Teaahedron Letters, Vol32, No 6, pp 739-742 1991 hinted m Great Bntam

004@4039/91 $3 Ml + PLrgaInon press plc

PROTECI-ION OF CARBOXAMIDE FUNCTIONS BY THE TRITYL RESIDUE. APPLICATION TO PEPTIDE SYNTHESISl

Peter Sleber and Bernhard Rmlker Pharmaceuticals Dlvislon, Ciba-Geigy Limxted, CH-4002 Basle, Switzerland

Abstract. Carboxamlde fun&Ions may be tntylated by an aad-catalyzed reaction wth tnphenylmethanol and acetic anhydnde m glaaal acetic acid The w-tntyl group of asparagme and glutamme 1s cleavable by TFA, but stable to strong rnmeral aads m aqueous solution, as well as to nucleophlles and bases In pepbde syntheses, It 1s Ideally sulted for combmatlon with slde-cham protectlons of the t butyl-type

The synthesis of peptides contammg asparagme or glutamine wth unprotected stde-chams IS known to be

fraught with different types of problems Reaction of the activated a-carboxyl group of asparagme mth the p-

carboxamlde function forms mtnles and succuunude denvatives llus IS often the cause of slow or incomplete couplmg

mth this ammo acid and of by-products wlch are hard to separate Under alkalme condlhons, asparagme-peptides

may be dearmdated, resulting m a nuxture of u- and P-aspartlc-acid denvatlves Finally, peptldes with asparagme

or glutamme frequently exhlblt low solublhty and aggregation phenomena caused by hydrogen bndges Thus may even

occur m resin-bound compounds and lead to unsahsfactory results of solid-phase syntheses Aunmg to Improve the

synthesis of peptldes with asparagme and glutamme restdues, various groups for the protechon of the w-carboxamlde

function have been proposed, for instance 9-xanthenyl (Xanj2, 2,4,6-tnmethoxybenzyl (Tmob) 3, 4,4’-dime-

thovybenzhydryl (Mbh) 4, and 2,2’4,4’-tetramethoxyberuhydryl (Tbh) 5 Their use has been reported for syntheses m

solution as well as by solld-phase methods, however, none of them has found a mde and general acceptance due to

shortcomings at certam stages of thclr preparahon or appllcatlon Currently, most peptide syntheses are still

performed with unprotected side-chams of asparagne and glutamme, although with sohd-phase methods an

increased tendency to use protecting groups can be observed m recent years

We consldered that the tntyl group should be a good protectmg group for w-carboxanude functions of asparagme

and glutarnme, for the followmg reasons Firstly, a comparison by Weygand et al of different N-subshtuents of

benzamlde has shown that m TFA soluhon, tntyl IS more labile than Tmob 3 This suggested that trltylated

carboxanudes could have a cleavage rate smular to the usual t butyl-type protectmg groups for peptldes Secondly,

our experience with the deprotectlon by TFA of peptides contammg tryptophan has shown that the presence of trltyl

groups does not lead to substitution products of the mdole moiety, m contrast to the four carboxanude protectmg groups

mentIoned m the introductory section

So far, no tntylanude denvatlves of ammo aads have been reported Our first expenments m this dnectlon, 1 e

acylatlon of trltylamme by the w-carboxyl funchon via actlvatlon by DCC/HOBt, as well as acid-catalyzed

trltylahon of the w-carboxanudc group, both failed to give useful amounts of the desired products However, the

second method was successful when the temperature was raised to 50 or 60” and acetlc anhydnde was added, m order

to trap the water formed m the reactlon The pathways leadmg to H-Asn(TrttOH (Za), H-GMT&OH (Zb), and

the-r Z-, Fmoc-, and Boc-denvatlves m Na are shown m the reachon scheme Za can be prepared m good reld

directly from H-Asn-OH 6, whle Zb, due to the lower solublllty of H-Gin-OH and to pyroglutanuc acid formation, 1s

obtained only m 35 % reld 2b 1s therefore best prepared via lb ‘, followed by catalytic hydrogenation 8 The Vu-

Fmoc derlvatlves 3a and 3b are preferably obtalned from 2a and 2b by reactlon with Fmoc-OSu 9 In our

739

CONt I Trl I

(YHJn

Boc NH -CH-COOH

CONH-Trl

hi*).

Z NH-AH-COOH

1

TrtOH I AyO

H+ I

t70NHz fyH&

Z NH-CH-COOH

/ TrlOH / AczO

H+

70NHz (YH,).

HZS04 NH,-CH-COOH

YoNH-Trl (FHZ)n

NH,-CH-COOH

2a ll=l 2b n=2

BOC,O

/

4

YoNtt2 (CH,)n

Fmoc NH-AH-COOH

I TrtOH / Ac,O H+

CONH Sri

AH&” Fmoc NH-iH-COOH

3

cxpcr~nce, the tntylatlon of Fmoc-Asn-OH and Fmoc-Gln-OH was more laborious, since unreacted startmg material

and by-products had to be removed by chromatography on &cage1 The acid-catalyzed tritylatlon wtth acetic

anhydride m acettc acid can also be applied with Z-ammoacld-a-carboxamtdes Similarly, Ac-Pro-Asn(Trt)-Gly-

Phe-Gly-OH was prepared directly from the pcntapeptlde with the unprotected P-carboxamldc group The

trltylatlon condlhons did not affect the enantiomeric purity of the ammoacids, as verlficd by GC m a ChlraslkVal

column 10 Tntyl denvatlves are much better soluble m orgamc solvents than the unsubstrtutcd amldes, and the simple

ammoand denvatlves generally have a good tcndcncy to crystalhse Table 1 summarises the w-tntyl derlvatlves of

asparagme and glutamme which we have prepared to date The three types of active esters were obtained m good

yields by reaction with DCC and the correspondmg hydroxy compounds m THF or ethyl acetate The structures of the

compounds were confn-med by standard spectroscopic methods and elemental analyses

Table 1 w-Tntyl derlvatnxs of asparagme and glutamine

Compound mol wt mP [q TLC a)

Z-Asn(Trt)-OH la 508 6 195-96” 210” MeOH 0 28 Z-Gln(Trt)-OH lb 522 6 161-62” - 45” MrOH 030 H-Asn(Trt)-OH . 0 5 Hz0 Za 383 5 >240” dec - 37” JN NaOH 0 02 H-Gln(Trt)OH l 0 5 H7.0 2b 397 5 >220” dec + 92” 1N NaOH 002 Fmoc-Asn(Trt)-OH 3a 596 7 211-13” - 147” Me011 032 Fmoc-Gln(Trt)-OH 3b 610 7 ca 155” - 12” Me01 1 033 Boc-Asn(Trt)-OH 4a 474 6 201 C?” - 177” MeOH 030 Boc-Gln(Trt)-OH 4b 488 6 113.150 - 20” McOH 031 Fmoc-Asn(Trt)-OTcp 7761 133-35” + 51 1” THF 082 Fmoc-Gln(Trt)-OTcp 790 1 176-78” - 16 1” THF 0 81 Fmnc-Asn(Trt)-OPfp 762 7 154-55” - 78” TlIF 080 c) Fmoc-Gln(Trt)-OPfp 7768 186-87” - 16 4” THF 079 c) Fmoc Asn(Trt)-ODhbt 741 8 ca 140”b) 40 8” THF 076 d) Fmoc-Gln(Trt)-ODhbt 755 a ca 135”b) - 53 7” TIIF 075 d)

a) Rf values in the solvent CIIW-MeOII-AcOH (94 5 1) on precoated slhca gel plates Merck 60 F 254 b) am~r hous powder c) slrght decomposltlon 011 the TLC plate to, tespectlvely, Fnmoc-Asn(Trt)-OH Fmoc-G nCTrt)-OH, theu methyl esters, and f1ODhbt d) partlal decomposltwn on the I LC platt, hke c) r

741

All reactlons of the activated a-carboxyl groups with the o-carboxamlde side-chams are ehmmated if the

latter are protected by trltyl As a consequence, the active esters are stable m DMF solution, whereas those of

asparagme (but much less so of glutamme) with a free side-cham form rmxtures of mtnle and cychc lmtde m base-

catalyzed reactions 11 When Fmoc-Asn(Trt)-OTcp and Fmoc-Gln(Trt)-OTcp were exposed to 1 HOBt and 1 5 DIEA for

2 h at 22O m DMF solution, we found 2 + 0 5 % and 0 %, respectively, of the D-enanttomers Under these catalyzed

condltlons, the acylatlon of u-ammo groups, even on s&d-phase resms, 1s usually complete m a few mmutes, thus

avoldmg the danger of racermzatlon m the practical apphcahon of fhese active esters There does not seem to be any

major retardation of the reachon by the bulk of a w-trltylamlde group at the C- or N-termmal end of a peptlde

coupling Fmoc-Asn(Trt)-OH and Fmoc-Gln(Trt)-OH can easily be esterlfled by the Z,&dlchlorobenzoyl method I2 to

benzylalcohol-type solid-phase resins

The w-trltyl protection of asparagme and glutamme IS rapidly cleaved by TFA at room temperature Table 2

compares the half-hves m TFA-dlchloroethane (1 1) of the tntyl groups m different compounds I3 The cleavage from

the carboxamlde group of glutamme was always found to be faster than of asparagme When Na-unprotected

asparagme 1s m the N-terminal positron of a peptlde, the cleavage of the side-cham trlty1 group IS much slower (t I/Z

ca 1 h m TFA-H20-EDT 90 5 5) due to the proximity of the protonated a-ammo-group Table 2 also illustrates the

higher lablhty of trltyl as compared with Tmob and Mbh In dlstmctlon to these protecting groups, however, trltyl

does not alkylate the mdole side-chain of tryptophan when ths ammoacid 1s present l4 In Table 3 the half-life of

the trltyl group m Fmoc-Asn(Trt)-OH 1s indicated m various common cleavage nuxtures, mcludmg some examples

contammg ethanedlthiol or dlmethylsulflde as cation scavengers l3 The cleavage rate decreases remarkably when

the TFA-concentration IS reduced from 50 % to 10 olo In contrast to NIm-trltyl of hlshdme 15, 90 % aqueous acetic acid

at 60” does not cleave the w-trityl protection of asparagme and glutamme

Table 2 Cleavage of the amide protection m TFA-dlchloroethane 1 1

Compound tmbd

Fmoc-Asn(Trt)-OH 8 Fmoc-Gln(Trt)-OH 2 Fmoc-Gln(Tmob)-OH 9 Fmoc-GlntMbh)-OH 27 Ac-Fro-Asn(Trt)-Gly-PheGly-OH 9

Table 3 Cleavage of the Trt group of Fmoc-Asn(I rt)-OH

Solvent (v/v) t l/2 bnl

TFA-H20 (95 5) 2 TFA-dlchloroethane (1 1) 8 TFA-H20-ethanedithiol (90 5 5) 2 TFA-H20-ethanedithiol (76 4 20) 3 TFA-dlmethyisulflde-ethanedithiol (77 20 3) 6

The rapid cleavage of trltyl by highly concentrated TFA is in a sharp contrast to its good stablhty towards HCl

m aqueous-organic solutions I6 An apphcatlon of this property 1s the selective removal of the Na-Boc group of

pcphdcs by a solution of 1 N HCl m 50 % lsopropanol or t butanol durmg 30 mm at 50” These conditions do not cleave

the trltyl moiety m the w-carboxarmde group of asparagme or glutamme

The w-trltyl group 1s completely stable to 20 % plpendme m DMF, condltlons which are of Importance m sohd-

phase syntheses by the Na-Fmoc method Further, being stable to strong aqueous alkah, it may be used to protect

against the cfeamldatlon of asparagme residues, a side reactlon resulting m the formatIon of a- and b-aspartic-acid

peptldes As an example, a solution of Ac-Pro-Asn(Trt)-Gly-Phe-Gly-OH m 0 1 N NaOH IS stable for 1 h at 22”,

whereas the compound wlthout trltyl on asparagne 1s transformed to a mixture of 23 % a- and 77 % P-peptlde

742

PeptIdes contammg asparagme or glutanune with o-tntyl protechons ~111 always have a higher solublllty m

orgamc solvents than their non-tntylated counterparts Apart from the prevention of side-reactlons, the trltyl groups

thus Improve the condlbons for couphng reactlons and purlfxatlon steps m class& solution-phase peptlde syntheses Selective removal of the followmg NQ-groups IS possible Fmoc (plperldme), Z (H2/Pd), Boc (1 N HCl m 50 %

t butanol), and Trt (80 % AcOH)

In solid-phase syntheses with the Na-Boc-method, the use of Boc-Asn(Trt)-OH and Boc-Gln(Trt)-OH or their

activated esters enables the unproblematic couplmg of these ammoaclds In the followmg cycles, the o-tntyl group IS

lost agam durmg the cleavage steps of the Na-Boc groups by TFA On the other hand, the o-carboxanude protechon

by trltyl IS ideally suited for solid-phase synthesis by the Na-Fmoc method Here Its cleavage by TFA takes place at

the end of a synthesis, together with the side-&am protections of the t butyl-type (Asp, Glu, Ser, Thr, Tyr, Lys), of

l’mc (Arg) 17, and of Trt (tills, Cys) In our laboratones, the use of the ti-trltyl protection for abparagme and glutamme

has become standard for solid-phase synthesis, and many examples and compansons have proven the superior quahty

of the pephdes obtamed this way

Acknowledgment

We wish to thank F Raschdorf {FAB-MS) and H Fuhrer (NMR) for the analytical work, and our coworkers V von

Arx, R Gattlen, J Seeberger, and R Wllle for then expert techmcal assltance

References and Notes 1

2 3 4 5 6

7

8

9

10 11

12 13 14

15 16 17

Part of this work was presented a the Symposium on Solid Phase Sythesls, Aug 29, 1989, in Oxford, England Patent apphcatlon filed on April 17, 1989, by Cuba-Geigy LImIted, Basle, Switzerland Y Shlmonlshl, S Sakaklbara, S Akabon, BuIl Chm Sot ]apan 1962, 35, 1966 F Weyand, W Steghch, J Blarnason, R Akhtar, N Chytll, Chem Ber 1968,101,3623 W Komg, R Geiger, Chem Ber 2970, 103, 2041 A Juhasz, S Balusz, Acta Chm Acad SCI Hung 1979, 102, 289 Preparation of H-Asn(Trt)-OH 0 5 H20 (2a) 13 2 g (0 1 mol) H-Asn-OH, 52 g (0 2 mol) Trt-OH, 18 9 ml (0 2 mol) Ac20, and 6 1 ml (0 115 mol) cone H2SO4 were suspended m 300 ml glacial acetic acid and heated to 60” for 75 nun The solution was then slowly added to 600 ml of cold H20 and adjusted to pH 6 by addition of ca 10 N NaOH After 1 h at 0”, the crystals were filtered and thoroughly washed with H20, then with toluene, and dned, yield 28 7 g = 75 % Preparation of Z-Gln(Trt)-OH (lb) 27 9 g (0 1 mol) Z-Gin-OH, 52 g (0 2 moi) Trt-OH, 18 9 ml (0 2 mol) Ac20, and 0 5 ml (0 009 mol) cone H2SO4 were suspended m 300 ml glacial acetic acid and stlrred for 1 h at 50” The solution was then slowly added to 3 1 cold H20, the precipitate filtered off, dissolved m 800 ml ethyl acetate, washed with H20, dried, evaporated and crystalhzed from ethyl acetate-hexane, yield 35 g = 67 % Preparation of H-Gln(Trt)-OH 0 5 Hz0 (Zb) 24 g (46 mmol) lb were hydrogenated in 240 ml of 90 % methanol and 46 ml 1 N HCl with 1 2 g I’d (10 %) on charcoal After after 90 mm, the startmg material was completely dissolved 1 h later, the catalyst was filtered off, the solution was concentrated to 200 ml, and 64 ml NEt3 added After shrnng for 1 h at 0”, the crystals were removed by flltratlon, washed with H20 and dried, yield 17g=93% P B W Ten Kortenaar, B G Van Dqk, J M Peeters, B J Raben, P J H Adams G I Tcsser, inf J Pept Prot Res 1986, 27, 398 H Frank G J NIcholson, E Bayer, J Chromat Sn 1977,15,174 A solution of Fmoc-Asn-OPfp in pure DMF had a half-hfe of cd 5 h, and m the presence of 1 eq HOBt and 1 5 eq DIEA of only a few mm P Sleber, Tefrah Lett 1987, 28, 6147 The t l/2 values were estnnated by TLC of soluhons, whose cleavage was quenched by the addltlon of methanol When FmocGln(Tmob)-OH or Fmoc-Gln(Mbh)-OH were treated rn TFA-H20-EDT (76 4 20), 1 h at 20” ln the presence of 1 eq of Ac-Trp-OH, the main product was shown to be Ac-Trp(Tmob)-OH or Ac-Trp(Mbh)-OH, respectlvcly P Sicber, B Rmlker, Tefrah Letf 1987, 28, 6031 A sm-~lar behaviour has been reported by Juhasz and Bapsz for the Tbh protectmg group of peptlde amldes 5 R Ramage, J Green, Tetrah L.&t 1987,28,2287

(Received m Germany 9 November 1990)