Immunochemical Studies on the Poly-γ-D-glutamyl Capsule of Bacillus anthracis. II. The Synthesis of Eight Dipeptides and Four Tripeptides of Glutamic Acid^*

Abstract

Eight dipeptides and four branched tripeptides of D-and L-glutamic acid were synthesized to be used as hapten inhibitors in the homologous immune system of capsular poly-y-D-glutamic acid isolated from Bacillus anthracis. N-Carbobenzoxy and benzyl esters were used as protective groups in the synthesis, and the coupling was achieved with a dicyclohexylcarbodiimide reagent. Protective groups were removed by catalytic hydrogenolysis in one step. The purity of the starting materials and products in each I nformation about the extent of the areas of antigen molecules involved in combining with antibodies (determinant groups) as well as the contributions of various structural features to specificity can be obtained by hapten inhibition of the precipitin reaction.In this investigation, the antigen studied was a capsular polypeptide isolated from a strain of Bacillus anthracis, to which rabbit antisera have been obtained. The polypeptide was shown to be essentially pure poly-7-0glutamic acid (Goodman and Nitecki, 1966), an antigen of relatively simple structure. The serological specificity of its reaction with rabbit antisera and hapten inhibition synthetic step was ascertained by thin layer chromatography on silica gel, paper chromatography, and high voltage electrophoresis. The free peptides and their hydrolysates reacted quantitatively with ninhydrin and 2,4-dinitrobenzene-l-sulfonic acid reagents and the peptide hydrolysates showed the expected specific optical rotations. Diastereomeric a-dipeptides and tripeptides were shown to be separable by paper chromatography in several solvents and by high voltage electrophoresis.of the homologous immune system using amino acids and synthetic di-and tripeptides of glutamic acid were investigated. The results of serological investigation are presented in another publication (Goodman and Nitecki, 1966).For this purpose eight dipeptides and four tripeptides of glutamic acid were synthesized. There are four possible a-dipeptides (1-4)l and four possible y-dipeptides (5-8). Four of the dipeptides have been synthesized before by a different method (Sachs and Brand, 1953a).In addition, four branched tripeptides have been synthesized in which one glutamic acid moiety is substituted in both a and y positions by glutamic acid units (9-12). In two of these, the central a,y-substituted glutamic acid is of opposite configuration to the other two glutamic acid moieties (9 and 10); the other two 1 The abbreviations used are described by Young (1962).

gamma-Glutamylamine cyclotransferase: specificity toward epsilon-(L-gamma-glutamyl)-L-lysine and related compounds.

“…The supernatant was extracted with ether and was freed of lysine by passage through a small column of Dowex 50-x8(H+) in water. * Detection was with a chlorine-tolidine reagent (14).…”

ChemInform Abstract: SYNTHESIS, BIOLOGICAL ACTIVITY, AND TRITIATION OF L‐3,4‐DEHYDROPROLINE‐CONTAINING PEPTIDES

“…Resin hydrolysates were carried out in 4 N methanesulfonic acid : propionic acid : water (1.0:2.4:0.6) in sealed, evacuated tubes for 24 h at 110". Solvent systems for thin-layer chromatography on silica gel G (Analtech, Inc.) were (A) n-propanol-water (70:30), (B) ethanol-water (70:30), (C) n-butanol-acetic acidpyridine-water (15:3:10:2), (D) n-butanolacetic acid-pyridine-water (1 5:3: 10: 12), and (E) methanol-chloroform (2: 1) and were developed by the fluorescamine spray procedure (Felix & Jimenez, 1974) or by the chlorine-tolidine procedure (Nitecki & Goodman, 1966). Melting points were determined on a Reichert hot stage apparatus and are uncorrected.…”

Synthetic Antigens with Sequential and Conformation‐Dependent Determinants Containing the Same l‐Tyrosyl‐l‐alanyl‐l‐glutamyl Sequence

“…An aliquot of the preparation was again submitted to paper electrophoresis using the same buffer and conditions as above. The developed electrophoretogram was scanned for radioactivity and then sprayed with both ninhydrin and with a peptide spray [51]. The presence of one peptide-spray positive, but ninhydrin negative, radioactive material indicated purity of the peptide.…”