Awadhesh N. Singh

Biosynthesis of isotetrandrine

Abstract The incorporation of (±)-coclaurine, (±)-N-methylcoclaurine, didehydro-N-methylcoclaurinium iodide, (+)-( S )-N-methylcoclaurine and (−)-( R )-N-methylcoclaurine into isotetrandrine in Cocculus laurifolius DC has been studied and specific utilization of (±)-, (+)-( S )- and (−)- R -N-methylcoclaurines and didehydro-N-methylcoclaurinium iodide demonstrated. The evidence supports intermolecular oxidative coupline of (+)-( S )- and (−)-( R )-N-methylcoclaurines to form isotetrandrine. Double labelling experiment with (±)-N- [ 14 C] methyl [1 - 3 H] coclaurine demonstrated that the hydrogen atom at the asymmetric centre in N-methylcoclaurine is retained in the bioconversion into isotetrandrine.

Biosynthesis of the bisbenzylisoquinoline alkaloid, tetrandrine

Abstract The incorporation of (±)-coclaurine, (±)-norcoclaurine, (±)- N -methylcoclaurine and didehydro- N -methyleoclaurinium iodide into tetrandrine in Cocculus laurifolius has been studied and specific utilization of (±)- N -ethylcoclaurine demonstrated. The evidence indicates that tetrandrine is formed in the plants by oxidative dimerization of N -methylcoclaurine. Double labelling experiment with (±)- N - [ 14 C]-methyl- [1- 3 H]-coclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into tetrandrine. Parallel feedings of (+)-( S )- and (−)-( R )- N -methylcoclaurines showed that the stereospecificity is maintained in the biosynthesis of tetrandrine from the 1-benzylisoquinoline precursor.

The structure, absolute configuration and biosynthesis of nortiliacorinine a

Abstract The incorporation of (±)-norcoclaurine, (±)-coclaurine, (±)-N-methylcoclaurine and dehydro-N-methylcoclaurine into nortiliacorinine A in Tiliacora racemosa colebr has been studied and specific utilisation of the (±)-coclaurine demonstrated. The evidence supports oxidative dimerization of two coclaurine units to give nortiliacorinine A. Experiments with (±)-N-methylcoclaurine and (±)-[1- 3 H, N- 14 CH 3 ]N-methylcoclaurine established that only one N-methylcoclaurine unit is specifically utilised to constitute that “half” of the base which had phenolic OH group in the benzylic portion and further demonstrated that the H atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into nortiliacorinine A. Double labelling experiment with (±)-[1- 3 H, 6,0- 14 CH 3 ]N-methylcoclaurine showed that O-Me function of the precursor is lost in the bioconversion into nortiliacorinine A. Parallel feedings of (+)-( S )- and (-)-( R )-N-methyl-coclaurines and (-)-( S )-, and ( + )-( R )-coclaurines revealed that the stereo-specificity is maintained in the biosynthesis of nortiliacorinine A from 1-benzylisoquinoline precursors and established ‘ S , S ’-configuration at the two asymmetric centres in nortiliacorinine A.

Biosynthesis of cocsulinin

The incorporation of (±)-norcoclaurine, (±)-coclaurine, and (±)-N-methylcoclaurine into cocsulinin in Cocculus laurifolius DC has been studied, and specific utilization of the (±)-N-methylcoclaurine has been demonstrated. The evidence supports oxidative dimerisation of two N-methylcoclaurine units to give cocsulinin. Experiments with (±)-N-[14C]methyl[1-3H]coclaurine have demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into cocsulinin. Parallel feedings of (+)-(S)- and (–)-(R)-N-methylcoclaurines showed that the configuration at C-1 is maintained in the biosynthesis of cocsulinin from the 1-benzylisoquinoline precursor.A double-labelling experiment with (±)-N-methyl[1-3H, 6-O-methyl-14C]coclaurine has shown that the 6-O-methyl group of an N-methylcoclaurine unit is lost in the biotransformation into cocsulinin. Incorporation of (+)-(S,S)-O-methylcocsulinin established that de-O-methylation is the terminal step in the biosynthesis of cocsulinin.

Biosynthesis of oxyacanthine

The incorporation of (±)-norcoclaurine, (±)-coclaurine, (±)-N-methylcoclaurine, didehydro-N-methylco-claurinium iodide, (+)-(S)-N-methylcoclaurine and (–)-(R)-N-methylcoclaurine into oxyacanthine in Cocculus laurifolius DC has been studied and specific utilization of (±)-N-methylcoclaurine, (+)-(S)-N-methylcoclaurine, (–)-(R)-N-methylcoclaurine, and didehydro-N-methylcoclaurinium iodide demonstrated. The evidence supports intermolecular oxidative coupling of (+)-(S)-N-methylcoclaurine and (–)-(R)-N-methylcoclaurine to give oxyacanthine. A double labelling experiment with (±)-[1-3H,N-14CH3]-N-methylcoclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into oxyacanthine.

Biosynthesis of the bisbenzylisoquinoline alkaloid cocsulin

The incorporation of (±)-coclaurine, (±)-norcoclaurine, (±)-N-methylcoclaurine, and didehydro-N-methylcoclaurinium iodide into cocsulin in Cocculus laurifolius DC has been studied, and specific utilization of the (±)-N-methylcoclaurine demonstrated. The evidence supports the occurrence of oxidative dimerization of two N-methylcoclaurine units to give cocsulin. Double labelling experiments with (±)-N-methyl[1-3H, methoxy-14C]coclaurine showed that the O-methyl function from one of the N-methylcoclaurine units is lost in the bioconversion into cocsulin. Experiments with (±)-N-[14C]methyl[1-3H]coclaurine demonstrated that the hydrogen atom at the asymmetric centre in the 1-benzylisoquinoline precursor is retained in the bioconversion into cocsulin. Parallel feedings of (+)-(S)- and (–)-(R)-N-methylcoclaurines showed that the stereospecificity is maintained in the biosynthesis of cocsulin from the 1-benzylisoquinoline precursor.

Biosynthesis of the morphinandienone alkaloid, sebiferine

The incorporation of (±)-nor-reticuline, (±)-reticuline, (±)-nor-orientaline, and (±)-laudanosine into sebiferine (O-methylflavinantine) in Cocculus laurifolius DC has been studied and the specific utilization of reticuline demonstrated. A double-labelling experiment involving the 4′-O-methyl group of (±)-nor-reticuline showed that the methoxy-function is retained in the bioconversion of the precursor into sebiferine and there was considerable loss of tritium at C-1. Parallel experiments with (+)- and (–)-reticulines showed that the stereospecificity is not maintained in the biosynthesis of sebiferine from 1-benzylisoquinoline precursors. Feeding experiments also demonstrated that the plants can also efficiently convert flavinantine into sebiferine.

Biosynthesis of the abnormal Erythrina alkaloids, cocculidine and cocculine

The incorporation of (±)-N-norprotosinomenine, (±)-N-nororientaline, (±)-N-nor-reticuline, (±)-norlaudanosoline, (±)-protosinomenine, and N-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-2-(4-hydroxyphenyl)ethylamine into cocculidine has been studied, and the specific utilization of the (±)-N-norprotosinomenine demonstrated. A double labelling experiment with (±)-[1-3H, 4′-methoxy-14C]-N-norprotosinomenine showed that the 4′-O-methyl group of the precursor is retained in the bioconversion and the erythrinan ring system is not formed by addition of the secondary amino function onto an ortho-quinone system. Parallel experiments with (+)- and (–)-N-norprotosinomenine demonstrated specific incorporation of the (+)-isomer into cocculidine. High incorporation of cocculidine into cocculine revealed that O-demethylation is the terminal step in the biosynthesis of the latter. Feeding experiments also revealed that the plants can convert isococculidine into cocculidine with very high efficiency.

Biosynthesis of reticuline

The incorporation of tyrosine, dopa, dopamine, and 4-hydroxy- and 3,4-dihydroxy-phenylpyruvic acids into reticuline in Litsea glutinosa has been studied, and it has been demonstrated that dopa and dopamine contribute only to the formation of the phenethylamine portion; the benzylic portion is biosynthesized from 3,4-dihydroxyphenylpyruvic acid not derived from dopa. Tyrosine and 4-hydroxy- and 3,4-dihydroxy-phenylpyruvic acids participate in the formation of both ‘halves’ of reticuline. Tracer experiments have shown the intermediacy of norlaudanosoline-1-carboxylic acid, norlaudanosoline, and didehydronorlaudanosoline in the biosynthesis of reticuline. Incorporation studies with (±)-4′-O-methylnorlaudanosoline, (±)-laudanosoline, (±)-6-O-methylnorlaudanosoline, and (±)-nor-reticuline have demonstrated that O-methylation prededes N-methylation and that there is no rigid selectivity of O-methylation in the biosynthesis of reticuline.

Biosynthesis of isococculidine

Tracer experiments prove that the Erythrina alkaloid, isococculidine, is biosynthesised in Cocculus laurifolius from (+)-norprotosinomenine.