ON OXIDATIVE DECARBOXYLATIONS WITH PERIODIC ACID*

BY DAVID B. SPRINSONt AND ERWIN CHARGAFF

(From the Department of Biochemistry, College of Physicians and Xurgeons, Columbia University, New York)

(Received for publication, March 29, 1946)

The behavior of hydroxypyruvic acid, CHzOH. CO. COOH, and some of its transformation products toward periodic acid, treated in detail in the preceding paper (I), suggested an orienting study of the action of this oxidant on glyoxylic acid, CHO. COOH, and other a-keto acids or on compounds giving rise to such keto acids, following oxidation by periodate.

While cr-glycols, a-ketols, a-diketones, and cY-keto aldehydes are oxidized by periodic acid, q-keto acids are assumed to be inert to this reagent under the conditions commonly applied. (Compare the survey of the literature in (2).) Tartaric acid is reported to consume only 1 mole of periodic acid (3, 4); the further oxidation of the resulting 2 moles of glyoxylic acid to formic acid and carbon dioxide is claimed to require 36 to 48 hours for com- pletion at room temperature (5). The very slow development of carbon dioxide (within 1 to 4 days) in the course of the oxidation of keto sugars by periodic acid has been attributed to the gradual breakdown of the glyoxylic acid arising as a primary product of the oxidation (6). Simi- larly, glyoxylic acid originating from the action of periodate on P-hydroxy- a-amino acids has been reported to be slowly attacked by the oxidant (7), though this action was shown to proceed at a faster rate in the presence of weak alkali (8). Pyruvic acid likewise is degraded slowly (9).

The following discussion will attempt to illustrate the extent to which the oxidative decarboxylation by periodate of a-keto acids and compounds giving rise to them may be useful as a diagnostic method. Tartaric acid, as the most thoroughly investigated example of a polyhydroxy acid, will be taken up first, followed in turn by various cu-keto acids, hydroxyamino acids, polyhydroxy acids, keto sugars, polycarbonyl compounds, and malonic and tartronic acids.

Oxidation of d-Tartaric Acid

Periodate Consumption-A closer study of the oxidation of tartaric acid by periodate showed it to be dependent upon the concentration of the oxi-

* This work has been supported by a grant from the John and Mary R. Markle Foundation.

t This report is from a dissertation submitted by David B. Sprinson in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University.

433

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434 DECARBOXYLATION’S VITH PERIODIC ACID

dant, the pH of the reaction mixture, and the temperature. The results are summarized in Table I. Since the primary oxidation of tartaric acid as an cr-glycol is very rapid (5), these experiments furnish an indication of the rates at which glyoxylic acid is oxidized under various conditions.

TABLE I

Consumption of Periodate bq Tartar% Acid

Experiment I%.*

1 2 3 4 5

‘3 7t 8 9

10 11 12 13 14 15 16 17

Diluent

M NaHC03 “ “ ‘C ‘. “ “ “ “ “ it “ “

0.13 N &soa 0.13“ “ I-I?0

“ “ “ CL “ “ ‘<

Oxidant

HI04 “ “ “ “ ‘< “ <‘ “ “ “ “ “

NaIOa “ “ ‘<

11; I ./-

Initial molar atio of oxidant

to lartaric acid

2.2 2.2 4.3 4.3 4.3 2.2 2.2 2.2 2.2 2.2 2.2 2.2 4.3 1.7 3.6 3.6 3.6

Duration of oxidation

min. 25 45 20 30 60 10 20

5 10

5 15 30

120 30 30 60 90

n

-

Oxidant con- sumed per

lole of tartsric acid

-

moles

1.9 2.0 2.8 2.0 3.0 1.0 1.1 0.68 0.84 1.1 1.1 1.3 2.2 1.3 2.2 2.6 2.7

* 1 mole of tartariq acid requires 1 mole of periodate for the production, and addi- tional 2 moles for the oxidation, of glyoxylic acid. In each experiment 5 cc. of 0.05 M solution of tartaric acid, 15 cc. of the diluent indicated, and 1 or 2 cc. of 0.54 M HIO( or of 0.43 M NaI04 Jrere used. The oxidation was, with the except.ion of Ex- periments 6 and 7, carried out at room temperature. In Experiments 1 to 7 the excess periodate was determined by the method of Fleury and Lange (10). In Experiments 8 to 17 the excess periodate was determined in acid solution, according to Malapradc

(3). t In these experiments the oxidation \\-as carried out at 5”.

In sodium bicarbonate solution cont.aining a moderate excess of oxidant, tartaric acid consumed almost 3 moles of periodate at room temperature in 20 to 30 minutes (Table I, Experiments 3 to 5). Even with glyoxylic acid in excess, rapid oxidation occurred (Experiments 1 and 2). At 5” or in strongly acid solution (Experiments 6 to 13) the secondary oxidation was slowed down considerably, while in weakly acid solution (sodium periodate solutions ha,vc a pH of about 4) intermediate effects were ob- served (Experiments 14 to 17).

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D. B. SPRINSON AXD E. CHARGAFF 435

Carbon Dioxide Produclien-When the reaction is performed with a large excess of sodium periodate, tartaric acid can be made to yield,, in the Van Slyke-Neil1 manometric apparatus, 2 moles of carbon dioxide in 10 minutes (Table IV, Experiment 3).l The procedure described in the experimental part invariably yielded easily reproducible results. No carbon dioxide was liberated from formaldehyde and formic, glycolic, lactic, and oxalic acids under the conditions employed.

Oxidation of cY-Keto Acids by Periodic Acid

Experi- ment No.

1 2 3 4 5 6 7 8 9

10 11 12

Substance

Glyoxylic acid Pyruvic acid

“ “

Hydroxypyruvic acid3 Mesoxalic acids ol-Ketobutyric acid P-Hydroxy-ol-lretobut~~ric acid1 ol-Ketoglutaric acid 2-Keto-d-gluconic acids

<< <<

2-Keto-Z-gulonic acids “ “

I-

i

Consumption of period&e*

Initial Ill&I

ratio of oxidant to substance

1.8 (b) 1.8 ‘I

2.1 ‘I 1.5 (a) 2.0 (6) 1.7 “ 1.8 (( 5.7 (a) 9.2 (b) 5.7 (a) 9.2 (b)

luratior of oxi- dation

I( L ‘CC P

hidant msunx3 er mole of sub- stance

min. moles

60 0.97 120 0.28

30 1 .o 20 1.0

120 0.36 30 1 .o

120 0.28 90 3.5 90 4.0 90 3.4 90 4.0

i1 0

Decarboxylationt

hration f oxida-

tion

--

min.

10 10 30 10 10 10 10 10 10

10

P

Carbon dioxide formed er mole of sub- stance

mole

1 .o 0.26 0.53 0.05 1 .o 0.34 0.03 0.17 0.14

0.13

* The conditions under which the oxidations were carried out are indicated as follows: (a) = weakly acid (sodium periodate, about pH 4), (5) = weakly basic (periodic acid in excess sodium bicarbonate). For the details, also with respect to analytical procedures, see the experimental part.

t 1 cc. each of solution, of water, and of 0.43 M sodium periodate in the Van Slyke- Neil1 manometric apparatus.

$ Compare the preceding paper (1) for a detailed discussion. 0 Additional dat,a on the oxidation mechanism in the experimental part.

Oxidation of cu-Keto Acids (Table II)

The rates of the oxidative cleavage of cr-aldehydo and a-keto acids by periodic acid revealed no definite regularity. Two compounds stood out by the readiness with which they mere decarboxylated oxidatively ;

1 It should be clearly understood that the determinations of periodate consump- tion and carbon dioxide production, though placed side by side in Tables II to VII for reasons of brevity and convenience, represent two independent set,s of experi- ments.

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436 DECARBOXYLATIONS WITH PERlODIC AClD

namely glyoxylic and mesoxalic acids.2 The solution of glyoxylic acid employed, which xv-as 0.3 M with respect to its aldehyde content, consumed 0.29 mole of periodic acid and produced 0.28 mole of carbon dioxide. Mesoxalic acid, COOK. CO. COOH, likewise reacted very rapidly with the consumption of 1 mole of oxidant and the formation of 1 mole each of oxalic acid and carbon dioxide. Pyruvic, cr-ketobutyric, and a-ketoglutaric acids reacted much less readily with respect to both the consumption of periodate and the production of carbon dioxide; but it will be noted that the extent of decarboxylation of these substances still was greater than that of simple P-hydroxyketo acids (compare Experiments 2, 3, 6, Table II, with Experiments 4 and 7).

In an attempt at a general formulation of the oxidation of cr-keto acids by periodic acid, these compounds would, therefore, appear to behave as

Experi- ment

No.

TABLE III

Oxidation of&Hydroxy-or-amino Acids by Periodic Acid

Consumption of period&e* Decarboxylation’

Substance Ini;~k,in~jar Oxidant Duration of consumed

Duration of Carbon diox- lde formed

oxidant to oxidation per mole of oxidation per mole of substance substance substance

min. moles min. mole

dl-Serine 2.9 (a) 60 2.0 10 1 .o “ 3.8 (b) 60 1.9

dl-Threonine 2.9 (a) 60 2.0 10 1 .o “ 3.8 (b) 60 2.0

* See Table II for the explanation.

though they were a-diketones, giving rise to two acids by oxidative decar- boxylation :

R. CO. COOH =+ R. COOH + CO(OH)a

It is evident from what is known of this reaction that in substances possessing two or more functional groups that may compete for the oxidiz- ing agent, e.g. in /3-hydroxy-a-keto acids, the much more rapid rate at which the a-ketol structure is attacked will make this type of cleavage prevail. Hydroxypyruvic and fl-hydroxy-a-ketobutyric acids react as a-ketols (1). 2-Keto-d-gluconic and 2-keto-Z-gulonic acids consume 4 moles of periodate and give rise to only little carbon dioxide.

2 It is noteworthy that these keto acids are known to form stable hydrates. The oxidative cleavage of or-keto acids by lead tetraacetate in the presence of hy- droxyl-forming substances was studied by Baer (11).

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D. B. SPRlNSON AND E. CHARGAFF 437

Oxidation of ,&Hydroxy-or-amino Acids (Table III)

dl-Sernae and dl-threonine consumed 2 moles of periodate under weakly alkaline or weakly acid conditions with the production of 1 mole of carbon dioxide from the glyoxylic acid arising from the instantaneous cleavage of the amino acids.

TABLE IV Oxidation of Polyhydroxy Acids by Periodic Acid

Experi- ment No.

1

2 3 4 5 6 7 8 9

10 11

Substance

Glyceric acid “ “

Tartaric “ t Dihydroxymaleic acid1 d-Gluconic acid 5-Keto-d-gluconic acidi

“ “

d-Glucuronic acid$ <‘ “

Mu& acid d-Saccharic acid

Consumption of periodate*

Initial molar ratio of

oxidant to substance

3.5 (a) 4.4 (b) 4.3 “ 3.2 “ 7.3 “ 5.7 (a) 7.3 (b)

14.3 (a) 7.3 (b)

11.0 IL 8.6 (a)

D

-

( luration of oxi- dation P

__ _ min.

60 60 60 10 60 90 90 60 60 60 60

hidant con-

sumed er mole of sub- stance

moles

2.0 1.9 3.0 2.0 5.0 3.6 3.9 4.6 4.9 4.9 5.0

I:

-

Decarboxylation’

luration of oxi- dation

min. 10

10

10 10 208 10

120 10 10

- ’ ,

,

P ,

Carbon 3ioxide formed er mole ,f sub- stance

moles

1 .o

2.0

1.0 0.33 0.73 0.3 0.9 2.0 2.0

* See Table II for the explanation. t Compare Table I for more details on periodate consumption. $ Additional data on the oxidation mechanism in the experimental part. 8 1 cc. of the solution was treated for 10 minutes with 1 cc. of sodium periodate

solution. Then 1 cc. of 0.1 N sodium hydroxide was added and the reaction con- tinued for 10 more minutes. The value is corrected for a small alkali blank.

Oxidation of Polyhydroxy Acids (Table IV)

In order to avoid the complications brought about by the possible pres- ence of la&one bridges, the neutral solutions of the polyhydroxy acids were subjected to oxidation. Glyceric, mucic, d-saccharic, and d-gluconic acids consumed oxidant and produced carbon dioxide, in accordance with the number of molecules of glyoxylic acid formed in their oxidation, and at a rate similar to that observed with tartaric acid.

Dihydroxymaleic acid (I) consumed 2 moles of periodate very rapidly, yielding 2 moles of oxalic acid. 1 mole of the oxidant was presumably used for the production from the enediol (I) of the dicarbonyl compound (II) and another mole for its cleavage to oxalic acid.

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438 2ECARROXYLATIONS WITH PERIODIC ACID

COOH. COH=COII. COOII Ao%

(I)

COOH. CO. CO. COOH IO,’

-+ 2(COOH),

(II)

5-Keto-d-gluconic acid reacted in a manner which is difficult to interpret (Experiments G and 7, Table IV). It consumed about 4 moles of periodate and gave rise to no more than 0.33 mole of carbon dioxide under normal conditions. In the presence of alkali the extent of oxidative decarboxyla- tion was increased (Experiment 7). Following an oxidation for 24 hours, when 4.25 moles of periodate had been consumed, 0.18 mole of formalde- hyde and 0.46 mole of oxalic acid were pr,oduced. If this keto acid is formulated as having the open chain structure, it should have reacted either via a trihydroxyglutaric acid, consuming a total of 5 moles of oxidant to give rise to I mole of formaldehyde and 2 moles of carbon dioxide, or by the way of a cleavage to glycolic acid and tartaric acid semialdehyde, in which case the consumption of 4 moles of periodate should have been accompanied by t.he formation of 1 mole of carbon dioxide.

The experimental results, especially the production of oxalic acid, are in better agreement with the assumption that 5-keto-d-gluconic acid reacts in the main as a furanoid compound (III). The principal pathway of oxidative degradation, which entails the formation of oxalic acid, could be formulated as proceeding through a series of esters (IV, V). The postu- lated replacement of the active hydrogen (marked with an asterisk) in the glycolic acid ester (IV) by a hydroxyl is in agreement with the recent

r----o------? I COOH. AH. CHOH. CIIOH. &OH. CIIzOH

210,- ,

(III) r-o---1 I I

HCOOH + COOH. CH*. CIIO 104-

CO. CHCOH -------+

(IV) r---o-1

COOH. COH. CHO IO4-

CO. CH,OH --------+

(V)

(COOIT), + C&OH. COOH + HCOOH

observations of Huebner et al. (12) and with analogous findings of Hackett and collaborators in oxidation experiments with lead tetraacetate (13).

d-Glucuronic acid (VI) reduced about 5 moles of periodate and produced

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D. B. SPRINSON AND IL CHARGAFF 439

almost 1 mole of carbon dioxide, although rather slowly (Experiments 8 and 9, Table IV). Following an oxidation with sodium periodate for 24

r~--- - -+-----, I I

CHOH-CHOH.CHOH.CHOH.CH-COOH

(VI)

hours, 0.5 mole of oxalic acid was isolated. The yield of oxalic acid, lower than in the case of bornyl-d-glucuronide in which almost 1 mole was isolated (12), and the production of carbon dioxide furnish an indication that the replacement of an active hydrogen by a hydroxyl cannot be the

TABLE V

Oxidation OJ Kctoses by Periodic Acid

_-

-

T Consumption of periodate* ~___-

DecarboxylatimP

d-Fructose

“

Norbose “

meso-Inosose <‘ I‘

--

/

____---.-- i -~----’ --__

mzn. moles

3.9 4.5 3.s 4.8 5.5 5.6 6.1

-

1 Carbon diox- Duration of 1 ide formed

oxidation ; p;,rrk;f

min. ?tlOle

60 I 0.09 20t /

’ 0.73

60 0.17 2ot 0.80 10 0.6 60 0.8 2ot 0.8

* See Table II for the explanation. t 1 cc. of the solution was treat,ed for 10 minutes with 1 cc. of sodium periodate

solution. Then 1 cc. of 0.1 N sodium hydroxide was added and the reaction con- tinued for 10 more minutes, The value is corrected for a small alkali blank.

only pathway by which the oxidntive degradation of unconjugated glu- curonic acid is achieved. A portion of this acid obviously reacts in a different way, perhaps via a partial hydrolysis of the formic acid ester of tartronic acid semialdehyde, formed by the oxidation of VI, and further oxidation to glyoxylic acid. It cannot yet be decided whether the condi- tions under which the decarboxylation experiments are carried out in the Van Slyke-Neil1 manometric apparatus (large excess of oxidant, presence of mercury) favor the latter type of degradation.

Oxidation of Ketoses (Table V)

d-Fructose and l-sorbose consume 3.8 to 3.9 moles of oxidant, when ex- posed to aqueous sodium periodate, whereas 4.8 moles are reduced in the

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440 DECARBOXYLATIONS WITH PERIODIC ACID

same period if the oxidation is carried out in bicarbonate solution. Al- most no decarboxylation is observed under ordinary conditions, even by prolonged oxidation. In the presence of weak alkali, however, 0.7 and 0.8 moles of carbon dioxide are produced from fructose and sorbose respectively (Experiments 2 and 3, Table V). The production from fructose of 0.7 mole of carbon dioxide is in good agreement with the isolation of 1.7 moles of formaldehyde (14).

The various findings can best be reconciled if it is assumed that the major part (70 to 80 per cent) of the keto sugar (VII) is oxidized to the glyoxylic acid ester (VIII), which then gives rise to carbon dioxide. A possible side reaction (20 to 30 per cent of the ketose), involving the intermediary formation of a glycolic acid ester (IX), would explain the deficits observed

r----l CO. CHO CHO- CH2

(VIII)

F ---

I / /

CH,OH. COH . CHOH . CHOH . CHOH * CHs

(VII) \ \

I r----O I 1

CHtOH. CO CHO. CHs

(IX)

in the production of both carbon dioxide and formaldehyde and in the total consumption of periodate.

meso-Inosose (X) reduced 6 moles of periodate rapidly in bicarbonate

OHH OHH OH

I 1 I I i rc-C-C-C-C-

1 I I I I I!

H OHH OHH

-co----’

0;)

solution; in weakly acidic solution the reaction was less complete. The production of carbon dioxide (0.8 mole in 1 hour) was not affected by the addition of alkali.

Oxidation of Polycarbonyl Compounds (Table VI)

Rhodizonic acid (XI) consumed 4.35 moles of the oxidant and yielded 2.3 moles of oxalic acid and 1.15 moles of carbon dioxide. The very rapid reaction was accompanied by the instantaneous loss of color. The relative

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D. B. SPRINSON AND E. CHARGAFF 441

0= u -OH

II 0

cm

proportions of oxalic acid and carbon dioxide isolated and the amount of oxidant used are in agreement with the assumption that the intermediate

TABLE VI

Oxidaiion of PolycarbonrJ Compounds by Periodic Acid

Exper:- merit No.

Substance

min.

90

moles

Dipotassium rhodixonatet 6.5 (a) 1.15 “ “ 1.15

Dipotassium croconatet 5.8 “ 1.03 “ “ 7.3 (b) 1.03

Triketohydrindene hydrate 2.9 (a) 0 “ “ 2.9 “ 0 “ “ 2.9 (b)

* See Table II for the explanation. 1 Additional data on the oxidation mechanism in the experimental part. $ The potassium rhodizonate powder (0.02 m&r) was weighed into the cup of the

manometric apparatus, washed into the chamber with 2 cc. of water, and oxidized with 1 cc. of 0.43 M sodium periodate in the usual manner.

90 90 10 60 60

moles

4.35

4.0 4.1 1 .o 1 .o 1 .o

CID

-

-

min.

101

301 10 20 10 20

Consumption of periodate*

h-ation of oxi- dation

hidant mslmm er mole of sub- stance

1 .-

Decarboxylation*

uration of oxi- dation

Carbon dioxide formed ,er mole of sub- stance

triquinoyl is further degraded in two ways; namely, partly through mes- oxalic acid to oxalic acid and carbon dioxide, partly to oxalic acid directly.

Croconic acid (XII) reduced 4 moles of periodate and yielded 1.9 moles of oxalic acid and 1.03 moles of carbon dioxide. As in the reaction scheme previously discussed for compound I, the enediol apparently first consumed 1 mole of oxidant, accompanied by the.fading of the yellow color of XII, to form leuconic acid (XIII), which then was further degraded via mes- oxalic acid.

Triketohydrindene hydrate (XIV) consumed exactly 1 mole of periodate; no carbon dioxide was formed in the reaction. The oxidation presumably stopped with the formation of phthalonic acid.

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442 DECARBOXYLATIONS WITH PERIODIC ACID - J) ”

01-I

c

-OH

II 0 0

(XII) (XIII)

(COOHh + COOH. CO,COOH IO,

--+ (COOH)2 + co2

2 II 0

WV)

Oxidation of Malonic and Tartronic Acids (Table VII)

The treatment of these acids with periodate resulted in the production from both compounds of 2 moles of carbon dioxide in 2 hours. The con- sumption of periodate proceeded rather slowly. Malonic acid reduced 3 moles of the oxidant, tartronic (hydroxymalonic) acid 2 moles. Neither compound formed oxalic acid. A possible reaction mechanism could be based on the assumption that the replacement of an active hydrogen by a hydroxyl (12) resulted in the conversion of malonic to tartronic acid, which then was further degraded via glyoxylic acid.

COOHe CH?. COOH 104- IO4-

--+ COOH. CHOH. COOH F

COn + CHO. COOH 1oi-

p----f HCOOH + CO2

The findings presented in the preceding pages show the liberation of carbon dioxide in the course of oxidations with periodate to be an indication of the presence, or the formation, of glyoxylic or mesoxalic acid in the oxidation mixture. With many compounds the results are entirely un- equivocal. Difficulties of interpretation are, however, encountered occa- sionally in substances, such as the keto sugars, possessing stable oxygen bridges in which the esters resulting from the primary attack of the oxidant may be susceptible of multiple secondary degradation. Much additional work along these lines obviously is necessary. With respect to the detec- tion of glyoxylic acid formed by the action of periodic acid it should be understood that the concentration of periodate will, in certain cases, affect the rate of oxidation considerably. For instance, in the investigation of compounds, such as 2,3-dimethylmannosaccharic acid (15) or 2,3-di- methylmucic acid (IG), that require only 1 mole of, and react rapidly with, periodic acid, the glyoxylic acid which is produced appears to remain

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D. B. SPRINSON AND E. CHARGAFF 443

unattacked-in the absence of an excess of periodate. This is especially true of lactones that give rise to stable esters of glyoxylic acid following oxidation with periodate (17, 18).

EXPERIMENTAL

Material

The compounds listed here are given in the order in which they appear in Tables II to VII. All other substances were commercially available pure preparations.

Glyoxylic acid wa.s prepared in solution according to Benedict (19) by the reduction of oxalic acid by magnesium powder, followed by the extrac-

TABLE VII

Oxidation of Malonic and ‘I’artroGc Acids

Experiment x0. Substance

; ; “ “

Malonic acid

3 Tartronic acid 4 “ “

5 / “ ((

Consumption of periodnte* Decarboxylationt -

IIGlli&~l~~

oxidant to substance

min. ?noles

2.9 (a) 180 1.2 5.8 “ 1140 3.1 4.3 “ 120 1.5 4.3 “ 240 1.G 5.1 I‘ 1140 2.0

60 1.90 90 1.96

* See Table II for the explanation. t 1 cc. of solution and 2 cc. of 0.43 ‘M sodium periodate in the Van Slyke-Neil1

manomelric apparatus.

tion of the reaction mixture with ether. The resulting solution was 0.66 N with respect to total acid content (glyoxylic and glycolic acids) and 0.3 M with respect to aldehydo acid content (20). We ase indebted to Dr. P. K. Stumpf for this solution.

The preparation of hydroxypyruvic, a-ketobutyric, and P-hydroxy-oc-lceto- butyric acids has been discussed in the preceding paper (1).

ACesoxalic acid was obtained through the courtesy of Dr. D. E. Green. a-Ketoglutaric acid, m.p. 113-114”, was synthesized by a method soon to be presented by Martell and Herbst (private communication).

2-Keto-d-gluconic acid, employed as the sodium salt, was prepared from the corresponding calcium salt trihydrate.3

8 We are highly indebted to Dr. J. A. hesclilimann of Hoffmann-La Itoche, Inc., Sutley, New Jersey, for these specimens, as well as for samples of 2-lreto-Z-gulonic and d-glucuronic acids, potassium acid d-snccharate, and I-sorbose.

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444 DECARBOXYLATIONS WlTH PERIODIC ACID

Xoclium 5-keto-d-gluconate was prepared from the calcium salt (23H20)3 by prolonged shaking with the required amount of sodium oxalate. The conversion was complete, as was shown by the weight and oxidimetric titration value of the resulting calcium oxalate.

meso-Inosose was prepared by the action of Acetobacter suboxydans (American Type Culture Collection No. 621) on meso-inositol (21, 22) and purified through its phenylhydrasone (22), m.p. 182” (with decom- position). The purified meso-inosose melted with decomposition at 199-200”.

Dipotassium rhodizonate was prepared from meso-inositol (23).

C606Kg (246.3). Calculated, C 29.3, K 31.8; found, C 29.0, K 32.1

Dipotassium croconate was synthesized from rhodizonic acid (24). Tartronic acid was prepared from dihydroxytartaric acid (25). A sus-

pension of 5.6 gm. of sodium dihydroxytartrate in 60 cc. of water was kept at 80” with stirring for 1 hour, when complete solution had taken place. The calcium tartronate, precipitated with 24 cc. of M calcium chloride, had a dry weight of 3.0 gm. The solution of the dry material in 6 cc. of 20 per cent hydrochloric acid was extracted eight times with 20 cc. portions of ether (26). 1 gm. of crude tartronic acid was recovered from the ethereal solution; recrystallization from absolute ether yielded 0.3 gm. of tartronic acid, melting at 160-162” (with decomposition) ; molecular weight (by titration) 116; required by C3H405, 120.

Reagents

Nearly 0.54 M HI04 solutions were prepared by dissolving 12.5 gm. of crystalline HI04.2Hz0 per 100 cc. of solution. A similar amount of nearly 0.43 M XaI04 was prepared from 9.5 to 10.0 gm. of sodium meta- periodate. The solutions mere allowed to stand overnight or longer and filtered on a fritted glass funnel to remove a small precipitate. They were then standardized against 0.1 N arsenite solution.

Determination of Periodate Consumption

The procedures followed in the oxidation of tartaric acid are described in Table I.

In experiments with other substances (Tables II to VII, in which condi- tions and amounts employed in each case are indicated) 1 to 10 cc. of a 0.01 to 0.03 M solution were treated by one of the two following methods. In procedure (a) the oxidation was carried out with 0.43 M sodium periodate in the presence of 15 cc. of water; then a few cc. of M sodium bicarbonate

4 Paraperiodic acid and sodium metaperiodate were obtained from the G. Frederick Smith Chemical Company, Columbus, Ohio.

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D. B. SPRINSON AND E. CHARGAFF 445

were added, followed immediately by 1 cc. of 20 per cent potassium iodide. In procedure (b) after oxidation with 0.54 M periodic acid in the presence of 15 cc. of M sodium bicarbonate 1 cc. of 20 per cent potassium iodide was added. The liberated iodine was, in both procedures, titrated with 0.1 N arsenite (27, 28).

Determination of Carbon Dioxide Production

1 cc. of solution (0.03 or 0.01 M when 1 or 2 moles of carbon dioxide respectively are liberated) is measured into the chamber of the Van Slyke- Neil1 manometric apparatus. This is followed by 1 cc. of water and 1 cc. of NaI04 solution. The mercury is lowered to the bottom of the chamber, returned slowly into the chamber, and then lowered to the 50 cc. mark. After being shaken for 15 seconds, the chamber contents are allowed to stand for the stated time. The CO2 is now liberated with 1 cc. of 2 N acetic acid,5 absorbed by 0.5 cc. of 5 N NaOH, and the Pcoz determined in the usual manner (29, 30). Mg. of carbon were calculated by multi- plying by the factors given in (28). The values so obtained were used to calculate the moles of COS, which are reported in Tables II to VII. Al- though these factors have been arrived at under somewhat different experi- mental conditions, their adequacy for the present purpose was demon- strated in experiments with tartaric acid as a test substance. Each experiment was carried out at least in duplicate. The results invariably showed very good agreement,.

Additional Data on Oxidation Mechanisms; Determination of Oxalic Acid and of Formaldehyde

Determination of Oxalic Acid-For the determination of oxalic acid an aliquot of the oxidation mixture was acidified with 2 N hydrochloric acid, and 7.5 M hydriodic acid was added (31) in an amount sufficient to reduce both the remaining periodic and the formed iodic acids. Iodine was removed by filtration and, from the filtrate, by the necessary amount of M sodium thiosulfate. From the solutions, neutralized with coneen’- trated ammonia followed by acidification (to methyl red) with glacial acetic acid, oxalic acid was precipitated as the calcium salt. This salt then was either analyzed both gravimetrically (as the monohydrate) and oxidimetrically (with standard potassium permanganate) or dissolved directly in 2 N sulfuric acid and subjected to a permanganate titration.

Determination of Formaldehyde-An aliquot of the oxidation mixture was made alkaline with sodium bicarbonate and unused periodate de- stroyed with 2 M sodium arsenite. The solution was made acid to methyl red with acetic acid and the formaldehyde precipitated and estimated as

6 The use of 2 N acetic acid was found preferable to other methods of acidification.

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446 DECARBOXYLATIONS WITH PERIODIC ACID

t,he dimedon derivative, a 0.4 per cent solution of the reagent being used (32). Removal of both iodate and periodate with arsenite under acidic conditions (14) gave the same result.

Mesoxalic Acid (Experiment 5, Table II)--To 20 cc. of a 0.03 PI solution of sodium mesoxalate, C3H206Ya2, 3 cc. of mater and 2 cc. of 0.43 M so-

dium periodate were added. After 20 minutes, a total consumption of 0.6 rnM of periodate was found, as shown by the titration of a 2 cc. aliquot. The production of oxalic acid amounted to 0.597 mM.

%Keto-d-gluconic and %Kic,to-l-gulonic Acids (Experiments 9 to 12, Table II)-2 KIM of the sodium salts were oxidized with 25 cc. of 0.43 M

sodium periodate in a final volume of 100 cc. After 20 hours the consump- tion of oxidant, as was shown by titration of a 5 cc. aliquot, amounted to 3.5 moles per mole of keto acid; i.e., the same value as that shown after 90 minutes (Table II, Experiments 9, and 11). The addition of 2 gm. of sodium bicarbonate caused the oxidation to proceed to the 4 mole level in 1 additional hour, after which 2-keto-d-gluconic acid gave rise to 0.82 mole and 2-keto-Z-gulonic acid to 0.85 mole of oxalic acid. Exactly 1 mole of formaldehyde per mole of keto acid was isolated as the dimedon derivative (m.p. and mixed m.p. 190-192”).

Dihydroxymaleic Acid (Experiment 4, Table IV-To a solution of 148 mg. of the dihydrate, ChH406.2H20 (0.8 mM), in 35 cc. of water and 5 cc. of M sodium bicarbonate, 6 cc. of 0.43 M sodidm periodate were added dropwise, in order t,o avoid the liberation of iodine. The mixture was adjusted to a volume of 50 cc. The titration of 5 cc. aliquots, removed 10 and 30 minutes after the start of the experiment, showed in both cases a total consumption of periodate corresponding to 1.6 mM. The remainder of the oxidation mixt,ure served for the isolation of oxalic acid, which was obtained in an amount corresponding to a total production of 1.54 mM.

5-Keto-d-gluconic Acid (Experiments 6 and 7, Table IV)-An aqueous suspension of 0.944 gm. of calcium 5-keto-d-gluconate (23HzO) was warmed slightly with a solution of 254 mg. of oxalic acid dihydrate. After 5 hours with occasional shaking the calcium oxalate was filtered off and 336 mg. of sodium bicarbonate were added to the filtrate, which had a volume of about 100 cc. Following the addition of 50 cc. of a 0.43 M sodium periodate solution, the volume of the mixture was adjusted to 200 cc. The titration of a 5 cc. aliquot 24 hours later showed that 4.25 moles of oxidant had been consumed per mole of keto acid. (At t.his point no change in periodate consumption was observed when a portion of the solution was made alkaline for an additional 90 minutes by the addition of an equal volume of M

sodium bicarbonate.) For the determination of the oxalic acid formed, 100 cc. of the oxidation mixture were treated as described above. The calcium oxalate monohydrate (0.141 gm.) required 18.2 cc. of 0.1 N KMn04.

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D. 13. SPRIXSON AND E. CHARGAFF 447

1 mole of the keto acid, therefore, yielded 0.46 mole of oxalic acid. From a 75 cc. aliquot formaldehyde was isolated as the dimedon derivative, melting at 189-191”. The product~ion of formaldehyde amounted to 0.18 mole per mole of keto acid.

In view of the probable formation of glycolic acid as one of the oxidat,ion products of 5-keto-d-gluconic acid, which was discussed before, it should be mentioned that glycolic acid, both in weakly acid and in alkaline solu- tion, proved inert to periodate.

d-Glucuronic izcid (Experiments 8 and 9, Table IV)-The oxidation of 1 rnaf of sodium glucuronate with 15 cc. of 0.425 i\l KaI04 in a total volume of 50 cc. for 24 hours showed a consumption of 5.09 moles of oxidant per mole of acid. The same value was obtained on an aliquot which was treated for l+ hours longer with an equal volume of M sodium bicarbonate. The analysis of a 40 cc. aliquot gave 61 mg. of calcium oxalate monohy- drate, requiring 8.0 cc. of 0.1 N KMnO+ i.e. 0.5 mole of oxalic acid per mole of glucuronic acid.

Dipotassium Rhodizonate (Experiments 1 and 2, Table VI)-A total of 1 m&f (0.246 gm.) of the salt was mixed in small portions with 90 cc. of 0.07 N sodium periodate in such a manner as to avoid too great a temporary excess of either oxidant or substrate. After about one-third of the rho- dizonate had been stirred into 30 cc. of oxidant, another 30 cc. portion of periodate wa,s added and this procedure was repeated once more. The color of the salt faded immediately on contact with t,he oxidant. The mixture was brought to a volume of 100 cc.; the titration of 5 cc. aliquots after 13, 43, and 24 hours showed a consumption of a total of 4.35 to 4.40 mM of periodate. Oxalic acid, produced in this oxidation experiment, amounted to 2.3 mM. In another experiment with 1 m&l of dipotassium rhodizonate 2.15 mM of oxalic acid were isolated.

Dipotassium Croconate (Experiments 3 and /t, Table VI)-To an aqueous solution of 109 mg. (0.5 mM) of t.he salt 5 cc. of 0.43 h\~ NaIOe were added and the mixture was adjusted with wat,er to a volume of 50 cc. 3 hours later, the titration of a 5 cc. aliquot indicated the consumption of 3.9 moles of oxidant per mole of substrate. The determination of oxalic acid shoIved the product.ion of 1.9 moles per mole of the salt.

The conditions and products of the oxidative decarboxylation by periodic acid of cr-keto, P-hydroxy-a-amino, and polyhydroxy acids, ketoses, polycarbonyl compounds, and malonic and tartronic acids are discussed in detail.

Outstanding in the speed of oxidation are glyoxylic and mesoxalic acids, which are decarboxylated quantitatively in 10 minutes. Compounds

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448 DECARBOXYLATlONS WITH PERIODIC ACID

giving rise to these keto acids after oxidation with periodate likewise pro- duce the theoretical amount of carbon dioxide.

Polycarbonyl compounds possessing an enediol structure (rhodizonic and croconic acids) similarly react with periodate, most likely with the intermediary formation of mesoxalic acid.

The results obtained with malonic and tartronic acids agree with the assumption that malonic acid first is hydroxylated to tartronic acid, which then is oxidized to glyoxylic acid and carbon dioxide. Tartronic acid is the only monohydroxy acid encountered which is oxidized.

The influence exerted by stable oxygen bridges is exemplified by the apparent multiplicity of mechanisms underlying the oxidative degradation of d-glucuronic acid and of keto sugars.

d-Glucuronic acid yields carbon dioxide and oxalic acid. It probably reacts through several pathways, partly by the way of glyoxylic acid as an intermediate, partly via hydroxylation of the hydrogen next to the carboxyl.

5-Keto-d-gluconic acid produces small amounts of formaldehyde and carbon dioxide and about 0.5 mole of oxalic acid, results best reconciled by the postulation of a furanoid structure for this compound.

Keto sugars do not yield carbon dioxide, except after alkalization of the oxidation mixture. Reaction mechanisms, which take into account the consumption of periodate and the production of carbon dioxide and for- maldehyde, are discussed.

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David B. Sprinson and Erwin ChargaffWITH PERIODIC ACID

ON OXIDATIVE DECARBOXYLATIONS

1946, 164:433-449.J. Biol. Chem. 

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