Cysteine promoted C-terminal hydrazinolysis of native peptides and proteins.
Cysteine promoted C-terminal hydrazinolysis of native peptides and proteins.
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DOI：
10.1002/anie.201304997
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发表时间：
2013-12-02
影响因子：
16.6
通讯作者：
Macmillan, Derek
中科院分区：
文献类型：
--
作者：
Adams, Anna L.;Cowper, Ben;Morgan, Rachel E.;Premdjee, Bhavesh;Caddick, Stephen;Macmillan, Derek
1998年，Ramage小组证明了合成肽的C-末端酰肼在重氮化为相应的酰基叠氮化合物后，可以转化为有用的功能化多肽，包括硫代酯。[1]缺点是某些氨基酸残基需要保护，但刘和他的同事最近证明，在优化的条件下，肽和蛋白质酰肼可以转化为硫酸酯，用于天然化学连接(NCL)，而不需要保护基团。[2]蛋白质C-末端肼本身是有用的产品，并允许通过唯一具有活性的C-末端对蛋白质进行选择性修饰。[4]蛋白质C端肼只能通过肼分解内含素融合前体获得。[2，3]因此，补充途径是可取的，特别是对于那些不期望表达为可溶的和折叠的内含素融合蛋白的蛋白质。S在没有内含子的情况下的酰基转移。[6]我们认为，作为一种有效的亲核剂，肼可以作为一种合适的添加剂，用于这一过程，该过程通过瞬时硫酯的肼分解生成C-端肼。由于酰肼在反应条件下不会发生水解性反应，这可能会提供一条更可靠的、尽管不那么直接的合成蛋白质硫代酯的途径。此外，当硫酯在原位经历NCL时，酰肼转化为硫酯似乎基本上不会被水解。[2]我们首先研究了模型肽[6]，并在pH为5.8(最终pH约为7)的磷酸二氢钠缓冲液(0.1m)中使用醋酸肼作为肼来源，[7]在存在和不存在MESNa的情况下(表1)。对反应产物进行的高效液相色谱和液质联用分析表明，在N2H4·HOAc(5%w/v)存在下，His-Cys基序的联氨裂解反应是有效的。在608℃下，反应在24小时内几乎完成(表1，条目1)，这证实了联氨对硫酯中间体的高效截留，使我们能够研究较低的反应温度(条目2和3)。反应进行得非常顺利，只观察到1和2个，除了：1)当反应在没有醋酸肼的情况下进行(条目4)，其中MESNa硫酯是主要产物，以及2)在没有MESNa的情况下(条目7)，通过LC-MS分析观察到样品的质量随着时间的推移而显著恶化。有趣的是，尽管在醋酸肼(5%w/v)存在下，LC-MS或13CNMR分析没有观察到硫酯中间体，但当使用13C标记的甘氨酸-半胱氨酸终止前体时(支持信息，图S1)，MESNa似乎是必需的。在没有MESNa的情况下，1二聚化和蛋氨酸氧化是最常见的反应。与硫酯的形成相反，延长反应时间并不会加剧产物的水解。酰肼2被分离出来，
In 1998, the Ramage group demonstrated that a C-terminal hydrazide of a synthetic peptide could, following diazotization to the corresponding acyl azide, be transformed into usefully functionalized peptides, including thioesters.[1] A drawback was that certain amino acid residues needed to be protected, but Liu and co-workers more recently showed that peptide and protein hydrazides could be converted into thioesters for use in native chemical ligation (NCL) under optimized conditions without protecting groups.[2] Protein C-terminal hydrazides are useful products in their own right and allow selective modification of the protein through the uniquely reactive C-terminus.[3] Until recently,[4] a protein C-terminal hydrazide had only been obtained by hydrazinolysis of intein fusion precursors.[2, 3] Consequently, it is desirable that complementary routes become available, particularly for proteins that are not anticipated to express as soluble and folded intein fusion proteins.[5] Previously, we demonstrated that native peptides and proteins could undergo thioester formation across Xaa–Cys motifs by N! S acyl transfer in the absence of inteins.[6] We reasoned that, as an efficient nucleophile, hydrazine could be a suitable additive for this process, which leads to C-terminal hydrazides by hydrazinolysis of a transient thioester. This may provide a more robust, albeit less direct, route to protein thioesters, as the acyl hydrazide would not hydrolyze under the reaction conditions. Furthermore, conversion of the hydrazide into the thioester appears to be essentially free from hydrolysis when the thioester undergoes NCL in situ.[2] We first examined a model peptide,[6] and employed hydrazinium acetate as the hydrazine source in sodium phosphate buffer (0.1 m) at a pH of 5.8 (final pH of ca. 7),[7] in the presence and absence of MESNa (Table 1). HPLC and LC-MS analysis of the reaction mixture indicated that hydrazinolysis of a His–Cys motif proceeded efficiently when using N2H4· HOAc (5% w/v). The reaction was nearly complete within 24hours at 608C (Table 1, entry 1), which confirms that hydrazine was highly effective at intercepting thioester intermediates, allowing us to investigate lower reaction temperatures (entries2 and 3). The reaction proceeded very smoothly, and only 1 and 2 were observed, except for: 1) when the reaction was conducted in the absence of hydrazinium acetate (entry4), where the MESNa thioester was the major product, and 2) in the absence of MESNa (entry 7), where significant deterioration in the quality of the sample was observed over time by LC-MS analysis. Interestingly, although thioester intermediates were not observed in the presence of hydrazinium acetate (5% w/v) by either LC-MS or 13CNMR analysis, when using a 13Clabeled Gly–Cys terminated precursor (Supporting Information, Figure S1), MESNa appeared to be required. In the absence of MESNa, dimerization of 1 and methionine oxidation were the most prevalent reactions. In contrast to thioester formation, a prolonged reaction time did not exacerbate product hydrolysis. Hydrazide 2 was isolated and
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影响因子：
4.7
作者：
Thom, Jennifer;Anderson, David;Cotton, Graham
通讯作者：
Cotton, Graham
影响因子：
15
作者：
Valiyaveetil, FI;MacKinnon, R;Muir, TW
通讯作者：
Muir, TW
DOI：
10.1034/j.1399-3011.1999.00075.x
发表时间：
1999-06-01
期刊：
JOURNAL OF PEPTIDE RESEARCH
影响因子：
--
作者：
Wang, P;Layfield, R;Ramage, R
通讯作者：
Ramage, R
DOI：
10.1039/b815888f
发表时间：
2009-01-28
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Kang J;Richardson JP;Macmillan D
通讯作者：
Macmillan D
DOI：
10.1073/pnas.1202762109
发表时间：
2012-05-08
影响因子：
11.1
作者：
Brailsford, John A.;Danishefsky, Samuel J.
通讯作者：
Danishefsky, Samuel J.