On the Role of Hydrogen Migrations in the Taxadiene System

Abstract

Taxa-4,11-diene is made by the taxa-4,11-diene synthase (TxS) from Taxus brevifolia. The unique reactivity of the taxane system is characterised by long distance hydrogen migrations in the biosynthesis. This study demonstrates that selective long range hydrogen migrations also play a role in the high energy process of the EI-MS fragmentation of taxa-4,11-diene. A TxS enzyme variant was generated that produces cyclophomactene, a compound that is formed through a concerted process involving a long range proton shift and a ring closure that can also be described as the addition of a methylcarbinyl cation to an olefin. Based on a previous computational study the cyclisation mechanism towards taxa-4,11-diene was suggested to involve two long distance proton migrations instead of one direct transfer. A substrate analog with a shifted double bond was converted with TxS to obtain experimental evidence for this proposal.

Keywords: biosynthesis; enzymes; natural products; substrate analogs; terpenoids.

Scheme 1

Taxa‐4,11‐diene biosynthesis. A) Mechanism for the cyclisation of GGPP to 1 and biosynthetically related products by TxS and its enzyme variants. For isolated products the source enzymes are given in boxes. The numbers in boxes are computed energies (in kcal/mol) of intermediates relative to A (set to 0.0 kcal/mol, blue), reaction barriers (Gibbs free energies of activation at 298.15 K, black) and Gibbs free reaction energies (red) relative to each preceeding intermediate. All structures were computed using the mPW1PW91/6‐311+G(d,p)//B97D3/6‐31 G(d,p) method (298 K). Asterisks indicate conformational changes needed between the product of one transformation and the starting structure of the next step. All carbon numbers follow GGPP numbering, which is different to the accepted carbon numbering of taxanes.

Scheme 2

Products obtained with TxS from GGPP derivatives.

Scheme 3

EIMS fragmentation mechanism for the base peak ion m/z 122 of 1. Blue dots indicate carbons for which a substitution with ¹³C leads to an increase of the base peak ion to m/z 123. The numbers in boxes are computed energies (in kcal/mol) of intermediates relative to A (set to 0.0 kcal/mol, blue), reaction barriers (Gibbs free energies of activation at 298.15 K, black) and Gibbs free reaction energies (red) relative to each preceeding intermediate. All structures were computed using the mPW1PW91/6‐311+G(d,p)//B97D3/6‐31 G(d,p) method (298 K).

Figure 1

Site‐directed mutagenesis of TxS. A) Active site residues targeted by site‐directed mutagenesis in this study (based on the X‐ray structure of TxS, PDB 3P5R). Enzyme variants shown in green were catalytically active, those shown in red were inactive. B) Relative production of enzyme variants. The sum of compounds produced by the wild‐type (WT) was set to 100 %. Bars represent means from triplicates. For standard deviations cf. Table S8.

Scheme 4

Computed kinetic isotope effects (ratio of rate constants k _H/k _D) for long distance hydrogen migrations in the biosynthesis of 1 and 8, determined with H/D exchange of only the migrating hydrogen. Kinetic isotope effects for A) the unusual cyclisation reaction in the biosynthesis of 8 and B–D) the 1,5‐proton shifts in the biosynthesis of 1.

Scheme 5

Mechanism for the cyclisation of iso‐GGPP I to 12 by TxS. The numbers in boxes are computed energies (in kcal/mol) of intermediates relative to A* (set to 0.0 kcal/mol, blue), reaction barriers (Gibbs free energies of activation at 298.15 K, black) and Gibbs free reaction energies (red) relative to each preceeding intermediate. All structures were computed using the mPW1PW91/6‐311+G(d,p)//B97D3/6‐31 G(d,p) method (298 K). Asterisks indicate conformational changes needed between the product of one transformation and the starting structure of the next step. All carbon numbers follow GGPP numbering (Scheme 1), which is different to the accepted carbon numbering of taxanes.