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BENZOTRIAZOLE INTERMEDIATES FOR HETEROCYCLES AND PHARMACEUTICALS

By

MEGUMI YOSHIOKA-TARVER

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2009

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© 2009 Megumi Yoshioka-Tarver

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I dedicate this work to my father Hitoshi Yoshioka, my mother Kazuyo Yoshioka, my brother Yuta Yoshioka, my sister Yukari Yoshioka, and finally my wonderful husband Dr. Matthew R.

Tarver. I would have never accomplished any of this works without their love, support, and understanding.

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ACKNOWLEDGMENTS

I greatly appreciated many people who have helped me in the preparation of this

dissertation. First, I would like to thank my advisor, Dr. Alan R. Katritzky for the opportunity,

great understanding, and support during my Ph.D. program. I also thank my committee members,

Dr. Margaret O. James, Dr. So Hirata, Dr. Sukwon Hong, and Dr. Ion Ghiviriga, for their helpful

suggestions and instructions. I also appreciate all of Katritzky group members; especially Dr. C.

Dennis Hall, Dr. Niveen M. Khashab, Dr. Tamari Narindoshvili, Dr. Anamika Singh, Dr.

Danniebelle N. Haase, Dr. Geeta Meher, Mr. Bahaa El-Dien M. El-Gendy, Ms. Longchuan

Huang Ms. Claudia El Nachef, Ms. Janet Cusido, and Ms. Judit Kovacs.

Special thanks go to Dr. Ben Smith, Dr. Tammy Davidson, Dr. Katsu Ogawa, Dr. Jodie

Johnson, Dr. Peter Steel, Mr. Alfred Chung, Ms. Lori Clark, Ms. Elizabeth Sheppard, Ms.

Elizabeth Cox, Ms. Gwen McCann, and all my fellow Gators at the University of Florida.

I also thank my parents, Hitoshi and Kazuyo Yoshioka, and my brother, Yuta Yoshioka,

my sister, Yukari Yoshioka, my parents-in-law, Dr. Robert and Karen Tarver, for their support.

Finally, I thank my husband, Dr. Matthew R. Tarver, for supporting, understanding, and loving

me continuously.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS.................................................................................................................... 4

LIST OF TABLES................................................................................................................................ 8

LIST OF FIGURES .............................................................................................................................. 9

LIST OF SCHEMES .......................................................................................................................... 11

LIST OF ABBREVIATIONS ............................................................................................................ 13

ABSTRACT ........................................................................................................................................ 17

CHAPTER

1 GENERAL INTRODUCTION .................................................................................................. 19

1.1 General Introduction of Benzotriazole ................................................................................ 19 1.2 General Methods ................................................................................................................... 24

2 C-AMINOIMIDOYLATION AND C-THIOCARBAMOYLATION OF SULFONES AND KETONES ......................................................................................................................... 26

2.1 Introduction ........................................................................................................................... 26 2.2 Results and Discussion ......................................................................................................... 28

2.2.1 Synthesis of 1-(Alkyl/arylthiocarbamoyl) benzotriazoles 2.4a-e and Benzotriazole-1-carboxamidine 2.5a-c .......................................................................... 28

2.2.2 C-Aminoimidoylation and C-Thiocarbamoylation of Sulfones .............................. 29 2.2.3 C-Aminoimidoylation and C-Thiocarbamoylation of Ketones ............................... 29 2.2.4 Compound Characterization and Tautomeric Structures ......................................... 32

2.3 Conclusion ............................................................................................................................. 37 2.4 Experimental Section ............................................................................................................ 38

2.4.1 General Procedure for the Preparation of Compounds 2.6 and 2.8......................... 38 2.4.2 General Procedure for the Preparation of Compounds 2.9a–d and 2.11a–d ......... 39 2.4.3 General Procedure for the Preparation of Compounds 2.12a–c .............................. 41

3 PEPTIDE SYNTHESIS UTILIZING (N-FMOC-PROTECTED-AMINOACYL)-BENZOTRIAZOLES AND (N-FMOC-PROTECTED-DIPEPTIDOYL)BENZOTRIAZOLES ..................................................................................... 43

3.1 Introduction ........................................................................................................................... 43 3.2 Results and Discussion ......................................................................................................... 45

3.2.1 Preparation of (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2......................... 45

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3.2.2 Preparation of (LL)-Dipeptides 3.4a-f and Diastereomeric Mixtures (3.4a+3.4a’), (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) Using (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-d ................................................. 46

3.2.3 Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f .................. 49 3.2.5 General Peptide Syntheses by Segment Condensation ............................................ 49

3.3 Conclusion ............................................................................................................................. 52 3.4 Experimental Section ............................................................................................................ 52

3.4.1 General Procedure for the Preparation of 3.1a-g ..................................................... 52 3.4.2 General Procedure for the Preparation of 3.4a-f, (3.4a+3.4a’), (3.4b+3.4b’)

(3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’)................................................................... 54 3.4.3 General Procedure for the Preparation of 3.5a-f ...................................................... 58 3.4.5 HPLC Results of Peptide 3.6-3.10 ............................................................................ 62

4 COUMARIN LABELING OF PEPTIDES ON SOLID PHASE............................................. 65

4.1 Introduction ........................................................................................................................... 65 4.2 Results and Discussion ......................................................................................................... 69

4.2.1 Preparation of Nα-Fmoc-Nε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (Nα-Fmoc-L-Lys(Mca)-OH) 4.4 and Its Benzotriazole Derivative 4.6 ............................... 69

4.2.2 Preparation of Nα-Fmoc-Nε-(coumarin-3-ylcarbonyl)-L-lysine Benzotriazolide (Nα-Fmoc-L-Lys(Cc)-Bt) 4.9 and Nα-(Coumarin-3-ylcarbonyl)-Nε-Fmoc-L-lysine Benzotriazolide (Nα-Cc-L-Lys(Fmoc)-Bt) 4.11 ............................................................. 70

4.2.3 Solid Phase Fluorescent Labeling with 4.6, 4.9, 4.11 to Synthesize Labeled Peptides 4.12-4.17 ............................................................................................................ 70

4.2.4 Solid Phase Fluorescent Labeling with 4.7 to Synthesize Labeled Dipeptide (Cc)-L-Leu-L-Leu-NH2 4.18 and Labeling with 4.5 to Synthesize Labeled Dipeptide (Mca)-L-Leu-L-Leu-NH2 4.19 ....................................................................... 72

4.2.5 Fluorescence Measurements of Peptides 4.12-4.19 ................................................. 73 4.3 Conclusion ............................................................................................................................. 75 4.4 Experimental Section ............................................................................................................ 75

4.4.1 Preparation of (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamido)pentanoic acid (Nα-Fmoc-L-Lys(Mca)-OH) 4.4 ........................................................................................................... 75

4.4.2 General Procedure for the Preparation of 4.5, 4.6, 4.9, 4.11 ................................... 76 4.4.3 General Procedure of Solid Support Peptide Synthesis ........................................... 78 4.4.4 HPLC Profiles of Peptide 4.12-4.19 ......................................................................... 79

5 DESIGN AND SYNTHESIS OF PH SENSITIVE GFP CHROMOPHORE ANALOGUES............................................................................................................................. 84

5.1 Introduction ........................................................................................................................... 84 5.2 Results and Discussion ......................................................................................................... 87

5.2.1 Synthesis of Imidazolinone Chromophores 5.9a-f and Their Fluorescent Activity ............................................................................................................................. 87

5.2.2 Synthesis of Imidazolinone Chromophore 5.14a-b and Their Fluorescent Activity ............................................................................................................................. 93

5.2.3 Absorption and Emission Measurement of Chromophores 5.9b-f and 5.14a-b .... 95

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5.3 Conclusion ............................................................................................................................. 96 5.4 Experimental ......................................................................................................................... 96

5.4.1 General Synthesis for the Preparation of Azalactone 5.8 ........................................ 96 5.4.2 General Synthesis for the Preparation of Imidazolinone 5.9 ................................... 98 5.4.3 Synthesis of 2-(2-Naphthamido)acetic acid 5.10 ................................................... 100 5.4.4 Synthesis of N-(2-Chloroacetyl)benzamide 5.11 ................................................... 100 5.4.5 Synthesis of N-(2-Azidoacetyl)benzamide 5.12 ..................................................... 101 5.4.6 Synthesis of 2-Phenyl-1H-imidazol-5(4H)-one 5.13 ............................................. 101 5.4.7 General Procedure for the Preparation of Imidazolinone 5.14 .............................. 101

6 SUMMARY OF ACHIEVEMENTS ....................................................................................... 103

LIST OF REFERENCES ................................................................................................................. 105

BIOGRAPHICAL SKETCH ........................................................................................................... 112

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LIST OF TABLES

Table page 2-1 1-(Alkyl/arylthiocarbamoyl)benzotriazoles 2.4a-e and benzotriazole-1-

carboxamidine 2.5a-c ............................................................................................................. 29

2-2 C-Aminoimidoylation and C-thiocarbamoylation of ketones 2.10a–d to give 2.9a–d and 2.11a–d ............................................................................................................................ 30

2-3 Formation of 1,3-oxazolidine-2-thione 2.12......................................................................... 31

3-1 (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-g ........................................................ 46

3-2 Preparation of (LL)-dipeptides 3.4a-f and diastereomeric mixtures (3.4a+3.4a’), and (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) from (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-d .......................................................................................... 48

3-3 Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazole 3.5a-f................................ 49

3-4 Synthesized peptides 3.6-3.10 by solid phase segment condensation ................................ 51

3-5 MS/MS Sequence of peptide 3.9 ........................................................................................... 64

4-1 Preparation of fluorescent peptides 4.12-4.17 ...................................................................... 72

4-2 Preparation of fluorescent peptides 4.18 and 4.19 ............................................................... 73

4-3 Absorption and fluorescence data of fluorescent labeled peptides 4.12-4.19 .................... 74

4-4 MS/MS Sequence of peptide 4.16 ......................................................................................... 82

5-1 Synthesis of GFP modified fluorophore 5.9a-f .................................................................... 88

5-2 Imidazolinone chromophore 5.14a-b .................................................................................... 93

5-3 Quantum yields and excitation coefficients of 5.9b-f and 5.14a-b..................................... 95

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LIST OF FIGURES

Figure page 1-1 Various functions of the benzotriazole groups ..................................................................... 19

1-2 Coumarin(carboxyl/acetyl)benzotriazole 1.16, 1.17 and coumarin-labeled-Fmoc-L-Lys-Bt 1.18-1.20 .................................................................................................................... 23

1-3 GFP modified unnatural amino acids.................................................................................... 23

1-4 GFP modified chromophore analogues ................................................................................ 24

2-1 X-ray structure of 2.12a ......................................................................................................... 31

2-2 Relevant 1H and 13C chemical shifts in compounds 2.6 and 2.8 ......................................... 32

2-3 1H and 13C Chemical shifts in the major and minor isomers of compound 2.9a ............... 34

2-4 Relevant 1H and 13C chemical shifts in compound 2.9 ........................................................ 34

2-5 1H and 13C Chemical shifts and the tautomers/rotamers for compound 2.11a................... 36

2-6 Rotamers of C-aminoimidoylation product 2.9 and C-thiocarbamoylation product 2.11 .......................................................................................................................................... 36

2-7 1H and 13C Chemical shifts for the tautomers of compound 2.11c in CDCl3 and acetone-d6 ............................................................................................................................... 37

3-1 Structure of coupling reagents ............................................................................................... 44

3-2 1H NMR of 3.4a and (3.4a+3.4a’). a) 1H NMR of 3.4a, methyl group, b) 1H NMR of (3.4a+3.4a’), methyl groups .................................................................................................. 48

3-3 13C NMR of 3.4a and (3.4a+3.4a’).c) 13C NMR of 3.4a, carbonyl groups, d) 13C NMR of (3.4a+3.4a’), carbonyl groups ................................................................................ 49

3-4 HPLC Profile of peptide 3.6 .................................................................................................. 62

3-5 HPLC Profile of peptide 3.7 .................................................................................................. 62

3-6 HPLC Profile of peptide 3.8. ................................................................................................. 63

3-7 HPLC Profile of peptide 3.9 .................................................................................................. 63

3-8 HPLC Profile of peptide 3.10. ............................................................................................... 64

4-1 Structure of Mca and Dnp moiety ......................................................................................... 67

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4-2 Absorption spectra of 4.12-4.17 ............................................................................................ 74

4-3 Absorption spectra of 4.18-4.19 ............................................................................................ 74

4-4 Emission spectra of 4.12, 4.17, 4.19 ..................................................................................... 75

4-5 HPLC Profile of peptide 4.12. ............................................................................................... 79

4-6 HPLC Profile of peptide 4.13 ................................................................................................ 80

4-7 HPLC Profile of peptide 4.14 ................................................................................................ 80

4-8 HPLC Profile of peptide 4.15 ................................................................................................ 81

4-9 HPLC Profile of peptide 4.16. ............................................................................................... 81

4-10 HPLC Profile of peptide ........................................................................................................ 82

4-11 HPLC Profile of peptide 4.18.. .............................................................................................. 82

4-12 HPLC Profile of peptide 4.19 ................................................................................................ 83

5-1 The proposed GFP-based lysin (Lys), asparagine (Asn), and glutamine (Gln) analogues 5.4-5.6.................................................................................................................... 86

5-2 Prevention of photoisomerization of imidazolinonyl compound 5.9a................................ 89

5-3 Absorption (left) and emission spectra (right) of 5.9b ........................................................ 91

5-4 Absorption (left) and emission spectra (right) of 5.9c ......................................................... 92

5-5 Absorption (left) and emission spectra (right) of 5.9d ........................................................ 92

5-6 Absorption (left) and emission spectra (right) of 5.9f ......................................................... 92

5-7 15N NMR study of 5.9c .......................................................................................................... 93

5-8 Absorption (left) and emission spectra (right) of 5.14a....................................................... 94

5-9 Absorption (left) and emission spectra (right) of 5.14b ...................................................... 95

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LIST OF SCHEMES

Scheme page 1-1 Protonation equilibria............................................................................................................. 20

1-2 Benzotriazole-mediated nucleophilic addition ..................................................................... 20

1-3 Displacement of benzotriazole derivatives by different nucleophiles ................................ 21

1-4 C-Aminoimidoylation and C-thiocarbamoylation of sulfones and ketones ....................... 21

1-5 Conversion of Nα-protected amino acid into Nα-protected(aminoacyl)-benzotriazole 1.12 and Nα-protected(benzopeptidoyl)benzotriazole 1.14 ................................................. 22

2-1 Literature method of the preparation of sulfonyl amidine 2.2............................................. 26

2-2 Literature methods for the preparation of ketene aminals 2.3 ............................................. 27

2-3 Preparation of mono-N-hydroxy- and N-aminothiourea from 1-(alkyl/arylthio- carbamoyl)benzotriazoles 2.4 ................................................................................................ 28

2-4 N-Aminoimidoylation with benzotriazole-1-carboxamidines 2.5....................................... 28

2-5 Synthesis of benzotriazole-1-carboxamidine 2.4a-e and 1-(alkyl/arylthio carbamoyl)benzotriazoles 2.5a-c .......................................................................................... 28

2-6 Preparation of C-aminoimidoylation product 2.6 and C-thiocarbamoylation product 2.8 from sulfone 2.7 ............................................................................................................... 29

2-7 Preparation of the C-aminoimidoylation products 2.9a–d and C-thiocarbamoylation products 2.11a–d from ketones 2.10a–d .............................................................................. 30

2-8 Formation of 1,3-oxazolidine-2-thione 2.12......................................................................... 31

2-9 Possible mechanism of 1,3-oxazolidine-2-thione 2.12a-c formation ................................. 31

2-10 Mechanism of isomeric mixtures formation ......................................................................... 34

3-1 General procedure of segment condensation peptide synthesis .......................................... 43

3-2 Epimerization mechanism during segment condensation.................................................... 44

3-3 Preparation of (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-g ............................... 46

3-4 Preparation of Nα-Fmoc-protected-dipeptides 3.4a-f, (3.4a+3.4a’), and (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) ......................................................................... 47

3-5 Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f .............................. 49

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3-6 General procedure of peptide synthesis by segment condensation ..................................... 51

4-1 Mechanism of FRET .............................................................................................................. 66

4-2 Reported synthesis of Nα-(fluoren-9-ylmethoxycarbonyl)-Nε-[(7-methoxycoumarin-4-yl)acetyl-L-lysine 4.4 ......................................................................................................... 68

4-3 Preparation of Nα-Fmoc-L-Lys(Mca)-Bt 4.6 ........................................................................ 69

4-4 Preparation of Nα-Fmoc-L-Lys(Cc)-Bt 4.9 and Nε-Cc-L-Lys(Fmoc)-Bt 4.11 ................... 70

4-5 Synthesis of coumarin-labeled dipeptide 4.12...................................................................... 71

4-6 Preparation of peptide 4.18 .................................................................................................... 73

5-1 Intramolecular biosynthesis of imidazolinonyl chromophore in wild-type GFP ............... 85

5-2 Literature examples ................................................................................................................ 86

5-3 Synthesis of GFP modified fluorophore 5.9a-d ................................................................... 87

5-4 Synthesis of GFP modified fluorophore 5.9e-f .................................................................... 87

5-5 Prevention of photoisomerization of imidazolinonyl compound 5.9a by six-membered ring intramolecular hydrogen bond .................................................................... 90

5-6 Expected protonation of GFP analogues 5.9b, 5.9c, 5.9e, 5.9f ........................................... 91

5-7 Expected protonation of GFP analogues 5.9d ...................................................................... 91

5-8 Synthesis of imidazolinone chlomophore 5.14a-b............................................................... 93

5-9 Possible mechanism of fluorophore 5.14a............................................................................ 94

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LIST OF ABBREVIATIONS

AcOH Acetic acid

Ac2O Acetic anhydride

Ala (A) Alanine

Anal Analytical

aq Aqueous

[α]D Optical rotation

BAL 2, 3-Dimercaproptopanol

Bn Benzyl

Boc tert-Butyl dicarbonyl

br s Broad shinglet

BtH 1H-Benzotriazole

Bt Benzotriazol-1-yl

Bu Buthyl

Calcd Calculated

Cbz Carboxybenzyl

CDCl3 Deutrated chloroform

CFP Cyan fluorescent protein

°C Celsius degree

δ Chemical shift

d Doublet

D (11 point) Dextrorotary

DCM Methylene chloride

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DMF Dimethyl formamide

DMSO Dimethyl sulfoxide

DMSO-d6 Deutreted dimethyl sulfoxide

Et Ethyl

et al And others

Et3N Triethyl amine

EtOAc Ethyl acetate

equiv Equivalent(s)

Fmoc Fluorenylmethyloxycarbonyl

g Gram(s)

GFP Green fluorescent protein

gHMBC Heteronuclear multiple bond correlation

Gln Glutamine

Gly (G) Glycine

h Hour

HCl Hydrochloric acid

His Histidine

HMBA Hexamethylene bisacetamide

HPLC High purity liquid chromatography

HRMS High resolution mass spectrometry

Hz Hertz

H2O Water

J Coupling constants

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L (11 point) Levorotary

Leu (L) Leucine

Lit Litterature

Lys (K) Lysine

m Multiple

M Molar

Me Methyl

MeCN Acetonitrile

MeOH Methanol

Met (M) Methionine

min Minute

MgSO4 Magnesium sulfate

mp Melting point

Na2CO3 Sodium carbonate

NaOH Sodium hydroxide

NMR Nuclear magnetic resonance

nOe’s Nuclear overhauser effect

NOESY Nuclear overhauser effect spectroscopy

OH Hydroxyl group

p Para

Pg Protecting group

Ph Phenyl

Phe (F) Phenylalanine

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Pro (P) Proline

Proj. Project

q Quartet

RT Room temperature

s Singlet

SOCl2 Thionyl chloride

t Triplet

t (tert) Tertiary

t-BuOK Potassium tert-butoxide

TFA Trifluoroacetic acid

TIPS Triisopropylsilane

THF Tetrahydrofuran

TMS Tetramethylsilane

Tol 4-Methyl-phenyl

tR Retention time

Trp (W) Tryptophane

Tyr Tyrosine

UV Ultraviolet

W Watt(s)

YFP Yellow fluorescent protein

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

BENZOTRIAZOLE INTERMEDIATES FOR HETEROCYCLES AND

PHARMACEUTICALS

By

Megumi Yoshioka-Tarver

December 2009

Chair: Alan R. Katritzky Major: Chemistry

The focus of this work is to expand the utility of 1H-benzotriazole (BtH) in organic

synthesis. Benzotriazole is a synthetic auxiliary and had been utilized in many reactions

previously. The application of BtH is descrived as an excellent leaving group in the synthesis of

various pharmaceuticals and heterocycles. Chapter 1 describes the properties of BtH and its

previous applications.

In Chapter 2, C-aminoimidoylation and C-thiocarbamoylation of sulfones, and ketones

from benzotriazole-1-carboxamidines and 1-(alkyl-or-arylthiocarbamoyl)benzotriazoles are

reported in the formation of new C-C bond respectively.

Chiraly pure Nα-Fmoc-protected-dipeptides are readily synthesized in solution phase

from commercially available Nα-Fmoc-protected-amino acids. In Chapter 3 and 4, small peptides

were synthesized using solid phase technique and benzotriazole methodology. Nα-Fmoc-

protected(aminoacyl)benzotriazoles are converted into Nα-Fmoc-protected(dipeptidoyl)benzotri-

azoles, which are used under mild microwave irradiation in solid phase peptide segment

condensations syntheses to give tri-, tetra-, penta-, hexa-, and heptapeptides as isolated pure

peptides (Chapter 3). In Chapter 4, Nα-Fmoc-Nε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (Nα-

Fmoc-L-Lys(Mca)-OH) is conveniently prepared by benzotriazole methodology. N-

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Acylbenzotriazoles: Mca-Bt, Nα-Fmoc-L-Lys(Mca)-Bt, coumarin-3-ylcarbonyl (Cc)-Bt, Nα-

Fmoc-L-Lys(Cc)-Bt, and Nα-(Cc)-L-Lys(Fmoc)-Bt enable efficient microwave-enhanced solid-

phase fluorescent labeling of peptides.

Chapter 5 presents eight green fluorescent protein (GFP) chromophore-modified

analogues, which were designed to act as pH sensors. Syntheses of these analogues were carried

out via two to four steps from commercially available compounds. The fluorescence and pH

dependency of analogues were studied. Finally, a summary of this work is presented in Chapter

6.

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CHAPTER 1 GENERAL INTRODUCTION

1.1 General Introduction of Benzotriazole

Benzotriazole is a well studied synthetic auxiliary and benzotriazole methodology has been

utilized for a wide variety of organic syntheses. 1H-Benzotriazole (BtH) has useful properties

such as its leaving group ability 1.1, its electron-donating character 1.2, or its activation of the

CH bond toward proton loss 1.3 (Figure 1-1). [98CR409] BtH is non-toxic, insensitive to

moisture, and commercially available at low cost. Moreover, it has high solubility in common

organic solvents such as MeOH, benzene, chloroform, toluene, and DMF. BtH behaves as a

weak base (pKa = 1.6) as well as a weak acid (pKa = 8.3), [48JCS2240, 00JACS5849] which

allows reactions to occur in both acidic and basic media (Scheme 1-1). Thus BtH can be easily

removed from reaction mixtures by a simple acid or base work-up during purification. My

research has focused on the utilization of BtH as an activating reagent in the synthesis of several

biological targets.

Figure 1-1. Various functions of the benzotriazole groups

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Scheme 1-1. Protonation equilibria

A recent review summarized the important uses of benzotriazolyl intermediates and the

displacement of Bt by nucleophilic attack. [94CSR363] Because of its ability as a leaving group,

benzotriazole can be easily displaced by the lone pair of a heteroatom, followed by reaction with

a nucleophile (Scheme 1-2).

Scheme 1-2. Benzotriazole-mediated nucleophilic addition

Benzotriazole methodology has been used in our research group for more than two

decades, and the utility of BtH has been exemplified in reactions such as alkylations,

[94CSR363] acylation, [03JOC4932, 03JOC5720, 05S1656] imine acylation, [00S2029]

guanylation, [06NGIR] and imidoylation. [06NGIR] Displacement of benzotriazole derivatives

have been utilized by different nucleophilic atoms such as C, S, N, or O (Scheme 1-3).

Benzotriazole methodology has also been utilized in Mannich reactions, [94JHC917] Michael

reactions, [01BCSJ2133] and Grignard reactions. [07S3141]

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Scheme 1-3. Displacement of benzotriazole derivatives by different nucleophiles

In Chapter 2, the C-aminoimidoylation and C-thiocarbamoylation of sulfones and ketones

using the 1-(alkylthiocarbamoyl)benzotriazoles 1.5 and benzotriazole-1-carboxamidine 1.6 were

presented (Scheme 1-4). [07JOC6742] These compounds were prepared from bis(benzotriazoyl)

methanethione 1.4 in two steps. Highly toxic and moisture sensitive thiophosgene was easily

converted to bis(benzotriazoyl)methanethione 1.4 [78JOC337] which is non-toxic and moisture

insensitive.

Scheme 1-4. C-Aminoimidoylation and C-thiocarbamoylation of sulfones and ketones

Using benzotriazole methodology also results in a facile preparation of peptides.

Carboxylic acids are activated by BtH in the presence of SOCl2. Benzotriazole-activated-N-

22

protected amino acids react with the -NH2 group of free amino acids to form longer peptide

chains without the necessity to protect C-termini. [05JOC4993, 05S397, 09A47] My research

utilized N-Fmoc-protected amino acid benzotriazole derivatives 1.12 and 1.14 in peptide

synthesis both in solution phase and solid phase with retention of the original chirality (Chapter

3, Scheme 1-5). [07CBDD465, 08CBDD181] Compounds 1.12 and 1.14 couple on solid resin

under microwave irradiation in 3 to 10 min. Because of C-terminus activation by benzotriazole,

the method does not require additional coupling reagents. In some sequences, peptide synthesis

during segment condensation using 1.14 did not cause epimerization, which is known to be a

major problem during segment condensation. [08CBDD181]

Scheme 1-5. Conversion of Nα-protected amino acid into Nα-protected(aminoacyl)-benzotriazole 1.12 and Nα-protected(benzopeptidoyl)benzotriazole 1.14

Currently, the focus is on fluorogenic peptide synthesis and fluorescent-labeled peptides

using benzotriazole methodology. Three coumarin-labeled-Fmoc-L-Lys-OH derivatives (Figure

1-2) were synthesized in an average yield of 52% in two steps and their benzotriazole derivatives

1.18-1.20 and coumarin(carboxyl/acetyl)benzotriazoles 1.16 and 1.17 were utilized in

fluorogenic peptide synthesis. [08OBC4582]

23

Figure 1-2. Coumarin(carboxyl/acetyl)benzotriazole 1.16, 1.17 and coumarin-labeled-Fmoc-L-Lys-Bt 1.18-1.20

Recently, I designed green fluorescent peptide (GFP) modified unnatural amino acids as

potential pH sensors. [Proj#1979] The GFP chromophores are subject to a photoisomerization

which decreases their fluorescence activity. The project is designed to inhibit photoisomerization

by intramolecular hydrogen bonding under acidic conditions. The designed chromophores are

able to tag Nα-protected natural amino acids such as lysine, asparagine, or glutamine for the

preparation of fluorogenic peptides (Figure 1-3). In order to optimize the fluorescent activity, I

synthesized GFP chromophore analogues (Figure 1-4, Chapter 5) and reported their fluorescent

activity under different pH conditions.

Figure 1-3. GFP modified unnatural amino acids

24

Figure 1-4. GFP modified chromophore analogues

1.2 General Methods

Reagents were purchased from Peptides International, Across, or Aldrich and used without

further purification. Rink-amide-MBHA resin (200–400 mesh, 0.35 meq/g) was purchased from

Peptide International (Louisville, KY, USA). Melting points were determined on a capillary

point apparatus equipped with a digital thermometer. NMR spectra were recorded in CDCl3 or

DMSO-d6 with TMS for 1H (300 MHz) and 13C NMR (75 MHz) as an internal reference.

Absorption and fluorescence measurements were recorded on Cary 100 UV-Vis and FluoroMax

spectrophotometers respectively. Optical rotation values were measured with the use of sodium

D line. Column chromatography was performed on silica gel (200-425 mesh) or basic alumina

(60-325 mesh). Elemental analyses were performed on a Carlo Erba-1106 instrument. MALDI

analyses were performed on Bruker Reflex II TOF mass spectrometer retrofilled with delayed

extraction.

Analytical reversed-phase HPLC was performed on a Rainin HPXL system with a Vydac

C-18 (5 m, 2.1 × 250 mm) silica column at a 1ml/min flow rate. Peptides were eluted using a 10–

80 % gradient of solvent B (0.1 % TFA in MeCN) vs solvent A (0.1% TFA in H2O) and peaks

were detected at a wavelength of 214 nm. The identification of the products was achieved by

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF, ABI

4700 Proteomics Analyzer) with -cyano-4-hydroxy cinnamic acid as the matrix. Synthesis of the

peptides was performed in a Discover BenchMate peptide synthesizer from CEM (Matthews,

25

NC, USA). The conditions for a variety of coupling steps were optimized to increase rate and

eliminate epimerization. Single mode irradiation with monitoring of temperature, pressure, and

irradiation power versus time was used throughout, making the procedure highly reproducible.

MS/MS peptide fragmentation was obtained on the crude peptides by way of low resolution MS

and tandem mass spectrometry (MSn) data obtained via HPLC/UV/(+)ESI-MS and –MSn on a

ThermoFinnigan (San Jose, CA) LCQ Classic quadruple ion trap mass spectrometer in

electrospray ionization (ESI) mode. High resolution mass spectrometry (HRMS) via flow-

injection positive [(+)ESI]-time of flight (TOF) was obtained on an Agilent 1200 series

spectrometer.

26

CHAPTER 2 C-AMINOIMIDOYLATION AND C-THIOCARBAMOYLATION OF SULFONES AND

KETONES1

2.1 Introduction

C-Acylation and C-imidoylation are widely used for the preparation of biologically active

compounds. Many acylating [47JACS119, 03JOC1443, 59JACS4882] and imidoylating reagents

[97TL6771, 99OL977, 02JOC4667] have been reported to give C-acylation and C-imidoylation;

by comparison, C-aminoimidoylation and C-thiocarbamoylation have both been relatively

unexplored synthetically. Literature examples of compounds that could conceptually have been

made by C-aminoimidoylation and C-thiocarbamoylation have generally been accessed by

multistep synthesis. [79S343, 83LAC290, 00JOC1583, 04JOC188]

A substructure search showed no example of C-aminoimidoylation by C-C bond

formation; one possible product of such a reaction (2.2) was reported by the reaction of an alkyl

β,β-dichlorovinyl sulfone 2.1 with 4-methoxyaniline (Scheme 2-1). [79ZOK2349]

Scheme 2-1. Literature method of the preparation of sulfonyl amidine 2.2

N,N´-Disubstituted ketene aminals 2.3 are used in many syntheses, especially in the

construction of heterocyclic compounds. [04JOC188] Structurally, ketene aminals can be

converted to the tautomers of C-aminoimidoylated ketones. They have been prepared (Scheme 2-

1Reproduced in part with permission from The Journal of Organic Chemistry, 2007, 72, 6742.

Copyright © 2009 American Chemical Society

27

2) starting from (i) activated methylene compounds and isothiocyanates, [04JOC188] (ii)

oxoketene N,S-acetals and lithiated secondary amines or aniline, [00JOC1583] or (iii)

tris(dimethylamino)ethoxymethane and simple ketones. [79S343, 83LAC290] Methods (i)-(iii)

(Scheme 2-2) each consist of multi-steps with average overall yields of ca 30%. Methods (i) and

(ii) are related to C-aminoimidoylation but no such reaction of a ketone was located in a

literature search.

Scheme 2-2. Literature methods for the preparation of ketene aminals 2.3

Recently, our group synthesized a) di- and tri-substituted thioureas, [04JOC2976] N-

hydroxythioureas, and thiosemicarbazides [06A226] using novel 1-(alkyl/arylthiocarbamoyl)-

benzotriazole thiocarbamoylating reagents 2.4 (Scheme 2-3) and b) 1,2,3-tri-substituted

guanidines, [05HCA1664] N-hydroxy-, and N-amino-guanidines [06JOC6753] using novel

benzotriazole-1-carboxamidine-N-aminoimidoylating reagents 2.5 (Scheme 2-4). I have

demonstrated C-aminoimidoylation and C-thiocarbamoylation of sulfones and ketones using 1-

(alkyl/arylthiocarbamoyl)benzotriazoles 2.4a–e and benzotriazole-1-carboxamidines 2.5a–c by a

simple benzotriazole procedure.

28

Scheme 2-3. Preparation of mono-N-hydroxy- and N-aminothiourea from 1-(alkyl/arylthio-

carbamoyl)benzotriazoles 2.4

Scheme 2-4. N-Aminoimidoylation with benzotriazole-1-carboxamidines 2.5

2.2 Results and Discussion

2.2.1 Synthesis of 1-(Alkyl/arylthiocarbamoyl) benzotriazoles 2.4a-e and Benzotriazole-1-carboxamidine 2.5a-c

Treatment of bis(benzotriazoyl)methanethione [78JOC337] with amines afforded 1-

(alkyl/arylthiocarbamo-yl)benzotriazoles 2.4a-e, [04JOC2976] which were converted into

benzotriazole-1-carboxamidines 2.5a-c [05HCA1664] by treatment with triphenylphosphine

ylides (Scheme 2-5, Table 2-1).

Scheme 2-5. Synthesis of benzotriazole-1-carboxamidine 2.4a-e and 1-(alkyl/arylthio carbamoyl)benzotriazoles 2.5a-c

29

Table 2-1. 1-(Alkyl/arylthiocarbamoyl)benzotriazoles 2.4a-e and benzotriazole-1-carboxamidine 2.5a-c

Entry R1 Yield (%)a R Yield (%)a 1 2.4a Cyclohexyl 95 - - - 2 2.4b n-Bu 98 2.5a p-Tol 92 3 2.5b p-ClC6H4 67 4 2.4c -(CH2)Ph 85 2.5c Ph 87 5 2.4d Bn 98 - - - 6 2.4e t-Bu 87 - - - aIsolated yield

2.2.2 C-Aminoimidoylation and C-Thiocarbamoylation of Sulfones

Reaction of 2.0 equiv of sulfones 2.7 with 2.5 equiv of potassium tert-butoxide in THF at

room temperature for 0.5 h followed by the addition of the appropriate benzotriazole reagent

(2.4a or 2.5a) afforded compounds 2.6 and 2.8 in yields of 30 and 40% respectively (Scheme 2-

6). The reaction was also carried out with methyl phenyl sulfone or ethyl phenyl sulfone and

compound 2.4 or 2.5, but the desired product did not form in a similar manner.

Scheme 2-6. Preparation of C-aminoimidoylation product 2.6 and C-thiocarbamoylation product 2.8 from sulfone 2.7

2.2.3 C-Aminoimidoylation and C-Thiocarbamoylation of Ketones

Reaction of the enolates from ketones 2.10a–d, with 2.4 or 2.5 after 0.5-4 h gave the C-

aminoimidoylation and C-thiocarbamoylation products 2.9a–d and 2.11a–d (27–65% yields,

Table 2-2) (Scheme 2-7) as monitored by TLC. For compounds 2.9a and 2.11a-d, 2D NMR

correlation experiments were carried out by Dr. Ion Ghiviriga to assign the 1H and 13C chemical

shifts.

30

Scheme 2-7. Preparation of the C-aminoimidoylation products 2.9a–d and C-thiocarbamoylation products 2.11a–d from ketones 2.10a–d

Table 2-2. C-Aminoimidoylation and C-thiocarbamoylation of ketones 2.10a–d to give 2.9a–d and 2.11a–d

Reagent R R1 Ketone R2 R3 Product Yield(%)a

2.5c Ph (CH2)2Ph 2.10a Ph H 2.9a 32

2.5a p-Tol n-Bu 2.10b 2-Thienyl H 2.9b 27

2.5c Ph (CH2)2Ph 2.10c Me Me 2.9c 27

2.5b p-ClC6H4 n-Bu 2.10a Ph H 2.9d 31

2.4b - n-Bu 2.10a Ph H 2.11a 50

2.4c - (CH2)2Ph 2.10d Ph Ph 2.11b 65

2.4b - n-Bu 2.10b 2-Thienyl H 2.11c 51

2.4e - t-Bu 2.10c Me Me 2.11d 40

aIsolated yield

Reaction of ketones 2.10a-c with 2.4a (R1= Bn) afforded the isomeric 1,3-oxazolidine-2-

thiones 2.12 instead of the expected C-thiocarbamoylation product (Scheme 2-8, Table 2-3).

Structures 2.12a–c were verified by NMR and in the case of 2.12a by X-ray crystallography

(Figure 2-1). 1,3-Oxazolidine-2-thione were previously prepared by cycloaddition of α-metalated

alkyl isothiocyanates to carbonyl compounds. [76CB3047] Compounds 2.12a-c were assumed to

form via a similar mechanism after isothiocyanate formation from 2.4a in the presence of excess

potassium tert-butoxide (Scheme 2-9).

31

Scheme 2-8. Formation of 1,3-oxazolidine-2-thione 2.12

Table 2-3. Formation of 1,3-oxazolidine-2-thione 2.12 Entry Compound R1 R2 Yield (%)a 1 2.12a Me 2-Thienyl 51 2 2.12b Me Ph 27 3 2.12c Me Et 40 aIsolated yield

Figure 2-1. X-ray structure of 2.12a

Scheme 2-9. Possible mechanism of 1,3-oxazolidine-2-thione 2.12a-c formation

32

Due to the weak basicity of potassium tert-butoxide, it allows the deprotonation of NH

group of 2.4a for isothiocyanate formation and cyclization to 1,3-oxazolidine-2-thione 2.12a-c.

This formation of 1,3-oxazolidine-2-thione might not have occurred in the presence of stronger

base to forbid to negate formation.

2.2.4 Compound Characterization and Tautomeric Structures

The tautomeric structure of a heterocyclic [or indeed any] compound can profoundly

influence its physical properties [e.g. boiling point, solubility] and chemical properties [e.g.

acid/base, electron distribution, reactivity]. It was, therefore, of interest to investigate the

tautomeric structures of the parent compounds, and in particular, to know whether they existed in

the enol or keto forms.

Figure 2-2. Relevant 1H and 13C chemical shifts in compounds 2.6 and 2.8

Compounds 2.6 and 2.8 exist in CDCl3 at 25 °C solely as the imino and thiocarbonyl forms,

respectively (Figure 2-2). The alpha protons of R3 phenyl group (5.20 ppm for 2.6 and 5.57 ppm

for 2.8) couple with the NH proton (6.11 ppm for 2.6 and 9.08 ppm for 2.8) in both compounds.

Compound 2.9a was present in CDCl3 as a mixture of two compounds in a 7:1 ratio both

with the same hydrocarbon skeleton, as revealed by the gHMBC spectra (Figure 2-3). Exchange

33

peaks in the NOESY spectrum between protons such as 5.43 ppm with 5.35 ppm, 7.88 ppm with

7.67 ppm, 7.03 ppm with 6.97 ppm, 7.13 ppm with 7.26 ppm, 2.89 ppm with 3.00 ppm, and 3.49

ppm with 3.60 ppm indicated that these species were interconverting. The chemical shift of the

carbonyl carbon in 2.9a is closer to that expected for an aromatic ketone than for an enol. Thus,

the tautomeric structure of 2.9a differs from the compounds of structure 2.6. These chemical

shifts suggest that both isomers of 2.9a are keto-enamine forms. Of the exchange cross-peaks at

11.77 ppm, the largest is 4.76 ppm, indicating that the two isomers differ in the amine hydrogen

facing the carbonyl group. In the major isomer of 2.9a, nOe’s between 5.43 ppm and 3.49 ppm,

and between 4.76 ppm and 7.03 ppm demonstrate that in this isomer the aniline NH is involved

in the hydrogen bond and that the alkyl group on the other nitrogen is anti to the aniline nitrogen,

as depicted in Figure 2-3. For the minor isomer, the nOe’s offered no information on the syn/anti

geometry of the aniline group, because they were mainly transferred nOe’s. It is reasonable

however to assume the same geometry as in the major isomer. Exchange peaks between 5.43

ppm, 5.35 ppm and all the four NH protons indicate that the keto tautomer is present in the

equilibrium, although it was not detected in the proton spectra. Another transient species displays

a signal at 13.26 ppm, in exchange with all of the exchangeable protons in both isomers. The

isomerization of 2.9a occurs due to the keto-enamine-enol-imine tautomerization (Scheme 2-10).

Compounds 2.9b and 2.9d displayed equilibria similar to that for 2.9a. The species with

the aniline NH involved in hydrogen bonding is ca. 7 times more abundant than the other isomer.

Compound 2.9c is solely in the imidamide form in CDCl3 at 25 °C, as indicated by the coupling

between the protons at 3.70 ppm and 1.13 ppm (Figure 2-4). The difference in tautomerism

between 2.9c and 2.9a, 2.9b, 2.9d may be explained by the steric hindrance in the enol form of

2.9c, where the two methyl groups are syn-periplanar.

34

Figure 2-3. 1H and 13C Chemical shifts in the major and minor isomers of compound 2.9a

Scheme 2-10. Mechanism of isomeric mixtures formation

Figure 2-4. Relevant 1H and 13C chemical shifts in compound 2.9

35

Compound 2.11a in CDCl3 at 25 °C displays an equilibrium between three species, in a

ratio 44 : 5 : 1. The interconversion of these species was demonstrated by exchange peaks in the

NOESY spectrum. The major compound is the keto tautomer, while the other two are enol

tautomers, as demonstrated by the chemical shift of the carbon bearing the oxygen atom. The

significant deshielding of the NH proton in the keto form as compared to the enol forms suggests

a hydrogen bond in the keto form, as in Figure 2-5. The spectra were repeated in acetone-d6, a

hydrogen bond acceptor that would compete with the carbonyl group of 2.11a for hydrogen

bonding. The chemical shift of the NH proton in the enol moved downfield by 2 ppm, while for

the keto form the change was only 0.07 ppm, demonstrating intramolecular hydrogen bonding in

the ketone. The two enol species have to be the Z and E isomers resulting from rotation about the

C-N bond in the thioamide. Thioamides are known to prefer the Z configuration and this is the

configuration in the intramolecular hydrogen-bonded keto species. [96JMS45] The similarity of

the proton chemical shifts at the -NHCH2- in the ketone form (3.71 ppm) and in the major enol

form (3.67 ppm) indicates that the latter is also in the Z configuration. The E configuration is

present in the enol tautomer and not in the ketone because it strengthens the resonance-assisted

hydrogen bonding (RAHB) in the enol. This is demonstrated by the higher chemical shift value

of the proton involved in the RAHB in the minor E enol form (14.86 ppm) as compared to the Z

enol (14.54 ppm). The rotamers form of C-thiocarbamoylation product 2.11a can be explained

with Newman projection shown in Figure 2-6. The 2.11-rotamer 1 and 2.11-rotamer 2 were

favored in chloroform due to less steric hinderance of the R group. However, the disappearance

of 2.11-rotamer 2 can be explained because of favored intermolecular hydrogen bonding with

acetone and NH group of 2.11-totamer 1.

36

Figure 2-5. 1H and 13C Chemical shifts and the tautomers/rotamers for compound 2.11a

Figure 2-6. Rotamers of C-aminoimidoylation product 2.9 and C-thiocarbamoylation product 2.11

Compound 2.11c in CDCl3 at 25 °C occurs solely as the keto tautomer. The chemical shift

of the NH proton is 9.22 ppm, indicating an intramolecular hydrogen bond. In acetone-d6

solution, both tautomers are present, and the keto : enol ratio is 1 : 0.44 (Figure 2-7). For

compound 2.11a also, the proportion of the enol was larger in acetone (keto : enol = 45 : 55) than

in chloroform (keto : enol = 88 : 12). As with the case of 2.11a, no E rotamer of the enol form

was detected in acetone-d6.

37

Figure 2-7. 1H and 13C Chemical shifts for the tautomers of compound 2.11c in CDCl3 and acetone-d6

Compounds 2.11b and 2.11d in CDCl3 at 25 °C display the keto tautomer only. The enol

tautomer is higher in energy when R3 is not H, due to steric repulsion between R2 and R3 which

have to be syn-periplanar in the enol.

2.3 Conclusion

Successful C-aminoimidoylations and C-thiocarbamoylations of sulfones and ketones were

achieved in yields of 27-65 % under mild reaction conditions. The method provides an easy

access to interesting classes of compounds for further transformations. However, higher yields of

those products may be achieved using stronger bases such as sodium hydride (pKa = 42), n-butyl

lithium (pKa = 48), or lithium diisopropylamide (pKa = 36) due to the weak acidity of ketones

2.10. Thus, incomplete deprotonation of ketones and formation of isothiocyanates can be

explained by unexpected products, 1,3-oxazolidine-2-thione 2.12. In future work, the reactions

should be repeated using stronger base than potassium tert-butoxide for more efficient C-

aminoimidoylation and C-thiocarbamoylation reaction, which may yield the desired C-

thiocarbamoyl product with compound 2.4a (R1 = Bn).

38

2.4 Experimental Section

2.4.1 General Procedure for the Preparation of Compounds 2.6 and 2.8

To a solution of the desired ester or sulfone (2.0 mmol) in THF (15 mL), potassium tert-

butoxide (2.5 mmol) was added. After stirring for 30 min, the desired reagent 2.4 or 2.5 (1.0

mmol) (Scheme 2-6) was added to the reaction mixture. The progress of the reaction was

monitored by TLC. Upon completion, water (20 mL) was added to quench the reaction followed

by extraction with DCM (3 x 30 mL). The combined extracts were dried over magnesium sulfate

and the solvent removed under vacuum. The crude mixture was purified by gradient column

chromatography over silica gel (EtOAc/hexanes) to give the desired products.

N-Butyl-N’-(4-methylphenyl)-2-phenyl-2-(phenylsulfonyl)ethanimidamide 2.6: White

microcrystals (30 %); mp 117.6 − 119.2 °C; 1H NMR (CDCl3) δ 7.66 (t, J = 8.4 Hz, 3H), 7.51 (t,

J = 7.1 Hz, 2H), 7.53−7.35 (m, 1H), 7.31−7.30 (m, 7H), 6.92 (d, J = 7.8 Hz, 2H), 6.11 (s, 1H),

6.11 (d, J = 8.1 Hz, 2H), 5.20 (s, 1H), 3.39−3.36 (m, 2H), 1.73−1.67 (m, 2H), 1.52−1.45 (m, 2H),

1.00 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3) δ 148.5, 146.5, 137.4, 134.2, 131.8, 130.8, 130.1,

129.4, 129.1, 128.9, 128.8, 128.7, 121.8, 68.4, 41.4, 31.0, 20.8, 20.4, 13.9. Anal. Calcd for

C25H28N2O2S: C, 71.40; H, 6.71; N, 6.66; found: C, 71.55; H, 6.96; N, 6.52.

N-Cyclohexyl-2-phenyl-2-(phenylsulfonyl)ethanethioamide 2.8: White microcrystals

(40 %); mp 148.9 − 149.7 °C; 1H NMR (CDCl3) δ 9.08 (br s, 1H), 7.76−7.73 (m, 2H), 7.67−7.62

(m, 1H), 7.52−7.47 (m, 2H), 7.41−7.30 (m, 5H), 5.57 (s, 1H), 4.38−4.35 (m, 1H), 2.06 (br s, 2H),

1.78−1.65 (m, 2H), 1.58 (br s, 3H), 1.50−1.34 (m, 3H); 13C NMR (CDCl3) δ 190.0, 137.2, 134.5,

129.7, 129.3, 129.2, 129.0, 128.7, 81.7, 54.8, 31.1, 30.8, 25.4, 24.4. Anal. Calcd for

C20H23NO2S2: C, 64.31; H, 6.21; N, 3.75; found: C, 63.96; H, 6.25; N, 3.62.

39

2.4.2 General Procedure for the Preparation of Compounds 2.9a–d and 2.11a–d

To a solution of the desired ketone (2.0 mmol) in THF (20 mL), potassium tert-butoxide

(2.5 mmol) was added, followed by the appropriate reagent 2.4 or 2.5 (1.0 mmol) (Scheme 2-7).

The mixture was stirred at room temperature until full conversion of starting materials (0.5-4.0 h)

was observed by TLC. The crude reaction mixture was then evaporated under reduced pressure,

washed with water (30mL x 3), and finally extracted with diethyl ether (30 mL x 3). Evaporation

of the organic fraction followed by flash column chromatography on silica gel afforded 2.9a–d

or 2.11a–d.

3-Oxo-N-phenethyl-N',3-diphenylpropanimidamide 2.9a: White microcrystals (32 %);

mp 156.2 − 157.7 °C; 1H NMR (CDCl3) δ 13.06 (s, 1H) 7.88 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.1

Hz, 2H), 7.41 (t, J = 7.1 Hz, 1H), 7.30 (t, J = 6.6 Hz, 2H), 7.30 (t, J = 7.6Hz, 2H), 7.25 (t, J =

7.6Hz, 1H), 7.13 (d, J = 7.6 Hz, 2H), 7.03 (d, J = 6.6 Hz, 2H), 5.43 (s, 1H), 4.76 (br s, 1H), 3.49

(q, J = 5.6 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H); 13C NMR (CDCl3) δ 185.6, 159.6, 141.8, 138.1,

136.5, 136.5, 130.2, 129.1, 129.0, 127.2, 126.9, 126.3, 125.4, 124.4, 76.0, 43.6, 35.3. Anal.

Calcd for C23H24N2O2: C, 76.64; H, 6.71; N, 7.77; found: C, 76.61; H, 6.32; N, 7.64.

N-Butyl-N'-(4-methylphenyl)-3-oxo-3-(2-thienyl)propanimidamide 2.9b: Yellow oil

(27 %); 1H NMR (CDCl3) δ 11.32 (s, 1H), 7.53 (d, J = 3.7 Hz, 1H), 7.39 (d, J = 4.9 Hz, 1H),

7.21 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 8.1 Hz, 2H), 7.06 (t, J = 3.8 Hz, 1H), 5.32 (s, 1H), 4.67 (br s,

1H), 3.20 (q, J = 6.9 Hz, 2H), 2.36 (s, 3H), 1.59-1.52 (m, 2H), 1.36 (q, J = 7.1 Hz, 2H), 0.95 (t, J

= 7.3 Hz, 3H); 13C NMR (CDCl3) δ 178.0, 159.5, 148.2, 136.2, 133.4, 130.5, 128.2, 127.4, 125.3,

121.6, 74.9, 42.1, 31.2, 21.0, 20.0, 13.7. HRMS Calcd for [C18H22N2OS+ H]+, 315.1554; found

315.1564.

2-Methyl-3-oxo-N-phenethyl-N'-phenylbutanimidamide 2.9c: Yellow oil (27 %); 1H

NMR (CDCl3) δ 7.40 − 7.20 (m, 7H), 7.01 (t, J = 7.3 Hz, 1H), 6.76 (d, J = 7.8 Hz, 2H), 4.47 (br

40

s, 1H), 3.70 (q, J = 7.0Hz, 1H), 3.61 (quintet, J = 6.9 Hz, 2H), 2.92−2.90 (m, 2H), 2.02 (s, 3H),

1.13 (d, J = 7.0 Hz, 3H); 13C NMR (CDCl3) δ 207.2, 154.7, 150.8, 139.2, 129.1, 128.8, 128.5,

126.4, 122.2, 122.0, 47.3, 41.9, 35.0, 29.0, 15.0. Anal. Calcd for C19H22N2O: C, 77.52; H, 7.53;

N, 9.52; found: C, 77.12; H, 7.73; N, 9.22.

(Z)-N-Butyl-N'-(4-chlorophenyl)-3-hydroxy-3-phenyl-2-propenimidamide 2.9d:

Yellow oil (31 %); 1H NMR (CDCl3) δ 7.89−7.86 (m, 2H), 7.43−7.31 (m, 5H), 7.20-7.18 (m,

2H), 5.41 (s, 1H), 4.61 (br s, 1H), 3.23 (q, J = 5.5 Hz, 2H), 1.61−1.54 (m, 2H), 1.42−1.34 (m,

2H), 0.96 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ 185.6, 159.4, 141.4, 135.1, 130.1, 130.0,

128.1, 126.6, 126.5, 82.4, 75.8, 42.2, 31.1, 20.1, 13.7. HRMS Calcd for [C19H21ClN2O+ H]+,

329.1404; found 329.1404.

N-Butyl-3-oxo-3-phenylpropanethioamide 2.11a: Brown oil (50 %); 1H NMR (CDCl3) δ

9.28 (br s, 1H), 8.04 (d, J = 7.3 Hz, 2H), 7.63 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 7.5 Hz, 2H), 4.50

(s, 2H), 3.67 (m, 2H), 1.70 (m, 2H), 1.44 (m, 2H), 0.97 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ

197.2, 194.2, 136.1, 134.5, 129.2, 128.9, 52.7, 46.5, 30.1, 20.4, 14.0. Anal. Calcd for

C13H17NOS: C, 66.35; H, 7.28; N, 5.95; found: C, 66.62; H, 7.41; N, 5.52.

3-Oxo-N-phenethyl-2,3-diphenylpropanethioamide 2.11b: White microcrystals (65 %);

mp 110.8 − 112.4 °C; 1H NMR (CDCl3) δ 9.01 (br s, 1H), 7.98−7.95 (m, 2H), 7.88−7.53 (m, 1H),

7.46−7.40 (m,2H), 7.34−7.18 (m, 8H), 7.13 (d, J = 7.3 Hz, 2H), 6.33 (s, 1H), 4.01−3.87 (m, 2H),

3.01−2.92 (m, 2H); 13C NMR (CDCl3) δ 197.7, 183.1, 135.9, 134.9, 134.0, 129.4, 129.0, 128.8,

128.7, 128.7, 128.4, 127.8, 126.6, 67.0, 47.5, 33.6. Anal. Calcd for C23H21NOS: C, 76.85; H,

5.89; N, 3.90; found: C, 77.05; H, 5.89; N, 3.84.

N-Butyl-3-oxo-3-(2-thienyl)propanethioamide 2.11c: Yellow oil (51 %); 1H NMR

(CDCl3) δ 9.36 (br s, 1H), 8.00 (d, J = 3.8 Hz, 1H), 7.57 (d, J = 4.9Hz, 1H), 7.25 (m, 1H), 4.39 (s,

41

2H), 3.68 (m, 2H), 1.68 (q, J = 7.4 Hz, 2H), 1.43 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H); 13C NMR

(CDCl3) δ 194.9, 187.0, 144.0, 134.1, 128.7, 127.0, 55.7, 45.8, 30.1, 20.1, 0.94. Anal. Calcd. for

C11H15NOS: C, 54.74; H, 6.26; N, 5.80; found: C, 55.06; H, 6.48; N, 5.60.

N-(Tert-butyl)-2-methyl-3-oxobutanethioamide 2.11d: White microcrystals (40 %); mp

108.3 − 110.2 °C; 1H NMR (CDCl3) δ 8.17 (br s, 1H), 3.96 (q, J = 7.1 Hz, 1H), 2.29 (s, 3H), 1.54

(s, 9H), 1.45 (d, J = 6.2 Hz, 3H); 13C NMR (CDCl3) δ 209.7, 198.7, 64.0, 55.6, 29.6, 27.3, 19.2.

Anal. Calcd for C9H17NOS: C, 57.71; H, 9.15; N, 7.48; found: C, 57.60; H, 9.56; N, 7.41.

2.4.3 General Procedure for the Preparation of Compounds 2.12a–c

To a solution of the desired ketone (2.0 mmol) in THF (20 mL), potassium tert-butoxide

(2.5 mmol) was added. After stirring the mixture for 30 min, 2.5a (1.0 mmol) (Scheme 2-8) was

added and the mixture was stirred at room temperature for 0.3-4 h. The reaction was stopped and

the solvent evaporated under vacuum. The crude product was washed with water and then

extracted with diethyl ether. Evaporation of the organic fraction followed by flash column

chromatography on basic alumina afforded 2.12a-c in moderate yields.

5-Methyl-4-phenyl-5-(2-thienyl)-1,3-oxazolidine-2-thione 2.12a: White microcrystals

(51 %); mp 157.3 − 158.8 °C; 1H NMR (CDCl3) δ 7.39-7.37 (m, 1H), 7.27−7.26 (m, 2H),

7.15−7.14 (m, 1H), 7.06−7.03 (m, 2H), 5.23 (s, 1H), 1.43 (s, 3H); 13C NMR (CDCl3) δ 188.7,

145.9, 134.3, 129.4, 129.1, 127.2, 126.8, 126.1, 124.7, 91.7, 70.8, 23.8. Anal. Calcd for

C14H13NOS2: C, 61.06; H, 4.76; N, 5.09; found: C, 61.40; H, 4.78; N, 5.09. Crystal data for

C14H13NOS2, MW 275.37, monoclinic, space group P2/n, a = 14.3023(4), b = 5.7750(2), c =

17.5354(5) Å, β = 112.690(1) o, V = 1336.26(7) Å3, F(000) = 576, Z = 4, T = -180 oC, μ (MoKα)

= 0.385 mm-1, Dcalcd = 1.369 g.cm-3, 2θmax 55 o (CCD area detector, MoKα radiation), GOF =

1.05, wR(F2) = 0.076 (all 3065 data), R = 0.028 (2981 data with I > 2δI).

42

5-Methyl-4,5-diphenyloxazolidine-2-thione 2.12b: White microcrystals (27 %); mp

178.3 − 179.4 °C; 1H NMR (CDCl3) δ 7.45−7.38 (m, 9H), 7.32−7.30 (m, 2H), 5.05 (s, 1H), 1.34

(s, 3H); 13C NMR (CDCl3) δ 189.0, 143.6, 135.1, 129.4, 129.2, 129.0, 128.4, 127.0, 124.1, 93.7,

70.8, 23.9. Anal. Calcd for C16H15NOS: C, 71.34; H, 5.61; N, 5.20; found: C, 71.13; H, 5.61; N,

5.15.

5-Ethyl-5-methyl-4-phenyloxazolidine-2-thione (mixture of stereo-isomers) 2.12c:

White microcrystals (40 %); mp 119.0 − 121.4 °C; 1H NMR (CDCl3) δ 7.62 (br s, 1H), 7.49-7.42

(m, 3H), 7.32−7.27 (m, 2H), 2.05−1.96 (m, 2H), 1.72 (s, 2H), 1.68 (s, 1H), 1.56 (m, 1H), 1.16 (t,

J = 7.4 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H); 13C NMR (CDCl3) δ 189.0, 135.7, 129.1, 129.0, 128.9,

126.9, 126.7, 94.3, 93.8, 69.6, 67.0, 33.9, 28.9, 24.3, 21.3, 7.8. Anal. Calcd for C24H30N2O2S2: C,

65.12; H, 6.83; N, 6.33; found: C, 65.50; H, 6.61; N, 6.36.

43

CHAPTER 3 PEPTIDE SYNTHESIS UTILIZING (N-FMOC-PROTECTED-AMINOACYL)-

BENZOTRIAZOLES AND (N-FMOC-PROTECTED-DIPEPTIDOYL)BENZOTRIAZOLES1

3.1 Introduction

Segment condensation (or convergent/fragment condensation) peptide synthesis

[05FSPPS215] is the construction of a polypeptide target by the assembly of several intermediate

segments and is useful for the synthesis of complex peptides and small proteins. Such convergent

synthesis often allows flexibility in the choice of protecting groups and coupling methods.

Usually, protected peptide fragments of up to 15 amino acids in length are used because they are

simpler to purify by reverse phase-HPLC compared with the longer peptides (Scheme 3-1).

Scheme 3-1. General procedure of segment condensation peptide synthesis

A fundamental drawback of convergent synthesis is epimerization at the C-terminal

residue of an N-segment during the condensation reaction with the C-segment (Scheme 3-2).

[99JACS1636, 05B238, 06JPS116] There are several possible mechanisms for the

racemization/epimerization procedures during peptide synthesis as shown in Scheme 3-2; a)

formation of an oxazolone and b) tautomerization. [70JACS5792] Poor solubility of large

protected intermediate segments can also impede this approach. [05FSPPS215] The use of

1Reproduced in part with permission from Chemical Biology & Drug Design, 2008, 72, 181.

Copyright © 2009 Wiley-Blackwell

44

dicyclohexylcarbodiimide (DCC) coupling additives such as 1-hydroxybenzotriazole (HOBt),

and reagents such as (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-

phosphate) (HBTU) or (2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-

phosphate) (HATU) can minimize these disadvantages (Figure 3-1). [77BCSJ1999, 84S572,

93JACS4397] Promising results in the suppression of racemization during peptide segment

condensation in solution were obtained with new copper (II) complexes in conjunction with

other additives [01P]. Recently disclosed segment condensations using the ‘O-acyl isopeptide

method’ have provided racemization–free syntheses of small (2–5 amino acid units) peptides.

[06TL7905]

Scheme 3-2. Epimerization mechanism during segment condensation

Figure 3-1. Structure of coupling reagents

45

Early attempts to synthesize oligomers with repeating units e.g. (Tyr-Ala-Glu)n (n = 1-4)

using Boc/benzyl protection strategy and polystyrene resin were plagued with failure sequences

and low yields. [94IJPPR118] An attempted synthesis of the polymer (Tyr-Gly-Glu)6 using

Fmoc/tert-butyl protection strategy also failed after the 10th amino acid was coupled to the

growing chain. However McMurray et al. [94IJPPR118] developed segment condensation on

solid support to eliminate failure sequences caused by single amino acid deletions. Other tactics

such as enzymatic condensation suffered problems of secondary hydrolysis during the synthesis

and the instability of the enzyme in the condensation media. [93JACS7912, 93JPPS405, 01RJBC

306]

(Nα-Protected-aminoacyl)benzotriazoles have been widely used for the preparation of

chiral di- and tri-peptides by stepwise and tetra-peptides by segment condensation in solution

phase. [05S397, 06CBDD326, 06S411] However, the benzotriazole methodology in solution

phase peptide synthesis does not allow for the extension of tri-peptides or tetra-peptides.

Recently, (Nα-protected-aminoacyl)benzotriazoles were utilized as efficient coupling reagents for

solid phase peptide synthesis. [07CBDD465] In this chapter, segment condensation syntheses of

five peptides from diverse (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2 and (Nα-Fmoc-

protected-dipeptidoyl)-benzotriazoles 3.5 using mild microwave conditions are reported. The

aim of this project was to minimize or prevent common epimerization problems during segment

condensation using our benzotriazole methodology.

3.2 Results and Discussion

3.2.1 Preparation of (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2

I prepared (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-g in 72-90% yield by the

reaction of Nα-Fmoc-protected amino acids 3.1a-g with 4 equiv of BtH and 1 equiv of SOCl2 in

46

DCM at room temperature for 2 h following a published procedure (Scheme3-3, Table 3-1).

[05S397, 06CBDD326, 06S411]

Scheme 3-3. Preparation of (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-g

Table 3-1. (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-g

Reagents Products Yields (%)a mp (°C) [α]D24 b

Fmoc-L-Phe-OH 3.1a Fmoc-L-Phe-Bt 3.2a 84 159.1-160.2 +5.7c Fmoc-L-Met-OH 3.1b Fmoc-L-Met-Bt 3.2b 90 122.7-123.3 +74.4d Fmoc-L-Trp-OH 3.1c Fmoc-L-Trp-Bt 3.2c 90 92.5-93.6 +15.0e Fmoc-L-Leu-OH 3.1d Fmoc-L-Leu-Bt 3.2d 88 121.0-122.8 +88.6f Fmoc-Gly-OH 3.1e Fmoc-Gly-Bt 3.2e 85 160.9-161.5 -g Fmoc-L-Ala-OH 3.1f Fmoc-L-Ala-Bt 3.2f 72 160.0-160.3 -101.4h Fmoc-L-Pro-OH 3.1g Fmoc-L-Pro-Bt 3.2g 88 163.0-165.0 -100.9i aIsolated yields, bc = 1.5 in DMF, cLit. [α]D

24 = +35.6 (c 1.6 in DMF) [05JOC4993], dLit. [α]D

24 = +74.4 (c 1.5 in DMF) [05S397], eLit. [α]D24 = +12.7 (c 1.5 in DMF)

[05S397], fLit. [α]D24 = +53.1 (c 1.5 in DMF) [08A47], gNot chiral, hLit. [α]D

24 = -96.8 (c 1.6 in DMF)[05JOC4993], iLit. [α]D

24 = -60.5 (c 1.5 in DMF) [08A47]

3.2.2 Preparation of (LL)-Dipeptides 3.4a-f and Diastereomeric Mixtures (3.4a+3.4a’), (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) Using (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-d

I coupled readily available (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-d [05A36,

05S397, 07CBDD465] with unprotected chiral amino acids L-Ala 3.3a, L-Met 3.3b, L-Phe 3.3c

and racemic mixtures DL-Ala (3.3a+3.3a’) and DL-Met (3.3b+3.3b’) in aqueous MeCN (MeCH

: H2O = 2 : 1) in the presence of Et3N at 20 °C for 1 h to afford Nα-Fmoc-protected-dipeptides

3.4a-f, (3.4a+3.4a’), (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) (77-93 %)

isolated by simple recrystalization in EtOAc/hexanes (Scheme 3-4, Table 3-2).

47

NMR analysis of dipeptides 3.4a-f showed no detectable epimerization (<5 %). 1H NMR

analysis for each LL-dipeptide 3.4a,c,e,f derived from L-Ala 3.3a showed a clear doublet of

methyl protons ranging from 1.28–1.34 ppm. However for the corresponding diastereomeric

mixture (3.4a+3.4a’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) the signal for the methyl

protons was observed as two sets of doublets. Each of the LL-dipeptides 3.4b-d derived from L-

Met 3.3b showed singlets for the methyl group (-S-CH3) at 2.03, 2.04 and 2.02 ppm respectively,

while each of the corresponding diastereomeric mixtures (3.4b+3.4b’) showed two singlets. The

13C NMR spectra of diastereomeric mixtures (3.4a+3.4a’), (3.4b+3.4b’), (3.4c+3.4c’),

(3.4e+3.4e’), and (3.4f+3.4f’) revealed duplication of almost all aliphatic and carbonyl carbon

signals, but for 3.4a-f, the 13C NMR spectra showed no such duplication of the carbon signals

(Figure 3-2, 3-3).

The enantiopurity of the dipeptides 3.4a-f was supported by HPLC analysis using a

Chirobiotic T column (detection at 220 nm, flow rate 0.7 mL / min, and MeOH as eluent). For

each of the LL-dipeptides 3.4a-f, the HPLC results showed a single peak. In contrast, two peaks

of equal intensity were observed for the diastereomeric mixtures (3.4a+3.4a’) and (3.4b+3.4b’)

(Table 3-2).

Scheme 3-4. Preparation of Nα-Fmoc-protected-dipeptides 3.4a-f, (3.4a+3.4a’), and (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’)

48

Table 3-2. Preparation of (LL)-dipeptides 3.4a-f and diastereomeric mixtures (3.4a+3.4a’), and (3.4b+3.4b’), (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’) from (Nα-Fmoc-protected-aminoacyl)benzotriazoles 3.2a-d

Reactant Amino Acid Product Yield (%)a mp (o C) tR (min)b [α]D

24 c

Fmoc-L-Phe-Bt 3.2a L-Ala 3.3a Fmoc-L-Phe-L-Ala-OH

3.4a 85 208.7−210.6 5.4 -16.9

Fmoc-L-Phe-Bt 3.2a L-Met 3.3b Fmoc-L-Phe-L-Met-OH

3.4b 82 186.0−186.5 5.8 -27.4

Fmoc-L-Met-Bt 3.2b L-Ala 3.3a Fmoc-L-Met-L-Ala-OH

3.4c 77 169.9−171.7 2.4 -9.0d

Fmoc-L-Met-Bt 3.2b L-Phe 3.3c Fmoc-L-Met-L-Phe-OH

3.4d 89 186.0−187.0 5.7 -6.3

Fmoc-L-Trp-Bt 3.2c L-Ala 3.3a Fmoc-L-Trp-L-Ala-OH

3.4e 83 119.0−121.0 5.7 -15.8e

Fmoc-L-Leu-Bt 3.2d L-Ala 3.3a Fmoc-L-Leu-L-Ala-OH

3.4f 87 179.0−179.8 2.3 -10.9

Fmoc-L-Phe-Bt 3.2a

DL-Ala (3.3a+3.3a’)

Fmoc-L-Phe-DL-Ala-OH (3.4a+3.4a’) 82 149.9−151.0 5.6, 6.0 -21.8

Fmoc-L-Phe-Bt 3.2a

DL-Met (3.3b+3.3b’)

Fmoc-L-Phe-DL-Met-OH (3.4b+3.4b’) 74 156.9−158.5 5.4, 6.6 -21.6

Fmoc-L-Met-Bt 3.2b

DL-Ala (3.3a+3.3a’)

Fmoc-L-Met-DL-Ala-OH (3.4c+3.4c’) 93 115.0-117.0 N/Af N/Af

Fmoc-L-Trp-Bt 3.2c

DL-Ala (3.3a+3.3a’)

Fmoc-L-Trp-DL-Ala-OH (3.4e+3.4e’) 78 146.3-147.1 N/Af -20.4

Fmoc-L-Leu-Bt 3.2d

DL-Ala (3.3a+3.3a’)

Fmoc-L-Leu-DL-Ala-OH (3.4f+3.4f’) 89 163.0-164.0 N/Af -13.2

aIsolated yield, btR = retention time, cc = 1.5 in DMF, dLit. [α]D24 = -9.7 (c 1.5 in DMF) [05S397], eLit. [α]D

24 = -15.7 (c 1.5 in DMF) [05S397], fNot determined

Figure 3-2. 1H NMR of 3.4a and (3.4a+3.4a’). a) 1H NMR of 3.4a, methyl group, b) 1H NMR of (3.4a+3.4a’), methyl groups

49

Figure 3-3. 13C NMR of 3.4a and (3.4a+3.4a’).c) 13C NMR of 3.4a, carbonyl groups, d) 13C

NMR of (3.4a+3.4a’), carbonyl groups

3.2.3 Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f

I treated Nα-Fmoc-protected dipeptides 3.4a-f in DCM with 4 equiv of BtH and 1 equiv

of SOCl2 at –15 °C for 3 h to give the novel dipeptidoylbenzotriazoles 3.5a-f (69-87 %) (Scheme

3-5, Table 3-3), fully characterized by 1H and 13C NMR spectroscopy and elemental analysis.

Scheme 3-5. Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f

Table 3-3. Preparation of (Nα-Fmoc-protected-dipeptidoyl)benzotriazole 3.5a-f Reactant Product Yield (%)a mp (o C) [α]D

24 b Fmoc-L-Phe-L-Ala-OH 3.4a Fmoc-L-Phe-L-Ala-Bt 3.5a 83 155.2-156.9 -72.2 Fmoc-L-Phe-L-Met-OH 3.4b Fmoc-L-Phe-L-Met-Bt 3.5b 73 159.0-161.0 -53.8 Fmoc-L-Met-L-Ala-OH 3.4c Fmoc-L-Met-L-Ala-Bt 3.5c 70 147.2-149.0 -50.1 Fmoc-L-Met-L-Phe-OH 3.4d Fmoc-L-Met-L-Phe-Bt 3.5d 87 197.5-199.0 -52.5 Fmoc-L-Trp-L-Ala-OH 3.4e Fmoc-L-Trp-L-Ala-Bt 3.5e 69 153.8-155.0 -74.7 Fmoc-L-Leu-L-Ala-OH 3.4f Fmoc-L-Leu-L-Ala-Bt 3.5f 81 153.0-154.7 -60.9 aIsolated yield, bc 1.5 in DMF

3.2.5 General Peptide Syntheses by Segment Condensation

I used (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f and (Nα-Fmoc-protected-

aminoacyl)benzotriazoles 3.2c-e for the synthesis of peptides 3.6-3.10 (Table 3.4) by solid phase

segment condensation under mild microwave irradiation.

50

Following standard Fmoc solid phase peptide synthesis (SPPS) strategy (Scheme 3-6),

approximately 120 mg of N-Fmoc-protected Rink-Amide MBHA Resin (200-400 mesh, 0.35

meq/g), (0.05 mmol) was placed in a reaction vessel. The resin was allowed to swell for 1 h in

DCM (5 mL) followed by the removal of Fmoc group using 20 % piperidine in DMF (5 mL x 2,

5 min and 10 min). The resin-NH2 was coupled to 5.0 equiv of the benzotriazole derivative

(3.2c-e or 3.5a-f) in 1.5 mL of solvent (DMF for 3.2c-e and DMSO for 3.5a-f) under microwave

irradiation (30 °C, 10 min, 30 W). When a negative Kaiser (ninhydrin) test verified completion

of coupling (10 min), the solid resin was washed with DMF (5 mL x 3) and DCM (5 mL x 3).

The Fmoc group was removed and another coupling was started. After the desired number of

coupling steps, the peptide was deprotected and the desired peptide was cleaved from the resin

using cleavage cocktails: i) TFA : anisole : thioanisole : BAL (90 : 2 : 3 : 5) (for peptide

sequences including Trp or Met) or ii) TFA : water : TIPS (95 : 2.5 : 2.5) (for the other peptide

sequences) at 20 °C for 2 h. The resin was filtered off, and the crude mixture was concentrated;

diethyl ether was added at –20 °C to afford the crude peptide as a precipitate. Precipitates were

filtered off and dried to yield peptides 3.6-3.10 in crude yields of 50-90 %. The crude peptides

were purified by reverse-phase HPLC to give the pure peptides 3.6-3.10 isolated in yields of 20

to 68 % (Table 3-4). Each peptide was characterized by HRMS.

HPLC analysis of crude tripeptide 3.6 and heptapeptide 3.10 revealed peaks for the desired

products with no racemized by-product (Table 4-3). For the peptides 3.7-3.9 formation of one

by-product in yields of 19 to 33 % derived from epimerization was detected. The HPLC profiles

of 3.6-3.10 are given in the experimental section.

51

Scheme 3-6. General procedure of peptide synthesis by segment condensation

Table 3-4. Synthesized peptides 3.6-3.10 by solid phase segment condensation

Reactants coupled to Rink resin-NH2 Product Structure (N to C terminus)

Pure yielda/ Purityb (%)

tR (min)

HRMS [M+H]+ e

3.2d+3.5f 3.6 H-L-Leu-L-Ala-L-Leu-NH2 68/99 8.49 315.2401

3.5c+3.5e 3.7 H-L-Trp-L-Ala-L-Met-L-Ala-NH2 26c/99 9.38 477.2291

3.5f+3.5b+3.2c 3.8 H-L-Trp-L-Phe-L-Met-L-Leu-L-Ala-NH2 21c/99 15.45 666.3432

3.5b+3.5d+3.5f 3.9 H-L-Leu-L-Ala-L-Met-L-Phe-L-Phe-L-Met- NH2

20d/72 16.43 758.3707

3.2e+3.5a+3.5c+3.5f 3.10 H-L-Leu-L-Ala-L-Met-L-Ala-L-Phe-L-Ala-Gly-NH2

30/99 11.29 679.3589

aIsolated yield after HPLC purification.; bPurity after HPLC purification.; cIsolated yield of the major peptide peak; dIsolated yield of the major peak and an impurity not derived from racemization (refer Figure 5.4).; eFor the calculated values, see the experimental section.

During segment condensations, epimerization frequently occurs at the C-terminus residue

because of the activation of the carboxylic acid function (Scheme 3-2a). [06TL7905] Gly and

Pro are widely used as C-terminus residues to prevent epimerization. [05FSPPS215] Peptide 3.6

(H-L-Leu-L-Ala-L-Leu-NH2) was synthesized using 3.2d and 3.5f, which contains Ala at C-

terminus in the fragment.With DMF as solvent, at 70 °C (60 W), for 3 min under microwave

irradiation followed by reported procedure, [07CBDD465] 50% of epimerization was observed.

DMF is generally avoided as a solvent due to higher possibility of epimerization during segment

52

condensation. The reactions were attempted in DMSO at 50 oC, and epimerization was again

observed. The epimerization is more likely at higher temperature due to equilibrium mechanism

(Scheme 3-2a). Coupling in DMSO at 30 °C (30 W) for 10 min provided 3.6 (13 min total

coupling time) and also 3.10 (33 min total coupling time) without epimerization in isolated

yields of 68 and 30 % respectively after HPLC purification. Thus, from the sequences from the

peptides synthesized, an L-Ala residue at the C-terminus (3.5a, 3.5c, 3.5f) prevents epimerization

by benzotriazole methodology. Although the epimerization site of peptide 3.7-3.9 is still

unknown, partial epimerization was probably caused at 3.5e, 3.5b, or 3.5d for 3.7-3.9 under the

same coupling conditions. After HPLC purification yields of isolated optically pure 3.7-3.9 were

still 20 to 26 %.

3.3 Conclusion

In conclusion I have prepared tri-, tetra-, penta-, hexa-, and heptapeptides in average

isolated yields of 33 % by solid phase segment condensation under microwave irradiation.

Benzotriazole intermediates, (Nα-Fmoc-protected-dipeptidoyl)benzotriazoles 3.5a-f, are air and

moisture insensitive acylation reagents enabling solid phase segment condensation without the

use of other coupling reagents or additives. After modification of couplind conditions,

epimerization was not observed in some sequences (3.6 and 3.10), but partial epimerization was

observed in other sequences (3.7-3.9). Temperature during the coupling is the crutial factor in

preventing epimerization at the C-terminus. Thus, low-temperature microwave synthesis may be

able to solve epimerization problems and offer a short coupling time.

3.4 Experimental Section

3.4.1 General Procedure for the Preparation of 3.1a-g

SOCl2 (1.0 mmol) was added to a solution of BtH (4.0 mmol) in DCM (16 mL), and the

reaction mixture was stirred for 30 min. The appropriate Fmoc-protected amino acid 3.1a-g was

53

added in one portion, and the reaction mixture was stirred at room temperature for 2 h. The

solute was then washed with 5 % Na2CO3 aq, extracted by DCM, and dried over MgSO4.

Evaporation of the solvent followed by recrystallization from DCM/hexanes to gave (Nα-Fmoc-

protected-aminoacyl)benzotriazoles 3.1a-g (Scheme 3-3).

(S)-(9H-Fluoren-9-yl)methyl-1-(1H-benzo[d][1,2,3]triazol-1-yl)-4-methyl-1-

oxopentan-2-ylcarbamate (Fmoc-L-Leu-Bt) 3.2d: White microcrystals (88 %); mp 121.0 −

122.8 °C; [α]D24 = +88.6 (c 1.5, DMF); 1H NMR (CDCl3) δ 8.27 (d, J = 8.2 Hz, 1H), 8.16 (d, J =

8.1 Hz, 1H), 7.21 (d, J = 7.3 Hz, 2H), 7.69 (t, J = 7.1 Hz, 1H), 7.61−7.52 (m, 3H), 7.41 (t, J = 7.0

Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 5.85 (t, J = 7.8 Hz, 1H), 5.50 (br s, 1H), 4.45 (d, J = 7.0 Hz,

2H), 4.25 (t, J = 6.6 Hz, 1H), 1.88 (br s, 2H), 1.77 (t, J = 10.2 Hz, 1H), 1.61 (s, 2H), 1.11 (d, J =

4.9 Hz, 3H), 0.99 (d, J = 5.4 Hz, 3H); 13C NMR (CDCl3) δ 172.4, 156.1, 146.0, 143.8, 141.3,

131.1, 130.7, 127.7, 127.0, 126.5, 125.0, 120.3, 120.0, 114.4, 67.1, 53.0, 47.1, 41.9, 25.2, 23.2,

21.3. Anal. Calcd for C27H26N4O3: C, 71.35; H, 5.77; N, 12.33; found: C, 71.19; H, 6.06; N,

12.21.

(S)-(9H-Fluoren-9-yl)methyl-2-(1H-benzo[d][1,2,3]triazole-1-carbonyl)pyrrolidine-1-

carboxylate (Fmoc-L-Pro-Bt, mixture of rotomers) 3.2g: White microcrystals (88 %); mp

163.0 − 165.0 °C; [α]D24 = -100.9 (c 1.5, DMF); 1H NMR (CDCl3) δ 8.29 (d, J = 8.2Hz, 1H),

8.20 (d, J = 8.2Hz, 1H), 8.14 (d, J = 8.1Hz, 2H), 7.78 (d. J = 7.5Hz, 2H), 7.73-7.49 (m, 8H),

7.44-7.30 (m, 10H), 7.21 (t, J = 6.0Hz, 3H), 7.09 (t, J = 6.7Hz, 2H), 6.89−6.78 (m, 3H), 5.87 (dd,

J = 4.1Hz, 1H), 5.43 (dd, J = 3.4Hz, 1H), 4.61−4.53 (m, 2H), 4.48−4.45 (m, 1H), 4.35-4.28 (m,

2H), 4.02 (t, J = 4.9Hz, 1H), 3.89−3.82 (m, 1H), 3.76−3.58 (m, 4H), 2.68−2.61 (m, 1H),

2.53−2.43 (m, 1H), 2.28-2.20 (m, 1H), 2.18-2.02 (m, 4H), 1.99−1.90 (m, 3H), 1.67 (br s, 2H);

13C NMR (CDCl3) δ 171.0, 170.6, 154.9, 154.1, 146.0, 144.0, 143.8, 143.5, 141.3, 141.0, 140.8,

54

131.2, 131.2, 130.5, 130.5, 127.7, 127.4, 127.1, 126.9, 126.8, 126.5, 126.4, 126.4, 125.2, 125.1,

124.1, 124.0, 120.2, 120.2, 120.0, 119.7, 119.4, 114.6, 114.5, 67.7, 66.5, 60.0, 59.2, 47.2, 47.0,

47.0, 31.6, 30.7, 24.5, 23.2. Anal. Calcd for C26H22N4O3: C, 71.22; H, 5.06; N, 12.78; found: C,

71.16; H, 5.03; N, 13.12.

3.4.2 General Procedure for the Preparation of 3.4a-f, (3.4a+3.4a’), (3.4b+3.4b’) (3.4c+3.4c’), (3.4e+3.4e’), and (3.4f+3.4f’)

(Nα-Fmoc-aminoacyl)benzotriazoles 3.2a-d (0.5 mmol) were added at room temperature to

a solution of unprotected-α-amino acid 3.3a-c, (3.3a+3.3a’), or (3.3b+3.3b’) (0.5 mmol) in

aqueous MeCN (MeCN : H2O = 7 mL : 3 mL) in the presence of Et3N (0.5 mmol). The reaction

mixture was then stirred at 20 °C until 3.2 was consumed. Then 6M HCl aq (1 mL) was added,

and the solution was concentrated under reduced pressure. The residue was extracted with

EtOAc, washed with 6 M HCl aq, brine, and the organic layer was dried over MgSO4.

Evaporation of the solvent followed by recrystallization from DCM/hexanes gave the desired

dipeptides 3.4a-f, (3.4a+3.4a’), (3.4b+4.4b’), (3.4c+3.4c’), (3.4e+3.4e’), (3.4f+3.4f’) (Scheme

3-4).

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phenylpropanamido)-

propanoic acid (Fmoc-L-Phe-L-Ala-OH) 3.4a: White microcrystals (85 %); mp 208.7 − 210.6

°C; [α]D24 = -16.9 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.60 (s, 1H) 8.38 (d, J = 7.1Hz, 1H),

7.87 (d, J = 7.4Hz, 2H), 7.70−7.56 (m, 2H), 7.45−7 .14 (m, 10H), 4.46−4.18 (m, 2H), 4.18−4.02

(m, 3H), 3.10−2.96 (m, 1H), 2.83−2.70 (m, 1H), 1.32 (d, J = 7.1Hz, 3H); 13C NMR (DMSO-d6)

δ 174.1, 171.5, 155.8, 143.8, 140.6, 138.2, 129.3, 128.0, 127.6, 127.1, 126.2, 125.4, 120.1, 65.6,

55.9, 47.5, 46.6, 37.4,17.2. Anal. Calcd for C27H26N2O5: C, 70.73; H, 5.72; N, 6.11; found: C,

70.45; H, 5.82; N, 5.93.

55

2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phenylpropanamido)-

propanoic acid (Fmoc-L-Phe-DL-Ala-OH) (3.4a+3.4a’): White microcrystals (82 %); mp

149.9 − 151.0 °C; [α]D24 = -21.8 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.64 (br s, 1H), 8.43-

8.35 (m, 1H), 7.91 (d, J = 7.4Hz, 2H), 7.68-7.65 (m, 2H), 7.50−7.16 (m, 10H), 4.36−4.24 (m,

2H), 4.22−4.07 (m, 3H), 3.09−2.97 (m, 1H), 2.86−2.75 (m, 1H), 1.35 (d, J = 7.1 Hz, 1.5 H), 1.26

(d, J = 7.1 Hz, 1.5 H); 13C NMR (DMSO-d6) δ 175.0, 174.9, 172.4, 172.1, 156.7, 156.6, 144.7,

144.7, 141.6, 139.2, 139.0, 130.2, 130.2, 129.0, 128.6,128.0, 127.2, 126.3, 121.0, 66.6, 56.8,

47.5, 38.9, 38.4, 18.4, 18.1. Anal. Calcd. for C27H26N2O5: C, 70.73; H, 5.72; N, 6.11; found: C,

70.39; H, 5.81; N, 5.95.

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-phenylpropanamido)-4-

(methylthio)butanoic acid (Fmoc-L-Phe-L-Met-OH) 3.4b: White microcrystals (82 %); mp

186.0 − 186.5 °C; [α]D24 = -27.4 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.78 (br s, 1H), 8.34 (d,

J = 7.7Hz, 1H), 7.87 (d, J = 7.4Hz, 2H), 7.70−7.58 (m, 2H), 7.46−7.36 (m, 3H), 7.36−7.22 (m,

6H), 7.22−7.13 (m, 1H), 4.46−4.24 (m, 2H), 4.24−4.04 (m, 3H), 3.09−2.97 (m, 1H), 2.85−2.71

(m, 1H), 2.59−2.40 (m, 3H), 2.38-2.56 (m, 2H), 2.03 (s, 3H), 2.00−1.83 (m, 2H); 13C NMR

(DMSO-d6) δ 173.2, 171.9, 156.8, 143.8, 140.7, 138.2, 129.3, 128.1, 127.6, 127.1, 126.3, 125.4,

120.1, 66.6, 56.0, 51.0, 46.6, 37.3, 30.8, 29.6, 14.6. Anal. Calcd for C29H30N2O5S: C, 67.16; H,

5.83; N, 5.40; found: C, 67.07; H, 5.95; N, 5.31.

2-{[(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-(1H-indol-3-yl)propanoyl]

amino}propanoic acid (Fmoc-L-Phe-DL-Met-OH) (3.4b+3.4b’): White microcrystals (74 %);

mp 156.9 − 158.5 °C; [α]D24 = -21.6 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.77 (br s, 1H), 8.41

(d, J = 8.0Hz, 0.5H), 8.36 (d, J = 8.0Hz, 0.5H), 7.91 (d, J = 7.4Hz, 2H), 7.77−7.64 (m, 2H),

7.53−7.39 (m, 3H), 7.39−7.26 (m, 6H), 7.23−7.21 (m, 1H), 4.48−4.22 (m, 3H), 4.17 (s, 3H),

56

3.10−2.95 (m, 1H), 2.89−2.74 (m, 1H), 2.66−2.46 (m, 1H), 2.46−2.32 (m, 1H), 2.06 (s, 1.5H),

2.04 (s, 1.5H), 2.02−1.82 (m, 2H). 13C NMR (DMSO-d6) δ 173.2, 171.8, 171.5, 155.8, 155.7,

143.8, 143.7, 140.7, 140.9, 138.2, 137.9, 129.3, 128.1, 127.6, 127.1, 126.3, 125.4, 125.3, 120.1,

65.7, 65.6, 56.0, 51.0, 50.9, 46.6, 38.1, 37.3, 30.8, 30.8, 29.6, 29.5, 14.6, 14.5. Anal. Calcd for

C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40; found: C, 67.22; H, 6.04; N, 5.26.

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-(methylthio)butanamido)

propanoic acid (Fmoc-L-Met-L-Ala-OH) 3.4c: White microcrystals (77 %); mp 169.9 − 171.7

°C (Lit. 155−156 °C); [α]D24 = -9.0 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.58 (br s, 1H),

8.29−8.21 (m, 1H), 7.89 (d, J = 7.4Hz, 2H), 7.68−7.78 (m, 2H), 7.62−7.54 (m, 1H), 7.47−7.38

(m, 2H), 7.38−7.28 (m, 2H), 4.34−4.05 (m, 5H), 2.56−2.44 (m, 2H), 2.04 (s, 3H), 1.98−1.74 (m,

2H), 1.28 (d, J = 7.1Hz, 3H); 13C NMR (DMSO-d6) δ 170.1, 171.3, 155.9, 143.9, 143.8, 140.7,

127.7, 127.1, 125.4, 120.1, 65.6, 53.5, 47.5, 46.6, 31.8, 29.5, 17.0, 14.6. Anal. Calcd for

C23H26N2O5S: C, 62.42; H, 5.92; N, 6.33; found: C, 62.12; H, 6.02.; N, 6.27.

2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-(methylthio)butanamido)-

propanoic acid (Fmoc-L-Met-DL-Ala-OH) (3.4c+3.4c’): White microcrystals (93 %); mp

115.0 − 117.0 °C; 1H NMR (DMSO-d6) δ 12.64 (br s, 1H), 8.24 (d, J = 7.4Hz, 0.5H), 8.18 (d, J

= 7.4Hz, 0.5H), 7.91 (d, J = 7.4Hz, 2H), 7.77.-7.68 (m, 2H), 7.55 (t, J = 8.2Hz, 1H), 7.42 (t, J =

7.4Hz, 2H), 7.32 (t, J = 7.1Hz, 2H), 4.33-4.05 (m, 3H), 2.60-2.37 (m, 2H), 2.11-1.98 (m, 3H),

1.95-1.77 (m, 2H), 1.33-1.20 (m, 3H); 13C NMR (DMSO-d6) δ 174.0, 173.9, 171.1, 155.9, 143.9,

143.7, 140.7, 127.6, 127.1, 125.3, 120.1, 65.6, 53.7, 53.5, 48.0, 46.6, 31.8, 29.5, 17.4, 17.0, 14.6,.

Anal. Calcd for C23H26N2O5S: C, 62.42; H, 5.92; N, 6.33; found: C, 62.38; H, 5.95.; N, 6.19.

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-(methylthio)butanamido)-

3-phenylpropanoic acid (Fmoc-L-Met-L-Phe-OH) 3.4d: White microcrystals (86 %); mp

57

186.0 − 187.0 °C; [α]D24 = -6.3 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.77 (br s, 1H), 8.15 (d, J

= 7.7 Hz, 1H), 7.89 (d, J = 7.7 Hz, 2H), 7.78−7.68 (m, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.42 (t, J =

7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.26−7.13 (m, 6H), 4.48−4.38 (m, 1H), 4.30−4.16 (m, 3H),

4.15−4.04 (m, 1H), 3.10−3.00 (m, 1H), 2.97−2.87 (m, 1H), 2.42 (t, J = 7.4 Hz, 2H), 2.02 (s, 3H),

1.90−1.71 (m, 2H); 13C NMR (DMSO-d6) δ 172.8, 171.4, 155.8, 143.9, 143.7, 140.7, 137.4,

129.1, 128.1, 127.6, 127.1, 126.4, 125.3, 120.1, 65.6, 53.7, 53.3, 46.6, 36.5, 31.8, 29.5, 14.6.

Anal. Calcd for C29H30N2O5S: C, 67.16; H, 5.83; N, 5.40; found: C: 67.20; H, 5.96; N, 5.38.

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-3-(1H-indol-3-yl)propan-

amido)propanoic acid (Fmoc-L-Trp-L-Ala-OH) 3.4e: Yellow microcrystals (83 %); mp 119.0

− 121.0 °C (Lit. 155.0−156.0 °C); [α]D24 = -15.8 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.58 (br

s, 1H), 10.86 (s, 1H), 8.42 (d, J = 6.9Hz, 1H), 7.90 (d, J = 6.9Hz, 2H), 7.74 (d, J = 7.7Hz, 1H),

7.66 (t, J = 7.7Hz, 2H), 7.55 (d, J = 8.5Hz, 1H), 7.42 (d, J = 6.3Hz, 2H), 7.39−7.33 (m, 2H),

7.30−7.23 (m, 2H), 7.10 (t, J = 7.7Hz, 1H), 7.03−6.99 (m, 1H), 4.37−4.26 (m, 2H), 4.17 (s, 3H),

3.19−3.14 (m, 1H), 3.01−2.92 (m, 1H), 1.34 (d, J = 7.4Hz, 3H); 13C NMR (DMSO-d6) δ 175.1,

172.9, 156.8, 144.7, 141.6, 128.6, 128.0, 126.3, 124.9, 121.8, 121.0, 119.6, 119.1, 112.2, 66.6,

56.1, 48.5, 47.5, 28.8, 18.1. Anal. Calcd for C29H27N3O5: C, 70.01; H, 5.47; N, 8.45; found: C,

69.68; H, 5.59; N, 8.14.

2-{[(2S)-2-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}-3-(1H-indol-3-yl)propanoyl]-

amino}propanoic acid (Fmoc-L-Trp-DL-Ala-OH) (3.4e+3.4e’): White microcrystals (78 %);

mp 146.3 − 147.1 °C; [α]D24 = -20.4 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.63 (br s, 1H),

10.86 (s, 1H), 8.42 (d, J = 7.1Hz, 0.5H), 8.35 (d, J = 7.7Hz, 0.5H), 7.90 (d, J = 7.1Hz, 2H), 7.79-

7.60 (m, 2H), 7.60-7.51 (m, 1H), 7.51-7.14 (m, 5H), 7.14-6.94 (m, 2H), 4.42-4.07 (m, 4H), 3.20-

3.05 (m, 2H), 3.05-2.82 (m, 2H), 1.35 (d, J = 7.1Hz, 1.5H), 1.24 (d, J = 7.1Hz, 1.5H); 13C NMR

58

(DMSO-d6) δ 174.1, 174.0, 171.9, 171.6, 155.8, 155.7, 143.8, 140.7, 136.1, 127.6, 127.3, 127.1,

125.4, 124.0, 120.8, 120.1, 118.7, 118.2, 111.3, 110.3, 110.2, 100.0, 65.7, 55.4, 55.2, 47.6, 46.6,

28.2, 27.8, 17.4, 17.2. Anal. Calcd for C29H27N3O5: C, 70.01; H, 5.47; N, 8.45; found: C, 69.72;

H, 5.44; N, 8.29.

(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-methylpentanamido)

propanoic acid (Fmoc-L-Leu-L-Ala-OH) 3.4f: White microcrystals (84 %); mp 179.0 − 179.8

°C; [α]D24 = -10.9 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.49 (s, 1H), 8.17 (d, J = 7.1 Hz, 1H),

7.89 (d, J = 7.4 Hz, 2H), 7.77−7.69 (m, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.42 (t, J = 7.1 Hz, 2H),

7.37−7.28 (m, 2H), 4.32−4.15 (m, 4H), 4.15−4.02 (m, 1H), 1.73−1.58 (m, 1H), 1.53−1.39 (m,

2H), 1.27 (d, J = 7.4 Hz, 3H), 0.98−0.80 (m, 6H); 13C NMR (DMSO-d6) δ 174.0, 172.2, 155.9,

144.0, 140.7, 127.6, 127.0, 125.3, 120.1, 65.5, 52.7, 47.4, 46.7, 24.1, 23.2, 21.4, 17.1. Anal.

Calcd for C24H28N2O5: C, 67.91; H, 6.65; N, 6.60; found: C, 68.06; H, 6.77; N, 6.51.

2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-4-methylpentanamido)-

propanoic acid (Fmoc-L-Leu-DL-Ala-OH) (3.4f+3.4f’): White microcrystals (89 %); mp 163.0

− 164.0 °C; [α]D24 = -13.2 (c 1.5, DMF); 1H NMR (DMSO-d6) δ 12.56 (br s, 1H), 8.23-8.19 (m,

1H), 7.92 (d, J=7.4Hz, 2H), 7.82-7.70 (m, 1H), 7.57-7.40 (m, 3H), 7.40-7.29 (m, 2H), 4.48-4.07

(m, 5H), 1.77-1.59 (m, 1H), 1.59-1.38 (m, 2H), 1.32-1.28 (m, 3H), 0.94-0.89 (m, 6H); 13C NMR

(DMSO-d6) δ 174.1, 174.0, 172.2, 172.2, 155.9, 155.9, 144.0, 143.8, 140.7, 127.7, 127.1, 125.4,

120.1, 65.6, 52.9, 52.7, 47.5, 46.7,24.2, 24.1, 23.2, 24.4, 17.4, 17.1. Anal. Calcd for C24H28N2O5:

C, 67.91; H, 6.65; N, 6.60; found: C, 67.78; H, 6.80; N, 6.52.

3.4.3 General Procedure for the Preparation of 3.5a-f

SOCl2 (1.0 mmol) was added to a solution of BtH (4.0 mmol) in DCM (16 mL), and the

reaction mixture was stirred for 30 min, then cooled to -15 °C. The appropriate dipeptide 3.4a-f

was added in one portion, and the mixture was stirred at -15 °C for 3 h. The reaction mixture

59

was diluted with DCM, washed with 5 % Na2CO3 aq, and dried with MgSO4. Evaporation of the

solvent followed by recrystallization from DCM/hexanes afforded the desired benzotriazole

derivatives 3.5a-f (Scheme 3-5).

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-

ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate (Fmoc-L-Phe-L-Ala-Bt) 3.5a: White

microcrystals (83 %); mp 155.2 − 156.9 °C; [α]D24 = -72.2 (c 1.5, DMF); 1H NMR (CDCl3) δ

8.23 (d, J = 7.4 Hz, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 7.0 Hz, 2H), 7.69 (t, J = 7.1 Hz,

1H), 7.60−7.50 (m, 2H), 7.40 (m, 2H), 7.30 (t, J = 7.4 Hz, 3H), 7.24−7.18 (m, 4H), 7.18−7.10

(m, 2H), 6.64 (br s, 1H), 5.88 (quin, J = 7.1 Hz, 1H), 5.43 (m 1H), 4.58−4.41 (m, 4H), 4.20 (t, J

= 7.0 Hz, 1H), 3.20−2.97 (m, 2H), 1.60 (d, J = 7.0Hz, 3H); 13C NMR (CDCl3) δ 171.1, 170.5,

156.0, 146.0, 143.6, 141.3, 136.1, 131.1, 130.8, 129.3, 128.7, 127.7, 127.1, 126.6, 125.0, 120.4,

120.0, 114.3, 67.1, 56.0, 49.1, 47.0, 38.5, 18.7. Anal. Calcd for C33H29N5O4: C, 70.83; H, 5.22;

N, 12.51; found: C, 70.77; H, 5.23; N, 12.44.

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-4-(methylthio)-

1-oxobutan-2-ylamino)-1-oxo-3-phenylpropan-2-ylcarbamate (Fmoc-L-Phe-L-Met-Bt) 3.5b:

White microsrystals (73 %); mp 159.0 − 161.0 °C; [α]D24 = -53.8 (c 1.5, DMF); 1H NMR

(DMSO-d6) δ 8.96 (d, J = 6.5 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.88 (d,

J = 7.7 Hz, 2H), 7.82 (t, J = 7.7 Hz, 1H), 7.76−7.58 (m, 4H), 7.46−7.14 (m, 9H), 5.88−5.75 (m,

1H), 4.48−4.33 (m, 1H), 4.21−4.08 (m, 3H), 3.11−3.00 (m, 1H), 2.86−2.58 (m, 3H), 2.40−2.10

(m, 2H), 2.05 (s, 3H); 13C NMR (DMSO-d6) δ 172.4, 171.1, 155.9, 145.4, 143.8, 140.7, 138.0,

131.1, 130.7, 129.2, 128.1, 127.6, 127.1, 126.8, 126.3, 125.3, 120.2, 120.1, 114.1, 65.7, 55.7,

52.0, 46.5, 37.3, 30.0, 29.5, 14.4. Anal. Calcd for C35H33N5O4S: C, 67.83; H, 5.37; N, 11.30;

found: C, 67.99; H, 5.44; N, 10.98.

60

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-

ylamino)-4-(methylthio)-1-oxobutan-2-ylcarbamate (Fmoc-L-Met-L-Ala-Bt) 3.5c: White

microcrystals (70 %); mp 147.2 − 149.0 °C; [α]D24 = -50.1 (c 1.5, DMF); 1H NMR (CDCl3) δ

8.25 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.3 Hz, 2H), 7.68 (t, J = 7.3 Hz,

1H), 7.60 (d, J = 7.6 Hz, 2H), 7.54 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.7

Hz, 2H), 7.00 (d, J = 6.6 Hz, 1H), 5.95 (t, J = 7.1 Hz, 1H), 5.59 (d, J = 7.7 Hz, 1H),

4.58−4.47(m, 1H), 4.43 (d, J = 6.9 Hz, 2H), 4.23 (t, J = 6.7 Hz, 1H), 2.67 (t, J = 6.5 Hz, 2H),

2.15 (s, 3H), 2.13−1.99 (m, 2H), 1.71 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3) δ 171.5, 171.0,

156.1, 146.0, 143.7, 143.6, 141.3, 131.1, 130.8, 127.7, 127.1, 126.6, 125.0, 120.4, 120.0, 114.4,

67.2, 53.3, 49.2, 47.1, 31.5, 29.9, 18.5, 15.1. Anal. Calcd for C29H29N5O4S: C, 64.07; H, 5.38; N,

12.88; found: C, 64.16; H, 5.43; N, 12.69.

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxo-3-

phenylpropan-2-ylamino)-4-(methylthio)-1-oxobutan-2-ylcarbamate (Fmoc-L-Met-L-Phe-

Bt) 3.5d: White microcrystals (87 %); mp 197.5 − 199.0 °C; [α]D24 = -52.5 (c 1.5, DMF); 1H

NMR (CDCl3) δ 8.23 (d, J = 8.1 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 7.4 Hz, 2H), 7.69

(t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.4 Hz, 3H), 7.41 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H),

7.20 (t, J = 7.3 Hz, 3H), 7.16−7.09 (m, 2H), 7.01−6.92 (m, 1H), 6.28−6.18 (m, 1H), 5.49 (d, J =

8.2 Hz, 1H), 4.50−4.32 (m, 3H), 4.21 (t, J = 6.7 Hz, 1H), 3.54−3.43 (m, 1H), 3.28−3.17 (m, 1H),

2.63−2.54 (m, 2H), 2.14−1.92 (m, 5H); 13C NMR (CDCl3) δ 171.0, 170.2, 155.9, 146.0, 143.7,

143.7, 141.3, 134.8, 131.0, 130.9, 129.2, 128.8, 127.7, 127.5, 127.1, 126.6, 125.0, 120.4, 120.0,

114.3, 100.2, 67.1, 54.2, 53.2, 47.0, 38.3, 31.2, 29.9, 15.0. Anal. Calcd for C33H35N5O4S: C,

67.83; H, 5.37; N, 11.30; found: C, 68.21; H, 5.69; N, 10.96.

61

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-

ylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-ylcarbamate (Fmoc-L-Trp-L-Ala-Bt) 3.5e:

Yellow microcrystals (69 %); mp 153.8 − 155.0 °C; [α]D24 = -74.7 (c 1.5, DMF); 1H NMR

(DMSO-d6) δ 10.90 (s, 1H), 9.01 (d, J = 5.5Hz, 1H), 8.33−8.21 (m, 2H), 7.90−7.71 (m, 4H),

7.71−7.65 (m, 3H), 7.45−7.19 (m, 6H), 7.08−6.93 (m, 2H), 5.66 (t, J = 6.0Hz, 1H), 4.50−4.35

(m, 1H), 4.14 (s, 2H), 4.10−3.95 (m, 1H), 3.22−3.11 (m, 1H), 3.03−2.90 (m, 1H), 1.61 (d, J =

7.1Hz, 3H); 13C NMR (DMSO-d6) δ 172.6, 171.9, 155.9, 145.4, 143.7, 140.7, 136.1, 131.1,

130.7, 127.6, 127.2, 127.1, 125.4, 125.3, 124.1, 120.8, 120.2, 120.1, 118.7, 118.2, 114.0, 111.3,

110.1, 65.7, 55.0, 48.7, 46.6, 27.8, 16.7. HRMS Calcd for [C35H30N6O4+H]+, 621.2221; found,

621.2232.

(9H-Fluoren-9-yl)methyl(S)-1-((S)-1-(1H-benzo[d][1,2,3]triazol-1-yl)-1-oxopropan-2-

ylamino)-4-methyl-1-oxopentan-2-ylcarbamate (Fmoc-L-Leu-L-Ala-Bt) 3.5f: White

microcrystals (81 %); mp 153.0 – 154.2 °C; [α]D24 = -60.9 (c 1.5, DMF); 1H NMR (CDCl3) δ

8.82 (d, J = 5.5 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.4 Hz,

2H), 7.80 (t, J = 8.0 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.64 (quin, J = 7.7 Hz, 1H), 7.55 (d, J =

8.5 Hz, 1H), 7.41 (t, J = 7.1 Hz, 2H), 7.31 (t, J = 7.1 Hz, 2H), 5.58 (t, J = 6.3 Hz, 1H), 4.32−4.13

(m, 4H), 1.75-1.62 (m, 1H), 1.56 (d, J = 6.9 Hz, 3H), 1.53−1.42 (m, 2H), 0.91 (t, J = 6.9 Hz,

6H); 13C NMR (CDCl3) δ 172.9, 171.9, 156.0, 145.3, 143.9, 143.8, 140.7, 131.1, 130.6, 127.6,

127.0, 126.7, 125.4, 120.2, 120.1, 65.5, 52.3, 48.6, 48.6, 46.7, 24.1, 23.1, 21.4, 16.5. Anal. Calcd

for C30H31N5O4: C, 68.55; H, 5.94; N, 13.32; found: C, 68.59; H, 5.94; N, 13.16

62

3.4.5 HPLC Results of Peptide 3.6-3.10

Figure 3-4. HPLC Profile of peptide 3.6. Bottom; the profile of crude peptide 3.6 (H-L-Leu-L-Ala-L-Leu-NH2). Top; The profile of peptide 3.6 after purification. HRMS Calcd for [C15H30N4O3+H]+, 315.2391; found, 315.2401.

Figure 3-5. HPLC Profile of peptide 3.7. Bottom; the profile of crude peptide 3.7 (H-L-Trp-L-Ala-L-Met-L-Ala-NH2). Two diastereoisomers were obtained in ratio 2.3 : 1 (9.5 min : 10.2 min). Top; The profile of peptide 3.7 after purification. HRMS Calcd for [C22H32N6O4S+H]+, 477.2279; found, 477.2291.

63

Figure 3-6. HPLC Profile of peptide 3.8. Bottom; the profile of crude peptide 3.8 (H-L-Trp-L-Phe-L-Met-L-Leu-L-Ala-NH2). Two diastereoisomers were obtained in ratio 4 : 1 (15.5 min : 16.1 min). Top; The profile of peptide 3.8 after purification. HRMS Calcd for [C34H47N7O5S+H]+, 666.3432; found, 666.3445.

Figure 3-7. HPLC Profile of peptide 3.9. Bottom; the profile of crude peptide 3.9 (H-L-Leu-L-Ala-L-Met-L-Phe-L-Phe-L-Met-NH2). Two diastereoisomers were obtained in ratio 2.5 : 1 (16.4 min : 16.9 min). Top; The profile of peptide 3.9 after purification. HRMS Calcd for [C37H55N7O6S2+H]+, 758.3728; found, 758.3707.

64

Table 3-5. MS/MS Sequence of peptide 3.9 L-A-M-F-F-M(NH2) MW = 757.4 [M+H]+ = 758.4 a-ions-NH3 69.10 140.1 271.1 418.2 565.3 696.3

a-ions (loss of CO) 86.1 157.1 288.2 435.2 582.3 713.3 C-term

b-ion N-term 114.1 185.1 316.2 463.2 610.3 741.3 753.4 Residue H L A M F F M -NH2 Residue mass 1.0 113.1 71.0 131.0 147.1 147.1 131.0 16.0 y-ions 758.4 645.3 574.3 443.2 296.2 149.1 Loss of NH3 741.4 628.3 557.3 426.2 279.2 132.1 Highlighted numbers were determined during analysis

Figure 3-8. HPLC Profile of peptide 3.10. Bottom; the profile of crude peptide 3.10 (H-L-Leu-L-Ala-L-Met-L-Ala-L-Phe-L-Ala-Gly-NH2). Top; The profile of peptide 3.10 after purification. HRMS Calcd for [C31H50N8O7S+H]+, 679.3596; found, 679.3589.

65

CHAPTER 4 COUMARIN LABELING OF PEPTIDES ON SOLID PHASE1

4.1 Introduction

Proteins modified with fluorescent dyes, enzymes, and other reporter groups are valuable

tools with widespread uses in immunology and biochemical research. Protein-capture

microarrays have become a promising tool for protein analysis in drug discovery, diagnostics,

and biological research. [04DDT24] Fluorescent derivatives of biologically active peptides are

useful experimental tools for studying biological structure and function and for visualization of

intracellular processes or molecular interactions. [95B3972, 97JPR444]

Matrix metalloprotease (MMP) proteins are implicated in many diseases, including

arthritis, periodontal disease, tumor cell invasion, and metastasis. [93CROBM197] The detection

of a protein bound by a specific capture agent is key to microarray-based methods, for traditional

immunoassays and biosensor applications. [02PS2655, 03CB53, 05CBC1043] Synthetic peptide-

based assays can differentiate enzyme types and monitor their activity. [89JBC4227, 91AB137,

93BJ601] Fluorogenic substrates can be monitored continuously and utilized at low

concentrations, thus providing a particularly convenient enzyme assay method. [07AHC131]

Fluorescent biosensors are composed of a binding molecule, such as an antibody or an

enzyme, derivatized with a single fluorescent probe, which is sensitive to changes in the local

environment. [02JMB429, 02JOC3120, 02PS2655] Fluorogenic groups can often be attached at

the cleavage sites of proteases and esterases. However, this simple approach is not applicable if

the enzyme requires binding interactions on both sides of the cleavage site. For such cases,

quenched fluorescent peptides are designed as short sequences of amino acids, containing

1Reproduced in part with permission from Organic & Biomolecular Chemistry, 2008, 6, 4582.

Copyright © 2009 The Royal Society of Chemistry

66

enzyme-cleavable specific sites, with fluorescent donor and acceptor probes linked at the N- and

C-termini. [93BC537, 93BJ601, 94JBC20952] In other biosensors [00BC71, 02JMR311] the

binding molecule is labeled with two fluorophores, suitable for Fӧster (fluorescence) resonance

energy transfer (FRET). The acceptor-quencher pair enables nonradiative energy transfer

between an excited donor fluorophore to a proximal acceptor fluorophore. [05ACIE2642]

Usually, the donor fluorescence is quenched by the acceptor without subsequent fluorescence

emission. Donor and acceptor groups, attached to a synthetic peptide undergo FRET, producing a

unique fluorescence spectrum. Enhanced donor fluorescence indicates proteolysis accompanied

by the loss of FRET as a result of separation of the donor and acceptor groups (Scheme 4-1).

Scheme 4-1. Mechanism of FRET

Many donor and acceptor groups have been incorporated into quenched fluorogenic

substrates. [94JBC20952, 97FEBS379, 03AB141] Coumarins have extensive and diverse

applications as fluorescent probes or labels; [97CR1515, 04CR3059] since they exhibit an

extended spectral range, are photostable and have high emission quantum yields. 7-Methoxy-

coumarin-4-ylacetyl (Mca, ε325 = 14500 M-1cm-1 and Фf = 0.49) functional group was proposed

as a fluorophore for thimet peptidase, pitrilysin and MMP substrates (Figure 4-1). [92FEBS263,

67

93BJ601, 94JBC20952] The combination of Mca as a fluorophore and a 2,4-dinitrophenyl (Dnp)

group as a quencher has several advantages over the more common fluorogenic substrate pair of

Trp/Dnp; the Mca residue is more fluorescent and chemically more stable than Trp, and Mca is

efficiently quenched by Dnp.

Figure 4-1. Structure of Mca and Dnp moiety

Fluorescently-labeled peptides might be created by the reaction of the peptide in solution

with an activated form of the fluorophore but a potentially more effective approach is to

assemble the peptide chain on solid phase and incorporate the fluorophore into the peptide whilst

attached to the solid support. [98BMCL597, 04TL6079]

For the solid phase peptide labeling by Mca, the N-termini of peptide–resins are acylated

with 7-methoxycoumarin-4-ylacetic acid using standard synthetic cycles. [92FEBS263,

93BJ601, 94JBC20952] However, inefficient acylation of the peptide–resin led Malkar and

Fields to incorporate Mca into Nα-Fmoc-lysine molecules by a 4-step method providing Nα-

Fmoc-L-Lys(Mca)-OH 4.4 (17 % overall) (Scheme 4-2). [01LPS263]

Coumarin-labeled lysines are of considerable interest for the design and synthesis of

fluorogenic triple-helical substrates for the analysis of MMP family members. [01B5795,

03AB105, 05JSS1812] Thus, Nε-coumarin-labeled-Nα-Fmoc lysines allow the successful labeling

of peptide substrates by solid phase peptide synthesis for an extracellular MMP and represent a

powerful tool for monitoring proteolysis. [01B5795, 03AB105, 05JSS1812]

68

Scheme 4-2. Reported synthesis of Nα-(fluoren-9-ylmethoxycarbonyl)-Nε-[(7-methoxycoumarin-4-yl)acetyl-L-lysine 4.4

Our group has reported the extensive use of N-acylbenzotriazoles for N-acylation,

[00JOC8210, 02A39, 08JOC511] C-acylation, [00JOC3679, 03JOC4932, 03JOC5720] and O-

acylation [04CCA175, 07BC994] reactions. (Nα-Fmoc-aminoacyl)benzotriazoles and their Boc-

and Cbz- analogs enabled the preparation of chiral di-, tri- and tetrapeptides in average yields of

88% from natural amino acids in solution phase. [02A134, 04S2645, 09A47]

The efficient fluorescent labeling of peptides on solid phase by acylation with

benzotriazole-activated derivatives of (i) coumarin-3-ylcarboxylic acid, 7-methoxycoumarin-4-

ylacetic acid, (ii) coumarin-3-ylcarbonyl (Cc) and Mca-labeled lysines are reported below.

69

4.2 Results and Discussion

The preparation of benzotriazole-activated fluorogenic substrates 4.5, 4.6, 4.7, 4.9, 4.11

and their utilization as useful reagents for the efficient fluorescent labeling of peptides by the

solid phase method have been demonstrated.

4.2.1 Preparation of Nα-Fmoc-Nε-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (Nα-Fmoc-L-Lys(Mca)-OH) 4.4 and Its Benzotriazole Derivative 4.6

7-Methoxycoumarin-4-ylacetic acid 4.2 was converted into crystalline, stable 4-

(benzotriazole-1-ylacetyl)-7-methoxycoumarin 4.5 (78 %) by reaction with BtH and SOCl2 in

DCM at 20 °C (Scheme 4-3) Compound 4.5 was then coupled with Nα-Fmoc-L-lysine in aqueous

MeCN in the presence of Et3N for 20 min to afford Nα-Fmoc-L-Lys(Mca)-OH 4.4 (overall 51

%). Compared with the recent literature procedure [01LPS263] for the preparation of Nα-Fmoc-

L-Lys(Mca)-OH 4.4, my two-step methodology using Nα-Fmoc-L-lysine, offers simple

preparative and workup procedures, short times to completion, the use of inexpensive reagents

and high yields. Conventional benzotriazole activation of 4.4 gave Nα-Fmoc-L-Lys(Mca)-Bt 4.6

(70 %).

Scheme 4-3. Preparation of Nα-Fmoc-L-Lys(Mca)-Bt 4.6

70

4.2.2 Preparation of Nα-Fmoc-Nε-(coumarin-3-ylcarbonyl)-L-lysine Benzotriazolide (Nα-Fmoc-L-Lys(Cc)-Bt) 4.9 and Nα-(Coumarin-3-ylcarbonyl)-Nε-Fmoc-L-lysine Benzotriazolide (Nα-Cc-L-Lys(Fmoc)-Bt) 4.11

3-(Benzotriazole-1-ylcarbonyl)chromen-2-one 4.7 [08BC1471] was coupled with

commercially available Nα-Fmoc-L-lysine and Nε-Fmoc-L-lysine in aqueous MeCN at 20 °C in

the presence of Et3N to provide lysine-scaffold based fluorescent building blocks 4.8

[08BC1471] and 4.10 (87 and 79 % respectively), that were converted into the corresponding N-

acylbenzotriazoles 4.9 and 4.11 (87 and 71 %) (Scheme 4-4).

Scheme 4-4. Preparation of Nα-Fmoc-L-Lys(Cc)-Bt 4.9 and Nε-Cc-L-Lys(Fmoc)-Bt 4.11

4.2.3 Solid Phase Fluorescent Labeling with 4.6, 4.9, 4.11 to Synthesize Labeled Peptides 4.12-4.17

Solid phase peptide synthesis, microwave-assisted as optimized previously in our group,

[07CBDD465] enables efficient acylation of NH2- groups on solid phase by benzotriazole-

activated fluorogenic substrates 4.5, 4.6, 4.7, 4.9, 4.11. Compounds 4.5 and 4.7 were used to

71

couple the fluorophore directly to diverse peptides. Alternatively, 4.6, 4.9, and 4.11 were used to

couple the fluorophore already attached to a lysine moiety to the peptides. Coumarin-labeled

peptides were synthesized as C-terminal amides using Fmoc solid-phase methodology under

microwave irradiation.

The utility of 4.6 for fluorescent labeling on solid phase was demonstrated for a model

dipeptide H-L-Ala-L-Lys(Nε-Mca)-NH2 4.12 (Scheme 4-5). Peptide 4.12 was synthesized using

microwave-assisted SPPS conditions. [07CBDD465] After initial removal of the Fmoc

protecting group, free Rink resin-NH2 was coupled with 4.6 in DMF under microwave

irradiation for 10 min at 70 °C. The second coupling was performed with the (Nα-Fmoc-

aminoacyl)benzotriazole reagent derived from Fmoc-L-Ala. Finally the desired peptide was

cleaved from the resin to produce peptide amide H-L-Ala-L-Lys(Nε-Mca)-NH2 4.12 (26 %)

(Table 4-1). Conditions were optimized to maximize the rate while avoiding epimerization.

Scheme 4-5. Synthesis of coumarin-labeled dipeptide 4.12

In a similar manner, microwave-assisted SPPS was effected (3 min coupling time for each

step) with 4.9 to obtain the fluorescently labeled di-, tri-, tetra-, and hexapeptides: H-L-Ala-L-

Lys(Nε-Cc)-NH2 4.13, H-L-Pro-L-Phe-L-Lys(Nε-Cc)-NH2 4.14, H-L-Trp-L-Lys(Nε-Cc)-L-Met-L-

72

Phe-NH2 4.15, H-L-Lys(Nε-Cc)-L-Pro-Gly-L-Leu-L-Met-L-Trp-NH2 4.16 in yields of 18–45 %

(after HPLC purification) (Table 4-1). The successive coupling steps utilized the appropriate N-

acylbenzotriazoles derived from Fmoc-L-Met, Fmoc-L-Trp, Fmoc-L-Phe, Fmoc-L-Leu, Fmoc-L-

Pro and Fmoc-Gly prepared by our published procedures. [05S397, 07CBDD465] The mild

synthetic conditions allowed utilization of the unprotected indole-NH of L-Trp, and no

complications were observed with L-Met or any other of the amino-N-protected amino acids

used.

I achieved fluorescent labeling by coupling to Nα-coumarin-attached lysine 4.11 through its

free Nε-position to prepare labeled tripeptide H-L-Phe-L-Leu-L-Lys(Nα-Cc)-NH2 4.17 (35 %)

under microwave-assisted SPPS (3 min coupling time for each step) and Fmoc strategy (Table 4-

1).

Table 4-1. Preparation of fluorescent peptides 4.12-4.17

Labeled Peptide Structure (N to C terminus) Yield

(%)a Purity (%)b

tR (min)c

HRMS [M+H]+ d

4.12 H-L-Ala-L-Lys(Nε-Mca)-NH2 26 99 9.10 433.2103 4.13 H-L-Ala-L-Lys(Nε-Cc)-NH2 45 99 9.82 389.1825 4.14 H-L-Pro-L-Phe-L-Lys(Nε-Cc)-NH2 23 99 13.38 562.2680 4.15 H-L-Trp-L-Lys(Nε-Cc)-L-Met-L-Phe-NH2 18 94 18.02 782.3328

4.16 H-L-Lys(Nε-Cc)-L-Pro-Gly-L-Leu-L-Met-L-Trp-NH2

20 99 17.00 902.4212

4.17 H-L-Phe-L-Leu-L-Lys(Nα-Cc)-NH2 35 99 14.28 578.2987 aIsolated yields after HPLC purification; bPurity after HPLC purification; ctR = retention time. For condition see the experimental section. dFor the calculated values, see the experimental section.

4.2.4 Solid Phase Fluorescent Labeling with 4.7 to Synthesize Labeled Dipeptide (Cc)-L-Leu-L-Leu-NH2 4.18 and Labeling with 4.5 to Synthesize Labeled Dipeptide (Mca)-L-Leu-L-Leu-NH2 4.19

I also demonstrated fluorescent labeling with benzotriazole-activated coumarin-3-

ylcarboxylic acid 4.7 by the preparation of Cc-labeled dipeptide (Cc)-L-Leu-L-Leu-NH2 4.18

(Scheme 4-6). After initial removal of the Fmoc protecting group from Rink amide resin, I

73

utilized Fmoc-L-Leu-Bt for each of two successive coupling steps. After final coupling with 4.7

(10 min coupling time), the desired fluorescent-labeled peptide 4.18 (29 %) (Table 4-2) was

cleaved from the resin.

Scheme 4-6. Preparation of peptide 4.18

Fluorescent labeling with benzotriazole activated 7-methoxycoumarin-4-ylacetic acid 4.5

was used to prepare Mcal-labeled dipeptide (Mca)-L-Leu-L-Leu-NH2 4.19 (26 %) (Table 4-2),

under similar conditions to those utilized for the preparation of 4.18.

Table 4-2. Preparation of fluorescent peptides 4.18 and 4.19

Labeled peptide Structure (N to C terminus)

Yield (%)a

Purity (%)b

tR (min)c

HRMS [M+H]+ d

4.18 (Cc)-L-Leu-L-Leu-NH2 29 >99 20.67 438.2223 4.19 (Mca)-L-Leu-L-Leu-NH2 26 >99 17.47 460.2455 aIsolated yields after HPLC purification; bPurity after HPLC purification; ctR = retention time. For condition see the experimental section. dFor the calculated values, see the experimental section.

4.2.5 Fluorescence Measurements of Peptides 4.12-4.19

Absorption (λAbs) and fluorescence (λEm) wavelength maxima were recorded for fluorescent

peptides 4.12–4.19 (Table 4-3).

74

Table 4-3. Absorption and fluorescence data of fluorescent labeled peptides 4.12-4.19 Entry 1 2 3 4 5 6 7 8 Peptide 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 λAbs.[nm]a 323 294 295 289 290 299 295 322 λEm. [nm]a 383 409 407 407 405 407 413 383 aDetermined in 95% methanol

0

0.2

0.4

0.6

0.8

1

1.2

220 270 320 370 420Wavelength (nm)

Abs

orpt

ion

Inte

nist

y

4.124.134.144.154.164.17

Figure 4-2. Absorption spectra of 4.12-4.17

0

0.2

0.4

0.6

0.8

1

200 250 300 350 400 450

Wavelength (nm)

Aps

orpt

ion

Inte

nsity

4.184.19

Figure 4-3. Absorption spectra of 4.18-4.19

75

0

0.2

0.4

0.6

0.8

1

1.2

350 400 450 500 550

Wavelength (nm)

Emis

sion

Inte

nsity

4.124.174.19

Figure 4-4. Emission spectra of 4.12, 4.17, 4.19

4.3 Conclusion

In conclusion a convenient and efficient preparation in solution phase of a variety of

coumarin fluorescent probes is described, including Cc and Mca labeled lysines as fluorogenic

substrates. Their benzotriazole derivatives are appropriate materials for peptide labeling thus

enabling efficient peptide α-amino group acylation under microwave irradiation on solid phase

without the use of coupling agents or additives and without side reactions or epimerization.

4.4 Experimental Section

4.4.1 Preparation of (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamido)pentanoic acid (Nα-Fmoc-L-Lys(Mca)-OH) 4.4

Compound 4.5 (1.1 mmol) was added in one portion to a solution of Nα-Fmoc-L-lysine

(1.8 mmol) in MeCN : H2O (24 mL : 5 mL) in the presence of Et3N (5.4 mmol) (Scheme 4-3).

The reaction mixture was stirred at 20 °C for 15 min. A solution of 6M HCl aq (2 mL) was then

added and MeCN was removed under reduced pressure. The residue was extracted with EtOAc

(100 mL), and the organic extract was washed with 6M HCl aq (50 mL x 2), brine (50 mL) and

dried over MgSO4. Evaporation of the solvent gave 4.4, which was recrystallized from

EtOAc/hexanes to give yellow microcrystals (65 %); mp 184.9 – 185.7 °C; 1H NMR (DMSO-d6)

76

δ 8.31–8.24 (m, 1H) 7.93 (d, J = 7.4Hz, 2H), 7.76 (d, J = 7.4Hz, 2H), 7.74–7.64 (m, 2H), 7.45 (t,

J = 7.2Hz, 2H), 7.36 (t, J = 7.2Hz, 2H), 7.04–6.97 (m, 2H), 6.28 (s, 1H), 4.36–4.20 (m, 3H),

3.98–3.90 (m, 1H), 3.88 (s, 3H), 3.70 (s, 2H), 3.15–3.02 (s, 2H), 1.80–1.46 (m, 2H), 1.46-1.20

(m, 4H); 13C NMR (DMSO-d6) δ 174.0, 167.4, 162.4, 160.1, 156.2, 154.9, 151.2, 143.9, 143.8,

140.7, 127.7, 127.1, 126.5, 125.3, 120.1, 112.7, 112.6, 100.9, 65.6, 55.9, 53.8, 46.6, 30.4, 28.5,

23.1. Anal. Calcd for C33H32N2O8: C, 67.80; H, 5.52; N, 4.79; found: C, 67.55; H, 5.60; N, 4.40.

4.4.2 General Procedure for the Preparation of 4.5, 4.6, 4.9, 4.11

SOCl2 (1.2 mmol) was added to a solution of BtH (4.0 mmol) in dry DCM (15 mL) at 20

°C and the reaction mixture was stirred for 20 min (Scheme 4-3 and 4-4). Compounds 4.2, 4.4,

4.8, 4.10 (1.0 mmol) were each added separately to the above reaction mixture, and each mixture

was stirred for 2 h at 20 °C. The white precipitate which formed in each case was filtered, the

filtrate diluted with additional DCM (80 mL) and the solution washed with 6M HCl aq (50 mL x

3) (for 4.2, 4.8, 4.10), with 10 % Na2CO3 aq (50 mL x 3) (for 4.4), brine (50 mL), and dried over

MgSO4. Removal of the solvent under reduced pressure gave 4.5, 4.6, 4.9, 4.11 that were each

recrystallized from DCM/hexanes.

4-(2-Benzotriazol-1-yl-2-oxoethyl)-7-methoxy-chromen-2-one (Mca-Bt) 4.5: Yellow

microcrystals (78 %); mp 125.0 − 126.0 °C; 1H NMR (CDCl3) δ 8.24 (d, J = 8.2 Hz, 1H) 8.17 (d,

J = 8.2 Hz, 1H), 7.74−7.65 (m, 1H), 7.63−7.52 (m, 2H), 6.92−6.84 (m, 2H), 6.41 (s, 1H), 4.87 (s,

2H), 3.88 (s, 3H); 13C NMR (CDCl3) δ 167.3, 163.0, 160.5, 155.6, 147.0, 146.4, 131.0, 130.9,

126.8, 125.5, 120.5, 114.7, 114.3, 112.8, 112.3, 101.2, 55.8, 38.4. HRMS Calcd for

[C18H13N3O4+Na]+, 358.0798; found, 358.0784.

(S)-(9H-Fluoren-9-yl)methyl1-(1H-benzo[d][1,2,3]triazol-1-yl)-6-(2-(7-methoxy-2-oxo-

2H-chromen-4-yl)acetamido)-1-oxohexan-2-ylcarbamate (Nα-Fmoc-L-Lys(Mca)-Bt) 4.6:

77

Yellow microcrystals (70 %); mp 144.0 − 146.0 °C; 1H NMR (DMSO-d6) δ 8.32−8.19 (m, 3H)

7.89 (d, J = 7.4 Hz, 2H), 7.81 (t, J = 7.7 Hz, 1H), 7.72 (d, J = 7.4 Hz, 2H), 7.66 (d, J = 8.7 Hz,

2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.1 Hz, 2H), 6.90−7.00 (m, 2H), 6.23 (s, 1H),

4.29−4.38 (m, 2H), 4.28−4.18 (m, 1H), 3.90−3.83 (m, 1H), 3.83 (s, 3H), 3.65 (s, 2H), 3.12−3.00

(m, 2H), 2.00−1.78 (m, 2H), 1.37−1.53 (m, 4H); 13C NMR (DMSO-d6) δ 172.2, 167.5, 162.4,

160.2, 156.5, 155.0, 151.2, 145.4, 143.8, 140.8, 131.3, 130.5, 127.7, 127.1, 126.9, 126.5, 125.3,

120.3, 120.2, 114.0, 112.8, 112.6, 112.1, 100.9, 65.1, 55.9, 55.9, 54.3, 46.6, 38.6, 30.2, 28.4,

23.1. HRMS Calcd for [C39H35N5O7+Na]+, 708.2428; found, 708.2455.

{(S)-1-(Benzotriazole-1-carbonyl)-5-[(2-oxo-2H-chromene-3-carbonyl)-amino]-

pentyl}-carbamic acid 9H-fluoren-9-ylmethyl ester (Nα-Fmoc-L-Lys(Cc)-Bt) 4.9: White

microcrystals (82 %); mp 113.0 − 115.0 °C; 1H NMR (DMSO-d6) δ 8.80 (s, 1H) 8.70 (t, J = 5.5

Hz, 1H), 8.32−8.28 (m, 2H), 8.23 (d, J = 9.6 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 6.7

Hz, 2H), 7.82−7.64 (m, 4H), 7.61 (t, J = 7.4Hz, 1H), 7.52−7.46 (m, 2H), 7.46−7.28 (m, 3H),

7.35−7.28 (m, 2H), 5.53−5.42 (m, 1H), 4.42−4.38 (m, 2H), 4.38−4.18 (m, 1H), 3.40−3.22 (m,

2H), 2.08−1.82 (m, 2H), 1.68−1.49 (m, 4H); 13C NMR (DMSO-d6) δ 172.1, 161.1, 160.3, 156.4,

153.8, 147.3, 145.3, 143.7, 143.7, 140.7, 134.0, 131.2, 130.6, 130.2, 127.6, 127.1, 126.8, 125.3,

125.1, 120.2, 119.0, 118.5, 116.1, 114.0, 65.9, 54.3, 46.6, 30.3, 28.4, 23.1. Anal. Calcd for

C37H31N5O6: C, 69.26; H, 4.87; N, 10.91; found: C, 69.01; H, 4.76; N, 11.03.

(S)-6-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-2-(2-oxo-2H-chromene-3-

carboxamido)hexanoic acid (Nα-(Cc)-L-Lys(Fmoc)-OH) 4.10: Solid 4.7 (0.5 mmol) was added

in one portion to a solution of Nε-Fmoc-L-lysine (0.5 mmol) in MeCN : H2O (5 mL : 3 mL), in

the presence of Et3N (0.5 mmol) (Scheme 4-4). The reaction mixture was stirred at 20 °C for 30

min, 6M HCl aq (2 mL) was added and the MeCN was removed under vacuum. The residue was

78

dissolved in DCM (50 mL), and the organic extract was washed with 6M HCl aq (50 mL), brine

(50 mL), and dried over MgSO4.Evaporation of the solvent gave 4.10 which was recrystallized

from DCM/hexanes to give white microcrystals (79 %); mp 87.9 − 89.9 °C; 1H NMR (DMOS-

d6) δ 13.00 (br s, 1H) 9.07 (d, J = 7.4 Hz, 1H), 8.89 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.85 (d. J =

7.1 Hz, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.65 (d, J = 7.1 Hz, 2H), 7.23−7.46 (m, 7H), 4.50 (q, J =

5.5Hz, 1H), 4.24−4.38 (m, 2H), 4.15−4.20 (m, 1H), 2.92−3.02 (m, 2H), 1.71−1.95 (m, 2H),

1.26−1.49 (m, 4H); 13C NMR (DMSO-d6) δ 172.9, 160.7, 160.6, 156.1, 154.0, 148.0, 143.9,

140.7, 134.3, 130.4, 127.6, 127.0, 125.2, 125.1, 120.1, 118.4, 118.2, 116.2, 65.2, 52.3, 46.7, 31.2,

29.0. Anal. Calcd for C31H28N2O7: C, 68.88; H, 5.22; N, 5.18; found: C, 68.59; H, 5.57; N, 4.97.

(S)-(9H-Fluoren-9-yl)methyl-6-(1H-benzo[d][1,2,3]triazol-1-yl)-6-oxo-5-(2-oxo-2H-

chromene-3-carboxamido)hexylcarbamate (Nα-(Cc)-L-Lys(Fmoc)-Bt) 4.11: White

microcrystals (71 %); mp 106.9 – 108.9 °C; 1H-NMR (DMOS-d6) δ 9.40 (d, J = 6.9 Hz, 1H)

8.92−8.84 (m, 1H), 8.36−8.19 (m, 2H), 7.96 (d, J = 7.7 Hz, 1H), 7.89−7.71 (m, 4H), 7.71−7.56

(m, 3H), 7.56−7.22 (m, 7H), 6.01−5.89 (m, 1H), 4.29−4.11 (m, 3H), 3.09−2.94 (m, 2H),

2.25−1.97 (m, 2H), 1.62−1.41 (m, 4H); 13C NMR (DMSO-d6) δ 170.9, 161.6, 160.5, 156.1,

154.0, 148.2, 145.4, 140.7, 134.5, 131.2, 130.7,130.5, 127.6, 127.0, 126.9, 125.3, 125.1, 120.3,

120.1, 118.4, 118.1, 116.3, 114.0, 65.2, 53.0, 46.7, 31.1, 31.0, 28.9, 22.4. HRMS Calcd for

[C37H31N5O6+Na]+, 664.2167; found, 664.2125.

4.4.3 General Procedure of Solid Support Peptide Synthesis

Labeled peptides were synthesized using Fmoc solid-phase methodology as C-terminal

amides utilizing Rink-amide-HMBA resin (200-400 mesh, 0.35 meq/g). Standard removal of the

Fmoc protecting group of rink amide resin (0.05 mmol) gave unprotected resin. Resin-NH2 was

coupled with 5 equiv of Nα-Fmoc-protected(aminoacyl)benzotriazole reagent derived from

79

Fmoc-protected amino acids, prepared following previously published procedures (Scheme 4-5

and 4-6). [09A47] When complete coupling was verified by a negative Kaizer (ninhydrin) test

(10 min), the solid resin was washed with DMF (5 mL x 3) and DCM (5 mL x 3) followed by

another coupling. After the coupling step, the desired peptide was cleaved from the resin using

cleavage cocktails: i) TFA : anisole : thioanisole : BAL (90 : 2 : 3 : 5) (for peptide sequences

including Trp or Met) or ii) TFA : water : TIPS (95 : 2.5 : 2.5) (for the other peptide sequences)

at 20 °C for 2 h. The resin was filtered, the cocktail was concentrated under nitrogen and cold

diethyl ether was added to achieve precipitated peptide (4.12-4.19), under conditions optimized

to increase rate but avoid epimerization.

4.4.4 HPLC Profiles of Peptide 4.12-4.19

Figure 4-5. HPLC Profile of peptide 4.12. Bottom; the profile of crude peptide 4.12 (H-L-Ala-L-Lys(Nε-Mca)-NH2). Top; The profile of peptide 4.12 after purification. HRMS Calcd for [C21H28N4O6+H]+, 433.2082; found, 433.2103.

80

Figure 4-6. HPLC Profile of peptide 4.13. Bottom; the profile of crude peptide 4.13 (H-L-Ala-L-Lys(Nε-Cc)-NH2). Top; the profile of peptide 4.13 after purification. HRMS Calcd for [C21H28N4O6+H]+, 389.1819; found, 389.1825.

Figure 4-7. HPLC Profile of peptide 4.14. Bottom; the profile of crude peptide 4.14 (H-L-Pro-L-Phe-L-Lys(Nε-Cc)-NH2). Top; the profile of peptide 4.14 after purification. HRMS Calcd for [C30H35N5O6+H]+, 562.2660; found, 562.2680.

81

Figure 4-8. HPLC Profile of peptide 4.15. Bottom; the profile of crude peptide 4.15 (H-L-Trp-L-Lys(Nε-Cc)-L-Met-L-Phe-NH2). Top; The profile of peptide 4.15 after purification. HRMS Calcd for [C41H47N7O7S+H]+, 782.3300; found, 782.3328.

Figure 4-9. HPLC Profile of peptide 4.16. Bottom; the profile of crude peptide 4.16 (H-L-Lys(Nε-Cc)-L-Pro-Gly-L-Leu-L-Met-L-Trp-NH2). Top; the profile of peptide 4.16 after purification. HRMS Calcd for [C45H59N9O9S+H]+, 902.4229; found, 902.4212.

82

Table 4-4. MS/MS Sequence of peptide 4.16 K(der)-P-G-L-M-W(NH2) MW = 901.4 [M+H]+ = 902.4 b-ions-H2O 681.3 867.4 a-ions (loss of CO) 273.1 370.2 427.2 540.3 671.3 587.4 b-ion N-term 301.1 398.2 455.2 568.3 699.3 885.4 C-term Residue H K(der) P G L M W -NH2 Residue mass 1.0 300.1 97.1 223.1 113.1 131.0 186.1 16.0 y-ions 902.4 602.3 505.3 448.3 335.2 204.1 Loss of NH3 885.4 585.3 488.3 431.3 318.2 187.1 Highlighted numbers were determined during analysis

Figure 4-10. HPLC Profile of peptide 4.17. Bottom; the profile of crude peptide 4.17 (H-L-Phe-L-Leu-L-Lys(Nα-Cc)-NH2). Top; the profile of peptide 4.17 after purification. HRMS Calcd for [C31H39N5O6+H]+, 578.2973; found, 578.2987.

Figure 4-11. HPLC Profile of peptide 4.18. Bottom; the profile of crude peptide 4.18 ((Cc)-L-Leu-L-Leu-NH2). Top; The profile of peptide 4.18 after purification. HRMS Calcd for [C22H29N3O5+H]+, 416.2180; found, 416.2223.

83

Figure 4-12. HPLC Profile of peptide 4.19. Bottom; the profile of crude peptide 4.19 ((Mca)-L-Leu-L-Leu-NH2). Top; The profile of peptide 4.19 after purification. HRMS Calcd for [C24H33N3O6+Na]+, 460.2442; found, 460.2455.

84

CHAPTER 5 DESIGN AND SYNTHESIS OF PH SENSITIVE GFP CHROMOPHORE ANALOGUES

5.1 Introduction

Fluorescent peptide labeling is useful for monitoring biological activity: a fluorophore in a

peptide or a protein enables ligands, inhibitors, and antigens to be detected at low concentration

by introducing a fluorophore into a peptide or a protein. [06S217, 07AHC131] Natural aromatic

amino acids (Phe, His, Trp, and Tyr) play key roles in the recognition of receptors; they have

frequently been replaced by unnatural highly fluorescent amino acids in specific positions by

bioactive peptides. [00B118, 01AMB274, 04PS1489, 06TA2393]

Despite many commercially available fluorophores, new examples are required (i) small

enough to avoid misfolding of the protein or blockage of the binding site and (ii) that absorb

above 320 nm to avoid interference from Trp residues. [09OBC627] New fluorophores with

wavelengths of absorption and emission that vary with media properties, e.g. the polarity and/or

pH, are needed for a wide range of applications. [06OBC4265] Enhanced sensitivity of a

fluorophore in the pH range of 5 to 9 is important because most tumors develop a

microenvironment characterized by low oxygen tension, high lactate concentration and/or low

extra-cellular pH. [01CRLC295, 06CR6699] Green fluorescent protein (GFP) and similar

proteins (CFP or YFP) are well established as fluorescent markers for monitoring biological

activity because they have high light emission (quantum yields up to Φf = 0.8, Scheme 1) and

work well in vitro and in living mammalian cells. [97PNAS230, 98B509, 07B9865] However,

the large size (up to 238 amino acids) of the GFP chromophore can cause misfolding or other

structural changes in target proteins.

85

Scheme 5-1. Intramolecular biosynthesis of imidazolinonyl chromophore in wild-type GFP

Unlike the chromophore of wild-type GFP, which is surrounded by its protein sequence

(1-64 and 68-238) and stabilized as the Z-isomer, [97B9759, 07B9865] the GFP model

chromophores of type 5.1 show only low fluorescence at 20 °C due to Z-E photoisomerization at

the exo-methylene group (Scheme 5-2a). [03FEBS35, 06CP358]. Hydrogen bonding can control

photochemical isomerization: Arai et al. demonstrated hemi-indigo derivatives 5.2 [98CL1153]

which exist as the Z-isomers stabilized by six-membered ring intramolecular hydrogen bonding

prevent or minimize photoisomerization (Scheme 5-2b). The Z-isomer of the GFP chromophore

analogue 5.3 stabilized by boron ligation showed high fluorescent activity (Φf = 0.89) compared

to low fluorescence of the boron ligated E-isomer (Scheme 5-2c). [08JACS4089]

The present work sought to construct new, pH-sensitive chromophores 5.4-5.6 (Figure 5-

1) modeled on GFP and connected to lysine, glutamine, and asparagine in which Z-E

photoisomerization would be controlled by pH, thus allowing monitoring of pH in cells under

natural and pathological conditions. Each chromophore contains five- or six-membered

heterocyclic rings, such as furyl, thienyl, or pyridyl groups, attached to the imidazolinone ring.

Heteroatoms (O, S, or N) in aromatic rings on position-2 have an important role in prevention of

E-Z photoisomerization. In acidic media, the basic heteroatom (the imidazolinone nitrogen if

thienyl/furyl containing chromophore or the pyridyl nitrogen) gets protonated to form more

stable six-membered intramolecular hydrogen bonding. To assess the utility of such

86

fluorophores, compounds 5.9a-f and 5.14a-b were synthesized and their fluorescence recorded

spectra over the pH range 1-7.

Scheme 5-2. Literature examples

Figure 5-1. The proposed GFP-based lysin (Lys), asparagine (Asn), and glutamine (Gln) analogues 5.4-5.6

87

5.2 Results and Discussion

5.2.1 Synthesis of Imidazolinone Chromophores 5.9a-f and Their Fluorescent Activity

Azalactones 5.8a-c were each synthesized by reaction of hippuric acid 5.7 with the

appropriate aldehyde in the presence of sodium acetate and acetic anhydride (Scheme 5-3).

[07SC1709, 05AJC576] Compounds 5.8a-c reacted under microwave conditions with p-toluidine

to give 5.9a and with N,N-dimethylethylenediamine to give 5.9b-d in yields of 33-81% (Table 5-

1).

Scheme 5-3. Synthesis of GFP modified fluorophore 5.9a-d

Similally, 5.8e-f were obtained by reactions of 5.10 (prepared from 2-naphthoyl chloride

and glycine) with the chosen aldehyde followed by treatment with N,N-dimethylethylenediamine

under MW conditions to give 5.9e and 5.9f in yields of 51 and 30 % (Scheme 5-4, Table 5-1).

Scheme 5-4. Synthesis of GFP modified fluorophore 5.9e-f

88

Table 5-1. Synthesis of GFP modified fluorophore 5.9a-f

The exo-methylene group in GFP chromophores photoisomerises to the E-isomer under

UV irradiation, but reverts to the Z-isomer on heating. [07S1103, 08JACS4089] Fluorophore

5.9a was isolated as the Z-isomer as revealed by 1H NMR (Figure 5-2a) which showed on

upfield resonance at 6.8 ppm for the olefinic proton compared to 5.3-Z and 5.3-E. [08JACS4089]

After 1.5-5.5 h under UV light (365 nm) a solution of 5.9a in DMSO-d6, the formation of

increasing amounts of the E-isomer was observed between 7.9 ppm to 8.2 ppm (Figure 5-2b,c).

In the presence of concentrated HCl, the NMR spectrum showed only the Z-isomer even after 16

h under UV irradiation (Figure 5-2d) demonstrating stabilization of the Z-isomer by

intramolecular hydrogen bonding (Scheme 5-5). The slightly upfield shift in the presence of HCl

is explained by formation of intramolecular hydrogen bonding by the protonated Z-isomer

(Figure 5-2d).

The absorption and emission spectra of the fluorophores 5.9b-d at 10-5 M in Britton-

Robinson Buffer solution [31JCS458] were recorded over the pH range 1-7; the 1H NMR data

indicate that protonation restricts 5.9a to the cis configuration (Figure 5-2). From pH 7 to pH 3

there is virtually no change in the absorption spectra of 5.9b-f but a distinct decrease in the

R1 R2 Yield (%)a mp (°C) R3 Yield

(%)a

5.8a Ph 2-Thienyl 69 180.0-182.0 5.9a p-(CH3)C6H4 56

5.9b (CH2)2NMe2 33

5.8b Ph 5-Methyl-2-furyl 59 150.5-152.0 5.9c (CH2)2NMe2 81

5.8c Ph 2-Pyrryl 35 155.5-156.5 5.9d (CH2)2NMe2 55

5.8d Naphth-2-yl 2-Thienyl 50 212.0-214.0 5.9e (CH2)2NMe2 51

5.8e Naphth-2-yl 5-Methyl-2-furyl 40 161.8-162.5 5.9f (CH2)2NMe2 30 aIsolated yield

89

intensity of the emission spectra by factors of between 2 and 4 (Figure 5-3 to 5-6). The origin of

the fluorescence intensity decrease is clearly not associated with the furyl, thienyl or pyrryl units

of 5.9b-f or to protonation of N3 of the imidazolinone ring (pKa ca 1.4). Compounds 5.9b and

5.9e, both containing the 2-thienyl group, gave virtually identical absorption and emission

spectra (shown only for 5.9b Figure 5-3) that revealed a small (ca 20nm) bathochromic shift of

the absorption spectra below pH 2.5 but no change in the emission spectra. The results are best

explained by protonation of N3 of the imidazolinone ring but only weak hydrogen bonding with

sulfur that may twist the thiophene ring out of planarity with the imidazolinone system.

Figure 5-2. Prevention of photoisomerization of imidazolinonyl compound 5.9a. (a) Isolated product 5.9a (in DMSO-d6), (b) UV irradiation (365 nm) after 1.5 h, (c) UV irradiation after 5.5 h, (d) addition of HCl, UV irradiation after 16 h

(d)

(c)

(b)

(a)

90

Scheme 5-5. Prevention of photoisomerization of imidazolinonyl compound 5.9a by six-membered ring intramolecular hydrogen bond

With compounds 5.9c and 5.9f containing the 5-methyl-2-furyl unit, the absorption

spectra again showed bathochromic shifts (ca 40nm) below pH 2.5 but this was accompanied by

significant increases of intensity in the emission spectra by factors of 7 (for 5.9c) and 10 (for

5.9f). Clearly, protonation of N3 and hydrogen bonding with furyl oxygen enforces planarity on

the cis configuration and the ensuing higher degree of conjugation enhances both the

fluorescence wavelength and intensity (Scheme 5-6). Finally, compound 5.9d containing the

pyrrole system, shows similar behavior to that of 5.9c and 5.9f with emission intensity increasing

5 fold between pH 2.5 and 1 (Figure 5-5). This fluorescence increase is probably due to

protonation of the carbonyl group in the imidazolinone ring since the N3 is already occupied

with existing intramolecular hydrogen bonding with the –NH of the pyrryl group (Scheme 5-7).

Protonation of the imidazolinone ring at low pH was supported by the 15N NMR shift of

N3 in 5.9c from 241.7 ppm in CDCl3 to 151.2 ppm in TFA-d6. The bathochromc shift in the

absorption and emission spectra of 5.9c (Figure 5-4), may be due to protonation of the C=O

91

oxygen (Scheme 5-6), but the NMR date do not provide unambiguous evidence for the

hypothesis (Figure 5-7).

Scheme 5-6. Expected protonation of GFP analogues 5.9b, 5.9c, 5.9e, 5.9f

Scheme 5-7. Expected protonation of GFP analogues 5.9d

0

0.5

1

200 250 300 350 400 450 500 550

Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

0

50000

100000

150000

200000

250000

400 450 500 550 600 650 700

Wavelentgh (nm)

Fluo

resc

ence

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

Figure 5-3. Absorption (left) and emission spectra (right) of 5.9b (10-5 M in Britton-Robinson

Buffer from pH 1-7, Raman peak of water has been fixed.)

92

0

0.5

1

200 250 300 350 400 450 500 550 600Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

400 450 500 550 600 650 700

Wavelength (nm)

Fluo

resc

ence

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

Figure 5-4. Absorption (left) and emission spectra (right) of 5.9c (10-5 M in Britton-Robinson Buffer from pH 1-7, Raman peak of water has been fixed.)

0

0.5

1

200 250 300 350 400 450 500 550Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1

pH1.3

pH1.6

pH2

pH3

pH4

pH6

pH7

0

100000

200000

300000

400000

500000

600000

430 480 530 580 630 680Wavelength (nm)

Fluo

resc

ence

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

Figure 5-5. Absorption (left) and emission spectra (right) of 5.9d (10-5 M in Britton-Robinson Buffer from pH 1-7, Raman peak of water has been fixed.)

0

0.5

1

200 250 300 350 400 450 500 550 600Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1pH1.3

pH1.6pH2pH2.5

pH4pH5pH6

pH7

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

400 450 500 550 600 650Wavelength (nm)

Fluo

resc

ence

Inte

nsity

pH1pH1.3pH1.6pH2pH4pH5pH6pH7

Figure 5-6. Absorption (left) and emission spectra (right) of 5.9f (10-5 M in Britton-Robinson Buffer from pH 1-7, Raman peak of water has been fixed.)

93

Figure 5-7. 15N NMR study of 5.9c

5.2.2 Synthesis of Imidazolinone Chromophore 5.14a-b and Their Fluorescent Activity

Compound 5.14a and 5.14b were synthesized by a different route from commercially

available benzamide and chloroacetyl chloride (Scheme 5-8, Table 5-2). [08JACS4089]

Scheme 5-8. Synthesis of imidazolinone chlomophore 5.14a-b

Table 5-2. Imidazolinone chromophore 5.14a-b Entry R Yield (%)a

1 5.14a 6-Methyl-2-pyridyl 18

2 5.14b 5-Methyl-2-furyl 29

aIsolated yield

94

The absorption and emission spectra of 5.14a and 5.14b were recorded in 10-5 M in

Britton-Robinson Buffer (Figure 5-8 and 5-9). Fluorescence of 5.14a increased below pH 6

initially due to protonation of the pyridine nitrogen to 5.14a′ followed by tautomerism of the

amide group to 5.14a″ (Scheme5-9) as indicated by the red shift in both absorption and emission

spectra. This tautomerization at high pH can be explained by the free NH group of the

imidazolinone ring and stronger intramolecular hydrogen bonding between protonated pyridyl

nitrogen and the imidazolinone N3. However, fluorescence of 5.14b increased only below pH

2.5 thus exhibiting similar behavior to that of 5.9c and 5.9f.

Scheme 5-9. Possible mechanism of fluorophore 5.14a

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

200 250 300 350 400 450 500

Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

0

500000

1000000

1500000

2000000

2500000

3000000

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400 450 500 550 600 650

Wavelength (nm)

Fluo

resc

ent I

nten

sity

pH1pH1.3pH1.6pH2pH2.5pH3pH4pH5pH6pH7

Figure 5-8. Absorption (left) and emission spectra (right) of 5.14a (10-5 M in Britton-Robinson

Buffer from pH 1-7, Raman peak of water has been fixed.)

95

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

200 250 300 350 400 450 500 550 600

Wavelength (nm)

Abs

orpt

ion

Inte

nsity

pH1

pH1.3

pH1.6

pH2

pH2.5

pH3

pH5

pH6

0

50000

100000

150000

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250000

300000

350000

400000

390 440 490 540 590 640 690Wavelength (nm)

Fluo

resc

ence

Inte

nsity

pH1

pH1.3

pH1.6

pH2

pH2.5

pH4

pH5

pH6

pH7

Figure 5-9. Absorption (left) and emission spectra (right) of 5.14b (10-5 M in Britton-Robinson Buffer from pH 1-7, Raman peak of water has been fixed.)

5.2.3 Absorption and Emission Measurement of Chromophores 5.9b-f and 5.14a-b

The absorption and emission spectra of 5.9b-f and 5.14a-b were also measured in Britton-

Robinson Buffer and quantum yields (Φf) at pH 1 and excitation coefficients (ε = M-1nm-1) were

evaluated (Table 5-3). Quantum yields were calculated relatively to Coumarin 30 as a reference

compound [85JPC294] according to the follwing equation (Eq.), where Aref, Sref, nref and Asample,

Ssample, nsample represent the absorption at the exited wavelength, the integrated emission band

area, and the solvent regractive index of the standard and the sample. [92JL269]

Φf = Φref (Ssample/Sref) (Aref/Asample) (n2sample/n2

ref) (Eq.)

The best potential fluorescence marker, 5.14a, provides only 3% of the fluorescence

intensity of GFP and then only at pH 1, a condition that is highly unlikely to be of value in vivo.

Table 5-3. Quantum yields and excitation coefficients of 5.9b-f and 5.14a-b

Entry Compound λabs max ε (M-1cm-1) λem max Φf

pH 1 pH 7 pH 1 pH 1 pH 7 pH 1 1 5.9b 409 394 24313 488 478 0.0009 2 5.9c 444 411 22376 502 484 0.0036 3 5.9d 462 423 45580 494 492 0.0018 4 5.9e 410 - 26913 512 - 0.0008 5 5.9f 451 416 28617 511 485 0.0060 6 5.14a 405 380 20343 526 469 0.0304 7 5.14b 449 420 20046 503 484 0.0072

96

5.3 Conclusion

GFP modified pH sensitive chromophores were synthesized and their fluorescence

activities were measured in Britton-Robinson Buffer over the pH range 1-7. Chromophores

containing five-membered heterocyclic rings (5.9a-f and 5.14b) showed an increase in

fluorescence below pH 2.5 and fluorescence increase below pH 6 for six-membered heterocyclic

ring 5.14a, but the best quantum yield (observed with 5.14a) was only 3% of the fluorescence

value at pH 1. The results, however, demonstrate that high fluorescence intensity requires a cis

configuration of the double bond exocyclic to the imidazolinone ring that is enforced by

hydrogen bonding between the protonated N3 of the imidazolinone ring and nitrogen or oxygen

atoms of the adjacent hetero-ring. The protonation mechanism of 5.9d remains unconfirmed

5.4 Experimental

5.4.1 General Synthesis for the Preparation of Azalactone 5.8

A mixture of hippuric acid (5.0 mmol) (for 5.8a-c) or 5.10 (5.0 mmol) (for 5.8d-e) and

sodium acetate (5.0 mmol) in acetic anhydride (3.0 mL) was stirred at room temperature for 30

min. Aldehyde (5.0 mmol) was added at room temperature and stirred for 30 min, and the

reaction mixture was then heated at 80 °C for 2 h. The reaction mixture was cooled to room

temperature and water (20 mL) was added. The precipitate was filtered off, washed with water,

dissolved in DCM, and the DCM solutes were washed wish brine. The organic solvent was

concentrated by reduced pressure and recrystallized from MeOH/DCM (5.6c was purified by

silica gel column chromatography using DCM) to yield stereoisomeric mixture of azalactone

5.8a-c (Scheme 5-3).

2-Phenyl-4-(thiophen-2-ylmethylene)oxazol-5(4H)-one 5.8a: Yellow microcrystal (68 %); mp

180.0 − 182.0 °C (Lit. [50JOC81] 174.5 – 175.5 °C); 1H NMR (CDCl3) δ 8.20−8.12 (m, 2H),

7.73 (d, J = 5.1Hz, 1H), 7.64 (d, J = 3.9 Hz, 1H), 7.62−7.57 (m, 1H), 7.57−7.52 (m, 2H),

97

7.52−7.48 (m, 1H), 7.19−7.14 (m, 1H); 13C NMR (CDCl3) δ 167.0, 162.5, 137.6, 135.4, 135.0,

133.2, 130.9, 128.9, 128.3, 127.9, 127.7, 125.6, 124.9. Anal. Calcd for C14H9NO2S: C, 65.87; H,

3.55; N, 5.49. Found: C, 65.70; H, 3.43; N, 5.45.

4-((5-Methylfuran-2-yl)methylene)-2-phenyloxazol-5(4H)-one 5.8b: Yellow microcrystal (59

%); mp 150.5 − 152.0 °C (Lit. [06JICS98] 139 – 141 °C)); 1H NMR (CDCl3) δ 8.18−8.10 (m,

2H), 7.62−7.54 (m, 1H), 7.54−7.46 (m, 3H), 7.12 (s, 1H), 6.30 (d, J = 3.3 Hz, 1H), 2.45 (s, 3H);

13C NMR (CDCl3) δ 167.4, 162.1, 158.1, 149.3, 132.9, 128.9, 128.8, 128.1, 125.7, 122.4, 118.5,

111.0, 14.2. Anal. Calcd for C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 70.90; H, 4.33; N,

5.57.

4-((1H-Pyrrol-2-yl)methylene)-2-phenyloxazol-5(4H)-one 5.8c: Yellow microcrystal (35 %);

mp 155.5 − 156.5 °C (Lit. [60DE1095833] 143 – 144 °C); 1H NMR (CDCl3) δ 12.10 (br s,

0.5H), 10.81 (br s, 1H), 8.11 (dd, J = 1.2 & 8.2 Hz, 2H), 8.03 (dd, J = 1.2 & 8.0 Hz, 1H),

7.62−7.45 (m, 5H), 7.43 (s, 0.5H), 7.25 (s, 1H), 7.19 (s, 1H), 6.87−6.81 (m, 0.5H), 6.81−6.75 (m,

1H), 6.46−6.40 (m, 0.5H), 6.41−6.36 (m, 1H); 13C NMR (CDCl3) δ 169.9, 167.0, 134.5, 132.7,

132.1, 130.6, 128.9, 128.9, 128.5, 127.7, 127.3, 127.2, 126.8, 123.0, 121.4, 120.6, 112.7, 111.9.

Anal. Calcd for C14H10N2O2: C, 70.58; H, 4.23; N, 11.76. Found: C, 70.25; H, 4.08; N, 11.59.

2-(Naphthalen-2-yl)-4-(thiophen-2-ylmethylene)oxazol-5(4H)-one 5.8d: Yellow microcrystal

(50 %); mp 212.0 − 214.0 °C; 1H NMR (CDCl3) δ 8.64 (s, 1H), 8.28−8.22 (m, 1H), 7.98 (t, J =

7.8 Hz, 2H), 7.91 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 5.1 Hz, 1H), 7.66 (d, J = 3.6 Hz, 1H), 7.64-

7.58 (m, 2H), 7.52 (s, 1H), 7.21−7.16 (m, 1H); 13C NMR (CDCl3) δ 167.0, 162.6, 137.7, 135.6,

135.3, 134.9, 132.7, 131.1, 129.8, 129.3, 128.9, 128.6, 128.0, 128.0, 127.1, 124.7, 123.7, 122.8.

Anal. Calcd for C18H11NO2S: C, 70.80; H, 3.63; N, 4.59. Found: C, 70.52; H, 3.82; N, 4.50.

98

4-((5-Methylfuran-2-yl)methylene)-2-(naphthalen-2-yl)oxazol-5(4H)-one 5.8e: Yellow

microcrystal (40 %); mp 161.8 − 162.5 °C; 1H NMR (CDCl3) δ 8.62 (s, 1H), 8.25−8.17 (m, 1H),

8.02−7.85 (m, 3H), 7.65−7.52 (m, 3H), 7.14 (s, 1H), 6.33 (d, J = 3.3 Hz, 1H), 2.47 (s, 3H); 13C

NMR (CDCl3) δ 167.4, 162.3, 158.1, 149.4, 135.5, 132.8, 129.6, 129.3, 129.2, 129.0, 128.8,

128.8, 128.5, 128.3, 128.0, 127.1, 127.0, 124.0, 123.6, 123.2, 123.0, 122.4, 118.4, 111.3, 111.0,

14.2. Anal. Calcd for C19H13NO3: C, 75.24; H, 4.32; N, 4.62. Found: C, 75.03; H, 4.39; N, 4.57.

5.4.2 General Synthesis for the Preparation of Imidazolinone 5.9

Unsym-N,N-dimethylmethylenediamine (3.0 mmol) was added to a solution of 5.8 (1.0

mmol) and sodium acetate (2.0 mmol) in acetic acid (2 mL) at 25 °C, and the reaction mixture

was then heated at 70 °C under microwave irradiation (100 W). After 0.5-2.5 h, the reaction

mixture was cooled to room temperature, and 10% Na2CO3 aq was added to the reaction

mixture. The precipitate was purified by silica gel column chlomatography (0% to 5% MeOH in

DCM) to yield stereoisomeric mixtures of 5.9a-f (Scheme 5-3).

1-Methyl-2-phenyl-4-(thiophen-2-ylmethylene)-1H-imidazol-5(4H)-one 5.9a: Yellow

microcrystals (56 %); mp 193.0 − 195.0 °C; 1H NMR (CDCl3) δ 7.67 (d, J = 4.8 Hz, 1H),

7.65−7.55 (m, 4H), 7.46−7.38 (m, 1H), 7.36−7.26 (m, 2H), 7.26−7.19 (m, 2H), 7.14 (t, J = 4.4

Hz, 1H), 7.10−7.04 (m, 2H), 2.39 (s, 3H); 13C NMR (CDCl3) δ 169.9, 159.1, 138.4, 138.2, 136.4,

134.7, 134.2, 132.1, 131.2, 130.1, 129.2, 128.8, 128.3, 127.6, 127.1, 122.5, 21.2,. Anal. Calcd for

C21H16N2OS: C, 73.23; H, 4.68; N, 8.13. Found: C, 73.01; H, 4.56; N, 8.01.

1-(2-(Dimethylamino)ethyl)-2-phenyl-4-(thiophen-2-ylmethylene)-1H-imidazol-5(4H)-one

5.9b: Yellow microcrystals (33 %); mp 103.0 – 105.0 °C; 1H NMR (CDCl3) δ 7.86−7.81 (m,

2H), 7.62 (d, J = 5.4 Hz, 1H), 7.57 (d, J = 3.9 Hz, 1H), 7.56−7.49 (m, 3H), 7.48 (s, 1H),

7.13−7.08 (m, 1H), 4.00 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H); 13C NMR

(CDCl3) δ 171.0, 170.8, 162.2, 161.3, 151.1, 146.1, 138.0, 136.7, 136.1, 134.5, 133.9, 131.2,

99

131.1, 129.9, 129.8, 128.8, 128.8, 128.5, 128.4, 127.5, 122.1, 119.2, 115.6, 113.3, 57.4, 57.3,

45.5, 45.4, 40.0, 39.9.

1-(2-(Dimethylamino)ethyl)-4-((5-methylfuran-2-yl)methylene)-2-phenyl-1H-imidazol-

5(4H)-one 5.9c: Yellow microcrystals (81 %); mp 77.0 − 79.0 °C; 1H NMR (CDCl3) δ 7.82−7.76

(m, 2H), 7.56−7.47 (m, 3H), 7.38 (d, J = 3.3 Hz, 1H), 7.11 (s, 1H), 6.21 (d, J = 3.3 Hz, 1H), 3.88

(t, J = 7.1 Hz, 2H), 2.46−2.38 (m, 5H), 2.13 (s, 6H); 13C NMR (CDCl3) δ 171.0, 161.1, 157.1,

149.9, 134.9, 131.0, 130.0, 128.7, 128.4, 121.3, 116.0, 110.5, 57.4, 45.4, 39.9, 14.2.

4-((1H-Pyrrol-2-yl)methylene)-1-(2-(dimethylamino)ethyl)-2-phenyl-1H-imidazol-5(4H)-

one 5.9d: Yellow sticky solid (55 %); 1H NMR (CDCl3) δ 13.20 (br s, 0.6H), 11.10 (br s,

0.4H),7.76−7.71 (m, 1.2H), 7.71−7.65 (m, 0.8m), 7.59−7.49 (m, 3H), 7.41 (s, 0.4H), 7.22−7.18

(m, 1H), 7.15−7.10 (m, 0.6H), 6.83−6.79 (m, 0.4H), 6.74-6.70 (m, 0.6H), 6.44−6.40 (m, 0.4H),

6.54−6.50 (m, 0.5H), 2.71 (t, J = 7.4 Hz, 2H), 2.34 (s, 6H); 13C NMR (CDCl3) δ 176.0,170.1,

131.1,130.7, 129.6, 129.5, 128.9, 128.4, 128.3, 128.2, 126.1, 126.0, 121.7, 119.2, 112.4, 111.3,

56.1, 44.3, 44.2, 38.9, 38.4, 21.1. HRMS Calcd for [C18H20N4O+H]+, 309.1710; found,

309.1717.

1-(2-(Dimethylamino)ethyl)-2-(naphthalen-2-yl)-4-(thiophen-2-ylmethylene)-1H-imidazol-

5(4H)-one 5.9e: Yellow microcrystals (51 %); mp 154.8 − 158.0 °C; 1H NMR (CDCl3) δ 8.30 (s,

1H), 7.93−7.78 (m, 4H), 7.58−7.44 (m, 4H), 7.42 (s, 1H), 7.03 (t, J = 4.1 Hz, 1H), 3.90 (t, J =

6.8 Hz, 2H), 2.42 (t, J = 6.8 Hz, 2H), 2.07 (s, 6H); 13C NMR (CDCl3) δ 170.9, 161.2, 138.2,

136.9, 134.4, 134.4, 133.9, 132.7, 128.6, 128.9, 128.6, 127.8, 127.5, 127.1, 126.9, 125.1, 122.0,

57.5, 45.5, 40.2.

1-(2-(Dimethylamino)ethyl)-4-((5-methylfuran-2-yl)methylene)-2-(naphthalen-2-yl)-1H-

imidazol-5(4H)-one 5.9f: Yellow oil (30 %); 1H NMR (CDCl3) δ 8.28 (s, 0.72H), 8.24 (d, J =

100

3.6 Hz, 0.28H), 8.21 (s, 0.28H), 7.92−7.51 (m, 4H), 7.56−7.45 (m, 2H), 7.35 (d, J = 3.3 Hz,

0.72H), 7.19 (s, 0.28H), 7.06 (s, 0.72H), 6.20 (d, J = 3.3 Hz, 0.28H), 6.15 (d, J = 3.3 Hz, 0.72H),

3.94-3.84 (m, 2H), 2.50-2.37 (m, 2H), 2.34 (s, 3H), 2.09 (s, 1.7H), 2.06 (s, 4.3H); 13C NMR

(CDCl3) δ 171.1, 161.0, 157.1, 150.0, 134.3, 132.8, 128.8, 128.8, 128.7, 128.6, 128.5, 127.9,

127.7, 127.6, 127.3, 126.9, 125.0, 124.8, 122.8, 121.5, 121.3, 115.9, 110.8, 110.5, 57.5, 45.5,

40.2, 14.2. HRMS Calcd for [C23H23N3O2+H]+, 374.1863; found, 374.1872.

5.4.3 Synthesis of 2-(2-Naphthamido)acetic acid 5.10

2-Naphthoylchloride (0.57g, 3.03mmol) and glycine (0.25g, 3.03 mmol) were mixed and

stirred in the presence of NaOH (0.12 g, 3.03 mmol) in aqueous MeCN (MeCN : H2O = 2mL :

5mL) at room temperature. After 1 h, the organic solvent was evaporated under reduced

pressure, washed with 6M HCl aq, and extracted with EtOAc. The organic layer was then

washed with brine, and dried with MgSO4 to yield 5.10 as white microcrystals followed by

recrystallization from EtOAc/hexanes (Scheme 5-4). (70%); mp 150.0 − 151.0 °C; 1H NMR

(DMSO-d6) δ 12.64 (br s, 1H), 9.03 (t, J = 5.8 Hz, 1H), 8.49 (s, 1H), 8.05-7.50 (m. 4H), 7.65-

7.55 (m, 2H), 3.99 (d, J = 5.7 Hz, 2H); 13C NMR (DMSO-d6) δ 171.4, 166.8, 134.3, 132.2,

131.3, 129.0, 128.1, 127.8, 127.7, 127.7, 126.9, 124.1, 41.4. Anal. Calcd for C13H11NO3: C,

68.11; H, 4.84; N, 6.11. Found: C, 67.78; H, 4.88; N, 6.49.

5.4.4 Synthesis of N-(2-Chloroacetyl)benzamide 5.11

Chloroacetyl chloride (2.9 mL, 0.04 mol) was added dropwise to a solution of benzamide

(4.0 g, 0.03 mol) in toluene (80 mL). The reaction mixture was heated under reflux for 1.5 h, and

then cooled to room temperature. The organic solvent was concentrated under reduced pressure.

The residue was recrystallized in DCM/hexanes to yield N-(2-chloroacetyl)benzamide 5.11 as

white microcrystals (Scheme 5-7). (70 %); mp 153.0 − 155.0 °C; 1H NMR (CDCl3) δ 9.69 (br s,

1H), 7.93 (d, J = 7.8 Hz, 2H), 7.70−7.60 (m, 1H), 7.60−7.45 (m, 2H), 4.79 (s, 2H); 13C NMR

101

(CDCl3) δ 168.8, 165.7, 133.6, 131.8, 129.0, 128.0, 45.4. Anal. Calcd for C9H8ClNO2: C, 54.70;

H, 4.08; N, 7.09. Found: C, 54.71; H, 4.03; N, 7.01.

5.4.5 Synthesis of N-(2-Azidoacetyl)benzamide 5.12

A mixture of 5.11 (1.0 g, 5.06 mmol) and sodium azide (0.7 g, 10.12 mmol) in DMSO (4.5

mL) was stirred at room temperature for 12 h, and ice-cold water was then added. The precipitate

was filtered off, and recrystallized from EtOAc/hexanes to yield 5.12 (Scheme 5-7). White

microcrystals (66 %); mp 125.0 − 126.0 °C; 1H NMR (CDCl3) δ 9.62 (br s, 1H), 7.96 (d, J = 7.3

Hz, 2H), 7.68-7.60 (m, 1H), 7.54 (t, J = 7.1 Hz, 2H), 4.60 (s, 2H); 13C NMR (CDCl3) δ 171.6,

165.8, 133.8, 131.5, 129.1, 127.9, 54.3. Anal. Calcd for C9H8N4O2: C, 52.94; H, 3.95; N, 27.44.

Found: C, 53.08; H, 4.09; N, 27.46.

5.4.6 Synthesis of 2-Phenyl-1H-imidazol-5(4H)-one 5.13

Triphenylphosphine (1.67 g, 6.37 mmol) was added to a solution of N-(2-azidoacetyl)-

benzamide (1.0 g, 4.90 mmol) in toluene (50 mL). The mixture was stirred at room temperature

for 3 h, then evaporated under reduced pressure. The residue was washed with ether and

recrystallized from DCM/hexanes to yield 5.13 (Scheme 5-7). Red microcrystals (66 %); mp

147.0 − 148.0 °C; 1H NMR (CDCl3) δ 10.59 (br s, 1H) 7.92 (d, J = 7.8 Hz, 2H), 7.40−7.60 (m,

3H), 4.42 (s, 2H); 13C NMR (CDCl3) δ 184.8, 161.3, 132.1, 129.0, 128.4, 126.7, 60.2. Anal.

Calcd for C9H8N2O: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.16; H, 5.00; N, 17.22.

5.4.7 General Procedure for the Preparation of Imidazolinone 5.14

A solution of the appropriate aldehyde (0.85 mmol) and imidazolin-4-one 5.13 (0.94

mmol) in piperidine (9 mL) was stirred at room temperature for 1.0-1.5 h (Scheme 5-7). H2O

was added and the mixture was extracted with DCM, washed with brine, and dried with MgSO4.

The solvent was removed under reduced pressure. The crude mixture was purified by neutral

102

alumina chromatography (2% MeOH in DCM) to yield isomeric mixtures of imidazolinone

chromophore 5.14 followed by recrystalization in MeOH.

4-((6-Methylpyridin-2-yl)methylene)-2-phenyl-1H-imidazol-5(4H)-one 5.14a: White

microcrystals (18 %); mp 189 °C (decomposed); 1H NMR (CDCl3) δ 11.65 (br s, 0.67H) 10.87

(br s, 0.33H), 8.68 (d, J = 7.8 Hz, 0.33H), 8.10−7.90 (m, 2H), 7.60−7.40 (m, 3.66H), 7.15 (d, J =

7.5 Hz, 0.67H), 7.11 (s, 0.67H), 6.99 (d, J = 7.7 Hz, 1.34H), 6.75 (s, 0.33H), 2.55 (s, 2H), 2.46

(s, 1H); 13C NMR (CDCl3) δ 180.1, 172.6, 158.6, 158.3, 153.6, 142.2, 137.5, 136.5, 134.5, 134.2,

132.9, 129.3, 129.1, 128.5, 128.3, 127.8, 127.6, 127.4, 125.1, 124.4, 123.4, 122.8, 113.2, 25.1,

24.5. Anal. Calcd for C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C, 72.75; H, 4.85; N,

15.66.

4-((5-Methylfuran-2-yl)methylene)-2-phenyl-1H-imidazol-5(4H)-one 5.14b: Yellow

microcrystals (29 %); mp 230.0 °C (decomposed); 1H NMR (DMSO-d6) δ12.05 (br s, 1H), 8.14

(dd, J = 7.9 & 1.4 Hz, 2H), 7.65-7.52 (m, 3H), 7.49 (d, J = 3.3 Hz, 1H), 6.80 (s, 1H), 6.44 (d, J =

1.8 Hz, 1H), 2.39 (s, 3H); 13C NMR (DMSO-d6) δ 171.2, 159.0, 156.1 ,149.5, 136.7, 132.2,

128.9, 128.0, 127.2, 120.0, 112.3, 110.7, 13.7. Anal. Calcd for C15H12N2O2: C, 71.41; H, 4.79; N,

11.10. Found: C, 71.54; H, 4.77; N, 10.87.

103

CHAPTER 6 SUMMARY OF ACHIEVEMENTS

Synthetic organic chemistry plays an important role in the fields of material science,

pharmaceuticals, agricultures, and food chemicals. The present work helps in the essential pursit

of more efficient and environmently-friendly intermadiates.

Chapter 1 provides an overview of the importance of BtH in organic syntheses. Previously,

our group reported the synthesis of novel benzotriazole intermediates, 1-(alkyl/arylthio-

carbamoyl) benzotriazoles and benzotriazole-1-carboxamidine, which were utilized in the

synthesis of guanidine- and thiourea-analogues. [04JOC2976, 06A226, 06JOC6753] In Chapter

2, new C-C bond forming C-aminoimidoylation and C-thiocarbamoylation reactions with

sulfones and ketones were achieved under mild reaction conditions. [07JOC6742] This approach

provides easy access to interesting classes of compounds for further transformations.

Furthermore, reactions of ketones with N-benzyl-1H-benzo[d][1,2,3]-triazole-1-carbothioamide

surprisingly gave the isomeric 1,3-oxazolidine-2-thione followed by formation of isothiocyanates

and cyclization due to deprotonation of benzyl protons.

In Chapter 3, tri-, tetra-, penta-, hexa-, and heptapeptides were prepared by solid phase

segment condensation assisted by microwave irradiation. [08CBDD181] (Nα-Fmoc-protected-

dipeptidoyl)benzotriazoles were synthesized, in which the original chirality was maintained. (Nα-

Fmoc-protected-dipeptidoyl)benzotriazoles are air and moisture insensitive acylation reagents

which enable solid phase segment condensation without the use of other coupling reagents or

additives. Side reactions and epimerization were not observed in these sequences, some of which

were previously reported as difficult during peptide synthesis by segment condensation.

In Chapter 4, a convenient and efficient preparation in solution phase of a variety of

coumarin fluorescent probes is described with coumarin-3-ylcarboxyl (Cc) and 7-

104

methoxycoumarin-4-ylacetyl (Mca) labeled lysines as fluorogenic substrates. [08OBC4582]

Their benzotriazole derivatives are appropriate materials for peptide labeling thus enabling

efficient peptide α-amino group acylation under microwave irradiation on solid phase and

without the use of coupling agents and/or additives, avoiding side reactions, and epimerization.

In Chapter 5, GFP modified pH sensitive chromophores were synthesized and their

fluorescent activities were measured in Britton-Robinson Buffer between pH 7 and pH 1.

[Proj#1976] Chromophores containing five-membered aromatic rings showed increase of

fluorescence below pH 2.5, but the chromophore containing 2-pyridyl group was more sensitive

at higher pH (below pH 6) due to a strong intramolecular hydrogen bonding with imidazolinone

nitrogen. This may be useful in the determination of the acidity of biological systems.

Many novel reactions using benzotriazole methodology have been carried out by previous

members of the Katritzky group, and my work has expanded their studies and presented further

applications of BtH in organic synthesis.

105

LIST OF REFERENCES

The reference citation system employed throughout this research report is from “

Comprehensive Heterocyclic Chemistry Ⅱ” (vol.1); Pergamon Press: New York, 1996 (Eds.

Katritzky, A. R.; Rees, C. W.; Scriven, E.). Each time a reference is cited, a number-letter code

is designated to the corresponding reference with the first two or four if the referencec is

before1910’s number indicating the year followed by the letter code of the journal and the page

number in the end.

Additional notes to this reference system are as follows:

1) Each reference code is followed by conventional literature citation in the ACS style.

2) Journals which are published in more than one part including in the abbreviation cited the appropriate part.

3) Less commonly used books and journals are still abbreviated as using initials of the journal name.

4) The list of the reference is arranged according to the designated code in the order of (i) year, (ii) journal/book in alphabetical order, (iii) part number or volume number if it is included in the code, and (iv) page number.

5) Project number is used to code the unpublished results.

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[94JBC20952] Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem. 1994, 269, 20952.

[94JHC917] Katritzky, A. R.; Galuszka, B.; Rachwal, S.; Black, M. J. Het. Chem. 1994, 31, 917.

[95B3972] Turcatti, G.; Vogel, H.; Chollet, A. Biochem. 1995, 34, 3972.

[96JMS45] Hansen, P. E.; Duus, F.; Bolvig, S.; Jagodzinski, T. S J. Mol. Struct. 1996, 378, 45.

[97B9756] Wachter, R. M.; King, B. A.; Heim, R.; Kallio, K.; Tsien, R. Y.; Boxer, S. G.; Remington, S. J. Biochem. 1997, 36, 9759.

[97CR1515] Silva, A. P. D.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev., 1997, 97, 1515.

[97FEBS379] Gulnik, S. V.; Suvorov, L. I.; Majer, P.; Collins, J.; Kane, B. P.; Johnson, D. G.; Erickson, J. W. FEBS Lett. 1997, 413, 379.

[97JPR444] Cowley, D. J.; Schulze, A. J. Pept. Res. 1997, 49, 444.

[97PNAS2306] Brejc. K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien, R. Y.; Ormӧ, M.; Remington, S. J. Proc. Natl. Acad. Sci. USA 1997, 94, 2306.

[97TL6771] Fustero, S.; Pina, B.; Simón-Fuentes, A. Tetrahedron Lett. 1997, 38, 6771.

[98B509] Tsien, R. Y. Annu. Rev. Biochem., 1998, 67, 509.

[98BMCL597] Weber, P. J. A.; Bader, J. E.; Folkers, G.; Beck-Sickinger, G. Bioorg. Med. Chem. Lett. 1998, 8, 597.

[98CL1153] Arai, T.; Hozumi, Y. Chem. Lett. 1998, 1153.

[98CR409] Katritzky, A. R.; Lan, X.; Yang, J. Z.; Denisco, O. V. Chem. Rev. 1998, 98, 409.

[99JACS1636] Hamuro, Y.; Scialdone, M. A.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 1636.

[99OL977] Fustero, S.; Pina, B.; Garcia de la Torre, M.; Navarro, A.; Ramírez de Arellano, C.; Simón A. Org. Lett. 1999, 1, 977.

[00B118] Murakami, H.; Hohsaka, T.; Ashizuka, Y.; Hashimoto, K.; Sisido, M. Biomacromol. 2000, 1, 118.

[00B4423] Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochem. 2000, 39, 4423.

[00BC71] Geoghegan, K. F.; Rosner P. J.; Hoth, L. R. Bioconjugate Chem. 2000, 11, 71.

108

[00JACS5849] Wang, H.; Burda, C.; Persy, G.; Wirz, J. J. Am. Chem. Soc. 2000, 122, 5849.

[00JOC1583] Barun, O.; Ila, H.; Junjappa, H. J. Org. Chem. 2000, 65, 1583.

[00JOC3679] Katritzky, A. R.; Pastor, A. J. Org. Chem. 2000, 65, 3679.

[00JOC8210] Katritzky, A. R.; He, H. Y.; Suzuki, K. J. Org. Chem. 2000, 65, 8210.

[00S2029] Katritzky, A. R.; Fang, Y.; Donkor, A.; Xu. L. Synthesis 2000, 14, 2029.

[01AMB274] Sisido, M.; Hohsaka, T. Appl. Microbiol. Biotechnol. 2000, 57, 274.

[01B5795] Lauer-Fields, J. L.; Broder, T.; Sritharan, T.; Chung, L.; Nagase, H.; Fields, G. B. Biochem. 2001, 40, 5795.

[01BCSJ2133] Mashraqui, S. H.; Kumar, S.; Mudaliar, C. D. Bull. Chem. Soc. Jpn. 2001, 74, 2133.

[01CRLC295] Metzler, D. E. ‘Biochem: The Chemical Reactions of Living Cells’, 2nd Edition, Harcourt/Academic Press 2001, 1, 295.

[01LPS263] Malkar, N. B.; Fields, G. B. Lett. Peptide Sci. 2001, 7, 263.

[01RJBC306] Kolobanova, S. V.; Filippova, I. Y.; Lysogorskaya, E. N.; Bacheva, A. V.; Oksenoit, E. S.; Stepanov, V. M. Rus. J. Bioorg. Chem. 2001, 27, 306.

[01P] Califano, J. C.; Devin, C.; Shao, J.; Blodgett, J. K.; Maki, R. A.; Funk, K. W.; Tolle, J. C. ‘Peptide 2000, Proceedings of the European Peptide Symposium, 26th Montpellier, France’, 2001.

[02A39] Katritzky, A. R.; Yang, H.; Zhang, S.; Wang, M. ARKIVOC 2002, xi, 39.

[02JMB429] Renard, M.; Belkadi, L.; Hugo, N;. England, P.; Altschuh, D.; Bedouelle, H. J. Mol. Biol. 2002, 318, 429.

[02JMR311] Wei A. P.; Herron, J. N. J. Mol. Recognit. 2002, 15, 311.

[02JOC3120] Enander, K.; Dolphin, G. T.; Andersson, L. K.; Liedberg, B.; Lundstrom, I.; Baltzer, L. J. Org. Chem. 2002, 67, 3120.

[02JOC4667] Fustero, S.; Pina, B.; Salavert, E.; Navarro, A.; Ramírez de Arellano, M. C.; Simón Fuentes, A. J. Org. Chem. 2002, 67, 4667.

[02PS2655] Lorimier, R. M. D.; Smith, J. J.; Dwyer, M. A.; Looger, L. L.; Sali, K. M.; Paavola, C. D.; Rizk, S. S.; Sadigov, S.; Conrad, D. W.; Loew, L.; Hellinga, H. W. Protein Sci. 2002, 11, 2655.

109

[03AB105] Lauer-Fields, J. L.; Kele, P.; Sui, G.; Nagase, H.; Leblanc, R. M.; Fields, G. B. Anal. Biochem. 2003, 321, 105.

[03AB141] Yan, Z. H.; Ren, K. J.; Wang, Y.; Chen, S.; Brock, T. A.; Rege, A. A. Anal. Biochem. 2003, 312, 141.

[03CB53] Takahashi, M.; Nokihara, K.; Mihara, H. Chem. Biol. 2003, 10, 53.

[03FBES35] He, X.; Bell. A. F.; Tonge, P. J. FEBS Lett. 2003, 549, 35.

[03JOC1443] Katritzky, A. R.; Abdel-Fattah, A. A. A.; Wang, M. J. Org. Chem. 2003, 68, 1443.

[03JOC4932] Katritzky, A. R.; Abdel-Fattah, A. A. A.; Wang, M. J. Org. Chem. 2003, 68, 4932.

[03JOC5720] Katritzky, A. R.; Suzuki, K.; Singh, S. K.; He, H. Y. J. Org. Chem. 2003, 68, 5720.

[04CCA175] Katritzky, A. R.; Suzuki, K.; Singh, S. K. Croat. Chem. Acta 2004, 77, 175.

[04CR3059] Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem. Rev., 2004, 104, 3059.

[04DDT24] Stoll, D.; Bachmann, J.; Templin, M. F.; Joos, T. O. DDT: Targets 2004, 3, 24.

[04JOC188] Shi, Y.; Zhang, J.; Grazier, N.; Stein, P. D.; Atwal, K. S.; Traeger, S. C.; Callahan, S. P.; Malley, M. F.; Galella, M. A.; Gougoutas, J. Z. J. Org. Chem. 2004, 69, 188.

[04JOC2976] Katritzky, A. R.; Ledouux, S.; Witek, R. M.; Nair, S. K. J. Org. Chem. 2004, 69, 2976.

[04PS1489] Filippis, V. D.; Boni, S. D.; Dea, E. D.; Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489.

[04S1806] Katritzky, A. R.; Shestopalov, A. A.; Suzuki, K. Synthesis 2004, 11, 1806.

[04S2645] Katritzky, A. R.; Suzuki, K.; Singh, S. K. Synthesis 2004, 2645.

[04TL6079] Fernandez-Carneado, J.; Giralt, E. Tetrahedron Lett. 2004, 45, 6079.

[05AJC576] Saravanan, V. S.; Kymar, S. P. V.; De, B.; Gupta, J. K. Asian J. Chem. 2005, 17, 269.

[05ACIE2642] Tinnefeld, P.; Sauer, M. Angew. Chem. Int. Ed. 2005, 44, 2642.

110

[05B238] Makino, T.; Matsumoto, M.; Suzuki, Y.; Kitajima, Y.; Yamamoto, K.; Kuramoto, M.; Minamitake, Y.; Kangawa, K.; Yabuta, M. Biopolymers 2005, 79, 238.

[05CBC1043] Engfeldt, T.; Renberg, B.; Brumer, H.; Nygren, P. A.; Karlström, A. E. Chem. Bio. Chem. 2005, 6, 1043.

[05FSPPS215] Chan, W. C.; White, P. D. ‘Fmoc Solid Phase Peptide Synthesis: A practice approach’, Orxford University Press Inc., 2005, 9, 215.

[05HCA1664] Katritzky, A. R.; Khashab, N. M.; Bobrov, S. Helvetica Chim. Acta. 2005, 88, 1664.

[05JOC4993] Katritzky, A. R.; Jiang, R.; Suzuki, K. J. Org. Chem. 2005, 70, 4993.

[05JSS1812] Shi, Y.; Xiang, R.; Horváth, C.; Wilkins, J. A. J. Sep. Sci. 2005, 28, 1812.

[05S397] Katritzky, A. R.; Angrish, P.; Hur, D.; Suzuki, K. Synthesis 2005, 3, 397.

[05S1656] Katritzky, A. R.; Suzuki, K.; Wang, Z. Synlett 2005, 11, 1656.

[06A226] Katritzky, A. R.; Khashab, N. M.; Gromova, A. V. ARKIVOC 2006, 3, 226.

[06CBDD326] Katritzky, A.R.; Meher, G.; Angrish, P. Chem. Biol. Drug Des. 2006, 68, 326.

[06CP358] Nifosì, R.; Tozzini, V. Chem. Phys. 2006, 323, 358.

[06CR6699] Rofstad, E. K.; Mathiesen, B.; Kindem, K.; Galappathi, K. Cancer Res. 2006, 66, 6699.

[06JICS98] Salehi, P.; Dabiri, M.; Khosropout, A. R.; Roozbehniya, P. J. Iranian Chem. Soc., 2006, 3, 98.

[06JOC6753] Katritzky, A. R.; Khashab, N. M.; Bobrov, S.; Yoshioka, M. J. Org. Chem. 2006, 71, 6753.

[06JPS116] Goulas, S.; Gatos, D.; Barlos, K. J. Peptide Sci. 2006, 12, 116.

[06NGIR] Khashab, N. M. ‘Novel guanylating and imidoylating reagents’, University of Florida, 2006.

[06OBC4265] Wilson, J. N.; Kool, E. T. Org. Biomol. Chem., 2006, 4, 4265.

[06S411] Katritzky, A. R.; Angrish, P.; Suzuki, K. Synthesis 2006, 3, 411.

[06S217] Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217.

111

[06TA2393] Royo, S.; Jiménez, A. I.; Cativiela, C. Tetrahedron: Asymmetry 2007, 17, 2393.

[06TL7905] Yoshiya, T.; Sohma, Y.; Kimura, T.; Hayashi, Y.; Kiso, Y. Tetrahedron Lett. 2006, 47, 7905.

[07AHC131] Suzuki, T.; Matsuzaki, T.; Hagiwara, H.; Aoki, T.; Takata, K. Acta Hiscochem. Cytochem. 2007, 40, 131.

[07B9865] Malo, G. D.; Pouwels, L. J.; Wang, M.; Weichsel, A.; Montfort, W. R.; Rizzo, M. A.; Piston, D. W.; Wachter, R. M. Biochem. 2007, 46, 9865.

[07BC994] Katritzky, A. R.; Angrish, P.; Narindoshvili, T. Bioconjugate Chem. 2007, 18, 994.

[07CBDD465] Katritzky, A. R.; Khashab, N. M.; Yoshioka, M.; Haase, D. N.; Wilson, K. R.; Johnson, J. V.; Chung, A.; Haskell-Luevano, C. Chem. Biol. Drug Des. 2007, 70, 465.

[07JOC6742] Katritzky, A. R.; Khashab, N. M.; Haase, D. N.; Yoshioka, M. Ghiviriga, I.; Steel, P. J. J. Org. Chem. 2007, 72, 6742.

[07S1103] Prüger, B.; Bach, T. Synthesis 2007, 7, 1103.

[07S3141] Katritzky, A. R.; Le. K. N. B.; Mohapatra. P. P. Synthesis 2007, 20, 3141.

[07SC1709] Jursic, B. S.; Sagiraju, S.; Ancalade, D. K.; Clark, T.; Stevens, E. D. Synthetic Comm. 2007, 37, 1709.

[08CBDD181] Katritzky, A. R.; Yoshioka, M.; Narindoshvili, T.; Chung, A.; Khashab, N. M. Chem. Biol. Drug Des. 2008, 72, 181.

[08JOC511] Katritzky, A. R.; Narindoshvili, T.; Draghici, B.; Angrish, P. J. Org. Chem. 2008, 73, 511.

[08JACS4089] Wu, L.; Burgess, K. J. Am. Chem. Soc. 2008, 130, 4089.

[08OBC4582] Katritzky, A. R.; Yoshioka, M.; Narindoshvili, T.; Chung, A.; Johnson, J. V. Org. Biomol. Chem. 2008, 6, 4582.

[09A47] Katritzky, A. R.; Singh, A.; Haase, D. N.; Yoshioka, M. ARKIVOC 2009, viii, 47.

[09OBC627] Katritzky, A. R.; Narindoshvili, T. Org. Biomol. Chem. 2009, 7, 627.

[Proj#1979] Katritzky, A. R.; Yoshioka-Tarver, M.; El-Gendy, B. E. M.; Hall, C. D. In progress.

112

BIOGRAPHICAL SKETCH

Megumi Yoshioka-Tarver, first daughter of Hitoshi and Kazuyo Yoshioka, was born in

Japan. She received her Bachelor of Engineering from University of Fukui, Japan, in March

2004. During her senior year, she worked as an undergraduate researcher in a synthetic organic

chemistry lab focused on supramoleculer chemistry, under the direction of Dr. Yuji Tokunaga.

Upon graduation, she continued her education at the Department of Chemistry, University of

Florida, from August 2005. Her doctorate-level research focused on synthesis of heterocycles

and peptides supervised by Dr. Alan R. Katritzky.