From Analysis to Synthesis

Sustainability in chemistry and the concept of green chemistry has become an important topic in modern synthetic applications over the last two decades. Nowadays, the chemical industry checks their production processes with respect to sustainability, and several companies are seeking novel and sustainable solutions in synthesis.

One important and widely used approach in this development is the application of biocatalyzed reactions to synthesize organic compounds. The biocatalysts are easily available from microbial growth and combine a couple of properties that are important for performing sustainable chemical synthesis. Notably, they distinctly reduce activation energy in the catalyzed reactions and allow product formation with an excellent chemo- and stereo-selectivity at ambient temperature. Ordinary water is the solvent of choice in these transformations, and consequently the synthetic processes do not lead to formation of problematic side products, avoiding formation of excessive waste.

However, biocatalysts evolved through a long process of mutations before human civilization came to our planet, and so enzymes are not at all optimized for biosynthetic applications in chemical synthesis of artificial products. The development of usable biocatalyzed synthetic processes demands the selection of a suitable enzyme as well as optimization of the molecular interaction between enzymes, co-factors, substrates and the desired product. Indeed, we must apply a complex combination of wet chemical synthesis and analysis in addition to in silico simulation to guide such a process. During development, it is necessary to gain detailed analytical information about the catalytic process at a molecular level, including kinetics of the biotransformation as well as concentrations and structural data of all molecules involved in the catalytic event. Furthermore, detailed knowledge of the binding processes is indispensable.

Such data can be recorded using several labor-intensive and costly analyses. However, NMR spectroscopy is an alternative versatile analytical method that allows fast and accurate determination of reliable information about catalysis at a molecular level. In particular, 1H-NMR is good at monitoring biocatalyzed reactions, as the proton is ubiquitous in organic molecules and has a high sensitivity of the NMR active nuclei. To that end, on-line monitoring can be performed to measure spectra in situ at any stage of enzyme catalyzed reactions without sampling the reaction mixture, which makes manipulating the biotransformation much more accessible. All the data about molecular structures and compound concentrations are taken directly from the spectra. And H2O or HDO signals can be managed using known pulse sequences. Most importantly, the results gained are not influenced by extraction, separation, chromatography or derivatization caused by additional sample preparation and separate analysis.

In addition, STD NMR spectroscopy can be applied as a time and labor efficient technique to determine complete mapping of ligand/enzyme binding in a global as well as in-site specific way. The determined binding patterns allow analysis of the specific substrate, intermediate and product binding to enzymes on a molecular basis. And a concomitant specific co-substrate and co-enzyme binding can be determined, including detailed analysis of ternary enzyme/co-enzyme/substrate complexes. All of the resulting binding patterns can be studied during catalysis as well as under binding-only conditions, meaning that this NMR-based analytical technique is particularly suited to performing a comparison of wild type and corresponding mutant enzyme binding.

In summary, the combination of both STD NMR and in situ 1H NMR techniques enables comprehensive studies of mechanistic details of biocatalyzed reactions, which can be supported and visualized by in silico molecular docking. Indeed, these powerful techniques facilitate sustainable and target-oriented optimization of synthetic approaches that allow us to benefit from – and reduce our impact on – the natural resources around us.