Synthesis of l-indospicine, [5,5,6-(2)H3]-l-indospicine and l-norindospicine.

Indospicine is a non-proteogenic amino acid that accumulates as the free amino acid in livestock grazing Indigofera plant species and causes both reproductive losses and hepatotoxic effects. An efficient synthetic route to l-indospicine from l-homoserine lactone is described. The methodology is applicable for the synthesis of both deuterium labelled isotopomers and structural analogues for utilisation in biological studies. The key steps are a zinc mediated Barbier reaction with acrylonitrile and a Pinner reaction that together introduce the target amidine moiety.

Synthesis of stable isotope-labelled monolysyl advanced glycation endproducts.

Advanced Glycation Endproducts (AGEs) are modified amino acids that form on proteins and are known to be implicated in the pathogenesis of diabetes and related diseases. Ready access to synthetic stable isotope-labelled AGEs allows for quantitative mass spectrometry studies to be undertaken, providing key insights into the roles AGEs play in the progression of such diseases. However, the majority of current syntheses of these compounds suffer from poor yields and lengthy procedures and are not suitable for the purposes required here. Here, we report robust syntheses of stable isotope-labelled monolysyl AGEs, N(ε)-(carboxymethyl)lysine, N(ε)-(carboxyethyl)lysine and pyrraline, that provide straightforward access to these compounds for quantitative amino acid analysis. This work will facilitate future investigations with these compounds and lead to a better understanding of the roles they play in diabetes and related diseases.

Enantiopure synthesis of side chain-modified α-amino acids and 5-cis-alkylprolines.

A short, concise synthesis of enantiopure, side chain-modified α-amino acids such as 4-oxo-L-norvaline, 6-oxo-L-homonorleucine, and 5-cis-alkyl prolines is described. Knoevenagel condensation of l-aminocarboxylate-derived ß-ketoesters with aldehydes followed by reductive decarboxylation results in unnatural α-amino acids in good yield. A fluorescent amino acid is synthesized using a similar protocol. These studies show that aminocarboxylate-derived ß-ketoesters are very useful intermediates and the method employed is both general and practical for the preparation of γ(δ)-oxo α-amino acids and alkylprolines.

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form.

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[(3)H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.

Novel 2-oxoamide inhibitors of human group IVA phospholipase A(2).

A novel class of potent human cytosolic phospholipase A(2) (GIVA PLA(2)) inhibitors was developed. These inhibitors were designed to contain the 2-oxoamide functionality and a free carboxyl group. Among the compounds tested, a long-chain 2-oxoamide containing L-gamma-norleucine was the most potent inhibitor, causing a 50% decrease in GIVA PLA(2) activity at 0.009 mole fraction.

Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances.

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity. Chiral intermediates and fine chemicals are in high demand, both from the pharmaceutical and agrochemical industries, for the preparation of bulk drug substances and agricultural products. Biocatalytic processes have been described for the synthesis of chiral intermediates for beta3- and beta2-receptor agonists, antihypertensive drugs, antiviral agents, melatonin receptor agonists, anticholesterol and anticancer drugs, and drugs to treat Alzheimer's disease.

Enzymatic synthesis of L-6-hydroxynorleucine.

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.

Synthesis of pyrraline reference material.

A simplified and improved method is described for the preparation of pyrraline, a lysine derivative from the advanced Maillard reaction and potential indicator for heat treatment of foods. The compound was obtained in a high degree of purity and with a yield of 31% from N alpha-t-butyloxycarbonyl-L-lysine after heating with 3-deoxy-D-erythro-hexos-2-ulose for 2 h at 70 degrees C in the dry state, preparative fractionation of the resulting N alpha-t-butyloxycarbonyl pyrraline with reverse-phase liquid chromatography and final deprotection of the intermediate compound with acetic acid.

Renin inhibitors. III. Synthesis and structure-activity relationships of transition-state inhibitors containing dihydroxyethylene isostere at the P1-P1 site.

The design, synthesis and structure-activity relationships of transition-state inhibitors containing the dihydroxyethylene isostere at the scissile site are described. The compounds with (2S,3R,4S)-4-amino-5-cyclohexyl-1-morpholino-2,3-pentanediol at the P1-P1 site are potent renin inhibitors. (2S,3R,4S)-4-[N-[(2S)-3-Ethylsulfonyl-2-(1-naphthylmethyl)propiony l]-L- norleucyl]amino-5-cyclohexyl-1-morpholino-2,3-pentanediol (2) (BW-175), which is the most potent inhibitor (IC50: 3.3 nM against human renin) in this series, poorly inhibits cathepsin D (IC50: 26000 nM) and pepsin (IC50: > 100000 nM), and thus it is specific for renin. Compound 2 contains only one amino acid and showed an oral bioavailability of 2.8% at 10 mg/kg and 9.7% at 30 mg/kg in rats. The interaction between renin and inhibitor 2 is discussed on the basis of molecular modeling studies.