synthesis and anticancer screening of combinatorial

Synthesis and Anticancer Screening of

Combinatorial Libraries of Cyclic Peptides

and Other Compounds

Thesis Submitted for the Partial Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY

By

SAMREEN ASHRAF

H. E. J. Research Institute of Chemistry

International Center for Chemical and Biological Sciences,

University of Karachi, Karachi-75270, Pakistan

(2017)

i

Certificate

To Whom It May Concern

This is to certify that the thesis entitled, “Synthesis and Anticancer Screening of

Combinatorial Libraries of Cyclic Peptide and Other Compounds”, has been

submitted by Ms. Samreen Ashraf to the Board of Advance Studies and Research,

University of Karachi, for the award of the degree of Ph.D. in Chemistry. This thesis, in

full on in parts, has not been submitted to any other institute or university for the award of

any degree or diploma.

Prof. Dr. Farzana Shaheen

Research Supervisor

Contents

ACKNOWLEDGMENTS ...................................................................................................... i

PERSONAL INTRODUCTION............................................................................................ ii

SUMMARY ..........................................................................................................................iii

Urdu Khulasa...………………………………………………………………………….…vi

List of Figures ...................................................................................................................... vii

List of Tables ........................................................................................................................ ix

List of Schemes ...................................................................................................................... x

CHAPTER-1

INTRODUCTION

1.1. Drug Discovery and Development ................................................................................. 1

1.1.1. The Lead Discovery Process ............................................................................ 1

1.2. Peptides: An Attractive Source of New Lead Molecules ............................................... 3

1.2.1. Peptide Hormones as Drugs ............................................................................. 5

1.2.2. Peptide as Radionuclide Carrier....................................................................... 6

1.2.3. Peptide as Vaccines.......................................................................................... 8

1.2.4. Peptides in Targeted Drug Delivery ................................................................ 9

1.2.5. Anti-Tumor Peptides ...................................................................................... 11

1.2.6. Cell Penetrating Peptides ............................................................................... 11

1.2.7. Anti-inflammatory Peptides ........................................................................... 12

1.2.7.1. Duanbanhuains (A–C) .................................................................... 13

1.2.7.2. Cyclomarins .................................................................................... 13

1.2.7.3. Cyclosquamosin .............................................................................. 14

1.2.7.4. Anti-inflammatory Peptides from Marine Sponges ........................ 15

1.3. Cyclic Peptides ............................................................................................................. 16

1.3.1. Stability of Cyclic Peptides ............................................................................ 17

1.3.2. Strategies to Develop Peptide Libraries ......................................................... 18

1.3.2.1. Combinatorial Library Approach .................................................... 19

1.3.2.2. Phage-Display Peptide Library Method .......................................... 19

1.3.2.3. Parallel Library Method .................................................................. 20

1.3.2.4. Combinatorial Library Methods Requiring Deconvolution ............ 20

1.3.2.5. Affinity Selection Method .............................................................. 21

1.3.2.6. Self-Assembled PNA-Encoded Chemical Microarrays .................. 21

1.3.2.7. OBOC Combinatorial Library Method ........................................... 21

1.3.3. Cyclization Strategies for Synthetic Cyclic Peptides ..................................... 22

1.3.4. Analysis of Cyclic Peptide through Mass Spectrometry ............................... 25

1.3.5. Conformational Analysis of Cyclic Peptides ................................................. 27

1.4. Importance of Cationic Cyclic Peptides ....................................................................... 28

1.5. Cancer Targeting Strategies .......................................................................................... 29

1.6. Inflammation ................................................................................................................. 33

1.6.1. Mediators of Inflammation ............................................................................ 36

1.6.1.1. Role of Interlukin-1 (IL-1) .............................................................. 36



1.6.1.4. Role of tumor necrosis factor-alpha (TNF-α) ................................. 38

1.6.1.5. Role of Nitric Oxide (NO.) ............................................................. 38

1.6.1.6. Role of Reactive Oxygen Species ................................................... 39

1.7. Solid-phase Peptide Synthesis ...................................................................................... 39

1.7.1. Solid Supports for Peptide Synthesis ............................................................. 40

1.7.2. Linkers used in Solid-phase Peptide Synthesis (SPPS) ................................. 41

1.7.2.1. Kenner’s Safety-catch Linker ......................................................... 41

1.7.2.2. Wang Linker ................................................................................... 42

1.7.2.3. Rink-amide Linker .......................................................................... 42

1.7.3. Protecting Groups Used in Solid-phase Peptide Synthesis ............................ 43

1.7.4. Coupling Reagents ......................................................................................... 44

1.8. Side-reactions in Peptide Bond Formation ................................................................... 45

1.8.1. Racemization: ................................................................................................ 45

1.8.2. Synthesis of Diketopiperazine During Peptide Chain Elongation ................. 45

1.9. Targets of Current Study ............................................................................................... 46

Chapter - 2

Result and Discussions

2.1. Design, Synthesis and Screening of Cyclic Peptide Library ........................................ 47

2.2. Structural Studies of Anticancer Peptides from Library 1 ............................................ 50

2.2.1. Cyclic Peptide 2 ............................................................................................. 50

2.2.2. Cyclic Peptide 14 ........................................................................................... 57

2.2.3. Cyclic Peptide 15 ........................................................................................... 62

2.2.4. Cyclic Peptide 16 ........................................................................................... 68

2.2.5. Anticancer Activities of Cyclic Peptides. ...................................................... 73

2.3. Studies on Stylissatin A Analogues .............................................................................. 81

2.3.1. Cyclic Peptide 26 ........................................................................................... 84

2.3.2. Cyclic Peptide 27 ........................................................................................... 90

2.3.3. Cyclic Peptide 28 ........................................................................................... 97

2.3.4. Cyclic Peptide 29 ......................................................................................... 105

2.3.5. Cyclic Peptide 30 ......................................................................................... 112

2.3.6. Cyclic Peptide 31 ......................................................................................... 118

2.4. Immunomodulatory Activities of Stylissatin A Analogues ........................................ 125

2.5. Conclusion .................................................................................................................. 126

Chapter - 3

EXPERIMENTAL PROCEDURES

3.1. General Experimental Details ..................................................................................... 128

3.2. General Synthesis Procedure for Cyclic Peptide Library I ......................................... 128

3.3. Synthesis of Biologically Active Compounds ............................................................ 129

3.3.1. Synthesis of Cyclic Peptide 2 ...................................................................... 129

3.3.1.1. Characterization Data of Peptide 2 ............................................... 130

3.3.2. Synthesis of Cyclic Peptide 14 .................................................................... 130

3.3.2.1. Characterization Data of Peptide 14 ............................................. 131


3.3.3.1. Characterization of Peptide 15 ...................................................... 132


3.3.4.1. Characterization of Peptide 16 ..................................................... 133

3.4. Synthesis Procedure for Stylissatin A Analogues ....................................................... 133




3.4.2.1. Characterization Data of Peptide 27 ............................................ 135

3.4.3 Synthesis of Cyclic Peptide 28. .................................................................... 136








3.5. Screening Protocol ...................................................................................................... 140

3.5.1. MTT (3- (4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide)

Assay……………………………………………………………………………..140

3.5.2. Chemiluminescence Assay .......................................................................... 140

3.5.3. Nitric Oxide Screening Protocol: ................................................................. 140

3.5.4. IL-2 Production and Quantification: ............................................................ 141

References .......................................................................................................................... 142

Glossary ............................................................................................................................. 168

Abbreviations……………………………………..……………………………………………..172

i

ACKNOWLEDGEMENTS

First of all I present my sincere gratitude to the Allah (SWT), who is the most beneficent and merciful

and Hazrat Muhammad (PBUH), whose teachings enlighten our souls and minds.

I present my honor to the pioneer of this prestigious institute Prof. Dr. Salim-uz-Zaman Siddiqui FRS

whose dedication makes this institute the home of great research. I also present a note of thanks to

revolutionary scientist Prof. Dr. Atta-ur-Rahman FRS. His dynamic leadership and guidance has driven

this country towards prosperity and development. I am also thankful to honorable Prof. Dr.

Muhammad Iqbal Choudhary H.I. S.I. T.I., Director of ICCBS. He is always a source of inspiration

throughout my Ph. D. studies. I wish to express my deepest gratitude for his expert guidance, sincere

advice and continuous support through the period of this research study.

I expressed my profound gratitude to my research supervisor Prof. Dr. Farzana Shaheen. Her endless

efforts, encouragements and attention allowed me to finally complete my Ph.D. work. I am highly

indebted to her for the contribution of experience and knowledge being shared and conveyed to me.

I am very thankful to Dr. Almas Jabeen for the biological evaluation of compounds reported in this

thesis.

I cordially express my thanks to all faculty members especially Dr. Shabana Simjee for their guidance,

cooperation and assistance and thanks are to all technical and non-technical staff of the institute who

have helped me in the completion of my work in so many ways.

It cheers me up to remember all my lab colleagues, Dr. Zafar Ali Shah, Comail Abbas, Asad Ziaee,

Muhammad Nadeem-ul-Haque, Salma Nazir, Attiya Hameed, Anila Bashir and Hira Shehzed and Lab.

attendant Syed Muhammad Nasar.

I also want to acknowledge the financial support for this study from Pakistan Science Foundation

(PSF) project PSF/NSLP(290).

I am also pleased to present my utmost gratitude to my family, parents, my husband, and my mother-in-

law, their patience, trust and prayers kept me going ahead widening the path and making ways to

achieve my goal.

Samreen Ashraf

Karachi, 2017

https://www.google.com.pk/search?biw=1024&bih=653&q=Prof.+Dr.+Salim-UZ-zaman+Siddiqui&spell=1&sa=X&ei=GALdUt3kF4Kt0QXInoHYBQ&ved=0CCUQBSgA

ii

PERSONAL INTRODUCTION:

On 17th of June, 1985, it was a hot afternoon of summer when ALLAH

bestowed HIS blessing on my parents and I open my eyes in this world. I

have been very fortune to be in the “City of Lights” Karachi, Pakistan, where

number of educational institutes is enlightening the lives of people with

knowledge and skills. After schooling I completed my higher secondary

education from Govt. Degree Science College Malir Cantt. I pursued my

Master’s degree from University of Karachi with organic chemistry as major

subject.

The thirst of knowledge was still not satisfied, and on March 2010, I joined the H.E.J. Research

Institute of Chemistry, International Center for Chemical and Biological Sciences, University of

Karachi. I have been very fortunate to carry out my research studies at one of the best institutions of

the region. I worked on the “Synthesis and Anticancer Screening of Combinatorial Libraries of Cyclic

Peptide and Other Compounds” for my Ph.D. studies under the kind and able supervision of Prof. Dr.

Farzana Shaheen.

At H.E.J. Institute, I received generous guidance by highly experienced and dedicated faculty members

who always helped me to broaden my scientific knowledge and skills. I learnt different ways of tackling

complex situations, which not only enhanced my self-confidence but also improved my capacity of

critical thinking. In the area of research my focus remains the peptide synthesis. In years to come, I

would like to take part in the development of theoretical chemistry and to correlate it with the peptide

synthesis for the development of biologically active molecules against diseases like cancer, diabetes,

tuberculosis, etc. I have a dream of serving the humanity through relevant science all over the world

generally, and Pakistan particularly by contributing in the development of field laboratories to promote

the scientific knowledge, and ultimately contribute towards national development.

iii

SUMMARY

The structural features of cyclic peptides make them good drug candidates, and up till now,

several cyclic peptides have been developed for clinical use. Many structurally important

cyclic peptides have been developed with different synthetic approaches. In recent years,

there has been an increasing interest in the synthesis of biologically active cyclic peptides.

The current study was focused on the synthesis and identification of novel biologically

active compounds from libraries based on cyclic peptide scaffold. During the synthesis of

cyclic peptides, different cyclization strategies were adopted to develop diverse forms of

cyclic peptides. These cyclic peptides were designed to contain some unusual building

blocks, such as phenyl glycine, naphthyl alanine, ornithine, 2-amino benzoic acid, D amino

acids as well as cationic residues at different positions. The synthesis of all cyclic peptides

was accomplished by macrocyclization strategy using standard Fmoc-peptide synthesis

protocol. The cyclic peptide libraries were screened against four human cancer cell lines

MCF-7, NCI-H460, and DoHH2 and HeLa cell lines to identify active compounds.

Detailed structural studies were performed on anticancer peptides discovered from the

peptide libraries.

Compound 16 was found very potent anticancer peptide during the screening of peptide

library against DoHH2 (IC50 4.4 µM) and MCF-7 (IC50 1.1 µM) cell lines, while

compounds 2 and 15 were found active against NCI-H460 (IC50 62.6 µM and IC50 41.9

µM) respectively. Compound 15 also showed some activity against HeLa cell lines (IC50

92.4 µM). Compound 14 was found active against MCF-7 with IC50 29.6 µM. All

compounds were found non-cytotoxic to 3T3 normal cell lines.

In the second part of this study, a small library was synthesized around a naturally

occurring cyclic peptide stylissatin A. stylissatin A is reported as a natural inhibitor of

nitric oxide. The solid- phase total synthesis of this natural product was also accomplished

by our research group. The current study was focused on the synthesis, structure

elucidation, and SAR study of a small library of stylissatin A. All compounds from this

library were tested against different cancer cell lines but all of them are found inactive.

iv

These all compounds were tested for immunomodulatory activity. The Synthetic analogue

27 inhibited ROS on whole blood, as well as neutrophils. Related analogues of stylissatin

A were identified as more potent inhibitors of interleukin 2 than peptide stylissatin A.

Potent Anti-cancer Cyclic Peptide 16

MCF-7 (IC50 = 1.1 ± 0.07 uM)

DoHH2 (IC50 = 4.4 ± 0.06 uM)

v

SAR Studies of Stylissatin A

vi

خالصہ

حلقئ پیپٹائیڈز اپنی ساختی خصوصیات کی بنیاد پر بہترین طبی کارکردگی کے حامل ہیں اور اب

تک کئی حلقئ پیپٹائیڈز طبی استعمال کیلئے منظور کئے جا چکے ہیں۔

کئی ساختی اعتبار سے اہم حلقئ پیپٹائیڈز مختلف ترکیبی طریقہ کار کے تحت تیار کئے جا چکے

وں میں حیاتیاتی سرگرمی رکھنے والے حلقئ پیپٹائیڈز کی تالیف پر بہت زیادہ ہیں۔ گذشتہ چند سال

توجہ اور تحقیق کی گئی ہے۔

زیر مطالعہ تحقیقی مقالے میں حیاتیاتی سرگرمی کے حامل نادر مرکبات کی پہچان اور تالیفی

ہ کار کو تجربات پر بحث کی گئی ہے۔ ان حلقئ مرکبات کی تالیف کے دوران مختلف حلقوی طریق

استعمال کیا گیا ہے۔ یہ حلقئ پیپٹائیڈز کچھ غیر معمولی لحمی ترشوں کو بنیادی اکائی کے طور پر

Fmocاستعمال کرتے ہوئے بنائے گئے ہیں۔تمام پیپٹائیڈزکو ایک بڑے حلقے میں ڈھالنے کیلئے۔

پیپٹائیڈ تالیفی طریقہ کار استعمال کیا گیا ہے۔

یری کوچار مختلف کینسرسیل الئینزکے خالف ضد سرطانی سرگرمی ان حلقوی پیپٹائیڈز کی الئبر

پیپٹائیڈ الئبریری سے دریافت ہونے والے سب سے زیادہ ضد معلوم کرنے کیلئے جانچا گیا ہے۔

۔سرطانی صالحیت رکھنے والے مرکبات کا تفصیلی ساختی مطالعہ کیا گیاہے

DoHH2 سے زیادہ کینسر مدافعتی نے سب 16سیل الئینز کی جانچ کے دوران مرکب

سیل الئینز کے خالف بالترتیب )آئی NCI-H460 15اور 2سرگرمی ظاہر کی ہے۔ جبکہ مرکب

ملی مولر(کے مطابق سرگرم پائے گئے ہیں۔ 41.9 50ملی مولر(اور )آئی۔سی 62.6 50۔سی

HELAملی مولر( جبکہ مرکب 29.6 50سیل الئینز کے خالف )آئی۔سی MCF-7 14مرکب

پر غیر خلیہ (3T3)سیل الئینز کے خالف بھی سرگرم پایا گیا ہے۔ یہ تمام مرکبات عام خلیوں 15

پاش پائے گئے ہیں۔ اس تحقیقی مقالے کے دوسرے حصے میں قدرتی طور پر پائے جانے والے

کے مماثالت کی ایک چھوٹی الئبریری کی تالیف پر بحث کی گئی Stylissatin Aحلقئ پیپٹائیڈ

کی پیداوار کی مزاحمت کرتا ہے۔ زیر بحث مقالے (NO)ہ قدرتی پیپٹائیڈ نائٹریک اکسائیڈ ی ہے۔

میں اسکے مماثالت کی تالیف، ساختی جانچ اور ساختی وسرگرمی کے باہمی تعلقات کا مظاہرہ

کے خالف زیادہ سرگرم پائے گئے ہیں جبکہ تالیفی Inteeleukin-2کیا گیا ہے۔ یہ تمام مماثالت

کے خالف سب سے زیادہ سرگرم پایا گیا ہے۔ ROS 27 مماثل

vii

List of Figures

Figure No Title Page No.

Figure-1: Lead Discovery Process ................................................................................... 2

Figure-2: Role of Peptides in Different Possible Ways of Cancer Treatment ................. 5

Figure-3: Binding and killing mechanism of radio-labeled peptide ................................ 8

Figure-4: Schematic representation of the four possible ways for peptide

macrocyclization. ........................................................................................................... 23

Figure-5: Peptide Fragmentation Notation Roepstorrff and Fohlman ........................... 27

Figure-6: Peptidyl-Prolyl conformation of Cis/Trans Isomers. ..................................... 28

Figure-7: Process of Inflammation ................................................................................ 35

Figure-8: The General Scheme for Solid-Phase Peptide Synthesis ............................... 40

Figure-9: Resin for SPPS ............................................................................................... 41

Figure-10: Wang Linker ................................................................................................ 42

Figure-11: Rink-amide Linker ....................................................................................... 43

Figure-12: Protecting Groups used in SPPS .................................................................. 43

Figure-13: Coupling Reagents used in SPPS ................................................................. 44

Figure-14: Diketopiperazine Formation ........................................................................ 46

Figure-15: Analytical HPLC Profile of Cyclic Peptide 2 .............................................. 52

Figure-16: Key HMBC Interaction of Cyclic Peptide 2 ................................................ 53

Figure-17: Key COSY Correlations of Cyclic Peptide 2 ............................................... 54

Figure-18: Key NOESY Correlation of Cyclic Peptide 2 .............................................. 54

Figure-19: Key HMBC and COSY Correlations of Cyclic Peptide 14 ......................... 60

Figure-20: Key HMBC Interactions of Cyclic Peptide 15............................................. 65

Figure-21: Key COSY Correlations of Cyclic Peptide 15 ............................................. 65


Figure-23: Analytical HPLC Profile of Cyclic Peptide 26 ............................................ 86


viii



Figure-27: Analytical HPLC Profile of Cyclic Peptide 27 ............................................ 93



Figure-30: Key NOESY correlations of Cyclic Peptide 27 ........................................... 96

Figure-31: Analytical HPLC Profile of Cyclic Peptide 28 .......................................... 100

Figure-32: Key HMBC Correlation of Cyclic Peptide 28 ........................................... 101

Figure-33: Key COSY Correlation of Cyclic Peptide 28 ............................................ 102

Figure-34: Key NOESY Correlation of Cyclic Peptide 28 .......................................... 103


Figure-36: Key HMBC Interactions of Cyclic Peptide 29........................................... 108

Figure-37: Key COSY Correlations of Cyclic Peptide 29 ........................................... 109

Figure-38: Key NOESY Correlation of Cyclic Peptide 29 .......................................... 110



Figure-41: Key COSY Correlation of Cyclic Peptide 30 ............................................ 116

Figure-42: Key NOESY Correlations of Cyclic Peptide 30 ........................................ 117

Figure-43: HPLC Profile of Cyclic Peptide 31 ............................................................ 121


Figure-45: Key COSY Correlations of Cyclic Peptide 31 ........................................... 123

Figure-46: Key NOESY Correlations of Cyclic Peptide 31 ........................................ 123

ix

List of Tables

Table No. Title Page No.

1(a) Cyclic Peptide Library containing Unusual and Cationic

Residues.

48

1(b) Cyclic Peptide Library Based on Stylissatin A 49

2 NMR Data of Cyclic Peptide 2 55




6 Cytotoxicity of New Cyclic Peptide 81

7 Alanine Substituted Analogues of Stylissatin A 83

8 NMR Spectroscopic Data of Cyclic Peptide 26 91






14 Mass Spectroscopic Analysis and [α]D of Peptides 26-31. 126

15 Effect of Peptides 26-31 on Oxidative Burst, Nitric Oxide

(NO.) and IL-2 Production.

126

x

List of Schemes

Scheme No. Caption Page No.

1 Kenner’s Safety-catch Principle 42

2 Racemization during Peptide Bond Formation in SPPS 45

3 Synthesis of Cyclic Peptide 2 51




7 Solid-Phase Synthesis of Cyclic Peptides (26-31) 84







CHAPTER-1

INTRODUCTION

1

1.1. Drug Discovery and Development:

The drug discovery process is initiated from the search of new lead molecule either from

natural sources or by high-throughput screening of synthetic libraries. The new lead

molecules can be further developed as new drug for the requisite therapeutic effect

(Panchagnula & Thomas, 2000). Drug discovery is a time consuming and laborious

process which comprises of different phases such as disease target identification, target

validation, high-throughput identification of hits and leads, lead optimization, preclinical

and clinical evaluation (Gombar, Silver, & Zhao, 2003). From high throughput screening

or from the primary screening of the natural molecules, thousands of compounds may be

potential candidates for development as a new drug, but most of them are filtered out at

next stages of development and only a small number of compounds successfully go

through development stages for new drug.

In development process, several experiments are conducted to evaluate the efficacy along

with safety/ toxicity of new candidate molecules. The efficient and safest (least toxic or

non-toxic) molecule is further tested for the absorbency, distribution, metabolism and

excretion in the body. Figure-1represents the stages of drug discovery and development

process, this whole process requires a huge investment of money and time. It is estimated

that out of ten candidate molecule, only one enters into clinical development, which costs

approximately 10 – 15 years in time domain and US $ 500-800 million from pre-clinical

stage to marketing (Pastan, Hassan, FitzGerald, & Kreitman, 2006).

1.1.1. The Lead Discovery Process:

The molecule which exhibit desired and reproducible activity against disease in a

compound screening is called the “Lead” or “Hit” compound. A variety of screening

procedures exist to identify hit molecules. There are several procedures that can provide

potential candidate to become the lead compound, such as, isolation of compounds from

the natural sources like plant, lower terrestrial or marine organisms, or from synthesis of

libraries of compounds by using computer aided drug design and/or combinatorial

synthesis (Panchagnula & Thomas, 2000). High throughput screening or in vitro screening

assays are used to identify “Hit” molecule from different sources.

2

When hits are identified, their optimization is the next step. Empirical and semi empirical

structure-activity relationship (SAR) studies are used to modify the structure in order to

improve the effectiveness and lessen the toxicity of molecule. However, good in vitro

activity does not guarantee a good in vivo activity; it depends on good bioavailability of

drug and a desirable duration of action.

When “Hit” compound passes through in vivo screening, optimization of ADMET

(Absorption, Distribution, Metabolism, Excretion and Toxicity) is also performed to

improve the degree of potency, which has already been achieved (Y. Yang, Adelstein, &

Kassis, 2009), (Hughes, Rees, Kalindjian, & Philpott, 2011), (Caldwell, Yan, Tang,

Dasgupta, & Hasting, 2009). In vivo screening involves testing on a suitable animal model

to evaluate its pharmacokinetic and toxico-kinetic profile. This whole process is a part of

pre-clinical studies in drug discovery and development. Once the candidate drug molecule

passes this phase, an investigation IND (Investigational New Drug) is filed to the drug

regulating authorities. After IND application approval, the clinical phase starts which

involves testing in human subjects. Although the small group of these therapies has

significant curative preclinical results for cancer, very fewer have progressed toward

commercialization (Wolinsky, Colson, & Grinstaff, 2012).

Figure-1: Lead Discovery Process

3

1.2. Peptides: An Attractive Source of New Lead Molecules

Due to recent advancement in the peptide-based lead discovery process, they are

considered as fascinating therapeutic agents. Peptides have acquired an important place in

the field of medicine and are being used individually or in conjugation with drugs or nano-

materials of all reported compositions. Over the years, peptides have been reported for

treatment of diabetes, cancer, congestive heart diseases and inflammation. Nearly, 60

peptide drugs have been marketed and results in an annual sale of more than $13 billion

(Thayer, 2011). Three out of four marketed peptide drugs are being used in the direct

treatment of cancer or in the episodes of treatment that is associated with certain tumors

(leuprolide, goserelin, and octreotide) and are generating global sales of more than $1

billion.

From 2000 till present, hundreds of peptide candidate entered in the clinic and pre-clinic

development, out of which mostly are used for the cancer detection and therapy (18%) and

metabolic disorders (17%) (Borghouts, Kunz, & Groner, 2005), (Reichert, Pechon, Tartat,

& Dunn, 2010).

Some anti-microbial peptides like, a few cathelicidin-derived AMPs, such as LL-37,

indolicidin (IN), bactenecin, BMAP-27, and β-defensin, can potentially inhibit LPS-

induced cellular cytokine and/or NO. release by binding directly to LPS or by blocking the

binding of LPS to LBP (Bowdish, Davidson, Scott, & Hancock, 2005), (Zughaier, Shafer,

& Stephens, 2005).

4

Peptide hybrids also can be a good choice for the inflammation therapy. Yi-Fan Liu et al

studied the effect of hybrid peptide and found that hybrid peptides have a potential to

inhibit the expression of LPS-induced pro-inflammatory cytokines and chemokines (Y.

Liu, Xia, Xu, & Wang, 2013).

At present, many anti-inflammatory peptides have been reported from various natural

sources and successfully synthesized. Cyclomarin A isolated from a culture of Streptomyce

showed cytotoxicity towards cancer cells but its anti-inflammatory properties were also

significant (Renner et al., 1999). Solomonamide A, a peptide, with a unique chemical

structure was isolated from the marine sponge Theonella swinhoei. It showed significant

reduction of inflammation in the carrageenan-induced paw edema model in rats. These two

compounds showed promising results as potential non-steroidal anti-inflammatory drugs.

5

Peptides can be used for the treatment of cancer in several different ways, including tumor

targeting agents that deliver cytotoxic drugs specifically to the tumor site, peptides

angiogenesis inhibitors, radionuclides, hormones, and vaccines (Aina, Sroka, Chen, &

Lam, 2002), (Meng et al., 2012), (X.-X. Zhang, Eden, & Chen, 2012).Figure-3 describes

the various plausible ways of treatment for cancer using peptides. Peptides have excellent

binding affinity with various receptors and are involved in several biochemical pathways

this is the reason that peptides are being used in diagnosis and as biomarkers in cancer

progression. There is continues progress in various other fields, such as development of

peptide-vaccine and peptide angiogenesis inhibitors. Several clinical trials are in progress

which will provide better options to millions of cancer patients (Lee et al., 2008).

Figure-2: Role of Peptides in Different Possible Ways of Cancer Treatment (Thundimadathil,

2012)

1.2.1. Peptide Hormones as Drugs:

For the first time, peptides were used for cancer therapy by Schally et al. He used LHRH

(luteinizing hormone-releasing hormone) agonists as prostate cancer therapy. A major

increase in different formulations for the LHRH agonists has been observed. The

mechanism of action of LHRH agonists is the down-regulation of the LHRH receptors in

the pituitary glands which results in the obstacle in release of follicle stimulating hormone

6

(FSH) and LH. Reduction in concentration of these hormones also reduces the associated

testosterone production. Ultimately, it helps in suggesting a new therapy of androgen

deficiency in prostate cancer patients.

There are other effective LHRH antagonists which are in clinical use nowadays. The first

LHRH antagonist available in market is cetrorelix (Debruyne, Bhat, & Garnick, 2006).

Many other potent LHRH antagonists such as abarelix and degarelix are now approved for

human use (Broqua et al., 2002), (D. Kwekkeboom, Krenning, & de Jong, 2000).

1.2.2. Peptide as Radionuclide Carrier:

Cancer diagnosis is equally important as its treatment. In the diagnosis of cancer, apart

from several other diagnostic tools, peptides are also implicated at this step. Specifically,

peptides associated with radionuclide can work as a diagnostic and as well as radio-

therapeutic agents. For instance, somatostatin hormone, which consists of fourteen amino

acid residues and present in delta (δ) cells of the pancreas, hypothalamic and other

7

gastrointestinal cells. It is normally employed in Peptide Receptor Radionuclide Therapy

(PRRT). In this therapy, radio-peptides or radio-labeled somatostatin analogues were

formed by the combination of a radionuclide and somatostatin analogue which finally

forms highly specialized molecules (D. J. Kwekkeboom et al., 2005), (Krenning et al.,

1999), (Esser et al., 2006), (Nicolas, Giovacchini, Müller-Brand, & Forrer, 2011),

(Grozinsky-Glasberg, Shimon, Korbonits, & Grossman, 2008).

This radio-labeled somatostatin analogue connects to tumor cells containing receptors for

somatostatin. Now the site of radioactivity or tumor cells connected with radio-labeled

somatostatin analogue can be detected using a radiation-measuring device ultimately clear

the sites of tumor cells in the body. Among many effective analogues of somatostatin,

[111In-DTPA]-octreotide (Octreoscan) is the first available radio-labeled analogue that is

used in the diagnosis of sst-positive NETs (Somatostatin positive Neuro-Endocrine

Tumors) (Virgolini et al., 2002), (Strowski & Blake, 2008). After binding with the tumor

cells, radiations are emitted from the radio-peptides which kill the tumor cells. On the

other hand, 111In-coupled peptides have not shown efficiency against PRRT; because after

emission, Auger electrons travelled very short distance. Hence, for effective tumoricidal

behavior of 111In, decay of the nuclei has to take place close to the tumor cells (Krenning et

al., 1999), (D. J. Kwekkeboom et al., 2005), (Saltz et al., 1993). At this time, five sub-

types of somatostatin receptor are identified (sst1-5) (Van de Wiele et al., 2008). Their

density is much higher in comparison to non-tumor tissues. Hence, radio-labeled

somatostatin analogues are commonly used for supply of radioactive somatostatin. In

8

recent years, few other receptors like gastrin-releasing peptide/bombesin (GRP) and

cholecystokinin (CCK) have been involved in tumor imaging and PRRT (H. Zhang et al.,

2004), (Ginj et al., 2006). Radio-labeled receptor antagonists are also developing as

substitutes in this field (Wild et al., 2011), (Henderson, Mossman, Nairn, & Cheever,

2005).

Figure-3: Binding and killing mechanism of radio-labeled peptide.(Thundimadathil, 2012)

1.2.3. Peptide as Vaccines:

Like many other diseases, cancer could also be treated by active immunization. Many

studies have been conducted on immune cells related to tumor or immune molecule.

(Berzofsky, Ahlers, & Belyakov, 2001), (Hareuveni et al., 1990). The host immune system

(T-cells) can easily recognized Tumor-associated antigens (TAAs) which are expressed by

tumor cell (Coulie, Hanagiri, & Takenoyama, 2001), (Eisenbach, Bar-Haim, & El-Shami,

2000). These TAAs can work through the procedure known as active immunotherapy or

vaccination. In this process the host’s immune system is either activated de novo or re-

stimulated to mount an effective, tumor-specific immune reaction that may ultimately lead

9

to tumor regression. The TAAs is injected into the patient’s body which induces a systemic

immune response that eradicates cancer cells from the body tissues. Tumor antigen can be

protein or peptide in nature that has abnormal structure due to mutation of the concerned

gene (Parmiani et al., 2002), (Beck et al., 2007), (Knutson, Schiffman, & Disis, 2001).

Several peptide vaccines have cleared phase I/II/III clinical trials and exhibited excellent

results in immunological as well as clinical responses. These peptide based vaccines

include HER-2 / new immune dominant peptide tested for breast, lung, or ovarian tumor

(Sole, 2006), (Hueman et al., 2005), (Ramanathan et al., 2005), mucin-1 peptide (MUC-1,

stimuvax), (breast or colon cancer) (YAMAMOTO et al., 2005), (J. Wang et al., 2012),

carcino-embryonic antigen (colorectal, gastric, breast, pancreatic and non-small-cell lung

cancers) (Weber et al., 2011), (Grunnet & Sorensen, 2012), prostate-specific membrane

antigen (prostate cancer) (Garetto et al., 2009), (Akhtar, Pail, Saran, Tyrell, & Tagawa,

2011), (Muderspach et al., 2000), HPV-16 E7 peptide (cervical cancer) (Khleif et al.,

1999), rasonco protein peptide (colorectal and pancreatic carcinomas) (Gjertsen &

Gaudernack, 1998), (Abrams, Hand, Tsang, & Schlom, 1996), (Kaufman, 2012), and

melanoma antigens (Melanoma) (Markowicz et al., 2012), (J. C. Yang, 2011). GV-1001,

an injectable form of promiscuous MHC class II peptide, which is in phase III trial for

adenocarcinomas (Nava-Parada & Emens, 2007), (Reubi, 2007).

The use of peptide as vaccine is synthetically economical and does not have problem of

batch to batch variation, having definite structure that is why easy to manipulate.

Drawback of peptide vaccine is their low immunogenicity. Different strategies are being

employed to improve the immunogenicity and efficacy of peptide vaccines (YAMAMOTO

et al., 2005), (J. Wang et al., 2012), (Garetto et al., 2009).

1.2.4. Peptides in Targeted Drug Delivery

In traditional drug delivery system, drug releases instantaneously, that result in the peak

concentration at the start of the dose administration that is also toxic to the normal tissues.

Particularly in cancer, drug distribution is not so specific and can cause severe side effects

to the other tissue than the tumor. Matte et al first published the delivery of ligand-directed

10

drug to leukemic cells. Since then many successful targeting systems have been studied

that effectively directed drugs to the specific site of action.

Peptides are also a better option for transferring drug to the targets. Peptide-drug

conjugates can be easily prepared and can carry the drug to the specific receptors. Several

peptides; known as cell targeting peptides have the ability to target cells expressing their

receptors. Studies showed that many peptide receptors can be excellent targets in cancer

therapy (Van de Wiele et al., 2008), (Wild et al., 2011), (Reubi, 2007), (Gotthardt et al.,

2006).

A number of researches have been conducted on the cancer treatment through peptides.

During recent years, several targeting peptides are discovered through phage display

technique that selectively binds to various diseased tissues. The RGD and NGR motif are

the first generation of targeting peptides. Studies demonstrate that these peptides bind to

the tumor, independent of its type, which indicates the up-regulation of receptors of these

peptides during angiogenesis (Zitzmann, Ehemann, & Schwab, 2002).

A model study indicates that RGD peptide not only identifies angiogenic vessels in general

but also has good affinity towards the αv integrin receptors in the angiogenic vasculature.

The NGR peptide was first recognized as a cell adhesion motif by an in vivo screening on

human breast carcinoma xenografts and specifically target angiogenic blood vessels

(Temming, Schiffelers, Molema, & Kok, 2005), (Burg, Pasqualini, Arap, Ruoslahti, &

Stallcup, 1999), (Porkka, Laakkonen, Hoffman, Bernasconi, & Ruoslahti, 2002). Arap et

al. reported in vivo effects of neo-vasculature targeting peptides RGD-4C and NGR

conjugated with doxorubicin in a mouse xenograft (Arap, Pasqualini, & Ruoslahti, 1998).

Ellerby et al. reported that hybrid peptides composed of D-amino acid pro-apoptotic

peptide, NGR motif and doxorubicin selectively target and destroy angiogenic endothelial

cells in mice (Ellerby et al., 1999). Chen et. al. reported tumor retardation by RGD-peptide

coupled to tachyplesin (Chen et al., 2001). Various chemotherapeutic drugs were also

studied in combination with TNF-NGR peptide and it was found that targeted delivery of

low doses of NGR-TNF-α to tumor vasculature increased the efficacy of various drugs

acting via different mechanisms (Curnis et al., 2000), (Curnis et al., 2002).

11

1.2.5. Anti-Tumor Peptides:

A large number of peptides having anti-tumor activity are reported to date. These peptides

work through a variety of mechanisms to eliminate or restrict the tumor, such as by

angiogenesis inhibition, protein-protein interactions, signal transduction pathways, or gene

expression (V Rosca et al., 2011), (Karagiannis & Popel, 2008), (Kritzer, Stephens,

Guarracino, Reznik, & Schepartz, 2005), (Mochly-Rosen & Qvit, 2009). Peptide

antagonists also behave as antitumor peptides and preferentially bind to a known receptor

(Cornelio, Roesler, & Schwartsmann, 2007), (Sotomayor et al., 2010). “Pro-apoptotic”

peptides induce apoptosis in tumors (Ellerby et al., 1999), (Smolarczyk et al., 2005),

(Walensky et al., 2004). Angiogenesis is the process which involves growth, migration and

differentiation of endothelial cells of blood vessels. A number of studies are conducted to

discover peptide based angiogenesis inhibitors for the safest treatment of cancer (V Rosca

et al., 2011). Recently it is reported that angiotensin-(1–7) inhibit growth of bronchogenic

carcinoma in mice by restricting blood vessel development (Soto-Pantoja, Menon,

Gallagher, & Tallant, 2009). An RGD peptide derivative cilengitide is found selective for

αv integrins and behaves as an anti-angiogenic agent (Alghisi, Ponsonnet, & Rüegg, 2009),

(Hariharan et al., 2007), (Reardon et al., 2008), (K. Park et al., 2008). This derivative is

now in phase II clinical study for the therapy of glioblastoma and refractory brain tumors

in children.

1.2.6. Cell Penetrating Peptide:

Efficient passage through the cellular plasma membrane remains a major hurdle for some

drugs, particularly molecules that are large, ionized or highly bound to plasma protein

(Peck & Hill, 2014). Prochiantz et al. proposed the concept of cell penetrating peptide

(CPP) which can go across the cellular boundary and facilitates drug internalization

(Derossi, Joliot, Chassaing, & Prochiantz, 1994). These peptides deliver various high

molecular weight cargos across plasma membrane (Schwarze & Dowdy, 2000). Although

the general mechanism of CPP uptake is not clearly understood but there is consensus that

CPP interact with plasma membrane through electrostatic interaction with proteoglycans.

The mechanism of interaction and deliverance not only depends on structure of the CPP

12

but nature of cargo and its concentration also play a key role (El-Andaloussi, Holm, &

Langel, 2005).

Endosomal escape is being considered now a days as efficient pathway for CPP-cargo

conjugates internalization (Wadia, Stan, & Dowdy, 2004), (Magzoub, Pramanik, &

Gräslund, 2005). CPP can conjugate with cargo through two main strategies: through

covalent linkage with cargo or by forming non-covalent stable complexes. CPP conjugated

to nano carriers or cargos have been extensively studied, this method appeared very

advantageous in in vivo testing. This procedure is reproducible, rationalized and

stoichiometry of CPP-cargo can be controlled. (Torchilin, 2008), (Mäe & Langel, 2006),

(Gupta, Levchenko, & Torchilin, 2005). However, alteration in the chemical activity of the

cargo is limited as it can alter the biological response of cargo (Juliano, Alam, Dixit, &

Kang, 2008).

Short amphiphilic peptide carriers that carries both hydrophobic and hydrophilic

characteristics usually works through non-covalent strategy. Primary or secondary

structure of these short peptides gives them an amphiphilic character (Deshayes, Morris,

Divita, & Heitz, 2005). The hydrophobic domain forms complex with hydrophobic cargos

and used for membrane anchoring, while the hydrophilic domain forms complex with

hydrophilic negatively charged molecules. Mostly the amphiphilic peptide folds to α

helical structure and the hydrophilic and hydrophobic domains arranges on one side of α

helix. The non-covalent strategy was initially designed for gene delivery. Peptide of HA2

subunit of influenza hemaglutinin (Lear & DeGrado, 1987), (Parente, Nadasdi, Subbarao,

& Szoka Jr, 1990) and histidine-rich peptides (Kichler, Mason, & Bechinger, 2006),

(Midoux, Kichler, Boutin, Maurizot, & Monsigny, 1998), (Kichler, Leborgne, März,

Danos, & Bechinger, 2003) have been described as potent gene delivery systems

1.2.7. Anti-inflammatory Peptides:

Peptides always appear as a valuable solution for several disease treatments. Cyclic

peptides have an extra advantage of fixed conformation compared to linear peptides.

Several cyclic peptides are being used as drug for different diseases. Different classes of

13

cyclic peptides are reported to behave as potential anti-inflammatory agents. Some of them

are discussed below.

1.2.7.1. Duanbanhuains (A–C):

Brachystemma calycinum, is a plant used as folk medicine in China to treat diseases related

to inflammatory conditions. Its roots contain three anti-inflammatory cyclic peptides called

duanbanhuains (A–C). These peptides showed inhibition of MCP-1, IL-6, collagen IV and

reactive oxygen species (ROS) against high-glucose-stimulated cells and was non-toxic.

Duanbanhuain A and B were more potent against the production of IL-6, collagen IV and

ROS at the concentration of 10 µM. while, duanbanhuain A showed inhibition against

collagen IV 1 µM (Cheng, Zhou, Yan, Chen, & Hou, 2011).

1.2.7.2. Cyclomarins:

Cyclomarins (A-C) were isolated from cultured marine actinomycetes. Among the three,

cyclomarin A, was found as major metabolite and found as potent anti-inflammatory

compound in the phorbol ester (PMA)-induced mouse ear edema assay. Structurally, this

compound is attractive due to the presence of four unique amino acids which are 5-

hydroxyleucin, β-methoxyphenylalanine, tert-prenylated β-hydroxytryptophan and 2-

amino-3,5-dimethyl-4-hexenoic acid. This compound is also effective against

Mycobacterium tuberculosis and Plasmodium falciparum (Renner et al., 1999), (Schmitt et

al., 2011).

14

1.2.7.3. Cyclosquamosin:

Cyclosquamosin is the family of cyclic peptide obtained from the seeds of Annona

squamosa. After the isolation of first peptide annosquamosin A, a number of cyclic peptide

such as cyclosquamosin A and B, cyclosquamosin H and I, squamin A and B,

cyclosquamosin D and E, and cherimolacyclopeptide B have been isolated from this plant.

Cyclosquamosin D appears as a major cyclic peptide constituent among all the cyclic

peptides isolated from the plant. It behaves as inhibitor of TNFα and IL-6 production in a

dose dependent manner (Y.-l. Yang et al., 2007). The first total synthesis of

cyclosquamosin D and parallel synthesis of its analogues was reported by Dellai et. al. It

was found that its analogues effectively suppressed IL-6 secretion but had no effect on

TNFα (Dellai et al., 2010).

15

1.2.7.4. Anti-inflammatory Peptides from Marine Sponges:

Marine organisms are the most important source of anti-inflammatory cyclic peptide.

Many peptides have been isolated from different marine sponges like, perthamide C and

perthamide D of the genus Theonella swinhoei (Festa et al., 2009). Solomonamides A and

B are effective anti-inflammatory cyclic peptides isolated from the same genus.

Stylissa is another genus of the marine sponges that is a source of various bioactive cyclic

peptides. To-date, there have been 13 cyclic peptides reported from sponges in this genus.

These include: stylissamides A - H, isolated from S. caribica (X. Wang, Morinaka, &

Molinski, 2014), (Schmidt, Grube, & Köck, 2007), (Cychon & Ko ck, 2010), stylissamide

X from Stylissasp (Arai, Yamano, Fujita, Setiawan, & Kobayashi, 2012), stylisins 1 and 2

from S. caribica (Mohammed, Peng, Kelly, & Hamann, 2006), phakellistatin 13 from S.

16

caribica (Mohammed et al., 2006) and stylissatin A from S. massa (Kita, Gise, Kawamura,

& Kigoshi, 2013). Interestingly, all of the peptides isolated are heptapeptide except

stylissamide X which is an octapeptide.

Stylissatin A, a heptapeptide was isolated from the Papua New Guinean marine sponge

Stylissa massa. Stylissatin A exhibited inhibitory effect on nitric oxide production in LPS-

stimulated murine macrophage RAW264.7 cells with an IC50 value of 87 µM (Kita et al.,

2013). Total synthesis of this peptide was also reported earlier by the combination of

solution and solid-phase synthesis (Akindele, Gise, Sunaba, Kita, & Kigoshi, 2015). Its

first total solid-phase synthesis was also reported by our research group (Shaheen et al.,

2016).

1.3. Cyclic Peptides:

Cyclic peptides represent a large privileged and yet under exploited category of molecules

for drug discovery. These macrocycles forms a chemical space where the biological

expertise collaborates with chemical approaches in search of leads against different targets.

Cyclic peptides are well known for broad spectrum of biological activities. Structurally,

polypeptides chain adopts cyclic form by connecting one terminal of the peptide with

another through amide bond or any other chemically stable bond such as lactone, thioether,

ether, and disulfide and so on. Many biologically active cyclic peptides are formed by

head-to-tail cyclization. The group of cyclic peptide is continuously growing and

thousands of cyclic peptide have been isolated or synthesized and many of them are used

medicinally. For example tyrocidine, gramicidin, vancomycin, polymixin and colistin are

used as antibiotics, cyclosporin A is an immunosuppressive agent, and octreotide is used in

radio therapy, calcitonin in hypercalcemia and osteoporosis, nisin as food preservatives.

Peptide cyclization has many advantages; peptide molecule attains fixed geometry by

cyclization which helps them to bind with their targets more efficiently. Even if the

receptors have several subtypes; it is believed that definite conformation of these peptides

will be selective to particular receptor subtypes (Roxin & Zheng, 2012). Cyclic peptides

can adopt limited molecular conformation in solution this is the reason; when these

molecules are used as ligands to target disease biomarkers, their limited conformations

17

allow effective binding to the different isoforms or subtypes of specific receptors (Deem &

Bader, 1996).

Cyclic peptides attain receptor selectivity and enhanced binding by losing entropy when

they interact with the target which results in the reduction of free energy of target-ligand

complex. Cyclic peptides lack both free carboxyl and amino terminal which makes them

less susceptible to hydrolysis by exo as well as endo-peptidases (Liskamp, Rijkers, &

Bakker, 2008).

The main goal of cyclization is to induce structural constrain in the peptide chain but the

site of cyclization greatly affect the binding affinity. Kumar et al. investigated a linear

sequence Ac-CIYKYY and reported the comparison of 20 linear peptides in which side

chain of amino acid has been modified and 11 cyclic analogues of original sequence (A.

Kumar et al., 2006).

The cyclization was performed by using different strategies such as head to tail cyclization,

head to side chain cyclization and side chain to side chain cyclization. The results showed

that the head to tail cyclized peptide was 62.5 times more effective than the parent linear

peptide. This suggests that mode of cyclization and the orientation of the cyclized amide

bond greatly alter the biological response.

Cyclic peptides have structural features that allow the easy passage of molecule across the

plasma membrane. For example, presence of intramolecular hydrogen bonding keeps the

hydrophobic groups on the surface of the molecule and facilitates its transport across the

membrane (Mandal, Nasrolahi Shirazi, & Parang, 2011).

1.3.1. Stability of Cyclic Peptide:

In solution, small linear peptides exist in fast equilibrium of easily interconvertible

conformations. From these conformations very few adopts the orientation that matches the

receptors active site while some conformers also fit in the active site of the proteolytic

enzymes that cause their degradation. Cyclization was performed to minimize the number

of possible conformations that enhance the receptors selectivity and avoid the

susceptibility of degradation by proteolytic enzymes (Gilon, Halle, Chorev, Selincer, &

18

Byk, 1991). Generally cyclization increases stability of peptides also that directly prolong

their biological activity by resisting the enzymatic degradation (Liskamp et al., 2008).

Bogdanowich-Knipp et. al. studied the cyclo-(1,6)-Ac-CRGDF-Pen-NH2 (Pen = 8,8-

dimethylcysteine) peptide and its linear analogue NH2-RGDF-OH and found that the cyclic

form is 30 time more stable than linear in the solution of pH from 3 to 7. The disulfide

bond is more prone to reduction at high pH that ultimately facilitates the degradation of

cyclic RGD (Gilon et al., 1991). This result suggests that the disulfide bond has key role in

the stabilization of peptide.

Besser et al. studied the comparison between cyclization through disulfide bond and

cyclization through amide bond. This study was conducted on SRIF derivatives

Somatostatin-14, an endogenous disulfide-cyclized peptide with the sequence AG

(CKNFFWKTFTSC) and nine cyclized derivatives of somatostatin receptor-binding

octapeptide with the sequence cyclo (fFYwKV)FT-NH2 in which cyclization was done

through D-phenylalanine and valine residues but the orientation of cyclized bond is

different and extended linkers were also used (L. Yang et al., 1998). It is found that the

amide cyclized peptide was stable for 15 hours while the disulfide-cyclized peptide

hydrolyzed within 1 hour. This study showed that receptor binding affinity might improve

by disulfide cyclization but due to enzymatic reduction its use is limited. Both targets

stability and selectivity might achieve by amide bond cyclization (Besser et al., 2000).

1.3.2. Strategies to Develop Peptide Libraries.

Peptides are considered as attractive drug lead molecules due to several reasons, that is

why scientists are making continuous efforts to develop medicinally important compound

based on peptides structure. Peptides can be synthesized by two methods, Genetic or

Synthetic. In genetic method, peptide generation is limited to 20 ribosomal amino acids but

the sequence determination is straight forward. Synthetic method is more versatile, many

natural and unnatural amino acids can be incorporated into the cyclic peptide chain. Some

of the common methods that are used for peptide generation are described below.

19

1.3.2.1. Combinatorial Library Approach:

Combinatorial library synthesis is the simple, economical and versatile method for the

synthesis of peptide libraries. These libraries can be synthesized through biological or

chemical synthesis approaches. Biological method is generally limited to naturally

occurring amino acids while in synthetic approach; versatility is achieved by incorporating

D-amino acids, unnatural amino acids, specific secondary structure or scaffold that can

enhance the biological activity as well as small organic molecules and biological building

blocks such as monosaccharides. These combinatorial peptide libraries can be screened

against a variety of biological targets for the rapid discovery of ligands and substrate and

inhibitors (Pandeya & Thakkar, 2005). Since its discovery the combinatorial chemistry has

become a powerful tool for the drug discovery and molecular recognition. There are six

general procedures for the synthesis and screening of combinatorial libraries:

(i) The biological peptide library method (phage-display peptide library, bacterial peptide

display library system (Salmon et al., 1996), FliTrx, polysome library, (Jayawickreme et

al., 1999), (Silen et al., 1998) or plasmid peptide library (Aina et al., 2007).

(ii) The spatially addressable parallel library method (Frank, 1992).

(iii) Combinatorial library methods requiring deconvolution (Houghten et al., 1991),

(Brown, Wagner, & Geysen, 1995)

(iv) Affinity selection method (Songyang et al., 1995)

(v) One-bead-one-Compound (OBOC) combinatorial library method (Lam et al., 1991),

(Lam, Lebl, & Krchnák, 1997)

(vi) Self-assembled peptide nucleic acid (PNA) encoded chemical microarrays (Winssinger

et al., 2004).

1.3.2.2. Phage-Display Peptide Library Method:

Smith et al introduced phage display in 1985. In this method, each phage particle displays

unique peptide on its surface and hit molecules was decided on the basis of binding with

the target. Usually, peptides are displayed on the N-terminus, middle, or C-terminus of coat

proteins, and DNA sequence of the phage directs the peptide sequence this allows the easy

determination of sequence. Repeated screening called bio-pinning can be done until the

20

DNA molecules are preserved. Cyclic peptides having even number of cysteine units can

be synthesized (Koivunen et al., 1993). The phage particles are exposed to oxygen rich

periplasmic space of bacteria, which forced the two Cys units to form disulfide bond. 1 ×

108 to 1 × 109 different phages are routinely generated through this method. The first main

drawback is, only 20 natural amino acids can be incorporated in the library; secondly

complex bicyclic, branched structures, or molecules with special chemistry of cyclization

cannot be synthesized; and only binding study and some functional assays can be achieved.

1.3.2.3. Parallel Library Method:

Parallel peptide libraries are generated through solid- phase peptide synthesis by using

manual or automated high-throughput synthetic methods. The amino acid sequence of each

of these compounds are known (STRYER, t AMY, & SOLAS, 1991). This method forms

the basis of many other techniques such as SPOT synthesis, (Frank, 1992) multipin

technology, NanoKan, peptide microarray, multi-syringe system, and the 96 dee p-well

plate system. Screening of parallel peptide library can be done by direct solid-phase

binding assay or by a solution-phase releasable assay. The main disadvantage of this

method is that limited number of peptides can synthesized and therefore the library size is

rather small (Lam, 1997), (R. Liu & Lam, 2008).

1.3.2.4. Combinatorial Library Methods Requiring Deconvolution:

This strategy is based on the synthesis of mixture of compounds and their biological

screening. This synthetic approach is the basis of many other methods such as the iterative

method (Houghten et al., 1991), (Geysen, Rodda, & Mason, 1986) positional scanning

method (Dooley & Houghten, 1993), orthogonal partition method (Deprez et al., 1995),

recursive deconvolution method (Erb, Janda, & Brenner, 1994)and the dual recursive

deconvolution method. A large peptide library (1 × 106 to 1 ×108) can be generated and

screening can be done through many existing biological assays including functional assays.

This method is very suitable if target protein has only one predominant motif. Targets with

multiple binding motifs likely lead to scramble and un-interpretable results.

21

1.3.2.5. Affinity Selection Method:

This method is based on the affinity of ligand with receptors. Peptides from solution

mixture library are passed through affinity column with immobilized receptors that

separates ligands from library. This mixture library is usually synthesized by split-pool

method (Furka, SEBESTYÉN, ASGEDOM, & DIBÓ, 1991), (Lam et al., 1991),

(Houghten et al., 1991) The bounded ligands elute by extensive washing of column and

their structure is elucidated. This method has been successfully used for the synthesis of

combinatorial libraries of oligo deoxynucleotide. Libraries greater than 10000 peptides are

difficult to generate. Main concerns about this approach are nonspecific binding to

receptors and un-interpretable results are obtained if more than one predominant motif is

present in the mixture.

1.3.2.6. Self-Assembled PNA-Encoded Chemical Microarrays:

In this technique, split-pool method is used to generate the peptide library. This library is

cleaved from resin and an encoded solution phase library is formed in which each

compound is the PNA-encoded peptide or small molecule, the library compound is linked

to a PNA code via a hydrophilic linker (Winssinger et al., 2004). The library is then mixed

with the target protein and later exposed to a planar oligonucleotide microarray of

predetermined sequences. The identity of the positive library compound that interacts with

the target protein can be determined by knowing the nucleic acid sequences of the

oligonucleotide microarrays. Like the OBOC method, a whole cell binding assay can also

be applied to the encoded planar chemical microarrays.

1.3.2.7. OBOC Combinatorial Library Method:

One-bead-one-compound (OBOC) library is synthesized by using split pool method

through solid-phase-peptide synthesis. In this library, every single 80–100 μm bead

contains approximately 100 pmol of single chemical compound(Lam et al., 1991), (Lam et

al., 1997), this OBOC library is then subjected to high throughput screening. On bead

binding assays as well as releasable solution phase assays can be used(Lam, Liu,

22

Miyamoto, Lehman, & Tuscano, 2003).On-bead binding assays includes standard enzyme-

linked colorimetric assays, fluorescent assays, or radionuclide assays. Color probe can also

be used for the screening. The positive beads are identified by color change. OBOC

method has given very good outcome in whole-cell binding assay and successfully

employed in the identification of cell surface binding ligands. The procedure includes the

incubation of library beads with living cells; then the beads with monolayer of cells are

considered as positive beads, these are picked and characterized. OBOC library method has

also been useful in the identification of protease substrate and inhibitors (Meldal,

Svendsen, Breddam, & Auzanneau, 1994), (Meldal, 2002), (Olivos, Bachhawat‐Sikder, &

Kodadek, 2003), (Juskowiak, Stachel, Tivitmahaisoon, & Van Vranken, 2004). For this

assay highly porous resin were used for the peptide library synthesis so that enzyme can

access to the bead interior. OBOC library can also be screened by solution-phase assay. In

situ releasable approach is used in which library beads are immobilized on a thin layer of

agar the compound from the beads is released in the vicinity of bead in the semi-solid

matrix (Salmon et al., 1996), (Jayawickreme et al., 1999), (Silen et al., 1998). The OBOC

method is advantageous because a large number (1 × 106 to 1 × 108) of compounds can be

rapidly synthesized and screened. Secondly it does not require deconvolution and multiple

peptide ligands with completely different motifs can often be identified in a single screen.

Usually the library compound is linked to the solid support via linker that may cause steric

hindrance between library compound and cellular receptor, but mostly linkers play a role

of convenient handle that links the ligand to the therapeutic payload.

1.3.3. Cyclization Strategies for Synthetic Cyclic Peptides:

Cyclic peptides are a very valuable class in the modern drug development and a large

number of bioactive cyclic peptides are reported but there are some major challenges re-

main for the efficient synthesis of cyclic peptides. The peptide can be cyclized through

lactamization (Parenty, Moreau, & Campagne, 2006), lactonization (Lundquist IV &

Pelletier, 2002) or disulfide bridge formation (X.-Y. Wang, Wang, Huang, Wang, & Yu,

2006). Synthesis of some cyclic peptides are difficult and the success of the synthetic

approach often largely depends on the amino acid constituents of the cyclic peptide, the

23

specific site of ring-closure, the ring-closing strategy and the desired ring-size (Roxin &

Zheng, 2012). These issues have stimulated the development of novel efficient methods for

peptide cyclization. Various strategies have been reported for peptide cyclization,

depending on its functional groups, on the application of the cyclic peptides and/ or on the

desired technique for conjugation to other molecules, there are four different methods for

peptide cyclization: head-to-tail (C-terminus to N-terminus), head-to-side chain, side

chain-to-tail or side chain-to-side chain. Cyclization can take place via classical amide-

bond formation reactions (lactamization), disulfide-formation, or via the use of orthogonal

ligation methods (Horton, Bourne, & Smythe, 2000)

Figure-4: Schematic representation of the four possible ways for peptide macrocyclization.

Peptide macrocyclization reactions can be carried out in solution or on the solid support

and especially the field of solid-supported macrocyclizations has been actively explored in

recent years. The macrocyclizations on solid-supported is advantageous because the

standard washing and filtration procedures used in solid phase peptide synthesis are often

enough for purification. Macrocyclizations in solution are usually best performed in very

dilute conditions to minimize unwanted intermolecular reactions. Although these

conditions increase the selectivity of the reaction, they generally slow down the reaction

speed, thereby increasing reaction times and the risk on side product-formation. In

addition, macrocyclization in solution is usually performed on partially protected peptides,

and the solubility of these peptides is unpredictable and often poor (White & Yudin, 2011).

24

The interactions between protein binding sites often do not involve backbone-interactions

between large peptide- or protein-fragments, but mostly rely on certain contact residues

that are present in the epitopes. When cyclic peptides are used for mimicry of these

epitopes, proper positioning of the main contact residues is of great importance, and

peptide conformation is a crucial feature with respect to its biological activity. Therefore,

the site and method for macrocyclization must be carefully selected, since these factors can

strongly influence the peptide conformation

In addition, alternative approaches have been developed that make use of orthogonal

ligation methods. An important advantage of the use of orthogonal ligation methods over

amide-bond formation for macrocyclization reactions is that cyclization can be performed

on side chain-unprotected peptides to improve the solubility of peptides (Kimmerlin &

Seebach, 2005).

The demand for a detailed understanding of complex biological processes has stimulated

the development of highly advanced chemical tools, in order to improve the efficiency of

peptide cyclization. Ideally, peptide cyclization should be sequence independent, the

cyclization strategy should be applicable on both short and longer peptides and the strategy

should only require easily accessible functionalities or chemical moieties. A variety of

chemo-selective ligation methods have been described for this purpose.

Kent and co-workers first reported the chemo-selective ligation methods, like the thiol-

mediated intermolecular native chemical ligation (NCL) of peptide segments have been

heavily exploited for both existing and novel purposes (Shao, Lu, & Kent, 1998). The

feasibility of this method for peptide cyclization via intramolecular trans thio esterification

and ring contraction was first demonstrated by Tam and Zhang. This method for the

synthesis of head-to-tail cyclized peptides has also been extended to a solid-phase based

approach. An obvious limitation of NCL is the necessity of a cysteine residue at the site of

cyclisation. In answer to that, several research efforts have focused on circumventing this

requirement, resulting in a number of methods that has been reported for peptide

cyclization mediated through removable sulfur-containing auxiliaries (L. Zhang & Tam,

1997).

25

Another powerful method for the introduction of ring-shaped constraints is ring-closing

metathesis (RCM). The chemistry of RCM, relying on the action of ruthenium-based

catalysts, has been applied for the first time to rigidify amino acids and peptides by Grubbs

and co-workers (Miller, Blackwell, & Grubbs, 1996).

The copper catalyzed azide-alkyne cycloaddition as developed by Sharpless has effectively

been translated to the macrocyclization of peptides. In addition to the high efficiency and

regioselectivity of this reaction, this triazole-forming reaction appeared to be of special

value in the synthesis of the small cyclic peptides that are usually difficult to synthesize.

The conformational restrictions imposed by the resulting triazole-ring can positively

influence the formation of these small macrocycles (Rostovtsev, Green, Fokin, &

Sharpless, 2002).

Whereas above-mentioned methods for the synthesis of cyclic peptides make use of main

chain or (modified) side chain functionalities of linear peptide precursors or involve

removable auxiliaries, peptide cyclization can also be achieved using scaffold molecules.

A successful example of the use of a synthetic scaffold for conformational fixation of

peptides is CLIPS-technology (Chemical Linkage of Peptides onto Scaffolds).A first

example of this approach involves the immobilization of a dicysteine-containing linear

peptide to an α,α’-dibromoxylene-scaffold, resulting in a scaffolded cyclic peptide. After

the introduction of CLIPS-technology for the generation of single peptide loops, the

technology has been extended for the synthesis of multicyclic peptides towards the

generation of mimics of more complex protein binding sites(Horton et al., 2000).

1.3.4. Analysis of Cyclic Peptide through Mass Spectrometry:

Mass spectrometry has gained signification attention and much development has been done

since last three decades. Mass spectrometry is also considered as an effective tool for the

identification and characterization of biological macromolecules. With the use of different

ionization methods such as field desorption (FD) (Beckey, 2016), plasma desorption (PD)

(Tsarbopoulos et al., 1994) and fast atomic bombardment (FAB) mass spectrometers can

detect ~40kDa. Recently matrix assisted laser desorption ionization technique (MALDI)

has emerged as a promising tool for the fast and accurate determination of number of

26

biomolecules (Yergey et al., 2002). This technique has been employed for the detection

and characterization of several biopolymers such as protein, polysaccharides, peptides and

many organic macromolecules like dendrimers and other synthetic polymers.

Peptide sequencing through mass spectrometry is not that much easy task as it may seems,

cyclic peptides are even more difficult. The reason is during CID multiple ring opening

reactions occurs that make the interpretation difficult. Some amino acids direct the

pathway of fragmentation and generate specific fragmentation patterns that make the

identification easy. For example, proline generates a specific fragmentation pattern that

occurs at the peptidyl-prolyl (Xaa-Pro) amide bond, leading to a linear peptide C- ended by

an acylium ion (bn), which upon further fragmentation gives a series of acylium ions which

leads to the structure determination.

Peptides can be fragmented through the dissociation of three types of bond; CO-NH,CO-

CH and CH-NH, as a result of each dissociation one neutral and one charged specie is

formed but only positively charged species are detected (Figure-5). For each amino acid

six fragment ions are possible that can be divided into two categories. If the N- terminus

retained the charged the ions formed will be labeled as a, b and c ions and if the charge

retain on the C-terminus the x, y and z ions will be formed. The CO-NH bond is the most

labile bond in the peptide chain and gives rise to “b” ions with the formation of ammonium

ion and “y” ion with the formation of acylium ion. These ions give characteristic peaks and

their difference indicates the loss of particular amino acid residue from the peptide chain.

The peptide identification can be made simpler by MS/MS scan. Hence the mass

spectrometry is a valuable tool for sequencing all synthetic and natural peptides including

both cyclic and linear peptide.

27

Figure-5: Peptide Fragmentation Notation Roepstorrff and Fohlman (1984)

1.3.5. Conformational Analysis of Cyclic Peptide:

The structural features of proteins have been extensively studied for the better

understanding of their functions. Conformation of proteins is important structural feature

that play a key role in their binding to different receptors. For the conformational studies of

proteins cyclic peptides are often used as model (Takahashi, Hayano, & Suzuki, 1989).

Cyclic peptides can be both rigid and flexible and can adopt many stable conformations.

Different physical and chemical factors such as polarity of solvent, intermolecular forces

and the nature of the side chain of amino acids can effect these conformations. Extensive

studies have been done on the small and large cyclic peptide to determine their

conformational preferences.

Proline has a significant position in peptide conformation due to its cyclic structure. The

free energy barrier between cis/trans conformers is very small that cause rapid inter-

conversion of these isomers. These changes affect the whole protein or peptide

conformations that ultimately alter their function. The real mechanism of this cis/trans

isomerism is still under investigation, several theoretical studies have been conducted to

understand the phenomenon. Recently a study has been conducted that uses the empirical

energy function and ab-initio molecular orbital calculations. Molecular dynamics

simulation techniques have also been used to demonstrate proline isomerism.

28

Figure-6: Peptidyl-Prolyl conformation of Cis/Trans Isomers.

1.4. Importance of Cationic Cyclic Peptide:

Structurally diverse libraries of cyclic peptides are important sources of new medicinal

agents. Therefore, the development of diverse forms of new and novel compounds is an

important area of research in medicinal chemistry. Cationic Peptides (CPs) (also called as

amphiphilic peptides or antimicrobial peptides) have been discovered in nearly all species

from micro-organism to human being (Zasloff, 2002), (Lehrer, 2004), (Giuliani, Pirri, &

Nicoletto, 2007). Despite of having extensive variety in their sequences and structures,

these peptides share some common characteristics. The chain length of naturally occurring

CPs generally includes 12-50 amino acids having a net positive charge, as basic residues

especially lysine and arginine and sometime histidine are in excess as compared to acidic

residues and having around 50% hydrophobicity (Zasloff, 2002), (Hancock, 2001).

Most of naturally occurring cationic peptides contain two main types of cationic amino

acids, lysine and arginine, while most synthetic cationic peptides are lysine (K) rich

peptides. Very few synthetic arginine rich (R) peptides are reported except the RW-rich

peptides (Papo, Shahar, Eisenbach, & Shai, 2003), (Li et al., 2011).

Morris et al reported that, Pep-1; a Lys rich peptide consists of three domains: a

tryptophan-rich hydrophobic region, a flexible linker and a lysine-rich domain. In addition,

the ends of the peptide are capped by acetyl and cysteamide groups (Morris, Deshayes,

Heitz, & Divita, 2008). Pep-1 is considered as cell penetrating peptide and believed to

work by first forming a complex with its cargo through electrostatic and/or hydrophobic

29

interactions and delivers it directly into the cytoplasm, even at 4 °C, where energy-

dependent endocytosis. However, endocytosis is the predominant transport mode at 37 °C.

Pep-1 induces pore-like defects in bilayers that are clearly visible in ionic current

measurements (Lein, Sgolastra, Tew, & Holden, 2015). Anjaneya.et al reported lysine rich

cyclotides isolated from Australasian violaceae, Malicytus chathamicus and M. latifolius.

Cyclotides are plant-derived disulfide-rich mini-proteins ranging from 28 to 37 amino

acids in size (Ravipati et al., 2015), (Craik, Daly, Bond, & Waine, 1999). Structurally these

are consists of head-to-tail cyclic backbone as part of their characteristic cyclic cystine

knot (CCK) that is formed by three disulfide bonds. This unique structural motif provokes

cyclotides with exceptional resistance to thermal, chemical, or enzymatic degradation

(Craik, Daly, & Waine, 2001), (Colgrave & Craik, 2004).These lysine rich cyclotides are

able to bind with the lipid bilayer, but showed very low binding affinity to neutral 1-

palmitoyl-2-oleoylphosphatidylcholine (POPC) and 1-palmitoyl-2-oleoylphosphatidyl

glycerol (POPG) bilayer compared to the negatively charged POPC/POPE/POPG

membrane. 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) improves the affinity

of lysine-rich cyclotides to the membrane. Binding of cationic peptides to lipid bilayer is

greatly affected by overall hydrophobicity, net charge, and location of charges (Henriques

et al., 2012), (Ravipati et al., 2015). The current study was focused on the synthesis of

diverse forms of cyclic peptide libraries containing various cationic, anionic residues and

unusual building blocks and structural motif of naturally occurring bioactive peptides to

identify new lead molecules against cancer and inflammation.

1.5. Cancer Targeting Strategies:

Cancer is not a disease it is rather a group of diseases characterized by the uncontrolled

growth and proliferation of abnormal cells from the site of origin to the distant organs that

may ultimately lead to death. Several factors are involved in the progression of cancer,

such as exposure to UV radiation, pollution smoking habits, Viruses, and an unhealthy

diet. Moreover many chronic infections like hepatitis B and C (liver cancer), human

papilloma (cervical and ano-genital cancers) and Helicobacter pylori (stomach cancer) also

convert into cancerous situation. It may also be caused by genetic mutations, hormones,

30

and immunological disorder etc. One or all of these factors may involve in the progression

of disease. Several therapies including surgery, hormone therapy, radiation, immune

therapy, chemotherapy and targeted therapy (administration of the drugs that specifically

inhibit cancer cell growth) are being used. Cancer that is caused by unhealthy life style,

overweight or obesity, physical inactivity, poor nutrition, by smoking and/or excess

alcohol consumption could be completely obstructed by abandon smoking or alcohol

intake (Petti, 2009).

Cancer can be detected earlier befor the appearance of symptoms which usually results in

less extensive treatment and better outcomes (Anand et al.). One of the common and major

approaches for the cancer is chemotherapy. This treatment is done by delivering cytotoxic

drugs to the site of cancer. This is mostly administered to the intermediate and late stages

cancers patients and can be provided with or without radiation and before or after surgical

resection. The drugs that are administered during chemotherapy are not targeted and

selective towards cancer cells. These drugs also accumulate in normal tissues and distrub

their function. For example, some unwanted effects such as hypersensitivity reactions,

myelo-suppression, and neurotoxicity has been reported against paclitaxel (PTX) a drug

which is used against lung, ovarian, and breast cancers (S. Kumar et al., 2010). Another

drug doxorubicin (DOX) that is being used in chemotherapy has also been described to

have cardio toxic side effects (Prados et al., 2012). Low aqueous solubility is another issue

associated with most of chemotherapeutic drugs that prevents intravenous administration.

Those drugs are then chemically modified as pro-drugs (e.g. Irinotecan) or formulated in a

surfactant containing solution. These strategies enhance the aqueous solubility but the

bioavailability is compromised and also leads to sensitization reaction and other secondary

side effects (Kakde, Jain, Shrivastava, Kakde, & Patil, 2011), (Gelderblom, Verweij,

Nooter, & Sparreboom, 2001).

31

The limitation of intravenous systemic chemotherapy is, it is not specifically targeted to

the tumor, not only it is difficult to achieve therapeutic levels of drug within or adjacent to

the tumor, significant concentrations of drug also accumulate in normal tissue, that cause

dose-limiting toxicity and severe side effects. Moreover, acquired chemo resistance is also

a limitation of chemotherapy (Thayer, 2011).

The treatment of cancer is expensive because either radiation therapy or chemotherapy or

both, has high material costs and require specialist personnel for delivery of therapy,

appropriate monitoring, and treatment of side effects. These treatments are time consuming

for both patients and personnel, requiring frequent visits over months of therapy.

To cut the long story short, drug resistance, bio-transformation, altered bio-distribution,

drug clearance and non-specific drug delivery collectively urge the search of new localized

anti-tumor drug lead or ligand molecule to improve the efficacy of the therapy.

The main goal of cancer therapy is to abolish cancer cells, while sparing normal tissues.

This can achieve only by the selective targeting of cancer cells at the site of malignancy.

32

Certain tumor can be distinguished by their specific gene expression patterns as numerous

genes are over expressed in tumors comparative to normal tissue and expressions of

membrane proteins produced specifically by tumor cells (Alizadeh, Ross, Perou, & Van De

Rijn, 2001), (Nielsen & Marks, 2000). Due to this over expression these proteins or genes

appeared as extremely useful markers for targeting cancer (Shadidi & Sioud, 2003).

Previously, number of active anti-tumor agents and methods has been employed for the

targeted drug delivery. Macromolecules such as protein ligands and monoclonal antibodies

(mAbs) have also been tested clinically as therapeutics for targeted delivery (Allen, 2002),

(Pastan et al., 2006), (Thorpe, 2004), but there is still two major limitations: due to their

large size these are not specifically delivered to tumors and passive diffusion across

endothelial cell membranes in capillaries is also difficult; and dose-limiting toxicity to the

liver and bone marrow due to nonspecific uptake by the liver and the reticuloendothelial

system (RES) (Aina et al., 2002), (Mori, 2004), (Reff, Hariharan, & Braslawsky, 2002).

Another approach is the polymer-based drug delivery depots. This method has been

studied to improve the systemic morbidities and unspecified tumor targeted treatments.

These exist in various forms to facilitate the drug delivery to the effected site for the long

period and in controlled manner. According to their mechanism of action and mode of

administration polymer based approach can be classify into two groups;

1) Systemic administration, 2) Controlled release drug delivery system.

Systemic administration includes polymer liposomes, nanoparticles and dendrimers.

Localization of these nano-meterial is difficult due to uptake of these by the

reticuloendothelial system. The controlled release drug delivery system includes various

form- factors such as drug-eluting films, rods, wafers, gels and particles that usually

implanted intra-tumorally or adjacent to the effected tissues. The polymer used in this

technique can be natural or synthetic. Natural polymer including polysaccharides (X. Liu,

Heng, Li, & Chan, 2006), (Abe et al., 2008), (Bouhadir, Alsberg, & Mooney, 2001),

dextran (Al-Ghananeem et al., 2009) and chitosan (Gerber et al., 2011), (Li et al., 2011),

(Ampollini et al., 2010), hyaluronic acid (Vassileva, Grant, De Souza, Allen, & Piquette-

Miller, 2007), polypeptides including albumin (Davidson et al., 1995), (Almond et al.,

33

2003), collagen (Kakinoki & Taguchi, 2007),elastin (McDaniel, Callahan, & Chilkoti,

2010),and gelatin (Konishi et al., 2003), (Stuart, Stokes, Jenkins, Trey, & Clouse, 1993).

1.6. Inflammation:

A vigilant defence system against attack and injury is vital for the survival of living

organisms. The innate immune system continuously surveys the body for the presence of

invaders. When tissue damage by an invasion of pathogenic organism is encountered, it

voluntarily sets in motion a discrete, localized inflammatory response to thwart most

pathogenic threat (K. Tracey, 2002). Inflammation is a normal host defence mechanism

that is initiated by a complex series of interactions involving several chemical mediators.

This defence response not only protects the host from infection and other insults but also

restores hemostasis at damaged or infected areas (Barrientos-Salcedo, Rico-Rosillo,

Giménez-Scherer, & Soriano-Correa, 2009). The magnitude of inflammatory response is

critical: insufficient responses are caused by immunodeficiency that leads to serious

infections; excessive responses also causes diseases such as Crohn’s disease, rheumatoid

arthritis, diabetes and many others, but generally it is well regulated, self-limiting and

resolves rapidly. This self-regulation involves the activation of negative feedback

mechanisms such as the secretion of anti-inflammatory cytokines, inhibition of pro-

inflammatory signaling cascades, shedding of receptors for inflammatory mediators, and

activation of regulatory cells (K. J. Tracey, 2002), (Calder, 2009).

Inflammation is classified into two categories: (1) acute and (2) chronic ones (Lawrence,

Willoughby, & Gilroy, 2002), (Ahmed, 2011). Acute inflammation is a body’s short-term

responses that result mostly in healing and repairing damaged tissues and cells. Chronic

inflammation, on the other hand, is a prolonged inflammatory response that can cause

damage to the tissues. The prolonged and persistent inflammation is uncontrolled and it is

associated with many chronic human diseases such as cancer, multiple sclerosis, arthritis,

and allergy. An uncontrolled inflammatory response can be harmful to the host either in an

acute setting, such as during an infection, or in a chronic context, such as arthritis

(Mantovani, Allavena, Sica, & Balkwill, 2008).

34

Inflammation includes a series of complex reactions that involves cytokines, enzymes,

growth factors, and nitric oxide (NO) produced by macrophage cells. The onset of

infections and inflammation leads to an increased level of enzymes and signal proteins in

the affected area. This biological response, triggered and promoted by cytokines, is a

protective mechanism where the injurious stimuli, pathogens, irritants, and infections will

be removed via phagocytosis to protect the cells or tissues and initiate the healing process

(Huang et al., 2013). During inflammation, cell signaling proteins known as cytokines play

an important role by controlling the host’s responses to infections and injuries as

inflammatory or anti-inflammatory reactions. Some cytokines promote or make infections

and diseases to escalate and are referred as pro-inflammatory cytokines. Other cytokines

control and reduce the level of inflammation and promote healing processes, and are

known as anti-inflammatory cytokines (Dinarello, 2000), (Opal & DePalo, 2000).

Almost all gram-negative bacteria have lipopolysaccharide (LPS; endotoxin) as the major

outer surface membrane component which acts as an extremely strong stimulator of

mononuclear phagocytes (monocytes and macrophages), which are part of the innate

immune system of various eukaryotic species ranging from insects to humans (P. Wang et

al., 2010). The activation mechanism of macrophages by LPS starts when LPS binds with

the LPS-binding protein (LBP), accelerating the binding of CD14, which is the primary

receptor for LPS and is expressed mainly on macrophages (Rosenfeld, Papo, & Shai,

2006).The LPS–LBP–CD14 complex initiates intracellular signaling by interacting with

the trans-membrane protein Toll-like receptor-4 (TLR-4), and triggers, activation of

nuclear factor kappa B (NF-κB). The interactions of the receptors results in transmitting

signals to nucleus where the activation of a selective set of genes takes place through

transcriptional and posttranscriptional mechanisms. For example, the activated NF-κB will

be detached from the inhibitory protein kappa B (IκB) and released into the cell nucleus

and binds onto the deoxyribonucleic acid (DNA). The attachment of NF-κB to the DNA

will induce enzymes such as inducible nitric oxide synthase (iNOS) andcyclooxygenase 2

(COX-2) to synthesize prostaglandin, thromboxane and nitric oxide, respectively as an

inflammatory response (S. Y. Park et al., 2011), (Tabas & Glass, 2013). During the onset

of inflammatory responses, cells also release pro-inflammatory cytokines to help the

35

process which sometimes results in chronic condition causing cell death and tissue

damages. To control this condition, the immune system will try to balance the

inflammation process by releasing anti-inflammatory cytokines that regulate and lower

inflammation level where the infections are cured and cells are repaired (Figure-7). After

the onset of inflammation, whether it is chronic or acute, cytokines play an important role.

For example, tumor necrosis factor α (TNF- α) and interleukin 1 (IL-1) are pro-

inflammatory cytokines. They cause inflammation, fever, tissue destruction, and

sometimes results in shock and death.

Figure-7: Process of Inflammation

On the other hand, IL-4, IL-10, and IL-13 are potent anti-inflammatory cytokines. Anti-

inflammatory cytokines have the ability to suppress and inhibit the synthesis of pro-

inflammatory cytokines resulting in tissue recovery and healing. Over the years, a lot of

research has been carried out to better understand and cure the diseases associated with

inflammation. Though inflammation is very critical for survival, over production of

inflammatory cytokines and mediators can cause infection and diseases. Many of the anti-

inflammatory drugs inhibit the synthesis of inflammatory mediators such as eicosanoids.

Eicosanoids are signaling molecules that play a vital role in an inflammatory response,

which are produced as a result of oxidation of arachidonic, eicosapentanoic, and dihomo-

gamma-linolenic acids (fatty acids containing 20 carbons). The phospholipase A2 (PLA2)

36

catalyzes the conversion of glycerol phospholipids from the cell membrane to arachidonic

acid, and their inhibition could potentially block the synthesis of all eicosanoids (Rosenfeld

et al., 2006). It is very important to identify anti-inflammatory drug candidates that could

selectively inhibit the activation and synthesis of enzymes and signaling molecules that

promote inflammation.

1.6.1. Mediators of Inflammation:

Usually, the clinically relevant drug targets for the treatment of inflammation are the

cytokines and interleukins. There are two types of cytokines, 1) pro-inflammatory

cytokines, which increases inflammation, e.g., IL-1, IL-2, IL-6, TNF-α, 2) anti-

inflammatory cytokines, which decrease inflammation and increase healing process, e.g.,

IL-3 and IL-4 (Sugano et al., 1991).

1.6.1.1. Role of Interlukin-1 (IL-1):

Interleukin-1(IL-1) is a proinflammatory, multifunctional cytokine, that regulates the

expression of more than 150 genes associated with inflammation and immunity(Fitzgerald

& Luke, 2000).It was first focused for its ability to cause fever, but later it was found to

perform various biological functions including neutrophil recruitment, lymphocyte

activation, induction of inflammatory mediators, induction of hepatic acute-phase proteins,

and up-regulated prostanoid synthesis(Mills & Dunne, 2009).

IL-1 is a member of the IL-F or sometimes called IL-1 family. At present 11 members of

this family are known, but the most prominent are the two forms of IL-1 i-e IL-1α and IL-

1β(Geddes, Magalhães, & Girardin, 2009). These two forms of the IL-1 are encoded by

distinct genes and both are translated as 31kDa molecules present in the cytoplasm and do

not have signal peptide. At the amino acid level, these both forms are only 23%

homologous but the tertiary structure of both forms consists of barrel like structure and 12

β-pleated sheets.The physiologically active form is IL-1α but the precursor form of IL-1β

is activated by intracellular cysteine protease caspase-1 (Murphy, Robert, & Kupper,

2000), (Mizutani, Black, & Kupper, 1991). IL-1α is an intracellular cytokine which

performs both as a cytokine and as a transcription factor while IL-1β performs a part in the

37

fundamental inflammatory response(Dinarello, 2006). Excessive production of IL-1β

caused the repeated episodes of fever and systemic inflammation in autoinflammatory

diseases such as familial cold autoinflammatory syndrome (FCAS), Muckle-Wells

syndrome (MWS), and gout (Masters, Simon, Aksentijevich, & Kastner, 2009).


IL-2 was originally known as T cell growth factor (TCGF). It is a 15 kDa glycoprotein. It

is mainly produced by activated T helper cells and acts as a growth factor/activator for T

cells, B cells and NK cells. It has a critical role in regulation of cellular and hormonal

chronic inflammatory responses. Binding of IL-2 to the IL-2 receptor on T lymphocytes

leads to cell proliferation, increased lymphokine secretion (IFN-γ, lymphotoxin, IL-4, IL-3,

IL-5, GM-CSF), and increases the expression of class II MHC molecules (Feghali &

Wright, 1997).


Interleukin-6 (IL-6) is a phosphorylated four-helical glycoprotein comprises of 184 amino

acids and a molecular weight of 26 kDa (Ataie-Kachoie, Pourgholami, & Morris, 2013). It

is a T-cell derived β cell stimulating factor. The cDNA of IL-6 was cloned in 1986 and it

belongs to a large cytokines family, sharing the four-helical protein topology (Rose-John,

2012).It is a pro inflammatory cytokine, mainly involved in inflammation by controlling

differentiation, migration, proliferation, and apoptosis of target cells. This is also involved

in regulation of immune system and plays role in multiple processes such as metabolism,

liver regeneration, embryonic development and memory consolidation. Increased levels of

IL-6 of malfunctioning of complex regulatory cytokine network may cause autoimmune or

chronic inflammatory diseases or neoplastic (Scheller, Garbers, & Rose-John, 2014),

(Alonzi et al., 1998), (Ohshima et al., 1998). Therefore, suppression or inhibition of IL-6

seems an attractive therapy for wide range of diseases (Heikkilä, Ebrahim, & Lawlor,

2008).

38

1.6.1.4. Role of tumor necrosis factor-alpha (TNF-α):

Tumour necrosis factor (TNF-α), a cytokine with a relative molecular mass of 17,000 (17

KDa), is produced by activated macrophages in response to pathogens and other injurious

stimuli, and is necessary mediator of local and systemic inflammation (K. J. Tracey et al.,

1986), (H. Wang et al., 1999). It is essential for the complete expression of inflammation

during invasion, and self-limiting inflammation is normally characterized by decreasing

TNF-α activity. Low amounts of TNF-α can contribute to host defence by limiting the

spread of pathogenic organisms into the blood stream, initiating coagulation to localize the

invader, and stimulating the growth of damaged tissues. The duration and magnitude of

TNF-α release is limited, its beneficial and protective activities predominate, and it is not

released systemically (K. Tracey, Vlassara, & Cerami, 1989).

1.6.1.5. Role of Nitric Oxide (NO.):

Nitric oxide is a short lived, gaseous free radical that acts as a signaling molecule and

regulates various physiological and pathophysiological responses in the human body that

includes circulation and blood pressure, platelet function, host defense, and

neurotransmission in central nervous system and in peripheral nerves (Korhonen, Lahti,

Kankaanranta, & Moilanen, 2005). During inflammation process NO. is the mediator of a

number of regulatory responses such as infection control, regulation of signaling cascades

and transcription factors, regulation of vascular responses, and regulation of leukocyte

rolling, migration, cytokine production, proliferation and apoptosis (Knowles & Moncada,

1994), (Marletta, 1994), (Alderton, Cooper, & Knowles, 2001).

Large amounts of "inflammatory NO." from myeloid cells are usually generated side by

side with large amounts of superoxide anion (O2-). These two can form peroxynitrite

(ONOO-) which mediates the cytotoxic effects of NO, such as DNA damage, LDL

oxidation, isoprostane formation, tyrosine nitration, inhibition of aconitase and

mitochondrial respiration (Channon & Guzik, 2002), (Guzik, West, Pillai, Taggart, &

Channon, 2002), (Ischiropoulos & Al-Mehdi, 1995). Nitric oxide is also involved in the

regulation of the release of hormones which have been shown to control inflammatory

39

process centrally, For example, inhibition of CRH-induced ACTH secretion and inhibition

of corticosterone secretion (Givalois, Li, & Pelletier, 2002). Nitric oxide may also regulate

mast cell functions that are considered to be a key player in the initiation of inflammatory

responses (Nathan, 2002). Nitric oxide may act like a double edged sword, on one hand it

may act as a mediator of inflammatory responses to mast cell derived histamine, on the

other hand NO was shown to inhibit mast cell activation, mediate inhibitory effects of IFN-

g, or inhibit vascular adhesion molecule expression thus limiting allergic inflammation.

1.6.1.6. Role of Reactive Oxygen Species:

Reactive oxygen species (ROS) production plays an important role in the modulation of

inflammatory reactions. Major ROS produced within the cell are superoxide anion,

hydrogen peroxide and hydroxyl radical (Salvemini, Ischiropoulos, & Cuzzocrea, 2003).

Extracellular release of large amounts of superoxide, produced as respiratory burst in

leukocytes, is an important mechanism of pathogen killing and also leads to endothelial

damage resulting in an increased vascular permeability as well as cell death (Tiidus, 1998).

These ROS production plays an important role in modulation or release of other mediators

of inflammation. They also increase chemokine and cytokine expression (Kimura et al.,

2003), (Brzozowski et al., 2003).

1.7. Solid-Phase Peptide Synthesis:

The solution-phase methodology of peptide synthesis was firstly introduced by Emil

Fischer but he encountered the major issue of carboxyl and amino group protection of the

two amino acids so that they undergo side reactions (Fischer & Fourneau, 1901). Then

carbobenzoxy group was introduced as a protecting agent (Bergmann & Zervas, 1932).

The mode of peptide synthesis was laborious and time consuming because each step

requires purification of the product.

R. B. Merrifield revolutionized the peptide synthesis in 1963 by giving the concept of solid

phase synthesis. In this technique, the peptide chain was tethered on a solid support and

amino acids were linked until the required sequence was obtained (Merrifield, 1963)

(Figure--6).

40

Figure-8: The General Scheme for Solid-Phase Peptide Synthesis

In solid-phase-synthesis, usually high equivalents of reagents are used to force the reaction

to completion. The excess reagents are just washed off in between the steps of synthesis

and the final cleaved product is carefully purified and characterized. The success of SPS is

incomplete without the advancement in purification and characterization techniques such

as HPLC, capillary electrophoresis, mass spectrometry, NMR spectrometry and automated

amino acid analysis.

1.7.1. Solid Supports for Peptide Synthesis:

Chemically, the solid support is an insoluble polymer having sufficient mechanical

stability desirable physiochemical properties. These physiochemical properties includes

constant shape and size, chemically inert to reaction conditions, according to desired

peptide sequence, suitable functionality should be present in the polymer (Miranda &

Alewood, 2000). The most commonly used solid supports in SPPS are resins. These are

small polymeric beads cross linked with polystyrene (Figure--22), polyacrylamide and

polyethylene glycol (PEG).

41

Figure-9: Resin for SPPS

1.7.2. Linkers used in Solid-phase Peptide Synthesis (SPPS):

Linker acts as a connecting medium between peptide and resin bead. Chemically linkers

should be stable against the reaction condition and make the attachment of the peptide with

resin easy. Some commonly used linkers are discussed below.

1.7.2.1. Kenner’s Safety-catch Linker

Safety-catch linker usually inert toward the reaction condition used for the chain

elongation until it is chemically activated for the cleavage. The use of this linker was

introduced by Kenner in 1971. He employed N- acylsulfonamide linkage for the

preparation of carboxylic acids and primary amides(Kenner, McDermott, & Sheppard,

1971). Utilization of this linker is associated with some major concerns that are poor

reactivity, racemization and poor loading efficiency. A large stoichiometric excess of

reagent have to use to overcome these issues (Backes, Virgilio, & Ellman, 1996).

Consequently more reactive linker was developed by replacing diazomethane with halo

acetonitrile (Backes & Ellman, 1999) (Scheme-5).

42

Scheme-1: Kenner’s Safety-catch Principle

1.7.2.2. Wang Linker:

The mostly used linker for solid phase peptide synthesis is the Wang linker (S.-S. Wang,

1973). This linker is used in the synthesis of acids, alcohols, phenols and peptide. Peptide

synthesis was performed by using the Fmoc / t-Bu protection strategy (Guillier, Orain, &

Bradley, 2000). The Wang linker (4-(hydroxymethyl) phenoxymethyl), is attached to a

resin through an aryl benzyl ether bond (Figure--8).

Figure-10: Wang Linker

1.7.2.3. Rink-Amide Linker:

A variety of supports are available which have rink amide linker (4-(2,4-dimethoxyphenyl)

(Fmoc-amino) methyl) phenoxy methyl) in both forms protected with Fmoc or

unprotected. This resin is used for the synthesis of peptide carboxamide (Atherton, Clive,

& Sheppard, 1975); (Albericio et al., 1990). These linkers are acid labile and release

43

peptides on treatment with 95 % trifluoroacetic acid (TFA). The exposure to high

concentration of TFA sometime results in the formation of side product in the last cleavage

step.

Figure-11: Rink-amide Linker

1.7.3. Protecting Groups Used in Solid Phase Peptide Synthesis:

There are two types of protecting groups that can be used in the synthesis i.e., temporary

and permanent (Figure--12). The “temporary” protecting group is used for the amine

functionality of amino acid. These protecting groups are removed after linking the amino

acid with the linker or with another amino acid. The side chain protection of tri-functional

amino acid is done with the permanent protecting groups. These groups are removed only

when the whole synthesis is complete.

Figure-12: Protecting Groups used in SPPS

44

1.7.4. Coupling Reagents:

Coupling agents are used in the synthesis for two reasons; firstly, in N terminal to C

terminal amino acid synthesis, it is necessary to activate the carboxylic group of amino

acid that further reacts with the amine part of the another amino acid residue. Coupling

agents does this job by converting the carboxylic acid in more reactive state. Secondly,

there are some side-reactions (such as racemization or Diketopiperazine formation)

occurring during the amide bond formation or sometimes incomplete coupling takes place.

Coupling agents are used to avoid the formation of such side products and to ensure the

complete coupling.

Different types of coupling reagents (Figure-13), are used, e.g., benzotriazol-1-yl-

oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), ethyl 2-cyano-2-

(hydroxyimino) acetate (Oxymapure), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-

uroniumhexafluorophosphate (HBTU), hydroxybenzotriazole (HOBT),

diisopropylcarbodiimide (DIC) and dicyclohexylcarbodiimide (DCC) etc. Oxymapure

showed clear superiority to HOBt / DIC or carbodiimide alone in terms of purity and yield

(El-Faham, Al Marhoon, Abdel-Megeed, & Albericio, 2013; STEINAUER, CHEN, &

LEO BENOITON, 1989).

Figure-13: Coupling Reagents used in SPPS

45

1.8.Side-reactions in Peptide Bond Formation:

1.8.1. Racemization:

Racemization is one of the main issues usually encountered during peptide synthesis. This

reaction results in the formation of aza-lactone (Scheme-2). This reaction can be suppress

by incorporation of urethane-type blocking groups, coupling agents that also suppress

racemization (such as oxyma pure) and auxiliary nucleophiles that minimize the chances of

racemization during peptide chain elongation (STEINAUER et al., 1989).

Scheme-2: Racemization during Peptide Bond Formation in SPPS

1.8.2. Synthesis of Diketopiperazine during Peptide Chain Elongation:

Diketopiperazine (DKP), a side product which results in the poor yield of final product.

Due to its formation during the cleavage process hydroxyl sites are generated on the

polymer which results in undesired side reactions (Giralt, Eritja, & Pedroso, 1981).

46

Figure-14: Diketopiperazine Formation

1.9.Targets of Current Study:

This study is designed by considering the emerging importance of peptides in cancer and

inflammatory diseases. We designed and synthesized diverse cyclic peptide library and

screened the compounds against different cancer cell lines which has led to discovery of

four cyclic peptides which were active against different cell lines.

It is very important to develop new anti-inflammatory agents that can meet the growing

need of effective anti-inflammatory drug. In the current study, we synthesized a series of

closely related analogues of anti-inflammatory natural product, stylissatin A. The

immunomodulatory effect of all analogues was studied and compared with the natural

stylissatin A in order to find the more potent and least toxic anti- inflammatory compound.

Chapter # 2

RESULTS

AND

DISCUSSION

47

2.1. Design, Synthesis and Screening of Cyclic Peptide Library:

The current study was focused on the identification of new and novel biologically active

compounds from randomly developed as well as focused libraries based on cyclic peptide

scaffold. A 25-member cyclic peptide library was synthesized using Fmoc-solid phase

chemistry to get collection of exceedingly diverse forms of cyclic peptides. In the synthesis

of diverse cyclic peptide library, some unusual building blocks such as phenyl glycine,

naphthyl alanine, 3-nitro tyrosine, 2-amino benzoic, D-phenylalanine, D-alanine residues

as well as cationic and anionic residues were used. The cyclic peptide library was screened

against different cancer cell lines, DoHH2, MCF-7, NCI-H460, HeLa and 3T3 cell lines

using MTT assay. From this library, four cyclic peptides 2, 14, 15, 16, were identified as

active compounds against different cancer cell lines. The detailed structural and biological

studies of 2, 14, 15 and 16 are presented in the following section 2.2.

In the second part of current study, a focused library around naturally occurring cyclic

peptide stylissatin A was synthesized. Stylissatin A is reported as a natural inhibitor of

nitric oxide. Its first solid phase total synthesis was also carried out by our research group.

The current study focused on the design, synthesis, structure elucidation and SAR study of

six member library of stylissatin A which has been discussed in this dissertation in section

2.3.

48

Table-1(a): Cyclic Peptide Library containing Unusual and Cationic Residues.

49

Table-1(b): Cyclic Peptide Library Based on Stylissatin A

50

2.2. Structural Studies of Active Peptides from Library I:

2.2.1. Cyclic Peptide 2:

The cyclic peptide 2 was designed to contain cationic residues like Lysine, Ornithine and

D-amino acid residues. Peptide 2 was synthesized on Ring amide AM resin by on-resin

macrocyclization strategy using standard Fmoc-peptide synthesis protocol. The first amino

acid phe (D- phenylalanine) was loaded onto the Rink amide resin by using the coupling

agent ethyl hydroxyiminocyanoacetate (oxyma pure) and diisopropyl carbodiimide (DIC).

The next residues Ile and phe were sequentially coupled followed by coupling with Fmoc-

Glu-Allyl-OH under standard Fmoc peptide synthesis conditions. After the removal of

Fmoc group of glutamic acid, the sequence of cyclic part of peptide 2 was constructed by

using two consecutive coupling with Fmoc-Lys (Boc)-OH followed by coupling with

Fmoc-Orn(Boc)-OH, Fmoc-phe-OH, and Fmoc-Lys(Alloc)-OH. Finally, palladium

catalyst was used to remove the protecting groups (allyl group of Glu and alloc group of

Lys residues) to allow head-to-side chain cyclization between -acid group of Glu and

amino group of Lys residue of peptide 2. TFA cocktail was used to cleave the peptide

from Rink resin (Scheme-3). The crude peptide product was purified by RP-HPLC using

the isocratic solvent system 60% ACN in water H2O. The pure peptide 2 was obtained as a

yellow solid material with an overall yield of 40% and [α]D25 +9.97 (c 0.00341, 60% ACN:

H2O). The high resolution electron spray ionization (HR-ESI) technique exhibited

51

molecular ion (M+H)+ at 1020.6325 (calc. 1020.6314) corresponding to molecular formula

C51H82 N13O9. IR spectrum on KBr disc showed absorptions at 3422.6, 3085.8, 2930.8,

1671.1, 1198.7 and 1133.3 cm-1.

Scheme-3: Synthesis of Cyclic Peptide 2

52

Figure-15: Analytical HPLC Profile of Cyclic Peptide 2

Conditions: C4 RP-analytical column/ 60% ACN: H2O

Deuterated solvent d6-DMSO was used for recording NMR spectra because it allowed the

clear detection of NH protons of peptide. All 1HNMR assignments were confirmed by 2D

NMR experiments. The phe1 residue exhibited clear signals for α proton (δH 4.41 / δC 54.1)

and CH2 group (δH 2.69, 3.08 / δC 37.2). The other prominent signals of phe1 were

observed for aromatic protons (δH 7.15 to δH 7.23), carbonyl carbon (δC172.2) and NH

proton (δH 8.40). The terminal free NH2 protons (δH7.42) were also observed. Val2 residue

exhibited signals for α proton, (δH 3.96 / δC 58.4), β CH (δH 1.68, δC 29.9), the gem-

dimethyl group (δH 0.47 / δC 18.9), (δH 0.57 / δC 18.2) and NH proton (δH 7.86). α and γ

acidic carbonyl groups of Glu3, exhibited signals at δC 171.0 and δC 171.1 respectively.

While, β CH2and γ CH2 groups (δH 1.49, 1.33, δC 26.7), (δH 2.58, 2.72, δC 38.0) and NH

proton (δH 8.15) of Glu3 were also observed. The signals for Lys4, Lys7 and Lys8 were

overlapped. The overlapping signals of CH proton of Lys4, Lys7 and Lys8 were observed

in the range of δH 4.20-4.23. For Orn5 residue, α CH (δH 4.41 / δC 54.1), CH2 groups (δH

1.51 / δC 30.5), (δH 1.49, 1.59 / δC 23.4), (δH 2.58, 2.73 / δC 38.8) and NH (δH 8.41) were

assigned. The aromatic residue phe6 exhibited signals for α proton (δH 4.71 / δC 54.1), β

CH2 (δH 2.64. 3.04 / δC 38.7) aromatic protons (δH 7.15- 7.23), NH proton (δH 8.31) and

carbonyl carbon (δC 173.3). All NMR assignments of peptide 2 were established by 2D

NMR techniques including HSQC, HMBC, COSY and NOESY interactions. The HMBC

showed very prominent interaction of α proton (δH 4.41) of phe1 with β CH2 (δC 37.2), C-1

(δC 138.2) and carbonyl carbon (δC 172.2). The terminal free NH2 protons (δH 7.42)

53

showed interaction with CO group of phe1, while the NH proton (δH 8.31) was found to

interact with α carbon (δC 54.1) and β carbon (δC 37.2) of phe1. α proton (δH 3.96) of Val2

residue exhibited interactions with β CH (δC 29.9), γ CH3 (δC 18.2), and carbonyl carbon

(δC 171.1), while NH proton showed interaction with α CH (δC 58.4), β CH (δC 29.9) of

Val2 and carbonyl carbon (δC 171.1) of the next amino acid i.e. Glu3. α proton of Glu3 (δH

4.23) showed the signals with β CH2 (δC 26.7) and carbonyl carbon (δC 171.1) while

interaction of γ protons (δH 2.58, 2.72) with β carbon was also observed. Similarly, α

proton (δH 4.41) of Orn5 showed correlation with β CH2 (δC 30.5), γ CH2 (δC 23.4), and NH

proton (δH 8.4) had interaction with α CH (δC 54.1), β CH2 (δC 30.5) and carbonyl carbon

(δC 171.0) of Orn5. The CH proton (δH 4.71) of phe6 was correlated with β CH2 (δC 38.7),

C-1’ (δC 137.7) of the phenyl ring and the carbonyl carbon (δC 173.3), while the correlation

of NH proton (δH 8.31) was observed with α CH (δC 53.5) and β CH2 (δC 38.7).

Figure-16: Key HMBC Interaction of Cyclic Peptide 2

The COSY spectrum showed the cross peaks among CH α of all residues with adjacent NH

protons. Thus α CH proton (δH 4.41) of phe1 was correlated to the β CH2 (δH 2.69, 3.08)

and NH (δH 8.32). α proton (δH 3.96) of Val2 exhibited the correlation with NH proton (δH

7.86), while β CH proton (δH 1.68) showed the cross peaks with gem-dimethyl (δH 0.47,

0.57). α proton (δH 4.23) of Glu3 was correlated with NH proton (δH 8.15), while β CH2

protons (δH 1.49, 1.33) were correlated with the γ CH2 protons (δH 2.58, 2.72). The NH

protons (δH 8.32) of the Lys4 and (δH 8.15) of Lys7 was correlated with their adjacent α

54

protons (δH 4.23), while, α proton (δH 4.41) of Orn5 showed interaction with NH proton (δH

8.40). The CH α proton (δH 4.71) of phe6 showed cross peaks with β CH2 (δH 2.68, 3.08)

and NH proton (δH 8.31).

Figure-17: Key COSY Correlations of Cyclic Peptide 2

The NOESY spectrum of cyclic peptide 2 showed the NH/NH interaction among residues,

Phe1/ Val2, Glu3/ Lys8, Lys4/ Orn5.

Figure-18: Key NOESY Correlation of Cyclic Peptide 2

55

Table-2: NMR Data of Peptide 2 (d6 DMSO, 600 MHz for 1H and 150 MHz for 13C)

57


The cyclic peptide 14 was synthesized on Ring amide AM resin by linking the side chain

of Fmoc-Glu-O-Allyl to Rink linker by using standard coupling procedure. After

successful loading of first residue glutamic acid, all amino acid were linked in a sequence

containing tyr, three units of Arg, Lys followed by the deprotection of both Glu α allyl and

alloc group of Lys side chain to produce a cyclic peptide 14 by head-to-side chain

cyclization on solid-phase (Scheme-4). The unusual amino acid Nal was linked to cyclic

structure through terminal amino acid Lys followed by its acylation with p-

methoxybenzoic acid. TFA cocktail was used to cleave the crude peptide from resin. The

crude peptide product was purified by RP-HPLC using the isocratic solvent system 60%

ACN in water. The pure peptide 14 was obtained as a yellow viscous material with an

overall yield of 41.9 %. [α]D25 + 5.85 (c 0.0022, 60% ACN: H2O). The high resolution

electron spray ionization (HR-ESI) technique exhibited molecular ion [M+2H]+2 at

610.3445 corresponding to molecular formula C59H84N18O11. IR spectrum on KBr disc

showed absorptions at 3348.8, 2932.6, 1662.7, 1543.1, 1444.6, 1253.0, 1179.2 and 1106.6

cm-1. Deuterated solvent d6-DMSO was used for recording NMR spectra because it

allowed the detection of NH protons of all six amino acid residues of peptide 14.

58


The 1HNMR showed the presence of α proton ranging between δH 4.01- 4.84. The Gln1

residue was recognized by signal of two methylene groups (δH 1.65, 1.82 / δC 27.4), (δH

2.79, 2.61 / δC 36.6), γ acidic carbonyl (δC 173.7) and NH proton (δH 8.10). Signals for tyr2

were prominent and identified for overlapping signals of H-2 / H-6 (δH 6.96 / δC 130.1) and

59

H-3 / H-5 (δH 6.62 / δC 114.9), the downfield resonance of hydroxyl proton (δH 9.18) along

with βCH2 (δH 2.92, 3.15 / δC 38.1), NH (δH 8.53) and CO (δC 172.1) groups. The 1HNMR

spectrum showed the overlapping signals of methylene groups of Arg3, Arg4, Arg5 and

Lys6 in the range of δH 1.42 to δH 3.12. However the NAL7 residue was fully characterized

by downfield signals of CH2 (δH 3.56, 3.66 / δC 34.0), while signals for naphthyl protons,

H-2ʹ (δH 7.73 / δC 126.9), H-3ʹ (δH 7.35 / δC 125.3), H-4ʹ (δH 7.49 / δC 127.2), H-5ʹ (δH 8.24 /

δC 123.7), H -6ʹ (δH 7.57 / δC 126.0), H-7ʹ (δH 7.85 / δC128.6), H-8ʹ (δH 7.49 / δC125.5) were

identified. While quaternary carbons C-9ʹ and C-10ʹ were found at δC134.3 and δC 131.6

respectively. The carbonyl carbon of Nal7 residue appeared at δC171.7, while amide proton

appeared at δH 8.60. p-Methoxy benzoic acid residue was identified by the sharp singlet of

methoxy group (δH 3.76 / δC 55.3) and aromatic protons (δH 7.71 / δC 129.2) H-2” / H-6”,

(δH 6.92 / δC 113.4) H-3” / H-5”, C-1” (δC 161.6) and C-4” (δC 156.9). The structure was

further confirmed by the HMBC (Figure-19).

The HMBC interactions of Arg3, Arg4, Arg5 and Lys6 residues of compound 14 were not

very clear due to extensive overlapping signals of these residues. The H-2 / H-6 (δH 6.96)

of tyr2 exhibited the interaction with β CH2 (δC 38.1) and C-4 (δC 155.7) of the ring. The

overlapping signals of H-3 and H-5 (δH 6.62) were correlated with the C-1 (δC 127.7) and

C-4 (δC 155.7) of the ring, while hydroxyl proton (δH 9.18) showed correlation with C-4

(δC 155.7) C-3 and C-5 (δC 114.9) of the phenyl ring. In case of NAL7 residue, the H-2’ (δH

7.73) and H-3’ (δH 7.35) exhibited the correlation with C-1’ (δC 127.7), C-9’ (δC 134.3) and

C-10’ (δC 131.6). While H-4’ (δH 7.49) was correlated with C-5’ (δC 123.7) and C-9’ (δH

134.3). H-5’ (δH 8.24) was correlated with C-4’ (δC 127.2), C-9’ (δH 134.3) and C-10’ (δC

131.6). H-6’ (δH 7.57) showed correlation with C-7’ (δC 128.6) and C-10’ (δC 131.6). H-7’

(δH 7.87) displayed correlation with C-9’ (δC 134.3) and C-10’ (δC 131.6). H-8 (δH 7.49)

was correlated to the β CH2 (δC 34.0) and with C-9’ (δC 134.3). The correlations for the H-

2” / H-6” (δH 7.71) of p-methoxybenzoic acid was observed with C-1” (δC 161.6) and

carbonyl carbon (δC 165.8) of the acid. H-3” and H-5” displayed the correlation with C-1”

(δC 161.6) and C-4” (δC 156.9), while the methoxy protons (δH 3.76) showed the

correlation with C-4” (δC 156.9) of the ring.

60

The clear COSY cross peaks were observed between α proton (δH 4.01) and NH proton

(δH8.10) of Gln1 residue. The CH proton in position (δH4.10) of tyr2 was correlated with

the β CH2 proton (δH 2.92, 3.15) and NH proton (δH 8.53), while aromatic protons H-2 / H-

6 (δH 6.96) were correlated with H-3 / H-5 (δH 6.62). The α proton of Arg3, (δH 4.04), Arg4

(δH 4.24) and Arg5 (δH 4.15) were correlated with the adjacent NH protons (δH 8.10), (δH

8.08) and (δH 7.94) respectively. The α proton (δH 4.21) of Lys6 was found to have

correlation with vicinal NH proton (δH 8.48). Nal7 had clear COSY interaction of α proton

(δH 4.84) with vicinal NH proton (δH 8.63). The aromatic protons H-2’ (δH 7.73), H-3’ (δH

7.35) and H-4’ (δH 7.49) of Nal7 residue were correlated with each other. H-5’ (δH 8.24),

H-6’ (δH 7.57), H-7’ (δH 7.49) and H-8’ (δH 7.87) were correlated with each other in the

same spin system. Protons of p-methoxybenzoic group, H-2”, H-6” (δH 7.71) and H-3”, H-

5” (δH 6.92) were correlated with each other.

Figure-19: Key HMBC and COSY Correlations of Cyclic Peptide 14

61

Table-3: NMR Data for Peptide 14 (d6 DMSO, 600 MHz for 1H, 150 MHz for 13C)

62


The cyclic peptide 15 was synthesized on Ring amide AM resin by linking the side chain

of glutamic acid residue to the Rink linker under standard coupling procedure. After

successful loading of glutamic acid on the resin, all amino acid were sequentially linked by

Asn, Lys, Arg, Lys, Lys, followed by the deprotection of α-allyl and alloc group of Glu

and Lys residues to allow cyclic peptide 15 formation by head-to-side chain cyclization

under cyclization coupling conditions (Scheme-5). After the completion of cyclic sequence

of peptide 15, tyr and Ile were linked to the N-terminal after the removing Fmoc of the

terminal Lys residue. TFA cocktail was used to cleave the crude peptide from resin. The

crude peptide product was purified by RP-HPLC using the isocratic solvent system 60%

ACN: H2O. The pure peptide 15 was obtained as a yellow viscous material with an overall

yield of 45 %. [α]D25 +4.25 (c 0.00282, 60% ACN: H2O). The high resolution electron

spray ionization (HR-ESI) technique exhibited molecular ion (M+H)1+ at m/z 1059.6481

corresponding to molecular formula C48H83N16O11. IR spectrum on KBr disc showed

absorptions at 3421.9 cm-1, 2925.6 cm-1, 1672.9 cm-1, 1629.8 cm-1, 1439.2 cm-1, 1207.2

cm-1, 1134.1 cm-1. Deuterated solvent d6-DMSO was used for recording NMR spectra

because it allowed the detection of NH protons of all eight amino acid residues of peptide

15. The α protons of all residues showed resonance ranging from δH 3.59 - δH 4.57. The

Gln1residue was identified by the presence of two CH2 (δH 1.68, 1.52 / δC 29.1) (δH 2.22,

2.19 / δC 39.6) and γ CO (δC 174.5) was identified through HMBC correlation with γ CH2.

63


The downfield signal of CH2 (δH 2.49, 2.51 / δC 37.4) was assigned to Asn2. The signals for

Lys3, Lys5, Lys6 methylene groups were overlapped and found in the range of δH 1.42 –

2.90. The β CH2 (δH 1.21, 1.30 / δC 28.9), γ CH2 (δH 1.45 / δC 25.0), δ (δH 3.08 / δC 40.2)

and CO (δC 174.0) were assign to Arg4. The aromatic residue tyr7 was fully characterized

64

by resonances of H-2 / H-6 (δH 7.03 / δC 130.2), H-3 / H-5 (δH 6.62 / δC 115.4) and OH

proton (δH 9.20). The carbon shifts (δC 127.2), (δC 155.9), (δC 171.3) were assigned to

quaternary carbon C-1, C-4 and CO respectively. The proton shift (δH 8.19) was given to

NH proton. Ile8 was characterized by β CH (δH1.77 / 36.8), γ CH2 (δH 1.03, 1.05 / 22.3), γ’

CH3 (δH 0.84 / 14.8), δ CH3 (δH 0.78 / δC11.2), carbonyl carbon (δC 167.9) and NH2

protons (δH 1.50).

The evaluation of HMBC interactions of compound 15 was difficult due to overlapping

methylene groups of Lys3, Lys5 and Lys6 residues. The amide protons exhibited interaction

with next carbonyl functionality of each residue (Figure-20). The NH proton (δH 8.05) of

Gln1 showed interaction with α CO (δC 171.5) of adjacent Asn2 residue. The NH proton (δH

8.01) of Asn2 showed interaction with CO group (δC 171.5) of Lys3. Similarly the amide

proton (δH 8.25) of Lys3, Lys5 and Lys6 showed the HMBC correlation with their adjacent

carbonyl groups of Arg4 (δC 174.0), Lys6 (δC 171.3) and tyr7 (δC 171.3) respectively. The H-

2 / H-6 (δH 7.03) of tyr7 exhibited the interaction with β CH2 (δC 38.2), C-3/C-5 (δC 114.9)

and C-4 (δC 155.9) of the ring. The β methylene protons (δH 2.73, 2.81) showed interaction

with the C-2 / C-6 (δC 130.1). The overlapping signal of H-3 and H-5 (δH 6.62) was

correlated with the C-1 (δC 127.2) and C-4 (δC 155.9) of the ring, while hydroxyl proton

(δH 9.18) showed correlation with C-4 (δC 155.9) C-3 and C-5 (δC 114.9) of the phenyl

ring. The β proton (δH 1.77) of Ile8 showed interaction with carbonyl carbon (δC 167.9), the

γ’ protons (δH 0.84) exhibited the correlation with α-carbon (δC 56.2) and γ carbon (δC 22.3)

while δ protons (δH 0.78) of Ile8 were correlated with β carbon (δC 36.8) and γ carbon (δC

22.3).

The clear COSY cross peaks were observed between α proton (δH 3.62) and NH proton (δH

8.05) of Glu1 residue. While α proton (δH 4.15) of Asn2 was correlated with the β CH2

proton (δH 2.41, 2.50). α proton of Lys3 (δH 4.57) was correlated with vicinal NH proton

(δH 8.25). While α proton of Arg4, (δH 4.05), was correlated with adjacent NH protons (δH

8.1) and β methylene protons (δH 1.21, 1.30).The α proton (δH 4.20) of Lys5 was found to

have correlation with vicinal NH proton (δH 8.0).

65

Figure-20: Key HMBC Interactions of Cyclic Peptide 15

COSY interaction of α proton (δH 4.21) with adjacent NH proton (δH 8.19) was observed

for Lys6 residue. α proton (δH 4.19) of tyr7 was correlated with the NH proton (δH 8.53),

while aromatic protons H-2 / H-6 (δH 6.96) were correlated with H-3 / H-5 (δH 6.62). The

correlation of δ protons (δH 0.78) of Ile8 was found with the vicinal methylene protons (δH

1.03, 1.05) (Figure-21).


66

Table-4: NMR Data for Peptide 15 (d6 DMSO, 600 MHz for 1H, 150 MHz for 13C)

68


The novel cyclic peptide 16 was a cyclic derivative of an anticancer linear peptide

temporin ICEa (C. Wang, Li, Li, Tian, & Shang, 2012) (Scheme-6). It was synthesized on

Ring amide AM resin by linking the side chain of Glu residue to Rink linker by using

standard coupling procedure. After successful coupling of glutamic acid, all amino acid

were sequentially linked by using Asn, Lys, Arg, Lys Lys followed by the deprotection of

allyl group of Glu residue and alloc group of Lys to produce a cyclic peptide by head-to-

side chain cyclization. The two residues tyr and Ile were linked to cyclic structure after

removal of Fmoc group of Ile. TFA cocktail was used to cleave the crude peptide from

resin. The crude peptide product was purified by RP-HPLC using the isocratic solvent

system 60% ACN: H2O. The pure peptide 16 was obtained as a whitish yellow solid

material with an overall yield of 35%. The high resolution matrix assisted laser desorption

ionization (MALDI) technique exhibited molecular ion (M+H)+ at 2775.6401

corresponding to molecular formula C131H215N35O31. Deuterated solvent d6-DMSO was

used for recording NMR spectra because it allowed the detection of NH protons of all

amino acid residues. The α protons of all residues appeared in the range of δH 3.59 - δH

4.57. The Glu1residue was identified by two CH2 (δH 1.50, 1.68 / δC 26.7) (δH 2.44, 2.49 /

δC 39.6), γ CO (δC 172.3), α CO (δC172.1), NH proton (δH 8.15) and terminal NH2 (δH

7.41).

69


The downfield signals of CH2 (δH 2.45, 2.52, δC 38.4), γ CO (δC 171.32), γ NH2 (δH 7.94),

α CO (δC 170.67), α NH (δH 6.96) were assigned to Asn2 residue. The overlapped signals

for Lys3,Lys5, Lys6, Lys19, Lys18 methylene groups were found in the range of δH 1.40 –

2.75. The aromatic amino acid tyr7 was fully characterized by protons signals H-2 / H-6

(δH 6.96 / δC 130.2), H-3 / H-5 (δH 6.62 / δC 115.3) and OH proton (δH 9.21). The carbon

shifts δC 133.8, 156.2, 169.2, were assigned to quaternary carbon C-1, C-4 and CO,

respectively, while the proton shift at δH 7.01 was given to NH proton. Ile8was identified

70

by β CH (δH 1.74 / 36.5), γ CH2 (δH 1.37, 1.50 / 24.2), γ’ CH3 (δH 0.64 / 15.3), δ CH3 (δH

0.66 / δC 11.1) and carbonyl carbon (δC 171.0). The Phe9 residue has signals for aromatic

protons H-2’ / H-6’ (δH 7.31 / δC 128.3), H-3’ / H-5’ (δH 7.19 / δC 129.9) and H-4’ (δH 7.14

/ δC 126.2). Signals for β CH2 (δH 2.93, 3.04 / δC 37.1) C-1 (δC 134.7), and CO (δC 169.2)

were also observed. Ile10, Ile13, Ile14, Ile17 showed overlapping signals of β CH from δH

1.58 – 1.74, γ CH2 δH 1.36 – 1.52, γ’ CH3 and δ CH3 δH 0.57 – 0.81. The most downfield

methylene signal of CH2 (δH 3.49, 4.41 / δC 61.7) were assigned to Ser11 residue. The NMR

spectrum also showed the overlapping downfield signals of β CH2 (δH 2.66 – 2.78) of

Asn12 and Asn15. Signals for α-carbonyl carbon (δC 170.6), γ CO (δC 171.0), α-NH (δH

6.96) were also observed. Ala16 was recognized by its CH3 signal (δH 1.17 / δC 18.26)

along with NH signal (δH 8.55) and CO signal (δC 171.9). The NMR shifts for β CH2 (δH

2.38, 2.41 / δC 40.87), γ CH (δH 1.36 / δC 24.1), δ CH3 (δH 0.78 / δC 21.4), δ’CH3 (δH 0.82 /

δC 23.34), NH proton (δH 7.21) and carbonyl group (δC 170.9) were assign to Leu20. The

Asp21 were assigned β CH2 shift (δH 2.67, 2.71 / δC 38.8), NH proton (δH 8.42) and α-CO

(δC 170.1) and γ-CO of acid functionality (δC 170.4). The signals for gem dimethyl (δH 0.82

/ δC 18.4), (δH 0.84 / δC 19.1) CH (δH 1.90 / δC 31.3), NH (δH 8.10) and CO (δC 167.8) were

assign to Val22. The Phe23 were characterized by aromatic protons H-2” / H-6” (δH 7.18 /

δC 129.9), H-3” / H-5” (δH 7.21 / δC 128.4), H-4” (δH 7.24 / δC 127.3) and carbonyl carbon

(δC 169.2).

The assignment of NMR shifts was most arduous due to overlapping signals of methyl

groups common in various amino acid residues. However, HMBC correlation helped us to

correctly assign the resonance to different residues. α proton of Asn2 (δH 4.58) exhibited

interaction with β CH2 (δC 38.4), while β protons (δH 2.45, 2.52) showed correlation with α

carbon (δC 49.4) and γ carbonyl group (δC 171.3). The γ protons (δH 1.42- 1.74) of Lys3,

Lys5, Lys6, Lys18, Lys19 were giving interaction with δ carbon (δC 26.7), while the δ protons

(δH 1.50) showed interaction with γ carbon (δC 23.6) and ϵ carbon (δC 40.0). α proton (δH

4.41) of tyr7 showed correlation with C-2 (δC 130.2), C-3 (δC 115.3), while β protons (δH

2.91, 3.03) were correlated with carbonyl carbon (δC 167.8). The overlapping signals of H-

2 / H-6 (δH 6.96) showed interaction with β carbon (δC 37.1), C-3 / C-5 (δC 115.3) and C-4

(δC156.2). H-3 / H-5 exhibited interaction with C-4 (δC 156.2) and OH proton (δH 9.21)

showed interaction with C-4 (δC 156.2). In case of residue at position 8, α proton (δH 4.13)

71

of Ile8 showed correlation with γ carbon (δC 24.2), while δ CH3 (δH 0.66) were correlated

with α carbon (δC 57.2), β carbon (δC 36.5), γ carbon (δC 24.2). The CH α proton (δH 4.12)

of Phe9 exhibited interaction with β carbon (δC 37.1), while β methylene protons (δH 2.93,

3.04) showed correlation with carbonyl carbon (δC 167.8), H-2’ / H-6’ (δH 7.31) showed

correlation with C-3’ / C-5’ (δC 129.9), H-3’ / H-5’ (δH 7.19) was correlated with C-2’ / C-

6’ (δC 128.3) and C-4’ (δC 126.4). The distinct α proton (δH 4.52) of Asn12 and Asn15

showed correlation with β carbon (δC 38.8), while β protons (δH 2.66, 2.72) were interacted

with α carbon (δC 49.4) and γ carbonyl carbon (δC 171.0). The CH3 protons (δH 1.17) of

Ala16 exhibited the interaction with α carbon (δC 48.2) and carbonyl carbon (δC 171.9). δ

CH3 protons (δH 0.78) of Leu20 were correlated with α carbon (δC 56.9), β carbon (δC 40.8)

and δ’ carbon (δC 23.3). α proton (δH 4.55) of Asp21showed interaction with β carbon (δC

38.8), while, CH2 β protons (δH 2.67, 2.69) showed interaction with α carbon (δC 49.8) and

γ-acidic group (δC 170.4). α proton (δH 4.24) of Val22 was interacted with β carbon (δC

31.3) and carbonyl carbon (δC 167.8), while β protons (δC 171.9) were interacted with α

carbon (δC 57.3), γ carbon (δC 18.4) and γ’carbon (δC 19.1). γ CH3 protons (δH 0.82)

showed interaction with γ’ carbon (δC 19.1). α proton (δH 4.16) of Phe23 exhibited

interaction with β carbon (δC 37.1), β methylene protons (δH 2.94, 3.05) showed interaction

with carbonyl carbon (δC 167.8), H-2” / H-6” (δH 7.18) exhibited correlation with C-1” (δC

137.2) and C-4” (δC 127.3), H-3” / H-5” (δH 7.21) were correlated with C-1” (δC 137.2).

72


73

Table 5: NMR Data for Peptide 16 (d6 DMSO, 600 MHz for 1H, 150 MHz for 13C)

78

2.2.5. Anticancer Activities of Cyclic Peptides.

Cancer is a group of diseases characterized by the uncontrolled growth and proliferation of

abnormal cells from the site of origin to the distant organs that may ultimately lead to

death. Several factors are involved in the progression of cancer, such as exposure to UV

radiation, pollution smoking habits, viruses, and an unhealthy diet. Moreover many chronic

infections like hepatitis B and C (liver cancer), human papilloma (cervical and ano-genital

cancers) and Helicobacter pylori (stomach cancer) also convert into cancerous situation. It

may also be caused by genetic mutations, hormones, and immunological disorder etc.

Several therapies including surgery, hormone therapy, radiation, immune therapy,

chemotherapy, and targeted therapy (administration of the drugs that specifically inhibit

cancer cell growth) are being used (Morris et al., 2008).

Breast cancer is about one third of the all malignancies found in females. This arises from

breast epithelial elements and can be grouped into in situ carcinomas and invasive

carcinomas. In situ carcinomas confined in one place and may arise from ductal or lobular

epithelium, while invasive carcinomas extend from ductal or lobular epithelium from

basement membrane of epithelial border. Radiation therapy was suggested but with care

taken to avoid damage to the heart and lungs. Systemic adjuvant chemotherapy is never

recommended for non-invasive cancer. For invasive cancer standard adjuvant

chemotherapy includes adriamycin, cyclophosphamide, taxol, methotrexate, fluorouracil,

epirubicin drugs. Hormonal therapy is also used to inhibit estrogen that is believed to

stimulate cancer cell growth. Tamoxifen is most commonly used in hormonal therapy.

Peptide therapeutics is considered promising for the advancement of breast cancer drug

target development. Several peptides based vaccines have been developed and studied

against this disease (Mittal, Kaur, Gautam, & Mantha, 2017). Chemist and biologist are

making continuous efforts to develop peptide based therapy to fight against breast cancer

http://www.hindawi.com/journals/jaa/2012/967347/

79

effectively. Keeping the same in mind the cyclic peptide library 1 was screened against

MCF-7 cancer cell line. The results of the screening are summarized in table-6

Lung cancer is considered as the most common human cancer. It is reported that non-small

cell lung cancer (NSCLC) accounts for 80 % of all lung cancer cases and five-year survival

rate of this cancer is less than 15% despite recent advancement made in surgery,

radiotherapy and chemotherapy. In chemotherapy, the cisplatin is widely used

chemotherapeutic agents for NSCLC treatment. This is considered unsatisfactory due to

drug resistance and severe side effects. Thus, identification of new and more effective

medicinal agents is utmost requirement for treatment of this disease. For the screening of

cyclic peptide library, NC-H460 cell line was used to find new anticancer compound

against non-small cell lung cancer (NSCLC) (Xu et al., 2013).

Non-Hodgkin lymphomas are the most frequent human cancers (Siegel et al, 2014).

Despite the noticeable progress in their treatment, they still face reversion after a first

complete remission (Dreyling et al, 2013; Ghielmini et al, 2013), thus demand new

therapeutic agents for the complete cure of disease. In the current study, cyclic peptide

library was also screened against non-Hodgkin lymphoma cell line DOHH2.

In the current study, anticancer screening results showed that the novel peptide 16 was a

strong inhibitor of MCF-7 and DOHH2 cell lines. This peptide 16 was designed as a novel

cyclic analogue of an anticancer linear peptide temporin-1CEa. Temporin-1CEa is an

antimicrobial peptide reported from the skin secretions of the Chinese brown frog (Rana

chensinensis). It is reported that temporin-1CEa-induced rapid cytotoxicity on MCF-7

(IC50 54.9 µM). In the current study, peptide 16 more potently inhibited MCF-7 cancer cell

line (IC50 1.1µM) compared to temporin-1CEa and also strongly inhibited the DOHH2 cell

line (IC50 4.4 µM). The peptides 15 exhibited inhibitions against NCI-H460 and HeLa cell

lines with IC50 41.9 µM, and 92.4 M, respectively, While the peptide, 2 was active against

NCI-H460 (IC50 62.6 µM) and compound 14 was active against MCF-7 cell line (IC50 29.6

µM). All four compounds were found inactive against normal 3T3 cell lines till 100 M.

http://onlinelibrary.wiley.com/doi/10.1111/bjh.13595/full#bjh13595-bib-0024



80

Table-6: Cytotoxicity of New Cyclic Peptides.

*Not tested

81

2.3. Studies on Stylissatin A Analogues:

The cyclic heptapeptide stylissatin A has been isolated from the Papua New Guinean

marine sponge Stylissa massa. The natural peptide had an inhibitory effect on nitric oxide

production in LPS-stimulated murine macrophage RAW264.7 cells with an IC50 value of

87 µM (Kita et al., 2013). The completed total synthesis of natural product was reported by

our research group. Here we report the complete solid-phase synthesis, structural studies

and immunomodulatory activities of different related analogues of stylissatin A (26-31).

Inflammation is an immediate cellular response of body against harmful foreign elements

and tissue injury. Upon first exposure to pathogens or other stimuli phagocytes, mainly

neutrophils and macrophages, releases various mediators including reactive oxygen (ROS)

and nitrogen species (RNS), chemokines and cytokines. The release of these mediators

progresses the processes of inflammation and activation of cell mediated immune

responses (Jadhav, Singh, & Bhutani, 2003).

Reactive oxygen species (ROS) and nitric oxide (NO.) released by inflammatory cells are

involved directly and indirectly in the advancement of oxidative damage (Valko et al.,

2007). Nitric oxide (NO.) has various physiological roles including maintenance of vessel

homeostasis, however the excessive production of NO. in inflammatory condition has

damaging effect on cells and organs.

T lymphocytes are the main cells involve in the generation of adaptive immune response.

The activation and proliferation of various immune cells depends on the cytokines secreted

by T cells. IL-2 is an important immunomodulatory cytokine, secreted by T cells. Along

with the effect on production of other cytokines, it is known to activate T cells in an

autocrine manner. Activated T cells have high affinity receptors for IL-2 on their

surface(Cantrell, Collins, & Crumpton, 1988). Inhibition of IL-2 provides strong

immunosuppressive approach by preventing the activation and proliferation of T cells in

transplantation rejection and other autoimmune diseases (Waldmann, 1993).

Different strategies for the synthesis of cyclic peptides are reported. Among them, the on-

resin cyclization approach has been successfully employed in the synthesis of several

82

biologically active natural peptides in good yields with minimum side products (Kumarn,

Chimnoi, & Ruchirawat, 2013), (Shaheen et al., 2012).

Six analogues 26-31 of stylissatin A were synthesized on Wang resin via on-resin

cyclization approach. Linear precursors of peptide analogues were anchored to the Wang

resin through the side chain of glutamic acid by Fmoc-peptide synthesis. Peptide sequences

of 26-31 were made different by Ala substitution of amino acids residues of original

peptide (Table-7). After the synthesis of linear chain, the allyl group of the side chain of

the Glu was removed by using palladium catalyst (Scheme 7).

Table-7: Alanine-Substituted Analogues (26-31) of Stylissatin A

The Fmoc group of terminal amino acid residue was removed by 4-methylpiperidine in

DMF. On-resin cyclization was carried out by using oxymapure / DIC. TFA cocktail was

used to cleave final product from resin. Structures of analogues were confirmed by

MALDI (Table-7) and NMR studies. Purification of all peptides was achieved by using

preparative recycling HPLC.

83

Scheme 7: Solid-Phase Synthesis of Cyclic Peptides (26-31).

84


The cyclic peptide 26 contain 7 amino acid residues, all having “L” configuration. The

peptide 26 was synthesized by substitution of Pro4 and Pro6 residues of original peptide

sequence of stylissatin A with Ala4 and Glu6 respectively as shown in scheme-8.

The pure peptide 26 was obtained as a yellowish white solid. UV spectra were recorded in

DMSO and showed λmax 229 nm. IR (KBr disc) showed absorptions at 3400, 3019, 2400,

1706, 1219, 791, 628 cm-1. [α]D25was −21.8 (c 0.055, MeOH). The melting point was

determined to be 155 0C. The high resolution matrix assisted laser desorption ionization

(MALDI) technique exhibited pseudo-molecular ion (M+Na)+1 at 906.4372 corresponding

to molecular composition C47H61N7O10Na. Deuterated solvent d6-DMSO was used for

recording NMR spectra which allowed the detection of all seven NH protons of analogue

26. The 1HNMR showed seven amide protons, resonating at δ 8.06 (1H, Phe3-NHCO), δ

7.99 (1H, NHCO), δ 7.90 (1H, Ala4-NHCO), δ 7.85 (1H, Tyr1-NHCO), δ 7.71 (1H,

NHCO), δ 7.1 (1H, Ile5-NHCO), δ 7.10 (1H, Glu6-NHCO). NMR shifts for α proton of

constituent amino acids of 26 appeared in the range of (δH 3.80 - 4.51). Tyr1 has prominent

signals for its aromatic protons δH 6.95 (H-2, H-6) and δH 6.57 (H-3, H-5). Ile2 and Ile5

exhibited the characteristic resonances for methine, methylene and two groups of gem-

dimethyl (Table-8).

85


86



The CH2 groups of Phe3 and Phe7 appeared at (δH 2.74, 2.93, δC 36.5) and (δH 2.82, 3.15,

δC 36.9) respectively. Ala4 residue signals were appeared at (δH 1.13, δC 18.2) and CH2

groups of Glu6 were observed at (δH 2.21, 2.32, δC 30.1) and (δH 1.90, 1.70, δC 28.5). The

HMBC spectrum showed the correlation of α methine proton of Tyr1 (δH 4.42) with β

methylene carbon (δC 37.4), while β CH2 protons were further correlated with α CH (δC

53.5) of Tyr1, as well as with C-1 (δC 137.9), C-2 and C-6 (δC 129.9) of aromatic ring of

Tyr1 residue. The HMBC correlation of the H-2 and H-6 (δH 6.95) with the aromatic

carbons (δC 114.8, 155.7) of Tyr1 residue were observed. The hydroxyl proton (δH 9.1) of

Tyr1 residue showed the HMBC interaction with the aromatic carbon C-3 and C-5 (δC

114.8) and NH proton (δH 7.85) showed interaction with the carbonyl carbon (δC 170.45)

of the next residue Ile2.

HMBC correlation of α CH of Ile2 (δH 4.12) was observed with the β CH (δC 36.1), γ CH2

carbon (δC 23.9) and carbonyl carbon (δC 170.4). The β-methine proton (δH 1.59) was

correlated with methylene carbon (δC 23.9). The NH proton (δH 7.76) of Ile2 showed

correlation with the carbonyl carbon (δC 171.9) of next residue Phe3. The β CH2 protons of

Phe3 (δH 2.71, 2.80) exhibited HMBC correlation with α carbon (δC 53.5) and with C-1ʹ of

the phenyl ring (δC 137.6). The H-3ʹ, H-5ʹ (δH 7.15) of the phenyl ring were correlated with

C-1ʹ (δC 137.6), C-2ʹ, C-6ʹ (δC 127.9) and C-4ʹ(δC 126.11) of the ring. The amide proton (δH

8.06) of Phe3was correlated with the carbonyl carbon (δC 171.5) of the next residue i.e.,

Ala4. The HMBC correlation was observed for α proton (δH 4.25) with methyl carbon (δC

18.2) and carbonyl carbon (δC 172.6) of Ala4 unit. The NH proton (δH 7.90) of Ala4 was

87

further correlated with the carbonyl carbon (δC 170.4) of Ile5 residue. HMBC correlation of

α proton (δH 3.84) of Ile5 residue was seen with the β methine carbon (δC 36.6), γ

methylene carbon (δC 24.3) and with carbonyl carbon (δC 170.4). The NH proton (δH 7.1)

of Ile5 was correlated with the carbonyl carbon (δC 166.2) of the Glu6 residue. The CH β

proton (δH 2.21, 2.32) of Glu6 residue showed HMBC interaction with the carbonyl carbon

(δC 173.8), and with γ carbon (δC 28.5). The NH proton of Glu6 residue showed correlation

with the carbonyl carbon (δC 166.2) of the Phe7. The HMBC correlation of α proton of

Phe7 residue (δH 4.49) was observed with the β CH2 (δC 36.9) and with carbonyl carbon (δC

166.2) of Phe7. β CH2 of Phe7 (δH 2.82, 2.99) showed correlation with α carbon (δC 53.1)

and with C-1ʹʹ (δC 137.5) of the phenyl ring. The correlation of the H-4ʹʹ (δH 7.12) with C-

1ʹʹ (δC 137.5), C-3ʹʹ, C-5ʹʹ (δC 129.99) and C-2ʹʹ, C-6ʹʹ (δH 126.22) of aromatic ring was also

observed. Furthermore, the NH proton (δH 7.87) of the Phe7 showed HMBC correlation

with the carbonyl carbon (δC 170.75) of the adjacent Tyr1 residue.


In the COSY spectrum of Tyr1 residue, α proton (δH 4.42) was correlated with the β

methylene protons (δH 2.65, 2.82) and amide proton (δH 7.85). The aromatic protons were

also found to have correlation with each other. COSY correlations of Ile2 residue were

clearly observed between α proton and NH proton (δH 7.71) and between β methine proton

88

(δH 1.60) and γ’ methyl protons (δH 0.65). Prominent cross peaks were also observed

between γ methylene protons (δH 1.03, 1.35) and σ methyl group (δH 0.76).

COSY correlations were observed for α proton (δH 4.51) of Phe3 residue with the NH

proton (δH8.06). The correlation was also seen between α proton resonance (δH 4.25) and

methyl protons (δH 1.13) and the NH group (δH 7.90) of Ala4 unit. α proton of Ile5showed

cross peaks with the NH proton (δH 7.1). The β methine proton (δH 1.65) is correlated with

that of γ’ methyl protons (δH 0.75). COSY correlation of γ methylene protons (δH 1.26,

1.30) was seen with the δ methyl (δH 0.74). COSY correlation of α proton of Phe7 was

observed with β methylene protons (δH 2.82, 3.15) and NH proton (δH 7.99).


The key NOESY interaction observed in the cyclic peptide 26 were between Tyr1 Hα /

Phe7 NH, Ile2 Hα / Tyr1 NH, Ile2 Hα / Phe3 NH, Ala4 NH / Ile5 Hα, Glu NH / Phe7 Hα.

89


90

Table-8: NMR Data of Peptide 26 (d6 DMSO, 500 MHz for 1H and 150 MHz for 13C)

91


The cyclic peptide 27 was composed of seven amino acids building blocks all having “L”

configuration. The peptide 27 was synthesized by substitution of Pro4 and Pro6 residues of

stylissatin A with Glu4 and Ala6 respectively as shown in scheme-9.

The pure peptide was obtained as a yellow solid. UV spectra were recorded in DMSO and

showed λmax 229 nm. IR (KBr disc) showed absorptions at 3283, 3074, 2400, 1706, 1219,

791, 628 cm-1. [α]D25 − 47.60(c 0.055, MeOH). Melting point was found to be 175.2 0C.

The molecular formula of analogue 27 was found to be C47H61N7O10Na on the basis of

pseudo-molecular ion (M+Na)+1 at 906.43 in the high-resolution matrix assisted laser

desorption ionization (MALDI) technique. Deuterated solvent d6-DMSO was used for

recording NMR spectra which facilitated the detection of all seven NH protons of peptide

27. The 1HNMR showed of all seven NH protons resonating at δ 8.15 (1H, Ala6-NHCO), δ

8.07 (1H, Phe7-NHCO), δ 7.91 (1H, Ile2-NHCO), δ 7.9 (1H, Ile5-NHCO), δ 7.89 (1H, Tyr1-

NHCO), δ 7.85 (1H, Glu4-NHCO), δ 7.78 (1H, Phe3-NHCO). The signals of α proton for

amino acid residues of peptide 27 were present in the range of δH 4.14 – 4.52. The

aromatic moiety of Tyr1 residue was characterized by the overlapped signals of H-2 / H-6

(δH 6.99, δC 130.0) and H-3 / H5 (δH 6.62, δC 114.7). The presence of two signals for each

of β CH proton (δH 1.58, δC 36.8) (δH 1.61, δC 36.9), γ CH2 (δH 0.95, 1.28, δC 24.0) (δH

1.27, 1.32, δC 24.2) and presence of four sets of gem-dimethyl (Table-9.) confirmed the

presence of two isoleucine residues.

92


93



The methylene protons (β CH2) of Tyr1, Phe3 and Phe7 were seen in the range of δH 2.59 –

2.79. The NMR signals for β protons (δH 2.60, 2.81, δC 29.8) of Glu4 were also observed.

Ala6 unit was easily identified by α proton shift (δH 4.34, δC 48.0) and methyl shift (δH 1.13,

δC 18.1). The 13C resonances were assigned to all the amino acid residues with the help of

broad band and DEPT-135 spectra. The structure was further confirmed by HMBC and

COSY correlation. The HMBC correlation of α proton of Tyr1 residue (δH 4.44) was

observed with the β CH2 protons (δH 2.59, 2.79), which were further correlated with the α

carbon (δC 53.8), C-1 (δC 135.9) and C-2, C-6 (δC 130.0) of the aromatic ring. H-2, and H-6

(δH 6.94) showed correlation with the β CH2 (δC 37.2). While H-3, H-5 (δH 6.56) of

Tyr1were correlated with C-2, C-6 and C-4. The NH proton (δH 7.89) showed interaction

with the carbonyl carbon (δC 170.4) of Ile2.

The CH α of Ile2 (δH 4.14) showed the HMBC correlation with the β carbon (δC 36.8), γ

carbon (δC 24.0) and carbonyl carbon (δC 170.54) of Ile2 itself. The γ’ methyl protons (δH

0.70) were related with α CH (δC 56.7), β CH (δC 36.8) and with γ CH2 (δC 24.0). The NH

proton (δH 7.91) signal was correlated with the carbonyl carbon (δC 170.9) of Phe3.

The α CH proton (δH 4.18) of Phe3residue exhibited interaction with the β CH2 (δC 36.5),

which was further correlated with C-1ʹ (δC 137.5) and C-2ʹ, C-6ʹ (δC 128.2) of the ring. The

H-2ʹ, H-6ʹ protons (δH 7.21) were correlated with the C-3ʹ, C-5ʹ (δC 127.8), with C-1ʹ (δC

137.5) and C-4ʹ (δC 127.5) of the ring. The NH proton (δH 7.78) showed interaction with

the carbonyl carbon (δC 173.0) of Glu4. The correlation of β proton (δH 2.60, 2.81) of Glu4

residue with α CH (δC 52.5) and with carbonyl carbon of acid group (δC 174) were

94

identified. For Ile5 residue, α proton (δH 4.16) was correlated with β CH (δC 36.0). The γ’

methyl protons (δH 0.67) showed the cross peaks with α CH (δC 56.7), β CH (δC 36.0), and

γ CH2 (δC 24.2). Furthermore HMBC interaction of the σ methyl protons (δH 0.75) are

observed with γ CH2 (δC 24.2). The NH proton (δH 8.0) showed interaction with the

carbonyl carbon (δC 172.5) of Ala6. The Ala6 unit was identified from the HMBC

correlation between α CH (δH 4.34), methyl (δC 18.1) and carbonyl carbon (δC 172.5) of

Ala6. The NH proton (δH 8.15) was correlated with the carbonyl carbon of the Phe7.

In COSY spectrum, clear cross peaks of α CH of Phe7 (δH 4.54) were observed with β CH2

(δH 2.79, 2.99) and NH proton (δH 8.07). The β methylene protons displayed the HMBC

correlation with α carbon (δC 53.4), with C-1ʹʹ(δC 138.4) and C-2ʹʹ, C-6ʹʹ (δC 129.0) of the

ring. H-2ʹʹ, H-6ʹʹ (δH 7.22) were observed with the C-1ʹʹ (δC 138.4), C-3ʹʹ, C-5ʹʹ (δC 129.18)

and C-4ʹʹ (δC 128.29).


The COSY correlations α CH proton of Tyr1 was observed with β CH2 protons (δH 2.59,

2.79) and amide proton (δH 7.88). H-2, H-6 (δH 6.94) were correlated with the H-3, H-5 (δH

6.56). α proton of Ile2 (δH 4.14) was correlated with the NH proton (δH 7.91), while β CH

proton (δH 1.55) showed interaction with the γ’ CH3 (δH 0.66). The CH2 γ (δH 0.95, 1.28)

showed interaction with the σ CH3 (δH 0.78). COSY correlation of α proton of Phe3 with

the β proton (δH 2.99, 2.78) and amide proton (δH 7.78) was observed. On the other hand,

CH α proton of Glu4 (δH 4.4) was correlated with the amide proton (δH 7.78) and β

methylene protons (δH 2.6, 2.8). CH α proton of Ile5 (δH 4.16) was correlated with the

95

amide proton (δH 7.9) and β CH proton was correlated with the γ’ methyl (δH 0.67). The

COSY correlation observed for CH resonance (δH 4.34) with the methyl (δH 1.18) and

NH group (δH 8.15) identified the Ala6 unit, While α CH proton of Phe7 displayed the

correlation with the β CH2 protons (δH 2.99, 2.78) and amide proton (δH 7.78).


The NOESY spectrum showed the cross peaks of Tyr1 Hα / Phe7 NH, Ile2 Hα / Tyr1 NH,

Phe3 Hα / Ile2 NH, Ala6 NH / Phe7 Hα.

96

Figure-30: Key NOESY correlations of Cyclic Peptide 27

97

Table-9: NMR Data of Peptide 27 (d6-DMSO, 500 MHz for 1H and 150 MHz for 13C)

98


The peptide 28 was synthesized by substitution of Pro6and Phe7 residues with Glu4 and

Ala6 respectively, as shown in scheme-10.

The pure peptide was obtained as a white solid. UV spectra were recorded in DMSO and

showed λmax 230 nm. IR (KBr disc) showed absorptions at 3207, 3074, 2400, 1729, 1219,

791, 628 cm-1. [α]D25 −77.3 (c 0.052, MeOH). Melting point was found to be 171.7 0C. The

molecular formula of analogue 28 was found to be C43H59N7O10Na on the basis of high

resolution matrix assisted laser desorption ionization (MALDI) technique, which gave the

pseudo-molecular ion (M+Na)+1 856.4216. Deuterated solvent d6-DMSO was used for

recording 1HNMR which helped in clear detection of seven amide protons of peptide 28.

According to 1HNMR, the NH protons were present at δ 8.16 (1H, Tyr1-NHCO), δ 8.16

(1H, Glu6-NHCO), δ 8.14 (1H, Ala7-NHCO), δ 8.01 (1H, Phe3-NHCO), δ 7.88 (1H, Ile2-

NHCO), δ 7.72 (1H, Ile5-NHCO), The Tyr1 residue was identified by aromatic moiety

resonances of (δH 6.99, δC 129.1,C-1, C-6) and (δH 6.58, δC 114.7,C-3, C-5). The methyl

group of Ala2 residue appeared at (δH 1.09, δC 18.2).The presence of two CH2 in the shift

range (δH 2.78 – 3.01, δC 37.30, 37.97) confirmed the two phenylalanine residues which

were present at 3 and 7 positions. The presence of CH2 signals at (δH 1.68, 1.83, δC 29.0)

(δH 1.48, 1.49, δC 24.1) and CH2 at (δH 3.49, 3.68, δC 47.1) characterized the Pro4 residue.

99

The Ile5 was confirmed by the CH signal at (δH 1.70, δC 36.0), CH3 signals (δH 0.82, δC

14.9) and (δH 0.78, δC 10.8).


100



The structure was confirmed by HMBC and COSY correlation. In the HMBC spectrum, α

CH of Tyr1 residue (δH 4.42) displayed the correlation with the β CH2 (δC 35.5). While β

CH2 protons (δH 2.78, 2.94) were correlated with C-1 (δC 137.6) and C-2, C-6 (δC 129.1) of

the ring. The aromatic protons H-2 / H-6 were correlated with the C-3, C-5 (δC 114.8) and

C-4 (δC 115.8). The hydroxyl proton (δH 9.18) showed the relation with C-2, C-4, and C-6.

The HMBC correlation of NH proton (δH 8.16) of Phe6 residue with the carbonyl carbon of

Tyr1 (δC 172.8) further supported their linkage. In case of Ile2 residue, α CH (δH 4.10)

exhibited the correlation with the β CH (δC 36.3), γ CH2 (δC 24.0), γ’ CH3 (δC 10.82) and

carbonyl carbon (δC 170.5). The γ’ methyl protons (δH 0.70) were correlated with α carbon

(δC 56.6) and NH proton (δH 7.88) was correlated with the carbonyl carbon (δC 167.69) of

next Phe3 residue.

The correlation of α CH proton (δH 4.33) with the β CH2 (δC 28.5) and β protons with C-1’

(δC 137.6), C-2’, C-6’, HMBC relation of the H-2’, H-6’ (δH 7.19) with the C-3’, C-5’ (δC

127.9) and C-4’ (δC 126.1) and correlation of NH proton with the carbonyl carbon (δC

170.6) of Pro4 provided the full characterization of Phe3 residue. For the Pro4 residue, the

correlation of α proton (δH 4.26) was observed with β carbon (δC 29.2) and carbonyl carbon

(δC 170.6). Another correlation of CH2 γ protons (δH 3.69, 3.44) with α carbon (δC 51.3)

was also present.

For Ile5 residue, CH α proton (δH 4.26) was correlated to β CH2 (δC 36.0), γ CH2 protons

(δH 1.79, 1.93) and carbonyl carbon (δC 169.0). Further, γ’ CH3 protons (δH 0.75) and σ

CH3 (δH 0.71) showed correlation with β CH (δC 36.0) and γ CH2 (δC 23.8). The NH proton

101

(δH 7.72) was correlated with the carbonyl carbon of the next Glu6 residue. For the Glu6

residue, α CH proton resonating at (δH 4.32) exhibit correlation with adjacent carbonyl

carbon (δC 167.69). Furthermore, β CH2 protons (δH 2.37, 2.38) were correlated with the γ

CH2 (δC 27.7). The NH proton was correlated with the CO group of the next Ala7 residue.

The correlation observed between the CH α proton at δH 3.80 with the methyl group at δC

47.2 and carbonyl carbon (δC 172.0) identified the Ala7 unit. The cis and trans

configuration around the Pro4 residue was assign on the basis of 13C chemical shift

difference as the 13C chemical shifts for Pro residue in cis and trans configurations vary

significantly both in small peptides and proteins. The signals of the β-C and γ-C atoms are

more separated in cis-Pro than trans-Pro peptide bond. If the 13C NMR chemical shift

difference between β-C and γ-C of proline is small then the Pro-peptide bond would be

trans and larger 13C chemical shifts difference of β-C / γ-C indicates the cis geometry. The

cyclic peptide 28 has proline residue at position 4 which was found to have trans Ile5-

Pro4 peptide bonds, respectively, as revealed by 13CNMR chemical shift differences of

Pro2 Cβ − Cγ i.e., (Pro4, Δδ Cβ (23.7) − Cγ (29.2) = 5.5. this trans geometry was further

confirmed by NOESY interaction (Figure-34).

Figure-32: Key HMBC Correlation of Cyclic Peptide 28

COSY correlations were observed for α proton (δH 4.42) with the β CH2 protons (δH 2.78,

2.94) and amide proton (δH7.95) of Tyr1 residue. In case of Ile2 residue, α proton (δH 4.10)

102

was correlated with the β methine proton (δH 1.62) and NH proton (δH 7.72). β CH proton

was further correlated with the adjacent methyl protons (δH 0.70). Phe3 residue had α

proton (δH 4.33) which was correlated with amide proton (δH 8.01). In case of Pro4 residue,

COSY correlation were observed between α CH proton (δH 4.26) and β CH2 protons (δH

1.03, 1.46), γ protons (δH 1.96, 1.86) and protons (δH 3.69, 3.44). Ile5 residue exhibited

correlation between α CH proton (δH 4.26) and β CH2 proton (δH 1.65), and γ’ CH3 proton

(δH 0.75), γ CH2 proton (δH 1.79, 1.93) and σ CH3 protons (δH 0.71). The COSY

correlations of Glu6α proton (δH 4.32) were observed with the amide proton (δH 7.72), and

β CH2 protons (δH 2.73, 2.38). The COSY correlations were also observed among CH

proton (δH 3.85) with the methyl group (δH 1.25) and NH group at (δH 8.14).

Figure-33: Key COSY Correlation of Cyclic Peptide 28

The NOESY spectrum showed correlation between Tyr1 Hα / Ile2 Hα, Tyr1 NH / Ile2 NH,

Ile2 Hα / Try1 NH, Pro4 Hδ / Ile5 Hα, Glu6 NH / Ala7 Hα. The presence ofcross peak

between Pro4 Hδ / Ile2 Hα confirmed the trans geometry of Pro4 / Ile5 peptide bond.

103


104

Table 10: NMR Data of Peptide 28 (d6-DMSO, 500 MHz for 1H and 150 MHz for 13C)

105

2.3.4.Cyclic Peptide 29:

The cyclic peptide 29 had “7” L-amino acids linked by peptide bond in a cyclic form. The

peptide 29 was synthesized by substitution of Phe3 and Pro6 residues of stylissatin A with

Ala3 and Glu6 respectively, as shown in scheme-11.

The pure peptide was obtained as a white solid. IR (KBr disc) showed absorptions at 3297,

3063, 2400, 1731, 1219, 791, 628 cm-1. [α]D25 −49.5 (c 0.047, MeOH). Melting point was

found to be 185.2 0C. The molecular formula of cyclic peptide 29 was found to be

C43H59N7O10Na by means of high resolution matrix assisted laser desorption ionization

(MALDI) technique, which gave the pseudo-molecular ion [M+Na]+ at 856.4216 (calcd.

833.43) Deuterated solvent d6-DMSO was used for recording NMR spectra which revealed

the clear shifts of seven amide protons of peptide 29. The 1HNMR showed the protons

resonating at δ 8.18 (1H, Tyr1-NHCO), δ 8.07 (1H, Ile5-NHCO), δ 8.01 (1H, Ala3-NHCO),

δ 8.0 (1H, Phe7-NHCO), δ 7.80 (1H, Glu6-NHCO), δ 7.6 (1H, Ile2-NHCO). Tyr1 unit was

identified by aromatic signals (H-2 / H-6, δH 6.92, δC 129.1) (H-3 / H-5, δH 6.56, δC 114.7),

β CH2 (δH 2.82, 3.10, δC 37.9). The presence of two Ile residues were evident from CH

signals (δH 1.59, δC 36.5) (δH 1.73, δC 36.1) and CH2 (δH 0.91, 1.23, δC 24.0) (δH 1.50, 1.78,

δC 24.2) along with 4 CH3 signals (δH 0.62, δC 10.8) (δH 0.70, δC 15.1) (δH 0.81, δC 11.0) (δH

0.88, δC 14.9). Ala3 residue was identified by the β CH3 signal (δH 1.13, δC 17.5).

106


107



Pro4 residue was characterized by δ proton (δH 3.52, 3.72, δC 47.1). β and γ CH2 methylene

groups (δH 1.03,1.23 δC 28.6) and (δH 1.79, 1.82, δC 24.3) respectively. The β CH2 (δH 2.22,

2.26 δC 30.2) and γ CH2 (δH 1.68, 1.98, δC 28.5) of Glu6 were also observed. Phe7 residue

showed the signals of aromatic moiety (H-2’ / H-6’, δH7.12, C-2’/ C-6’, δC 130.4), (H-3’ /

H-5’, δH 7.23, C-3’ / C-5’, δC 128.3) and (H-4’ δH 7.17, δC 126.7).

The structure was further confirmed by HMBC and COSY correlation. In HMBC spectra,

α proton of Tyr1 residue (δH 4.18) showed the correlation with the β methylene carbon (δC

37.9), C-1 (δC 135.9) and carbonyl carbon (δC 167.0). The H-2 / H-6 protons (δH 6.92) were

correlated with β carbon (δC 37.9), C-3 / C-5 (δC 114.7) and C-4 (δC 155.7). The hydroxyl

proton (δH 9.10) showed signal with the C-3, C-5 and C-4 of the ring. The NH proton (δH

8.18) showed correlation with the carbonyl carbon (δC 170.54) of the adjacent Ile2. α

proton of Ile2 residue (δH 4.02) showed correlation with β CH (δC 36.9), γ CH2 (δC 24.0), σ

CH3 (δC 15.1) and carbonyl carbon (δC 170.54) of its own. The δ methyl proton (δH 0.62)

were related with α CH (δC 56.2), β CH2 (δC 36.9) and γ CH2 (δC 24.0). The γ’ methyl (δH

0.70) also showed signal with β CH2 and γ CH2. The NH proton (δH 7.6) had correlation

with the carbonyl carbon (δC 172.0) of the adjacent Ala3.

The HMBC correlation observed for α proton (δH 4.18) with methyl group (δC 17.5) and

carbonyl carbon (δC 172.0) supported the Ala3 unit. While the amide proton (δH 8.01)

showed correlation with the carbonyl carbon (δC 166.3) of the next residue i-e Pro4.

The HMBC correlation supported the assignment of Pro4 residue. HMBC correlations were

observed from α proton (δH 3.65) with β carbon (δC 28.4) and carbonyl carbon (δC 166.3). γ

108

CH2 protons (δH 1.79, 1.82) exhibited correlation with β CH2 (δC 28.4) and α CH (δC 52.8).

While CH2 protons (δH 3.52, 3.72) showed correlation with β carbon (δC 28.47). The

HMBC correlation further supported the assignments of Ile5 residue. α proton (δH 4.28)

exhibited interaction with β CH (δC 36.1) and carbonyl carbon (δC 170.0). γ’ CH3 (δH 0.81)

showed correlation with β CH (δC 36.1) and γ CH2 (δC 24.2). The correlation of methyl

with α CH (δC 54.9), β CH (δC 36.1), and γ CH2 as well as correlation of NH proton (δH

8.07) with carbonyl carbon (δC 172.0) supported the Ile5 residue. For the Glu6 residue,

HMBC correlation of NH proton with carbonyl carbon (δC 172.0) was observed. The

HMBC correlation of α CH proton of Phe7 (δH 4.51) with β CH2 (δC 37.9) were observed.

The β CH2 (δH 2.81, 3.15) also showed the correlation with the C-1’ (δC 135.9) and C-2’ /

C-6’ (δC 130.2) of the phenyl ring. HMBC correlation of the H-2’/ H-6’ (δH 7.12) with the

β CH2, C-3’/ C-5’ (δC 127.9) and C-4’ (δC 126.7), while H-4’ (δH 7.16) interaction with C-

2’/ C-6’ (δC 130.2) was also observed. The peptide 29 also has proline residue at position

4 so the 13CNMR chemical shift differences of Pro4 Cβ − Cγ i.e., (Pro4, Δδ Cβ (28.9) −

Cγ (24.3) = 4.6 indicated the trans peptide bond between Ile5- Pro4.


The COSY interaction of α CH proton (δH 4.18) with β CH2 protons (δH 2.82, 3.10) and

amide protons (δH 8.18) along with the interaction of H-2 / H-6 (δH 6.92) with H-3 / H-5

(δH 6.56) supported Tyr1 residue. α proton (δH 4.08) of Ile2 displayed the COSY correlation

with the CH proton (δH 1.59) and also with amide proton (δH 7.65). The methine proton

109

further had correlation with γ’ methyl (δH 0.62). The COSY correlation observed for the

proton resonance (δH 4.20) with both the methyl group (δH 1.13) and the NH group at (δH

8.19) supported the Ala3 unit. α proton (δH 3.65) of Pro4 was correlated with the β CH2

protons (δH 1.03, 1.23). While α proton (δH 4.31) of Ile5was correlated with the β CH2

proton (δH 1.73). The methine proton further showed correlation with γ’ methyl (δH 0.81).

α proton showed correlation with amide proton (δH 8.07). The correlation between γ

protons (δH 1.50, 1.78) and δ CH3 protons (δH 0.88) were also observed. The COSY

correlations for Glu6 residue between α CH proton (δH 4.37) and amide proton (δH 7.80)

were also observed. α proton of Phe7 showed the correlation with the β CH2 protons (δH

2.81, 3.12) and amide proton (δH 8.0).


The trans geometry of Ile5-Pro4 amide bond was further confirmed by NOESY correlation

spectrum that showed prominent cross peaks of Pro4 Hδ / Ile5 Hα. Other NOESY

correlations were observed among Tyr1 Hα / Phe7 NH, Ile2 NH / Ala3 Hα, Ile5 Hα / Glu6

Hα, Glu6 NH / Phe7 NH, Tyr1 Hα / Ile2 NH, Ile5 NH / Glu6 NH.

110


111


112


The cyclic peptide 30 had “7” amino acids all having “L” configuration. . The peptide 30

was synthesized by substitution of Tyr1 and Pro6 residues of stylissatin A with Ala1 and

Glu6 respectively as shown in scheme-12.

The pure peptide was obtained as a white solid. IR (KBr disc) showed absorptions at 3303,

3067, 2400, 1706, 1219, 791, 628 cm-1. [α]D25 −71.1 (c 0.06, MeOH). Melting point was

found to be 171.5 0C. The molecular formula was found to be C43H59N7O9Na by high

resolution matrix assisted laser desorption ionization (MALDI) technique, which gave the

pseudo-molecular ion [M+Na]+ at 840.4266. Deuterated solvent d6-DMSO was used for

recording NMR spectra because the amide protons signals appeared very clear in DMSO.

1HNMR showed seven amide protons, resonating at δ 7.99 (1H, overlap, Ala1-NHCO), δ

7.80 (1H, overlap, Ile2-NHCO), δ 7.89 (1H, m, Phe3-NHCO), δ 8.01 (1H, b, Ile5-NHCO), δ

8.01 (1H, b, Glu6-NHCO), δ 7.89 (1H, m, Phe7-NHCO). α protons of all constituent amino

acid appeared between δH 4.12 and δH 4.48. In analogue 30, Ala1 was used to substitute

Tyr1 of original sequence of stylissatin A, and it was evident by the appearance of CH3

signal at δH1.12, δC 18.1.

113


114



The two CH signals (δH 1.65, δC 36.1) and (δH 1.68, δC 36.1) along with the two CH2

signals (δH 1.35, 1.01, δC 24.1), (δH 1.71, 1,68, δC 24.1) and four methyl signals (δH 0.73, δC

14.9), (δH 0.72, δC 10.8), (δH 0.81, δC 15.1), (δH 0.84, δC 10.9) confirmed the presence of

two isoleucine residues. The presence of two phenyl alanine residues was evident by the

aromatic proton signals, for Phe3, H-2 / H-6 (δH 7.12, δC 130.3), H-3 / H-5 (δH 7.19, δC

129.1) H-4 (δH 7.14, δC 126.1), β CH2 (δH 2.77, 2.83 δC 37.2), while for Phe7 residue the

signals of aromatic protons observed at (δH 7.14, δC 127.9) for H-2’ / H-6’, (δH 7.19, δC

129.2) for H-3’ / H-5’ and (δH 7.18, δC 126.1) for H-4’. For Glu6 the CH2 resonances (δH

2.22, 2.25, δC 29.7) (δH 1.72, 1.85, δC 28.6) were observed. The structure was further

confirmed by the HMBC and COSY correlation. The correlation of α proton (δH 4.29) of

Ala1 residue to the β methyl carbon (δC 18.1) and carbonyl carbon (δC 172.8) was observed.

The correlation of amide proton (δH 7.99) with carbonyl carbon (δC 170.6) of next Ile2 was

also present. For the Ile2 residue, α proton (δH 4.12) displayed the correlation with the β

CH (δC36.1), γ CH2 (δC 24.1) and γ’ CH3 (δC 14.9). The γ CH2 protons (δH 1.35, 1.01)

showed the correlation with the δ CH3 (δC 10.8). The γ’ CH3 protons (δH 0.73) was

correlated to β CH (δC 36.1) and α carbon (δC 56.6). The NH proton of Ile2 is correlated

with carbonyl carbon (δC 171.8) of adjacent Phe3 residue.

In the Phe3 residue, the correlation of α proton (δH 4.48) with β- methylene carbon (δC

37.2) and with the carbonyl carbon (δC 171.8) was detected. The β methylene protons

showed the correlation with α carbon (δC 53.5), C-1 (δC 137.5) and C-2 / C-6 (δC 130.3) of

the phenyl ring. The NH proton is correlated with the carbonyl carbon (δC 170.3) of Pro4.

115

For the Pro4 residue, correlation of α proton (δH 4.30) is seen with the β CH2 (δC 28.5) and

γ CH2 (δC 24.2). β proton (δH 1.77, 1.92) and γ protons (δH 1.48, 1.13) were correlated to

the δ carbon (δC 47.12). For the Ile5 residue, the correlation of α proton (δH 4.30) with the β

CH (δC 36.1), γ CH2 (δC 24.1) and with the carbonyl carbon (δC 170.3) of Ile5 was

observed. Correlation of amide proton (δH 8.01) is observed with the carbonyl carbon (δC

170.3) of the adjacent amino acid. For the Glu6 residue, α proton (δH 4.34) had correlation

with carbonyl carbon (δC 170.7). The correlation of β protons (δH 2.22, 2.25) was observed

with the carbonyl carbon (δC 173.8) of acidic group and γ carbons (δC 28.6), while the NH

proton (δH 8.01) showed the correlation with the carbonyl carbon (δC 171.3) of adjacent

Phe7. For Phe7 residue, the HMBC correlation of α proton was observed with the β

methylene carbon (δC 37.9) and carbonyl carbon (δC 171.3). The correlation of β protons

with the C-1’ (δC 137.5), with C-2’ / C-4’ (δC 127.9) and with α carbon (δC 4.48) were also

present and the NH proton (δH 7.89) showed the signals with the carbonyl carbon of the

Ala1 (δC 172.8). The peptide 30 has proline residue at position 4 and the 13CNMR chemical

shift differences of Pro4 Cβ − Cγ i.e., (Pro4, Δδ Cβ (28.5) − Cγ (24.2) = 4.3 which indicated

its trans geometry around the Ile5-Pro4 amide bond.


116

In COSY spectrum α proton (δH 4.29) of Tyr1 showed the cross peaks with β proton (δH

1.12) and NH proton (δH 7.99). For Ile2 residue, α proton had the correlation with the β CH

(δH 1.65) and NH proton (δH 7.80). β CH further correlated with the γ’ CH3 (δH 0.72). γ

CH2 exhibited the relation with the δ CH3 (δH 0.75). For Phe3 residue, α proton (δH 4.48)

showed the correlation with β protons (δH 2.77, 2.83) and with the NH proton (δH 7.89).

For Pro4, α proton (δH 4.34) was correlated with the β protons (δH 1.77, 1.92). For Ile5

residue, α proton (δH 4.30) was correlated with the β proton (δH 1.68) and NH proton (δH

8.04). β proton is further correlated with the γ’ CH3 (δH 0.81). The γ methylene protons (δH

1.73, 1.84) are further correlated to the δ methyl protons (δH 0.84). For Glu6 COSY

correlation of α proton (δH 4.30) with the NH (δH 8.01), β protons (δH 2.22, 2.25) with the γ

methylene protons (δH 1.72, 1.85) was observed. For Phe7, α proton (δH 4.48) showed the

relation with the β protons (δH 2.78, 2.96) and NH proton (δH 7.89).

Figure-41: Key COSY Correlation of Cyclic Peptide 30

117

The NOESY spectrum indicated the key correlation of Ala1 NH / lle2 Hα, Ile2 Hα / Phe3

NH, Ile2 NH / Phe3 Hα, Phe3 NH / Pro4 Hα, Pro4 Hδ / Ile5 Hα, Glu6 NH / Phe7 Hα, Phe7 Hα

/ Ala1 NH.

Figure-42: Key NOESY Correlations of Cyclic Peptide 30

118

Table-12: NMR Data of Peptide 30 (d6-DMSO, 500 MHz for 1H and 150 MHz for 13C)

119


The cyclic peptide consists of 7 amino acids building blocks all having “L” configuration.

The peptide 31 was synthesized by substitution of Ile2 and Pro6 residues with Ala2 and Glu6

respectively, as shown in scheme-13.

The pure peptide was obtained as a yellow solid. IR (KBr disc) showed absorptions at

3302, 3062, 2400, 1732, 1245, 752, 628 cm-1. [α]D25 −60.36 (c 0.055, MeOH). Melting

point was found to be 172.6 0C. The molecular formula of analogue 31 was found to be

C46H57N7O10Na by high resolution matrix assisted laser desorption ionization (MALDI)

technique, which exhibited the pseudo-molecular ion [M+Na]+ at m/z 890.4059 (calcd.

866.4214) Deuterated solvent d6-DMSO was used for recording NMR spectra. 1HNMR

showed seven amide protons, resonating at δH 8.02 (1H, overlap, Tyr1-NHCO), δH 7.90

(1H, overlap, Ala2-NHCO), δH 7.88 (1H, m, Phe3-NHCO), δH 8.07 (1H, b, Ile5-NHCO), δ

8.07 (1H, b, Glu6-NHCO), δ 7.8 (1H, m, Phe7-NHCO), Pro4 does not contain any amide

proton. The α proton of all constituent amino acid appears between (δH 4.17 and δH 4.51).

Tyr1 residue had the β CH2 at (δH 2.62, 2.85 δC 37.32) and aromatic protons, H-2 / H-6 at

(δH 6.92, δC 130.03), H-3 / H-5 at (δH 6.58, δC 114.78).

120


121

Figure-43: HPLC Profile of Cyclic Peptide 31


Ala2 was confirmed by CH3 signal (δH 1.09, δC 18.2). The two phenyl alanine present at

position “3” and “7” were confirmed by the aromatic proton signals for Phe3, H-2ʹ / H-6ʹ

(δH 7.10, δC 130.2), H-3ʹ / H-5ʹ (δH 7.25, δC 129.2) H-4ʹ (δH 7.28, δC 128.0), CH2 (δH 2.78,

2.99 δC 37.3), while for Phe7 the signals of aromatic protons located at (δH 7.14, δC 130.0)

for H-2ʹʹ / H-6ʹʹ, (δH 7.07, δC 128.5) for H-3ʹʹ / H-5ʹʹ and (δH 7.15, δC 127.9) for H-4ʹʹ and

CH2 was present at (δH 2.83, 3.01, δC 37.97). The CH signal at (δH 1.70, δC 36.0) along with

CH2 signal at (δH 1.05, 1.48, δC 24.3) and two methyl signals at (δH 0.78, δC 10.8), (δH 0.82,

δC 14.9) confirmed the presence of isoleucine residue at position “5”. Glu6, had two CH2

(δH 2.26, 2.28, δC 29.8) and (δH 1.75, 1.86, δC 28.2). This structure was further confirmed

by the HMBC and COSY interactions.

The HMBC correlation of α proton (δH 4.34) of Tyr1 residue was observed with the β

methylene (δC 37.32), and with its carbonyl carbon (δC 170.4). While β methylene protons

were related with C-1 (δC 135.9) and C-2 / C-6 (δC 129.19) of the phenyl ring. H-2 / H-6

(δH 6.92) showed correlation with C-3 / C-5 (δC 114.7), and C-4 (δC 155.7). The hydroxyl

proton was related with C-4 and C-3 / C-5 of ring. Ala2 unit was identified by HMBC

correlation of α proton (δH 4.17, δC 48.6) with both the methyl group (δH 1.09, δC 18.2) and

with the carbonyl carbon (δC 172.8). For Phe3 residue, α proton (δH 4.38) was correlated

with the β CH2 (δC 37.3) and with its carbonyl carbon (δC 169.9). The β protons (δH 2.76,

2.99) were further related with α carbon (δC 54.0), with the C-1ʹ of the phenyl ring (δC

135.9) and with the C-2ʹ / C-6ʹ (δC 130.2). For Pro4, no signal is observed. For Ile5 residue,

signal of α proton (δH 4.29) was correlated with the β methine (δC 36.0), γ methylene (δC

122

24.3), γ’ methyl (δC 14.9) and with its carbonyl carbon (δC 170.6), β CH was also

correlated with the γ CH2. For Phe7 interaction of α proton (δH 4.51) was observed with the

β CH2 (δC 37.97) and with its carbonyl carbon (δC 170.8). The β CH2 of phe7 (δH 2.83, 3.01,

δC 37.97) was correlated with α carbon (δC 53.4) and with the C-1 of the phenyl ring (δC

135.9). The peptide 31 has proline residue at position 4 and the 13CNMR chemical shift

differences of Pro4 Cβ − Cγ i.e., (Pro4, Δδ Cβ (29.0) − Cγ (24.1) = 4.9 which indicates the

trans orientation of the Pro4-Ile5 peptide bond.


For Tyr1, the COSY correlation of α proton (δH 4.34) was observed with the β protons (δH

2.62, 2.83) and NH proton (δH 8.02). The correlations between H-2 / H-6 (δH 6.92) and H-3

/ H-5 (δH 6.58) were also observed. α proton (δH 4.17) of Ala2 was correlated with both the

methyl (δH 1.09) and NH proton (δH 7.9). α proton (δH 4.38) of Phe3 was observed with

that of NH proton (δH 7.88). α proton of Ile5 exhibited relation with the methine proton (δH

1.70) and NH proton (δH 8.07), while CH2 protons (δH 1.05, 1.48) showed interaction with

δ CH3 protons (δH 0.78). For glu6 residue, COSY correlation between β protons (δH 2.26,

2.28) and γ protons (δH 1.75, 1.86) was present. For Phe7, residue COSY correlation of α

proton (δH 4.51) was observed with NH proton (δH 7.8).

123


The NOESY spectrum showed cross peaks between Tyr1 Hα / Ile2 Hα, Tyr1 NH / Ile2 NH,

Tyr1 NH / Ile2 Hα, Pro4 Hδ / Ile5 Hα and Glu6 NH / Ala7 Hα.

Figure-46: Key NOESY Correlations of Cyclic Peptide 31

124


125

Table-14: Mass Spectroscopic Analysis and [α]D of Cyclic Peptides 26-31.

Table-15: Effect of Peptides on Oxidative Burst, Nitric Oxide (NO.) and IL-2

Production.

2.4. Immunomodulatory Activities of Stylissatin A analogues:

To evaluate the structure activity relationship, six analogs 26–31 were synthesized

(scheme-7) and evaluated for their anti-inflammatory potential by investigating different

immune parameters including effects on production of NO, intracellular ROS, and IL-2

126

cytokine (Table-15). The natural product was good inhibitor of NO (IC50 63.0 ± 5.4 μM) as

compared to the standard drug (IC50 97.5 ± 3.2 μM) (Kita et al., 2013).

The analogues 26-31 showed weak inhibitory activity of NO. and more potent inhibition of

interleukin 2 production compared to parent peptide. It also appeared that by substitution

of both proline residues (Pro4 and Pro6) of natural peptide stylissatin A, with Ala and Glu

respectively, the resulting peptide analogue 26 was found to be a strong inhibitor of ROS

with no inhibition of NO.. It potently inhibited ROS from neutrophils as well as strongly

inhibited the release of cytokine interleukin 2 (IC50 13.4 ± 0.1 μM). All analogues were

found more potent inhibitor of IL-2 production compared to the synthetic natural product.

This indicated that both proline residues are involved in the mechanism of NO. inhibition.

The peptide analogue 31, in which Ile2 and Pro6 were replaced by Ala and Glu,

respectively, was found to be the most potent inhibitor of interleukin 2 (IC50 6.0 ± 0.9 μM)

releases among the peptides 26-31. The results obtained from current studies showed

significant anti-inflammatory potential of analogues 26-31. All peptides were also

evaluated against different cell lines including MCF-7, NCI-H460, DoHH2, HeLa as well

as 3T3 cell lines. It was releaved that the stylissatin A analogues 26 – 31 were non

cytotoxic to all standard cell lines

2.5. Conclusion:

The synthesis and anticancer screening of cyclic peptide libraries has led to discovery of

many hit compounds including anticancer compounds 2, 14, 15, 16 and anti-inflammatory

peptides 26-31. All these peptides were found non cytotoxic to normal cell lines (3T3

normal mouse cell line), thus they have potential as more safe new medicinal agents

against cancer and inflammation. gfggfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfgfff

Chapter # 3

EXPERIMENTAL

PROCEDURES

128

3.1. General Experimental Details:

The protected amino acids, resins, coupling reagents and solvents were purchased from

Sigma Aldrich, Novabiochem and Chem- impex. Chromatographic solvents were synthetic

grade and were filtered through 0.45 µm and degassed by sonication prior to use. RP-

HPLC was done on (LC-900 Japan). C-18 Column Jaigel ODS-MAT 80 with a flow rate

of 4 ml / min using ACN: H2O (60: 40). IR spectra were recorded on shimadzu® Japan,

FTIR-8900 Fourier Transform infrared spectrophotometer. Optical rotation was

determined on P-2000 polarimeter JASCO® Japan. UV-Visible spectra were recorded at

Thermoscientific® Evolution 300 UV-visible spectrophotometer. NMR spectra were

recorded at Bruker® and Avance AV-600 Nuclear Magnetic Resonance Spectrometers were

used for recording 1H (600 MHz) and 13C NMR spectra (150 MHz), operating NMR

solvent was d6- DMSO. Chemical shifts were reported in parts per million (ppm). Matrix-

assisted laser desorption ionization (MALDI) mass spectra were recorded on Ultraflex III

TOF/TOF (Bruker Daltonics, Bremen, Germany).

3.2. General Synthesis Procedure for Cyclic Peptide Library I:

Peptide 1 to 25 was synthesized by solid-phase chemistry by using Fmoc protocol. The

sequences of all peptides are shown in Table 1 (a). The resin was soaked in synthetic grade

DCM for 2 hours for thorough exposure of reaction sites. The first amino acid was loaded

by using the oxyma pure and DIC as the coupling agents. The reaction mixture was kept

under gentle shaking for two hours at room temperature. The coupling was confirmed by

the ninhydrin test. The Fmoc deprotection was achieved by treating peptide-bound resin

with 20% 4-methylpiperidine in DMF for 30 min. Peptide chain was further elongated by

using Fmoc protected amino acids. Each cyclic peptide was composed of eight residues.

As the linear sequence was synthesized, the acid terminal having allyl protecting group and

amine terminal having alloc group were deprotected by a mixture of palladium catalyst

[tetrakistripehnylphosphine palladium (0)] in DCM with acetic acid and N-methyl

morpholine (37: 2: 1) respectively. The macrocyclization was carried out between free

acid and amino groups by using oxyma pure and DIC. After the deprotection of terminal

Fmoc group by 20% 4-methylpiperidine, the resin was treated with 90% TFA cocktail

(TFA: Phenol: H2O: TIPS, 90: 4: 4: 2) for simultaneous cleavage of peptide from resin and

129

removal of side chain protecting groups. Extensive washing was carried out after each

coupling and deprotection step by DMF, DCM, MeOH, and DCM, respectively. The crude

reaction mixture was treated with cold ether and peptide was obtained in the form of

precipitate.

3.3. Synthesis of Biologically Active Compounds:

3.3.1. Synthesis of Cyclic Peptide-2:

Compound 2 was synthesized by solid-phase chemistry using Fmoc protocol on Rink

amide AM resin (130 µm, 2.5 g, 0.51 mmol / g). The resin was washed and soaked in

solvent as described in the general procedure. Fmoc-D-phe-OH (100.7 mg, 0.25 mmol)

was loaded by using the oxyma pure (36.9 mg, 0.25 mmol) and DIC (0.037 ml, 0.26

mmol). The reaction mixture was kept on shaking for two hours and reaction progress was

monitored by the ninhydrin test. The Fmoc deprotection was done by passing a solution of

20% 4-methylpiperidine in DMF (30 min) into the reaction vessel containing peptide-

bound resin. Peptide chain was further elongated with Fmoc-Val-OH (88.2 mg, 0.25

mmol), Fmoc-Glu-α-allyl-OH (106.4 mg, 0.25 mmol), Fmoc-Lys(Boc)-OH (121.8 mg,

0.25mmol), Fmoc-Orn(Boc)-OH (118.1mg, 0.26 mmol) Fmoc-D-phe-OH (100.7 mg, 0.25

mmol), Fmoc-Lys(Boc)-OH (121.8mg , 0.25 mmol) and the peptide sequence was

completed with Fmoc-Lys (Alloc)-OH (117.6 mg, 0.25 mmol). The side chain NH2 group

of lysine and α acidic group of glutamic acid was deprotected by palladium (0) catalyst in

DCM with acetic acid and N-methylmorpholine (37: 2: 1). Cyclization was achieved

between acid group of glutamic acid and amino group of lysine side chain by using oxyma

pure and DIC. The resin was extensively washed with DMF, DCM, MeOH, and DCM

respectively, after each coupling / deprotection step. The Fmoc group of terminal residue

Lys was removed by 20% 4-methylpiperidine and resin bound peptide was treated with

90% TFA cocktail (TFA: phenol: H2O: TIPS, 90: 4: 4: 2) for two hours for simultaneous

cleavage of side chain protecting groups and release of crude peptide from the resin. The

peptide was precipitated out with cold ether and further purified by HPLC using isocratic

solvent system of 60% acetonitrile: water. The structure of purified product 2 was

elucidated by interpretation of spectral data.

130

3.3.1.1. Characterization Data of Peptide 2:

3.3.2. Synthesis of Cyclic Peptide 14

Compound 14 was prepared by solid-phase synthesis by using Fmoc chemistry on Rink

amide AM resin (130 µm, 2.5 g, 0.51 mmol / g). After soaking of resin in DCM, the first

amino acid Fmoc-Glu-α-allyl-OH (106.4 mg, 0.25 mmol), was loaded using oxyma pure

(36.9 mg, 0.25 mmol) and DIC (0.037 ml, 0.26 mmol) as coupling agents. The reaction

was gently shaked for two hours. The coupling reactions were driven to completion and

confirmed by the ninhydrin test. The Fmoc deprotection was carried out by passing a

solution of 20% 4-methylpiperidine in DMF (30 min) into the reaction vessels containing

peptide-bound resin. Peptide chain was further elongated by coupling with Fmoc-D-tyr

(OtBu)-OH (119.4 mg, 0.25 mmol), and three consecutive couplings of Fmoc-Arg(Pbf)-

OH (168.68 mg, 0.25 mmol for each coupling), followed by coupling with Fmoc-

Lys(Alloc)-OH (117.65 mg, 0.25 mmol). Alloc group of lysine and allyl group of glutamic

acid were removed by using palladium (0) catalyst in the presence of DCM, acetic acid

and N-methylmorpholine (37: 2: 1). Cyclization was carried out between acid group of

glutamic acid and amino group of lysine side chain by using oxyma pure and DIC. The

resin was thoroughly washed after each coupling / deprotection with DMF, DCM, MeOH,

and DCM. Finally, Fmoc group of Lys was removed by 20% 4-methylpiperidine and resin

bound peptide was treated with 90 % TFA mixture (TFA: phenol: H2O: TIPS, 90: 4: 4: 2)

for two hours for simultaneous deprotection of side chain protecting groups and release of

crude cyclic peptide from the resin. The peptide was precipitated out with cold ether and

131

further purified by HPLC using isocratic solvent system of 60% acetonitrile: water. The

structure of purified product 14 was elucidated by interpretation of spectral data.


3.3.3. Synthesis of Cyclic Peptide 15:



amino acid Fmoc-Glu-α-allyl-OH (106.4 mg, 0.25 mmol), was loaded using oxyma pure

(36.94 mg, 0.25 mmol) and DIC (0.037 ml, 0.26 mmol). The reaction was gently shaked

for two hours. The coupling reaction was driven to completion and confirmed by the

ninhydrin test. The Fmoc deprotection was achieved by passing a solution of 20% 4-

methylpiperidine in DMF into the reaction vessels containing peptide-bound resin. Peptide

chain was further extended with Fmoc-Asn-OH (155.14 mg, 0.25 mmol), Fmoc-Lys(Boc)-

OH (121.3 mg, 0.25 mmol), Fmoc-Arg(Pbf)-OH (168.6 mg, 0.25 mmol), Fmoc-Lys(Boc)-

OH (121.3 mg, 0.25 mmol), Fmoc-Lys(Alloc)-OH (117.6 mg, 0.25 mmol), Fmoc-D-

tyr(tBu)-OH (119.47 mg, 0.25 mmol) and Fmoc-D-ile-OH (91.88 mg, 0.25 mmol). Allyl

protecting groups of lysine and glutamic acid were deprotected by palladium (0) catalyst in

DCM with acetic acid and N-methylmorpholine (37: 2: 1). Cyclization was carried out

between -acidic functionality of glutamic acid and ϵ amino group of lysine side chain by

using oxyma pure and DIC. The resin was washed after each coupling / deprotection step.

The terminal NH2 group of D- ile was deprotected by 20% 4-methylpiperidine and resin

132

bound peptide was treated with 90% TFA cocktail (TFA: phenol: H2O: TIPS, 90: 4: 4: 2)

for two hours for the simultaneous cleavage of peptide from the resin and side chain

deprotection. The peptide was precipitated out with cold ether and further purified by

HPLC using isocratic solvent system of 60% acetonitrile in water. The structure of purified

product 15 was elucidated by interpretation of spectral data.

3.3.3.1. Characterization of Peptide 15:




amino acid Fmoc-Glu-α-allyl-OH (106.4 mg, 0.25 mmol), was loaded by using PyBop

(benzotriazol-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluoro-phosphate) (81.1 g, 0.156

mmol) and N,N-diisiopropylethylamine (DIEA, 44.5 µl, 0.26 mmol. The reaction was

gently shaked for two hours. The coupling of amino acid was confirmed by ninhydrin test.

The Fmoc deprotection was performed by passing a solution of 20% 4-methylpiperidine in

DMF for 30 min into the reaction vessels containing peptide-bound resin. Peptide chain

was further extended by coupling with Fmoc-Asn-OH (2.37g, 3.9 mmol), Fmoc-Lys(Boc)-

OH (1.86 g, 3.9 mmol) Fmoc-Arg(Pbf)-OH (2.58 g, 3.9 mmol), Fmoc-Lys(Boc)-OH (1.86

g, 3.9 mmol), Fmoc-Lys(Alloc)-OH (1.80 g, 3.9 mmol), Fmoc-D-tyr(tBu)-OH (1.82g, 3.9

mmol), and Fmoc-d-ile-OH (1.40g, 3.9 mmol). ϵ NH2 group of lysine and α acid group of

glutamic acid was deprotected by palladium (0) catalyst in DCM with acetic acid and N-

133

methylmorpholine (37: 2: 1). Cyclization occurs between acid group of glutamic acid and

NH2 group of lysine side chain using oxyma pure and DIC. The Fmoc group of D-ile was

removed by 20% 4-methylpiperidine and peptide conjugation with temporin ICea peptide

was initiated by coupling with Fmoc-Phe-OH (1.80 g, 3.9 mmol), Fmoc-Ile-OH( 1.40 g,

3.9 mmol), Fmoc-Ser(tBu)-OH (1.52 g, 3.9 mmol), Fmoc-Asn(trt)-OH (2.37 g, 3.9 mmol),

Fmoc-Ile-OH (1.40 g, 3.9 mmol), Fmoc-Ile-OH (1.40 g, 3.9 mmol), Fmoc-Asn(trt)-OH

(2.37 g, 3.9 mmol), Fmoc-Ala-OH (1.23 g, 3.9 mmol), Fmoc-Ile-OH (1.40 g, 3.9 mmol),

Fmoc-Lys(Boc)-OH (1.86 g, 3.9 mmol), Fmoc-Lys(Boc)-OH (1.86 g, 3.9 mmol), Fmoc-

Leu-OH (1.40 g, 3.9 mmol), Fmoc-Asp(tBu)-OH (1.63 g, 3.9 mmol), Fmoc-Val-OH (1.35

g, 3.9 mmol), Fmoc-Phe-OH (1.80 g, 3.9 mmol). The NH2 group of Phe residue was

deprotected and the resin was treated with 90% TFA mixture (TFA: EDT: H2O: TIPS, 94:

2.5: 2.5: 1) for the simultaneous cleavage of peptide from resin and removal of all side

chain protecting groups. The cold diethyl ether was used for precipitation of peptide and

further purified by HPLC using isocratic solvent system of 60% acetonitrile in water. The

structure of purified product 16 was elucidated by interpretation of spectral data.

3.3.4.1. Characterization of Peptide 16:

3.4. Synthesis Procedure for Library 2 (stylissatin A Analogues):

Peptide 26 to 31 was synthesized by solid-phase chemistry by using Fmoc protocol on 4-

benzyloxybenzyl alcohol resin (Wang resin). The detail synthetic procedure was discussed

below.


Peptide analogue 26 was synthesized by using Fmoc solid-phase peptide synthesis protocol

on 4-benzyloxybenzyl alcohol resin (Wang resin, 100 – 200 µm, 3 g, 1.2 mmol / g).

134

(Chem-impex Int. INC. USA). The properly soaked resin was loaded with first amino acid

Fmoc-Glu-α-allyl (3.3 g, 8.0 mol) in the presence of coupling agents, oxyma pure (1.91g,

13.4 mmol) and DIC (2.113 ml, 13.5 mmol) along with 4-dimethylamino pyridine (32.9

mg, 0.269 mmol) as a catalyst. The reaction was gently shaked for two hours and coupling

was confirmed by ninhydrin test. After Fmoc deprotection with a solution of 20% 4-

methylpiperidine in DMF, the next amino acids, Fmoc-Phe-OH (2.0 g, 5.39 mmol), Fmoc-

Tyr(tBu)-OH (2.6 g, 5.4 mmol), Fmoc-Ile-OH (1.9 g, 5.4 mmol), Fmoc-Phe-OH (2.0 g,

5.39 mmol), Fmoc-Ala-OH (1.6 g, 5.1 mmol), Fmoc-Ile-OH (1.9 mg, 5.4 mmol) were

sequentially attached to the first residue Glu. The deprotection of allyl group of glutamic

acid was carried out by using palladium (0) in DCM, acetic acid and N-methylmorpholine

(37: 2: 1) for 24 hours. The terminal NH2 group of Ile residue was deprotected by 20 % 4-

methylpiperidine and cyclization was allowed between α acidic group of glutamic acid and

amino group of Ile by using oxyma pure and DIC. The resin was thoroughly washed after

each coupling / deprotection step. The cyclized product was cleaved from resin by 95%

TFA in DCM / TIS (few drops). The cleaved product was precipitated out by cold diethyl

ether and precipitate was further washed with ether and purified by Recycling HPLC using

isocratic solvent system of acetonitrile/ water (ACN: H2O 6: 4) 0.1% TFA. The structure

of pure peptide 26 was elucidated by interpretation of spectral data.


135


Peptide analogue 27 was synthesized by using Fmoc solid-phase peptide synthesis protocol







methylpiperidine in DMF, the next amino acids, Fmoc-Ile-OH (1.9 g, 5.4 mmol), Fmoc-

Ala-OH (1.6 g, 5.1 mmol), Fmoc-Phe-OH (2.0 g, 5.39 mmol), Fmoc-Tyr(tBu)-OH (2.6 g,

5.4 mmol), Fmoc-Ile-OH (1.9 mg, 5.4 mmol) and Fmoc-Phe-OH (2.0 g, 5.39 mmol). Acid

group of glutamic acid was deprotected by using palladium (0) in DCM, acetic acid and N-

methylmorpholine (37: 2: 1) for 24 hours. The terminal NH2 group of Phe residue was

deprotected by 20% 4-methylpiperidine and cyclization was allowed between α acidic

group of glutamic acid and amino group of Phe using oxymapure and DIC. The resin was

thoroughly washed after each coupling / deprotection step. The cyclized product was

cleaved from resin by 95% TFA in DCM with TIS (few drops). The cleaved product was

precipitated out by cold diethyl ether and precipitate was further washed with ether and

purified by Recycling HPLC using isocratic solvent system of acetonitrile/ water (ACN:

H2O 6: 4) 0.1% TFA. The structure of pure peptide 27 was elucidated by interpretation of

spectral data.


136

3.4.3 Synthesis of Cyclic Peptide 28.

Peptide 28 was synthesized by using Fmoc solid-phase peptide synthesis protocol on 4-

benzyloxybenzyl alcohol resin (Wang resin, 100 – 200 µm, 3 g, 1.2 mmol / g). (Chem-

impex Int. INC. USA). The properly soaked resin was loaded with first amino acid Fmoc-

Glu-α-allyl (3.3 g, 8.0 mol) in the presence of coupling agents, oxyma pure (1.91g, 13.4

mmol) and DIC (2.113 ml, 13.5 mmol) along with 4-dimethylamino pyridine (32.9 mg,

0.269 mmol) as a catalyst. The reaction was gently shaked for two hours and coupling was

confirmed by ninhydrin test. After Fmoc deprotection with a solution of 20% 4-

methylpiperidine in DMF, the next amino acids, Fmoc-Ala-OH (1.6 g, 5.1 mmol), Fmoc-

Tyr(tBu)-OH (2.6 g, 5.4 mmol), Fmoc-Ile-OH (1.9 g, 5.4 mmol), Fmoc-Phe-OH (2.0 g,

5.39 mmol), Fmoc-Pro-OH (1.8 g, 5.3 mmol) and Fmoc-Ile-OH (1.9 g, 5.4 mmol). Acid


methylmorpholine (37: 2: 1) for 24 hours. The terminal NH2 group of Ile residue was


group of glutamic acid and amino group of Ile using oxymapure and DIC. The resin was






spectral data.


137










Tyr(tBu)-OH (2.6 g, 5.4 mmol), Fmoc-Ile-OH (1.9 g, 5.4 mmol), Fmoc-Ala-OH (1.6 g, 5.1

mmol), Fmoc-Pro-OH (1.8 g, 5.3 mmol) and Fmoc-Ile-OH (1.9 g, 5.4 mmol). Acid group

of glutamic acid was deprotected by using palladium (0) in DCM, acetic acid and N-



group of glutamic acid and amino group of Ile using oxyma pure and DIC. The resin was






spectral data.

3.4.4.1. Characterization Data of Peptide 29

138









methylpiperidine in DMF, the next amino acids, Fmoc-Phe-OH (2.0 g, 5.39 mmol),Fmoc-

Ala-OH (1.6 g, 5.1 mmol), Fmoc-Ile-OH (1.9 g, 5.4 mmol), Fmoc-Phe-OH (2.0 g, 5.39

mmol), Fmoc-Pro-OH (1.8 g, 5.3 mmol) and Fmoc-Ile-OH (1.9 g, 5.4 mmol). Acid group

of glutamic acid was deprotected by using palladium (0) in DCM, acetic acid and N-









spectral data.


139


Cyclic peptide 31 was synthesized by using Fmoc solid-phase peptide synthesis protocol








Tyr(tBu)-OH (2.6 g, 5.4 mmol), Fmoc-Ala-OH (1.6 g, 5.1 mmol), Fmoc-Phe-OH (2.0 g,

5.39 mmol), Fmoc-Pro-OH (1.8 g) (5.3 mmol) and Fmoc-Ile-OH (1.9 g) (5.4 mmol). Acid










spectral data.


140

3.5. Screening Protocol:

3.5.1. MTT (3- (4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide) Assay

Cytotoxicity of compounds was determined using (3- (4, 5-Dimethylthiazol-2-yl)-2, 5-

Diphenyltetrazolium Bromide) reagent (Hansen et al., 1989). Cell lines were purchased

from ATCC (USA) and used in the present study were NCI-H460 (lung cancer cells), and

MCF-7 (breast cancer cells). The NCI-H460 cell were cultured at the concentrations of 2 x

104 /ml, and MCF-7 were cultured at 3 x 104 /ml. Plates were incubated overnight at 37°C

with 5% CO2. Following incubation, cells were treated for 48 hrs. with 25, 50, 100 and 200

µM with the test compounds. Since the test compounds were soluble in DMSO (0.5%)

therefore a vehicle control was also set. Following 48 hr. treatment, wells were emptied

and MTT dye (0.5 mg / mL final concentration in each well) along with the fresh medium

was added. Plates were allowed to stand for 4 hours in sterile environment. Then,

incubation medium was removed and formazan crystals were dissolved in DMSO with

gentle shaking. Plates were read at 550 nm filter of photometer (Thermofisher Scientific)

and percent inhibition was calculated using the following formula:

3.5.2 Chemiluminescence Assay

Luminol or lucigenin enhanced chemiluminescence assays were performed to study the

effect of compounds on ROS from phagocytes. This assay was performed as described by

Mesaik et al (Mesaik et al., 2012).

3.5.3. Nitric Oxide Screening Protocol:

Mouse macrophage cell lines (J774.2) (European Collection of Cell Cultures, UK) was

used to carry out the nitric oxide assay. The sample peptide was used in three different

concentrations (2, 10, and 50μM) and the protocol was used as described in Shaheen et al

(Shaheen et al., 2014), (Shaheen et al., 2016).

% inhibition = [(OD of the treated well- OD of untreated well) / OD of the untreated well] x 100

141

3.5.4. IL-2 Production and Quantification:

The IL-2 production and quantification was performed on Jurkat (human T lymphocyte

leukemia) cells. The protocol used was reported in Shaheen et al (Shaheen et al., 2016) and

according to manufacturer’s instructions (Manger, Hardy, Weiss, & Stobo, 1986).

3.4.12. T-cell Proliferation Assay:

Cell proliferation assay performed by standard thymidine, incorporation assay (Tabudravu,

Morris, Kettenes-van den Bosch, & Jaspars, 2002). The complete procedure was reported

in Shaheen et al (Shaheen et al., 2016).

142

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Glossary

Anticancer/ Antitumor Agents

Agents that inhibit the formation of tumor or to stop the irregular cell division are known

as anticancer or antitumor agents.

Bioassay

The comparison of test and standard compound in-vitro and in-vivo analysis is known as

bioassay.

Broad-Band Decoupled 13C-NMR Spectrum

It is a fully decoupled 13C-NMR spectrum, which gives information about the number of

carbon atoms in a molecule and their electronic environment.

Chemical Shifts (δ)

The difference between the precession frequencies of a nucleus to standard (TMS) is called

chemical shift value. It is expressed in ppm (Parts per million).

Chromatography

It is a separation technique in which the constituents of the mixture are separated between

the two phases (Stationary and mobile phases).

COSY-45° Spectrum

It is a homonuclear two-dimensional NMR spectroscopic technique, used for the

determination of vicinal and geminal 1H-1H couplings.

Coupling Constant (J)

The magnitude of splitting of NMR signal is known as coupling constant. It is a constant

number which is employed to determine the scalar coupling between adjacent protons and

other nuclei. It also determines the strength of interaction of a proton with a non-equivalent

proton on same carbon (geminal coupling or 1, 2 coupling) or on adjacent carbon (vicinal

coupling or 1,3 coupling). It is expressed in cycle/second or Hz.

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Distortionless Enhancement by Polarization Transfer (DEPT)

A one-dimensional 13C-NMR spectroscopic technique, employed to differentiate the

multiplicity among CH3, CH2, and CH. The DEPT spectra is recoded at different pulse

angles,i.e. = 45°, 90°, and 135°.

Electrospray Ionization Mass Spectrometry (ESI-MS)

The low resolution mass spectrum in which the ionization and fragmentation of the

compound is brought about by the bombardment of electron beam with 70 ev energy. This

gives an important clue about the structure of molecule through fragmentation pattern.

Elute

The process in which the solute molecules is carrying away by solvent in a

chromatographic operation is called elute.

Eluent

Mobile phase which is used in chromatographic technique for the separation of compounds

from mixture is called eluent.

Elution

The process by which the dissolved constituents in column were carried by mobile phase is

called elution.

Flow Rate

The time required for mobile phase to pass through the column is called flow rate.

Gradient Elution

The process in which the polarity of mobile phase changed with time is called gradient

elution. It is used to decrease the separation time by increasing the polarity of mobile phase

over time in chromatography. It is also known solvent programming system. Gradient

elution may be continuous or stepwise. In routine, HPLC based separation technique,

binary, tertiary and quaternary gradients solvent are used.

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Heteronuclear Multiple Bond Connectivity (HMBC)

It is a heteronuclear two-dimensional NMR technique, which determines the long-range

couplings between carbons and hydrogens (1J, 2J, 3J and 4J in conjugated system).

Heteronuclear Single Bond Coherence (HSQC)

It is an inverse heteronuclear two-dimensional NMR spectroscopy, which is used to

determinethe 1H/13C one bond correlations.

High-resolution Electron Impact Mass Spectrum (HREI-MS)

It is a mass spectrometry technique which is used directly for the determination of

elemental composition of a compound through exact mass of the molecule. The HREI-MS

is recorded by a double focusing mass spectrometer.

Heteronuclear Multiple Bond Connectivity (HMBC)

It is an inverse two-dimensional heteronuclear NMR experiment, which determines the

long-range coupling between proton and carbon (1H/13C, 1J, 2J, 3J and 4J in conjugation).

Heteronuclear Single Quantum Coherence (HSQC)

It is an inverse two-dimensional NMR technique used to deduce the direct linkage of

proton to carbon (1H/13C) through one-bond connectivity.

Inflammation

The body’s adaptive and immediate to tissue and cell damage by pathogens stimuli, or

physical injuries is called Inflammation.

Pharmacokinetic

The process by which a drug is absorbed, distributed, metabolized and eliminated by the

body.

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Infrared Spectroscopy

It is a spectroscopic technique, which is used for the determination of functionalities in

molecule. The infrared spectra are generated by the absorption of infrared radiations in

region ranging from 320-4000 cm-1 by a substance.

Isocratic solvent system

The process in which the polarity of mobile phase remains same throughout the operation

in column chromatography is known as isocratic solvent system.

Mobile Phase

The solvent (liquid, gas) used to elute the solute in column chromatography is called

mobile phase.

Optical Rotation

It is a physical property of a molecule that rotates the plane polarized light either towards

the right or towards the left. Its presence in a molecule indicates the chiral nature of the

molecule.

Proton-NMR Spectrum (1H-NMR)

A one-dimensional NMR technique which gives information about the electronic

environment of a proton in a molecule.

TOCSY

TOCSY is a total correlation spectroscopy, which is also called HOHAHA (Homonuclear

Hartmann Hahn). It provides couplings of all the protons in an isolated spin system.

172

Abbreviation

ACPs Anticancer peptides

Boc tert-Butoxycarbonyl

Cbz Benzyloxycarbonyl

DCC Dicyclohexylcarbodiimide

DIC Diisopropylcarbodiimide

DIPEA Diisopropylethylamine (Hünig’s base)

DMF Dimethylformamide

Fmoc Fluoren-9-ylmethyloxycarbonyl

PyBOP Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluoro

phosphate

HATU N-[(Dimethylamino)-1H-1, 2, 3-triazole [4, 5-b]pyridin-1- yl methy

lene]-N-methylmethanaminiumhexafluorophosphate N-oxide

HBTU N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylene]-N-methyl

methan-aminium hexafluorophosphate N-oxide

HOAt 1-Hydroxy-7-azabenzotriazole

HOBt 1-Hydroxybenzotriazole

HPLC High performance liquid chromatography

IC50 Inhibitor concentration giving 50 % inhibition

MALDI – TOF Matrix Assisted Laser Desorption/Ionization Time-of-Flight

NMM N-Methylmorpholine

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NMP N-Methyl-2-pyrrolidone

NMR Nuclear magnetic resonance (spectroscopy)

Pbf 2, 2, 4, 6, 7-Pentamethyldihydrobenzofuran-5-sulfonyle

SAR Structure–activity relationship

TBTU O-(Benzotriazol-1-yl)-N, N, N', N'-tetra methyl uranium tetra fluoro

borate

TFA Trifluoroacetic acid

TIS Triisopropylsilane

Ala (L) Alanine

ala (D) Alanine

BNZ 2-Amino-1- methyl benzimidazole

Gly Glycine

iPr Isopropyl

Ileu Isoleucine

Lys Lysine

2-Nal 2-Naphthyl alanine

Thr Threonine

Trp Triptophane

Tyr Tyrosine

synthesis and anticancer screening of combinatorial

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