Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy; Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, 28040 Madrid, Spain.
Department of Chemistry, Sapienza University of Rome, 00185 Rome, Italy.
Talanta. 2017 May 15;167:126-133. doi: 10.1016/j.talanta.2017.01.072. Epub 2017 Jan 29.
The objective of the present work is to demonstrate a rational way to prepare selective sorbents able to extract simultaneously several structural analogs. For this purpose the binding specificity of two hexapeptides computationally designed (VYWLVW and YYIGGF) versus four synthetic cannabinoids Naphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH 018), naphthalen-1-yl-(1-butylindol-3-yl)methanone (JWH 073), (R)-(1-((1-methylpiperidin-2-yl)methyl)-1H-indol-3-yl)(naphthalen-1-yl)methanone (AM 1220) and (R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55) was computationally studied and then experimentally tested by solid-phase extraction (SPE) clean-up and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The two peptides were chosen using a semi combinatorial virtual technique by generating 4 cycles of peptide libraries (around 2.3×10 elements). To select the two peptides, the simulated binding scores between synthetic cannabinoids and peptides was used by maximizing the recognition properties of amino acid motif between the two JWH and the other synthetic cannabinoids. In particular, the peptide YYIGGF, having also affinity for AM 120, was selected as control because it was the only one without tryptophan residues within the best peptides obtained from simulation. Experimentally, the two hexapeptides were tested as SPE sorbent using nanomolar solutions of the four drugs. After optimization of best retentions the binding constants were calculated by loading synthetic cannabinoids solutions at different concentrations. The results indicated a strong interaction between hexapeptide VYWLVW and JWH 018 (15.58±2.03×10M), 3-fold and 40-fold larger compared to the analog JWH 073 and both AM 1220 and the WIN 55. Similar trend was observed for the hexapeptide YYIGGF but the binding constants were at least three times lower highlighting the key role of the tryptophan. To demonstrate the hexapeptides specific interaction with only synthetic cannabinoids, a cross-reactivity study was carried out using other drugs (cocaine, morphine, phencyclidine and methamphetamine) in the same SPE condition. Finally the practical utility of these peptide modified sorbent materials was further demonstrated by detecting the synthetic cannabinoids in real samples using hair matrix.
本工作的目的是展示一种合理的方法来制备能够同时提取几种结构类似物的选择性吸附剂。为此，我们通过计算设计了两种六肽（VYWLVW 和 YYIGGF），并对其与四种合成大麻素萘烷-1-基-（1-戊基吲哚-3-基）甲酮（JWH 018）、萘烷-1-基-（1-丁基吲哚-3-基）甲酮（JWH 073）、（R）-（1-（1-甲基哌啶-2-基）甲基）-1H-吲哚-3-基）（萘烷-1-基）甲酮（AM 1220）和（R）-（+）-[2,3-二氢-5-甲基-3-（4-吗啉基甲基）吡咯并[1,2,3-de]-1,4-苯并恶嗪-6-基]-1-萘基甲酮（WIN 55）的结合特异性进行了计算研究，然后通过固相萃取（SPE）净化和超高效液相色谱-串联质谱（UHPLC-MS/MS）分析进行了实验验证。这两种肽是使用半组合虚拟技术选择的，通过生成 4 个肽文库循环（约 2.3×10 个元素）。为了选择这两种肽，使用模拟结合分数来最大化两种 JWH 和其他合成大麻素之间的氨基酸模体识别特性。特别是，由于肽 YYIGGF 也对 AM 120 具有亲和力，因此被选为对照，因为它是从模拟中获得的最佳肽中唯一不含色氨酸残基的肽。实验中，使用四种药物的纳摩尔溶液测试了这两种六肽作为 SPE 吸附剂。在优化最佳保留后，通过在不同浓度下加载合成大麻素溶液来计算结合常数。结果表明，六肽 VYWLVW 与 JWH 018 之间存在强烈相互作用（15.58±2.03×10M），与类似物 JWH 073 相比，其相互作用强度增加了 3 倍，与 AM 1220 和 WIN 55 的相互作用强度增加了 40 倍。对于六肽 YYIGGF 也观察到了类似的趋势，但结合常数至少低了三倍，突出了色氨酸的关键作用。为了证明六肽与合成大麻素的特异性相互作用，我们在相同的 SPE 条件下使用其他药物（可卡因、吗啡、苯环利定和甲基苯丙胺）进行了交叉反应研究。最后，我们通过使用毛发基质检测实际样品中的合成大麻素，进一步证明了这些肽修饰吸附材料的实际应用。
