Decarboxylation of a-Keto Acids R. W. Hanson Department of Biological Sciences, Plymouth Polytechnic, Plymouth. Devon, UK It is well established that some of the most important reactions that a-keto acids, RCOCOzH (I), undergo during metabolism involve decarhoxylation ( I ) . For example pyruvic acid (1, R = CH3) is decarhoxylated nonoxidatively during alcoholic fermentation PD
CH,COCO,
+ Hi * CH,CHO + CO,
(1)
(PD = pyruvate decarhoxylase) and in the biosyntbesis of acyloins such as acetolactate,
be drawn from this is that the nonenzymic decarboxylation of a-keto acids is difficult to accomplish and has not been investigated extensively.' At least two authors, however, make statements which contradict this. Thus March (3) includes wketo acids in a table of "some acids which undergo decarhoxylation fairly easily", while Finar (4) is more specific, stating that "pyruvic acid is easily decarboxylated with warm dilute sulphuric acid" and suggests that the reaction proceeds via a carhene (eq 5),
ALS
2CH,COC02
+ Ht = CH,COC(CH),(OHICO,- + CO,
(2)
(A1.S = acetolacrate synrhetase~.Additionally, pvruvir arid nn(l n - k p t w l ~ ~ t n racid i c r l . R = -O,CCI-IqCH?) . . undwzo oxidative dec~rhoxylationwhen they-are converted to acetylcoenzyme A and succinylcoenzyme A, respectively, CH,COCO,-
+ NADt + HSCoA PDH + CH,COSCoA
~O,CCH,CH,COCO,-
-
a-KGDH
+ NADH + CO,
(3)
+ NADt + HSCoA -O,CCH,CH,COSCoA
+ NADH + CO,
(4)
(PDH = pyruvate dehydrogenase complex; a-KGDH = a ketoglutarate dehydrogenase complex). The enzymes that catalyze reactions 1-4 all have an absolute requirement for thiamine pyrophosphate (TPP),
as coenzyme. Almost every undergraduate textbook of hiochemistry contains an explanation of how T P P is involved in the decarboxylation of a-keto acids, hut the explanation is rarely accompanied by a comparative discussion of analogous nonenzymic reactions, which is essential if the student is to gain a proper understanding of the chemical role of the coenzyme. Unfortunately, information regarding the nonenzymic decarhoxylation of ru-keto acids is not readily available. The recent review of the synthesis and properties of a-keto acids by Cooper, Ginos, and Meister ( 2 ) might he expected to provide such information. Three of the four papers cited in the review under the heading "thermal decarboxylation" refer, however, to decarhoxylations catalyzed by metal ions (the fourth refers to catalysis by cyanide, below). The mechanisms of these reactions are unclear and therefore provide no insight into the mode of action of TPP. The situation regarding textbooks is even worse. Most textbooks of organic chemistry ignore a-keto acids entirely or make no specific reference to their decarhoxylation. The logical conclusion to
As will be shown subsequently available evidence does not support these statements. The object of this article is to rectify the lack of information concerning the nonenzymic decarhoxylation of a-keto acids found in many textbooks and to correct errors in others. Nonoxidative Decarboxylatlon Thermal Decarboxylation Laneenbeck and Hutschenreuter ( 5 ) found that heatine pyruvic acid under an atmosphwr of nitrogen for 15 min a t 13: O C ~ r o d u c e dno acetaldehvde lai the u-nitronhmvlhvdrazon;). The pyrolysis of hen&ylf&mic &id (phenyl&yokcylic acid, I, R = CsH5) under an atmosphere of carhon dioxide or of nitrogen a t temperatures between 250 OC and 300 OC was investigated by Hurd and Raterink (61, who found that the identifiable products were carbon monoxide (20%),carhon dioxide (50%),benzaldehyde (-22%) and heuzoic acid (-55%). Phenylpyruvic acid (1, R = C6H5CH2)was found to give carbon monoxide (-60%), carhon dioxide (-44%), and phenylacetic acid (-35%), but no aldehyde. In a more recent study Ahmad and Spencer (7) found that 10 a keto acids were not decarboxylated when each was heated in boiling water for 25 min under nitrogen. The conditions used in these studies would cause rapid and complete decarhoxylation of p-keto acids (8). Indeed, the conditions used by Hurd and Raterink would decarhoxylate most types of carboxylic acid, and it is clear that, in relative terms, a-keto acids are thermally stable. This conclusion is reinforced by t h e observation t h a t , if a dicarboxylic acid, such as 2-oxalopropionic acid (I, R = HOZCCH(CHJ)),in which one carboxyl group is a and the other 0 to a carhonyl group, is heated, i t is the 8-carhoxyl group that is lost (9).
' Cf.,the easy decarboxylation of p-keto acids, which is discussed
in virtually every textbook of organic chemistry.
Volume 64
Number 7 July 1987
591
Decarboxylationin Acid Solotion
In 1884 Beilstein and Wiegand (10) reported that acetaldehyde and carhon dioxide were produced when pyruvic acid was heated with dilute sulfuric acid. This report has subsequently been quoted by a number of authors and presumably is the basis of Finar's statement that pyruvic acid is easily decarboxylated. Reference to Beilstein and Wiegand's original paper reveals, however, that the reactants were heated in a sealed tube for 5 h a t 150 O C and that the yield of products was not determined. Instead, the reaction mixture was made weakly alkaline and distilled; the distillate "gab eine sehr starke Reaction auf Aldehyd". Because the conditions used were vigorous, rather than mild, and because a comparatively small amount of aldehyde could give a "very strong" reaction, it is clear that the results of the experiment do not warrant the conclusion that pyruvic acid is easily decarboxylated by warm, dilute, sulfuric acid. At the lower temperature of 100 OC many other a-keto acids are demonstrably stable in aqueous acid as evidenced by the fact that they can be synthesized by routes that involve acid-catalyzed hydrolysis of the immediate precursor of the a-keto acid in question ( 2 , I I ) .
The free acid undergoes decarboxylation approximately 53 times faster than its anion ( 8 ) despite the fact that the presence of a proton on the carboxylate group would he expected to hinder loss of carhon dioxide. In this case it appears that the proton is probably transferred to the oxygen of the P-carbonyl group via a low-energy, formally neutral, six-membered transition state that collapses to an enol, (10).
Mechanism of Decarboxylation
The resistance of a-keto acids to thermal decarhoxylation is explicable in terms of the mechanism that is currently accepted for the reaction (8, 12). Many carboxylic acids undergo decarboxylation by unimolecular heterolysis of the anion,
R?
:a
C4
= R:- + COz
The intermediate produced during the reaction is a carbanion; decarhoxylation would, therefore, be expected to be facilitated by the presence of a group, within the side chain of the acid, capable of stabilizing the carhanion by charge delocalization, and this is found to be so. Thus, whereas sodium acetate must he fused with soda lime to effect its decarboxylation
CO,
+ HO- = HCO;
Other carhoxylic acids which are easily decarboxylated due to the presence of unsaturation p to the carboxyl group are cyanoacetic acid,
and 2- and 4-pyridylacetic acid and other similar heterocyclic acids. From the fact that the last two acids are so easily decarboxylated as to he difficult to isolate, i t is clear that protonated nitrogen, either in the Bposition or conjugated to it, is a very effective electron sink. The reactive species in the case of 4-pyridylacetic acid is thought to he the zwitterion,
nitroacetic acid is rapidly and quantitatively decarboxylated by heating an aqueous solution of the sodium salt at 80 "C,
A suitably placed carbonyl group is also very effective in promoting decarboxlation as evidenced by the chemistry of 0-keto acids such as acetoacetic acid. In this case both the anion and the free acid loose carhon dioxide. Decarhoxylation of the anion is thought to proceed via a resonancestabilized enolate, 592
Journal of Chemical Education
while, in the case of the 2-pyridyl acid, the proton chelate may he involved. As pointed out by Walsh, (13)the carhonyl group is an a-keto acid is in a position where i t cannot delocalize negative charge arising from decarboxylation. The anion of an a-keto acid would generate an acyl anion 3,
while an internally hydrogen bonded free acid would give rise to a formally neutral intermediate akin to an ylide, 4.
Both would he high-energy species because delocalization of change to oxygen would create a partially electron deficient carbon atom and hence would be severely restricted, making decarboxylation difficult. Intermediate 4 would also be expected to be produced during decarhoxylation in acidic solution (eq 5). Here, an additional harrier to reaction would he the low basicity of oxygen. Catalysis of Nonoxldatlve Decarboxylatlon
Primary amines that possess a p-carhonyl group were found to he much more effective than simple amines as catalysts 'for the decarhoxylation of a-keto acids. The most active compounds of this type are derivatives of 3-amino-2-oxindole,
The parent compound is claimed to he about 650 times more active than ethylamine. Again, catalysis is thought to be effected by formation of a Schiff base. In these derivatives, however, the p-carbonyl group is thought to promote a prototropic shift, possibly via a cyclic transition state. The product of the shift (8) contains an unsaturated system B to the carhoxyl group, and it is this structural feature that facilitates decarboxylation:
Catalysis by Amines (14, 15) The extensive studies of Langenheck and his co-workers, and of others. have shown that ~ r i m a r vamines facilitate the decarhoxylation of a-keto acids in aqueous solution, or in anhvdrous nhenol. a t tem~eraturesbetween 50 'C and 170 "C. Simple primary amines, ahirh are poor carnlystli, probahlv orodure s raitterionir Schiff hase in which the posirivrly chaiged nitrogen forms an effective electron sink, ~~
~~
5
RCHO
5
+ HxNRL
8
6
+ RCHO
+ RCHO
Because nitrogen is considerably more basic than oxygen, the concentration of the nitrogen ylide produced would he considerably larger than the concentration of the analogous oxygen ylide (4), thus accounting for the catalytic effect of the amine. In one sense, however, the term catalytic is inappropriate because, regardless of whether the reaction is performed in water or in phenol, the amine is quickly consumed via side reactions. Support for the involvement of a nitrogen ylide in the catalysis is provided by the fact that species of this type have been trapped during the decarhoxylation of a number of heterocyclic acids in which the carboxyl group is P to a nitrogen atom, the Hammick reaction (81,
- RCHO
In comparison with primary amines the effect of secondary and tertiary amines on the decarhoxylation of a-keto acids has not been extensively investigated. Brown (16) claims that secondary amines produce catalysis hut cites no supporting evidence. It has long been known that quinoline catalyzes the decarboxylation of many types of carboxylic acid (17). Clark and others (18) have shown that the tertiary amine promotes the decarhoxylation of both a- and 0-keto acids and their anions. The results of thestudies suggest that the base facilitates loss of carbon dioxide by formation of a bimolecular complex with the carbon atom of the carboxyl group of the acid (9),
Catalysis by Cyanide (14, 15) Catalysis of the decarhoxylation of a-keto acids, and of their conversion to acyloins, by cyanide was first investigatVolume 64 Number 7 July 1987
593
ed by Franzen and Fikentscher. Cyanide is a good nucleo~ h i l and e readily adds to the carbonyl group - of the acid. The an electron s i n k i n a position 6 to the adduct carboxyl group and hence is susceptible to easy decarboxylation. Finally, cyanide is a good leaving group; the aldehyde which results from loss of cyanide can react with the carbanion (lo), to give an acyloin, or can he trapped as the dimedone derivative:
dimedone derivative
'V CKCHO
(i) dimedone, aq. dioxan. 10%
CHRCOCH(OH) CHJ + CNCatalysis by Thiamine Pyrophosphate-Dependent Enzymes (13-15) The studies of the nonenzymic decarboxylation of a-keto acids reviewd in P r e ~ ~ i o t ~ ~ indicateihat s r ~ ~ t i ~ ~11) n ~this type ut acid is resistant tar thermal decarboxylation and (2) the process can be faciliated by introducing & electron sink into the acid, preferably bound to a 0 carbon by a multiple bond.% It appears that because none of the 20 amino acid residues commonly found in enzymes are capable of generating a suitable electron sink. evolution has nroduced a snecific coenzyme, T P P , to do so. Studies with'^^^-dependent holo: enzvmes. " . with T P P itself. and with model thiazolium salts suggest that the coenzyme functions in a manner analogous to cyanide, as shown in the reaction,
R;
, RS
The hydrogen atom a t position two in T P P has been shown to be weakly acidic and the evidence that is available suggests that the coenzyme is present in the active site of its apoenzymes as the 2-anion (11).This anion is a good nucleophile and, under the catalytic influence of the apoenzyme, adds to the carbonyl group of the a-keto acid. The adduct (12), which possesses an electron sink (positively charged nitrogen) attached by a double bond to the carbon @ t othe carhoxyl group, undergoes rapid enzymr-catalyzed derarb o x h t i o n . l'hr resonanre-stabilized carbanmn (13) that results from h s s of eurhon dioxide can then have at least three different fates depending on the specificity of the apoenzyme (or, in cases where the reaction is catalyzed by an enzyme complex, on the enzyme composition of the c&plex), viz.: (1) The carbanion may be protonated, and the 2-anion of TPP may
? An interesting variation of this prlncnpie is described in Ahmad and Spencer's paper (71These workers foundthat the oximes of 10 reoresenlarives o-keto acmdswere oecarooxvlaled to aive carbon triies in excellent yield when heated for 25 min in boilingwater. They suggest that the zwitterions react as shown,
the negative change resulting from decarboxylation being acccommodated by the leaving group. This type of reaction may have bioiogical significance in view of the fact that, recently, both Chlorella and spinach leaves have been shown to possess an enzyme that catalyzes the production of hydrogen cyanide from glyoxylate oxime (R = H) (2).
594
Journal of Chemical Education
then act as a leaving group, allowing the aldehydic decarboxylation product to diffuseaway from the enzyme (i, eq 6). (2) The apoenzyme may catalyze the addition of the intermediate carbanion to a carbanvl cram oresent in either the substrate akrro arid. ~healdehydir'drt~~rl;t>xyinric,n produrr, or an aldehvdir second st~bsrntr;rhar is, the carhanion moy pnrticipole in the formnrioll ofd!lv acyluin such as acelolactate ri, eq GI. (3) The carbanion may be protonated and the protonated form may then undergo two-electron oxidation as described in the next section. Oxldative Decarboxylatlon ( 13) As evidenced by the n u m l m of papers cited in the re\,ieu. hy Cooper. Cinos, and hleister (21 the nonenzymic oxidnttur decarhoxylation ufwketu acids has received more attention from researchers than has nonoxidative decarboxylation. Several mild oxidants, including ceric sulfate, lead tetraacetate, potassium permanganate, peroxyphthalic acid, and hydrogen peroxide (the last either alone or in the presence of base or of ferrous ions) are known to cause rapid, quantitative decarboxylation of the title compounds.
It would he interesting to relate the mechanisms of these nonenzymic reactions with the mechanisms of correspondine.. enzvme-catalvzed reactions as was done in the previous , iectiun for nonohidative decarhouylatiun. This, however, is not Dossible beca11.i~ the mechanisms hy which the nonen'ymic oxidative reactions occur appear, in many cases, to be complex and cannot be specified in detail. In contrast, the mechanism by which the enzyme-catalyzed oxidative decarhoxylation of a-keto acids occurs appears to he well authenticated. The two reactions (eqs 3,4) given in the first section that involve oxidation are catalyzed by similar enzyme complexes. Both contain three catalytic components, viz., a decarboxylase, a dihydrolipoamide transacvlase. The TPP. . and a dihvdrolivamide dehvdropenase. . dependent decarboxylase components catalyze decarboxylation of their substrates as described in the previous section. The carbanions (13) that are produced arethen protooated and the protonated materials (14) act as the substrates of the appropriate transacylase. The transacylases both utilize lipoic acid
0 R H~ ~ J
+
CoASH
RC0ShA
11
+
R1, R2, R3, R4.m in eq 6: R5 = (CH2)&ONHtransacylase; CoASH = coenzyme A Literature Clted 1. Strver. L. Biachemistrv. 2nd od.: Freeman; Ssn Frsneixo. 1975: Lehnineu, A. ~ i o ~ h r m i s l rW& y:
New York, 1970.
L.
2. Cooiler,A. J. L.: Cinoa, J. 2.; Meirter, A. Chom. Reus. 1983,83,321. 3. ~ a r e hJ., Orgonie Chemiaiy. 3rd ed.:Wiloy: New York. 1985; p 563. 4. Finar, I. L. Organic Chemistry, 6th ed.:Longmans: London, 1973; p 305. 5. Laneenheck. W.: Hutschenreuter.R. 2.Anorn Alleam. Chemie 1930.186.1.
as coenzyme; the cyclic disulfide is hound to the apoenzymes by an amide bond involving the 6-amino group of a lysine residue. In both cases the carbanion (13) is regenerated and the transacylase catalyzes its addition to lipoic acid. Expulsion of the T P P anion from the adduct generates an Sacyldihydrolipoamide and completes two-electron oxidation of the decarboxylated a-keto acid. Finally, the acyl group is transferred from the dihydrolipoamide to coenzyme A (eq 7). The remaining component of each enzyme complex, the dehydrogenase, is concerned with reoxidation of reduced lipoamide.
7. Ahrnad, A.:Speneer.I.D.Con. J. Cham. 1961.39.1340. 3. Brown. B. R.Quwt.Re". Chom. Soc. 1951.5,131. 9. Kubsia.G.: Marteii. A.E.J.Am. Chem Soe. 1981.103.7M9. . . 10. Beilst& F.: wie&d,E, Ber. 1884. 17,841. 11. Water.K.L. Chem.Reua. 1947.42,585. 12. Could, E. S.Mechanism and Structure in Organic Chemistry; Halt: London, 1959: p 3"a "7".
13. Wslsh, C . EnrymoticRmction Mechanism; heeman: San Franeirreo. 1979:p 683. 14. Bruiee, T. C.; Benkovie, S. Bioorgonic Mechonirms: Benjamin: Now Vork, 1966; Val. 11. Chaptor 8. 15. Bender, M . L. Meehonianu of Homogrnsous Catalysis from Protons to Pmfeim: Wiloy: New York, 1971: Chapter 6. 16. Brown, J. M. In Comprehenaiua Organic Chamisfry; Barton. D.: Ollis, W . D.. Eda.; Pergsmon: Oxford, 1979. 17. Fi8ser.L. F.;Fieser, M . Raogenfsfor OigonicSynfhasis: Wiley: New Y a k , 1962~975. 13. Clark. L. W.In The Chemistry of Carborylic Acids ond Esters; Patai. S..Ed.: Interscience: London. 1969:p 539.
Volume 64
Number 7
July 1967
595
