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N alpha-9-Fluorenylmethoxycarbonyl-N epsilon-4=methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N epsilon-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.

Inability to increase the yield of reaction between 2,4 dihydroxybenzaldehyde and gelatin beyond 55 and 60% has led to an extensive investigation of reductive alkylation with free lysine. Even with free lysine, the extent of reaction was about 60%. Since this could be attributed to the electron donation by the phenolic hydroxyls, reductive alkylation was performed between o-, m-, and p-nitrobenzaldehydes and free lysine; o- and m-nitrobenzaldehyde were ineffective in increasing the yield while with p-nitrobenzaldehyde a yield of 72% was achieved. The unreacted 28% of the lysines are susceptible to epichlorohydrin. These results suggest that the slow, reversible first step in reductive alkylation, the formation of the Schiff's base, is responsible for the low yield.

In order to obtain a readily derivatized analog of the ionophore antibiotic valinomycin, [1-lysine] valinomycin (Lys-VAL) was synthesized. The compound was built up on a polystyrene support by stepwise segment condensation and was cyclized in solution. The segments used were didepsipeptides protected by the t-butyloxycarbonyl and p-nitrobenzyloxycarbonyl groups. Derivatives prepared by acylation of the epsilon-amino group of Lys-VAL included [14C]acetyl-Lys-VAL, dansyl-Lys-VAL, palmitoyl-Lys-VAL and dithiodiglycoyl-bis-Lys-VAL. These derivatives had a high potassium binding capacity but were in general much less active than VAL in mediating ion transport in membranes.

4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group.