Veronika Hahn

Synthesis of model morpholine derivatives with biological activities by laccase-catalysed reactions.

The efficient enzyme‐catalysed reaction of morpholines as model structures for bioactive compounds with para‐dihydroxylated aromatic systems was carried out using the oxidoreductase laccase and atmospheric oxygen to produce eight novel morpholine‐substituted aromatics. The laccase of Myceliophthora thermophila was used for cross‐linking morpholines containing primary or secondary amino groups with para‐dihydroxylated laccase substrates. We demonstrate that not only primary amino groups, but also secondary amino groups, are able to couple with para‐dihydroxylated aromatic systems in laccase‐catalysed reactions. The resulting model products (yields up to 80%) were isolated, structurally characterized and tested for their antibacterial, antifungal and cytotoxic activities. Four of the eight products showed low to moderate growth inhibition against several Gram‐positive and ‐negative bacterial strains and against the yeasts Candida maltosa and Candida albicans. The antibacterial and antifungal activities were determined by an agar disc diffusion test and a modified method according to the EUCAST discussion document E.Dis 7.1 [Rodríguez‐Tudela et al. (2003) Clin. Microbiol. Infect. 9, i–viii] for the evaluation of MIC (minimal inhibitory concentration). Differences in cytotoxicity against the human urinary bladder carcinoma cell line 5637 are discussed.

Laccase-catalyzed carbon–nitrogen bond formation: coupling and derivatization of unprotected l-phenylalanine with different para-hydroquinones

Unprotected l-phenylalanine was derivatized by an innovative enzymatic method by means of laccases from Pycnoporus cinnabarinus and Myceliophthora thermophila. During the incubation of l-phenylalanine with para-hydroquinones using laccase as biocatalyst, one or two main products were formed. Dependent on the substitution grade of the hydroquinones mono- and diaminated products were detected. Differences of the used laccases are discussed. The described reactions are of interest for the derivatization of amino acids and a synthesis of pharmacological-active amino acid structures in the field of white biotechnology.

Enzymatic cyclizations using laccases: Multiple bond formation between dihydroxybenzoic acid derivatives and aromatic amines

Oxidative C–N bond formation followed by cyclization of dihydroxybenzoic acid derivatives with aromatic and heteroaromatic amines was catalyzed in the presence of oxygen by laccases [E.C. 1.10.3.2] from the white rot fungi Pycnoporus cinnabarinus and Myceliophthora thermophila. The laccase-catalyzed formation of cycloheptenes, cyclooctenes, diazaspiro cyclohexenes, and phenazines was investigated for the first time with regard to the ring size and substituents of the aromatic amines as well as to the substitution patterns of the substrates. Differences to C–N bond formation without cyclization are discussed.

Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]10, dihydroxylated aromatics and laccase are discussed.

Derivatization of the azole 1‐aminobenzotriazole using laccase of Pycnoporus cinnabarinus and Myceliophthora thermophila: influence of methanol on the reaction and biological evaluation of the derivatives

The laccases of Pycnoporus cinnabarinus and Myceliophthora thermophila are extracellular enzymes with high protein stability. They were used for the ‘one pot’ synthesis of azole derivatives from 1‐aminobenzotriazole together with the p‐dihydroxylated laccase substrates 2,5‐dihydroxybenzoic acid methyl ester and 2,5‐dihydroxybenzoic acid ethyl ester. The reactions yielded heteromolecular dimers (in yields of up to 34%). Methanol was used as the co‐solvent to determine the influence of solvent concentration on the course of reaction. The resulting products were isolated, structurally characterized and tested for their antibacterial, antifungal and cytotoxic activities. The products showed low antimicrobial activity and low cytotoxicity compared with commercial available standard compounds but these variables exceeded those of the initial reactants used for the synthesis. In addition to the synthesis of heteromolecular dimers, oligomers were formed and structurally characterized by LC/MS (liquid chromatography/MS).

Metabolism of alkenes and ketones by Candida maltosa and related yeasts

Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known. In this study we show the degradation of the aliphatic ketone dodecane-2-one by Candida maltosa and the related yeasts Candida tropicalis, Candida catenulata and Candida albicans as well as Trichosporon asahii and Yarrowia lipolytica. One pathway is initiated by the formation of decyl acetate, resulting from a Baeyer-Villiger-oxidation of this ketone. Beyond this, an initial reduction to dodecane-2-ol by a keto reductase was clearly shown. In addition, two different ways to metabolize dodec-1-ene were proposed. One involved the formation of dodecane-2-one and the other one a conversion leading to carboxylic and dicarboxylic acids. Furthermore the induction of ketone degrading enzymes by dodecane-2-one and dodec-1-ene was shown. Interestingly, with dodecane no subterminal degradation products were detected and it did not induce any enzymes to convert dodecane-2-one.

Biotransformation of 4-sec-butylphenol by Gram-positive bacteria of the genera Mycobacterium and Nocardia including modifications on the alkyl chain and the hydroxyl group

The environmental pollutant 4-sec-butylphenol (4-sec-BP) which possesses estrogenic properties was transformed by the aerobic Gram-positive bacteria Mycobacterium neoaurum and Nocardia cyriacigeorgica into three main products (P1–P3) which were isolated and structurally characterized in detail. Two of them were products of a process resembling anaerobic metabolism of alkylphenols based on modifications of the alkyl side chain of 4-sec-BP. The first product (P1) was identified as 4-(2-hydroxy-1-methylpropyl)-phenol. The second product P2 was isolated as a mixture of at least four structures which could be identified as I 4-sec-butylidenecyclohexa-2,5-dienone; II 4-(1-methylenepropyl)-phenol; III 4-(1-methylpropenyl)-phenol; and IV 4-(1-methylallyl)-phenol. In contrast to P1 and P2, the third product (P3) resulted from a modification of the hydroxyl group of 4-sec-BP. This product was only formed by M. neoaurum and was identified as the glucoside conjugate 4-sec-butylphenol-α-d-glucopyranoside. Since in general, fungi synthesize sugar conjugates to detoxify hazardous pollutants, the formation of this conjugate is a peculiarity of M. neoaurum. Thus, altogether, six products were formed from 4-sec-BP and different transformation pathways are introduced. The hydroxylating and glucosylating capacity of the characterized bacteria open up applications in environmental protection.

DIN SPEC 91315: A First Attempt to Implement Mandatory Test Protocols for the Characterization of Plasma Medical Devices

The continuous development of novel plasma sources for therapeutic applications is leading to a growing demand for test protocols that allow a standardized physical characterization and the estimation of biological efficacy. These data are mandatory for estimating the biological performance of a given plasma source, and to serve as a tool that enables a rough comparison of different plasma sources. Additionally, data can be used for the technical improvement of plasma devices, and regarded as a preliminary stage for the licensing of such sources as medical devices. Thus, a DIN SPEC 91315:2014-06 was implemented to characterize plasma sources in terms of their biological performance including risk and safety potential for the therapeutic use of plasma in medicine.