Laccase-Catalyzed Derivatization of Antibiotics with Sulfonamide or Sulfone Structures

Trametes spec. laccase (EC 1.10.3.2.) mediates the oxidative coupling of antibiotics with sulfonamide or sulfone structures with 2,5-dihydroxybenzene derivatives to form new heterodimers and heterotrimers. These heteromolecular hybrid products are formed by nuclear amination of the p-hydroquinones with the primary amino group of the sulfonamide or sulfone antibiotics, and they inhibited in vitro the growth of Staphylococcus species, including multidrug-resistant strains.


Introduction
The evolution of antibiotic resistances amongst microorganisms is a global problem [1][2][3]. Because of this, novel antimicrobial substances and synthesis routes are needed. For this, enzymatic catalysis may be an alternative to conventional chemical synthesis processes [4,5]. The oxidoreductase laccase [E.C. 1.10.3.2, benzenediol:dioxygen oxidoreductase] possesses a number of advantages. The reactions can be performed under mild and environmentally friendly conditions, such as atmospheric pressure and room temperature. Additionally, no cofactors such as a coenzyme or NADPH are needed.
These diverse reaction types resulted in the synthesis or derivatization of antitumor [22][23][24], antioxidative [25][26][27], or estrogenic [28,29] compounds. Additionally, the synthesis of antibiotics by homomolecular and heteromolecular reactions have also been described. Thus, the laccase-catalyzed dimerizations of different ortho-aminophenol derivatives by C=N and C−O bond formations resulted in phenoxazinone pigments proposed as antitumor antibiotics [30][31][32][33][34][35]. Additionally, the catalytic derivatization of β-lactam antibiotics has been of particular interest. Agematu et al. [36] described the laccase-mediated synthesis of homomolecular dimers from different esters of penicillin X. The dimers of the penicillin X methyl ester showed hardly any antibacterial activity. In contrast, the ortho-ortho coupling product formed from penicillin X pivaloyloxymethyl ester represented a precursor (prodrug) of the penicillin X dimer. However, the minimum inhibitory concentration was lower than that of penicillin X sodium salt [36]. The laccase-catalyzed oxidation of a cephalosporanic acid resulted in the formation of two diastereomers, which differed in

Determination of Antibacterial Activity
An agar diffusion method described by Mikolasch et al. [39] was used to determine the antibacterial activity in the range from 29 to 1490 nmol. In brief, sterile Mueller-Hinton II-Agar in petri dishes (Becton Dickinson Microbiology systems, Cockeysville, Maryland, U.S.A.) was inoculated with 200 µL of bacterial solution (bacterial cell suspension 15 × 10 7 cells). The bacterial strains Staphylococcus aureus ATCC6538/DSM799, northern German epidemic MRSA (methicillin-resistant S. aureus; Norddeutscher Epidemiestamm), and the multidrug-resistant strain isolated from patients S. epidermidis 99847 were used. The test substances were applied on sterile paper discs (Sensi-Disc, 6 mm diameter, Becton Dickinson Microbiology systems). Petri dishes were kept for 3 h in a refrigerator (for prediffusion) and were then incubated for 24 h at 37 • C. Average inhibition zone diameters were calculated from 3 replicates.
After separation of 3a 1 and 3b 1 by solid phase extraction, LC/MS analyses in negative and positive mode showed molecular masses of these products attributed to the formation of heteromolecular hybrid dimers each consisting of a structural part of a 2,5dihydroxybenzene derivative (1a or 1b) coupled to 2a accompanied by the loss of four hydrogen atoms. These couplings were confirmed by the presence of characteristic signals for 1a or 1b and for 2a in the 1 H NMR spectra of 3a 1 and 3b 1 as well as by the presence of all carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the products, but two signals in the range of 180 ppm indicated a quinoid character of these products. The number of CH proton signals of 1a and 1b changed from three in the substrate, to two signals in the products. The multiplicity of the proton signals H−4 and H−5 indicated an additional substituent at the C−2 atom and the loss of a proton. The chemical shift to lower field of the H−4 and H−5 signals confirmed the presence of an electron-withdrawing group. Signals for phenolic hydroxyl groups could not be measured, but instead additional amine protons were detected. All analytical data confirmed the oxidation of the p-hydroquinone structure of 1a and 1b to a quinone. The heteromolecular hybrid dimers 3a 1 and 3b 1 were formed by nuclear amination of the p-hydroquinones 1a or 1b at the C−6 position with the primary amino group of 2a ( Figure 1).
Educt 1c reacted with 2a to a heteromolecular hybrid dimer 3c 2 , which was only analyzed by LC/MS in positive mode in a mixture together with a heteromolecular hybrid trimer 4c 1 . From the LC/MS data of 3c 2 we deduced a dimer with hydroquinoid structure. The product 4c 1 was described by LC/MS and 1 H NMR analyses. All data showed a heteromolecular hybrid trimer consisting of a structural part of 1c coupled to two molecules of 2a accompanied by the loss of six hydrogen atoms. The number of CH proton signals of 1c changed from three in the substrate, to one singlet in the product. The data of this proton signal H−4 indicated an additional substituent at the C−2 atom and a second substituent at the C−5 atom of the product. The chemical shift of the H−4 signal verified the presence of electron-withdrawing groups. Furthermore, the two amine protons detected confirmed the coupling of two molecules of 2a to one molecule of 1c accompanied by the formation of a heteromolecular hybrid trimer with quinoid structure 4c 1 .

11'
1 not detected by the measurements used.
After separation of 3a1 and 3b1 by solid phase extraction, LC/MS analyses in negative and positive mode showed molecular masses of these products attributed to the formation of heteromolecular hybrid dimers each consisting of a structural part of a 2,5-dihydroxybenzene derivative (1a or 1b) coupled to 2a accompanied by the loss of four hydrogen atoms. These couplings were confirmed by the presence of characteristic signals for 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as by the presence of all carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the products, but two signals in the range of 180 ppm indicated a quinoid character of these products. The number of CH droxybenzene derivatives (1a-1c) and sulfonamides (2a, 2b).

Quinoid Trimers
Hydro quinoi Trimer After separation of 3a1 and 3b1 by solid phase extraction, LC/MS analyses in nega and positive mode showed molecular masses of these products attributed to the forma of heteromolecular hybrid dimers each consisting of a structural part of a 2,5-d droxybenzene derivative (1a or 1b) coupled to 2a accompanied by the loss of four hy gen atoms. These couplings were confirmed by the presence of characteristic signal 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as by the presence o carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the products, but two signa the range of 180 ppm indicated a quinoid character of these products. The number of droxybenzene derivatives (1a-1c) and sulfonamides (2a, 2b).

Hydroquinoid Dimers
Qu Tri After separation of 3a1 and 3b1 by solid phase extraction, LC/MS and positive mode showed molecular masses of these products attrib of heteromolecular hybrid dimers each consisting of a structural droxybenzene derivative (1a or 1b) coupled to 2a accompanied by th gen atoms. These couplings were confirmed by the presence of cha 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as b carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the produc the range of 180 ppm indicated a quinoid character of these product nd nd 1b 8 9 10 R 1 =NHCH 2 CH 2 OH 2b 3e 1 8 9 10 R 1 =NHCH 2 CH 2 OH R 2 = Microorganisms 2021, 9, x FOR PEER REVIEW 7     After separation of 3a1 and 3b1 by solid phase extraction, LC/MS analyses in nega and positive mode showed molecular masses of these products attributed to the forma of heteromolecular hybrid dimers each consisting of a structural part of a 2,5-d droxybenzene derivative (1a or 1b) coupled to 2a accompanied by the loss of four hy gen atoms. These couplings were confirmed by the presence of characteristic signal 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as by the presence o carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the products, but two signa the range of 180 ppm indicated a quinoid character of these products. The number of   After separation of 3a1 and 3b1 by solid phase extraction, LC/MS and positive mode showed molecular masses of these products attrib of heteromolecular hybrid dimers each consisting of a structural droxybenzene derivative (1a or 1b) coupled to 2a accompanied by th gen atoms. These couplings were confirmed by the presence of cha 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as b carbons of 1a or 1b and of 2a in the 13 C−NMR spectra of the produc the range of 180 ppm indicated a quinoid character of these product     Like the reactions of 1a or 1b with 2a, the reactions of 1a or 1b with sulfamerazine (2b) also showed the same reaction patterns. Whereas 1a or 1b each reacted with 2a to yield a heteromolecular hybrid dimer with clear quinoid structure (3a 1 and 3b 1 ), the reaction of 1a or 1b with 2b each resulted in a heteromolecular hybrid dimer, which yielded the hydroquinoid structures 3d 2 and 3e 2 by 1 H NMR and 13 C NMR, and as the quinoids 3d 1 and 3e 1 by LC/MS.
From the reaction of 1c with 2b only a product mixture could be isolated and measured by LC/MS. Data for heteromolecular hybrid dimers with quinoid 3f 1 and hydroquinoid 3f 2 structure and for heteromolecular hybrid trimers with quinoid 4f 1 and hydroquinoid 4f 2 structure were detected. In this case, pure substances could not be isolated.

Educts Products Quinoid Dimers
Hydroquinoid Dimers  1 not detected by the measurements used.
After separation of 3a1 and 3b1 by solid phase extraction, LC/MS analyses in negative and positive mode showed molecular masses of these products attributed to the formation of heteromolecular hybrid dimers each consisting of a structural part of a 2,5-dihydroxybenzene derivative (1a or 1b) coupled to 2a accompanied by the loss of four hydrogen atoms. These couplings were confirmed by the presence of characteristic signals for 1a or 1b and for 2a in the 1 H NMR spectra of 3a1 and 3b1 as well as by the presence of all 13 5c 1 R 1 =H R 2 = Microorganisms 2021, 9, x FOR PEER REVIEW Table 1. Heterodimers and heterotrimers produced by laccase-mediated r droxybenzene derivatives (1a-1c) and sulfonamides (2a, 2b).

Hydroquinoid Dimers
Quin Trim   1 not detected by the measurements used.
After separation of 3a1 and 3b1 by solid pha and positive mode showed molecular masses of of heteromolecular hybrid dimers each consi droxybenzene derivative (1a or 1b) coupled to 2 gen atoms. These couplings were confirmed by 1a or 1b and for 2a in the 1  1 not detected by the measurements used.
In contrast, the product formation of 1d or 1e with sulfamerazine (2b) was comparable to that of 1a or 1b. Thus, the respective heteromolecular hybrid dimers with quinoid and hydroquinoid structures were detected in all four reactions. The only difference is that 1d or 1e reacted with 2b only to a product mixture from which no pure substance could be isolated and only LC/MS measurements were possible whereas from the reaction of 1a or 1b with 2b products were isolated and analyzed both by LC/MS and by NMR. But all of these four reactions are expected to follow the same reaction mechanism. In general, the reactions of 1d or 1e with 2a or 2b did not lead to isolated pure substances whereas the reactions with 1a or 1b resulted in products, which could be isolated. resulting in heteromolecular products, which were detected by characteristic UV-Vis spectra on HPLC for both substances (Figure 3 and Table 3).

Biological Activity of Biotransformation Products
The products 3a 1 and 3b 1 were those with the best supported structural data, and they were stable over a longer time period. Because of this, they were selected for assays of biological activity. The agar diffusion test was chosen for the determination of the antibacterial activity of the substances. Thereby, the size of the respective inhibition zone is a measure of the antibacterial efficacy. The educts 1a and 2a were not active against the strains tested, 1b showed only very low activity with the highest concentration used. This indicates that products with antimicrobial activity can be produced by laccase-mediated reactions from two initially very poorly active or inactive compounds.
HPLC measurements demonstrated good product stability for the compounds 3a 1 and 3b 1 , which had been stored in solid form at 4 • C for several weeks. However, incubation of the compounds 3a 1 and 3b 1 in aqueous solutions at 30 • C resulted in decomposition within some hours. For these reasons, the survey of their antimicrobial effects was restricted to the agar diffusion test.

Discussion
Because of increasing bacterial resistance to therapeutically used drugs, there is an urgent need to develop new antimicrobial agents. The laccase-mediated reaction of penicillin and cephalosporanic acid derivatives resulted in a dimerization by C−C or C−O bond formation and the synthesis of diastereomers, respectively [36,37]. These homomolecular products showed antimicrobial activity. Additionally, the laccase-mediated C−N bond formation between different partners resulted in heteromolecular products with antibacterial efficacy [48,49]. Such aminations are consequently a suitable green method for the synthesis of novel antibiotics. Thus, penicillins [38,40,41], cephalosporins [39,40,42], and its basic structures [46] have been structurally altered to achieve advanced antibacterial activities, especially against multidrug-resistant organisms. Furthermore, derivatives of the isolated antibacterial agents corollosporine from the marine fungus Corollospora maritima [50] and the ganomycins from Ganoderma pfeifferi [51] were transformed by one pot laccase-mediated reactions to improve their biological activity [38,42,52].
The sulfonamides and sulfones are antibacterial drugs that have been used for a long time [45,46], but whose value has progressively declined due to the emergence of resistant strains [53][54][55]. We have here derivatized antimicrobial sulfonamides and sulfones by laccase. Heterodimers and heterotrimers were synthesized by laccase-catalyzed reactions of sulfonamide antibiotics or dapsone with 2,5-dihydroxybenzene derivatives via nuclear amination. The formation of dimers (3a-3f, 5a-d) was determined for all reactions of 2a, 2b with 1a-1e. Trimers (4f, 6a, 6b) were only detected in the reactions of 2a, 2b with 1c and for 2c with 1a, 1b. The structure of these trimers differed depending on whether the starting compound possessed one (2a, 2b) or two free amino groups (2c) accessible for a reaction with the p-hydroquinones (1a-c). Thus, the trimers formed with 2a or 2b consisted of one molecule 1c and two molecules of 2a or 2b, respectively, whereas the trimers with 2c were formed from two molecules of 1a or 1b and only one molecule of 2c.
The dimers and trimers were detected in hydroquinoid or quinoid form, as described previously for heterodimers as products of p-hydroquinones with amino compounds such as aminopyrazole carboxamide [56] or aminothiophenol [21]. Interestingly, for the trimers with 2c, also a mixed quinoid-hydroquinoid trimer was formed with one molecule of 1a or 1b as hydroquinone and one quinoid molecule of 1a or 1b.
The results regarding the biological activity showed that, with the exception of compound 1b, the educts showed no inhibiting effect against the tested Staphylococcus species. Compound 1b had low activity only at the highest concentration tested (1.27 µmol). In contrast, the products 3a 1 and 3b 1 were effective at concentrations between 0.14 to 0.74 µmol. This increase in antimicrobial activity of these products of laccase-catalyzed reactions is remarkable, given that previous attempts to use laccase-mediated transformation of β-lactam antibiotics (such as ampicillin, amoxicillin) did not lead to an increase in antibiotic efficacy [36][37][38].
The increased antibacterial efficacy for the products 3a 1 and 3b 1 in comparison to the educts as well as the inhibition of different Staphylococcus species, in particular the multidrug-resistant northern German epidemic MRSA, without toxicity for mammalian cells underlines the high potential of laccase-mediated reactions for the synthesis of novel antibiotics.