Martin Nathaniel I, Woodward Joshua J, Winter Michael B, Beeson William T, Marletta Michael A
Departments of Chemistry, Molecular and Cellular Biology, and Division of Physical Sciences, Lawrence Berkeley National Laboratory, University of California, Berkeley, Berkeley, California 94720-1460, USA.
J Am Chem Soc. 2007 Oct 17;129(41):12563-70. doi: 10.1021/ja0746159. Epub 2007 Sep 25.
The role of nitric oxide (NO) as a biological signaling molecule is well established. NO is produced by the nitric oxide synthases (NOSs, EC 1.14.13.39), a class of heme proteins capable of converting l-arginine to NO and l-citrulline. Despite the large body of knowledge associated with the NOSs, mechanistic details relating to the unique oxidative chemistry performed by these enzymes remain to be fully elucidated. Furthermore, a number of disease states are associated with either the over- or underproduction of NO, making the NOS pathway an attractive target for the development of therapeutics. For these reasons, molecular tools capable of providing mechanistic insights into the production of NO and/or the inhibition of the NOSs remain of interest. We report here the stereospecific synthesis and testing of a number of new l-arginine analogues bearing a minimal substitution, methylation at position 5 of the amino acid side chain (such analogues have not been previously reported). The synthetic approach employed a modified photolysis procedure whereby irradiation of the appropriate diacylperoxide precursors at 254 nm gave access to the required unnatural amino acids in good yields. A heme domain construct of the inducible NOS isoform (iNOSheme) was used to assess the binding of each compound to the enzyme active site. The compounds were also investigated as either inhibitors of, or alternate substrates for, the inducible NOS isoform. The results obtained provide new insight into the steric and stereochemical tolerance of the enzyme active site. These findings also further support the role of a conserved active site water molecule previously proposed to be necessary for NOS catalysis.
一氧化氮（NO）作为一种生物信号分子的作用已得到充分确立。NO由一氧化氮合酶（NOSs，EC 1.14.13.39）产生，这是一类能够将L-精氨酸转化为NO和L-瓜氨酸的血红素蛋白。尽管与NOSs相关的知识很多，但这些酶所执行的独特氧化化学的机制细节仍有待充分阐明。此外，许多疾病状态与NO的过量或不足产生有关，使得NOS途径成为治疗药物开发的一个有吸引力的靶点。出于这些原因，能够提供有关NO产生和/或NOSs抑制机制见解的分子工具仍然备受关注。我们在此报告了一些新的L-精氨酸类似物的立体定向合成和测试，这些类似物在氨基酸侧链的5位带有最小取代基甲基化（此类类似物以前未被报道过）。合成方法采用了改良的光解程序，即在254 nm波长下照射适当的二酰基过氧化物前体，从而以良好的产率获得所需的非天然氨基酸。诱导型NOS同工型的血红素结构域构建体（iNOSheme）用于评估每种化合物与酶活性位点的结合。这些化合物还作为诱导型NOS同工型的抑制剂或替代底物进行了研究。获得的结果为酶活性位点的空间和立体化学耐受性提供了新的见解。这些发现还进一步支持了先前提出的保守活性位点水分子对NOS催化所必需的作用。
