Allylic diphosphate

Following this initial SN1 reaction, loss of the pro-R hydrogen gives geranyl diphosphate, itself an allylic diphosphate that dissociates a second time. Reaction of the geranyl carbocation with water in a second S>jl reaction, followed by loss of a proton, then yields geraniol. 

The reactions catalyzed by prenyltransferases are unique and interesting from a mechanistic viewpoint. The reaction starts with elimination of the diphosphate ion from an allylic diphosphate (APP) to form an allylic cation, which is attacked by the IPP molecule, with stereospecific removal of a proton to form a new C-C bond and a new double bond in the product. By repeating this type of condensation between IPP and the allylic prenyl diphosphate product, prenyltransferase can synthesize a prenyl diphosphate with a certain length and stereochemistry fixed by ifs specificify (seeFig. 10) [252]. 

In the 1970s the biosynthesis of cannabinoids was investigated with radiolabeling experiments. 14C-labeled mevalonate and malonate were shown to be incorporated into tetrahydrocannabinolic acid and cannabichromenic acid at very low rates (< 0.02%). Until 1990 the precursors of all terpenoids, isopentenyl diphosphate and dimethyl-allyl diphosphate were believed to be biosynthesized via the mevalonate pathway. Subsequent studies, however, proved that many plant terpenoids are biosynthesized via the recently discovered deoxyxylulose phosphate pathway (Eisenreich et al., 1998 Rohmer, 1999). It was shown that the Cio-terpenoid moiety of cannabinoids is biosynthesized entirely or predominantly (>98%) via this pathway (Fellermeister et al., 2001). The phenolic moiety is generated by a polyketide-type reaction sequence. 

Having now reached a precursor that would enable our final approach to the lipid I target, our first task was to identify a mild method for installation of the chemically sensitive allylic diphosphate linkage. Initially, we considered the methods shown below and illustrated with representative examples [Scheme 8],... 

Figure 10.7 All terpenes are derived from allylic diphosphates which are polymers of repeating isopentyl units (IPP) put together by the action of prenyltransferases. In plants, IPP can be derived from the mevalonate biosynthetic pathway (a cytoplasmic pathway) or the methyl erythritol phosphate pathway (a plastidic pathway). Monoterpenes are then derived from the CIO precursor geranyl diphosphate (GPP), sesquiterpenes from the C15 precursor famesyl diphosphate (FPP), and diterpenes from the C20 precursor geranylgeranyl diphosphate (GGPP) by the action of terpene synthases or cyclases, which divert carbon into the specific branch pathways.

Figure 1.2 Main pathways leading to secondary metabolites. Abbreviations IPP, isopentenyl diphosphate DMAPP, dimethyl allyl diphosphate CAP, glyceraldehyde-3-phosphate NPAAs, non-protein amino acids AcCoA, acetyl coenzyme A. (See Plate 1 in colour plate section.)...

Isopentenyl diphosphate isomerase (IPI E.C. 5.3.3.2) catalyzes the isomerization of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMADP), a previous and mandatory step to create the electrophilic allylic diphosphates needed for the condensation reaction generating geranyl diphosphate, Fig. (6). Thus, IPI is an essential enzyme in organisms which synthesize IPP through the mevalonic acid pathway as archaea, eukaryota and some Gram-positive bacteria [275, 305]. 

Elongation consists of the sequential condensation of IPP and allylic diphosphate through the action of the prenyl chain elongating enzyme, commonly called prenyl transferase. The reactions catalyzed by prenyltransferases start with the formation of allylic cations after the elimination of pyrophosphate ions, to form allylic prenyl diphosphate. This is followed by addition of an IPP with stereo-specific removal of the proton at the 2-position. This is the key reaction which determines cis- or rrans-configuration of the double bonds contained in the linear isoprenoid chain. Cis-trans isomerism is dependent on the nature of the prenyltransferase involved in elongation catalysis. A comprehensive review of cis- and traAW-prenyltransferases was recently undertaken by Takahashi and Koyama. CA-prenyltransferases are much less well known than their trawi-homologues. 

Phase 1 the formation of short-chain allylic diphosphates where geranyl-PP (14), farnesyl-PP (15) and geranylgeranyl-PP (16) are formed by the action of trara-prenyltransferases. 

Phase 2 the traws -short-chain allylic diphosphates obtained are then employed as allylic primer substrates for additional IPP condensation with trans- or cA-configuration. ... 

The synthesis site for allylic diphosphate primers and cw-polyisoprene is largely assumed to occur on the surface of pre-existing rubber particles, but rubber biosynthesis activity has also been localized in the membrane of non-rubber particles from the bottom fraction after ultracentrifugation of latex. " The latter authors " presumed that previous localization of rubber biosynthesis on rubber particles was due to an artefact resulting from the rapid deterioration of bottom fraction (BF) particles after tapping, which led to the migration of rubber synthesis machinery from BF particles to rubber particles. 

Geranyl diphosphate is an allylic diphosphate and, like dimethylallyl diphosphate, can react with isopentenyl diphosphate. A 15-carbon carbocation is formed, which on deprotonation gives farnesyl diphosphate. Hydrolysis of farnesyl diphosphate gives the sesquiterpene farnesol. 

The enzyme-catalyzed reactions that lead to geraniol and farnesol (as their diphosphate esters) are mechanistically related to the acid-catalyzed dimerization of alkenes discussed in Section 6.21. The reaction of an ally lie diphosphate or a carbocation with a source of 7T electrons is a recurring theme in terpene biosynthesis and is invoked to explain the origin of more complicated structural types. Consider, for example, the formation of cyclic monoterpenes. Neryl diphosphate, formed by an enzyme-catalyzed isomerization of the E double bond in geranyl diphosphate, has the proper geometry to form a six-membered ring via intramolecular attack of the double bond on the allylic diphosphate unit. 

The biosynthesis of rubber may be divided into three steps (1) initiation, which requires an allylic diphosphate molecule, (2) elongation, in which IPP units are added to a Z-l,4-polyisoprene chain, and (3) termination, in which the polymer is released from the rubber transferase enzyme (Cornish, 1993). In plants, the elongation of Z-l,4-polyiso-prene (natural rubber) requires a small -allylic diphosphate initiator (less than or equal to C20). Famesyl pyrophosphate (FPP) is an effective initiator of polyisoprene biosynthesis (Light et al, 1989) further, because only one molecule of FPP is needed for each molecule of rubber formed, small traces of this substance that are inadvertently present complicate biosynthetic studies. The E-allylic diphosphates are hydrophilic cytosolic compounds, whereas Z-l,4-polyisoprene is hydrophobic and compartmentalized in subcellular rubber particles. A soluble E-prenyl transferase from the latex of Hevea brasiliensis serves as a famesyl diphosphate synthase and plays no direct role in elongation of Z-l,4-polyisoprene (Cornish, 1993). Because the hydro-phobic rubber molecule is produced inside a rubber particle but is formed from hydrophilic precursors from the cytoplasm, the polymerization reaction must take place at the particle surface. 

The Z-1,4-prenyl transferase is associated with rubber particles in Hevea brasiliensis and other rubber-producing plants. This enzyme is membrane bound (Cornish, 1993). This enzyme requires Mg or Mn ", but will not synthesize mbber unless a second component of the system, an allylic diphosphate, is present (Fig. 18.7). 

Biosynthesis. The initiating species in C. biosynthesis in Hevea brasiliensis is a derivative of trans-tram-famesyl diphosphate (i.e. a C15 allylic diphosphate), which is presumably modified at the methyl carbon of the dimethylallyl group. [Y. Tanaka etal. Initiation of Rubber Biosjmthesis in Hevea brasiliensis Characterization of Initiating Species by Structural Analysis Phytochemistry 41 (19 ) 1501-1505]... 

A central role in the biosynthesis of isoprenoids is filled by the isopentenyl diphosphate-dimethylallyl diphosphate isomerase (IDl) that catalyzes the interconversion of IPP and DMAPP. The necessity for such an enzyme was suggested in the 1950s when only IPP was known as a monomeric isoprenoid precursor, but an allylic diphosphate such as DMAPP was assumed to have the higher intrinsic reactivity for polyisoprenoid synthesis [22, 88, 89]. The first enzymatic isomerization of IPP to DMAPP was observed in 1959 from a cell-free extract of baker s yeast [90, 91]. Two types of IDI with essentially no amino acid sequence or structural similarities are able to catalyze this interconversion by completely different enzyme mechanisms. The well-known IDI-I have been identified in animals, plants, fungi, and bacteria, whereas the IDI-II can be found mainly in archaea but also in some bacteria [92, 93]. 

Upon the synthesis of IPP (C5) and its isomer DMAPP (C5) by either MVA or DXP pathways, the next step is chain elongation. The carbonium ion is a potent alkylating agent that can react with IPP (three molecules of IPP converted into one molecule of geranyl diphosphate (GPP) by condensation reaction catalyzed by enzyme geranyl diphosphate synthase), to yield geranyl diphosphate (GPP). Famesyl pyrophosphate synthase (EPS) is the prenyltransferase that catalyzes l -4 condensation reactions of IPP with the allylic diphosphates, dimethylallyl diphosphate (DMAPP), to produce famesyl pyrophosphate (FPP). 

The rubber biosynthesis was found to take place on the rubber surface of rubber particles by the incorporation of IPP into a terminal allylic diphosphate group of rubber molecules. It was assumed that the growing hydrocarbon chain of rubber diffuses into the rubber particles, while the hydrophilic diphosphate end group remains in the serum phase where it can react with IPP bound to the active site of the rubber transferase, as shown in Figure 2.2. It has been confirmed that the rubber transferase is tightly bound to the Hevea rubber particles even after purification by washing. 

---