Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents
Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents
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DOI：
10.1021/ja210335f
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发表时间：
2012-01-25
影响因子：
15
通讯作者：
Haddleton, David M.
中科院分区：
文献类型：
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作者：
Jones, Mathew W.;Strickland, Rachel A.;Haddleton, David M.
一系列二溴马来酰亚胺已被证明在插入肽二硫键方面非常有效。这种缀合在缓冲溶液中的试剂的化学计量平衡下在少于15分钟内进行，以得到离散的产物，同时保持二硫键和因此的肽构象。通过使用水溶性膦三（2-羧乙基）膦（TCEP）的二硫化物还原引发插入，其允许随后通过生成的硫醇取代两个马来酰亚胺溴化物。鲑鱼降钙素（sCT）与2，3-二溴马来酰亚胺（1.1过量）在TCEP（1.1当量）存在下在pH 6.2的水溶液中的反应给出了不需要后处理的单一缀合物的完全产生。通过Mitsunobu反应将线性甲氧基聚（乙二醇）（PEG）官能化，并用于sCT的成功位点特异性和快速聚乙二醇化。该反应在15分钟内发生，具有小的化学计量过量的聚乙二醇化剂，以在二硫化物处产生插入，HPLC显示单一产物，并且MALDI-ToF确认缀合。尝试在功能性ATRP聚合引发剂中使用该基团导致聚合抑制。因此，为了制备一系列官能聚合物，选择经由叠氮化物和苯胺官能引发剂的间接途径，所述叠氮化物和苯胺官能引发剂经由适当的反应转化为2，3_二溴马来酰亚胺。例如，叠氮化物官能聚合物通过Huisgen CuAAC点击反应反应生成炔官能2，3-二溴马来酰亚胺。这种新试剂允许合成sCT与衍生自PEG甲基丙烯酸单体的梳形聚合物的缀合物，其另外给出适当的浊点。该反应代表了一种高效的聚合物缀合方法，其避免了通常由于必须使用大量过量的缀合物而产生的纯化问题。此外，肽的三级结构得到有效保持。
A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while maintaining the disulfide bridge and thus peptide conformation. The insertion is initiated by disulfide reduction using a water-soluble phosphine, tris(2-carboxyethyl)phosphine (TCEP) which allows for subsequent substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary structure of the peptide is efficiently maintained.