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首页> 外文期刊>Chemistry: A European journal >Binding Site Optimisation for Artificial Enzymes by Diffusion NMR of Small Molecules
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Binding Site Optimisation for Artificial Enzymes by Diffusion NMR of Small Molecules

机译：小分子扩散核磁共振的人工酶结合位点优化

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摘要

A binding site optimisation protocol for the design of artificial enzymes based on "small molecule-small molecule" binding studies by difusion NMR is presented. Since the reaction chosen was the hydrolysis of reaction chosen was the hydrolysis of ester 1 ([4-(4-carboxyl-1-oxobutyl)-ami-nobenzyl]-phenethyl ester), an analogous phosphonate ester 2 ([4-(4-car-boxy-1-oxobutyl)-aminobenzyl]-phos-phonic phenethyl ester) was selected as a suitable transition state analogue (TSA). The key objective of the NMR studies was to find a unit with functional groups capable of binding to the acidic sites of the TSA. Nine dipeptides, mainly with basic and hydroxyl groups, were used and their affinity to the TSA was studied by measuring the change in the diffusion coefficient, D_pep, upon binding by p9ulse field gradient NMR. The value of D_ped at 298 K in D_2O at pD 5, 7 and 10 was measured both in free solution, and mixtures containing one dipeptide and the TSA. As both components are low molecular weight species with M<500, a TSA-to-dipeptide ratio of 10:1 was used to detect significant changes in D_pdp. The detect significant changes in D_ped. The results revealed that dipeptides with basic residues show higher affinity to the TSA than those with hydroxyl or aliphatic side chains in aqueous solutions. The dipeptide showing the most significant relative change in D_pep was H-Arg-Arg-OH, and the binding constant was estimated to be 86 LM~(-1) by measuring D_pep at varying concentrations of the TSA. In addition binding of the TSA to a new water-soluble polymer with a polyallylamine backbone and randomly distrihuted Arg-Arg binding sites was examined and the binding constant was estimated to be >=1500 LM~(-1). As confirmed by further catalytic activity tests, polymers containing Arg-Arg as a binding site are capable of significant rate accelerations in the hydrolysis of ester 1.
机译：提出了一种结合位点优化方案，用于通过扩散NMR基于“小分子-小分子”结合研究设计人工酶。因为选择的反应是水解，所以选择的水解是酯1（[4-（4-羧基-1-氧丁基）-氨基-无苄基]-苯乙基酯），类似的膦酸酯2（[4-（4 （-羧基--1-氧代丁基）-氨基苄基]-膦酸苯乙酯）作为合适的过渡态类似物（TSA）。 NMR研究的主要目标是找到一个带有能够与TSA酸性位点结合的官能团的单元。使用了九个主要具有碱性和羟基基团的二肽，并通过测量p9ulse场梯度NMR结合后扩散系数D_pep的变化，研究了它们对TSA的亲和力。在游离溶液以及含有一种二肽和TSA的混合物中，分别在pD 5、7和10的D_2O中测量298 K下D_ped的值。由于这两种成分均为M <500的低分子量物质，因此TSA与二肽之比为10：1可检测D_pdp的显着变化。检测D_ped中的显着变化。结果表明，在水溶液中，具有碱性残基的二肽对TSA的亲和力高于具有羟基或脂族侧链的二肽。在D_pep中表现出最显着相对变化的二肽是H-Arg-Arg-OH，通过在不同浓度的TSA中测量D_pep，结合常数估计为86 LM〜（-1）。另外，检查了TSA与具有聚烯丙基胺主链和随机分散的Arg-Arg结合位点的新的水溶性聚合物的结合，并且结合常数估计为> = 1500 LM〜（-1）。如进一步的催化活性测试所证实的那样，含有Arg-Arg作为结合位点的聚合物能够在酯1的水解中显着加快速率。

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