SynopsisTo exemplify the usefulness of the S-tert-butylthio group for a reversible blocking of the cysteine thiol function in peptide synthesis, fully protected dihydrosomatostatin was prepared by the fragment-condensation procedure. The experimental results confirm the excellent stability of the asymmetric disulfide under the normal conditions of peptide synthesis and prove that the selective, acid-catalyzed nucleophil removal-as well as by mercaptans-of the 2-nitrophenylsulfenyl group proceeds smoothly in the presence of this thiol protection.Thus, the strategy of overall acid-labile side-chain protection in combination with the N"-2-nitrophenylsulfenyl group for the chain-elongation steps can be successfully applied to the synthesis of cysteine-containing peptides using their S-tert -butylthio derivatives. Removal of the acid-labile groups, followed by reductive cleavage of the asymmetric disulfides and successive air oxidation, allowed a clean conversion of protected dihydrosomatostatin into somatostatin a t a high degree of purity and in good yields. INTRODUCTIONThe great demand for new thiol-protecting groups, highly stable in the normal operating conditions of peptide synthesis and removable by mild and, in regard to other known cysteine derivatives, selective methods led Wunsch and Spangenberg' to propose the S-tert-butylthio function as a group fulfilling above requirements. This asymmetric disulfide is, in fact, resistant to acid-and base-induced disproportionation, and it is cleaved by mild reagents such as mercaptans.' Because of our positive experiences (Ref. 2, p. 792, and unpublished results) with this protecting group, a new synthesis of the clinically important hypothalamic hormone s~rnatostatin:~-~ was performed to further investigate the potential properties of the Stert-butylthio group in the course of peptide synthesis.* Some of the results of this paper were presented in a preliminary communication a t the Second FRG-USSR Symposium on Chemistry of Peptides and Proteins, Grainau-Eibsee/ Bavaria, May [24][25][26][27] 1978: Moroder, L., Musiol, d., Scharf, R., and Wunsch, E. (1978) in Proceedings, pp. 24-25.t To whom correspondence should be addressed.Biopolymers, Vol. 20.17-37 (1981 EXPERIMENTALMelting points were determined on a Tottoli capillary melting-point apparatus and are uncorrected. Optical rotations were measured in a jacketed 1-dm cell on a Perkin Elmer model 141 polarimeter. The acid hydrolyses were conducted in 6M HC1 a t 110°C for 20 hr in the presence of thioglycolic acid for Trp-containing peptides6 in evacuated, sealed ampules. D-Amino acid oxidase (Boehringer, Mannheim, Lot No. 1419221) digestion was carried out according to the procedure of Larson et al.7 Aminopeptidase M (Sigma, L-6007, Lot No. 67C-85001) digestion was performed in Tris buffer, pH 7.8, a t 37°C for 20 hr. Amino acid analyses were obtained on the Beckman amino acid analyzer equipped with automatic digital integrator (models 120 B and 125). The purity tests for the single compounds (25-50 pg) were car...

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd ‫ס‬ 1 M) for each other. However, the affinity of HslU for HslV fivefold increased (Kd ∼ 0.2 M) after binding with the MBP∼SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP∼SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP∼SulA with 10 −9 M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd ‫ס‬ 0.2 M) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP∼SulA stimulates a conformational change in HslU to a high-affinity form for HslV.

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1. Das Hydrazid (I) (Secretin‐Sequenz 3‐8) wird ausgehend von an Polyäthylenglykol gebundenem tert.‐Butyloxycarbonyl‐O‐benzyl‐Lrserin durch kontrollierten schrittweisen Aufbau nach der Kristallisationslnethode der Flüssigphasenpeptidmethode erhalten; unter milden Hydrazinolysebedingungen läßt sich (I) in hoher Reinheit vom Träger abspalten.