Machine learning guided rational design of a non-heme iron-based lysine dioxygenase improves its total turnover number.

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based machine-learning algorithm with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40% improvement in the TTN (218±3) as compared to WT LDO (TTN = 160±2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies.

Rational Design of Formate Dehydrogenase for Enhanced Thermal Stability and Catalytic Activity in Bioelectrocatalysis.

Formate dehydrogenase can be utilized as a biocatalyst in the bioelectrocatalysis of converting CO2 into formic acid. However, its industrial application has been hindered by limited thermal stability. This study successfully obtained a mutant (D533S/E684I) with enhanced thermal stability and catalytic activity through the rational design of flexible regions. The mutant exhibited a half-life (t1/2) 1.5 times longer than the wild type (WT) at 35 °C, along with a specific enzyme activity 7.46 times higher than that of the WT. Additionally, the catalytic efficiency (kcat/Km value) of the mutant toward the substrate was 2.72 s-1·mM-1, representing a 19.4-fold increase compared to the WT (0.14 s-1·mM-1). Formic acid production reached 53.4 mM through bioelectrocatalysis after 10 h, utilizing the mutant as the biocatalyst. Molecular dynamics simulations and structural analysis were employed to investigate the molecular mechanisms behind the enhanced thermal stability and activity. The displacement of a highly flexible region in the mutant may counteract the stability-activity trade-off. This study proposed a method for improving both thermal stability and activity in enzyme evolution.

Improving the activity of an inulosucrase by rational engineering for the efficient biosynthesis of low-molecular-weight inulin.

Inulin, a widely recognized prebiotic, has diverse applications across various industrial sectors. Although inulin is primarily produced through plant extraction, there is growing interest in enzymatic synthesis as an alternative. The enzymatic production of inulin from sucrose, which yields polymers with degrees of polymerization similar to those of plant-derived inulin, shows potential as a viable replacement for traditional extraction methods. In this study, an inulosucrase from Neobacillus bataviensis was identified, demonstrating a non-processive mechanism specifically tailored for synthesizing inulin with polymerization degrees ranging from 3 to approximately 40. The enzyme exhibited optimal activity at pH 6.5 and 55 °C, efficiently producing inulin with a yield of 50.6%. Ca2+ can improve the activity and thermostability of this enzyme. To enhance catalytic total activity, site-directed and truncated mutagenesis techniques were applied, resulting in the identification of a mutant, T149S, displaying a significant 57% increase in catalytic total activity. Molecular dynamics simulations unveiled that the heightened flexibility observed in three surface regions positively influenced enzymatic activity. This study not only contributes to the theoretical foundation for inulosucrase engineering but also presents a potential avenue for the production of inulin.

Precise Immobilization Strategy Combined with Rational Design to Improve ß-Agarase Stability.

Recently, the orientational immobilization of enzymes has attracted extensive attention. In this study, we report the development of a strategy combined with rational design to achieve precise site-specific covalent immobilization of ß-agarase. We first rationally screened six surface sites that can be mutated to cysteine by combining molecular dynamics simulation and energy calculation. Site-specific immobilization was successfully achieved by Michael addition reaction of mutant enzymes and maleimide-modified magnetic nanoparticles (MAL-MNPs). The enzyme activity retention rate of R66C-MAL-MNPs and K588C-MAL-MNPs was greater than 96%. The thermal deactivation kinetics study revealed that the site-specific immobilization strategy significantly improved the thermal stability of Aga50D, resulting in a substantial increase in its antidenaturation activity at elevated temperatures, and the highest t1/2 of the immobilized mutant enzymes was increased by an impressive 21.25-fold at 40 °C. The immobilized mutant enzymes also showed significantly enhanced tolerance to metal ions and organic reagents. For instance, all of the immobilized enzymes maintained over 90% of their enzymatic activity in the 50% (v/v) acetone/water solution. The present work may pave the way for the design of precisely immobilized enzymes, which can help promote green manufacturing.

Channel Engineering of a Glutamate Exporter.

Mechanosensitive channel MscCG2 is involved in glutamate excretion in most C. glutamicum strains. Improving the excretion efficiency of MscCG2 is beneficial to the production of glutamate. In this study, structure-based rational design was carried out to obtain an improved efflux ability of exporter MscCG2 and its mechanistic advance via two strategies: widening the channel entrance for smoother entry of glutamate and reducing the electronegativity at the entrance of the channels to minimize the rejection of negatively charged glutamate entry. The designed variants were found to enhance glutamate excretion by 2 to 3.3-fold in the early phase and 1.1-fold to 1.5-fold in the late phase of fermentation. The enhanced glutamate excretion was further confirmed by using glutamate toxic analog 4-fluoroglutamate (4-FG) and Glu-Glu peptide uptake and glutamate export assay. Molecular dynamic (MD) simulations revealed that the amino acid substitutions indeed enlarged the channel entrance and reduced the repulsion of glutamate when entering the channel. The finding of this study is important for understanding the underlying structure-function relationship and the mechanism of glutamate secretion to improve glutamate efflux efficiency of glutamate exporter.

A computational mechanistic study on the formation of aryl sulfonyl fluorides via Bi(III) redox-neutral catalysis and further rational design.

Sulfonyl fluorides hold significant importance as highly valued intermediates in chemical biology due to their optimal balance of biocompatibility with both aqueous stability and protein reactivity. The Cornella group introduced a one-pot strategy for synthesizing aryl sulfonyl fluorides via Bi(III) redox-neutral catalysis, which facilitates the transmetallation and direct insertion of SO2 into the BiC(sp2) bond giving the aryl sulfonyl fluorides. We report herein a comprehensive computational investigation of the redox-neutral Bi(III) catalytic mechanism, disclose the critical role of the Bi(III) catalyst and base (i.e., K3PO4), and uncover the origin of SO2 insertion into the Bi(III)C(sp2) bond. The entire catalysis can be characterized via three stages: (i) transmetallation generating the Bi(III)-phenyl intermediate IM3 facilitated by K3PO4. (ii) SO2 insertion into IM3 leading to the formation of Bi(III)-OSOAr intermediate IM5. (iii) IM5 undergoes S(IV)-oxidation yielding the aryl sulfonyl fluoride product 4 and liberating the Bi(III) catalyst for the next catalytic cycle. Each stage is kinetically and thermodynamically feasible. Moreover, we explored other some small molecules (NO2, CO2, H2O, N2O, etc.) insertion reactions mediated by the Bi(III)-complex, and found that NO2 insertions could be easily achieved due to the low insertion barriers (i.e., 17.5 kcal/mol). Based on the detailed mechanistic study, we further rationally designed additional Bi(III) and Sb(III) catalysts, and found that some of which exhibit promising potential for experimental realization due to their low barriers (<16.4 kcal/mol). In this regard, our study contributes significantly to enhancing current Bi(III)-catalytic systems and paving the way for novel Bi(III)-catalyzed aryl sulfonyl fluoride formation reactions.

Genome Mining and Biological Engineering of Type III Borosins from Bacteria.

Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA's self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology.

Theoretical study on the design of allosteric inhibitors of diabetes associated protein PTP1B.

The protein tyrosine phosphatase 1B (PTP1B) is a critical therapeutic target for type 2 diabetes mellitus (T2DM). Many PTP1B inhibitors have been reported, however, most of them lack high specificity and have adverse effects. Designing effective PTP1B inhibitors requires understanding the molecular mechanism of action between inhibitors and PTP1B. To this end, molecular dynamics (MD) simulations and molecular mechanics Poisson Boltzmann Surface Area (MM-PB/SA) methods were used to observe the binding patterns of compounds with similar pentacyclic triterpene parent ring structures but different inhibition abilities. Through structure and energy analysis, we found that the positions of cavities and substituents significantly affect combining capacity. Besides, we constructed a series of potential inhibitor molecules using LUDI and rational drug design methods. The ADMET module of Discovery Studio 2020 was used to predict the properties of these inhibitor molecules. Lastly, we obtained compounds with low toxicity and significant inhibitory activity. The study will contribute to the treatment of T2DM.

Enhancing catalytic efficiency of Bacillus subtilis laccase BsCotA through active site pocket design.

BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 105 M-1 s-1 to 1.18 × 106 M-1 s-1, without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA's ability to accommodate the 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced kcat. Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases' active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. KEY POINTS: • Active site pocket design that enhanced BsCotA laccase efficiency • 7.7-fold improved in catalytic rate • All tested variants retain thermal stability.

Rational design of short-chain dehydrogenase/reductase for enantio-complementary synthesis of chiral 1,2-diols by successive hydroxymethylation and reduction of aldehydes.

Enantiopure 1,2-diols are widely used in the production of pharmaceuticals, cosmetics, and functional materials as essential building blocks or bioactive compounds. Nevertheless, developing a mild, efficient and environmentally friendly biocatalytic route for manufacturing enantiopure 1,2-diols from simple substrate remains a challenge. Here, we designed and realized a step-wise biocatalytic cascade to access chiral 1,2-diols starting from aromatic aldehyde and formaldehyde enabled by a newly mined benzaldehyde lyase from Sphingobium sp. combined with a pair of tailored-made short-chain dehydrogenase/reductase from Pseudomonas monteilii (PmSDR-MuR and PmSDR-MuS) capable of producing (R)- and (S)-1-phenylethane-1,2-diol with 99% ee. The planned biocatalytic cascade could synthesize a series of enantiopure 1,2-diols with a broad scope (16 samples), excellent conversions (94%-99%), and outstanding enantioselectivity (up to 99% ee), making it an effective technique for producing chiral 1,2-diols in a more environmentally friendly and sustainable manner.

From natural to synthetic: Promoter engineering in yeast expression systems.

Synthetic promoters are particularly relevant for application not only in yeast expression systems designed for high-level heterologous protein production but also in other applications such as metabolic engineering, cell biological research, and stage-specific gene expression control. By designing synthetic promoters, researcher can create customized expression systems tailored to specific needs, whether it is maximizing protein production or precisely controlling gene expression at different stages of a process. While recognizing the limitations of endogenous promoters, they also provide important information needed to design synthetic promoters. In this review, emphasis will be placed on some key approaches to identify endogenous, and to generate synthetic promoters in yeast expression systems. It shows the connection between endogenous and synthetic promoters, highlighting how their interplay contributes to promoter development. Furthermore, this review illustrates recent developments in biotechnological advancements and discusses how this field will evolve in order to develop custom-made promoters for diverse applications. This review offers detailed information, explores the transition from endogenous to synthetic promoters, and presents valuable perspectives on the next generation of promoter design strategies.

Rational Design and Modification of NphB for Cannabinoids Biosynthesis.

The rapidly growing field of cannabinoid research is gaining recognition for its impact in neuropsychopharmacology and mood regulation. However, prenyltransferase (NphB) (a key enzyme in cannabinoid precursor synthesis) still needs significant improvement in order to be usable in large-scale industrial applications due to low activity and limited product range. By rational design and high-throughput screening, NphB's catalytic efficiency and product diversity have been markedly enhanced, enabling direct production of a range of cannabinoids, without the need for traditional enzymatic conversions, thus broadening the production scope of cannabinoids, including cannabigerol (CBG), cannabigerolic acid (CBGA), cannabigerovarin (CBGV), and cannabigerovarinic acid (CBGVA). Notably, the W3 mutant achieved a 10.6-fold increase in CBG yield and exhibited a 10.3- and 20.8-fold enhancement in catalytic efficiency for CBGA and CBGV production, respectively. The W4 mutant also displayed an 9.3-fold increase in CBGVA activity. Molecular dynamics simulations revealed that strategic reconfiguration of the active site's hydrogen bonding network, disulfide bond formation, and enhanced hydrophobic interactions are pivotal for the improved synthetic efficiency of these NphB mutants. Our findings advance the understanding of enzyme optimization for cannabinoid synthesis and lay a foundation for the industrial-scale production of these valuable compounds.

Enhancing the imidase activity of BpIH toward 3-isobutyl glutarimide via semi-rational design.

(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6-9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (kcat/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein's affinity for the substrate and improving catalytic efficiency. KEY POINTS: • 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution • Semi-rational design of BpIH based on conservativity with homologous enzymes • Mutant with enzyme activity of sixfold and product ee value of 99.9.

Rational Design of Untranslated Regions to Enhance Gene Expression.

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5' UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5' and 3' UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5' UTR is 25-30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, "Corn", in the 3' UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5' UTRs and 3' UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3' UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

Functional and Mechanistic Dissection of Protein Glutaminase PG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar Proteins.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65 °C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Elucidating acceptance and clinical indications to support the rational design of drug-eluting contact lenses.

The advent of drug-eluting contact lenses (DECLs) has opened up new avenues for the treatment of eye diseases. DECLs is expected to partially overcome the shortcomings of eye drops due to single-dose packaging, accurate dosing, prolonged drug elution behavior, and simplified dosing procedures. Currently, a significant proportion of the DECLs design effort has been directed towards enhancing the compatibility of contact lenses with drugs. The appropriate elution time for the drug remains unclear. Additionally, it is ambiguous for which ophthalmic diseases DECLs offers the greatest therapeutic advantage. To rationally design DECLs in practice, it is necessary to understand the acceptance of DECLs by patients and practitioners and to clarify the indications for DECLs. This review will first focus on the acceptance of DECLs by different patients and practitioners and discuss the factors that influence its acceptance. Secondly, this review presents an overview of the current effectiveness of DECLs treatments in animals and in the clinical phase, with a particular focus on the suitability of DECLs for the treatment of ophthalmic diseases. Overall, patients and practitioners expressed positive attitudes towards DECLs. However, this is related to factors such as DECLs' treatment cycle, safety, and price. In addition, DECLs has good application prospects for ocular wound healing, postoperative management, and treatment of contact lenses-related complications. Furthermore, chronic diseases such as glaucoma that necessitate long-term medication and intraocular diseases that require implants or injections represent additional potential applications for DECLs. It is hoped that this review will facilitate a deeper understanding of DECLs acceptance and indications, thereby supporting the rational design of DECLs. At the same time, this review provides a reference for the design of other drug-device combination products.

The influence of amphiphilic quaternary ammonium palmitoyl glycol chitosan (GCPQ) polymer composition on oil-loaded nanocapsule architecture.

HYPOTHESIS: Predicting the exact nature of the self-assembly of amphiphilic molecules into supramolecular structures is of utmost importance for a variety of applications, but this is a challenge for nanotechnology. The amphiphilic drug delivery polymer-N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) self-assembles in aqueous media to form nanoparticles. EXPERIMENT: This work aimed to develop a systematic predictive mathematical model on the eventual nature of oil-loaded GCPQ-nanoparticles and to determine the main independent variables that affect their nanoarchitecture following self-assembly. GCPQ polymers were produced with varying degree of palmitoylation (DP, 5.7-23.8 mol%), degree of quaternization (DQ, 7.2-22.7 mol%), and molecular weight (MW, 11.2-44.2 kDa) and their critical hydrophilic-lipophilic balance (cHLB) optimized to produce oil-loaded nanocapsules. FINDINGS: Non-linear mathematical models (Particle size (nm) = 466.05 - 5.64DP - 6.52DQ + 0.13DQ2 - 0.03 MW2 - 14.48cHLB + 0.48cHLB2) were derived to predict the nanoparticle sizes (R2 = 0.998, R2adj = 0.995). Smaller nanoparticle sizes (148-157 nm) were obtained at high DP, DQ, and cHLB values, in which DP was the main independent variable responsible for nanoparticle size. Single or multiple-oil cores with small particles stabilizing polymer shells could be observed depending on the oil volume. Nanoparticle architectures, especially the nature of the oil-core(s), were driven by the DP, DQ, cHLB, and oil concentration. Here, we have developed a predictive model that may be applied to understand the nanoarchitecture of oil-loaded GCPQ-nanoparticles.

Rational design of Ag-doped MnFe2O4/HNTs for peroxidase-mimicking activity and colorimetric sensing of uric acid in human serum.

Mimicking enzyme have significantly advanced sensing assays by replicating native enzyme functions, yet achieving both high catalytic activity and easy recyclability remains a challenge. In this study, Ag-doped MnFe2O4/halloysite nanotubes (HNTs) were rationally designed as a novel nanozyme by depositing in-situ Ag and MnFe2O4 nanoparticles onto HNTs. The resulting nanocomposite exhibited excellent peroxidase-like activity along with magnetic properties. Leveraging these features, a highly efficient and sensitive colorimetric system for detecting uric acid (UA) was developed. The Ag-doped MnFe2O4/HNTs catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of H2O2, causing a color change from colorless to blue. The system showed a linear absorbance response to UA concentrations ranging from 1 to 20 µM, with a detection limit of 59 nM. Mechanistic studies revealed that reactive oxygen species intermediates (1O2) were generated through the decomposition of H2O2, leading to peroxidase-like activity in the Ag-doped MnFe2O4/HNTs. The assay was successfully applied to detect UA in human serum with recoveries over 99.68 %. This study indicates the successful application of Ag-doped MnFe2O4/HNTs for colorimetric UA detection in human serum. This research introduces a novel approach for designing recyclable, high-performance mimicking enzyme and establishes an effective colorimetric sensing platform for UA detection in human serum.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Rational Design of Non-Covalent Imprinted Polymers Based on the Combination of Molecular Dynamics Simulation and Quantum Mechanics Calculations.

Molecular imprinting is a promising approach for developing polymeric materials as artificial receptors. However, only a few types of molecularly imprinted polymers (MIPs) are commercially available, and most research on MIPS is still in the experimental phase. The significant limitation has been a challenge for screening imprinting systems, particularly for weak functional target molecules. Herein, a combined method of quantum mechanics (QM) computations and molecular dynamics (MD) simulations was employed to screen an appropriate 2,4-dichlorophenoxyacetic acid (2,4-D) imprinting system. QM calculations were performed using the Gaussian 09 software. MD simulations were conducted using the Gromacs2018.8 software suite. The QM computation results were consistent with those of the MD simulations. In the MD simulations, a realistic model of the 'actual' pre-polymerisation mixture was obtained by introducing numerous components in the simulations to thoroughly investigate all non-covalent interactions during imprinting. This study systematically examined MIP systems using computer simulations and established a theoretical prediction model for the affinity and selectivity of MIPs. The combined method of QM computations and MD simulations provides a robust foundation for the rational design of MIPs.