Yan Kejia, Triana Vivian, Kalmady Sunil Vasu, Aku-Dominguez Kwami, Memon Sharyar, Brown Alex, Greiner Russell, Derda Ratmir
Department of Chemistry, University of Alberta Edmonton AB T6G 2G2 Canada
Department of Computer Science, University of Alberta Alberta AB T6G 2E8 Canada.
Chem Sci. 2021 Oct 21;12(42):14301-14308. doi: 10.1039/d1sc04146k. eCollection 2021 Nov 3.
The Wittig reaction can be used for late stage functionalization of proteins and peptides to ligate glycans, pharmacophores, and many other functionalities. In this manuscript, we modified 160 000 N-terminal glyoxaldehyde peptides displayed on phage with the Wittig reaction by using a biotin labeled ylide under conditions that functionalize only 1% of the library population. Deep-sequencing of the biotinylated and input populations estimated the rate of conversion for each sequence. This "deep conversion" (DC) from deep sequencing correlates with rate constants measured by HPLC. Peptide sequences with fast and slow reactivity highlighted the critical role of primary backbone amides (N-H) in accelerating the rate of the aqueous Wittig reaction. Experimental measurement of reaction rates and density functional theory (DFT) computation of the transition state geometries corroborated this relationship. We also collected deep-sequencing data to build structure-activity relationship (SAR) models that can predict the DC value of the Wittig reaction. By using these data, we trained two classifier models based on gradient boosted trees. These classifiers achieved area under the ROC (receiver operating characteristic) curve (ROC AUC) of 81.2 ± 0.4 and 73.7 ± 0.8 (90-92% accuracy) in determining whether a sequence belonged to the top 5% or the bottom 5% in terms of its reactivity. This model can suggest new peptides never observed experimentally with 'HIGH' or 'LOW' reactivity. Experimental measurement of reaction rates for 11 new sequences corroborated the predictions for 8 of them. We anticipate that phage-displayed peptides and related mRNA or DNA-displayed substrates can be employed in a similar fashion to study the substrate scope and mechanisms of many other chemical reactions.
维蒂希反应可用于蛋白质和肽的后期功能化，以连接聚糖、药效基团和许多其他功能基团。在本论文中，我们在仅使文库群体的1%功能化的条件下，使用生物素标记的叶立德，通过维蒂希反应修饰了展示在噬菌体上的160000个N端乙醛肽。对生物素化群体和输入群体进行深度测序，估计每个序列的转化率。这种来自深度测序的“深度转化率”（DC）与通过高效液相色谱法测量的速率常数相关。具有快速和慢速反应性的肽序列突出了主链酰胺（N-H）在加速水相维蒂希反应速率中的关键作用。反应速率的实验测量和过渡态几何结构的密度泛函理论（DFT）计算证实了这种关系。我们还收集了深度测序数据以建立结构-活性关系（SAR）模型，该模型可以预测维蒂希反应的DC值。利用这些数据，我们基于梯度提升树训练了两个分类模型。在确定一个序列在反应性方面是否属于前5%或后5%时，这些分类器在ROC（接收器操作特征）曲线下的面积（ROC AUC）分别达到了81.2±0.4和73.7±0.8（准确率为90-92%）。该模型可以预测具有“高”或“低”反应性的从未在实验中观察到的新肽。对11个新序列的反应速率进行实验测量，证实了其中8个序列的预测结果。我们预计，展示在噬菌体上的肽以及相关的mRNA或DNA展示底物可以以类似的方式用于研究许多其他化学反应的底物范围和机制。
