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Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91– 97

Contents lists available at SciVerse ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

jo u rn al hom epa ge: www.elsev ier .com/ locate /molcatb

accase-mediated synthesis of 2-methoxy-3-methyl-5-(alkylamino)- and-methyl-2,5-bis(alkylamino)-[1,4]-benzoquinones

usanne Hertera,c,∗, Dirk Michalikb, Annett Mikolaschc, Marlen Schmidtd, Roland Wohlgemuthe,we Bornscheuerd, Frieder Schauerc

Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UKLeibniz Institute for Catalysis, Albert-Einstein-Str. 29a, 18059 Rostock, GermanyInstitute of Microbiology, Department of Applied Microbiology, University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, 17487 Greifswald, GermanyInstitute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Felix-Hausdorff-Str. 4, 17487 Greifswald, GermanySigma–Aldrich Chemie GmbH, Industriestr. 25, 9470 Buchs, Switzerland

r t i c l e i n f o

rticle history:eceived 12 December 2012eceived in revised form 23 January 2013ccepted 23 January 2013vailable online 8 February 2013

eywords:yceliophthora thermophila

ycnoporus cinnabarinus

a b s t r a c t

The synthesis of 5-alkylamino- and 2,5-bis(alkylamino)-[1,4]-benzoquinones, showing structural sim-ilarity to natural mitomycins, was performed through coupling of 2-methoxy-3-methylhydroquinonewith primary amines such as n-octylamine, geranylamine and cyclooctylamine using laccases from Myce-liophthora thermophila (MtL) and Pycnoporus cinnabarinus SBUG-M 1044 (PcL). Product spectra of laccasereactions differ due to reaction systems pH values (pH 7.0 for MtL and pH 5.0 for PcL) applied to assureenzymes optimal catalytic efficiency. The MtL- and PcL-mediated formation of monoaminated productswas achieved at equimolar reactant concentrations with amine coupling at the meta-position to benzo-quinones methyl group. Increased formation of diaminated products occurred in PcL-mediated reactions

iocatalysis N couplingitomycin

and generally when the amine was supplied in excess. Diamination entailed elimination of the benzo-quinone methoxy group (amination in para-position to the first amine substituent). Six products weresynthesised and characterised by NMR and HR-MS analysis. The laccase-mediated amine coupling to2-methoxy-3-methylhydroquinone confers two of the essential pharmaceutical active motifs from mit-omycins: (i) a stable 1,4-benzoquinoic parent structure and (ii) a biological active alkylation function( NH).

. Introduction

The enzyme class of laccases (para-diphenol:dioxygen oxidore-uctases, EC 1.10.3.2) finds increasing significance in a variety ofrganic synthetic methodologies and the manufacture of syntheticuilding blocks for fine chemistry due to a broad substrate scope,he variety of reactions catalysed, and in particular the high resis-ance towards harsh reaction conditions [1–4]. The versatility ofaccases enables a broad range of applications from the productionf polymers and food additives to the synthesis of fragrance com-ounds, and cosmetic and pharmaceutical precursors in the field ofhite biotechnology [5–7].

The family of mitomycins comprised pharmaceutical active,ntineoplastic compounds. Mitomycins provide a broad spectrum

f efficacy due to their antibiotic and cytotoxic characteristics andherefore selected mitomycins are used in control of infectious dis-ases caused by Gram positive as well as Gram negative bacteria,

∗ Corresponding author. Tel.: +49 3834 864204; fax: +49 3834 864202.E-mail address: susanne.herter@manchester.ac.uk (S. Herter).

381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molcatb.2013.01.020

© 2013 Elsevier B.V. All rights reserved.

and also some viruses [8,9]. They are also employed as chemother-apeutics in the treatment of several types of cancer, includingsolid carcinomas [10,11]. The biological activity of mitomycins hasbeen related to certain biologically active structure motifs such asa para-benzoquinoidic parent structure, a strong alkylating func-tion, especially that of a secondary amino-group, and partially aurethane-function (Fig. 1A) [12].

Approaches for the chemical synthesis of mitomycins arechallenging due their demanding stereochemistry, the difficultworkability of quinones, and often requiring complex reactionsteps [13–15]. With respect to the aforementioned difficulties, inparticular quinone instability, the use of the biocatalyst laccaseaffords mild reaction conditions and can catalyse the transforma-tion of dihydroxylated compounds into the corresponding stablebenzoquinones. Previously, it was shown that laccases can medi-ate a wide range of heteromolecular reactions (e.g. C C, C O, C N,C S coupling) at which the enzyme substrate radical is linked

with diverse coupling agents yielding stable hybrid molecules[16–19]. In most of the published laccase-mediated C N cou-pling reactions, simple substituted para-hydroquinones acting asnative laccase substrates due to position of hydroxyl groups were

92 S. Herter et al. / Journal of Molecular Cata

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ig. 1. Model structure for selected naturally occurring mitomycins (A). Laccase-ubstrate 1 and primary amines 3a–c (coupling agents) used in reactions focusedn the enzyme-mediated synthesis of mitomycin-like compounds (B).

pplied in assays containing aromatic amines [20–22]. It wasemonstrated that product formation in such laccase-mediated

N coupling results in satisfying yields and is equivalent or bet-er when compared to synthetic routes, as for example use of theoupling-mediator sodium iodate [23,24].

These findings encouraged us to study the laccase-mediatedynthesis of mitomycin-like compounds. The 2-methoxy-3-ethylbenzoquinone represents in its protonated form a suitable

accase substrate (1) and was hence investigated in the cou-ling with aliphatic (n-octylamine 3a and geranylamine 3b) asell as cyclic (cyclooctylamine 3c) primary amines using laccases

rom Myceliophthora thermophila (MtL) and Pycnoporus cinnabar-nus SBUG-M 1044 (PcL) (Fig. 1B). The influence of pH values ofuffered reaction systems, as well as reactant ratios, on productields and formation of mono- or diaminated products was studied.

. Experimental

.1. Chemicals

All chemicals were of reagent grade or better and purchasedrom Sigma–Aldrich (Steinheim, Germany) and Merck (Darmstadt,ermany).

.2. Enzymes

M. thermophila laccase (MtL) was commercially obtained fromovozymes® (Bagsvaerd, Denmark). Laccase from the white-rot

ungus P. cinnabarinus SBUG-M 1044 was produced as described byerter et al. [25] according to methods of Kordon et al. [26]. Laccasectivity was determined by monitoring the oxidation of 2,2′-zino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammoniumABTS) spectrophotometrically at 420 nm (ε = 3.6 × 104 M−1 cm−1)n sodium acetate buffer (100 mM, pH 5) at 25 ◦C as described ear-ier. One unit (1 U) of activity is defined as the amount of enzyme,

hich catalyzes the conversion of 1 �moL mL−1 min−1 substrate at5 ◦C.

.3. Laccase-mediated heteromolecular coupling of-methoxy-3-methylhydroquinone with primary amines

Analytical assays were conducted in sealed brown 6-mL-glassasks with a total reaction volume of 5 mL at room temperaturend shaking at 200 rpm. Reactions were performed either in

lysis B: Enzymatic 90 (2013) 91– 97

phosphate-citrate buffer (pH 7.0) with M. thermophila laccase(1 �moL mL−1) or in sodium acetate buffer (20 mM, pH 5.0) withP. cinnabarinus SBUG-M 1044 laccase (1 �moL mL−1). Educts wereprepared as 50 mM methanolic stock solutions and biocatalysisconducted either in equimolar concentration or with an excessof the primary amines. Product formation was followed by ana-lytical HPLC at regular intervals. Preparative scale reactions wereperformed in 100-mL-Erlenmeyer flasks protected against light,containing 50 mL of the reaction mixture using the same conditionsdescribed above. Monoaminated products were obtained usinga 2:2 mM ratio between 2-methoxy-3-methylhdroquinone 1 andthe primary amines 3a–c from MtL-catalysed reactions. Diami-nated products were enriched using a 2:5 mM ratio under the sameconditions as described for enrichment of monoaminated products.

2.4. Analytical HPLC

Reaction batches were sampled at regular intervals over 24 hand analyzed via HPLC (Shimadzu, Duisburg, Germany) consistingof a SCL-10A VP control unit, a LC-A FCV-10AL VP fluid chromato-graph and a SPD-10A VP photodiode array detector. Grading ofanalytes was accomplished with a LiChroCart 125-4 RP-18 end-capped column of 5-�m particle size (Merck, Darmstadt, Germany).The mobile phase consisted of an aqueous component A (0.1% phos-phoric acid) and a methanol component B at an initial ratio of 30%B to 70% A, simultaneously raised up to 100% B within 14 min.The flow rate was always adjusted to 1 mL min−1 with an injectionvolume of 40 �L.

2.5. Isolation of heteromolecular coupling products

Products were isolated by solid reverse phase extraction witha Strata C18-E silicagel column (60 mL Giga Tubes, 10 g absorbentmaterial, Merck Darmstadt, Germany), progressively charged with50 mL of the reaction mixture. Residual starting material andlow-molecular by-reaction products were eluted with 60 mL of asolution composed of 60% methanol and 40% bidistilled water. Themonoaminated products were obtained with 60 mL of a mixtureconsistent of 85% methanol and 15% acetic acid (0.1%, v/v in bidis-tilled water), followed by elution of the corresponding diaminatedproducts due to addition of pure methanol (40 mL). The methanolconcentration of samples was initially reduced via rotary evapo-ration at 30 ◦C. Afterwards, samples were diluted with bidistilledwater to reach a final methanol concentration of 5% and subse-quently frozen at −20 ◦C for 24 h, followed by freezing at -70 ◦Cfor further 4 h. Products were obtained as solids via lyophilisation(20 ◦C).

2.6. Structural characterisation

HR-MS measurements were conducted on an ESI-TOF/MS (Agi-lent Technologies, Böblingen, Germany) in positive ion mode([M+H]+, [M+Na]+) by direct injection of methanolic sampleslacking a previous chromatographic column separation usinga MeOH/0.1% acetic acid mobile phase in a 9:1 ratio (v/v).1H, 13C, DEPT and two-dimensional NMR spectra (1H,1H COSY,1H,1H NOESY, 13C,1H HMBC, 13C, 1H HSQC) were recorded onBruker Avance spectrometers AV 600, AV 500, and AV 250, Karl-sruhe, Germany, in deuterated methanol (MeOH-d4, ı(1H) = 3.31,ı(13C) = 49.1).

2.6.1. 2-Methoxy-3-methyl-5-(octylamino)-[1,4]-benzoquinone(5a)

Red solid. HPLCRt 14.9 min, UV-vis (MeOH) �max 212, 309,498 nm. HR-MS (ESI): calcd for C16H26NO3 [M+H]+ 280.19072;

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S. Herter et al. / Journal of Molecul

ound 280.19068 (error: −0.15 ppm); calcd for C16H25NNaO3M+Na]+ 302.17266; found 302.17257 (error: −0.33 ppm).

1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.26 (s, 1H, H-6); 4.04s, 3H, H-1′′); 3.14 (t, 1H, 3J = 7.3 Hz, H-1′); 1.86 (s, 3H, H-2′′); 1.62m, 2H, H-2′); 1.33 (m, 10H, H-7′, H-6′, H-5′, H-4′, H-3′); 0.90 (t, 3H,J = 7.2 Hz, H-8′). 13C NMR (150.9 MHz, MeOH-d4) ı (ppm) = 185.0C-4); 182.8 (C-1); 159.8 (C-2); 149.2 (C-5); 124.3 (C-3); 61.8 (C-1′′);3.3 (C-1′); 32.7 (C-6′); 28.8 (C-2′); 28.0 (C-3′); 30.1, 30.1 (C-4′,5′);3.4 (C-7′); 14.3 (C-8′); 8.1 (C-2′′).

.6.2. 3-Methyl-2,5-bis-octylamino-[1,4]-benzoquinone (6a)Brown-ochre solid. HPLCRt 17.0 min, UV–vis (MeOH) �max

17, 346, 508 nm. HR-MS (ESI): calcd for C23H41N2O2 [M+H]+

77.31625; found 377.3165 (error: 0.66 ppm); calcd for23H40N2NaO2 [M+Na]+ 399.30014; found 399.30018 (error:.54 ppm).

1H NMR (500.13 MHz, MeOH-d4) ı (ppm) = 5.23 (s, 1H, H-6); 3.60t, 1H, 3J1 ′′

,2′′ = 7.2 Hz, H-1′′); 3.18 (t, 1H, 3J1′ ,2′ = 7.2 Hz, H-1′); 2.04

s, 3H, H-1′′′); 1.63 (m, 4H, H-2′, H-2′′); 1.40–1.27 (m, 20H, H-3′--7′, H-3′′-H-7′′); 0.90 (m, 6H, H-8′, H-8′′). 13C NMR (125.8 MHz,eOH-d4) ı (ppm) = 180.5, 179.9 (C-1, C-4); 153.3 (C-5); 150.2 (C-

); 102.9 (C-3); 92.1 (C-6); 45.9 (C-1′′); 43.6 (C-1′); 33.0, 33.0 (C-6′,-6′′); 31.9, 29.3 (C-2′, C-2′′); 30.4, 30.4 (3) (C-4′, C-4′′, C-5′, C-5′′);8.1, 27.8 (C-3′, C-3′′); 23.8, 23.8 (C-7′, C-7′′); 14.5 (2) (C-8′, C-8′′);0.5 (C-1′′′).

.6.3. 5-[[(2E)]-3,7-dimethylocta-2,6-dienyl]-amino]-2-ethoxy-3-methyl-[1,4]-benzo-quinone (5b)

Red solid. HPLCRt 14.5 min, UV–vis (MeOH) �max 212, 308,88 nm. HR-MS (ESI): calcd for C18H26NO3 [M+H]+ 304.19072;ound 304.19013 (error: −1.95 ppm); calcd for C18H25NNaO3M+Na]+ 326.17266; found 326.1736 (error: 2.87 ppm).

1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.22 (s, 1H, H-6); 5.19t, 1H, 3J = 6.5 Hz,H-2′); 5.08 (t, 3J = 7.0 Hz, 1H, H-6′); 4.04 (s, 3H, H-′′); 3.76 (d, 2H, 3J = 6.5 Hz,H-1′); 2.12 (m, 2H, H-5′); 2.05 (m, 2H,-4′); 1.86 (s, 3H, H-2′′); 1.72 (s, 3H, H-10′); 1.65 (s, 3H, H-9′); 1.59

s, 3H, H-8′). 13C NMR (150.9 MHz, MeOH-d4) ı (ppm) = 184.9 (C-); 181.8 (C-1); 159.6 (C-2); 149.1 (C-5); 140.8 (C-3′); 132.3 (C-7′);24.4 (C-6′); 124.2 (C-3); 119.7 (C-2′); 95.7 (C-6); 61.5 (C-1′′); 41.1C-1′); 40.1 (C-4′); 26.9 (C-5′); 25.4 (C-9′); 17.5 (C-8′); 16.1 (C-10′);.9 (C-2′′).

.6.4. 2,5-bis[[2E]-3,7-dimethylocta-2,6-dienyl]amino]-3-ethyl-[1,4]-benzoquinone (6b)

Brown-ochre solid. HPLCRt 16.7 min, UV–vis (MeOH) �max

17, 347, 508 nm. HR-MS (ESI): calcd for C27H41N2O2 [M+H]+

25.31625; found 425.3165 (error: 0.57 ppm); calcd for27H40N2NaO2 [M+Na]+ 447.2982; found 447.29853 (error:.74 ppm).

1H NMR (600.13 MHz, MeOH-d4) ı (ppm) = 5.29 (m, 1H, H-2′′);.21 (m, 1H, H-2′); 5.20 (s, 1H, H-6); 5.08 (m, 2H, H-6′,6′′); 4.20 (d,H, 3J = 6.3 Hz,H-1′′); 3.80 (d, 2H, 3J = 6.8 Hz,H-1′); 2.12 (m, 4H, H-′,5′′); 2.07 (m, 4H, H-4′,4′′); 2.05 (s, 3H, H-1′′′); 1.73 (m, 3H, H-10′);.70 (m, 3H, H-10′′); 1.66 (m, 3H), 1.65 (m, 3H), (H-9′,9′′); 1.60 (br, 3H), 1.59 (br s, 3H), (H-8′,8′′). 13C NMR (150.9 MHz, MeOH-d4)

(ppm) = 180.7 (C-1); 180.3 (C-4); 153.0 (C-5); 150.0 (C-2); 141.5C-3′); 141.1 (C-3′′); 132.8 (2) (C-7′,7′′); 125.0 (2) (C-6′,6′′); 122.6 (C-′′); 120.1 (C-2′); 103.3 (C-3); 92.6 (C-6); 43.8 (C-1′′); 41.5 (C-1′);0.6, 40.6 (C-4′,4′′); 27.4, 27.4 (C-5′,5′′); 26.0, 26.0 (C-9′,9′′); 17.9 (2)C-8′,8′′); 16.6, 16.6 (C-10′,10′′); 10.4 (C-1′′′).

.6.5. 2-Methoxy-3-methyl-5-(cyclooctylamino)-[1,4]-enzoquinone (5c)

Red solid. HPLCRt 13.7 min, UV–vis (MeOH) �max 212, 309,85 nm. HR-MS (ESI): calcd for C16H24NO3 [M+H]+ 278.17507;

lysis B: Enzymatic 90 (2013) 91– 97 93

found 278.17553 (error: 1.65 ppm); calcd for C16H23NaNO3[M+Na]+ 300.15701; found 300.15741 (error: 1.33 ppm).

1H NMR (250.13 MHz, MeOH-d4) ı (ppm) = 5.19 (s, 1H, H-6); 4.04(s, 3H, H-1′′); 3.45 (m, 1H, H-1′); 1.85–1.53 (m, 14H, H-2′, H-3′, H-4′,H-5′); 1.85 (s, 3H, H-2′′). 13C NMR (62.9 MHz, MeOH-d4) ı = 185.3(C-1); 182.8 (C-4); 160.0 (C-2); 147.8 (C-5); 124.5 (C-3); 96.1 (C-6); 62.0 (C-1′′); 53.9 (C-1′); 32.6 (C-2′); 28.0, 25.1 (C-3′, C-4′); 27.0(C-5′); 8.5 (C-2′′).

2.6.6. 3-Methyl-2,5-bis-cyclooctylamino-[1,4]-benzoquinone (6c)Brown-ochre solid. HPLCRt 16.5 min, UV-vis (MeOH) �max

216, 348, 508 nm. HR-MS (ESI): calcd for C23H37N2O2 [M+H]+

373.28495; found 373.28542 (error: 1.25 ppm); calcd forC23H36N2NaO2 [M+Na]+ 395.26671; found 395.26671 (error:−0.48 ppm).

1H NMR (500.13 MHz, MeOH-d4) ı (ppm) = 5.20 (s, 1H, H-6);4.19 (s, 3H, H-1′′); 3.52 (m, 1H, H-1′); 2.03 (s, 3H, H-1′′′); 1.98–1.53(m, 28H, H-2′,2′′,3′,3′′,4′,4′′,5′,5′′). 13C NMR (125.8 MHz, MeOH-d4)ı = 180.6 (C-1); 180.0 (C-4); 151.5 (C-5); 148.4 (C-2); 102.9 (C-3);92.5 (C-6); 54.8 (C-1′′); 53.9 (C-1′); 34.4 (C-2′′); 32.6 (C-2′); 28.3,28.2, 25.0, 24.4 (C-3′,3′′,4′,4′′); 26.9, 26.8 (C-5′,5′′); 10.7 (C-1′′′).

3. Results and discussion

3.1. Laccase-mediated coupling reactions: product formation andrecovery

Prior to heteromolecular coupling reactions which targetedthe synthesis of mitomycin-like compounds, the laccase-mediatedhomomolecular reaction (reaction without amines 3a–c) of theenzyme substrate 2-methoxy-3-methylhydroquinone 1 (1 mM)was studied. For all reactions, phosphate-citrate buffer (pH 7.0) wasused for M. thermophila laccase (MtL) and sodium acetate buffer (pH5.0) for P. cinnabarinus laccase (PcL). The aforementioned reactionsystems and pH values were chosen to ensure the optimal catalyticefficiency of both laccases [27–29].

In a homomolecular reaction, enzyme substrate 1 was com-pletely oxidized within 60 min using MtL and 100 min using PcL.Laccase-mediated oxidation of 1 led to simultaneous formation ofthe corresponding 2-methoxy-3-methylbenzoquinone 2a (HPLCRt6.0 min, �max 283, 381 nm; Scheme 1), appearing in each case as themain product with yellow colouration of the initially uncolouredreaction mixture. In reactions with MtL, concomitant formation ofone further main product 2b (HPLCRt 5.7 min, �max 208, 294 nm)and a minor product 2c (HPLCRt 8.4 min, �max 254, 324 nm)appeared together with decrease of benzoquinone 2a. Products 2band 2c were not detected in the PcL-catalyzed reaction of 1, how-ever, formation of two minor products 2d (HPLCRt 4.9 min, �max

221, 267 nm) and 2e (HPLCRt 13.5 min, �max 272 nm) occurred.The structures of the homomolecular products were not examinedwithin this study, but additional data is available in SupplementaryMaterial (Table S1). The observed differences in the spectrum andquantities of homomolecular products are reasoned with differ-ent pH values of the reactions rather than with laccase catalysedactivity. Referring to this, the benzoquinone 2a showed a higherreactivity in a phosphate-citrate buffer at pH 7.0 (used for MtL) andwhich also resulted in an increased formation of by-products whencompared to a sodium acetate buffer system at pH 5.0 (PcL). Thisis in accordance with a general characteristic of para- as well asortho-benzoquinones, which possess at neutral to alkaline pH val-ues an increased affinity for side reactions due to increased redox

potentials [30,31].

In order to obtain mitomycin-like compounds by laccase-mediated transformation reactions, the heteromolecular couplingbetween 1 and amines n-octylamine 3a, geranylamine 3b and

94 S. Herter et al. / Journal of Molecular Cata

1

3 45

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Scheme 1. Laccase-mediated synthesis of mono- (5a–c) and diaminated (6a–c)mitomycin-like compounds by heteromolecular coupling of 2-methoxy-3-methylhydroquinone 1 with primary amines 3a–c at RT and 200 rpm. Reactionand conditions: (a) Oxidation of 1 by MtL (phosphate–citrate buffer, pH 7.0) or PcL(sodium acetate buffer, pH 5.0) yielding benzoquinone 2a. (b) Monoamination of 2aby coupling of one molecule of amines 3a–c. (c) Diamination of 5a–c by coupling ofai

cfutmHba

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methanol). Therefore, at relatively low amine concentrations, an

FlP

second molecule of amines 3a–c. Postulated structure for products 4a–c formedn reaction with MtL is shown in brackets.

yclooctylamine 3c was analysed (Fig. 1B). As already describedor homomolecular reactions, a different spectrum of heteromolec-lar products was obtained depending upon reaction pH, andhe laccase used. Generally, formation of up to three hetero-

olecular main products occurred with each of the amines used.eteromolecular coupling reactions are described for the reactionetween 1 (1 mM) and n-octylamine 3a (5 mM) catalysed by MtLnd PcL (Fig. 2A and B, respectively).

In both reaction systems, enzymatic oxidation of 1 wasssociated with the primary generation of the corresponding ben-oquinone 2a as already described. In the PcL-catalysed reactionpH 5.0), benzoquinone 2a accumulated up to 60 min. A subse-uent decrease of 2a was related to the formation of product 5aHPLCRt 14.9 min, �max 212, 309, 498 nm), generally appearing onlyn low concentrations, and of the main product 6a (HPLCRt 17.0 min,max 217, 346, 508 nm) (Fig. 2B). Products 5a and 6a were later

dentified as mono- and diaminated compounds, respectively. In

ontrast to the reaction with PcL, in the MtL-catalysed reaction (pH.0), decreasing concentrations of benzoquinone 2a were partiallyelated to an increase of by-product 2b which was already detected

0

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ig. 2. Decrease of 2-methoxy-3-methylhydroquinone 1 (1 mM, �) within formation of theike products 4a (�), 5a ( ), and 6a ( ) in heteromolecular coupling reactions with n-occL (B) in sodium-acetate buffer (pH 5.0). Enzyme activities[ABTS] 1.0 �moL mL−1, 200 rpm

lysis B: Enzymatic 90 (2013) 91– 97

in the homomolecular reaction (Fig. 2A). A further difference tothe PcL-mediated reaction was observed in the number as well asquantities of heteromolecular coupling products. Besides the minorproduct 4a (HPLCRt 14.5 min, �max 300, 498 nm), which was notformed in the PcL-reaction, the monoaminated 5a was observed asthe main product in the MtL-mediated reaction. Products 4a and5a were generated in a 1:2 ratio based upon their HPLC peak areaswithin the first 2 h of reaction (Fig. 2A). With regard to productretention time and UV/VIS-spectra, the presence of monoaminatedregioisomers was highly presumed, although not examined withinthis study. Proceeding reaction led to a decrease of monoaminatedproduct 5a together with concomitant increase of the diaminatedproduct 6a (Fig. 2A).

In summary, the following conclusions for heteromolecular cou-pling reactions between 2-methoxy-3-methylhydroquinone 1 andn-octylamine 3a at pH 5.0 and pH 7.0, can be made: (i) In the PcL-catalyzed system (pH 5.0) the diaminated product 6a is formed asthe main product, the monoaminated regioisomer 5a occurred onlyin negligible concentrations. (ii) In the MtL-catalyzed system (pH7.0) two monoaminated regioisomeric products 4a and 5a weresimultaneously generated, whereby 5a presented the main prod-uct and proceeding incubation led to increased formation of thediaminated product 6a. (iii) The formation of heteromolecular cou-pling products in MtL- and PcL-mediated systems is dependent onassay pH.

As observed in the MtL-catalysed reaction, in each case theproducts 4a–6a were formed, and their generation in relationto concentration of the amine donor 3a was analysed. Duringproduct isolation only the main products 5a (monoaminated)and 6a (diaminated) could be enriched in sufficient quantitiesand purities. Therefore, data given in Table 1 are restrictedto these products. At an equimolar reactant ratio, and with a2 mM excess of 3a, formation of monoaminated product 5a wasachieved with yields up to 32.9% (Table 1). Generation of thediaminated product 6a occurred only at marginal rates. Althoughequal concentrations of 1 and amine 3a led predominantly toformation of monoaminated product 5a (and 4a), the concen-tration of by-product 2b was still remarkable and correlatedwith comparable yields of 5a. Even though the structure of by-product 2b was not elucidated within this study, we suggest acompeting reaction for the same position at the benzoquinone2a between the amine and solvent molecules (e.g. water or

advanced coupling of water or methanol to benzoquinone 2ais highly presumed, and as described previously for aminationreactions of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide or

0

1000000

2000000

3000000

4000000

5000000

6000000

0 10 0 20 0 30 0 40 0

200

400

600

0

Pea

kar

ea[m

Aux

104 ;

220

nm]

0 200 300 400100Rea ction time [min]

B

0

1000000

2000000

3000000

4000000

5000000

6000000

0 10 0 20 0 30 0 40 0

200

400

600

0

Pea

kar

ea[m

Aux

104 ;

220

nm]

0 200 300 400100Rea ction time [min]

B

2-methoxy-3-methylbenzoquinone 2a (©), by-product 2b ( ) and the mitomycin-tylamine 3a (5 mM) catalysed by MtL (A) in phosphate-citrate buffer (pH 7.0) and, RT (mAu = milliabsorption unit).

S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91– 97 95

Table 1Influence of reactant ratios on the formation of monoaminated product 5a and diaminated product 6a. MtL-catalysed reactions between the 2-methoxy-3-methylhydroquinone 1 and n-octylamine 3a in a 20-mL-reaction scale in phosphate-citrate buffer (pH 7.0). Enzyme activities[ABTS] 1 �moL mL−1, 200 rpm, RT.

Ratio of 1 to amine 3a [mM] Concentration of products 5a and 6a [mM] Formation of 5a and 6a [%]a

1 h 6 h 6 h

5a 6a 5a 6a 5a 6a

1:1 0.043 0.039 0.207 0.045 20.7 4.5

poun

3e6tsdrmwird

ot3Mim4�tpt

T1

l

M

b

1:2 0.183 0.047

2:5 0.254 0.854

a Yields of product formation [%] compared to a 100% conversion into desired com

-methylcatechol [19,25]. Furthermore, amine 3a applied in anxcess (5:2 mM) resulted in formation of the diaminated producta with a HPLC-yield of 78.8%. This again supports the aforemen-ioned hypothesis of a competing reaction between the amine andolvent molecules as an increased amine concentration causedecreased formation of by-product 2b. Therefore, at almost equaleactant ratios, the reaction could be directed to the formation ofonoaminated product (together with a distinct presence of 2b),hereby a higher amine concentration favours diamination. An

ncreased formation of diaminated products in nuclear aminationeactions with excessively applied amine donors was alreadyemonstrated [22,32].

As described in detail for the reaction between 1 and n-ctylamine 3a, and with respect to the pH of the reaction systems,he same results were obtained in experiments with geranylamineb and cyclooctylamine 3c as amine donors. With amine 3b in antL-catalysed reaction system, formation of the minor monoam-

nated product 4b (HPLCRt 14.1 min, �max 289, 488 nm) and theajor monoaminated product 5b (HPLCRt 14.5 min, �max 212, 308,

88 nm) as well as the diaminated product 6b (HPLCRt 16.7 min,

max 217, 347, 508 nm) occurred. MtL-catalysed coupling reac-ions with cyclooctylamine 3c led to formation of monoaminatedroduct 4c (HPLCRt 13.2 min, �max 299, 485 nm) in low concentra-ions and monoaminated product 5c (HPLCRt 13.7 min, �max 212,

able 2H and 13C NMR assignments and HMBC correlations for the monoaminated product 5b iamine 3b.a .

NH

O

O

Me

HeO 12

3

4 5

6

2"

1"

1`

2`3`

10`

4`

5`

6`7` 8`

9`

Pos. 13C 1H 1H,13C correlations (HM

4 184.9 – –1 181.8 – –2 159.6 – –5 149.1 – –3′ 140.8 – –7′ 132.3 – –6′ 124.4 5.08, t, 1H, (7.0) C-8′ (17.5), C-9′ (25.4)3 124.2 – –2′ 119.7 5.19, m, 1H, (6.5) C-10′ (16.1), C-4′ (40.16 95.7 5.22, s, 1H C-5 (149.1), C-2 (159.61′′ 61.5 4.04, s, 3H C-2 (159.6)1′ 41.1 3.76, d, 2H, (6.5) C-10′(16.1), C-5′(26.9)4′ 40.1 2.05, m, 2H C-10′(16.1), C-5′(26.9)5′ 26.9 2.12, m, 2H C-4′ (40.1), C-6′ (124.49′ 25.4 1.65, s, 3H C-8′ (17.5), C-6′ (124.48′ 17.5 1.59, s, 3H C-9′ (25.4), C-6′ (124.410′ 16.1 1.72, s, 3H C-4′ (40.1), C-1′ (41.1)2′′ 7.9 1.86, s, 3H C-1′′ (61.5), C-3 (124.21′ 41.1 3.76, d, 2H, (6.5) C-10′(16.1), C-5′(26.9)

a Chemical shifts are expressed in ı (ppm) referred to TMS = 0 (calibration was perforrackets.b Correlation with low intensity.

0.329 0.045 32.9 4.50.279 1.69 12.9 78.8

ds 5a and 6a, respectively, estimated via HPLC-analysis after 6 h.

309, 485 nm) at higher concentrations. The diaminated product6c (HPLCRt 16.5 min, �max 216, 348, 508 nm) was also formed. Inthe PcL-catalyzed heteromolecular reactions using a 1:5 mM ratiobetween 1 and amines 3b and 3c respectively, the diaminatedproducts 6b (yield[6h,HPLC] 84.7%) and 6c (yield[6h,HPLC] 89.8%) werealways identified as the main products. The monoaminated prod-ucts 5b and 5c were only transiently detected, acting as precursorsfor diamination reaction.

3.2. Structural characterisation of heteromolecular couplingproducts

The monoaminated products 5a–c were obtained from MtL-catalysed reaction batches (6 h) in which educts were used in a2:2 mM ratio: 16.3 mg 5a (and 1.4 mg 6a, 4 mL × 50 mL); 20.7 mg5b (and 2.6 mg 6b, 4 mL × 50 mL); 22.4 mg 5c (and 3.1 mg 6c,7 mL × 50 mL). Diaminated products 6a–c were obtained using a2:5 mM ratio at the same conditions described above: 6.7 mg 6a,9.3 mg 6b, and 6.6 mg 6c each with 6 mL × 50 mL. The solids were

stable at room temperature as well as 4 ◦C within the analytic periodof 3 months. HPLC analysis of products dissolved in methanolrevealed no decay over 24 h. Structural characterisation of themonoaminated products 5a–c is shown for compound 5b (Table 2).

solated from a reaction between 2-methoxy-3-methylhydroquinone 1 and gerany-

BC)

, C-5′ (26.9), C-4′ (40.1)

), C-1′ (41.1)), C-1 (181.8), C-4 (184.9)

, (C-4′ (40.1))b, (C-6 (95.7))b, C-2′ (119.7), C-3′ (140.8), C-5 (149.1), (C-4 (184.9))b

, C-1′ (41.1), C-2′ (119.7), C-6′ (124.4), C-3′ (140.8)), C-7′ (132.3), (C-3′ (140.8))b

), C-7′ (132.3)), C-7′ (132.3), C-2′ (119.7), C-3′ (140.8)), C-2 (159.6), C-4 (184.9), (C-4′ (40.1))b, (C-6 (95.7))b, C-2′ (119.7), C-3′ (140.8), C-5 (149.1), (C-4 (184.9))b

med on solvent signals: ı (MeOH-d4) = 3.31 (1H), 49.1 (13C)). J values are in Hz in

96 S. Herter et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 91– 97

Table 31H and 13C NMR assignments and HMBC correlations for the diaminated product 6b isolated from a reaction between 2-methoxy-3-methylhydroquinone 1 and geranylamine3b.a .

NH

NH

O

Me

H

O

1 2

3

45

6

1`` `

1`

2`3`

10`

4`

5`

6`7`8`

9`

1``

2``3``

4``

10``

5``

6``7`` 8``

9``

Pos. 13C 1H 1H,13C correlations (HMBC)

1 180.7 – –4 180.3 – –5 153.0 – –2 150.0 – –3′ 141.5 – –3′′ 141.1 – –7′ ,7′′ 132.8 (2) – –6′ ,6′′ 125.0 (2) 5.08, m, 2H C-9′ ,9′′ (26.9), C-8′ ,8′′ (17.9), C-5′ ,5′′ (27.4), C-4′ ,4′′ (40.6)2′′ 122.6 5.29, m, 1H C-10′′ (16.6), C-4′′ (40.6), C-1′′ (43.8)2′ 120.1 5.21, m, 1H C-10′ (16.6), C-4′ (40.6)3 103.3 – –6 92.6 5.20, s, 1H (C-3 (103.3))b, C-2 (150.0), C-4 (180.3)1′′ 43.8 4.20, d, 2H,

(6.3)C-5′′ (27.4), C-4′′ (40.6), C-2′′ (122.6), C-3′′ (141.1), C-2 (150.0)

1′ 41.5 3.80, d, 2H,(6.8)

C-5′ (27.4), (C-4′ (40.6))b, C-2′ (120.1), C-3′ (141.5), C-5 (153.0)

4′ ,4′′ 40.6 (2) 2.07, m, 4H C-10′ ,10′′ (16.6), C-5′ ,5′′ (27.4), C-2′ (120.1), C-2′′ (122.6), C-3′ (141.5), C-3′′ (141.1)5′ ,5′′ 27.4 (2) 2.12, m, 4H C-4′ ,4′′ (40.6), C-6′ ,6′′ (125.0), C-7′ ,7′′ (132.8), C-3′′ (141.1), C-3′ (141.5)9′ ,9′′ 26.0 (2) 1.66, s, 3H

1.65, s, 3HC-8′ ,8′′ (17.9), C-4,4′′ (40.6), C-6′ ,6′′ (125.0), C-7,7′′ (132.8)

8′ ,8′′ 17.9 (2) 1.60, br s, 3H1.59, br s, 3H

C-9′ ,9′′ (26.0), (C-4′ ,4′′ (40.6))b, C-6′ ,6′′ (125.0), C-7′ ,7′′ (132.8)

10′ 16.6 1.73, s, 3H C-4′ (40.3), C-2′ (120.1), C-3′ (141.5)10′′ 16.6 1.70, s, 3H C-4′′ (40.6), C-2′′ (122.6), C-3′′ (141.1)1′ ′ ′ 10.4 2.05, s, 3H C-3 (103.3), C-2 (150.0), C-4 (180.3)5′ ,5′′ 27.4 (2) 2.12, m, 4H C-4′ ,4′′ (40.6), C-6′ ,6′′ (125.0), C-7′ ,7′′ (132.8), C-3′′ (141.1), C-3′ (141.5)9′ ,9′′ 26.0 (2) 1.66, s, 3H

1.65, s, 3HC-8′ ,8′′ (17.9), C-4,4′′ (40.6), C-6′ ,6′′ (125.0), C-7,7′′ (132.8)

8′ ,8′′ 17.9 (2) 1.60, br s, 3H1.59, br s, 3H

C-9′ ,9′′ (26.0), (C-4′ ,4′′ (40.6))b, C-6′ ,6′′ (125.0), C-7′ ,7′′ (132.8)

10′ 16.6 1.73, s, 3H C-4′ (40.3), C-2′ (120.1), C-3′ (141.5)1′ ′ ′ 10.4 2.05, s, 3H C-3 (103.3), C-2 (150.0), C-4 (180.3)

perforb

cHars(cHarawbus1tsimH−f2

a Chemical shifts are expressed in ı (ppm) referred to TMS = 0 (calibration was

rackets.b Correlation with low intensity.

The signal appearing at 5.22 ppm in the 1H NMR spectrum of 5bould be assigned to H-6. A signal of the ortho-neighboured proton-5 did not occur. Therefore, initial consideration was given formine coupling at the C-5-atom. Signals at 4.04 ppm and 1.86 ppmespectively could be related to methyl protons of the methoxyubstituent (H-1′′) and the protons of the methyl substituentH-2′′). A coupling of geranylamine 3b to C-5-position was clearlyonfirmed by the correlation of the proton H-1′ with C-5 in theMBC spectrum, whereas additional relatively weak correlationsppeared also with C-6 and C-4. All further signals in the aliphaticange could be assigned to geranylamine 3b. Presence of either

quinoidic, hydroquinoidic or quinone imine parent structureas obvious in 13C and HMBC data. As the carbonyl-groups of

enzoquinones possess low-field resonance signals (180 ppmp to 190 ppm), compound 5b was identified as a 1,4-quinoidictructure according to 13C signals at 181.8 ppm for C-1 and84.9 ppm for C-4. Moreover, the coupling between the H-2′′ pro-ons with the C-4 as well as between H-6 and C-4 and C-1 (weak)trengthened the presence of a 1,4-benzoquinoidic structuren 5-[[(2E)]-3,7-dimethylocta-2,6-dienyl]-amino]-2-methoxy-3-

ethyl-[1,4]-benzoquinone (5b). Additionally, data obtained from

R-MS measurement of product 5b ([M+H]+ 304.19013 (error:1.95 ppm); [M+Na]+ 326.1736 (error: 2.87 ppm), molecular

ormula: C18H26NO3) reconfirmed presence of a monoaminated-methoxy-3-methylbenzoquinone.

med on solvent signals: ı (MeOH-d4) = 3.31 (1H), 49.1 (13C)). J values are in Hz in

Consequently, monoamination reactions on 1 led to 1,4-benzoquinoid coupling products 5a–c, revealing amine coupling inpara-position to benzoquinone’s methoxy substituent and in meta-position to the methyl substituent, respectively (Scheme 1). Withregard to lower-concentration regioisomer products 4a–c, aminecoupling at benzoquinones C-6-atom could be assumed, howeverthis was not examined within this study.

Structural characterisation of diaminated products 6a–c isexplained as with compound 6b obtained from a reaction between1 and geranylamine 3b (Table 3). HR-MS recorded in positive modegave for the pseudo-molecule ion [M+H]+ a mass of 425.3165 amu(error: 0.57 ppm) and a molecular formula of C27H41N2O2. The cor-responding sodium adduct [M+Na]+ (447.29853, error 0.74 ppm,molecular formula: C27H40N2NaO2) was also detected. With ref-erence to pseudo-molecule ions masses and molecular formulas,a diamination by coupling of two geranylamine molecules wasclearly proven as product 6b revealed presence of two nitrogenatoms. However, only two oxygen atoms appeared in the analytemolecule indicating loss of one of the oxy-functionalised groupsof 2-methoxy-3-methylbenzoquinone 2a, either belonging to thecarbonyl groups or the methoxy substituent. With interpretation

of the 1H NMR spectrum of compound 6b, the signal appearingat 5.20 ppm was dedicated to H-6. Furthermore, the signal formethyl protons of the 2a methyl substituent at 2.05 ppm was alsodetected. However, a signal for methyl protons of the methoxy

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S. Herter et al. / Journal of Molecul

ubstituent did not appear as determined for the monoaminatedroduct 5b (cf. Table 2). Therefore, loss of the methoxy substituentue to coupling of a geranylamine molecule was indicated. More-ver, besides HMBC correlation between the H-1′ protons of therst coupled geranylamine with C-5 (monoamination), additionalorrelations of the H-1′′ protons of the second coupled amineolecule with C-2 appeared. Presence of carbonyl-groups due to

iamination and therefore existence of a 1,4-benzoquinoidic ringas again clearly observed by the low-field resonance signals at

80.7 ppm (C-4) and 180.3 ppm (C-4). Hence, diamination reactionn 2-methoxy-3- methylbenzoquinone 2a entailed a replacementeaction of benzoquinone’s methoxy group (Scheme 1) and thusccurred in para-position to the first amine substituent. Sim-lar to the herein determined nucleophilic replacement of the

ethoxy group of 2b due to diamination reactions, Schäfer andguado [33] previously described comparable mechanisms for

he sodium-iodate catalysed diamination reaction on alkylsubsti-uted para-hydroquinones. In an earlier study we have alreadyescribed a comparable replacement reaction for the monoam-

nation of ortho-dihydroxylated 3-methylcatechol, in which aethoxy-activated radical intermediate was identified as pre-

ursor for a following amination, entailing the replacement of theethoxy substituent [25]. Furthermore, our results obtained for

tructural characterisation of diaminated mitomycin-like productsa–c stand in accordance with other works focused on diaminationf para-hydroquinones whereby coupling of the second amine tohe monoaminated compound usually appeared para to the firstmine substituent [22,34,35].

. Conclusions

The reactions presented herein provide a convenient approacho amino-functionalised para-benzoquinones with structural alike-ess to known mitomycins. The laccase-mediated amine couplingo 2-methoxy-3-methylhydroquinone 1 confers two of the essen-ial pharmacologically active motifs from mitomycins: (i) a stable,4-benzoquinoic parent structure and (ii) a biological active alkyl-tion function ( NH). Reactions can be driven to the formationf monoaminated mitomycin-like products in particular in a MtL-atalysed reaction system at pH 7.0, and within this, by using equalatios of enzyme substrate and amine. Diaminated mitomycin-likeroducts are obtained in high yields in the PcL-catalysed reactionpH 5.0) and in general when amine donors are applied in excess.

onflict of interest statement

The authors declare that they have no conflicts of interest.

cknowledgments

Financial support by the Deutsche Bundesstiftung Umwelt

Osnabrück, Germany, grant no. AZ13191) is gratefully acknowl-dged. S. H. is also grateful to the government of Mecklenburg-orpommern, Germany, for financial support in the frameworkf Landesgraduiertenförderung. We thank M. L. Thompson

[[[

lysis B: Enzymatic 90 (2013) 91– 97 97

(Manchester Institute of Biotechnology, University of Manchester)for help in preparing the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.molcatb.2013.01.020.

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