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7.8: Biological Substitution Reactions

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    227079
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    Objective

    After completing this section, you should have an appreciation that SN1 and SN2 mechanisms exist and are well-known in biological chemistry.

    Leaving Groups in Biochemical Reactions

    In biological reactions, we do not often see halides serving as leaving groups (in fact, outside of some marine organisms, halogens are fairly unusual in biological molecules). More common leaving groups in biochemical reactions are phosphates, water, alcohols, and thiols. In many cases, the leaving group is protonated by an acidic group on the enzyme as bond-breaking occurs. For example, hydroxide ion itself seldom acts as a leaving group – it is simply too high in energy (too basic). Rather, the hydroxide oxygen is generally protonated by an enzymatic acid before or during the bond-breaking event, resulting in a (very stable) water leaving group.

    Mechanism showing water as a leaving group.

    More often, however, the hydroxyl group of an alcohol is first converted enzymatically to a phosphate ester in order to create a better leaving group. This phosphate ester can take the form of a simple monophosphate (arrow 1 in the figure below), a diphosphate (arrow 2), or a nucleotide monophosphate (arrow 3).

    image077.png

    Due to resonance delocalization of the developing negative charge, phosphates are excellent leaving groups.

    Mechanism showing phosphate as the leaving group.

    Here’s a specific example (from DNA nucleotide biosynthesis) that we will encounter in more detail in section 11.5:

    Ribofuranose becomes PRPP which becomes ATP,GTP after many steps.

    Here, the OH group on ribofuranose is converted to a diphosphate, a much better leaving group. Ammonia is the nucleophile in the second step of this SN1-like reaction.

    We will learn much more about phosphates in chapter 10. What is important for now is that in each case, an alcohol has been converted into a much better leaving group, and is now primed for a nucleophilic substitution reaction.

    SAM Methyltransferases

    Some of the most important examples of SN2 reactions in biochemistry are those catalyzed by S-adenosyl methionine (SAM) – dependent methyltransferase enzymes. We have already seen, in chapter 6 and again in chapter 8, how a methyl group is transferred in an SN2 reaction from SAM to the amine group on the nucleotide base adenosine:

    image001.png

    (Nucleic Acids Res. 2000, 28, 3950).

    Another SAM-dependent methylation reaction is catalyzed by an enzyme called catechol-O-methyltransferase. The substrate here is epinephrine, also known as adrenaline.

    image002.png

    Notice that in this example, the attacking nucleophile is an alcohol rather than an amine (that’s why the enzyme is called an O-methyltransferase). In both cases, though, a basic amino acid side chain is positioned in the active site in just the right place to deprotonate the nucleophilic group as it attacks, increasing its nucleophilicity. The electrophile in both reactions is a methyl carbon, so there is little steric hindrance to slow down the nucleophilic attack. The methyl carbon is electrophilic because it is bonded to a positively-charged sulfur, which is a powerful electron withdrawing group. The positive charge on the sulfur also makes it an excellent leaving group, as the resulting product will be a neutral and very stable sulfide. All in all, in both reactions we have a reasonably good nucleophile, an electron-poor, unhindered electrophile, and an excellent leaving group.

    Because the electrophilic carbon in these reactions is a methyl carbon, a stepwise SN1-like mechanism is extremely unlikely: a methyl carbocation is very high in energy and thus is not a reasonable intermediate to propose. We can confidently predict that this reaction is SN2. Does this SN2 reaction occur, as expected, with inversion of stereochemistry? Of course, the electrophilic methyl carbon in these reactions is achiral, so inversion is not apparent. To demonstrate inversion, the following experiment has been carried out with catechol-O-methyltransferase:

    image005.png

    Here, the methyl group of SAM was made to be chiral by incorporating hydrogen isotopes tritium (3H, T) and deuterium (2H, D). The researchers determined that the reaction occurred with inversion of configuration, as expected for an SN2 displacement (J. Biol. Chem. 1980, 255, 9124).

    Substitution by Electrophilic Addition/Elimination

    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).

    image074.png

    This is a nice example of an electrophilic addition/elimination mechanism, which we saw in general form in section 15.1:

    image076.png

    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.

    image078.png

    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).

    image080.png

    How do we know that these are indeed SN1-like mechanisms with carbocation intermediates, rather than concerted SN2-like mechanisms? First of all, recall that the question of whether a substitution is dissociative (SN1-like) or associative (SN2-like) is not always clear-cut - it could be somewhere in between, like the protein prenyltransferase reaction (section 9.3). 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.

    image082.png

    This difference in the identity of the nucleophilic species would lead one to predict that the chain elongation reaction has more SN1-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 SN2 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 SN1-like than SN2-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 (see section 9.3B).

    image084.png

    If the reaction is SN1-like, the electron-withdrawing fluorines should destabilize the allylic carbocation intermediate and thus slow the reaction down considerably. If the mechanism is SN2-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 SN1-like displacement.

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