FMOC METHODOLOGY - Solid phase peptide synthesis on a beaded cellulose support : a thesis prese

9 1 CBAP'l'ER 5 SOLm PRASE PEPTIDE SYN'l'BES :rS ON PERLOZA : I'MOC METHODOLOGY

5 . 1 rNTRODUCT:rON

The 9 -fluorenylmethoxycarbonyl ( Fmoc ) amine protecting group, int roduced by Ca rp i n o a n d Han ( 1 9 7 0 , 1 9 7 2 ) , i s c le a ved ( F i g u r e 5 . 1 ) by base p romoted p- e l imination . P iperidine in DMF is most frequently used for c leavage o f t he Fmoc group (Fields and Noble , 1 9 9 0 ) .

Use o f the Fmoc group for temporary Na-amino protect ion in solid phase pept ide s yn t hes i s al lowed an o rt hogona l s ynthes i s methodo logy to be e s tablished . An orthogonal system has been defined a s

. . . a set of completely independent cl asses o f protecting groups, s uch that ea ch class of groups can be removed in any order and in the presence of all other classes ( Kneib-Cordonie r et a I , 1 9 9 0 ) .

T h e o r t h o go n a l n a t u re o f t h e Fmo c me t h odo l o gy de r ives f rom the d i f f e r e n c e b e t w e e n the c l e a v a g e me c h a n i sm of the Fmoc Na-amino protection ( base catalysed P el imi nat ion ) , a nd the acidolyt ic cleavage o f the s ide chain protect ing groups and pept ide-resin link . With use of the Fmoc group f o r Na-amino protect ion, it was pos sible t o use side cha in protect ing groups and peptide-resin linkages more acid-labile than those required for the Boc methodology . :rt was found, for example , that TFA- I ab i l e pept ide - r e s i n l inkages of t he p-al koxybe n zyl e st e r type ( Wa ng , 1 9 7 3 ) could be used in con junct ion with t e rt i a ry butyl ( tBu ) b a s ed s ide c h a in protec t i on f o r S P P S when us ing Fmoc f o r Na-amino p r o t e c t i o n . A d i a g r am o f t h e Fmo c S P P S me t h o d o l o gy i s g i ven a s F igure 5 . 2

The Fmo c / t B u s t rategy , a s it i s s omet imes known, a l lowed synthesis of pept ides u s i n g much milder cond i t i ons than those used for the Boc /Bzl s t r a tegy . Whe reas the Boc methodo logy requi red repetit ive acidolytic c le a vage o f the Boc group us ing TFA, followed by peptide cleavage with st rong acid ( fo r example l iquid HF) , the Fmoc method used a much gent ler p i p e r i d i n e t re a tme nt f o r c l e a vage o f t h e Fmoc t emp o r a r y Na-amino p r o t e c t ion , f o l lowed by a s i ng le TFA t rea tment for c leavage' of the pept ide f rom the support .

Figure 5 . 1 Mechanism of cleavage of the Fmoc Na-amino protecting group Fmoc-aminoacyl-Support � 11 I 11

��

0 R 0 CH-CHrO-C-NH-CH-C-O-CH2-Support � , � ...: - 11 I 11

��

0 R 0 C-CH2-0-C-NH-CH-C-O-CHrSuppon � \ � Dibenzofulvene-piperidine adduct + + Piperidine + + Hr

O

o R 0 - 11 O-C- upport R 0 11

H-O

Figure 5 . 2 Fmoc SPPS methodology illustrated by the synthesis of a dipeptide

Fmoc-amino acyl-HM.P A-tinker-Suppon

RI 0 0

FmOC-NH-

t

H-

�

-O-CH2

-o

0-CHr

�

-NH-Support

20% Piperidine/DMF (Deprotect) RI O

1

0 11

11

O-CHrC-NH-Suppon R2 0 I 11 Fmoc-NH-CH-C-Y

(Activated Fmoc-amino acid)

R2 0 RI 0

-0 11

0 � !J O-CH2-C-NH-Suppon R2 0 RI 0 + I 11 I Dipeptide i) 20% Piperidine/DMF

ii) 95% TFA (Cleave)

I1

The f i r s t s o l id phase pept ide syntheses us ing the Fmoc group for Na-

amino prot e c t ion were reported in 1 9 7 8 . Chang and Meienhofer fl97 8 )

used 1 % divinylbenzene cross linked polystyrene a s a support for SPPS of the 1 4 amino a c id residue pept ide dihydrosomatostatin . Atherton et al ( 1 9 7 8 a , b ) s ynthe s i sed the decapept ide 65-74 sequence o f acyl ca rrie r p r o t e i n ( ACP 6 5 -7 4 ) , f o l lowed by the 3 1 - re s i due pept ide p-endorph in, u s i n g a be a de d po lyamide s upport . F rom these beginnings, the Fmoc method has been developed and ref ined to the point where it is widely used f o r SPP S . Excellent sources o f information on the Fmoc SPPS method include Athe rton and Sheppard ( 1 9 8 9 ) , and Fields and Noble ( 1 9 9 0 ) .

As wel l a s the polystyrene and polyamide mat rices previously mentioned, o t h e r s uppo rt s which have been demonst rated a s adequate for the Fmoc me t h o d include c ontrol led po re g l a s s ( Albe r i c io et a I , 1 9 8 7 , 1 9 8 9 ) , polyaroide-kieselguhr composite (Atherton et a I , 1 9 8 1b ) , cellulose paper

( F rank and Doring 1 9 8 8a , b; Eichler et a l , 1 9 8 9 ) , cellulose cotton ( Lebl

a n d E i c h l e r , 1 9 8 9 ; E i c h l e r e t a I , 1 9 9 0 , 1 9 9 1 ) , a n d P o lyh i pe™ poly ( st yrene-co-divinylbenzene ) ( Small and She r rington, 1 9 8 9 ) .

B o t h d i s cont i n u o u s bat chwi se and cont inuous f low aut omated pept ide s ynthes isers may be used for syntheses by the Fmoc method . The type of s ynthes iser used depends on the solid support . For example, a batchwise s ynthes i ser such as the AB! 4 3 0 A is used for beaded polyamide and beaded 1 % divinyl-benzene cross linked polystyrene support s , because if they are u s ed i n f l o w s ystems t he mat r ix packs down and exc e s s ive ly high back pre s s u re s a re generated (Atherton et al, 1 9 8 1b) . A polyamide-kieselguhr c ompos ite was developed for use in cont inuous f low synthesisers such as

t he LKB Biolyn x 4 17 5 . The inorganic kieselguhr se rves to support the

p o ly am ide in t he c o lumn , without gene r a t ion o f high back pressures . Howeve r , the p o lyamide -kieselguh r support cannot be used in a shaken bat chwise synthe s iser because the f ragile kieselguhr suffers attrit ion and generates f ines which clog the filters of the reaction vessel .

A c ont inuous f low system for SPPS was seen as des irable ( Sheppard, 1 9 8 3 ) because i t offe red s ignif ic ant advantages over a batchwise system .

Th e remov a l o f excess re a c t a n t s b y a c on t i n u o u s s o l vent fl ow through a col umn bed is inherently more effici en t , economical, and

r ap i d t h a n ba t ch wi se wa sh in g . Th i s i s pa rt i c u l a rly t rue for gel a t in o u s s o l i ds wi th a very h i gh l i quid re t ention . ( Sheppa rd, 1 9 8 3 ) .

P o lyamide-kiese lguhr and beaded polyamide suppo rt s , used in a flow and b a t c h s y s t em respectively, were used for comparat ive syntheses of an octadecapept ide ( Atherton et a I , 1 9 8 3 a ) . Little dif ference , if any, was f ound in the efficiency of synthesis on the two polyamide supports us ing t he t wo dif ferent synthesisers .

One a dvant a ge o f the continuous f low s ystem was that it of fered the p o s s ib i l i t y o f r e a l - t ime mon i t o r i ng o f dep r o t ect ion and coupling r e a c t i o n s by inserting a UV f low cell into the rec irculation ci rcuit , a nd r e c o r d i n g t h e s o lu t i o n a b s o rbance w i t h t ime ( S heppard, 1 9 8 8 ; Athe rton and Sheppard, 1 9 8 9 )

T h e s o l id s uppo rt i s usua l l y func t i o n a l i sed w i t h amine o r hydroxyl group s . The p - a lkoxy benzyl ester TFA c leavable linkage between the s upport and the C-terminal Fmoc -amino acid may be int roduced in one of t h ree general ways .

1 ) T h e h y d r o xy�� thyl l in k e r mo l e c u l e may be c oupled t o an amine f u nc t i o n a l i sed s �pport di rectly, or t he linker may already be present f rom a different route (Wang, 1 97 3 ) . The C-terminal Fmoc -amino acid may t he n be e s t e r i f ie d t o the hydroxyl g roup . For example , Chang and Me ienho f e r ( 1 9 7 8 ) used DCC in the presence o f DMAP to esterify Fmoc Cys ( S -t Bu ) t o a p-ben zyloxybenzyl a lcoho l resin . P reformed symmetrical a nh yd r i de s of Fmoc - amino a c ids h a ve a l s o been used f o r loading t o hydroxyl s uppo rt s us ing DMAP catalyst . Howeve r , i t was reported that s i g n i f i c a n t r a c e mi s a t ion r e s u l t ed when DMAP was used a s c a t a l y s t ( Athe r t on et a I , 1 9 8 1 a ) . P rocedu res t o minimi se racemi sat ion during l o a d i ng of t he Fmoc-amino acid to hydroxyl suppo rts involving use of HOBt (a racemisat ion suppress ant ) have been described (van Nipsen et a I , 1 9 8 5 ) . Another problem with DMAP catalysed esterificat ion of Fmoc-amino a cids t o hydroxyl funct iona l ised support s is premature Fmoc removal by DMAP , resulting in dipept ide format ion ( Pedroso et a I , 1 9 8 3 ) . Dipeptide f o rma t ion may also be reduced by adding HOBt during the loading reaction ( Van Nipsen et a l , 1 9 8 5 ) . A ful l discus s ion of t he problems encountered

in loading Fmoc-amino acids to hydroxyl funct ionalised supports is given by F i e lds and Noble ( 1 9 9 0 ) .

2 ) The Fmoc-amino acid may be coupled t o the linker molecule prior to attachment of the l inker t o the resin . The preformed Fmoc-amino acyl l inker compound is then coupled to an amine-funct ionalised support . For e xamp l e , Bernatowic z et al ( 1 9 8 9a ) synthe s ised Fmoc-aminoac y l - 4 -oxy met h y lphenoxy ac e t i c a c id 2 , 4 -di c h l o rophenyl e s t e r s f o r coup l i ng t o amine- funct iona lised suppo rt s . The 2 , 4 -dichlo ropheny l ester served to a c t ivate the l inker c a rboxyl for coupling to the amine groups o f the s uppo rt . pyridine wa s used t o catalyse the reaction . Danie ls et al ( 1 9 9 1 ) u s ed a s im i l a r p r o c e du re e xcept a comb ina t i on of N-methyl morphol ine and HOBt were used t o catalyse the coupling reaction .

3 ) A halomethyl derivat ive of the linker molecule may be coupled to an amine- funct ionalised support , followed by displacement of the halogen by t he c e s i um s a l t o f t he Fmoc-amino a c id . For example , Colombo et al ( 1 9 8 3 ) c o u p l e d 4 - c h l o r ome t h y l p h e n o xya c e t i c a c i d to aminome t hyl poly s tyrene o r norleucyl-poly ( dimethylacry lamide ) res in to yield a 4- chloromet hylphenoxyacetyl functionalised support . The chlorine was then d i s p l a ce d by t he c e s i um s a lt o f t h e Fmo c - amino a c i d t o r e s u l t in forma t ion o f an ester bond between t he Fmoc-amino acid and the linker . One ma j o r advantage o f t h i s method i s t ha t i t res u l t s in negligible racemisat ion of the amino a cids (Mergler et a I , 1 9 8 9 ) .

A numbe r of linkers , usually of the acid labile p-alkoxybenzyl alcohol t ype ( Wang, 1 9 7 3 ) , have been used f o r Fmoc syntheses . The 4 -hydroxy methyl phenoxyacetyl ( HMPA) l inker ( Atherton et a I , 1 97 8a ) is commonly used f o r s ynthes i s o f peptide acids . Pept ide amides may be synthesised us ing l inkers which furnish the amide on cleavage with TFA, a number of these a re discussed by Bernatowicz et al ( 1 9 8 9b) . The p- [ (R, S ) -a- ( 9H f l u o re n - 9 -y l ) -me t h o x y f o rmamido } - 2 , 4 -dimet h oxyben zyl ] -phenoxyacetyl l inke r (Figure 5 . 3 ) , purchased f rom Novabiochem, was used in this study for the synthesis o f peptide amides .

Figure 5 . 3 The p- [ (R, s ) -a- ( 9H-fluoren-9-yl) -methozyformamido -

2 , 4-dimethozybenzyl] -phenozyacetyl linker for synthesis of peptide amides

NH-Fmoc

T h e Fmo c me t h od a l l owed u s e o f TFA- l a b i l e amino a c i d s i de c h a i n protect ing groups . The protect ing groups commonly used are : tBu ethers for the hydroxyls of serine , threonine and tyros ine ; t Bu esters for the c a rboxyl s ide cha ins o f a spa rt i c and glut amic ac ids , Boc for the NE g roup of lys ine ; t riphenylmethyl ( T rt ) for t he Nim of h ist idine ; Trt for the s u l f hydryl of cyste ine ; and NG- 4 -methoxy-2 , 3 , 6 -t r imethyl-benzene s ulphonyl (Mt r ) or NG-2 , 2 , 5 , 7 , 8 -pentamethylchroman- 6 -sulphonyl (Pmc ) for

the NG o f a rg i n ine . In addition , the amide s ide cha ins of asparagine and g lut amine may be protected, for example by the T rt g roup .

Fmo c - am i n o a c i d s may be act ivated f o r coup l ing i n SPP S by met hods including HOBt /DIe (Fields et a I , 1 9 8 9 ) , symmetrical anhydride (Athe rton et a I , 1 9 7 8 a ) , pent a f luo rophenyl act ive e s ter ( K i s fa ludy and Schon , 1 9 8 3 ) , 2 - ( lH -benzotriazo l - 1 -yl ) - 1 , 1 , 3 , 3 , -tet ramethylu ronium hexafluoro- pho s p h a t e ( HB T U ) ( F i e lds et a I , 1 9 9 1 ) , 2 - ( l H -ben z o t r i a z o l - l - yl ) - 1 , 1 , 3 , 3 , -tet ramethyluronium tetraf luoroborate ( TBTU ) (Beck-Sickinger et a I , 1 9 9 1 ) and ben zot r i a z o - 1 -y l - oxy-t r i s - ( dimethyl amino ) phosphon ium hex a f luo rophosphate ( BOP ) ( Hudson , 1 9 8 8 ) .

The e xtent o f complet ion o f coup l i ng may be moni t o red by withdrawing s amp l e s f o r t h e qu a l i t a t ive TNBS ( H a n c o c k and B a t t e r sby, 1 9 7 6 ) or quant itative ninhydrin test s ( Sa r in et a I , 1 9 8 1 ) . Both o f these methods r e q u i r e r emo v a l o f re s i n s amp l e s . R e a l t ime mo n i t o r i ng ma y be

a c c omp l i s h e d b y a dd i ng a sma l l amount o f bromophen o l blue t o the s o l u t i on o f a c t i vated Fmoc-amino acid . The blue colour fades as the re s in bound amine g roups a re a c y l a t ed ( K rchnak et a I , 1 9 8 8 ) The

cont inuous flow Fmoc met hod also a llowed monitoring of the deprotection a n d coupling s teps by insert ion of a UV flow cell into the c i rcuit . H o we ve r , t h e me t hod i s n o t s e n s i t i ve e nough t o dete rmine whet he r quant itat ive coupling of the Fmoc-amino acid has taken place .

Pept ides made by t he Fmoc method a re usually c leaved from the support w i t h T F A . S c a ve nge r s a re i n c l uded t o t r ap react ive c a rbocat ions generated during s ide chain c leavage . The scavengers used depend on the t ype o f s ide c h a i n protect ion employed a nd t he amino a c ids in the pept i de ( Appl ied Biosystems , Inc . , 1 9 9 0 a ) . Typical cleavage mixtures a r e g iven in Sect ion 5 . 2 . 8 . of this Chapter ( see also Van Wandelen et a I , 1 9 8 9 ) .

Two groups have reported the use of carbohydrate supports for SPPS using t he F m o c me t hodo l ogy . F r a n k a n d Do ring ( 1 9 8 8 a ) used Whatman 3MM c e l l u l o se pape r , in a continuou s f low s ynthe s i se r , a s a s upport for pep t i de s ynthesis us ing the Fmoc methodology . The paper was funct ional

In document Solid phase peptide synthesis on a beaded cellulose support : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University (Page 120-170)