Nα-Fmoc-Nω-(2,2,4,6,7-Pbf-5-sulfonyl)-L-arginine 4-alkoxy-benzyl-alcohol resin

A strain of streptomycete isolated from a soil sample was found to produce a novel amino acid metabolite. The compound was purified from the culture fluid by chromatography, using cation exchange resin, a synthetic adsorbent, and finally by preparative HPLC with a reverse-phase column. The structure of the compound was established as N(delta)-(5-methyl-4-oxo-2-imidazolin-2-yl)-L-ornithine on the basis of an analysis of the spectral data and chemical degradation. This was confirmed by comparing the NMR spectrum of the metabolite with that of the compound synthesized by treating methylglyoxal and N(alpha)-acetyl-L-arginine. The substance did not show any antimicrobial activity against bacteria, fungi and yeasts by the agar plate method, but exhibited a weak preventive effect on cucumber mildew disease in a pot test.

An improved method has been developed for the determination of L-arginine and its methylated metabolites, N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetric DMA, ADMA) and N(G),N(G)'-dimethyl-L-arginine (symmetric DMA, SDMA) in biological samples. Extraction of these compounds with a strong cation-exchange resin AG50W-X8 with L-homoarginine (2-amino-6-guanidinohexanoic acid) as an internal standard gave a recovery of more than 70% except for SDMA from plasma samples. After extracted samples were converted to fluorescent derivatives with o-phthalaldehyde (OPA) in an alkaline medium, the following high-performance liquid chromatographic separation with a ODS column (wide-pore size, 300 A) was successfully performed with an isocratic mobile phase system. The method permits quantitative determination of L-arginine and its methylated metabolites at concentrations as low as 4 microM and 0.18 microM, respectively. Using this method, the levels of L-arginine, L-NMMA, ADMA and SDMA in human plasma, urine and rat tissue were determined.

To make the native LHRH immunogenic, a multimer of LHRH interspersed with T non-B peptides (r-LHRH-d2) was expressed as recombinant protein in Escherichia coli. The expression level of the recombinant protein was around 15% of the total cellular protein and it aggregated as inclusion bodies. Inclusion bodies from the bacterial cells were isolated and purified to homogeneity. Instead of high concentrations of chaotropic agents, r-LHRH- d2 was solubilized in 50 mM citrate buffer at pH 3 containing 2 M urea. The protein was refolded by 5-fold dilution (pulsatile) with cold 10 mM citrate buffer at pH 6 in presence of 0.3 M L-arginine. Purification of r-LHRH-d2 was carried out by successive passages on CM-Sepharose column at pH 6.0 which retained extraneous proteins and pH 4.8 at which r-LHRH-d2 bound to the resin. The elution was carried out by using linear salt gradient (0.1-1 M NaCl). The overall yield of the purified r-LHRH-d2 was 40% of the initial inclusion body proteins. The purity and homogeneity were confirmed by a single homogeneous peak on analytical HPLC eluting out at 29.51 min and by single band on SDS-PAGE reactive with polyvalent anti-LHRH antibodies. Mass spectroscopic analysis indicated the protein to be of 16.6 kDa which equals the theoretically expected mass. The N-terminal amino acid analysis of r-LHRH-d2 showed the sequence which corresponded to the designed protein. The CD spectrum of the refolded r-LHRH-d2 showed that the multimer has considerable beta sheet structure like the monomeric LHRH protein.