 (2 s)], [63.99, 63.92 (2 d, JC,P =

5.9 Hz, OCH2CH3a)], [63.8, 63.7 (2 d, JC,P = 5.5 Hz, OCH2CH3b)], [54.3, 54.0 (2 s, α)],

[50.5, 50.4 (2 s, )], 46.9 (C-9'), 16.11 (d, JC,P = 6.0 Hz, OCH2CH3a), 16.07 (d, JC,P =

175

5.2 Hz, OCH2CH3b). 31

P NMR (120 MHz, CDCl3): δ 4.54. MS (ESI) m/z: 605 [M+H]+,

627 [M+Na]+. HRMS (ESI): observed, 605.2168, [C31H33N4O7P+H]

+ requires,

605.2160. [D = +2.3° (c 1.0, CHCl3).

Cu(I)-catalysed

(S)-Benzyl-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-

(diethoxyphosphoryl)-1H-1,2,3-triazol-1-yl)propanoate (181)




diluted with water (20 mL) and extracted with EtOAc (3 × 100 mL). The organic extract


residue was subjected to flash chromatography. Elution with EtOAc gave 181 as a white

solid (116 mg, 85%), mp 100–101 C. Rf = 0.10 (1:1 EtOAc/hexanes). IR (thin film)

cm–1

: 3271 (NH), 1700-1750 (br, C=O + NC=O). 1H NMR (500 MHz, CDCl3): 8.10

(s, 1H, triazolyl), 7.73 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 7.0 Hz, 2H), 7.22-7.39 (m, 9H),

6.31 (br d, J = 7.5 Hz, 1H, NH), 5.17 (br s, 2H, OCH2Ph), 4.90 (m[apparent br d], 2H,

-H), 4.83 (m, 1H, -H), 4.33 (m[apparent t], 2H, H-10'), 4.09-4.22 (m, 5H, H-9' + 2 ×

OCH2CH3), 1.26-1.32 (m, 6H, 2 × OCH2CH3). The 13

C NMR spectra showed the

presence of the rotamers. 13

C NMR (125 MHz, CDCl3): δ 168.3 (CO2), 155.7 (NCO2),

143.3, 141.0, 137.2 (d, JC,P = 239.1 Hz, CP), 134.4 (ArC), 131.8 (d, JC,P = 33.5 Hz,

triazolyl CH), 128.5 (ArH), 128.46 (ArH), 128.3 (ArH), 127.6, [126.92, 126.88 (2 s)],

[124.93, 124.85 (2 s)], 119.8, 68.0, 67.2, 62.9 (d, JC,P = 5.7 Hz, OCH2CH3), 54.0 (α),

50.4 (), 46.8 (C-9') 16.03 (br s, OCH2CH3a), 15.98 (d, JC,P = 1.0 Hz, OCH2CH3b). 31

P

NMR (120 MHz, CDCl3): δ 7.56. MS (ESI) m/z: 605 [M+H]+, 627 [M+Na]

+. HRMS

176

(ESI): observed, 627.1975, [C31H33N4O7P+Na]+ requires, 627.1979. [D = +10.4º (c

1.0, CHCl3).

~Chapter 6~

N

NHI

4-Iodopyrazole (201)

This known compound was synthesised according to the method of Casida with slight

modification.97

Ceric ammonium nitrate (CAN) (5.65 g, 10.3 mmol) was added to a solution of

pyrazole (1.17 g, 17.2 mmol) and iodine (2.61 g, 10.3 mmol) in MeCN (50 mL) at room

temperature. The solution was stirred at room temperature for 5 h, then diluted with

10% aq. Na2SO3 (50 mL) and extracted with EtOAc (3 × 50 mL). The organic extract



201 as a white solid (3.11 g, 93%). 1H NMR (500 MHz, CDCl3): 11.95 (br s, 1H,

NH), 7.61 (br s, pyrazolyl, 2H). 13

C NMR (125 MHz, CDCl3): δ 138.7 (pyrazolyl CH),

56.4 (CI). MS (ESI) m/z: 194 [M+H]+. The

1H and

13C NMR data are similar to those

reported.97

N

NTsI

4-Iodo-1-tosyl-1H-pyrazole (203)

This known compound was synthesised according to the method of Obala with slight

modification.98

Tosyl chloride (3.33 g, 17.5 mmol) was added to a solution of 201 (3.05 g, 15.7 mmol)

and pyridine (4.80 mL, 59.5 mmol) in DCM (10 mL) at room temperature. The solution

was stirred at room temperature for 3 h, then diluted with 1M HCl (20 mL) and

177

extracted with DCM (3 x 40 mL). The organic extract was washed with brine (20 mL),

dried and concentrated under reduced pressure. The residue was recrystallised from

EtOH (50 mL), and 203 was obtained as a white solid (3.44 g, 63%). 1H NMR (500

MHz, CD3OD): ; 8.15 (s, 2H, pyrazolyl), 7.70 (d, J = 6.5 Hz, 2H, aromatic), 7.23 (d, J

= 8.0 Hz, 2H, aromatic), 2.36 (s, 3H, CH3). 13

C NMR (125 MHz, CD3OD): δ 143.3,

141.9, 140.3, 129.9, 126.9, 58.9, 21.3. MS (ESI) m/z: 348 [M+H]+. The

1H NMR data

are similar to those reported.98

N

NTsP

O

EtO

EtO

Diethyl-1-tosyl-1H-pyrazol-4-ylphosphonate (204)

Palladium acetate (6 mg, 10 mol%) was added to a solution of 203 (100 mg, 0.29

mmol), diethyl phosphite (80 L, 0.63 mmol), triethylamine (80 L, 0.58 mmol) and

triphenyl phosphine (23 mg, 30 mol%) in EtOH (10 mL) under N2. The resulting

solution was heated at reflux for 6 h, then diluted with sat. aq. NH4Cl (10 mL) and

extracted with EtOAc (3 × 30 mL). The organic extract was washed with brine (20 mL),


chromatography. Elution with 1:1 EtOAc/hexanes gave 204 as a pale yellow oil (52 mg,

50%). Rf = 0.15 (1:1 EtOAc/hexanes). IR (thin film) cm–1

: 1386 (S=O). 1H NMR (600

MHz, CDCl3): 8.40 (br s, 1H, pyrazolyl), 7.87 (d, J = 5.5 Hz, aromatic), 7.80 (d, J =

1.0 Hz 1H, pyrazolyl), 7.31 (d, J = 5.5 Hz 2H, aromatic), 4.06 (m, 4H, 2 × OCH2CH3),

2.37 (s, 3H, CH3), 1.26 (dt, J = 5.5, 2.0 Hz, 6H, 2 × OCH2CH3). 13

C NMR (150 MHz,

CDCl3): δ 146.6 (ArC), 145.8 (d, JC,P = 13.4 Hz, pyrazolyl), 135.9 (d, JC,P = 23.4 Hz,

pyrazolyl), 132.8 (ArC), 130.1 (ArH), 128.4 (ArH), 111.40 (d, JC,P = 217.0 Hz, CP),

62.4 (d, JC,P = 5.6 Hz, OCH2CH3), 21.6 (CH3), 16.1 (d, JC,P = 6.5 Hz, OCH2CH3). 31

P

178

NMR (120 MHz, CDCl3): δ 11.74. MS (ESI) m/z: 359 [M+H]+. HRMS (ESI): observed,

359.0828, [C14H19N2O5PS+H]+ requires, 359.0825.

N

NHP

O

EtO

EtO

Diethyl-1H-pyrazol-4-ylphosphonate (202)

(Method A: Removal of the tosyl group)

KOH (160 mg, 2.9 mmol) was added to a solution of 204 (200 mg, 0.56 mmol) in 1:1

dioxane/H2O (10 mL) at room temperature. The solution was heated at reflux for 6 h,

and then the solvents were evaporated. The residue was diluted with sat. aq. NH4Cl (30

mL) and extracted with EtOAc (3 × 50 mL). The residue was subjected to flash

chromatography. Elution with EtOAc gave 202 as a white solid (61 mg, 53%), mp 85–

87 C. Rf = 0.10 (EtOAc). IR (thin film) cm–1

: 3152 (NH); 1H NMR (300 MHz, CDCl3):

7.81 (s, 2H, pyrazolyl), 3.97 (m, 4H, CH2), 1.20 (t, J = 6.9 Hz, 6H, CH3). 13

C NMR

(75 MHz, CDCl3): δ 138.2 (d, JC,P = 17.1 Hz, CP), 107.6 (pyrazolyl CH), 104.6

(pyrazolyl CH), 62.5 (d, JC,P = 5.5 Hz, CH2), 16.4 (d, JC,P = 6.5 Hz, CH3). 31

P NMR

(120 MHz, CDCl3): δ 16.49. MS (ESI) m/z: 205 [M+H]+, 227 [M+Na]

+. HRMS (ESI):

observed, 205.0735, [C7H13N2O3P+H]+ requires, 205.0737.

(Method B: Direct phosphonation of 4-iodopyrazole)

Palladium acetate (6 mg, 10 mol%) was added to a solution of 201 (56 mg, 0.29 mmol),

diethyl phosphite (80 L, 0.63 mmol), triethylamine (80 L, 0.58 mmol) and triphenyl

phosphine (23 mg, 30 mol%) in EtOH (10 mL) under N2. The resulting solution was

heated at reflux for 6 h, then diluted with sat. aq. NH4Cl (10 mL) and extracted with



179

chromatography. Elution with EtOAc gave 202 as a white solid (49 mg, 47%). Identical

in every respect with the material described above.

BocNOMe

O

OTs

H

(S)-Methyl-2-(tert-butoxycarbonylamino)-3-(tosyloxy)propanoate (208)

This known compound was synthesised according to the method of Silks105

with slight

modifications.


and pyridine (4.50 mL, 55.6 mmol) in DCM (20 mL) at 0 C. The solution was allowed

to warm to room temperature, stirred for 40 h and then diluted with sat. aq. NH4Cl (50

mL). The layers were separated, and the aqueous phase was extracted with DCM (3 ×



with 1:9 EtOAc/hexanes gave 208 as a white solid (4.45 g, 87%). 1H NMR (500 MHz,

CDCl3): 7.72 (d, 2H, J = 8.5 Hz, ), 7.32 (d, 2H, J = 8.0 Hz, ), 5.3 (br d, 1H, J = 8.0

Hz, NH), 4.45-4.50 (m, 1H, -H), 4.35 (dd, 1H, J = 10.5, 3.0 Hz, -Ha), 4.25 (dd, 1H, J

= 10.0, 3.0 Hz, -Hb), 3.65 (s, 3H, OCH3), 2.41 (s, 3H, CCH3), 1.38 (s, 9H, t-Bu). 13

C

NMR (125 MHz, CDCl3): δ 168.9 (CO2), 154.9 (NCO2), 145.1, 132.2, 129.8, 127.9,

80.3 [OCCH3)3], 69.4 (-H), 52.8 (OCH3), 52.7 (-H), 28.1 [C(CH3)3], 21.5 (CCH3).

MS (ESI) m/z: 396 [M+Na]+. []D = +3.3 (c 1.0, MeOH) [lit.

130 +3.0 (c 2.0, MeOH)].

The 1H and

13C NMR data are similar to those reported.

105

180

O

OBocNH

(S)-tert-Butyl 2-oxooxetan-3-ylcarbamate (218)

This known compound was synthesised according to the method of Levesque with

slight modifications.131

DEAD (0.950 mL, 6.02 mmol) was added to a solution of 172 (1.00 g, 4.87 mmol) and

triphenylphosphine (1.55 g, 5.92 mmol) in THF (20 mL) at –78 ºC. The solution was

allowed to warm to room temperature and stirred for 5 h, and then volatile components

were removed under reduced pressure. The residue was subjected to flash

chromatography. Elution with 1:4 EtOAc/hexanes gave the title lactone in a slightly

impure state. Further purification was achieved by recrystallisation from CHCl3,

affording 218 as a white solid (410 mg, 45 %). 1H NMR (500 MHz, CDCl3): ; 5.63 (br

d, 1H, J = 6.5 Hz, NH), 5.06 (m [apparent q], 1H, -H), 4.45-4.43 (m, 2H, -H), 1.42

(s, 9H, t-Bu). 13

C NMR (125 MHz, CDCl3): δ; 169.7 (CO2), 154.6 (NCO2), 81.2

[OC(CH3)3], 66.5 (-H), 59.3 (-H), 28.1 [CCH3)3]. MS (ESI) m/z: 188 [M+H]+, 210

[M+Na]+. []D = 27.1 (c 1.0, MeCN) [lit.

132 27.0 (c 1.0, MeCN)]. The

1H NMR

data are similar to those reported.131

N N N

OO

N

Azodicarboxylic acid dipiperidide (223)

This known compound was synthesised according to the method of Kanety-Londner.113

Piperidine (1.96 g, 23.0 mmol) was added to a solution of DEAD (1.00 g, 5.74 mmol) in

Et2O (10 mL), and the resulting mixture was stirred at room temperature for 18 h. The

precipitate was filtered, washed with Et2O and then recrystallised from CHCl3,

providing 223 as an orange solid (1.01 g, 70%). 1H NMR (500 MHz, CD3OD): 3.65

181

(m, 4H), 3.46 (m, 4H), 1.71 (m, 8H), 1.58 (m, 4H). 13


162.1 (CO), 46.7, 45.6, 27.0, 26.5, 25.0. MS (ESI) m/z: 275 [M+Na]+.

NHN

I

4(5)-Iodoimidazole (226)

This known compound was synthesised according to the method of Still with slight

modifications.114

A solution of iodine (6.71 g, 26.5 mmol) in 20 % aqueous potassium iodide (50 mL)

was added to a solution of imidazole (1.00 g, 14.7 mmol) in 2.0 M aqueous NaOH (100

mL). The resulting mixture was stirred at room temperature for 18 h and then

neutralised by acetic acid. The produced precipitate was filtered and then washed with

water. The product was dried and used for the next step without further purification. A

mixture of the crude product and sodium sulfate (8.40 g, 66.7 mmol) was heated at

reflux in 30 % aqueous EtOH (100 mL) for 18 h, and then the EtOH was removed under

reduced pressure. The remaining mixture was filtered, and the filtrate was extracted

with Et2O (3 × 100 mL). The organic extract was washed with brine (50 mL), dried and

concentrated under reduced pressure. The residue was recystallised from EtOH,

providing 226 as a white solid (1.71 g, 60%). 1H NMR (500 MHz, CD3OD): 7.62 (d, J

= 1.0 Hz, 1H, imidazolyl), 7.19 (d, J = 1.5 Hz, 1H, imidazolyl). 13

C NMR (125 MHz,

CD3OD): δ 138.9 (pyrazolyl), 124.8 (CI). MS (ESI) m/z: 194 [M+H]+. The

1H NMR

data are similar to those reported.114

182

NTsN

I

4-Iodo-1-p-toluenesulfonylimidazole (229)

This known compound was synthesised according to the method of Still with slight

modifications.114


and triethylamine (1.50 mL, 10.8 mmol) in THF (20 mL). The resulting solution was

stirred at room temperature for 18 h, then diluted with water and extracted with EtOAc

(3 × 50 mL). The organic extract was washed with brine (50 mL), dried and

concentrated under reduced pressure. The residue was recrystallised from EtOH,

providing 229 as a white solid (2.51 g, 70%). 1H NMR (500 MHz, CD3OD): 8.17 (d, J

= 1.5 Hz, 1H, imidazolyl), 7.95 (d, J = 8.5 Hz, 2H, aromatic), 7.73 (d, J = 1.5 Hz, 1H,

imidazolyl), 7.47 (d, J = 8.5 Hz, 2H, aromatic), 2.44 (s, 3H, CH3). 13

C NMR (125 MHz,

CD3OD): δ 148.6, 140.0, 135.6, 131.8, 130.1, 128.9, 124.5, 21.7. MS (ESI) m/z: 348

[M+H]+. The


114

184

References

1. Burnett, G.; Kennedy, E. P. J Biol Chem 1954, 211, 969-980.

2. Mandell, J. W. Am. J. Pathol 2000, 163, 1687-1698.

3. Besant, P. G.; Tan, E.; Attwood, P. Int J Biochem Cell Biol 2003, 35, 297-309.

4. Weigel, N. L.; Moore, N. L. Nucl. Recept. Signaling 2007, 5, 1-13.

5. Attwood, P. V.; Besant, P. G.; Zu, X. L.; Piggott, M. J. Amino Acids 2007, 32, 145-

156.

6. Hultquist, D. E.; Moyer, R. W.; Boyer, P. D. Biochemistry. 1966, 5, 322-331.

7. Pirrung, M. C. Chem. Biol. 1999, 6, 167–175.

8. Besant, P. G.; Attwood, P. V. Biochim. Biophys. Acta 2005, 1754, 281-290.

9. Severin, S. E.; Yudelovich, R. Y. Biokhimiya 1947, 12, 105-110.

10. Muller, T.; Rathlev, T.; Rosenberg, T. Biochim. Biophys. Acta 1956, 19, 563-564.

11. Klumpp, S.; Krieglstein, J. Eur. J. Biochem. 2002, 269, 1067-1071.

12. Stock, A. M.; Rovinson, V. L.; Goudreau, P. N. Annu. Rev. Biochem 2000, 69, 183-

215.

13. Matsushita, M.; Janda, K. D. Biorg. Med. Chem. 2002, 10, 855-867.

14. Saito, H. Chem. Rev. 2001, 101, 2497-2509.

15. Barrett, J. F.; Goldschmidt, R. M.; Lawrence, L. E.; Foleno, B.; Chen, R.; Demers,

J. P.; Johnson, S.; Kanojia, R.; Fernandez, J.; Bernstein, J.; Licata, L.; Donetz, A.;

Huang, S.; Hlasta, D. J.; Macielag, M. J.; Ohemeng, K.; Frechette, R.; Frosco, M. B.;

Klaubert, D. H.; Whiteley, J. M.; Wang, L.; Hoch, J. A. Proc. Natl. Acad. Sci. USA.

1998, 95, 5317–5322.

16. Steeg, P. S.; Palmieri, D.; Ouatas, T.; Salerno, M. Cancer Lett. 2001, 190, 1-12.

17. Schneider, B.; Norda, A.; Karlsson, A.; Veron, M.; Deville-Bonne, D. Protein Sci.

2002, 11, 1648-1656.

18. Tan, E.; Besant, P. G.; Zu, X. L.; Turck, C. W.; Bogoyevitch, M. A.; Lim, S. G.;

Attwood, P. V.; Yeoh, G. C. Carcinogenesis 2004, 11, 2083-2088.

19. Stock, J. B.; Stock, A. M.; Mottonen, J. M. Nature. 1990, 344, 395-400.

20. Modro, T. A. ACS Symp. Ser. 1981, 171, 619–622.

21. Duclos, B.; Marcandier, S.; Cozzone, A. J. Methods Enzymol. 1991, 210, 10-21.

22. Hultquist, D. E. Biochim. Biophys. Acta 1968, 153, 329-340.

23. Boyle, W. J.; Geer, P. V. D.; Hunter, T. Methods Enzymol. 1991, 201, 110-148.

24. Besant, P. G.; Attwood, P. V. Int J Biochem Cell Biol 2000, 32, 243-253.

25. Zu, X. L.; Besant, P. G.; Imhof, A.; Attwood, P. V. Amino Acids 2007, 32, 347–357.

26. Napper, S.; Kindrachuk, J.; Olson, D. J. H.; Ambrose, S. J.; Dereniwsky, C.; Ross,

A. R. S. Anal. Chem. 2003, 75, 1741-1747.

27. Sites, D. P. Basic & Clinical Immunology 1976.

28. Alberts, D. Molecular Biology of the Cell 1983.

29. Lipman, N. S.; Jackson, L. R.; Trudel, L. J.; Weis-Garcia, F. ILAR Journal 2005, 46,

258-268.

30. Köhler, G.; Milstein, C. Nature. 1975, 256, 495-497.

31. Sell, S. Immunology, Immunopathology and Immunity 1987.

32. Lasker, M.; Bui, C. D.; Besant, P. G.; Sugawara, K.; Thai, P.; Medzihradszky, G.;

Turck, C. W. Protein Sci. 1999, 8, 2177-2185.

33. Pirrung, M. C.; James, K. D.; Rana, V. S. J. Org. Chem. 2000, 65, 8448-8453.

34. Dr Paul Besant. Personal Communication. School of Biomedical, Biomolecular and

Chemical Sciences, The University of Western Australia.

35. Truel, I.; Mohamed-Hachi, A.; About-Jaudet, E.; Colligoron, N. Synth. Commun.

1997, 27, 1165.

36. Schenkles, C.; Emi, B.; Reymond, J. L. Bioorg. Med. Chem. Lett. 1999, 9, 1443-

1446.

185

37. Schneider, H.; Sigmund, G.; Schricker, B.; Thirring, K.; Bemer, H. J. Org. Chem.

1993, 58, 683.

38. Schricker, B.; Thirring, K.; Berner, H. Bioorg. Med. Chem. Lett. 1992, 5, 387.

39. Prof. Paul Attwood. Personal Communication. School of Biomedical, Biomolecular

and Chemical Sciences, The University of Western Australia.

40. Kee, J.-M.; Villani, B.; Carpenter, L. R.; Muir, T. W. J. Am. Chem. Soc. 2010, 132,

14327–14329.

41. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057-3064.

42. Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless, K. B.;

Fokin, V. V.; Jia, G. J. Am. Chem. Soc. 2005, 127, 15998-15999.

43. McAllister, T. E.; Nix, M. G.; Webb, M. E. Chemical Communications (Cambridge)

2010.

44. Boger, D. L.; Honda, T.; Menezes, R. F.; Colletti, S. L.; Dang, Q.; Yang, W. J. Am.

Chem. Soc. 1994, 116, 82-92.

45. Trost, B. M.; Rudd, M. T. Org. Lett. 2003, 5, 4599-4602.

46. Mitsunobu, O. Synthesis 1981, 1-28.

47. Lipshutz, B. H.; Chung, D. W.; Rich, B.; Corral, R. Org. Lett. 2006, 8, 5069-5072.

48. Dondoni, A.; Giovannini, P. P.; Massi, A. Org. Lett. 2004, 6, 2929-2931.

49. Xun, S.; LeClair, G.; Zhang, J.; Chen, X.; Gao, J. P.; Wang, Z. Y. Org. Lett. 2006,

8, 1697-1700.

50. Cohen, S. G.; Zand, R.; Steel, C. J. Am. Ceram. Soc. 1961, 83, 2895-2900.

51. Vuilhorgne, M.; Malpart, J.; Mutti, S.; Mignani, S. J. Heterocycl. Chem. 2003, 40,

159-162.

52. Steinberg, G. M. J. Org. Chem. 1950, 15, 637-647.

53. Yamauchi, Y.; Yuki, M.; Tanabe, Y.; Miyake, Y.; Inada, Y.; Uemura, S.;

Nishibayashi, Y. J. Am. Chem. Soc. 2008, 130, 2908-2909.

54. Saalfrank, R. W.; Welch, A.; Haubner, M.; Bauer, U. Liebigs Annalen 1996, 2, 171-

181.

55. Balthazor, T. M.; Grabia, R. C. J. Org. Chem. 1980, 45, 5425-5426.

56. Yao, Q.; Levchik, S. Tetrahedron Lett. 2006, 47, 277-281.

57. Hägele, G.; Goudetsidis, S.; Wilke, E.; Seega, J.; Blum, H.; Murray, M.

Phosphorus, Sulfur Silicon Relat. Elem. 1990, 48, 131-140.

58. Jacobsen, M. F.; Andersen, C. S.; Knudsen, M. M.; Gothelf, K. V. Org. Lett. 2007,

9, 2851-2854.

59. Patrick, D. A.; Bakunov, S. A.; Bakunova, S. M.; Kumar, E. V. K. S.; Lombardy, R.

J.; Jones, S. K.; Bridges, A. S.; Zhirnov, O.; Hall, J. E.; Wenzler, T.; Brun, R.; Tidwell,

R. R. J. Med. Chem. 2007, 50, 2468-2485.

60. Boyall, D.; Lopez, F.; Sasaki, H.; Frantz, D.; Carreira, E. M. Org. Lett. 2000, 2,

4233-4236.

61. Huisgen, R. Angew. Chem. 1963, 75, 604.

62. Gothelf, K. V.; Jørgensen, K. A. Chem. Rev. 1998, 98, 863-909.

63. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int.

Ed. 2002, 41, 2596-2599.

64. Johnson, J. A. Org. Lett.

65. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128-1137.

66. Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.;

Fokin, V. V. J. Am. Chem. Soc. 2008, 130, 14900.

67. Kirkley, J. E.; Goldstein, A. L.; Naylor, P. H. Immunobiology 2001, 203, 601-615.

68. Lesma, G.; Meschini, E.; Recca, T.; Sacchetti, A.; Silvani, A. Tetrahedron 2007, 53,

5567-5578.

69. Coeffard, V.; Beaudet, I.; Evain, M.; Grognec, E. L.; Quintard, J. P. Eur. J. Org.

Chem. 2008, 19, 3344-3351.

186

70. Angione, M. C.; Miller, S. J. Tetrahedron 2006, 62, 5254-5261.

71. Lee, S. J.; Kim, E.; Seo, M. L.; Do, Y.; Lee, Y.-A.; Lee, S. S.; Jung, J. H.; Kogiso,

M.; Shimizu, T. Tetrahedron 2008, 64, 1301-1308.

72. Feenstral, R. W.; Stokkmgreef, E. H. M.; Relchwem, A. M.; Lousberg, W. B. H.;

Ottenheqm, H. C. J. Tetrahedron 1990, 46, 1745-1756.

73. Mak, C. C.; Brik, A.; Lerner, D. L.; Elder, J. H.; Morris, G. M.; Olsonb, A. J.;

Wong, C. H. Biorg. Med. Chem. 2003, 11, 2025–2040

74. Stocking, E. M.; Schwarz, J. N.; Senn, H.; Salzmann, M.; Silks, L. A. Journal of the

chemical society, Perkin Transactions 1997, 1, 2443-2447.

75. Sims, J. W.; Schmidt, E. W. J. Am. Chem. Soc. 2008, 130, 11149-11155.

76. Davoli, P.; Forni, A.; Moretti, I.; Prati, F. Tetrahedron: Asymmetry 1995, 6, 2011-

2016.

77. Maruyama, K.; Hashimoto, M.; Tamiaki, H. J. Org. Chem. 1992, 57, 6143-6150.

78. Angione, M. C.; Miller, S. J. Tetrahedron 2006, 62, 5254-5261.

79. Conchillo, A.; Camps, F.; Messeguer, A. J. Org. Chem. 1990, 55, 1728-1735.

80. Schmittberger, T.; Cotte, A.; Waldmann, H. Chemical Communications

(Cambridge) 1998.

81. Levengood, M. L.; Donk, W. A. Nat. Protoc. 2006, 6, 3001-3010.

82. Brinkley, M. Bioconjugate Chem. 1992, 3, 2-13.

83. Brandon, D. L.; Binder, R. G. Food and Agricultural Immunology 2006, 17, 53-61.

84. Helling, S. A. F.; Zhang, S. O. S.; Lloyd, S. H. I. K. O.; Livingston, P. O. Cancer

Immunol Immunother 1995, 41, 185-192.

85. Lai, J. H.; Marsilje, T. H.; Choi, S.; Nair, S. A.; Hangauer, D. G. J.Pept.Res. 1998,

51, 271-281.

86. Zhang, H.; Duan, Z.; Wang, L.; Zhang, Y.; Wang, S. J. Agric. Food. Chem. 2006,

54, 4499-4505.

87. Hermanson, G. T. Bioconjugate Techniques 1996.

88. Galibert, M.; Dumy, P.; Boturyn, D. Angew. Chem. Int. Ed. 2009, 48, 2576 –2579.

89. Gilmour, R.; Foster, E.; Sheng, Q.; McClain, J. R.; Riley, A.; Sun, P.-M.; Ng, W.-

L.; Yan, D.; Nicas, T. I.; Henry, K.; Winkler, M. E. J. Bacteriol., 187, 8196–8200.

90. Stephenson, K.; Yamaguchi, Y.; Hoch, J. A. J. Biol. Chem. 2000, 275, 38900.

91. Bayardon, J.; Sinou, D. Tetrahedron: Asymmetry 2005, 16, 2965-2972.

92. Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214.

93. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154.

94. Huang, Y.; Dey, S.; Zhang, X.; nnichsen, F. S.; Garner, P. J. Am. Chem. Soc. 2004,

126, 4626-4640.

95. Gajda, T.; Andrzej Zwierzak Synthesis 1976, 4, 243-244.

96. Brown, Z. Z.; Schafmeister, C. E. Org. Lett. 2010, 12, 1436-1439.

97. Sammelson, R. E.; Casida, J. E. J. Org. Chem. 2003, 68, 8075-8079.

98. Heinisch, G.; Holzer, W.; Obala, C. Monatsh. Chem. 1988, 119, 253--262.

99. Felding, J.; Kristensen, J.; Bjerregaard, T.; Sander, L.; Vedsø, P.; Begtrup, M. J.

Org. Chem. 1999, 64, 4196-4198.

100. Gelman, D.; Jiang, L.; Buchwald, S. L. Org. Lett. 2003, 5, 2315-2318.

101. Bessmertnykh, A.; Douaihy, C. M.; Muniappan, S.; Guilard, R. Synthesis 2008, 10,

1575-1579.

102. Liu, Y.; Shen, L.; Prashad, M.; Tibbatts, J.; Repic, O.; Blacklock, T. J. Org.

Process Res. Dev. 2008, 12, 778-780.

103. Shirley, D. A.; Roussel, P. A. J. Am. Chem. Soc. 1953, 75, 375.

104. Roth, S.; Thomas, N. R. Synthesis 2010, 4, 607-609.

105. Stocking, E. M.; Schwarz, J. N.; Senn, H.; Salzmann, M.; Silks, L. A. Journal of

the chemical society, Perkin Transactions 1997, 1, 2443-2447.

106. Spetzler, J. C.; Jensen, T. H. J. Pept. Sci. 1999, 5, 582-592.

187

107. Amold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem. Soc. 1985, 107, 7105-

7109.

108. Paradis-Bleau, C.; Beaumont, M.; Sanschagrin, F.; Voyerb, N.; Levesqu, R. C.

Biorg. Med. Chem. 2007, 15, 1330-1340.

109. Nakatani, S.; Hidaka, K.; Ami, E. i.; Nakahara, K.; Sato, A.; Nguyen, J.-T.;

Hamada, Y.; Hori, Y.; Ohnishi, N.; Nagai, A.; Kimura, T.; Hayashi, Y.; Kiso, Y. J.

Med. Chem. 2008 51, 2992-3004.

110. Walczak, K.; Wamberg, M.; Pedersen, E. B. Helv. Chim. Acta 2004, 87, 469-478.

111. Tsunoda, T.; Yamamiya, Y.; Ito, S. Tetrahedron Lett. 1993, 34, 1639.

112. Tsunoda, T.; Yamamiya, Y.; Kawamura, Y.; Ito, S. Tetrahedron Lett. 1995, 36,

2529.

113. Kosower, E. M.; Londnerle, H. K. J. Am. Chem. Soc. 1976, 98, 3001-3007.

114. Panosyan, F. B.; Still, I. W. J. Can. J. Chem. 2001, 79, 1110-1114.

115. Turner, R. M.; Lindell, S. D. J. Org. Chem. 1991, 56, 5739-5740.

116. Carver, D. S.; Lindell, S. D.; Saville-Stones, E. A. Tetrahedron 1997, 53, 14481-

14496.

117. Vaswani, R. G.; Chamberlin, A. R. J. Org. Chem. 2008, 73, 1661-1681.

118. Yeager, A. R.; Finney, N. S. J. Org. Chem. 2004, 69, 613-618.

119. Xun, S.; LeClair, G.; Zhang, J.; Chen, X.; Gaoa, J. P.; Wang, Z. Y. Org. Lett. 2006,

8, 1697–1700.

120. Cohen, S. G.; Zand, R.; Steel, C. J. Am. Chem. Soc. 1961, 83, 2895-2900.

121. Lecercle´, D.; Sawicki, M.; Taran, F. d. r. Org. Lett. 2006, 8, 4283-4285.

122. Lee, S. J.; Kim, E.; Seo, M. J.; Do, Y.; Lee, Y. A.; Lee, S. S.; Jung, J. H.; Kogiso,

M.; Shimizu, T. Tetrahedron 2008, 2008, 1301-1308.

123. Bergel, F.; Peutherer, M. A. J. Chem. Soc. 1964, 3973-3981.

124. Wolfrom, M. L.; Remieux, R. U.; Olin, S. M. J. Am. Chem. Soc. 1949, 71, 2870-

2873.

125. Davoli, P.; Forni, A.; Moretti, I.; Prati, F. Tetrahedron 1995, 6, 2011-2016.

126. Cai, J.; Li, X.; Yue, X.; Taylor, J. S. J. Am. Chem. Soc. 2004, 126, 16324-16325.

127. Marcantoni, E.; Massaccesi, M.; Torregiani, E. J. Org. Chem. 2001, 66, 4430-

4432.

128. Nakata, T.; Nakatani, M.; Takahashi, M.; Okai, J.; Kawaoka, Y.; Kouge, K.; Okai,

H. Bull. Chem. Soc. Jpn. 1996, 69, 1099-1106.

129. Tummatorn, J.; Albiniak, P. A.; Dudley, G. B. J. Org. Chem. 2007, 72, 8962-8964.

130. Jackson, R. F. W.; Perez-Gonzalez, M. Organic Syntheses. 2005, 81, 77-88.

131. Paradis-Bleau, C.; Beaumont, M.; Sanchagrin, F.; Voyer, N.; Levesque, R. C.

Biorg. Med. Chem. 2007, 15, 1330-1340.

132. Solorzano, C.; Antonietti, F.; Duranti, A.; Tontini, A.; Rivara, S.; Lodola, A.;

Vacondio, F.; Tarzia, G.; Piomelli, D.; Mor, M. J. Med. Chem. 2010, 53, 5770-5781.

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