Acid-PEG4-mono-methyl ester

For the conjugation of the trihydroxamate bifunctional chelating agent N-[tris[2-[[N-(benzyloxy)amino]-carbonyl]ethyl]methyl]succinamic acid (trisuccin, 1) to antibodies, we originally used the corresponding 2,3,5,6-tetrafluorophenyl active ester followed by the postconjugation removal of the benzyl protecting groups by catalytic hydrogenation. It was of interest to us to design a conjugation protocol capable of incorporating deblocked hydroxamates into peptides and proteins. Reported procedures that were expected to be compatible with the functionalities present in trisuccin were used with no success, as judged by the lack of ability of the products to radiolabel with 188Re. A simple conjugation method was then developed utilizing the o-nitrophenol (ONP) activated ester of the unprotected trisuccin, N-[tris[2-[(N-hydroxyamino)carbonyl]ethyl]methyl]succinamic acid, 3, which eliminates the need for the postconjugation deblocking. An assay for indirect estimation of the active ester content, based on the concentration of its decomposition byproduct, ONP-OH, was developed. Comparison of the indirectly estimated concentrations with those obtained directly from purified products showed > 90% accuracy for this assay. This procedure has the advantage of rapidly using the unpurified active ester, eliminating the possibilities of its decomposition through solvolysis or self-condensation by the unprotected hydroxamate functions. A colorimetric assay was developed for estimation of the number of ligands per molecule of protein. This assay and the fact that all conjugates consistently radiolabeled with 188Re show that this procedure conjugated the unprotected hydroxamate ligands to the CC49 monoclonal antibody. These results indicate the potential applicability of this technique to conjugation of unprotected hydroxamate derivatives with other proteins and peptides.

The objective of this study was to identify the Fatty Acid Methyl Ester (FAME) from Neurospora intermedia N-1 that isolated from Indonesian red peanut cake (oncom). FAME profiles have been used as biochemical characters to study many different groups of organisms, such as bacteria and yeasts. FAME from N. intermedia N-1 was obtained by some stages of extraction the orange spores and fractination using a chromatotron. The pure compound (1) was characterized by 500 mHz NMR (1H and 13C), FTIR and LC-MS. Summarized data's of 1H and 13C NMR spectra of compound 1 contained 19 Carbon, 34 Hydrogen and 2 Oxygen (C19H34O2). The position of the double bonds at carbon number 8 and 12 were indicated in the HMBC spectrum (2D-NMR). LC-MS spectrum indicates molecular weight of the compound 1 as 294 which is visible by the presence of protonated molecular ion [M+H] at m/z 295. Methyl esters of long chain fatty acids was presented by a 3 band pattern of IR spectrum with bands near 1249, 1199 and 1172 cm(-1). We suggested that the structure of the pure compound 1 is methyl octadeca-8,12-dienoate. The presence methyl octadeca-8,12-dienoate in N. intermedia is the first report.

Cyclopropane FA occur in nature in the phosphoplipids of bacterial membranes, in oils containing cyclopropene FA, and in Litchi sinensis oil. Dihydrosterculic acid (2-octyl cyclopropaneoctanoic acid) and its methyl ester were selected for 1H and 13C NMR analysis as compounds representative of cyclopropane FA. The 500 MHz 1H NMR spectra acquired with CDCl3 as solvent show two individual peaks at -0.30 and 0.60 ppm for the methylene protons of the cyclopropane ring. Assignments were made with the aid of 2D correlations. In accordance with previous literature, the upfield signal is assigned to the cis proton and the downfield signal to the trans proton. This signal of the trans proton is resolved from the peak of the two methine protons of the cyclopropane ring, which is located at 0.68 ppm. The four protons attached to the two methylene carbons alpha to the cyclopropane ring also show a split signal. Two of these protons, one from each methylene moiety, display a distinct shift at 1.17 ppm, and the signal of the other two protons is observed at 1.40 ppm, within the broad methylene peak. The characteristic peaks in the 13C spectra are also assigned.