Chapter 9: A return to the carbonyl

As we have seen carbonyl compounds undergo both acid and base catalyzed reactions involving nucleophilic attack at the carbonyl carbon (or at the beta carbon of conjugated carbonyls). This reaction, in its many guises, can produce an impressive range of products from the formation of an ester from an acid to the production of alcohols from carbonyls. While these reactions may seem superficially different, if you understand the underlying mechanism involved, it is possible to make plausible predictions for the outcome for literally thousands of reactions. By this time, you should have come to understand such processes. Now, it is time to reconsider carbonyl groups in light of the fact that there is a completely different set of reactions that involve the reactivity of the alpha carbon of the carbonyl groups. An image of a reaction of ketone and enol.Carbonyl compounds typically exist in two tautomeric forms: the keto and enol forms. The keto form is usually the major tautomer and there is always some enol present as well.

The structure of the enol form can provide clues about its different reactivity, which is distinct from that of the keto form. The enol form consists of an alcohol directly attached to a C that is involved in a double bond. An image of a reaction of enol.As we know, alkenes are electron-rich and tend to undergo electrophilic attack; the presence of an attached –OH group makes such an electrophilic attack even more likely. Just like an –OH group on an aromatic ring, the OH can donate electrons through resonance with the –C=C– and make the enol more reactive. Carbonyl groups can react, through the small percentage of the enol form present, to undergo electrophilic attack at the alpha carbon.

An image of a reaction of tautomerisation of carboxylic acid.We have already seen that aldehydes and ketones exist as keto-enol tautomers, but, in fact, carboxylic acids, esters, and other acid derivatives also have the potential to exist in the corresponding enol form.

Another implication of the alcohol-nature of an enol is that we expect it to be acidic—and indeed it is. The conjugate base of the enol is called the enolate ion and it is resonance-stabilized so that the negative charge is delocalized on both the oxygen and on the alpha carbon.

An image of a reaction of resonance stabalized enolate anion.

The pKa of acetone is 19—somewhat higher than a typical alcohol (pKa ~15). In the enolate form, the majority of the charge sits on the more electronegative oxygen, but a significant proportion of the negative charge is associated with the alpha carbon: the enolate ion is a stabilized carbanion. The enolate anion is often written in its carbanion form because this is the form that produces most of the interesting chemistry. Treatment of a carbonyl compound with a base, such as an alkoxide, results in the reversible formation of a small amount of the enolate ion (although the equilibrium lies on the side of the unprotonated form). Similarly, many carbonyl compounds can be deprotonated to give the corresponding enolate anion. For example, esters can be treated with a base to give the corresponding enolate anion.

An image of a reaction of ethyl acetate.

Ethyl acetate has a pKa of around 25 (less acidic than acetone: pKa 19), but still well within reach of many of the strong bases. For example, sodium amide (NaNH2), the conjugate base of ammonia (pKa 33), is strong enough to deprotonate the ester. In fact, we typically use what is known as a hindered base, such as lithium di-isopropylamide (LDA), which is similar to sodium amide but the nitrogen has two bulky isopropyl groups attached to it.[1]An image of lithium di-isopropylamide (LDA).

Since LDA is such a strong base, treatment of most carbonyl compounds with LDA produces essentially 100% of the corresponding enolate anion. However, there are exceptions. Any carbonyl compound that has a more acidic proton than the H associated with the alpha carbon will not undergo this reaction. For example, treatment of carboxylic acids with LDA will merely result in the loss of the acidic proton from the carboxylic acid OH group.

Most carbonyl compounds have pKa‘s between 19 and 25. Compounds that have carbonyl groups that are beta to each other (that is, separated by an intervening carbon), have significantly lower pKa‘s (around 9), because the resulting anion can be stabilized on both carbonyl oxygens.

An image of a reaction of enolate anion (EtO-).

They can be easily deprotonated by bases such as sodium ethoxide or sodium hydroxide.

Reactions of enols and enolates

The keto and enol forms of carbonyl compounds can undergo completely different reactions. The carbonyl (keto) form undergoes nucleophilic attack at the carbonyl carbon and the enol/enolate form undergoes electrophilic attack, usually at the alpha carbon (although the O is also reactive). For example, aldehydes and ketones can be halogenated at the alpha carbon just by treatment with a solution of the halogen, either with acid or base catalysis. The first step is enolization, which produces the very electron-rich alkene that attacks the bromine (just like the first step of addition to a normal alkene). This intermediate then loses a proton to give to the halogenated compound and HBr.

An image of a reaction of enolization.

The reaction can also be done in a base via the enolate, but in this case the reaction is difficult to stop after one halogen has added and, typically, all alpha positions will end up brominated. Such a reaction is analogous to the first step of addition of halogens to an alkene, but the second step involves the regeneration of the carbonyl. Just as reversible nucleophilic addition to the carbonyl typically produces the sp2 hybridized product, these enol/enolate forms also end up as substitutions rather than additions.

An image of a reaction of LDA/THF.

A reaction of an alpha carbon that has no analogy in alkene chemistry involves their acting as a nucleophile in an SN2 reaction. An image of a three LDA/THF structures.The reaction occurs via the enolate anion, which then attacks any appropriate alkyl halide via an SN2 reaction.

If the ketone undergoing such a reaction has the possibility of forming two different enolates, and therefore producing two different alpha alkylation products, the enolate that has the most substituted double bond is the most stable and is thermodynamically favored. Typically, the enolate formed from the least-hindered carbon is formed fastest (it has the lowest activation energy). An image of a reaction of LDA/THF in kinetic and thermodynamic product.It is therefore possible to control the product of such a reaction by carefully controlling the reaction conditions. At very low temperatures, the kinetic product is formed, while at higher temperatures thermodynamic product is formed.

The Aldol reaction

As we have previously discussed, the carbonyl group has a kind of split personality. The carbonyl group is susceptible to nucleophilic attack at the carbonyl carbon and carbonyl compounds can be nucleophiles at the alpha carbon. Therefore, we should not be too surprised to learn that carbonyl compounds can (and do) react with themselves. For example, when acetaldehyde, the simplest carbonyl compound that is capable of forming an enol, is treated with a reversible base such as NaOH, it will form a small amount of the enolate anion, which can then react with the carbonyl of another molecule as shown. This reaction is known as the aldol reaction. As the enolate is used up in the reaction, more is formed and more aldol reaction occurs. The product is a beta-hydroxy aldehyde.

An image of a reaction of -OH giving a product of beta hydroxyaldehyde.

Typically, aldehydes undergo this reaction readily and the aldol product is formed in good yield. The reaction is reversible, however, and ketones often do not give good yields of the aldol product. The reverse reaction is called a “retro aldol” and occurs via deprotonation of the alcohol and loss of the enolate anion as shown.

An image of a reaction of -OH giving a reaction of retro aldol.

In fact, the aldol reaction rarely ends at the simple addition of one carbonyl and its corresponding enolate. Usually, the reaction is heated and, under these conditions, the alcohol that is formed undergoes an elimination to form the alpha, beta unsaturated carbonyl. When this happens, the reaction is called an aldol condensation (the term “condensation” is usually used for reactions in which water is lost).

An image of a reaction of aldol condensation.

An image of a reaction of NaOH.Aldol condensation is often used to synthesize rings via an intramolecular aldol condensation. In these cases, although there may be the potential to form different ring sizes, typically only the most stable rings are produced: that is, five- or six-membered rings.

The Claisen Condensation

As we have noted, all carbonyl compounds are capable of forming enols and enolate anions, and just as aldehydes and ketones undergo condensation reactions with each other, so then to esters. The ester version of the aldol is called a Claisen condensation, but the essential details are very similar in terms of the initial mechanistic sequence.

An image of a reaction of Claisen condensation.

The enolate anion of the ester attacks the carbonyl of another molecule and the resulting tetrahedral intermediate collapses back to the carbonyl by regenerating the ethoxide anion that was used to initiate enolate formation. The difference between the Claisen and the aldol reactions is that the Claisen product is a β-ketoester, which can be a useful species in its own right. Claisen condensations can also form rings via intramolecular condensations (which are known as Dieckmann cyclizations).

An image of a reaction of Dieckmann cyclization.

Both Aldol and Claisen condensations can be carried out between two different carbonyl compounds: however, if both are capable of forming enolates, there is the possibility of forming four different products. Consider two carbonyl compounds A and B, if enol A reacts with carbonyl B, we get product AB, but if enol B reacts with carbonyl A we get product BA (which would have a different structure). A can also condense with another A to form product AA, and similarly we could get BB. Therefore, it is important to control the reaction conditions carefully: for example, by using an irreversible base such as LDA to form the enolate first. This precludes the possibility of the enolate reacting with itself. Then, the other component can be added slowly to the reaction mixture and the condensation can be carried out.

An image of a reaction of LDA an irreversible formation of enolate that gives a product of a mixed Claisen.

β-Ketoacids Decarboxylate

All of these Claisen condensation reactions produce a difunctional compound in which a carbonyl group is located on the beta position of an ester. There is a useful reaction that can be carried out if the ester is hydrolyzed to the corresponding acid. If the resulting β-keto acid is heated, it decarboxylates (loses CO2) in a pericyclic reaction that involves the cyclic rearrangement of six electrons as shown below.

An image of a reaction of Beta-ketoester "acetoacetic ester."

The β-ketoester here is known as acetoacetic ester. The CH2 group between the two carbonyls is easily deprotonated, and the resulting anion can do a nucleophilic attack on any susceptible substrate: for example, an alkyl halide. A subsequent hydrolysis and decarboxylation results in a compound that has three more carbons in it than the original alkyl halide, as shown below.

An image of a reaction of alkyl halide.

The “acetoacetic ester” synthesis is a powerful way of adding a 3-carbon unit. A similar reaction involves malonic ester (below), which can be used to add a 2-carbon unit.

An image of a reaction of malonic ester.

In the decarboxylation step, only one of the carboxylic acids decarboxylates and the alkyl group is extended by two carbon atoms. Interestingly, fatty acids (long chain carboxylic acids) are synthesized by a mechanism that is analogous to this malonic ester synthesis.

Biosynthesis of Fatty Acids:

As we move forward, we will discuss some biosynthetic pathways. It is not our intention that you reproduce these pathways, complete with enzymes and co-factors, but that you understand the underlying organic chemistry behind them. As you will see, most biochemical reactions are quite simple: it is their surroundings (the other parts of the molecules, enzymes, and co-factors) that make it possible for these reactions to occur (generally around room temperature) in a crowded aqueous environment; these pathways appear complex.

An image of a reaction of malonyl.Fatty acids are built two carbons at a time by the following mechanism: the two-carbon unit is provided by a malonyl unit that is formed from malonyl-CoA, (a thioester) which is attached to a carrier protein for recognition purposes. It is formed from acetyl CoA (co-enzyme A)—which is a product of the breakdown of glucose (glycolysis) by reaction with bicarbonate. The result of adding two carbon units is that most biological fatty acids have an even number of carbons. They are synthesized by a sequence of reactions that is highly analogous to the malonic ester synthesis. The syntheses of fatty acids is one of the mechanisms that the body uses to “store” energy and to generate various membranes. Fatty acids often occur as esters of glycerol and are therefore called triglycerides.

An image of a reaction of acetyl CoA, malonyl CoA, and malonyl ACP.
An image of a reaction of malonyl ACP and two chained carbons.

Acetyl CoA is transferred to the acyl carrier protein (ACP) and is then attacked by the malonyl anion with loss of the S-ACP group (this is analogous to the SN2 reaction on an alkyl halide). Decarboxylation produces a new beta-keto thioester, extended by two carbons.

The next step is reduction of the carbonyl (using NADPH—which is analogous to NADH—and delivers hydride ion), elimination, and reduction to the fully saturated chain. The system then cycles around to add two more carbons from malonyl ACP.

An image of a reaction of NADPH reduction.

All of these reactions are very similar to those we have learned throughout the course. They seem more complex because of the biological “bits” that control the direction of reaction and activation of the functional groups, but once you understand organic chemistry, biochemistry makes much more sense!

Michael Reactions.

Recall that aldol condensations result in α,β-unsaturated carbonyl compounds, a functionality that we have already discussed at some length.

An image of a reaction of aldol condensation.

An image of "hard" and "soft" nucleophiles.These conjugated carbonyl groups can undergo nucleophilic attack at either the carbonyl carbon or at the β carbon, depending on the nature of the nucleophile. For example: highly reactive (or “hard”) nucleophiles like Grignards or alkyl lithiums tend to react at the carbonyl carbon, while less reactive (soft) nucleophiles like dialkyl cuprates or reversible nucleophiles like amines or alcohols tend react at the β carbon.

Anions formed from β-diketones are relatively unreactive (they are stabilized) and, therefore, we might predict that they will attack the conjugated carbonyl at the β position – and we would be correct! This reaction is called the Michael reaction.

An image of Micheal reaction.

In fact, we can condense the same β-diketone with a different conjugated ketone (not formed from an aldol condensation) to produce an intermediate that can then undergo an intramolecular aldol condensation as shown below. This two-step procedure is called the Robinson Annulation.

An image of a reaction of Robinson Annulation.

An image of Stork enamine synthesis.Unfortunately, this reaction only works well with beta-diketones; a simple ketone does not attack the conjugated system. However, there is a relatively simple solution to this, which is to modify the ketone to form an enamine, which will then react as shown below. This variation is known as the Stork enamine synthesis.

Glycolysis

Glycolysis is the name we give to the group of reactions that result in the splitting of a C-6 glucose molecule into two C-3 units, which is accompanied by the overall production of ATP, the molecule that can be used to provide energy to drive unfavorable chemical reactions such as building up biopolymers like peptides, nucleic acid polymers (DNA and RNA), and production of fats. This process is usually depicted schematically, particularly in biology texts, but every step of the process is a relatively simple organic reaction that can be understood in terms of the principles that we have learned over the past year. The overall schematic for glycolysis is given below[2], but our intent here is not that you memorize each step so that it can be regurgitated; it’s to allow you to understand how and why these reactions occur the way that they do (or at least the way they do in biological systems).

An image of Glycolysis.

We will be treating these reactions from an organic chemistry perspective, but it is important to note that in the body all of these reactions are mediated by enzymes and co-factors that lower the activation energy for each reaction. The first part of the glycolysis pathway involves the conversion of the sugar glucose, to a different sugar, fructose. Therefore we will begin by looking briefly at the structure and properties of sugars.

Glucose:

While glucose looks complex, we have already investigated the functional groups present and all the reactions that are important here. Glucose belongs to the family of compounds called sugars part of a larger group, known as carbohydrates, denoted by the suffix -ose. An image of a hexose lewis structure.Since it has six carbons, glucose is known as hexose (similarly, a five-carbon sugar would be known as a pentose). Glucose can exist in several forms; both open and closed chain. We consider the open-chained form first. The easiest way to represent sugars is by using a Fischer projection (in fact these representations were invented for just this purpose). Remember that in a Fischer projection, the horizontal bonds are pointing out of the plane and are all eclipsed. The naturally occurring form of glucose is D-Glucose [latex]\rightarrow[/latex].

Note that there are four chiral centers in glucose, and therefore there are 16 (24) possible stereoisomers, many of which do occur naturally. An image of Glucose in 3-D.The D designation has to do with the stereochemistry at position 5 and does not refer to the direction of the rotation of plane-polarized light. (While it is possible to designate R or S for each chiral center[3], it is not possible to designate R or S for the molecule as a whole). Note that glucose also has an aldehyde group (at position 1) and, therefore, also belongs to the class of sugars called aldoses.

In its open chain form, glucose has an aldehyde group and five hydroxyl groups and, as you might expect, there is great potential here for reactions, both inter- and intra-molecular. In a solution, glucose commonly exists in the hemiacetal form, in which the OH group on C-5 has attacked the carbonyl group to form a six-membered ring which is referred to as the pyranose form (pyran is a six-membered heterocyclic ring with an oxygen atom in it). The pyranose form is usually drawn in the chair form as shown below.

Images of hemiacetal formations.Hemiacetal formation produces two possible configurations, but rather than calling them R and S, we label them alpha and beta. In alpha form, the OH on carbon 1 is on the opposite side of the ring from the CH2OH (C-6 of the original chain), the beta form has the OH group on the same side as the CH2OH. These two forms are stereoisomers because they have the opposite configuration at C-1, but unlike typical stereoisomers, they can be interconverted by ring-opening of the hemiacetal and reclosure of the ring. Images of alpha furanose form and beta form.They are called anomeric forms and C-1 is referred to as the anomeric carbon. Such carbons can be identified by the fact that they have two oxygens attached to them. In an aqueous equilibrium solution of glucose, less than 1% is present in the open chain form, but since there is always an equilibrium concentration present, the alpha and beta forms can and will interconvert via this open-chain form. As might be expected, the beta form is more stable (because the OH is equatorial) and at equilibrium there is about 64% beta. For sugars such as glucose, there is also the possibility that a five-membered ring can form by the reaction of the alcohol at C-4 with the carbonyl. Again, two forms (alpha and beta) are possible, which can be interconverted via ring-opening. This five-membered ring form is called the furanose form (furan is a five-membered ring with one oxygen).

Fructose: Fructose is another six-carbon sugar. It differs from glucose in that it that has a ketone rather than an aldehyde at C-2; for this reason, it is called a ketose (rather than an aldose). Fructose usually exists in a five-membered hemiacetal ring formed by reaction of the OH at C-5 with the ketone carbonyl at C-2. The resulting five-membered ring is called the furanose form (furan is a five-membered ring with one oxygen) and typically furanose rings are depicted using yet another structural representation: the Haworth projection, shown here.An image of glucose fructose in alpha and beta forms. In this structure, the ring is drawn as a plane (although it isn’t, of course), and the substituents are either above or below the plane of the ring. In the same way as glucose, fructose can exist in either the alpha or beta forms.

Glycolysis: From glucose to fructose.

The first steps of the biological cycle known as glycolysis involve the conversion of glucose to fructose. As we will see, this sets up the reverse aldol reaction that results in the splitting of glucose into two 3-carbon fragments—but we will get to that shortly. The first step in glycolysis is the phosphorylation of the OH group at C-6. An image of adenosine triphosphate(ATP).This reaction is analogous to the formation of a carboxylate ester, the only difference being that the attack occurs at a phosphorus rather than a carbon. The source of phosphate is adenosine triphosphate (ATP) and, in this case, it is the terminal phosphate that is transferred to the glucose and the leaving group is ADP (adenosine diphosphate). The reaction is mediated by an enzyme (a kinase): this reaction is highly thermodynamically favorable (ΔG is negative).

The consequences of glucose phosphorylation reaction are twofold: First, it serves to keep the concentration of glucose in the cell low, so that glucose can continuously diffuse into the cell (through membrane channel proteins). Glucose-6-phosphate is highly charged (the oxygens on the phosphate group are almost entirely ionized at physiological pH) and cannot diffuse back out of the cell (through the channel). The second consequence is that the hydroxyl group on C-6 has been activated. The phosphate is an excellent leaving group.

An image of a reaction of glucose into fructose.

The next step is the transformation of glucose into fructose. In organic chemistry terms, this is a simple tautomerization as fructose is a structural isomer of glucose. The enzyme that regulates this conversion is known as an isomerase.

The glucose aldehyde undergoes enolization as shown, followed by another tautomerization to produce the fructose ketone. All of these reactions are entirely analogous to the keto-enol interconversions that we have seen in simpler systems.

An image of Glucose-6-P, enol, and Fructose-6-P.The next step is another phosphorylation reaction at C-1 to produce fructose-1,6-diphosphate; it involves the same reaction mechanism that produced phosphorylation at C-6 using ATP as the source of phosphate. You might be asking, how is it that these reactions occur in this particular sequence and why don’t the other oxygens undergo phosphorylation? The answer to this lies in the fact that these reactions are mediated by enzymes that guide the substrates into the correct (pre-established, evolutionarily) orientations. Reactions in solution depend entirely on random collisions with enough energy to surmount the activation energy barrier and in the correct orientation. In biological systems, the substrate first collides with, and its orientation is constrained through interactions with, the enzyme; limiting subsequent aspects (steps) of the reaction. While there is potential for other hydroxyls to be phosphorylated (and it would certainly be difficult to control the site of attack if the molecules were free in solution, the reactant-enzyme complex favors certain ones dramatically over others. Remember the enzyme itself is result of evolutionary mechanisms. Given the ubiquity of this process, it was likely established early, and present in the last common ancestor of life.

The reverse aldol: The system is now set up for the cleavage of the six-carbon sugar into two three-carbon species via a reverse aldol reaction as shown.

An image of a reaction of reverse aldol.

The result is the production of two molecules of glyceraldehyde-3-phosphate from one molecule of glucose. Up to this stage, glycolysis has involved the use of two ATP molecules associated with the two phosphorylation reactions.

 

In the next stage of glycolysis, another phosphate is added to each glyceraldehyde-3-phosphate molecule; this phosphate is not derived from ATP, but from inorganic phosphate. The mechanism involves an addition of a phosphate unit to the aldehyde carbonyl, followed by oxidation as the tetrahedral intermediate collapses, with loss of the hydride ion that adds to NAD+, to form NADH.

An image of two reactions of Glyceraldehyde-3-phosphate and 1,3,-diphosphoglycerate.

The resultant species now contains two phosphate groups, but they are different chemically: the phosphate attached to the newly oxidized carbon is much more reactive than the other. Attack by a nucleophile at the phosphorus preferentially expels a carboxylate anion from the intermediate, rather than an alkoxide. In the next step of the reaction, the highly reactive phosphate is transferred to ADP through an attack by the O on the terminal phosphate of ADP onto the P of the 1,3-diphosophoglycerate, with subsequent loss of 3-phosphoglycerate (because the carboxylate is a good leaving group).

An image of a reaction of 3-phosphoglycerate.

This reaction produces two ATP molecules (and 2 reduced NADH molecules) from one original glucose (because there are now two three-carbon units), so, at this stage, the net production of ATP = 0. The transfer of the other phosphate unit from C-3 to another ADP does not occur because the leaving group (an alkoxide) is not thermodynamically favorable. Instead, the phosphate group is transferred from C-3 to C-2 by an intermolecular nucleophilic attack that forms a five-membered ring intermediate and subsequent elimination of water.

An image of a reaction of transferring of phosphate.

The product of this elimination reaction is called phosphoenol pyruvate (PEP). In essence, it is an enol that has been trapped by esterification with the phosphate. The enol is an excellent leaving group (since it is really a carbonyl group) and, therefore, PEP will also undergo attack by ADP to produce ATP and pyruvate, resulting in a in a net +2 ATP molecules for the overall glucose to pyruvate reaction (plus the two NADH molecules).

An image of reaction of phosphenol pyruvate.

Once again, our intent here is not to have you memorize this long sequence of reactions, but rather to recognize that even in systems that appear highly complex, when each reaction is viewed at the molecular level, it is recognizable as the same reactions that we have been studying. In fact, the mechanisms of a large majority of reactions in biological systems can be understood in relatively simple terms. Many of the reactions are the same: attack at carbonyl (or phosphate) groups, aldol and retro aldol condensations, and keto-enol tautomerizations.


  1. Recall that we have used hindered bases before. In that case, it was to prevent nucleophilic attack when we wanted to bring about an E2 elimination reaction and tertiary butoxide (KOC(CH3)3, as our hindered base.
  2. Diagram from Wikipedia https://en.wikipedia.org/wiki/File:Glycolysis_metabolic_pathway_3_annotated.svg#file licensed under a creative commons license.
  3. In fact the name of the most common form of glucose is (2R,3R,4S,5S,6R)-6(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol. There is no need to remember this!

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OCLUE: Organic Chemistry, Life, the Universe & Everything by Melanie Cooper and Michael Klymkowsky is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.

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