Department of Physical Chemistry, Institute of Chemistry, Martin Luther University Halle-Wittenberg, D-06099 Halle/Saale, Germany; Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle/Saale, Germany.
Department of Physical Chemistry, Institute of Chemistry, Martin Luther University Halle-Wittenberg, D-06099 Halle/Saale, Germany.
J Pharm Sci. 2020 Feb;109(2):981-991. doi: 10.1016/j.xphs.2019.10.052. Epub 2019 Nov 2.
Protein-polymer conjugates have been used as therapeutics because they exhibit frequently higher stability, prolonged in vivo half-life, and lower immunogenicity compared with native proteins. The first part of this report describes the enzymatic synthesis of poly(glycerol adipate) (PGA(M)) by transesterification between glycerol and dimethyl adipate using lipase B from Candida antarctica. PGA(M) is a hydrophilic, biodegradable but water insoluble polyester. By acylation, PGA(M) is modified with 6-(Fmoc-amino)hexanoic acid and with hydrophilic poly(ethylene glycol) side chains (mPEG12) rendering the polymer highly water soluble. This is followed by the removal of protecting groups, fluorenylmethyloxycarbonyl, to generate polyester with primary amine groups, namely PGA(M)-g-NH-g-mPEG12. H NMR spectroscopy, FTIR spectroscopy, and gel permeation chromatography have been used to determine the chemical structure and polydispersity index of PGA(M) before and after modification. In the second part, we discuss the microbial transglutaminase-mediated conjugation of the model protein dimethylcasein with PGA(M)-g-NH-g-mPEG12 under mild reaction conditions. SDS-PAGE proves the protein-polyester conjugation.
蛋白质-聚合物缀合物已被用作治疗剂，因为与天然蛋白质相比，它们通常表现出更高的稳定性、更长的体内半衰期和更低的免疫原性。本报告的第一部分描述了使用南极假丝酵母脂肪酶通过甘油和己二酸二甲酯之间的酯交换反应酶促合成聚（甘油己二酸酯）（PGA（M））。PGA（M）是一种亲水性、可生物降解但不溶于水的聚酯。通过酰化反应，PGA（M）用 6-（芴甲氧羰基氨基）己酸和亲水性聚（乙二醇）侧链（mPEG12）进行修饰，使聚合物高度水溶性。然后去除保护基团芴甲氧羰基，生成具有伯胺基团的聚酯，即 PGA（M）-g-NH-g-mPEG12。NMR 光谱、傅里叶变换红外光谱和凝胶渗透色谱已用于确定修饰前后 PGA（M）的化学结构和多分散指数。在第二部分中，我们讨论了在温和的反应条件下微生物转谷氨酰胺酶介导的模型蛋白二甲基酪蛋白与 PGA（M）-g-NH-g-mPEG12 的缀合。SDS-PAGE 证明了蛋白质-聚酯的缀合。
