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A biosynthesis of dimethylpyruvic acid
A Biosynthesis of Dimethylpyruvic Acid 1 K. Ramachandran and T. K. Walker of Manchester, Manchester, England ReceivedOctober12, 1950
From the College of Technology, University
Hida (7) reported that in presence of sodium sulfite and ammonium chloride young preformed mycelial felts of Aspergihs niger produce pyruvic acid (PA) and dimethylpyruvic acid (DMPA) from glucose or from sucrose. The production of the former acid under these conditions is due probably to zymasis, but the sequence of processeswhich constitutes this phenomenon cannot account in a direct manner for the formation of dimethylpyruvic acid, and Hida’s experiments fail to throw light on this point. During the course of studies of acid formation in mold fungi, the authors found it necessary to carry out some experiments on the lines adopted by Hida, and confirmation of the latter’s results was obtained in trials with a representative selection of strains of A. niger. Omission of ammonium chloride from the medium left the yield of pyruvic acid unaffected but caused that of dimethylpyruvic acid to fall to a trace. This suggested that ammonium chloride acts catalytically to effect a condensation involving a carbonyl group. Examples from organic chemistry can be cited in support of this idea; for instance, the use of ammonium chloride as a catalyst for the condensation of R. CHO with 2R’.OH, yielding an acetal R-CH(OR’)2 (6), and the use of ammonium acetate or piperidine acetate to promote condensation of R-CO-R’ with CN-CH,COOGHs, to yield R-C(R’) = C(CN)COOGHs. Small amounts of ammonia or of certain secondary or tertiary bases are also employed to promote condensation of -CO-CH2-COwith aldehydes (8). Bearing this in mind and taking into account the facts that most strains of A. niger can form citric acid from sugar and that the action of A. niger on citric acid can give a mixture of acetonedicarI a-Oxoisovaleric acid. 224
DIMETHYLPYRUVIC
ACID
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boxylic acid, acetone, and malonic acid (3), it seemed probable that the first stage in the synthesis of dimethylpyruvic acid might consist of a condensation between acetonedicarboxylic acid (or acetone) and a substance containing an activated -CHzgroup, for example, pyruvie acid, acetaldehyde, or malonic acid. In favor of this view were the the additional facts that pyruvic acid may occur as one of the products of the action of A. niger on sodium citrate (authors’ unpublished observation) and that pyruvic acid is produced along with dimethylpyruvic acid under the conditions adopted by Hida. Much experimental work failed, however, to furnish evidence that citric acid can act, either alone or in the presence of pyruvate, ethanol or acetate, as a precursor of dimethylpyruvic acid; further, experiments in which mixtures of acetonedicarboxylic acid (or acetone) with pyruvate, ethanol, or acetate, were submitted to the action of the mold, were likewise fruitless. The possibility that amino acids from autolyzed mycelial felt might be oxidatively deaminated, or transdeaminated, to yield dimethylpyruvic acid, was then tested. In separate experiments nn-valine, nn-alanine and sodium glutamate were exposed to the action of the mold under appropriate conditions. These three amino acids correspond, respectively,’ to dimethylpyruvic, pyruvic, and a-ketoglutamic acids. In each case the result was negative. Similarly, an experiment in which the substrate was a mixture of nn-valine with the sodium salts of glutamic and pyruvic acids, failed to yield dimethylpyruvic acid. In the meantime we found in other experiments that a mixture of pyruvic and dimethylpyruvic acids could be obtained when xylose was substituted for glucose under Hida’s experimental conditions. This indicated that a condensation involving a triose might initiate the sequence of changes giving rise to dimethylpyruvic acid, because there is evidence that pentoses can undergo enzymic scission to molecules containing, respectively, two and three carbon atoms (1,9,11,13). Since glycerol can be converted to dihydroxyacetone by A. niger (2) and to a mixture of dihydroxyacetone, pyruvic acid, and ethanol by the mold Fusarium lini Bolley (5), experiments were performed with glycerol as substrate and these afforded pyruvic and dimethylpyruvic acids in greater yields than did xylose. Finally, it was observed in a single experiment with sodium malonate that the yields of both acids were improved very slightly when this salt, at a certain concentration, was added to the glycerol medium. Subsequently, it was discovered that very considerable increases occurred in the yields of both acids when sodium acetate
226
K. RAMACHANDRAN
AND T. I(. WALKER
was used in place of the malonate. While, in the absence of additional evidence, the slight increase in yields obtained in the single experiment in which malonate was added must be regarded as of doubtful significance, no such reservation is necessary with respect to the verdict on the influence of acetate, reproducibility of results being noted in four experiments. EXPERIMENTAL
(a) Particulars
of the Strains and of the Cultural Conditions Employed
The strains of A. nfger used in these experiments were designated Nl, AC. 1, Tl, T2, B7, B18, N.R.R.L. 67, N.R.R.L. 599, and N.R.R.L. 607, and all had shown capacity to produce citric acid from sugar. They were grown in the aqueous solution of inorganic salts recommended by Currie (4) with addition of glucose (10 g./lOO ml.). This medium, unless otherwise stated, was contained in conical flasks each.of X00-ml. capacity. These had necks of 40 mm. diameter and the volume of liquid per flask was 250 ml. and the depth was 25 mm. After sterilization in steam for 20 min. on three successive days the contents of the flasks were sown with spores and incubated at 30” for a period of time varying, according to the rate of growth, from 48 to 72 hr. When it was noted that sporulation was commencing, usually after 72 hr., the liquid was poured away from beneath the mycelial felts and was replaced, under aseptic conditions, first by sterile water for 12 hr. and, afterwards, by Hida’s inorganic medium to which had been added an appropriate quantity of sugar or other source of carbon. Hida’s medium contains: 1 g. NH&l, 2 g. H&Pod, and 5 g. Na80a, in water to 100 ml. It was pasteurized at 70” for 15 min. and was introduced beneath the felts by means of a sterile pipet. The time (12 hr.) during which the young felts were allowed to stand over water, before the latter was replaced by Hida’s medium, was found adequate to ensure that the glucose in the cells was metabolized. Control experiments, in which felts were incubated over sterile water, were always performed, however, to ensure that the results observed, when replacing the original glucose medium by substrates other than sugar, were not due to traces of sugar retained in the cells of the mold. Incubation of these preformed felts over the substrates undergoing test was carried out at 30”, aliquots being withdrawn daily and tested with a solution of 2, 4dinitrophenylhydrazine in 2 N HCI. By this test it was possible to judge approximately the time at which production of ketonic products was optimal, and precipitation of the maximum quantities of ketonic products, by this reagent, occurred usually on or about the sixth day. At this stage the contents of the flasks were worked up.
(b) Method of Isolating
and Estimating
the Products
To an aliquot of the filtered medium, half its volume of dilute HCI was added and the whole was heated in a boiling water bath for 2 min. to destroy excess sulfite and to break up its addition compounds. The flask containing the sample was then cooled and an excess of the dinitrophenylhydrazine reagent was added. After 30 min. the precipitate was collected on a Btichner funnel. Separation of the mixed derivatives was effected by taking advantage of the fact that their solubilities in NazCOs solution
DIMETHYLPYRUVIC
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227
depend on the temperature. Thus, the derivative of pyruvic acid (PA) was found to be completely soluble in a dilute aqueous solution of Na&03 at lo”, while the derivative of dimethylpyruvic acid (DMPA) dissolved much less readily in this reagent at this temperature. Above 70”, however, the DMPA derivative dissolved freely in the carbonate solution. The mixed derivatives were triturated with cold 2 N Na2C03 and filtered with suction; the insoluble part was washed with further portions of cold carbonate solution. The filtrates, after mixing, were acidified and the 2, 4dinitrophenylhydrazone of the PA, which separated as a yellow substance, was collected, washed with dilute HCI, then with water, and crystallized from ethanol. The insoluble material left on the filter was mixed with more Na&03 solution and heated on the water bath, yielding a brown solution which, after filtering from any insoluble residue, was acidified. The derivative of the DMPA then separated and, after washing with dilute HCl, it was recrystallized from ethanol. The melting point of the 2, 4-dinitrophenylhydrazone of PA was 218”, alone or mixed with a specimen prepared from authentic material. The melting point of the 2, 4-dinitrophenylhydrazone of DMPA was 1945’, alone or mixed with an authentic specimen prepared from dimethylpyruvic acid which had been synthesized by the method of Ramage and Simonsen (10). When working up larger volumes of metabolism solution and when carrying out estimations of the yields of the keto acids in such larger volumes, the following procedure was adopted. The liquid was filtered and its volume was ascertained, after which it was evaporated at 50”, under reduced pressure, to about one-fifth of its original volume. Ketonic substances were not detected in the distillate and this was rejected. To the concentrated liquid there was then added sufficient 5 N H&O, to provide a 10% excess over the quantity required to decompose the sulfite initially present in the medium. A careful distillation under reduced pressure was then performed and the distillate was collected through a water condenser. The bulk of the DMPA passed over before the PA but efficient separation of the two acids could not be attained in this way, and at the end of the distillation a little water was added to the dry residue in the flask and was, in turn, distilled over and added to the first distillate. The two keto acids in this distillate were then precipitated with 2, 4-dinitrophenylhydrazine and separated as already described. The derivatives, after washing and drying at llO”, were weighed. The residual dry mixture of salts left in the distillation flask invariably contained a little PA. This residue was therefore taken up in the least volume of distilled water necessary to dissolve it, and the solution, after filtration, was treated with a fresh quantity of the dinitrophenylhydrazine reagent. The PA and any other ketonic bodies present formed derivatives, but since that of PA was deposited within 2 hr. while other material required longer to deposit, the precipitate of the PA derivative was filtered off after 2 hr., and was purified and estimated along with the main quantity of that compound. This method allowed a rough estimate to be made of the quantities of the two keto acids in the mixed product. The method was checked in experiments with artificial mixtures of the two acids and the results showed that, for the purposes of the present exploratory work, it could be considered satisfactory. In the course of the preliminary work it was found that if the ammonium chloride was omitted from the mixture of salts recommended by Hida the yield of PA was undiminished but that of DMPA fell to a very low level.
228
K.
RAMACHANDRAN
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WALKER
RESULTS
In descending order of capacity to form the keto acids from glucose, the different strains of A. niger fell into the following grading: Nl, AC. 1, B7, B18, N.R.R.L. 607. The results obtained by use of Tl, T2 and N.R.R.L. 599 were moderate only, while N.R.R.L. 67 proved unreliable. Strain AC. 1 gave mainly PA with traces only of DMPA. The other strains formed more of the latter acid than of PA. In most of the experiments Nl was employed. Formation of Pyruvic and Dimethylpyruvic
Acids from Xylose
In this case, and in all the other experiments now to be recorded, felts of the mold (Nl) were first developed on the solution of glucose in Currie’s medium of inorganic salts. The replacement solution consisted in this case of xylose dissolved at a concentration of 4 g./lOO ml. in the solution of Hida’s salts. Test portions withdrawn from the flasks showed maximum concentration of products at 6 days from the time when the xylose was placed beneath the felts. Two acidic products were isolated and yielded 2, Pdinitrophenylhydrazones which melted at 218” and 194”, respectively. The derivative of m.p. 218’ also melted at this temperature when in admixture with the 2, 4-dinitrophenylhydrazone of authentic pyruvic acid and, similarly, a mixture of the derivative of m.p. 194’ with authentic 2, 4-dinitrophenylhydrazone of dimethylpyruvic acid also melted at 194“. (Found, for derivative of m.p. 218’: N, 20.7. Calcd. for C&HsOeNd: N, 2O.9o/o.Found, for derivative of m.p. 194’: N, 18.7. Calcd. for CUH~~O~NGN, 18.8yo.) Formation of Pyruvic and Dimethylpyruvic Acids from Glycerol and Identijkation of Dihydroxyacetone as an Intermediate Product
The culture medium to be placed beneath the preformed felts was prepared by adding glycerol (5 g./lOO ml.) to Hida’s solution of salts. Several experiments were performed in flasks of different sizes and the quantities of medium varied in the several cases from 200 ml. to 1000 ml., but the depth of medium in each flask was always 25 mm. The contents of the different flasks were worked up for isolation of products at periods varying from 6 to 9 days from the time of replacement of the medium, the time chosen being at the stage of optimum yield of prod-
DIMETHYLPYRUVIC
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ucts as shown by daily tests on aliquots of medium. In all cases both PA and DMPA were found to be present as shown by the isolation of 2, 4-dinitrophenylhydrazones of m.p. 218” and 194-5”, respectively, and in each case the melting point was unchanged in admixture with authentic material. (Found, for derivative of m.p. 218’: N, 21.2. Calcd. for CsHsOeNd: N, 20.9%. Found, for derivative of m.p. 194-5”: N, 19.2. Calcd. for C,lH1206N4: N, l&8%.) During the isolation of these two acids as their 2, 4-dinitrophenylhydrazones it was found that the distillate contained traces of a substance giving with 2, 4-dinitrophenylhydrazine a derivative which was insoluble in sodium carbonate solution. It was insoluble also in ethanol and, after being washed by this solvent and dried, its color was a dull yellow and it melted at 279-81”. The quantity was insufficient to permit further purification. More of the substance was found to be present in the distilling flask after all the PA had passed over. The substance reduced Fehling’s solution. As these observations indicated the presence of dihydroxyacetone (or glyceraldehyde) the residue in the distillation flask was lixiviated with a little water and, after filtration, the solution was treated with an excess of a solution of 2, 4-dinitrophenylhydrazine in hydrochloric acid. A red precipitate settled and was filtered off and washed, when it was found to be partially soluble in hot ethanol. The crystals obtained from the ethanol were in two crops. The first of these melted at 250-5” and the second crop melted over the range 155-180”. These two substances were set aside for future examination. The major portion of the red precipitate was insoluble in ethanol but it dissolved readily in a mixture of pyridine and glacial acetic acid, giving a dark red solution from which, on cooling, minute, orange-red crystals were deposited. After further recrystallizations these had m.p. 282’. A specimen of Merck’s dihydroxyacetone was converted to its 2, 4-dinitrophenylosazone and this had m.p. 283”. When it was mixed with the derivative obtained from the cultures the m.p. was 282”. (Found, for derivative of m.p. 282”, from the cultures: N, 25.6. Calcd. for GHrzOeNs: N, 25.0s.) Hence, dihydroxyacetone is formed during the conversion of glycerol to a mixture of PA and DMPA. This identification of dihydroxyacetone was repeated in subsequent experiments with glycerol as substrate. In one of these the 2,4-dinitro-
230
K.
RAMACHANDRAN
AND
T.
K.
WALKER
phenylosazone separated from the mixture of pyridine and acetic acid in crystals which melted sharply at 265”. It was presumed that this might be the triose-osazone in a different form, because Goepfert and Nord [Zoc. cit., Ref. (5)] also obtained this osazone in crystals which melted at 264-5”. Formation
of Pyruvic and Dimethylpyruvic Acids from Mixtures Glycerol and Sodium Acetate and from a Mixture of Glycerol and Sodium Malonate
of
In five experiments the substrates consisted of glycerol and acetate in the molecular ratios of 3 : 1, 5: 2, 2: 1, 3 : 2, and 1: 1, respectively. In each experiment the concentration of glycerol in the solution of Hida’s salts was 5 g./lOO ml., while in the several cases the weight of acetate taken was such as to afford the required ratio of glycerol to acetate. In one of these experiments the effect of sodium malonate was TABLE
I
The Effects of Acetate and of Malunate on the Fornudm of PA and of DMPA from Glycerol, at 30’
=
=
Products Substrate PA
_.
.-
W./l.
Glycerol Glycerol and acetate
I
2 (4
04 .-
__-
Glycerol Glycerol and acetate Glycerol Glycerol and acetate Glycerol Glycerol and acetate Glycerol and malonate Glycerol Glycerol and acetate Glycerol Glycerol and acetate
5
-
_.
_. -. -
??a*.11.
56 1100
310 734
5:2
32 240 60 860
180 516 319 603
2:l 2:l
70 1000 81
236 560 260
3:2
trace 262
392 337
1:l
50 nil
300 nil
3:l 5:2
_.
DMPA
DIMETHYLPYRUVIC
ACID
231
tested. Preliminary tests had already shown that felts of the mold placed on Hida’s solution with addition solely of acetate or of malonate as the source of carbon, failed to form either PA or DMPA. In every experiment a “control” with glycerol alone (5 g./lOO ml.) was also set up. In each experiment 1500 ml. of medium was distributed equally between six conical flasks, each of 1500 ml. capacity, and the contents were always worked up on the sixth day. The results have been collected in Table I, scrutiny of which shows that acetate over a certain range of concentrations effects very considerable increases, from eightfold to 20-fold, in the yield of PA. Simultaneously, the yield of DMPA is increased, though not to the same degree, the highest yield being nearly three times that given by the control. It will be noted that the yields of PA and DMPA, respectively, in Expt. 2(a) are all relatively low in comparison with those in Expts. 1 and 3. When, however, Expt. 2(a) was repeated, a different result was obtained [see Expt. 2(b)]. The low values in Expt. 2(a) are probably attributable to the fact that in this instance the metabolism solutions were not incubated for a sufficiently long time. The quantitative aspect of experiments such as these should not be stressed too strongly, since a difference of only a few hours in the times at which the samples are withdrawn will sometimes result in a very considerable difference in the yield of PA and of DMPA, because these are not final, stable products but are labile, intermediate metabolites, the respective concentrations of which, in the medium, are constantly undergoing change. DISCUSSION
It seems probable that the conversion of glycerol to DMPA occurs in a sequence of phases, the first of which consists of oxidation of glycerol to dihydroxyacetone, a conversion which A. niger can bring about (2), and the second of which is a condensation of the last-named substance with acetic acid or with some active 2-carbon metabolit,e closely related to acetic acid. Possibly in the mold the actual reactants are phosphorylated derivatives of the components; or one component may react as such. When the substrate is glycerol alone, it is to be presumed that the necessary 2-carbon component is formed from a proportion of the glycerol molecules by way of triose and of pyruvic acid [compare Ref. (5)]. In our view the series of reactions might be represented tentatively by a scheme such as the following one, the successive phases
232
K.
RAMACHANDRAN
AND
T.
K.
WALKER
of which are visualized as consisting of addition of the aldol type, removal of the elements of water, reduction of a polarized double bond, shifting of a double bond, glycol formation at a double bond and enolization: CHz.OH
THaeoH
HO.CHz-CO -Hz0 d
+ CHz.COOH + HO.CHs-C(OH)-CHt.COOH CH, . OH
CHt. OH
+2H
HO.CHz-C=CH.COOH
-
HO.CHB-CH-CHz.COOH
CHZ
-HpO II HO. CH2-C-CHs .
COOH + HO. CHs-C=CH
CHs
+2H __+
C&
-Hz0 -
HO.CH*-CH-CHs.COOH CHa
-+ CHS-C=CH
CHa CH,=C-CH2.
COOH
CHa
glycol
CH&OH)-CH(OH) 1COOH formation -He0 A
. COOH
CHs CH,-C=C(OH)
.COOH
CHa .COOH + CHa-CH-CO.
COOH
All the changes which this phase sequence involves are of types encountered frequently in organic chemistry, and there is ample evidence of the occurrence of similar reactions under the influence of enzymes. The well-known equilibria between the 4-carbon dicarboxylic acids in living cells, the interconversion of crotonic and fl-hydroxybutyric acids in animal tissue, and some of the oxidations and reductions studied by Wieland (12) can all be cited in this connection. Experiments to test these suggestions respecting the intermediate stages in the formation of DMPA, are now in progress in these laboratories. SUMMARY
1. Out of nine strains of Aspergillus niger which were tested, eight proved capable of forming pyruvic and dimethylpyruvic acids when their pre-formed mycelial felts were allowed to act upon glucose in the presence of NH&l, KH~POI, and NaaOs, in the proportions recommended by Hida (Zoc.cit.). When NH&l was omitted from the medium the yield of pyruvic acid was undiminished, but that of dimethylpyruvic acid fell to a negligible amount.
DIMETHYLPYRUVIC
ACID
233
2. In Hida’s medium both pyruvic and dimethylpyruvic acids were also formed by A. niger when n-xylose and glycerol, respectively, were used in place of glucose as substrate. 3. In one experiment, addition of sodium malonate to the medium when glycerol was the substrate caused a slight increase in the yield both of pyruvic acid and of dimethylpyruvic acid, but in each case this increase was so small as to render its significance doubtful. Addition of sodium acetate to the glycerol medium caused a very large increase in the yield of pyruvic acid and a substantial increase also in the yield of dimethylpyruvic acid, when the molecular ratio of glycerol to acetate lay between 3 : 1 and 2: 1. When acetate was added in excess of the molecular ratio 1: 1, neither pyruvic acid nor dimethylpyruvic acid was formed. 4. Tentative suggestions are advanced respecting the mechanisms by which dimethylpyruvic acid may be derived from glycerol. REFERENCES 1. CWALLENQER, F., KLEIN, L., AND WALKER, T. K., J. Chem. Sot. 1929, 1499. 2. CNALLENQER, F., KLEIN, L., AND WALKER, T. K., 1. Chem. Sot. 1931, 16. 3. CNALLENQER, F., SUBRAIKANIAM, V., AND WALKER, T. K., J. Chem. SIX. 1927,
200,3044. 4. CURRIE, J. N., J. Biol. Chem. 31, 15 (1917). 5. GOEPFERT, G. J., AND NORD, F. F., Arch. B&hem. 1, 292, 299 (1942). 6. HAWORTH, R. D., AND LAPWORTH, A., J. Chem. Sot. 121, 79 (1922). 7. HIDA, T., J. Shanghai Sci. Inst. Sect. IV, 1, 201 (1935). 8. KNOEVENAQEL, E., Ber. 31, 2585 (1898); ibid., 37, 4461 (1904). 9. PETERSON, W. H., FRED, E. B., AND ANDERSON, J. A., J. Bad. 8, 277 (1923). 10. RAMAQE, G. R., AND SIMONSEN, J. L., J. Chem. Sot. 1936, 534. 11. WALKER, T. K., Advances in Enzymol. 9, 579 (1949). 12. WIELAND, H., Ann. 436, 229 (1924). 13. WIRTH, J. C., AND NORD, F. F., Arch. Biochem. 1, 143 (1942).
From the College of Technology, University
Hida (7) reported that in presence of sodium sulfite and ammonium chloride young preformed mycelial felts of Aspergihs niger produce pyruvic acid (PA) and dimethylpyruvic acid (DMPA) from glucose or from sucrose. The production of the former acid under these conditions is due probably to zymasis, but the sequence of processeswhich constitutes this phenomenon cannot account in a direct manner for the formation of dimethylpyruvic acid, and Hida’s experiments fail to throw light on this point. During the course of studies of acid formation in mold fungi, the authors found it necessary to carry out some experiments on the lines adopted by Hida, and confirmation of the latter’s results was obtained in trials with a representative selection of strains of A. niger. Omission of ammonium chloride from the medium left the yield of pyruvic acid unaffected but caused that of dimethylpyruvic acid to fall to a trace. This suggested that ammonium chloride acts catalytically to effect a condensation involving a carbonyl group. Examples from organic chemistry can be cited in support of this idea; for instance, the use of ammonium chloride as a catalyst for the condensation of R. CHO with 2R’.OH, yielding an acetal R-CH(OR’)2 (6), and the use of ammonium acetate or piperidine acetate to promote condensation of R-CO-R’ with CN-CH,COOGHs, to yield R-C(R’) = C(CN)COOGHs. Small amounts of ammonia or of certain secondary or tertiary bases are also employed to promote condensation of -CO-CH2-COwith aldehydes (8). Bearing this in mind and taking into account the facts that most strains of A. niger can form citric acid from sugar and that the action of A. niger on citric acid can give a mixture of acetonedicarI a-Oxoisovaleric acid. 224
DIMETHYLPYRUVIC
ACID
225
boxylic acid, acetone, and malonic acid (3), it seemed probable that the first stage in the synthesis of dimethylpyruvic acid might consist of a condensation between acetonedicarboxylic acid (or acetone) and a substance containing an activated -CHzgroup, for example, pyruvie acid, acetaldehyde, or malonic acid. In favor of this view were the the additional facts that pyruvic acid may occur as one of the products of the action of A. niger on sodium citrate (authors’ unpublished observation) and that pyruvic acid is produced along with dimethylpyruvic acid under the conditions adopted by Hida. Much experimental work failed, however, to furnish evidence that citric acid can act, either alone or in the presence of pyruvate, ethanol or acetate, as a precursor of dimethylpyruvic acid; further, experiments in which mixtures of acetonedicarboxylic acid (or acetone) with pyruvate, ethanol, or acetate, were submitted to the action of the mold, were likewise fruitless. The possibility that amino acids from autolyzed mycelial felt might be oxidatively deaminated, or transdeaminated, to yield dimethylpyruvic acid, was then tested. In separate experiments nn-valine, nn-alanine and sodium glutamate were exposed to the action of the mold under appropriate conditions. These three amino acids correspond, respectively,’ to dimethylpyruvic, pyruvic, and a-ketoglutamic acids. In each case the result was negative. Similarly, an experiment in which the substrate was a mixture of nn-valine with the sodium salts of glutamic and pyruvic acids, failed to yield dimethylpyruvic acid. In the meantime we found in other experiments that a mixture of pyruvic and dimethylpyruvic acids could be obtained when xylose was substituted for glucose under Hida’s experimental conditions. This indicated that a condensation involving a triose might initiate the sequence of changes giving rise to dimethylpyruvic acid, because there is evidence that pentoses can undergo enzymic scission to molecules containing, respectively, two and three carbon atoms (1,9,11,13). Since glycerol can be converted to dihydroxyacetone by A. niger (2) and to a mixture of dihydroxyacetone, pyruvic acid, and ethanol by the mold Fusarium lini Bolley (5), experiments were performed with glycerol as substrate and these afforded pyruvic and dimethylpyruvic acids in greater yields than did xylose. Finally, it was observed in a single experiment with sodium malonate that the yields of both acids were improved very slightly when this salt, at a certain concentration, was added to the glycerol medium. Subsequently, it was discovered that very considerable increases occurred in the yields of both acids when sodium acetate
226
K. RAMACHANDRAN
AND T. I(. WALKER
was used in place of the malonate. While, in the absence of additional evidence, the slight increase in yields obtained in the single experiment in which malonate was added must be regarded as of doubtful significance, no such reservation is necessary with respect to the verdict on the influence of acetate, reproducibility of results being noted in four experiments. EXPERIMENTAL
(a) Particulars
of the Strains and of the Cultural Conditions Employed
The strains of A. nfger used in these experiments were designated Nl, AC. 1, Tl, T2, B7, B18, N.R.R.L. 67, N.R.R.L. 599, and N.R.R.L. 607, and all had shown capacity to produce citric acid from sugar. They were grown in the aqueous solution of inorganic salts recommended by Currie (4) with addition of glucose (10 g./lOO ml.). This medium, unless otherwise stated, was contained in conical flasks each.of X00-ml. capacity. These had necks of 40 mm. diameter and the volume of liquid per flask was 250 ml. and the depth was 25 mm. After sterilization in steam for 20 min. on three successive days the contents of the flasks were sown with spores and incubated at 30” for a period of time varying, according to the rate of growth, from 48 to 72 hr. When it was noted that sporulation was commencing, usually after 72 hr., the liquid was poured away from beneath the mycelial felts and was replaced, under aseptic conditions, first by sterile water for 12 hr. and, afterwards, by Hida’s inorganic medium to which had been added an appropriate quantity of sugar or other source of carbon. Hida’s medium contains: 1 g. NH&l, 2 g. H&Pod, and 5 g. Na80a, in water to 100 ml. It was pasteurized at 70” for 15 min. and was introduced beneath the felts by means of a sterile pipet. The time (12 hr.) during which the young felts were allowed to stand over water, before the latter was replaced by Hida’s medium, was found adequate to ensure that the glucose in the cells was metabolized. Control experiments, in which felts were incubated over sterile water, were always performed, however, to ensure that the results observed, when replacing the original glucose medium by substrates other than sugar, were not due to traces of sugar retained in the cells of the mold. Incubation of these preformed felts over the substrates undergoing test was carried out at 30”, aliquots being withdrawn daily and tested with a solution of 2, 4dinitrophenylhydrazine in 2 N HCI. By this test it was possible to judge approximately the time at which production of ketonic products was optimal, and precipitation of the maximum quantities of ketonic products, by this reagent, occurred usually on or about the sixth day. At this stage the contents of the flasks were worked up.
(b) Method of Isolating
and Estimating
the Products
To an aliquot of the filtered medium, half its volume of dilute HCI was added and the whole was heated in a boiling water bath for 2 min. to destroy excess sulfite and to break up its addition compounds. The flask containing the sample was then cooled and an excess of the dinitrophenylhydrazine reagent was added. After 30 min. the precipitate was collected on a Btichner funnel. Separation of the mixed derivatives was effected by taking advantage of the fact that their solubilities in NazCOs solution
DIMETHYLPYRUVIC
ACID
227
depend on the temperature. Thus, the derivative of pyruvic acid (PA) was found to be completely soluble in a dilute aqueous solution of Na&03 at lo”, while the derivative of dimethylpyruvic acid (DMPA) dissolved much less readily in this reagent at this temperature. Above 70”, however, the DMPA derivative dissolved freely in the carbonate solution. The mixed derivatives were triturated with cold 2 N Na2C03 and filtered with suction; the insoluble part was washed with further portions of cold carbonate solution. The filtrates, after mixing, were acidified and the 2, 4dinitrophenylhydrazone of the PA, which separated as a yellow substance, was collected, washed with dilute HCI, then with water, and crystallized from ethanol. The insoluble material left on the filter was mixed with more Na&03 solution and heated on the water bath, yielding a brown solution which, after filtering from any insoluble residue, was acidified. The derivative of the DMPA then separated and, after washing with dilute HCl, it was recrystallized from ethanol. The melting point of the 2, 4-dinitrophenylhydrazone of PA was 218”, alone or mixed with a specimen prepared from authentic material. The melting point of the 2, 4-dinitrophenylhydrazone of DMPA was 1945’, alone or mixed with an authentic specimen prepared from dimethylpyruvic acid which had been synthesized by the method of Ramage and Simonsen (10). When working up larger volumes of metabolism solution and when carrying out estimations of the yields of the keto acids in such larger volumes, the following procedure was adopted. The liquid was filtered and its volume was ascertained, after which it was evaporated at 50”, under reduced pressure, to about one-fifth of its original volume. Ketonic substances were not detected in the distillate and this was rejected. To the concentrated liquid there was then added sufficient 5 N H&O, to provide a 10% excess over the quantity required to decompose the sulfite initially present in the medium. A careful distillation under reduced pressure was then performed and the distillate was collected through a water condenser. The bulk of the DMPA passed over before the PA but efficient separation of the two acids could not be attained in this way, and at the end of the distillation a little water was added to the dry residue in the flask and was, in turn, distilled over and added to the first distillate. The two keto acids in this distillate were then precipitated with 2, 4-dinitrophenylhydrazine and separated as already described. The derivatives, after washing and drying at llO”, were weighed. The residual dry mixture of salts left in the distillation flask invariably contained a little PA. This residue was therefore taken up in the least volume of distilled water necessary to dissolve it, and the solution, after filtration, was treated with a fresh quantity of the dinitrophenylhydrazine reagent. The PA and any other ketonic bodies present formed derivatives, but since that of PA was deposited within 2 hr. while other material required longer to deposit, the precipitate of the PA derivative was filtered off after 2 hr., and was purified and estimated along with the main quantity of that compound. This method allowed a rough estimate to be made of the quantities of the two keto acids in the mixed product. The method was checked in experiments with artificial mixtures of the two acids and the results showed that, for the purposes of the present exploratory work, it could be considered satisfactory. In the course of the preliminary work it was found that if the ammonium chloride was omitted from the mixture of salts recommended by Hida the yield of PA was undiminished but that of DMPA fell to a very low level.
228
K.
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AND
T.
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RESULTS
In descending order of capacity to form the keto acids from glucose, the different strains of A. niger fell into the following grading: Nl, AC. 1, B7, B18, N.R.R.L. 607. The results obtained by use of Tl, T2 and N.R.R.L. 599 were moderate only, while N.R.R.L. 67 proved unreliable. Strain AC. 1 gave mainly PA with traces only of DMPA. The other strains formed more of the latter acid than of PA. In most of the experiments Nl was employed. Formation of Pyruvic and Dimethylpyruvic
Acids from Xylose
In this case, and in all the other experiments now to be recorded, felts of the mold (Nl) were first developed on the solution of glucose in Currie’s medium of inorganic salts. The replacement solution consisted in this case of xylose dissolved at a concentration of 4 g./lOO ml. in the solution of Hida’s salts. Test portions withdrawn from the flasks showed maximum concentration of products at 6 days from the time when the xylose was placed beneath the felts. Two acidic products were isolated and yielded 2, Pdinitrophenylhydrazones which melted at 218” and 194”, respectively. The derivative of m.p. 218’ also melted at this temperature when in admixture with the 2, 4-dinitrophenylhydrazone of authentic pyruvic acid and, similarly, a mixture of the derivative of m.p. 194’ with authentic 2, 4-dinitrophenylhydrazone of dimethylpyruvic acid also melted at 194“. (Found, for derivative of m.p. 218’: N, 20.7. Calcd. for C&HsOeNd: N, 2O.9o/o.Found, for derivative of m.p. 194’: N, 18.7. Calcd. for CUH~~O~NGN, 18.8yo.) Formation of Pyruvic and Dimethylpyruvic Acids from Glycerol and Identijkation of Dihydroxyacetone as an Intermediate Product
The culture medium to be placed beneath the preformed felts was prepared by adding glycerol (5 g./lOO ml.) to Hida’s solution of salts. Several experiments were performed in flasks of different sizes and the quantities of medium varied in the several cases from 200 ml. to 1000 ml., but the depth of medium in each flask was always 25 mm. The contents of the different flasks were worked up for isolation of products at periods varying from 6 to 9 days from the time of replacement of the medium, the time chosen being at the stage of optimum yield of prod-
DIMETHYLPYRUVIC
ACID
229
ucts as shown by daily tests on aliquots of medium. In all cases both PA and DMPA were found to be present as shown by the isolation of 2, 4-dinitrophenylhydrazones of m.p. 218” and 194-5”, respectively, and in each case the melting point was unchanged in admixture with authentic material. (Found, for derivative of m.p. 218’: N, 21.2. Calcd. for CsHsOeNd: N, 20.9%. Found, for derivative of m.p. 194-5”: N, 19.2. Calcd. for C,lH1206N4: N, l&8%.) During the isolation of these two acids as their 2, 4-dinitrophenylhydrazones it was found that the distillate contained traces of a substance giving with 2, 4-dinitrophenylhydrazine a derivative which was insoluble in sodium carbonate solution. It was insoluble also in ethanol and, after being washed by this solvent and dried, its color was a dull yellow and it melted at 279-81”. The quantity was insufficient to permit further purification. More of the substance was found to be present in the distilling flask after all the PA had passed over. The substance reduced Fehling’s solution. As these observations indicated the presence of dihydroxyacetone (or glyceraldehyde) the residue in the distillation flask was lixiviated with a little water and, after filtration, the solution was treated with an excess of a solution of 2, 4-dinitrophenylhydrazine in hydrochloric acid. A red precipitate settled and was filtered off and washed, when it was found to be partially soluble in hot ethanol. The crystals obtained from the ethanol were in two crops. The first of these melted at 250-5” and the second crop melted over the range 155-180”. These two substances were set aside for future examination. The major portion of the red precipitate was insoluble in ethanol but it dissolved readily in a mixture of pyridine and glacial acetic acid, giving a dark red solution from which, on cooling, minute, orange-red crystals were deposited. After further recrystallizations these had m.p. 282’. A specimen of Merck’s dihydroxyacetone was converted to its 2, 4-dinitrophenylosazone and this had m.p. 283”. When it was mixed with the derivative obtained from the cultures the m.p. was 282”. (Found, for derivative of m.p. 282”, from the cultures: N, 25.6. Calcd. for GHrzOeNs: N, 25.0s.) Hence, dihydroxyacetone is formed during the conversion of glycerol to a mixture of PA and DMPA. This identification of dihydroxyacetone was repeated in subsequent experiments with glycerol as substrate. In one of these the 2,4-dinitro-
230
K.
RAMACHANDRAN
AND
T.
K.
WALKER
phenylosazone separated from the mixture of pyridine and acetic acid in crystals which melted sharply at 265”. It was presumed that this might be the triose-osazone in a different form, because Goepfert and Nord [Zoc. cit., Ref. (5)] also obtained this osazone in crystals which melted at 264-5”. Formation
of Pyruvic and Dimethylpyruvic Acids from Mixtures Glycerol and Sodium Acetate and from a Mixture of Glycerol and Sodium Malonate
of
In five experiments the substrates consisted of glycerol and acetate in the molecular ratios of 3 : 1, 5: 2, 2: 1, 3 : 2, and 1: 1, respectively. In each experiment the concentration of glycerol in the solution of Hida’s salts was 5 g./lOO ml., while in the several cases the weight of acetate taken was such as to afford the required ratio of glycerol to acetate. In one of these experiments the effect of sodium malonate was TABLE
I
The Effects of Acetate and of Malunate on the Fornudm of PA and of DMPA from Glycerol, at 30’
=
=
Products Substrate PA
_.
.-
W./l.
Glycerol Glycerol and acetate
I
2 (4
04 .-
__-
Glycerol Glycerol and acetate Glycerol Glycerol and acetate Glycerol Glycerol and acetate Glycerol and malonate Glycerol Glycerol and acetate Glycerol Glycerol and acetate
5
-
_.
_. -. -
??a*.11.
56 1100
310 734
5:2
32 240 60 860
180 516 319 603
2:l 2:l
70 1000 81
236 560 260
3:2
trace 262
392 337
1:l
50 nil
300 nil
3:l 5:2
_.
DMPA
DIMETHYLPYRUVIC
ACID
231
tested. Preliminary tests had already shown that felts of the mold placed on Hida’s solution with addition solely of acetate or of malonate as the source of carbon, failed to form either PA or DMPA. In every experiment a “control” with glycerol alone (5 g./lOO ml.) was also set up. In each experiment 1500 ml. of medium was distributed equally between six conical flasks, each of 1500 ml. capacity, and the contents were always worked up on the sixth day. The results have been collected in Table I, scrutiny of which shows that acetate over a certain range of concentrations effects very considerable increases, from eightfold to 20-fold, in the yield of PA. Simultaneously, the yield of DMPA is increased, though not to the same degree, the highest yield being nearly three times that given by the control. It will be noted that the yields of PA and DMPA, respectively, in Expt. 2(a) are all relatively low in comparison with those in Expts. 1 and 3. When, however, Expt. 2(a) was repeated, a different result was obtained [see Expt. 2(b)]. The low values in Expt. 2(a) are probably attributable to the fact that in this instance the metabolism solutions were not incubated for a sufficiently long time. The quantitative aspect of experiments such as these should not be stressed too strongly, since a difference of only a few hours in the times at which the samples are withdrawn will sometimes result in a very considerable difference in the yield of PA and of DMPA, because these are not final, stable products but are labile, intermediate metabolites, the respective concentrations of which, in the medium, are constantly undergoing change. DISCUSSION
It seems probable that the conversion of glycerol to DMPA occurs in a sequence of phases, the first of which consists of oxidation of glycerol to dihydroxyacetone, a conversion which A. niger can bring about (2), and the second of which is a condensation of the last-named substance with acetic acid or with some active 2-carbon metabolit,e closely related to acetic acid. Possibly in the mold the actual reactants are phosphorylated derivatives of the components; or one component may react as such. When the substrate is glycerol alone, it is to be presumed that the necessary 2-carbon component is formed from a proportion of the glycerol molecules by way of triose and of pyruvic acid [compare Ref. (5)]. In our view the series of reactions might be represented tentatively by a scheme such as the following one, the successive phases
232
K.
RAMACHANDRAN
AND
T.
K.
WALKER
of which are visualized as consisting of addition of the aldol type, removal of the elements of water, reduction of a polarized double bond, shifting of a double bond, glycol formation at a double bond and enolization: CHz.OH
THaeoH
HO.CHz-CO -Hz0 d
+ CHz.COOH + HO.CHs-C(OH)-CHt.COOH CH, . OH
CHt. OH
+2H
HO.CHz-C=CH.COOH
-
HO.CHB-CH-CHz.COOH
CHZ
-HpO II HO. CH2-C-CHs .
COOH + HO. CHs-C=CH
CHs
+2H __+
C&
-Hz0 -
HO.CH*-CH-CHs.COOH CHa
-+ CHS-C=CH
CHa CH,=C-CH2.
COOH
CHa
glycol
CH&OH)-CH(OH) 1COOH formation -He0 A
. COOH
CHs CH,-C=C(OH)
.COOH
CHa .COOH + CHa-CH-CO.
COOH
All the changes which this phase sequence involves are of types encountered frequently in organic chemistry, and there is ample evidence of the occurrence of similar reactions under the influence of enzymes. The well-known equilibria between the 4-carbon dicarboxylic acids in living cells, the interconversion of crotonic and fl-hydroxybutyric acids in animal tissue, and some of the oxidations and reductions studied by Wieland (12) can all be cited in this connection. Experiments to test these suggestions respecting the intermediate stages in the formation of DMPA, are now in progress in these laboratories. SUMMARY
1. Out of nine strains of Aspergillus niger which were tested, eight proved capable of forming pyruvic and dimethylpyruvic acids when their pre-formed mycelial felts were allowed to act upon glucose in the presence of NH&l, KH~POI, and NaaOs, in the proportions recommended by Hida (Zoc.cit.). When NH&l was omitted from the medium the yield of pyruvic acid was undiminished, but that of dimethylpyruvic acid fell to a negligible amount.
DIMETHYLPYRUVIC
ACID
233
2. In Hida’s medium both pyruvic and dimethylpyruvic acids were also formed by A. niger when n-xylose and glycerol, respectively, were used in place of glucose as substrate. 3. In one experiment, addition of sodium malonate to the medium when glycerol was the substrate caused a slight increase in the yield both of pyruvic acid and of dimethylpyruvic acid, but in each case this increase was so small as to render its significance doubtful. Addition of sodium acetate to the glycerol medium caused a very large increase in the yield of pyruvic acid and a substantial increase also in the yield of dimethylpyruvic acid, when the molecular ratio of glycerol to acetate lay between 3 : 1 and 2: 1. When acetate was added in excess of the molecular ratio 1: 1, neither pyruvic acid nor dimethylpyruvic acid was formed. 4. Tentative suggestions are advanced respecting the mechanisms by which dimethylpyruvic acid may be derived from glycerol. REFERENCES 1. CWALLENQER, F., KLEIN, L., AND WALKER, T. K., J. Chem. Sot. 1929, 1499. 2. CNALLENQER, F., KLEIN, L., AND WALKER, T. K., 1. Chem. Sot. 1931, 16. 3. CNALLENQER, F., SUBRAIKANIAM, V., AND WALKER, T. K., J. Chem. SIX. 1927,
200,3044. 4. CURRIE, J. N., J. Biol. Chem. 31, 15 (1917). 5. GOEPFERT, G. J., AND NORD, F. F., Arch. B&hem. 1, 292, 299 (1942). 6. HAWORTH, R. D., AND LAPWORTH, A., J. Chem. Sot. 121, 79 (1922). 7. HIDA, T., J. Shanghai Sci. Inst. Sect. IV, 1, 201 (1935). 8. KNOEVENAQEL, E., Ber. 31, 2585 (1898); ibid., 37, 4461 (1904). 9. PETERSON, W. H., FRED, E. B., AND ANDERSON, J. A., J. Bad. 8, 277 (1923). 10. RAMAQE, G. R., AND SIMONSEN, J. L., J. Chem. Sot. 1936, 534. 11. WALKER, T. K., Advances in Enzymol. 9, 579 (1949). 12. WIELAND, H., Ann. 436, 229 (1924). 13. WIRTH, J. C., AND NORD, F. F., Arch. Biochem. 1, 143 (1942).