Optimization of the Reaction Conditions for the Synthesis of Neoglycoprotein-AZT Monophosphate Conjugates

• Published in: Journal of medicinal chemistry Vol. 38; no. 6; pp. 883 - 889
• Main Authors: Kuipers, Mirjam E, Swart, Pieter J, Hendriks, Margriet M. W. B, Meijer, Dirk K. F
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.1995
• Subjects: Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Antiviral Agents - administration & dosage; Antiviral Agents - chemical synthesis; Biological and medical sciences; Dideoxynucleotides; Drug Carriers - chemical synthesis; Glycoproteins - chemical synthesis; human immunodeficiency virus 1; Humans; Lactose - chemistry; Medical sciences; Pharmacology. Drug treatments; Phosphorylation; Prodrugs - chemical synthesis; Serum Albumin - chemistry; Thymine Nucleotides - administration & dosage; Thymine Nucleotides - chemistry; Zidovudine - administration & dosage; Zidovudine - analogs & derivatives; Zidovudine - chemistry; Process conditions; Prevention; Dideoxynucleoside; Polymerization; Conjugated compound; Modeling; Pyrimidine nucleoside; Computer aid; Optimization; Chemical coupling
• ISSN: 0022-2623; 1520-4804
• DOI: 10.1021/jm00006a005

The coupling of the monophosphate derivative of 3-azido-2,3-dideoxythymidine (AZTMP) to glycoproteins by water soluble carbodiimide (1-ethyl-3-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) was greatly improved, relative to a recently reported method, by using also N-hydroxysulfosuccinimide (NHS) in the conjugation reaction. The hydrolysis of the activated AZTMP intermediate, responsible for the low degree of conjugation in the earlier method, could be delayed considerably if the activated phosphate group was converted into an activated ester by addition of NHS. In order to minimize the use of compounds needed for the preparation of AZTMP-protein conjugates, the present study was undertaken to determine if the reaction conditions could be optimized such that a conjugate with 2 AZTMP molecules/mol of neoglycoprotein would result. In addition a low proportion of cross-linked conjugates was desired. Optimization was achieved studying the shape of three-dimensional response surfaces, in which the degree of AZTMP coupling and the percentage of monomeric conjugates were regarded as the relevant responses. It appeared that the optimal conditions for coupling 1-2 mol of AZTMP to 1 mol of glycoprotein were an incubation time of 30 h, an AZTMP amount of 4 mg, an NHS amount between 8 and 15 mg, and a glycoprotein amount of 50 mg.