B. M errifi el d S OLI D P H ASE SY NT HESIS

Nobel lect ure, 8 Dece mber, 1984 b y B R U C E M E R R I F I E L D

The Rockefeller University, 1230 York Aven ue, Ne w York, N. Y. 10021-6399

T h e pr ot ei ns, as t h e Gr e e k r o ot of t h eir n a m e i m pli es, ar e of first r a n k i n li vi n g s yste ms, a n d t heir s maller relati ves, t he pe pti des, ha ve no w also bee n disco v- er e d t o h a v e i m p ort a nt r ol es i n bi ol o g y. A m o n g t h eir m e m b ers ar e m a n y of t h e hor mo nes, releasi ng factors, gro wt h factors, io n carriers, a ntibiotics, toxi ns, a n d ne uro pe pti des. My p ur pose to day is to describe t he c he mical sy nt hesis of pe pti des a n d protei ns a n d to disc uss t he use of t he s y nt hetic a p proac h to a ns wer vario us biological q uestio ns. T he st or y be gi ns wit h E mil Fisc her (1) at t he t ur n of t his ce nt ur y w he n he sy nt hesize d t he first pe pti de a n d coi ne d t he na me. T he ge neral c he mical req uire ments were to block the carboxyl gro u p of one a mino aci d an d the a mi no gro u p of t he seco n d a mi no aci d. T he n, b y acti vatio n of t he free car box yl gro u p the pe pti de bon d co ul d be for me d, an d selective re moval of the t wo protecti ng gro u ps wo ul d lea d to t he free di pe pti de. Fisc her hi mself was ne ver a bl e t o fi n d a s uit a bl e r e v ersi bl e bl o c ki n g gr o u p f or t h e a mi n e f u n cti o n, b ut his for mer st u dent Max Berg mann, with Zervas, was s uccessf ul (2). Their design of t he carbobe nzox y gro u p us here d i n a ne w era. W he n I be ga n wor ki n g o n t he s y nt hesis of pe pti des ma n y years later t his sa me ge neral sc he me was u ni versal- l y i n us e a n d w as v er y eff e cti v e, h a vi n g le d, f or e x a m pl e, t o t h e first s y nt h esis of a pe pti de hor mo ne by D u Vig nea u d i n 1953 (3). It soo n beca me clear to me, h o w e v er, t h at s u c h s y nt h es es w er e diffi c ult a n d ti m e c o ns u mi n g a n d t h at a n e w a p proac h was nee de d if large n u mbers of pe pti des were req uire d or if larger a n d more co m plex pe pti des were to be ma de.

SY NT HESIS O N A S OLI D M ATRIX O n e d a y I h a d a n i d e a a b o ut h o w t h e g o al of a m or e effi ci e nt s y nt h esis mi g ht b e ac hie ve d. T he pla n (4) was to asse mble a pe pti de c hai n i n a ste p wise ma n ner w hile it was attac he d at o ne e n d to a soli d s u p port. Wit h t he gro wi n g c hai n co vale ntl y a nc hore d to a n i nsol uble matrix at all sta ges of t he s y nt hesis t he pe pti de wo ul d also be co m pletely i nsol uble a n d, f urt her more, wo ul d be i n a s uitable p hysical for m to per mit ra pi d filtratio n a n d was hi ng. T herefore, after co m pletio n of eac h of t he s y nt hetic reactio ns t he mixt ure co ul d be filtere d a n d thoro ughly washe d to re move excess reactants an d by- pro d ucts. The inter me- 1 5 0 C h e mi str y 1 9 8 4 di at e p e pti d es i n t h e s y nt h esis w o ul d t h us b e p urifi e d b y a v er y si m pl e, r a pi d proce d ure rat her t ha n by t he us ual te dio us crystallizatio n met ho ds. W he n a m ultiste p pr ocess, s uc h as t he pre parati o n of a l o n g p ol y pe pti de or pr otei n is c o nt e m pl at e d, t h e s a vi n g i n ti m e a n d eff ort a n d m at eri als c o ul d b e v er y l ar g e. T he fact t hat all of t he ste ps j ust descri be d are heter o ge ne o us reacti o ns bet wee n a sol uble reage nt i n t he liq ui d p hase a n d t he gro wi ng pe pti de c hai n i n t he i ns ol u bl e s oli d p h as e l e d t o t h e i ntr o d u cti o n of t h e n a m e “s oli d p h as e p e pti d e s y nt hesis”. T h e g e n er al s c h e m e f or s oli d p h as e s y nt h esis is o utli n e d i n Fi g. 1. It b e gi ns

Fi g. 1. T h e g e n er al s c h e m e f or s oli d p h as e s y nt h esis. B. M errifi el d 1 5 1

Fig. 2. Mono mer units for solid phase synthesis wit h a n i ns ol u bl e p arti cl e, i n di c at e d b y t h e l ar g e cir cl es, w hi c h is f u n cti o n ali z e d wit h a gr o u p, X. T he first m o n o mer u nit, s mall circles, is bl oc ke d at o ne e n d a n d at t he reacti ve si de c hai n gr o u ps ( blac k d ots) a n d a nc h ore d t o t he s u p p ort b y a sta ble c o vale nt b o n d. T he α protecting gro u p is then re move d an d the s e c o n d m o n o m er u nit is a d d e d t o t h e first b y a s uit a bl e r e a cti o n. I n a si mil ar way t he s ubseq ue nt u nits are co mbi ne d i n a ste p wise ma n ner u ntil t he e ntire p ol y m eri c s e q u e n c e h as b e e n ass e m bl e d. Fi n all y, t h e b o n d h ol di n g t h e c h ai n t o t he s oli d s u p p ort is selecti vel y clea ve d, t o get her wit h t he si de c hai n pr otecti n g gro u ps, a n d t he pro d uct is liberate d i nto sol utio n. S uc h a syste m offers fo ur mai n a d va ntages: it si m plifies a n d accelerates t he m ultiste p sy nt hesis beca use it is p ossi bl e t o c arr y o ut all t h e r e a cti o ns i n a si n gl e r e a cti o n v ess el a n d t h er e b y a v oi d t h e m a ni p ul ati o ns a n d att e n d a nt l oss es i n v ol v e d i n t h e r e p e at e d tr a nsf er of materials; it also a voi ds t he large losses w hic h nor mally are e nco u ntere d d uri ng t he isolatio n a n d p urificatio n of i nter me diates; it ca n res ult i n hig h yi el ds of fi n al pr o d u cts t hr o u g h t h e us e of e x c ess r e a ct a nts t o f or c e t h e i n di vi d- ual reactio ns to co m pletio n; a n d it i ncreases sol vatio n a n d decreases aggrega- ti o n of t h e i nt er m e di at e pr o d u cts. It o nl y r e m ai n e d t o tr a nsl at e t h e g e n er al i d e a i nt o a w or ka ble set of reacti o ns. 1 5 2 Che mistry 1984

Alt ho u g h t he pla n was ori gi nall y co ncei ve d as a wa y to s y nt hesize pe pti des, t he ge neral sc he me does not s pecify t he nat ure of t he mo no mer u nits. It soo n beca me a p pare nt t hat t he tec h niq ue s ho ul d be a p plicable to u nits ot her t ha n a mi n o a ci ds, s u c h as t h os e s h o w n i n Fi g. 2. W e e xt e n d e d it t o t h e s y nt h esis of de psi pe pti des (5) a n d ot her laboratories s uccee de d i n sy nt hesizi ng polya mi des (6), poly n ucleoti des (7) a n d polysacc hari des (8). I n pri nci ple t he mo no mer may be a ny bif u nctio nal co m po u n d t hat ca n be selecti vely blocke d at o ne e n d a n d a cti v at e d at t h e ot h er. I n a d diti o n, t h e s oli d s u p p ort i d e a c a n b e a p pli e d t o a v ari et y of c o n v e nti o n al r e a cti o ns i n or g a ni c c h e mistr y t o ai d i n dir e cti n g t h e c o urs e of t h e r e a cti o n or i n t h e s e p ar ati o n of t h e pr o d u cts fr o m r e a g e nts a n d b y- pr o d u cts. It als o l e d t o t h e s oli d p h as e s e q u e n ci n g t e c h ni q u e.

S OLI D P H ASE PEPTI DE SY NT HESIS A d et ail e d s c h e m e f or t h e s y nt h esis of p e pti d es is s h o w n i n Fi g. 3. E a c h of t h e ste ps has been mo difie d in many ways, b ut the che mistry sho wn here has ser ve d well a n d has bee n a p plie d t o t he s y nt hesis of lar ge n u m bers of pe pti des (9). T he carboxyter mi nal a mi no aci d is blocke d at t he a mi no e n d by a tert- b utyloxycarbo nyl ( Boc) gro u p a n d is covale ntly attac he d to t he resi n s u p port as a b e n z yl est er vi a t h e c hl or o m et h yl gr o u p. Si d e c h ai n f u n cti o n al gr o u ps m ust

A N C H O R

D E P R O T E C T

COUPLE

C L E A V E

P U R I F Y

Fi g. 3. A s c h e m e f or s oli d p h as e p e pti d e s y nt h esis. B. M errifi el d 1 5 3 also be blocke d, us ually wit h be nzyl-base d derivatives. T he sy nt hesis de pe n ds o n t he differe ntial se nsiti vit y of t hese t w o classes of pr otecti n g gr o u ps t o aci d, w hic h is greater t ha n 1000: 1. T he Boc gro u p is co m pletel y re mo ve d wit h 50 % trifl uoroacetic aci d in dichloro methane, with mini mal loss of the anchoring bo n d or of t he ot her protecti n g gro u ps. T he res ulti n g α a mi n e s alt is n e utr al- ize d wit h a tertiar y a mi ne s uc h as diiso pro p ylet h yla mi ne a n d t he free a mi ne of t he resi n-bo u n d a mi no aci d is t he n rea dy to co u ple wit h a seco n d Boc-a mi no aci d. T he latter m ust be acti vate d f or t he reacti o n t o occ ur. T he si m plest a n d most ofte n use d proce d ure is activatio n wit h dicyclo hexylcarbo dii mi de (10) as s ho w n, b ut active esters (11), a n hy dri des (12), a n d ma ny ot her activate d deri vati ves ha ve bee n s uccessf ull y a p plie d. All of t hese reacti o ns ar e carrie d o ut un der non-aq ueo us con ditions in organic solvents that s well the resin an d accelerate the rates. Dichloro methane an d di methylfor ma mi de ar e t he s ol ve nts of c h oi c e. To exte n d t he pe pti de c hai n t he de protectio n, ne utralization an d co u pling ste ps are re peate d for eac h of t he s uccee di ng a mi no aci ds u ntil t he desire d seq uence has been asse mble d. Finally, the co m plete d pe pti de is de protecte d a n d cl e a v e d fr o m t h e s oli d s u p p ort. Wit h t h e c h e mistr y d es cri b e d h er e, t his is acco m plis he d by treat me nt wit h a stro ng a n hy dro us aci d s uc h as HF (13). T he free pe pti de is t he n p urifie d b y s uita ble pr oce d ures. It is v er y i m p ort a nt t h at t h e r e p etiti v e st e ps pr o c e e d r a pi dl y, i n hi g h yi el ds, a n d wit h mi ni mal si de reactio ns i n or der to preve nt t he acc u m ulatio n of excessi ve a mo u nts of by- pro d ucts. M uc h of o ur effort has bee n directe d to war d develo pi ng a n d eval uati ng t hese req uire me nts.

S OLI D P H ASE N UCLE OTI DE SY NT HESIS Si mil ar s c h e m es f or t h e s oli d p h as e s y nt h esis of oli g o n u cl e oti d es h a v e n o w b e e n de velo pe d w hic h are ra pi d a n d gi ve relati vel y hi g h yiel ds (14, 15). T he y e m ploy protecte d n ucleoti des as mo no mer u nits a n d make use of eit her p hos- p hotriester or p hos p hite triester c he mistry. O ne s uc h proce d ure is o utli ne d i n Fi g ur e 4. T h e r esi n is first f u n cti o n ali z e d wit h a n a mi n o m et h yl gr o u p a n d t h e n u cl e oti d e d eri v ati v e is c o u pl e d t o it, t hr o u g h a s p a c er, b y a st a bl e a mi d e b o n d. I n t his e x a m pl e t h e 5’ h y dr o x yl is est erifi e d t o t h e s p a c er a n d t h e 3’ h y dr o x yl is te m porarily blocke d with a di methoxytrityl ( D M T) gro u p. The latter is re- mo ve d wit h aci d or Z n Br 2 a n d t h e c h ai n is e xt e n d e d at t h e 3’ e n d b y c o u pli n g wit h t he next protecte d n ucleoti de by acti vatio n wit h 1-( mesityle ne-2-s ulfo nyl)- 3- nitr o-1, 2, 4-triaz oli de ( MS N T). T h e c o m pl et e d oli g o n u cl e oti d e is fi n all y clea ve d fro m t he soli d s u p port a n d de protecte d by treat me nt wit h a base s uc h a s N H 4 O H or tetra met hylg ua ni di ne a n d t he n wit h hot acetic aci d. T he pro- d ucts are rea dily p urifie d by io n exc ha nge c hro matogra p hy or by electro p hore- sis w h er e t h e d esir e d pr o d u ct al w a ys h as t h e gr e at est n e g ati v e c h ar g e. I will n ot deal f urt her wit h pol y n ucleoti des a n d t heir use i n site directe d m uta ge nesis or wit h s y nt hetic ge nes i n t his prese ntatio n b ut will co nce ntrate i nstea d o n pe p- ti des a n d pr otei ns. 1 5 4 Che mistry 1984

Fi g. 4. A s c h e m e f or s oli d p h as e n u cl e oti d e s y nt h esis.

T HE S UPP ORT T he first req uire me nt for t he de velo p me nt of soli d p hase sy nt hesis was a s uitable s u p port. After exa mi natio n of ma ny pote ntial s u p ports it was fo u n d t hat t he most satisfactory o ne was a gel pre pare d by s us pe nsio n co poly meriza- tio n of styre ne a n d 1 % of di vi nylbe nze ne as crossli nki ng age nt (4). T he res ulti n g s p herical bea ds ( Fi g. 5) are a b o ut 50 µ m i n dia meter w he n dr y, b ut i n or ga nic sol ve nts s uc h as dic hloro met ha ne t he y s well to 5 or 6 ti mes t heir origi nal vol u me. F urt her more, as pe pti de c hai ns gro w t he dry vol u me i ncreases to acco mo date the a d de d mass an d, most i mportantly, the s wollen volu me B. M errifi el d 1 5 5

Fig. 5. Co poly(styrene-1 %- divinylbenzene) resin.

co nti n ues to i ncrease. Val ues u p to 25 fol d have bee n meas ure d a n d calc ula- ti o ns i n di c at e t h at t h e m a xi m u m e x p a nsi o n s h o ul d b e a b o ut 2 0 0 f ol d ( 1 6). T his mea ns t hat t he pol yst yre ne matrix a n d t he pe n da nt pe pti de are hi g hl y sol vate d d uri ng t he c he mical reactio ns a n d are freely accessible to diff usi ng reage nts. T he reactio ns occ ur not o nl y at t he s urface of t he bea d b ut, i n major part, wit hi n t he i nterior of t he crossli nke d poly meric matrix. T his co ul d be de mo n- strate d b y a utora dio gra p h y of a cross sectio n of a bea d co ntai ni n g a s y nt hetic triti u m-labele d pe pti de (17). At t his resol utio n t he sil ver grai ns were locate d u nifor mly t hro ug ho ut t he bea d, alt ho ug h t he distrib utio n is not k no w n at t he m olec ular le vel. Beca use of t he s ol vati o n a n d s welli n g of t he bea ds, t he reac- 1 5 6 Che mistry 1984 ti o ns ar e v er y f ast, wit h h alf ti m es i n t h e or d er of s e c o n ds f or b ot h t h e c o u pli n g a n d t he de protectio n ste ps. C urre nt efforts to e val uate t he effects of mass tra nsfer a n d diff usi o n i n dicate t hat t he y are n ot rate li miti n g. We belie ve t he soli d matrix not o nly does not ha ve detri me ntal effects o n t he sy nt hesis b ut act uall y has be neficial effects i n certai n i nsta nces. O ne of t he well rec o g nize d diffic ulties wit h t he classical s y nt hesis i n ho mo ge neo us sol utio n is i nsol u bilit y of so me i nter me diates. T his proble m ca n be o verco me i n ma ny cases by t he use of soli d s u p ports, w here t he pe pti de c hai n a n d t he li g htl y crossli n ke d pol y mer chain beco me inti mately mixe d an d exert a m ut ual solvating effect on one a not her. It beco mes t her mo dy na mically less favorable for t he pe pti de to self a g gre gate a n d it re mai ns a vaila ble f or reacti o n. F or t his t o occ ur t he s ol vate d st at e of t h e b o u n d p e pti d e n e e ds o nl y t o b e f a v or a bl e r el ati v e t o t h e a m or p h o us u nsol vate d state wit hi n t he pe pti de-resi n matrix (16). Si milar sol ubilizi ng pro perties of linear poly mers for covalently attache d co m ponents are kno wn, b ut t he effect will be greater for a li g htl y cross-li n ke d pol y mer net wor k. T he p he no me no n ca n be ill ustrate d by t he sy nt hesis of oligoisole uci nes (18). T he sta n dar d s ol uti o n s y nt hesis faile d after t he tetra pe pti de sta ge beca use of a g gre- gatio n a n d i nsol ubilit y, w hereas t he c hai n co ul d be exte n de d u p to 8 resi d ues on linear polyethyleneglycol. A soli d phase synthesis procee de d s moothly at least as far as t he d o deca mer, w here t he ex peri me nt was st o p pe d. T here is ver y sig nifica nt poly mer c hai n motio n i n t hese crossli nke d polystyre ne resi ns. Bot h 1 H a n d 1 3 C N M R meas ure ments (19) have sho wn that the motional rates for t he aro matic gro u ps a n d t he ali p hatic backbo ne ato ms i n C H 2 CI 2 are hi g h a n d eq ui vale nt to li near sol uble polystyre ne T h e α car bo n 1 3 C reso na nces of mo del resi n-s u p porte d pe pti des were as s har p as t he sol ve nt peak i n C H 2 Cl 2 or di methylfor ma mi de an d si milar to s mall molec ules in sol ution sec). A variety of che mical ex peri ments also have sho wn poly mer flexibility. For exa m ple s hort resi n-bo u n d pe pti des t hat were too far a part o n a v er a g e t o r e a c h o n e a n ot h er if t h e r esi n w er e ri gi d c o ul d b e s h o w n t o r e a ct t o t he exte nt of 99.5 % i n dicati ng co nsi derable motio n of t he polystyre ne seg- m e nts wit hi n t h e m atri x ( 2 0). Ma ny ot her soli d s u p ports have also bee n exa mi ne d a n d several have bee n satisfactory for pe pti de synthesis. These have incl u de d poly methyl methacry- late, pol ysacc hari des, p he nolic resi ns, silica, poro us glass, a n d pol yacr yla- mi des, b ut o nl y t he latter ha ve see n wi des prea d use (21). Co m parati ve st u dies with polystyrene an d polyacryla mi de have sho wn that they can be eq ually effecti ve, e ve n wit h diffic ult pe pti des.

AUTO MATION T he a bilit y to p urif y after eac h reactio n b y si m ple filtratio n a n d was hi n g a n d t he fact t hat all reactio ns co ul d be co n d ucte d wit hi n a si n gle reactio n vessel a p peare d to le n d t he mselves i deally to a mec ha nize d a n d a uto mate d process. I nitially, a si m ple ma n ually o perate d a p parat us was co nstr ucte d (Fig. 6). T his syste m was first use d to work o ut t he met ho dology a n d to sy nt hesize bra dy- ki ni n (22) a n gi ote nsi n (23) oxytoci n (24), a n d ma ny ot her s mall pe pti des. I n or der to accelerate t he process we u n dertook t he desig n a n d co nstr uctio n (25) B. M errifi el d 1 5 7

Fi g. 6. A m a n u all y o p er at e d s y nt h e si s a p p ar at u s

of t he a uto mate d i nstr u me nt s ho w n i n Fig. 7. T he esse ntial feat ures were t he reactio n vessel, co ntai ni ng t he resi n wit h its gro wi ng pe pti de c hai n, a n d t he necessary pl u mbi ng to e nable t he a p pro priate solve nts a n d reage nts to be pu mpe d in, mixe d, an d re move d in the proper sequence. These mechanical e ve nts were u n der t he co ntrol of a si m ple ste p pi n g dr u m pro gra m mer a n d a set of ti mers. I n t he past fe w years ma ny co m mercial i nstr u me nts have bee n c o nstr ucte d i n se veral c o u ntries. T he y differ c o nsi dera bl y i n detail, partic ularl y i n t he so p histicatio n of t he electro nic progra m mec ha nis ms b ut are desig ne d to carr y o ut t he sa me c he mistr y.

T HE SY NT HESIS OF RIB O N U CLE ASE A The i dea of che mically synthesizing an enzy me m ust have occ urre d to many peo ple o ver t he years. T here was a ti me w he n s uc h a t ho u g ht wo ul d ha ve bee n unacce ptable even on philoso phical gro un ds, b ut fro m the perio d when en- zy mes were sho wn to be proteins an d proteins were sho wn to be discrete or g a ni c m ol e c ul es it w as a g o al t h at c h e mists c o ul d b e gi n t o t hi n k a b o ut. If a n e nzy me co ul d be ma de i n t he laboratory, t he n it s ho ul d beco me possible to lear n ne w t hi ngs abo ut ho w t hese large a n d very co m plex molec ules f u nctio n. S pecific c ha nges co ul d be ma de i n t heir str uct ures t hat co ul d not be ma de rea dily by alteri ng t he native protei n a n d data s ho ul d be fort hco mi ng t hat wo ul d s u p ple ment the infor mation alrea dy obtaine d fro m the nat ural enzy mes t he msel ves. I n t his re gar d, a q u otati o n fr o m Fisc her i n 1906 (26) is perti ne nt: “ W hereas ca utio us professio nal colleag ues fear t hat a ratio nal st u dy of t his class of co m po u n ds [ protei ns], beca use of t heir co m plicate d str uct ure a n d t heir 1 5 8 Che mistry 1984

Fi g. 7. An auto mated peptide synthesizer.

hig hly i nco nve nie nt p hysical c haracteristics, wo ul d to da y still u nco ver i ns ur- mo u ntable diffic ulties, ot her o pti mistically e n do we d observers, a mo ng w hic h I will c o u nt m ys elf, ar e i n cli n e d t o t h e vi e w t h at a n att e m pt s h o ul d at l e ast b e ma de to besie ge t his vir gi n fortress wit h all t he ex pe die nts of t he prese nt; b e c a us e o nl y t hr o u g h t his h a z ar d o us aff air c a n t h e li mit ati o ns of t h e a bilit y of o ur met ho ds be ascertai ne d.” B. M errifi el d 1 5 9

Wit h t he develo p me nt of soli d p hase pe pti de sy nt hesis a n d its a uto matio n t he ti me see me d ri g ht to atte m pt t he total s y nt hesis of a n e nz y me. Dr. Ber n d G utte a n d I selecte d bo vi ne pa ncreatic ribo n uclease A beca use it was a s mall stable protei n of k no w n a mi no aci d seq ue nce (27), a n d t he t hree di me nsio nal str uct ure was k no w n fro m X-ray diffractio n st u dies (28). M uc h of t he detaile d mechanis m by which this enzy me hy drolyzes an d depoly merizes ribonucleic aci d was also k no w n. T he p ur pose of a c he mical sy nt hesis of t his 124-resi d ue m ol e c ul e w as, first, si m pl y t o d e m o nstr at e t h at a pr ot ei n wit h t h e hi g h c at al yti c acti vit y a n d s pecificit y of a nat urall y occ urri n g e nz y me co ul d be s y nt hesize d i n t h e l a b or at or y. F or t h e l o n g r a n g e t h e m or e i m p ort a nt p ur p os e w as t o pr o vi d e a ne w a p proac h to t he st u dy of e nzy mes. We believe d it s ho ul d be possible to m o dif y t h e str u ct ur e a n d t o alt er t h e a cti vit y a n d t h e s u bstr at e s p e cifi cit y of t h e enzy me. T he sy nt hesis (29) was carrie d o ut o n a co poly(styre ne-1 %- divi nylbe nze ne)- resi n s u p port usi ng t he ge neral met ho ds describe d above. T he C-ter mi nal Boc- Val was a nc h ore d t o t he s oli d matrix b y a be nz yl ester b o n d, t he us ual be nz yl- base d si de c hai n protecti ng gro u ps were use d a n d t he Boc gro u p pro vi de d t he r e v ersi bl e protectio n. T he de protectio n ste ps were wit h trifl uoroacetic aci d a n d t he co u pli ng reactio ns were wit h dicyclo hexylcarbo dii mi de activatio n. Fig. 8 s h o ws t h e fi n al pr ot e ct e d d eri v ati v e of ri b o n u cl e as e. It c o nt ai n e d a t ot al of 6 7 si de c hai n protecti ng gro u ps a n d ha d a molec ular weig ht of 19,791. T he s y nt hesis is s u m marize d i n Ta ble I. T he o verall yiel d after se veral p urificati o n proce d ures was abo ut 3 % base d o n t he origi nal a mo u nt of vali ne attac he d to t he resi n. T here was a lar ge (83 %) 1 o s s of c h ai ns d uri n g t h e ass e m bl y of t h e pe pti de c hai n d ue t o partial i nsta bilit y of t he a nc h ori n g b o n d, a n d t he acc u m u- late d losses d uri n g H F clea va ge fro m t he resi n a n d t he p urificatio n ste ps were a not her 80 %. T he cr u de cleave d pro d uct was air oxi dize d to for m t he fo ur dis ulli de bo n ds a n d t he mo no mer fractio n was isolate d by gel filtratio n. T he mo no mers wit h i ncorrect dis ulli de pairi n g or i ncorrect fol di n g were di geste d b y tr y psi n a n d t h e s m all fr a g m e nts w er e s e p ar at e d fr o m t h e st a bl e pr ot ei n wit h t h e correct str uct ure. A n a m mo ni u m s ulfate fractio natio n gave t he fi nal p urifie d enzy me possessing a p proxi mately 80 % s pecific activity co m pare d with native ribo n uclease A. We co ul d not clai m t hat o ur pro d uct was co m pletely p ure or t hat t he sy nt hesis co nstit ute d a str uct ure proof for R Nase, o nly t hat t he molec ule s ho we d a close c he mical a n d p hysical rese mbla nce to t he native protei n a n d t hat it was a tr ue e nzy me. T he c he mical a n d p hysical co m pariso ns were base d on a mino aci d analysis, enzy me digestions, pe pti de ma ps, pa per electr o p h oresis, gel filtrati o n, ion-exchange chro matography, and antibody ne utralizatio n. At t hat ti me we di d not have H P L C or a n affi nity c hro mato- gra p hy syste m. Ta ble II s u m marizes t he acti vit y data at vari o us sta ges of p urificati o n of t he sy nt hetic e nzy me. Bot h t he s pecific acti vity a n d t he total n u mber of u nits of R Nase i ncrease d as t he p urificatio n procee de d, i n dicati n g eit her t hat i n hi bitor y i m p urities were being re move d or that the molec ule was gra d ually refol ding i nto a co nfor matio n t hat more closely rese mble d t he native str uct ure. T he s ubstrate s pecificity of t he sy nt hetic e nzy me was co nsiste nt wit h t hat to be

B. M errifi el d 1 6 1

1 0 0

1 7

1 2

3 7 3 6 . 4

2 5 6 4 . 4

1 6 9 2 . 9

T a bl e II 1 6 2 Che mistry 1984 e x p e ct e d f or R N as e A: it w as a bl e t o cl e a v e b ot h l ar g e ( R N A) a n d s m all ( C > p) s ubstrates an d therefore to catalyze both the trans phos phorylation an d the h y dr ol ysis ste ps; it was s pecific f or D-ri b ose i nstea d of D- de ox yri b ose a n d f or a p yri mi di n e i nst e a d of a p uri n e at t h e 3’ p ositi o n of t h e p h os p h o di est er s u bstr at e ( Table III). T he K m val ues to war d R N A were also t he sa me for t he nat ural a n d sy nt hetic e nzy mes. T he p urifie d R Nase A was co m pare d o n a C M-cell ulose col u m n wit h nat ural R Nase A an d with re d uce d an d reoxi dize d nat ural R Nase A. They were i d e nti c al b y t his crit eri o n, w hi c h w as t h e o n e first us e d b y W hit e ( 3 0) t o s h o w t hat R Nase A after re d uctio n a n d reoxi datio n of t he dis ulfi de bo n ds was i n disti ng uis hable fro m t he native e nzy me. His was t he de mo nstratio n t hat le d to t he h y pot hesis t hat t he pri mar y str uct ure of t he protei n deter mi ne d its tertiar y str uct ure (31). O ur s y nt hesis pr o vi de d a ne w ki n d of e vi de nce f or t his h y pot hesis. T he fact t hat t he o nl y i nfor matio n p ut i nto t he s y nt hesis was t he li near seq ue nce mea ns t hat t he pri mary str uct ure m ust be s ufficie nt to direct t h e fi n al f ol di n g of t h e m ol e c ul e i nt o its a cti v e t erti ar y str u ct ur e. T h e s y nt h esis of a n active e nzy me co ntai ni ng no s ubstit ue nts exce pt a mi no aci ds also pro- vi de d a ne w proof for t he no w well establis he d belief t hat e nz y matic acti vit y ca n be attrib ute d to a si m ple protei n co ntai ni ng no ot her co m po ne nts.

T able III. S ubstr ate S pecificity of Sy nt hetic Ribo n ucle ase A

S u bstrate A cti vit y

STRUCTURE-FU NCTI O N STUDIES O N RIB O NUCLEASE T he sy nt hesis of ribo n uclease provi de d a ns wers to several f u n da me ntal q ues- tio ns a n d lai d t he fo u n datio n for ne w st u dies o n t he relatio n of str uct ure to f u nctio n i n t he e nzy me. T he classic S- pe pti de /S- protei n syste m discovere d by Ric har ds (32) pro vi de d a n i deal way to st u dy s uc h relatio ns beca use a s mall pe pti de (resi d ues l-20) a n d a large protei n co m po ne nt (resi dues 21-124) co ul d be co mbine d noncovalently with regeneration of nearly f ull enzy matic activity. T he exte nsive work fro m t he Hof ma n n (33) a n d Scoffo ne (34) labora- t ories o n t he s y nt hesis of t he S- pe pti de a n d its c o m bi nati o n wit h t he nat ural S- protei n ha d alrea dy pro vi de d a great a mo u nt of i nfor matio n abo ut t he role of i n divi d ual resi d ues i n t he N-ter mi nal regio n of t he e nzy me. We u n dertook to st u d y t his re gi o n of R Nase b y t otal s y nt hesis ( Fi g. 9). D uri n g t he i nitial synthesis we re move d sa m ples after co u pling Cys 2 6 a n d a gai n after Ser 2 1 a n d i n t hat way pre pare d sy nt hetic S- protei n (21-124) a n d S- protei n (26-124). T he B. M errifi el d 1 6 3

Fig. 9. The S-peptide/ S-protein syste m

partiall y p urifie d protei ns were re d uce d at t heir 4 dis ulfi de bo n ds, eac h mixe d wit h sy nt hetic S- pe pti de, reoxi dize d a n d assaye d for e nzy matic activity. Eac h of t he cr u de mixt ures was fo u n d to ha ve as m uc h acti vity as t he pro d uct deri ve d fro m nati ve S- protei n by t he sa me treat me nt. Fro m t hese data it was co ncl u de d first, t hat S- protei n ha d bee n sy nt hesize d a n d seco n d, t hat t he li ve resi d ues 2 l-25 were defi nitel y not necessar y for t he bi n di n g a n d reacti vatio n t o occ ur. Earlier X-ra y data (35) ha d pre dicte d t hat t he seri nes at p ositi o ns 21, 22 a n d 23 wo ul d probably not be necessary, b ut As n 2 4 a n d T yr 2 5 a p peare d to b e i n v ol v e d i n a t ot al of 5 h y dr o g e n b o n ds i n R N as e S a n d it w as e x p e ct e d t h at t hey mig ht be necessary for t he for matio n of a n acti ve co m plex. T he sy nt hetic st u di es s h o w e d t h at t h e y w er e n ot. Se veral years earlier I ha d bee n i ntereste d i n t he q uesti o n of w het her or n ot a p e pti d e fr o m t h e c ar b o x yl e n d of R N as e mi g ht f u n cti o n i n a m a n n er si mil ar t o t hat of t he S- pe pti de at t he a mi no e n d. Co nseq ue ntly, t he R Nase 111- 124 tetra deca pe pti de was synthesize d an d p urifie d. R Nase was inactivate d by c ar b o x y m et h yl ati o n of t h e i mi d a z ol e ri n g of His 1 1 9 Atte. m pts to reacti vate t he e nzy me by a d ditio n of t he sy nt hetic pe pti de were u nifor mly u ns uccessf ul. So me w hat later i n a se parate st u dy Li n et al. (36) s uccee de d i n pre pari ng a series of shortene d R Nases. They ma de R Nase 1-120, R Nase l-l19 and 1 6 4 Che mistry 1984

R Nase 1-118 by e nzy matic digestio n. W he n t he sy nt hetic pepti de 111-124 was assa ye d i n t he prese nce of t hese i nacti ve pr otei ns, hi g h e nz y matic acti vit y was ge nerate d (37) a n d it beca me clear t hat a s yste m existe d at t he C-ter mi n us of R N as e t h at w as si mil ar t o t h e o n e at t h e N-t er mi n us. We t he n ma de t he i nteresti ng discovery t hat t he C-ter mi nal pe pti de 111- 124 co ntai ni ng His 1 1 9 , t he N-ter mi nal pe pti de l-20 co ntai ni ng His 1 2 , a n d t h e central protein co m ponent 21-118 containing Lys 4 1 co ul d be mixe d toget her no n-covale ntly a n d ribo n uclease activity wo ul d be ge nerate d. T herefore, t hree co m po ne nts eac h co ntai ni ng o ne of t he k no w n resi d ues req uire d for e nzy matic acti vity co ul d bi n d toget her a n d for m t he s pecific well or dere d str uct ure necessar y for s u bstrate bi n di n g a n d catal ytic acti vit y. A s eri es of s y nt h eti c st u di es w as t h e n u n d ert a k e n t o d efi n e t h e r ol es of s o m e of t h e i n di vi d u al r esi d u es i n t h e C-t er mi n al r e gi o n. T h es e c a n b e s u m m ari z e d a n d disc usse d by referri ng to Fig. 10. W he n pe pti des s horter t ha n 111-124 were pre pare d an d co mbine d with R Nase 1-118, both the bin ding constant an d the activity were progressi vely decrease d a n d pe pti de 117-124 was i nactive, i n dicati ng t hat eac h resi d ue co ntrib ute d to t he bi n di ng e nergy (38). Ho wever, eve n i n t he co m plex 1-118 + 116-124 t here were 3 over- la p pi n g resi d ues. It was t he n fo u n d t hat t he co m plex 1-115 + 116-124, i n w hic h t here were no overla p pi ng resi d ues, ha d a bi n di ng co nsta nt 100 ti mes larger (39). I n t hese ex peri me nts it co ul d also be s ho w n t hat Tyr 1 1 5 was n ot necessary for e nzy matic acti vity.

Fig. 10. A 3-di mensional representation of ribonuclease frag ments l-20, 21-118 and 111-124 su m marizing the synthetic structure-function studies. B. M errif el d 1 6 5

P h e n yl al a ni n e- 1 2 0 w as s h o w n t o b e i m p ort a nt i n st a bili zi n g t h e ri b o n u cl e as e str uct ure by tra nsitio n te m perat ure st u dies a n d to i nteract wit h t he pyri mi di ne s ubstrate by X-ray a n d N M R st u dies. O ur sy nt hetic a nalog work o n t he 1-118 + 111-124 syste m sho wed that replace ment of Phe 1 2 0 b y L e u 1 2 0 o r ll e 1 2 0 re d uce d the bin ding by 5 an d 17 fol d an d re d uce d the maxi m u m enzy matic a cti vit y t o 1 0 % a n d 5 % r es p e cti v el y, i n di c ati n g t h at t h e ar o m ati c si d e c h ai n of phenylalanine was of consi derable i m portance in bin ding the pe pti de to the protei n (40). It co ul d o nly partially be re place d by a hy dro p hobic ali p hatic c hai n, i n dicati n g a n i nexact ali g n me nt of t he catal ytic site. T he s mall resi d ue Al a 1 2 0 a n d t he b ulky aro matic resi d ue Tr p 1 2 0 were i nacti ve. Re place me nt of P h e 1 2 0 b y a n ar o matic resi d ue of si milar size, T yr 1 2 0 , i n t he 111-124 pe pti de ga ve a co m plex wit h 1-118 t hat was f ull y acti ve to war d C > p as s u bstrate a n d 190 % as acti ve to war d U > p (41). A se misynthetic enzy me with enhanced a cti vit y w as a n o v el fi n di n g. K m a n d Ki d at a l e d t o t h e c o n cl usi o n t h at P h e 1 2 0 does not ha ve a u ni q ue role i n t he bi n di n g of s u bstrate b ut is i m porta nt for stabilizi ng t he pe pti de- protei n co m plex a n d t he nati ve e nzy me itself. Ne vert he- less, t he prese nce of s ubstrate i ncrease d t he bi n di ng co nsta nt bet wee n 1-118 a n d 111-124 b y a fact or of 50. Si milar ex peri me nts wit h t he as partic aci d resi d ue at positio n 121 ha ve s ho w n t hat it ca n be re place d partially ( ~20 %) by gl uta mic aci d, b ut t he A s n 1 2 1 a n d Ala 1 2 1 a nalogs di d not s ho w meas urable bi n di ng. Re moval of Va1 1 2 4 fro m R Nase A does not affect t he e nz y matic acti vit y a n d re mo val of Va1 1 2 4 fro m S- protei n does not re d uce t he activity of t he co m plex wit h S- pe pti de. I n co ntrast, o missio n of Val 1 2 4 fro m the C-ter minal tetra decapepti de pro duce d an esse ntially i nactive co m plex wit h R Nase 1-118, i n dicati ng a n i m porta nt hy- dro p hobic i nteractio n necessary for pe pti de- protei n bi n di ng. T he s maller ali- p hatic resi d ue Ala 1 2 4 c o ul d o nl y r est or e h alf of t h e bi n di n g e n er g y ( 4 1). X-ray data (42) i n dicate t hat t he uracil a n d cytosi ne resi d ues of R N A a n d t he cyclic n ucleoti des probably bi n d to ribo n uclease t hro ug h t he series of hy droge n bo n ds s ho w n i n Fig. 11. For uracil t he hy droxyl of T hr 4 5 i s a hy drogen acce ptor an d for cytosine it is a hy drogen donor. Conversely the h y dr ox yl of Ser 1 2 3 is a do nor for uracil a n d a n acce ptor for c ytosi ne. We reaso ne d t hat if t hese t wo hy droxyls were blocke d as met hyl et hers t hey co ul d o nl y be h y dro ge n acce ptors a n d if re place d b y Ala t he y co ul d be neit her do nor nor acce ptor. A s uitable co mbi natio n of t hese resi d ues i n re place me nt a nalogs mi g ht, t heref ore, lea d t o a s y nt hetic ri b o n uclease wit h altere d s u bstrate s peci- ficity. S uc h a nalogs have bee n ma de for Ser 1 2 3 ( 4 3). The tetra deca pe pti de containing Ala 1 2 3 gave a co m plex with R Nase 1-118 t hat s ho we d a p preciable selectivity for s ubstrates co ntai ni ng cytosi ne relative t o t h os e c o nt ai ni n g ur a cil ( eit h er t h e 2’, 3’ c y cli c n u cl e oti d es or p ol y n u cl e oti d es) ( Table I V). Re place me nt wit h 0- met hylseri ne di d not res ult i n differe ntial s ubstrate s pecificity. It was co ncl u de d t hat a hy droge n bo n d bet wee n t he h y drox yl of Ser 1 2 3 a n d t h e C 4 a mi no gro u p of c ytosi ne is not i m porta nt for s ubstrate bi n di ng a n d catalytic activity, b ut t hat t he hy droge n bo n d bet wee n t he hy droxyl of Ser 1 2 3 a n d t h e C 4 car bo n yl of uracil co ntri b utes si g nifica ntl y to t h e bi n di n g a n d a cti vit y; w h e n S er is r e pl a c e d b y Al a t h e H- b o n d is a bs e nt a n d 1 6 6 Che mistry 1984

U R I D I N E

R i b o s e

H

Fig. II. Proposed hydrogen bonding of uracil and cytosine s u bstrates t o ribonuclease.

t h e acti vit y is re d uce d. The corres pon ding st u dies with re place ment of Thr 4 5 b y Al a 4 5 an d Ser( Me) 4 5 i nvolve total sy nt hesis of t he e nzy me a n d t hese m uc h more diffic ult ex peri me nts have not yet bee n co m plete d. We believe t hat t he s ub- strate bi n di n g at T hr 4 5 is m uc h ti g hter t ha n at Ser 1 2 3 a n d t hat c ha n ges at t his resi d ue will lea d t o m uc h greater s u bstrate selecti vit y.

T a ble I V. S u bstr ate Selectivity of ( Al a 1 2 3 ]- R Nase Co mplex

Enzy me

RECE NT I MPR O VE ME NTS I N S OLI D P H ASE PEPTI DE SY NT HESIS Alt h o u g h t h e e arli er s oli d p h as e c h e mistr y w as v er y us ef ul f or t h es e st u di es o n ribo n uclease, it was clear t hat t here was a nee d for i m prove me nt i n several ar e as. O ne was t he mo de of attac h me nt of t he pe pti de to t he resi n. If t he str at e g y of diff er e nti al st a bilit y t o w ar d a ci d f or t h e a n d groups was to be B. M errifi el d 1 6 7

Fig. 12. Acyloxy methyl-Pa m-resin

co nti n ue d, a more aci d stable a nc hori ng bo n d was nee de d. We pre dicte d t hat t he i nsertio n of a n aceta mi do met hyl gro u p bet wee n t he be nzyl ester a n d t he p ol yst yr e n e m atri x w o ul d i n cr e as e t h e st a bilit y of t h e b e n z yl est er t o trifl u or o- acetic aci d b y a fact or of a p pr oxi matel y 25 t o 400 ti mes. W he n s uc h a li n ka ge was fi nall y co nstr ucte d it was fo u n d to be 100 ti mes more sta ble (44). A ne w synthesis of a mino methyl-resin was first develo pe d in which N-hy droxy methyl- p ht hali mi de a n d polystyre ne resi n were reacte d u n der aci d catalysis wit h

F 3 C S O 3 H, H F, or S n Cl 4 (45). T his pro d uct was t he n co u ple d wit h a derivative of t h e C-t er mi n al a mi n o a ci d. T h us, aci d was pre pare d an d activate d with dicyclohexylcarbo dii mi de for the reac- tion. The product was the acyloxy methylphenylaceta mido methylcopoly(styr- ene-1 %- divinylbenzene) resin (acyloxy methyl-Pa m-resin) (Fig. 12). This ne w pr e p ar ati o n h as t h e a d v a nt a g es t h at it is m or e a ci d st a bl e, a n d it is m a d e fr o m p urifie d, well c haracterize d i nter me diates, w hic h give a clea ner pro d uct wit h fe wer si de reactio ns. It is free of c hloro met h yl gro u ps t hat ca n gi ve rise to q uater nizatio n a n d io n exc ha nge reactio ns a n d is free of hy droxyl gro u ps t hat ca n lea d to pe pti de c hai n ter mi natio ns via trifl uoroacet ylatio n (46). A n alter native protecti ng gro u p strategy is to make use of a n ort hogo nal s yste m (47) i n w hic h t he a n d si de chain gro u ps re present three differe nt classes of co m po u n ds t hat are clea vable by t hree differe nt ki n ds of reactio ns. I n t hat wa y a n y o ne of t he f u nctio nal gro u ps ca n be selecti vel y r e m o v e d i n t h e pr es e n c e of t h e ot h er t w o. Fi g ur e 1 3 ill ustr at es s u c h a s yst e m i n w hic h t he a nc hori ng o- nitrobe nzyl ester is p hotolabile b ut stable to aci d or n ucle o p hiles, t he si de c hai n gr o u ps are base d o n tert- b ut yl deri vati ves t hat are v er y a ci d l a bil e b ut st a bl e t o li g ht or n u cl e o p hil es, a n d t h e protecti ng gro u p is t he dit hias ucci noyl gro u p w hic h is re move d by n ucleo p hilic t hiols b ut is

Fi g. 1 3. An orthogonal protecting group sche me 1 6 8 Che mistry 1984 st a bl e t o a ci d a n d p h ot ol ysis. T his s c h e m e h as r e c e ntl y b e e n p ut t o t h e t est a n d f o u n d t o gi v e e x c ell e nt r es ults ( 4 8). Anhy dro us hy drogen fl uori de, the us ual cleavage reagent for soli d phase pe pti de s y nt hesis, is a ver y str o n g aci d ( H 0 -10.8) a n d is k no w n to pro mote a n u m ber of si de reactio ns. I n partic ular it lea ds to t he for matio n of car bo ni u m ions which then can alkylate tyrosine, try pto phan, methionine an d cysteine resi d ues of t he pe pti de. I n a d ditio n, HF ca n proto nate a n d de hy drate t he si de c hai n carboxyl of gl uta mic aci d resi d ues wit h for matio n of t he very reactive a c yli u m i o n, w hi c h h as b e e n s h o w n t o a c yl at e t h e ar o m ati c ri n gs of a nis ol e a n d ot her scave ngers prese nt i n t he mixt ure. Activate d gl uta mic resi d ues ca n also for m pyrroli done ( pyrogl uta mic)containing pro d ucts. As partyl resi d ues can close i n H F to t he as parti mi de deri vati ve a n d s ubse q ue ntl y o pe n to pro d uce β − as part yl resi d ues. All of t hese u n desire d reactio ns res ult fro m t he S N l mec ha- nis m of t he cleavage reactio n u n der t he us ual co n ditio ns (90 % HF + 10 % a nis ol e, 0º C, 1 hr). W e r e as o n e d t h at if c o n diti o ns c o ul d b e f o u n d t h at w o ul d c h a n g e t h e r e a cti o ns t o a n S N 2 m e c h a ni s m i n w hi c h t h e a ci d ol y si s is ai d e d b y a n ucleo p hile a n d car bocatio n is ne ver for me d ( Fi g. 14) it s ho ul d be possi ble to mi ni mize or a voi d t hese proble ms. Dr. Ja mes Ta m a n d W.F. Heat h, a gra d uate st u dent, have s uccee de d in develo ping s uch con ditions an d in de monstrating marke d i m prove me nts i n soli d p hase pe pti de sy nt hesis (49). T he proble m was to fi n d a s uitable weak base w hic h wo ul d re d uce t he aci dity f u nctio n of t he HF b ut w hic h wo ul d re mai n largely u n proto nate d a n d n ucleo p hilic u n der t he res ulti n g aci dic co n ditio ns. It s ho ul d be a wea ker base t ha n t he gro u ps to be clea ve d so t hat t hey wo ul d be largely proto nate d u n der the sa me con ditions. Di methylsulfi de ( D MS) w as f o u n d t o b e a n i d e al b as e f or t his p ur p ose. It has a p Ka of -6.8 c o m pare d wit h val ues of -2 t o -5 f or t he be nz yl et hers, esters a n d car ba mates t o be clea ve d. It is a g o o d s ol ve nt f or H F a n d it is v ol atil e a n d e asil y r e m o v e d fr o m t h e r e a cti o n mi xt ur e. A 1: 1 m ol ar

0 R- ?- O H B. Merrifiel d 1 6 9 m i x t u r e o f H F a n d D M S ( 1 : 3 b y v o l u m e ) w a s d e t e r m i n e d b y H a m m e t t in dicators to have an He bet ween -4.6 an d -5.2. The mechanis ms of re moval of various benzyl-base d protecting groups by HF/ D MS mixtures were teste d by ki netic a n d pro d uct a nalysis ex peri me nts. Base d o n earlier work wit h

H 2 S O 4 h y d r o l y s i s o f a l k y l a c e t a t e s ( 5 0 ) , a s h a r p u p w a r d b r e a k i n t h e r a t e constant was ex pecte d when the aci d concentration was increase d. At the break

point the mechanis m change d fro m S N 2 t o S N 1. A si milar c ha n ge was f o u n d i n t h e c l e a v a g e o f O - b e n z y l s e r i n e b y H F / D M S m i x t u r e s ; a b o v e 5 0 % H F b y vol u me t he rate i ncrease d ra pi dly, i n dicati ng t he c ha nge i n mec ha nis m. Pro d- uct a nal ysis f or t he de pr otecti o n of t yr osi ne be nz yl et her as a f u ncti o n of H F co nce ntratio n is s ho w n i n Fig. 15. Above 15 % HF i n D MS t he yiel d (after 1 hr, 0º C) of t yrosi ne was q ua ntitati ve a n d t he ot her pro d uct was t he be nz yl- di methyls ulfoni u m salt. In the range of 40-50 % HF the a mo unt of s ulfoni u m salt bega n to decrease a n d t he level of t he u n desirable by pro d uct, 3-be nzyltyro-

si ne, i ncrease d. A gai n, t here was a c ha n ge fro m t he S N 2 t o t h e S N l m e c h a n i s m a r o u n d 4 0 - 5 0 % H F . T h e r e a c t i o n s w e r e a c c e l e r a t e d i n t h e p r e s e n c e o f 5 - 1 0 % o f c r e s o l . W e s e l e c t e d 2 5 % H F / 6 5 % D M S / 1 0 % c r e s o l a s t h e b e s t rea ge nt a n d refer t o it as “l o w H F”. T his reage nt was also effecti ve i n pre ve nti ng acyli u m io n for matio n i n gl uta myl a n d as partyl pe pti des a n d avoi de d t he acylatio n a n d i mi de si de reactio ns. It was also fo u n d to be ver y effecti ve i n co n verti n g met hio ni ne

Fi g. 1 5. Pro d uct analysis for the de protection of tyrosine benzyl ether in mixt ures of HF an d di met hyls ulfi de. 1 7 0 Che mistry 1984 s ulfoxi de to methionine, with for mation of di methyls ulfoxi de as co pro d uct. F urt her more, i n t he prese nce of 5 % of t hiol s uc h as t hiocresol a nearl y q u a ntit ati v e r e m o v al of t h e f or m yl pr ot e cti n g gr o u p fr o m t h e i n d ol e nitr o g e n of tryptophan was possible. The byproduct was H C(S R) 3 . T h e l ast t w o r e a cti o ns do not occ ur at hi g h (90 %) H F d ue to proto natio n of t he rea ge nts.

The derivatives Arg( Tos), Arg( N O 2 ), Cys(4- Me Bzl), or As p( Oc Hex) will not be de protecte d u n der t he lo w HF co n ditio ns, a n d pe pti des co ntai ni ng t hese a n d certai n ot her resi d ues m ust be retreate d i n hi g h H F (90 %) after re m o val of t he D MS. Ho wever, si nce most of t he pote ntial carbo ni u m io ns i n t he pe pti de will alr e a d y h a v e b e e n tr a p p e d as t h e l ess r e a cti v e di m et h yls ulf o ni u m s alt, t h e b y pr o d ucts of t he reacti o n are still greatl y re d uce d.

T HE NEE D T O P AY ATTE NTI O N T O DET AILS I ca n not e m p hasize e no ug h ho w i m porta nt it is to be atte ntive to eve n t he s mallest of details if o ne ex pects t o s y nt hesize a pe pti de of hi g h q ualit y. T he pri nci pal b y pro d ucts of soli d p hase pe pti de s y nt hesis ca n be classifie d as ter mi natio n, deletio n, or mo dificatio n pe pti des. M uc h effort has go ne i nto i dentifying these proble ms, develo ping ways to q uantitate the m, an d fin ding w a ys t o eli mi n at e t h e m. First of all, it is i m p ort a nt t o b e gi n wit h cl e a n, w ell c haracterize d resi ns, clea n a mi no aci d deri vati ves, a n d clea n sol ve nts. Most of t he k no w n si de reactio ns ca n no w be eli mi nate d or greatly mi ni mize d if t he pro per co u pli ng met ho ds a n d co n ditio ns are selecte d (51). It is i m porta nt to mo nitor co u pli ng reactio ns to deter mi ne t hat t hey have procee de d to co m ple- ti o n s o t h at d el eti o n p e pti d es missi n g o n e or m or e r esi d u es will b e a v oi d e d. T h e q ua ntitati ve ni n h y dri n reactio n (52) is usef ul for t hat p ur pose a n d ca n detect t he prese nce of 0.1 % u nreacte d c hai ns (i.e. 99.9 % co u pli ng). After a pe pti de c hai n has bee n asse mble d it ca n be a nalyze d by soli d p hase seq ue nci ng met h o ds (53) t o q ua ntitate t he le vels of pre vie w a n d t heref ore of deleti o n se q ue nces (54). Exce pt for s pecial cases, race mizatio n is not us uall y a pro ble m i n ste p wise soli d p hase sy nt hesis, b ut se nsiti ve met ho ds for its detectio n are a v ail a bl e ( 5 5). If t h e v ari o us pr e c a uti o ns all u d e d t o h er e ar e t a k e n, s atisf a ct or y res ults ca n be ex pecte d i n m ost i nsta nces.

S O ME RECE NT SY NT HESES OF PEPTI DES Very large n u mbers of pe pti des ha ve bee n sy nt hesize d i n rece nt years by t he tec h niq ues t hat have bee n disc usse d a n d I ca n not begi n to cover t he m here. Fro m o ur o w n laboratory we have re porte d rece nt sy nt hetic st u dies o n a pa mi n (56), t h y mosi n (57) gl uca g o n (58), a n d cecr o pi n A (59, 60). F or t his disc ussi o n I ha ve selecte d exa m ples of s y nt heses t hat ser ve t o ill ustrate certai n are as of i nterest. A n excelle nt exa m ple of a s y nt hetic pe pti de st u d y lea di n g t o usef ul dr u gs is t hat of Ma n ni ng a n d Sa wyer o n develo p me nt of vaso pressi n a nalogs wit h hig h a nti di uretic acti vit y a n d esse ntiall y no re mai ni n g pressor acti vit y for treat me nt of diabetes i nsi pi d us (61). T he best was 1- dea mi no-[4-vali ne, 8- D-argi ni nelva- so pressi n. T hey have also discovere d, t hro ug h sy nt hesis, argi ni ne vaso pressi n a nal o gs t hat are str o n g i n hi bit ors of b ot h a nti di uretic a n d press or acti vit y f or B. M errifi el d 1 7 1

use i n patie nts wit h h y p o natre mia d ue t o excessi ve rete nti o n of water (62). T he b est w as [ cyclopenta methylenepropionic acid), 2- D-pheny- lalanine, 4-valine]-arginine-vaso pressin. I n a fe w i nsta nces soli d p hase s y nt heses ha ve bee n scale d u p for co m mercial p ur poses. A goo d exa m ple is sal mo n calcito ni n (63). It has bee n pre pare d i n 50-100 g batc hes of hi g hl y p urifie d pe pti de. T his 32-resi d ue hor mo ne is hi g hl y effecti ve f or t he treat me nt of Pa get’s disease a n d ot her c o n diti o ns of h y percal- c a e mi a. T he area of greatest c urre nt i nterest a n d acti vit y is u n d o u bte dl y t he s y nt he- sis of pe pti des for t he el uci datio n of t he i m m u no ge nic deter mi na nts of protei ns a n d f or t he de vel o p me nt of s y nt hetic vacci nes a gai nst viral a n d ot her i nfecti o us diseases. T he work fro m Ler ner’s laboratory (64) has give n a n i m porta nt i m p et us t o t his fi el d. S y nt h eti c a nti g e ns ar e als o us ef ul f or t h e d e v el o p m e nt of dia g nostics a n d for t he pro d uctio n of a ntibo dies as ai ds i n detecti n g a n d isolati ng u ni de ntifie d ge ne pro d ucts. As a n ill ustratio n of my e m p hasis o n t he i m porta nce of ne w c he mistry a n d t he nee d to pay atte ntio n to t he details w he n utilizi ng soli d p hase pe pti de synthesis I wo ul d mention so me ne w work on the epi der mal gro wth factor ( E G F) b y Bill Heat h (65). E G F sti m ulates cell ular pr oliferati o n, i n hi bits gastric aci d secretio n a n d plays a role i n e mbryo nic de velo p me nt. T he str uc- t ure of t his 53-resi d ue pe pti de (66) is s h o w n i n Fi g. 1 6. It is a h y dr o p h o bi c, hig hly crossli nke d, co m pact molec ule t hat ot hers have fo u n d very diffic ult to sy nt hesize i n t he past. By usi ng t he ne wly develo pe d Pa m-resi n s u p port, several ne w protecti ng gro u ps, p ure reage nts, t he q ua ntitative mo nitori ng proce d ures, t he ne w HF cleavage met ho ds, a n d by taki ng all t he ot her k no w n preca utio ns agai nst si de reactio ns, he s uccee de d i n obtai ni ng a n esse ntially q ua ntitati ve asse mbly of t he pe pti de c hai n a n d a 97 % clea vage yiel d, lea di ng t o a cr u de u n p urifie d pr o d uct t hat c o ntai ne d 65 % of t he desire d E G F. It c o ul d

b e r e a dil y is ol at e d i n a hi g hl y p urifi e d f or m w hi c h el ut e d fr o m a C 1 8 H P L C

Fi g. 1 6. Str uct ure of mo use e pi der mal gro wt h factor. 1 7 2 Che mistry 1984 c ol u m n at e x a ctl y t h e s a m e ti m e as n at ur al E G F ( Fi g. 1 7). I n t h e s e nsiti v e a n d discri minating Ley dig cell gro wth assay the synthetic an d nat ural E GF ha d i d e nti c al a cti vit y.

Fig. 17. H P L C analysis of synthetic E G F. The arro w indicates the position of natural E G F.

Fro m t he acc u m ulate d data prese nte d, we co ncl u de t hat t he soli d p hase sy nt hesis of pe pti des u p to 50 or so me w hat more resi d ues ca n be rea dily a c hi e v e d i n g o o d yi el d a n d p urit y; a n d t his is a f ar b ett er sit u ati o n t h a n I c o ul d ha ve ex pecte d w he n t his tec h niq ue was first pro pose d. As a n e x a m pl e of a s y nt h esis of a pr ot ei n I h a v e s el e ct e d o ur r e c e nt st u di es o n interferon. The seq uence of h u man le ucocyte interferon was first de d uce d fro m t he D N A seq ue nce of t he clo ne d ge ne (67). It co ntai ns 166 a mi no aci ds wit h 5 cystei ne resi d ues (Fig. 18). T he a mi no aci d seq ue nce of t he isolate d protei n of h u ma n le ucoc yte i nterfero n w as als o d et er mi n e d ( 6 8) a n d f o u n d t o ha ve o nly 155 resi d ues. T here is a hig h degree of ho mology bet wee n t he t wo, b ut t he latter has o ne deleti o n at As p 4 4 a n d i s mi s si n g t h e l a st 1 0 r e si d u e s pre dicte d fro m the D N A seq uence (Fig. 18). We have synthesize d these t wo pr otei ns a n d als o t heir Ser 1 a nalogs a n d p urifie d t he m by re d uctio n, gel filtr ati o n, r e o xi d ati o n, g el filtr ati o n, a n d affi nit y p urifi c ati o n o n a c ol u m n of s u p porte d polyclonal antibo dies to h u man le ucocyte interferon (69). The sy nt hetic protei ns a n d t he nat ural a n d reco mbi na nt i nterfero n all ha d 10 8 t o 1 0 9 u nits / mg i n a ntiviral assays agai nst a broa d s pectr u m of cell li nes. T he develo p me nt a n d d uratio n of t he a ntiviral state were also si milar. Sy nt hetic an d nat ural H u- Le-IF N- α s ho we d si milar gro wt h i n hibitio n of K 5 6 2 c ells. an d nat ural H u- Le-IF N- α ca use d a si milar i ncrease of nat ural killer cell acti vit y w hereas s y nt hetic ca use d a decrease. All fo ur s y nt hetic i nterfero ns bi n d to a n d are el ute d fro m pol yclo nal a nti- H u- Le-IF N- α a ntibo dies u n der si milar co n ditio ns. B. M errifi el d 1 7 3

Fig. 18. Sequences of leucocyte interferons α 1 a n d α 2

T hese res ults are e nco uragi ng, b ut m uc h more nee ds to be do ne to ass ure t h at e v e n s m all pr ot ei n s c a n b e s y nt h e si z e d r e a dil y i n hi g h yi el d a n d p urit y. I t hi n k w e c a n b e o pti misti c a b o ut t h e f ut ur e.

A C K N O W L E D G E M E N T S I o we a ver y s pecial de bt of gratit u de t o m y teac hers, Dr. M. S. D u n n of U. C. L. A. a n d Dr. D. W. Woolley of T he Rockefeller U ni versity. Se veral of t he past a n d prese nt me mbers of m y laborator y ha ve bee n referre d to here, b ut to the many others who have not been s pecifically mentione d I a m eq ually gratef ul beca use t he y all ha ve c o ntri b ute d t o t he pr o gress of o ur w or k. Fi nall y, I wish to ackno wle dge the contin uing s u p port of The Rockefeller University a n d of t he Nati o nal I nstit utes of Healt h of t he U nite d States. 1 7 4 Che mistry 1984

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