Papeo Gianluca, Giordano Patrizia, Brasca Maria Gabriella, Buzzo Ferdinando, Caronni Dannica, Ciprandi Franco, Mongelli Nicola, Veronesi Marina, Vulpetti Anna, Dalvit Claudio
Chemistry Department, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano, Milano, Italy.
J Am Chem Soc. 2007 May 2;129(17):5665-72. doi: 10.1021/ja069128s. Epub 2007 Apr 7.
Two novel series of polyfluorinated amino acids (PFAs) were designed and synthesized according to a very short and scalable synthetic sequence. The advantages and limitations of these moieties for screening purposes are presented and discussed. The potential applications of these PFAs were tested with their incorporation into small arginine-containing peptides that represent suitable substrates for the enzyme trypsin. The enzymatic reactions were monitored by 19F NMR spectroscopy, using the 3-FABS (three fluorine atoms for biochemical screening) technique. The high sensitivity achieved with these PFAs permits a reduction in substrate concentration required for 3-FABS. This is relevant in the utilization of 3-FABS in fragment-based screening for identification of small scaffolds that bind weakly to the receptor of interest. The large dispersion of 19F isotropic chemical shifts allows the simultaneous measurement of the efficiency of the different substrates, thus identifying the best substrate for screening purposes. Furthermore, the knowledge of KM and Kcat for the different substrates allows the identification of the structural motifs responsible for the binding affinity to the receptor and those affecting the chemical steps in enzymatic catalysis. This enables the construction of suitable pharmacophores that can be used for designing nonpeptidic inhibitors with high affinity for the enzyme or molecules that mimic the transition state. The novel PFAs can also find useful application in the FAXS (fluorine chemical shift anisotropy and exchange for screening) experiment, a 19F-based competition binding assay for the detection of molecules that inhibit the interaction between two proteins.
根据一个非常简短且可扩展的合成序列，设计并合成了两个新型的多氟氨基酸（PFAs）系列。介绍并讨论了这些部分用于筛选目的的优点和局限性。通过将这些PFAs掺入含精氨酸的小肽中来测试其潜在应用，这些小肽是胰蛋白酶的合适底物。使用3-FABS（用于生化筛选的三个氟原子）技术，通过19F NMR光谱监测酶促反应。这些PFAs实现的高灵敏度允许降低3-FABS所需的底物浓度。这在基于片段的筛选中利用3-FABS来鉴定与感兴趣的受体弱结合的小支架时具有重要意义。19F各向同性化学位移的大分散性允许同时测量不同底物的效率，从而确定用于筛选目的的最佳底物。此外，了解不同底物的KM和Kcat有助于确定负责与受体结合亲和力的结构基序以及那些影响酶催化化学步骤的结构基序。这使得能够构建合适的药效团，可用于设计对酶具有高亲和力的非肽类抑制剂或模拟过渡态的分子。新型PFAs还可在FAXS（用于筛选的氟化学位移各向异性和交换）实验中找到有用的应用，这是一种基于19F的竞争结合测定法，用于检测抑制两种蛋白质之间相互作用的分子。
