Total Synthesis of K777: Successful Application of Transition-Metal-Catalyzed Alkyne Hydrothiolation toward the Modular Synthesis of a Potent Cysteine Protease Inhibitor.

We report the total synthesis of K777 and a series of analogues via alkyne hydrothiolation catalyzed by Wilkinson's complex (ClRh(PPh3)3). The alkyne hydrothiolation reactions proceeded with excellent regio- and diastereoselectivity to generate the desired E-linear vinyl sulfides in high yield. The use of Ellman's auxiliary generates the requisite propargyl amines in excellent enantiomeric excess (ee) and obviates the use of L-homophenylalanine, an expensive unnatural amino acid. The vinyl sulfone derivatives exhibit a large difference in rate toward Michael addition. Kinetic data are consistent with rate-limiting nucleophilic attack to generate the carbanion intermediate.

Inhibition of aberrant complement activation by a dimer of acetylsalicylic acid.

We here report synthesis for the first time of the acetyl salicylic acid dimer 5,5'-methylenebis(2-acetoxybenzoic acid) (DAS). DAS inhibits aberrant complement activation by selectively blocking factor D of the alternative complement pathway and C9 of the membrane attack complex. We have previously identified aurin tricarboxylic and its oligomers as promising agents in this regard. DAS is much more potent, inhibiting erythrocyte hemolysis by complement-activated serum with an IC50 in the 100-170 nanomolar range. There are numerous conditions where self-damage from the complement system has been implicated in the pathology, including such chronic degenerative diseases of aging as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and age-related macular degeneration. Consequently, there is a high priority for the discovery and development of agents that can successfully treat such conditions. DAS holds considerable promise for being such an agent.

Structure and mechanism of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway.

PhnP is a phosphodiesterase that plays an important role within the bacterial carbon-phosphorus lyase (CP-lyase) pathway by recycling a "dead-end" intermediate, 5-phospho-α-d-ribosyl 1,2-cyclic phosphate, that is formed during organophosphonate catabolism. As a member of the metallo-ß-lactamase superfamily, PhnP is most homologous in sequence and structure to tRNase Z phosphodiesterases. X-ray structural analysis of PhnP complexed with orthovanadate to 1.5 Å resolution revealed this inhibitor bound in a tetrahedral geometry by the two catalytic manganese ions and the putative general acid residue H200. Guided by this structure, we probed the contributions of first- and second-sphere active site residues to catalysis and metal ion binding by site-directed mutagenesis, kinetic analysis, and ICP-MS. Alteration of H200 to alanine resulted in a 6-33-fold decrease in k(cat)/K(M) with substituted methyl phenylphosphate diesters with leaving group pK(a) values ranging from 4 to 8.4. With bis(p-nitrophenyl)phosphate as a substrate, there was a 10-fold decrease in k(cat)/K(M), primarily the result of a large increase in K(M). Moreover, the nickel ion-activated H200A PhnP displayed a bell-shaped pH dependence for k(cat)/K(M) with pK(a) values (pK(a1) = 6.3; pK(a2) = 7.8) that were comparable to those of the wild-type enzyme (pK(a1) = 6.5; pK(a2) = 7.8). Such modest effects are counter to what is expected for a general acid catalyst and suggest an alternate role for H200 in this enzyme. A Brønsted analysis of the PhnP reaction with a series of substituted phenyl methyl phosphate esters yielded a linear correlation, a ß(lg) of -1.06 ± 0.1, and a Leffler α value of 0.61, consistent with a synchronous transition state for phosphoryl transfer. On the basis of these data, we propose a mechanism for PhnP.

Structure of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway for phosphonate degradation.

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the beta-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2',3'-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn(2+) and Ni(2+) for catalysis, whereas Zn(2+) afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn(2+) ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn(2+) binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.