A combined resin solvation-peptide chain motion and kinetics of coupling reaction approach was applied to monitor details of the synthesis of TM-34, a 34-residue transmembrane segment of the bradykinin receptor. The dynamics of resinbound peptide fragments attached to a stable free radical amino acid were examined by EPR spectroscopy. In agreement with an abrupt decrease (from 83 to 43%) in peptide purity occurring in the 12−16 region when DMF was used, a much more strongly immobilized chain population was detected, especially at the 12-mer stage. Conversely, faster

This work proposes a combined swelling-electron paramagnetic resonance (EPR) approach aiming at determining some unusual polymer solvation parameters relevant for chemical processes occurring inside beads. Batches of benzhydrylamine-resin (BHAR), a copolymer of styrene-1% divinylbenzene containing phenylmethylamine groups were, labeled with the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amine-4-carboxylic acid (TOAC), and their swelling properties and EPR spectra were examined in DCM and DMF. By taking into account the BHARs labeling degrees, the corresponding swelling values, and some polymer structural characteristics, it was possible to calculate polymer swelling parameters, among them, the volume and the number of sites per bead, site-site distances and site concentration. The latter values ranged from 17 to 170 A and from 0.4 to 550 mM, respectively. EPR spectroscopy was applied to validate the multistep calculation strategy of these swelling parameters. Spin-spin interaction was detected in the labeled resins at site-site distances less than approximately 60 A or probe concentrations higher than approximately 1 x 10(-2) M, in close agreement with the values obtained for the spin probe free in solution. Complementarily, the yield of coupling reactions in different resins indicated that the greater the inter-site distance or the lower the site concentration, the faster the reaction. The results suggested that the model and the experimental measurements developed for the determination of solvation parameters represent a relevant step forward for the deeper understanding and improvement of polymer-related processes.

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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 estabilidade foi: benzidrilamino-resina>p-metilbenzidrilamino-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 m in 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. The clas sic hy dro ly sis pro ce dure for quan ti fi ca tion of resin-bound aminoacyl and peptidyl groups with 12 N HCl: propionic acid was re eval u ated by study ing the in flu ence of the na ture of the resin and the resin-bound group. Their sta bil ity dur ing acid hy dro ly sis was de pend ent on th e C-terminal amino acid, and the or der of acid sta bil ity was Phe > Val > Gly. Oth er wise, the dip eptides Ala-Gly, Ala-Val, and Ala-Phe dis played en hanced rates of hy dro ly sis of the resin if com pared with their par ent aminoacyl groups. Amongthe res ins as sayed, the or der of acid sta bil ity was: benzhydrylamine-resin >p-methylbenzhydrylamine-resin ≅ 4-(oxymethyl)-phenylacetamidomethyl-resin > chloromethyl-copolymer of styrene-1%-divinylbenzene. Im por tant for pep tide syn the sis method, the find ings dem on strate th at lon ger hy dro ly sis times than pre vi ously rec om mended in the lit er a ture (1 h at 130 °C and 15 min at 160 °C for pep tides at tached to the chloromethyl-copolymer of sty rene-1%-divinylbenzene) are nec es sary for the quan ti ta tive acid-catalyzed cleav age of some resin-bound groups. The ob ser ved broad range of hy dro ly sis time var ied from less than 1 h to about 100 h.

Tachyphylaxis, defined as the acute loss of response of some smooth muscles upon repeated stimulations with angiotensin II (Ang II), has been shown to be dependent mainly on the N-terminal region of the ligand. To further study the structural requirements for the induction of tachyphylaxis we have synthesized Ang II analogs containing the bulky and very lipophilic substituents 9-fluorenylmethyloxycarbonyl (Fmoc) and 9-fluorenylmethyl ester (OFm) at the alpha-amino (Nalpha-Fmoc-Ang II) or the beta-carboxyl ([Asp(OFm)1]-Ang II) groups of the Asp1 residue, respectively. In binding assays with Chinese hamster ovary cells transfected with the AT1 Ang II receptor, Nalpha-Fmoc-Ang II bound with high affinity, whereas [Asp(OFm)1]-Ang II showed lower affinity. In biological assays, these two analogs were full agonists and showed 30 and 3%, respectively, of the Ang II potency in contracting the guinea-pig ileum smooth muscle. The two analogs induced tachyphylaxis, in spite of the lack of a free amino group in Nalpha-Fmoc-Ang II. Thus, analogs with Fmoc- or OFm-type groups coupled to the Asp1 residue, whether at the amino or carboxyl functions, induce tachyphylaxis through an unreported mechanism. Based in these findings and those available from the literature, an alternate molecular interaction mode between Ang II N-terminal portion and the AT1 receptor is proposed to explain the tachyphylactic phenomenon.