1,5-di- 3-phos- 2-carboxy-3-ketopentitol-1,5- - PNAS · thesis in green plants is the combination of carbon dioxide with ribulose 1,5-di- phosphate followed byhydrolysis of the product

CHEMISTRY: MOSES AND CALVIN

about 0.9 keal/mole when the hydrogens are trans (assuming negligible delocaliza-tion bonding in other configurations).Another interesting example (called to our attention by Professor R. E. Powell)

is the trans effect of Chernyaev.5 A group trans to an electronegative or otherlabilizing group is weakened in its attachment relative to cis groups. As we havesaid above in a different way, an electronic orbital behaves like a fluid of high sur-face tension.6 Thus we are led to an interpretation of such phenomena as the higherdissociation constants of the trans forms of compounds such as Pt(NH3)2(&H)2 and[Pt(NH3)2(H20)2]++. The potential "hole" left by the removal of one of the nega-tive groups allows the "fluid" orbital to flow into the vacancy. The square planarconfiguration is such that there is less orbital curvature when the potential hole istrans than when it is cis, thereby stabilizing the trans dissociation product to agreater extent than the cis product.

In this light, the trans effect is attributed, not to the labilizing of the trans bonds,but rather to a greater stabilizing of the activated complex in solvolysis as transelimination proceeds, as compared with cis elimination.The trans effect is not restricted to the metal chelates. The same principles can

be applied to the elimination reactions of organic chemistry, which usually involveelements which are trans to each other. Effect 3 also makes readily understandablethe usually greater stability of trans- over cis-disubstituted ethylenes, planarity ofhydrocarbon chains, the greater stability of the chair form of cyclohexane, andother phenomena. A more detailed discussion is in preparation.

1 N. S. Bayliss, J. Chem. Phys., 16, 287, 1948.2E. B. Wilson, Jr., these PROCEEDINGS, 43, 816, 1957.3 R. S. Mulliken, J. Chem. Phys., 7, 339, 1939.4H. Eyring, J. Am. Chem. Soc., 54, 3191, 1932; E. Gorin, J. Walter, and H. Eyring, J. Am.

Chem. Soc., 61, 1876, 1939.6 For discussion and original references see J. V. Quagliano and L. Schubert, Chem.. Rev., 50,

201, 1952; F. Basolo, Chem. Rev., 52, 459, 1953.6 H. Eyring, G. H. Stewart, and R. B. Parlin, Can. J. Chem., 36, 72, 1958.

THE PATH OF CARBON IN PHOTOSYNTHESIS. XXI.THE IDENTIFICATION OF CARBOXY-KETOPENTITOLDIPHOSPHATES AS PRODUCTS OF PHOTOSYNTHESIS*

BY V. MOSESt AND M. CALVIN

RADIATION LABORATORY AND THE DEPARTMENT OF CHEMISTRY, UNIVERSITY OF CALIFORNIA,BERKELEY

Communicated January 13, 1958

INTRODUCTION

It has been well established that the initial carboxylation reaction of photosyn-thesis in green plants is the combination of carbon dioxide with ribulose 1,5-di-phosphate followed by hydrolysis of the product to form two molecules of 3-phos-phoglyceric acid.'-5 Calvin6' 7 has suggested that the reaction proceeds via a six-carbon sugar keto acid diphosphate intermediate, 2-carboxy-3-ketopentitol-1,5-

260 Pitoc. N. A. S.


diphosphate (2-phosphohydroxymetlyl-3-ketopentonic acid-5-phosphate), whichhas, however, not been identified in experiments involving photosynthesis in thepresence of radioactive carbon dioxide, hitherto reported.The standard methods used in this laboratory for the analysis of the products of

short-term photosynthesis experiments performed in the presence of C14 02 involvekilling the cells at the desired time by rapidly pouring the cell suspension into fourvolumes of boiling ethanol. The cell residue is then centrifuged and re-extractedwith boiling 20 per cent ethanol, followed by a final extraction with water. Thepooled extracts are evaporated to a small volume in vacuo at temperatures not ex-ceeding 40°, and aliquots are chromatographed on sheets of oxalic acid-washedWhatman No. 4 filter paper. The solvents (phenol-water in the first dimensionand butanol-propionic acid-water in the second dimension) are normally allowed torun only to the bottom edge of the paper (8-10 hours at 250), or at most twice thatfar, before the papers are dried. The locations of radioactive substances on thechromatograms are determined by radioautography.'These techniques, while very valuable for general purposes, suffer from at least

two disadvantages. First, the extraction of the cells by boiling ethanol-water mix-tures is liable to cause breakdown of heat-labile substances, of which 2-carboxy-3-ketopentitol-1,5-diphosphate (being a f3-keto acid) may be expected to be one.Second, on chromatograms run for the standard lengths of time, the area near theorigin containing principally the sugar phosphates, and particularly the sugar di-phosphates, is not well separated; it is known that the apparently single spots seenin this area are often mixtures containing several diphosphate esters of the relevantsugars.9The present communication reports attempts which have been made to overcome

both these difficulties in an effort to isolate and identify the intermediates proposedfor the reaction between ribulose diphosphate and carbon dioxide to form phos-phoglyceric acid. Two substances of the carboxy-ketopentitol diphosphate typehave now been tentatively identified.

METHODS

Cells of Chlorella jyrenoidosa for experimental purposes were grown in the con-tinuous culture apparatus described by Holm-Hansen et al.'0 The cells were har-vested, washed twice with distilled water, and 400 ,l. of wet-packed cells were re-suspended in 10 ml. of distilled water in a "lollipop" of suitable size. To obtain thecells in a steady metabolic state, the cell suspension was flushed for 30 minutes withair containing 1 per cent (v/v) CO2, the lollipop being immersed in a water bath atroom temperature and illuminated from both sides with 150-watt RS-P-2 photospotlamps. After 30 minutes of flushing, the gas flushing tube was removed from thelollipop, and 1 minute later 0.25 ml. of NaHC"403 (100 ,C.; 6.6 Amoles) was added.The lollipop was stoppered and shaken, and the cells were allowed to photosynthe-size between the light sources for 3 minutes. The cell suspension was then pouredrapidly into 40 ml. of ethanol (precooled to - 15°) and was extracted overnight at- 150. After centrifugation at this temperature, the residue was extracted over-night with 20 ml. of 20 per cent ethanol at 00 and again with 10 ml. of 20 per centethanol at 00 overnight. The cell residue was finally extracted with 10 ml. of 20

VOL. 44, 1958 261


per cent ethanol at 600 for 30 minutes. The extracts were pooled and evaporatedto a small volume in vacuo at room temperature.

Aliquots of the concentrated extract were spotted onto oxalic acid-washed What-man No. 4 filter paper, using a current of air at room temperature to dry the spots;the chromatograms were developed in the first dimension with phenol-water and inthe second dimension with n-butanol-propionic acid-water, the solvents being al-lowed to run for 48 hours in each direction. Radioactive materials were located byradioautography, using Dupont blue-sensitive single-coated X-ray film 507E.

RESULTS

Chromatography for 48 hours in each dimension of the ethanol- and water-soluble components of algal cells after 3 minutes of photosynthesis in the presenceof C1402 showed the presence of three distinct radioactive spots in the diphosphatearea of the chromatogram (Fig. 1). Each of these spots was cut out, eluted, phos-

J- Al

~OPOR0NIC*CIO-WATER

Is~~~~~~~~~~~~~~~~~~~~~~4 #5

'S.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

FIG. l.-Radioautogram of ethanol-water extract of Chiorella after 3 min-utes of photosynthesis in the presence of C1402. The solvents were allowed torun for 48 hour~s in each dimension.

phatased, and rechroinatographed. The spot labeled "RuDP" contained, afterphosphatasing, predominantly ribulose, smaller quantities of glyceric and glycollicacids, together with twelve or more weakly radioactive substances which were notidentified. The spot labeled "FDP and other Di-P's" contained, after phosphatas-ing,' mainly fructose, glucose, and sedoheptulose, together with small quantities ofglyceric and glycollic acids and one unidentified compound. The third spot ("Ketoacid Di-P's"), never previously observed, ran more slowly in the butanol-propionicacid solvent than did the other two. It seemed possible that this spot might con-tain a component more acidic than the other sugar diphosphates and steps weretaken to determine the nature of this substance.

262 PROC. N. A. S.

VOL. 44, 1958 CHEMISTRY: MOSES AND CALVIN 263

After treatment with purified Polidase SI' or with human seminal acid phospha-tase'2 overnight in approximately 0.02 M acetate buffer, pH 5, a number of spotswere seen on subsequent chromatography and radioautography (Fig. 2). Two ofthese appeared of particular interest, and they have now been tentatively identified(see "Discussion") as 2-carboxy4-ketopentitol (CH20H COH(COOH) CHOH -CO CH20H) and the lactone of 2-carboxy-3-ketopentitol (CH,2OH. COH(COOH)CO CHOH CH20H). These substances will henceforth be referred to as the 'y-keto acid and the /3-keto acid, respectively.

SUPETD '~KTO ACID LACTSM.Nj CIi

~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~. .......

.....H S)

MIUWOSEWAE

SUSP'ECTED-KETO AMD

UNKNOWN ..PHOS RATEAFTER TRE TMENT WITHACID PH SPH ATASE

PHENOL~-*ATEP 1 OURS *-

FIG. 2".-Radioautogram of spot labeled "Keto acid Di-P's" from Fig. 1, afterphosphatasing.

Investigation of the 'y-Keto Acid.-A number of tests have been applied to deter-mine the structure of this compound, the results of which are consistent with itsbeing 2-carboxy-4-ketopentitol. At no time have more than tracer amounts ofeither the f3- or the -y-keto acid been available, and all investigations have dependedon following the radioactivity after various treatments.

a) Electrophoresis and Chromatography with Known Substances. Electrophoresison oxalic acid-washed W~hatman No. 4 filter paper in 0.1I M ammonium acetatebuffer, pH 9, for 3 hours at 600-700 volts, together with authentic samples of glu-conic acid, 2-ketogluconic acid, 5-ketogluconic acid, and hamamelonic acid, showedthat all the available known six-carbon acids and the unknown substance ran thesame distance along the paper and could be separated from five-carbon and seven-carbon sugar acids. The known six-carbon sugar acids could not be separated fromeach other or from the unknown substance when chromatographed in the solventsystems mentioned above, suggesting that the unknown had a generally similarstructure, i.e., a polyhydroxymonocarboxylic six-carbon sugar acid.


b) Lactonization. Treatment of the -y-keto acid with 0.07 M HCl at 1000 for 30minutes produced no lactone (Fig. 3), suggesting that it is not a simple aldonic acid.Polyhydroxymonocarboxylic sugar acids all readily lactonize in acid solution.However, the keto acids-2-ketoluconic acid and 5-ketoluconic acid-undergodirect acid lactonization with much greater difficulty than the parent hydroxy acid,gluconic acid. 13

c) Enolization and Epimerization by Alkali.-The y-keto acid was allowed to standfor 9 hours at room temperature at pH 11-12 (NaOH), and was then acidified at 00with HCl to pH 3. Chromatography showed the formation, by such treatment, ofsome ribulose and a trace of glyceric acid, while most of the original material re-mained apparently intact (Fig. 4); there thus appeared to be no stable epimerformed by alkali. The double spot of the 7-keto acid resulting from this treat-ment was probably due to the presence of sodium chloride. The question ofenolization will be discussed below in the section on the ,3-keto acid (paragraph a).

d) Treatment with 2,4-Dinitrophenylhydrazine.-The y-keto acid was treated withan excess of 2,4-dinitrophenylhydrazine in 2 M HCl for 30 minutes at 37°. Carrier5-ketogluconic acid was also present. The hydrazones were extracted from the re-action mixture and chromatographed in one dimension on Whatman No. 1 filterpaper, using n-butanol saturated with 6 per cent (w/v) aqueous ammonia.'4 Two2,4-dinitrophenylhydrazones were produced in low yield, which ran with the hydra-zones of the added carrier. This suggested the presence of a carbonyl grouping.

e) Reduction with Borohydride.15' 16 Reduction of the y-keto acid by approxi-mately 0.0075 Ml potassium borohydride for 2 hours at room temperature produceda substance capable of undergoing acid-lactone interconversion at appropriate pHlevels (Figs. 5 and 6). This implies the reduction of a keto group to a hydroxylgroup, which is then capable of lactonization.The product found after reduction with borohydride chromatographed very

nearly, but not identically, with hamamelonic acid and hamamelonic lactone. Twosamples of "hamamelonic acid" were available; one, made by Schmidt and Heintz,"'was obtained by the action of hydrogen cyanide on d-ribulose, followed by hydrol-ysis of the nitrile and separation of the epimeric acids produced, by fractionalcrystallization of their phenylhydrazide derivatives.'8 The other sample preparedby Rabin et al.,'9 was synthesized by treating ribulose diphosphate with potassiumcyanide, with simultaneous hydrolysis of the nitrile to hamamelonic acid diphos-phate, followed by enzymatic removal of the phosphate groups. This sample, pre-sumably, contained hamamelonic acid and its epimer in unknown proportions.The y-keto acid, after reduction with potassium borohydride, chromatographed

more nearly with Rabin et al's. sample of mixed hamamelonic acid and epimer (ortheir lactones) (Figs. 7 and 8) than with Schmidt and Heintz's pure hamamelonicacid or lactone (Figs. 5 and 6). Before reduction with borohydride, the -y-keto acidran quite distinctly from the hamamelonic acid of Schmidt and Heintz (Fig. 9), andno lactonization could be observed (Fig. 3). The explanation for these findings maybe that the -y-keto acid is reduced by borohydride to a mixture of two epimers of thehamamelonic acid type, neither of which is hamamelonic acid itself, but one of whichis the same as the epimer of hamamelonic acid present in Rabin et al's. sample.

f) Treatment with Acid. On heating with 0.07 Ml HCl for 30 minutes at 1000,some 35-45 per cent of the oy-keto acid was converted into xylulose, presumably by

PROC. N. A. S.264

CHEMISTRY: MOSES ANDCALVIN2

p

.o...I.F-A.I...CXYLULOE

BUTANOL-

PROPIONIC AIWATER(8 HOURS)

u

Y-KETO ACID

* . .: :::::. .: . :* .: . :: :: : ... . . .:..::

*:

::..::... ......... .. : .*:*.:

:*: ::: .:: ....... ...

...........::. : ...... ..* .. :: ::.

,l. . ... ...... .t4> "LED FOR.S:.Sl" elrH o.XT N <l

:: .:

.......:

*. :::.......

*: ::

::s:

::.

W.z-- I PHENOL-WATER (S HOURS)

FIG. 3.-Radioautogram of -y-keto acid after treatment with 0.07 M HClfor 30 minutes at 1000.

SUTAfOL-FOROROMC AOCIDWATER

(8 HOURS(: .:̂.

GWCERl:G# . ^ tr: t i:* * st ,> _ X w3

7I PHENOL-MTER (S HOURS

FIG. 4.-Radioautogram of the y-keto acid after being maintained at pH11-12 at room temperature for 9 hours, followed by acidification to pH 3 at0.

J0

VOL . 44, 1958 265

:1

ORIGNALY-KETO

CHEMISTRY: MIIOSES AND CALVIN PROC. N. A. S.

1)~~~~~~~~~~~pA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..'4CHK .;

A'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*Se*4ikN..\

AUTHAMA DE AC.

FiG. 5.-Radioautogram of the -y-keto acid, reduced with potassium boro-hydride and co-chromatographed with the hamamelonic acid sample ofSchmidt and Heintz (Ann., 515, 77, 1935), spotted from alkaline solution (pH13). The outline of the marker spot of authentic hamamelonic acid has beendrawn in.

"U.!-4 .:J.4:

7-KETO ACID REDUCED WITHKBH4 AND CHROMATOGRAPHEDWITH HAMAMELONIC LACTONEOF SCHMIDT ANV HEINTZ;SPOTTED FROM AID SOLUTION

FIG. 6.-Radioautogram of the -y-keto acid, reduced with potassiumborohydride, and co-chromatographed with the hamamelonic acid sampleof Schmidt and Heintz [Ann., 515, 77, (1935)], spotted from acid solution(pH 1). The outline of the marker spot of authentic hamamelonic lactonehas been drawn in.

266


r

v

BUTANOL- tPROPIONC ACID-

WATER(5 HOURS)

-t

2r-nrO AC> REOUCED WITH

KBK, AND CHROMATOGRAPHEDWITH HAMAMELONIC ACV AND-ISOMERS OF RIN r AL.

SPOED FROM ALKALINE SOLUTION

REDUCED Q OUTLINE OF MARKERY-KETO ACI o*SPOT OF HAMAMLONIC

ACID ISOMERS

I PHENOL-WATER IS HOURS$

FIG. 7.-Radioautogram of the y-keto acid, reduced with potassium boro-hydride and co-chromatographed with the hamamelonic acid sample ofRabin et al. (J. Am. Chem. Soc. [in press]), spotted from alkaline solution (pH13). The outline of the marker spot of authentic hamamelonic acid (andepimer) has been drawn in.

9A

SJTA-L-PROPIOMN ACID-WATERHOURS)

LACTONE OF OUTLINE Of MARKER SPOT

REDUCED Y-KETO AQDO&F HAMAMELONsC LACTON SOvMERS

st

Y-KETO ACID REDUCED WITHK&H4 AND CHROMATOGRAPHEDWITH HAMAMELONIC LACTONEOF RABIN CTr Al SPOTTEDFROM ACID SOLUTION

WAT;' R ( isf l5)FIG. 8.-Radioautogram of the y-keto acid, reduced with potassium boro-

hydride and co-chromatographed with the hamamelonic acid sample ofRabin et al. (J. Am. Chem. Soc. [in press]), spotted from acid solution (pH 1).The outline of the marker spot of authentic hamamelonic Iactone (and epi-mer) has been drawn in.

VOL. 44, 1958 267


decarboxylation, leaving the rest of the original compound apparently unchanged(Fig. 3).

g) Treatment with Alkali.-On heating the y-keto acid with 0.015 M NaOH for30 minutes at 1000, several substances were produced, one of which was glycericacid (the others were not identified), and again some (about two-thirds) of theoriginal compound was left apparently unchanged.

65 ANOL-

i..K....... ..

pPPROONCC AC!D-

u8 HOUPS),

:UT:NE:A.KE SPOTOF HAMAMELONMC LACTONE

.... .... ... . .

::-::..: T.: ROMATOGRAPNED,

NAMAIEWN11 AC-I AND LAC0TNENOPSHNO AND ENERTZ:--W

SWOTEO FROM SOLUTION OF' pH

~~~~~~~~... ....

FIG. 9.-Radioautogram of the -y-keto acid prior to reduction, co-chroma-tographed with the authentic sample of hamamelonic acid and lactone ofSchmidt and Heintz (Ann., 515, 77, 1935), spotted from solution of pH 5.The outlines of the marker spots of hamamelonic acid and lactone have beendrawn in.

Investigation of the f3-Keto Acid. a) Possible Formation from the y,-Keto Acid.-Treatment of the -y-keto acid with weak alkali, followed by acidification (see para-graph c above) resulted in the formation of some glyceric acid and ribulose, whilemost (over 90 per cent) of the -y-keto acid remained unchanged (Fig. 4). Acidtreatment of the y-keto acid results in the partial conversion to xylulose, not ribulose(Fig. 3). It seems that at pH 11-12, some gl-keto acid is produced from the 'y-ketoacid by enolization, the former decomposing to glyceric acid in alkali and to ribulosein acid.

b) Lactonization.-Treatment of the 03-keto lactone with 10-4 M NaOH pro-duced a substance running chromatographically similar to the -y-keto acid (i.e.,just behind hamamelonic acid; see Fig. 9), together with a little of what appearedto be glyceric acid (Fig. 10). Alkali treatment of the 03-keto lactone apparently con-verted it partly into the i3-keto acid and also caused some breakdown to glycericacid (cf. paragraph g above).

c) Reduction with Borohydride.-When the f3-keto lactone was treated with0.0075 M potassium borohydride for 2 hours at room temperature (pH about 10),

PROC. N. A. S.268

4(CHEIIISTIRV: MOSES AND (-ALV1I2-

OUTLINE OF MARKER SPOT OFHAMAMELONIC LACTONE

(3-KET(

(3-KETO ACID: CHROMATOGRAPHED,

BEFORE REDUCTION, WITH

HAMAMELONIC ACID OF

SCHMIDT AND HEINTZ

BUTANOL-PROPIONIC ACID-WATER(8 HOURS)

GLYCERIC ACID

O LACTONE

II

OUTLINE OF MARKER SPOTOF HAMAMELONIC ACID

Ki KETO ACID

I PHENOL-WATER (8 HOURS) AFi(.. 10. Line (drawing posite Ia(lioautogram an lspae c hromatog ram of3-keortolactonle co-chromaltograiphedl w-ith ham~amelonic acid and lactone of Schmidt and Heintz (A no..

515, 77, 1935), spotted from a solution of pH 10. Solid tareas represent radioactive sul)staaces';outlines ale of authentic compounds.

BUTANOL-PROPIONICWATER(8 HOURS)

ACID-

iI

GLYCERIC ACID

OUTLINE OF MARKER SPOT OF

OF HAMAMELONIC LACTONE

(3-KETO ACID REDUCED WITHKBH4 AND CHROMATOGRAPHEDWITH HAMAMELONIC ACID OF

SCHMIDT AND HEINTZ

REDUCED /9--KETO ACID

,_OUTLINE OF MARKERSPOT OF HAMAMELONICACID

4- I PHENOL-WATER (8 HOURS) AFici. I 1. Line d(rawinig of composite iadioautogram ani(I spiraved chromatogramI of 0-keto

lactone after re(ldction with )otassillmn orohd(lricle and( chromatographe1 wvith hamnainelonic acidand lactone of Schhmidt and Heintz (,Anni., 515, 77, 1935), spotted from solution of pH 10. Solidareas rel)resent rad(ioactive sullbstances outlines are of authentic compounds.

apparently some glyceric acid and a substance which ran chromatographically to-gether wvith hlamamelonic acid wN-ere formed (Fig. 11). However, the radioactivitvw-as too weak to determine the exactitude of coincidence wvith haimamelonic acid.A careful investigaition of each of the three diphosphate spots shown in Figure 1

has not revealed the presence of any other substances either of this type (keto acid)or of the reduced form (hamamelonic acid and epimers).

VO(L. 44, 1958 26,9

_ _


DISCUSSION

While the final identification of the two materials under investigation must awaitco-chromatography with authentic samples, the evidence accumulated here per-mits a tentative assignment of structure, with some degree of confidence. Theposition of the original spot on the chromatogram (Fig. 1) is fairly definite evidenceof its diphosphate character. Its susceptibility to the enzyme phosphatase, re-sulting in a material apparently free from phosphate, may be taken as additionalevidence for its phosphate ester character. Most of the evidence available hasbeen obtained with the more plentiful and the more stable of the two new com-pounds, and the course of the argument for the structure of this compound willfirst be outlined, followed by inferences from this substance and additional evidencefor the structure of the second and more unstable compound.The chromatographic and electrophoretic behavior of the dephosphorylated

material seems to establish its character as a hydroxy sugar acid; beyond this, itslocation in the region of gluconic acid is fairly definite evidence of its weight, cor-responding to that of a six-carbon polyhydroxy acid. While the possibility of aheptonic acid is not eliminated on chromatographic grounds alone, the other evi-dence to be mentioned later appears to preclude this possibility. Electrophoreti-cally, the material moves exactly parallel with gluconic acid as well as with hama-melonic, 5-ketogluconic, and 2-ketogluconic acids. The demonstrated ability ofpaper electrophoresis to separate the pentonic acids from a variety of penta-oxy-hexonic acids makes the value of the mobility of the unknown substance a signifi-cant quantity in establishing its penta-oxyhexonic character.The failure of the compound to lactonize under acid conditions which lead to

lactone formation in all other simple aldonic acids, such as gluconic, hamamelonic,ribonic, and similar acids, indicates that the material is not a simple aldonic acid.Reduction by potassium borohydride produces a material which does exhibit thefacile acid lactone interconversion so characteristic of the aldonic acids. This in-formation, taken together with the fact that the two keto-aldonic acids available tous-2-ketogluconic and 5-ketogluconic-are also sluggish in their lactonizationunder the conditions used in this work, suggests that the compound is originally aketo-aldonic acid. The co-chromatography of the reduced material, both in thefree acid and in the lactone form, with hamamelonic acid and its lactone is so closeas to confirm the idea that the material after reduction is indeed a six-carbon al-donic acid and therefore, before reduction, a six-carbon keto-aldonic acid. Co-chromatography of the unreduced substance with hamamelonic acid clearly dem-onstrated its difference from the latter. The treatment of the original keto acidwith 0.07 M HCl for 30 minutes at 100° yielded about 40 per cent conversion toxylulose, the remaining 60 per cent being recovered as the original acid. Thisstability eliminates the serious possibility that the unknown substance is a f3-ketoacid.The nearest analogy available to us is ,B-ketogluconic acid, which has been postu-

lated as an intermediate in the conversion of 6-phosphogluconic acid to ribulose 5-phosphate.20'2' The inability to isolate a sample of this material (6-phospho-3-ketogluconate) has been taken as evidence of its gross instability. The chemicalanalogies about which we do have direct information include acetoacetic acid, a,a-dimethylacetoacetic acid, and dihydroxymaleic acid (a-hydroxyoxaloacetic acid).22

270 PROC. N. A. S.


The substitution of either methyl or of hydroxyl on the a-carbon atom of a f-ketoacid increases its lability considerably. Neither of the latter two acids could survivethe 1000 treatment for more than a few seconds to minutes. We can therefore beconfident that the substitution of both methyl and hydroxyl on an a-carbon atom ofa /3-keto acid such as is here proposed would lead to an extremely labile compound.The possibility that we might be dealing with a 2-keto-3-carboxy pentitol stabilizedagainst decarboxylation by a 2,5-ketol (furanoside) formation is eliminated on thegrounds that such a furanoside would not exhibit the great susceptibility to borohy-dride reduction that this compound does. Beyond this, the appearance of glycericacid in the degradation of the compound speaks against the carboxyl group beingon the three-carbon atom of a five-carbon chain.The clean and quantitative formation of xylulose under the very mild conditions

of acid treatment described above eliminates both the a- and the a-keto acids aspossibilities since these acids would be unchanged under the mild conditions usedhere. 13 2-Ketogluconic and 5-ketogluconic acids were treated with 0.07 M HC1for 30 minutes at 100° and chromatographed in the usual manner: they were foundto be unchanged, and no neutral material (lactones or sugars) appeared. Instronger acid (5 M HCl) both the 2- and the 5-ketogluconic acid can be convertedvia the corresponding ascorbic acids to decarboxylation products such as furfural.'3The likelihood thus remains that the compound is a 4-(or y-)keto acid.

Steric considerations apart, three six-carbon 'y-keto sugar acids are possible (I,II, and III):

COOH COOH COOH

C-(OH)-CH20H CHOH CHOH

CHOH CHOH C-(OH)-CH20H

C=:O _C=O C O

CH20H CHOH CH20H

CH20HI II III

Structure III can probably be excluded on the grounds that decarboxylation ofsuch a branched-chain sugar acid, if it occurred at all, would most probably result ina branched-chain pentose, or a tetrose, whereas xylulose was actually the decarboxyl-ation product. Structures I and II cannot be unequivocally distinguished, thoughthe available evidence favors I. Decarboxylation of II would be expected to bemore difficult than that of I, since it would require the elimination of a secondaryhydroxyl group in II rather than a tertiary one, as in I. Furthermore, the alkalinedecomposition of the unknown substance (presumably via epimerization to the ,B-keto acid) to produce some glyceric acid is more in keeping with I than with II.We must now provide a route for the formation of xylulose (IX) from such a

'y-keto acid (IV) under the conditions in which we know this transformation occurs.Unfortunately, we have no single model compound incorporating all the structuralfeatures of this one, viz., an a-tertiary hydroxyl group, a fl-hydroxyl group, and a-y-ketone function, which seem pertinent to an understanding of this transformation.

VOL. 44, 1958 271


o CH20H

0- -C-OH

HO0H-_-OH (IV)y-Keto acid

&H2OH+H+

0.07 M HC1 for 30 min. at 1000

CH20H

H 11OH (V)

IH20H

transelimination of waterfrom tertiary alcohol(rate-limiting step)

-H+ -H20

1-0

CH20H

H-C-OH

HO-A-H (IX)l=0 Xylulose (racemic)C=0

CH2O-Httransaddition of water

H2C H

0 C.- - - -- OH (above the plane)L I11 C .-----H (below the plane)

II /H2 (VIII)H CH2i

Enol stabilized by inter-O--H nal hydrogen bonds (one

t 5-ring; one 6-ring)

decarboxylation of3,,y-diketo acid

k I

C--t-V -H + (from below the plane)WH~~~~l A/ o~~~~~~~ H2 (above the paper plane)+H 0c~O

0 C==O H-, OH (below the paper plane/ ketonization

HI2C |C=-O

O-H 1H2C

(VI) O-H (VII)

FIG. 12.-Proposed mechanism for the decarboxylation of the -y-keto acid to xylulose.

One can, however, formulate it in terms of a sequence of three reactions, all ofwhich are known to take place rapidly under the conditions here required. Theseare (1) the transelimination of water from V to form the a,#3-unsaturated, f3-hy-droxy, y-keto acid (VI), which ketonizes to form the fl,,y-diketo acid (VII); (2)the decarboxylation of this ,3-keto acid to form an enolic ketone (VIII); and (3)the transaddition of water to this enolic ketone, yielding xylulose (racemic) (IX)-(Fig. 12). The rates of all these reactions in separate model cases are known to besufficiently rapid to account for this sequence. The absence of any other inter-mediates on the chromatogram (Fig. 3) would require that the over-all rate-limitingstep be the transelimination of water (V to VI). The uncertainty lies in the pos-sible simultaneous occurrence of the decarboxylation (VII to VIII) and the trans-

272 PROC. N. A. S.

e)


C02

H20H

HOOC-A-OH

H-&--OH

-0(XV)&H20H

KBH4

I!

CH20H

HO-C-COOH

H-C-OH

HO-C-H

XI20H

t (XII)

AH2OHHOOC-b-OH

H-A-OH

HO-I-H

&20H

(XI)

CH20H

H & OH Ribulose

H-C-OH

JH20H

KCN

\UH20H H20H

HOOC- !-OH HO-2-COOH

H-&-OH H-X-OH

H-s-OH H-s-OH

&H20H bH20H

(XIII) I (XIV)J.HOi Hamamelonic

KBH4 Acid

HO-Y--COOH

H-s--OH

6H20H (XVI)FIG.13. -The steric relationships between the two possible diastereomers of the y-keto acid

and its reduction products, epimers of hamamelonic acid.

addition of water (VIII to IX). One can rationalize a stabilized enolic ketone(VIII) in terms of internal hydrogen bonds which would give it a lifetime longenough to provide the configurational requirement needed for the transaddition toproduce xylulose. The possibility that the series of transformations may beginby the formation of the f,,y-enol, -y-lactone of IV is recognized and considered un-likely in view of the ease of the tertiary hydroxyl elimination to form VI.The question of the steric configuration of the 'y-keto acid poses a further consid-

eration. Figures 5-8 show that, after reduction with borohydride, the y-ketoacid co-chromatographs very closely, though not identically, with the two samplesof hamamelonic acid available. Figure 13 shows the steric relationship betweenthe two possible diastereomers of the -y-keto acid (XV and XVI) and its reductionproducts, epimers of hamamelonic acid (XI to XIV). The sample of hamamelonicacid obtained by Schmidt and Heintz"7 contained only one isomer, hamamelonic

273VOL. 44,1958


acid itself, structure XIV, while that of Rabin et al.,19 prepared by the action ofpotassium cyanide on ribulose diphosphate, presumably contained structures XIIIand XIV. Two possible epimeric forms of the y-keto acid can be produced by car-boxylation of ribulose, structures XV and XVI, which on reduction would result insubstances XI and XIII and XV, and XII and XIV from XVI. Experimentally itwas found that-the reduced 7y-keto acid co-chromatographed more closely with themixture of XIII and XIV than with pure XIV itself. It could be argued from thisthat reduction of the -y-keto acid produced XI and XIII, neither of which was iden-tical with the hamamelonic acid of Schmidt and Heintz (XIV)17 and which there-fore did not co-chromatograph precisely with this sample. However, one form ofthe reduced y-keto acid (XIII) would correspond with one epimer in Rabin et al's.'9sample. The lack of exact co-chromatography in this case would then result fromthe presence of radioactive XI derived from the y-keto acid by reduction and of car-rier XIV present in the mixture of Rabin et al.The )-keto acid thus tentatively appears to be best represented by structure XV.

It is interesting to note that transelimination from this stereoisomer would producedirectly the proper geometric form of the enolic ketone VI (Fig. 12). The structureXV is consistent with the inability to lactonize, as six-membered lactone rings con-taining a ketone group represent strained configurations and tend not to be formed.The evidence for the structure of the ,3-keto acid (XVII) is less extensive. fl-Keto

acids are known to be very unstable,22 as this substance has been found to be

CH20H

C-(OH)-COOH

CHORICH20H XVII

while the ability of the substance to lactonize under acid conditions (indeed, it wasfirst observed as a lactone) would, in this case, involve a five-membered lactone ringcontaining a ketone group, which is a less sterically strained configuration. Undermild alkaline conditions the lactone ring is easily opened to produce the free acid,and the substance readily decomposes to form glyceric acid, by hydrolysis betweencarbon atoms 2 and 3. The free acid, like the -y-keto acid, does not co-chromato-graph with hamamelonic acid, though, after reduction with borohydride, co-chro-matography is very close to and perhaps identical with hamamelonic acid (Figs. 10and 11).

Decarboxylation of the B-keto acid was not directly demonstrated, since the en-tire supply was used in the reduction experiment. However, treatment of the 'y-keto acid with mild alkali, followed by acidification at 00 to pH 3, conditions whichmight be expected to produce some of the ,B-keto acid from the y-keto acid by enoli-zation, produced instead ribulose and glyceric acid (Fig. 4). The latter could re-sult from hydrolytic cleavage of the j#-keto acid at alkaline pH, and the ribulose bydecarboxylation at acid pH. In this case decarboxylation would involve the migra-tion of the carbonyl group (Fig. 14). No evidence is available as to the steric con-

274 PROC. N. A. S.


o CH20H CH20H-C--OH Decarboxylation of IOH

/l-keto acid 2:3Enediol

0 H-0-O (XIX)v-1 ~~~H--OHHOH2&H20H

(XVIII) Assymetricallyinduced

ketonization

&20H

H--OtH Ribulose

H-k|H (active)

&H20H (XX)

FIG. 14.-Suggested mechanism for the decarboxylation of the ,-keto acid to ribulose.

figuration of the #-keto acid, save that the 5-carbon atom should have the sameconfiguration as the original ribulose from which it was formed.

Biological Significance.-While the f-keto acid diphosphate has been predictedtheoretically in the photosynthetic carboxylation of ribulose diphosphate,6' I therehas been, to our knowledge, no previous demand for the-existence of the 'y-keto acid,and hence no role is immediately apparent for it. It may, indeed, be an artifactresulting from the procedures of killing and extracting the cells and from the subse-quent chromatography and thus be produced non-enzymatically from the 3-ketoacid or from another compound. However, it seems that, using identical killingand chemical techniques, more radioactive -y-keto acid appears to be formed fromlabeled carbon dioxide in cells which have been allowed to carry on photosynthesisin the presence of 0.001 M NH4NO3 than in cells suspended in distilled water. The-y-keto acid may accordingly be involved in nitrogen metabolism.

It is of interest in this connection to recall the findings of Done and Fowden,23who observed that in peanuts the most important free amide present in these plantswas y-methylene-glutamine (XXI), while the relative amount of glutamine in rela-tion to asparagine was very much reduced. y-Methylene-glutamic acid (XXII)and 'y-methylene-a-ketoglutaric acid (XXIII) were also subsequently found inpeanuts,24' U and it was suggested24 that these compounds may be the ones mostintimately concerned with nitrogen transport in these plants. The similarity be-

CONH2 COOH COOH

C=CH2 C=CH2 C=-CH2

CH2 CH2 OH2

CHNH2 CHNH2 C=O

OOH COOH GOGHXXI XXII XXIII

VOL. 44, 1958 275


CH2-OH CH2-OH CH2-OH

HOOC-C-O-H HOOC- A-S0H HOOC-b

H-C-O-H +H20 H4-0-oH -H20 A-0-Hb=0 -4(H)>ie7'&H20H bOOH OOH

(IV) (XXIV) (XXV)+2(H)

CH2-OH CH2-OH CH2-OH

HOOC-b-H HOOC- HOOC-C-H

H-Y-H +2(H) C H -H20 H-C-0-H

C=OC==0 C=0

bOOH OOH bOOH(XXVIII) (XXVII) (XXVI)-H20

OOH COOH CONH2

b=CH2 L=CH2 b=CH2

&H2 transamination &H2 &H2do &HNH2 &HNH2OOH OOH OOH(XXIII) (XXII) (XXI)

FIG. 15.-Hypothetical scheme for the conversion of the y-keto acid into y-methyleneglutamicacid.

tween the skeletal structures of the y-keto acid (IV) and 'y-methylene-glutamic acidand amide (XXI and XXII) are immediately apparent, and it would not be diffi-cult to conceive a metabolic pathway for the production of 'y-methylene-glutamicacid from the y-keto acid. Such a hypothetical pathway, involving alternate de-hydration and reduction following an initial oxidation, is illustrated in Figure 15,though many other schemes would be possible; it is of interest to note that the -keto acid and y-methylene a-ketoglutaric acid are isoximeric.Another possible route for the further metabolism of the y-keto acid might be an

enzymatic inverse aldolase split to hydroxypyruvate and dihydroxyacetone. Thehydroxypyruvate could give rise by transamination to serine, a substance known toincorporate radiocarbon very rapidly from carbon dioxide during photosynthesis.The dihydroxyacetone, during the initial stages of photosynthesis, would not in-corporate isotopic carbon from labeled carbon dioxide, and such a dilution oflabeled dihydroxyacetone, formed from 3-phosphoglyceric acid, by this unlabeleddihydroxyacetone might in part account for some of the anomalous labeling whichhas been reported in the hexoses during photosynthesis.26Treatment of all the three diphosphate spots shown in Figure 1 with phosphatase,

followed by further chromatography, has confirmed the findings of Rabin et al,19that hamamelonic acid diphosphate is not formed during photosynthesis in the ab-

276 PROC. N. A. S.


sence of inhibitors. Apart from the #- and y-keto acids which form the subject ofthis communication, no other acids moving chromatographically in the area ofhamamelonic acid or its lactone have been found in any of the three diphosphatespots of the original chromatogram.

SUMMARY

Two new compounds, tentatively identified, have been isolated from chromato-grams of ethanol-water extracts of cells of Chlorella pyrenoidosa which had been al-lowed to carry on photosynthesis for 3 minutes in the presence of NaHC'403.Chemical investigation suggested that the two compounds were 1,5 diphosphateesters of 2-carboxy4-ketopentitol and of the lactone of 2-carboxy-3-ketopentitol.The latter has been postulated as an intermediate in the photosynthetic carboxyla-tion of ribulose-1,5-diphosphate to yield two molecules of 3-phosphoglyceric acid.While the former might be an artifact resulting from the techniques used to kill, ex-tract, and analyze the cells, possible biological roles for it have been suggested.

* The work described in this paper was sponsored in part by the U. S. Atomic Energy Com-mission and in part by the Department of Chemistry, University of California, Berkeley, Cali-fornia.

t Postgraduate Traveling Fellow of the University of London, 1956-57.1 J. A. Bassham, A. A. Benson, L. D. Kay, A. Z. Harris, A. T. Wilson, and M. Calvin, J. Am.

Chem. Soc., 76, 1760, 1954.2 J. R. Quayle, R. C. Fuller, A. A. Benson, and M. Calvin, J. Am. Chem. Soc., 76, 3610, 1954.3 A. Weissbach, B. L. Horecker, and J. Hurwitz, J. Biol. Chem., 218, 795, 1956.4 J. Mayaudon, A. A. Benson, and M. Calvin, Biochim. et Biophys. Acta, 23, 342, 1957.5 E. Racker, Arch. Biochem. and Biophys., 69, 300, 1957.6 M. Calvin, Fed. Proc., 13, 697, 1954.7 M. Calvin, J. Chem. Soc., 1895, 1956.8 A. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas, and W. Stepka, J. Am.

Chem. Soc., 72, 1710, 1950.9 A. A. Benson, in Modern Methods of Plant Analysis (Berlin-Gottingen-Heidelberg: Springer-

Verlag, 1955, 2, 113.10 0. Holm-Hansen, P. Hayes, and P. Smith, University of California Radiation Laboratory

Report UCRL 3595, p. 56 (October, 1956).11S. S. Cohen, J. Biol. Chem., 201, 71, 1953.12 A gift from Professor H. A. Barker.13 P. P. Regna and B. P. Caldwell, J. Am. Chem. Soc., 66, 246, 1944.14 V. Moses, J. Gen. Microbiol., 13, 235, 1955.15 p. D. Bragg and L. Hough, J. Chem. Soc., 4347, 1957.16 M. Abdel-Akher, J. K. Hamilton, and F. Smith, J. Am. Chem. Soc., 73, 4691, 1951.17 0. T. Schmidt and K. Heintz, Ann., 515, 77, 1935.18 We are indebted to Professor 0. T. Schmidt for samples of the ammonium salt and of the

phenylhydrazide of hamamelonic acid: the latter was converted by N. G. Pon to free hama-melonic acid by refluxing with cupric sulfate and removal of excess copper and sulfate with hydro-gen sulfide and barium hydroxide, respectively.

19 B. R. Rabin, D. F. Shaw, N. G. Pon, J. Anderson, and M. Calvin, J. Am. Chem. Soc. (in press)(1958).

20 B. L. Horecker and P. Z. Smyrniotis, Arch. Biochem., 29, 232, 1950.21 J. C. Gunsalus, B. L. Horecker, and W. A. Wood, Bacteriol. Revs., 19, 79, 1955.22 B. R. Brown, Quart. Revs. (London), 5, 131, 1951.23 J. Done and L. Fowden, Biochem. J., 49, xx, 1951.24 J. Done and L. Fowden, ibid., 51, 451, 1952.25 L. Fowden and J. A. Webb, ibid., 59, 228, 1955.26 0. Kandler and M. Gibbs, Plant Physiol., 31, 411, 1956.

VOL. 44, 1958 277

Documents

1,5-di- 3-phos- 2-carboxy-3-ketopentitol-1,5- - PNAS · thesis in green plants is the combination of carbon dioxide with ribulose 1,5-di- phosphate followed byhydrolysis of the product