- Alpha-carbon chemistry questions
- Aldol reactions in metabolism
- Keto-enol tautomerization (by Jay)
- Enolate formation from aldehydes
- Enolate formation from ketones
- Kinetic and thermodynamic enolates
- Aldol condensation
- Mixed (crossed) aldol condensation
- Mixed (crossed) aldol condensation using a lithium enolate
- Retro-aldol and retrosynthesis
- Intramolecular aldol condensation
Aldol and retro-aldol are two important classes of chemical reactions, occasionally encountered in biochemistry too.
As illustrated above, you will witness aldol and retro-aldol reaction mechanisms in the following biochemical reactions:
- The first step of Kreb’s cycle (commonly also referred to as the ‘citric acid cycle’ or the tricarboxylic acid (TCA) cycle) involves the addition of oxaloacetate to acetyl CoA. This reaction follows an aldol reaction mechanism.
- In gluconeogenesis (sugar building) biochemical pathway, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) condense together to form fructose 1,6-bisphosphate via an aldol reaction.
- The cleavage of the 1,6 phosphate diester of fructose in glycolysis (sugar burning) pathway is an example of a retro-aldol reaction.
Before we get into the details of the above biochemical reactions, let us first revise what an aldol reaction is.
Aldol reaction: its chemistry and mechanism
Typically, aldehydes and ketones undergo an aldol reaction. This reaction involves 3 basic steps:
Step 1: Electrophilic attack of a base at the alpha-carbon (C
) of the aldehyde or ketone to generate an “enolate” carbanion. The carbon atom adjacent to the carbonyl carbon is referred to as alpha-carbon.
Step 2: Nucleophilic attack of the above enolate at the electrophilic carbonyl carbon of a second molecule of aldehyde or ketone.
Step 3: Protonation of the product formed.
If heat is applied then an irreversible fourth step can take place:
Step 4: A dehydration step to yield a αβ-unsaturated aldehyde or ketone.
Just a clarification, if the mechanism involves steps 1 to 3 only, then it is termed as an aldol addition reaction (or simply an aldol reaction). But if step 4 takes place, then it becomes an aldol condensation reaction (because a water molecule is lost in the event).
Aldol reactions can be base-catalyzed or acid-catalyzed. For our discussion, we will restrict ourselves to base catalyzed aldol reactions. Now let’s walk through a real example. Let’s pick the simplest aldehyde, acetaldehyde (CH
Step 1: Formation of enolate intermediate of acetaldehyde after abstraction of an alpha-hydrogen by a base (the carbon atom adjacent to the carbonyl carbon is referred to as an α-carbon, and α-hydrogens are hydrogens bonded to this α-carbon).
The enolate carbanion is resonance stabilized as shown below.
Step 2: The enolate of acetaldehyde (formed in step 1) attacks the electrophilic carbonyl carbon of the second molecule of acetaldehyde.
Step 3: Protonation of the product formed in step 2 to form an aldol product.
As you can see, the above product contains both an aldehyde and an alcohol functional group, thus the name 'aldol reaction'. Also, an aldol reaction always leads to the generation of a new carbon-carbon bond.
As mentioned earlier if the reaction is taking place under elevated temperatures, then an additional step 4 will take place.
Step 4: Loss of a water molecule (H+ and OH-) to form the final product, a αβ-unsaturated aldehyde.
Aldol reactions in metabolism
In biochemistry, enzymes act as catalysts for any chemical reaction; and the class of enzyme that catalyzes aldol reactions is called, quite intuitively, 'aldolase'. Let’s now discuss the first step of Kreb’s cycle: acetyl coenzyme A (acetyl CoA) condenses with oxaloacetate to produce (S)-Citryl CoA through an aldol mechanism. Here, instead of an aldehyde or a ketone, a thioester acts as the nucleophilic partner as illustrated below in the reaction mechanism.
The net reaction is:
Step 1: Base (B:) abstracts a α-proton to form enolate of acetyl CoA.
Step 2: The enolate attacks the electrophilic carbonyl carbon of oxaloacetate ion.
Step 3: Protonation of the above product to form the final product, (S)-citryl CoA.
As you can see, the aldol reaction leads to the generation of a new carbon-carbon bond and a new stereo-center. This stereo-carbon happens to have an ‘S’ configuration. In fact in biochemistry, stereocenter is created by the specific stereo-requirements of the enzyme’s active site where the reaction takes place.
Now let’s discuss a particular reaction that takes place in the gluconeogenesis (sugar building) biochemical pathway. It’s the reaction between two 3-carbon containing sugars, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), to form a six-carbon product, fructose 1,6-bisphosphate. This particular reaction also follows an aldol reaction mechanism, and is catalyzed by the enzyme, fructose 1,6-bisphosphate aldolase. This enzyme belongs to 'Class II' aldolase, in which a metal cation - generally Zn
, is bound in the active site of the enzyme. This helps to stabilize the negative charge of the enolate intermediate formed (as will be illustrated in the mechanism below).
The net reaction is:
Let’s chalk out a mechanism for the above reaction. We simply have to reiterate the steps of an aldol reaction once again!
Step 1: An α-proton of DHAP is abstracted by a base (B:), leading to the formation of an enolate intermediate of DHAP.
The above carbanion enolate intermediate is stabilized by resonance, and the negative charge on the enolate is further stabilized by the aldolase enzyme-bound zinc cation (Zn
) as shown below.
Step 2: The nucleophilic attack of the enolate carbanion on the electrophilic carbonyl carbon of glyceraldehyde-3-phosphate (GAP).
Step 3: Protonation of the product formed in step 2 to form the final product, fructose 1,6-bisphosphate. Source of proton donor is water (H2O).
As you must have already noticed, in this case also the aldol reaction leads to the generation of a new carbon-carbon bond and a new stereocenter. This stereo-carbon has an ‘R’ configuration. This enzyme-catalyzed reaction, not surprisingly, is completely stereospecific: the DHAP substrate is positioned in the active site such that the attack of the GAP carbonyl group, leads to an R configuration at the new stereocenter.
Now let’s change gears and talk about ‘retro-aldol’ reactions. As the name suggests, retro-aldol is exactly the reverse of an aldol reaction. Here, a carbon-carbon bond is broken to form two fragments.
It is important to emphasize that aldol reactions are highly reversible in nature; in most cases, the energy levels of reactants and products are not very different. Thus, depending on the metabolic conditions, aldolases can also catalyze retro-aldol reactions (i.e. the reverse of aldol reactions, in which a carbon-carbon bond is broken). As a typical example; fructose 1,6-bisphosphate aldolase is involved in two divergent pathways.
- the sugar synthesis (gluconeogenesis) pathway, as we have already discussed
- the sugar breakdown (glycolysis) pathway
In the latter glycolytic pathway, the zinc bound aldolase enzyme catalyzes the retro-aldol cleavage of fructose 1,6-bisphosphate into DHAP and GAP as shown below.
Mechanism of the above reaction:
Step 1: Abstraction of a proton by base (B:). Notice how the electrons move around leading to breakage of the carbon-carbon bond, generating glyceraldehyde-3-phosphate (GAP). The enolate ion serves as the leaving group as depicted below.
The enolate intermediate is stabilized by resonance, as shown below. The negative charge on the enolate is further stabilized by the Zn
bound to the active site of fructose 1,6-bisphosphate aldolase enzyme.
Step 2: Protonation of the enolate carbanion resulting in the formation of dihydroxyacetone phosphate (DHAP).