Asymmetric Synthesis of Chiral 2-Cyclohexenones with Quaternary Stereocenters via Ene-Reductase Catalyzed Desymmetrization of 2,5-Cyclohexadienones.

Stereoselective synthesis of quaternary stereocenters represents a significant challenge in organic chemistry. Herein, we describe the use of ene-reductases OPR3 and YqjM for the efficient asymmetric synthesis of chiral 4,4-disubstituted 2-cyclohexenones via desymmetrizing hydrogenation of prochiral 4,4-disubstituted 2,5-cyclohexadienones. This transformation breaks the symmetry of the cyclohexadienone substrates, generating valuable quaternary stereocenters with high enantioselectivities (ee, up to >99%). The mechanistic causes for the observed high enantioselectivities were investigated both experimentally (stopped-flow kinetics) as well as theoretically (quantum mechanics/molecular mechanics calculations). The synthetic potential of the resulting chiral enones was demonstrated in several diversification reactions in which the stereochemical integrity of the quaternary stereocenter could be preserved.

Understanding the riddle of amine oxidase flavoenzyme reactivity on the stereoisomers of N-methyl-dopa and N-methyl-tyrosine.

Enzymes are usually stereospecific against chiral substrates, which is commonly accepted for the amine oxidase family of enzymes as well. However, the FsqB (fumisoquin biosynthesis gene B) enzyme that belongs to the family of sarcosine oxidase and oxidizes L-N-methyl-amino acids, shows surprising activity for both enantiomers of N-methyl-dopa. The aim of this study is to understand the mechanism behind this behavior. Primary docking experiments showed that tyrosine and aspartate residues (121 and 315 respectively) are located on the ceiling of the active site of FsqB and may play a role in fixing the N-methyl-dopa via its catechol moiety and allowing both stereoisomers of this substrate to be in close proximity of the N5 atom of the isoalloxazine ring of the cofactor. Three experimental approaches were used to prove this hypothesis which are: (1) studying the oxidative ability of the variants Y121F and D315A on N-methyl-dopa substrates in comparison with N-methyl-tyrosine substrates; (2) studying the FsqB WT and variants catalyzed biotransformation via high-performance liquid chromatography (HPLC); (3) molecular dynamics simulations to characterize the underlying mechanisms of the molecular recognition. First, we found that the chemical characteristics of the catechol moiety of N-methyl-dopa are important to explain the differences between N-methyl-dopa and N-methyl-tyrosine. Furthermore, we found that Y121 and D315 are specific in FsqB and not found in the model enzyme sarcosine oxidase. The on-bench and theoretical mutagenesis studies show that Y121 residue has a major role in fixing the N-methyl-dopa substrates close to the N5 atom of the isoalloxazine ring of the cofactor. Simultaneously, D315 has a supportive role in this mechanism. Jointly, the experimental and theoretical approaches help to solve the riddle of FsqB amine oxidase substrate specificity.

Simple, fast and inexpensive quantification of glycolate in the urine of patients with primary hyperoxaluria type 1.

In primary hyperoxaluria type 1 excessive endogenous production of oxalate and glycolate leads to increased urinary excretion of these metabolites. Although genetic testing is the most definitive and preferred diagnostic method, quantification of these metabolites is important for the diagnosis and evaluation of potential therapeutic interventions. Current metabolite quantification methods use laborious, technically highly complex and expensive liquid, gas or ion chromatography tandem mass spectrometry, which are available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with glyoxylate generated from glycolate using recombinant mouse glycolate oxidase (GO) and glycine leads to the formation of a stable dihydroquinazoline double aromatic ring chromophore with specific peak absorption at 440 nm. The urinary limit of detection and estimated limit of quantification derived from eight standard curves were 14.3 and 28.7 µmol glycolate per mmol creatinine, respectively. High concentrations of oxalate, lactate and L-glycerate do not interfere in this assay format. The correlation coefficient between the absorption and an ion chromatography tandem mass spectrometry method is 93% with a p value < 0.00001. The Bland-Altmann plot indicates acceptable agreement between the two methods. The glycolate quantification method using conversion of glycolate via recombinant mouse GO and fusion of oABA and glycine with glyoxylate is fast, simple, robust and inexpensive. Furthermore this method might be readily implemented into routine clinical diagnostic laboratories for glycolate measurements in primary hyperoxaluria type 1.

The scope of flavin-dependent reactions and processes in the model plant Arabidopsis thaliana.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized as coenzymes in many biochemical reduction-oxidation reactions owing to the ability of the tricyclic isoalloxazine ring system to employ the oxidized, radical and reduced state. We have analyzed the genome of Arabidopsis thaliana to establish an inventory of genes encoding flavin-dependent enzymes (flavoenzymes) as a basis to explore the range of flavin-dependent biochemical reactions that occur in this model plant. Expectedly, flavoenzymes catalyze many pivotal reactions in primary catabolism, which are connected to the degradation of basic metabolites, such as fatty and amino acids as well as carbohydrates and purines. On the other hand, flavoenzymes play diverse roles in anabolic reactions most notably the biosynthesis of amino acids as well as the biosynthesis of pyrimidines and sterols. Importantly, the role of flavoenzymes goes much beyond these basic reactions and extends into pathways that are equally crucial for plant life, for example the production of natural products. In this context, we outline the participation of flavoenzymes in the biosynthesis and maintenance of cofactors, coenzymes and accessory plant pigments (e. g. carotenoids) as well as phytohormones. Moreover, several multigene families have emerged as important components of plant immunity, for example the family of berberine bridge enzyme-like enzymes, flavin-dependent monooxygenases and NADPH oxidases. Furthermore, the versatility of flavoenzymes is highlighted by their role in reactions leading to tRNA-modifications, chromatin regulation and cellular redox homeostasis. The favorable photochemical properties of the flavin chromophore are exploited by photoreceptors to govern crucial processes of plant adaptation and development. Finally, a sequence- and structure-based approach was undertaken to gain insight into the catalytic role of uncharacterized flavoenzymes indicating their involvement in unknown biochemical reactions and pathways in A. thaliana.

The family of sarcosine oxidases: Same reaction, different products.

The subfamily of sarcosine oxidase is a set of enzymes within the larger family of amine oxidases. It is ubiquitously distributed among different kingdoms of life. The member enzymes catalyze the oxidization of an N-methyl amine bond of amino acids to yield unstable imine species that undergo subsequent spontaneous non-enzymatic reactions, forming an array of different products. These products range from demethylated simple species to complex alkaloids. The enzymes belonging to the sarcosine oxidase family, namely, monomeric and heterotetrameric sarcosine oxidase, l-pipecolate oxidase, N-methyltryptophan oxidase, NikD, l-proline dehydrogenase, FsqB, fructosamine oxidase and saccharopine oxidase have unique features differentiating them from other amine oxidases. This review highlights the key attributes of the sarcosine oxidase family enzymes, in terms of their substrate binding motif, type of oxidation reaction mediated and FAD regeneration, to define the boundaries of this group and demarcate these enzymes from other amine oxidase families.

The catalytic machinery of the FAD-dependent AtBBE-like protein 15 for alcohol oxidation: Y193 and Y479 form a catalytic base, Q438 and R292 an alkoxide binding site.

Monolignol oxidoreductases are members of the berberine bridge enzyme-like (BBE-like) protein family (pfam 08031) that oxidize monolignols to the corresponding aldehydes. They are FAD-dependent enzymes that exhibit the para-cresolmethylhydroxylase-topology, also known as vanillyl oxidase-topology. Recently, we have reported the structural and biochemical characterization of two monolignol oxidoreductases from Arabidopsis thaliana, AtBBE13 and AtBBE15. Now, we have conducted a comprehensive site directed mutagenesis study for AtBBE15, to expand our understanding of the catalytic mechanism of this enzyme class. Based on the kinetic properties of active site variants and molecular dynamics simulations, we propose a refined, structure-guided reaction mechanism for the family of monolignol oxidoreductases. Here, we propose that this reaction is facilitated stepwise by the deprotonation of the allylic alcohol and a subsequent hydride transfer from the Cα-atom of the alkoxide to the flavin. We describe an excessive hydrogen bond network that enables the catalytic mechanism of the enzyme. Within this network Tyr479 and Tyr193 act concertedly as active catalytic bases to facilitate the proton abstraction. Lys436 is indirectly involved in the deprotonation as this residue determines the position of Tyr193 via a cation-π interaction. The enzyme forms a hydrophilic cavity to accommodate the alkoxide intermediate and to stabilize the transition state from the alkoxide to the aldehyde. By means of molecular dynamics simulations, we have identified two different and distinct binding modes for the substrate in the alcohol and alkoxide state. The alcohol interacts with Tyr193 and Tyr479 while Arg292, Gln438 and Tyr193 form an alkoxide binding site to accommodate this intermediate. The pH-dependency of the activity of the active site variants revealed that the integrity of the alkoxide binding site is also crucial for the fine tuning of the pKa of Tyr193 and Tyr479. Sequence alignments showed that key residues for the mechanism are highly conserved, indicating that our proposed mechanism is not only relevant for AtBBE15 but for the majority of BBE-like proteins.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h-1 L-1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition.

Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or "slow" substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors.

Enantioselective Oxidative Aerobic Dealkylation of N-Ethyl Benzylisoquinolines by Employing the Berberine Bridge Enzyme.

N-Dealkylation methods are well described for organic chemistry and the reaction is known in nature and drug metabolism; however, to our knowledge, enantioselective N-dealkylation has not been yet reported. In this study, exclusively the (S)-enantiomers of racemic N-ethyl tertiary amines (1-benzyl-N-ethyl-1,2,3,4-tetrahydroisoquinolines) were dealkylated to give the corresponding secondary (S)-amines in an enantioselective fashion at the expense of molecular oxygen. The reaction is catalyzed by the berberine bridge enzyme, which is known for CC bond formation. The dealkylation was demonstrated on a 100 mg scale and gave optically pure dealkylated products (ee>99 %).

Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism.

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family.

Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites.

Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

UNLABELLED: The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. DATABASE: Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF.

Deracemization by simultaneous bio-oxidative kinetic resolution and stereoinversion.

Deracemization, that is, the transformation of a racemate into a single product enantiomer with theoretically 100% conversion and 100% ee, is an appealing but also challenging option for asymmetric synthesis. Herein a novel chemo-enzymatic deracemization concept by a cascade is described: the pathway involves two enantioselective oxidation steps and one non-stereoselective reduction step, enabling stereoinversion and a simultaneous kinetic resolution. The concept was exemplified for the transformation of rac-benzylisoquinolines to optically pure (S)-berbines. The racemic substrates were transformed to optically pure products (ee>97%) with up to 98% conversion and up to 88% yield of isolated product.

The structure of glycerol trinitrate reductase NerA from Agrobacterium radiobacter reveals the molecular reason for nitro- and ene-reductase activity in OYE homologues.

In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated C=C bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20 %, but they exhibit significant structural similarities with the adoption of a conserved (αß)(8)-barrel fold. Some OYEs have been shown not only to reduce C=C double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X-ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4-hydroxybenzaldehyde and with 1-nitro-2-phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro-containing substrates can bind to OYEs in different binding modes, one of which leads to C=C double bond reduction and the other to nitro group reduction.

Unusual C=C bond isomerization of an α,ß-unsaturated γ-butyrolactone catalysed by flavoproteins from the old yellow enzyme family.

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (k(cat) =0.076 s(-1) ) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cß, followed by hydride abstraction from endo-Cß', which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.

Inverting the regioselectivity of the berberine bridge enzyme by employing customized fluorine-containing substrates.

Fluorine is commonly applied in pharmaceuticals to block the degradation of bioactive compounds at a specific site of the molecule. Blocking of the reaction center of the enzyme-catalyzed ring closure of 1,2,3,4-tetrahydrobenzylisoquinolines by a fluoro moiety allowed redirecting the berberine bridge enzyme (BBE)-catalyzed transformation of these compounds to give the formation of an alternative regioisomeric product namely 11-hydroxy-functionalized tetrahydroprotoberberines instead of the commonly formed 9-hydroxy-functionalized products. Alternative strategies to change the regioselectivity of the enzyme, such as protein engineering, were not applicable in this special case due to missing substrate-enzyme interactions. Medium engineering, as another possible strategy, had clear influence on the regioselectivity of the reaction pathway, but did not lead to perfect selectivity. Thus, only substrate tuning by introducing a fluoro moiety at one potential reactive carbon center switched the reaction to the formation of exclusively one regioisomer with perfect enantioselectivity.

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme.

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2=O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.

Biocatalytic organic synthesis of optically pure (S)-scoulerine and berbine and benzylisoquinoline alkaloids.

A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C-C bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4-8 linear steps using either a Bischler-Napieralski cyclization or a C1-Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5-9 linear steps.