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Biosynthesis of chelidonic acid I. Preliminary observation on the precursors of chelidonic acid in Convallaria majalis L.
ARCHIVES OF BlOClIEMIS'l'RY AND BJOPHYS!CS
115, 181-186 (1966)
Biosynthesis of Chelidonic Acid I. Preliminary Observation on the Precursors of Chelidonic Acid in Convollaria maio/is L. BRUCE A. BOIIlVP Department of Pharmacognosy, University of Rhode Islancl, Kingston, Rhode Island
Received August 26, 1965 The biosynthesis of chelidonic acid ('Y-pyrone-2,6-dicarboxylic acid) has been studied in mature, flowering Convallaria ma;jaJis L. plants. Glucose-U.L.-140 and ribose-I-He were incorporated into the chelidonic acid molecule with dilutions of 720 and 940, respectively, while the dilution for acetate-2- 14C was 11,000. Shikimate-G-14C was not incorporated. Chemical degradation of chelidonio acid obtained from feeding glucose-f-v'C showed 75% of the label in carbons 3, 4, and 5 and 25% in carbons 1, 2, 6, and 7. These results would preclude formation of chelidonic acid from sedoheptulose as a major pathway.
The ')'-pyrone ring structure occurs in a variety of well-known and often studied compounds, e.g., chromones, xanthones, and some f1.avonoids. There also exists a small group of naturally occurring compounds which are derivatives of v-pyrone per se. In his chapter on naturally occurring pyrones and pyrans, Dean (1) lists less than a dozen of these derivatives of -y-pyrone.
similarity of the structure of kojic acid (I) to that of glucose was discussed. Experiments with specifically labeled glucose in Aspergillus species led them to conclude that a very large percentage of kojic acid arises by means of a direct conversion of glucose. A minor pathway involving threecarbon intermediates also appears to function.
HO -if . HoD OH ~_~CH20H ~_ ~CH3 o
nooc
0
Whereas the more complex molecules 111.entioned above have been examined with regard to their biosynthesis, considerably less is known about the formation of the 'Y'pyrone ring system in the simpler derivatives. Today, the only evidence concerning biosynthesis of -v-pyrones based upon 140 tracer studies is the excellent series of papers by Arnstein and Bentley (2). In this work, the 1 Present address: Department of Biology and Botany, University of British Columbia, Vancouver 8, B. C., Canada.
The intermediacy of simple carbohydrates in the formation of other 'j'·pyrones in bacteria has been discussed by Terada and his co-workers. 3-Hydroxykojic acid has been isolated (3) from cultures of Gluconobacter cerinue supplied with fructose as a carbon source. These workers (4) have also observed the formation of 5-ketofructose during the synthesis of koj ie acid from sorbose in Acetobacter species. It was also demonstrated (5) that Acetobacter species could utilize fructose for the synthesis of 5-hydroxymaltol 181
182
BOI-IM
(II). The form ation of 5-ketofruct{)se was observed by t his group (6) to occur in five species of Acetooacier and t wo species of Gluconobacier, The formation of 5-ketofruct oso in Gluconobacter cerinus has recently been confirmed (7-9). Although this work h as not been supported by isoto pic labeling experiments, it does serv e to indic at e that simpl e sugars play a significant part in t he form at ion of -v-pyrones in bacteri a. Several very interesting -y-pyro ne deriv at ives occur naturally in h igher plants. Perh aps t he most widespread of these is chelidonic acid" (III), which R amst ad (10) reported present in 688 species out of 1143 tested from 9 families. Nothing is known, how ever, about the formation of molecules of this sort in higher plants. The structural similarit y of chelidonic acid to common carbohydrates su ch as glucose or p erhaps sedohep tulose, prompted me to under take an investigat ion of the biosynthesis of th is molecule in Convallaria m ajolie L. It is the purpose of this paper to st udy t he hypothesis that chelidonic acid is a product of carbohyd rate metabolism in higher pl ants. If t his proves to be the case, it will be of interest to compar e th e biochemistry of this general group of com pounds in h igher plants with tha t of kojic ac id an d r elated compounds in the lower organ isms. EXPERIlVrENTAL PROCE DUR E
Source of plant mat eri al. The Con uallaria maj alis L. (Liliaceae) pla nts use d for t he exper im ent s were obtained from thre e sources . Those used for experiments 1-4 were ob tained fr om a local florist. These plants had been maintained u nder refrigeration and limited light to hold them back for E aster sales. Before being us ed, they were allowed to grow in a greenhouse for several days under n ormal illumination and temperatur e (about 75° F ). Pla nts u sed for experiment s 5 and 6 were taken from my home flow er garden . Plants used for experim ents 7 and 8 were t ak en fr om the de partment al Drug Plan t G arden . All exper ime nts 2 The number ing of t he chelidon ic acid m olecul e as presented in t he abstr act of t his p ap er r elies up on 'Y -p yr one as t he p ar en t compound; he nce , t h e number 1 is as sig ned to the h et ero cyclic oxyg en. For the purposes of di scu ssing degradation of ch elid onic a cid, t h e ca rb on a t oms will be number ed from 1 t o 7.
were conducted wit h plan ts which had b egun to flower. Descri ption of feedin g e.lJperiments . Pl a n ts with the flower st alk were cut wi thin one-h alf inch of the ground with a razor bla de, and the cu t end was immedi ately pu t int o a beak er of dis tilled water . The st ems were then out a second ti me , but t h is cu t- t ing was done b eneath t he su r face of t he water to insure t he integri ty of t he transpiration s t r eam . The stem, with a bead of wa te r adheri ng to t he cut end , was then quick ly transferred t o the feedi ng solution. Glucose and ribose were dissolved in di st illed wat er and sodium acetate an d shiki mic acid were dissolved in 0.1 M phosph at e buffer (pH 7.5). After the major portion of th e sol ution had been t aken up by the pla n t , t he remain der was washed in with dis tilled water added 1 ml at a time. After two or three such was hings, the plant was transferred to a b eak er of dis tilled water and allowed to metabolize for a total of 24 hour s . In t he case of the time-dep end ent exp eriments , times of 12, 24 , 36, and 48 hou rs were used . All feed ing experime nts were conducted with the plant materi al located a t a dis tance of 10 inches fro m a ban k of t hree 20 W whi te fluorescent lamps. The t empera t ure during the se experimen ts was n ot controlled but was approxim a tely 25°. In all cas es , t ho pla nt mat eri al appeare d very h ealthy a t t he te rmina tio n of t he feeding experimen ts . S ource of chemi c(tls. The radioactively labeled glucose , r ibose, and sodium aceta te were ob t aine d fr om commercial sources and were used without further purificatio n. Labeled shikimic aci d was prepared fr om Ginkgo biloba shoots by t he procedure of Weinstein et al. (11). Chelido ni c acid was purchas ed from Aldrich Chemical Comp any and w as purified by recrystallization , first fro m wat er and t hen from ethanol. I solati on and analysis of chelidonic acid . At t he end of the clesir ed metabolism period , the plant material was immediately extracted with boiling 80% eth an ol containing 1% hydrochl oric acid. He-extract ion s with this solvent were run un til the pl an t mater ial was no long er colored . T he ext racts t hus obtained were combined and diluted to a known volum e with 80% ethanol, and duplica te aliquo ts were used for determina tion of t otal ethanol sol uble radioac tivi ty . The so lvent was than blown off under a str eam of dry air an d t he resid ue was extracted with boili ng water. After filtr a tion t hr ough a bed of Celite, t he aq ueo us extract was ad j usted t o pH 6.0 with dilute sodium h ydrox ide and passed thro ugh a colum n of Amberli te CG -400 resin (OH -form) . N eutral and basic com ponen ts of the extract were removed fro m the
BIOSYNTHESIS OF CHELIDONIC ACID column by continuous wa shing with distilled water. The total acidic fract ion was eluted with a mixture of ethanol, water, and concentrated HOI in the ratio of -10: 50: 10. The eluate, a pale yellow color , W M repeatedly extracted with ether, which removed t h e chel idonic ac id from the aqueous ph ase . The ether extracts were oombined an d blown t o dryness under a stream of air, and the res idue was dissolved in a little 80% ethanol and applied t o sheets of Whatman No. 1 paper as a narrow band . The papers were run in the system developed by Kwasniewski (12) consisting of ethanol, ooncentrated NH,OII, and water (80:4: 16). I n ea oh case, a sam ple of t he p urified chelidonie acid was run as a standard to assist in locating the desired band. Ch elidonic acid has an R» of 0.55 in this system (at about 25°) and can be detected by its quenching of ultraviolet light of wavelength 2537 A. The bands were cut out and eluted with 80% ethanol, and the combined eluates were blown down to a small volume and reapplied to sheets of Whatman No.1 paper. The sheets were rerun in the same solvent system. On "Vhe second paper, the chelidonic acid ran as a very compact b and without t a ilin g. No other bands were observed under ultraviolet light of 2537 or 3660 A 01' under visible light. To check the possibility t hat some impurity might be running with the same R F as that of chelidonie acid, a sample of the naturally derived material was sub] ected t o chromatography in several other solvent systems: isopropanol-concentrated NH.IOH-water (10: 1: 1), R F 0.13-0 .14; benzene-acetic acid -water (10: 7: 3) , O.Oj and n-bu tanol-pyridine-wat er (14:3:3); R F 0.0. In all these systems, no other substance was present under the lighting conditions mentioned above. Af ter elution from the second banding, the chelidonic acid solution was diluted to a known volume from which aliqucts were taken for radioactivity and ultraviolet spectral measurements. The concentration of chelidonio acid was measured at 277 mJ! (Ema~ 8830) on the Beckman DU spectrophotometer. Radioactivity measurements were made on a vibrating reed elect r om et er system made by Nuclear Chicago (Dynacon (000). In all cases , appropriate blank and background measurements were performed. Deqroslation. of chelulonic acid . Chelidonic acid was degraded according to the procedure of Wilde (13). Chelidonic acid was suspended in a small volume of wa ter, a nd bromine was adde d until liquid bromine could be seen in the bottom of the react ion flask. The re action mix ture was he ated to about 40-50° on a steam bath with gentle agitation from time to time. More bromine was a dded as that in the reaction flask vaporized. Generally,
u,
183
in about 45 minutes most of the chelidonic acid had dissolved and a crystalline product could be seen in the reaction mixture. At this point, bromine was again added and the react ion was allowed to continue for anot her half hour. Heating was continued until all color of bromine was dissipated. The mixture was then cooled to about 1 ° for several hours and filtered. After drying on the filter for a short time, the solid was washed with small portions of chloroform. The chloroform washings were combined and evaporated to dryness under an air jet to yield crude pentabromoacetone. The pentabromoaeetone was recrystallized from ace tic acid to which a few drops of water had been added. The product thus obtained was identical with an authentic sample of pentabromoacetone. The crude oxalic acid remaining on the filter was recrystallized from a small volume of water. A test for the completion of the bromine oxidation was performed on the recrystallized oxalic acid. A few crystals of the acid were placed in contact with a drop of 20% potassium hydroxide solution on a spot plate. The development of a yell ow color would indicate the presence of ehelidonic acid. The color is due to the formation of the open ring xanthochelidonate system. Contaminating chelidonic acid can be removed by oxidation with bromine as described above. Radioactivity meaSUrements from degradation. st udy. Compounds involved in the degradation were counted on a Nuclear Chicago model 720 liquid scintillation counter. Pentabromoacetone was dis solved in tolu ene which cont ained the necessary scintillators. Chelidonic acid and oxalic acid were dissolved in Nuclear Chicago NCS solubifizer and then diluted with toluene-scintillator solution. The solubilieer, which is a strongly basic quaternary ammonium hydroxide, caused oonsiderable degradation of chelidonic acid to the yellow xanthochelidonate system. The The colored solution was responsible for serious quenching, which prevented reliable counts being obtained for the parent compound. No difficulty was encountered when pentabromoacetone and oxalic acid were counted.
RESULTS AND DISCUSSION
The results of the feeding experiments are presented in T able 1. Dilution values were calculated as t he r at io of specific activity of precursor t o specific activity of product. The percentage in corporation in these experim ents was calcul ated by comparing the activity in chelidonic acid with the total aC-
184
BOHM TABLE I RESULTS
OF PREOURSOR FEEDING EXPERIMENTS WITH
Conoallaria majalie L.
Chelidonic Acid
Expt,
Precursor !-,M
1 2 3 4 5 6 7 8a 8b 8e 8d
Glucoae-U .L.- 14C Ribose-1-l 4C Sodium acetate-z-l-C Sodium shikimato-Gvwfl Glucose-U .L.-14C/ribose Glucose-6-14C Sodium acetate-2- 14C Glucose-U.L.-HC (12 hours) Glucose-U.L.-14C (24 hours) Glueose·U.L.-14C (48 hours) Glueose-U.L.-14C (72 hours)
X 10-'
3.3 4.3 2.1 1.7 3.3/40 2.0 2.1 3.3
Sp. act. a
3.05 2.33 48.7 0.11 3.05 5.0 48.7 3.05
pM
X 10-'
!-'C
Sp, act. X 10' a
%
InCOI'P.b
Diluticn"
0.53 1.90 1.29 0.87 27.1 29.9 19.0 21.7
2.25 4.7 5.6 0.0 23.0 27.8 2.51 6.75
4.2.5 2.48 4.34
0.47 0.12 0.15
720 940 11,000
0.85 0.93 0.13 0.31
1.13 1.05 0.09 0.11
3,600 5,400 375,000 10,000
3.3
3.05
40.7
22.7
0.56
0.49
5,500
3.3
3.05
54.3
32.9
0.61
0.84
5,100
3.3
3.05
32.6
27.9
0.85
0.74
3,600
Sp. act. = mC/mmole % Incorp, = ,tiC in chelidonie acid/fLC in initial EtOH extract. c Dilution = Sp. act. of preoursor/sp. act. of product. a
b
tivity in the corresponding initial 80 % ethanol extract. The first four experiments were run in order to gain an insight into the general area of metabolism from. which chelidonic acid is derived. In general, this can be accomplished by studying the incorporation of glucose, acetate, and shikimate into the compound of interest. In the present case, these three precursors along with ribose were examined. No incorporation of label from shikimate was observed. This indicates that chelidonic acid is not a product of aromatic metabolism. The conversion of shikimate to chelidonie acid would entail a difficult to picture series of transformations, the like of which has not been observed with shikimate or its close relatives (14). Dilution values of 720 and 940 were observed with glucose and ribose, respectively. Considering the metabolic activity of these carbohydrates, these dilutions are reasonably low. Acetate was incorporated with a dilution of 11,000, which is 10-15 times less than the simple sugars. It would be incorrect to attribute much significance to the difference between the glucose and ribose dilutions on the basis of the experiments run so far. However, a real difference ap-
pears to exist between these compounds and acetate. This conclusion is supported by the results of experiments 6 and 7, where a difference of almost 70 was observed in favor of glucose over acetate. The overall dilutions were greater in these experiments due to the considerably larger amount of chelidonic add present in the plants used. These results suggest that the point of origin of chelidonic acid lies closer to a carbohydrate pathway than it does to one primarily involving acetate. The manner in which these precursors are converted to the acid is not known at this time, but it seems unlikely that chelidonie acid is derived from the "head-to-tail" condensation normally associated with acetate-derived natural products. The incorporation of glucose into chelidonio acid agrees with the observation of Arnstein and Bentley (2) that glucose was readily incorporated into the related ')'-pyrone, kojic acid. It was pointed out that kojic acid bears a strong resemblance to the glucose molecule. In fact, Arnstein and Bentley (2) concluded that glucose was converted to kojic acid without ring opening. Following their example, one can suggest a resemblance between chelidonic acid and sedoheptulose. When the cyclic form of sedoheptulose and cheli-
185
BIOSYNTHESIS OF CHELIDONIC ACID
donie acid are written side by side, the similarity becomes apparent.
o HOOC { ) C O O H
o The incorporation of label from glucose and ribose into chelidonic acid also leads one to the t empting conclusion that the acid might be a side product of the pentose phosphate pathway in some plants. Two tests of the intermediacy of sedoheptulose were done in the present work. These experiments and their implications are discussed below. Sedoheptulose (as the 7-phosphate) is formed via the pentose phosphate pathway which can be considered to employ glucose 6-phosphate as a starting material. The seven-carbon. sugar arises by means of transfer of a two-carbon. fragmen t to ribose 5phosphate. If ehelidonic acid does derive from sedoheptulose, then a large amount of unlabeled ribose administered concurrently with glu cose-t-O would be expected to trap the label from glucose before it reached sedohep tulose. In experiment 5, glucoseU.J.J.-HC was administered along with a 12""1 excess of unlabeled ribose. In a companion experiment (N o. 6), glucose-f-' -C was administer ed alone. Examination of per centage incorporation and dilution values would suggest that the ribose had no effect on the incorporation of label fr0111 glucose into chelidonic acid. Although ribose does appear to be a relatively good precursor for chelidonie acid, the trapping experiment would seem to preclude a pathway through sedoheptul ose. In order to get a much clearer pi cture of the pro cesses involved in the biosynthesis of chelidonic acid, data on the distribution of label from the precursors would be necessary. To this end, ribo se-Lt-C and glucose0_140 were fed to plan ts and the chelidonic acid was isolated in each case for degradation. Unfor tunately, the ribose feeding product was lost. The degradation procedure chosen for the present st udy was based upon a met hod
described by Wilde (13) in which bromine oxidatively cleaves chelidonic acid into one OH
HO~OH HOCHZ
~n~CHZOH OH 0
equivalent of pentebromoacetone and two equivalents of oxalic acid. Pentabromoacetone is derived from carbons 3, 4, and 5 of chelidonic acid; the two equivalents of oxalic acid are derived from carbons 1 and 2 and carbons 6 and 7 of chelidonie acid. The fragments obtained were counted in toluene solution in a liquid scintillation apparatus after the necessary solubilization of oxalic acid had been accomplished. The solubilizer caused exten sive degradation of chelidonic acid resulting in quen ching; consequently, no valid activity could be recorded for the parent molecule. Results of the degr adation are shown in T able II. If chelidonic acid is formed from sedoheptulose, then a characteristic pattern of labeling would be expected from each member of the pentose phosphate pathway. In the pres ent case, t he labeling from glueose-d -l'C would be found as 0-7 in sedoheptulose. Inso far as chelidon ic acid is a symmetrical molecule, sedoheptulose-7_140 (and, hence, glu cose-Bv'C) would be expected to label the acid in carbons 1 and 7. Exclusive formation from sedoheptulose-f-t -C would yield oxalic acid with 100 % of the 14 0 and pentabromoacetone with none. However, only about 25 % of the chelidonic acid label was found in oxalic acid in the present case; TABLE II DEGRADATIO N OF CHELlDONIC A CID FROM G LUCOSE-G- 140 FEEDING
Compound
dcm/mmole"
%"
Pentabromoacetone Oxalic acid Chelidoni c acid
3620 1240 4850"
75 25
• Disintegra tions per minute per millimole . b To nearest five. c T a ken as to tal of t he degrad ation fr agments.
BOHM
186
hence, the major pathway of its formation does not involve sedoheptulose. A lesser contribution from sedoheptulose is possible, a possibility which will be examined in more detail in a later publication. The present results have opened the door to investigation of the part played by smaller carbohydrate molecules in the formation of chelidonic acid. Experiment 8 was performed in order to look at the formation of chelidonic acid as a function of time. Equal amounts of glucoseU.L._14C were administered to four sets of plants which were allowed to metabolize for 12, 24, 36, and 48 hours in the light. The largest increase in specific activity of chelidonie acid occurred between 12 and 24 hours, during which time the value almost doubled. The 48- and 72-hour values were higher in each case than the proceeding ones, but the differences were not as striking as between the 12- and 24-hour values. No experiments were performed to examine the possibility that an equilibrium situation was being approached. It is possible to conclude, however, that a net synthesis of chelidonic acid was occurring over the period of time involved in the present studies. ACKNOWLEDGMENTS
I wish to express my appreciation to the N ational Science Foundation for support of this work through grant GB-2200. Mr. C. W. Glennie's help with the spectral measurements and Mr. S. Max'
assistanoe with the liquid scintillation equipment were appreciated. I am particularly indebted to one of the referees for his most helpful criticisms and for calling my attention to the publications of the Japanese workers. REFERENCES 1. DEAN, F. M., in "Naturally Occurring Oxygen Ring Compounds." Butterworths, London (1963.) 2. ARNSTEIN, H. R. V., AND BENTLEY, R., Biochem. J. 54, 493, 508, 517 (1953). 3. TERADA, 0., SUZUKI, S., A.ND KINOSHITA., S., Agr. Bioi. Chern. (Tokyo) 25, 80Z (1961). 4. TERADA, 0., SUZUKI, S., AND KINOSHITA, S., Agr. Bioi. Chern. (Tokyo) 25, 871 (1961). 5. TERADA, 0., SUZUKI, S., AND KINOSHITA, S., Agr. Bioi. Chern. (Tokyo) 25, 939 (1961). 6. TERADA, 0., TOMIZAWA, K., SUZUKI, S., AND I{INOSIiITA, S., Bull . .I1gr. Chern. Soc. Japan 24, 535 (1960). 7. AVIGAD, G., AND ENGLARD, S., J. Biol. Chern. 240, 2290 (1965). 8. El';GLARD, S., AND AVIGAD, G., J. Biol. Chern. 240, 2297 (1965). 9. ENGLARD, S., AVIGAD, G., AND PROSKY, L., J. Bioi. Chern. 240, 2302 (1965). 10. RAMSTAD, E., Pharm. Acta Helv. 28, 45 (1953). 11. WEINSTEIN, L. H., PORTER, C. A., AND LAuRENCOT, H. J., JR., Contrib, Boyce Thompson Inst, 21, 439 (1962). 12. KWASNIEWSKI, V., Areneimittelforscli, 5, 90 (1955). 13. WILDE, C., Ann. Chern. 127, 167 (1963). 14. BOHM, B. A., Chern. Rev. 65, 435 (1965).
115, 181-186 (1966)
Biosynthesis of Chelidonic Acid I. Preliminary Observation on the Precursors of Chelidonic Acid in Convollaria maio/is L. BRUCE A. BOIIlVP Department of Pharmacognosy, University of Rhode Islancl, Kingston, Rhode Island
Received August 26, 1965 The biosynthesis of chelidonic acid ('Y-pyrone-2,6-dicarboxylic acid) has been studied in mature, flowering Convallaria ma;jaJis L. plants. Glucose-U.L.-140 and ribose-I-He were incorporated into the chelidonic acid molecule with dilutions of 720 and 940, respectively, while the dilution for acetate-2- 14C was 11,000. Shikimate-G-14C was not incorporated. Chemical degradation of chelidonio acid obtained from feeding glucose-f-v'C showed 75% of the label in carbons 3, 4, and 5 and 25% in carbons 1, 2, 6, and 7. These results would preclude formation of chelidonic acid from sedoheptulose as a major pathway.
The ')'-pyrone ring structure occurs in a variety of well-known and often studied compounds, e.g., chromones, xanthones, and some f1.avonoids. There also exists a small group of naturally occurring compounds which are derivatives of v-pyrone per se. In his chapter on naturally occurring pyrones and pyrans, Dean (1) lists less than a dozen of these derivatives of -y-pyrone.
similarity of the structure of kojic acid (I) to that of glucose was discussed. Experiments with specifically labeled glucose in Aspergillus species led them to conclude that a very large percentage of kojic acid arises by means of a direct conversion of glucose. A minor pathway involving threecarbon intermediates also appears to function.
HO -if . HoD OH ~_~CH20H ~_ ~CH3 o
nooc
0
Whereas the more complex molecules 111.entioned above have been examined with regard to their biosynthesis, considerably less is known about the formation of the 'Y'pyrone ring system in the simpler derivatives. Today, the only evidence concerning biosynthesis of -v-pyrones based upon 140 tracer studies is the excellent series of papers by Arnstein and Bentley (2). In this work, the 1 Present address: Department of Biology and Botany, University of British Columbia, Vancouver 8, B. C., Canada.
The intermediacy of simple carbohydrates in the formation of other 'j'·pyrones in bacteria has been discussed by Terada and his co-workers. 3-Hydroxykojic acid has been isolated (3) from cultures of Gluconobacter cerinue supplied with fructose as a carbon source. These workers (4) have also observed the formation of 5-ketofructose during the synthesis of koj ie acid from sorbose in Acetobacter species. It was also demonstrated (5) that Acetobacter species could utilize fructose for the synthesis of 5-hydroxymaltol 181
182
BOI-IM
(II). The form ation of 5-ketofruct{)se was observed by t his group (6) to occur in five species of Acetooacier and t wo species of Gluconobacier, The formation of 5-ketofruct oso in Gluconobacter cerinus has recently been confirmed (7-9). Although this work h as not been supported by isoto pic labeling experiments, it does serv e to indic at e that simpl e sugars play a significant part in t he form at ion of -v-pyrones in bacteri a. Several very interesting -y-pyro ne deriv at ives occur naturally in h igher plants. Perh aps t he most widespread of these is chelidonic acid" (III), which R amst ad (10) reported present in 688 species out of 1143 tested from 9 families. Nothing is known, how ever, about the formation of molecules of this sort in higher plants. The structural similarit y of chelidonic acid to common carbohydrates su ch as glucose or p erhaps sedohep tulose, prompted me to under take an investigat ion of the biosynthesis of th is molecule in Convallaria m ajolie L. It is the purpose of this paper to st udy t he hypothesis that chelidonic acid is a product of carbohyd rate metabolism in higher pl ants. If t his proves to be the case, it will be of interest to compar e th e biochemistry of this general group of com pounds in h igher plants with tha t of kojic ac id an d r elated compounds in the lower organ isms. EXPERIlVrENTAL PROCE DUR E
Source of plant mat eri al. The Con uallaria maj alis L. (Liliaceae) pla nts use d for t he exper im ent s were obtained from thre e sources . Those used for experiments 1-4 were ob tained fr om a local florist. These plants had been maintained u nder refrigeration and limited light to hold them back for E aster sales. Before being us ed, they were allowed to grow in a greenhouse for several days under n ormal illumination and temperatur e (about 75° F ). Pla nts u sed for experiment s 5 and 6 were taken from my home flow er garden . Plants used for experim ents 7 and 8 were t ak en fr om the de partment al Drug Plan t G arden . All exper ime nts 2 The number ing of t he chelidon ic acid m olecul e as presented in t he abstr act of t his p ap er r elies up on 'Y -p yr one as t he p ar en t compound; he nce , t h e number 1 is as sig ned to the h et ero cyclic oxyg en. For the purposes of di scu ssing degradation of ch elid onic a cid, t h e ca rb on a t oms will be number ed from 1 t o 7.
were conducted wit h plan ts which had b egun to flower. Descri ption of feedin g e.lJperiments . Pl a n ts with the flower st alk were cut wi thin one-h alf inch of the ground with a razor bla de, and the cu t end was immedi ately pu t int o a beak er of dis tilled water . The st ems were then out a second ti me , but t h is cu t- t ing was done b eneath t he su r face of t he water to insure t he integri ty of t he transpiration s t r eam . The stem, with a bead of wa te r adheri ng to t he cut end , was then quick ly transferred t o the feedi ng solution. Glucose and ribose were dissolved in di st illed wat er and sodium acetate an d shiki mic acid were dissolved in 0.1 M phosph at e buffer (pH 7.5). After the major portion of th e sol ution had been t aken up by the pla n t , t he remain der was washed in with dis tilled water added 1 ml at a time. After two or three such was hings, the plant was transferred to a b eak er of dis tilled water and allowed to metabolize for a total of 24 hour s . In t he case of the time-dep end ent exp eriments , times of 12, 24 , 36, and 48 hou rs were used . All feed ing experime nts were conducted with the plant materi al located a t a dis tance of 10 inches fro m a ban k of t hree 20 W whi te fluorescent lamps. The t empera t ure during the se experimen ts was n ot controlled but was approxim a tely 25°. In all cas es , t ho pla nt mat eri al appeare d very h ealthy a t t he te rmina tio n of t he feeding experimen ts . S ource of chemi c(tls. The radioactively labeled glucose , r ibose, and sodium aceta te were ob t aine d fr om commercial sources and were used without further purificatio n. Labeled shikimic aci d was prepared fr om Ginkgo biloba shoots by t he procedure of Weinstein et al. (11). Chelido ni c acid was purchas ed from Aldrich Chemical Comp any and w as purified by recrystallization , first fro m wat er and t hen from ethanol. I solati on and analysis of chelidonic acid . At t he end of the clesir ed metabolism period , the plant material was immediately extracted with boiling 80% eth an ol containing 1% hydrochl oric acid. He-extract ion s with this solvent were run un til the pl an t mater ial was no long er colored . T he ext racts t hus obtained were combined and diluted to a known volum e with 80% ethanol, and duplica te aliquo ts were used for determina tion of t otal ethanol sol uble radioac tivi ty . The so lvent was than blown off under a str eam of dry air an d t he resid ue was extracted with boili ng water. After filtr a tion t hr ough a bed of Celite, t he aq ueo us extract was ad j usted t o pH 6.0 with dilute sodium h ydrox ide and passed thro ugh a colum n of Amberli te CG -400 resin (OH -form) . N eutral and basic com ponen ts of the extract were removed fro m the
BIOSYNTHESIS OF CHELIDONIC ACID column by continuous wa shing with distilled water. The total acidic fract ion was eluted with a mixture of ethanol, water, and concentrated HOI in the ratio of -10: 50: 10. The eluate, a pale yellow color , W M repeatedly extracted with ether, which removed t h e chel idonic ac id from the aqueous ph ase . The ether extracts were oombined an d blown t o dryness under a stream of air, and the res idue was dissolved in a little 80% ethanol and applied t o sheets of Whatman No. 1 paper as a narrow band . The papers were run in the system developed by Kwasniewski (12) consisting of ethanol, ooncentrated NH,OII, and water (80:4: 16). I n ea oh case, a sam ple of t he p urified chelidonie acid was run as a standard to assist in locating the desired band. Ch elidonic acid has an R» of 0.55 in this system (at about 25°) and can be detected by its quenching of ultraviolet light of wavelength 2537 A. The bands were cut out and eluted with 80% ethanol, and the combined eluates were blown down to a small volume and reapplied to sheets of Whatman No.1 paper. The sheets were rerun in the same solvent system. On "Vhe second paper, the chelidonic acid ran as a very compact b and without t a ilin g. No other bands were observed under ultraviolet light of 2537 or 3660 A 01' under visible light. To check the possibility t hat some impurity might be running with the same R F as that of chelidonie acid, a sample of the naturally derived material was sub] ected t o chromatography in several other solvent systems: isopropanol-concentrated NH.IOH-water (10: 1: 1), R F 0.13-0 .14; benzene-acetic acid -water (10: 7: 3) , O.Oj and n-bu tanol-pyridine-wat er (14:3:3); R F 0.0. In all these systems, no other substance was present under the lighting conditions mentioned above. Af ter elution from the second banding, the chelidonic acid solution was diluted to a known volume from which aliqucts were taken for radioactivity and ultraviolet spectral measurements. The concentration of chelidonio acid was measured at 277 mJ! (Ema~ 8830) on the Beckman DU spectrophotometer. Radioactivity measurements were made on a vibrating reed elect r om et er system made by Nuclear Chicago (Dynacon (000). In all cases , appropriate blank and background measurements were performed. Deqroslation. of chelulonic acid . Chelidonic acid was degraded according to the procedure of Wilde (13). Chelidonic acid was suspended in a small volume of wa ter, a nd bromine was adde d until liquid bromine could be seen in the bottom of the react ion flask. The re action mix ture was he ated to about 40-50° on a steam bath with gentle agitation from time to time. More bromine was a dded as that in the reaction flask vaporized. Generally,
u,
183
in about 45 minutes most of the chelidonic acid had dissolved and a crystalline product could be seen in the reaction mixture. At this point, bromine was again added and the react ion was allowed to continue for anot her half hour. Heating was continued until all color of bromine was dissipated. The mixture was then cooled to about 1 ° for several hours and filtered. After drying on the filter for a short time, the solid was washed with small portions of chloroform. The chloroform washings were combined and evaporated to dryness under an air jet to yield crude pentabromoacetone. The pentabromoaeetone was recrystallized from ace tic acid to which a few drops of water had been added. The product thus obtained was identical with an authentic sample of pentabromoacetone. The crude oxalic acid remaining on the filter was recrystallized from a small volume of water. A test for the completion of the bromine oxidation was performed on the recrystallized oxalic acid. A few crystals of the acid were placed in contact with a drop of 20% potassium hydroxide solution on a spot plate. The development of a yell ow color would indicate the presence of ehelidonic acid. The color is due to the formation of the open ring xanthochelidonate system. Contaminating chelidonic acid can be removed by oxidation with bromine as described above. Radioactivity meaSUrements from degradation. st udy. Compounds involved in the degradation were counted on a Nuclear Chicago model 720 liquid scintillation counter. Pentabromoacetone was dis solved in tolu ene which cont ained the necessary scintillators. Chelidonic acid and oxalic acid were dissolved in Nuclear Chicago NCS solubifizer and then diluted with toluene-scintillator solution. The solubilieer, which is a strongly basic quaternary ammonium hydroxide, caused oonsiderable degradation of chelidonic acid to the yellow xanthochelidonate system. The The colored solution was responsible for serious quenching, which prevented reliable counts being obtained for the parent compound. No difficulty was encountered when pentabromoacetone and oxalic acid were counted.
RESULTS AND DISCUSSION
The results of the feeding experiments are presented in T able 1. Dilution values were calculated as t he r at io of specific activity of precursor t o specific activity of product. The percentage in corporation in these experim ents was calcul ated by comparing the activity in chelidonic acid with the total aC-
184
BOHM TABLE I RESULTS
OF PREOURSOR FEEDING EXPERIMENTS WITH
Conoallaria majalie L.
Chelidonic Acid
Expt,
Precursor !-,M
1 2 3 4 5 6 7 8a 8b 8e 8d
Glucoae-U .L.- 14C Ribose-1-l 4C Sodium acetate-z-l-C Sodium shikimato-Gvwfl Glucose-U .L.-14C/ribose Glucose-6-14C Sodium acetate-2- 14C Glucose-U.L.-HC (12 hours) Glucose-U.L.-14C (24 hours) Glueose·U.L.-14C (48 hours) Glueose-U.L.-14C (72 hours)
X 10-'
3.3 4.3 2.1 1.7 3.3/40 2.0 2.1 3.3
Sp. act. a
3.05 2.33 48.7 0.11 3.05 5.0 48.7 3.05
pM
X 10-'
!-'C
Sp, act. X 10' a
%
InCOI'P.b
Diluticn"
0.53 1.90 1.29 0.87 27.1 29.9 19.0 21.7
2.25 4.7 5.6 0.0 23.0 27.8 2.51 6.75
4.2.5 2.48 4.34
0.47 0.12 0.15
720 940 11,000
0.85 0.93 0.13 0.31
1.13 1.05 0.09 0.11
3,600 5,400 375,000 10,000
3.3
3.05
40.7
22.7
0.56
0.49
5,500
3.3
3.05
54.3
32.9
0.61
0.84
5,100
3.3
3.05
32.6
27.9
0.85
0.74
3,600
Sp. act. = mC/mmole % Incorp, = ,tiC in chelidonie acid/fLC in initial EtOH extract. c Dilution = Sp. act. of preoursor/sp. act. of product. a
b
tivity in the corresponding initial 80 % ethanol extract. The first four experiments were run in order to gain an insight into the general area of metabolism from. which chelidonic acid is derived. In general, this can be accomplished by studying the incorporation of glucose, acetate, and shikimate into the compound of interest. In the present case, these three precursors along with ribose were examined. No incorporation of label from shikimate was observed. This indicates that chelidonic acid is not a product of aromatic metabolism. The conversion of shikimate to chelidonie acid would entail a difficult to picture series of transformations, the like of which has not been observed with shikimate or its close relatives (14). Dilution values of 720 and 940 were observed with glucose and ribose, respectively. Considering the metabolic activity of these carbohydrates, these dilutions are reasonably low. Acetate was incorporated with a dilution of 11,000, which is 10-15 times less than the simple sugars. It would be incorrect to attribute much significance to the difference between the glucose and ribose dilutions on the basis of the experiments run so far. However, a real difference ap-
pears to exist between these compounds and acetate. This conclusion is supported by the results of experiments 6 and 7, where a difference of almost 70 was observed in favor of glucose over acetate. The overall dilutions were greater in these experiments due to the considerably larger amount of chelidonic add present in the plants used. These results suggest that the point of origin of chelidonic acid lies closer to a carbohydrate pathway than it does to one primarily involving acetate. The manner in which these precursors are converted to the acid is not known at this time, but it seems unlikely that chelidonie acid is derived from the "head-to-tail" condensation normally associated with acetate-derived natural products. The incorporation of glucose into chelidonio acid agrees with the observation of Arnstein and Bentley (2) that glucose was readily incorporated into the related ')'-pyrone, kojic acid. It was pointed out that kojic acid bears a strong resemblance to the glucose molecule. In fact, Arnstein and Bentley (2) concluded that glucose was converted to kojic acid without ring opening. Following their example, one can suggest a resemblance between chelidonic acid and sedoheptulose. When the cyclic form of sedoheptulose and cheli-
185
BIOSYNTHESIS OF CHELIDONIC ACID
donie acid are written side by side, the similarity becomes apparent.
o HOOC { ) C O O H
o The incorporation of label from glucose and ribose into chelidonic acid also leads one to the t empting conclusion that the acid might be a side product of the pentose phosphate pathway in some plants. Two tests of the intermediacy of sedoheptulose were done in the present work. These experiments and their implications are discussed below. Sedoheptulose (as the 7-phosphate) is formed via the pentose phosphate pathway which can be considered to employ glucose 6-phosphate as a starting material. The seven-carbon. sugar arises by means of transfer of a two-carbon. fragmen t to ribose 5phosphate. If ehelidonic acid does derive from sedoheptulose, then a large amount of unlabeled ribose administered concurrently with glu cose-t-O would be expected to trap the label from glucose before it reached sedohep tulose. In experiment 5, glucoseU.J.J.-HC was administered along with a 12""1 excess of unlabeled ribose. In a companion experiment (N o. 6), glucose-f-' -C was administer ed alone. Examination of per centage incorporation and dilution values would suggest that the ribose had no effect on the incorporation of label fr0111 glucose into chelidonic acid. Although ribose does appear to be a relatively good precursor for chelidonie acid, the trapping experiment would seem to preclude a pathway through sedoheptul ose. In order to get a much clearer pi cture of the pro cesses involved in the biosynthesis of chelidonic acid, data on the distribution of label from the precursors would be necessary. To this end, ribo se-Lt-C and glucose0_140 were fed to plan ts and the chelidonic acid was isolated in each case for degradation. Unfor tunately, the ribose feeding product was lost. The degradation procedure chosen for the present st udy was based upon a met hod
described by Wilde (13) in which bromine oxidatively cleaves chelidonic acid into one OH
HO~OH HOCHZ
~n~CHZOH OH 0
equivalent of pentebromoacetone and two equivalents of oxalic acid. Pentabromoacetone is derived from carbons 3, 4, and 5 of chelidonic acid; the two equivalents of oxalic acid are derived from carbons 1 and 2 and carbons 6 and 7 of chelidonie acid. The fragments obtained were counted in toluene solution in a liquid scintillation apparatus after the necessary solubilization of oxalic acid had been accomplished. The solubilizer caused exten sive degradation of chelidonic acid resulting in quen ching; consequently, no valid activity could be recorded for the parent molecule. Results of the degr adation are shown in T able II. If chelidonic acid is formed from sedoheptulose, then a characteristic pattern of labeling would be expected from each member of the pentose phosphate pathway. In the pres ent case, t he labeling from glueose-d -l'C would be found as 0-7 in sedoheptulose. Inso far as chelidon ic acid is a symmetrical molecule, sedoheptulose-7_140 (and, hence, glu cose-Bv'C) would be expected to label the acid in carbons 1 and 7. Exclusive formation from sedoheptulose-f-t -C would yield oxalic acid with 100 % of the 14 0 and pentabromoacetone with none. However, only about 25 % of the chelidonic acid label was found in oxalic acid in the present case; TABLE II DEGRADATIO N OF CHELlDONIC A CID FROM G LUCOSE-G- 140 FEEDING
Compound
dcm/mmole"
%"
Pentabromoacetone Oxalic acid Chelidoni c acid
3620 1240 4850"
75 25
• Disintegra tions per minute per millimole . b To nearest five. c T a ken as to tal of t he degrad ation fr agments.
BOHM
186
hence, the major pathway of its formation does not involve sedoheptulose. A lesser contribution from sedoheptulose is possible, a possibility which will be examined in more detail in a later publication. The present results have opened the door to investigation of the part played by smaller carbohydrate molecules in the formation of chelidonic acid. Experiment 8 was performed in order to look at the formation of chelidonic acid as a function of time. Equal amounts of glucoseU.L._14C were administered to four sets of plants which were allowed to metabolize for 12, 24, 36, and 48 hours in the light. The largest increase in specific activity of chelidonie acid occurred between 12 and 24 hours, during which time the value almost doubled. The 48- and 72-hour values were higher in each case than the proceeding ones, but the differences were not as striking as between the 12- and 24-hour values. No experiments were performed to examine the possibility that an equilibrium situation was being approached. It is possible to conclude, however, that a net synthesis of chelidonic acid was occurring over the period of time involved in the present studies. ACKNOWLEDGMENTS
I wish to express my appreciation to the N ational Science Foundation for support of this work through grant GB-2200. Mr. C. W. Glennie's help with the spectral measurements and Mr. S. Max'
assistanoe with the liquid scintillation equipment were appreciated. I am particularly indebted to one of the referees for his most helpful criticisms and for calling my attention to the publications of the Japanese workers. REFERENCES 1. DEAN, F. M., in "Naturally Occurring Oxygen Ring Compounds." Butterworths, London (1963.) 2. ARNSTEIN, H. R. V., AND BENTLEY, R., Biochem. J. 54, 493, 508, 517 (1953). 3. TERADA, 0., SUZUKI, S., A.ND KINOSHITA., S., Agr. Bioi. Chern. (Tokyo) 25, 80Z (1961). 4. TERADA, 0., SUZUKI, S., AND KINOSHITA, S., Agr. Bioi. Chern. (Tokyo) 25, 871 (1961). 5. TERADA, 0., SUZUKI, S., AND KINOSHITA, S., Agr. Bioi. Chern. (Tokyo) 25, 939 (1961). 6. TERADA, 0., TOMIZAWA, K., SUZUKI, S., AND I{INOSIiITA, S., Bull . .I1gr. Chern. Soc. Japan 24, 535 (1960). 7. AVIGAD, G., AND ENGLARD, S., J. Biol. Chern. 240, 2290 (1965). 8. El';GLARD, S., AND AVIGAD, G., J. Biol. Chern. 240, 2297 (1965). 9. ENGLARD, S., AVIGAD, G., AND PROSKY, L., J. Bioi. Chern. 240, 2302 (1965). 10. RAMSTAD, E., Pharm. Acta Helv. 28, 45 (1953). 11. WEINSTEIN, L. H., PORTER, C. A., AND LAuRENCOT, H. J., JR., Contrib, Boyce Thompson Inst, 21, 439 (1962). 12. KWASNIEWSKI, V., Areneimittelforscli, 5, 90 (1955). 13. WILDE, C., Ann. Chern. 127, 167 (1963). 14. BOHM, B. A., Chern. Rev. 65, 435 (1965).