Investigation of asymmetric synthesis of chiral amines by biological oxidoreductase enzymes by computational methods

Abstract

Chiral amines play a pivotal role as essential building blocks in the production of drugs, agricultural chemicals and some other special chemicals. Enantiopure chiral amines are of particular importance in pharmaceutical chemistry, as evidenced by their use as strong pharmacophores in various drugs, including pain relievers, tuberculosis drugs, Alzheimer's drugs and depression drugs. However, the chemical syntheses of these amines often entail significant challenges. These include the use of toxic chemicals, the formation of numerous by-products, and a multistage synthesis process, which has led to an increased focus on more environmentally friendly approaches. A promising solution that has gained prominence in recent studies is the use of chiral amine synthesis with the assistance of enzymes. It has been established that enzymes such as ꙍ-transaminases, monoamine oxidases, amine dehydrogenase, phenylalanine ammonium lyases, imine reductases (IREDs), lipases, p450 monooxygenases, Pictet-Spenglerases, and berberine bridge enzymes are employed in the synthesis of chiral amines. Of these, imine reductases, which are NADPH-dependent oxidoreductases, are of particular significance. These enzymes catalyze the asymmetric reduction of prochiral imines to the corresponding amines. Tetrahydroisoquinolines (THIQs) are a class of chemical compounds that has been identified in the structural frameworks of pharmaceutical drugs employed in the treatment of various medical conditions, including cancer, gout, pain management, and neurodegenerative diseases. These substituted tetrahydroisoquinolines are included in the structures of approved natural, synthetic, and semi-synthetic drugs. The present study investigates the stereoselectivity of the SnIR enzyme from Stackebrandtia Nassauensis on 1-methyl-3,4-dihydroisoquinoline substrate through in silico methods. In the experimental study by Li et al., it was reported that the aforementioned enzyme is highly S selective on the 1-methyl-3,4-dihydroisoquinoline substrate. The calculations were initiated with model work. The objective of the model study was to ascertain the critical distances and angles. The model study involved two distinct calculations: one with and one without the use of water. The barrier of the model study with water assistance was found to be considerably higher than that without water assistance. Consequently, it was determined that the subsequent analysis would be conducted without the use of water assistance. As it is known, it is extremely difficult to make quantum mechanical calculations with enzymes since these structures contain a large number of atoms. To address this challenge, a structure is devised to model the enzyme through the formation of a cluster, encompassing the critical residues and the reaction zone. The cluster that was utilized in the DFT calculations was formed using three principal elements, which were based on both empirical and computational evidence: 1. The crystal structure of SnIR 2. Experimental data including kinetics and mutations of IREDs in general 3. Residue interaction patterns from docking. For the cluster formation step, the IRED enzyme (PDB ID: 6JIT) was selected from the protein data bank. The relevant structure contains substrate, cofactor, and enzyme. Subsequently, enzyme-substrate and enzyme-cofactor interactions were evaluated in order to determine the critical residues in the cluster. Given that the substrate currently in the 6JIT structure differs from the substrate to be studied, molecular docking was performed for the new substrate by deleting the previous substrate from the structure. The molecular docking analysis of the new structure revealed the presence of the enzyme, 1-methyl-3,4-dihydroisoquinoline substrate, and the cofactor. Subsequently, an evaluation of the enzyme-substrate interactions within the novel structure was conducted to detect the critical residues. Additionally, a comprehensive review of the existing literature was conducted to determine the residues that should be included in the cluster model. Once the residues that were critical were determined, the cluster model was formed. Some of the residues used to construct the cluster for DFT calculations are included in their full form, while others are represented as backbones or side chains. The residues were generally cut from the Cα of their proceeding residue properly, ends capped with hydrogen atoms and the end atoms were frozen in their 3-dimensional geometry in the crystal structure of the enzyme during geometry optimization. The formed cluster model included 284 atoms and has a net charge of zero. In the quantum mechanical calculations, all geometry optimizations were executed with the 6-31G(d,p) basis set with the B3LYP functional. To refine the energies, the 6-311+G(2df,2p) basis set was employed with the wB97XD functional for single point energies. The polarizable continuum model (PCM) was selected as the solvent method, and calculations were executed with a dielectric constant of 4.0. The Gaussian 16 program was utilized for quantum mechanical calculations. Following optimization, a comparison was made between the non-optimized and the optimized clusters. An evaluation was conducted to ascertain whether a change in the position of critical residues had occurred. It was determined that no significant changes were present. During the conversion of imine to the amine structure, two hydrogens are needed to convert the double bond to single bond: One of these hydrogens is from a proton donor residue in the vicinity of the active site and the other is the hydride from the NADPH, which acts as a cofactor. The reaction was first considered as concerted and a corresponding mechanism was modelled where these hydrogens were transferred simultaneously. The barrier was found to be quite high and incompatible with the enzyme kinetics. Consequently, the stepwise reaction was examined. The stepwise reaction consists of two steps. The first step is the proton transfer from a proton donor residue. It is postulated that a protic residue in the vicinity of the active site of the enzyme may be involved in the iminium formation step. In S-type IREDs this proton donor residue was reported to be usually Tyr. In the second step of the reaction, hydride transfer takes place from NADPH to the formed iminium ion. In the first step, the proton transfer process was investigated, and the barriers were calculated for both scenarios: with and without the aid of water. The barriers were found to be very small and close to each other. Based on these findings, it was concluded that the initial step of the reaction could occur with or without the assistance of water. The second step was identified as both the rate-determining step, and the stereo selective step. The hydride transfer was subjected to comprehensive analysis through computational modelling. In constructing the input geometries for transition states, the residues and NADPH were held stationary in their crystal structure in the cluster and the substrate was positioned with respect to NADPH in a suitable geometry for a transition state structure for hydride transfer. All possible orientations of the substrate with respect to NADPH was spanned, without ignoring the crystal structure geometry for the substrate. All the transition states and the enzyme-substrate complexes were evaluated according to the position of the substrate nitrogen with respect to Tyr171, the corresponding barriers and the relative energies of the modelled enzyme-substrate complexes. The proximity of the substrate nitrogen to Tyr171, both in proximity and in its absence, has been demonstrated to effect interactions and the reaction barriers. The calculations presented in this study demonstrated that the hydrogen bonding (especially between the C=O oxygen of the amide group of NADPH and the substrate, which created a stabilization in the transition state structures) and steric effects (in particular, the overlap of the substrate isoquinoline and the NADPH's pyridine rings as a result of the hydride attack at Si vs Re faces of the substrate) and their complex interplay between the two are the most significant parameters that determine the stereoselectivity. A review of the literature revealed that Tyr is generally found to be important in terms of stereo selectivity for S-type IREDs, while Asp is found to be important for R-type IREDs. In this study, a mutation was introduced into Tyr171, the proton donor residue, converting it to Asp. Subsequent to this alteration, a recalculation of the barrier energies for the hydride transfer step revealed no discernible change in stereo selectivity. A thorough review of the study indicates that the reaction mechanism was meticulously examined, considering all its stages and potential outcomes. This detailed investigation led to the determination that the reaction occurred in two steps. The calculations performed on the hydride transfer step, which is the step that determines both the stereoselectivity and the rate, have demonstrated a preference for the S-product, in accordance with the experimental study conducted by Li et al. It is noteworthy that no mutation was introduced in the experimental study by Li and colleagues. The mutation study conducted exclusively through the in silico method in this thesis revealed no alteration in stereo selectivity. The present study has focused on the asymmetric synthesis of 1-Methyl-1,2,3,4-tetrahydroisoquinoline, which is of considerable importance to the pharmaceutical industry. The research has created a valuable opportunity to study the phenomenon of stereoselectivity in such a large system with an atomistic approach. It is asserted that, when considered in conjunction with other examples found in the existing literature on the subject, this study will facilitate an in-depth understanding of these systems and the development of approaches that will enable the tuning of selectivity in the desired direction. The comprehension of structure-activity relationships in asymmetric syntheses employing IREDs is anticipated to offer a considerable contribution to the long-term engineering of such processes.