We describe novel chemoenzymatic routes to (S)-benzylisoquinoline and (S)-tetrahydroprotoberberine alkaloids using the enzymes transaminase (TAm) and norcoclaurine synthase (NCS) in a one-pot, one-substrate ‘triangular’ cascade. Employment of up to two C–C bond forming steps allows for the rapid generation of molecular complexity under mild conditions

Hailes, HC

Lamming, ED

Lichman, BR

Pesnot, T

Smith, JM

Ward, JM

UCL Discovery

Green ChemistryCOMMUNICATIONCite this: Green Chem., 2015, 17, 852Received 25th November 2014,Accepted 12th December 2014DOI: 10.1039/c4gc02325kwww.rsc.org/greenchemOne-pot triangular chemoenzymatic cascades forthe syntheses of chiral alkaloids from dopamine†B. R. Lichman,a E. D. Lamming,b T. Pesnot,b J. M. Smith,a H. C. Hailes*b andJ. M. Ward*aWe describe novel chemoenzymatic routes to (S)-benzylisoquino-line and (S)-tetrahydroprotoberberine alkaloids using the enzymestransaminase (TAm) and norcoclaurine synthase (NCS) in a one-pot, one-substrate ‘triangular’ cascade. Employment of up to twoC–C bond forming steps allows for the rapid generation of mole-cular complexity under mild conditions.IntroductionIn order to minimise waste products and reduce the usage offinite resources, chemistry must find ‘green’ alternatives to tra-ditional synthetic methods.1 The employment of enzymes ascatalysts oﬀers significant potential in this regard as enzymesoften demonstrate exquisite chemo- and enantio-selectivity,and operate in mild conditions.2 Furthermore, using enzymesin one-pot cascades can enable the formation of highlycomplex compounds from cheap starting materials, frequentlywithout the requirement of intermediate isolation or func-tional group protection strategies.3Benzylisoquinoline alkaloids (BIAs) are a large, diversefamily of natural products found in both plants4 and animals.5Many BIAs have pharmacological activities—these includeanalgesic,6 antimicrobial7 and antitumor8 eﬀects—and conse-quently are common synthetic targets.9 A key step in the syn-thesis of many of these compounds is the formation of thetetrahydroisoquinoline (THIQ) moiety via a Pictet–Spenglercondensation.10 Recently, a mild one-pot biomimetic approachto the formation of THIQs was developed, using phosphatebuﬀer as a catalyst.11 There has also been recent progressmade in the enzymatic synthesis of diverse chiral THIQs usingthe plant enzyme norcoclaurine synthase (NCS).12Norlaudanosoline (tetrahydropapaveroline) 1a is a BIAfound in mammals: it is the precursor to ‘endogenous’ mor-phine,5 and also has a number of neuronal eﬀects includingroles in Parkinson’s disease13 and drug addiction.14 Syntheti-cally, 1a is a versatile BIA precursor, and consequently it hasbeen employed in synthetic biology/metabolic engineeringroutes to various BIAs.15 BIA 1a can be accessed by a Pictet–Spengler condensation between dopamine 2a and 3,4-dihydoxy-phenylacetaldehyde 3a.16Tetrahydroprotoberberine alkaloids (THPBs, berbines) are asubgroup of the BIAs found in both plants and animals, andinclude compounds such as canadine17 and spinosine.18a TheTHPB moiety can be accessed from 1a via a Pictet–Spenglerreaction with formaldehyde.18 The two compounds directlyformed by this reaction (4 and 5) have been shown to havepotential in cancer chemotherapeutics.19 The regioselectivityof this chemical Pictet–Spengler reaction (major product:10,11-dihydroxy 4) contrasts with the regioselectivity of theplant berberine-bridge enzyme (BBE), which typically forms9,10-dihydroxy-THPBs from a N-methylated BIA precursor.20Thus chemical approaches to THPBs provide a complementarymethod to the reported BBE approach.Triangular cascade designIn this work, we present one-pot two-enzyme one-substratesyntheses of (S)-1a and (S)-1b using the enzymes transaminase(TAm) and NCS. The reaction involves in situ generation andutilisation of reactive aldehyde species. We also demonstrate aone-pot, two-enzyme, three-step chemoenzymatic synthesis ofthe THPB (S)-4. This synthesis uses the same cascade route asfor (S)-1a, but involves the sequential addition of formal-dehyde to trigger a second Pictet–Spengler cyclisation(Scheme 1).Previous attempts to achieve this type of cascade in vitroand in vivo have utilised a monoamine oxidase (MAO) as thecatalyst in the first step, converting 2a to 3a. Several MAOapproaches have focused on forming rac-1a through a spon-taneous Pictet–Spengler reaction (typically phosphate cata-lysis).16 A number of in vivo metabolic cascades forming†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4gc02325kaDepartment of Biochemical Engineering, University College London, Bernard KatzBuilding, Gordon Street, London, WC1H 0AH, UK. E-mail: j.ward@ucl.ac.ukbDepartment of Chemistry, University College London, Christopher-Ingold Building,20 Gordon Street, London, WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk852 | Green Chem., 2015, 17, 852–855 This journal is © The Royal Society of Chemistry 2015(S)-reticuline report the use of recombinant NCS as the Pictet–Spengler catalyst. However, it is not clear that NCS is active inthese systems: any chirality reported seems to be the result of(S)-selective enzymes downstream of NCS.15a–dHere, we employ a TAm for the conversion of 2a to 3a (step1), which then combine to form 1a (step 2). TAms have anadvantage over MAOs in this system: the conversion of 2a to 3awith TAms can be controlled by stoichiometrically limiting thequantity of co-substrate (in this instance, pyruvate) available.Furthermore, we avoid the problems associated with drivingTAm reactions to completion, as the Pictet–Spengler reactionremoves equal quantities of molecules from both sides of theTAm equilibrium. This three-sided ‘triangular’ cascade designresults in a system with high atom economy.3Results and discussionThe first step towards establishing the one-pot cascade systemwas the identification of a TAm with good activity towards 2a.The screening reaction was conducted in phosphate buﬀer,which enabled the in situ formation of rac-1a from 2a and 3a(Table 1). The screen identified two TAms with activity towards2a at pH 7.5: CV2025 (Chromobacterium violaceum)21 andPP_3718 (Pseudomonas putida), providing 21% and 15% con-version of 1a from 50 mM 2a respectively. Trace activities werefound in PP_0596 (P. putida), SaV_2612 (Streptomyces avermiti-lis) and VF_JS17 (Vibrio fluvialis)22 (Table 1, ESI† Fig. S1). Weselected the best performing TAm, CV2025, for use in furtherstudies.In order to form (S)-1a from 2a two aspects of the previousTAm screening reaction conditions were modified. Changingthe buﬀer from phosphate to HEPES removed most of thebackground chemical reaction11 and adding purifiedΔ29TfNCS enabled catalytic formation of the chiral product(S)-1a from 2a. Optimum reaction conditions were determinedby varying the concentrations of 2a, NCS and TAm present. Abalance between reaction components was crucial to ensurethe rates of the two steps were matched; a build-up of 3awould cause an increase in undesirable side-reactions includ-ing the non-enzymatic Pictet–Spengler condensation, whichwould reduce the final enantiomeric excess (ee) of the product.BIA (S)-1a was produced from 2a with very good conversionsand excellent enantioselectivity. In all cases, an increase inTAm concentration resulted in a greater consumption of 2a,Scheme 1 Overview of the biocatalytic and non-enzymatic cascadespresented in this work, including the ‘triangular’ cascade.Table 1 TAm mediated synthesis of rac-1a from 2aaEntry TAm Organism Uniprot entry name Gene name Conv.b (%)1 Empty vector n.d.c2 BSU_09260 Bacillus subtilis YHXA_BACSU yhxA n.d.3 CV_202521 Chromobacterium violaceum Q7NWG4_CHRVO CV_2025 214 Dgeo_1416 Deinococcus geothermalis Q1IYH3_DEIGD argD/lysJ n.d.5 KPN_00255 Klebsiella pneumoniae A6T537_KLEP7 gabT n.d.6 PP_0596 Pseudomonas putida Q88Q98_PSEPK PP_0596 Trace7 PP_3718 Pseudomonas putida Q88GK3_PSEPK PP_3718 148 SaV_2612 Streptomyces avermitilis Q82JZ2_STRAW SAV_2612 Trace9 SaV_4551 Streptomyces avermitilis Q82ER2_STRAW SaV_4551 n.d.10 VF_JS1722 Vibrio fluvialis F2XBU9_VIBFL JS17 Tracea 50 mM 2a, 25 mM pyruvate, 1 mM PLP, 10% v.v−1 TAm, 37 °C, 4 h. b Concentration of rac-1a, determined by analytical HPLC. c n.d. = notdetected. See ESI Fig. S1 for reaction time course.Green Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2015 Green Chem., 2015, 17, 852–855 | 853generally leading to an increase in product formation(Table 2). However, at lower concentrations of 2a (20 mM) theuse of 20% v.v−1 CV2025 lysate, rather than 30%, was optimal.The enantioselectivity was excellent under all conditions. Interms of conversion, the eﬀect of NCS concentration was moreapparent with higher concentrations of dopamine. The bestconditions identified were those with 20 mM 2a, 500 µg mL−1NCS and 20% v.v−1 CV2025 lysate, which provided an 87% con-version of (S)-1a from 2a with an ee of 99%.To demonstrate the versatility of this system, it was used tosynthesise (S)-1b from 2-(3-hydroxyphenyl)ethylamine 2b. Thepara-hydroxyl group of 2a is not required for the Pictet–Spengler condensation, and thus 2b is a suitable substrate forNCS.12b Using the optimum conditions determined previouslyfor 2a, (S)-1b was formed from 2b with a fair conversion of56% and a high ee of 90%. The lower conversion and ee com-pared to (S)-1a is possibly reflective of a poorer aﬃnity ofTfNCS towards for 2b compared to the natural substrate 2a.In order to form THPBs (S)-4 and (S)-5, a one-pot synthesisof (S)-1a was conducted (as described above) and formal-dehyde was added to the reaction after 3 hours. This triggeredthe second Pictet–Spengler condensation and resulted in theformation of (S)-4 and (S)-5 in a ratio of approximately 7 : 1(Scheme 2). This cascade features 2 enzymes, 3 steps and theformation of 4 bonds, including 2 C–C bonds. The overall reac-tion occurred with good conversion: the second Pictet–Spen-gler step alone (from (S)-1a) provided 74% conversion (64%and 9% for (S)-4 and (S)-5 respectively), which translates as anoverall 64% conversion (56% and 8%) from 2a. As the chiralityof the system is established by NCS, the subsequent additionof formaldehyde does not aﬀect the ee: only the (S)-enantiomerof the major product 4 was observed by chiral HPLC.The major regioisomer 4 formed by this method has 10,11-dihydroxy regiochemistry. BBE, a plant enzyme which catalysesthe formation of (S)-THPBs en route to berberine, typicallyforms products with 9,10-dihydroxy regioselectivity.20 Thisenzyme has recently been used in a novel in vitro deracemisa-tion cascade to produce (S)-THPBs.20b The synthesis presentedhere and the BBE catalysed cascade can be seen as comp-lementary methods, providing eﬃcient chemoenzymaticroutes to 10,11-dihydroxy-(S)-THPBs and 9,10-dihydroxy-(S)-THPBs respectively.Finally, to demonstrate the preparative potential of our cas-cades, we performed syntheses of (S)-1a and (S)-4 on a0.5 mmol scale. The concentrations of components in thesecascades were the same as those used in the micro-scale cas-cades (Scheme 2). The synthesis scaled with no complications:conversion of 2a to (S)-1a was achieved in 2 hours with 86%conversion and 62% isolated yield (>95% ee). (S)-4 was formedfrom 2a in 2.5 hours with 47% conversion and 42% isolatedyield (>95% ee). The identities of these products were verifiedby NMR.ConclusionsIn summary, we have developed one-pot syntheses of chiralalkaloids, using the enzymes TAm and NCS in a ‘triangular’cascade. BIA (S)-1a was formed from 2a with very good conver-sion and excellent enantioselectivity in only 2 hours. (S)-1bwas formed in a similar manner with fair conversion and ahigh ee. A chemical extension to the cascade enabled the for-mation of (S)-4 in a one-pot three-step reaction from the low-cost starting material 2a, again with excellent enantio-selectivity and an overall reaction time of less than 3 hours.The cascades presented here exhibit high atom economy,the in situ generation of reactive intermediates, and the rapidaccumulation of molecular complexity through C–C bondforming steps. Overall, these syntheses demonstrate theTable 2 One pot synthesis of (S)-BIAsaAmine[mM]CV2025(v.v−1) [%]NCS[µg mL−1]Conv.b[%]eec[%]2a 20 10 100 74 (74) 992a 20 20 100 86 (89) 992a 20 30 100 82 (93) 992a 20 10 500 77 (82) 992a 20 20 500 87 (92) 992a 20 30 500 84 (95) 982a 50 10 100 36 (42) 982a 50 20 100 55 (66) 972a 50 30 100 70 (78) 992a 50 10 500 42 (48) 982a 50 20 500 65 (73) 982a 50 30 500 72 (85) 962b 20 20 500 56 (57) 90aGeneral reaction conditions: 2 equivalents 2a or 2b, 1 equivalentpyruvate, purified Δ29TfNCS and CV2025 lysate, 50 mM HEPES pH7.5, 37 °C, 3 h. bDetermined by analytical HPLC. Conversions inbrackets refer to depletion of primary amine. cDetermined by chiralHPLC of the crude product.Scheme 2 One-pot chemoenzymatic synthesis of (S)-4 and (S)-5.Reaction conditions: (a) 20 mM 2, 10 mM sodium pyruvate, 500 µg mL−1NCS and 20% v.v−1 CV2025 lysate, 50 mM HEPES pH 7.5, 37 °C, 3 h. (b)40 mM formaldehyde, 1 M sodium phosphate, pH 6, 30 min, 37 °C.Communication Green Chemistry854 | Green Chem., 2015, 17, 852–855 This journal is © The Royal Society of Chemistry 2015remarkable potential of in vitro biocatalysis for the formationof complex chiral compounds.We gratefully acknowledge the Wellcome Trust for student-ship funding to B. R. L., EPSRC and GlaxoSmithKline for stu-dentship funding to E. D. L., and the Biotechnology andBiological Sciences Research Council (BBSRC) (BB/G014426/1)for funding T. P.Notes and references1 (a) J. H. Clark, Green Chem., 1999, 1, 1–8; (b) R. A. Sheldon,Pure Appl. Chem., 2000, 72, 1233–1246.2 B. M. Nestl, S. C. Hammer, B. A. Nebel and B. Hauer,Angew. Chem., Int. Ed., 2014, 53, 3070–3095.3 For reviews see: (a) E. Ricca, B. Brucher andJ. H. Schrittwieser, Adv. Synth. Catal., 2011, 353, 2239–2262;(b) R. C. Simon, N. Richter, E. Busto and W. Kroutil, ACSCatal., 2014, 4, 129–143.4 J. M. Hagel and P. J. 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One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine

Green ChemistryCOMMUNICATIONCite this: Green Chem., 2015, 17, 852Received 25th November 2014,Accepted 12th December 2014DOI: 10.1039/c4gc02325kwww.rsc.org/greenchemOne-pot triangular chemoenzymatic cascades forthe syntheses of chiral alkaloids from dopamine†B. R. Lichman,a E. D. Lamming,b T. Pesnot,b J. M. Smith,a H. C. Hailes*b andJ. M. Ward*aWe describe novel chemoenzymatic routes to (S)-benzylisoquino-line and (S)-tetrahydroprotoberberine alkaloids using the enzymestransaminase (TAm) and norcoclaurine synthase (NCS) in a one-pot, one-substrate ‘triangular’ cascade. Employment of up to twoC–C bond forming steps allows for the rapid generation of mole-cular complexity under mild conditions.IntroductionIn order to minimise waste products and reduce the usage offinite resources, chemistry must find ‘green’ alternatives to tra-ditional synthetic methods.1 The employment of enzymes ascatalysts oﬀers significant potential in this regard as enzymesoften demonstrate exquisite chemo- and enantio-selectivity,and operate in mild conditions.2 Furthermore, using enzymesin one-pot cascades can enable the formation of highlycomplex compounds from cheap starting materials, frequentlywithout the requirement of intermediate isolation or func-tional group protection strategies.3Benzylisoquinoline alkaloids (BIAs) are a large, diversefamily of natural products found in both plants4 and animals.5Many BIAs have pharmacological activities—these includeanalgesic,6 antimicrobial7 and antitumor8 eﬀects—and conse-quently are common synthetic targets.9 A key step in the syn-thesis of many of these compounds is the formation of thetetrahydroisoquinoline (THIQ) moiety via a Pictet–Spenglercondensation.10 Recently, a mild one-pot biomimetic approachto the formation of THIQs was developed, using phosphatebuﬀer as a catalyst.11 There has also been recent progressmade in the enzymatic synthesis of diverse chiral THIQs usingthe plant enzyme norcoclaurine synthase (NCS).12Norlaudanosoline (tetrahydropapaveroline) 1a is a BIAfound in mammals: it is the precursor to ‘endogenous’ mor-phine,5 and also has a number of neuronal eﬀects includingroles in Parkinson’s disease13 and drug addiction.14 Syntheti-cally, 1a is a versatile BIA precursor, and consequently it hasbeen employed in synthetic biology/metabolic engineeringroutes to various BIAs.15 BIA 1a can be accessed by a Pictet–Spengler condensation between dopamine 2a and 3,4-dihydoxy-phenylacetaldehyde 3a.16Tetrahydroprotoberberine alkaloids (THPBs, berbines) are asubgroup of the BIAs found in both plants and animals, andinclude compounds such as canadine17 and spinosine.18a TheTHPB moiety can be accessed from 1a via a Pictet–Spenglerreaction with formaldehyde.18 The two compounds directlyformed by this reaction (4 and 5) have been shown to havepotential in cancer chemotherapeutics.19 The regioselectivityof this chemical Pictet–Spengler reaction (major product:10,11-dihydroxy 4) contrasts with the regioselectivity of theplant berberine-bridge enzyme (BBE), which typically forms9,10-dihydroxy-THPBs from a N-methylated BIA precursor.20Thus chemical approaches to THPBs provide a complementarymethod to the reported BBE approach.Triangular cascade designIn this work, we present one-pot two-enzyme one-substratesyntheses of (S)-1a and (S)-1b using the enzymes transaminase(TAm) and NCS. The reaction involves in situ generation andutilisation of reactive aldehyde species. We also demonstrate aone-pot, two-enzyme, three-step chemoenzymatic synthesis ofthe THPB (S)-4. This synthesis uses the same cascade route asfor (S)-1a, but involves the sequential addition of formal-dehyde to trigger a second Pictet–Spengler cyclisation(Scheme 1).Previous attempts to achieve this type of cascade in vitroand in vivo have utilised a monoamine oxidase (MAO) as thecatalyst in the first step, converting 2a to 3a. Several MAOapproaches have focused on forming rac-1a through a spon-taneous Pictet–Spengler reaction (typically phosphate cata-lysis).16 A number of in vivo metabolic cascades forming†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4gc02325kaDepartment of Biochemical Engineering, University College London, Bernard KatzBuilding, Gordon Street, London, WC1H 0AH, UK. E-mail: j.ward@ucl.ac.ukbDepartment of Chemistry, University College London, Christopher-Ingold Building,20 Gordon Street, London, WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk852 | Green Chem., 2015, 17, 852–855 This journal is © The Royal Society of Chemistry 2015Open Access Article. Published on 18 December 2014. Downloaded on 1/3/2019 12:32:28 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issue(S)-reticuline report the use of recombinant NCS as the Pictet–Spengler catalyst. However, it is not clear that NCS is active inthese systems: any chirality reported seems to be the result of(S)-selective enzymes downstream of NCS.15a–dHere, we employ a TAm for the conversion of 2a to 3a (step1), which then combine to form 1a (step 2). TAms have anadvantage over MAOs in this system: the conversion of 2a to 3awith TAms can be controlled by stoichiometrically limiting thequantity of co-substrate (in this instance, pyruvate) available.Furthermore, we avoid the problems associated with drivingTAm reactions to completion, as the Pictet–Spengler reactionremoves equal quantities of molecules from both sides of theTAm equilibrium. This three-sided ‘triangular’ cascade designresults in a system with high atom economy.3Results and discussionThe first step towards establishing the one-pot cascade systemwas the identification of a TAm with good activity towards 2a.The screening reaction was conducted in phosphate buﬀer,which enabled the in situ formation of rac-1a from 2a and 3a(Table 1). The screen identified two TAms with activity towards2a at pH 7.5: CV2025 (Chromobacterium violaceum)21 andPP_3718 (Pseudomonas putida), providing 21% and 15% con-version of 1a from 50 mM 2a respectively. Trace activities werefound in PP_0596 (P. putida), SaV_2612 (Streptomyces avermiti-lis) and VF_JS17 (Vibrio fluvialis)22 (Table 1, ESI† Fig. S1). Weselected the best performing TAm, CV2025, for use in furtherstudies.In order to form (S)-1a from 2a two aspects of the previousTAm screening reaction conditions were modified. Changingthe buﬀer from phosphate to HEPES removed most of thebackground chemical reaction11 and adding purifiedΔ29TfNCS enabled catalytic formation of the chiral product(S)-1a from 2a. Optimum reaction conditions were determinedby varying the concentrations of 2a, NCS and TAm present. Abalance between reaction components was crucial to ensurethe rates of the two steps were matched; a build-up of 3awould cause an increase in undesirable side-reactions includ-ing the non-enzymatic Pictet–Spengler condensation, whichwould reduce the final enantiomeric excess (ee) of the product.BIA (S)-1a was produced from 2a with very good conversionsand excellent enantioselectivity. In all cases, an increase inTAm concentration resulted in a greater consumption of 2a,Scheme 1 Overview of the biocatalytic and non-enzymatic cascadespresented in this work, including the ‘triangular’ cascade.Table 1 TAm mediated synthesis of rac-1a from 2aaEntry TAm Organism Uniprot entry name Gene name Conv.b (%)1 Empty vector n.d.c2 BSU_09260 Bacillus subtilis YHXA_BACSU yhxA n.d.3 CV_202521 Chromobacterium violaceum Q7NWG4_CHRVO CV_2025 214 Dgeo_1416 Deinococcus geothermalis Q1IYH3_DEIGD argD/lysJ n.d.5 KPN_00255 Klebsiella pneumoniae A6T537_KLEP7 gabT n.d.6 PP_0596 Pseudomonas putida Q88Q98_PSEPK PP_0596 Trace7 PP_3718 Pseudomonas putida Q88GK3_PSEPK PP_3718 148 SaV_2612 Streptomyces avermitilis Q82JZ2_STRAW SAV_2612 Trace9 SaV_4551 Streptomyces avermitilis Q82ER2_STRAW SaV_4551 n.d.10 VF_JS1722 Vibrio fluvialis F2XBU9_VIBFL JS17 Tracea 50 mM 2a, 25 mM pyruvate, 1 mM PLP, 10% v.v−1 TAm, 37 °C, 4 h. b Concentration of rac-1a, determined by analytical HPLC. c n.d. = notdetected. See ESI Fig. S1 for reaction time course.Green Chemistry CommunicationThis journal is © The Royal Society of Chemistry 2015 Green Chem., 2015, 17, 852–855 | 853Open Access Article. Published on 18 December 2014. Downloaded on 1/3/2019 12:32:28 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinegenerally leading to an increase in product formation(Table 2). However, at lower concentrations of 2a (20 mM) theuse of 20% v.v−1 CV2025 lysate, rather than 30%, was optimal.The enantioselectivity was excellent under all conditions. Interms of conversion, the eﬀect of NCS concentration was moreapparent with higher concentrations of dopamine. The bestconditions identified were those with 20 mM 2a, 500 µg mL−1NCS and 20% v.v−1 CV2025 lysate, which provided an 87% con-version of (S)-1a from 2a with an ee of 99%.To demonstrate the versatility of this system, it was used tosynthesise (S)-1b from 2-(3-hydroxyphenyl)ethylamine 2b. Thepara-hydroxyl group of 2a is not required for the Pictet–Spengler condensation, and thus 2b is a suitable substrate forNCS.12b Using the optimum conditions determined previouslyfor 2a, (S)-1b was formed from 2b with a fair conversion of56% and a high ee of 90%. The lower conversion and ee com-pared to (S)-1a is possibly reflective of a poorer aﬃnity ofTfNCS towards for 2b compared to the natural substrate 2a.In order to form THPBs (S)-4 and (S)-5, a one-pot synthesisof (S)-1a was conducted (as described above) and formal-dehyde was added to the reaction after 3 hours. This triggeredthe second Pictet–Spengler condensation and resulted in theformation of (S)-4 and (S)-5 in a ratio of approximately 7 : 1(Scheme 2). This cascade features 2 enzymes, 3 steps and theformation of 4 bonds, including 2 C–C bonds. The overall reac-tion occurred with good conversion: the second Pictet–Spen-gler step alone (from (S)-1a) provided 74% conversion (64%and 9% for (S)-4 and (S)-5 respectively), which translates as anoverall 64% conversion (56% and 8%) from 2a. As the chiralityof the system is established by NCS, the subsequent additionof formaldehyde does not aﬀect the ee: only the (S)-enantiomerof the major product 4 was observed by chiral HPLC.The major regioisomer 4 formed by this method has 10,11-dihydroxy regiochemistry. BBE, a plant enzyme which catalysesthe formation of (S)-THPBs en route to berberine, typicallyforms products with 9,10-dihydroxy regioselectivity.20 Thisenzyme has recently been used in a novel in vitro deracemisa-tion cascade to produce (S)-THPBs.20b The synthesis presentedhere and the BBE catalysed cascade can be seen as comp-lementary methods, providing eﬃcient chemoenzymaticroutes to 10,11-dihydroxy-(S)-THPBs and 9,10-dihydroxy-(S)-THPBs respectively.Finally, to demonstrate the preparative potential of our cas-cades, we performed syntheses of (S)-1a and (S)-4 on a0.5 mmol scale. The concentrations of components in thesecascades were the same as those used in the micro-scale cas-cades (Scheme 2). The synthesis scaled with no complications:conversion of 2a to (S)-1a was achieved in 2 hours with 86%conversion and 62% isolated yield (>95% ee). (S)-4 was formedfrom 2a in 2.5 hours with 47% conversion and 42% isolatedyield (>95% ee). The identities of these products were verifiedby NMR.ConclusionsIn summary, we have developed one-pot syntheses of chiralalkaloids, using the enzymes TAm and NCS in a ‘triangular’cascade. BIA (S)-1a was formed from 2a with very good conver-sion and excellent enantioselectivity in only 2 hours. (S)-1bwas formed in a similar manner with fair conversion and ahigh ee. A chemical extension to the cascade enabled the for-mation of (S)-4 in a one-pot three-step reaction from the low-cost starting material 2a, again with excellent enantio-selectivity and an overall reaction time of less than 3 hours.The cascades presented here exhibit high atom economy,the in situ generation of reactive intermediates, and the rapidaccumulation of molecular complexity through C–C bondforming steps. Overall, these syntheses demonstrate theTable 2 One pot synthesis of (S)-BIAsaAmine[mM]CV2025(v.v−1) [%]NCS[µg mL−1]Conv.b[%]eec[%]2a 20 10 100 74 (74) 992a 20 20 100 86 (89) 992a 20 30 100 82 (93) 992a 20 10 500 77 (82) 992a 20 20 500 87 (92) 992a 20 30 500 84 (95) 982a 50 10 100 36 (42) 982a 50 20 100 55 (66) 972a 50 30 100 70 (78) 992a 50 10 500 42 (48) 982a 50 20 500 65 (73) 982a 50 30 500 72 (85) 962b 20 20 500 56 (57) 90aGeneral reaction conditions: 2 equivalents 2a or 2b, 1 equivalentpyruvate, purified Δ29TfNCS and CV2025 lysate, 50 mM HEPES pH7.5, 37 °C, 3 h. bDetermined by analytical HPLC. Conversions inbrackets refer to depletion of primary amine. cDetermined by chiralHPLC of the crude product.Scheme 2 One-pot chemoenzymatic synthesis of (S)-4 and (S)-5.Reaction conditions: (a) 20 mM 2, 10 mM sodium pyruvate, 500 µg mL−1NCS and 20% v.v−1 CV2025 lysate, 50 mM HEPES pH 7.5, 37 °C, 3 h. (b)40 mM formaldehyde, 1 M sodium phosphate, pH 6, 30 min, 37 °C.Communication Green Chemistry854 | Green Chem., 2015, 17, 852–855 This journal is © The Royal Society of Chemistry 2015Open Access Article. Published on 18 December 2014. Downloaded on 1/3/2019 12:32:28 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlineremarkable potential of in vitro biocatalysis for the formationof complex chiral compounds.We gratefully acknowledge the Wellcome Trust for student-ship funding to B. R. L., EPSRC and GlaxoSmithKline for stu-dentship funding to E. D. L., and the Biotechnology andBiological Sciences Research Council (BBSRC) (BB/G014426/1)for funding T. P.Notes and references1 (a) J. H. Clark, Green Chem., 1999, 1, 1–8; (b) R. A. Sheldon,Pure Appl. Chem., 2000, 72, 1233–1246.2 B. M. Nestl, S. C. Hammer, B. A. Nebel and B. Hauer,Angew. Chem., Int. Ed., 2014, 53, 3070–3095.3 For reviews see: (a) E. Ricca, B. Brucher andJ. H. 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One-pot triangular chemoenzymatic cascades for the syntheses of chiral alkaloids from dopamine

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