Justas Vaitekūnas

The development of safer and more efficient synthetic approaches to insensitive high-energy materials remains important because conventional synthesis may require hazardous reagents and relatively long reaction times. In this study, we aimed to develop improved methods for the synthesis of picrylamino derivatives and evaluate their applicability to the preparation of high-energy materials. Microwave-assisted synthesis was applied to previously known reactions and compared with conventional synthetic approaches. The purity of the synthesized compounds was confirmed by LC-MS analysis, while their impact sensitivity was qualitatively evaluated by bullet impact tests. In addition, the structure of 3-picrylamino-1H-1,2,4-triazole was analyzed to provide insight into its stability. The proposed microwave-assisted approaches substantially reduced reaction times and enabled the use of less hazardous reagents while providing products with good yields and high purity. One of the investigated methods is particularly suitable for the synthesis of 3-picrylamino-1H-1,2,4-triazole because of its slightly higher yield and reduced consumption of harmful picryl chloride; moreover, substitutions in the benzene or triazole ring can be introduced without additional synthetic steps, and the resulting compounds exhibit improved stability and/or detonation performance. These results demonstrate that the proposed methods provide a practical approach to the synthesis of insensitive high-energy materials. The developed methodology may facilitate the preparation and further development of safer, more stable, and high-performance energetic materials.

In this paper, we present new methods for the synthesis of picrylamino derivatives. Improvements over existing methods are demonstrated, including significantly reduced reaction times and the use of less hazardous reagents. One of these methods is recommended for the synthesis of 3-picrylamino-1H-1,2,4-triazole due to its slightly higher yield and reduced use of harmful picryl chloride. The proposed methods provide a practical approach for producing insensitive high-energy materials with good yields and high purity. The purity of the synthesized materials was confirmed by LC-MS or FT-IR spectra analysis. It is also shown that introducing substitutions into the benzene or triazole ring does not require additional synthetic steps, while the resulting compounds exhibit improved stability and/or detonation performance. The proposed methods provide a practical approach for producing insensitive high-energy materials with good yields and high purity. The stability of the picrylamino derivatives was confirmed by bullet impact tests. In addition, the structure of picrylamino-1,2,4-triazole is analyzed to provide insight into its stability. The results establish a practical and scalable approach to the development of safer, more stable, and high-performance energetic materials.

A plethora of di‐ and oligosaccharides isolated from the natural sources are used in food and pharmaceutical industry. An enzymatic hydrolysis of fungal cell wall β‐glucans is a good alternative to produce the desired oligosaccharides with different functionalities, such as the flavour enhancer gentiobiose. We have previously identified PsGly30A as a potential yeast cell wall degrading β‐1,6‐glycosidase. The aim of this study is to characterise the PsGly30A enzyme, a member of the GH30 family, and to evaluate its suitability for the production of gentiobiose from β‐1,6‐glucans. An endo‐β‐1,6‐glucanase PsGly30A encoding gene from Paenibacillus sp. GKG has been cloned and overexpressed in Escherichia coli. The recombinant enzyme has been active towards pustulan and yeast β‐glucan, but not on laminarin from the Laminaria digitata, confirming the endo‐β‐1,6‐glucanase mode of action. The PsGly30A shows the highest activity at pH 5.5 and 50 °C. The specific activity of PsGly30A on pustulan (1262±82 U/mg) is among the highest reported for GH30 β‐1,6‐glycosidases. Moreover, gentiobiose is the major reaction product when pustulan, yeast β‐glucan or yeast cell walls have been used as a substrate. Therefore, PsGly30A is a promising catalyst for valorisation of the yeast‐related by‐products.

Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an amino donor to a keto substrate. ATs are promising biocatalysts that are replacing traditional chemical routes for the production of chiral amines. In this study, an in silico-screening of a metagenomic library isolated from the Curonian Lagoon identified 11 full-length fold type IV aminotransferases that were successfully expressed and used for substrate profiling. Three of them (AT-872, AT-1132, and AT-4421) were active toward (R)-methylbenzylamine. Purified proteins showed activity with L- and D-amino acids and various aromatic compounds such as (R)-1-aminotetraline. AT-872 and AT-1132 exhibited thermostability and retained about 55% and 80% of their activities, respectively, even after 24 h of incubation at 50 °C. Active site modeling revealed that AT-872 and AT-4421 have an unusual active site environment similar to the AT of Haliscomenobacter hydrossis, while AT-1132 appeared to be structurally related to the AT from thermophilic archaea Geoglobus acetivorans. Thus, we have identified and characterized PLP fold type IV ATs that were active toward both amino acids and a variety of (R)-amines.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2′-deoxycytidine to uridine and 2′-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N ⁴ -acyl-cytidines, N ⁴ -alkyl-cytidines, and N ⁴ -alkyloxycarbonyl-cytidines, and S ⁴ -alkylthio-uridines and O ⁴ -alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.

Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-β-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli. The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear β-1,3-glucans—curdlan and pachyman but had no effect on hydrolysis of β-1,3/β1,6-branched glucans—laminarin and yeast β-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases.

We recently discovered a [Fe‐S]‐containing protein with in vivo thiouracil desulfidase activity, dubbed TudS. The crystal structure of TudS refined at 1.5 Å resolution is reported; it harbors a [4Fe‐4S] cluster bound by three cysteines only. Incubation of TudS crystals with 4‐thiouracil trapped the cluster with a hydrosulfide ligand bound to the fourth non‐protein‐bonded iron, as established by the sulfur anomalous signal. This indicates that a [4Fe‐5S] state of the cluster is a catalytic intermediate in the desulfuration reaction. Structural data and site‐directed mutagenesis indicate that a water molecule is located next to the hydrosulfide ligand and to two catalytically important residues, Ser101 and Glu45. This information, together with modeling studies allow us to propose a mechanism for the unprecedented non‐redox enzymatic desulfuration of thiouracil, in which a [4Fe‐4S] cluster binds and activates the sulfur atom of the substrate.

The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-bornepyrgene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism ofN-heterocyclic compounds in nature.

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichia coli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-(N-formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathway.