Remarks on the Biosynthesis of Gramine

832

A. BRECCIA AND A. M. CRESPI

U m w andlung in Elym oclavin zwei M öglichkeiten: oxydativer R ingschluß zwischen der « V ständ ig en M ethylgruppe u nd dem A m inostickstoff oder R in g schluß zwischen der H ydroxym ethylgruppe und dem A m inostickstoff nach v o rh erig er ciV^rans-Isom erisieru n g . M it dem Nachweis, daß C hanoclavin-I auch in A groclavin übergeht, — und zw ar m it gerin g erer biologischer V erdünnung als in Elym oclavin —, ist gezeigt, daß w ahrscheinlich die zweite der beiden M öglichkeiten zutrifft. Es konnte gezeigt w erden, daß d e r M utterkornpilz alle Schritte d er R eaktionsfolge T ry ptophan-*- Chanoc la v in -I-> A g r o c la v in ^ E ly m o clav in -> L ysergsäureD erivate au sfü h ren kann. Ob diese Sequenz der H auptw eg d er E rgolin-B iosynthese ist, das C hano clavin-I also ein obligatorisches Zw ischenprodukt darstellt, oder ob daneben noch eine R eaktionsfolge vom T ry p to p h an zu den E rgolinen existiert, die nicht über das Chanoclavin-I fü h rt, läßt sich nach

den bisherigen V ersuchen nicht ein d eu tig entscheiden. Obwohl die z. T. seh r hoh en E in b a u ra ten bei den einzelnen Schritten d a fü r sprechen, d aß es sich um den H auptw eg d er B iosynthese h an d elt, w ird m an doch die M öglichkeit im A uge beh alten m üssen, daß das Chanoclavin-I n u r in einem N ebenw eg d er B io synthese liegt o der daß es m it einem der Zwischen p ro d u k te des H auptw eges im Gleichgewicht steht. V ersuche zur K läru n g dieser F ra g e und der nach dem M echanism us d er Iso m erisieru n g des Chanoclavin-I sind gegenw ärtig im Gange. W ir danken den H erren Dr. A . H o f m a n n und Dr. B r a c k , Basel, für die Überlassung von R einalka loiden und Pilzm aterial. H errn Dr. W. A c k l i n , Zürich, sind wir für die M itteilung unveröffentlichter A rbeits vorschriften zur chromatographischen Trennung der Chanoclavin-Isomere zu großem Dank verpflichtet. Einer der Autoren (H. G. F.) dankt der D e u t s c h e n F orschungsgemeinschaft für die Bereit stellung einer Sachbeihilfe.

A.

Remarks on the Biosynthesis of Gramine A l b e r t o B r e c c ia

and

A n n a M a r ia C r e s p i

Istituto Chimico “G. Ciamician” Universitä di Bologna Centro Nazionale Chim. Rad. del C.N.R. Sez. Ill, Bologna, Italy (Z. Naturforschg. 21 b, 832— 835 [1966]; eingegangen am 2. Ju n i 1966)

The biosynthesis of gramine was tested by growing the germinating barley in media containing pyruvic, 2-14C acid, A^-benzyloxycarbonyl-tryptophan ß - u C and tryptophan, ß - u C. Iso-nicotinylhydrazide was also added in a series of experiments to inhibit the cleaving action of pyridoxal phosphate. The presence of the inhibitor reduces the total uptake of tryptophan and benzyloxycarbonyl-tryptophan by a factor of 15 —20, while the uptake of pyruvic acid and the total pro duction of gramine were reduced by a factor of only 0.5. This fact suggests an alternative route for the biosynthesis of gramine, when the path via tryptophan is blocked.

Recent investigations ca rrie d out by L e e t e et a l. 2 have confirm ed that 3-m ethyleneindole nucleus derived from try p to p h an is utilized entirely fo r the synthesis of gram ine, an indole alkaloid present in barley sprouts. The 3-m ethyleneindole nucleus is thus a direct p recu rso r of gram ine. F o rm atio n of the latter is suggested to involve the rem oval of the am ine group from try p to p h an by p yridoxal phos phate. T here is, how ever, some disagreem ent about the n atu re of precu rso rs and the extent to which they are incorporated in the gram ine form ed. T his is

p artly due to the fact th at different au th o rs used different culture m e d ia 1 -4 . N ot well stated results have also been p u blished on the period of the in vivo form ation of g r a m in e 6. These facts have prom pted us to re-exam ine the m echanism of the gram ine synthesis. B arley sprouts w ere grow n from g rain s on the presence of labelled p rec u rso rs such as try p to p h an , ß -u C, /V -benzyloxycarbonyl-tryptophan, ß -xiC, and pyruvic acid, 2-14C. Iso-nicotinyl h y drazide was a d ded in one series of experim ents to in h ib it specifi cally the cleaving action of p y rid o x al p h o sp h ate;

and E. L e e t e , J. Amer. diem. Soc. 85, 461 [1963]. 2 B. G . G o w e r and E. L e e t e , J. Amer. chem. Soc. 85, 3683 [1963],

3 A. B r e c c ia and L . M a r i o n , Can. J. Chem. 37, 1066 [1959]. 4 F. W h i g h tm a n , M . D. C h is o lm , and A. C . N e is h , Phyto chemistry 1, 30 [1961].

1 D . 0 . D onovan

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REMARKS ON THE BIOSYNTHESIS OF GRAMINE

Tryptophan, /?-14C

Precursors

N -Benzyloxy carbonyl

tryptophan, ß-14C 1* 2*

2*

Amount administered mmole

833

0.55

0.15

9.0 X 107

9.0 X 107

9.0 X 107

5.5 X 107

total activity administered disint. min.-1

1.35 x 107

1.3 x 107

1.3 x 107

3.0 X 107

Spec, activity of gramine disint, min.-1 mmole-1

2.5

x 104

1.2 x 104

1.9 x 104

6.2 X 102

total activity incorporated disint. min.-1

1.6 X 104

2.4 X 104

9.3 X 102

Percentage incorporation

23.0 X 10-2

12.0 x 10-2

10.0 x 10-2

3.0 x IO-3

X

Spec, activity of precursors disint. min.-1 mmole-1

oj-

0.15

to

0.15

Pyruvic acid, 2-14C 1*

Table 1.

* See Table 2.

Tryptophan, /S-14C 1* 2*

Precusors in the presence of the inhibitor

N -benzyloxy carbonyl tryptophan, ß - l4C n

Pvruvic acid, 2-14C 1*

0.18

0,20

0.15

0.55

Spec, activity of precursors disint. min-1 mmole-1

9.0 x 107

9.0 x 107

9.0 X 107

5.5 X 107

total activity administered disint. min.-1

1.6 x 107

1.8 X 107

1.3 x 107

3.0 x 107

Spec, activity of gramine disint. min.-1 mmole-1

1.1 x 103

3.1 X IO»

1.6 x 103

3.7 x 102

total activity incorporated disint. min.-1

8.0 x 102

2.1 x 103

1.0 x 103

2.6 x 102

percentage incorporation

5.1 X IO-3

Amount administered mmole

10 X IO-3

8 X IO-3

0.86 X IO-3

Table 2. * Experiments 1 refer to precursor fed through the roots of whole seedlings. Experiments 2 refer to precursor fed directly into the shoots.

Inhibitor (feeding day) J

^>re" 7th day)

1st —

—

5th

—

“

~

Gramine yield at 5th day mg for g of dry tissue 1.37 mg/g 2.73 mg/g —

—

Ratio of Gramine gramine yield with and yield at without 11th day inhibitor —

— 1.10 mg/g 2.30 mg/g

Inhibitor (feeding day)

0.5

5th

0.5

5th

—

Precursors (fed at 7th day)

Percentage incorporation

3.0 x 10- 3 pyruvate-214C 0.86 X IO-3 pyruvate-214C tryptophan-/?- 14C 23.0 x 10-2 tryptophan-/5-14C 1.0 x 10-2

Ratio of incorporation with and without inhibitor ~ 0.30 0.04

T a b .3 b

T ab. 3 a also 100 mg of A T P w ere fed to the culture for each experim ent. Owing to its established function in the biosynthe sis of gram ine, trypto p h an served as a reference

m a teria l; its A^-benzyloxycarbonyl derivative was chosen to investigate the effect of p rotecting the am ine g ro u p in try p to p h an , while pyruvic acid was used to establish w hether or not the biosynthesis

5 B. A. Bohm, Chem. Reviews 65, 447 [1965].

6 K.

B ow den

and L.

M a r io n ,


Can. J. Chem. 29, 1037 [1951].

834

A. BRECCIA AND A. M. CRESPI

follows an alternative ro u te; since pyruvate is know n to be involved in the biosynthesis of shikim ic acid and an th ran ilic acid °, and hence in the b io synthesis of the indole nucleus. The results are shown in T ables 1, 2 and 3 a —b. O ther possible precursors, such as acetic-2-14C acid, succinic acid-2.3-3H, glycine-2-3H and mevalolactone-2-14C were also tested, but their uptake in the biosynthesis of gram ine was not appreciable (it was obtained a 2.5 X 1 0 -4 percentage incorporatio n for acetic acid, 2.6 X 1 0 ~ 4 for succinic acid, 1.0 x 1 0 ~ 3 for glycine, nil for m evalolactone). In addition to known d a t a 6 it has now been found that a considerable am ount of gram ine is present in barley sprouts as early as the fifth day of sprouting, the m ean quantity being 2.7 mg per gram of dry plant tissue as com pared to 2.3 m g/g found on the eleventh sprouting day (see Table 3 a ) . On the basis of the radioactivity of gram ine, the protection of the am ine group in tryptophan by the benzyloxycarbonyl group did not cause a significant difference in the incorporation of the p recu rso r, the results being sim ilar to those obtained w ith tryptophan. It appears therefore that proteolytic enzymes can cleave the am inoblocked derivative once it has been incorporated in the tissue. O utside the tissues, the benzyloxycarbonyl derivative is highly stable in the pn range of the grow th m edium . The presence of the inhibitor (iso-nicotinyl hydrazide) reduces the specific and the total uptake of the tryptophan and its benzyloxycarbonyl derivative by a factor of 15 —20 (see T ables 2 and 3 b ) . The fact that a certain incorporation does take place suggests that we do not fully know the effect of isonicotinyl hydrazide on the transam inases in p lan t tissues, especially the tim e and the am ount of this com pound needed for com plete inhibition. It rem ains to be explained why the am ount of gram ine extracted from barley sprouts is not so much different as aspected w hether the plant is grow n in the presence or the absence of the in h i bitor. In Table 3 a it is shown the yield of gram ine in the presence or not of the inhibitor at different tim e of germ ination of barley sprouts; while in T able 3 b it is shown the ratio of precursors incorporatio n in the presence or not of the in h ib ito r; this ratio is to be com pared w ith the gram ine yield. These analytical data show a ratio of about 1: 2 as com pared to the ratio of 1: 20 obtained from

radiochem ical m easurem ents via tryptophan. This discrepancy may be p artly explained by the speci fic and total in co rp o ratio n of pyruvic acid, both in the presence and the absence of the inhibitor. In fact pyruvate could form the gram ine via shickimic acid, 3-indolil-glycerolphosphate and hence 3-methyleneindole (as p rin cip al ste p s). This w ould suggest an alternative route for the synthesis of gram ine, when the steps via tryptophan is blocked. G ram ine form ed in the presence of labelled p y ru vic acid was subjected to d eg rad atio n to indole and to indol-3-carboxylic acid. The specific activity of the latter was found to be about the sam e as that of the p aren t gram in e (6 X 102 d isin t./m in ./m M ). The activity of the indole obtained from 8.0 mg of gram ine was 2.3 d is in t./m in .; this shows that the indole nucleus was radioactive. Experimental A. P r e p a r a t i o n o f l a b e l l e d c o m p o u n d s The labelled compounds used were supplied by the Radiochemical Centre at Amersham, except for iV-benzyloxycarbonyl-tryptophan-/?-14C, prepared from trypto phan-/?-1^ in the following m anner: tryptophan-/?-14C (2.18 mg = 0.1 mC) was diluted with 500 mg of un labelled tryptophan. 300 mg of this mixture were with drawn and dissolved in 3 ml of I N NaOH. Benzyl oxycarbonyl chloride (0.25 ml) and 1 ml of NaOH were added, with constant stirring. The reaction mixture was stirred for 4 hr, after which the excess benzyloxy carbonyl chloride was extracted with small amount of diethyl ether and the solution was acidified with 1 N HC1. The precipitate was filtered off, dried and recrystal lized from ethanol/petroleum ether. The yield of JV-benzyloxycarbonyl-tryptophan was 395 mg, m.p. 124 to 126 °C, specific activity 9.0 x 107 disint./min./mM. The yield percentage was 80% respect to tryptophan. B. C u l t i v a t i o n o f b a r l e y a n d t h e e x t r a c t i o n of g r a m i n e 2 Kg of barley (Hordeum vulgare) was grown on a thin layer of glass wool in small plastic dishes, and each dish received every day about 50 ml of distilled water. The tem perature was kept at about 25 °C during the day with the aid of an IR lamp. The temperature at night was 20 °C. The labelled compounds were ad ministered in suitable solution on the seventh day of germination. Iso-nicotinyl hydrazide (0.14 g dissolved in 40 ml of distilled water) was added to each dish on the first or fifts day of germination in cases when the test was to be carried out in the presence of the inhi bitor (see Tables 3 a, b).


REMARKS ON THE BIOSYNTHESIS OF GRAMINE

The sprouts were cut off on the eleventh day, dried in an oven to eliminate all moisture, and extracted with methanol for 48 hr. The solution was filtered, concen trated to about 200 ml, and extracted three times with 30° —60° petroleum ether. The ethereal extracts were discarted and the methanolic solution was evaporated to dryness. The residue was dissolved in 40 ml of 1 N H2S 0 4 and extracted four times with diethyl ether. The ether extracts were again discarted, the remaining solution was cooled with ice, and 20 ml of 10 M NaOH were added to it. The alkaline mixture was extracted with diethyl ether, the ether extracts were washed with water, dried over Na2S 0 4 , filtered, and evaporated. Sublimation of the residue under a pressure of 1 mm Hg gave pure gramine, m.p. 134 °C. C. D e g r a d a t i o n o f t h e g r a m i n e A 80 mg of radioactive gramine were dissolved in 30 ml of 20% alcoholic solution of KOH containing a small amount of K N 0 3 . The solution was refluxed for 20 hrs. Most of the alcohol was then evaporated and the remaining solution was extracted several times with an equal volume of diethyl ether. The ether ex tracts were concentrated and subjected to thin layer chromatography to separate gramine and indole. The remaining aqueous solution was acidified and extracted with diethyl ether. This ether extract was concentrated and used for the T. L. Chromatographic separation of indol-3-carboxylic acid.

835

Conditions of the chromatographic analysis A suspension of 3 g of B r o c k m a n - type alumina (ex Merck) in 5 ml of distilled water was applied to two 5 x 20 cm polished glass plates. 1) Eluents: i-PrOH : H 20 : HC1 ( 8 : 1 : 0.1) (for separating gramine and indole), 2) i-PrOH : H 20 : NH3 ( 8 : 1 : 1) (for separating indole-3-carboxylic ac id ). Developer: E h r 1 i c h ’s reagent, obtained by dis solving 2 g of p-dimethylaminobenzaldehyde in a mix ture of 80 ml of 95° ethanol and 20 ml of 6 N HC1. The following /fy-values were obtained : Eluents 1 2 indole 0.85 — gramine 0.58 0.94 indole-3-carboxylic acid — 0.34 D. D e t e r m i n a t i o n o f t h e r a d i o a c t i v i t y Radioactivity of the gramine samples was deter mined with the aid of a liquid scintillation counter supplied by S.E.L.O. The toluene-based liquid scintil lator was composed of 5 g of diphenyloxazole (PPO) as the prim ary scintillator and 0.5 g of 1.4-bis-2-(5phenyloxazolyl) benzene (POPOP) as the secondary scintillator. The accuracy of the counter was deter mined with the aid of known samples of 14C-n-hexadecane and 3H-/i-hexadecane.
