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James M Cook - One of the best experts on this subject based on the ideXlab platform.

Joachim Stöckigt - One of the best experts on this subject based on the ideXlab platform.

  • Acetyltransfer in natural product biosynthesis--functional cloning and molecular analysis of vinorine synthase.
    Bioorganic & medicinal chemistry, 2004
    Co-Authors: Anja Bayer, Joachim Stöckigt
    Abstract:

    Abstract Vinorine synthase (EC 2.3.1.160) catalyses the acetyl-CoA- or CoA-dependent reversible formation of the alkaloids vinorine (or 11-methoxy-vinorine) and 16-epi-Vellosimine (or gardneral). The forward reaction leads to vinorine, which is a direct biosynthetic precursor along the complex pathway to the monoterpenoid indole alkaloid ajmaline, an antiarrhythmic drug from the Indian medicinal plant Rauvolfia serpentina. Based on partial peptide sequences a cDNA clone was isolated and functionally expressed in Escherichia coli. The Km values of the native enzyme for gardneral and acetyl-CoA were determined to be 7.5 and 57 μM. The amino acid sequence of vinorine synthase has highest level of identity (28–31%) to that of Papaver salutaridinol acetyltransferase, Fragaria alcohol acyltransferase, and Catharanthus deacetylvindoline acetyltransferase involved in morphine, flavor, and vindoline biosynthesis, respectively. Vinorine synthase is a novel member of the BAHD superfamily of acyltransferases. Site-directed mutagenesis of 13 amino acid residues provided clear evidence that both, His160 and Asp164 of the consensus sequence HxxxD belong to the catalytic center. The mutations also showed that an amino acid triad is not characteristic of vinorine synthase. The experiments demonstrated the importance of the conserved motif SxL/I/VD near the N-terminus and the consensus sequence DFGWG near the C-terminal.

  • In vivo monitoring of alkaloid metabolism in hybrid plant cell cultures by 2D cryo-NMR without labelling.
    Bioorganic & medicinal chemistry, 2003
    Co-Authors: Christiane Hinse, Christian Richter, Alessandro Provenzani, Joachim Stöckigt
    Abstract:

    Abstract Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne were carried out. When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13C. Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800). Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe™ that the alkaloid enters two metabolic routes. Vinorine is intracellularly transformed on route I through Vellosimine and 10-deoxysarpagine into sarpagine. On route II, the alkaloid is converted by hydroxylation through vomilenine into the glucoside raucaffricine. Intracellular alkaloid concentrations of ∼500 μM are measurable in vivo with cryogenic NMR technology.

  • Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis
    European journal of biochemistry, 2002
    Co-Authors: Emine Mattern-dogru, Joachim Hartmann, Heinz Decker, Joachim Stöckigt
    Abstract:

    In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-Vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the α/β hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the α/β hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the α/β hydrolase enzyme superfamily.

  • The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of theα/β hydrolase super family
    European journal of biochemistry, 2000
    Co-Authors: Emine Dogru, Heribert Warzecha, Frank Seibel, Sophie Haebel, Friedrich Lottspeich, Joachim Stöckigt
    Abstract:

    The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-Vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.

Wenyuan Yin - One of the best experts on this subject based on the ideXlab platform.

Tao Wang - One of the best experts on this subject based on the ideXlab platform.

  • General Approach for the Synthesis of Sarpagine Indole Alkaloids. Enantiospecific Total Synthesis of (+)-Vellosimine, (+)-Normacusine B, (−)-Alkaloid Q3, (−)-Panarine, (+)-Na-MethylVellosimine, and (+)-Na-Methyl-16-epipericyclivine
    The Journal of organic chemistry, 2003
    Co-Authors: Tao Wang, Xiaoxiang Liu, Jeffrey R. Deschamps, Judith L. Flippen-anderson, Xuebin Liao, James M Cook
    Abstract:

    The first total synthesis of (+)-Na-methyl-16-epipericyclivine (9) was completed [from d-(+)-tryptophan methyl ester] in an overall yield of 42% (eight reaction vessels). The optical rotation [[α]D +22.8 (c 0.50, CHCl3)] obtained on this material confirmed that the reported optical rotation [[α]D 0 (c 0.50, CHCl3)]47 was biogenetically unreasonable. The total syntheses of (+)-Vellosimine, (+)-normacusine B, (−)-alkaloid Q3, (−)-panarine, and (+)-Na-methylVellosimine are also described. Moreover, a mixed sample (1:1) of synthetic (−)-panarine and natural (−)-panarine yielded only one set of signals in the 13C NMR; this indicated that the two compounds are identical and further confirmed the correct configuration of (+)-Vellosimine, (+)-normacusine B, and (−)-alkaloid Q3. In this approach, the key templates, (−)-Na-H,Nb-benzyltetracyclic ketone 15a and (−)-Na-methyl,Nb-benzyltetracyclic ketone 43 were synthesized on multihundred gram scale by the asymmetric Pictet−Spengler reaction and a stereocontrolled Di...

  • general approach for the synthesis of sarpagine indole alkaloids enantiospecific total synthesis of Vellosimine normacusine b alkaloid q3 panarine na methylVellosimine and na methyl 16 epipericyclivine
    Journal of Organic Chemistry, 2003
    Co-Authors: Tao Wang, Xiaoxiang Liu, Jeffrey R. Deschamps, Xuebin Liao, Judith L Flippenanderson, James M Cook
    Abstract:

    The first total synthesis of (+)-Na-methyl-16-epipericyclivine (9) was completed [from d-(+)-tryptophan methyl ester] in an overall yield of 42% (eight reaction vessels). The optical rotation [[α]D +22.8 (c 0.50, CHCl3)] obtained on this material confirmed that the reported optical rotation [[α]D 0 (c 0.50, CHCl3)]47 was biogenetically unreasonable. The total syntheses of (+)-Vellosimine, (+)-normacusine B, (−)-alkaloid Q3, (−)-panarine, and (+)-Na-methylVellosimine are also described. Moreover, a mixed sample (1:1) of synthetic (−)-panarine and natural (−)-panarine yielded only one set of signals in the 13C NMR; this indicated that the two compounds are identical and further confirmed the correct configuration of (+)-Vellosimine, (+)-normacusine B, and (−)-alkaloid Q3. In this approach, the key templates, (−)-Na-H,Nb-benzyltetracyclic ketone 15a and (−)-Na-methyl,Nb-benzyltetracyclic ketone 43 were synthesized on multihundred gram scale by the asymmetric Pictet−Spengler reaction and a stereocontrolled Di...

Tanja Gaich - One of the best experts on this subject based on the ideXlab platform.

  • Total Syntheses of Vellosimine,N-MethylVellosimine, and 10-MethoxyVellosimine and Formal Synthesis of 16-Epinormacusine B through a [5+2] Cycloaddition
    European Journal of Organic Chemistry, 2016
    Co-Authors: Sebastian Krüger, Tanja Gaich
    Abstract:

    To date, more than 100 members of the sarpagine alkaloid family have been isolated. Their structural variations originate from oxidative transformations of the carboskeleton and the presence of both absolute configurations at the C-16 atom, which is established in the course of their biosynthesis. More than 40 sarpagine alkaloids belong to the either the 16-regular or 16-epi subgroups, depending on the stereochemistry at C-16. Herein, we report the formal synthesis of 16-epinormacusine B, a member of the 16-epi group, by using our well-established generalized strategy for the total synthesis of these alkaloids. Furthermore, we provide the synthetic details and pitfalls of the asymmetric total syntheses of Vellosimine, N-methylVellosimine, and 10-methoxyVellosimine, all members of the 16-regular group.

  • Enantioselective, Protecting‐Group‐Free Total Synthesis of Sarpagine Alkaloids—A Generalized Approach
    Angewandte Chemie (International ed. in English), 2014
    Co-Authors: M. Sc. Sebastian Krüger, Tanja Gaich
    Abstract:

    A generalized synthetic access to sarpagine alkaloids through a joint synthetic sequence has been accomplished. Its applicability is showcased by the enantioselective total syntheses of Vellosimine (1), N-methylVellosimine (3), and 10-methoxyVellosimine (8). The synthetic sequence is concise (eight steps) from known compound 13, and requires no protecting groups. The indole heterocycle was introduced in the last step. This strategy allows access to sarpagine alkaloids through a shared synthetic route leading to precursor 10, which we term “privileged intermediate”. Starting from this intermediate, all sarpagine alkaloids can be synthesized using phenylhydrazines with different substitution patterns (15–17). Our approach brings about the advantage, that synthesis optimization only needs to be performed once for many natural products. The key features of the synthesis are a [5+2]-cycloaddition and a ring enlargement.