Reversible binding of peptide aldehydes to papain. Structure-activity relationships.

The hydration of eleven peptide and hipuryl aldehydes has been measured as a function of temperature by means of NMR spectroscopy. In all cases the aldehydes were strongly hydrated (i.e., 90-95%) in aqueous solution. Dehydration of the hydrates was strongly endothermic, but this was partly offset by a positive entropy for dehydration. The binding of the aldehydes to papain was measured by fluorescence titration, and from these data dissociation constants for the hemithioacetal enzyme adducts were derived. Binding of N-Ac-L-PheNHCH2CHO (1) was particularly tight (Kd,corr = 0.00043 micro M) whereas that of its D-enantiomer (2) was 300-fold weaker (Kd,corr = 0.129 microM). The binding constants of the eleven aldehydes correlated with those for the reversible covalent binding of the analogous nitriles according to the equation log Kd(CHO) = -2.687 +/- 1.016 log K d(CN) (r = 0.99), lending support to previous suggestions that both peptide aldehydes and peptide nitriles behave as transition-state- or reactive intermediate analogs for papain. This finding is particularly striking in view of the obvious differences in hybridization (sp2 vs. sp3) and geometry (trigonal vs. tetrahedral) at the reactive P1 carbon center in their covalent adduct forms (thioimidate ester vs. hemithioacetal, respectively). A model for the binding of substrates, their transition states and analogs thereof is proposed. A key feature of the model is an obligatory covalent (or developing covalent) interaction between Cys-25-SH and the carbonyl or equivalent carbon of P1, augmented by intermolecular P1NH--OC(Asp-158), P2CO--HN(Gly-66) and P2NH--OC(Gly-66) hydrogen bonds and a hydrophobic P2-S2 interaction. The latter three interactions are optimum or nearly optimum when P2 is a hydrophobic L-amino acid with an N-acyl substituent. Data presented suggest that hippuryl derivatives are relatively non-specific substrates or inhibitors for papain and, consequently, are of diminished value as probes for binding and catalytic studies.

Comparison of the toxicity of methylcyclopentadienyl manganese tricarbonyl with that of its two major metabolites.

Pneumotoxicity and lethality resulting from administration of methylcyclopentadienyl manganese tricarbonyl (MMT) and its 2 major metabolites in rats were compared. Following i.p. injection, MMT was found to have an LD50 value of 12.1 mg/kg. Neither of the metabolites appeared to have significant acute toxicity even when doses as high as 250 mg/kg were given. This impressive difference in toxicity may be due in part to changes in solubility of the metabolites, allowing for (1) decreased distribution into the central nervous system, coupled with (2) a more rapid excretion rate. This suggests that the oxidative metabolism of MMT that results in the formation of these metabolites is an important detoxifying pathway.

A novel frequency distribution selection method for efficient plate layout of a diverse combinatorial library.

A deterministic method (frequency distribution method) for selecting compounds from a partitioned virtual combinatorial library for efficient synthesis is presented here. The method is based on reagent frequency analysis and can be applied to any library of molecules distributed in any given partitioned chemical space (cluster, cell-based, etc.). Compound selection by reagent frequency distribution can produce a unique, diverse set of molecules that adequately represents the library while requiring the least amount of compounds to be synthesized and minimizing the number of different reagents that must be used. This method also provides a practical solution to the configuration of plate layout. Because the method essentially identifies "expensive" regions in the chemical space to synthesize for a desired diversity or similarity coverage, decisions concerning the necessity to synthesize these compounds can be addressed. Minimum compound generation and efficient plate layout results in savings both in time of synthesis and cost of materials. This method always results in a discrete solution, which can be used for any given library size as well as any combination of reagents and is also readily adaptable to robotic automation.