Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.
Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.
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DOI：
10.1002/cbic.201100603
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发表时间：
2012-01-02
期刊：
影响因子：
3.2
通讯作者：
Caddick, Stephen
中科院分区：
文献类型：
--
作者：
Moody, Paul;Smith, Mark E. B.;Ryan, Chris P.;Chudasama, Vijay;Baker, James R.;Molloy, Justin;Caddick, Stephen
溴代马来酰亚胺最近已被证明是作为小的，通用的支架硫醇化生物分子的控制组装。[1-4]它们呈现三个化学连接点，从而允许多功能生物缀合物的模块化构建。溴代马来酰亚胺支架没有向最终构建体引入新的手性中心，并且与二硫键相反，溴代马来酰亚胺可以用于连接两个相同浓度的未活化的生物分子，同时形成最少的同二聚体副产物。[2]溴代马来酰亚胺加合物已被证明在还原剂存在下在体外解离以释放复合硫醇。[2]细胞的细胞质含有1-10 mM还原型谷胱甘肽，[5]因此提高了溴代马来酰亚胺结合物在体内可裂解的可能性。如果这一点能够得到证实，那么可以设想将联合收割机的体内切割与多点支架连接相结合的许多医学和学术应用。然而，许多因素可能抑制细胞质裂解，特别是pH依赖性[3]和蛋白酶催化的酰胺键水解。使用一系列溴马来酰亚胺连接的绿色荧光蛋白（GFP）-罗丹明缀合物，设计为FRET对，我们在此证明，溴马来酰亚胺连接的生物缀合物在哺乳动物细胞的细胞质中裂解。罗丹明-马来酰亚胺衍生物4-6通过相关马来酸酐与共同中间体3的缩合产生（方案1）。野生型超折叠GFP中的两个天然半胱氨酸，[6] C48和C70，在我们的反应条件下被证明是马来酰亚胺官能化不可接近的（参见支持信息中的第4节）。通过将S147 C突变引入超级折叠GFP中来产生具有接近其荧光团的游离的、可接近的巯基的GFP（GFP-SH）。突变的GFP-SH产生与野生型超折叠GFP相似的发射光谱。折叠的蛋白质显示对二硫化物介导的二聚化具有抗性，因此允许GFP-SH与马来酰亚胺缀合而不需要还原剂（参见支持性信息中的第5节）。如支持性信息第5节中所述，将化合物4-6连接至GFP-SH。通过质谱法证实了罗丹明-马来酰亚胺的化学计量加成。所得构建体7-10的发射光谱如图1所示。在每种情况下，罗丹明-马来酰亚胺的添加显示导致GFP荧光的有效淬灭。在590 nm处的发射几乎没有增加。通过GFP和rhoda的双通道测量体外监测生理相关浓度的还原型谷胱甘肽（1 mM）对化合物7-10的切割-方案1。罗丹明-溴代马来酰亚胺的合成。a）（COCl）2，208 ℃，15 h; B）哌啶-4-基氨基甲酸叔丁酯（10.4当量），CsCO 3（10.4当量），CH 2Cl 2，208 ℃，24 h，71%（2步）; c）TFA/CH 2Cl 2（1：1），208 ℃，5 h，100%; d）马来酸酐（1.4当量），AcOH，1208 ℃，5小时，40%; e）溴代马来酸酐（1.4当量），AcOH，1208 ℃，5小时，66%; f）二溴代马来酸酐（1.4当量），AcOH，1208 ℃，5小时，66%。
Bromomaleimides have recently been shown to act as small, versatile scaffolds for the controlled assembly of thiolated biomolecules.[1–4] They present three points of chemical attachment, thereby allowing the modular construction of multifunctional bioconjugates. The bromomaleimide scaffold introduces no new chiral centres to the final construct and, in contrast to disulfide linkages, bromomaleimides can be used to link two unactivated biomolecules of equal concentration with minimal formation of homodimeric side products.[2] Bromomaleimide adducts have been shown to dissociate in vitro in the presence of reducing agents to liberate the composite thiols.[2] The cytoplasm of cells contains 1–10 mM reduced glutathione,[5] thus raising the possibility that bromomaleimide conjugates could be cleavable in vivo. If this could be demonstrated, then a number of medical and academic applications can be envisaged that combine in vivo cleavage with multiple points of scaffold attachment. However, a number of factors might inhibit cytoplasmic cleavage, in particular pH-dependant [3] and protease-catalysed amide bond hydrolysis. Using a series of bromomaleimidelinked green fluorescent protein (GFP)–rhodamine conjugates, designed as FRET pairs, we demonstrate herein that bromomaleimide-linked bioconjugates cleave in the cytoplasm of mammalian cells. Rhodamine–maleimide derivatives 4–6 were generated by condensation of the relevant maleic anhydride with a common intermediate, 3 (Scheme 1). The two native cysteines in wild-type superfolder GFP,[6] C48 and C70, were shown to be inaccessible to maleimide functionalisation under our reaction conditions (see Section 4 in the Supporting Information). A GFP with a free, accessible thiol close to its fluorophore (GFP-SH) was generated by introducing an S147C mutation into superfolder GFP. The mutated GFP-SH produces a similar emission spectrum to wild-type superfolder GFP. The folded protein was shown to be resistant to disulfide-mediated dimerisation, thus allowing GFP-SH to be conjugated to maleimides without the need for reducing agents (see Section 5 in the Supporting Information). Compounds 4–6 were attached to GFP-SH as described in the Supporting Information, Section 5. Stoichiometric addition of rhodamine–maleimide was confirmed by mass spectrometry. The emission spectra of the resultant constructs 7–10 are illustrated in Figure 1. In each case, addition of rhodamine–maleimide was shown to result in efficient quenching of GFP fluorescence. Little increase in emission at 590 nm was seen. Cleavage of compounds 7–10 by a physiologically relevant concentration of reduced glutathione (1 mM) was monitored in vitro by dual-channel measurement of the GFP and rhoda-Scheme 1. Synthesis of rhodamine-bromomaleimides. a)(COCl) 2, 208C, 15 h; b) piperidin-4-yl carbamic acid tert-butyl ester (10.4 equiv), CsCO3 (10.4 equiv), CH2Cl2, 208C, 24 h, 71%(2 steps); c) TFA/CH2Cl2 (1: 1), 208C, 5 h, 100%; d) maleic anhydride (1.4 equiv), AcOH, 1208C, 5 h, 40%; e) bromomaleic anhydride (1.4 equiv), AcOH, 1208C, 5 h, 66%; f) dibromomaleic anhydride (1.4 equiv), AcOH, 1208C, 5 h, 66%.
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影响因子：
4.7
作者：
Schumacher, Felix F.;Nobles, Muriel;Ryan, Chris P.;Smith, Mark E. B.;Tinker, Andrew;Caddick, Stephen;Baker, James R.
通讯作者：
Baker, James R.
影响因子：
4.9
作者：
Tedaldi, Lauren M.;Smith, Mark E. B.;Baker, James R.
通讯作者：
Baker, James R.
影响因子：
46.9
作者：
Pédelacq, JD;Cabantous, S;Waldo, GS
通讯作者：
Waldo, GS
DOI：
10.1039/c1cc11114k
发表时间：
2011-05-21
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Ryan CP;Smith ME;Schumacher FF;Grohmann D;Papaioannou D;Waksman G;Werner F;Baker JR;Caddick S
通讯作者：
Caddick S
影响因子：
3.8
作者：
Smith, CV;Jones, DP;Lauterburg, BH
通讯作者：
Lauterburg, BH