Biocatalysis with Laccases: an Updated Overview

catalysts

Review Biocatalysis with Laccases: An Updated Overview

Ivan Bassanini, Erica Elisa Ferrandi, Sergio Riva * and Daniela Monti *

Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” (SCITEC), CNR, Via Mario Bianco 9, 20131 Milano, Italy; [email protected] (I.B.); [email protected] (E.E.F.) * Correspondence: [email protected] (S.R.); [email protected] (D.M.)

Abstract: Laccases are multicopper oxidases, which have been widely investigated in recent decades thanks to their ability to oxidize organic substrates to the corresponding radicals while producing water at the expense of molecular oxygen. Besides their successful (bio)technological applications, for example, in textile, petrochemical, and detoxifications/bioremediations industrial processes, their synthetic potentialities for the mild and green preparation or selective modification of fine chemicals are of outstanding value in biocatalyzed organic synthesis. Accordingly, this review is focused on reporting and rationalizing some of the most recent and interesting synthetic exploitations of laccases. Applications of the so-called laccase-mediator system (LMS) for alcohol oxidation are discussed with a focus on carbohydrate chemistry and natural products modification as well as on bio- and chemo-integrated processes. The laccase-catalyzed Csp2-H bonds activation via monoelectronic oxidation is also discussed by reporting examples of enzymatic C-C and C-O radical homo- and hetero-couplings, as well as of aromatic nucleophilic substitutions of hydroquinones or quinoids. Finally, the laccase-initiated domino/cascade synthesis of valuable aromatic (hetero)cycles, elegant strategies widely documented in the literature across more than three decades, is also presented.

Keywords: laccase; biocatalysis; oxidation; stereoselectivity; laccase-mediator systems; Csp2-H bonds activation; radical C-O and C-C couplings; cycloadditions

Citation: Bassanini, I.; Ferrandi, E.E.; Riva, S.; Monti, D. Biocatalysis with 1. Introduction Laccases: An Updated Overview. Catalysts 2021, 11, 26. Laccases are enzymes belonging to the blue multicopper oxidase family that catalyze https://doi.org/10.3390/ the oxidation of a wide range of substrates, such as phenols and aromatic or aliphatic catal11010026 amines, thereby reducing molecular oxygen to water. They are widely distributed in nature as they have been described in bacteria, fungi, higher plants and insects [1]. In eukaryotes, Received: 4 December 2020 laccases are usually glycosylated and the carbohydrate component seems to ensure lac- Accepted: 23 December 2020 case conformational stability and to preserve enzymes from inactivation by radicals and Published: 28 December 2020 proteolysis [2]. These enzymes exist in various forms. In fact, although most of them are monomeric, Publisher’s Note: MDPI stays neu- some have been reported to be homodimeric, heterodimeric, and multimeric, with molecu- tral with regard to jurisdictional claims lar mass ranging from 50 to 140 kDa, including their sugar component. Their amino acid in published maps and institutional sequence could span from 220 to 800 amino acids and may contain 2 or 3 cupredoxin-like afﬁliations. domains depending on the organism they belong to. These domains bind copper centers involved in inter-molecular electron transfer reactions and constitute the catalytic core of laccases. Despite the fact that laccases from each species exhibit peculiar catalytic character-

Copyright: © 2020 by the authors. Li- istics and sequences, their molecular architecture is common for all multicopper oxidases censee MDPI, Basel, Switzerland. This showing a simple 3D structure mainly constituted of beta sheets and turns [2,3]. article is an open access article distributed Since the discovery in 1896 of the ﬁrst fungal laccase in a mushroom of the Boletus under the terms and conditions of the genus [2], laccases have been found in more than 60 fungal strains, and fungal laccases Creative Commons Attribution (CC BY) represent the most signiﬁcant group of the blue multicopper oxidase family with regard to license (https://creativecommons.org/ the number and extent of characterization [1,4,5]. Typical fungal laccases are 60–70 kDa licenses/by/4.0/).

Catalysts 2021, 11, 26. https://doi.org/10.3390/catal11010026 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, 26 2 of 30

monomeric glycoproteins containing three cupredoxin-like domains that bind four copper atoms [3]. The catalytic mechanism of laccases has been well characterized and involves the formation of radical species that can follow different pathways giving either dimers or polymers of their parent substrates by oxidative coupling reactions or yielding dead end products through intramolecular rearrangements. Furthermore, radical species may play as mediators by oxidizing non-phenolic compounds, and thus causing, i.a., bond cleavage [6]. In general, the physiological function of these enzymes is indeed related to their ability to catalyze polymerization or depolymerization processes. For example, in fungi these biocatalysts are involved in lignin degradation, in plants are key in the lignification process and cell wall formation [5], while in insects they seem to participate in the sclerotization of the cuticle [7]. Both the possibility to use laccases in synthetic or degradative processes and their wide substrate specificity make these enzymes suitable “green tools” for a plethora of applications in different fields, such as textile, paper, food, pharmaceutical and cosmetics [6,8]. In particular, their main technological applications are in the textile industry, e.g., for textile properties improvement and in processes related to fiber bleaching and dyeing, in the pulp and paper industries for the delignification of woody fibers, and in the food industry for food improvement [1,6,9]. The industrial interest toward laccases is also well documented by the large number of patents filed during the last years, as recently reviewed [10]. Besides these applications, laccases have gained wide attention in recent years for the one-pot synthesis of complex scaffolds, the selective modification of natural products by oxidation, as well as the biocatalytic activation of normally inert Csp2-H bonds. Presently, to the best of our knowledge, they have not found large scale (industrial) applications. However, as it is nowadays well documented in the scientific literature, these enzymes have indeed high potential for a significant impact in organic synthesis. In this review, we will describe the numerous reaction outcomes that can derive from their catalytic action.

2. Laccases Catalytic Cycle and Their Use in Non-Conventional Reaction Systems The catalytic cycle of laccases starts with the mono-electronic oxidation of four equiva- lent suitable reducing substrates, such as phenols or aromatic and aliphatic amines to form organic radicals at the expense of molecular oxygen which is eventually reduced to two molecules of water. A four-membered copper cluster represents the catalytic machinery of these enzymes, being also the site of oxygen coordination and reduction as well as of water formation and release. In fungal laccases, three different sites are typically identified in the copper cluster depending on their role in the catalytic cycle: the so-called “blue site” or Type 1 (T1), “normal site” or Type 2 (T2) and the “binuclear site” or Type 3 (T3) (Scheme1 ). Specifically, the T1 copper is involved in substrate oxidation, while T2 and T3 coppers, which define a trinuclear sub-cluster, catalyze the reduction in molecular oxygen [1,6,11]. From a chemical perspective, the action of laccases in synthetic processes is generally described by one of the simplified mechanisms reported below. All of them relay on the redox activity of the copper cluster located in the active site (Scheme2). When the substrate of interest possesses the proper redox potential, laccases can be used to directly oxidize it to the corresponding organic radical(s) (Scheme2a). Instead, when a direct oxidation in not achievable due to steric hindrance/active site penetration and/or redox potentials incompatibility, the use of the so-called “chemical mediators”, acting as redox intermediates in a nature mimicking-fashion strategy (e.g., the cytochrome chain), is generally applied with success. Accordingly, the free radicals originating from the oxidations of these chemical mediators can both interact with bulky or high redox-potential substrate targets (Scheme2b). This versatile redox apparatus, known in the literature by the name of “laccase-mediator-system” (LMS), represents one of the most reported laccase application both in organic synthesis, i.e., for the oxidation of activated hydroxyl groups to the corresponding carbonyls or acids, and in (bio)technological and manufacturing processes [12–16]. Moreover, the LMS can be also applied for cofactor regeneration in Catalysts 2021, 11, 26 3 of 30

coupled, multienzymatic redox transformations (Scheme2c): Haltrich and co-workers exploited the LMS to efﬁciently regenerate the FAD cofactor in a multienzymatic redox process [17]. Ferrandi et al., instead, applied LMS, in the form of laccases from Trametes pubescens and the redox intermediate Meldola Blue, to the regeneration of NAD+ in the Catalysts 2021, 11, x FOR PEER REVIEW 3 of 34 multigram, biocatalytic preparation of 7-keto derivatives of cholic acid catalyzed by a 7α-hydroxysteroid dehydrogenase [18]. More recently, Hanefeld and coworkers expanded this topic using the LMS in the biocatalytic oxidation of the 12α-OH group of hydroxysteroids for the preparation of bile acids derivatives by employing a 12α-hydroxysteroid Catalysts 2021, 11, x FOR PEER REVIEW 3 of 34 dehydrogenase in combination with laccases of different origins and a plethora of redox mediators [19].

Scheme 1. Schematic representation of the active site of the laccase from Trametes versicolor and of the reactions catalyzed in a redox cycle [1].

From a chemical perspective, the action of laccases in synthetic processes is generally SchemeSchemedescribed 1. 1. SchematicSchematic by one representation representationof the simplified of of the the mechanisms active active site site of the reported laccase below. from TrametesTrametes All of versicolor versicolorthem relayand and of onof the the thereactionsredox reactions activity catalyzed catalyzed of in the ain redox acopper redox cycle cyclecluster [1]. [1]. located in the active site (Scheme 2).

From a chemical perspective, the action of laccases in synthetic processes is generally described by one of the simplified mechanisms reported below. All of them relay on the redox activity of the copper cluster located in the active site (Scheme 2).

SchemeScheme 2. 2.Laccase-catalyzed Laccase-catalyzed redox redox cycles cycles for for substrate substrate oxidation: oxidation: (a )(a direct) direct substrate substrate oxidation; oxidation; (b ()b substrate) substrate oxidation oxidation in in thethe presence presence of of a chemicala chemical mediator; mediator; (c )(c cofactor) cofactor regeneration regeneration in in multienzymatic multienzymatic coupled coupled redox redox biotransformations biotransformations [1]. [1].

FromWhen a practical the substrate point of view,interest the possesses synthetic applicationsthe proper redox of enzymes potential, very laccases often require can be Scheme 2. Laccase-catalyzednon-conventional redoxused cycles to directly for substrate oxidize reaction oxidation: it systems, to the (a )correspond direct including substrateing those organicoxidation; applying radical(s) (b) organicsubstrate (Scheme solventsoxidation 2a). orin Instead, other the presence of a chemical mediator;mediawhen capable (ac) directcofactor of oxidationdissolving regeneration in hydrophobic not in multienzymatic achievable substrates, due coupled to steric as redox well hindrance/active as biotransformations those where thesite [1]. biocatalystpenetration isand/or immobilized/compartmentalized redox potentials incompatibility, and thethe biotransformationuse of the so-called is carried“chemical out mediators”, in either batch-based or ﬂow-based bioprocesses. actingWhen as theredox substrate intermediates of interest in a possessesnature mimicking-fashion the proper redox strategy potential, (e.g., laccases the cytochrome can be usedchain), to directly is generally oxidize applied it to the with correspond success. Accordingly,ing organic radical(s) the free radicals (Scheme originating 2a). Instead, from whenthe aoxidations direct oxidation of these in chemical not achievable mediators due cato nsteric both hindrance/active interact with bulky site orpenetration high redox- and/orpotential redox substrate potentials targets incompatibility, (Scheme 2b). the This use versatile of the so-calledredox apparatus, “chemical known mediators”, in the lit- actingerature as redox by the intermediates name of “laccase-mediator-syste in a nature mimicking-fashionm” (LMS), strategy represents (e.g., one the ofcytochrome the most re- chain),ported is generallylaccase application applied with both success. in organic Accordingly, synthesis, the i.e., free for radicals the oxidation originating of activated from thehydroxyl oxidations groups of these to thechemical corresponding mediators carbonyls can both or interact acids, andwith in bulky (bio)technological or high redox- and potentialmanufacturing substrate processes targets (Scheme [12–16]. 2b). Moreover, This versatile the LMS redox can apparatus,be also applied known for in cofactor the lit- re- eraturegeneration by the in name coupled, of “laccase-mediator-syste multienzymatic redoxm” transformations (LMS), represents (Scheme one of2c): the Haltrich most re- and ported laccase application both in organic synthesis, i.e., for the oxidation of activated hydroxyl groups to the corresponding carbonyls or acids, and in (bio)technological and manufacturing processes [12–16]. Moreover, the LMS can be also applied for cofactor regeneration in coupled, multienzymatic redox transformations (Scheme 2c): Haltrich and

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Since laccases substrates show in most cases a poor solubility in water, the use of water-miscible organic cosolvents is very common and generally well-tolerated by these enzymes. A systematic evaluation of the effects of various organic solvents on the activity of the plant laccase from Rhus vernicifera, in either free or immobilized form, was performed in 2010 by Wan and coauthors [20]. The results showed that the amounts of organic cosolvents that could be applied without a significant decrease in laccase performances vary depending on the specific solvent, but could be up to 50% v/v. A very good tolerance toward different water-miscible cosolvents, e.g., ethanol, methanol, dimethyl sulfoxide and acetone, was shown also by the alkaline laccase from Bacillus licheniformis LS04 [21]. Several examples showing the use of organic cosolvents and of water-organic solvent biphasic systems in biotransformations catalyzed by fungal laccases, such as the laccase from Myceliophthora thermophila [22–24], Trametes versicolor [25–27], and Agaricus bisporus [28–30], have been reported in the literature as well. Recently, the possible application in laccase-catalyzed biotransformations of the so- called natural deep eutectic solvents (NADES), alternative non-toxic reaction media com- posed by mixtures of hydrogen bond donors, e.g., polyols, and hydrogen bond acceptors, e.g., ammonium salts, has been investigated by different research groups. Some preliminary studies on the influence of various NADES on the activity and stability of microbial laccases [31,32], showed that both choline- or betaine-based media could be applied as “green solvents” in place of organic solvents. Further investigations highlighted the need of a careful choice of NADES components. For instance, a strong inhibitory effect was observed when using choline chloride as hydrogen bond acceptor, but it could be easily overcome by replacing the chloride anion with either dihydrogen citrate or dihydrogen phosphate anions [33]. In addition, the possible effect of NADES on the overall reaction environments, e.g., on the reaction pH, has to be carefully taken into account since both laccase activity and stability could be significantly affected [34]. As recently reviewed [35], as far as laccase immobilization concerns, a wide number of supports, either inorganic, organic, or hybrids, have been investigated during the last few years, mostly prompted by the technological application of this class of enzymes in bioremediation and in the textile industry. The use of immobilized laccases in organic synthesis is documented as well [1,36]. For example, in 2005, immobilized samples of the M. thermophila laccase were obtained by adsorption on glass beads or celite and used in the oxidation of the model substrate 5,6,7,8-tetrahydronaphthlen-2-ol, thus studying the influence of different organic solvents on enzyme selectivity [23]. More recently, the T. versicolor laccase was instead covalently immobilized onto magnetic fibrous silica-based nanopar- ticles, carriers showing high surface/volume ratio and good mechanical properties [37]. The immobilized biocatalyst showed excellent stability by retaining about 80% of its initial activity after 15 reaction cycles and was successfully applied, in the presence of suitable chemical mediators, in the oxidation of different aromatic alcohols to the corresponding aldehydes, as well as in the one-pot synthesis of a set of chromene derivatives. Alternatively, the same laccase has been recently immobilized using, as support, a magnetic-graphene oxide-based nanocomposite [38]. The performances of this heterogeneous biocatalyst were tested in the synthesis of various sulfonamide derivatives, which could be obtained with up to 90% yields. Moreover, the immobilized laccase could be reused for eight reaction cycles, at the end of which the residual activity was about 85% of the starting one, thus showing quite good operational stability. Surprisingly, despite the growing interest toward the use of immobilized enzymes in flow systems, i.e., the so-called flow biocatalysis [39,40], the examples with laccases in this field are still scarce and showing quite preliminary results. For instance, a flow bioreactor was recently prepared by immobilizing the T. versicolor laccase on gold-impregnated poplar wood samples and tested in continuous flow biocatalysis experiments [41]. The immobilized biocatalyst showed a remarkable stability by retaining about 90% of its starting activity after 25 cycles. However, the bioreactor performances were tested only in the oxidation of the artificial chemical mediator ABTS [2,20-azino-bis(3-ethylbenzothiazoline-6- Catalysts 2021, 11, 26 5 of 30

sulfonic acid)] and not in other target reactions of synthetic interest. In another very recent example, laccases were included in a study of 3D-printed continuous flow bioreactors [42]. Unfortunately, when compared to other types of enzymes, e.g., alkaline phosphatase and glucose dehydrogenase, the results obtained with laccases showed very high levels of experimental error that hampered a definitive evaluation of this flow system. As stated by the authors, this variability could be possibly due to different oxygen diffusion rates in the flow system, thus suggesting the need of its further implementation to achieve a better control of reaction kinetics.

3. Synthetic Exploitation of Laccases 3.1. Laccase-Mediator Systems: From Alcohols Bio-Oxidation to Integrated Chemoenzymatic Systems A selective and precise manipulation of the oxidation state of hydroxyl and carbonyl groups represents a versatile tool to access to (chiral) synthetic intermediates or to achieve late-stage modiﬁcations in complex (semi)synthetic compounds or natural products derivatives. Allowing the selective introduction of ketones or aldehydes in the framework of complex molecular skeletons (i.e., natural compounds and/or polyhydroxylated substrates) while working in mild and “green” conditions, the biocatalytic oxidation of primary and secondary alcohols is among the most documented enzymatic procedures applied in organic synthesis [43,44]. Among the plethora of different redox biocatalysts available to a synthetic chemist, the LMS offers the possibility of easily converting primary alcohols and activated (i.e., allylic and benzylic) secondary alcohols into the corresponding aldehydes, acids or ketones. A large number of reports are available in the literature on this topic and they have been collected in numerous review articles published through the years [5,12–16,36,45–47]. Some interesting applications of the LMS focusing on the preparation of bioactive compounds as well as the selective oxidation of valuable and bioactive naturally occurring compounds have also been described [48–50]. In addition, LMS has been successfully applied for the thermodynamically disfavored oxidation of β,β-dihalogenated secondary alcohols (i.e., 2,2-dihalo-1-phenylethanol derivatives) working in a biphasic medium and providing the corresponding ketones [51]. Given their ability to oxidize lignin or lignin models [52–54], more than one hun- dred compounds have been tested as component of LMS; among them, some of the most synthetically employed are reported in Figure1. Both natural, i.e., small molecules involved in laccase-catalyzed lignin degradation in fungi such as 3-hydroxyanthranilic acid (HAA, Figure1a ), and artiﬁcial chemical mediators, e.g., ABTS [2,20-azino-bis(3- Catalysts 2021, 11, x FOR PEER REVIEW 6 of 34 ethylbenzothiazoline-6-sulfonic acid] or TEMPO (2,2,6,6-tetramethylpiperidine) (Figure1b,c , respectively), have been employed in the LMS for alcohol oxidation [45,54–56].

Figure 1. Examples of chemical mediators employed in laccase-mediator system (LMS). Figure 1. Examples of chemical mediators employed in laccase-mediator system (LMS). (a) 3-Hy- droxyanthranilic(a) 3-Hydroxyanthranilic acid (HAA); (bacid) 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic (HAA); (b) 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); acid) (c) 2,2,6,6-tetramethyl(ABTS); (c) 2,2,6,6-tetramethyl piperidine-1-yloxy piperidine-1-yloxy (TEMPO); (d) N-hydroxybenzo-triazole (TEMPO); (d) N-hydroxybenzo-triazole (HBT); (e) vio- (HBT); luric(e acid) violuric (VLA); acid (f) methyl (VLA); ester (f) methylof 4-hydroxy-3,5-dime ester of 4-hydroxy-3,5-dimethoxy-benzoicthoxy-benzoic acid (syringic acid). acid (syringic acid).

The exploitation of TEMPO or ABTS and laccases for the oxidation of primary alcohols and even benzylic secondary alcohols was reported years ago [57,58]. Later on, the laccase from T. pubescens, together with TEMPO, was used to catalyze the regioselective oxidation of the primary hydroxyl groups in a panel of sugar derivatives to the corresponding carboxylic acids via the intermediate aldehydes (Scheme 3a). The efficiency of this system was initially tested with mono- and disaccharides (i.e., phenyl β-D-glucopyranoside), and then this LMS approach was also exploited to achieve the partial oxidation of a water soluble cellulose sample [59]. The same approach was exploited for the efficient and mild oxidation of more complex bioactive glycosides, e.g., thiocolchicoside (Scheme 3b) [48].

Scheme 3. Exploitation of laccase-mediator systems for the regioselective oxidation of primary hydroxyl groups. (a) Oxi- dation of glucosides to the corresponding glucuronic acids; (b) oxidation of the bioactive glucoside thiocolchicoside [48].

The oxidation of natural polysaccharides by TEMPO (regenerated in situ by different oxidants) is an old reaction and has been recently reviewed [60]. In comparison to other approaches, the use of laccases for the reoxidation of TEMPO proved to be very mild and, promoting a lower degree of oxidations, resulted in the formation of soft gels. The prop-

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Figure 1. Examples of chemical mediators employed in laccase-mediator system (LMS). (a) 3-Hy- droxyanthranilic acid (HAA); (b) 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); (c) 2,2,6,6-tetramethyl piperidine-1-yloxy (TEMPO); (d) N-hydroxybenzo-triazole (HBT); (e) violuric acid (VLA); (f) methyl ester of 4-hydroxy-3,5-dimethoxy-benzoic acid (syringic acid). Catalysts 2021, 11, 26 6 of 30 The exploitation of TEMPO or ABTS and laccases for the oxidation of primary alcohols and even benzylic secondary alcohols was reported years ago [57,58]. Later on, the laccaseThe from exploitation T. pubescens of TEMPO, together or ABTS with andTEMPO, laccases was for used theoxidation to catalyze of primarythe regioselective alcohols andoxidation even benzylic of the primary secondary hydroxyl alcohols groups was reported in a panel years of ago sugar [57, 58derivatives]. Later on, to the the laccase corre- fromspondingT. pubescens carboxylic, together acids with via the TEMPO, intermediat was usede aldehydes to catalyze (Scheme the regioselective 3a). The efficiency oxidation of ofthis the system primary was hydroxyl initially tested groups with in a mono- panel and of sugar disaccharides derivatives (i.e., to phenyl the corresponding β-D-glucopy- carboxylicranoside), acidsand then via the this intermediate LMS approach aldehydes was also (Scheme exploited3a). to The achieve efﬁciency the partial of this oxidation system wasof a initiallywater soluble tested cellulose with mono- sample and [59]. disaccharides The same approach (i.e., phenyl was exploitedβ-D-glucopyranoside), for the efficient andand then mild this oxidation LMS approach of more wascomplex also exploited bioactive toglycosides, achieve the e.g., partial thiocolchicoside oxidation of (Scheme a water soluble3b) [48]. cellulose sample [59]. The same approach was exploited for the efﬁcient and mild oxidation of more complex bioactive glycosides, e.g., thiocolchicoside (Scheme3b) [48].

Scheme 3. Exploitation of laccase-mediator systems for the regioselective oxidation of primary hydroxyl groups. (a) Oxida- tionScheme of glucosides 3. Exploitation to the correspondingof laccase-mediator glucuronic systems acids; for (theb) oxidationregioselective of the oxidation bioactive of glucoside primary thiocolchicosidehydroxyl groups. [48 (a].) Oxi- dation of glucosides to the corresponding glucuronic acids; (b) oxidation of the bioactive glucoside thiocolchicoside [48]. Catalysts 2021, 11, x FOR PEER REVIEW The oxidation of natural polysaccharides by TEMPO (regenerated in situ by7 of differ- 34 ent oxidants)The oxidation is an oldof natural reaction polysaccharides and has been by recently TEMPO reviewed (regenerated [60]. Inin situ comparison by different to otheroxidants) approaches, is an old the reaction use of and laccases has been for therecently reoxidation reviewed of TEMPO[60]. In comparison proved to beto veryother mildapproaches, and, promoting the use of a lowerlaccases degree for the of reoxid oxidations,ation of resulted TEMPO in proved the formation to be very of mild soft gels. and, erties of these materials, specifically those obtained by the LMS oxidation of guar polygal- Thepromoting properties a lower of these degree materials, of oxidations, speciﬁcally resulted those obtainedin the formation by the LMS of soft oxidation gels. The of guarprop- actomannanes,polygalactomannanes, was recently was investigated recently investigated by Galante by and Galante coworkers and coworkers and reported and in reported a se- riesin of a seriespublications of publications [61–67]. [61–67]. TheThe redox redox mechanism mechanism of ofTEMPO TEMPO usually usually prev preventsents the the oxidations oxidations of secondary of secondary alco- alco- hols.hols. Exceptions Exceptions are are given given by bybenzylic, benzylic, allylic allylic and and propargylic propargylic secondary secondary alcohols. alcohols. In Inan an earlyearly example example [68], [68 Passarella,], Passarella, Riva Riva and and cowo coworkersrkers described described the the enzyme enzyme assisted assisted enanti- enantios- oselectiveelective synthesis ofof thethe alkaloidalkaloid (+)-aloperine(+)-aloperine exploiting exploiting the the LMS LMS for for the the oxidations oxidations of of two twoalcoholic alcoholic intermediates, intermediates, as as shown shown in in Scheme Scheme4: 4:

SchemeScheme 4. Exploitation 4. Exploitation of LMS of LMS in the in the synthesis synthesis of the of the alkaloid alkaloid (+)-aloperine (+)-aloperine [68]. [68 ].

The biocatalytic production of enantiomerically enriched alcohols is also of great synthetic interest. Clearly, laccases and LMS by themselves cannot fulfill this task, but the deracemization of chiral alcohols could be obtained by designing suitable multienzymatic and cascade processes as well as by exploiting integrated chemoenzymatic approaches [69–72]. For example, by sequentially applying the LMS-catalyzed alcohol oxidation and a stereoselective enzymatic transformation of the obtained prochiral carbonyl intermediate, racemic alcohols could be converted into enantiomerically enriched compounds, as shown in Scheme 5. In both cases TEMPO was the mediator used in combination with the laccase from T. versicolor. Martínez-Montero et al. were able to convert 17 differently substituted racemic (hetero)aromatic sec-alcohols into enantio-enriched amines. Enantioselec- tivity and conversions were up to 99%, depending on the substrate, by building a one- pot/two-step process combining (R)- or (S)-selective aminotransferases (ATAs) with the laccase (Scheme 5a) [73]. In a second example, González-Granda et al. combined the oxy- radical TEMPO-based LMS with a small library of NAD(P)H-dependent alcohol dehydrogenases (ADHs) for the deracemization of 1-arylprop-2-yn-1-ols through, again, a sequential one-pot two-step process. Propargylic ketones were obtained in quantitative conversions (87–99% yields), thus demonstrating the efficiency of LMS in comparison with traditional chemical oxidants, and their stereoselective reduction easily allowed the access to both (R) or (S) alcohol enantiomers depending on the aromatic pattern substitution (97– 99% e.e.) (Scheme 5b) [74].

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The biocatalytic production of enantiomerically enriched alcohols is also of great synthetic interest. Clearly, laccases and LMS by themselves cannot fulﬁll this task, but the deracemization of chiral alcohols could be obtained by designing suitable multienzymatic and cascade processes as well as by exploiting integrated chemoenzymatic approaches [69–72]. For example, by sequentially applying the LMS-catalyzed alcohol oxidation and a stereoselective enzymatic transformation of the obtained prochiral carbonyl intermediate, racemic alcohols could be converted into enantiomerically enriched compounds, as shown in Scheme5. In both cases TEMPO was the mediator used in combination with the laccase from T. versicolor. Martínez-Montero et al. were able to convert 17 differently substituted racemic (hetero)aromatic sec-alcohols into enantio-enriched amines. Enantioselectivity and conversions were up to 99%, depending on the substrate, by building a one-pot/two- step process combining (R)- or (S)-selective aminotransferases (ATAs) with the laccase (Scheme5a )[73]. In a second example, González-Granda et al. combined the oxy-radical TEMPO-based LMS with a small library of NAD(P)H-dependent alcohol dehydrogenases (ADHs) for the deracemization of 1-arylprop-2-yn-1-ols through, again, a sequential one- pot two-step process. Propargylic ketones were obtained in quantitative conversions (87–99% yields), thus demonstrating the efﬁciency of LMS in comparison with traditional Catalysts 2021, 11, x FOR PEER REVIEWchemical oxidants, and their stereoselective reduction easily allowed the access to both8 (ofR 34)

or (S) alcohol enantiomers depending on the aromatic pattern substitution (97–99% e.e.) (Scheme5b) [74].

Scheme 5. Applications of LMS in multienzymatic and cascade processes aimed at the preparation of enantioenriched aminesScheme (route 5. Applications (a)) or alcohols of LMS (route in (bmultienzymatic)) [73,74]. and cascade processes aimed at the preparation of enantioenriched amines (route (a)) or alcohols (route (b)) [73,74]. The synthesis of active pharmaceutical ingredients (APIs) is a fertile ground for the exploitationThe synthesis of multienzymatic of active pharmaceutical synthetic methodologies. ingredients (APIs) A three-step is a fertile biocatalytic ground proce-for the dureexploitation for the conversion of multienzymatic of methyl synthetic and ethyl meth cyclopentene-odologies. A and three-step cyclohexene-carboxylates biocatalytic proce- intodure both for thethe enantiomersconversion of of methyl the corresponding and ethyl cyclopentene- chiral 3-oxoesters, and cyclohexene-carboxylates useful intermediates en routeinto both to APIs, the enantiomers was described of bythe Brenna corresponding et al. in 2017chiral [75 3-oxoesters,]. In the designed useful intermediates synthetic path- en way,route the to laccase/TEMPOAPIs, was described system by Brenna was applied et al. toin oxidize2017 [75]. the In intermediate the designed allylic synthetic alcohols path- (obtainedway, the fromlaccase/TEMPO the allylic hydroxylation system was applied of starting to oxidize cycloalkene the intermediate carboxylates allylic catalyzed alcohols by Rhizopus(obtained oryzae fromresting the allylic cells hydroxylation entrapped in alginate of starting beads) cycloalkene to the corresponding carboxylates unsaturatedcatalyzed by ketones.Rhizopus The oryzae obtained resting products cells entrapped were then in subjected alginateto beads) an ene to reductase the corresponding (ERED)-mediated unsatu- hydrogenation of the alkene bond in the same reaction vessel in a sequential mode working rated◦ ketones. The obtained products were then subjected to an ene reductase (ERED)- atmediated 30 C in acetatehydrogenation buffer (Scheme of the 6alkene). bond in the same reaction vessel in a sequential modeIn working this framework, at 30 °C Mart in acetateínez-Montero buffer (Scheme et al. also 6). exploited the T. versicolor laccase and TEMPO to easily convert a series of racemic allylic sec-alcohols into the corresponding α,β-unsaturated ketones. These intermediate products could then be reduced by different commercially available EREDs, in concert with a glucose/glucose dehydrogenase (GDH) system for NADPH cofactor regeneration, into the corresponding saturated ketones as enantio-enriched species (Scheme7)[76].

Scheme 6. Multienzymatic synthesis of chiral cyclic γ-oxoesters by (i) allylic hydroxylation catalyzed by Rhizopus oryzae whole cells, (ii) oxidation of the allylic alcohols by LMS, and (iii) alkenes reduction by ene reductases [75].

In this framework, Martínez-Montero et al. also exploited the T. versicolor laccase and TEMPO to easily convert a series of racemic allylic sec-alcohols into the corresponding α,β-unsaturated ketones. These intermediate products could then be reduced by different commercially available EREDs, in concert with a glucose/glucose dehydrogenase (GDH) system for NADPH cofactor regeneration, into the corresponding saturated ketones as enantio-enriched species (Scheme 7) [76].

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Scheme 5. Applications of LMS in multienzymatic and cascade processes aimed at the preparation of enantioenriched amines (route (a)) or alcohols (route (b)) [73,74].

The synthesis of active pharmaceutical ingredients (APIs) is a fertile ground for the exploitation of multienzymatic synthetic methodologies. A three-step biocatalytic procedure for the conversion of methyl and ethyl cyclopentene- and cyclohexene-carboxylates into both the enantiomers of the corresponding chiral 3-oxoesters, useful intermediates en route to APIs, was described by Brenna et al. in 2017 [75]. In the designed synthetic pathway, the laccase/TEMPO system was applied to oxidize the intermediate allylic alcohols (obtained from the allylic hydroxylation of starting cycloalkene carboxylates catalyzed by Rhizopus oryzae resting cells entrapped in alginate beads) to the corresponding unsaturated ketones. The obtained products were then subjected to an ene reductase (ERED)- Catalysts 2021, 11, 26 8 of 30 mediated hydrogenation of the alkene bond in the same reaction vessel in a sequential mode working at 30 °C in acetate buffer (Scheme 6).

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Scheme 6. Multienzymatic synthesis of chiral cyclic γ-oxoesters by (i) allylic hydroxylation catalyzed by Rhizopus oryzae Scheme 6. Multienzymatic synthesis of chiral cyclic γ-oxoesters by (i) allylic hydroxylation catalyzed by Rhizopus oryzae wholewhole cells, cells, ( ii(ii)) oxidation oxidation of of the the allylic allylic alcohols alcohols by by LMS, LMS, and and (iii (iii) alkenes) alkenes reduction reduction by by ene ene reductases reductases [75 [75].].

Scheme 7. Use of LMS in the multienzymatic synthesis of enantiomerically enriched saturated ketones from the correspond- Scheme 7. Use of LMS in the multienzymatic synthesis of enantiomerically enriched saturated ketones from the corre- ingsponding racemic racemic allylic secallylic-alcohols sec-alcohols [76]. [76]. By combining chemical and biocatalytic strategies, integrated chemo-enzymatic pro- By combining chemical and biocatalytic strategies, integrated chemo-enzymatic processes offer virtually an endless resource of synthetic potency subjected to a chemist’s cesses offer virtually an endless resource of synthetic potency subjected to a chemist’s fan- fantasy. The LMS catalyzed oxidation of allylic alcohols, when conducted on properly tasy. The LMS catalyzed oxidation of allylic alcohols, when conducted on properly de- designed substrates, can trigger a series of subsequent structural rearrangements which signed substrates, can trigger a series of subsequent structural rearrangements which al- allow to easily manipulate the carbon skeleton of a molecule via the promotion of in- low to easily manipulate the carbon skeleton of a molecule via the promotion of intramo- tramolecular reactions. In 2018, Brenna et al. proposed a chemo-enzymatic processes tolecular prepare reactions. enantio-enriched In 2018, Brennacis or ettrans al. pr3-methylcyclohexan-1-oloposed a chemo-enzymatic starting processes from to the prepare corre- spondingenantio-enriched endocyclic cis or allylic trans tertiary 3-methylcyclohexan-1-ol alcohol. Specifically, starting the following from the catalytic corresponding steps were en- performeddocyclic allylic in a series:tertiary (i) alcohol. a laccase-triggered Specifically, [1the,3]-oxidative following catalytic rearrangement; steps were (ii) a performed reduction ofin C=Ca series: double (i) a bond laccase-triggered catalyzed by the[1,3]-oxidative ene reductase rearrangement; OYE1; (iii) a ADH (ii) a catalyzed reduction carbonyl of C=C reductiondouble bond(Scheme catalyzed8 )[77 by]. the The ene feasibility reductase of OYE1; the LMS (iii) to a catalyze ADH catalyzed the [1,3]-oxidative carbonyl reduc- rear- rangementtion (Scheme using 8) [77]. Bobbitt’s The feasibility salt (TEMPO of the+ BF4LMS-) into combinationcatalyze the [1,3]-oxidative with T. versicolor rearrange-laccase + - wasment successfully using Bobbitt’s demonstrated salt (TEMPO and studiedBF4 ) in incombination detail. Two with reaction T. versicolor media were laccase engineered: was suc- acessfully homogeneous demonstrated aqueous and system, studied highly in detail. efficient Two for reaction the transposition media were of substratesengineered: devoid a ho- ofmogeneous electron withdrawing aqueous system, groups highly (EWGs), efficient and fo ar heterogeneousthe transposition system of substrates exploiting devoid an im- of mobilizedelectron withdrawing laccase preparation groups (EWGs), to perform and thea heterogeneous reaction on EWG-containing system exploiting macrocyclic an immo- alkenolsbilized laccase or tertiary preparation alcohols into acetonitrile. perform the reaction on EWG-containing macrocyclic alkenolsInterestingly, or tertiary the alcohols LMS was in acetonitrile. also successfully applied by the Beifuss group in 2013 to catalyze the first-ever reported enzymatic Achmatowicz reaction, a rearrangement which transforms furfuryl alcohols into dihydropyrans (Scheme9a) [ 78]. In detail, LMS was used to oxidize bulky, disubstituted (5-alkylfuran-2-yl)carbinols using aerial oxygen and TEMPO, and selectively afforded 6-hydroxy-(2H)-pyran-3(6H)-ones with yields up to 90% (Scheme9b). In addition, starting from suitably substituted furan-2-yl carbinols as substrates, this procedure allowed the efficient preparation of (2H)-pyran-2,5(6H)-diones in a single step.

Scheme 8. Chemoenzymatic preparation of enantio-enriched cis or trans 3-methylcyclohexan-1-ols from a starting tertiary allylic alcohol: (i) [1,3]-oxidative rearrangement by a laccase/TEMPO+BF4- system; (ii) stereoselective reduction of the activated ketone by an ene reductase; (iii) ADH-catalyzed stereoselective carbonyl reduction. Alternative cascade processes were developed by coupling the ene reductase with either a pro(R)-ADH (iv) or a pro(S)-ADH (v) [77].

Interestingly, the LMS was also successfully applied by the Beifuss group in 2013 to catalyze the first-ever reported enzymatic Achmatowicz reaction, a rearrangement which transforms furfuryl alcohols into dihydropyrans (Scheme 9a) [78]. In detail, LMS was used to oxidize bulky, disubstituted (5-alkylfuran-2-yl)carbinols using aerial oxygen and TEMPO, and selectively afforded 6-hydroxy-(2H)-pyran-3(6H)-ones with yields up to

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Scheme 7. Use of LMS in the multienzymatic synthesis of enantiomerically enriched saturated ketones from the corresponding racemic allylic sec-alcohols [76].

By combining chemical and biocatalytic strategies, integrated chemo-enzymatic processes offer virtually an endless resource of synthetic potency subjected to a chemist’s fantasy. The LMS catalyzed oxidation of allylic alcohols, when conducted on properly designed substrates, can trigger a series of subsequent structural rearrangements which allow to easily manipulate the carbon skeleton of a molecule via the promotion of intramolecular reactions. In 2018, Brenna et al. proposed a chemo-enzymatic processes to prepare enantio-enriched cis or trans 3-methylcyclohexan-1-ol starting from the corresponding endocyclic allylic tertiary alcohol. Specifically, the following catalytic steps were performed in a series: (i) a laccase-triggered [1,3]-oxidative rearrangement; (ii) a reduction of C=C double bond catalyzed by the ene reductase OYE1; (iii) a ADH catalyzed carbonyl reduction (Scheme 8) [77]. The feasibility of the LMS to catalyze the [1,3]-oxidative rearrangement using Bobbitt’s salt (TEMPO+ BF4-) in combination with T. versicolor laccase was successfully demonstrated and studied in detail. Two reaction media were engineered: a homogeneous aqueous system, highly efficient for the transposition of substrates devoid of electron withdrawing groups (EWGs), and a heterogeneous system exploiting an immo- Catalysts 2021, 11, 26 bilized laccase preparation to perform the reaction on EWG-containing macrocyclic9 of 30 alkenols or tertiary alcohols in acetonitrile.

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SchemeScheme 8. 8.Chemoenzymatic Chemoenzymatic preparation preparation of of enantio-enriched enantio-enrichedcis cisor ortrans trans3-methylcyclohexan-1-ols 3-methylcyclohexan-1-ols from from a a starting starting tertiary tertiary + - allylic alcohol: (i) [1,3]-oxidative rearrangement by a laccase/TEMPO+ BF- system; (ii) stereoselective reduction of the allylic alcohol: (i) [1,3]-oxidative90% (Schemerearrangement 9b). Inby addition,a laccase/TEMPO startingBF from4 system;4 suitably (ii) stereoselective substituted reductionfuran-2-yl of carbinolsthe acti- as activated ketone by an ene reductase; (iii) ADH-catalyzed stereoselective carbonyl reduction. Alternative cascade processes vated ketone by an ene reductase;substrates, (iii) ADH-catalyzedthis procedure stereoselective allowed the effici carbonylent preparationreduction. Alternative of (2H)-pyran-2,5(6H)-diones cascade processes werewere developed developed by by coupling coupling the the ene ene reductase reductase with with either either a pro(a pro(R)-ADHR)-ADH (iv (iv) or) or a pro(a pro(S)-ADHS)-ADH (v ()[v)77 [77].]. in a single step. Interestingly, the LMS was also successfully applied by the Beifuss group in 2013 to catalyze the first-ever reported enzymatic Achmatowicz reaction, a rearrangement which transforms furfuryl alcohols into dihydropyrans (Scheme 9a) [78]. In detail, LMS was used to oxidize bulky, disubstituted (5-alkylfuran-2-yl)carbinols using aerial oxygen and TEMPO, and selectively afforded 6-hydroxy-(2H)-pyran-3(6H)-ones with yields up to

Scheme 9. (a) The Achmatowicz reaction; (b) LMS-catalyzed oxidation of furan-2-yl carbinols [78]. Scheme 9. (a) The Achmatowicz reaction; (b) LMS-catalyzed oxidation of furan-2-yl carbinols [78].

2 3.2.3.2. LaccasesLaccases asas GreenGreen CspCsp2-H-H BondsBonds ActivatorsActivators ModernModern syntheticsynthetic chemistry aims at at proposing proposing novel novel methods methods to to construct construct chemical chemi- calbonds bonds in the in theframework framework of complex of complex molecular molecular structures structures with with economical, economical, efficient, efﬁcient, and and environmentally benign approaches. Since their outbreak discovery, transition-metal environmentally benign approaches. Since their outbreak discovery, transition-metal cat- catalyzed organic reactions have been used in the construction of a variety of chemical alyzed organic reactions have been used in the construction of a variety of chemical bonds bonds since they can minimize pre-functionalization steps and waste formation via the since they can minimize pre-functionalization steps and waste formation via the catalytic catalytic functionalization of unreactive C–H bonds [79–83]. In fact, the direct oxidation of functionalization of unreactive C–H bonds [79–83]. In fact, the direct oxidation of an organic substrate to afford an organic radical is of particular interest. Organic radicals are indeed highly reactive species which undergo complex quenching mechanisms in the process of dissipating the excess in energy derived from the homolytic bond-breaking at the basis of their creation. By controlling the different quenching destines available to these unstable species, products of diverse chemical nature can theoretically be obtained. In the framework of this review, focused on the role and application of laccases in organic synthesis for the preparation of fine chemicals, the reaction pathway previously described in Scheme 2a, that is the activation of normally inert Csp2-H bonds, is of particular interest. In this scenario, laccases offer a “green” alternative to transition-metal catalyzed organic reactions, allowing one to operate in a one-step reaction under mild, eco- friendly conditions.

Catalysts 2021, 11, 26 10 of 30

an organic substrate to afford an organic radical is of particular interest. Organic radicals are indeed highly reactive species which undergo complex quenching mechanisms in the process of dissipating the excess in energy derived from the homolytic bond-breaking at the basis of their creation. By controlling the different quenching destines available to these unstable species, products of diverse chemical nature can theoretically be obtained. In the framework of this review, focused on the role and application of laccases in organic synthesis for the preparation of ﬁne chemicals, the reaction pathway previously Catalysts 2021, 11, x FOR PEER REVIEW 2 11 of 34 Catalysts 2021, 11, x FOR PEER REVIEWdescribed in Scheme2a, that is the activation of normally inert Csp -H bonds, is of11 par- of 34 ticular interest. In this scenario, laccases offer a “green” alternative to transition-metal catalyzed organic reactions, allowing one to operate in a one-step reaction under mild, eco-friendlyAccordingly, conditions. different products can be obtained depending on the molecular skeleton of Accordingly,theAccordingly, radicals and different different the functional products products groups can can be be obtainedin obstalledtained dependingon depending them and on substituentson the the molecular molecular as skeletonwell skele- as theofton thereaction of radicals the radicals media, and conditions theand functional the functional and groups partners groups installed (Scheme installed on 10). them on them and substituentsand substituents as well as well as the as reactionthe reaction media, media, conditions conditions and partnersand partners (Scheme (Scheme 10). 10).

Scheme 10. Schematic representation of the possible destinies of laccase-formed organic radicals. SchemeScheme 10. 10.Schematic Schematic representationrepresentation of of the the possible possible destinies destinies of of laccase-formed laccase-formed organic organic radicals. radicals. Specifically, when aromatic amines or phenols (substrates generally characterized by Specifically,Specifically, when when aromatic aromatic amines amines or or phenols phenols (substrates (substrates generally generally characterized characterized by by redoxredox potentials potentials suitable suitable to to be be directly directly processed processed by by laccase) laccase) are are biocatalytically biocatalytically oxidized, oxidized, redox potentials suitable to be directly processed by laccase) are biocatalytically oxidized, radicalradical formation formation occurs, occurs, at atfirst, first, on onthe the electr electronegativeonegative heteroatoms, heteroatoms, i.e., i.e.,nitrogen nitrogen or ox- or radical formation occurs, at first, on the electronegative heteroatoms, i.e., nitrogen or oxygen.oxygen. Thanks Thanks to mesomeric to mesomeric resonance, resonance, the the form formeded radicals radicals are are then then delocalized delocalized along along all all ygen. Thanks to mesomeric resonance, the formed radicals are then delocalized along all thethe carbonaceous carbonaceous skeleton skeleton of of the the fully fully conjugated conjugated ππ-system-system (i.e., (i.e., the the group group of of molecules molecules the carbonaceous skeleton of the fully conjugated π-system (i.e., the group of molecules shownshown in in Scheme Scheme 1111),), dede factofacto activating activating different different ring ring positions positions and and Csp Csp2-H2-H bonds bonds toward to- shown in Scheme 11), de facto activating different ring positions and Csp2-H bonds to- wardthe formation the formation of new of C-Cnew orC-C C-Het or C-Het bonds. bond Thes. useThe of use specific, of specific, ad hoc ad designed hoc designed substrates sub- ward the formation of new C-C or C-Het bonds. The use of specific, ad hoc designed sub- stratesand the and proper the proper engineering engineering of the of operative the operative reaction reaction conditions conditions allow allow us tous “select” to “select” the strates and the proper engineering of the operative reaction conditions allow us to “select” thequenching quenching processes processes of theof the reactive reactive species species promoting promoting different different reaction reaction mechanisms mechanisms and the quenching processes of the reactive species promoting different reaction mechanisms andthe the formation formation of different of different classes classes of products. of products. and the formation of different classes of products.

Scheme 11. Csp2-H radical activation of fully conjugated aromatic phenols and anilines. Scheme 11. Csp2-H radical activation of fully conjugated aromatic phenols and anilines. Scheme 11. Csp2-H radical activation of fully conjugated aromatic phenols and anilines. The use of laccase-catalyzed Csp2-H bonds activation for the synthesis of complex 2 molecularThe useskeletons of laccase-catalyzed is widely documented Csp -H bonds in the activation literature for thanks the synthesis to the ofsynthetic complex knowledgemolecular acquiredskeletons through is widely the documentedyears in the infields the ofliterature laccase-mediated thanks to formationthe synthetic of biarylknowledge compounds acquired [84–87], through as well the as years of formal in the nucleophilic fields of laccase-mediated aromatic substitutions formation [88– of 94].biaryl Selected compounds examples [84–87], will be as di scussedwell as ofin formalthe following. nucleophilic aromatic substitutions [88– 94]. Selected examples will be discussed in the following. 3.2.1. Radical C-O and C-C Couplings 3.2.1. Radical C-O and C-C Couplings The development of innovative and, most importantly, mild catalytic synthetic entries toThe biaryl development compounds of is innovativeamong the mostand, mostinvestigated importantly, research mild field catalytic of methodological synthetic en- organictries to chemistry biaryl compounds [95–97]. Beingis among potential the most axially investigated stereogenic research and chiralfield of due methodological to atropoi- organic chemistry [95–97]. Being potential axially stereogenic and chiral due to atropoi-

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The use of laccase-catalyzed Csp2-H bonds activation for the synthesis of complex molecular skeletons is widely documented in the literature thanks to the synthetic knowledge acquired through the years in the ﬁelds of laccase-mediated formation of biaryl compounds [84–87], as well as of formal nucleophilic aromatic substitutions [88–94]. Selected examples will be discussed in the following.

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The development of innovative and, most importantly, mild catalytic synthetic entries to biaryl compounds is among the most investigated research field of methodological somery,organic chemistrybiaryl compounds [95–97]. Being find potentialin fact wide axially (bio)technological stereogenic and and chiral biological due to atropoiso- applica- tions,mery, which biaryl compoundshas made them find versatile in fact wide and (bio)technological highly cherished andsynthetic biological targets. applications, Thus, the applicationwhich has made of enzymatic them versatile protocols and to highly enter cherishedbiaryl compounds synthetic is targets. of great Thus, interest the and applica- rep- resentstion of enzymaticone of the protocolsmost logical, to enter even biaryl if not compounds trivial at all, is ofsynthetic great interest exploitation and represents of direct laccaseone of theoxidation mostlogical, of anilines even and if notphenols trivial (see, at all,for syntheticinstance ref exploitation [22]). of direct laccase oxidationIn 2005, of anilinesCiecholewski and phenols et al. prepared (see, for a instance small library ref [22 of]). biaryl compounds via laccase- catalyzedIn 2005, radical Ciecholewski C-C homo-couplings et al. prepared (Schem a smalle library12). A difficult of biaryl control compounds over viathe laccase- regio- chemistrycatalyzed radicalof the C-Ccoupling homo-couplings and over the (Scheme reaction 12). Aproceedings difficult control itself over(a high the regiochem-degree of polymerizationistry of the coupling was observed) and over thewere reaction identified proceedings as two harming itself (a issues high degreeconnected of polymer- to some ofization the processes was observed) they investigated. were identified As it asis twonow harminggenerally issues well documented, connected to the some substitu- of the processes they investigated. As it is now generally well documented, the substitution tion pattern of the to-be-coupled aromatic rings appeared to be crucial, as the presence, pattern of the to-be-coupled aromatic rings appeared to be crucial, as the presence, for ex- for example, of electron-donating or withdrawing groups (EDGs, EWGs) in specific posi- ample, of electron-donating or withdrawing groups (EDGs, EWGs) in specific positions tions guides the process towards one of the possible C-H derivatized products by selec- guides the process towards one of the possible C-H derivatized products by selectively tively stabilizing one activated radical over another [84]. stabilizing one activated radical over another [84].

Scheme 12. Overview of the oxidative homocoupling of salicylic esters conducted by Ciecholewski [84]. Scheme 12. Overview of the oxidative homocoupling of salicylic esters conducted by Ciecholewski [84]. Highlighting the potentialities of laccase-mediated biaryl synthesis, Beifuss and coworkersHighlighting provided the an potentialities elegant entry of tolaccase-mediated 3-tert-butyl-1H-pyrazol-5(4H)-one biaryl synthesis, Beifuss containing and coworkersbiaryl compounds provided from an theelegant bio-oxidation entry to 3-te of catecholsrt-butyl-1H-pyrazol-5(4H)-one which proceeded with containing very high biarylisolated compounds yields (Scheme from 13 the)[ 98bio-oxidation]. of catechols which proceeded with very high isolated yields (Scheme 13) [98].

Catalysts 2021, 11, x FOR PEER REVIEW 13 of 34

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Scheme 13. The 3-tert-butyl-1H-pyrazol-5(4H)-one containing biaryl compounds obtained by laccase-catalyzed oxidation Scheme 13. The 3-tert-butyl-1H-pyrazol-5(4H)-one containing biaryl compounds obtained by laccase-catalyzed oxidation of of catechols [98]. catechols [98]. Moreover, Ncanana et al. demonstrated how good control over the reaction engineer- Moreover, Ncanana et al. demonstrated how good control over the reaction engineering can promote high degrees of selectivity in the laccase-catalyzed coupling of bioactive, ing can promote high degrees of selectivity in the laccase-catalyzed coupling of bioactive, natural products. The diterpene totarol was in fact successfully subjected to homocou- natural products. The diterpene totarol was in fact successfully subjected to homocoupling pling reactionsreactions in which inthe which use of the different use of solvents, different modulating solvents, modulating the stabilizing the stabilizing effects of effects of the the C* radicalsC* in place radicals of the in placephenolic of the O* phenolicradicals, promoted O* radicals, the promoted selective isolation the selective of the isolation of the target C-C dimertarget in excellent C-C dimer yield in excellentreducing yield the formation reducing of the the formation C-O dimeric of the byproduct C-O dimeric byproduct (Scheme 14) [99].(Scheme 14)[99].

Scheme 14. C-O and C-C dimers isolated from laccase-catalyzed oxidation of totarol [99]. Scheme 14. C-O and C-C dimers isolated from laccase-catalyzed oxidation of totarol [99]. A significant and unexpected solvent influence on the selectivity of laccase-catalyzed coupling of tetrahydro-2-naphthol derivatives was also previously observed [23].

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RegardlessA significant ofthe and presentedunexpected solvent examples influence and on the the number selectivity of of reports laccase-catalyzed available in the coupling of tetrahydro-2-naphthol derivatives was also previously observed [23]. literature so far [84–87,98,99], the use of laccases for the preparation of biaryl compounds Regardless of the presented examples and the number of reports available in the lit- is stillerature a challenging so far [84–87,98,99], synthetic the application, use of laccases which for the appears preparation difficult of biaryl to be compounds generalized is as well as fullystill rationalized.a challenging synthetic As exemplified application, by which the appears work of difficult Constantin to be generalized and coworkers as well in 2012, laccase-mediatedas fully rationalized. bio-oxidations As exemplified are quiteby the prone work of to Constantin results in and the formationcoworkers in of 2012, unexpected and unpredictedlaccase-mediated products bio-oxidations since subtle are quite differences prone to results in the in structure the formation of the of oxidizedunexpected substrates can largelyand unpredicted influence products the outcome since subtle of a differ reaction.ences Inin the this structure specific of work, the oxidized sesamol, sub- a natural compoundstrates can component largely influence of sesame the outcome seeds, ofwas a reaction. subjected In this to specific laccase work, oxidation sesamol, aiminga at natural compound component of sesame seeds, was subjected to laccase oxidation aiming isolatingat isolating the corresponding the corresponding C-C C-C dimer. dimer. Instead, Instead, thethe authors authors isolated isolated and and characterized characterized for the firstfor the time first a previouslytime a previously unknown unknown trimeric trimeric compound compound whichwhich was was obtained obtained in inhigh high yield in a singleyield in step a single (Scheme step (Scheme 15)[85 15)]. [85].

SchemeScheme 15. Formation 15. Formation of a of trimeric a trimeric C-C C-C adduct adduct from sesamol sesamol in inthe the presence presence of laccase of laccase [85]. [85].

EvenEven if they if they are are often often unpredictableunpredictable by by na nature,ture, radical radical couplings, couplings, when run when of struc- run of struc- turallyturally complex complex and and sterically sterically hindered hindered molecules, molecules, can can allow allow highly highly selective selective and valua- and valuable ble late-stage derivatizations normally precluded to classical organic chemistry. As re- late-stage derivatizations normally precluded to classical organic chemistry. As reported by ported by Sagui et al. [100], laccase catalysis could be efficiently applied to promote the SaguiC-C et al.coupling [100], of laccase the indolic catalysis alkaloids could catharanthine be efﬁciently and appliedvindoline to which promote resulted the in C-C the coupling of theformation indolic alkaloidsof a eniminium catharanthine cationic intermediate and vindoline (Scheme which 16). Following resulted NaBH in the4 reduction, formation of a en- iminiumthe synthetically cationic intermediate useful dimer anhydrovinblastine (Scheme 16). Following (AVBL) was NaBH isolated4 reduction, in a 56% yield the synthetically and usefulfully dimer characterized. anhydrovinblastine The practicability (AVBL) of this bioconversion was isolated on in this a 56%class yieldof bioactive and alka- fully charac- loids was further confirmed through the condensation of catharanthine with the vindoline Catalysts 2021, 11, x FOR PEERterized. REVIEW The practicability of this bioconversion on this class of bioactive alkaloids15 of 34 was furtheranalogue conﬁrmed 11-methoxy-dihydrotabersonine. through the condensation of catharanthine with the vindoline analogue 11-methoxy-dihydrotabersonine.

Scheme 16.SchemeLaccase-based 16. Laccase-based chemoenzymatic chemoenzymatic entry entry to to anhydrovinblastine anhydrovinblastine (AVBL) (AVBL) from from catharan catharanthinethine and vindoline and vindoline [100]. [100].

3.2.2. Nucleophilic Aromatic Substitution Scheme 17 depicts the operating mechanism, i.e., a 1,4-conjugate addition to a quinoid species formed in situ by enzymatic bio-oxidation, at the basis of laccase-catalyzed aromatic nucleophilic substitutions.

Scheme 17. Operating mechanism for the laccase-mediated aromatic nucleophilic substitution reactions.

Different examples of laccase-catalyzed nucleophilic aromatic substitution dealing with different quinoid precursor as substrates and nucleophiles (nitrogen, oxygen and sulfur containing) can be found in the literature. In the framework of this review, some of the most interesting ones, either dealing with the manipulation of complex molecular skeletons or of bioactive compounds, will be discussed. A first report of the use of aliphatic and, most interestingly, aromatic amines as nucleophiles to obtain derivatized substituted p-hydroquinones via nuclear amination was presented by Niedermeyer et al. in 2005 (Scheme 18) [88]. As stated in the previous paragraph, the yields and regioselectivities of the reaction presented in this study were strictly correlated to the substitution of the aromatic rings. The investigation on this topic was then widened to a systematic and mechanistic level in 2015 [101].

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3.2.2. Nucleophilic Aromatic Substitution Scheme 17 depicts the operating mechanism, i.e., a 1,4-conjugate addition to a quinoid species formed in situ by enzymatic bio-oxidation, at the basis of laccase-catalyzed aromatic nucleophilic substitutions.

Scheme 17. Operating mechanism for the laccase-mediated aromatic nucleophilic substitution reactions.

Different examples of laccase-catalyzed nucleophilic aromatic substitution dealing with different quinoid precursor as substrates and nucleophiles (nitrogen, oxygen and sulfur containing) can be found in the literature. In the framework of this review, some of the most interesting ones, either dealing with the manipulation of complex molecular skeletons or of bioactive compounds, will be discussed. A ﬁrst report of the use of aliphatic and, most interestingly, aromatic amines as nucleophiles to obtain derivatized substituted p-hydroquinones via nuclear amination Catalysts 2021, 11, x FOR PEER REVIEWwas presented by Niedermeyer et al. in 2005 (Scheme 18)[88]. As stated in the previous16 of 34

Catalysts 2021, 11, x FOR PEER REVIEWparagraph, the yields and regioselectivities of the reaction presented in this study16 were of 34

strictly correlated to the substitution of the aromatic rings. The investigation on this topic was then widened to a systematic and mechanistic level in 2015 [101].

Scheme 18. Representative examples of laccase-mediated nuclear amination with anilines [101]. Scheme 18. Representative examples of laccase-mediated nuclear amination with anilines [101]. Scheme 18. Representative examples of laccase-mediated nuclear amination with anilines [101]. In subsequent works, laccase-catalyzed nuclear amination was exploited by Hahn In subsequent works, laccase-catalyzed nuclear amination was exploited by Hahn and coworkerIn subsequent to prepare works, bioactive laccase-catalyzed morpholine nuclear derivatives amination [92,93], was and exploited by Mikolasch by Hahn et al. and coworker to prepare bioactive morpholine derivatives [92,93], and by Mikolasch andto efficiently coworker synthetize to prepare novel bioactive antibacterial morpholine derivatives derivatives from [92,93], corollosporine and by Mikolasch [90] and peni-et al. et al. to efﬁciently synthetize novel antibacterial derivatives from corollosporine [90] and topenicillinscillins efficiently [89,91,102], [89 synthetize,91,102 as ],shown as novel shown in antibacterial Scheme in Scheme 19. derivatives19. from corollosporine [90] and penicillins [89,91,102], as shown in Scheme 19.

1 Scheme 19. Laccase-mediated preparation of penicillin derivatives [89,91,102]. Scheme 19. Laccase-mediated preparation of penicillin derivatives [89,91,102]. Scheme 19. Laccase-mediated preparation of penicillin derivatives [89,91,102]. Several examples can be found in the literature describing an analogous synthetic sequenceSeveral to produceexamples sulfoquinones can be found via in thethe bi liteo-oxidationrature describing of hydroquinones an analogous followed synthetic by a sequence1,4-conjugate to produce Michael sulfoquinones addition. via the bio-oxidation of hydroquinones followed by a 1,4-conjugateIn 2012, WellingtonMichael addition. et al. reported on the efficient and selective one-pot preparation of 1,4-naphthoquinone-2,3-bis-sulfideIn 2012, Wellington et al. reported from on the 1,4-dihydroxy-2-naphthoic efficient and selective one-pot acid (Schemepreparation 20) of[94]. 1,4-naphthoquinone-2,3-bis-sulfide from 1,4-dihydroxy-2-naphthoic acid (Scheme 20) [94].

Scheme 20. Laccase-catalyzed synthesis of 2,3-bis-sulfides derivatives [94]. Scheme 20. Laccase-catalyzed synthesis of 2,3-bis-sulfides derivatives [94].

Catalysts 2021, 11, x FOR PEER REVIEW 16 of 34

Scheme 18. Representative examples of laccase-mediated nuclear amination with anilines [101].

In subsequent works, laccase-catalyzed nuclear amination was exploited by Hahn and coworker to prepare bioactive morpholine derivatives [92,93], and by Mikolasch et al. to efficiently synthetize novel antibacterial derivatives from corollosporine [90] and penicillins [89,91,102], as shown in Scheme 19.

Catalysts 2021, 11, 26 Scheme 19. Laccase-mediated preparation of penicillin derivatives [89,91,102]. 15 of 30

Several examples can be found in the literature describing an analogous synthetic sequence to produce sulfoquinones via the bio-oxidation of hydroquinones followed by a 1,4-conjugateSeveral examples Michael addition. can be found in the literature describing an analogous synthetic sequenceIn 2012, to produce Wellington sulfoquinones et al. reported via theon the bio-oxidation efficient and of hydroquinonesselective one-pot followed preparation by a 1,4-conjugateof 1,4-naphthoquinone-2,3-bis-sulfide Michael addition. from 1,4-dihydroxy-2-naphthoic acid (Scheme 20) [94]. In 2012, Wellington et al. reported on the efﬁcient and selective one-pot preparation of 1,4-naphthoquinone-2,3-bis-sulﬁde from 1,4-dihydroxy-2-naphthoic acid (Scheme 20)[94].

Catalysts 2021, 11, x FOR PEER REVIEW 17 of 34

Scheme 20. Laccase-catalyzed synthesis of 2,3-bis-sulﬁdes derivatives [94]. Scheme 20. Laccase-catalyzed synthesis of 2,3-bis-sulfides derivatives [94]. The 1,4-dihydroxy-2-naphthoic acid was a clever substrate ad hoc designed by the authorsThe to 1,4-dihydroxy-2-naphthoic guarantee high selectivity toward acid was the a formation clever substrate of the target ad hoc di-sulfurate designed by prod- the ucts.authors Two to reactive guarantee positions high selectivity of the quinone toward are the information fact blocked of theby the target conjugation di-sulfurate with prod- the ucts. Two reactive positions of the quinone are in fact blocked by the conjugation with the second aromatic ring, the presence of a EWG in the ortho position stabilizes and facilitates second aromatic ring, the presence of a EWG in the ortho position stabilizes and facilitates the formation of a radical in position 3 of the ring while the transient formation of a β-keto the formation of a radical in position 3 of the ring while the transient formation of a β-keto acid intermediate shifts the equilibrium toward a second step of 1,4-conjugate addition by acid intermediate shifts the equilibrium toward a second step of 1,4-conjugate addition by irreversible decarboxylation (Scheme 21). irreversible decarboxylation (Scheme 21).

Scheme 21. Operating mechanism for the laccase-mediated preparation of 2,3- bis-sulﬁdes 1,4-dihydroxy-2-naphthoic acid Scheme 21. Operating mechanism for the laccase-mediated preparation of 2,3- bis-sulfides 1,4-dihydroxy-2-naphthoic acid sulphurated derivatives [94]. sulphurated derivatives [94]. An interesting and outbreaking example of laccase-mediated quinone manipulation wasAn presented interesting by Cannatelliand outbreaking et al. inexample 2015 in of the laccase-mediated form of an enzymatic quinoneα manipulation-arylation of wasbenzoyl presented acetonitrile by Cannatelli with ortho et-quinones. al. in 2015 Speciﬁcally,in the form of the an authors enzymatic were α able-arylation to selectively of ben- zoylinstall acetonitrile a novel C-C with bond ortho between-quinones. an aliphatic Specifically, Csp 3theand authors an aromatic were able Csp 2to, thus selectively obtaining in- 3 2 stalluseful a novel benzylic C-C nitriles bond (Schemebetween 22an)[ aliphatic87]. Csp and an aromatic Csp , thus obtaining useful benzylic nitriles (Scheme 22) [87].

Scheme 22. Laccase-based entry to benzylic nitriles from 1,4-hydroxyquinones [87].

3.2.3. Csp2-H Bond Activation for Ring Closing Reactions The laccase-mediated generation of reactive radical intermediates has been successfully applied to obtain domino, cascade and one-pot ring closure reactions for the synthesis of (hetero)cyclic compounds. Accordingly, by using rationally designed substrates and working under engineered and optimized reaction conditions, (pseudo)quinones for- mations, nucleophilic aromatic substitutions and C-O/C-C radical couplings can be merged to obtain laccase-mediated ring closure reactions [103]. The neat results of these transformations can be described as formal oxidative homo- and/or hetero-coupling couplings, that is, ring closing reactions that involve two molecules of the same substrate or two different partners. It is noteworthy that no expensive, toxic and/or hazardous metal-based chemical catalysts, which usually require inert atmosphere and specific experimental conditions, are needed to perform this biocatalytic Csp2-H bond activation. • Homocouplings

Catalysts 2021, 11, x FOR PEER REVIEW 17 of 34

The 1,4-dihydroxy-2-naphthoic acid was a clever substrate ad hoc designed by the authors to guarantee high selectivity toward the formation of the target di-sulfurate products. Two reactive positions of the quinone are in fact blocked by the conjugation with the second aromatic ring, the presence of a EWG in the ortho position stabilizes and facilitates the formation of a radical in position 3 of the ring while the transient formation of a β-keto acid intermediate shifts the equilibrium toward a second step of 1,4-conjugate addition by irreversible decarboxylation (Scheme 21).

Scheme 21. Operating mechanism for the laccase-mediated preparation of 2,3- bis-sulfides 1,4-dihydroxy-2-naphthoic acid sulphurated derivatives [94].

An interesting and outbreaking example of laccase-mediated quinone manipulation was presented by Cannatelli et al. in 2015 in the form of an enzymatic α-arylation of ben- zoyl acetonitrile with ortho-quinones. Specifically, the authors were able to selectively in- Catalysts 2021, 11, 26 stall a novel C-C bond between an aliphatic Csp3 and an aromatic Csp2, thus obtaining16 of 30 useful benzylic nitriles (Scheme 22) [87].

Scheme 22. Laccase-based entry to benzylic nitriles from 1,4-hydroxyquinones [87]. Scheme 22. Laccase-based entry to benzylic nitriles from 1,4-hydroxyquinones [87].

3.2.3.3.2.3. Csp Csp2-H2-H Bond Bond Activation Activation for for Ring Ring Closing Closing Reactions Reactions TheThe laccase-mediated laccase-mediated generation generation of of reacti reactiveve radical radical intermediates intermediates has has been been success- success- fullyfully applied applied to to obtain obtain domino, domino, cascade andand one-potone-pot ringring closureclosure reactionsreactions for for the the synthesis synthe- sisof of (hetero)cyclic (hetero)cyclic compounds. compounds. Accordingly, Accordingly, by by using using rationallyrationally designeddesigned substrates and and workingworking under engineered andand optimizedoptimized reaction reaction conditions, conditions, (pseudo)quinones (pseudo)quinones forma- for- mations,tions, nucleophilic nucleophilic aromatic aromatic substitutions substitutions and C-O/C-Cand C-O/C-C radical radical couplings couplings can be mergedcan be mergedto obtain to laccase-mediatedobtain laccase-mediated ring closure ring reactionsclosure reactions [103]. The [103]. neat The results neat of results these transfor-of these transformationsmations can be describedcan be described as formal as formal oxidative oxidative homo- homo- and/or and/or hetero-coupling hetero-coupling couplings, cou- Catalysts 2021, 11, x FOR PEER REVIEWplings, that is, that ring is, closing ring closing reactions reactions that involve that involve two moleculestwo molecules of the of same the same substrate substrate18 orof 34 two or twodifferent different partners. partners. It is noteworthy that no expensive, toxic and/or hazardous metal-based chemical Catalysts 2021, 11, x FOR PEER REVIEW It is noteworthy that no expensive, toxic and/or hazardous metal-based chemical18 ofcat- 34 alysts,catalysts, which which usually usually require require inert inert atmosphere atmosphere and andspecific speciﬁc experimental experimental conditions, conditions, are The first example of the preparation of heterocycles2 by laccase oxidation was the for- neededare needed to perform to perform this thisbiocatalytic biocatalytic Csp2 Csp-H bond-H bond activation. activation. mation of phenoxazinones from 3-hydroxyanthranilic acid (3-HAA, Scheme 23), de- •• scribedHomocouplingsThe Homocouplingsby Eggertfirst example et al. in of 1995 the [55].preparation of heterocycles by laccase oxidation was the for- mationThe of ﬁrst phenoxazinones example of the preparationfrom 3-hydroxya of heterocyclesnthranilic by acid laccase (3-HAA, oxidation Scheme was the 23), forma- de- scribedtion of phenoxazinonesby Eggert et al. in from 1995 3-hydroxyanthranilic [55]. acid (3-HAA, Scheme 23), described by Eggert et al. in 1995 [55].

Scheme 23. Laccase-catalyzed oxidation of 3-hydroxyanthranilic acid (3-HAA) to the corresponding phenoxazinone [55].

Scheme 23. Laccase-catalyzed oxidation of 3-hydroxyanthranilic acid (3-HAA) to the corresponding phenoxazinone [55]. Scheme 23. Laccase-catalyzed Regardlessoxidation of 3-hydroxyanthranilicof the preliminary natureacid (3-HAA) of this to study the corresponding and the fact phenoxazinone that phenoxazinone [55]. formation was attested only by UV/Vis spectroscopy, this approach was soon validated Regardless of the preliminary nature of this study and the fact that phenoxazinone by formationthe Regardlesssynthesis was attested ofof the preliminaryantiproliferative only by UV/Vis nature spectroscopy,mo oflecule this study actinocin this and approach theby factOsiadacz was that soon phenoxazinone et validatedal. [104] by (Schemeformationthe synthesis 24). was of attested the antiproliferative only by UV/Vis molecule spectr actinocinoscopy, bythis Osiadacz approach et al.was [104 soon](Scheme validated 24). by the synthesis of the antiproliferative molecule actinocin by Osiadacz et al. [104] (Scheme 24).

Scheme 24. Laccase-catalyzed synthesis of actinocin by oxidative homocoupling [104]. Scheme 24. Laccase-catalyzed synthesis of actinocin by oxidative homocoupling [104]. The synthetic utility of this protocol to obtain nitrogenous homocycles was further The synthetic utility of this protocol to obtain nitrogenous homocycles was further Schemedemonstrated 24. Laccase-catalyzed by Sousa et synthesis al., who of published actinocin by three oxidative papers homocoupling dealing with [104]. the rationally demonstrateddesigned biosynthesis by Sousa et of al., substituted who published phenazines, three papers phenoxazinones dealing with and the benzocarbazoles rationally de- by signedusing Thebiosynthesis the synthetic laccase fromof utility substitutedBacillus of this subtilis phenazinprotocol(spore toes, obtain coatphenoxazinones protein nitrogenous A, CotA, and homocycles Schemebenzocarbazoles 25 was)[105 further– 107by ]. usingdemonstrated the laccase by from Sousa Bacillus et al., subtilis who published (spore coat th proteinree papers A, CotA,dealing Scheme with the 25) rationally [105–107]. designed biosynthesis of substituted phenazines, phenoxazinones and benzocarbazoles by using the laccase from Bacillus subtilis (spore coat protein A, CotA, Scheme 25) [105–107].

Catalysts 2021, 11, 26 17 of 30

Scheme 25. Substituted phenazines, phenoxazinones (a) obtained from laccase from Bacillus subtilis (spore coat protein A, CotA). Mechanistic insights on phenazines (b) and benzocarbazoles (c) formation catalyzed by laccases CotA. [105–107].

A domino process can be built in order to afford oxygen-containing heterocyclic compounds, by exploiting the oxidative (homo)coupling of phenolic substrates [108–110]. Specifically, vinyl phenols and stilbenoids, molecules structurally related to laccases natural substrates, that is the mono-lignols composing lignin, can be mainly converted into three different groups of oxygenated heterocycles, as shown in Scheme 26: 2,3-dihydrobenzofurans (DHB, route i), dioxanes (route ii), and pinoresinol-like bicyclic compounds (hexahydrofuro[3,4- c]furans, route iii). The formation of one of the proposed structures (which in some cases are obtained in mixture) is, again, guided by the structural features of the reacted substrates; all the novel stereocenters are built with no control over their absolute configuration, while, due to steric hindrance and thermodynamics, 2,3-DHBs and dioxanes are trans-configured.

2 Catalysts 2021, 11, 26 18 of 30

In general, when R is both an alkyl or an aryl substituent and R’ is a second phenolic moiety (cathecol derivative as a substrate), a dioxane ring (structure ii) is usually obtained as the major product. Catalysts 2021, 11, x FOR PEER REVIEW In the presence of an allylic alcohol (R = OH) and of an alkyl substituent20 of as 34 R’, the formation of racemic hexahydrofuro[3,4-c]furans (iii), the core of the natural product pinoresinol, is preferred [47,111].

SchemeScheme 26. 26. OxygenatedOxygenated heterocyclic heterocyclic products products thatthat can can be be obtained obtained via via laccase-mediated laccase-mediatedhomocouplings homo- . couplings. Finally, 2,3-DHBs (i, β-5 type dimers in lignin framework) can be selectively ob- tainedThe fromformation ad hoc of designed one of the substrates. proposed In struct theseures molecules, (which in R’ some is a “spectator cases are group”obtained (i.e., in hydrogen,mixture) is,alkyl again, chains, guided protected by the phenols)structural and features the R substituentof the reacted is eithersubstrates; an alkyl all orthe an novelaryl stereocenters group [27,110 are,112 built–115 with]. no control over their absolute configuration, while, due Catalysts 2021, 11, x FOR PEER REVIEW From a mechanistic point of view, as shown in Scheme 27, the ring closure21 occurs of 34 to steric hindrance and thermodynamics, 2,3-DHBs and dioxanes are trans-configured. via a sequence of phenol oxidation, C-C/O radical coupling, and 1,4-conjugate addition. In general, when R is both an alkyl or an aryl substituent and R’ is a second phenolic Two novel stereocenters are formed in this domino process. moiety (cathecol derivative as a substrate), a dioxane ring (structure ii) is usually obtained as the major product. In the presence of an allylic alcohol (R = OH) and of an alkyl substituent as R’, the formation of racemic hexahydrofuro[3,4-c]furans (iii), the core of the natural product pinoresinol, is preferred [47,111]. Finally, 2,3-DHBs (i, β-5 type dimers in lignin framework) can be selectively obtained from ad hoc designed substrates. In these molecules, R’ is a “spectator group” (i.e., hydrogen, alkyl chains, protected phenols) and the R substituent is either an alkyl or an aryl group [27,110,112–115]. From a mechanistic point of view, as shown in Scheme 27, the ring closure occurs via a sequence of phenol oxidation, C-C/O radical coupling, and 1,4-conjugate addition. Two novel stereocenters are formed in this domino process.

Scheme 27. Domino process to 2,3-DHBs via formal oxidative homocoupling. Scheme 27. Domino process to 2,3-DHBs via formal oxidative homocoupling.

These easy-to-handle enzymatic entries to complex oxygenated heterocycles were exploited to build a small library of substituted benzofurans, which was tested in vitro as potential modulators of the molecular chaperone Hsp90 in the quest for novel antiproliferative drugs [116]. More recently, this approach was successfully used to transform 4-hydroxy-chalcones of synthetic origin into the corresponding 2,3-DHB derivatives. Due to the presence of the conjugated system with the fully conjugated π-carbonyl group characterizing chalcones, a C-O dimer was obtained in mixture with the 2,3-DHB based products (Scheme 28) [117].

Scheme 28. Laccase-catalyzed dimerization of 4-hydroxy-chalcones.

Nature has solved the lack of enantioselectivity in laccase-mediated synthesis of 2,3- DHBs by the action of the so-called “dirigent proteins” [118,119]. Since these enantiopure scaffolds represent valuable synthons for the preparation of the plethora of different naturally occurring bioactive compounds [120], alternative synthetic approaches have been investigated. An elegant strategy was proposed by Navarra et al. in two consecutive reports. The racemic mixtures of 2,3-DHB obtained from laccase catalysis were submitted to lipase-catalyzed kinetic resolutions in organic solvents. By remote center discrimination

Catalysts 2021, 11, x FOR PEER REVIEW 21 of 34

Catalysts 2021, 11, 26 Scheme 27. Domino process to 2,3-DHBs via formal oxidative homocoupling. 19 of 30

These easy-to-handle enzymatic entries to complex oxygenated heterocycles were exploited to build a small library of substituted benzofurans, which was tested in vitro as potentialThese modulators easy-to-handle of the molecular enzymatic chaper entriesone to Hsp90 complex in the oxygenated quest for novel heterocycles antiprolif- were erativeexploited drugs to [116]. build a small library of substituted benzofurans, which was tested in vitro as potentialMore recently, modulators this of approach the molecular was chaperonesuccessfully Hsp90 used in to the transform quest for 4-hydroxy-chal- novel antiprolifer- conesative of drugs synthetic [116 ].origin into the corresponding 2,3-DHB derivatives. Due to the presence of the conjugatedMore recently, system this with approach the fully was conjugated successfully π used-carbonyl to transform group characterizing 4-hydroxy-chalcones chalcones,of synthetic a C-O dimer origin was into obtained the corresponding in mixture 2,3-DHB with the derivatives. 2,3-DHB based Due to products the presence (Scheme of the 28)conjugated [117]. system with the fully conjugated π-carbonyl group characterizing chalcones, a C-O dimer was obtained in mixture with the 2,3-DHB based products (Scheme 28)[117].

Scheme 28. Laccase-catalyzed dimerization of 4-hydroxy-chalcones. Scheme 28. Laccase-catalyzed dimerization of 4-hydroxy-chalcones. Nature has solved the lack of enantioselectivity in laccase-mediated synthesis of 2,3-DHBsNature byhas the solved action the of lack the so-calledof enantioselecti “dirigentvity proteins” in laccase-mediated [118,119]. Since synthesis these enantiop- of 2,3- DHBsure scaffoldsby the action represent of the valuableso-called synthons “dirigentfor proteins” the preparation [118,119]. of Since the plethorathese enantiopure of different scaffoldsnaturally represent occurring valuable bioactive synthons compounds for the [120 preparation], alternative of syntheticthe plethora approaches of different have nat- been urallyinvestigated. occurring Anbioactive elegant compounds strategy was [120], proposed alternative by Navarrasynthetic et approaches al. in two have consecutive been investigated.reports. The An racemic elegant mixtures strategy of was 2,3-DHB proposed obtained by Navarra from laccase et al. catalysisin two consecutive were submitted reports.to lipase-catalyzed The racemic mixtures kinetic of resolutions 2,3-DHB inobtained organic from solvents. laccase By catalysis remote center were submitted discrimination to lipase-catalyzedin alcoholysis reactionskinetic resolutions both enantiomers in organic of 2,3-DHB solvents. could By remote be obtained center with discriminatione.e. up to the 98 % [121,122]. In an alternative approach, Gavezzotti et al. reported on the laccase-mediated Csp2-H activation, followed by the hydrolytic action of glycosidases and preparative RP-HPLC, to access both enantiomers of δ-viniferin, a natural 2,3-DHB-based polyphenol endowed with pharmaceutical potentialities, in a fully enzymatic approach starting from piceid, the α-glucoside of resveratrol [113]. The biological activities of these pure enantiomers were then carefully evaluated [123]. As largely highlighted during this discussion, laccase-mediated processes are far from being easily rationalized and the outcomes of the reactions they catalyze can often lead to unpredicted yet valuable chemical skeletons. As a noteworthy example, the oxidative homocoupling of tyrosol resulted in the unexpected formation of a 2-hydrobenzofuran- based tetracyclic, polyoxygenated product. As shown in Scheme 29, after a step of radical C-C coupling promoted by laccase oxidation, the obtained adduct immediately rearranged to form a C3 tetra-substituted benzofuran. This intermediate, due to the presence of a nucleophilic primary alcohol near to an electrophilic enone, spontaneously evolved to the tetracyclic product isolated in virtue of an intramolecular Michael addition [110]. • Heterocouplings The aromatic substitution reactions discussed in the previous paragraph allowed us to obtain substituted quinones with different degrees of regioselectivity depending on both the hydroxyquinone partner and the nucleophile applied. When conducting Catalysts 2021, 11, x FOR PEER REVIEW 22 of 34

in alcoholysis reactions both enantiomers of 2,3-DHB could be obtained with e.e. up to the 98 % [121,122]. In an alternative approach, Gavezzotti et al. reported on the laccase-mediated Csp2- H activation, followed by the hydrolytic action of glycosidases and preparative RP-HPLC, to access both enantiomers of δ-viniferin, a natural 2,3-DHB-based polyphenol endowed with pharmaceutical potentialities, in a fully enzymatic approach starting from piceid, the α-glucoside of resveratrol [113]. The biological activities of these pure enantiomers were then carefully evaluated [123]. As largely highlighted during this discussion, laccase-mediated processes are far Catalysts 2021, 11, 26 from being easily rationalized and the outcomes of the reactions they catalyze can often 20 of 30 lead to unpredicted yet valuable chemical skeletons. As a noteworthy example, the oxidative homocoupling of tyrosol resulted in the unexpected formation of a 2-hydrobenzofuran-based tetracyclic, polyoxygenated product. As shown in Scheme 29, after a step of radicalthe mentioned C-C coupling processes promoted in by the laccase presence oxidation, of a the bidentate obtained nucleophile, adduct immediately cyclic structures rearrangedcan be obtained to form avia C3 tetra-substituted a sequence of onebenzofuran. inter- and This one intermediate, intra-molecular due to the laccase-mediated pres- ence1,4-additions of a nucleophilic. As an primary example, alcohol in 2015, near Cannatelli to an electrophilic et al. reported enone, spontaneously a biocatalyzed domino evolvedsynthesis to the of tetracyclic 2,3-ethylenedithio-1,4-quinones product isolated in virtue basedof an intramolecular on the laccase-activation Michael addition of the C2 and [110].C3 positions in the presence of 1,2-ethanedithiol (Scheme 30)[124].

Catalysts 2021, 11, x FOR PEER REVIEW 23 of 34

Scheme 29. Laccase-catalyzed oxidation of tyrosol [110]. Scheme 29. Laccase-catalyzed oxidation of tyrosol [110].

• Heterocouplings The aromatic substitution reactions discussed in the previous paragraph allowed us to obtain substituted quinones with different degrees of regioselectivity depending on both the hydroxyquinone partner and the nucleophile applied. When conducting the mentioned processes in the presence of a bidentate nucleophile, cyclic structures can be obtained via a sequence of one inter- and one intra-molecular laccase-mediated 1,4-additions. As an example, in 2015, Cannatelli et al. reported a biocatalyzed domino synthesis of 2,3-ethylenedithio-1,4-quinones based on the laccase-activation of the C2 and C3 positions in the presence of 1,2-ethanedithiol (Scheme 30) [124].

Scheme 30. Laccase-mediated cyclization reaction in the presence of 1,2-ethanedithiol [124]. Scheme 30. Laccase-mediated cyclization reaction in the presence of 1,2-ethanedithiol [124]. Hahn and coworkers studied the applicability of a domino process based on the Hahn and coworkers studied the applicability of a domino process based on the laccase-activationlaccase-activation of structurally of complex structurally hydroquinoid complex hydroquinoid substrates to obtain substrates poly(hetero)cy- to obtain poly(hetero)cyclic clic structuresstructures in the presence in the presence of different of different sulfurate sulfurateor nitrogenous or nitrogenous nucleophiles nucleophiles (Scheme (Scheme 31). An extensive and very detailed analytical study was performed in order to characterize the 31). An extensive and very detailed analytical study was performed in order to characterize the complexcomplex mixture mixture of products; of products; difficulties difﬁculties in controlling in controlling the regiochemical the regiochemical outcome outcome of the of the reactionsreactions was washighlighted highlighted by the byauthors. the authors. Again, the Again, substitution the substitution pattern of the pattern sub- of the substrates strates appearedappeared as crucial as crucial [101,103]. [101, 103].

Scheme 31. Poly(hetero)cyclic structures obtained via laccase-catalysis [101,103].

Beifuss and coworkers have been studying the laccase-catalyzed domino/cascade synthesis of oxygenated heterocycles since 2005. Outstanding examples are represented by their works regarding the use of aromatic 1,3-dicarbonyls as carbon nucleophiles in combination with laccase-activated 1,2-dihydroquinones (Scheme 32) [125–127].

Catalysts 2021, 11, x FOR PEER REVIEW 23 of 34

Scheme 30. Laccase-mediated cyclization reaction in the presence of 1,2-ethanedithiol [124].

Hahn and coworkers studied the applicability of a domino process based on the laccase-activation of structurally complex hydroquinoid substrates to obtain poly(hetero)cyclic structures in the presence of different sulfurate or nitrogenous nucleophiles (Scheme 31). An extensive and very detailed analytical study was performed in order to character- Catalysts 2021, 11, 26 ize the complex mixture of products; difficulties in controlling the regiochemical outcome 21 of 30 of the reactions was highlighted by the authors. Again, the substitution pattern of the substrates appeared as crucial [101,103].

Scheme 31. Poly(hetero)cyclic structures obtained via laccase-catalysis [101,103]. Scheme 31. Poly(hetero)cyclic structures obtained via laccase-catalysis [101,103]. Beifuss and coworkers have been studying the laccase-catalyzed domino/cascade Beifuss and coworkers have been studying the laccase-catalyzed domino/cascade Catalysts 2021, 11, x FOR PEER REVIEWsynthesis of oxygenated heterocycles since 2005. Outstanding examples are represented24 of 34 synthesis of oxygenated heterocycles since 2005. Outstanding examples are represented by theirby their works works regarding regarding the use the of usearomatic of aromatic 1,3-dicarbonyls 1,3-dicarbonyls as carbon nucleophiles as carbon nucleophiles in in combinationcombination with laccase-activated with laccase-activated 1,2-dihydroquinones 1,2-dihydroquinones (Scheme 32) (Scheme [125–127]. 32)[ 125–127].

Scheme 32. Aromatic 1,3-dicarbonyls as carbon nucleophiles toward laccase-activated Scheme 32. Aromatic 1,3-dicarbonyls as carbon nucleophiles toward laccase-activated 1,2-dihy- droquinones1,2-dihydroquinones [125–127]. [ 125–127]. In their investigations, and in related publications from other authors [128], the possi- In their investigations, and in related publications from other authors [128], the pos- bility of using sterically demanding, hetero- and carba-cyclic/acyclic 1,3-dicarbonyls as sibility of using sterically demanding, hetero- and carba-cyclic/acyclic 1,3-dicarbonyls as nucleophiles in these biocatalyzed processes has been investigated and validated. The struc- nucleophiles in these biocatalyzed processes has been investigated and validated. The turally diverse benzofurans obtained by the authors are shown in Figure2: structurally diverse benzofurans obtained by the authors are shown in Figure 2:

Figure 2. Example of oxygenated heterocycles obtained by Beifuss and coworkers [125–128].

Beifuss’ investigation was also expanded to the preparation of sulphurated and nitrogenous poly(hetero)cycles by the clever exploitation of S- and N-containing synthetic equivalents of 1,3-dicarbonyls. Their elegant biocatalytic entry to pyrimidinyl benzothiazoles, which was based on the use of 2-thioxo pyrimidine as nucleophiles, is presented in Scheme 33 [28]. Target products were obtained as enriched mixtures of regioisomers.

Catalysts 2021, 11, x FOR PEER REVIEW 24 of 34

Scheme 32. Aromatic 1,3-dicarbonyls as carbon nucleophiles toward laccase-activated 1,2-dihydroquinones [125–127].

In their investigations, and in related publications from other authors [128], the possibility of using sterically demanding, hetero- and carba-cyclic/acyclic 1,3-dicarbonyls as Catalysts 2021, 11, 26 nucleophiles in these biocatalyzed processes has been investigated and validated. The22 of 30 structurally diverse benzofurans obtained by the authors are shown in Figure 2:

Figure 2. Example of oxygenated heterocycles obtained by Beifuss and coworkers [125–128]. Figure 2. Example of oxygenated heterocycles obtained by Beifuss and coworkers [125–128]. Catalysts 2021, 11, x FOR PEER REVIEW 25 of 34 Beifuss’ investigation was also expanded to the preparation of sulphurated and ni- trogenousBeifuss’ investigation poly(hetero)cycles was also by theexpanded clever exploitationto the preparation of S- and of Nsulphurated-containing and synthetic ni- Catalysts 2021, 11, x FOR PEER REVIEWtrogenousequivalents poly(hetero)cycles of 1,3-dicarbonyls. by the Their clever elegant exploitation biocatalytic of S- entry and N to-containing pyrimidinyl synthetic benzothia-25 of 34

equivalentszoles, which of 1,3-dicarbonyls. was based on the Their use elegant of 2-thioxo biocatalytic pyrimidine entry as to nucleophiles, pyrimidinyl is benzothia- presented in zoles,Scheme which 33 was[28]. based Target on products the use wereof 2-thioxo obtained pyrimidine as enriched as nucleophiles, mixtures of regioisomers. is presented in Scheme 33 [28]. Target products were obtained as enriched mixtures of regioisomers.

Scheme 33. Biocatalytic entry to pyrimidinyl benzothiazoles [28].

SchemeSchemeThe 33. 33. BiocatalyticcomplexBiocatalytic regiochemical entry entry to pyri to pyrimidinylmidinyl outcome benzothiazoles benzothiazolesof this domino [28]. [28 process ]. was further investigated by using cathecol and a sterically demanding 2-thioxo pyrimidine, as shown in Scheme The complex regiochemical outcome of this domino process was further investigated 34. The complex regiochemical outcome of this domino process was further investigated by using cathecol and a sterically demanding 2-thioxo pyrimidine, as shown in Scheme 34. by using cathecol and a sterically demanding 2-thioxo pyrimidine, as shown in Scheme 34.

Scheme 34. Laccase-initiated domino process to access pyrimidinyl benzothiazoles [28]. Scheme 34. Laccase-initiated domino process to access pyrimidinyl benzothiazoles [28].

•• Laccase-catalyzed cycloadditions Scheme 34. Laccase-initiated Laccase-catalyzed domino process cycloadditions to access pyrimidinyl benzothiazoles [28]. WhenWhen quinones quinones areare generatedgenerated by by the the laccase-catalyzed laccase-catalyzed oxidation oxidation of 1,4-dihydroquinones of 1,4-dihydroqui- • nonesandLaccase-catalyzed catechols and catechols in the presencein cycloadditions the presence of a diene, of a Diels-Alder diene, Diels-Alder cycloadditions cycloaddi can occur.tions can Ragauskas occur. Ra- and gauskasWhen andquinones coworkers are generated extensively by theinvestigated laccase-catalyzed this topic oxidation and published of 1,4-dihydroqui- two reports nonesdealing and with catechols the one-pot in the presencesynthesis ofof a1,4-naphthoquinones diene, Diels-Alder cycloaddi (Schemetions 35) [129,130]. can occur. Ra- gauskas and coworkers extensively investigated this topic and published two reports dealing with the one-pot synthesis of 1,4-naphthoquinones (Scheme 35) [129,130].

Catalysts 2021, 11, 26 23 of 30

Catalysts 2021, 11, x FOR PEER REVIEW 26 of 34 coworkers extensively investigated this topic and published two reports dealing with the one-pot synthesis of 1,4-naphthoquinones (Scheme 35)[129,130].

Scheme 35. Overview of the obtained cyclic products from laccase-initiated Dies-Alder cycloadditions. Scheme 35. Overview of the obtained cyclic products from laccase-initiated Dies-Alder cycloadditions.3.3. Miscellanea

3.3. MiscellaneaScant reports have described the oxidation of aromatic methyl groups [57,131], alkenes [132], andScant even reports dibenzyl have ethers described [58], tothe the oxidation corresponding of aromatic aldehydes methyl groups catalyzed [57,131], by the al- previously kenesdescribed [132], and LMS. even However, dibenzyl with ethers the [58], last to two the corresponding groups of compounds, aldehydes thecatalyzed conversions by were thegenerally previously low. described In Table LMS.1, representative However, with examples the last two of thegroups oxidation of compounds, of benzyl the alcohols con- to the versionscorresponding were generally benzaldehydes low. In Table are 1, reported. representative examples of the oxidation of benzyl alcohols to the corresponding benzaldehydes are reported. Table 1. Representative examples of the oxidation of benzyl alcohols to the corresponding benzalde- Tablehydes 1. viaRepresentative laccase/ABTS examples catalysis. of the oxidation of benzyl alcohols to the corresponding benzaldehydes via laccase/ABTS catalysis. Substrate Product Yield (%) Substrate Product Yield (%) toluenetoluene benzaldehydebenzaldehyde 92 92 p-nitrotoluenep-nitrotoluene p-nitrobenzaldehydep-nitrobenzaldehyde 98 98 mm-chlorotoluene-chlorotoluene m-chlorobenzaldehydem-chlorobenzaldehyde 89 89 3,4-dimethoxytoluene 3,4-dimethoxybenzaldehyde 90 3,4-dimethoxytoluene 3,4-dimethoxybenzaldehyde 90

AnAn interesting interesting report report in which in which the use theof laccases use of in laccases peptide insynthesis peptide has synthesis been pro- has been posedproposed deserves deserves a mention. a mention. In this study, In this laccases study, were laccases used were for the used selective for the removal selective of removal phenylhydrazide-protectingof phenylhydrazide-protecting groups in groups a mild inprocess a mild that process caused neither that caused oxidative neither mod- oxidative ificationmodiﬁcation nor destruction nor destruction of methionine of methionine or tryptophan or tryptophan side chains [133]. side chains [133]. Laccase-mediated C-H bond activation was also successfully employed in the selec- Laccase-mediated3 C-H bond activation was also successfully employed in the selective tive hydroxylation of Csp3 -H aliphatic bonds. In 2012, Chirivì et al. were able to unexpect- edlyhydroxylation but conveniently of Csp mono-hydroxylate-H aliphatic bonds. a panel In 2012,of ergot Chiriv alkaloidsì et al. at were the C-4 able benzylic to unexpectedly positionbut conveniently via laccases mono-hydroxylatecatalysis (Scheme 36) a [134 panel]. Quite of ergot notably, alkaloids the proposed at the C-4 protocol benzylic al- position lowed,via laccases for the first catalysis time, the (Scheme regio- and 36 )[stereoselective134]. Quite C-4 notably, functionalization the proposed of the protocol ergoline allowed, skeleton.for the ﬁrst When time, a C-10 the methoxy regio- and substituent stereoselective was not C-4 present, functionalization hydroxylation of was the stereose- ergoline skeleton. lective,When thus a C-10 furnishing methoxy C-4α substituent OH derivatives. was not present, hydroxylation was stereoselective, thus furnishing C-4α OH derivatives.

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Catalysts 2021, 11, x FOR PEER REVIEW 27 of 34

Scheme 36. Example of the laccase-mediated stereoselective hydroxylation of an Ergot alkaloid [134]. Scheme 36. Example of the laccase-mediated stereoselective hydroxylation of an Ergot alkaloid [134]. 3.4. Innovative Perspectives in Laccases Synthetic Exploitation The last example of our overview on laccases synthetic applications is an interest- 3.4.Scheme Innovative 36. Example Perspectives of the laccase-mediatedin Laccases Synthetic stereoselect Exploitationive hydroxylation of an Ergot alkaloid [134].ing work by Sulci et al. in which laccase-mediated C-H activation was coupled with a stereoselectiveThe last example organocatalytic of our overview process on [ 135laccases]. The synthetic authors exploited applications a prolinol-based is an interesting amine- work3.4.based Innovative by Sulci organocatalyst, et Perspectives al. in which i.e., in Laccaseslaccase-mediat the Hayachi-Jorgensen Syntheticed ExploitationC-H activation catalyst, suitablewas coupled for the with activation a stere- of oselectivethe α-position organocatalytic of carbonyl process compounds [135]. The [136 authors,137], coupled exploited with a prolinol-based the previously discussedamine- The last example of our overview on laccases synthetic applications is an interesting basedlaccase-mediated organocatalyst, activation i.e., the Hayachi-Jorgensen of 1,2-hydro quinones catalyst, to prepare suitable enantiomericallyfor the activation enrichedof the work by Sulci et al. in which laccase-mediated C-H activation was coupled with a stere- ⍺-position2,3-DHBs of incarbonyl a organo-enzymatic compounds [136,137], integrated coupled process with (Scheme the previously37). discussed lac- oselective organocatalytic process [135]. The authors exploited a prolinol-based amine- case-mediatedAs shown activation in Scheme of 1,2-hydro 37, the describedquinones to multi-catalytic prepare enantiomerically system gave enriched products 2,3- with based organocatalyst, i.e., the Hayachi-Jorgensen catalyst, suitable for the activation of the DHBshigh in isolated a organo-enzymatic yields and with integrated moderate process to high (Scheme (65–92%) 37).e.e ., while it failed in promoting ⍺-position of carbonyl compounds [136,137], coupled with the previously discussed lac- enantioselection in the case of the only reported example of tetrasubstituted stereocenter case-mediated activation of 1,2-hydro quinones to prepare enantiomerically enriched 2,3- in position C3. The authors also proposed a catalytic mechanism, which is reported in DHBs in a organo-enzymatic integrated process (Scheme 37). Scheme 38.

Scheme 37. Laccase-initiated organocatalytic synthesis of enantioenriched 2,3-DHB derivatives.

As shown in Scheme 37, the described multi-catalytic system gave products with Scheme 37. Laccase-initiated organocatalytic synthesis of enantioenriched 2,3-DHB derivatives. highScheme isolated 37. Laccase-initiated yields and with organocatalytic moderate to synthesi high (65–92%)s of enantioenriched e.e., while it 2,3-DHB failed in derivatives. promoting enantioselection in the case of the only reported example of tetrasubstituted stereocenter As shown in Scheme 37, the described multi-catalytic system gave products with in position C3. The authors also proposed a catalytic mechanism, which is reported in high isolated yields and with moderate to high (65–92%) e.e., while it failed in promoting Scheme 38. enantioselection in the case of the only reported example of tetrasubstituted stereocenter in position C3. The authors also proposed a catalytic mechanism, which is reported in Scheme 38.

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SchemeScheme 38. 38.CatalyticCatalytic cycle cycle for forthe the laccase-initiated laccase-initiated asymmetric asymmetric 2,3-DHB 2,3-DHB formation. formation.

ThisThis pioneering pioneering research research paves paves the the way way to toa wide a wide range range of ofsynthetic synthetic applications applications for for thethe laccase-mediated laccase-mediated C-H C-H activation activation assumingassuming thatthat the the following following issues issues might might be addressedbe ad- when assembling any integrated multi-catalysts chemo-enzymatic strategies: (1) reaction dressed when assembling any integrated multi-catalysts chemo-enzymatic strategies: (1) media compatibility (pH, solvents), (2) enzyme stability and activity, (3) stability and reaction media compatibility (pH, solvents), (2) enzyme stability and activity, (3) stability turnover of the organic catalyst, and (4) control over possible cross-reactivity. and turnover of the organic catalyst, and (4) control over possible cross-reactivity. 4. Conclusions and Perspectives 4. Conclusions and Perspectives As shown by the last presented example, the limits and the complete potentiali- tiesAs of shown laccase-mediated by the last presented synthetic example, chemistry the is limits still to and be fullythe complete disclosed. potentialities These enzymes of laccase-mediatedrepresent in fact synthetic an oxidative chemistry toolbox is ofstill extraordinary to be fully disclosed. convenience These in termsenzymes of mild repre- reac- senttion in mediafact an and oxidative environmental toolbox of impact. extraordinary Being robust convenience and solvent-compatible in terms of mild biocatalysts, reaction medialaccases and allowenvironmental one to access impact. complex Being molecular robust and skeletons solvent-compatible in one-pot, chemo-integrated biocatalysts, lac- or casesmultistep/cascade allow one to access redox complex processes molecular by consuming skeletons the oxidizing in one-pot, power chemo-integrated of molecular oxygen or multistep/cascadeand producing waterredox inprocesses exchange. by Byconsuming deeply understanding the oxidizing power the redox of molecular features of oxy- these gen“radical and producing forges” and water by learningin exchange. how By to properlydeeply understanding combine them the with redox other features chemo- of and thesebiocatalysts “radical forges” in more and complex by learning reaction how systems, to properly the number combine of them possible with laccase other chemo- synthetic andapplications biocatalysts will in bemore limited complex only reaction by chemist systems, imagination. the number of possible laccase synthetic applications will be limited only by chemist imagination. Author Contributions: All authors (I.B., E.E.F., S.R., and D.M.) have equally contributed to literature Authorsearch Contributions: and analysis and All toauthors manuscript (I.B., preparationE.E.F., S.R., and editing.D.M.) have All authorsequally havecontributed read and to agreedlitera- to turethe search published and analysis version ofand the to manuscript. manuscript preparation and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. InstitutionalInformed ConsentReview Board Statement: Statement:Not applicable. Not applicable. InformedData Availability Consent Statement: Statement: NotNot applicable. applicable. DataConflicts Availability of Interest: Statement:The authors Not applicable. declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References References1. Riva, S. Laccases: Blue enzymes for green chemistry. Trends Biotechnol. 2006, 24, 219–226. [CrossRef][PubMed] 2. Arregui, L.; Ayala, M.; Gómez-Gil, X.; Gutiérrez-Soto, G.; Hernández-Luna, C.E.; Herrera de los Santos, M.; Levin, L.; 1. Riva,Rojo-Dom S. Laccases:ínguez Blue, A.; enzymes Romero-Mart for greenínez, chemistry. D.; Saparrat, Trends M.C.N.; Biotechnol. et al. Laccases:2006, 24, 219–226, Structure, doi:10.1016/j.tibtech.2006.03.006. function, and potential application in 2. Arregui,water bioremediation.L.; Ayala, M.; Gómez-Gil,Microb. Cell X.; Fact. Gutiérrez-Soto,2019, 18, 200. G.; [CrossRef Hernández-Luna,][PubMed] C.E.; Herrera de los Santos, M.; Levin, L.; Rojo- 3. Domínguez,Janusz, G.; A.; Pawlik, Romero-Martínez, A.; Swiderska-Burek,´ D.; Saparra U.;t, M.C.N.; Polak, J.;et al. Sulej, Laccases: J.; Jarosz-Wilkołazka, Structure, function, A.; and Paszczyński,A. potential application Laccase in properties, water physiological functions, and evolution. Int. J. Mol. Sci. 2020, 21, 966. [CrossRef][PubMed]

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