An approach to the semisynthesis of acyl carrier protein : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University

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AN APPROnCH TO THE SEMISYNTHESI S

OF

ACYL CnRRIER PROTEIN

A ^thesis pres�ntcd in partial fulfilmGPt of the requirements for the ^degree

of Doctor of Phi^losophy in Chemistry at Massey University

Ross Leonard Prestidge

1 9 7 7

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ABSTRACT

The pu rpose of thi s s tudy wa s to prepa r e seve r a l

s e m i syn the t i c analogues o f the a c y l carrier prote in o f

�·

^{co l}

�

^,

u s i n g the Merr i f ie ld s o l i d - phase me thod for the prepa r a t ion o f the syn the t i c l - 6 he xapept i de i n the prote c te d form . I t w a s hoped tha t the syn the s i s o f the se ana logue s , and the i r eva l ua t ion b y the 1 �C02 a s say a n d b y othe r method s , mi ght cont r i bute to the s tudy o f pro t e i n s truc ture . and fun c t ion i n gene r a l , and those of ACP in par t i c u l ar .

The s tr a tegy chosen for the synthe s i s o f the protec ted 1 - 6 hexapeptide was to employ a c i d - s table protec t i n g groups for g lutami c , a sparti c , and arg i n ine re s id ue s , and to c le ave the completed peptide from the r e s i n in a protected form u s i n g HBr in ace t i c a c i d . The E-n i troben z y l g roup wa s

cho s en for the protec t ion o f ac i d ic amino a c i d s id e - cha iris , a s th i s group has often been s tated to be s tab l e to H B r in ace t i c a c i d .

I n the course o f thi s work , howeve r , it became obvious that the stab i l i ty of the E-n i troben z y l group to HBr i n

ace t i c a c i d , wh i l e greater than that o f other ben z y l e s te r s , wa s n o t suf fic i e n t to a l low the c onven ient preparat i on o f pep t i d e s pro t e c ted with this b lo c k ing group . C le avage o f the prote c t i n g group o c curred t o a n apprec i ab l e extent , and th u s the prod u c t was contaminated wi th a mixture o f

depro te c ted pep t ide s .

Other disadvantages o1 ^the E-nitrobe�zyl group ^were a l s o e n c oun tered dur i n g th i s s tudy . I n part i c u l a r , prob l ems

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o f i n s tabi l i ty wer� encountered i n the prepa r a ti o n o f

protected amino a c id i n t e rmed i a t e s for pep t i d e synthe s i s . The

E_- n i troben z y l group vJa s a l so found to g ive lmv coup l i n g y i e l d s d u r i n g synthe s i s , and poor s o l ub i l i ty d�r i n g pur i f i c a t ion , to amino a c id s and pep t i d e s protected w i th i t . I t i s c l e a r that the problems of i n s tab i l i ty and i n solub i l i ty a s s o c ia t e d with E_- n i trobenzyl e s ter pro t e c t ion were aggravated by the f a c t th a t man y o f t h e target pep t i d e s bore two E_-n i trobe n z y l groups pe r mo l e cu l e .

The coup l i n g o f a crude h�xape ptide to the n a t i ve 7-77 pep t i d e y i e l ded a produc t wh i ch gave some a c t iv i ty i n the

1 4C0 2 a s s ay a f ter exte n s ive pur i f i cation o f the semi synthe t i c

prote i n . Th i s r e s u l t , toge ther w i th amino a c i d ana l y s i s o f

the c r ude pe ptide , impl i e s that the d e s ired protected hexa

pep t i d e composed a s i gn i f i c ant proport ion o f the peptid.e a f t e r c l e avage from t h e re s in . The i n s o l ub i l i ty c o n f erred by t h e ^·I

E- n i troben z y l prote c t i n g group , howeve r , pre sumab l y caused the t a r g e t peptide to be s e l e c t ive ly l o s t from the mixture dur ing the pur i f i c a t i on procedur e s , in favour o f deprot e c ted

pep t i de s and d e l e t ion pep t i d e s having greater s o l ub i l i ty . For thi s rea son , the ma j or produc ts wh i c h were pur i f i e d

from t h e crude c le aved peptid e s were pentape p t i d e s l ac k i ng one g l u tami c acid r e s idue b e a r i n g the E- n i troben z y l pro t e c t i n g group .

The se pentapeptid e s , when coup l e d to the n a t i ve 7-77 pept i d e , gave products wh i c h were i na c t ive i n the 1 4C 0 2 as say . Th i s resu l t is interest i n g, i n tha t i t s ugge sts that both G l u4 and G l u 5 are e s s e n t i a l to the i n te r a c t i on ^between

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the 1 - 6 and 7 - 7 7 pept i d e s wh ich mai n ta i n s the a c t ive

con f o rmat i on of ACP . Howeve r , these d ata mus t be treated a s t e n t a t ive un t i l an a c tive s emisyn thetic ^ACP s a t i sfy i ng ana l y t i c a l c r i te r i a i s prepa red by th i s method . The

re s u l t s ob tained by a s s ay in g the pur i f i e d semi s y n thet i c ACP prepared from a crude hcxapept i de sugge s t that a f u l l y

a c t i ve s em i s y n the t i c ACP c o u l d be prepared by thi s me thod , but they cannot be regarded a s conc l u s ive i n the ab sence o f suppo r t i n g a n a l y t i c a l d ata .

The approach to s emi s yn the s i s c hosen for th i s study appear s to be bas i c a l l y sound , but new acid- s t a b l e

pro te c t i n g groups f o r c a rbox y l functions a r e c le arly req u i r e d i f the me thod i s to become gener a l l y u s e fu l .

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ACKNOWLEDGEMENTS

I would l i k e to thank my supervi sor , Dr W.S . Han cock , fo r h i s advice and encourageme n t throughout the cour s e o f t h i s p ro j e c t . I am also grate ful to many other memb e r s o f the s ta f f o f the Depar tmen t of Chem i s try , B iochem� s t ry and B iophy s i c s for the i r help . I n par t i cular , I � i sh to thank Dr G .. G . Midwin ter for per formin� nume rous amino a c i d an a l y se s , Pro f e s s o r R . Hodg e s for ma s s spe c t r al determi n a t ion s , and

Dr C . H . Moore for c r i t i c al comments on a dra f t of t h i s t h e s i s .

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TABLE OF CONTENTS

ABS TRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS L I S T OF FIGURES INTRODUCTION SECTION 1

Sub s t i tuted Ben z y l E s te r s a s S id e -Cha i n Prote c t i n g Groups in S o l i d - Ph a s e Pept ide

( i i )

( v i ) ( v i i i )

1

Synth e s i s 2 1

Part A - Synth e s i s o f the D i e s ter s of Glutami c

Ac i d 2 9

Part B - Syn th e s i s o f t h e y-Mon o e s te r s o f

Glutamic Acid 3 1

Part C - Syn the s i s of t-Boc Der i vative s 3 0 Part D - Stabi l i ty o f S ub s t ituted Benzyl

Monoe s ters o f Glu tum i c A c i d to

Ac ido l j t i c C l e av a g e 4 3 Part E - Synth e s i s o f Prote c te d Tr ipep t i d e s 4 8 SECTION 2

Synth e s i s and Pur i f i c a t i on o f th e Fu lly

Deprotected Ac t ive E s t e r De r ivative s o f the ACP l- 6 Hexapeptide and Seve ra l Aha logue s

Introduction 5 4

Part A - Syn the s i s o f the t - Boc Arg (N0 2 ) Re s i n 6 1 Part B - Synthe s i s o f the Prote c te d ACP 1 - 6

Hexapeptide and C l e avage f rom the

Re s i n 6 3

Par t C ^- Purification b� the Pro�ected ACP

1 - 6 Ile xapcptj_de 6 9

Part ^D - Prepa r a t i on o f An a l o gues o f the

Protected ACP 1-G H exapcpt ide 8 7

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Part E ^- Preparat i on and Pur i f i c a t ion o f the Penta chlorophen y l Ac t ive E s te r s

o f t h e Protec ted Hexape p t i des 8 9 Part F - Depro t e c t ion o f the Protected

Pep t i de Active E s t e r s 9 0 S ECTION 3

I s o l a t i on o f t h e N a tive 7-77 Peptide ,

Semi s y n t h e t i c Coup l in g , and As s a y o f Produc t s In trod u c t ion

Part A - Growth of E. Co l i Stra i n B and S t r a i n H-99

Part B - I s o l a t i on and Puri f i c a t ion o f Acy l Car r i e r Prote i n

Part C - Ace ty l a t ion and Trypt i c C l eavage Part ^D - Sem i synthetic Coup l i n g

P a r t E The 1 4 C02 Exchange As s ay C ONCLUSION

9 4

9 9

101 105 110 114 123

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REFERENCES 124.

.I

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F igure 1 F i gure 2

F igure 3 F i gure 4 F i gure 5 F igure 6

F igure 7 F igure 8

LIST OF F I GURES

S impli f ied Outl i n e o f F r a gment Synthe s i s and Semi synthe s i s

The U se o f Block ing G roups in Fragmen t Synthe s i s and Semi synthe s i s to Ach ieve Spec i f i c Peptide Bond F o rma t i on

4

6 Me rr i f i eld S olid - ph a se Pe p t i d e Syn th e s i s 7 The Re a c t i o n s o f Fatty Ac i d Syn the s i s 1 1 The Amino Ac id Sequence o f the Acyl

Carr i e r Prote i n o f E. Co l i 1 3 The Ac t iv i t

i

o f Synth e t i c An a logues o f

ACP i n the 4C 0 2 As s ay 1 7

The Sem i syn the s i s o f ACP 19

S impl i f i e d Outl i n e of the Syn thesis o f Substituted y-Monoe s t e r s o f Glutam i c Acid by the Coppe r - c a t a l y s e d Hydrolysi s o f the

Corre spond ing D i e s te r s 23

F igure 9 Copper^-cat a lysed Hyd roly s i s o f Glu tami c

Ac i d Diesters 32

F i gure 10 The Ra t e s of Copper---c atalysed Hydrolysis for Sub s titu ted Ben zyl E s te r s o f Glu tami c

Ac i d 3 6

F igure 11 F avoured and Non -Favoured Comple x e s

B e tween Cu ( I I ) and Glu tamic and Aspa r t i c

Ac i d s D i e s te r s 3 8

F�gure 12 H i gh-Pre s sure Liquid Chromatogr aphy o f Glu tam i c Ac id y-Benzyl E s te r , and the Rate of Hydroly s i s of Glu ( ODzl ) and Glu ( OBzl

£- C l ) by Tr i f luoroac e t i c Ac i d ⁴⁵ F igure 1 3 The Stab i l i ty o f the S ub s t i tuted Ben z y l

E s te r s o f Glutam i c Ac i d a n d o f G lutamy l

P e p t id e s to HBr - Ac e t i c Ac i d 4 6 F i gure 1 4 HBr^-Ac e t i c Acid C l e avage o f Protec ted

Tr i pepti d e s from the Re s in 48

F igure 1 5 HBr -Ace tic Ac id Cleava0e of H-Gly-Glu {ODzl E_-NO:> )-Ala-OH from the Re s in . Paper Electro- phore s i s- o f C l e avage Produc t s 5 1

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F i gure 1 6 Synthe s i s of the Pro t e c ted Form o f the

ACP 1-6 Hexapep t i d e 6 4

F i gure 1 7 Typ i c a l Coup l i n g Cyc l e for Addi tion o f One Ami n o ^Acid Re s id u e t o t he Re s in·-J3oun d

P ept i d e Cha i n 65

F igure 18 _Picric A c i d Ana ly s i s o f the Amoun t of F r e e Amino Groups on a ne s in -Bound Ami n o Ac i d

o r Pep t ide 67

F i gure 1 9 Procedure f o r C l e avage o f.a Protec te d P e p t i d e from t h e Re s i n u s i n g HBr-Acetic

Ac id 68

F i gure 2 0 Ge l F i l t r a t ion a n d P a r t i t io n Ch roma t

ography o f the ^Protected Form o f the ACP

1 -6 Hexapeptide 7 2

F i gure 2 1 S e phadex LH-20 Chroma tography o f the

Prote c t e d Form of the ACP l-6 Hexape pt i d e .

· Typ i c a l U n s a t i s f a c to r y S eparat ion s 7 4 F i gure 2 2 Se phad ex LII- 2 0 Chromatography o f the

P r o te c ted Form of the ACP l-6 Hexapep t i d e , a n d An a lyt i c a l H PLC o f the Major Pept ide

Peak 76

Figure 2 3 An a lyt i c a l H PLC of Protected Pept ide s .

Un suc c e s sful Separat i o n Cond ition s 78 F i g ure 2 4 The E f f e ct o f Recyc l i ng on the Re s o l u t i o n

o f the C o l umn S e t U s ed f o r Prepa r a t ive

H P LC 80

F i gure 2 5 Pur i f ic a t ion o f a Pro t e c t e d Pep t i d e by

P r epar a t i ve HPLC 86

F i gure 26 The U . V . Spec trum o f P e n t ach lorophe n o l

Dur i n g C a t a l y t i c Hydrogen a t ion 92 F igure 2 7 I6n-Ex c h ange Chromatog�aphy o f ( 1 4 C ) -

l ab e l l e d ACP 103

F i gure 28 Ge l Fi l tr ation and Ion- Exchange

C hroma tography on the ACP 7 - 7 7 P e p t i d e 1 06 F i gure 2 9 Ami no Ac i d Compos i ti o n o f the ACP 7- 7 7

P e p t ide 108

F i gure 3 0 1\..m ino Acid Compos i t i on o f Semisynthetic

ACP 1 1 2

Fi gure 31 1 ''C02 Assay of JI.CP and Acet.y lated ACP 1 1 9

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F i gure 3 2 1�C02 As s ay of ACP ( 7-7 7 ) and o f ACP i n

the p r e s e n c e o f ACP ( 7-7 7 ) 1 20 F i gure 3 3 1 4 C02 A s s a y of ACP , ACP Der iva t ive s , and

Semisyn the t i c ACP 1 2 2

L I ST OF TABLES

Tab l e I The Syn the s i s o f G lu t am i c Acid D i e s t e r s 3 0 Tabl e II Y i e ld o f Asp and G l u Mon oe s ters Prepared

by the Coppe r-c a t a l y s e d Hyd roly s i s o f

Corre s ponding Di e s te r s 3 4

Table Ill Rate Constants for the Cu ( II ) -Cata lysed

Hyd ro l y s i s of G l u t am i c Ac id Diesters 35 TABLE IV Semi syn the tic Coupl ing Y i e l d s Us ing

Di f fe ren t Coupling S o l v e n t s 113 ( x )