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Abstract

In order to develop glycosylated cytokine, recombinant human IL-1alpha was chemically modified with N-acetylneuraminic acid (NANA). NANA with C9 spacer, 8-(hydrazinocarbonyl)octyl 5-acetamido-3, 5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo-pyranosidonic acid potassium salt (6), was synthesized by glycosylation of C9 spacer, 8-[2-N-(benzyloxycarbonyl)hydrazinocarbonyl]octanol, with methylthio glycoside derivatives of NANA in the presence of molecular sieves 3A and methyl (methylthio)sulfonium trifrate in propionitrile, followed by separation of a and beta anomers with a column chromatography and deprotection. Compound 6 was coupled to IL-1alpha by the acyl azide method. The glycosylated IL-1 was purified by anion-exchange chromatography, and NANA coupled to IL-1 was confirmed by oxidation with NaIO4. Based on the molecular weight average number of carbohydrate molecules introduced per molecule of IL-1alpha was estimated to be 2.9.

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“…Acyl azide derivative of NeuAc with C9 spacer was synthesized and introduced to rhIL-1a as described previously [18]. The NeuAc-IL-1a was puri®ed by anion-exchange chromatography, and the NeuAc-coupling was con®rmed by oxidation with NaIO 4 , the increase of its molecular weight on SDS-PAGE and time of¯ight mass spectroscopy (TOF-MS) analysis.…”

“…D-Gal conjugated IL-1a manifested a similar decrease in biological activities in vitro and receptor binding af®nity [15,16], however, the magnitude of its decrease in activities in vivo was less than that in vitro [17]. To study the effect of sialic acid modi®cation on IL-1a activity, we coupled N-acetylneuraminic acid (NeuAc), a major member of sialic acid, to rhIL-1a [18]. NeuAc-IL-1a exhibited a reduction in biological activities to about 1a10 of IL-1a in all the activities assayed in vitro and IL-1 receptor binding af®nity [19].…”