Citric Acid, the well known Krebs cycle intermediate for which the cycle is also known, is normally produced under enzymatic conditions in the mitochondria of cells. However, the non-enzymatic route to citric acid was unknown until 1977 when Lever Brothers, a division of Unilever, was granted two patents for its chemical production via reaction of the alkaline earth metal salts, e.g., either calcium isocitrate/alloisocitrate or calcium aconitate at pH's greater than 11.6 and temperatures of 160°-200°C. This paper will depict the reactions employed and propose the reaction mechanism.
The reactions listed herein are actually the reverse of the Krebs cycle and are described in US4056567 V. Lamberti, E. Gutierrez and US4324906 E. Gutierrez, V. Lamberti . The starting materials employed for US4056567 patent can be either the diastereoisomer mixture of isocitrate/alloisocitrate, the dl mixture of both alone, or cis/trans aconitic acids. US4324906 patent employs the esters of a halogenated acetotricarballylate (ATC), (the structure of chloro ATC is shown below). This halogenated compound can be converted directly to mixtures of cis/trans aconitate and isocitrates at high temperatures/pressure via ketonic cleavage of the acetyl group, dehydrohalogenation and ester hydrolysis and where the isocitrates that form are dehydrated directly to aconitate (shown below). The cis aconitate that forms is subsequently converted to citrate salts all in the same pot.
Let us begin with the diastereoisomeric mixture of isocitrate. The reaction involves heating the alkaline earth metal, such as Ca+2, salts of the isocitrate mixture to 160°-200°C at a pH of 11.6 or higher. The first step in both reactions is the elimination of water to give trans aconitate or cis aconitate depending on whether the starting material is the erythrose or threose form as is shown below. Both mechanisms on the left show the concerted elimination of water by hydroxide (HO−) attack on the central hydrogen of either allo-isocitric (I) or isocitric (II) and regeneration of the HO− leaving group. Note that the Ca+2 ions are shown as binding two internal carboxylate groups on the same molecule, while the ½Ca+2 could be considered bonded to two carboxylate groups on different molecules.
It has been postulated that only isocitrate (I) produces cis aconitate, the presumed reactive intermediate. But since all of the aconitate in the reaction medium has been shown to proceed to citrate it can be further postulated that trans aconitate under the forced conditions of the reaction is isomerized to the cis form.
It can also be postulated that conversion may also occur through the partially reactive hydrogens of the CH2 group, which being both "allylic" and α to a carboxyl, is acidic enough to be removed by hydroxide (HO−) ion (step 1.) and placing a negative charge at one of the carbon atoms, followed by abstraction of hydrogen from water (step 2.) and regeneration of hydroxide as shown below. Alkaline earth metal hydroxides especially calcium hydroxide, supply the requisite cation and are basic enough to generate the right basic conditions required for the reactions to proceed. For instance, Ca+2 ions appear to be just the right size and are known for forming stable metal chelates of polycarboxylates. Consequently, in order for the hydration of cis isocitrate to occur, it would be ideal if the two cis carboxylates are chelated and held tightly by Ca+2 on the same side of the double bond forming the very reactive cis intermediate susceptible to a nucleophilic attack by HO−. In fact, by analogy, the calcium salts of maleate, a cis carboxylate, reacts quite rapidly with base to give malic acid salts.
But before we discuss the hydration to citrate, I will depict the resonance forms of cis aconitate and give an insight as to why the the hydroxide (HO−) anion chooses to attack the middle carbon 2 of cis aconitate and not carbon 1.
The resonance structures for cis aconitate are shown below.
If we remove a hydrogen of the CH2 group of cis aconitate (via attack of HO− anion) and then push electrons around we can generate structure (III)
which places a partial negative charge at carbon 3. If we push electrons back towards carbon 1 we get structure (IV) also having a negative partial charge.
This distributes the charge of (III) and (IV) to (V). In this last structure there is more negative charge at both the terminal
Let us continue to the actual hydration of cis aconitate to citrate as depicted below.
I mentioned that the reaction takes place at a pH of 11.6. Why this value? Mygel and Sychen (ZH. Khim. 1958 3, 314) have given the value of 11.6 for the pKa4 of the hydroxyl group of citrate. According to the Henderson-Hasselbach equation, if the concentration of the [citrate-4] is equal to that of [citrate-3], then the pH of the reaction mixture should equal to that of the pKa4. The calcium chelated species involving 3 carboxylate groups and one ionized hydroxyl is what probably gives citrate the added stability at this high pH. Normally alkali metal salts at high pHs cause decomposition of citrate under the high temperature and pressure conditions. On the other hand, Silva et al [Biometals(2009) 22:771-778] calculate, via C13 NMR, the pKa4 to be 14.4. However, they state that the substitution at the alpha carbon may modulate the ionization behavior.
Hydration of cis aconitic, therefore, follows the mechanistic scheme below. The HO− anion, acting as a nucleophile attacks the electrophilic carbon and places a negative charge at carbon 1 (step 1.) folloed by abstration of hydrogen from water (step 2.) and regeneration of the HO− group. The stability of chelated calcium citrate under the high temperature and high alkaline conditions employed appears to drive the reaction to the right. The stability of the chelated isocitrates at these same conditions are less robust and as shown by the mechanisms above, result in their conversion to the more stable citrate.
What may be considered a hybrid of (E) and (Z) aconitic follows a similar hydration step but produces a highly reactive non isolable product. It will also be shown how increasing the electrophilicity at carbon 2 (middle carbon) can result in an increase in reaction rates.
Compound VII is produced via halogenation followed by saponification of intermediate esters from US4146543 E. Gutierrez, derived from maleic anhydride and US4273935 E. Gutierrez, V Lamberti, derived from the monoalkyl ester salt of maleic acid both using calcium hydroxide as base. Under the reaction conditions shown compound VI (from the saponification esters) is dehydrated to form VII. This carboxylated aconitate being a hybrid of both (E) and (Z) aconitate is shown to hydrate at a lower pH and under atmospheric pressure to produce VIII (NMR analysis shows the disappearance of the CH2 group with time). However, VIII is not isolable and appears to decompose via the retro Aldol reaction into aceto acetate (IX) and malonate (X) both of which exchange hydrogen for deuterium during NMR analysis. Although the intermediate VIII unlike the parent citrate is not a stable entity, its possible formation, as is the case for citrate, points to a similar mechanism.
To keep the number of intermediates to a minimum, the intermediates VII and VIII are formed via a 2 step reaction first attack by hydroxide, followed by proton abstraction from water.
To continue to Part II discusses the non enzymatic preparation of erythro and threo isocitrates.
Go back to homepage.
© April 2015 (revised 2/25/2025) by Eddie N Gutierrez E-mail: enaguti1949@gmail.com