Comparative Time-Course Study of Aminoacyl-and Dipeptidyl-Resin Hydrolysis

O método clássico de hidrólise para a quantificação de grupamentos aminoacílicos e peptídicos ligados a resinas em HCl 12 N : ácido propiônico foi reavaliada, estudando-se a influência da natureza da resina e do grupamento ligado à mesma. A estabilidade frente à hidrólise ácida foi dependente do amino acido C-terminal e a ordem de estabilidade ácida foi Phe > Val > Gly. Por outro lado, os dipeptídeos Ala-Gly, Ala-Val e Ala-Phe apresentaram maiores velocidades de hidrólise da resina, se comparadas com a dos correspondentes grupos aminoacílicos. Dentre as resinas testadas, a ordem de estabil idade foi : benzidri lamino-resina>p-meti lbenzidri lamino-resina ≅4-(oximetil)-fenilacetamidometil-resina > copolímero de estireno clorometilado, contendo 1% de divinilbenzeno. Importante para a metodologia de síntese de peptídeos, os resultados demonstraram que tem pos de hidrólise mais longos do que os propostos há anos na literatura (1 h a 130 °C e 15 min a 160 °C para os peptídeos ligados ao copolímero de estireno clorometilado, contendo 1% de divinilbenzeno), são necessários para uma clivagem quantitativa de alguns grupos ligados à resina. A ampla faixa de tem pos de hidrólise observada variou de menos de 1 h até cerca de 100 h.

tion of the propionic acid-12 N HCl so lu tion (1:1, v/v) as the stan dard hy dro ly sis pro to col [5][6][7] , no ad di tional sys tematic stud ies have been car ried out on the ap pli ca bil ity of this pro ce dure to other res ins in tro duced later in the pep tide syn the sis tech nique.More over, the ex act role of other factors in this cleav age re ac tion, such as the na ture and the load ing of the aminoacyl or peptidyl groups bound to the resin ma trix, has not yet been thorougly in ves ti gated.
The goal of the pres ent re port is to in ves ti gate the in fluence of these fac tors and to pro pose an ap pro pri ate hy droly sis pro ce dure for each class of resin-bound group.Among the res ins stud ied, this pa per will fo cus on those em ployed in the Boc-α amino group pro tect ing the syn thesis strat egy 1,2 , i.e., the clas sic chloromethyl-copolymer of sty rene 1% divinylbenzene (CMSD) and the 4-(oxymethyl)-phenylacetamidomethyl-resin (PAMR) 8 em ployed for the syn the sis of C-terminal free α-car boxyl pep tides, and the benzhydrylamine-resin (BHAR) 9 and the p-methylbenzhydrylamine-resin (MBHAR) 10 used al terna tively for as sem bling α-carboxamide se quences.To avoid the risk of acid deg ra da tion which could ham per the pres ent quan ti ta tive time-course hy dro ly sis study, the acid-stable amino ac ids Gly, Val and Phe were cho sen as the resin-bound res i dues.The cor re spond ing resin-bound alanyl se quences (Ala-Gly, Ala-Val, and Ala-Phe) were se -lected not only as mod els of pep tide res ins, but also to evalu ate the in flu ence of the α-amino group acylation of the C-terminal res i due on their rate of acid cleav age from the resin.

Ma te rials and Methods
The tert-butyloxycarbonyl (Boc)-amino ac ids (Gly, Val, Phe, and Ala) were pur chased from Bachem, Cal i fornia.The aminoacyl-resins were ac quired from the fol lowing com pa nies: MBHAR, Sigma; PAMR, Bachem Cal i for nia, and CMSD, Bachem Cal i for nia and Calbiochem-Novabiochem. BHAR was ei ther syn the sized in the lab o ra tory or pur chased from Watanabe or Bachem Cal i for nia.Un less oth er wise stated, the amino acid load ing of res ins ranged from about 0.3 to 0.6 mmol/g.Sol vents and re agents were of an a lyt i cal grade and pur chased from Aldrich or Fluka.
Amino-acyl-or peptidyl-resins were man u ally syn thesized us ing the solid phase method 1,2 , and the ninhydrin assay 11 was used to check the ef fec tive ness of the cou pling re ac tions.The Boc-N α -pro tect ing group was re moved by trifluoroacetic acid treat ment (30% v/v, in di chloromethane for 30 min) prior to the acid cleav age study.Hydro ly ses of res ins were done with propionic acid:12 N HCl tef lon-coated screw caps (13 cm x 1 cm), and amino acid anal y ses were per formed in a Beckman 6300 amino acid an a lyzer sys tem.

Hy dro ly sis of aminoacyl-resins
Fig ure 1 dis plays the time course of the hy dro ly sis of the four aminoacyl-resins at 130 °C.As ex pected, re gard less of the resin, the small po lar Gly res i due was much more la bile than Val or Phe, in ac cor dance with the well-known higher acid sta bil ity of resin-bound hy dro pho bic res i dues 12 .As previ ously dis cussed 13 , the higher sta bil ity of Phe com pared to the Val res i due may be due to the elec tron with draw ing effect of its ben zene ring at the resin link age, re sult ing in a higher re sis tance to acidolytic cleav age.
The na ture of the poly meric sup port also af fected the rate of hy dro ly sis, ir re spec tive of the resin-bound amino acid.The de creas ing or der of resin-bound acid sta bil ity was: BHAR > MBHAR = PAMR > CMSD (Fig. l).To the best of our knowl edge, there are no pre vi ous re ports on such a compar a tive study of the hy dro ly sis of these res ins which are widely em ployed for pep tide syn the sis.The lower acid stabil ity of CMSD as com pared to PAMR, and of MBHAR as com pared to BHAR, has al ready been dem on strated based upon trifluoroacetic 14 and hy dro gen flu o ride 10 treat ments, re spec tively.The find ings con firmed the very high acid stabil ity of BHAR 9,10,15 , and also re vealed rather sim i lar acid sta bil ity prop er ties of the PAMR and MBHAR poly mers.
The re sults sug gest that the re ac tion times rec om mended in the lit er a ture for the hy dro ly sis pro ce dure, i.e., 2 h with the CMSD resin [5][6][7] or 18 h with the MBHAR/ BHAR resins 10 at 130 °C, are not ad e quate to achieve quan ti ta tive removal of resin-linked amino ac ids.The time re quired for the com plete cleav age of resin-bound amino ac ids var ied from a min i mum of ap prox i mately 15 h with the most la bile Gly-CMSD to a max i mum of 100 h with the very sta ble Phe-BHAR.
As de tailed in the Ma te rial and Methods sec tion, the aminoacyl-or peptidyl-containing res ins used in the pres ent re port were also dif fer ent re gard ing their load ing de gree or their com mer cial source.To de ter mine whether the de gree of resin load ing af fected the hy dro ly sis re sults, the hy dro lysis data of three Phe-BHAR batches con tain ing 0.44, 0.91, and 1.54 mmol/g of Phe were com pared.The two lat ter samples were ob tained from highly amino group-loaded BHAR batches syn the sized ac cord ing to a force ful syn thetic pro tocol 16 .No sig nif i cant dif fer ences in the acid sta bil ity prop erties of the three res ins were de tected (data not shown).A pos si ble in flu ence of the source of res ins on their hy dro ly sis be hav ior was also ruled out, since the two com mer cial Phe-BHAR batches (pur chased from Bachem Cal i for nia and Watanabe) showed sim i lar hy dro ly sis ki net ics when com pared to the above-described sam ples of lab o ratory-synthesized BHAR.Also, no dif fer ence was ob served be tween the Val-CMSD batches from dif fer ent com pa nies (Bachem Cal i for nia and Calbiochem-Novabiochem, data not shown).
Al ter na tive pro ce dures us ing tem per a tures higher than 130 °C have been pre vi ously in tro duced 7 to ac cel er ate the hy dro ly sis re ac tion.Ta ble 1 pres ents the time-course data of the hy dro ly sis of Gly-, Val-, and Phe-resins ob tained at 160 °C.As ex pected, hy dro ly ses faster than those at 130 °C were ob served, but the re ac tion time nec es sary to quan ti ta tively cleave the aminoacyl groups ranged from about 4 h (Gly-CMSD) to about 30 h (Phe-BHAR), lon ger than that rec om mended in the lit er a ture for 160 °C 7 .

The hy dro ly sis of dipeptidyl-resins
The mech a nism of the acid hy dro ly sis of an aminoacyl group bound to the resin ma trix ir re spec tive of the na ture of the link age (es ter or am ide in the case of CMSD/ PAMR or MBHAR / BHAR res ins, re spec tively) is pos si bly in fluenced by the free or acylated form of its α-amino group.Thus, to com pare with the Gly-, Val-, and Phe-resins, the cor re spond ing dipeptidyl Ala-Gly-, Ala-Val-and Ala-Phe-containing res ins were syn the sized and stud ied as to their sta bil ity in acid hy dro ly sis.To avoid the in flu ence of other fac tors, the same N-terminal acylation group (alanine) was de lib er ately em ployed in this com par a tive study.
Fig ure 2 shows the re sults of the hy dro ly sis ex per i ments with peptidyl-resins at 130 °C.Faster hy dro ly ses were observed with the peptidyl-resins when com pared with the parent aminoacyl-resins (Fig. l).For in stance, the hy dro ly sis time nec es sary for the to tal cleav age of the Gly res i due from the resin de creased from about 15 h for Gly-CMSD (Fig. 1A) to about 10 h for Ala-Gly-CMSD (Fig. 2A).In the case of Phe-containing res ins, the same trend was ob served and ranged from about 100 h (Phe-BHAR, Fig. 1D) to 70 h (Ala-Phe-BHAR, Fig. 2D).These find ings in di cate that the acylation of the α-amino group of the resin-bound amino acid fa cil i tates its hydrolytic re moval from the resin.
Con cern ing the in flu ence of the type of amino acid or resin, the de creas ing or der of acid sta bil ity for peptidyl-resins par al leled that ob served for aminoacyl-resins, be ing: Ala-Phe > Ala-Val > Ala-Gly and BHAR > MBHAR = PAMR > CMSD.
As ex pected, un der a more se vere con di tion (160 °C), the min i mum and max i mum hy dro ly sis times nec es sary to achieve com plete peptidyl cleav age var ied from less than 1 h to about 25 h for the la bile Ala-Gly-CMSD and the more stable Ala-Phe-BHAR, re spec tively (Ta ble 2).
More over, the pres ent time-course study also al lowed the eval u a tion of the hy dro ly sis time nec es sary for the complete cleav age of the pep tide bond be tween the alanyl res idue and Gly, Val, or Phe res i dues bound to the resin.The re sults in di cate that hy dro ly sis times of about 2 h (at 130 °C) and about 45 min (at 160 °C) are suf fi cient for the quan ti tative cleav age of these pep tide bonds.As these hy dro ly sis times are shorter than those nec es sary for quan ti ta tively cleav ing any resin-bound C-terminal amino ac ids un der the same con di tions (see Figs. 1 and 2 and Ta bles 1 and 2), the Ala/Gly, Ala/Val, and Ala/Phe mo lar ra tios found in amino acid anal y ses of dipeptidyl-resin hydrolyzates are usu ally higher than 1 .
Ta ble 3 sum ma rizes the re sults of this study show ing the ap prox i mate re ac tion times nec es sary to quan ti ta tively cleave the resin-bound C-terminal amino acid when alone or in a pep tide se quence at 130 °C and at 160 °C.As in di cated, this re ac tion time is strongly de pend ent on the na ture of the Ta ble 1.The ex tent of the hy dro ly sis (%) of aminoacyl-resins in propionic acid: 12  resin and its pen dant group and also on the hy dro ly sis temper a ture.There fore, it is im por tant to al ways be aware of the acid-sensitivity of each class of amino acid to hy dro lysis con di tions for any quan ti ta tive amino acid or pep tide con tent-de ter mi na tion in resin.Much lon ger re ac tion times than ini tially re ported in the field of pep tide chem istry are needed, de pend ing on the resin sam ple.The pres ent re port points to the need for fur ther research with al ter na tive pro to cols which might al low a reduc tion in resin hy dro ly sis times.Cleav age ex per i ments with other hydrolytic sys tems, such as po tas sium car bonate in the pres ence of a phase trans fer re agent and ul trasound 17 , triethanolamine, dimethylformamide and aque ous so dium hy drox ide 18 , methanosulfonic acid/propionic acid/wa ter 19 , and even the ap pli ca tion of the mi cro wave approach 20 , are cur rently been un der way in our lab o ra tory.
Ta ble 3. The ap prox i mate time (h) nec es sary for the com plete hy dro ly sis of resin-bound aminoacyl-and dipeptidyl-groups at 130 °C and 160 °C in propionic acid:12 N HCl (1:1, v/v).The ex tent of the hy dro ly sis (%) of dipeptidyl-resins in propionic acid:12 N HCl (1:1, v/v) at 160 °C