Reaction Mechanisms

Objectives

Reaction mechanisms

Understanding reaction mechanisms is useful for biologist because it allows you to understand how reactions occur, and therefore optimise the yield of a chemical in an biotechnological industrial process, understand catalysis by metals and by enzymes, and design inhibitors of enzymes, such as drugs and pesticides.

There are several ways of studying reaction mechanisms: we can look at the reaction kinetics, in particular, the effect of reactant concentrations, pH, temperature and inhibitors, and we can also use radiotracers and NMR to follow individual atoms and groups. The stereochemistry of products and the isolation/detection of intermediates also aid in our understanding of how a reaction actually occurs.

Bromomethane - click for Jmol version

To start with, we will take a simple example of a reaction mechanism: the hydrolysis of methyl bromide. Methyl bromide (CH3Br) is a horrible soil fumigant, which is toxic and contributes to ozone depletion. How is it degraded?

Mechanism notation involves drawing curly arrows to indicate where electrons go. That's 'all' there is to it! Here is the mechanism for the alkaline hydrolysis of methyl bromide by a hydroxide anion.

A hydroxide ion is attracted to the partial positive charge on the carbon atom of MeBr: as it approaches, its negative charge displaces electrons even further towards the Br until eventually the C-Br bond is broken, and a new HO-C bond is formed.

The steps I took to draw it were as follows:

For the methyl bromide example, we note that:

This sort of reaction is called a nucleophilic substitution: it's a substitution because the Br has been substituted by the OH, and it's nucleophilic because the attacking chemical species is a nucleophile, i.e. a chemical that seeks out positive charge.

You may be wondering why we didn't end up with [HO-CH3-Br]− as a product? The reason is that it is unstable: carbon doesn't make 5 bonds or ionise readily. This species may exist fleetingly as a reaction intermediate but will not be the final product. Other atoms are less fussy: although the valency of carbon is always 4, but the valency of other elements may vary.

The heterocyclic nitrogen in guanine can also attack MeBr.
It's not just hydroxyl ions that can attack methyl bromide. Here the lone pairs of a heterocyclic nitrogen attack the compound, forming an adduct.

Nucleophiles are chemical species that are electron rich (may have lone pairs) and seek out positive charge (nucleus is +). For example, they may be anions or or the δ- end of a polar molecule.

The opposite of a nucleophile is an electrophile, which are electron deficient, and therefore seek out negative charge (e.g. π bonds). They may be cations or the δ+ end of a polar molecule.

A substitution reaction occurs when a species substitutes another, like the OH replacing the Br in the mechanism above, i.e. something goes in and something else comes out.

On the other hand, an addition reaction is one where a product is formed by addition across a multiple bond: like the next example, where HCl adds across the double bond of an alkene.

HCl adds across a double bond in ethene: the partially positive H is attracted to the π bond of ethene. As it approaches, the H-Cl bond is broken, and the chloride ion produced then attacks the π-intermediate, forming chloroethane.

Something goes in, but nothing comes out. Be careful though: this thing below is not an addition! The epoxide is substituted by the amine group. No multiple bond is involved.

The attack of an epoxide by an amine is a substitution, since the epoxide group is substituted for by the amine group.

The rate limiting step of a reaction (i.e. the slowest of a series of reactions) can be used to describe the mechanism. Two common sorts of Nucleophilic Sustitution are SN1 and SN2 reactions. In an SN2 reaction, like the hydrolysis of methyl bromide, the rate limiting step involves two molecules:

SN2 reactions are nucleophilic substitutions where the rate limiting step is bimolecular.

The attack of a primary bromide (R-CH2-Br), or indeed any primary compound, by a nucleophile is usually SN2. However, secondary (R2-CH-Br), and especially tertiary (R3-C-Br) alcohols, bromides, etc., undergo SN1 reactions, where the rate limiting step involves just one molecule. This is due to the steric hindrance offered to the nucleophile by the bulky substituents on the δ+ carbon, and also on the electron donating ability of alkyl groups: alkyl groups force electrons onto groups to which they are attached, and hence tend to stabilise the short-lived carbocation intermediate:

SN1 reactions are nucleophilic substitutions where the rate limiting step is unimolecular.

Electron donating (alkyl, -OH) and withdrawing (-NO2) groups are important in determining the course of reaction of aromatic compounds with electrophiles, such as the nitronium cation (NO2+):

Electrophilic attack of benzene by the nitronium cation.

If electron withdrawing or donating groups are present, they influence which of the other positions are substituted. If we take phenol as an example:

When two groups are attached to a benzene ring, they are described as being ortho is there is not intervening carbon, meta if there is one, and para if there are two, and the groups are therefore on opposite sides of the ring.

We can see there are three different positions which can be substituted, ortho, para and meta (or positions 2, 4 and 3 in IUPAC nomenclature). Electron donating groups like -OH and -CH3 tend to push electrons on the ring, making it a more attractive site for electrophilic attack. That is, they activate the ring. In particular, they activate the ortho and para positions: the benzene ring can be though of as a resonance hybrid of the structures below, and you can see that the electron density (negative charge) is particularly localised at the 2, 4, 6 positions:

Electron donating groups tends to activate ortho and para because of the way in which resonance structures can be drawn.

As a result, the substitution products of phenol with the nitronium cation are far more likely to be 2-nitrophenol and 4-nitrophenol than 3-nitrophenol. Electron withdrawing groups on the other hand, deactivate the ring, making it a less ready site for nucleophilic attack. However, they particularly deactivate the ortho and para positions (note the positive charge, which will repel a positively charged electrophile), making the meta position the more likely site for substitution, hence nitrobenzene reacting with the nitronium cation is likely to produce mostly 1,3-dinitrobenzene as a product.

Electron withdrawing groups deactivate ortho and para.

Free radical mechanisms are not particularly common in biology, since cells take great pains to prevent their production, and mop them up safely with antioxidant vitamins and enzymes when they are produced. However, for the sake of completeness, here is such a mechanism: note that free radicals contain single unpaired electrons, and are produced by homolytic fission of molecules. This is represented by curly arrows with a half head, rather than a full head, indicating the movement of a single electron, rather than a pair of electrons:

Free radical mechanisms involve the formation of species with unpaired electrons: three steps, initiation, propagation and termination, are recognised.

Reactions of carbonyl compounds

Carbonyl groups are C=O, and are found in many biological molecules:

The C=O group is polar, with δ+ on C and δ- on O. Nucleophiles (Nu) are attracted to the slightly positive C, forcing electrons upon it. Electrophilic protons are attracted to the slightly negative O, which can donate electrons to it.

Carbonyl compounds are subject to nucleophilic attack.

The product may be unstable and react further, possibly by elimination of water. At low pH, the nucleophile (Nu) attacks protons (rather then the C=O) to form an unreactive conjugate acid Nu-H+. However, protons attack carbonyl groups too, to form extremely reactive conjugate acids (R2C=O-H+). The is an optimum between the pKas of the nucleophile and the carbonyl compound.

One of the most important carbonyl derivatives is the hemiacetal:

This is a rather complex example: the cyclisation of glucose, an intramolecular attack of an aldehyde group by an alcohol group). Make sure you can identify the parts of the molecule equivalent to the R and X in the aldehyde equation above.

Glucose undergoes internal nucleophilic attack of its aldehyde group by its hydroxyl groups, forming a ring.

Acetals are the nest step on from a hemiacetal: here we add two alcohols rather than one, then eliminated a molecule of water:

Imines are the amine analogue of the acetal reaction. Imines are biologically important: the amino acids proline and arginine are both imines:

The catalytic reaction mechanism of the enzyme amino acid racemase is basically the formation of an imine, which proceeds though an unstable carbinolamine intermediate, and we will cover it presently. Hydrazones are formed from the reaction of hydrazine (NH2-NH2) with carbonyl compounds.

2,4-dinitrophenylhydrazone derivatives are useful in the identification of aldehydes and ketones because they have characteristic sharp melting points.

The reaction mechanisms of carbonyl compounds are important in biology: the formation of imines in particular is widely found, for example in the isomerisation of the compound retinal, which is responsible for detecting light in the rod cells of the mammalian retina. Retinal is an aldehyde, and is nucleophilically attacked by the protein opsin, as follows:

The enzyme amino acid racemase, which converts optical isomers of amino acids into one another, also uses an imine reaction mechanism, which we will go through in steps. Bacteria use it to provide the D-alanine they need for their cell walls. Things to note about the reaction:

Amino acid racemase works by generating a planar imino intermediate from chiral amino acids, which then has a 50-50 chance of decaying to its enantiomeric form.

  1. Amino acid on left, pyridoxal phosphate on right. We'll keep the protons and everything else in the same position so it's easier to understand. Note that the R is wedged and the H is dotted. The reaction starts with a simple nucleophilic attack - lone pairs on nitrogen attack carbonyl group, with upshot that electrons are removed from the NH bond in the amine group and form a new bond to the carbon. Electrons in the CO group are forced away and form a bond with a proton.
  2. This is a carbinolamine -NH-CHOH- and is highly unstable. Note the proton on the left, produced when the electrons were stolen from its bond to the nitrogen to form the new N-C bond.The carbinolamine rearranges to form an imine, with the elimination of water. This is an example of the reaction mechanism by which imines are produced from the reaction of any carbonyl compound and an amine.
  3. This is the imine intermediate, which is also unstable, due to the way the enzyme holds the substrates, stressing them. This is often how enzymes work: they put stress on bonds that they 'want' to react. This is the complex bit, and the most important. The N in the imine pulls out electrons from the dotted C-H bond in the amino acid, and the N=C bond is attacked by a proton (electrophilic attack). That is, the imine is converted to its conjugate acid (by taking up a proton).
  4. The amino acid is now a doubly bonded imine conjugate, which is PLANAR: the stereochemistry of the amino acid has been destroyed. Again, this is unstable. The entire process now repeats itself backwards. First, the conjugate acid of the imine reconverts to the imine form.
  5. NOTE!!!! The stereochemistry has changed. H is now wedged and R is dotted. This is how bacteria make D-alanine. In fact, slow spontaneous racemisation can be used to date some biological samples if you know exactly what temperature they are at. Now we get hydrolysis of the imine. This occurs because the N=C bond is polar, and easily attacked by nucleophiles like water.
  6. Almost there. We're back to the unstable carbinolamine we started with (just in the opposite stereochemical form) which rearranges, basically undergoing nucleophilic attack in reverse.
  7. And it's finished. All back to what we start with, but with amino acid in other form. Run this long enough and you'll turn 100% R-isomer into 50/50 mix of R and S. It won't run to 100% S-isomer, because this isomer would also be attacked by the racemase enzyme. Enzymes run things to equilibrium, not to completion!

Reactions of esters

Esters are the reaction product of an alcohol and an acid.

Esters are organic salts. Simple ones often smell nice: ethyl ethanoate has a peachy smell. Note that esterification is reversible The alarm pheromone of bees makes them attack would be invaders. The pheromone is the ester 3-methylbutyl ethanoate, which also happens to be the principal component of banana oil. Don't eat bananas near a beehive.

3-methylbutyl_ethanoate - click for Jmol version
3-methylbutyl ethanoate

Acids catalyse esterifications, but esterifications are usually reversible, as you can see from this mechanism:

In acid deesterification, water nucleophilically attacks the ester carbonyl group, hydrolysing the ester. The protons are catalysts for this.

To get a high yield of ester, you need to remove water. Sulfuric acid is often a good choice, because it has both a catalytic effect and it removes water too.

Alkalis catalyse the degradation of esters back to their constituent acid and alcohol.

Alkaline deesterification is also a nucleophilic substitution, but the hydroxide ions are reactants rather than catalysts.

Esters can also swap their alcohol-derived group with another alcohol (this is not dissimilar to the alkaline degradation described above; the OH- group should just be replaced with a ROH). This is termed transesterification.

There are many biologically important esters, not least of which are the acyl glycerols used as fat stores. However, from a mechanistic point of view, a particularly interesting ester is acetylcholine, and the enzyme that breaks it down, acetylcholine esterase (AChE).

AChE is the enzyme responsible for the degradation of acetylcholine, an ester used as a neurotransmitter in vertebrates and insects. In the first step, the choline (N+) part of the molecule is held in place by ionic bonds to a negatively charged amino acid in the active site. A serine group in the active site then nucleophilically attacks the carbonyl group in what is effectively transesterification. After this, the acetyl group is hydrolysed off by water. This regenerates the enzyme, and the precursors acetic acid and choline, which can be recycled.

Acetylcholine esterase has a serine group that catalyses deesterification of acetylcholine: an acetylated enzyme is the intermediate, which is hydrolysed by water.

Phosphoric acid is tribasic (has three dissociable protons, as you saw in the titration practical) and can form many esters.

Phosphoric acid.
Phosphoric acid

Psilocybin - click for Jmol version
Psilocybin, a phosphate ester of psilocin.

Many of these esters are important biochemicals: ATP, UTP, Coenzyme-A, DNA and others are all phosphate esters. Phosphorylation of serine residues is also a common strategy for regulating enzyme activity. Many acetylcholine esterase inhibitors are phosphoryl esters (organophosphates). They are able to react with the serine in AChE irreversibly, and so inactive the enzyme. AChE inhibitors are widely used as nerve gases (Sarin, Tabun), and as insecticides (carbamates and organophosphates, such as malathion and permethrin).

Sarin is also attacked by acetylcholine esterase, but it forms a hydrolysis-resistant intermediate.
Sarin forms a stable adduct with AChE, destroying its activity. The adduct is resistant to hydrolysis.

Test yourself

  1. Complete the following diagram showing the mechanism by which NADH reduces a ketone to an alcohol by nucleophilic substitution. It is the electrons in the C-H bond at the top of the ring that attack the carbonyl group.

    NADH mechanism.

  2. In the following reactions, indicate which of the products named would be present in the greatest amount:
    • Phenol + nitronium cation (excess) → 2,4,6-trinitrophenol and 3,5-dinitrophenol
    • Benzene + nitronium cation (excess) → 1,3-dinitrobenzene and 1,2-dinitrobenzene
  3. Why does amino acid racemase not convert L-alanine entirely to D-alanine?
  4. Show how the following compound will react with the serine group of acetyl choline esterase, destroying its catalytic activity.

    Diisopropylphosphofluoridate.

Answers

Peer Review.
This page has been peer reviewed by 3 people. Thanks to Mike Hooper for his feedback and to Rebecca Newhouse for her correction.