I:Non-Enzymatic Chemical Synthesis of erythro and threo IsoCitrates
II:Theoretical View of Hydration of (E) and (Z) Aconitic Employing
HOMO-LUMO Bonding of a Substituted Ethene (Part II)

Picture of isocitric acids

"Once you eliminate the impossible, whatever remains, no matter how improbable, must be the truth"- Sherlock Holmes

Isocitric Acid Background

Isocitric Acid, an intermediate in the well known Krebs cycle , is normally produced under enzymatic conditions in the mitochondria of cells. However, a simple, non-enzymatic high yield route to a mixture of isocitric and alloisocitric acid was not available until 1977 when Lever Brothers, a division of Unilever, was granted a patent for the chemical production via the oxidation of compounds of 1,1,2,3 propane tetracarboxylic acid or esters. A theoretical treatise will be shown depicting the synthesis of these two compounds via HOMO-LUMO interactions of a substituted ethene (ethylene) 2 electron π system. Note that this is a theoretical since the reactions have not been performed. However, it helps to shed some light as to the reason why aconitic behaves the way it does.

The Synthesis of a Mixture of Isocitrates via a non-Krebs Cycle Reaction

Synthesis of Isocitrate and Alloisocitrate and Mechanisms of Formation

The reactions to produce erythro and threo isocitrics employ non-enzymatic non-Krebs cycle routes and are described in US Patent 4022803 (must be accessed through US patents database). The starting materials, 1,1,2,3,propane tetracarboxylic acid or its esters, are halogenated (by a hapohalite: chlorine, bromine or H2O2/catalytic iodine) to generate isocitrics when the starting material is the acid at a pH of about 5. Below is the reaction of the tetracarboxylic acid (I) with hypochlorite as the oxidizing agent via an oxidative decarboxylation of the methine carbon to form the mixture of isocitric acid lactones.

Picture of a mixtures of isocitrate/alloisocitrate

On the other hand, when the triester (II) is used (US patent 4275007) the chloro triester (III) as well as the four membered β-lactone (IV) are isolated with complete conversion to this lactone when pyridine is used. The presence of the two carboxylated ester in (I) appear to stabilize the four membered ring by forcing the ring shut. This idea that stability of the four membered lactone ring by the presence of carboxyesters is required for stabilization of the lactone is important during the HOMO-LUMO interaction of the ethene σ* orbitals. When the starting tetraester (V), (US patent 4022803) is employed, the isolable halo tetraester (VI), is isolated. Subsequent hydrolysis under acidic conditions of (III), (IV) and (V) results in the formation of both isocitrates.

Picture of a mixtures of isocitrate/alloisocitrate

Under none of these conditions is the alternative isomer citric acid formed. We have shown that it can be prepared in Part I but only during the hydration of cis aconitic under basic conditions. What I plan to discuss next is the HOMO-LUMO interaction between a mixture of (E) and (Z) aconitic acids. These synthesis have not been performed since its being a while (about 40 years) since these reactions were done, but since working recently on the HOMO-LUMO interactions for citric I decided to look into how aconitic can give rise to the isocitrates.

What HOMO-LUMO System Should we use?

Since the HOMO-LUMO interaction of aconitate under basic conditions shows that only citrate and not isocitrates are formed due to instability of these acids, I decided that the the allyl anion 4 π electron system is probably not the right choice to use. If we were to (a) operate at lower pHs of about 4.5-5.0 where the pKa2 of isocitrates and aconitics are in the range of 4.4-4.7, and (b) where the concentration of hydroxide ion ([OH-]) is 1x10-9, low enough so that no deprotonation of the hydrogens on the α carbon of aconitic can occur, then this might produce the desired result. The two CH2 hydrogens would then not be part of an extended π system such as is present in the allyl anion 4 π electron system with its extended π electrons. The configuration we should be looking at is the ethene or ethylene π electron system of aconitic without its two CH2 hydrogens included. This would reduce the number of π orbitals on ethylene to two, viz. the π bonding and the π* antibonding orbitals instead of the three π orbitals of the allyl anion 4 π electron system.

Resonance forms for Aconitatic under slightly acidic conditions

The resonance structures for cis and trans aconitic may be set up as shown below. The counter ion is left blank since both alkali metal and alkaline earth metal ions may work. The (H)s adjacent to the carboxy groups mean that either one of these is deprotonated. The middle carboxy being the more acidic is present as COO- but at a pH of about 4.5-5 either or both of the terminal COOH groups might be present as COO-. Since the original isocitrates were synthesized using alkali metal salts, the low acid conditions should also favor the hydration of both the (E) and (Z) forms of aconitics to produce threo and erythro isocitrates, just as both diastereomers were produced during the halogenation/decarboxylation step above. We can obtain two resonance forms for each, VIII and IX for the (E) aconitic and XI and XII for (Z) aconitic, where through resonance both the terminal and central carbons may have either both positive and negative charge for one resonance form or negative and positive charge for the other resonance form.

What these resonance forms show is that either carbon is susceptible to nucleophilic attack and we need a complementary, more powerful method capable of distinguishing between the reactivity of both carbons. It will be shown that HOMO-LUMO Interactions using ethene as the backbone for the aconitate molecules is able to distinguish between the two carbons even though both positions are susceptible to attack.

Picture of resonance forms

Picture of resonance forms

Reaction Mechanisms Description using HOMO-LUMO Interactions

Aconitic can be visualized as an ethylene group with multiple attachments. The CH2 groups at carbon 3 remain put since the basicity is not high enough to remove them and delocalizing the electrons as in the allyl anion. An MO description of this ethylene shows that there are two π orbitals, viz., π containing 2 electrons and π* containing none as depicted below:

Picture of allyl anion orbitals

HOMO-LUMO Interactions of (E) and (Z) Aconitate under near Neutral Weak Acid Conditions

In the previous paper we treated aconitate as a 4 π allyl anion system. Here aconitate will be treated as 2 π ethene system of either (E) or (Z) aconitic as the resonance forms above show . We will assume that both aconitics can react in a non stereo specific manner to give a mixture of diastereomers. Since this is a non enzymatic reaction both aconitics could be produced. Let's take a look at the HOMO and LUMO molecular orbitals, π and π*, of the ethene group of aconitate. In this case the LUMO of hydrogen σ* of water can interact with the lobes of both carbons 1 and 2. (Scheme I and Scheme II).

In a concerted manner the HOMO of the carboxylate anion on the adjacent CH2 can interact with the LUMO of the empty π* orbital of the ethene group via two routes. In Scheme I, if it interacts with the lobe on carbon 2 the result is the β-lactone of citric acid. It was mentioned above that the isocitrate β-lactone is stable when two carboxy esters are present. Such is not the case here and the formation of this 4 membered lactone is probably unlikely due to the high ring strain.

In Scheme II the HOMO of the carboxylate can interact with the lobe on carbon 1 to generate the γ-lactone, a highly stable product. This is probably the preferred route via a stable 5 membered ring closure to generate threo and erythro isocitrates, that are consistent with the halogenated route. So we have shown that the ethene HOMO-LUMO interaction with the terminal the CH2 carboxylate of the aconitates may proceed via a non stereospecific manner to generate the isocitrates, unlike citric acid which was produced via an alternative mechanism involving the allyl anion of cis aconitate.

Picture of resonance form allyl anion

Picture of resonance form allyl anion

So we may without a doubt say that aconitic has a split personality disorder - A Veritable Dr.Jeckyll and Mr. Hyde.

Summary of the Formation of cyclic isocitric lactones

We may summarize the hydration isocitric and alloisocitric salts as shown in the figure below. The cis aconitic affords threo isocitrate, while the trans aconitic affords erythro alloisocitrate. Since this is a non enzymatic reaction, both isomers as a mixture of dl pairs, are possible.

Picture of resonance form allyl anion

Go back to homepage.. To go to Part III Non-Enzymatic Citric Acid Dehydration.

Copyright © April 2015 by Eddie N Gutierrez E-mail: enaguti1949@gmail.com