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Synthesis and enzymatic studies of bisubstrate analogues for farnesyl diphosphate synthase

Ramamoorthy G, Pugh ML, Tian B, Phan RM, Perez LB, Jacobson MP, Poulter CD (2015) J Org Chem, 80, 3902-3913. PMID: 25734506

The production and use of substrate analogues sheds light on the functional capacity of a farnesyl diphosphate synthase being investigated by the Isoprenoid Synthase Bridging Project. The Computation Core is then integral at providing a molecular-level view of how these analogues are positioned within the synthase active site - and enlightening the group on the functional import of structural components of this IS family member.


Farnesyl diphosphate synthase catalyzes the sequential chain elongation reactions between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP) and between IPP and GPP to give farnesyl diphosphate (FPP). Bisubstrate analogues containing the allylic and homoallylic substrates were synthesized by joining fragments for IPP and the allylic diphosphates with a C-C bond between the methyl group at C3 in IPP and the Z-methyl group at C3 in DMAPP (3-OPP) and GPP (4-OPP), respectively. These constructs placed substantial limits on the conformational space available to the analogues relative to the two substrates. The key features of the synthesis of bisubstrate analogues 3-OPP and 4-OPP are a regioselective C-alkylation of the dianion of 3-methyl-3-buten-1-ol (5), a Z-selective cuprate addition of alkyl groups to an α,β-alkynyl ester intermediate, and differential activation of allylic and homoallylic alcohols in the analogues, followed by a simultaneous displacement of the leaving groups with tris(tetra-n-butylammonium) hydrogen diphosphate to give the corresponding bisdiphosphate analogues. The bisubstrate analogues were substrates for FPP synthase, giving novel seven-membered ring analogues of GPP and FPP. The catalytic efficiencies for cyclization of 3-OPP and 4-OPP were similar to those for chain elongation with IPP and DMAPP.

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Figure Abstract Image

Figure Scheme 1. Mechanism for Chain Elongation by FPP Synthase

FigureFigure 1. Structures of the bisubstrate analogues.

Figure Scheme 2. Retrosynthetic Analysis of the Bisubstrate Analogues

Figure Scheme 3. Synthesis of the a/b-Unsaturated Esters 11-OTHP and 12-OTHP

Figure Scheme 4. Synthesis of Bisdiphosphates 3-OPP and 4-OPP.

Figure Figure 2. 31P and 1H NMR spectra of 3-OPP and 4-OPP.

Figure Figure 3. 31P NMR spectra: (a) FPPase-catalyzed reaction of 4-OPP after overnight incubation; (b) incubation with alkaline phosphatase for 12 h.

Figure Scheme 5. FPP Synthase-Catalyzed Reactions of 3-OPP and 4-OPP

Figure Figure 4. Important COSY, HMBC, NOESY correlations for 17-OH.

Figure Figure 5. Important COSY, HMBC, and NOESY correlations for 18-OH.

Figure Figure 6. Mechanism for formation of 17-OPP and 18-OPP.

Figure Figure 7. Conserved aspartate residues in the X-ray structure of E. coli FPPase-IPP-thioDMAPP (a) and homology modeled/ docked structures for avian FPPase-3-OPP (b) and avian FPPase-4-OPP.

Reprinted with permission from Ramamoorthy et al. JACS. Copyright © 2015 American Chemical Society.