CA1321280C - Urethane-protected amino acid-n-carboxyanhydrides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride having the structure:

Description

~32~280 1~27-~

U~ET~ANE-P~OTECTED A~INO ACID-N-CA~BO~AN~ IDES

BACKGROUND OF THE INVENTION

I.) Field_of__he_I_venti_n This lnvent~on relate3 to a novel cla~s of N-protected aoino acid-N-carboxyanhydrides and thiocarboxyanhydrides, na~ely N-urethane protected a~ino acid-N-carboxyanhydride3 ~nd N-urethane protected N-thiocarboxyanhydrides, their preparatlon and their u e in peptide, poly pept~de and protein synthe~lR.

Il.) Prior_Art Cla~sically, polypeptides of a defined ~equence have been prepared by extreDely laborlou~ techniques Jherein the inter~ediate~ have been isolated after the addltion of each a~ino acld ~oiety. Thls has co~plicated the synthe~is and ~ade the preparatlon of long ehal~ polypeptldes and protelns ne~rly l~possible because of lo~ yields, race~ization, and/or other side reactions. In 1963, herrlfleld (J. A~. CheD. Sor., 8S, ~199i and Let~inger and Kornet (J. A~. Che~. Soc., 85, 2~453 suggested the use of ln~oluble poly~eric supports for the grouing pep~ide chaln. This proce~s, co~only referred to as a solid pha~e peptlde ~ynthesl~, per~ltted the "purificatlon" cf the grouing peptlde chain, ~itbout isolating the lnter~ediate.

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~32~2~

Heretofore, the uidely accepted ~ethods for both cla~slcal lliquid phase) and solid phase polypeptlde ~ynthe~is requlred the use of a coupling or actlvating a~ent ~o react Ylth the carboxyl group of an other~se N-protected a~ino acid to g~ve a carboxyl-activated N-protected a~ino acid. Th~s activated species ~ould then be used in several uays to pro~ote peptlde bond forxation. For exs~ple, the activated protected a~lno acld is allo~ed to react dlrectly wlth the free a~lno ~roup of an anlno acid, a~ino acld ester, or aai~o acld a~ide to for~ the peptlde bond. This has been the procedure of choice for preparing peptide~ for ~any year~. The sctlvation step c~n be accoDpanied by a nu~ber of poi~ible ~lde reactlons. For exa~ple, ~here dlcyclohexylcarbodilDldetDCC~ i8 tbe act~vatln~
rea~ent, the actlve ~olecule ~ay "re-arran~e" to an inactlve N-acyl urea.

Another disadvantage of the carbodli~lde procedure ls the for~atlon of lnsnluble urea~. Tbis i9 partlcularly troubleso~e in s41id pba~e ~yntbe~ls ~nd 18 vlrtually unacceptable i~ ~olid pha~e flow 8ysteo~ Tbese uress also cause difficult purlfication proble~ in ~olution phaqe reac~ion~.

Researchers have allevlated so~e of the proble~ a~ociated wlth in ~itu actlvation by fir~t reactlng tbe DCC actlv~ted N-protected aDiDo acid uith an alcohol or phenol tsuch as p-nitrophenol, pe~tachlorophenol, N-hydroxy-succini~ide, etc.) to for~ an "active e~ter" ~hich ~ay be i~olated and purlfied snd then alloued to couple ith the free a~ine of the next a~lno ' ,' ' ` ' ~ " ` ' ` ' 132~280 acid. This approach i~ not ~itbout lt3 i~hortcoDings, hoYever, because th~ liberated alco~ol or phenol aay be involved in or proLote other ~lde reaction~ and act~ve eqter couplingq tend to be slug~iqh and require long reaction ti~e~.

Another co~on procedure i8 to for~ a "sy~etrical anhydride" by allowing tuo equivalentq of N-protec~ed a~lno acid to react ~lth one equivslent of DCC, filtering the DC~ forDed and then allouing the "~yn~etrical anhydrlde" to couple ulth the free auine group of the next a~lno acld~ Thls procedure h2s the urea proble~ in additlon to requlring uie of twice a~ Duch of an expen~lve N-protected a~ino acid.

So~e researcbers have recently begun to ~e cerbodli~ldes that for~ ~oluble ureas after coupling, but the~e are still prone to re-arrange~ent to N-acyl urea~.

Varlous type3 of N-protecting groups have been proposed for u~e in peptlde synthe~i~, but the ~ost videly accepted cla~ of N-protectlng groups are the urethan~. Urethanei are broadly ac~nowledged tn provide a hi~b de8ree of protection, ~lni~lze race~iza~ion, are readily prepared, and are ~tabl~ to storage.
Urethane-protecting group~ can be prepared ~hlch are lablle to nild acid ~1.e. t-butyloxycarbonyl~, qtrong a ~d ~i~e., benzyloxyc&rbonyl), extre~ely ~ild acld 2(p-biphenylyl)-laopropyloxycarbonyl, anhydrous ba~e (l.e., 9-fluorenyl-~ethyloxycarbonyl), and 80 forth.

:; . : :, ; :
. .,;:. . ::
'' ` :""'` ''''" ' '``' ' ' :, ::

13212~

Urethane-protected anino acids are con~only prepared by reaction of an al~yl, aryl or aralkylchlorofor~a~e ~or other suitably activated for~ate or carbonate> vith the a~ino acid ln the pre3ence of alXali ~etal hydroxlde or carbonate in a ~lxed aqueousiorganic ~olvent sy~teD ti.e., Schotten-BauDann conditions~. After acidlfication of the reaction nlxture, th~
urethane-protected 8~1nO acid 1B extracted into an or~anlc solvent lea~ing all slde product~ in the aqueous phase. After crystallization, these co~pound3 are ~sed f~r peptlde bond for~tion a~ descrlbed abo~e.

A partlcularly lnteresting type of reactive del-lYat~ve of aD~no aclds for use in peptide bond for~atlon are th~ so-called N-carboxyanhydrldes or N-thiocarboxyanhydrides, such as:

H--N Z

2~

uhereln R, ~' are typically hydrogen or the 31de chaine Sor protected s~d~ chaln~) of tbe co~on a~ino acld~, and ~ 1 oxygen or sulfur.

Anlno acid-N-carb~xyanhydr~des ~it ii understood that the ter0 N-carboxyanhydride in thi~ specification and appended clai~3 i~ to lnclude the N-th1ocarboxyanhydrldes) are vell-kno~n and react readily ~lth nost free a~ines. ~ pri~ary advAntago of : ' . ' ~ ' ~: :

~ ~32128~

N-carboxyanhydrides (NCA's) and even protected NCA'q for use ln peptide bond fornat~on i~ the fact that they are potent acylatlng agents ~see Peptide~, Yol. 9, page 83). They al~o generally glve higher yields of peptides than DCC or N-hydroxysucclni~ide (OSu) ester coupling procedures. But NCA's have not found ~idespread u3e in polypeptide synthesls because of the lac~ of ability to control or li~it the coupling reaction. Once an NCA reacts ~ith the free anlne of an a~ino acid, carbon dioxide is ln~ediately liberated and a dlpeptide i~
for~ed uhich al90 contain~ a free asine. Thls a~ine vill sub3equently react uith another NCA to for~ a trlpeptide, and 80 on. Thl~ reactlon has alloued a~ino acld N-carboxyanhydrldes to find extenslve u~e ln the for~ation of poly ~-a~lno aclds but bas virtually precluded t~eir use tn sequentlal polypeptide ~or~atlon. Hlr~ch~ann, et al ~The Contro}led Synthesis of Peptides ln Aqueous MediuD. VIII.) The PreparatioD and Use of Novel a-A~ino Acid N-Carboxyanhydrides. J.A.C.S., 93:11, i971, pg. 27g6-2~74) have ~ucceeded ln uqing a~ino acid N-carboxyanhydrideY for ~he preparation of dl-and tripeptlde~ 1 ~queou~-organic solvent syqte~ by careful control o~
te~perature, pH, salt, ~nd organic solvent of the reaction ~lxture. Ho~ever, this procedure 1~ ed to s~all peptlde~
because of the cheni~try of NC~'~ de~cribed above. Further~ore, the products obtalned fro~ the~e 001ut~0n phaYe reacti~ns ~ust be exten~ively purlfied prlor to bein8 used for the preparation of larger peptldes.

A variety of N-sub~tltuted aDinO acid N-carboxyanhydride~

: ~ :

~ 3~28~

have been reported in the literature Ruch a3 N-~ethyl, N-ben~yl, N-acetyl, N-nitrophenylsulfenyl, N-xanthyl, 4,~'-dinethylbenzhydryl, trityl, and the li~e. Several of the~e subqtltut_d-NCA's have been propo~ed for u~e in ~equentlal peptide synthesi~ and particularly for solid pha~e pPptide jynthe~is, but none have ~ained acceptance for gener~l use by peptide che~iYts.

~ rlcheldorf ~Ange~. Che~. Acta 85, 86-87, (i978~ proposPd the u~e of o-nitrophenylYulfenyl ~NPS~ Yub~tituted NCA'~ for use ln ~equentlal polypeptide Yynthe~1~. TheYe vere prepared by the reaction of o-nitrophenylsulfenylchloride with a N-carboxyanhydride ln the presence of triethylanlne.
Subsequently, ~t has been ~houo tbat triethyla~ine proLote~ the race~ization of NPS- NCA' 9. In addition, oligo~erlzstion d~e to the action of triet~yla~ine on the NCA, requires t~at very s~ringent reactioD condition~ ~u~t be e~ployed li.e. teuper~ture <~ C. and very slou addition of trletbyla~lne to the rea~tlon ~ixture) during NPS-NCA ~ynthesls. H~ls~ro~l et 81, SZ.
Phy~lol. Cbe~. 35S, 82-B4, (19~4)) consequently propo~ed t~e ~ynthesis of NPS-NCA'Y by re~ctlon of pho~gene Yi~b the NPS-a~ino acid but the yield~ uere very low ~about 20X). Once prepared, NPS-NCAts are dlfficult to Ytore, and tend to "bleed off" the protecting ~roup d~ring condensation, giving rise to ~ultiple co~pllng, and other Yide reactlons. Al~o, the nitrogen of the reYulting NPS protected peptide pos~e~Y~ sub~tantlal nucleophillcity snd ~ay under~o addltional conden~at~on reaction~.

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aloc~ and Cox t"PeptldeY, Proc. of the Stb Europ. Sy~p., Oxford, Septe~ber 1~62". Per~anon Press 1963, Ed. G.T. Young, pp. 84-87.) propo3ed the UQe of N-trityl a~ino acid-N-carboxyanhydrideY for use in peptide synthesis, although they vere only able to prepare the Qi~plest N-tr~tylanino acid NC~' 8 ~ie. glycine and alanine). These co~pounds uere prepared by t~e reaction of a N ~rltyl-a~ino acld ~i~h pho~gene. By thiQ
procedure, they uere al30 able to prepare N-acetyl-glyclne-NCA.
TheQe reQearchers reco~nized the potential u~efulneQ~ of t-butyloxycarbonyl glyclne N-carboxyanhydride and benzyloxycarbonyl glycine N-carboxyanhydride but ~ere unsuccesYful in their atte~pt~ to prepare the~ and concluded that uret~ane-protected a~ino acld N-carboxyanhydride~ could not be ~ade! Even if all the ~ trityl-NCA's could be prepared lt 13 uell ~novn th~t the u~e of trityl protectlon of a~ino acidQ in varlou~ condensatlon ~ethods produce~ lou yieldY
because of the considerable steric hlndrance i~posed by the trltyl group. The trityl group is al~o extremely se~Ritive to acid vhich ~a~es the preparatio~ of the trltyl-NCA difficult and tend3 to "bleed off" during ~or~al solid pba~e ~aDipulations.

~ al~troe~ & Kovac~q ~C-~a-ch~l--sca~-~-avical-er:-B-l98~ ;
BYO~6~7_462-465 and U.S. Patent No. 4, 26~, 344) al80 recogni~ed the advantages and the potential u~efulnes~ of N-protected a~ino acld N-carboxyanhydride~ and ~ere able to prepare ~everal ~-sub~tituted NCA's uhlch they fel-t uould fulfill all the requlre~ents neceqsary for u3e ln peptide ~ynthe~is. They vere able to prepare a nu~ber of 9-xanthyl ~and related~ ~ubstltuted 132~L2~0 a~ino acld N-carboxyan~ydride3. These co~pounds ~ere clal~ed to be prepara~le by the direct condensation of xantbydrol ~lth the appropriate NC~ ln r~luxing benzene, toluene, xylene or other alkyl benzene. The water for~ed during condensation ua3 renoved a~eotropically. Thls procedure suffers fro~ the lrstabillty of NCA's to heat and to water, consequently leadlng to lou yields and potentially iDpure products. The3e co~pounds oay also be prepared by the reactlon of phosgene ~or phosgene equivalent) uith the corre~ponding 9-xanthyl-a~ino acid and, in fact, ~o~t of the substituted NCA'~ ln thi~ cla~ h~ve been prepared by this procedure.

~ hen used in peptide synthesis tbe 9-xanthyl-NCA's have been found to react sluggishly requirlng as long 8a S hours at S0C
in solution and 24 hours at 25 C in ~olid phase synthe3i~.
This 19 ll~ely due to the ~teric hindrance of the 9-xanthyl group and/or tbe deact~vating effect. Anotber proble~
associated wlth 9-xanthyl protection of a~ine ~roups i8 that the nitrogen ~to~ of the 9-xanthyl a~lno acid for~ed after the coupllng reaction i~ ~till nucleophllic and capsble of undergoing sub~equent condensation reaction~. Tbese group~
also tend to bleed off during D~nip~lation. CoDsequently, to date, subssltuted a~ino acid N-carboxyaDhydridP~ of this or any other type have not found ~idespread u3e in peptlde synthesis, particularly in solid phsse peptide synthesis.

~ richeldorf, (~aXr_Dol _C e~ Vol. 178, pp 90S-939, 197~) has de~crlbed a ~ethod for the preparation of ~ethoxycarbonyl , . - :, , , , , : ~ , 13212~

glycine NCA and ethoxycarbonyl glycine NCA. However, ~rlc~eldorf alYo report4 thst t~i~ procedure was lncapable of producing urethane-protected NCA's of a~ino acids havlng a side chsln other than hydrogen becau~e of steric hindrance.

It i8 an ob~ect of the lnventlon, therefore, to provide the heretofore unobtainable urethane-protected N-carboxy~nhydrldes and N-thiocarboxyanhydride4 of the higher A~lno acids.

Another obJect of the invention i~ to provide procedurez for the preparation of pure, crystalline, stable urethane-protected s~ino acid N-carboxyanhydrideq and urethane-protected N-tbiocarboxyanhydrlde~.

Yat another ob~ect of the invention ls to provide 8 ~ethod for the synthesis of polypeptlde~ utilizlng pure, cryntalline urethane-protected a~ino acid-~-carboxyanhydrides, vhich ~ynthe~is offers the fo~louing ~ajor advaDta~e~ oYer conventional ~ethods of polypeptide Yynthe9i~:

1) Pre-activstlon of the carboxyl ~roup to be coupled i8 unneces ary, thu~ eliDinating ~ide prod~cts gener~t~d by conventlonal actl~stlng ~olec~les.

2~ No addltlve~ 4uch a N-hydroxybenzotri~zole are needed to inhibit raceDization.

,. . . . :, ,, ~, . . .

~ 3212~

S~ The only co-product fro~ the coupling reaction i3 carbon dioxide.

4~ These N-protected carboxyl activated a~ino acids are stable, ~torable, crystAlline s~terials and, therefare, facili~ate and sl~plify both solld and liquid phase peptlde synthesis, especially in auto~ated peptide ~ynthesizers, by ell~lnating the need for activations, filtratlons, and couplings prior to the peptide bond fornlDg reaction. The purlficatlo~ of peptides prepared ln solution ~B greatly facilltatPd by the use o f t he s e nove 1 c o~pounds because of the lac~ of by-product 3 produced by coupling agents.

S~ The procedure ulll provide, after co~pling, the uidely accepted urethane-protecting groups on tbe anlno function of the gro~ing peptide chain ~hich ~ay then be ~anipulated by conventloD~l techniques.

1321 28~

SUM~ARY_OF_THE_INVENTlON

These objectQ are obtalne~ by a urethane-protected a~ino acld-N-carboxyanhydrlde or N-thiocarboxyanhydride having the struc e ure:

o o 11 It /\
R' o- c - N
Rt~ c~ )1~
R' o wherein R and R' are hydrogen, alkyl, cycloalkyl, ~ub~tituted alhyl qubstltuted cycloalkyl, aryl, or subotltuted aryl and at les~t one of ~ and R' is other than hydrogen; R" 18 alkyl, aryl, subst~tuted alkyl or subQtituted aryl; Z iq oxygen or sulf~r; and n is 0, 1, or 2.

The preferred R, R' and R" groups are alkyl groups, lncluding cycloalkyl group~, of i to 12 carbon ato~ or ~ore, aryl ~roups of 6 to 20 carbon ato~s or ~ore ineluding aral~yl and alXaryl gro~p3 of 7 to 2~ carbon ato~s or ~ore. Exe~pl~ry of ~uitable alXyl group~ are ~ethyl, ethyl, propyl 9 i~opropyl, iQobutyl, butyl, t-butyl~ hexyl, cyclopentyl, cyclohexyl, heptyl, octyl, and the li~e. lllustrat~ve of ~uitable aryl groups are phenyl, ~ethylphenyl~ ethylphenyl, naphthyl, Dethylnaphthyl, anthracyl ~nd the like. Exa~ples of sultable ar~l~yl groups incl~de benzyl, p-DethoxybeDzyl, 9-fluorenyl~ethyl, phenylethyl, and the like. Suitable alkaryl .

--li-- -.

, , . . ~ , . .
. ~

~32~

groups lnclude tDlyl, ethylphenyl, isopropylphenyl and the like.
The R, R' and R" groups nay also be ~ubYtituted with non-in~erferin8 ~roups such as fluoro, ~ethoxy, t-but~xy, carboxyl, s~ido, benzyloxy, hydroxy, substituted a~lno, ~ubstituted hydroxy, ~ulfur, 3ubstituted sulfur, chloro, broDo, and the li~e. R aDd R' sre typically the protected or unprotected groups attached to the a-carbon ato~ t3ide chains) of a~ino acids or analogueR thereof.

In ~o~t instances, one of R or R' i~ u~ually H ~hlle the other is tbe side chain on the a-car~on ato~ of of an a~no acid such a~ lysine~ leuciDe, arginine, serlne, aspartic acld, alanlne, asparaglne, cy~telne, cy~tine, gluta~ic acid, hl~tldlne, gluta~ine, isoleuclne, ~ethlonlne, norleucine, ornithine, phenylalanine, threonine, tryptophan, tyro.~ine, valine, ~-alanine, bo~o~erine and the like. Exeaplary of ~uch slde chains sre:

-(CH2)g-NU2 -(CH2~-COOH

-CH2-C~-CH3 ~1 CH3 -~CH2)- H

-(CH2)3-N~-C~ -~CH2)2-CONH~

-cH-cH2-cH3 , ~ :

~3212~0 -CH3 -~H2-cH2-s-c~}3 o ~ -(cH2~3-c~3 -~CH2~3-N~2 -CH

l~2 -CH2 S-S-CH2-C~-COOH -CH-CH3 0~1 -CH2-0~1 -CH2--C~2~0H

These slde chains ~ay be yrotected 89 required, u~ing co~on teehnlque~ and protectlng groups uel~ ~nouD to one sX~lled ln the art, ~uch as the o~only e~ployed a~ino, hydroxy, thiol and carboxy protecting groups.

Tbe co~poundi of the inventlon al~o include instances ubere both R and R' are side chain3 attached to the a carbon of an a~no acid as, for exa~ple, in the ca~e of lsovaline ~bere one of R
or R' is -CH2~H3 and tbe other i~ ~ethyl.

The co~poundY of the inventlon al90 lnclude lnstanceB uhere R and R' are part of a cyclic ~tructure as, for exaDple, ln t~e . ~,, ~ , :. .

, ~ 32~2~

ca-~e of l-a~ino-1-cyclohexane carboxylic acid.

The co~pounds of the lnventlon also include exanples ~uch as ortho-a~lno benzolc acld or l-a~ino-2-carboxy cyclohexane wherein carbon ato~s fro~ the :R or R' groups are part of a cyclic ring.

Another aspect of the invention involve~ sn l~prove~ent ln the synthe313 of a polypeptlde chain ~herein a N-protected a~ino acld conponent 1~ deprotected and the deprotected a~lno acid co~ponent 18 alloued to react uith a second ~ r or-dlsslLilar activated N-protected a~lno acid co~ponent and the proces~ repeated untll the de~ired polypeptide i~ obtained, said i~prove~ent co~pri~ing using a~ the act~vated N-pro~ected aDinO
acld conponent in a~ least one of said reaction~ a co~pound havlng the structure:

o ~
--o--c --~ z ~t~C~2~

~herein R, R', R", Z and n are as de~i~Dated above.

Yet another a~pect of the lnYention involves an i~proveRent in the ~olid pha~e ~ynthe~l~ of a polypeptlde chaln on an in~oluble ~olid ~upport wherein a N-proterted a~ino acld ,, , , :

l32~2sn ,~

co~ponent i~ co~pled by condensation reaction to an ln~oluble ~olid qupport contalning 3ubstituent group3 reactive uith the carboxyl ter~inu3 end of said a~ino acid co~ponent, the coupled N-protected a~ino acld co~ponent i9 deprotected, a second sinilar or di~qi~ilar activated N-protec~ed a~ino ac~d co~ponent i8 coupled to said deproeected a~ino acid conpound, and the proce s repeated until the deQired polypeptide is obtained, sald i~prove~ent co~pri~ing uslng as the activated N-protected a~ino acid co~ponent in at lea~t one of ~aid reactions a co~pound having the ~tructure:

o R" o ~ z R ~CH2)n I ~' o uherein R, R', Z and n are designated above.

Included as a further e~bodi~ent of the invent~on 19 the Dethod of prepar~ng the ~rethane-pro~cted a~ino acid-N-carboxyanhydrides of the lnvention ~hich conpri~e~ the reaction of an a~ino acld N-carboxyanhydrlde baving the ~tructure:

H--N Z
R J~ CH2 ~1~
K' o ;. ~ . `:

~ 3212~0 ~ hereln ~, R', Z and n are a3 designated above, uith a halofor~ate tor other suitably reactive fornate, ~ch as azldo fornate~ having the ~tructure:

R"- O - C - X

~ herein X is chlol-lne, bronine, fluorine, az~de, or the like, and ~" is alkyl, aryl, or aralkyl, in an lnert dlluen~, under anhydrous conditionY and in the presence of N-nethyl~orpholine. lt has been 3urprlqingly found that utllizing an inert diluent, anhydrous condition~ and selectin~ h-nethyl~orpholine as the base ln t~ls reaction avo~ds poly~erization of N-carboxyanhydrides and othervise enable~
the production of the heretofore unobtainable urethane-protected NCA'~ and NTA'~ of higher a~ino acids.

-~6-.

:
:, ; ,, ': ~ : ~

~2~L28~

DETAILED_DESCRIPTION_OF_THE_lNYENTIQN

The a~ino acid N-carboxyanhydrlde~ ~NCA'~) and N-thlocarboxyanhydrlde~ ~NTAt 8 ~ whlch serve as starting ~aterlals for the preparation of the N-urethane protected NCA's and NTA'~
of the lnventlon ~ay be prepared by a nu~b~r of procedures uell ~no~n to one 3killed ln the art. See for exa~ple: Fuller et.
al. Blopoly~er~ 15, No. 9, 1869-1871 ~1976~; Krlcbeldorf, Che~.
Ber. 104, ~7-~1 ~1971); and Halstro~ and ~ovac8, Acta Che~ics Scandinavica, B~0, 462-465 ~i986~.

~ hile urethane~ in general ~ay be u~ed as protectl~g groups for nucleophilic ato~s, only a feu have found ~ideQpread u~e ln peptide synthesis, for exa~ple, t-butyloxycarbonyl ~Boc);
benzyloxycarbonyl ~Cbz~; and 9-fluoreno~ethyloxycarbonyl ~Fnoc~.
Con~equeDtly, a~ino ac~d N-carboxyanhydrldes or N- ~ -thiocarboxyanhydrlde~ ~ub3tit~ted ~ith tbe~e protecting ~roups are of particular intere~t. ~ccordin~ly, very u~eful ~olecules for peptide ~yDthesi~ are the NCA'~ of L-~-a~ino acld~ prctected by one of the above~entloned protectln~ group~, ~uch a3~

O ' O
1113 ~
Clti3 C-- O-C--li Z ~C~12 -- C--N Z
CH3 H~ ~ \o _ ~ 7--''' . . ' ;` '~. ' ' . : ' `' ` ::

. ` : : , : ` , .. `

~3~2~

o 11 o 11 (~ C~12 O--C--~ Z ~CH2 O ~ Z

~ hereln R 19 the side chain of an -a~ino acld, Z is O or S, and X is ~ethoxy, chloro or the lihe.

As afore~en~ioned, the N-urethane protected NCA'R of the invention are unobta~nable by the react~on oÇ pho~ne ~lth the N-urethane protected s~ino ~cid a~ dPscribed by ~loc~ and Cox ~"Peptides, Proc. of the 5th Europ. Sy~p., Oxford, Septe~ber 1962". Perga~on Pre~s 1963, Ed. G.T Yo~lng, pp. 84-87.); nor are N-urethane protected NCA's Df the hi~her a~lno acld~
obtalnable by the syr)thesls descrlbed br Krlcheldorf ~ha~ro~ol.
Che~., Yol. 176, pp ~OS-939, ~77). It has been found that urethane-protected NCA's and NTA's ~ay be prepared by the resction of a prevloualy ~ynthe~ized NCR or NTA uith the ~ppropriate halofor~ate in an anhYdrous~ non-interfering solvent ~ith the u~e of a tertiary a~lne as ba~e. The reactlon la preferably carr~ed out beluv roo~ te~peratul-e. 11seful-solvents for the reactlon are tetrahydrofuran, ethyl acetate, ~ethy~ene chloride, toluene, benzene, dioxane, and the llhe.

Thv~, the novel urethane-pl-otected a~lno acld N-carboxyanhydrldes and N-thlocarbox~anhYdl-ldeR of the lnventi~n ~ay be prep~red by d~sol~in~ sn NCA in a non-interfelin~

,., ~ . , ~ , . .

, , . ~ : ~ ::

.

132128~

solvent (such as toluene) and cooling the resulting solution with stirring. The desired haloformate (e.g.
benzylchloroformate) is then added all at once. To this mixture is added a tertiary amine base (such as triethylamine, diisopropylethylamine, N-methylmorpholine, etc.) which promotes condensation and scavenges hydrochloric acid formed during the reaction.
Surprisingly, we have found that certain tertiary amine bases such as N-methylmorpholine and N-ethylmorpholine do not promote polymerization of NCA's. Such preferred bases are those with a pK low enough not to promote NCA
polymerization but hlgh enough to catalyze the reaction of an NCA with a haloformate. Consequently, when one of these preferred bases is used in the condensation reaction, polymerization is not initiated. Since there is no fear of polymerization, the base can be used in excess and the resulting urethane protected NCA's are easily isolated by crystallization.

As a result of these discoveries, virtually any urethane protected NCA (or NTA) can be prepared easily in high yield with only minimal precaution for exclusion of moisture. The process is readily scaled up and provides products which are highly crystalline, are readily purifiable by simple technlques (i.e. crystallization), and are stable to storage (completely stable at 25C for at least 6 months and probably much longer). Thus, these materials can be weighed, shipped, and stored for use in peptide synthesis without fear of decomposition.

~ ~7 ..

'; . ; : : ; :: ~
, ~32~0 The major advantage that the urethane-protected NCA's offer over other N-substituted NCA's is that after they are used to form a peptide bond, the resulting peptide is protected on the N-terminus by one of the widely accepted urethane protecting groups commonly used in peptide synthesis. These protecting groups are well known by those skilled in the art to provide the best available protection to the amine group of a growing peptide chaln.

Thus, the use of urethane-protected N-carboxyanhydrides will offer all the advantages of the unsubstituted NCA's (high reactivity, freedom from formation of undesired rearrangement products, and CO2 as the only by product) but with none of the disadvantages of the unsubstituted NCA's (i.e. instability, polymerization, and multiple condensations) which have limited their use to carefully controlled aqueous conditions. Consequently, the invention provides a storable, yet highly reactive, preactivated reagent, which yields minimal side products during peptide bond formation. The invention also provides the widely accepted, well understood, urethane protection on the nitrogen of the N-terminus of the peptide after the condensation reaction.

While the urethane-protected amino acid-N-carboxyanhydrides of the invention can be used in the synthesis of polypeptides by classical methods using a series of deprotection and coupling reactions, they undoubtedly will find more extensive use in solid phase polypeptide synthesis. It should be understood that ~, . :: , , : :: - : : . .

~ 3 ~ 0 the term "polypeptides" as used in the specification and appended claims is meant to include peptides and proteins.
Also, it should be understood that the present invention contemplates sequential peptide synthesis wherein N-protected amino acids other than the urethane-protected amino acid-N-carboxyanhydrides are employed as well as at least one urethane-protected NCA of the invention. In practice, however, the N-protected amino acid component used in each sequence will more than likely be the urethane-protected NCA's of the invention.

In solid phase polypeptide synthesis, an insoluble solid support or matrix, advantageously in bead form, is used. Such solid supports can be any of the solid-phase polymeric substrates conventionally employed for the synthesis of polypeptides. Typical of such polymeric resins are crosslinked polystyrene resins, glass beads, clays, celite, crosslinked dextran, polyacrylamides, polyamide resins and similar insoluble solid supports which either naturally contain reactive sites for coupling with the amino acid components or which can be provided with such reactive sites.

If desired, the solid phase polypeptide synthesis of the invention can be carried out in a flow reactor under pressure as described in U.S. Patent No. 4,192,~98, but the use of superatmospheric pressure is not essential.

,. .~
.,~", .. ,, ~ , , ; ~ , , - ~
.. . . . ..
: .
~' ' ' - ' ' ,. . .. ..

~2~2~

Several preliminary operations are necessary before the solid phase synthesis of a peptide can be started.
First, the supporting resin containing the C-terminal amino acid component of the proposed peptide chain must be prepared. This can be accomplished by any of a number of procedures known to one skilled in the art. Many of these N-protected amino acids, linked to various solid supports, are articles of commerce and may be purchased as desired.

The remaining synthesis to form the desired polypeptide sequence is carried out as follows. Before coupling of the second amino acid residue can take place, the first residue already on the support must be deprotected. Deprotection of the first amino acid residue on the resin as well as of each of the subsequently coupled amino acid residues can be carried out by contacting the protected amino acid residue with an appropriate deprotecting agent. The deprotecting agents employed for this purpose are well known to those of ordinary skill in the art of peptide synthesis and the particular deprotecting agent employed in any given instance will depend, of course, upon the protecting group on the amino acid/resin. For example, if the protecting group is t-butyloxycarbonyl, trifluoroacetic acid in dichloromethane or hydrochloric acid in a suitable solvent such as dioxane may be used. On the other hand, if the protecting group is 9-fluorenylmethyloxycarbonyl, basic conditions such as piperidine in DMF will be the preferred method. The concentrations of the particular deprotecting agent in the solvent will vary depending again upon the particular .,~;,~
~.~

- - : , . , -;

,, ., .~ : -~32~2~

protecting agent employed but will ordinarily range from about 5 to 50% by volume.

After the deprotecting step, the resin is washed with a suitable solvent in order to remove excess deprotecting agents. If the deprotecting agent is an acid the peptide on the resin must be neutralized by washing with an appropriate base such as triethylamine in a solvent such as dichloromethane. Any excess triethylamine and triethylammonium chloride or trifluoroacetate formed may be removed by repeated washings with a suitable solvent such as dichloromethane or dimethylformamide. The free amine, thus prepared, is now ready for coupling with the next N-protected amino acid.

If the next N-protected amino acid is a urethane-protected amino acid N-carboxyanhydride of the invention, it need not be activated and can be reacted directly with the support now containing an unprotected resin bound amino acid. If, however, the N-protected amino acid component is to be coupled by more conventional procedures, it will be necessary to first activate, that is, convert it into a reactive form, for instance, by converting the amino acid into an anhydride or by activation with dicyclohexylcarbodiimide, carbonyldiimidazole or other activating agents. In general, an excess of the activated N-protected amino acid component is employed in the reaction.

., , , , , ~ ~ , ,. . . .
; :~: . ~ . . ;
,. ., , , ~ .i . .

132~2~

After the coupling of the second protected amino acid component to the first amino acid component, the attached protected dipeptide is then deprotected, neutralized if necessary, and washed as described above before coupling of the next amino acid derivative is effected. This procedure is repeated until the desired sequence of amino acids has been assembled on the insoluble support.

Because of the lack of undesirable side reactions and byproducts (CO2 being the only one) in the urethane protected NCA coupling, and because of their stability, the excess urethane protected NCA used in the coupling reactions may be easily recovered, recrystallized and re-used, thus markedly increasing the cost effectiveness of these materials.

The completed peptide can be removed from the insoluble support by any of the standard methods as, for instance, by cleavage with anhydrous hydrogen fluoride, transesterification, aminolysis, etc.

After cleavage, the resulting peptide is found to be remarkably homogeneous and to require no or minimal purification. Because of the very low contamination of byproducts overall yields are found to be surprisingly high and whatever purification is necessary can be carried out with relative ease. Such purifications are preferably carried out by par-tition chromatography, ion exchange chromatography or a combination of both. Such procedures are well-known to one skilled in the art of peptide synthesis.

- 2~ -^r~
J~A~

:'. ) , ' ~ .. ' ' EXAMpLE I ~ 3 2 .~

N-Carboxyanhydride Decomposition as a Function of Base A. Valine N-carboxyanhydride (72 mg) was dissolved in dry, distilled tetrahydrofuran (2 mL) and triethylamine (30 ~1) added. The disappearance of the NCA was followed by infrared spectroscopy.

B. Valine N-carboxyanhydride (72 mg) was dissolved in tetrahydrofuran (2 mL) and N-methylmorpholine (25 ~1) added. The disappearance of the NCA was followed by infrared spectroscopy.

The results of A and B are shown in the graph of Figure 1:

EXAMPLE II

N-(9-~luorenylmethyloxycarbonylL~L=~lanine-N-Carboxyanhydride A. A mixture of L-alanine (40.3 g, 0.45 mol) and phosgene (275 mL of 3.3 M solution in tetrahydrofuran, 0.90 mol~ was stirred at 62-64 C for 4 hours. The resulting solution was allowed to cool to room temperature, filtered, and the volatiles removed under reduced pressure. The resulting oil was dissolved in 100 mL of ~etrahydrofuran and 300 mL of hexane was added with stirring, followed by cooling to -20 C. The yield of L-alanine N-carboxyanhydride was 35.79 g (6~%).

B. A solution of N-methylmorpholine (8.15 g, 80.5 mmol) in toluene (5U mL) was added to a o C mixture of L-alanine-N-carboxyanhydride (8.84 g, 76.8 mmol~ and 9-fluorenvlmethyloxycarbonyl chloride (19.9 g, 76.8 mmol) in toluene (200 m~). The xeaction mixture was stirred at 0 C
for 2 hours and filtered. The volume of solvent was ~r reduced to 20 mL and crystallization occurred upon ,~

- :

:

~3~128~
addition of 100 mL of hexane to give 21.4 g (82%) of crude 9-fluorenylmethyloxycarbonyl-L alanine-N-carboxyanhydride.
The product was purified by trituration with cold diisopropyl ether followed by recrystallization from ethyl acetate/hexane: mp 106-107 C; IR (CH2C12) 1870, 1801, 1740 cm~; NMR (CDCll) ~ 6.90-7.~0 (m, 8 l~), 3.95-q.55 (m, 4 H), 1.35 (d, J = 7 Hz, 3 ~l). Anal. Calcd for ClgHI5NO5: C, 67.65;
H, 4.48; N, 4.15. Found: C, 67.73; H, 4.65; N, 4.19.

EXAMPLE III

N-(9-Fluorç~ylmethylnxYrarbo~ L-~u-c---i-ne-N
Carboxyanhy ride A. L-Leucine-N-carboxyanhydride was prepared from L-leucine in 78% yield by the procedure outlined in Example IIA.

B. A mixture of L-leucine N-carboxyanhydride (9.2 g, 58.4 mmol) and 9-fluoroxylmethyloxycarbonyl chloride (15.1 g, 58.4 mmol) in toluene (125 mL) was cooled to 0 C and a solution of N-methylmorpholine (6.5 g, 64 mmol) in 20 mL
of toluene was added dropwise. The reaction mixture was stirred at 0 C for 2.5 h, filtered, and the volume of solvent reduced to 20 mL. Hexane (480 mL) was added, and the solution was cooled to -20 C overnightl to give 18.8 g (85%) of N-(9-fluorenylmethyloxycarbonyl)-L-leucine-N
-carboxyanhydride. An analytical sample was obtained by recrystallization from ether/methylene chloride/hexane:
mp 118~120 C; NMR (CCl4) ~ 7.35-7.91 (m, 8 ~); 4.72 (t/ J
= 7 Hz, 2 H~, 4.58 (m, 3 H~; 4.37 (t, J = 7 ~z, 1 H~; 2.05 (m, 2 H); 1.09 (t, J = 6 Hz, 6 }~). Anal. Calcd for C22H2~NOs: C, 69.64; H, 5.58; ~, 3.69. Found: C, 69.08; H, 5.97; N, 3.70.

, ~ . .

s~ : :
" , ., ;, , ~ . , ~ 32~2~
EXAMPLE IV

N-~-(9-Fluorenylmethyloxycarbonyl)-N-~-t-Butyloxycarbonyl-L-Lysine-N-Car~oxyanhvdride A. A mixture of N-~-t-butyloxycarbonyl-L-lysine (1.23 g, 5.0 mmol) and chlorotrimethylsilane (1.08 g, lO.0 mmol) in tetrahydrofuran t50 mL) was cooled to 0 C and a solution of triethylamine (1.01 g, 10.0 mmol) in 5 mL of tetrahydrofuran added dropwise. The mixture was stirred at 0 C for 2.5 hours, filtered, and added to a solution of phosgene (10 mmol) in 15 mL of tetrahydrofuran. The temperature was raised to 60 C and the solution was stirred for 2.0 hours, then overnight at ambient temperature. The volatiles were removed by rotary evaporation to give 0.79 g (58%) of N-~-t-butyloxycarbonyl-L-lysine N-carboxyanhydride:
IR(CH2Cl2) 1860, 1795, 1710 cm'~.

B. A mixture of N-~-t-bu~yloxycarbonyl-L-lysine N-carboxyanhydride (0.79 g, 2.90 mmol) and 9-fluorenylmethyloxycarbonyl chloride (0.75 g, 2.90 mmol) in toluene (25 ml) was cooled to 0 C and a solution of N-methylmorpholine (0.32 g, 3.2 mmol) in toluene (5 ml) added dropwise. ,The reaction was worked up as in Example IIIB to give 0.88 g (~6%) of N-~-(9-fluorenylmethyloxycarbonyl)-N-~-t-butyloxycarbonyl-L-lysine-N-carboxyanhydride: mp 81-85 C
(ethyl acetate/hexane); NMR (CDCl3) ~ 7.3-7.7 (m, 8 H), 4.11-4.58 (m 5 H), 2.95-3.20 ~m, 2 ll); 1.90-1.98 (m, H);
0.9-1.4 (m, 13 H). Anal. Calcd for C27H3~2G7: C, 65.57; H, 6.11; N, 5.67. Found C, 66.33; H, 6.3~; N, 5.67.

EXAMPLE V

N-Benzyloxycarbonyl-L-Alanine N-Carboxyanhydride A solution of N-methylmorpholine (1.06 g, 10.5 mmol) in ethyl acetate (20 mL) was added dropwise to a mixture ,, ,,~, , , .

~' " ' ,' ' ' '` .
. .

132~2~
of L-alanine-N-carboxyanhydride (from IIA) (0.81 g, 7.0 mmol) and benzyloxycarbonyl chloride (1.89 g, 10.5 mmol) in ethyl acetate (80 mL) at 0 C. The reaction mixture was stirred for 1.5 h at o C, filtered, and the volume of the solution was reduced to 75 mL. Hexane (75 mL) was added with stirring, followed by cooling to -20 C, to give 1.20 g (71%) of N-benzyloxycarbonyl-L-alanine-N-carboxyanhydride: mp 101-104 C; NMR (CDCl3) ~ 7.33 (s, 5 H), 5.27 ts, 2 H), 4.60 (q, J = 7 Hz, 1 H), 1.61 (d, J =
7 Hz, 3 H). Anal. Calcd for Cl2H~lNOs: C, 57.83; H, 4.45; N, 5.62. Found: C, 57.60; H, 4.50; N, 5.53.

EXAMPLE VI

N-Benzyloxycarbonyl~L-Leucine-N-Carboxyanhydrlde A solution of N-methylmorpholine (0.76 g, 7.50 mmol) in ethyl acetate (10 mL) was added dropwise to a solution of L-leucine N-carboxyanhydride (0.79 g, 5.0 mmol)(from IIIA) and benzyloxycarbonyl chloride (1.35 g, 7.50 mmol) in ethyl acetate (50 mL) at 0 C. The reaction mixture was stirred at 0 C for 1.25 h, filtered and the volume of the solution was reduced to 5 mL. Hexane (50 mL) waC added, followed by cooling to -20 C, to give 0.89 g (61%) of N-benzyloxycarbonyl-L-leucine-N-carboxyanhydride: mp 72-73.5 C (ether/hexane); NMR (CDCl3) ~ 7.40 (s, 5 H), 5.33 (s, 2 H), 4.71 (t, J = 6 Hz, 1 H), 1.80-2.04 (m, 3 H), 0.91 (m, 6 H). Anal. Calcd for C~5HI7NOs: C, 61.84; H, 5.88; N, 4.81. Found: C, 61.64; H, 6.02; N, 4.90.

- EXAMPLE VII

N-Phenyloxycarbonyl-L-Vallne-N=Carboxyanhydride A. L-Valine-N-carboxyanhydride wa~ prepared from L-valine in 75% yield by the procedure described in Example IIA.

B. Example V was repeated substituting , .

, ' 1 32:1 2~0 phenylchloroformate for benzylchloroformate and valine N-carboxyanhydride for leucine N-carboxyanhydride. The result was a 78~ yield of N-phenyloxycarbonyl-L-valine-N-carboxyanhydride: mp 105-106 C
(chloroform/hexane); NMR (CDC13) ~ 7.30 (m, 5 H), 4.70 (d, J = 3.5 Hz, l H), 2.60 (m, 1 H), 1.22 (d, J = 7 Hz, 3 H), 1.07 (d, J = 7 Hz, 3 H), 1.07 (d, J = 7 Hz, 3 H). Anal.
Calcd for Cl2HI3NOs: C, 59.31 ~l, 4.98; N, 5.32. Found: C, 59.09; H, 4.91; N, 5.49.

EXAMPLE VIII

N-Ethyloxycarbonyl-L-Alanine N-Carboxyanhydride Example V was repeated substituting ethyl chloroformate for benzyl chloroformate. The result was a 62% yield of N-ethyloxycarbonyl-L-alanine N-carboxyanhydride: mp 72-73.5 C (ethyl acetate/hexane);
NMR (CDCl3) ~ 4.73 (q, J = 7 Hz, 2 H), 4.33 (q, J = 7 Hz, 1 H), 1.70 (d, J = 7 Hz, 3 H), 1.33 (t, J = 7 Hz, 3 H).
Anal. Calcd for C7H9NO5: C, 44.92; H, 4.85~ N, 7.49. Found:
C, 45.08; H, 5.03; N, 7.33.

EXAMPLE IX

Benzyloxycarbonvl-L-Phenylalanine-N-Carboxyanhydride A. L-Phenylalanine-N-carboxyanhydride was prepared from L-phenylalanine in 53% yield by the procedure of Example IIA.
B. To a solution of L-phenylalanine-N-carboxyanhydride (2.5 g, 13 mmol) and benzylchloroformate (3.4 g, 20 mmole) in ethyl acetate (130 mL) was added dropwise a solution of N-methylmorpholine (2.0 g, 20 mmol~ in ethyl acetate (lO mL) at 0 C. The resulting mixture was stirred at 0 C for 2.5 h and worked up as described in Example V to give 2.0 g (48%) of N- benzyloxycarbonyl-L-phenylalanine-N-carboxyanhydride: mp 108-lO9 C; NMR (CDC13) S 7.35 (s, v~ --- .

13~2~
5 H), 7.00 (m, 5 H), 5.31 (s, 2 H), 4.83 (m, 1 ~), 3.~8 (m, 2 H), Anal. Calcd for C~8H~7NO5: C, 68.13; H, 5.40; N, 4.42. Found: C, 68.11; H, 5.38; N, 4.20.

EXAMPLE X

Phenyloxycarbonyl-L-~lanine-N-Thiocarboxyanhydride A. O-Ethyl-S-methylxanthate. To a solution of potassium ethylxanthate (16.0 g, 100 mmol) in water (50 mL) was added dropwise dimethyl sulfate (12.6g, 100 mmol) at 4 + 1 C. Upon completion of the addition, the reaction mixture was washed with dichloromethane (2 x 40 mL) and the combined organic fractions were dried (MgSO4) and concentrated. The oily residue was dissolved in methanol and concentrated to give O-ethyl-S-methylxanthate of sufficient purity for use in the next step.
.
B. Ethoxythiocarbonyl-L-alanine. To the O-ethyl-S-methylxanthate prepared above was added a solution of L-alanine (~.9 g, 100 mmol) and NaOH (4.0 g, 100 mmol) in water (100 mL) The solution was heated to 45 C for 2.3 h.
While being purged with N2, methanol (50 mL) was added and the mixture stirred at 45 C for an additional 0.7 h. The reaction mixture was allowed to cool to room temperature, washed with dichloromethane (3 x 25 mL3, acidified to pH
2.5 with concentrated HCl, and extracted with ethyl acetate (2 x 50 mL). The combined organic solutions were dried (MgSO4) and concentrated. Addition of hexane ko the resulting oil gave 9.5 g (54%) of ethyloxythiocarbonyl-L-alanine as a colorless solid which melted at 74-78 C. This material was further purified by recrystallization from ether/hexane: mp 77-79 C; IR (CCl~) 3397, 1716 cml.

C. ~-Alanine-N-thiocarhoxyanhydride. To a solution of ethyloxythiocarbonyl-L-alanine (3.0 g, 17 mmol) and imidazole (1.2 g, 17 mmol) in THF (20 mL) was added dropwise PBr3 (5.4 g, 20 mmol) at 20 C. Stirring was 3~

~ 32~2~
continued until the solid mass had broken up into a fine suspension. ~he reaction mixture was poured into a mixture of saturated NaHC03 ~200 mL) and ethyl acetate (150 mL). The organic layer was separated, washed with 1 M
~Cl(2 x 100 mL), saturated NaHC03 (100 mL), an~ brine (100 mL), dried (MgSO4), and concentrated. The resulting oil solidified on standing. Recrystalli2ation of the solid gave 0.75 g (34%) of L,-alanine-N-thiocarboxyanhydride: mp 91-92 C; IR (CCl4) 1750, 1695 cm-~.

D. Phenyloxycarbonyl-L-alanine-N-thiocarboxyanhydride. ~o a solution of L-alanine-N-thiocarboxyanhydride (0.~9 g, 3.8 mmol) in 50 mL of ethyl acetate was added phenyl chloroformate (0.95 g, 6.1 mmol) at 0 C, followed by dropwise addition of a solution of N-methylmorpholine (0.57 g, 5.6 mmol) in ethyl acetate (10 mL) at 0 C. ~he resulting mixture was stirred for 3 h at 0 C, filtered and concentrated to a white semi-solid. The semi-solid material was dissolved in 20 mL
of ethyl acetate, hexane (150 mL) was added~ and the mixture cooled to -20C to give 0.55 g (62%) of phenyloxycarbonyl-L-alanine-N-thiocarboxyanhydride:
mp 110-111 C~ NMR (CDCl3) ~ 7.18 (m, 5 H), 4.83 (q, 1 H, J
= 7 Hz), 1.71 ~d, 3 H, J = 7 Hz); IR (CH2C12) 1810, 1740 (doublet), ,1715 tShoulder). Anal. Calcd for C~H9NO4S: C, 52.58; H, 3.61; N, 5.58; S, 12.76. Found: C, 52.75; H, 3.72; N, 5.36; S, 12.98.

EXAMPLE XI

N-(9-Fluorenylm thyloxycarbonylj-O-t-Butyl-L-Threonine-N=
Carboxyanhydride A. 0-~-Butyl-L-threonine N carboxyanhydride was prepared from O-t-butyl-L-threonine in 57% yield using the trimethylsilyl procedure described in Example IVA~

B. To a solution of 0-t-butyl-L-threonine N~carboxyanhydride (0.80 g, 4.0 mmol) and .

- -!
, 13~2~
9-fluorenylmethyloxycarbonyl chloride (1.0 g, 4.0 mmol) in toluene (50 mL) was added dropwise a solution of N-methylmorpholine tO.49 g, 4.8 mmol) in 8 mL of toluene at 0 C. The reaction was stirred for 3 h at 0 C, filtered, and the volatiles removed under reduced pressure. The residue was crys-tallized from ether/hexane to give 1.0 g (60%) of N (9-fluorenylmethyloxycarbonyl)-0-t-butyl-L-threonine: mp 124-127 C; NMR (CDCl3) ~
7.08-7.78 (m, 8 H), 4.05-4.61 (m, 4 H), 1.18 (s, 9 H), 1.16 (d, 3 H, J= 7 llz). Anal. Calcd for C2~H2sNO6: C, 68.07; H, 5.95; N, 3.31. Found: C, 67.89; H, 5.96; N, 3.28.
EXAMPLE XII

Ethyloxycarbonyl-~-Aminoisobutyric Acid-N-Carboxyanhydride A. a-Aminoisobutyric acid N-carboxyanhydride was prepared in 67% by the procedure described in Example IIA.

B. Example VIIIB was repeated substituting -aminoisobutyric acid N-carboxyanhydride for L-alanine-N-carboxyanhydride to give a 16% yield of ethyloxycarbonyl-~-aminoisobutyric acid N-carboxyanhydride: mp 68-70 C (chloroform/hexane); NMR
(CCl4) ~ 4~59 tq, 2 H, J = 7 Hz), 2.00 (s, 6 ~l), 1.65 (t, 3 H, J = 7 Hz). Anal Calcd for C8HI~NOs: C, 47.76; H, 5.51;
N, 6.96. Found: C, 47.67; H, 5.51; N, 7.14.

EX~MPLE XIII

N-t-ButyLoxycarbonyl-L-Alanine N-Carboxyanhydride . To a solution of t-butyl alcohol (1.25 g, 16.9 mmol) and phosgene (3.4 mL o~ a 5 M solution in dioxane, 17 mmol) in B0 mL of ethyl acetate was add~d dropwise N-methylmorpholine (3.4 g, 34 mmol) at -50 C. The reaction mixture was stirred for 0.5 h.
L-Alanine-N-carboxyanhydride (0.23 g, 2.0 mmol) in ethyl acetate (10 mL) was added and the mixture stirred at -50 C

. , . " ~
. . ..

.

13212~0 for an additional 0.75 h. N-Methylmorpholine (1.0 ~, 10 mmol) was added, and stirring was continued for another 0.75 h at -50 C. The solids were removed by filtration, the solution was concentrated, and the product was obtained after trituration with hexane. ~ecrystallization from toluene gave 0.28 g (65%) of N-t-butyloxycarbonyl-h-alanine-N-carboxyanhydride. mp 103-104.5 C; NMR (CDC13) ~ 4.71 (q, 1 H, J = 7 ~Iz), 1.80 (d, 3 H, J = 7 Hz), 1.70 (s, 9 H). Anal. Calcd for C~HI3NO5: C, 50.23; H, 6.09; N, 6.5~. Found: C, 50.66; H, 6.36; N, 6.38.

EXAMPLE XIV

N-tt-Butyloxycarbonyl)-O-Benzyl-L-Serine-N-Carboxya hydride A. 0-Benzyl-L-serine-N-carboxyanhydride was prepared in 68% yield by the procedure of Example IIA.
.

B. Example XIII was repeatad substituting O-Benzyl-L-serine-N-carboxyanhydride for L-alanine-N-carboxyanhydride to give N-(t-butyloxycarbonyl)-O-benzyl-L-serine-N-carboxyanhydride in 52% yield: mp 98-99.5 C; NMR (CCl4) ~ 7.30 (m, 5 Il), 4.64 (m, 3 H, benzyl CH2 and NCA ring proton), 4.09 (dd, 1 H, J = 15, 5 Hz), 3.88 (dd, 1 H, J =
15~ 5 Hz), 1.65 (s, 9 H); Anal. Calcd for C~5H~9N06: C, 59.80; H, 5.96; N, 4.36. Found: C, 59.71; ~, 6.25; N, 4.05.
EXAMPLE XV

Phe~y~oxycarbo~y~ __ ion of l-Amino-l-Cyclohexanecarboxylic ACid N-Carboxyanhydride A. l-Amino-l-cyclohexanecarboxylic acid-N-carboxyanhydride was prepared from 1-amino-1-cyclohexanecarboxylic acid in 50% yield by the procedure described in Example IIA.

, ~ 32~2~
B. To a solution of the N-carboxyanhydride prepared in A (0.85 g, 5.0 mmol) and phenyl chloroformate (1.2 g, 7.5 mmol) in ethyl acetate (30 mL) at 0 C was added a solution of N-methylmorpholine (0.76 g, 7.5 mmol) in 8 mL
of ethylacetate. The reaction mixture was stirred for 2 h at 0 C, filtered, and concentrated. The white, semi-solid residue was recrystallized from ethyl ether/methylene chloride/hexane to give 0.92 g (66%) of the N-carboxyanhydride: mp 156.5-158 C; NMR (CDCl3) S 7.28 (m, 5 H), 1.20-3.10 (m, 10 H). Anal. Calcd for C~sH~5NO5: C, 62.27; H, 5.23; N, 4.~4. Found: C, 62.03; ~, 5.22; N, 4.77.

EXAMPLE XVI

N~(9-Fluorenylmethyloxycarbonyl~-L-Leucine-N-Carboxyanhydride A. L-Leucine-N-carboxyanhydride was prepared from L-leucine in 78% yield by the procedure outlined in Example IIA.

B. A mixture of L-leucine N-carboxyanhydride (9.2 gm, 58.4 mmole) in dry, distilled ethyl acetate (150 mL) was cooled to -10 C and a solution of triethylamine (9mL, 64 mmol) in 20 mL of ethyl acetate was added dropwise over 5 minutes. Cooling was discontinued and the reaction was allowed to stir for 15 minutes. A small amount of anhydrous hydrochloric acid in dioxane (4.4M, 5 ml~ was added in order to ensure that all of the triethylamine had been neutralized. The reaction was filtered and the solvent removed ~n=Y3ç~. The resulting crude product was taken up in ethyl ether, filtered, and the product obtained by cry~tallization after addition to hexane.

After three additional, careful crystallizations, N-(9-fluorenyl-methyloxycarbonyl)-L-leucine-N-carboxyanhydride was obtained in 16% yield (3.6 grams).
Analytical data was essentially identical to that obtained in Example III. ~ 3 2 ~

EXAMPLE XVII

L-Leucyl-L-Valine 9~Fluorenylmethyloxycarbonyl-L-valine esterified to p-alkoxybenzyl alcohol derivitized 2% crosslinked polystyrene (0.25 gm, 0.13 mmol valine) was placed in a solid phase peptide synthesis vessel. Dimethylformamide (5 mL) was added and the slurry was shaken for 30 min.
The dimethylformamide was removed and the swollen resin treated twice with 10% piperidine in dimethylformamide (5 mL for 5 min followed by 5 mI. for 15 min) to remove the 9-fluorenylmethyloxycarbonyl protecting group. The resin was washed with dimethylformamide (4 x 5 mL) and reacted with 9-fluorenylmethyloxycarbonyl-L-leucine-N-carboxyanhydride (145 mg, 0.38 mmol) in dimethylformamide (6 mL) ~or 45 min. The fluorenylmethyloxycarbonyl protecting group was removed as above and the resin washed with dimethylformamide (3 x 5 mL) and methylene chloride (3 x 5 mL). The resulting dipeptide was cleaved from the resin by treatment with methylene chloride/
trifluoroacetic acid (6 mL, 1/1) for 45 min. The solution was removed and the resin washed with methylene chloride (3 x 5 mL) and methanol (2 x 5 mL). The combined solution and washes were evaporated ln vacuo to a semi-solid, which was taken up in distill~d water and filtered. The aqueous solution was freeze-dried, the resulting solid triturated with ethert3X) to remove resin related contaminants, and dried under reduced pressure to give L-leucyl-L-valine in >90% yield. The identity o~ the dipeptide was confirmed by HPLC analysis (flow rate = 1.5 mL/min, detection at 215 nm, 30% methanol in 0.5 M perchloric acid) by co-elution with a known standard (Retn. time ~.~9 minutes). The purity was determined to be >97~, with all contaminants being traceable to the resin. No D-leucyl-L-valine (Retn.
time 32 minutes) could be detected (detection limits <O. 1%) .

\ ~ 35 .
., . ,, ,, , ~, , . -.
,, , .-.
" .

~, . , , :
,: , ~ , , ~3~2~
EXAMPLE XVIII

L-Leucyl-L-Valine Example XVI was repeated except that 9-fluorenylmethyloxycarbonyl-L-leucine-N-carboxyanhydride was reacted with the free amine of L-valine on the resin using methylene chloride (5 mL) instead of dimethylformamide as the solvent. The results were comparable to Example XVXI.

EXAMPLE XIX

L-Leucyl~L-Alanyl-L-Valine (FMOC Procedure) The procedure o~ Example XVII was used to prepare L-leucyl-L-alanyl-L-valine. After cleavage from the resin and ether washes the tripeptide was obtained in >88% yield as a white solid. HPLC analysis, using conditions d~scribed in Example XVI confirmed the identity of the product which co-eluded with a known standard. (Rtn. time 16.28 min.) Deletion sequences such as L-leucyl-L-valine and L-alanyl-L-valine were not detected (detection limits <O. 1%) -EXAMPLE XX

L-Leucyl-L-Alanyl-L-Valine (Boc Procedure) t-Butyloxycarbony-L-valine esterified to methylated 2%
crosslinked polystyrene (i.e. Merryfield resin) (0.50 gm, 0.23 mmol valine) was placed in a solid phase peptide synthesis vessel. Methylene chloride (5 mL) was added and t~e slurry shaken for 30 minutes. ~he solvent was removed and the resin treated with methylene chloride/tri~luoroacetic acid (6 mL of 1/1) for 30 min to remove the t-butyloxycarbonyl protecting group. ~he resin was washed with methylene chloride (3 x 5 mL) neutralized with 10% triethylamine in methylene chloride (5 mL), .. . ..

. .

~3~2~
washed with methylene chloride (3 x 5 mL) and then reacted with a solution of t-butyloxycarbonyl-L-alanine-N-carboxyanhydride (200 mg, 1.0 mmol) in methylene chloride (5 mL) for 45 min. The resulting protected dipeptide resin was washed with methylene chloride (3 x 5 mL). The resin was again deblocked, washed, neutralized and washed as described above. t-Butyloxycarbonyl-L-leucine-N-carboxyanhydride (240 mg, 1.0 mmol) in methylene chloride (6 mL) was added to the resin and the mixture shaken for 45 min. The solution was removed and the resin washed with methylene chloride (3 x 5 mL), methanol (3 x 5 mL) and methylene chloride (3 x 5 mL) and dried under high vacuum. The t-butyloxycarbonyl protected tripeptide resin was reacted with liquid hydroqen fluoride at 0 C for 30 minutes. The hydrogen fluoride was removed and the residue dried under high vacuum. The peptide was taken up in water and the resin removed by filtration. The solution was freeze-dried to give a nearly quantitative yield of L-leucyl-L-alanyl-L-valine. HPLC analysis results were comparable to Example XVIII.

EXAMPLE XXI

L-Alanyl-L-Phenylalanine (Via Full Protectlon Procedure) A. To L-phenylalanine benzyl ester p-toluenesulfonate (1.07 g, 2.5 mmol) in tetrahydrofuran (20 mL) at 0 C was added N-methylmorpholine (0.25 g, 2.5 mmol). The mixture was stirred for 0.5 h at 0 C and N-benzyloxycarbonyl-L-alanine-N-carboxyanhydride (0.50 g 2.0 mmol) wa~ added. The reaction mixture was stirred 2 h at 0 C and water (20 mL) and dichloromethane (50 mL) were added. The layers were separated and the aqueous layer washed with dichloromethane (25 mL). The combined organic fractions were washed with 0.5 M HCl (2 x 50 mL), 10%
sodium bicarbonate (50 mL), and water (2 x 50 mL~, dried (MgSO4), and concentrated. Crystallization occurred upon addition of hexane to give 0.66 q (72%) of "~jC
.~

:

.

132~2~
N-benzyloxycarbonyl-L-alanyl-L-phenylalanine benzyl es~er:
mp 118.5-119 C; N~R (CDCl3) ~ 7.64(s, 1 H), 6~82-7.39 (m, 6 H), 5.04 (s, 2 H), 5.00 (s, 2 H), 4,58-4.92 (m, 2 H~, 3.07 (d, J = 6 Hz, 2 H) 1,29 (d, J=7 Hz, 3 H).

B. A mixture of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine benzyl ester ~0.50 g, l.1 mmol) and 10% palladium on carbon (0.1 g) in ethyl alcohol (150 mL) was shaken on a Parr hydrogenatlon apparatus for 6.5 h at 20 C. The reaction mixture was filtered and the filtrate rinsed with water (lOOmL). The solution was concentrated to give 0.26 g (100~) of L-alanyl-L-phenylalanine. HPLC analysis showed ~99%
purity and no evidence of racemization, EXAMPLE XXII

L-Alanyl-L-Phenylalanine (Via Partlal Protectlon_Procedure A. To a solution of L-phenylalanine tO.33 g, 2.0 mmol) in 0.20 M potassium carbonate (20 mL) and acetonit~ile (30 mL) was added dropwise a solution of N-benzyloxycarbonyl-L-alanine N-carboxyanhydride (0.45 g, 1.8 mmol) in acetonitrile (5 mL) at O C. The mixture was stirred 40 min at O C and diluted with ethyl acetate (50 mL) and 1 M hydrochloric acid (10 mL). The layers were ~eparated and the aqueous layer extracted ~ith ethyl acetate (2 x 35 mL). The combined organic fractions were washed with brine (30 mL), 0.5 M hydrochloric acid (2 x 50 mL) and water (2 x 50 mL), dried (MgSO4) and concentrated.
The residue was recrystallized from chloroform/hexane to give 0.26 g (39%) of N-benzyloxycarbonyl-L-alanyl-L-phenylalanine: mp 121-122 C; NMR (DMSO-d6) ~ 12.71 (s, 1 H), 8.06 (m, 1 H), 7.30 (m, 5 H~, 5.01 (s, 2 H), 4.43 (m, 1 H), 4.06 (m, 1 H), 2.99 (m, 2 H), 1.19 (d, 3 H, J 7 Hz).

B. A mixture of N benzyloxycarbonyl-L-alanyl-L-phenylalanine (0.208 g, 0~562 mmol) and 10% Pd on carbon ~ r '~

.
.. . :. , : , .
, : , ~ ~2~2g~
(0.1 g) in 95% ethyl alcohol (50 mL) was shaken on a Parr hydrogenation apparatus for 16 h at 20 C. The reaction mixture was filtered and the filtrate rinsed with water (lOOmL~. The combined solutions were concentrated to give 0.122 g (92%) of L-alanyl-L-phenylalanine as a white solid. HPLC analysis showed >99.5% purity and no evidence of racemization.

:, .
, :

Claims (73)

1. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride having the structure:

wherein R and R' are hydrogen, alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, aryl, or substituted aryl and at least one of R and R' is other than hydrogen; R" is alkyl, aryl, substituted alkyl or substituted aryl, Z is oxygen or sulfur; and n is 0, 1 or 2;
and wherein any substituents in the groups R, R' and R" are chosen from fluoro, alkoxy, carboxyl, amido, aryloxy, hydroxy, sulfur containing groups, chloro, and bromo.

2. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 1 wherein R" is an alkyl of 1 to 20 carbon atoms.

3. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 2 wherein R" is lower alkyl or substituted lower alkyl.

4. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 3 wherein R" is t-butyl.

5. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 1 wherein R" is aryl or substituted aryl.

6. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 5 wherein R" is phenyl or substituted phenyl.

7. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 1 wherein R" is aralkyl or substituted aralkyl.

8. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 7 wherein R" is benzyl or substituted benzyl.

9. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 8 wherein R" is p-methoxybenzyl.

10. A urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 7 wherein R" is 9-fluorenylmethyl or substituted 9-fluorenylmethyl.

11. A urethane protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 1 through 10 wherein at least one of R or R' is the side chain of a protected or unprotected amino acid.

12. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of alanine.

13. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of arginine or suitably protected arginine.

14. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of aspartic acid or suitably protected aspartic acid.

15. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of asparagine or suitably protected asparagine.

16. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of cysteine or suitably protected cysteine.

17. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of cystine.

18. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of glutamic acid or suitably protected glutamic acid.

19. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of glutamine or suitably protected glutamine.

20. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of histidine or suitably protected histidine.

21. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of isoleucine.

22. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of a suitably protected lysine.

23. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of leucine.

24. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of methionine.

25. A urethane-protected amino acid N-carboxyanhydride or H-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of norleucine.

26. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of a suitably protected ornithine.

27. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of phenylalanine.

28. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of serine or suitably protected serine.

29. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of threonine or suitably protected threonine.

30. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of tryptophan or suitably protected tryptophan.

31. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of tyrosine or suitably protected tyrosine.

32. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of valine.

*

33. A urethane-protected amino acid N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 11 wherein R or R' is the side chain of homoserine or suitably protected homoserine.

34. N-9-fluorenylmethyloxycarbonyl-L-leucine-N-carboxyanhydride.

35. N-9-fluorenylmethyloxycarbonyl-L-alanine-N-carboxyanhydride.

36. N-.alpha.-(9-fluorenylmethyloxycarbonyl)-N-.epsilon.-t-butyloxycarbonyl-L-lysine-N-carboxyanhydride.

37. N-benzyloxycarbonyl-L-alanine-N-carboxyanhydride.

38. N-benzyloxycarbonyl-L-leucine-N-carboxyanhydride.

39. N-phenyloxycarbonyl-L-valine-N-carboxyanhydride.

40. N-ethyloxycarbonyl-L-alsnine-N-carboxyanhydride.

41. N-benzyloxycarbonyl-L-phenylalanine-N-carboxyanhydride.

42. N-phenyloxycarbonyl L-alanine-N-thiocarboxyanhydride.

43. N-(9-fluorenylmethyloxycarbonyl)-O-t-butyl-L-threonine-N-carboxyanhydride.

44. N-9-fluorenylmethyloxycarbonyl-.beta.-alanine-N-carboxyanhydride.

45. N-t-butyloxycarbonyl-L-alanine-N-carboxyanhydride.

46. N-(t-butyloxycarbonyl)-O-benzyl-L-serine-N-carboxyanhydride.

47. N-phenyloxycarbonyl-1-amino-1-carboxy cyclohexane-N-carboxyanhydride.

48. N-ethyloxylcarbonyl-.alpha.-aminoisobutyric acid-N-carboxyanhydride.

49. A method for the synthesis of a polypeptide chain wherein an amino acid component is allowed to react with a second similar or dissimilar amino acid component and the process repeated until the desired polypeptide is obtained, the improvement comprising using as the protected amino acid component in at least one of said reactions a compound having the structure:
wherein R and R' are hydrogen, alkyl, aryl, substituted alkyl, or substituted aryl; R" is alkyl, aryl, substituted alkyl or substituted aryl; Z is oxygen or sulfur; and n is 0, 1 or 2, and at least one of R and R' is other than hydrogen wherein any substituent present in the groups R, R' and R" is inert under the conditions of the polypeptide chain synthesis reaction.

50. A method according to Claim 49 wherein the protected amino acid component is a urethane-protected amino acid-N-carboxyanhydride or N-thiocarboxyanhydride according to Claim 1 wherein R" is an alkyl, or substituted alkyl of 1 to 20 carbon atoms.

51. A method according to Claim 50 wherein R" is lower alkyl or substituted lower alkyl.

52. A method according to Claim 50 wherein R" is t-butyl.

53. A nethod according to Claim 49 wherein R" is aryl or substituted aryl.

54. A nethod according to Claim 53 wherein R" is phenyl or subbtituted phenyl.

55. A method according to Claim 49 wherein R" is aralkyl or substituted aralkyl.

56. A method according to Claim 55 wherein R" is benzyl or substituted benzyl.

57. A method according to Claim 55 wherein R" is p-methoxybenzyl.

58. A method according to Claim 55 wherein R" is 9-fluorenylmethyl or substituted 9-fluorenylmethyl.

59. A method according to Claim 49 through 57 wherein at least one of R or R' is the side chain of a protected or unprotected amino acid.

60. A method for the solid phase synthesis of a polypeptide chain on a soluble or insoluble support wherein a protected amino acid component is coupled by condensation reaction to the support containing substituent groups reactive with the carboxyl terminus end of said amino acid component;
the coupled protected amino acid component is deprotected, neutralized if necessary, and a second similar or dissimilar protected amino acid component coupled to said deprotected amino acid compound, and the process repeated until the desired polypeptide is obtained, the improvement comprising using as the protected amino acid component in at least one of said reactions a compound having the structure:
wherein R and R' are hydrogen, alkyl, cycloakyl, substituted alkyl, substituted cycloalkyl, aryl, or substituted aryl; R" is alkyl, aryl, substituted alkyl or substituted aryl; Z is oxygen or sulfur; and n is 0, 1 or 2, and at least one of R and R' is other than hydrogen, and wherein any substituent present in the groups R, R' and R" is inert under the conditions of the coupling reaction.

61. A method according to Claim 60 wherein R" is an alkyl or substituted alkyl of 1 to 20 carbon atoms.

62. A method according to Claim 61 wherein R" is lower alkyl or substituted lower alkyl.

63. A method according to Claim 62 wherein R" is t-butyl.

64. A method according to Claim 60 wherein R" is aryl or substituted aryl.

65. A method according to Claim 64 wherein R" is phenyl or substituted phenyl.

66. A method according to Claim 60 wherein R" is aralkyl or substituted aralkyl.

67. A method according to Claim 66 wherein R" is benzyl or substituted benzyl.

68. A method according to Claim 66 wherein R" is p-methoxybenzyl.

69. A method according to Claim 66 wherein R" is 9-fluorenylmethyl or substituted 9-fluorenylmethyl.

70. A method according to Claims 60 through 69 wherein at least one of R or R' is the side chain of a protected or unprotected amino acid.

71. A method of preparing urethane-protected amino acid N-carboxyanhydrides or N-thiocarboxyanhydrides having the structure:

wherein R and R' are hydrogen, alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, aryl, or substituted aryl; R" is alkyl, aryl, substituted alkyl or substituted aryl; Z is oxygen or sulfur; and n is 0, 1 or 2, and at least one of R and R' is other than hydrogen, and wherein any substituents in the groups R, R' and R" are chosen from fluoro, alkoxy, carboxyl, amido, aryloxy, hydroxy, sulfur containing groups, chloro, and bromo, which method comprises reacting an amino acid N-carboxyanhydride or N-thiocarboxyanhydride having the structure:

wherein R, R', Z and n are as designated above, with a haloformate having the structure:

wherein X is halogen and R" is as designated above, in an inert diluent, under anhydrous conditions and in the presence of a tertiary amine base.

72. A method according to Claim 71 wherein the tertiary amine base is N-methylmorpholine.

73. A method according to Claim 71 wherein the urethane-protected amino acid N-carboxyanhydride reaction product is recovered by crystallization.