Synthetic and Conformational Studies of Active Site Peptides of Amino Acid Racemases

D-alanine，an essential constituent of bacterial cell wall pepti-deglycan，is biosynthesized from L-Alanine by the mediation of amino acid racemase。Amog the amino acid racemase，a thermostable alanine racemase from Bacillus stearothermophilus，two alanine racemases coded by dad B and dal genes fromSalmonella typhimurium，and amino acid racemase from Pseudomonas striata were relatively well studied enzymatically。The amino acid sequences around the lysine residue to which the cofactor pyridoxal phosphate linked were elucidated as follows.B.stearothermophilus：Ala-Val-Val-Lys-Asn-Asn-Ala-Tyr(former sequence：Ala-Pro-Pro-Lys-Ala-Asn-Ala-Tyr)；Salmonella typhimurium：dad B Val-Trp-Ser-Val-Val-Lys-Ala-Asn-Ala-Tyr-Gly-Lly-Lys-Ala-Lyr-Ala-Lys-Ala-Allysine。P.striata：Leu-Thr-Ala-Val-Leu-Lys-Ala-Ala-Asp-Ala-Try-Gly-His-Gly-Ile.In order to obtain the basic concept in developing new inhibitor(Antibacterial Agents)，We started the synthetic and conformational studies of active site peptides of these enzymes as model active site。At first，we compared the amino acid sequences and divided the sequences in several fragment peptides。The synthetic fragment peptides were then condensed to give protected two kinds of octapeptides and three kinds of tetradecapeptides。All the synthetic procedures were performed in the solution phase.After the N-protective group was converted to N-acetyl group，the protective groups were then removed by treatment of trimethylsilyltriflate-thioanisole system to give acetyl derivatives of two octa-and three tetradecapeptides corresponding to the amino acid sequences of active sites of above mentioned racemases。The conformational studies using NMR spectroscopy for Boc-Leu-Thr-Ala-Val-Leu-OMe and Boc-Ala-Pro-Pro-Lys(Z)-Ala-Asn-Ala-Tyr(Bzl)-OBzl were also performed。

D-alanine, an essential constituent of bacterial cell wall pepti-deglycan, is biosynthesized from L-Alanine by the mediation of amino acid racemase. Amog the amino acid racemase, a thermostable alanine racemase from Bacillus stearothermophilus, two alanine racemases coded by dad B and dal genes fromSalmonella typhimurium, and amino acid racemase from Pseudomonas striata were relatively well studied enzymatically. The amino acid sequences around the lysine residue to which the cofactor pyridoxal phosphate linked were elucidated as follows.B. stearothermophilus : Ala-ValーVal-Lys-Asn-Asn-Ala-Tyr (former sequence : AlaーPro-ProーLys-Ala-Asn-Ala-Tyr) ; Salmonella typhimurium : dad B Val-Trp-Ser-Val-ValーLys-Ala-Asn-Ala-Tyr-Gly-His-Gly-Ile, dal Leu-Val-Ala-Val-Val-Lys Ala-Asn-Ala-Tyr-Gly-His-Gly-Leu ; P. striata : Leu-Thr-Ala-Val-Leu-Lys-Ala-Ala-Asp-Ala-Try-Gly-His-GlyーIle.In order to obtain the basic concept in developing new inhibitor (antibacterial agents), We started the synthetic and conformational studies of active site peptides of these enzymes as model active site. At first, we compared the amino acid sequences and divided the sequences in several fragment peptides. The synthetic fragment peptides were then condensed to give protected two kinds of octapeptides and three kinds of tetradecapeptides. All the synthetic procedures were performed in the solution phase. After the N-protective group was converted to N-acetyl group, the protective groups were then removed by treatment of trimethylsilyltriflate-thioanisole system to give acetyl derivatives of two octa-and three tetradecapeptides corresponding to the amino acid sequences of active sites of above mentioned racemases. The conformational studies using NMR spectroscopy for Boc-Leu-Thr-Ala-Val-Leu-OMe and Boc-Ala-Pro-Pro-Lys (Z) -Ala-Asn-Ala-Tyr (Bzl) -OBzl were also performed.