Bio-Catalysis in Multicomponent Reactions

molecules

Review Bio-Catalysis in Multicomponent Reactions

Ndze Denis Jumbam and Wayiza Masamba *

Department of Chemical and Physical Sciences, Faculty of Natural Sciences, Walter Sisulu University, Nelson Mandela Drive, Mthatha 5117, South Africa; [email protected] * Correspondence: [email protected]

Academic Editors: M. Graça P. M. S. Neves and Gianfranco Favi Received: 13 November 2020; Accepted: 11 December 2020; Published: 15 December 2020

Abstract: Enzyme catalysis is a very active research area in organic chemistry, because biocatalysts are compatible with and can be adjusted to many reaction conditions, as well as substrates. Their integration in multicomponent reactions (MCRs) allows for simple protocols to be implemented in the diversity-oriented synthesis of complex molecules in chemo-, regio-, stereoselective or even speciﬁc modes without the need for the protection/deprotection of functional groups. The application of bio-catalysis in MCRs is therefore a welcome and logical development and is emerging as a unique tool in drug development and discovery, as well as in combinatorial chemistry and related areas of research.

Keywords: bio-catalysis; diversity-oriented chemistry; enzyme; multicomponent reactions; whole-cell catalyst

1. Introduction Recent advances in organic synthesis, fueled by the need for more efficient, environmentally compatible processes, have led to the development of new strategies. Multicomponent reactions (MCRs) are one such strategy. In these reactions, three or more components react together in one pot, generating a rapid assembly of high-complexity molecular architectures resulting in a single-molecular entity containing most, if not all, the starting materials components. Compared to standard organic synthesis wherein compounds are prepared individually and sequentially, consisting of isolation/purification steps that oftentimes generate waste products, MCRs allow for a fast construction of complex and diverse molecular structures in a convergent manner under mild conditions in a single step, leading, in many instances, to the formation of heterocyclic compounds [1–4]. A unique characteristic of MCRs is their simplicity and atom efficiency, high selectivity and low toxicity of readily available starting materials. Therefore, the general principles of green chemistry can easily be applied to MCRs [5]. Given the above characteristics, MCRs have experienced an exponential growth in recent years due to their numerous synthetic applications in pharmaceuticals in fine chemicals, as well as in drug discovery and optimization [6,7]. Multicomponent reactions have been known since a long time. The Strecker reaction is the first documented MCR whereby α-amino acids were prepared from an aldehyde or ketone, ammonia and hydrogen cyanide [8,9]. Since then, many other MCRs have been discovered, and there is a multitude of these reactions today, which include, among others, the Asinger reaction, Biginelli reaction, the Hantzsch pyridine synthesis, Mannich, Strecker and Ugi [10,11]. Most MCRs are mediated by a variety of catalysts [12–14]. Although many of them are important industrial organic reactions, the development of biocatalytic MCRs has only recently attracted the interest of organic chemists [15]. Few direct bio-catalyzed asymmetric MCRs have been reported in the literature.

Molecules 2020, 25, 5935; doi:10.3390/molecules25245935 www.mdpi.com/journal/molecules Molecules 2020, 25, 5935 2 of 19

MoleculesMolecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 22 ofof 2020 Many enzymes exhibit diverse catalytic activity that goes beyond their natural substrates. ThisManyMany phenomenon, enzymesenzymes knownexhibitexhibit diversediverse as enzyme catalyticcatalytic promiscuity activityactivity thth [16atat], goesgoes can beyondbeyond be easily theirtheir exploited naturalnatural substrates. andsubstrates. applied ThisThis in phenomenon,MCRs.phenomenon, Enzymes knownknown are ecofriendly, asas enzymeenzyme biodegradable promiscuitypromiscuity [16],[16], and theircancan bebe reactions easilyeasily exploitedexploited occur under andand mild appliedapplied conditions inin MCRs.MCRs. [17], EnzymesthusEnzymes outperforming areare ecofriendly,ecofriendly, most biodegradable traditionalbiodegradable chemical andand theirtheir catalysts. rereactionsactions When occuroccur immobilized underunder mildmild on conditionsconditions solid supports [17],[17], thusthus [18], outperformingtheyoutperforming can be recovered mostmost traditionaltraditional and reused chemicalchemical in multiple catalysts.catalysts. transformations. WhenWhen immobilizedimmobilized Enzymes, onon therefore, solidsolid supportssupports are becoming [18],[18], theythey a canbettercan bebe alternative recoveredrecovered and toand chemical reusedreused inin catalysts multiplemultiple and transformations.transformations. are ideal in MCRs. Enzymes,Enzymes, therefore,therefore, areare becomingbecoming aa betterbetter alternativealternative toto chemicalchemical catalystscatalysts andand areare idealideal inin MCRs.MCRs. 2. The Asinger Reaction 2.2. TheThe AsingerAsinger ReactionReaction The Asinger reaction involves the synthesis of 3-thiazoline derivatives originally from two moleculesTheThe AsingerAsinger of a ketone, reactionreaction one moleculeinvolvesinvolves the ofthe sulfur synthesissynthesis and oneofof 3-thiazoline3-thiazoline molecule of derivatives gaseousderivatives ammonia originallyoriginally [19 , 20fromfrom]; today, twotwo moleculesαmolecules-haloaldehydes ofof aa ketone,ketone, or α-haloketones, oneone moleculemolecule ammonia, ofof sulfursulfur NaSH andand oneone and moleculemolecule aldehydes ofof orgaseousgaseous ketones ammoniaammonia are usually [19,20];[19,20]; the starting today,today, αmaterialsα-haloaldehydes-haloaldehydes in the reaction oror αα-haloketones,-haloketones, [21–23], allowing ammonia,ammonia, for theNaSHNaSH simultaneous andand aldehydesaldehydes formation oror ketonesketones of the 3-thiazolineareare usuallyusually ring thethe startingstructurestarting materialsmaterials (Scheme 1inin). thethe reactionreaction [21–23],[21–23], allowingallowing forfor thethe simultaneoussimultaneous formationformation ofof thethe 3-thiazoline3-thiazoline ringring structurestructure (Scheme(Scheme 1).1). NN RR11 RR11 OO RR22 RR11 1/81/8 SS88 RR RR HH RR11 SS 22 22 HH OO NHNH33 RR22 RR11,, RR22 == alkyl,alkyl, aryl.aryl. SchemeScheme 1.1. AsingerAsinger reaction. reaction.

ThoughThough discovereddiscovered inin in 1956,1956, 1956, itsits its fullfull full syntheticsynthetic synthetic vava valueluelue waswas was recognizedrecognized recognized onlyonly only recentlyrecently recently thanksthanks thanks toto thethe to advancestheadvances advances mademade made inin MCRs. inMCRs. MCRs. TheThe The 3-thiazolidine3-thiazolidine 3-thiazolidine ringring ring isis isanan an interestinginteresting interesting scaffoldscaffold scaﬀold containedcontained contained inin many many bioactivebioactive moleculesmolecules [24,25][24,25] [24,25] andand and servesserves serves asas as anan an industrialindustrial industrial keykey key intermediateintermediate intermediate forfor for thethe the productionproduction production ofof D ofD-- d penicillaminepenicillamine-penicillamine [26][26] [26 andand] and representsrepresents represents aa astructuralstructural structural motifmotif motif inin in aa a rangerange range ofof of HIVHIV HIV proteaseprotease protease inhibitors inhibitors [27].[27]. [27]. ZumbrägelZumbrägel and and Gröger Gröger developed developed the the first ﬁrstfirst andand onlyonly bio-catalyzedbio-catalyzed enantioselectiveenantioselecenantioselectivetive synthesis synthesis of of this this ringring system system [28] [[28]28] and prepared prepared ( (SS)-2,2,3-trimethyl-1-thia-4-azaspiro[4.4]nonane)-2,2,3-trimethyl-1-thia-4-azaspiro[4.4]nonane via via aa one-potone-pot process,process, leadingleading toto to moderatemoderate moderate conversionconversion conversion andand and excellexcell excellententent enantioselectivitiesenantioselectivities enantioselectivities (99%(99% (99% ee)ee) usingusing ee) using EscherichiaEscherichiaEscherichia colicoli wholewhole coli wholecellscells asas cells thethe catalyst.catalyst. as the catalyst. TheThe reactionreaction The reaction proceededproceeded proceeded viavia aa 3-thiazolidine3-thiazolidine via a 3-thiazolidine ringring intermediateintermediate ring intermediate (Scheme(Scheme (Scheme 2),2), whichwhich2), whichwaswas subsequentlysubsequently was subsequently bio-catalyticallybio-catalytically bio-catalytically reducedreduced reduced byby anan by imineimine an imine reductasereductase reductase inin in combinationcombination combination withwith with aa a glucose glucose dehydrogenasedehydrogenase andand glucoseglucose forfor inin situsitu cofactorcofactor recycling.recycling.

OO OO aq.aq. NHNH33 HH ClCl NN E-coliE-coli NN ++ 26%26% yieldyield SS wholewhole cellcell SS NaSHNaSH 99%99% eeee

SchemeScheme 2. 2. AsingerAsinger synthesis synthesis of of ( (SS)-2,2,3-trimethyl-1thia-4-azaspiro[4.4]nonane.)-2,2,3-trimethyl-1thia-4-azaspiro[4.4]nonane.

AnAn overalloverall moderatemoderate moderate conversionconversion conversion ofof 26%26% of 26% isis duedue is duetoto thethe to limitedlimited the limited diffusiondiffusion diﬀ ofusionof thethe thiazolidine ofthiazolidine the thiazolidine inin thethe in AsingertheAsinger Asinger reaction.reaction. reaction. ThisThis Thislimitationlimitation limitation notnot notwithstandiwithstandi withstandingngng representsrepresents represents anan improvimprov an improvementementement comparedcompared compared toto thethe to the compartmentalizedcompartmentalized one-potone-pot one-pot processprocess process thatthat that affordedafforded aﬀorded onlyonly 13% only13% yield.yield. 13% yield.

3.3. TheThe Biginelli Reaction Reaction TheThe BiginelliBiginelli reactionreaction reaction waswas was initiallyinitially initially usedused used toto to allowallow allow anan anefficientefficient eﬃcient accessaccess access toto multi-functionalizedmulti-functionalized to multi-functionalized 3,4-3,4- dihydropyrimidin-2(13,4-dihydropyrimidin-2(1dihydropyrimidin-2(1HH)-ones)-onesH)-ones (DHPMs)(DHPMs) (DHPMs) [29][29] [ 29 fromfrom] from aldehyde-ureaaldehyde-urea aldehyde-urea derivativesderivatives derivatives ofof of aceto-aceto- aceto- andand oxaloaceticoxaloacetic acids.acids. TheThe The relativelyrelatively relatively harshharsh harsh acidicacidic acidic conditcondit conditionsionsions involvedinvolved involved inin the inthe the reactionreaction reaction ledled led thethe thesearchsearch search forfor greenerforgreener greener enzyme-catalyzedenzyme-catalyzed enzyme-catalyzed versions.versions. versions. TheThe reactionreaction The reaction ququicklyickly quickly becamebecame became oneone ofof onethethe majormajor of the MCRsMCRs major asas MCRs aa routeroute as toato route manymany to manyheterocyclicheterocyclic heterocyclic compounds,compounds, compounds, includingincluding including 4-dihydropyrimidin-2(14-dihydropyrimidin-2(1 4-dihydropyrimidin-2(1HH)-thiones,)-thiones,H)-thiones, asas as wellwell well asas intermediatesintermediates inin thethe HantzschHantzsch pyridinepyripyridinedine synthesissynthesis [[30,31][30,31]30,31] (Scheme(Scheme3 3).3).).

Molecules 2020, 25, x 5935 FOR PEER REVIEW 33 of of 20 19 Molecules 20202020,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 2020 OEt R O X OEt R O O X O NH R-CHO O OEt H2N NH2 36-98% O NH R-CHO N X R-CHO O OEt H2N NH2 36-98% 2 2 36-98% HN X R = Ph, 4-Me-Ph, 4-Cl-Ph, 4-MeO-Ph, 4-OH-Ph, 4-NO 2-Ph... N X R = Ph, 4-Me-Ph, 4-Cl-Ph, 4-MeO-Ph, 4-OH-Ph, 4-NO -Ph... X = O,H S, NH R = Ph, 4-Me-Ph, 4-Cl-Ph, 4-MeO-Ph, 4-OH-Ph, 4-NO22-Ph... X = O, S, NH Scheme 3. Preparation of different dihydropyrimidine derivatives via the general Biginelli reaction. Scheme 3. PreparationPreparation of of different different dihydropyrimidine derivativesderivativesrivatives viavia thethe generalgeneragenerall BiginelliBiginelli reaction.reaction. Since both urea and the β-ketoester are bifunctional groups, an MCR would lead to the formation Since both urea and the β-ketoester are bifunctional groups, an MCR would lead to the formation of fiveSince new both bonds urea in andthe endthe βproducts-ketoester-ketoester and areare constitute bifunctionalbifunctionals an groups,groups, efficient anan method MCRMCR wouldwould to access leadlead these toto thethe derivatives. formationformation of five new bonds in the end products and constitutes an efficient method to access these derivatives. Aof biocatalyticfive new bonds Biginelli in the three-component end products and reaction constitute wasss an andeveloped efficientefficient methodmethodfor the high-yield toto accessaccess thesethese synthesis derivatives.derivatives. of 3,4- A biocatalytic Biginelli three-component reaction was developed for the high-yield synthesis of dihydropyrimidine-2-(1A biocatalytic Biginelli three-componentH)-ones, consisting reaction of the condensationwas developed of ureafor the or high-yieldthiourea with synthesis a substituted of 3,4- 3,4-dihydropyrimidine-2-(1H)-ones, consisting of the condensation of urea or thiourea with a substituted benzaldehydedihydropyrimidine-2-(1 and a 1,3-ketoesterH)-ones,)-ones, consistingconsisting in aqueous ofof phosphatethethe condensationcondensation buffer ofofand ureaurea using oror thioureathiourea Saccharomyces withwith aa cerevisiae substitutedsubstituted as benzaldehyde and a 1,3-ketoester in aqueous phosphate buffer and using Saccharomyces cerevisiae abenzaldehyde biocatalyst [15]. and Baker’sa 1,3-ketoester yeast was in aqueous later found phosphate to successfully buffer and catalyze using Saccharomycesthe one-pot synthesis cerevisiae asofas as a biocatalyst [15]. Baker’s yeast was later found to successfully catalyze the one-pot synthesis dihydropyrimidinonesa biocatalyst [15]. Baker’s [32]. yeast The was Biginelli later foundreaction to ofsuccessfully acetoacetate, catalyze aromatic the one-potaldehyde synthesis and urea of of dihydropyrimidinones [32]. The Biginelli reaction of acetoacetate, aromatic aldehyde and urea (thiourea)dihydropyrimidinones using trypsin [32].from porcineThe Biginelli pancreas reaction as the catalystof acetoacetate, has also beenaromatic reported aldehyde [33] (Scheme and urea 4). (thiourea) using trypsin from porcine pancreas as thethe catalyst has also been reported [33] [33] (Scheme 4 4).). (thiourea) using trypsin from porcine pancreas as the catalyst has also been reportedOEt [33] (SchemeR 4). O o O Trypsin, 37 C OEt R O O oo O NH R-CHO + + O Trypsin, 37 C O OEt H2N NH2 5 mL of EtOH O NH R-CHO + + N O R = Ar O OEt + H2N NH2 5 mL of EtOH 2 2 5 mL82-96% of EtOH HN O R = Ar N O R = Ar H 82-96% H Scheme 4. Preparation of different dihydropyrimidinones via the enzyme-catalyzed Biginelli Scheme 4. Preparation of different dihydropyrimidinones via the enzyme-catalyzed Biginelli reaction. reaction.Scheme 4. Preparation of different dihydropyrimidinones via the enzyme-catalyzed Biginelli reaction. Basedreaction. on the results obtained, a general mechanism is proposed that starts with the activation Based on the results obtained, a general mechanism is proposed that starts with the activation of the carbonyl groups of the aceto ester and the aromatic aldehyde in the active site of the enzyme, of theBased carbonyl on the groups results of obtained,the aceto estera general and themechanism aromatic isisaldehyde proposedproposed in thatthatthe activestartsstarts withsitewith of thethe the activationactivation enzyme, followed by the aldol condensation with the aromatic aldehyde to generate the corresponding followedof the carbonyl by the groups aldol ofcondensation the aceto ester with and the the aromatic aromatic aldehydealdehyde into thegenerate active thesite ofcorresponding the enzyme, unsaturated β-ketoester. The latter undergoes a Michael addition of urea, followed by cyclization and unsaturatedfollowedfollowed byby β the-ketoester.the aldolaldol condensationcondensation The latter undergoes withwith thethe a Michael aromaticaromatic addi aldehydealdehydetion of urea, toto generategenerate followed the theby cyclizationcorrespondingcorresponding and dehydration to afford the product (Scheme5). dehydrationunsaturated βto-ketoester.-ketoester. afford the TheThe product latterlatter (Scheme undergoesundergoes 5). a a MichaelMichael addiadditiontion ofof urea,urea, followedfollowed byby cyclizationcyclization andand dehydration to afford the product (Scheme 5).

H H H H H H H H H H H H OO OO H H H H - H O OO OHO OO 2 O Aldol - H O OR1 + OHO OR1 - H22O O O Aldol OR1 R O H OR1 OR OR1 + OR11 R O R H OR1 ROH 1 R O ROH R ROH H2N NH2 H N NH H22N NH22 H H H H H H O HO O H H O OO O HO O HN OR OO 1 HN OR1 HN OR OR HN OR11 HN OR 1 - H2O HN OR11 H2N O N R O N R OR1 -- HH2O H2N 1 O HN R 2 O H 2 NH R O N R O H H NH R H O Scheme 5. Mechanism of the enzyme-catalyzed Biginelli reaction. Scheme 5. Mechanism of the enzyme-catalyzed Biginelli reaction. Scheme 5. Mechanism of the enzyme-catalyzed Biginelli reaction.

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Compared to some traditional methods [34–36], the above enzyme-catalyzed synthesis of 1,4-dihydropyridine (DHP) derivatives appears superior not only because the procedures are milder, simple in their operation and more environmentally friendly but, also, because they allow access to a wider variety of heterocycles in this category. The promiscuous activity of bovine serum albumin (BSA) was further demonstrated in the synthesis of DHPM and the corresponding thione derivatives by Sharma et al. [37], wherein a -gram scale synthesis of monastrol (1) (see Scheme6), a potent mitotic kinesin Eg5 inhibitor, was obtained in 67% yield. The plausible mechanism suggested is depicted in Scheme6 and is somewhat di fferent from the one described in Scheme5. In this case, the side chain amino acids of the enzyme play the role of the catalytic base and extract a proton from the active methylene compound to form a nucleophile, which reacts with an iminium ion arising from the reaction between the aldehyde and urea. Cyclization of this intermediate followed by dehydration, as described in Scheme5, leads to the formation of the products. The aldehyde components mostly used as partners in this MCR are aromatic aldehydes instead of acetaldehyde, because the latter is generally difficult to handle directly given its low boiling point, ease of polymerization and oxidation. In order to circumvent this drawback and expand the scope of the reaction, Wangand his group developed the first enzyme-catalyzed Biginelli reaction using acetaldehyde generated in situ by trypsin-catalyzed transesterification from vinyl acetate, combining two catalytic activities of one enzyme in a one-pot strategy for a two-step cascade reaction [38] (Scheme7). Urea and N-methylurea, as well as the corresponding thiourea derivatives, were tolerated in the reaction, affording a wide variety of heterocyclic compounds in high yields of up to 99%. The biocatalytic preparation of a series of novel dihydropyrimidin-2(1H)-ones was achieved by the proper combination of Rhizopus oryzae lipase in a deep eutectic solvent consisting of a mixture of choline chloride and urea [16,39], giving rise to the expected products in good yields. This lipase in the deep eutectic medium gave high isolated yields in a short period of time. In addition, both the solvent and catalyst could be recycled up to four times without any major loss of activity, thus making the process both economical and environmentally friendly. The versatility of the Biginelli reaction to rapidly generate biologically active heterocycles has been recently demonstrated in the synthesis of the benzopyran-connected pyrimidine (3) and benzopyran- connected pyrazole (2) derivatives via the reaction using Cu(II)-tyrosinase as a green catalyst [40] (see Scheme6). The authors noted that literature observations showed that these coumarin derivatives are generally prepared by conventional methods using an acid catalyst while affording low yields and long reaction times. This procedure is, therefore, ecofriendly and gives high yields of 86–98%. The enzyme quickly catalyzed the cyclization reaction to afford compounds whose biotests exhibited larvicidal and antifeedant activities. In a related study, a series of pyrimidine-thione derivatives with potential mosquito larvicidal activity were prepared via a Cu(II)-tyrosinase catalyzed Biginelli reaction in up to 90% yield [41]. When urea or, indeed, a bifunctional nucleophile such as 5-aminopyrazole is used as the nitrogen source in a Biginelli reaction [42], Hantzsch products can potentially result under appropriate reaction conditions, such as the use of urease as a biocatalyst, and divergence between the two reactions is possible, leading to potential mixtures of DHMP and 1,4-dihydropyridine (DHP) derivatives. This competition between the two reactions was studied by Tamaddon et al. using urease immobilized on magnetic micro/nanocellulose dialdehydes. The immobilized urease catalyzed the Biginelli reaction with 100% selectivity in 68–92% yield, while the nonimmobilized enzyme, known to catalyze the hydrolysis of urea to ammonia, exclusively shifted the reaction to the Hantzsch product [43,44] (Scheme8). The main novelty in the above procedure is the immobilization of the enzyme on cellulose, a cheap, natural, biocompatible biopolymer that can easily be recycled. In addition, the enzyme denaturation due to its sensitivity and instability can be avoided thanks to the mild conditions during immobilization. Molecules 2020, 25, x FOR PEER REVIEW 4 of 20

O O H O + BSA NH2 R H H2N NH2 - H O + 2 R N NH2 H O BSA 1 H R 2 NH R 2 R = p-ClPh, R1 = CH3, R2 = CO2Et

R O 2 R Products N NH2 1 H R O OH R R NH O NH HO O N O EtO NH O N R = Ar R = Ar N S Benzopyran-pyrimidinone derivatives (3) H Benzopyran-pyrazole derivatives (2) Monastrol (1)

H2N H2N O R O O 1 X CO R 2 X O 2 R O O N N O H 1 R = Ar, R1 = Me, Et, tBu R Polyhydroquinolines (4) X = CN, CO Et 2 X = CN, CO Et Spiroindoles (5) 2 Spiroacenaphthylenes (6) Molecules 2020, 25, x FOR PEERR REVIEW 5 of 20

N S The aldehyde components mostly used as partners in thisN MCR are aromaticO aldehydes instead N of acetaldehyde,N becauseN the latter is generally difficult to handle directly givenHO its low boiling point, N N N R H O ease of polymerizationH H H and oxidation. In order to circumvent this drawback and expand the scope of R = H, 2-Cl, 2-NO2, 2-OH, 4-OH... the reaction,R = H, 2-Cl, Wang 3-NO and2, 3-OH, his 4-Me...group developed the first enzyme-catalyzed Biginelli reaction using 2-Hydrazono-4-thiazolidinone derivatives (8) acetaldehydeTetrahydropyridipyrazoopyridines generated in situ by (7)trypsin-catalyzed transesterification from vinyl acetate, combining two catalytic activities of one enzyme in a one-pot strategy for a two-step cascade reaction [38] Scheme 6.6. Mechanism of the bovine serum albumin (BSA)-catalyzed(BSA)-catalyzed Biginelli reaction and some (Scheme 7). selected products accessibleaccessible byby thisthis method.method. X O O O R3 R4 OH NH Trypsin NH R2 N R4 O + H OO R1 NX R1 R2 R Trypsin 3 33-99% R1 = Me, n-Bu, Ph, OEt R2 = OMe, OEt, iPrO, iBuO, Me R3 = H, Me; R4 = H, Me, X = O, S Scheme 7. Enzyme in-situ generation of acetaldehyde in the Biginelli reaction.

Urea and N-methylurea, as well as the corresponding thiourea derivatives, were tolerated in the reaction, affording a wide variety of heterocyclic compounds in high yields of up to 99%. The biocatalytic preparation of a series of novel dihydropyrimidin-2(1H)-ones was achieved by the proper combination of Rhizopus oryzae lipase in a deep eutectic solvent consisting of a mixture of choline chloride and urea [16,39], giving rise to the expected products in good yields. This lipase in the deep eutectic medium gave high isolated yields in a short period of time. In addition, both the solvent and catalyst could be recycled up to four times without any major loss of activity, thus making the process both economical and environmentally friendly. The versatility of the Biginelli reaction to rapidly generate biologically active heterocycles has been recently demonstrated in the synthesis of the benzopyran-connected pyrimidine (3) and benzopyran-connected pyrazole (2) derivatives via the reaction using Cu(II)-tyrosinase as a green catalyst [40] (see Scheme 6). The authors noted that literature observations showed that these coumarin derivatives are generally prepared by conventional methods using an acid catalyst while affording low yields and long reaction times. This procedure is, therefore, ecofriendly and gives high yields of 86–98%. The enzyme quickly catalyzed the cyclization reaction to afford compounds whose biotests exhibited larvicidal and antifeedant activities. In a related study, a series of pyrimidine- thione derivatives with potential mosquito larvicidal activity were prepared via a Cu(II)-tyrosinase catalyzed Biginelli reaction in up to 90% yield [41]. When urea or, indeed, a bifunctional nucleophile such as 5-aminopyrazole is used as the nitrogen source in a Biginelli reaction [42], Hantzsch products can potentially result under appropriate reaction conditions, such as the use of urease as a biocatalyst, and divergence between the two reactions is possible, leading to potential mixtures of DHMP and 1,4-dihydropyridine (DHP) derivatives. This competition between the two reactions was studied by Tamaddon et al. using urease immobilized on magnetic micro/nanocellulose dialdehydes. The immobilized urease catalyzed the Biginelli reaction with 100% selectivity in 68–92% yield, while the nonimmobilized enzyme, known to catalyze the hydrolysis of urea to ammonia, exclusively shifted the reaction to the Hantzsch product [43,44] (Scheme 8).

Molecules 2020, 25, x FOR PEER REVIEW 6 of 20

O O O Ph H 2N NH2 O Ph OEt NH MoleculesPh-CHO 2020, 25,+ 5935 EtO EtO 6 of 19 water + Molecules 2020, 25, x FOR PEER REVIEW N O 6 of 20 OO catalyst N H H O OEt Hantzsch product (1,4-DHP) Biginelli product (DHPM) catalyst: O O Ph H 2N NH2 O Ph free urease, 100% 0% OEt NH Ph-CHO + EtO EtO water + catalyst N O OO immobilized urease, 0%N 100% H H OEt Hantzsch product (1,4-DHP) Biginelli product (DHPM) Scheme 8. Catalytic effect of freecatalyst: and immobilized urease, leading to Biginelli and Hantzsch products. DHP: 1,4-dihydropyridine andfree DHPM: urease, 3,4-dihydropyrimidin-2(1100%H)-one. 0%

The main novelty in the aboveimmobilized procedure urease, is the immobilization0% of the enzyme on100% cellulose, a cheap, natural, biocompatible biopolymer that can easily be recycled. In addition, the enzyme denaturationSchemeScheme 8. due 8.Catalytic Catalytic to its sensitivity e ffeffectect of of free free and and and instability immobilized immobilized can urease, urease, be avoided leading leading thanks to to Biginelli Biginelli to the and and mild Hantzsch Hantzsch conditions products. products. during immobilization.DHP:DHP: 1,4-dihydropyridine 1,4-dihydropyridine and and DHPM: DHPM: 3,4-dihydropyrimidin-2(1 3,4-dihydropyrimidin-2(1H)-one.H)-one. In another process, the synthesis of DHMP was catalyzed by a combination of the biocatalyst α- In another process, the synthesis of DHMP was catalyzed by a combination of the biocatalyst chymotrypsinThe main in noveltymicrowave in the irradiation above procedure at 55 °C in is a the circulating immobilization reaction of system the en [45],zyme demonstrating on cellulose, a α-chymotrypsin in microwave irradiation at 55 C in a circulating reaction system [45], demonstrating the thecheap, synergistic natural, effect biocompa of the microwavetible biopolymer and the ◦that enzy came,n easily thus greatly be recycled. reducing In theaddition, reaction the time enzyme and synergistic effect of the microwave and the enzyme, thus greatly reducing the reaction time and increasing increasingdenaturation the yield. due to its sensitivity and instability can be avoided thanks to the mild conditions during theimmobilization. yield. In another process, the synthesis of DHMP was catalyzed by a combination of the biocatalyst α- 4. The The Hantzsch Hantzsch Reaction Reaction chymotrypsin in microwave irradiation at 55 °C in a circulating reaction system [45], demonstrating The Hantzsch reaction, first reported in 1882, is a well-known protocol for the synthesis of 1,4- the Thesynergistic Hantzsch effect reaction, of the microwave first reported and in the 1882, enzy isme, a well-knownthus greatly protocolreducing forthe thereaction synthesis time and of dihydropyridines (DHPs) from ethyl acetoacetate, an aldehyde and ammonia. It has undergone many 1,4-dihydropyridinesincreasing the yield. (DHPs) from ethyl acetoacetate, an aldehyde and ammonia. It has undergone modifications and improvements and is now the method of choice for fast access to DHP derivatives, many modifications and improvements and is now the method of choice for fast access to DHP which are useful for various bioactivities such as calcium channel blockers and antihypertensive derivatives,4. The Hantzsch which Reaction are useful for various bioactivities such as calcium channel blockers and agents [10,46] but, also, as scaffolds in medicinal chemistry [14]. Under oxidative conditions, these antihypertensive agents [10,46] but, also, as scaffolds in medicinal chemistry [14]. Under oxidative so-calledThe Hantzsch Hantzsch esters reaction, can firstbe transformed reported in 1882,into the is acorresponding well-known protocol pyridines, for athe strategy synthesis used of to1,4- conditions, these so-called Hantzsch esters can be transformed into the corresponding pyridines, preparedihydropyridines single regio-isomer (DHPs) sfrom of poly-substituted ethyl acetoacetate, pyridines an aldehyde [47–49] and (Scheme ammonia. 9). It has undergone many amodifications strategy used toand prepare improvements single regio-isomers and is now the of poly-substitutedmethod of choice pyridines for fast access [47–49 to] (SchemeDHP derivatives,9). which are useful for various bioactivities such as calcium channel blockers and antihypertensive O R R agents [10,46] but, also, as scaffolds in medicinal chemistry1 [14]. Under oxidative conditions,1 these CO Me MeO C CO Me MeO C CO Me so-calledO HantzschR esters can2 be transformed2 into the corresponding2 pyridines,2 a strategy 2used to prepareR1 single regio-isomers of poly-substituted pyridines [47–49] (Scheme 9). NH OAc H 4 RRN 35.7-93.5% R NR R = CH3, Et; R1 = Aryl H O R1 R1 COSchemeMe 9. GeneralMeO HantzschC pyridineCO Me synthesis. MeO C CO Me O R Scheme2 9. General 2Hantzsch pyridine2 synthesis. 2 2 RA1 novel and efficient synthesis of Hantzsch DHPs in the presence of fermenting baker’s yeast H NH4OAc R NR appearedA novel in theand literatureefficient synthesis in 2005. of The Hantzsch acetaldehydeRR DHPsN in involved the presence35.7-93.5% in the of reaction fermenting resulted baker’s from yeast an R = CH , Et; R = Aryl H appearedin-situ carbohydrate in the literature fermentation3 in 2005.1 The [50]. acetaldehyde The same strategy involved was in later the usedreaction by Kumarresulted and from Maurya an in-situ [32] carbohydrateto expand the scopefermentation of the reaction [50]. The to moresame aldehydesstrategy was and laterβ-ketoesters, used by allowingKumar and for accessMaurya to various[32] to Scheme 9. General Hantzsch pyridine synthesis. expandpolyhydroquinoline the scope of derivativesthe reaction 4to (see more Scheme aldehydes6) via anand unsymmetrical β-ketoesters, allowing Hantzsch for reaction. access to various polyhydroquinolineThe source of derivatives ammonia 4 in (see the Scheme reaction 6) via can an unsymmetrical be ammonium Hantzsch acetate, reaction. liquid ammonia, A novel and efficient synthesis of Hantzsch DHPs in the presence of fermenting baker’s yeast magnesiumThe source nitride of ammonia [51] or urea. in the The reaction unprecedented can be ammoniumCandida antarcticaacetate, liqulipaseid ammonia, B-catalyzed magnesium (CAL-B) appeared in the literature in 2005. The acetaldehyde involved in the reaction resulted from an in-situ nitrideHantzsch [51] reaction or urea. using The acetamide unprecedented as the ammonia Candida source antarctica was developedlipase B-catalyzed by Lin et al.(CAL-B) [52], giving Hantzsch access carbohydrate fermentation [50]. The same strategy was later used by Kumar and Maurya [32] to reactionto an array using of 1,4-DHPs acetamide in as excellent the ammonia yields ofsource up to was 92%. developed On the other by Lin hand, et al when. [52], urea giving was access used asto thean expand the scope of the reaction to more aldehydes and β-ketoesters, allowing for access to various arraynitrogen of 1,4-DHPs source, the in reaction excellent was yields successfully of up to catalyzed 92%. On by the urease other leading hand, selectivelywhen urea under was used appropriate as the polyhydroquinoline derivatives 4 (see Scheme 6) via an unsymmetrical Hantzsch reaction. nitrogenreaction conditionssource, the to eitherreaction Biginelli was orsuccessfully Hantzsch products,catalyzed as by indicated urease earlierleading in Schemeselectively8. under The source of ammonia in the reaction can be ammonium acetate, liquid ammonia, magnesium appropriateSimilarly, reaction replacing conditions the aldehyde to either by isatin,Biginell ai one-potor Hantzsch papain-catalyzed products, as synthesisindicated ofearlier a series in nitride [51] or urea. The unprecedented Candida antarctica lipase B-catalyzed (CAL-B) Hantzsch Schemeof spiropyrazolo[3,4-b]pyridines, 8. starting from cyclic-1,3-diketones and 3-methyl-5-aminopyrazole, reaction using acetamide as the ammonia source was developed by Lin et al. [52], giving access to an was achieved, affording highly substituted derivatives in moderate yields [53] (Scheme 10). array of 1,4-DHPs in excellent yields of up to 92%. On the other hand, when urea was used as the nitrogen source, the reaction was successfully catalyzed by urease leading selectively under appropriate reaction conditions to either Biginelli or Hantzsch products, as indicated earlier in Scheme 8.

Molecules 2020, 25, x FOR PEER REVIEW 7 of 20 Molecules 2020, 25, x FOR PEER REVIEW 7 of 20 Similarly, replacing the aldehyde by isatin, a one-pot papain-catalyzed synthesis of a series of Similarly, replacing the aldehyde by isatin, a one-pot papain-catalyzed synthesis of a series of spiropyrazolo[3,4-b]pyridines, starting from cyclic-1,3-diketones and 3-methyl-5-aminopyrazole, spiropyrazolo[3,4-b]pyridines, starting from cyclic-1,3-diketones and 3-methyl-5-aminopyrazole, wasMolecules achieved,2020, 25 ,affording 5935 highly substituted derivatives in moderate yields [53] (Scheme 10). 7 of 19 was achieved, affording highly substituted derivatives in moderate yields [53] (Scheme 10). H HN H O N HN O O O N NH O O papain O N O NH2 papain O N O + 2 + N + NN + 48% O N NN 48% O H H N H H N H H Scheme 10. Papain-catalyzed Hantzsch synthesis of spiropyrazolo[3,4-b]pyridine. SchemeScheme 10. Papain-catalyzedPapain-catalyzed Hantzsch Hantzsch synthesi synthesiss of of spiropyrazolo[3,4-b]pyridine. spiropyrazolo[3,4-b]pyridine. A related reaction with isatin and acenaphthenequinone was bio-catalyzed by swim bladders A related reaction with isatin and acenaphthenequinone was bio-catalyzed by swim bladders obtained from Caspian Sea fish (isinglass), leading to the domino synthesis of spiro-oxindoles and obtained from Caspian Sea fish fish (isinglass), leading to the domino synthesis of spiro-oxindoles and spiroacenaphthylenes 6 in water (Scheme 6) [54]. This is the first time this nontoxic, biocompatible spiroacenaphthylenes 6 6 in in water water (Scheme 66))[ [54].54]. This This is is the first first time this nontoxic, biocompatible and reusable biocatalyst, which required no special handling precautions, was used. Moreover, the and reusable reusable biocatalyst, biocatalyst, which which required required no no special special handling handling precautions, precautions, was was used. used. Moreover, Moreover, the present protocol is superior to the previous ones in terms of reaction time, yields and general reaction presentthe present protocol protocol is superior is superior to the to previous the previous ones in ones terms in of terms reaction of reaction time, yields time, and yields general and reaction general conditions. conditions.reaction conditions. Several enzyme-catalyzed three-component “Hantzsch-type” reactions where the amine partner Several enzyme-catalyzed three-componentthree-component “Hantzsch-type” “Hantzsch-type” reactions reactions where where the the amine amine partner partner is is replaced by malonitrile, giving rise to several pyran derivatives, have appeared as an alternative isreplaced replaced by by malonitrile, malonitrile, giving giving rise rise to several to several pyran pyran derivatives, derivatives, have have appeared appeared as an alternativeas an alternative access access to this important class of compounds under mild, efficient and environment-friendly accessto this importantto this important class of compounds class of undercompounds mild, effiundercient andmild, environment-friendly efficient and environment-friendly conditions [55–57] conditions [55–57] (Scheme 11). conditions(Scheme 11 [55–57]). (Scheme 11).

OPh OO OPh OO Candida Rugosa CN Candida Rugosa EtO CN Ph-CHO+ OEt + NC CN EtO Ph-CHO+ OEt + NC CN lipase lipase ONH 90% ONH2 90% 2 Scheme 11.11. LipaseLipase from fromCandida Candida rugosa rugosa(CRL)-catalyzed (CRL)-catalyzed Hantzsch-type Hantzsch-type synthesis synthesis of 2-amino-4 of 2-amino-4H-pyrans.H- Scheme 11. Lipase from Candida rugosa (CRL)-catalyzed Hantzsch-type synthesis of 2-amino-4H- pyrans. pyrans.Recently,a Hantzsch-type four-component synthesis of tetrahydrodipyrazolo-pyridines 7 (Scheme6) from ethyl acetoacetate, an aldehyde, hydrazine and ammonium acetate was introduced. This ecofriendly Recently, a Hantzsch-type four-component synthesis of tetrahydrodipyrazolo-pyridines 7 protocolRecently, uses ovalbumin a Hantzsch-type obtained four-component from egg whites assynt anhesis environmentally of tetrahydrodipyrazolo-pyridines benign biocatalyst [58]. 7 (Scheme 6) from ethyl acetoacetate, an aldehyde, hydrazine and ammonium acetate was introduced. (SchemeThe 6) stereochemistry from ethyl acetoacetate, of most of an the aldehyde above Hantzsch, hydrazine reactions and ammonium was not reported,acetate was implying introduced. that This ecofriendly protocol uses ovalbumin obtained from egg whites as an environmentally benign Thisthe productsecofriendly obtained protocol may uses have ovalbumin been racemic obtained mixtures. from egg Yet, whites enzymes as an are environmentally well-known to benign exhibit biocatalyst [58]. biocatalystexcellent control [58]. of the reaction stereoselectivity [59], resulting in a predefined absolute configuration The stereochemistry of most of the above Hantzsch reactions was not reported, implying that of a specificThe stereochemistry stereocenter. of Since most di ffoferent the above enantiomers Hantzs ofch many reactions DHP was calcium not reported, blockers haveimplying different that the products obtained may have been racemic mixtures. Yet, enzymes are well-known to exhibit thepharmacokinetic products obtained properties may [have60–62 ],been the controlracemic of mi productxtures. enantioselectivityYet, enzymes are inwell-known Hantzsch synthesis to exhibit is excellent control of the reaction stereoselectivity [59], resulting in a predefined absolute configuration excellentyet to be developed.control of the reaction stereoselectivity [59], resulting in a predefined absolute configuration of a specific stereocenter. Since different enantiomers of many DHP calcium blockers have different of a specific stereocenter. Since different enantiomers of many DHP calcium blockers have different pharmacokinetic properties [60–62], the control of product enantioselectivity in Hantzsch synthesis pharmacokinetic5. The Strecker Reaction properties [60–62], the control of product enantioselectivity in Hantzsch synthesis is yet to be developed. is yetThe to be discovery developed. of the Strecker reaction (Scheme 12) made it possible to readily synthesize the 5.building The Strecker blocks thatReaction make up our proteins and enzymes. The first and interesting approach towards a 5. The Strecker Reaction biocatalytic asymmetric Strecker reaction was reported by Ramström and Vongvilai, in which they The discovery of the Strecker reaction (Scheme 12) made it possible to readily synthesize the combinedThe discovery transamination of the Strecker with imine-cyanation reaction (Scheme under 12) thermodynamicmade it possible control to readily and, synthesize subsequently, the building blocks that make up our proteins and enzymes. The first and interesting approach towards buildingcoupled inblocks a one-pot that make process up withour proteins a lipase-catalyzed and enzymes. transacylation The first and under interesting kinetic controlapproach [63 ].towards a biocatalytic asymmetric Strecker reaction was reported by Ramstrom̈ and Vongvilai, in which they a biocatalyticKawara etasymmetric al. reported Strecker the chemo–enzymatic reaction was reported catalyzed by synthesis Ramstrom̈ of and (R)-phenylglycine Vongvilai, in which derivatives they combined transamination with imine-cyanation under thermodynamic control and, subsequently, combinedin high yields transamination (79.5–97.8%) with and highimine-cyanation selectivity (86.5–98.7% under thermodynamic ee) from benzaldehyde, control and, ammonium subsequently, buffer coupled in a one-pot process with a lipase-catalyzed transacylation under kinetic control [63]. coupledand potassium in a one-pot cyanide process [64]. with They a usedlipase-catalyzed nitrilase AY487533 transacylation to catalyze under the kinetic reaction, control which [63]. led first to the formation of the corresponding racemic α-amino nitriles, whose R-enantiomer preferentially hydrolyzed to the acid than the S-enantiomer, which, under the equilibrium conditions of the Strecker reaction, reverts back to the starting materials. The forward reaction then regenerates the enzyme substrate, and the process is repeated until complete consumption of the starting materials.

Low concentrations of the reagents and strict temperature and pH control are key to the success of the reaction. Molecules 2020, 25, x FOR PEER REVIEW 8 of 20

R1 O R1 NH2 + HCN + NH4Cl R2 H R2 CO2H NH3 - H O + 2 H O H O - H 2 2 - NH3 + R NH R NH R NH 1 2 R1 NH2 1 2 OH 1 2 OH Molecules 2020, 25, 5935 8 of 19 C C MoleculesR 2020H , 25, x FOR PEER REVIEW C + R R 8 of 20 2 - R2 H NH 2 H + 2 H + C N NH2 HO NH3 R1 O R1 NH2 R , R = Alky, Aryl + HCN 1 + 2 NH4Cl R2 H R CO H Scheme 12. General Strecker reaction. 2 2 NH3 - H O + 2 H O H O - H Kawara et al. reported the chemo–enzymatic2 catalyzed synthesis2 of -( RNH)-phenylglycine3 + derivativesR NH in high yields (79.5–97.8%) and high selectivityR NH (86.5–98.7% ee) fromR benzaldehyde,NH 1 2 R1 NH2 1 2 OH 1 2 OH ammonium buffer and potassium cyanide [64]. They used nitrilase AY487533 to catalyze the reaction, C + C C whichR2 ledH first to the formationR of the correspondingNH racemicR2 H α-amino +nitriles, whoseR2 H R-enantiomer+ - 2 H NH NH preferentially hydrolyzedC N to the acid than the S-enantiomer, which, under2 the equilibriumHO conditions3 of the Strecker reaction, reverts back to the starting materials. The forward reaction then regenerates R1, R2 = Alky, Aryl the enzyme substrate, and the process is repeated until complete consumption of the starting materials. Low concentrations of Schemethe reagents 12. General and strictStrecker temperature reaction. and pH control are key to the success of the reaction. The rational design of an enzyme-catalyzed multicomponent Strecker reaction can lead to the TheKawara rational et aldesign. reported of an enzyme-catalyzedthe chemo–enzymatic multicomponent catalyzed Streckersynthesis reaction of (R )-phenylglycinecan lead to the asymmetric synthesis of α-amino acids [65–67], thus avoiding the classical multistep procedures asymmetricderivatives insynthesis high yields of α -amino(79.5–97.8%) acids and[65–67], high thus selectivity avoiding (86.5–98.7% the classical ee) multistep from benzaldehyde, procedures consisting, among others, of the resolution of racemic mixtures. Unfortunately, the above two consisting,ammonium amongbuffer andothers, potassium of the cyanide resolution [64]. of They racemic used nitrilasemixtures. AY487533 Unfortunately, to catalyze the the above reaction, two examples of bio-catalyzed Strecker reactions appear to be the only ones described to date and, therefore, exampleswhich led offirst bio-cata to the lyzedformation Strecker of the reactions corresponding appear racemic to be the α-amino only onesnitriles, described whose R-enantiomerto date and, clearly need further developments. therefore,preferentially clearly hydrolyzed need further to the developments. acid than the S-enantiomer, which, under the equilibrium conditions of the Strecker reaction, reverts back to the starting materials. The forward reaction then regenerates 6. The Mannich Reaction 6.the The enzyme Mannich substrate, Reaction and the process is repeated until complete consumption of the starting materials.The Mannich Low concentrations reaction is a powerfulof the reagents tool in organicand strict synthesis temperature that consists and pH of control an attack are of key a primary to the The Mannich reaction is a powerful tool in organic synthesis that consists of an attack of a orsuccess secondary of the aminoreaction. nitrogen on the carbonyl carbon of formaldehyde, resulting in an intermediate primary or secondary amino nitrogen on the carbonyl carbon of formaldehyde, resulting in an iminiumThe ionrational (pH ~design 4 to 5), of which an enzyme-catalyzed then subsequently multicomponent reacts with an enolisableStrecker reaction carbonyl can compound lead to the to intermediate iminium ion (pH ~ 4 to 5), which then subsequently reacts with an enolisable carbonyl generateasymmetricβ-aminoketone synthesis of derivatives,α-amino acids also [65–67], known asthus Mannich avoiding bases the [ 68classical,69]. To multistep exploit this procedures powerful compound to generate β-aminoketone derivatives, also known as Mannich bases [68,69]. To exploit syntheticconsisting, tool among and accessothers, more of the complex resolution molecular of racemic structures, mixtures. formaldehyde Unfortunately, is generally the above replaced two this powerful synthetic tool and access more complex molecular structures, formaldehyde is byexamples substituted of bio-cata benzaldehydeslyzed Strecker while reactions anilines replaceappear theto be amines. the only The ones first described lipase-catalyzed to date direct, and, generally replaced by substituted benzaldehydes while anilines replace the amines. The first lipase- three-componenttherefore, clearly need Mannich further reaction developments. from a ketone or aldehyde and an amine to form β-aminoketone catalyzed direct, three-component Mannich reaction from a ketone or aldehyde and an amine to form compounds was reported in 2009 by Yu et al. [70] (Scheme 13). β-aminoketone compounds was reported in 2009 by Yu et al. [70] (Scheme 13). 6. The Mannich Reaction Ar The Mannich reaction is a powerful tool in organic synthesis that consists of Phan attack of a Enzyme primary or secondary amino nitrogen on the carbOonyl carbon of formaldehyde, resultingNH in an Ph NH + intermediate iminium2 ion Ar(pH - CHO~ 4 to 5), which+ then subsequently reacts witho an enolisable carbonyl DMSO, 25 C O compound to generate β-aminoketone derivatives, also known as Mannich bases [68,69]. To exploit this powerful synthetic tool and access more complex molecular43.6-89.1% structures, formaldehyde is generally replaced by substitutedScheme benzaldehydes 13. Lipase-catalyzed whileMannich anilinesreaction. replace the amines. The first lipase- Scheme 13. Lipase-catalyzed Mannich reaction. catalyzed direct, three-component Mannich reaction from a ketone or aldehyde and an amine to form β-aminoketoneA series of compounds substrates were was exploredreported inin this2009 reaction, by Yu et resulting al. [70] (Scheme in moderate-to-good 13). yields using A series of substrates were explored in this reaction, resulting in moderate-to-good yields using various enzymes in water. However, lipase from Mucor miehei (MML) gave the best results, while lipase various enzymes in water. However, lipase from Mucor miehei (MML) gave the best results,Ar while CAL-B showed moderate catalytic activity. Nevertheless, no selectivity was reported in all cases Ph lipase CAL-B showed moderate catalytic activity. Nevertheless, Enzymeno selectivity was reported in all studied [71–73]. It was only a few years later that XueO et al. reported what is believed toNH be the first casesPh studiedNH [71–73].+ It was only a few years later that Xue et al. reported what is believed to be the truly enzyme-catalyzed,2 directAr - three-componentCHO + asymmetric Mannich reactiono using protease type XIV first truly enzyme-catalyzed, direct three-component asymmetricDMSO, Mannich 25 C reaction usingO protease from Streptomyces griseus [74]. The reaction was carried out in acetonitrile, affording up to 92% yield, type XIV from Streptomyces griseus [74]. The reaction was carried out in acetonitrile, affording up to 88% ee and 92:8 diastereoselectivities (syn:anti). Controlled43.6-89.1% experiments confirmed the enzyme 92% yield, 88% ee and 92:8 diastereoselectivities (syn:anti). Controlled experiments confirmed the catalytic action, mainly directing the stereoselective pathway of the reaction [16]. Encouraged by these results, Guan et al. studiedScheme the13. Lipase-catalyzed activity of another Mannich enzyme, reaction. acylase I (aminoacylase) from Aspergillus melleus, which catalyzes the hydrolysis of l-acyl-amino acids to produce the corresponding A series of substrates were explored in this reaction, resulting in moderate-to-good yields using l-amino acids [75]. This enzyme activity and stereoselectivity were improved by adjusting the solvent, various enzymes in water. However, lipase from Mucor miehei (MML) gave the best results, while pH, water content, temperature, molar ratio of substrates and enzyme loading. Enantioselectivities of lipase CAL-B showed moderate catalytic activity. Nevertheless, no selectivity was reported in all up to 89% ee, diastereoselectivities of up to 90:10 diastereoisomeric ratio (syn/anti) and yields of up to cases studied [71–73]. It was only a few years later that Xue et al. reported what is believed to be the 82% were achieved. first truly enzyme-catalyzed, direct three-component asymmetric Mannich reaction using protease type XIV from Streptomyces griseus [74]. The reaction was carried out in acetonitrile, affording up to 92% yield, 88% ee and 92:8 diastereoselectivities (syn:anti). Controlled experiments confirmed the

Molecules 2020, 25, x FOR PEER REVIEW 9 of 20 Molecules 2020, 25, x FOR PEER REVIEW 9 of 20 enzymeenzyme catalyticcatalytic action,action, mainlymainly directingdirecting thethe stereostereoselectiveselective pathwaypathway ofof thethe reactionreaction [16].[16]. EncouragedEncouraged byby thesethese results,results, GuanGuan etet alal.. studiedstudied thethe activityactivity ofof anotheranother enzyme,enzyme, acylaseacylase II (aminoacylase)(aminoacylase) fromfrom Aspergillus melleus, which catalyzes the hydrolysis of L-acyl-amino acids to produce the Aspergillus melleus, which catalyzes the hydrolysis of L-acyl-amino acids to produce the corresponding L-amino acids [75]. This enzyme activity and stereoselectivity were improved by corresponding L-amino acids [75]. This enzyme activity and stereoselectivity were improved by adjusting the solvent, pH, water content, temperature, molar ratio of substrates and enzyme loading. adjustingMolecules 2020 the, 25 solvent,, 5935 pH, water content, temperature, molar ratio of substrates and enzyme loading.9 of 19 EnantioselectivitiesEnantioselectivities ofof upup toto 89%89% ee,ee, diastereoseldiastereoselectivitiesectivities ofof upup toto 90:1090:10 diastereoisomericdiastereoisomeric ratioratio (syn/anti)(syn/anti) andand yieldsyields ofof upup toto 82%82% werewere achieved.achieved. InIn theirtheir effortsefforts efforts to to assess assess the the generality generality of of th thethee promiscuous promiscuous lipase-catalyzed lipase-catalyzed MannichMannich reaction,reaction, reaction, HeHe etet alal al... [71] [[71]71] identified identifiedidentified lipase lipase from from CandidaCandida rugosarugosa (CRL)(CRL) asas thethe mostmost activeactive forfor thethe reactionreaction ofof bothboth aromaticaromatic aldehydes,aldehydes, aliphatic aliphatic ketones ketones and and aniline. UnderUnder optimizedoptimized conditions,conditions, thethe MannichMannich adductsadducts couldcould bebe isolatedisolated inin 20–94%20–94% with with moderate moderate diastereoselectivity. diastereoselectivity. InIn eacheach each case,case, case, thethe the mechanismmechanism mechanism startsstarts starts withwith with thethe the formationformation formation ofof ofthethe the SchiffSchiff Schi basebaseff base fromfrom from thethe thereactionreaction reaction ofof thethe of amineaminethe amine andand aldehyde.aldehyde. and aldehyde. AtAt thethe At samesame the time,time, same thethe time, enolateenolate the enolateananionion isis anion stabilizedstabilized is stabilized byby thethe oxyanionoxyanion by the oxyanionhole,hole, followedfollowed hole, bybyfollowed thethe formationformation by the formation ofof thethe iminiumiminium of the iminium ionion inin thethe ion acidicacidic in the activeactive acidic sitesite active ofof the sitethe enzyme. ofenzyme. the enzyme. TheThe enolateenolate The enolate anionanion anion thenthen attacksattacksthen attacks thethe iminiumiminium the iminium ion,ion, ion,formingforming forming aa newnew a new carbon-carboncarbon-carbon carbon-carbon bond.bond. bond. H-atomH-atom H-atom transfertransfer transfer toto to thethe the O-atomO-atom O-atom fromfrom from acetoneacetone takestakes placeplace inin aa concertedconcerted process.process. Finally,Finally, Finally, thethe MannichMannich adductadduct isis relerele releasedasedased fromfrom thethe oxyanionoxyanion holehole (Scheme(Scheme 14).14).14).

1 1 O 1 N R O 1 N R R H2N R R R H2N R R HH H HH H R 1 R 1 1 1 NN RR RR H R N HH H H R - N H Hoxyanion hole H H H - H oxyanion hole H H H HH ProductsProducts O - O O - O OO H H H HH H H H Scheme 14.SchemeMechanism 14. Lipase-catalyzed of the Lipase-catalysed Mannich reaction. Mannich reaction. Scheme 14. Lipase-catalyzed Mannich reaction.

TheThe catalytic catalytic activity activity of of lipase lipase from from CandidaCandida antarctica antarctica lipaselipase B B (CAL-B) (CAL-B) in in Mannich Mannich reactions reactions was was studiedThe by catalytic the group activity of Zaharia, of lipase who from showed Candida that antarctica this enzyme lipase wasB (CAL-B) very e ﬀinective Mannich in the reactions synthesis was of studiedstudied byby thethe groupgroup ofof Zaharia,Zaharia, whowho showedshowed thatthat thisthis enzymeenzyme waswas veryvery effectiveeffective inin thethe synthesissynthesis ofof newnew Mannich basesbases derivedderived from from 2-phenylthiazole 2-phenylthiazole and and 2-phenylaminothiazole 2-phenylaminothiazole (Scheme (Scheme 15). 15). While While the newreaction Mannich worked bases well derived with acetone from 2-phenylthiazole as the nucleophile and component, 2-phenylaminothiazole aﬀording the (Scheme target compounds 15). While thethe reactionreaction workedworked wellwell withwith acetoneacetone asas thethe nuclnucleophileeophile component,component, affordingaffording thethe targettarget compoundscompounds with good good yields yields in in mild mild and and ecofriendly ecofriendly reaction reaction conditions, conditions, this enzyme this enzyme proved proved inactive inactive with other with withother good ketones yields [76 ,in77 mild]. and ecofriendly reaction conditions, this enzyme proved inactive with other ketonesketones [76,77].[76,77].

AcAc O N O N N CaL-B Ac NH N O CaL-B Ac NH O O + N NN O SS + N CH3CO2Na NH2.1/2H2SO4 CH3CO2Na R NH2.1/2H2SO4 S R 68-76% S 68-76% R = H, Me, OEt RR R = H, Me, OEt

Scheme 15. Synthesis of 2-phenylthiazole Mannich bases. Scheme 15. SynthesisSynthesis of 2-phenylthiazole Mannich bases.

MostMost reportedreported bio-catalyzedbio-catalyzed MannichMannich reactionsreactions useuse thethe samesame typetype ofof startingstarting materials:materials: ketones,ketones, ketones, arylaryl amines amines and and aromatic aromatic aldehydes.aldehydes. However,However, However, itit it cancan can sometimessometimes sometimes bebe be necessarynecessary necessary toto to preformpreform thethe intermediateintermediate ketamineketamine in in aa separate separate stepstep andand and complecomple completetete the the MannichMannich Mannich reactionreaction reaction withwith thethe the relevantrelevant relevant ketone ketone inin thethe the presencepresence presence ofof of thethe the biocatalyst.biocatalyst. biocatalyst. ThisThis This approachapproach approach waswas was usedused used byby bythethe the groupgroup group ofof WuWu of Wu [78],[78], [78 whowho], who studiedstudied studied thethe reactionreactionthe reaction ofof 3-phenyl-23-phenyl-2 of 3-phenyl-2HH-1,4-benzoxazine-1,4-benzoxazineH-1,4-benzoxazine withwith with acetoneacetone acetone inin inthethe the presencepresence presence ofof of thethe the cheapcheap cheap andand and readilyreadily

available wheat germ lipase (WGL). Under optimized conditions, they obtained the corresponding Mannich adducts in moderate yields (56%) with a good enantioselectivity (87% ee) (see Scheme 16). Molecules 2020, 25, x FOR PEER REVIEW 10 of 20

Moleculesavailable 2020 wheat, 25, x FOR germ PEER lipase REVIEW (WGL). Under optimized conditions, they obtained the corresponding10 of 20 Mannich adducts in moderate yields (56%) with a good enantioselectivity (87% ee) (see Scheme 16). available wheat germ lipase (WGL). Under optimized conditions, they obtained the corresponding MannichMolecules 2020 adducts, 25, 5935 in moderate yields (56%) with a good enantioselectivity (87% ee) (see Scheme10 16). of 19 O O O O Enzyme + o O DMSO, 25 C O O N O Enzyme N Ph + 56% o H DMSO, 25 C N N H Ph Scheme 16. Lipase-catalyzed Mannich reaction56% of 3-phenyl-2H-1,4-benzoxazine. Scheme 16. Lipase-catalyzed Mannich reaction of 3-phenyl-2H-1,4-benzoxazine. 7. The Ugi ReactionScheme 16. Lipase-catalyzed Mannich reaction of 3-phenyl-2H-1,4-benzoxazine. 7. TheIn Ugi its standard Reaction form, the Ugi reaction is a one-pot, four-component condensation reaction of a 7. The Ugi Reaction primaryIn its amine, standard a carbonyl form, the compound, Ugi reaction a carboxylic is a one-pot, acid four-componentand an isocyanide condensation to afford α-amino reaction acid of aderivatives primaryIn its standard amine, of a bis-amidea form, carbonyl the product Ugi compound, reaction with isthe a a carboxylic one-pot,release of four-component acid only and one an molecule isocyanide condensation of water to aff reaction ordas theα-amino onlyof a primaryacidbyproduct derivatives amine, [79,80]. a of carbonyl aDue bis-amide to compound,its simplicity product a with carboxylicand theapplications release acid and of in only an many isocyanide one fields, molecule tosuch afford of as water combinatorial,α-amino as the acid only derivativesbyproductmedicinal, [peptideof79 ,a80 bis-amide]. and Due peptidomimetics to product its simplicity with chemistry,the and releas applications ethis of MCRonly in onerapidly many molecule fields, developed suchof water to as become combinatorial, as the one only of byproductmedicinal,the most versatile peptide[79,80]. reactions andDue peptidomimeticsto itsin organicsimplicity synthesis chemistry,and applications [81]. this The MCR acid in component rapidly many developedfields, can suchbe replaced to as become combinatorial, byone an acidic of the medicinal,mostcatalyst versatile to peptideform reactions what and is peptidomimetics now in organic known synthesis as a chemistry,three- [81component]. The this acid MCR Ugi component rapidly reaction. developed canUnder be replacedthese to become circumstances, by an one acidic of thecatalystenzymes most toversatile are form good what reactions candidates is now in knownorganic as possible assynthesis a three-componentcatalysts. [81]. The acid Ugi component reaction. can Under be replaced these circumstances, by an acidic catalystenzymesThe to arefirst form good enzyme-catalyzed what candidates is now known as possibleUgi as reaction, a three- catalysts. componentmediated by Ugi Novozym reaction. 435, Under was these reported circumstances, in 2013 by Kzossowski [80], allowing for the synthesis of a new class of dipeptides in up to 87% yield (Scheme enzymesThe are first good enzyme-catalyzed candidates as possible Ugi reaction, catalysts. mediated by Novozym 435, was reported in 2013 by 17). KzossowskiThe first [80 enzyme-catalyzed], allowing for the Ugi synthesis reaction, of amediat new classed by of dipeptidesNovozym 435, in up was to 87% reported yield (Schemein 2013 by 17 ). Kzossowski [80], allowing for the synthesis of a new class of dipeptides in up to 87% yield (Scheme 17). NC R2 O R NH R Novozyme 435 NH R 1 2 + R2 CHO + 3 HN 1 Toluene, 24 hours R2 NH NCO O R O R NH R Novozyme43 - 87 435 % 1 NH R 1 2 + R2 CHO + 3 HN 1 R3 = OEt, OBn, NHPhToluene, 24 hours NH O O R Scheme 17. Enzyme-catalyzed43 Ugi - reaction.87 % 1 SchemeR = 17.OEt, Enzyme-catalyzed OBn, NHPh Ugi reaction. 3 Building on this experience, this same group replaced the aldehyde by a cyclic imine to developBuilding an elegant on this synthesis experience, ofScheme substituted this same17. Enzyme-catalyzed group pyrrolidines replaced and Ugi the piperidinesreaction. aldehyde by catalyzed a cyclic byimine Novozym to develop 435, therebyan elegant expanding synthesis the of activity substituted promiscuity pyrrolidines of this and enzyme piperidines [82]. Later catalyzed on, they by further Novozym demonstrated 435, thereby that theexpanding enzymeBuilding the catalyst on activity this selectivity experience, promiscuity was this dependent of same this groupenzyme on replaced the [82]. chain Later the length on,aldehyde they of the further by isocyanide a cyclic demonstrated imine ester, to indicating develop that the a anenzyme elegant catalyst synthesis selectivity of substituted was dependent pyrrolidines on theand chain piperidines length catalyzedof the isocyanide by Novozym ester, 435, indicating thereby a preference for short-chain isocyanoesters (C1 to C4) and switching the enzyme promiscuity between a expandingthree-componentpreference thefor activityshort-chain and apromiscuity four-component isocyanoesters of this MCR (Cenzyme1 to [83 C].4 [82].) and Later switching on, they the further enzyme demonstrated promiscuity betweenthat the enzymea three-componentThe catalyst mechanism selectivity and of a the four-component was reaction dependent is depicted MCR on the [83]. in chain Scheme length 18. of It the is based isocyanide on the ester, traditional indicating lipase- a preferencecatalyzedThe mechanism esterfor short-chain hydrolysis, of the isocyanoesters whichreaction involves is depicted (C1 theto C acylationin4) andScheme switching by 18. the It estertheis based enzyme of the on isocyanidepromiscuitythe traditional by between alipase- serine aresiduecatalyzed three-component of ester the catalytic hydrolysis, and a triad four-component which of the involves enzyme MCR the as [83]. theacylation first step. by the Activation ester of the by isocyanide acidic Asp by residues a serine of theresidue SchiThe ff ofmechanismbase the catalytic (formed of fromtriadthe reaction theof the reaction enzyme is depicted of as the the aminein fi Scrstheme onstep. the 18.Activation aldehyde) It is based by generates acidicon the Asp traditional the residues corresponding lipase- of the catalyzediminiumSchiff base ion,ester (formed which hydrolysis, isfrom attacked the which reaction by involves the isocyanideof the the am acylationine nucleophile. on the by aldehyde)the Theester subsequent of generates the isocyanide stepsthe corresponding are by similara serine to iminium ion, which is attacked by the isocyanide nucleophile. The subsequent steps are similar to residuethose of of the the classical catalytic Ugi triad reaction. of the enzyme The lack as ofthe stereoselectivity first step. Activation is attributed by acidic to Asp reversibility residues of of the the those of the classical Ugi reaction. The lack of stereoselectivity is attributed to reversibility of the Schiffreaction base between (formed the from isocyanide the reaction and the of iminiumthe amine ion. on the aldehyde) generates the corresponding reaction between the isocyanide and the iminium ion. iminiumThe aboveion, which protocol is attacked allowed by the the first isocyanide synthesis nucleophile. of dipeptide The derivatives subsequent in Scheme steps are17 under similar mild, to thoseecofriendly of the conditionsclassical Ugi in moderate-to-excellentreaction. The lack of stereoselectivity yields. is attributed to reversibility of the reaction between the isocyanide and the iminium ion.

Molecules 2020, 25, 5935 11 of 19 Molecules 2020, 25, x FOR PEER REVIEW 11 of 20

H2N R O + HO 1 R Asp O O H2O OEt HO O - + - R N Asp C + O-Ser 1 C N N R O HO Ser Products

R HO N Asp - 1 O R O H N R R N H 2 1 Asp R O O - C C + O-Ser O NH O N

O-Ser - R O - N H Asp R O 1 R O N H Asp 1 C + R O N O C HO N O O-Ser H2O O-Ser Scheme 18. Mechanism of the enzyme-catalyzed Ugi reaction.

8. MulticomponentThe above protocol No Namedallowed Reactionsthe first synthesis of dipeptide derivatives in Scheme 17 under mild, ecofriendlyThere are conditions a few multicomponent in moderate-to-excellent reactions catalyzedyields. by enzymes but do not have special names. These are classified under this section according to the number of reactants involved in the reaction. 8. Multicomponent No Named Reactions 8.1. Three-Component Reactions There are a few multicomponent reactions catalyzed by enzymes but do not have special names. TheseA are series classified of 4H-pyran under this derivatives section according similar to to those the number shown of in reactants Scheme involved11 were synthesizedin the reaction. by three-component reactions catalyzed by various enzymes [55,84–86]. 8.1. Three-ComponentIn their search for Reactions potential new antidiabetic agents containing the hydrazono-4-thiazolidinone scaffold 8 (Scheme6), Chavan et al. [ 87] designed an efficient and simple one-pot baker’s yeast catalyzed A series of 4H-pyran derivatives similar to those shown in Scheme 11 were synthesized by three- protocol for the synthesis of substituted derivatives of 2-hydrazono-4-thiazolidinone-5-acetic acids and component reactions catalyzed by various enzymes [55,84–86]. the corresponding pyrazole analogs. In their search for potential new antidiabetic agents containing the hydrazono-4-thiazolidinone The dihydropyrano[2,3-c]pyrazole skeleton (Scheme 19) can be accessed by a three-component scaffold 8 (Scheme 6), Chavan et al. [87] designed an efficient and simple one-pot baker’s yeast reaction of aldehyde/ketone or isatin, malononitrile and 3-methyl-1H-pyrazol-5(4H)-one, catalyzed by catalyzed protocol for the synthesis of substituted derivatives of 2-hydrazono-4-thiazolidinone-5- BSA. The reaction is described to take place at ambient temperature in a mixture of ethanol and water acetic acids and the corresponding pyrazole analogs. to afford the products in 76–94% yield [88]. The dihydropyrano[2,3-c]pyrazole skeleton (Scheme 19) can be accessed by a three-component The same group later on developed a new synthesis of ortho-aminocarbonitriles from aromatic reaction of aldehyde/ketone or isatin, malononitrile and 3-methyl-1H-pyrazol-5(4H)-one, catalyzed aldehydes, cyclohexanone and malononitrile using porcine pancreas lipase (PPL) as the catalyst [89]. by BSA. The reaction is described to take place at ambient temperature in a mixture of ethanol and In another report, a one-pot synthesis of spiropyrazolo[3,4-b]pyridine and spiro-oxindole scaffolds water to afford the products in 76–94% yield [88]. catalyzed, respectively, by papain and catalase, was carried out starting from isatin, 1,3-dicarbonyls and 3-methyl-5-amino pyrazole [90] (Scheme6). In addition to good-to-high yields, other advantages of this protocol include operational simplicity, simple filtration, no need for a chemical catalyst or activation and no column chromatographic purification and the biocatalyst tolerance to a wide range of substrates. The proposed reaction mechanism begins with the normal formation of an iminium ion from the aldehyde and the basic lysine residues of the enzyme. At same time, the removal of the proton from malononitrile by the free basic amino group of BSA forms a carbanion that, subsequently, undergoes a Knoevenagel condensation with the iminium ion. The reaction intermediate condenses with the

Molecules 2020, 25, x FOR PEER REVIEW 12 of 20

R4 R1 R2 O O R CN 4 N R N R1 R2 N CN N 3 R3 N O NH N + 2 BSA O R 1 2 R O CN BSA N CN R = H, Ph; R , R = Me, Et, Ph N ArCHO BSA O NH 80-95% H 2 R = H; R3 = H, Et, benzyl, 4-Clbenzyl; R4 = H, Cl, Br Ar 84-98% CN Molecules 2020, 25, 5935 N 12 of 19 N O NH2 H R = H activated pyrazalone in a Michael addition reaction to form an adduct whose cyclization aﬀords the 82-95% productMolecules 2020 (Scheme, 25, x FOR 20). PEER REVIEW 12 of 20 Scheme 19. BSA-catalyzed synthesis of dihydropyrano[2,3-c]pyrazoles and spiro[indoline-3,4′- pyrano[2,3-c]pyrazoles]. R4 R1 R2 O O R CN 4 N R N The same groupR1 lateR2 r on developedN a new synthesisCN of ortho-aminocarbonitriles from aromaticN 3 R N N + 3 aldehydes,O cyclohexanoneNH2 and malononitrile using porcine pancreas lipase (PPL) as the catalyst O[89]. BSA CN InR another report,1 2 a one-pot synthesisR O of spiropyrazolo[3,4-b]pyridineBSA and spiro-oxindoleN scaffoldsCN R = H, Ph; R , R = Me, Et, Ph N ArCHO BSA O NH catalyzed,80-95% respectively, by papain and catalase, was carried out starting from isatin, H1,3-dicarbonyls2 and 3-methyl-5-amino pyrazole [90] (Scheme 6). R = H; R3 = H, Et, benzyl, 4-Clbenzyl; R4 = H, Cl, Br In addition to good-to-high yields,Ar other advantages of this84-98% protocol include operational simplicity, simple filtration, no need for CNa chemical catalyst or activation and no column chromatographic purification andN the biocatalyst tolerance to a wide range of substrates. N The proposed reaction mechanism beginsO NH with2 the normal formation of an iminium ion from the H aldehyde and the basic lysine residuesR of = Hthe enzyme. At same time, the removal of the proton from malononitrile by the free basic amino82-95% group of BSA forms a carbanion that, subsequently, undergoes a Knoevenagel condensation with the iminium ion. The reaction intermediate condenses with the SchemeScheme 19.19. BSA-catalyzedBSA-catalyzed synthesis synthesis of of dihydropyran dihydropyrano[2,3-c]pyrazoleso[2,3-c]pyrazoles and and spiro[indoline-3,4 spiro[indoline-3,4′-0- activated pyrazalone in a Michael addition reaction to form an adduct whose cyclization affords the pyrano[2,3-c]pyrazoles].pyrano[2,3-c]pyrazoles]. product (Scheme 20).

The same group later on developed a new synthesis of ortho-aminocarbonitrilesBSA from aromatic BSA BSA + aldehydes, cyclohexanone and malononitrile using porcine pancreas lipaseN (PPL) as the catalyst [89]. In anotherNH report,O a one-pot synthesisH + of spiropyrazolo[3,4-b]pyridine1 and spiro-oxindole scaffolds 2 - H O N R R H catalyzed, respectively,1 by 2papain and catalase, was carried out startingH Hfrom isatin, 1,3-dicarbonyls R R 1 and 3-methyl-5-amino pyrazole [90]R (SchemeR 6). CN O In addition to good-to-high CNyields, other advantagesC C of this protocol include operationalH NH2 N N H BSA NH N simplicity,NH simple filtration, no need for a chemical catalyst or activation and Ono column 3 CN + CN N N chromatographic purificationNH3 and the biocatalyst tolerance to a wide range of substrates. BSA The proposed reaction mechanismBSA begins with the normal formation of an iminium ionH from the aldehyde and the basic lysine residues of the enzyme. At same time, the removal of the protonH2N from NH 1 malononitrile by the free basic amino group of BSA2 formsH R a carbanionR that, subsequently, undergoes a Knoevenagel condensationR 1 with the iminium ion. The reaction intermediate condensesBSA with the R N CN activated pyrazalone in aCN Michael addition reaction to form an adduct whose cyclization affords the cyclization N product (SchemeN 20). C H O N NH O NH2 H BSA BSA BSA H + H N + H NH O H 1 + 2 - H O N R R H 1 2 HN H R R 1 R R BSA CN O CN C C H Scheme 20. Mechanism of the BSA-catalyzedNH2 synthesisN ofN dihydropyrano[2,3-c]pyrazoles and SchemeH 20. Mechanism of the BSA-catalyzedBSA synthesis of dihydropyrano[2,3-c]pyrazolesNH andN NHspiro[indoline-3,40-pyrano[2,3-c]pyrazoles. O spiro[indoline-3,43 CN ′-pyrano[2,3-c]pyrazoles.+ CN N N NH3 8.2. Four-ComponentBSA Reactions BSA H Enzyme-catalyzed four-component reactions are not very common. The ﬁrst reactionH recorded2N NH2 1 in this category is the synthesis of dihydropyrano[2,3-c]pyrazolesH R R [91] (see Scheme 19), mediated by R 1 BSA Aspergillus niger lipase (ANL)R from a mixture of ethylN acetoacetate,CN hydrazine hydrate, aldehyde/ketone CN and malononitrile. The reaction wascyclization carried out atN room temperature and aﬀorded excellent yields of N C the expected products. This report was followed byH theO porcine pancreas lipase (PPL) MCR developed N NH O NH2 by Bihani et al.H [92] in what they termed the “pot, atom and step economic (PASE)” synthesis of H 5-monosubstituted barbiturates bearing biologically active pyrazaloneH cores (Scheme 21), which were H then screened against selected human pathogens with good pharmacological+ activity. N BSA

Scheme 20. Mechanism of the BSA-catalyzed synthesis of dihydropyrano[2,3-c]pyrazoles and spiro[indoline-3,4′-pyrano[2,3-c]pyrazoles.

Molecules 2020, 25, x FOR PEER REVIEW 13 of 20

8.2. Four-Component Reactions Molecules 2020, 25, x FOR PEER REVIEW 13 of 20 Enzyme-catalyzed four-component reactions are not very common. The first reaction recorded 8.2.in this Four-Component category is the Reactions synthesis of dihydropyrano[2,3-c]pyrazoles [91] (see Scheme 19), mediated by AspergillusEnzyme-catalyzed niger lipase four-component (ANL) from reactionsa mixture are notof veryethyl common. acetoacetate, The first hydrazine reaction recordedhydrate, inaldehyde/ketone this category is and the malononitrile.synthesis of dihydropyrano[2,3-c]pyrazoles The reaction was carried out at[91] room (see temperature Scheme 19), andmediated afforded by Aspergillusexcellent yields niger of lipasethe expected (ANL) products. from a This mixture report ofwas ethyl followed acetoacetate, by the porcine hydrazine pancreas hydrate, lipase aldehyde/ketone(PPL) MCR developed and malononitrile. by Bihani et Theal. [92] reaction in what was they carried termed out atth eroom “pot, temperature atom and step and economic afforded excellent(PASE)” synthesisyields of theof 5-monosubstitutedexpected products. barbituratesThis report wasbearing followed biologically by the activeporcine pyrazalone pancreas lipasecores (PPL)(Scheme MCR 21), developed which bywere Bihani then et alscreened. [92] in whatagainst they selectedtermed thhumane “pot, atompathogens and step with economic good (PASE)”pharmacological synthesis activity. of 5-monosubstituted barbiturates bearing biologically active pyrazalone cores (Scheme 21), which were then screened against selected human pathogens with good Molecules 2020, 25, 5935 H R O13 of 19 pharmacological activity. O NH O PPL HN O NH H R O + + H NNH .H O + N + X Et 2 2 2 O O R H O Ethanol, RT H - O O O N X O NH PPL HN H O H NH 77 - 94% X = O, S; + + H NNH .H O + N + X Et 2 2 2 R O= alkyl/benzyl O R H Ethanol, RT H - O O N X O Scheme 21. Porcine pancreas lipase (PPL)-catalyzedH 4-component77 - 94% synthesis of HpyrazaloneX = O, S; derivatives. R = alkyl/benzyl SchemeScheme 21.21. Porcine pancreas lipase (PPL)-catalyzed 4-component synthesis of pyrazalone The mechanismPorcine of pancreas this reaction lipase (PPL)-catalyzed was not investig 4-componentated, but synthesis the initial of pyrazalone possible formation derivatives. of a derivatives. pyrazaloneThe mechanism derivative of followed this reaction by its was condensation not investigated, with the but barbiturates the initial possible was ruled formation out by ofthe a pyrazaloneauthors.The mechanism derivative followedof this reaction by its condensation was not investig withated, the barbiturates but the initial was possible ruled out formation by the authors. of a In another proof of its catalytic promiscuity, BSA was also shown to efficiently catalyze the one- pyrazaloneIn another derivative proof of itsfollowed catalytic by promiscuity, its condensation BSA was with also the shown barbiturates to efficiently was catalyze ruled out the one-potby the pot four-component green synthesis of pyrano[2,3-c]pyrazoles (see Scheme 19). The catalyst could be four-componentauthors. green synthesis of pyrano[2,3-c]pyrazoles (see Scheme 19). The catalyst could be recovered and reused up to five consecutive times with minimal loss of activity [93]. recoveredIn another and reused proof of up its to catalytic five consecutive promiscuity, times BS withA was minimal also shown loss of to activity efficiently [93]. catalyze the one- pot four-component green synthesis of pyrano[2,3-c]pyrazoles (see Scheme 19). The catalyst could be 8.3. Five-Component Reactions 8.3.recovered Five-Component and reused Reactions up to five consecutive times with minimal loss of activity [93]. TheThe groupgroup ofof WuWu reportedreported anan aldol–Michaelaldol–Michael additionaddition cascade reaction leading to to the the formation formation 8.3. Five-Component Reactions ofof sixsix carbon–carboncarbon–carbon/carbon–nitrogen/carbon–nitrogen bonds and two ring systems in in a a single operation operation [94] [94] using using CandidaCandidaThe antarcticaantarctica group of lipase lipaseWu reported BB (CAL-B)(CAL-B) an asaldol–Michaelas thethe biocatalystbiocatalyst addi (Scheme(Schemetion cascade 2222).). reaction leading to the formation of six carbon–carbon/carbon–nitrogen bonds and two ring systems in a single operation [94] using Candida antarcticaCH3CONH lipase2 B (CAL-B) as the biocatalyst (Scheme 22). O2N NO2 O OCH3 + CAL-B CH3CONH2 2 O2N Ar1NO O O N 2 Ar = 4-MeO-phenyl 1 OCH Ar CHO 3 +2 2 Ar2 = 4-NOCAL-B2-phenyl Ar1 72% O N Ar1 = 4-MeO-phenyl Ar CHO Scheme2 22. Enzyme-catalyzed 5-componentAr2 = 4-NO 2-phenyl synthesis of spiro-oxazino derivatives. Scheme 22. Enzyme-catalyzed 5-component synthesis of spiro-oxazino derivatives. 72% The reaction involved a lipase/acetamide-catalyzed Michael addition of ketones to nitroalkenes, The reaction involved a lipase/acetamide-catalyzed Michael addition of ketones to nitroalkenes, affording spiro-oxazinoScheme 22. Enzyme-catalyzed derivatives. While 5-component the reaction synthesis worked of spiro-oxazino well with derivatives.p-nitrobenzaldehyde, affording spiro-oxazino derivatives. While the reaction worked well with p-nitrobenzaldehyde, low low yields were obtained with other aldehydes. The proposed reaction mechanism involved an yields were obtained with other aldehydes. The proposed reaction mechanism involved an initial initialThe aldol reaction condensation, involved a followed lipase/acetamide-catalyzed by a Michael addition Michael of the addition acetamide-activated of ketones to nitroalkenes, aldol to the aldol condensation, followed by a Michael addition of the acetamide-activated aldol to the nitroalkeneaffording spiro-oxazino [95,96]. Cyclization derivatives. of this While adduct the reaction and the additionworked well of a with second p-nitrobenzaldehyde, molecule of the ketone low nitroalkene [95,96]. Cyclization of this adduct and the addition of a second molecule of the ketone leadsyields to were the finalobtained product with as other a racemic aldehydes. mixture The (Scheme proposed 23). reaction mechanism involved an initial leads to the final product as a racemic mixture (Scheme 23). aldolBased condensation, on these preliminaryfollowed by results, a Michael the authors addition were of ablethe toacetamide-activated extend the scope ofaldol the reactionto the bynitroalkene using a preformed [95,96]. Cyclization aldol compound. of this adduct Under and these the new addition conditions, of a second CAL-B molecule catalyzed of the a two-step, ketone one-potleads to the multicomponent final product as synthesis a racemic of mixture spiro-oxazino (Scheme derivatives,23). starting from readily available aldehydes, activated olefins, cyclohexanone and acetamide [97]. The first step consisted of the PPL-catalyzed asymmetric aldol reaction, followed after filtration of the PPL by the addition of CAL-B and all other substrates in the same pot, giving acceptable yields with diastereoselectivity and enantioselectivity of up to 93% ee. This approach opened up the door to many potential applications in the synthesis of complex organic molecules hardly accessible by other protocols. Molecules 2020, 25, 5935 14 of 19 Molecules 2020, 25, x FOR PEER REVIEW 14 of 20

O O OH O O CAL-B NH + R R 2 H Acetamide CAL-B

OH HN NO OH NH2 2 R 1 - CH3COOH 1 R R R R OH HN CAL-B - H3NO2 1 R = 4-NO2-Ph; R = 4-OMe-Ph O

R 1 R O N

Scheme 23. MechanismMechanism of of the the enzyme-catalyzed enzyme-catalyzed 5-compon 5-componentent synthesis of spiro-oxazino derivatives. 9. Conclusions Based on these preliminary results, the authors were able to extend the scope of the reaction by usingThis a preformed short review aldol shows compound. that bio-catalysisUnder these cannew beconditions, successfully CAL-B applied catalyzed to a a wide two-step, variety one- of multicomponentpot multicomponent reactions synthesis and constitute of spiro-oxazino an ideal substitute derivatives, to traditional starting catalystsfrom readily in the viewpointavailable ofaldehydes, efficiency, activated selectivity, olefins, yield cyclohexanone and green sustainability. and acetamide The [97]. stereochemical The first step outcome consisted of theof the Asinger, PPL- Mannichcatalyzed and asymmetric Strecker reactions aldol reaction, was successfully followed after controlled, filtration leading of the to thePPL expected by the addition products of with CAL-B high eeand and all diastereoselectivity. other substrates in However, the same only pot, one giving example acceptable of enzyme-catalyzed yields with reaction diastereoselectivity has been reported and thusenantioselectivity far for both theof up Asinger to 93% and ee. This Strecker approach reactions, opened despite up the the door importance to many potential and versatility applications of the opticallyin the synthesis active intermediates of complex organic derived molecule from theses hardly reactions accessible in the production by other protocols. of various pharmaceuticals and fine chemicals. These bioactive molecules obtained under a greener protocol offered by enzymatic catalysis9. Conclusions is an attractive option for future scientific exploration. TheThis Biginellishort review reaction shows is, undoubtedly,that bio-catalysis the can MCR be mostsuccessfully investigated applied using to a enzyme wide variety catalysis. of Whilemulticomponent many Biginelli reactions reaction and productsconstitute can an beideal effi sucientlybstitute obtained to traditional using traditionalcatalysts in non the enzymaticviewpoint procedures,of efficiency, such selectivity, as under yield solvent-free and green and sustainability./or mechanochemical The stereo conditions,chemical outcome many of of these the methodsAsinger, useMannich more and expensive Strecker catalysts, reactions sometimes was successfully times in controlled, stoichiometric leading amounts, to the expected require longer products reaction with timeshigh ee and and generate diastereoselectivity. waste materials. However, The advantage only one of example bio-catalysis of enzyme-catalyzed in this MCR is the reaction accommodation has been ofreported a broader thus substrate far for both range the under Asinger mild and reaction Strecker conditions. reactions, despite the importance and versatility of theThe optically Hantzsch active reaction intermediates is closely relatedderived to from the Biginelli these reactions reaction, andin the both production lead to two of sca variousffolds withpharmaceuticals several common and structuralfine chemicals. features: These 1,4-DHP bioact (Hantzsch)ive molecules and obtained 3,4-DHMP under (Biginelli), a greener starting protocol from theoffered same by reagents enzymatic and catalysis with similar is an conditions. attractive option The use for of enzymesfuture scientific as catalysts exploration. in these reactions opens newThe possibilities Biginelli toreaction access is, the undoubtedly, two scaffolds the under MCR ecofriendly most investigated conditions. using The enzyme formation catalysis. of racemic While productsmany Biginelli in the tworeaction MCRs products and, indeed, can inbe many efficiently other reactionsobtained needsusing furthertraditional investigation non enzymatic to fully utilizeprocedures, the enzyme such as selectivity. under solvent-free and/or mechanochemical conditions, many of these methods use moreThe Ugi expensive reaction catalysts, is among sometimes the most used times diversity-oriented in stoichiometric MCRs amounts, applicable require in longer drug discovery, reaction α leadingtimes and to generate the formation waste of materials. peptide-like The advantage-acylaminoamides of bio-catalysis containing in this one MCR or is more the accommodation chiral centers in non-racemicof a broader forms,substrate which range are under otherwise mild di reactionfficult to conditions. obtain by conventional methods. The benefits from the well-recognizedThe Hantzsch reaction facial discrimination is closely rela broughtted to the about Biginelli by enzyme reaction, catalysis and both is herebylead to highlighted.two scaffolds with Multicomponentseveral common reactionsstructural with features: four or 1,4-DHP more components (Hantzsch) are and not 3,4-DHMP very widespread, (Biginelli), but progressstarting continuesfrom the same to be reagents made in theand development with similar conditions. of new MCRs, The and use they of enzymes can benefit as catalysts from the uniquein these versatility reactions ofopens enzymes new possibilities to accommodate to access various the substrates.two scaffolds under ecofriendly conditions. The formation of

Molecules 2020, 25, 5935 15 of 19

While bio-catalysis in many classical reactions, such as the Michael reaction, aldol condensations, carbonyl reductions and ester hydrolysis, is well-established, its application in MCRs is still underdeveloped, and much work is required to harness its full potential.

Author Contributions: Conceptualization, W.M.; formal analysis, W.M.; resources, W.M.; writing—original draft preparation, W.M.; writing—review and editing, W.M. and N.D.J. and funding acquisition, W.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by The Directorate of Research and Innovation at Walter Sisulu University. Acknowledgments: We thank the Directorate of Research and Innovation at Walter Sisulu University for financial assistance. Conflicts of Interest: The authors declare no conflict of interest.

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