Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids

International Journal of Molecular Sciences

Review Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids

Francesca Ghirga 1, Alessandra Bonamore 2,*, Lorenzo Calisti 2, Ilaria D’Acquarica 3,* ID , Mattia Mori 1, Bruno Botta 3 ID , Alberto Bofﬁ 2 and Alberto Macone 2,* ID

1 Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy; [email protected] (F.G.); [email protected] (M.M.) 2 Deaprtment of Biochemical Sciences “A.Rossi Fanelli”, Sapienza University of Rome, Pizzale Aldo Moro 5, 00185 Rome, Italy; [email protected] (L.C.); alberto.bofﬁ@uniroma1.it (A.B.) 3 Department of Chemistry and Technology of Drugs, Sapienza University of Rome, Pizzale Aldo Moro 5, 00185 Rome, Italy; [email protected] * Correspondence: [email protected] (A.B.); [email protected] (I.D.); [email protected] (A.M.); Tel.: +39-06-49910813 (A.M.)

Received: 6 November 2017; Accepted: 16 November 2017; Published: 20 November 2017

Abstract: Benzylisoquinoline alkaloids (BIAs) are among the most important plant secondary metabolites, in that they include a number of biologically active substances widely employed as pharmaceuticals. Isolation of BIAs from their natural sources is an expensive and time-consuming procedure as they accumulate in very low levels in plant. Moreover, total synthesis is challenging due to the presence of stereogenic centers. In view of these considerations, green and scalable methods for BIA synthesis using fully enzymatic approaches are getting more and more attention. The aim of this paper is to review fully enzymatic strategies for producing the benzylisoquinoline central precursor, (S)-norcoclaurine and its derivatives. Speciﬁcally, we will detail the current status of synthesis of BIAs in microbial hosts as well as using isolated and recombinant enzymes.

Keywords: alkaloids; benzylisoquinoline alkaloids; green synthesis; recombinant enzymes; microbial factories

1. Introduction Benzylisoquinoline alkaloids (BIAs) are a nitrogen-containing group of plant secondary metabolites with a centenary history of investigation [1]. The interest in the wide variety of BIA structures is paramount, due to their well-established and long-standing pharmacological proﬁles [2]. The most covered therapeutic areas by BIAs are analgesics [3,4], antimicrobial [5], antimalarial [6,7], anti-HIV [7–9], and anticancer agents [10–12], whereas new areas are being explored more recently for BIAs, such as their employment as cholesterol-lowering and protecting agents against the atherosclerotic disease [13–16] and as signaling suppressors in colon cancer cells [17]. In 2010, with the aim of providing comprehensive information about both the source and the medicinal properties of BIAs, there was created an ad hoc database of benzylisoquinolines compounds, called BIAdb, helpful for all those who are working in the ﬁeld of drug discovery, to explore the pharmacological potential of BIAs [18]. One of the most important features of BIAdb is that it provides information about 627 plant species as a source of benzylisoquinoline and 114 biological activities shown by these compounds. A lot of tools have been integrated in the database, which help users in discovering the full potential of the program itself. The general benzylisoquinoline skeleton (Figure1) can be mostly synthesized (with few exceptions) by a cyclization reaction, namely, the Pictet-Spengler (P-S) reaction, which has been the subject of a huge number of review articles [19–22] and in 2011 has entered its second century

Int. J. Mol. Sci. 2017, 18, 2464; doi:10.3390/ijms18112464 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2464 2 of 19

Int. J. Mol. Sci. 2017, 18, 2464 2 of 18 of lifeInt. [23 J. Mol.,24 ].Sci. Nowadays, 2017, 18, 2464 the P-S reaction is still one of the most employed synthetic tools2 of not 18 only [23,24]. Nowadays, the P-S reaction is still one of the most employed synthetic tools not only to yield Int. J. Mol. Sci. 2017, 18, 2464 β 2 of 18 to yield[23,24].tetrahydroisoquinolines tetrahydroisoquinolines Nowadays, the P-S (THIQs) reaction (THIQs) and is still tetrahydro- one and of tetrahydro- theβ most-carbolines employed-carbolines (THBCs), synthetic (THBCs),but tools also not to but onlybuild also to novelyield to build novel scaffolds for structure-activity relationship studies and/or for combinatorial libraries for drug tetrahydroisoquinolinesscaffolds[23,24]. Nowadays, for structure-activity the P-S (THIQs) reaction relationship and is stilltetrahydro- one stud of theiesβ-carbolines most and/or employed for (THBCs), combinatorial synthetic but toolsalso libraries notto buildonly for to novel drugyield discoveryscaffoldsdiscoverytetrahydroisoquinolines programs. forprograms. structure-activity (THIQs) relationship and tetrahydro- studiesβ-carbolines and/or for (THBCs),combinatorial but also libraries to build for noveldrug discoveryscaffolds programs.for structure-activity relationship studies and/or for combinatorial libraries for drug R discovery programs. R N RR R H N RR R H N RR R H RR R FigureFigure 1. The1. The benzylisoquinoline benzylisoquinoline skeleton (R (R = =H, H, OH, OH, OMe). OMe). Figure 1. The benzylisoquinolineR skeleton (R = H, OH, OMe). The P-S reaction is essentially an electrophilic aromatic substitution and can be described as a Figure 1. The benzylisoquinoline skeleton (R = H, OH, OMe). ThemodificationThe P-S P-S reaction reactionof the isMannich is essentially essentially reaction. anan electrophilicelectrophilicThe name comes aromatic aromatic from substitution the substitution chemists and Pictet andcan and be can describedSpengler, be described whoas a as a modiﬁcationmodificationsynthesizedThe P-Sof 1-methyl-1,2,3,4-tetrahydroisoquinoline ofreaction the the MannichMannich is essentially reac reaction.tion. an The electrophilic The name name comes aromatic (Scheme comes from thefromsubstitution1) inchemists 1911, the chemistsby andPictet cyclocondensation can and Pictetbe Spengler, described and ofwho Spengler, as β a- who synthesizedphenethylaminemodification 1-methyl-1,2,3,4-tetrahydroisoquinoline 1-methyl-1,2,3,4-tetrahydroisoquinolineof thewith Mannich acetaldehyde reaction. dimethyl The name acetal comes (Schemecatalyzed from (Scheme 1)the byin chemists 11911,HCl) in 1911,[25].by Pictet cyclocondensation With by and cyclocondensation the Spengler, exception of who βof- of β-phenethylaminephenethylamineformaldehyde,synthesized 1-methyl-1,2,3,4-tetrahydroisoquinoline any withwith aldehyde acetaldehydeacetaldehyde which isdimethyl dimethyl submitted acetal acetal to the catalyzed(Scheme catalyzedP-S reaction 1) by in byHCl1911, creates HCl [25]. by [acyclocondensation 25 Withnew]. With chiralitythe exception the centre exception of ofatβ- of formaldehyde,formaldehyde,thephenethylamine C-1 carbon any anyatom aldehyde with aldehyde of acetaldehydethe THIQ which which sk is eleton;is dimethyl submitted submitted this acetalis to tothe the the reasoncatalyzed P-S P-S reaction why reaction by the HClcreates stereoselectivity creates [25]. a newWith a new chirality the of chirality exceptionthe centre reaction centre atof at the C-1theinformaldehyde, the carbonC-1 synthesis carbon atom atom anyof of isoquinoline thealdehyde of the THIQ THIQ which skeleton; alkaloids skeleton; is submitted thishas this b is eenis the theto an reason thereason ancient P-S whyreactionwhy competition the createsstereoselectivity stereoselectivity for a organicnew chirality chemists.of of the the reactioncentre reaction at in in the synthesis of isoquinoline alkaloids has been an ancient competition for organic chemists. the synthesisthe C-1 carbon of isoquinoline atom of the alkaloids THIQ skeleton; has been this anis the ancient reason competition why the stereoselectivity for organic chemists.of the reaction in the synthesis of isoquinoline alkaloids has been an ancient competition for organic chemists.

Scheme 1. First appearance of the Pictet-Spengler (P-S) reaction. The asterisk denotes the chirality SchemeSchemecentre 1. atFirst 1. C-1. First appearance appearance of of the the Pictet-Spengler Pictet-Spengler (P-S (P-S)) reaction. reaction. The The asterisk asterisk denotes denotes the chirality the chirality centre at C-1. centreScheme at C-1.1. First appearance of the Pictet-Spengler (P-S) reaction. The asterisk denotes the chirality Despite the huge structural diversity, BIAs have the same biosynthetic precursor, namely (S)- centre at C-1. DespitenorcoclaurineDespite the the huge(Schemehuge structural structural 2), which diversity, diversity, is BIAsformed ha BIAsve bythe havesamecondensation biosynthetic the same of precursor, biosyntheticdopamine namely and precursor, (S4-)- norcoclaurinehydroxyphenylacetaldehyde (Scheme 2), (4-HPAA),which is withformed an ex cellentby condensation stereoselectivity of [26,27].dopamine The andenzyme 4- namely (DespiteS)-norcoclaurine the huge structural (Scheme diversity,2), which BIAs is ha formedve the same by biosynthetic condensation precursor, of dopamine namely (S)- and hydroxyphenylacetaldehydeinvolved in the biosynthesis is(4-HPAA), the norcoclaurine with an synthase excellent (NCS; stereoselectivity EC 4.2.1.78), which[26,27]. can The be enzymedefined 4-hydroxyphenylacetaldehydenorcoclaurine (Scheme 2), (4-HPAA),which is withformed an excellentby condensation stereoselectivity of dopamine [26,27]. Theand enzyme4- involvedas a “Pictet-Spenglerase”. in the biosynthesis One is the of norcoclaurinethe most fascinating synthase features (NCS; EC of 4.2.1.78),BIAs, in whichfact, is can that be theydefined are involvedhydroxyphenylacetaldehyde in the biosynthesis is the (4-HPAA), norcoclaurine with synthasean excellent (NCS; stereoselectivity EC 4.2.1.78), [26,27]. which canThe be enzyme deﬁned as assynthesizedinvolved a “Pictet-Spenglerase”. in inthe the biosynthesis plant as singleOne is theof enantiomers, thenorcoclaurine most fascinating as usuallysynthase featureshappens (NCS; EC offor BIAs, 4.2.1.78),natural in products, fact,which is canthat even be they definedthough are a “Pictet-Spenglerase”. One of the most fascinating features of BIAs, in fact, is that they are synthesized synthesizedthisas a is “Pictet-Spenglerase”. not always in the planttrue [28]. as single OneMoreover, ofenantiomers, the theremost arefascinating as casesusually where happensfeatures enantiomerically forof naturalBIAs, in products, oppositefact, is thateven metabolites they though are in thethisaresynthesized plant producedis not as always single in by the different true enantiomers,plant [28]. as genera single Moreover, asenantiomers,or species, usually there orare happens aseven cases usually they where for happenshave natural enantiomerically been for products,isolated natural from products, opposite even different though evenmetabolites portions though this is not alwaysareofthis thetrue produced is same not [28 always]. plant Moreover,by different [27]. true [28]. there genera Moreover, are or cases species, there where or are even enantiomericallycases they where have enantiomerically been oppositeisolated from metabolites opposite different metabolites areportions produced by differentofare the produced same genera plant by or different[27]. species, genera or even or species, they have or even been they isolated have been from isolated different from portionsdifferent portions of the same HO plantof [27 the]. same plant [27]. HO HO NH NCS HO H + NH HO O HO NH2 NCS HO HO + HO H O HO NH2 NH HO HO NCS HO + HO H O NH2 HO HO HO Scheme 2. Biosynthetic pathway for (S)-norcoclaurine alkaloid catalyzed by the norcoclaurine Schemesynthase 2.(NCS) Biosynthetic enzyme. pathway for (S)-norcoclaurine alkaloid catalyzedHO by the norcoclaurine synthaseScheme (NCS)2. Biosynthetic enzyme. pathway for (S)-norcoclaurine alkaloid catalyzed by the norcoclaurine SchemeThe 2. majorBiosynthetic strategies pathway that have for (Sbeen)-norcoclaurine developed alkaloidso far to catalyzed synthesize by the the biologically norcoclaurine active synthase (S)- synthase (NCS) enzyme. (NCS)enantiomerThe enzyme. major of 1-substitutedstrategies that THIQ have alkaloids been developed are the followingso far to synthesize two [29]: (i)the the biologically Bischler-Napieralski active (S)- enantiomercyclization/reductionThe major of 1-substituted strategies sequence that THIQ haveof dihydroisoquinolinealkaloids been developed are the following so intermediates;far to synthesizetwo [29]: and (i) the (ii)the biologically theBischler-Napieralski P-S condensation active (S)- Thecyclization/reductionofenantiomer β-arylethylamines major of strategies 1-substituted suchsequence that as THIQdopamine have of dihydroisoquinoline alkaloids been with developed aare variety the following of intermediates; so aldehydes far two to synthesize (Scheme[29]: and (i) (ii) the 3). the theBischler-Napieralski P-S biologically condensation active of β-arylethylamines such as dopamine with a variety of aldehydes (Scheme 3). (S)-enantiomer cyclization/reduction of 1-substituted sequence THIQ of dihydroisoquinoline alkaloids are the following intermediates; two [and29]: (ii) (i) the P-S Bischler-Napieralski condensation cyclization/reduction of β-arylethylamines sequence such as ofdopamine dihydroisoquinoline with a variety of intermediates; aldehydes (Scheme and (ii) 3). the P-S condensation of β-arylethylamines such as dopamine with a variety of aldehydes (Scheme3). Int. J. Mol. Sci. 2017, 18, 2464 3 of 19

Int. J. Mol. Sci. 2017, 18, 2464 3 of 18 In the ﬁrst case, it is the reduction step that governs the introduction of the chirality centre at theInt. C-1 J. Mol.In carbon the Sci. first 2017 atom; ,case, 18, 2464 it therefore, is the reduction chiral step hydride that gove reducingrns the introduction agents must of the be chirality employed, centre or at3 of elsethe 18 the hydrogenationC-1 carbon must atom; be therefore, carried outchiral in thehydride presence reducing of chiral agents catalysts. must be In employed, the last case, or theelse absolutethe In the first case, it is the reduction step that governs the introduction of the chirality centre at the conﬁgurationhydrogenation of the must catalyst be carried is a fundamental out in the presence tool to prepareof chiral anycatalysts. of the In desired the last alkaloidcase, the enantiomers.absolute C-1 carbon atom; therefore, chiral hydride reducing agents must be employed, or else the configuration of the catalyst is a fundamental tool to prepare any of the desired alkaloid enantiomers. Inhydrogenation the second must method, be carried the chirality out in the centre presence at C-1 of chiral is formed catalysts. during In the the last ring case, closure the absolute step, in a In the second method, the chirality centre at C-1 is formed during the ring closure step, in a oneone-potconfigurationInt. fashion. J. Mol. Sci. 2017 Asof the, 18 a, result,2464catalyst a is properly a fundamental added tool chiral to prepare auxiliary any of must the desired be capable alkaloid of enantiomers. transferring3 of 18 its pot fashion. As a result, a properly added chiral auxiliary must be capable of transferring its chirality chirality toIn the either second the method,β-arylethylamine the chirality orcentre the at aldehyde, C-1 is formed and during this meansthe ring that closure a diastereoselective step, in a one- to eitherIn thethe first β-arylethylamine case, it is the reduction or the aldehyde,step that gove andrns this the meansintroduction that a of diastereoselective the chirality centre synthesisat the synthesispot fashion. occurs. As a result, a properly added chiral auxiliary must be capable of transferring its chirality occurs.C-1 carbon atom; therefore, chiral hydride reducing agents must be employed, or else the to hydrogenationeither the β-arylethylamine must be carried or out the in aldehyde,the presence and of thischiral means catalysts. that Ina thediastereoselective last case, the absolute synthesis occurs.configuration of the catalyst is a fundamental tool to prepare any of the desired alkaloid enantiomers. In the second method, the chirality centre at C-1 is formed during the ring closure step, in a one- pot fashion. As a result, a properly added chiral auxiliary must be capable of transferring its chirality to either the β-arylethylamine or the aldehyde, and this means that a diastereoselective synthesis occurs. SchemeScheme 3. 3.The The two two general general enantioselectiveenantioselective method methodss for for the the preparation preparation of of1-substituted 1-substituted tetrahydroisoquinolinestetrahydroisoquinolines (THIQs) (THIQs) skeleton skeleton (R (R1= = alkylalkyl or aryl). aryl). Scheme 3. The two general enantioselective1 methods for the preparation of 1-substituted tetrahydroisoquinolinesThe Bischler-Napieralski (THIQs) cyclization/reduction skeleton (R1 = alkyl orsequence aryl). (Scheme 4) has been the most used The Bischler-Napieralski cyclization/reduction sequence (Scheme4) has been the most synthetic approach for the asymmetric production of THIQs [29]. According to it, a β-arylethylamide The Bischler-Napieralski cyclization/reduction sequence (Scheme 4) has been the most used usedundergoes synthetic approacha cyclization for theto asymmetric1-substituted productiondihydroisoquinoline of THIQs or [29a]. corresponding According to it, syntheticScheme approach 3. The for two the asymmetricgeneral enantioselective production method of THIQss for [29]. the Accordingpreparation to of it, 1-substituteda β-arylethylamide a β-arylethylamidedihydroisoquinolinium undergoes salt, afollowed cyclization by a to reduction 1-substituted to tetrahydroisoquinoline. dihydroisoquinoline Transition or a corresponding metals undergoestetrahydroisoquinolines a cyclization (THIQs) to skeleton1-substituted (R1 = alkyl ordihydroisoquinoline aryl). or a corresponding dihydroisoquinoliniumsuch as ruthenium (Ru), salt, followedrhodium (Rh), by a and reduction iridium to (Ir) tetrahydroisoquinoline. have been successfully Transitionemployed as metals chiral such dihydroisoquinolinium salt, followed by a reduction to tetrahydroisoquinoline. Transition metals as rutheniumcatalystsThe (Ru),in Bischler-Napieralski the rhodiumasymmetric (Rh), hydrogenation cyclization/reduction and iridium of (Ir) a havevariety sequence been of dihydroisoquinolines, successfully(Scheme 4) has employed been theresulting asmost chiral usedin high catalysts such as ruthenium (Ru), rhodium (Rh), and iridium (Ir) have been successfully employed as chiral enantiomericsynthetic approach excess for(ee) the values asymmetric with a growingproduction number of THIQs of substrates [29]. According [30–32]. to it, a β-arylethylamide in thecatalysts asymmetric in the hydrogenation asymmetric hydrogenation of a variety of dihydroisoquinolines,a variety of dihydroisoquinolines, resulting inresulting high enantiomeric in high excess (undergoesee) values witha cyclization a growing numberto 1-substituted of substrates dihydroisoquinoline [30–32]. or a corresponding enantiomericdihydroisoquinoliniumRO excess (ee) valuessalt,X followed with a growingROby a reduction number to oftetrahydroisoquinoline. substratesRO [30–32]. RO Transition metals + O such as rutheniumH2 N(Ru), rhodium (Rh), and iridiumNH (Ir) have been successfullyN employed asNH chiral RO XR1 RO RO RO catalysts in the asymmetric hydrogenation of aO variety of dihydroisoquinolines, resulting in high O + R1 R1 H2N NHR1 N NH enantiomericRO excess (ee) valuesR1 with a growingRO number of substratesRO [30–32]. RO O R R Scheme 4. Bischler-Napieralski cyclization/reductionR1 sequence for the synthesis1 of chiral isoquinoline1 RO X RO RO RO alkaloids. + O Scheme 4. Bischler-NapieralskiH2N cyclization/reductionNH sequence for the synthesisN of chiral isoquinolineNH Scheme 4.RO Bischler-NapieralskiR1 cyclization/reductionRO RO sequence for theRO synthesis of chiral O alkaloids. R R isoquinolineIn 2015, alkaloids. a general approach has been describedR1 to the enantioselective1 synthesis1 of 1-benzyl- 1,2,3,4-THIQs based on an asymmetric P-S reaction catalyzed by BINOL-phosphoric acid [(R)-TRIP] InScheme 2015, a4. generalBischler-Napieralski approach cyclization/reductionhas been described sequence to the for enantioselective the synthesis of chiral synthesis isoquinoline of 1-benzyl- [33]. The novelty of the method has been the introduction of a moderately strong electron- In1,2,3,4-THIQs 2015,alkaloids. a generalbased on an approach asymmetric has P-S been reaction described catalyzed toby BINOL-phosphoric the enantioselective acid [( synthesisR)-TRIP] of withdrawing group on the nitrogen atom of the phenylethylamine, i.e., the O-nitrophenylsulfenyl 1-benzyl-1,2,3,4-THIQs[33]. The novelty ofbased the method on an has asymmetric been the in P-Stroduction reaction of catalyzeda moderately by BINOL-phosphoricstrong electron- (Nps) substituent (Scheme 5). Many biologically active alkaloids have been synthesized thanks to this acidwithdrawing [(R)-TRIP]In 2015, [33group a ].general Theon theapproach novelty nitrogen has of atom been the ofdescribed method the phenylethylamine, to has the beenenantioselective the i.e., introduction the synthesis O-nitrophenylsulfenyl of of 1-benzyl- a moderately approach,1,2,3,4-THIQs such based as on(R )-crispine,an asymmetric (R )-coclaurine,P-S reaction catalyzed (R)-laudanosine, by BINOL-phosphoric and (R)-calycotomine. acid [(R)-TRIP] ( R)- strong(Nps) electron-withdrawing substituent (Scheme 5). Many group biologically on the active nitrogen alkaloids atom haveof been the synthesized phenylethylamine, thanks to this i.e., Norcoclaurine[33]. The novelty was obtainedof the method as well has as beenits hydrochloride the introduction salt of (HCl), a moderately with 89% strong ee, and electron- the same approach, such as (R)-crispine, (R)-coclaurine, (R)-laudanosine, and (R)-calycotomine. (R)- the Osynthetic-nitrophenylsulfenylwithdrawing protocol group with on the (Nps)the ( Snitrogen)-TRIP substituent catalystatom of might (Schemethe phenylethylamine, lead5 ).to the Many biologically biologically i.e., the relevant O-nitrophenylsulfenyl active (S)-enantiomer. alkaloids have Norcoclaurine was obtained as well as its hydrochloride salt (HCl), with 89% ee, and the same been synthesized(Nps) substituent thanks (Scheme to this5). Many approach, biologically such active as alkaloids (R)-crispine, have been (R)-coclaurine, synthesized thanks (R)-laudanosine, to this synthetic protocol with the (S)-TRIP catalyst might lead to the biologically relevant (S)-enantiomer. and (R)-calycotomine.approach, such (asR)-Norcoclaurine (R)-crispine, (R)-coclaurine, was obtained (R as)-laudanosine, well as its hydrochlorideand (R)-calycotomine. salt (HCl), (R)- with 89% ee, andNorcoclaurine the same synthetic was obtained protocol as well with as the its (Shydrochloride)-TRIP catalyst salt might(HCl), with lead 89% to the ee, biologicallyand the same relevant (S)-enantiomer.synthetic protocol with the (S)-TRIP catalyst might lead to the biologically relevant (S)-enantiomer.

Scheme 5. Asymmetric P-S reaction of 3-hydroxy-4-MOM-phenylethylamine as O-nitrophenylsulfenyl derivative (Nps) with arylacetaldehydes. (MOM = methoxymethyl; iPr = isopropyl). Scheme 5. Asymmetric P-S reaction of 3-hydroxy-4-MOM-phenylethylamine as O-nitrophenylsulfenyl derivative (Nps) with arylacetaldehydes. (MOM = methoxymethyl; iPr = isopropyl). Scheme Scheme 5. Asymmetric 5. Asymmetric P-S P-S reaction reaction of of 3-hydroxy-4-MOM-phenylethylamine 3-hydroxy-4-MOM-phenylethylamine as O-nitrophenylsulfenyl as O-nitrophenylsulfenyl derivativederivative (Nps) (Nps) with with arylacetaldehydes. arylacetaldehydes. (MOM = =methoxymethyl; methoxymethyl; iPr = iso iPrpropyl). = isopropyl).

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AmongAmong thethe numerousnumerous syntheticsynthetic effortsefforts toto easilyeasily accessaccess enantiomericallyenantiomerically purepure BIAsBIAs toto bebe testedtested asas novelnovel drugs,drugs, thethe stereochemicalstereochemical featurefeature ofof thesethese alkaloidsalkaloids isis currentlycurrently beingbeing studiedstudied toto assignassign thethe correctcorrect absoluteabsolute configuration configuration of someof some alkaloids alkaloids isolated isolated from from different different parts ofparts a plant. of a To plant. this purpose, To this 0 thepurpose, (R)- and the ( S(R)-diastereoisomers)- and (S)-diastereoisomers of norcoclaurine of norcoclaurine 4 -O-β-D-glucoside 4′-O-β-D-glucoside were synthesized were synthesized [34] and their [34] NMRand their and NMR HPLC and profiles HPLC were profiles compared were tocompared unequivocally to unequivocally assign the stereochemistryassign the stereochemistry of a previously of a isolatedpreviously product isolated [35 ].product [35]. Briefly,Briefly, the the Bischler-Napieralski Bischler-Napieralski cyclization/re cyclization/reductionduction sequence sequence of a proper of aamide proper (Scheme amide 6) (Schemeby employing6) by employingRuCl [R,R-TsDPEN( RuCl [ Rp,R-cymene)]-TsDPEN( asp-cymene)] the asymmetric as the catalyst asymmetric yielded catalyst the (S)- yieldedN-Cbz-6,7- the (di-S)-ON-benzylnorcoclaurine,-Cbz-6,7-di-O-benzylnorcoclaurine, which, after the which, attachment after theof a attachmentglucosyl moiety of a glucosylto the p-hydroxybenzyl moiety to the 0 punit-hydroxybenzyl and subsequent unit deprotection and subsequent gave deprotection the (S)-norcoclaurine-4 gave the (S)-norcoclaurine-4′-O-β-D-glucoside-O as-β -trifluoroacetateD-glucoside as trifluoroacetate(TFA) salt in 93% (TFA) yield salt and in 95% 93% diastereomeric yield and 95% diastereomericexcess. excess.

BnO BnO

NH NCbz BnO BnO O

OAllyl

NH.TFA HO

HO OH O OH O OH

0 SchemeScheme 6.6. SyntheticSynthetic pathway pathway to to ( (SS)-norcoclaurine-4)-norcoclaurine-4′--O--β--DD-glucoside-glucoside by by enantioselective enantioselective Bischler- NapieralskiNapieralski cyclization/reduction cyclization/reduction sequence sequ andence coupling and withcoupling 2,3,4,6-tetra- withO -acetyl-2,3,4,6-tetra-β-D-glucopyranosylO-acetyl-β-D- 2,2,2-trichlotoacetimidate.glucopyranosyl 2,2,2-trichlotoacetimidate. Each arrow of the designEach representsarrow of onethe stepdesign in therepresents synthetic one process. step in the synthetic process. Although many elegant syntheses of BIAs have been proposed, the development of more efficient Although many elegant syntheses of BIAs have been proposed, the development of more synthetic routes is still a great challenge to the chemists due to the intricate molecular architecture of efficient synthetic routes is still a great challenge to the chemists due to the intricate molecular these molecules. Usually, chemical synthesis of alkaloids is complex due to the presence of multiple architecture of these molecules. Usually, chemical synthesis of alkaloids is complex due to the stereogenic centres and requires multistep procedures, many of which involve the use of protective presence of multiple stereogenic centres and requires multistep procedures, many of which involve groups resulting in moderate to poor yields. the use of protective groups resulting in moderate to poor yields. As a matter of fact, one of the major limitations in the chemical synthesis of BIAs is the scale up of As a matter of fact, one of the major limitations in the chemical synthesis of BIAs is the scale up the process that, even if feasible, it still remains not economically convenient. of the process that, even if feasible, it still remains not economically convenient. Moreover, direct extraction of alkaloids from plants is often impractical, as they are often produced Moreover, direct extraction of alkaloids from plants is often impractical, as they are often in very small amounts by their natural source. For all these reasons, in this last decade new synthetic produced in very small amounts by their natural source. For all these reasons, in this last decade new processes have been developed joining the expertise of chemists with the new tools emerging in the synthetic processes have been developed joining the expertise of chemists with the new tools field of biotechnology [36]. These alternative pathways, involving microorganisms such as bacteria emerging in the field of biotechnology [36]. These alternative pathways, involving microorganisms and yeast as well as purified enzymes, allow rapid and stereospecific production of large quantities of such as bacteria and yeast as well as purified enzymes, allow rapid and stereospecific production of advanced intermediates, reducing at the same time waste products generated during the synthetic large quantities of advanced intermediates, reducing at the same time waste products generated process, thus making the overall process green and environment friendly. These topics will be described during the synthetic process, thus making the overall process green and environment friendly. These in Sections2 and3. topics will be described in Sections 2 and 3.

Int. J. Mol. Sci. 2017, 18, 2464 5 of 19

2. Working with Living Organisms: Biotechnological Approaches in Benzylisoquinoline Alkaloids (BIA) Production

2.1. BIA Production in Plants The growing interest in plant alkaloids and their use for therapeutic purposes has led to the development of different extraction methods. In fact, due to their complex chemical structures, when feasible BIAs are directly extracted from plants. However, very often these methods consist in expensive and time-consuming procedure and the isolation yields are generally too low. Moreover, the secondary metabolite patterns of plants grown at different times and places with different physical and chemical stimuli can vary greatly and this represents a major obstacle in using plants as a drug producing source. Hence, the production of plant derived drugs requires genetically uniformed monocultures grown in completely standardized conditions. BIA metabolism involves many enzymes belonging to a small number of protein families, including S-adenosylmethionine (SAM)-dependent N- and O-methyltransferases, cytochromes P450, NADPH dependent dehydrogenases/reductases, FAD-dependent oxidoreductases, O-acetyltransferases, and O-demethylase [1]. All these enzymes can be the target of metabolic engineering both for functional studies and to improve BIAs production in plant. To this purpose, biotechnological approaches such as knock-out, knock-down, or overexpression of selected genes encoding for key enzymes involved in alkaloid metabolism have been employed. Overexpression of genes encoding for rate-limiting enzyme, for instance, can favour the accumulation of end products; introduction of new genes from different species can create novel products; RNAi, virus-induced gene silencing (VIGS) or clustered regularly interspaced short palindromic repeats associated with Cas9 endonuclease (CRISPR/Cas9) can decrease undesired compounds or can help to accumulate valuable intermediates. Recently, it has been reported that opium poppy transformed with constitutively expressed gene for codeinone reductase, showed a higher content of morphinan alkaloid [37,38]. Coptis japonica was modified to improve alkaloid yields by overexpressing the 3-hydroxy-N-methylcoclaurine-4-O-methyltransferase (4-OMT) gene, one of the key enzymes in BIAs biosynthesis in this species [39,40]. Overexpression of the Papaver somniferum berberine bridge enzyme (BBE) in Eschscholzia californica led to the accumulation of the end products benzophenanthridine alkaloids compared to controls [41]. Overexpression of salutaridinol 7-O-acetyltransferase (SalAT), a key gene in morphinan alkaloids biosynthesis pathway, specifically resulted in increased morphinan alkaloid accumulation in transgenic hairy root lines from Papaver bracteatum [42,43]. RNAi has been also used to create transgenic poppies displaying high levels of reticuline rather than the narcotic morphine [44]. After gene silencing, the intermediate (S)-reticuline, which is produced several steps upstream of codeinone (see Scheme7), accumulated in transgenic plants at the expense of morphine, codeine, oripavine, and thebaine. Methylated derivatives of reticuline also accumulated. VIGS has been used as a functional genomic tool to investigate the regulation of morphine biosynthesis by reducing the levels of enzymes catalyzing the last six steps in morphine biosynthetic pathway [45]. Even though the study was focused on validating the physiological role of biosynthetic genes involved in morphinan branch pathway, the increase of reticuline, salutaridine, thebaine, and codeine concentrations could make this approach suitable for the generation of plant biofactories (Scheme7). CRISPR/Cas9 system is a new tool in molecular biology which provides a number of applications in metabolic engineering and molecular farming, characterized by higher accuracy and precision compared to other well-known genome editing tools [46]. Very recently, CRISPR/Cas9 system has been employed to knock-out the 40-OMT2 gene involved in the regulation of the biosynthesis of many BIAs including noscapine and morphine [47]. This study paves the way for further research aimed at addressing some issues connected to BIA biosynthesis. Int. J. Mol. Sci. 2017, 18, 2464 6 of 19

Although plant engineering has been used to induce secondary metabolites accumulation [40,48–50], it is not easy to achieve the desired products because their biosynthesis are tightly regulated. For example, in most pathways, overexpression of one enzyme results in making subsequent reactions more limiting. In the same way, blocking the expression of a selected enzyme very often results in a lower concentration of downstream metabolites, but not in their complete disappearance [47]. Nonetheless, modifications in alkaloid metabolism can impact primary metabolism. It has been shown, in fact, that suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE has the side effect of reducing the growth rate of the root cultures [41]. Similarly, the overexpression of 4-OMT in transgenic Coptis japonica plant resulted in an increase of berberine content, but also in a slower-growth phenotype [40]. Moreover, metabolic engineering of BIA biosynthetic pathway, besides the expected phenotype, often results in the accumulation of unknown/undesired metabolites [47] and this could be due to feedback regulation and metabolic channels. Finally, the effects of genetic modification can be strongly different in individual plants. This could be due to genetic or physiological heterogeneity that strongly influences the amount of specific alkaloids. For example, silencing of codeinone reductase (CoR) in opium poppy (Scheme7) resulted in the accumulation of reticuline, whose levels proved to vary in a wide range from plant to plant [45].

2.2. BIA Production in Microbial Systems: Bacteria and Yeast

2.2.1. Bacterial Factories Engineering microbial factories meet the quest for more sustainable and cheaper biotechnological processes for the production of alkaloids. As described in Section 2.1, alkaloids extraction from plants is characterized by low yields and requires many solvents; these problems are not completely overcome even when the engineering is directed towards the production of selected metabolites. In recent years, many steps forward have been made in reconstituting BIA biosynthesis into microbial systems. Plant natural product biosynthesis in microbial hosts offers strategic advantages such as the chance of choosing the appropriate codon usage, finely tuning enzyme expression levels and timing, and manipulating host metabolism to increase precursor and co-factor concentrations [51]. Now, many pathways in BIA biosynthesis have been extensively characterized, allowing the opportunity of moving entire branches into microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. Sometimes, the transplantation of a full metabolic pathway is not still enough to guarantee the desired results. For instance, it could be useful to increase the levels of the metabolic precursors of the desired BIAs reducing unwanted byproducts by metabolic flow modifications. This approach may lead to a reduction of the concentration of those enzymes associated with undesired microbial pathways. A good example of synthetic pathway transplantation is represented by a work that can considered as a proof-of-concept study for a technology that allows the transfer of a great number of genes [52]. They developed a fermentative production in E. coli of (S)-reticuline, one of the main branch-point intermediates in the biosynthesis of different of BIAs (Scheme7). Optimizing the production of (S)-reticuline could be useful for the synthesis of rare or non-natural BIAs to be tested for their potential physiological activities. The authors developed an E. coli strain overproducing tyrosine, the precursor of BIA backbone. This was accomplished by disrupting the tyrR gene (a repressor of aromatic amino acid biosynthesis), overexpressing genes of the shikimic acid pathway (feedback inhibition resistant aroG and tyrA), and introducing phosphoenolpyruvate synthetase (PEPS: ppsA) and transketolase (TKT: tktA) genes in order to hijack metabolic flow into the shikimic acid pathway (Scheme8). Int. J. Mol. Sci. 2017, 18, 2464 7 of 19 Int. J. Mol. Sci. 2017, 18, 2464 7 of 18

Scheme 7.7. Main pathways for the biosynthesisbiosynthesis of benzylisoquinolinebenzylisoquinoline alkaloids (BIAs). Light blue: pathway leading from Tyrosine toto ((SS)-Scoulerine;)-Scoulerine; greengreen line:line: pathwaypathway leading from (S)-Coclaurine to Papaverine; blue line: pathway leading from (S)-Reticuline toto Morphine; purplepurple line:line: pathway leading from ((S)-Scoulerine to Sanguinarine; brownbrown line: pathway leading from ( S)-Scoulerine to Noscapine; red line:line: pathway pathway leading leading from from (S)-Canadine (S)-Canadine to Berberine. to Berberine. The end-products The end-products of the metabolic of the metabolic pathways arepathways in full colorare in squares. full color The enzymessquares. catalyzingThe enzymes each ca reactiontalyzing step each are reaction reported step in correspondence are reported ofin thecorrespondence reaction arrow. of the reaction arrow.

These bacteria were effectively able to produce larger amounts of tyrosinetyrosine from simplesimple carboncarbon sources that that can can be be used used to generate to generate dopamine dopamine and 4-hydroxyphenyl and 4-hydroxyphenyl acetaldehyde acetaldehyde (4-HPAA) (4-HPAA) whose condensation to (S)-norcolaurine represents the first committed step in BIA biosynthesis. L-DOPA was efficiently synthesized from L-tyrosine by tyrosinase form Streptomyces castaneoglobisporus (TYR;

Int. J. Mol. Sci. 2017, 18, 2464 8 of 19 whose condensation to (S)-norcolaurine represents the first committed step in BIA biosynthesis. L-DOPAInt. J. Mol. was Sci. efficiently 2017, 18, 2464 synthesized from L-tyrosine by tyrosinase form Streptomyces castaneoglobisporus8 of 18 (TYR; EC 1.14.18.1); 4-HPAA produced by tyrosine decarboxylation was catalyzed by monoamine oxidaseEC 1.14.18.1); from Micrococcus 4-HPAA produced luteus (MAO; by tyrosine EC 1.4.3.4); decarboxylation (S)-norcoclaurine was catalyzed was generatedby monoamine by norcoclaurine oxidase from Micrococcus luteus (MAO; EC 1.4.3.4); (S)-norcoclaurine was generated by norcoclaurine synthase (NCS) from Coptis japonica. Finally, to convert (S)-norcoclaurine in (S)-reticuline three different synthase (NCS) from Coptis japonica. Finally, to convert (S)-norcoclaurine in (S)-reticuline three plant methyltransferases (6-OMT, CNMT (coclurine-N-methyltransferase), and 40-OMT) were also different plant methyltransferases (6-OMT, CNMT (coclurine-N-methyltransferase), and 4′-OMT) recombinantlywere also recombinantly expressed in expressed this strain. in Furtherthis strain optimization. Further optimization in the production in the production yields (conversionyields efficiency(conversion 14-fold efficiency higher) 14-fold was obtainedhigher) was by obtained fine tuning by fine the tuning molar the ratio molar of ratio MAO of MAO and NCS and NCS that both competethat both for compete dopamine. for dopamine. This was This realized was realized cloning cloning the genes the genes into into two two plasmids plasmids with with different different copy number,copy undernumber, the under control the of IPTGcontrol (isopropil- of IPTGβ -(isopropil-D-1-tiogalattopiranoside)β-D-1-tiogalattopiranoside) inducible inducible T7 promoters. T7 promoters.In 2017, an improved system to produce large amounts of (S)-reticuline combining in vivo tetrahydropapaverolineIn 2017, an improved production system into E.produce coli with largein amounts vitro enzymatic of (S)-reticuline synthesis combining of (S)-reticuline in vivo was developedtetrahydropapaveroline using crude extracts production of E. in coliE. colistrains with in expressing vitro enzymatic methyltransferases synthesis of (S)-reticuline [53]. The wasin vitro developed using crude extracts of E. coli strains expressing methyltransferases [53]. The in vitro reaction catalyzed by methyltransferases was more efficient than in vivo fermentation system, due to reaction catalyzed by methyltransferases was more efficient than in vivo fermentation system, due to the larger amounts of SAM that could be supplied. This experimental setting allows to obtain a further the larger amounts of SAM that could be supplied. This experimental setting allows to obtain a improvementfurther improvement in (S)-reticuline in (S)-reticuline yields (593 yields mg/L (593 isolated mg/L isolated product). product).

Pentose phosphate Glycolysis pathway

COOH PEP + 4EP DAHP corismate NH2 HO

TYR H3CO

NCH3 HO HO COOH H HO NH2 HO

H3CO (S)-Reticuline DODC

4'-OMT HO

H3CO H3CO HO NH2 HO NCH3 CNMT NH 6-OMT NH NCS HO HO HO H H H MAO HO HO HO

HOO HO HO HO

SchemeScheme 8. Metabolic 8. Metabolic pathways pathways leading leading to the synthesis synthesis ofof (S)-reticuline (S)-reticuline in engineered in engineered E. coliE. strains. coli strains. PEP:PEP: phosphoenolpyruvate; phosphoenolpyruvate; 4EP: 4EP: eritrose-4-phosphate; eritrose-4-phosphate; DAHP: DAHP: 3-deossi-arabinoeptulsonato-7-fosfato; 3-deossi-arabinoeptulsonato-7- fosfato; DODC: L-DOPA decarboxylase; MAO: monoamine oxidase; 6-OMT: 6-O-methyltransferase; DODC: L-DOPA decarboxylase; MAO: monoamine oxidase; 6-OMT: 6-O-methyltransferase; CNMT: CNMT: coclurine-N-methyltransferase; 4′-OMT: 4′-O-methyltransferase. coclurine-N-methyltransferase; 40-OMT: 40-O-methyltransferase. 2.2.2. Yeast Factories 2.2.2. Yeast Factories One of the limits of bacteria is that they are not suitable to stably express plant tailoring enzymes, suchOne as of the the endomembrane-localized limits of bacteria is that cytochrome they are not P450s, suitable which to stablythat are express essential plant in many tailoring branches enzymes, suchof as BIA the pathways. endomembrane-localized Eukaryotic hosts such cytochrome as S. cerevisiae P450s, and which P. pastoris that can are be essential used to overcome in many branchesthis of BIAlimitation. pathways. They are Eukaryotic very important hosts model such asorganismsS. cerevisiae for theirand capabilityP. pastoris to heterologouslycan be used toexpress overcome thiscomplex, limitation. branched, They aremulti-step very importantbiosynthetic model pathways organisms and challenging for their heterologous capability enzymes to heterologously such as cytochromes P450. They allow the production of a number of different BIAs with different express complex, branched, multi-step biosynthetic pathways and challenging heterologous enzymes biotechnological engineering. such as cytochromes P450. They allow the production of a number of different BIAs with different As an example, (S)-reticuline was produced in yeast starting from commercially available (R,S)- biotechnologicalnorlaudanosoline engineering. by using a combinations of enzymes both from plants (norcoclaurine 6-O-

Int. J. Mol. Sci. 2017, 18, 2464 9 of 19

As an example, (S)-reticuline was produced in yeast starting from commercially available (R,S)-norlaudanosoline by using a combinations of enzymes both from plants

(norcoclaurineInt. J. Mol. Sci.6- 2017O-methyltransferase, 18, 2464 (6-OMT), coclaurine-N-methyltransferase (CNMT)9 of 18 and 30-hydroxy-N-methylcoclaurine 40-O-methyltransferase (40-OMT)), and human (cytochrome P450 80B1 (CYP80B1))methyltransferase [54]. (6-OMT), coclaurine-N-methyltransferase (CNMT) and 3′-hydroxy-N- Amethylcoclaurine further optimization 4′-O-methyltransferase of the process (4 was′-OMT)), achieved and human by combining (cytochrome enzymes P450 80B1 from (CYP80B1)) plants, [54]. yeast, A further optimization of the process was achieved by combining enzymes from plants, yeast, mammals, and bacteria [55]. In this case, the first step was a modification of tyrosine metabolism to mammals, and bacteria [55]. In this case, the first step was a modification of tyrosine metabolism to direct its flux through BIA pathway. Once the concentration of tyrosine was increased, heterologous direct its flux through BIA pathway. Once the concentration of tyrosine was increased, heterologous expressionexpression of a mammalianof a mammalian tyrosine tyrosine hydroxylase hydroxylase (TyrH), (TyrH), bacterialbacterial DOPA DOPA decarboxylase decarboxylase (DODC), (DODC), and fourand enzymes four enzymes associated associated with with BH4 BH4 biosynthesis biosynthesis and and recycling recycling allowed to to obtain obtain 160 160 fold fold increase increase of (S)-noroclaurine,of (S)-noroclaurine, with with fed fed dopamine dopamine withoutwithout supply supplyinging exogenous exogenous tyrosine. tyrosine. As a final As astep, final five step, five enzymesenzymes from from plant sources sources were were incorporated incorporated (three (three methyltransferases, methyltransferases, a cytochrome a cytochrome P450, and its P450, and itsreductase) reductase) to toachieve achieve the theproduction production of reticuline of reticuline with a withtiter of a titer19.2 μ ofg/L 19.2 (Schemeµg/L 9). (Scheme 9).

COOH HO COOH HO TyrH DODC HO

NH2 NH2 NH2 HO HO HO NH NCS HO H

HO HO 6-OMT

H CO H3CO H3CO H3CO 3 CYP80B1 NCH CNMT NH NCH3 4'-OMT NCH3 CRP 3 HO HO HO HO H H H H HO HO

HO HO H3CO HO

SchemeScheme 9. 9. ReticulineReticuline biosynthesis biosynthesis in engineered in engineered yeast. TyrH: yeast. tyrosine TyrH: hydroxylase; tyrosine hydroxylase; DODC: L-DOPA DODC: L-DOPAdecarboxylase;decarboxylase; NCS: NCS: norcolaurine synthase;synthase; CYP80B1: CYP80B1: cytochrome cytochrome P450; P450; CRP: CRP: cytochrome cytochrome P450 reductase;P450 6-OMT:reductase; 6-O-methyltransferase; 6-OMT: 6-O CNMT:-methyltransferase; coclurine-N -methyltransferase;CNMT: coclurine- 4N0-OMT:-methyltransferase; 40-O-methyltransferase. 4′-OMT: 4′-O- methyltransferase.

OtherOther authors authors engineered engineered yeast yeast strains to to prod produceuce protoberberine protoberberine alkaloid alkaloid starting startingfrom (R,S)- from (R,S)-norlaudanosolinenorlaudanosoline adding adding more more steps steps to tothe the previously previously reported reported process process [56]. [They56]. Theyproduced produced S. S. cerevisiaecerevisiaeexpressing expressing seven seven heterologous heterologous enzymes enzymes includingincluding the the flavin-dependent ﬂavin-dependent oxidase oxidase berberine berberine bridgebridge enzyme, enzyme, the cytochrome the cytochrome P450 P450 canadine canadine synthase, synthase, and and a cytochrome a cytochrome P450 P450 reductase. reductase. As As a result, a canadineresult, was canadine obtained was with obtained a titer with up toa titer 1.8 mg/L.up to 1.8 mg/L. In 2016,In 2016, the gene the gene cluster cluster leading leading to to noscapine noscapine was was reconstitutedreconstituted in in S.S. cerevisiae cerevisiae achievingachieving the the microbial production of noscapine (titer of 1.64 ± 0.38 μM noscapine) and related intermediates [57]. microbial production of noscapine (titer of 1.64 ± 0.38 µM noscapine) and related intermediates [57]. Starting from canadine produced as described above, the authors developed yeast strains expressing Startingdifferent from genes canadine from P. produced somniferum as (tetrahydroprotoberberine- described above, thecis authors-N-methyltransferase developed (PsTNMT), yeast strains expressingseveral different cytochrome genes P450 from monooxygenasesP. somniferum (CYP82Y1,(tetrahydroprotoberberine- CYP82X1, CYP82X2),cis an-N acetyl-methyltransferase transferase (PsTNMT),(PsAT1), several a carboxylesterase cytochrome P450 (PsCXE1), monooxygenases a short-chain (CYP82Y1, dehydrogenase/reductase CYP82X1, CYP82X2), (PsSDR1), an acetyla transferasemethyltransferase (PsAT1), a carboxylesterase (Ps6-OMT)), and a (PsCXE1), cytochrome a short-chainP450 reductase dehydrogenase/reductase partner from Arabidopsis thaliana (PsSDR1), a methyltransferase(AtATR1) (Scheme (Ps6-OMT)), 10). and a cytochrome P450 reductase partner from Arabidopsis thaliana (AtATR1) (SchemeBased on 10 the). successful microbial production of (S)-reticuline, the main branch point in BIA Basedbiosynthesis, on the different successful research microbial groups production attempted ofto engineer (S)-reticuline, the biosynthetic the main pathway branch leading point in to BIA the alkaloid sanguinarine. One of these works proved the production of the metabolic intermediate biosynthesis, different research groups attempted to engineer the biosynthetic pathway leading to stylopine from (S)-reticuline by using a P. pastoris expression system, characterized by high the alkaloid sanguinarine. One of these works proved the production of the metabolic intermediate expression of recombinant proteins under the control of a strong inducible promoter [58]. Conversion stylopineof reticuline from (S)-reticuline to stylopine by was using achieved a P. pastoris by introducingexpression berberine system, bridge characterized enzyme, bycheilanthifoline high expression of recombinantsynthase (CYP719A5), proteins under and stylopine the control synthase of (CYP a strong719A2). inducible The authors promoter evaluated [58 two]. experimental Conversion of reticulinedesigns: to stylopine in the first was one achieved these three by introducinggenes were co-e berberinexpressed bridge in a single enzyme, cell; cheilanthifolinein the second one synthase each

Int. J. Mol. Sci. 2017, 18, 2464 10 of 19

(CYP719A5),Int. J. Mol. Sci. 2017 and, 18 stylopine, 2464 synthase (CYP719A2). The authors evaluated two experimental designs:10 of 18 in the ﬁrst one these three genes were co-expressed in a single cell; in the second one each gene was expressedgene was inexpressed a single cellin a and single the threecell and strains the werethree co-cultured.strains were This co-cultured. last approach This waslast moreapproach efﬁcient, was asmore it resulted efficient, in as the it production resulted in ofthe 150 production nmol of stylopine. of 150 nmol of stylopine. OtherOther authorsauthors were able to to go go further further through through th thisis metabolic metabolic pathway. pathway. They They transplanted transplanted a 10- a 10-genegene plant plant pathway pathway in in S.S. cerevisiae cerevisiae leadingleading to to sanguinari sanguinarinene and and dihydrosanguinarine synthesissynthesis [[59]59] (see(see Scheme Scheme7 ).7). Although Although only only trace trace amounts amounts of dihydrosanguinarineof dihydrosanguinarin weree were obtained, obtained, this this work work can becan reasonablybe reasonably considered considered the the most most complex complex plant plant alkaloid alkaloid biosynthetic biosynthetic pathway pathway ever ever reconstituted reconstituted in yeastin yeast and and shows shows the the potential potential of engineeringof engineering microbes microbes for for the the synthesis synthesis of of complex complex plant plant alkaloids. alkaloids. ReconstructionReconstruction of the the sanguinarine sanguinarine branch branch of the of BIA the pathway BIA pathway in S. cerevisiae in S. cerevisiae was also wasachieved. also achieved[60]. The [authors60]. The were authors able were to im ableprove to cheilanthifoline, improve cheilanthifoline, stylopine, stylopine,cis-N-methylstylopine,cis-N-methylstylopine, protopine, protopine,and sanguinarine and sanguinarine production production (see Scheme (see 7) Scheme by expressing7) by expressing multiple multipleplant cytochrome plant cytochrome P450 enzymes, P450 enzymes,choosing choosingthe best performing the best performing isoenzymes isoenzymes and partne andr reductases, partner reductases, and paying and particular paying attention particular to attentionthe best conditions to the best for conditions their expression for their expressionand activity. and activity.

Scheme 10. Noscapine biosynthesis in engineered yeast. TNMT: tetrahydroprotoberberine-cis-N- Scheme 10. Noscapine biosynthesis in engineered yeast. TNMT: tetrahydroprotoberberine-cis-N- methyltransferase; CYP82Y1, CYP82X1, CYP82X2: cytochrome P450 monooxygenases; PsAT: acetyl transferase, methyltransferase; CYP82Y1, CYP82X1, CYP82X2: cytochrome P450 monooxygenases; PsAT: acetyl PsCXE: carboxylesterase, PsSDR1: short-chain dehydrogenase/reductase; Ps6-OMT: methyltransferase; transferase, PsCXE: carboxylesterase, PsSDR1: short-chain dehydrogenase/reductase; Ps6-OMT: AtATR1: Arabidopsis thaliana cytochrome P450 reductase. methyltransferase; AtATR1: Arabidopsis thaliana cytochrome P450 reductase.

OneOne ofof thethe mostmost exciting challenges in in microbial microbial BIA BIA synthesis synthesis is is the the fermentative fermentative production production of ofthe the high high value value morphinan morphinan alkaloids alkaloids in inyeast, yeast, that that are are the the strongest strongest narcotic narcotic analgesics analgesics employed employed in pain treatment. Starting from 2008, researchers were able to create S. cerevisiae strains that can perform sections of the glucose-to-morphine pathway [54,59,61–63]. However, a single strain capable of executing the whole pathway is not yet available.

Int. J. Mol. Sci. 2017, 18, 2464 11 of 19 in pain treatment. Starting from 2008, researchers were able to create S. cerevisiae strains that can perform sections of the glucose-to-morphine pathway [54,59,61–63]. However, a single strain capable of executing the whole pathway is not yet available. It is important to underline that the ﬁrst committed step in the synthesis of morphine is the isomerizationInt. J. Mol. Sci. of 2017 (S)-reticuline, 18, 2464 to (R)-reticuline. A reticuline epimerase (a fusion between a cytochrome11 of 18 P450 and an aldo-keto reductase) was identiﬁed in opium poppy and in Papaver bracteatum [64]. This enzymeIt is important catalyzes to the underlineS-to-R epimerizationthat the first committed of reticuline step in via the 1,2-dehydroreticuline. synthesis of morphine is Anyway, the it cannotisomerization be excluded of ( theS)-reticuline existence to of ( alternativeR)-reticuline. pathways A reticuline for theepimerase production (a fusion of (R )-intermediatesbetween a involvingcytochrome enzymes P450 selective and an foraldo-keto the (R )-enantiomersreductase) was fromidentified the beginningin opium ofpoppy the reticulineand in Papaver synthesis pathwaybracteatum [65]. [64]. This enzyme catalyzes the S-to-R epimerization of reticuline via 1,2- dehydroreticuline. Anyway, it cannot be excluded the existence of alternative pathways for the The yeast-based plant pathway reconstitution is a valuable tool for understanding plant secondary production of (R)-intermediates involving enzymes selective for the (R)-enantiomers from the metabolismbeginning and of the has reticulin the advantagee synthesis of pathway building [65]. strains for the production of intermediates that can be consideredThe yeast-based lead compounds plant pathway for drug reconstitution discovery. Nowadays, is a valuable however, tool for the understanding isolated yields plant of BIAs producedsecondary with metabolism this approach and arehas ratherthe advantage low and of limitedbuilding to strains a lab for scale. the production of intermediates that can be considered lead compounds for drug discovery. Nowadays, however, the isolated yields 3. Norcolaurineof BIAs produced Synthase: with this The approach Gate of are BIA rather Chirality low and limited to a lab scale.

3.1. Chemo-Enzymatic3. Norcolaurine Synthase: Synthesis The of BIAsGate of BIA Chirality Among the different approaches for BIA production, in vitro biocatalysis is gaining more and 3.1. Chemo-Enzymatic Synthesis of BIAs more importance as it allows to produce complex THIQs in high stereoselectivities and under mild conditions.Among The the first different committed approaches step infor BIABIA production, biosynthesis in isvitro the biocatalysis Pictet-Spengler is gaining condensation more and of two simplemore importance molecules, as dopamineit allows to produce and 4-HPAA, complex to THIQs yield thein high nitrogen stereoselectivities heterocycle and (S )-norcoclaurineunder mild conditions. The first committed step in BIA biosynthesis is the Pictet-Spengler condensation of two (Scheme7). This reaction is catalyzed by the enzyme norcolaurine synthase that introduces a chiral simple molecules, dopamine and 4-HPAA, to yield the nitrogen heterocycle (S)-norcoclaurine center that can be considered of paramount importance as it is needed for all the stereoselective (Scheme 7). This reaction is catalyzed by the enzyme norcolaurine synthase that introduces a chiral enzymaticcenter reactionsthat can be characterizing considered of theparamount BIA biosynthetic importance pathways as it is needed [66–68 fo].r all the stereoselective Itenzymatic is not reactions surprising characterizing that many the efforts BIA biosynthetic have been pathways directed [66–68]. towards the full biochemical characterizationIt is not of surprising NCS because that itma pavesny efforts the wayshave tobeen new, directed otherwise towards non-viable the full syntheticbiochemical routes leadingcharacterization to benzylisoquinoline of NCS because alkaloids. it paves NCSthe ways activity to new, was otherwise isolated non-viable in protein synthetic extracts routes of opium poppyleading (Papaver to benzylisoquinoline somniferum) and alkaloids. some other NCS related activity specieswas isolated [66]. in Soon protein afterwards, extracts of theopium enzyme poppy form Thalictrum(Papaver flavum somniferumwas purified) and some to homogeneity other related starting species from [66].cell Soon culture afterwards, [68] and the then enzyme heterologously form expressedThalictrum in E. flavum coli [67 was,69 purified]. The first to homogeneity crystallographic starting structure from cell culture of NCS [68] from andT. then flavum heterologouslyin its complex expressed in E. coli [67,69]. The first crystallographic structure of NCS from T. flavum in its complex with dopamine and the non-reactive substrate analogue 4-hydroxybenzaldehyde was obtained by our with dopamine and the non-reactive substrate analogue 4-hydroxybenzaldehyde was obtained by researchour groupresearch [70 group] allowing [70] allowing to understand to understa NCSnd catalytic NCS mechanismcatalytic mechanism and kinetics and kinetics [71]. Afterwards, [71]. it wasAfterwards, demonstrated it was that demonstrated a dopamine that first a dopami mechanismne first governs mechanism the governs stereoselective the stereoselective production of (S)-norcoclaurineproduction of [(72S)-norcoclaurine,73] and showed [72,73] also and that showed the kinetic also behaviorthat the kinetic can explain behavior the can ineffectiveness explain the of recombinantineffectiveness NCS inof vivorecombinantsystems. NCS Nevertheless, in vivo systems. NCS Nevertheless, is a powerful NCS tool is a for powerful the in tool vitro forasymmetric the in synthesisvitro ofasymmetric (S)-norcolaurine synthesis andof (S derivatives.)-norcolaurine and derivatives. OurOur group group set upset up the the first first efficient, efficient, chemo-enzymatic chemo-enzymatic synthesis synthesis of of(S)-norcoclaurine (S)-norcoclaurine using using the the recombinantrecombinant NCS NCS enzyme, enzyme, starting starting from from cheap cheap substrates like like tyrosine tyrosine and and dopamine dopamine in a inone-pot, a one-pot, two-steptwo-step process process [74 ][74] (Scheme (Scheme 11 11).).

SchemeScheme 11. Chemo-enzymatic 11. Chemo-enzymatic synthesis synthesis of of( (SS)-norcolaurine)-norcolaurine starting starting from from tyrosine tyrosine and and dopamine dopamine in in the presencethe presence of norcoclaurine of norcoclaurine synthase synthase (NCS). (NCS).

In the first step, 4-HPAA was generated by the oxidative decarboxylation of tyrosine in the presence of equimolar amounts of sodium hypochlorite. In the second step, dopamine and NCS were added to the reaction mixture in the presence of ascorbate to avoid catechol moiety oxidation. The

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In the first step, 4-HPAA was generated by the oxidative decarboxylation of tyrosine in the presence of equimolar amounts of sodium hypochlorite. In the second step, dopamine and NCS were added to the reaction mixture in the presence of ascorbate to avoid catechol moiety oxidation. The process was carried out in phosphate buffer obtaining (S)-norcolaurinein 81% yield and 93% enantiomeric excess (ee). Later investigations [75] showed that the phosphate buffer itself was able to catalyze the non stereoselective synthesis of norcolaurine, thus the choice of the buffer proved to be crucial for the stereoselectivity of the reaction. Phosphates are able to catalyze the reaction and show a very good tolerance for the aldehydes. The use of phosphate buffer represents an alternative to previously reported chemical strategies to synthesize simple tetrahydroisoquinoline alkaloids with mild reaction conditions. Nevertheless, this approach leads to racemic mixtures that need further resolution. To avoid background activity of phosphate in NCS catalyzed reaction, other buffers such as HEPES and MOPS should be used. This shrewdness allows for enantiopure products to be obtained. The reaction catalyzed by the NCS from different sources was tested for synthetic purposes as the specificity towards aldehyde substrates is quite relaxed, in contrast with the requirement for the amine substrate, which is limited to dopamine and few other dopamine analogues, highlighting the requirement of a meta-hydroxy moiety in dopamine [70,71,76–78]. In the light of the above, different research groups developed chemo-enzymatic strategies for the production of BIAs coupling a chemical approach for the aldehyde synthesis with the enzymatic activity of NCS. One of these groups exploited the selectivity of T. flavum NCS towards fifteen chemically synthesized aldehydes including substituted phenylacetaldehydes, (hetero) aromatic substrates, aliphatic (hetero)cycles, and open chain aliphatic compounds [77]. These aldehydes, where not commercially available, were synthesized either by oxidation of the corresponding alcohols with Dess-Martin periodinane or by reduction of the corresponding Weinreb amide with lithium aluminium hydride (LAH). In the following step, these aldehydes were used as substrates in NCS catalyzed reaction. Although the synthesis was performed in lab scale and the enantiomeric excess of the reaction products was not investigated, this work paved the way to the chemo-enzymatic synthesis of non-natural THIQs. Another group, instead, investigated the substrate specificity of NCS from Coptis japonica (CjNCS2) towards both the amine and aldehydic substrates [78]. In this work, more than ten structurally diverse aldehydes and amines were tested. The aryl and hetero aromatic acetaldehydes were synthesized from either the corresponding terminal alkenes via ozonolysis or the corresponding alcohols via a Parikh–Doering oxidation, while the ethylamines were produced from the corresponding phenylacetonitriles by means of standard reduction reactions. With this kind of approach the authors afforded (S)-THIQs, including the non-natural compounds in good to excellent yield (56–99%) and with an ee of 0.95%.

3.2. Fully Enzymatic Synthesis of Substituted Tetrahydroisoquinolines A green improvement to the chemo-enzymatic methods for the production of BIAs described above is the employment of enzymes also for aldehyde production. Using enzymes in domino processes leads to the formation of highly complex compounds from cheap starting materials, often without intermediate isolation or functional group protection. In recent years, more and more attention has been devoted to these synthetic strategies. The ﬁrst attempt to perform a fully enzymatic synthesis of tetrahydroisoquinoline derivatives was achieved by coupling the activities of transaminase (TAm) and norcolaurine synthase in a one-pot, one-substrate “triangular” cascade (Scheme 12)[79]. Int. J. Mol. Sci. 2017, 18, 2464 13 of 19 Int.Int.Int. J.J. J. Mol.Mol. Mol. Sci.Sci. Sci. 2017 2017,, ,18 18,, ,24642464 2464 1313 of of 18 18

HO NH2 HHOO NHNH22

HO HO HHOO HHOO Pyruvate NH PyruvatePyruvate HO NHNH NCS HHOO H NCSNCS HH TAm HO TAmTAm HHOO AlanineAlanine HO HO O HO HHOO OO HO

HO HHOO Scheme 12. Fully enzymatic synthesis of norlaudanosoline involving transaminase (TAm). SchemeScheme 12. 12. FullyFully Fully enzymaticenzymatic enzymatic synthesissynthesis synthesis ofof of norlaudanosolinenorlaudanosoline norlaudanosoline involvinginvolving involving transaminasetransaminase transaminase (TAm).(TAm). (TAm).

AldehydeAldehydeAldehyde species speciesspecies were werewere generated generatedgenerated in inin situ situ by by tran transaminasetransaminasesaminase (TAm) (TAm) starting startingstarting from fromfrom dopamine dopaminedopamine that thatthat in inin itsitsits turn turnturn reacts reactsreacts with withwith the thethe newly newlynewly synthesized synthesizedsynthesized aldehyde aldehydealdehyde inin inin thethe thethe presencepresence presencepresence ofof ofof NCSNCS NCSNCS toto toto givegive givegive norlaudanosolinenorlaudanosoline norlaudanosolinenorlaudanosoline withwithwith good goodgood conversion conversion and and ex excellentexcellentcellent enantioselectivity. enantioselectivity. MoreMoreMore recently recentlyrecently the thethe aldehydes aldehydesaldehydes to toto be bebe used usedused in inin NCS NCSNCS catalyzed catalyzedcatalyzed reaction reactionreaction were werewere produced producedproduced by byby oxidative oxidativeoxidative deaminationdeaminationdeamination catalyzed catalyzed by by LathyrusLathyrusLathyrus cicera ciceracicera diaminediaminediamine oxidase oxidaseoxidase (LCAO) (LCAO)(LCAO) that thatthat shows showsshows a aa broad broadbroad substrate substrate specificityspeciﬁcityspecificity towards towardstowards several severalseveral ethyl ethylethyl amines aminesamines [80,81]. [[80,81].80,81]. Our OurOur group groupgroup set setset up upup a aa fully fully enzymatic enzymatic synthesis synthesis of ofof new new chiralchiralchiral THIQs THIQsTHIQs in inin two twotwo steps stepssteps starting startingstarting from from dopamine dopaminedopamine and and four four different different amines aminesamines by byby coupling coupling the the activity activity LCAOLCAOLCAO withwithwith T. T.T. ﬂavum flavumflavumnorcoclaurine norcoclaurinenorcoclaurinenorcoclaurine synthase synthasesynthasesynthase (Tf NCS) (((TfTfNCS)NCS) (Scheme (Scheme(Scheme 13). In 13).13). this InIn paper, thisthis fourpaper,paper, amines fourfour carrying aminesamines carryingmorpholinecarrying morpholine morpholine or piperazine or or piperazine piperazine moieties moieties weremoieties converted were were co co innvertednverted the corresponding in in the the corresponding corresponding aldehydes aldehydes aldehydes and then and and used then then for usedtheused production for for the the production production of novel of BIAs.of novel novel All BIAs. BIAs. the reaction All All the the reaction stepsreaction were steps steps performed were were performed performed in HEPES in in buffer. HEPES HEPES buffer. buffer.

Scheme 13. Fully enzymatic synthesis of substituted THIQs involving Lathyrus cicera amine oxidase SchemeScheme 13. 13. Fully FullyFully enzymatic enzymaticenzymatic synthesis synthesissynthesis of ofof substituted substitutedsubstituted THIQs THIQsTHIQs involving involvinginvolving Lathyrus Lathyrus cicera cicera amine amineamine oxidase oxidaseoxidase (LCAO) and norcolaurine synthase (NCS). (LCAO)(LCAO) and and norcolauri norcolaurinene synthase synthase (NCS). (NCS).

The first step was optimized removing H2O2 produced by LCAO adding catalase. Before the TheTheThe first ﬁrstfirst stepstep waswas optimizedoptimizedoptimized removingremovingremoving HHH222OO22 produced produced by by LCAO LCAO adding adding catalase. catalase. Before BeforeBefore the the secondsecondsecond step,step, LCAO LCAO waswas was inactivatedinactivated inactivated withwith with phenylhydrazine phenylhy phenylhydrazinedrazine and and and then then then NCS NCS NCS and and and dopamine dopamine dopamine were were were added added added to totheto thethe reaction reactionreaction pot. pot.pot. The The newlyThe newlynewly synthesized synthesizedsynthesized BIAs BIAs wereBIAs obtainedwerewere obtainedobtained in very inin good veryvery yields goodgood (>72%) yieldsyields and (>72%)(>72%) excellent andand excellentenantioselectivityexcellent enantioselectivity enantioselectivity (ee > 99%). ( (eeee > >> 99%). 99%).99%). TheTheThe firstfirst ﬁrst exampleexample example ofof of aa three-step athree-step three-step enzymaticenzymatic enzymatic casccasc cascadeadeade forfor the forthe stereoselectstereoselect the stereoselectiveiveive synthesissynthesis synthesis ofof 1,3,4-1,3,4- of trisubstituted-THIQ1,3,4-trisubstituted-THIQtrisubstituted-THIQ was was reported reported was reported in inin 2017 20172017 in [82] [82]2017[82] (Scheme (Scheme(Scheme [82] (Scheme 14). 14).14). 14).

OH OH OH OHOH OHOH OHOH HO HCHO HCHO 3 HO CH3 HCHO 3 HCHHCHOO HHOO CHCH HHOO O CL HCHO 33 TAm 33 33 OO CLCL TAmTAm NH NH O NHNH2 NHNH OO 22 NCS NCSNCS H HH

O OO

HO HHOO Scheme 14. Fully enzymatic synthesis of 1,3,4-trisubstituted-THIQ involving carboligase (CL), SchemeSchemeScheme 14. 14. FullyFullyFully enzymaticenzymatic synthesissynthesis of of 1,3,4-trisubstituted-THIQ 1,3,4-trisubstituted-THIQ involving involving carboligase carboligase (CL), (CL), transaminase (TAm), and norcolaurine synthase (NCS). transaminasetransaminasetransaminase (TAm), (TAm),(TAm), and and norc norcolaurinenorcolaurineolaurine synthasesynthase (NCS).(NCS).

Int. J. Mol. Sci. 2017, 18, 2464 14 of 19 Int. J. Mol. Sci. 2017, 18, 2464 14 of 18

The ﬁrstfirst reaction step was catalyzed by carbol carboligaseigase (CL) that converted 3-hydroxybenzaldehyde and pyruvate pyruvate in in acyloin. acyloin. In the In second the secondstep, a transaminase step, a transaminase (TAm) was (TAm) used to was form used 2-amino-1-(3- to form 2-amino-1-(3-hydroxyphenyl)hydroxyphenyl) propan-1-ol that propan-1-ol in its turn that reac inted its with turn phenylacethaldehyde reacted with phenylacethaldehyde in a NCS catalyzed in a NCSPictet-Spengler catalyzed Pictet-Spenglercyclization. The cyclization. 1,3,4-trisubstituted The 1,3,4-trisubstituted-THIQ-THIQ thus obtained was thus isolated obtained excellent was isolated yield excellent(92%). yield (92%). In the the same same year, year, TfTfNCSNCS variants variants with with improved improved ketone ketone tole tolerancerance that that were were able ableto accept to accept non- non-aldehydealdehyde carbonyl carbonyl substrates substrates were were identified identified [83]. [83 ].These These enzymes enzymes could could accept accept a a number number of unactivated ketonesketones for thefor synthesis the synthesis of new chiral of 1,10-disubstituted- new chiral 1,10-disubstituted- and spiro-tetrahydroisoqionolines and spiro- (Schemetetrahydroisoqionolines 15). (Scheme 15).

HO HO R 1 NCS + O NH2 NH HO R2 HO R1 R2

Scheme 15. 15. NorcolaurineNorcolaurine synthase synthase (NCS) (NCS) catalyzed catalyzed synthesis synthesis of chiral 1,10-disubstituted- of chiral 1,10-disubstituted- and spiro- andtetrahydroisoqionolines. spiro-tetrahydroisoqionolines.

The (chemo)-enzymatic methods here reported for the synthesis of natural and non-natural BIAs The (chemo)-enzymatic methods here reported for the synthesis of natural and non-natural BIAs demonstrate the great potential of in vitro biocatalysis for the formation of complex chiral demonstrate the great potential of in vitro biocatalysis for the formation of complex chiral compounds. compounds. The main advantage of these domino processes is that they do not require intermediate The main advantage of these domino processes is that they do not require intermediate isolation or isolation or functional group protection strategies. Moreover, all these reactions are performed in functional group protection strategies. Moreover, all these reactions are performed in aqueous media aqueous media and exhibit high atom economy. and exhibit high atom economy. However, the critical step is represented by the lab scale production that needs to be improved However, the critical step is represented by the lab scale production that needs to be improved at least to gram scale. This improvement can be in principle achieved by finely tuning the enzyme at least to gram scale. This improvement can be in principle achieved by finely tuning the enzyme activity and/or by increasing its stability and recycling. In fact, many enzymes lack of long-term activity and/or by increasing its stability and recycling. In fact, many enzymes lack of long-term stability under the experimental conditions and it is also difficult to recover and recycle them in order stability under the experimental conditions and it is also difficult to recover and recycle them in order to make the overall process economically and environmentally advantageous. Enzyme to make the overall process economically and environmentally advantageous. Enzyme immobilization immobilization can be a useful tool for enhancing stability, reducing costs and modulating catalytic can be a useful tool for enhancing stability, reducing costs and modulating catalytic properties. properties. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations Abbreviations BIAs Benzylisoquinoline alkaloids THIQsBIAs Benzylisoquinoline Tetrahydroisoquinolines alkaloids 4-HPAATHIQs Tetrahydroisoquinolines 4-hydroxyphenylacethaldehyde NCS4-HPAA 4-hydroxyphenylacethaldehyde Norcoclaurine synthase VIGSNCS Norcoclaurine Virus-induced synthase gene silencing CRISPRVIGS Virus-induced Clustered regularly gene silencing interspaced short palindromic repeats OMTCRISPR ClusteredO-Methyltransferase regularly interspaced short palindromic repeats CMNTOMT O-Methyltransferase Coclurine-N-methyltransferase TNMTCMNT Coclurine- Tetrahydroprotoberberine-N-methyltransferasecis -N-methyltransferase BBETNMT Tetrahydroprotoberberine- Berberine bridge enzymecis-N-methyltransferase DODCBBE BerberineL-DOPA bridge decarboxylase enzyme MAODODC L-DOPA Monoamine decarboxylase oxidase LCAOMAO MonoamineLathyrus cicera oxidaseamine oxidase CYPLCAO Lathyrus Cytochrome cicera amine P450monooxygenases oxidase TAmCYP Cytochrome Transaminase P450 monooxygenases TAm Transaminase References References 1. Hagel, J.M.; Facchini, P.J. Benzylisoquinoline alkaloid metabolism: A century of discovery and a brave new 1. Hagel, J.M.; Facchini, P.J. Benzylisoquinoline alkaloid metabolism: A century of discovery and a brave new world. Plant Cell Physiol. 2013, 54, 647–672. [CrossRef][PubMed] world. Plant Cell Physiol. 2013, 54, 647–672. 2. Shamma, M. The Isoquinoline Alkaloids: Chemistry and Pharmacology; Academic Press: New York, NY, USA, 1972; Volume 25.

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