Conversion of IPP to Terpenoids

The electrophilic double bond isomerization catalyzed by IPP isomerase is a highly reversible reaction, with an equilibrium IPP:DMAPP ratio of about 6:1. In the next step of isoprenoid biosynthesis, the two five-carbon isomers condense to form a 10-carbon isoprenoid product called geranyl diphosphate (GPP).

This is a nice example of an electrophilic addition/elimination mechanism:

The first step is ionization of the electrophile - in other words, the leaving group departs and a carbocation intermediate is formed. In this case, the pyrophosphate group on DMAPP is the leaving group, and the electrophilic species is the resulting allylic carbocation.

In the condensation (addition) step, the C₃-C₄ double bond in IPP attacks the positively-charged C₁ of DMAPP, resulting in a new carbon-carbon bond and a second carbocation intermediate, this time at a tertiary carbon. In the elimination phase, proton abstraction leads to re-establishment of a double bond in the GPP product. Notice that the enzyme specifically takes the pro-R proton in this step.

To continue the chain elongation process, another IPP molecule can then condense, in a very similar reaction, with C₁ of geranyl diphosphate to form a 15-carbon product called farnesyl diphosphate (FPP).

How do we know that these are indeed S_N1-like mechanisms with carbocation intermediates, rather than concerted S_N2-like mechanisms? First of all, recall that the question of whether a substitution is dissociative (S_N1-like) or associative (S_N2-like) is not always clear-cut - it could be somewhere in between, like the protein prenyltransferase reaction. The protein prenyltransferase reaction and the isoprenoid chain elongation reactions are very similar: the electrophile is the same, but in the former the nucleophile is a thiolate, while in the latter the nucleophile is a pi bond.

This difference in the identity of the nucleophilic species would lead one to predict that the chain elongation reaction has more S_N1-like character than the protein prenylation reaction. A thiolate is a very powerful nucleophile, and thus is able to push the pyrophosphate leaving group off, implying some degree of S_N2 character. The electrons in a pi bond, in contrast, are only weakly nucleophilic, and thus need to be pulled in by a powerful electrophile - ie. a carbocation.

So it makes perfect sense that the chain elongation reaction should more S_N1-like than S_N2-like. Is this in fact the case? We know how to answer this question experimentally - just run the reaction with fluorinated DMAPP or GPP substrates and observe how much the fluorines slow things down.

If the reaction is S_N1-like, the electron-withdrawing fluorines should destabilize the allylic carbocation intermediate and thus slow the reaction down considerably. If the mechanism is S_N2-like, the fluorine substitutions should not have a noticeable effect, because a carbocation intermediate would not be formed. When this experiment was performed with FPP synthase, the results were dramatic: the presence of a single fluorine slowed down the rate of the reaction by a factor of about 60, while two and three fluorines resulted in a reaction that was 500,000 and 3 million times slower, respectively (J. Am. Chem. Soc. 1981, 103, 3926.) These results strongly suggest indicate the formation of a carbocation intermediate in an S_N1-like displacement.

In this section, we will briefly examine the reaction catalyzed by an enzyme called squalene synthase, an important enzymatic transformation that involves some very interesting and unusual electrophilic additions, rearrangements, and reactive intermediates. This particular enzyme is also of interest because it represents a potential new target for cholesterol-lowering drugs.

Cholesterol, as we discussed earlier in this chapter, is derived from a 30-carbon isoprenoid molecule called squalene. Squalene, in turn, is derived from the condensation of two molecules of farnesyl diphosphate (FPP), a 15-carbon isoprenoid. You may recall that FPP is the product of the C₄ to C₁, or 'head to tail' electrophilic condensation of isoprenoid chains:

The condensation of two molecules of FPP to form squalene, however, is something different: this is a 'head to head' condensation, where C₁ of the first molecule forms a bond to C₁ of the second. The chemistry involved is quite a bit more complicated.

The first two steps are familiar: first, the pyrophosphate on one FPP molecule leaves (step 1), resulting in an allylic carbocation that is attacked by the C₂-C₃ π bond of the second molecule (step 2).

This results in a new carbon-carbon bond between the two FPP molecules, but with incorrect C₁ to C₂ connectivity (remember, the overall reaction is a C₁ to C₁ condensation). In step 3, a proton is abstracted and the electrons from the broken C-H bond bridge across a 2-carbon gap to form a cyclopropyl intermediate.

In the second stage of squalene synthesis, the second pyrophosphate group leaves, generating a cyclopropylcarbinyl cation (step 4). Because this is a primary carbocation, you probably are wondering about how stable it could be (and thus how likely an intermediate). As it turns out, such carbocations are remarkably stable, due to favorable interactions between the empty orbital and orbitals on the three-membered ring (the level of bonding theory needed to really understand this idea is beyond the scope of this text, but you may learn about it if you take a class in advanced organic chemistry). What occurs next is an alkyl shift leading to a tertiary carbocation (step 5).

Discussion of the final step (step 6) will need to be put off - this is a reduction with a hydride nucleophile derived from a coenzyme called NADPH. Although this may seem like an extremely convoluted (and perhaps unlikely!) mechanism, there is much experimental evidence to back it up.