N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP.

Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6). While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.

Stereoisomeric acylamidomorpholinium carnitine analogues: selective inhibitors of carnitine palmitoyltransferase I and II.

Acylamidomorpholinium carnitine analogues, 6-(tetradecanamidomethyl- and -hexadecanamidomethyl)-4,4-dimethylmorpholin-4-ium-2-a cetate, 1, synthesized as complete sets of stereoisomers, were assayed as inhibitors for isozymes of carnitine palmitoyltransferase (CPT). Microsomal CPT isoymes showed modest discrimination among the stereoisomers; while rat-liver mitochondrial CPT-I and CPT-II showed distinct differences. The tetradecanamidomethyl analogue of (2R,6S)-1 activated CPT-I but inhibited CPT-II.

Acylcarnitine analogues as topical, microbicidal spermicides.

Acylcarnitine analogues, (+)-6-Carboxylatomethyl-2-alkyl-4,4-dimethylmorpholinium (Z-n, where n = the number of carbons in the alkyl chain), synthesized in multi-gram quantities show in vitro activities as spermicides, anti-HIV agents, and inhibitors of the growth of Candida albicans. Activity improves with increasing chain length. Compound Z-15 is a candidate for further study as a topical, microbicidal spermicide.

Change in the mode of inhibition of acetylcholinesterase by (4-nitrophenyl)sulfonoxyl derivatives of conformationally constrained choline analogues.

A chiral, five-step synthesis of 2-(hydroxymethyl)-2,4-dimethylmorpholine (12) from (R)- and (S)-2-methylglycidols gives an overall yield of 63%. Morpholines (R)- and (S)-12 are converted into 2-(azidomethyl)-2,4-dimethylmorpholine (15) via 2,4-dimethyl-2-[[(4-nitrophenyl)sulfonoxy]methyl]morpholine (14). The tertiary morpholines 12, 14, and 15 are quaternarized to afford 2-(hydroxymethyl)-2,4,4-trimethylmorpholinum iodide (2), 2,4,4-trimethyl-2-[[(4-nitrophenyl)sulfonoxy]methyl]morpholinium iodide (3), and 2-(azidomethyl)-2,4,4-trimethylmorpholinium iodide (4), respectively, which all inhibit acetylcholinesterase (AChE). These morpholinium inhibitors are compared with conformationally constrained aryl hemicholinium AChE inhibitors. Enantiomers of 2 and 4 are reversible competitive inhibitors of AChE, with values of Ki = 360 +/- 30 microM for (S)-2, 650 +/- 90 microM for (R)-2, 450 +/- 70 microM for (S)-4, and 560 +/- 30 microM for (R)-4, respectively. Enantiomers of 3 are noncompetitive inhibitors of AChE with values of Ki = 19.0 +/- 0.9 microM for (S)-3 and 50 +/- 2 microM for (R)-3, respectively. AChE shows a 2-fold chiral discrimination in the case of inhibition by 2 and 3. Inhibition also changes from competitive to noncompetitive when (3-hydroxyphenyl)-N,N,N-trimethylammonium iodide (18) [Ki = 0.21 +/- 0.06 microM; Lee, B. H., Stelly, T. C., Colucci, W. J., Garcia, J. G., Gandour, R. D., and Quinn, D. M. (1992) Chem. Res. Toxicol. 5, 411-418] is converted into [3-[(4-nitrophenyl)sulfonoxy]phenyl]-N,N,N-trimethylammonium iodide (5), Ki = 6.0 +/- 0.5 microM. These results indicate that the 4-nitrobenzenesulfonyl group controls the mode of inhibition.