Oxalacetic acid

Structures Related to Oxalacetic Acid:

Chemical name	Structure
2-ketosuccinic acid, 2-oxosuccinic acid (1)
2-hydroxyfumaric acid (2), Z-2-hydroxy-but-2-enedioic acid; CAS names: 2-Butenedioic acid, 2-hydroxy-, (Z)- (9CI); Fumaric acid, hydroxy- (7CI,8CI); Hydroxyfumaric acid; trans-Enol-oxalacetic acid CAS: 6153-53-3
2-hydroxymaleic acid (3) E-2-hydroxy-but-2-enedioic acid hydroxybutenedioic acid CAS names: 2-Butenedioic acid, 2-hydroxy-, (2E)-; 2-Butenedioic acid, 2-hydroxy-, (E)-; Maleic acid, hydroxy- (6CI,7CI,8CI); Hydroxymaleic acid CAS number: 1115-67-9
2,2-dihydroxysuccinic acid (4)

Enolization

The keto (1) and enol (2) form in solution is in equilibrium. Previous reports on the existence of 2-hydroxymaleic acid (3) had not been confirmed, but rather proved the existence of enol form by 1H- and 13C-NMR studies and by tandem mass spectrometric studies [1].

In solid phase, the enol form is the dominant species based on IR investigations [2].

UV/VIS spectroscopic analysis and IR spectrum showed that in ethanol, water, and diethyl ether the enol form is the predominant species (and more stable). The energy barrier of the enolization reaction is calculated to be very high [3].

The enol form absorbs strongly in the 250-265 nm region where most keto- and amino-acids do not absorb. This provides the possibility for its direct quantitative determination in the presence of L-aspartic, L-glutamic, and alpha-ketoglutaric acids between pH 7-9 in phosphate and tris(hydroxymethyl)aminomethane) buffers. Calibration curves can be costructed. Absorbance at 260 nm was found to be linear over the concentration range of 1x10-5 - 5x10-3M [4].

Even though the energy barrier between the keto and the enol form is high on the basis of calculations [3], there are some evidence that enolization might occur during a hydration of the keto group.NMR and polarographic studies shown, that 87% of oxalacetic acid and its Et ester, and 93% of di-Et oxalacetate are in the hydrated form (4). Hydration and acid-base catalyzed dehydration results in pH-dependence of polarographed reduction currents. The rate of keto-enol equilibrium is much slower than the rate of hydration-dehydration. The stability of the 2,2-dihydroxysuccinic acid (4) can be rationalized on the basis of extensive hydrogen bonding inside the molecule [5].

In biological systems, special enzymes might be responsible for the quick and efficient transformation of the enol to the keto form. In additon to catalyzing the fumarase reaction, Fumarase A, a product of the fumA gene of Escherichia coli, catalyzes the isomerization of enol to keto oxalacetic acid. The kcat/Km for the isomerization was almost identical to that for the fumarase reaction. However, porcine fumarase, isopropylmalate isomerase, and dihydroxyacid dehydratase did not catalyze this isomerization [6].

Biological Role

Oxalacetic acid is an intermediate of the citric acid (Szent-Györgyi - Krebs) cycle and gluconeogenesis. In the discovery of this cycle Albert Szent-Györgyi, a Hungarian Scientist had a major role and won the Nobel Prize in Physiology or Medicine in 1937 for the discovery of Vitamic C, found in high concentration of the Hungarian paprika in its unripen stage.

Oxalacetic acid in the Szent-Györgyi - Krebs cycle combines with acetyl-coenzyme A to yield citric acid. It is also an important component of the intercyclic pathway between the Embden-Meyerhof cycle and the Szent-Györgyi - Krebs cycle.

For more information on what type of enzymes can make or transform oxalacetic acid see the biocyc.org website where a collection of 371 Pathway/Genome Databases can be found.

The decarboxylation of oxalacetic acid yields pyruvic acid and carbon dioxide. This reaction produces energy. Keto acids, like pyruvic and oxalacetic acid are frequently added to tissue culture media formulations to maintain maximum cell metabolism and open certain biologically shunted pathways.

For more information see the Wikipedia [7].

Physical data:

Hydroxyfumaric acid (trans enol form, Z)
Properties: Crystals from acetone and benzene, melting point: 184°C. pKa at 17°C: 2.76 x 10-3. Soluble in water, ethanol, ether. [8].
Can be converted into the lower-melting cis-form, mp 152°C, by dissolving the acid in water and re-isolating it as rapidly as possible.

Hydroxymaleic acid (cis enol form, E) [9]
Properties: Crystals from ethyl acetate + carbon tetrachloride, mp 152°C. pKa at 17°C: 2.505 x 10-3. Soluble in ethanol, acetone, ethyl acetate. Sparingly sol in ether.
Melting point: mp 152°C [8].

pKa: pK1=2.22; pK2=3.89; pK3=13.03(enolic OH) (25°C, 0.1 M KCl) [11];

Due to the thermal instability of oxalacetic acid, the melting point observed during the determination is strongly dependent on the heating rate of the capillary tube (average melting point determination). Therefore the published melting point data are difficult to compare.
The melting point of the commercial preparations are generally above 160°C, the range is typically between 165-175°C. The compound decomposes during the melting point determination, the rate of heating the the starting point of the melting point determination is crucial.

Condensed phase thermochemistry data can be obtained from the NIST website.

Spectra:

The FT-IR spectrum of oxalacetic acid liquid can be accessed from Sigma-Aldrich website from here.

The FT-Raman spectrum of oxalacetic acid can be downloaded from Sigma-Aldrich website from this link here.

The UV-VIS spectrum, the mass spectrum of oxalacetic acid is available from NIST.

Stability:

Oxalacetic acid is known to decompose in solution even at 0°C. The decomposition increases as the temperature increases. In food processing, even traces of oxalacetic acid does not remain in the precessed food [10]. This sensitivity is one the highest among the oxocarboxylic acids. pH, metal ions (Cu2+, Fe3+, Li+) and several type of amines have strong effect on the decomposition rate. Therefore the quality of the oxalacetic acid used in cell culture is essential.

The storage stability is largely depends on the purity and the contaminants present in oxalacetic acid. High quality oxalacetic acid is recommended to be stored and ship below 10°C, however, lower quality materials and long term storage term require storage temperatures below -10°C.

Characterization of Quality and Quantitative Determination of Oxalacetic Acid:

The sensitivity of the oxalacetic acid limits the techniques used for the analysis methods used in the purity determination.

• The titrimetric (acidimetric) assay sensitively indicates its purity since the primary decomposition route, decarboxilation, yields pyruvic acid.
• The water content of the oxalacetic acid is also a critical parameter. The sum of acidimetric assay and the water content should approach to 100%, otherwise the material is already decomposed. If Karl Fischer water determination is employed, the rate of the titration and the program may affect the results: iodine slowly reacts with the enol form which might yield higer water assay values.
• The color index of the solution (generally provided in Hazen) is a sensitive measure of the metal content and the progress of decomposition. By time and with decomposition (even in solid form) oxalacetic acid turns to be darker and darker, and the Hazen number increases.

Oxalacetic Acid Uses:

Synthetic uses:

• Oxaloacetic acid is a valuable intermediate in the synthesis of optically active benzylamino acids [12],
• different type of heterocyles [13], [14],
• can be selectively alkylated obtaining otherwise difficult-to-obtain carboxylic acids, like allyl derivatives [15].

In biochemistry and cell culture laboratories oxalacetic acid is an ingredient in assays and also a cell culture media component.

• A substrate for use in Malic Dehydrogenase UV assay [16] and several other type of assays.
• Oxalacetic acid is an increasingly important component of serum-free cell culture media especially in the medium of mammalian cells [17], [18], [19], [20].

See also what Sigma-Aldrich says about this compound in its product information sheet.

Qualities of oxalacetic acid

Oxalacetic Acid Manufacturers, Distributors

References:

1. a) Enol oxalacetic acid exists in the Z form in the crystalline state and in solution. Flint, Dennis H.; Nudelman, Abraham; Calabrese, Joseph C.; Gottlieb, Hugo E. Journal of Organic Chemistry (1992), 57(26), 7270-4. b) The intriguing behavior of (ionized) oxalacetic acid investigated by tandem mass spectrometry.     Fell, Lorne M.; Francis, James T.; Holmes, John L.; Terlouw, Johan K.   International Journal of Mass Spectrometry and Ion Processes  (1997),  165/166  179-194.
2. The vibrational spectra and structure of oxaloacetic acid. Schiering, David W.; Katon, J. E. Journal of Molecular Structure (1986), 144(1-2), 71-82.
3. DFT study of oxaloacetic acid condensation - The first step of the citric acid cycle. Delchev, V. B.; Delcheva, G. T. Journal of Structural Chemistry (2007), 48(4), 615-622.
4. Spectrophotometric determination of oxalacetic acid in the presence of keto- and amino-acids. Hussein, Wedad R.; Carr, DeAndre' M. Analytical Letters (1997), 30(7), 1267-1277.
5. a) Carbon-13 nuclear magnetic resonance spectra of the hydrate, keto and enol forms of oxalacetic acid. Buldain, Graciela; De los Santos, Carlos; Frydman, Benjamin.  Magnetic Resonance in Chemistry  23(6),  478-81 (1985).  b) Polarographic reduction of aldehydes and ketones. Part XXX. Effects of acid-base, hydration-dehydration and keto-enol equilibria on reduction of a-ketoglutaric and oxalacetic acid and their esters. Kozlowski, J.; Zuman, P. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 226(1-2), 69-102 (1987).
6. Escherichia coli fumarase A catalyzes the isomerization of enol and keto oxalacetic acid. Flint, Dennis H. Biochemistry 32(3), 799-805 (1993).
7. See the following Wikipedia articles: Oxalacetic acid; Szent-Györgyi - Krebs (citric acid) cycle; Vitamin C
8. Budavari, The Merck Index, 13th Edition, monograph number 6978, O'Neil,, M.J. The Merck Index, 14th edition, monograph number 6909.
9. Heidelberger, Biochem. Prepn. 3, 59 (1953).
10. Decomposition of organic acids during processing and storage. Chu, N. T.; Clydesdale, F. M.      Journal of Milk and Food Technology  39(7),  477-80 (1976).
11. Data for Biochemical Research, 3rd ed., Dawson,R. M. C., et al., Oxford University Press (New York, NY: 1986), pp. 46-47.
12.  The First Highly Enantioselective Homogeneously Catalyzed Asymmetric Reductive Amination: Synthesis of a -N-Benzylamino Acids.      Kadyrov, Renat; Riermeier, Thomas H.; Dingerdissen, Uwe; Tararov, Vitali; Boerner, Armin.    Journal of Organic Chemistry  68(10),  4067-4070 (2003). 
13.  A New Substrate for the Biginelli Cyclocondensation: Direct Preparation of 5-Unsubstituted 3,4-Dihydropyrimidin-2(1H)-ones from a b -Keto Carboxylic Acid.      Bussolari, Jacqueline C.; McDonnell, Patricia A.     Journal of Organic Chemistry  65(20),  6777-6779 (2000). 
14. Hexafluoroacetone as protecting and activating reagent: reactions of hexafluoroacetone with a -keto acids.      Spengler, Jan; Osipov, Sergej N.; Heistracher, Elisabeth; Haas, Alois; Burger, Klaus.   Journal of Fluorine Chemistry    125(6),  1019-1023 (2004).
15. The efficient allylations of 2-oxocarboxylic acids. Synthesis of 2-allyl derivatives of 2-hydroxycarboxylic acids.      Kumar, Subodh; Kaur, Pervinder; Chimni, Swapandeep Singh.      Synlett  (2002),   (4),  573-574.
16. A unique series of lymphomas related to the Ly-1+ lineage of B lymphocyte differentiation. Davidson WF, Fredrickson TN, Rudikoff EK, Coffman RL, Hartley JW, Morse HC 3rd. J Immunol. 133(2):744-53 (1984).
17. A fully defined, clear and protein-free liquid medium permitting dense growth of Neisseria gonorrhoeae from very low inocula.      Wade, Jeremy James; Graver, Michelle Angela.  FEMS Microbiology Letters  273(1),  35-37 (2007). 
18. Biosynthesis of pseudoisoeugenols in tissue cultures of Pimpinella anisum. Phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase activities. Reichling J, Kemmerer B, Sauer-Gürth H. Pharm World Sci. 17(4):113-9 (1995).
19. Investigation on response of the metabolites in tricarboxylic acid cycle of Escherichi coli and Pseudomonas aeruginosa to antibiotic perturbation by capillary electrophoresis. Peng Gao, Chunyun Shi, Jing Tian, Xianzhe Shi, Kailong Yuan, Xin Lu and Guowang Xu. Journal of Pharmaceutical and Biomedical Analysis Volume 44(1) , 180-187 (2007).
20. GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site. David Dishart, Nicole Schnur, Niko Klan, Oliver Werza, Dieter Steinhilber, Bengt Samuelsson and Olof Rådmark. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1738(1-3) 37-47 (2005).

This information is provided as a reference by Reanal Finechemical Private Ltd., one of the largest producer of high quality oxalacetic acid for biochemical and cell culture use for more than 20 years.

If you need further information, or would like to provide more information on the use, properties, physical, chemical behavior of oxalacetic acid, please please contact us at salesreanal.hu.