Research projects

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An investigation regarding biochemical synthesis of styrene and its derivatives

The versatility of the utilization of polymeric materials (i. e. packaging, gaskets, construction materials, tires) makes it hard to imagine todays' world entirely without plastics. Currently used production methods of polymers depend on the processing of distillation products from petroleum in energetically costly chemical reactions, leading to the intensified exploitation of non-renewable natural resources. In order to tackle this issue, there have been attempts to develop an environmentally friendly method to obtain one of the mostly constituent of polymeric materials - styrene. The main advantage of this novel method was the utilization of renewable resources (glucose) as the main reagent in the synthesis. The key role in this process belong to a bacterial enzyme UbiD and its fungal derivative Fdc1 - in the presence of a cofactor in a form of prenylated flavine (prFMN), they catalyze a reversible decarboxylation of aromatic compounds, making it possible to obtain a monomer accordingly to green chemistry rules. Interestingly, so far performed investigations indicate that this reaction is the first known enzymatic reaction that undergoes accordingly to 1,3 - dipolar cycloaddition mechanism, greatly broadening the knowledge regarding the catalysis in nature. The goal of this project is obtaining a detailed, atomic-scale knowledge about two enzymatic reactions: the decarboxylation of aromatic compounds that lead to the formation of styrene or its derivatives and the forming of prFMN, which requires the formation of bond between flavin and a dimethylallyl moiety in the presence of UbiX enzyme. Computational chemistry tools will be employed to shed light on those processes.

Revisiting the approaches for isotope effects prediction in condensed phase

The objective of the project is to develop the best theoretical framework to predict thermal rate constants in condensed phase (e.g. in homogenous solution). KIEs on selected model reactions are measured and calculated.
For precise heavy-atom isotope analysis (carbon, chlorine, and bromine) a recently developed method based on gas chromatography - isotope ratio mass spectrometry (GC-IRMS) is used in the collaboration with dr. Faina Gelman from the Geological Survey of Israel.  It is combined with modern electronic structure calculations together with dynamical methods used to reliably describe and interpret the experimental findings. Thorough test of available computational methods and tools at different stages of generating necessary input data to calculate isotope effects is carried out.

Rational Drug Design

The emergence of multi-drug resistant strains is one of the most challenging problems of modern pharmacology that prompts interdisciplinary studies in quest for new antibiotics. We participate in such studies in collaboration with the Medical University of Lublin. Within these studies a few families of heterocyclic molecules are evaluated for their biological activity, and their physico-chemical properties evaluated experimentally and theoretically are used to guide future rational synthesis of effective antibacterials. Quantum-chemical calculations are used to find a reasonably robust and inexpensive theory level that allows SAR-like descriptors such as geometries, partial atomic charges, ionization potentials, electron affinities, HOMO, LUMO, HOMO-LUMO gaps, logP, hydration energy, refractivity, and polarizability to be calculated for large number of compounds. Furthermore, in order to understand interactions of the synthesized inhibitors with the active site of target enzymes (such as for example topoisomerase and HIV reverse transcriptase) docking, MD, and QM/MM calculations are carried out. We also try to employ isotope effects, and in particular binding isotope effects in these studies.

The increasing resistance of pathogens to the drugs used induces constant search for new biologically active substances that could effectively replace them. The process of searching for such substances is extremely difficult and expensive, which is why it is increasingly preceded by in-depth scientific research, the purpose of which is to provide information on the mechanism of their operation to limit the range of searches to a group of highly promising compounds.

A large part of pharmaceuticals are compounds blocking the action of specific enzymes (so-called inhibitors), which consequently lead to inhibition of pathogen development. Therefore, research aimed at the development of new drugs focuses on the detailed recognition of the mechanisms of enzymatic reactions and understanding the interactions between the drug molecule and the enzyme. One of the most subtle kinetic methods of studying the mechanisms of enzymatic reactions is the observation of the impact on the dynamics of changes in the isotopic composition - these are the so-called isotopic effects. It is believed that the extraordinary efficiency of enzymes results mainly from the stabilization of the structure with the lowest energy necessary to react (this structure is referred to as the transition state). Interactions between the transition state and the place where the enzyme reacts in the so-called active site are responsible for stabilization. In recent years, however, frequently the second, no less important, source of enzymatic catalysis is suggested - the destabilization of the inhibitor as a result of binding to the enzyme. However, the use of isotopic effects to study the enzyme-inhibitor interactions has been significantly limited so far because the isotopic effects associated with this process are usually small and their measurement is difficult and expensive.

Our research has focused on the use of isotope effects on the inhibitor-enzyme binding for a detailed understanding of the interactions between them and on this basis for the rational design of compounds with high biological activity. As a model enzyme, we chose HIV-1 reverse transcriptase, one of the three enzymes responsible for the proliferation of this virus. This enzyme shows a high tendency to mutation, which makes it necessary to constantly search for new inhibitors.

In the initial phase, based on the known structures of this enzyme, we determined the binding strength of a given compound with the enzyme (this process is called docking) and then we performed a structure-activity relationship study in the tested class of compounds that allowed us to indicate the directions of beneficial structural changes. The next step was to perform theoretical calculations proposed on the basis of the above tests, which allowed to determine the molecular basis of the interaction between these compounds and the enzyme and to calculate the expected values ​​of isotopic effects. During the study it turned out that these inhibitors can bind to reverse transcriptase in more than one place. We performed theoretical calculations of the isotopic effects values ​​associated with these alternative binding sites and showed that the isotopic effect of the oxygen atom of the carbonyl group can allow to experimentally determine where the enzyme actually binds the inhibitor. In the final step, we developed a method to determine the experimental values of this isotope effect.

Our studies have demonstrated the usefulness of isotopic effects to determine the binding site of the inhibitor to the enzyme and the nature of interactions determining the binding strength of molecules by enzymes. In addition, we have synthesized a non-toxic inhibitor that exceeds in biological activity the compounds currently used in clinical practice and is characterized by significantly higher solubility, due to which it could be used in much lower doses. This compound is an excellent starting point for further exploration of an even more effective drugs for use in anti AIDS therapy.

Degradation of Nitroaromatics

Within the studies of environmental pollutants we work on isotopic fractionation of nitroaromatic compounds. These studies are carried out with a group at Eawag, Zurich which delivers experimental measurements of deuterium, carbon and nitrogen signatures which are predicted theoretically in our laboratory for the relevant chemical and enzyme-catalyzed processes.

Polish-Swiss Research Programme

CSI ITN project

Dehalogenations

Chlorine atoms present in a proportionally big amount in polychlorinated hydrocarbons are suspected to be a source of toxicity, carcinogenicity and other negative properties of these compounds. Taking into account the vast number of different sources of halogenated compounds, Nature has developed several mechanisms in order to reduce the levels of these toxic molecules. Many groups of enzymes are capable of degrading the halogenated compounds. For example, hydrolytic dehalogenases replace the halogen with a hydroxyl group, reductive dehalogenases replace the halogen with a hydrogen, and oxidative dehalogenases exchange the halogen for oxygen with concomitant substrate oxidation.

Our group has been exploring different aspects and faces of dehalogenation process since late 90s. The earlier studies comprise a number of hydrolytic dehalogenases and some model systems. The most important contributions have been collected and presented in the following publications: P. Paneth, Acc. Chem. Res. 2003; Y. Fang et al. Chem. Eur. J. 2003; A. Dybala-Defratyka et al., J. Org. Chem. 2004; P. Paneth in Isotope Effects in Chemistry and Biology, Chapter 35; A. Kohen, H.H. Limbach, Eds.; CRC Press, 2006. Most recently we have turned our attention to other mechanisms of halogen substituents removal.

Oxidative dehalogenation catalyzed by selected peroxidases

Two dehalogenating oxidoreductases and their activity toward chlorinated phenols are being explored with a use of experimentally and theoretically predicted chlorine kinetic isotope effects. The obtained results so far allowed us to make a working hypothesis that the peroxidase activity of the studied enzymes is mostly focused on a transformation of the toxic halogenated substrate into a cationic intermediate form which can easily expel a halogen substitutent to form a lesser toxic compound. For both HRP and DHP, the dehalogenation reaction takes place at an external site, and the QM/MM calculations suggest that the HRP- and DHP- catalyzed dehalogenation reactions happen in a very similar fashion as the same reaction in solution without the enzyme (MSHE, Poland).

Abiotic and biotic hydrolysis of s-triazines

s-Triazine anthropogenic compounds were introduced into the environment more than half century ago as dyes, resins and herbicides. Especially herbicides based on s-triazine ring have been used worldwide by direct application either to soils or plants. Their widespread presence and environmental accumulation leading to certain dysfunctions in vertebrate species or toxic conditions to other species present in the shared contaminated area have emerged many laboratory studies aiming at their biodegradation. In a collaboration with prof. Larry Wackett from University of Minnesota we are planning to provide a detailed description of transformation taking place in the polluted environment during herbicides biodegradation. The proposed investigation of TrzN and AtzA enzymes, their mutants and their catalytic activities with both terbutylazine and atrazine using an interdisciplinary approach should help us to elucidate underlying mechanism of reaction catalyzed by these enzymes and understand in depth transformation processes occurring at the contaminated sites (NCN, Poland 2011-2014).

Hexachlorocyclohexanes dehydrochlorination catalyzed by Lin enzymes

Within CSI-ITN project we attempt to theoretically predict isotopic fractionation on dehalogenation of HCH isomers catalyzed by LinA and LinB enzymes. Apart from providing detailed mechanistic insights we hope to explore whether chlorine isotope effects can be used as a mechanistic indicator for these systems and how the predicted values would fit to the existing data on chlorine fractionation accompanying elimination reaction.


Reductive dehalogenation of chloroethylenes

One of approaches used for removing chlorine substituents is reductive dehalogenation. Among metal-mediated reductive dehalogenation cobalt species have gathered special attention due to its wide presence in reductive dehalogenases - enzymes responsible for catalyzing degradation of chlorinated organic compounds during in situ biodegradation. Such systems contain cobalt complexes in a form of corrinoid-like molecule.

Due to their widespread use and top position among the most common groundwater contaminants worldwide chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) and the studies regarding their assessment and attenuation have received a great interest. Despite extensive efforts and numerous attempts directed to detailed description of possible degradation pathways of CEs a mechanism that would satisfactorily explain all experimental observations collected so far has not been proposed yet.

In order to shed some additional light onto this ongoing mechanistic debate theoretical investigation of carbon and chlorine isotope fractionation has been undertaken. Different operative pathways for reductive degradation of PCE in the presence of cobalt species have been explored with the use of quantum mechanical calculations (isoSoil).



Isotopic Food Authentification

Isotopic signature of materials can be employed in studies of their age, geographic origin, manufacturer etc. One of a very promising applications of isotopic analysis is food authentication. In collaboration with University of Nantes, France we are developing new methods of authentication of foodstuff and plan to extend it to drugs and pharmaceuticals. We have pending Polish patent application for isotopic authentication of coffee beans by carbon isotopic analysis.

Theoretical Calculations of Isotope Effects

Isoeff, one of the most popular programs for recalculation of isotope effects from results of quantum-calculation has been developed in our laboratory. It can also be used for analysis of the influence of small changes in force constants on isotope effects. The original program has been described in
„ISOEFF98. A Program for Studies of Isotope Effects Using Hessian Modifications” V.Anisimov, P.Paneth J. Mathem. Chem. 26, 75-86 (1999)
and is distributed upon request to authors. It can also be used interactively at app.isosoil.eu

Isotopic fractionation data base


An exhaustive data base of isotopic fractionations has been compiled in our laboratory and is being constantly updated. Selected entries are available at app.isosoil.eu