5. MARCO DE REFERENCIA
5.2. MARCO CONTEXTUAL
5.2.1. Responsabilidad Social Empresarial en Colombia
CHAPTER 3 SOLm PHASE PEPTmB SYNTHESIS ON PJ!:RLOZA : BOC CHJ!:MISTIlY
3 . 1 rNTRODUCTION
49
T h e t e rt i a r y b u t yloxyca rbonyl ( Bo c ) group w a s deve loped as a n acid l a b i l e amine p rotecting group ( C a rpino 1 9 5 7 a , 1 9 57b) and applied to s o lu t ion pept ide s ynthes is by McKay and Albertson ( 1 957 ) and Anderson and MacGrego r ( 1 9 5 7 ) . The Boc group was f i r s t u sed for s o l id pha se pept i de synthe s i s by Me rrifield ( 1 9 6 4 a , b ) , and has come to be the most c o mmo n l y u s ed a c id l a b i le Na-ami no p rotec t ing g roup ( E r ic ks on and Me r r i fie ld, 1 9 7 6 ) . The Boc group is c leaved by 2 5 % TFA in DCM, 4M HCL in d io x ane ( St ewa rt and Young, 1 9 8 4 ) , 1 0 % s u lphuric a c id in dioxane ( Houghten et a l , 1 9 8 6 ) , and boron t r i f luoride in acetic a c id ( Hiskey et a l , 1 9 7 1 ; Schnabel et a l , 1 97 1 ) . A diagram of the �oc method of SPPS i s g iven as Figure 3 . 1 .
T h e s o l i d s u pp o rt u s u a l ly u s e d f o r S P P S by t he Boc met hod i s 1 % divinylbenzene c ross linked polystyrene ( Erickson and Merrifield, 1 9 7 6 ) , a l t h o u g h many other s uppo rts have been inve st igated ( Ba rany et a l ,
1 9 8 7 ) . The polystyrene s upport was f i rst funct iona l ised for SPPS by
chloromethylation of pendant benzene rings by chlo romethyl methyl ether i n t he p r e s e n c e o f a Lew i s a c id c a t a lyst s u c h as SnC1 4 (Merrifield 1 9 6 3 ) . The c h l o ride was t hen displa ced by a Boc-amino acid, as i t s t ri e t hy l ammonium salt , to anchor the Boc amino acid t o the support via a ben zy l e s te r . Huwever , the benzyl e ster l i nkage was not found to be comp le t e ly s t able to the acid s olut ions used f o r Boc c leavage . For examp l e , Gutte and Me rrifie ld ( 1 97 1 ) noted an average 1 . 4 % c leavage of the ancho ring benzyl ester bond during each cycle in a synthes is of the
1 2 4 amino a c id res idue polypept ide Ribonuclease A . 5 0 % TFA/DCM was used f o r Boc cleavage .
In that synthesis
The phenylacetamidomethyl ( PAM) l inker was developed in response to the rep o r t e d cleavage of pept ide f rom the t radit ional benzyl ester during acidic Boc c leavage (Mitchell et a l , 1 9 7 6b) . The amino acyl-PAM ester linkage was found to be 1 0 0 times more stable to refluxing TFA than the ben z y l ester p revious ly used, and is now one of the mos t commonly used l i n k e r s for s o l id phase peptide synthe s i s by the Boc methodology . A PAM l i n k e d Boc-amino acid may be int roduced to aminomethyl polystyrene
( Mi t che l l e t a I , 1 9 7 6 a ) b y DCC mediated coupling of Boc-amino acyl - 4 - o x yme t h y l pheny l a cet i c a c i d ( Mit che l l e t a I , 1 9 7 6b , 1 9 7 8 ) . Alter nat ively, 4 -bromomethyl phenylacetic acid may be coupled to aminomethyl p o l y s t yrene and a Boc-amino a c id subs equent ly coupled either a s its t r iethylammonium salt (Mitche l l et aI, 1 9 7 6b ) , or it may be coupled in t he pre sence of potass ium f luoride ( Toth and Penke , 1 9 9 1 ) .
Figure 3 . 1 Boe SPPS methodology illustrated by the synthesis of a dipeptide
CO2 +
Boc-amino acyl-Pam linker-SuPP9rt
CH3 0 RI 0 0 I II I II CH3 + 1F A (Deprotect) OlEA (Neutralise) RI 0 0 I 11
-0
11 NH2-CH-C-O-CH2 � !J CH2-C-NH-Support R2 0 I 11 Boc-NH-CH-C-Y(Activated Boc-amino acid)
R2 0 RI 0 0
I II I II
-0
11Boc-NH-CH-C- NH-CH-C-O-CH2 � !J CH2-C-NH-Support
R2 0 RI 0 + I 11 I 11
J
!IF (aeave) o HrN-CH-C- NH-CH-C-OH + 11 Dipeptide +Sl
Reac t ive s i de chains of NQ-BoC amino acids must be protected during the cou r s e o f pept ide synthesis . For example , the s ide chain hydroxyls of s e r ine and threonine are protected as their benzyl ethers, and the side cha i n ca rboxy l s o f a spart ic and glutamic acids a re protected as their ben z y l e s t e r s . The �-amino o f lys ine is protected by the p-chloro c a rboben z y loxy (N£-pCl-CBZ ) g roup, the NG of Arg is protected by the t o s y l ( To s ) group , and the Nim of His is p rotected by the tosyl group ( i f HOBt is not to be used for coupling of any later Boc-amino acids ) or by the 2 , 4 -dinitrophenyl (Dnp ) group i f HOBt is to be used for following coupl i ng s . The s ide' chain thiol o f cyste ine i s protected by the p - me t h o xybe n z y l ( S -pMeO - B z I ) g r o up , and f i n a l l y t he Nin o f Trp i s p r o t e c t e d w i t h the f o rmyl ( Nin -CHO) group . A l l o f the s ide chain protect ing groups given a re cleaved by strong acid, for example liquid HF, e xcept the 2 , 4 -dinit rophenyl group on the Nim of His and the formyl group on the Nin o f Trp . The His Nim-Dnp group is generally removed, p r i o r to pept ide c leavage , by thiolysi s of the pept ide - resin, while the T r p N in- f o rmy l g roup i s cleaved, a f t e r pept ide c leavage , by aqueous pipe r idine .
A c u r rent ly popular method f o r coupling most Boc amino ac ids i s via the i r s ymmet r i c a l anhydrides , preformed in solution us ing DCC (Wieland et a I , 1 9 7 1 ; Hagenmaier and Frank, 1 9 72 ) . Boc-Asn and Boc-Gln cannot be c o u p l e d a s t h e i r s ymme t r i c a l a n h y d r ide s be c a u s e o f s i de c h a i n dehydration t o form the nit r i le . They a re instead coupled as their HOBt e st e r s ( Konig and Geige r , 1 9 7 0a , b, c ) . Boc-Arg (NG-TO s ) is also coupled a s i t s HOBt e s t e r instead of the anhydride to prevent int ramo lecular
l a c t am f o rmat i on . Othe r methods o f act i vat i ng Boc - amino ac ids for coup l i ng include TBTU (Reid and Simpson , 1 9 92 ) , HBTU ( Schnolzer et a I , 1 9 9 1 ) , a nd BOP ( Co s te e t a l , 1 9 8 9 ) . T h e p r o g re s s o r ext ent o f c omp l e t i on o f c o up l ing i s u s u a l l y mon i t o red b y t h e quant i t a t ive n inhydrin assay ( S a rin et a I , 1 9 8 1 ) .
Cleavage o f the pept ide from the resin support is usually accomplished by s t rong anhydrous acid. For example , l iquid hydrogen f luoride , at a Oc , c on t a i n i n g a n i s o l e a s s ca venge r , is commonly u sed for peptide
c l e avage ( S a ka iba ra et a l , 1 9 6 7 , 1 9 7 1 ) . The s cavenger is included to t ra p c a rbocations formed from cleavage of side chain protect ing groups . P ept i de c leava ge us ing l iquid HF must be c a rried out in a special HF
res i st ant a l l-teflon apparatus . Trifluoromethanesulphonic acid ( TFMSA) ( Be r got e t a I , 1 9 8 6 ) , with scavengers , may also be used for pept ide cleavage . One of the advantages of us ing TFMSA rather than liquid HF is t h a t the cleavage can be carried out in glass apparatus . This is not p o s s ible with HF because HF reacts with glass .
C a rbohydrate s upports that have been used for SPPS by Boc methodology include Sephadex LH2 0 (Vlasov and Bilibin , 1 9 6 9 ; Orlows ka et a I , 1 9 7 5 ; E r i c k s on and Merri field, 1 9 7 6 ) , cellulose paper ( E ichler et a I , 1 9 8 9 ) , and cellu lose cotton ( Eichler et a I , 1 9 9 1 ) .
Me r r ifield initially investigated Sephadex LH2 0 a s a support for solid phas e peptide synthesis in 1 9 5 9 , but really satisfactory conditions were not, found . The work was resumed in 1 9 6 5 , and the tetrapeptide LAGV was
s y n t h e s i s e d . � h e me t h o d w a s s t i l l n o t f o u n d t o be e n t i r e l y
s at i s factory, as inexplicable s ide chain terminat ion was observed during s ev e r a l s ep a r a t e s ynt h e s e s of the t e t r apept ide LAGV ( E r i c k s on and Me r r i f ield, 1 9 7 6 ) .
V l a s o v and B i l ibin ( 1 9 6 9 ) coupled Boc-glyc ine direct ly ont o Sephadex L H 2 0 u s i n g CO l , a mixed a n h yd r i de , o r Boc-glyc ine chlo r ide . Boc c l e a v a ge w i t h TFA, f o llowed by coupling of Boc-amino ac ids as their a c t i ve e s t e r s , and cleavage by hydro lys i s , furnished a pentapept ide . The n onapept ide bradykinin was synthes ised on a s imilar support us ing Dec f o r coupl ing and 1M p-toluenesulphonic acid in acetic acid for Boc c le a vage (Vlas ov et a l , 1 9 7 3 ) .
Orlowska et a l ( 1 9 7 5 ) synthesised a pentapept ide fragment of Substance P on LH2 0 u s ing Boc methodology . Boc-glycine was anchored to the support hydroxyl g roups in the presence of eD I to give an initial amino acid sub s t itut ion of 0 . 2 5 mmole/g . Boc cleavage was by 1M p-toluenesulphonic a c i d in a ce t ic acid . D i f f icult ies were enc ounte red in c leaving the pept ide f rom the support . Methanolic NaOH , O . 1M, was found to be the mo s t e f fect ive cleavage reagent .
E i c h l e r e t a l ( 1 9 8 9 ) anchored Fmo c - a l an ine t o Whatman 5 4 0 cel lulose pape r by react ion of the paper with Fmoc-alanine chloride , to ancho r the amino acid t o the paper via an ester bond . The Fmoc group was cleaved
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w i t h p iperidine in DMF , and three hexapeptides (YVPKXA, YEETXA, YKQlXA; X= 6 -aminohexanoi c acid) were then synthesised by a combination of Boc and Fmoc methodology . Fmoc-amino acids used were Fmoc-Lys (Boc ) , Fmoc-
P r o and Fmoc -Va l . The benzyl side chain protect ing groups of Thr and G l u we re c leaved us ing bo ron t r i s t r i f luoroacetate in TFA ( P less and Baue r , 1 9 7 3 ) . The pept ides were then c leaved f r om the cellulo se by a lk a l ine hydro lys i s .
Eichler et al ( 1 9 9 1 ) used cellulose cotton a s a support for the SPPS of a h e x a pept i de a n d e i ght hept apept i de s by t he Boc / B z l and Fmo c / tBu me t hods . The C - t e rm i n a l amino a c ids we re ancho red direct ly to the c o t t o n by N -me t h y l i m i da z o l e ( NM I ) c a t a l y s e d c a r b o d i i m i de / HOBt a c t i vat ion . Compa rison by HPLC of the product s produced by the Boc and Fmoc methods s howed that the pept ides made us ing the Boc /Bzl methodology we r e c o n s ide r ab l y mo r e hete rogeneous t han t hose made u s ing the Fmoc me t h o d o l o g y . T h i s w a s e xp l a i n ed by E i c h l e r e t a l in t e rms of infer i or ity o f the met hod used to cleave the peptides made us ing the Boc met h od o l ogy . I n c ompl e t e c le a v a ge o f pept i de s ide c h a i n protect ing groups by boron t r i s t rifluoroacetate in TFA, as well as subsequent s ide react i ons dur ing a lkal ine cleavage ( for example aspart imide format ion dur ing alkaline t reatment of Asp ( OBzI ) conta ining pept ide s ) , were seen as ma j o r problems with the methodology employed in the i r study .
The pub l ished results s howed that solid phase peptide s ynthesis could be c a r r ie d out on known c a rbohydrate supports us ing the Boc methodology . Howeve r , a s d i s c u s sed above , these supports each presented the i r own problems when used for SPPS with t he Boc methodology . Therefore , it was dec ide d to invest iga t e t he fea s ibility of u s ing the l e s s we l l known P e r l o z a be a de d c e l lu l o s e f o r S P P S by Boc me thodo l ogy . An init i a l a t t e mpt w a s made t o s ynthe s i s e the tet rapept ide LAGV directly onto
amin o p r opyl P e r lo z a . As the synthesis proved succe s s ful the work was
extended by anchoring a C-terminal Boc-amino acid to the support via a c l e avable l inke r , f o l lowed by s yntheses of short t e s t pept ides . The ba s e - labile glyco l amide l inker ( Baleux et a I , 1 9 8 4 ) was chosen as the anchoring group because of its low cost , st ra ight forward chemist ry, and ava i lability . The approach taken to anchor Boc-amino ac ids to Perloza was to react e ithe r a-chloroacetic or a-bromoacet ic acid anhydride with ami n op ropyl P e r lo z a , and to t hen displace t he hal ide with the cesiurn
s a lt o f a Boc- amino a c id . T h i s f u rn i s hed a glycolamide e s t e r link
be t we e n t he B o c - amino a c i d and t he P e r l o z a . P eptide synthesis was
c a r r ie d out by coupling Boc-amino a c ids as the i r HOBt esters . The pept i de s were c leaved by alkaline hydrolys is .
3 . 2 MATERXALS AND METHODS
3 . 2 . 1 Chemicals and
Aminopropyl Perloza beaded cellulose was made as described in Chapter 2 . Na-Bo c - L-amino a cids ( Ala, Gly, Leu , Phe , Tyr ( Bzl ) , Val ) were supplied
by B a c h em , T o r rance, Ca l i fo r n i a . purcha sed from Vega .
Aut hent i c leuc ine-enkephalin was
E le c t r o n i c grade acetic acid was f rom Rhone -Poulenc , NZ . Analyt ical reagent grade acet ic anhydride was obta ined f rom A j ax Chemica l s , Sydney . T r i fluoroacetic acid was from Halocarbon , N . J . , and was dist illed before u s e . Re agent grade a-bromoac e t i c a c i d and N-methylmorpho l ine (NMM)
we r e p urchased f rom BDH , NZ . N-methylmorphol ine ( NMM) was dis t i l led
f ro m b a ri um oxide . The a-chloroacet ic acid was f rom J . T . Baker Ltd .
D im et h ylaminopyridine (DMAP ) was f rom Riedel -de Haen . Reagent grade dicyclohexylcarbodiimide (DCC) , diisopropylcarbodiimide (DIC) , and 1-N hydroxyben zot riazole monohydrate ( HOBt . H20 ) were purchased from Aldrich . Meth anesulphonic acid (MSA) was f rom May and Bake r . Dimethylacetamide ( DMA ) was from BDH , NZ . All other reagents were from sources listed in
the p revious Chapter .
P e p t i de s yntheses were carried out manually us ing a Rototo rque wheel ( Co l e P a rmer, Chicago ) for end over end mixing, or semimanual ly us ing an LKB Biochrom Ltd . Biolynx 4 1 7 5 cont inuous flow pept ide s ynthesiser . A s c r e w c apped react ion vessel, f itted with a glass f rit and tef lon valve, was made for use with the Rototorque ( Figure 3 . 2 ) .