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The rapidly increasing problem of antimicrobial-drug resistance requires the development of new antimicrobial agents. The laccase-catalyzed amination of dihydroxy aromatics is a new and promising method to enlarge the range of currently available antibiotics. Thirty-eight potential 1,2- and 1,4-hydroquinoid laccase substrates were screened for their antibacterial and cytotoxic activity to select the best substrates for laccase-catalyzed coupling reaction resulting in potent antibacterial derivatives. As a result, methyl-1,4-hydroquinone and 2,3-dimethyl-1,4-hydroquinone were used as parent compounds and 14 novel cephalosporins, penicillins, and carbacephems were synthesized by amination with amino-β-lactam structures. All purified products were stable in aqueous buffer and resistant to the action of β-lactamases, and in agar diffusion and broth micro-dilution assays, they inhibited the growth of several Gram-positive bacterial strains including multidrug-resistant Staphylococcus aureus and Enterococci. Their in vivo activity and cytotoxicity in a Staphylococcus-infected, immune-suppressed mouse model are discussed.

Seven novel β-lactam antibiotics with activities against Gram-positive bacterial strains, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, were synthesized by amination of 2,5-dihydroxyphenylacetic acid in usable yields (30-60%). These products protected mice against an infection with S. aureus lethal to the control animals. The results show the usefulness of laccase for the synthesis of potential new antibiotics, in addition to the interdependence of the laccase substrates, the amino coupling partners, and the product formation, yield, and activity. The syntheses of β-lactam antibiotics with 2,5-dihydroxyaromatic acid substructures (para-substituted) are then compared with those of 3,4-dihydroxyaromatic acid substructures (ortho-substituted). Para-substituted laccase substrates were better reaction partners in these syntheses than ortho-substituted compounds., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

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 epsilon-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.

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).

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.

Novel cephalosporins, penicillins, and carbacephems were synthesized by amination of catechols with amino-beta-lactams like cefadroxil, amoxicillin, ampicillin and the structurally related carbacephem loracarbef using laccase from Trametes sp. All isolated monoaminated products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant Enterococci. Observed differences in the cytotoxicity and in vivo activity in a "Staphylococcus-infected, immune suppressed mouse" model are discussed.

Corollosporine isolated from the marine fungus Corollospora maritima and N-analogous corollosporines are antimicrobial substances. Owing to the basic structure of the N-analogous corollosporines, they have become an attractive target for laccase-catalyzed derivatisation. In this regard we report on the straightforward laccase-catalyzed amination of dihydroxylated arenes with N-analogous corollosporines. In biological assays the obtained amination products are more active than the parent compounds.

Sixteen novel cephalosporins were synthesized by amination of 2,5-dihydroxybenzoic acid derivatives with the aminocephalosporins cefadroxil, cefalexin, cefaclor, and the structurally related carbacephem loracarbef using laccases from Trametes sp. or Myceliophthora thermophila. All products inhibited the growth of several Gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. The products protected mice against an infection with Staphylococcus aureus lethal to the control animals. Cytotoxicity and acute toxicity of the new compounds were negligible. The results show the usefulness of laccase for the synthesis of potential new antibiotics. The biological activity of the new compounds stimulates intensified pharmacological tests.

Eight novel penicillins were synthesized by heteromolecular reaction of ampicillin or amoxicillin with 2,5-dihydroxybenzoic acid derivatives using a laccase from Trametes spec. All products inhibited the growth of several gram positive bacterial strains in the agar diffusion assay, among them methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. The products protected mice against an infection with Staphylococcus aureus lethal to the untreated animals. Cytotoxicity and acute toxicity of the new compounds were neglectable. The results show the usefulness of laccase for the synthesis of potential new antibiotics. The biological activity of the new compounds stimulates intensified pharmacological tests.