CHEMICAL MODIFICATION OF PEPTIDES CONTAINING GAMMA-CARBOXYGLUTAMIC ACID
CHEMICAL MODIFICATION OF PEPTIDES CONTAINING GAMMA-CARBOXYGLUTAMIC ACID
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DOI：
10.1021/jo00349a003
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发表时间：
1982-01-01
影响因子：
3.6
通讯作者：
HISKEY, RG
中科院分区：
文献类型：
--
作者：
BOGGS, NT;BRUTON, HD;HISKEY, RG
在酸性pH条件下，羧基谷氨酸(GLA)侧链上的质子发生快速交换。我们利用得到的烯醇形式的反应性来开发一种对肽结合的GLA残基进行化学修饰的方法。在pH为4.5的条件下，GLA多肽与吗啉-甲醛混合物反应生成Mannich碱加合物。Mannich碱在50%的DMF水溶液中迅速裂解，生成二氧化碳、吗啉和相应的亚甲基谷氨酰基残留物。结合我们对含有羧基谷氨酸的多肽和蛋白的研究，我们开发了一种潜在有用的方法来修饰GLA残基的丙二酸基侧链。Stenflo等人早期的核磁共振观测。5和Marki等人。6表明，GLA侧链上的-质子很容易与氚发生交换。因此，我们使用模型肽L-phenylalanyl-L-leucyl-LY-carboxyglutamyl-LY-carboxyglutamyl-L-leucine甲酯研究了交换的pH依赖性。在100 MHz下，五肽的GLA残基的质子共振落在以3.7ppm为中心的包络下。如图1所示，在给定的pH值下，可从半对数图中确定-质子的一阶损失。在几个pH值下，观察到的-质子交换速率如表I所示。在氢离子浓度类似增加的情况下，交换速率大约增加10倍。这一结果表明，正如酸催化所预期的那样，y-质子交换是线性依赖的；然而，由于-碳的烯醇化依赖于-羧基的电离状态，因此在较大的pH范围内不可能存在线性依赖。事实上，表一中的数据表明，在中等碱性条件下(pH 8.54，r=23h)-质子交换非常缓慢，而pH值向中性移动则显著增加了交换速率。
At acidic pH the-proton of the-carboxyglutamic acid (Gla) side chain undergoes rapid exchange. We have utilized the reactivity of the resulting enol form to develop a method for the chemical modification of peptide-bound Gla residues. Reaction of Gla peptides with a morpholine-formaldehyde mixture at pH 4.5 yields the Mannich base adduct. Fragmentation of the Mannich base occurs rapidly in 50% aqueous DMF to yield carbon dioxide, morpholine, and the corresponding-methyleneglutamyl residue.In connection with our studies on-carboxyglutamic acid containing peptides and proteins4 we have developed a potentially useful method of modifying the malonyl side chains of Gla residues. The earlier NMR observations of Stenflo et al. 5 and Marki et al. 6 indicated that the-proton of the Gla side chain exchanged readily with deuterons. Consequently, we examined the pH dependence of the exchange using the modelpeptide L-phenylalanyl-L-leucyl-LY-carboxyglutamyl-LY-carboxyglutamyl-L-leucine methyl ester. At 100 MHz the-proton resonance of the Gla residues of the pentapeptide falls under an envelope centered at about 3.7 ppm. At a given pH, the first-order loss of-protons can be determined from semilogarithmic plots as shown in Figure 1. The observed rates of-proton exchange for deuterium at several pH values are given in Table I. An approximate10-fold increase in exchange rate is observed over a similar increase in hydronium ion con-centration. This result suggests a linearly dependent y-proton exchange, as expected for acid catalysis; however, since enolization at the-carbon depends on the ionization state of the-carboxyl groups, a linear dependence is not expected over a larger pH range. Indeed, the data in Table I indicate that-proton exchange is very slow under mildly basic conditions (pH 8.54, r= 23 h) whereas pH values moving toward neutrality significantly increase the rate of exchange.