Studies in the biochemistry of micro-organisms—II

Tetrahedron.

1963. Vol.

19. pp. I I I to 115.

Pergamon

Prm

Ltd.

Printed

in Northern

Ireland

STUDIES IN THE BIOCHEMISTRY OF MICRO-ORGANISMS-II CONSTITUTION OF CURVULIN, CURVULINIC AND CURVULOL, METABOLIC PRODUCTS CURVVLARIA

ACID OF

SIDDIQUI

A. KAMAL,M. ALI KHAN and A. ALI QURE~HI Central Laboratories and West Regional Laboratories, Pakistan Council of Scientific and Industrial Research, Lahore (Received 12 June 1962) Abstract-Constitutions of curvulin, curvulinic acid and curvulol have been established as: Ethyl 2-acetyl-3,5-dihydroxyphenylacetate, 2-acetyl-3,5-dihydroxyphenylacetic acid and 4-acetyl-5-/Thydroxyethyl resorcinol respectively.

IN PARTI1 the isolation of four metabolic products was reported : curvulin Ci2H1.,05, curvulinic acid C,,H,,O,, non-acid fraction m.p. 204”, now named curvulol, and another acid m.p. 154”. Curvulol has since been found with molecular formula C,,H,,O,. When curvulin is hydrolysed with potassium hydroxide in the cold curvulinic acid is obtained which may be re-esterified with ethyl alcohol to curvulin.* This proves that curvulin is the ethyl ester of curvulinic acid. Methylation of curvulin with diazomethane’ gives dimethyl curvulin which was utilized without further purification in subsequent reactions and was characterized by its aklaline hydrolysis to dimethyl curvulinic acid, m.p. 121”, also obtained through methylation of curvulin and curvulinic acid. With four of five oxygen atoms accounted for and the relationship between curvulin and curvulinic acid established the next step was to orient the two phenolic oxygensr and to determine the function of the fifth oxygen. Although curvulin and curvulinic acid both fail to give ketone derivatives, curvulinic acid gives a positive iodoform test. Further, the IR spectrum of curvulinl shows a strong band at 1704 cm-’ (5.87 mp) indicating the presence of an unconjugated ester carbonyl system. When curvulin is heated with sulphuric acid (80 %) a dark-brown resinous product is obtained, but dimethyl curvulin, C,,H,,O,, on similar treatment yields an acid C,,H,,O, (m.p. 103”), which was identified as 3,5-dimethoxy-phenylacetic acid by comparison with an authentic sample. 2 This shows that hydrolysis of the ethyl ester group and removal of the -CO.CH, present as a substituent in the benzene ring has taken place. The mechanism of this elimination under very strong acid conditions is in accordance with the explanation by Birch et ~1.~for the elimination of the acetyl group in curvularin. The 3,5-dimethoxyphenylacctic acid on oxidation with alkaline potassium * In part I*, analysis gave OCH., 12.96%. Since curvulin is an ethyl ester, OCH. value when corrected to O.GH, comes to 18-S% against the theoretical value of 19.1 ‘A for l*OC,H, in the molecule of curvulin. 1 A. Kamal, N. Ahmad, M. A. Khan and I. H. Qureshi, Tetrahedron 18,433 (1962). 2 0. C. Musgrave, J. Chem. Sot., 1107 (1957). * A. J. Birch, 0. C. Musgrave, R. W. Richards and Herchell Smith, J. Gem. Sot., 3146 (1959). 8

111

A. KAMAL, M.

112

ALI

KHANand A. Au QURE~H~

permanganate gives ,3,5dimethoxybenzoic acid. Identification of this acid and its homo-acid, 3,5-dimethoxyphenylacetic acid, as the degradation products of dimethyl curvulin establishes the partial structure of curvulin as I. OH I -COCH, CH,COOC,H,

HO

The acetyl group may either be between the two phenolic hydroxyls’ or in the position ortho to the -CH,COOC,H, group. When curvulin is methylated two isomeric mono-methyl derivatives (m.p. 84” and 206”) are obtained.’ If the acetyl group were between the two phenolic groups which are meta to each other, only one mono-methyl derivative would be possible. Therefore, the acetyl group must be orrho to the -CH,COOC,H, group. O,O-Dimethylcurvulin (or O,O-dimethylcurvulinic acid) on oxidation with alkaline potassium permanganate gives an acid, C,,H,,O,, m.p. 213” (A,, at 309 rnpc; 275 rnp and 230 mp) identified as 2-carboxymethyl-4-6-dimethoxyphenyl-glyoxylic acid (II); m.p. 212” by comparison with an authentic sample prepared in accordance with the method of Musgrave. 2 Curvulin is, therefore, ethyl 2-acetyl-3,5_dihydroxyphenylacetate (III). OH OCH, COXH,

COCOOH

/’

CH,O

J

,/

/’ @

\

,/

/’ 0

\

HO

CH,COOC,H,

CH,COOC,H,

III

II

When curvulin is reduced a non-acid, C,,H,,O,, is obtained which is identical with natural curvulol m.p. 204’,’ and, therefore, the CH,COOC,H, group has been reduced to -CH,CH,*OH and the acetyl group unaffected. Though like curvulin, curvulol fails to give an oxime or a 2,4_dinitrophenylhydrazone, it gives a positive iodoform test. It gives an IR absorption band at 1636 cm-’ which is indicative of the presence of an acetyl group in the para position to phenolic OH.4 Again the reduced -CH(OH)CH, group if present in natural curvulol would confer optical activity on the molecule due to its asymmetric carbon. Asymmetry is, however, absent as it shows no optical rotation. Apart from slight shifting of bands the UV absorption spectra of both curvulol and curvulin closely resemble each other. All this evidence indicates the presence of an unreduced -COCH, group in curvulol which is, therefore, Cacetyl-5$hydroxyethylresorcinol (IV). In the light of the above findings it is now possible to postulate the structure of as 7-acetoxy-5-carbethoxymethyl-2-methyl-y-chromone. dehydrodiacetylcurvulin’ OH

HO”

‘I 0

CH,COO

COCH,

I

\(

‘CH 1’CH1’OH

Infrared Spectra

of Complex

l(“;;Hs C’

C,H,OOC!CH,

2 V

IV 4 &llamy,

0

Molecules

p.

120.Methuen (1954).

Studies in the biochemistry

of micro-organisms-11

113

To establish the relationship of the two mono O-methyl isomers (m.p. 173” and 104”) of curvulinic acid with the two mono-O-methyl isomers of curvulin (i: m.p. 84”; and ii: m.p. 206”),’ 0-methyl-curvulinic acid (m.p. 172”) was esterfied. The resulting ethyl ester was identical with the mono-0-methylcurvulin m.p. 84”,l and hence the relationship of the remaining two isomers can be inferred. Biosynthesis Birch et aL3 clearly established the ‘acetate’ as the fundamental unit-with eight such units forming a head-to-tail linkage in the biosynthesis of curvularin. In the case of curvulin a similar biosynthetic route may be formulated :

:’ i z

(C-C)?

\

C EXPERIMENTAL AU m.ps. are corrected. UV absorption spectra were determined in 95 % ethanol with a Beckman Spectrophotometer Model D.U. IR absorption spectra were determined with Leitz IR Spectrograph No. 105. cuwui01 Product m.p. 204” isolated from the tail fractions in the crystallization of curvulin and assigned the name “Curvulol”’ gave the following analysis: (Found: C, 61.10; H, 6.51; 0, 3260x, M.W. 199; C,,H,,O, (M.W. 194); requires: C, 61.21; H, 6.17; 0,32*62x). Curvulol is very soluble in acetone and ethylacetate, moderately in ether and methyl and ethyl alcohols, slightly soluble in chloroform and insoluble in benzene and light-petroleum (60-80”). Curvulol gives a brownish-red colour with ferric chloride, and a pink colour changing to red with cont. or oxime but gives a positive iodoform sulphuric acid. It does not give a 2&dinitrophenylhydrazone test. Curvulol fails to show any optical rotation and shows UV absorption bands at I.,, 275 rnp (log E 3.77); 285 rnp (log E 3.96); Rmlo255 rnp (log E 3.65); 280 rnp (log E 3.38). IR spectrum shows (CHICO p to OH),’ 3600 cm-l main absorption peaks at 1600 cm-l (6.17 cc); 1636 cm-l (6.16 FCC) (2.78 p); in chloroform and at 1636 cm-l (6.16 p) (CH,COp to OH): 3400 cm-’ (2.94 p) in nujol Mull.

Acetylation of cuwulol A mixture of curvulol(500 mg), acetic anhydride (5 ml) and anhydrous sodium acetate (200 mg) was heated on a water-bath (5 hr). Addition of water and removal of acetic acid in vacua gave diacetyl curvulol which crystallized from ether-light petroleum (60-80’) in tiny prisms, m.p. 99-100”. (Found: C, 6056; H, 5.73; 0. 33.71%; Calc. for C,,H,,O,: C, 59.99; H, 5.75; 0, 34.25%). fiterifcation of curvulinic acid with ethyl alcohol Curvulinic acid (150 mg) in absolute ethyl-alcohol (7 ml) containing cone sulphuric acid (0.3 ml) was refluxed on a steam bath (11 hr). After removal of ethyl alcohol in vacua dilution with water and extraction with ether, the extract was washed with an aqueous bicarbonate solution followed byacidulated water, dried (NaSO,) and evaporated, leaving an oil which crystallized on standing (90 mg). The product recrystallized from water, m.p. 145”. undepressed on admixture with natural curvulin (m.p. 145”). The colour tests and the UV and IR Spectra are identical with that of curvulin. Hydrolysis of curvulin (a) With alcoholicpotassium hydroxti. Curvulin (100 mg) in alcoholic potassium hydroxide (10 %; 2.5 ml) was allowed to stand (2.5 hr) at room temp, (16-lS”). Removal of ethyl alcohol (in vacua),

114

A. KAMAL, M. ALI KHAN and A. Au QURE~HI

acidification with dil. HaSOd and extraction with ethyl acetate gave a crystalline material which was recrystallized from water; 65 mg; m.p. 216” (dec) undepressed m.p. with curvulinic acid m.p. 218”. (b) Reaction ofuqueouspotussium hydroxide on curuulin. Curvulin (100 mg) in aqueous potassium hydroxide (10%; 2.5 ml) was allowed to stand (2.5 hr), acidified with dil. H,SO, and extracted with ethyl acetate. The crystalline product was recrystallized from water m.p. 218” (dec), mixed m.p. with curvulinic acid was undepressed and the IR spectra identical. (Found: C, 57.50; H, 4.89; 0,37.88 %, Calc. for C,,,H,,O,: C, 57.14; H, 4.80; 0, 38.06%). O,O-Dimethylcuroulin Methylation of monomethyl curvulin isomers with diazomethane. Either of the two mono-O-methyl curvulin isomers (i: m.p. 84”, and ii: m.p. 206”)’ (100 mg) in dry ether was treated with excess of diazomethane (prepared from 1.5 g of N-nitrosomethylurea) in ether and left to stand (96 hr). Excess of diazomethane was decomposed and after drying and removal of ether, O,O-dimethylcurvulin as a reddish-brown viscous liquid (IOOmg) was used without further purification. It was however, characterized by hydrolysis to the acid: O,O-dimethylcurvulinic acid, m.p. 121”. Complete

methylation

of cur&in

with diazomethane

Curvulin (100 mg) was treated with an excess diazomethane (prepared from 3.5 g N-nitrosomethylurea) in ether and allowed to stand (96 hr). Excess diazomethane was decomposed and the ether removed leaving a reddish-brown thick viscous liquid (100 mg), which failed to crystallize. It was however, characterized by hydrolysis to O,O-dimethylcurvulinic acid. Hydrolysis

of O,O-dimethylcuroulinin

0,0-Dimethylcurvulin (100 mg) in alcoholic potassium hydroxide (10%; 3.0 ml) was allowed to stand 2 hr. Ethyl alcohol was removed in uacuo and the residue acidified with dil. HCI. The separated oil was repeatedly extracted with ether and the ether extract shaken with a saturated aqueous solution of sodium bicarbonate. The bicarbonate extract was acidified and extracted with ether. The ethereal extract gale, on removal of the solvent, O,O-dimethyl curoulinic acid as a reddish-brown oil which crystallized on standing and was recrystallized from water, 70 mg, m.p. 121”. (Found: C, 60.29; H, 5.97; 0, 33.87; OCH,, 25.54; CCH,, 6.20; Calc. for CIPHISOL; C, 6050; H, 5.92; 0, 33.58; 2 x OCH,, 2604; IC-CH,, 6.3 %). Methy&z!ion q/ curoulinic acid

Curvulinic acid (400 mg) was dissolved in aqueous potassium hydroxide (10%; 8.0 ml) and dimethyl sulphate (800 mg) added drop by drop with vigorous shaking and occasional cooling, then boiled (1 hr), cooled, acidified with dil. HCI and extracted with ether. Removal of ether gave a crystalline solid which recrystallized from ethyl acetate in prisms, 320 mg; m.p. 121” and was undepressed on admixture with O,O-dimethylcurvulinic acid described above. Partial methylation

of cur&in

with dimethyl

sulphate

Curvulin (500 mg) was dissolved in aqueous potassium hydroxide (10%; IO.0 ml) and dimethyl sulphate (1.0 ml) added dropwise with cooling with vigorous shaking (1 hr) and then heated on a sand-bath (10 min), acidified with dil. HCl and extracted with ether. The ethereal extract when dried gave an oil (500 mg) which solidified on standing. The product was crystallized from ether-light petroleum (60-80”) and the first crop of crystals (320 mg) recrystallized as colourless prismatic needles from aqueous methyl alcohol; m.p. 173” (I). (UV absorption bands at A,., 270 rnp (log E 4.09), 220 m,r4(log c: 440), &I, 290 rnp (log E 4.91) and 247 m/t (log E 3.78). The second crop (135 mg) recrystallized from ether-light pertoleum (6&80”) in microscopic needles, m.p. 104” (II). (UV absorption bands at d,.x 267 rnp (log E 4.25), 217 rnp (log E 4.56), and &in 284 rnp (log E 4%) and 245 my (log E 4.02). Both I and II are mono O-methyl isomers of curvulinic acid. Found: For I: (m.p. 173”) C, 58.66; H, 5.67; 0, 35.69; l.OCH,-13.7%; CCH,-5.81; M.W. (Rast) 236. For II; (m.p. 104”) C, 58.70; H, 5.46; 0, 3540-OCH,, 13.97; Calc. for C,iH,,O,; (M.W. 226): C, 58.92; H, 540; 0, 35.68; l.OCHs, 13.8; ICCHS, 6.7%). Reaction

of sulphuric acid on O,O-dimethyl curoulin

O,O-Dimethylcurvulin (1.0 g) was treated with cont. H,SO, (80%; 2.0 ml) and left overnight. The mixture was diluted with water (0.5 ml), heated on a water-bath (3 hr), left overnight, extracted with ether, charcoaled and dried, colourless needles were deposited on standing (590 mg). The product

Studies in the biochemistry

of micro-organisms--II

115

recrystallized from ether-light petroleum (6tX30”), m.p. 103”, and was undepressed by 3,5-dimethoxyphenylacetic acid, m.p. 104105”. (Found: C, 61.34; H, 6.26; 0, 31.96%; M.W. (Rast) 221; C, 61.34; H, 6.21; 0,32.25x; M.W. (Rast) 218; Calc. for C,,H,,O, (M.W. 194): C, 61.21; H, 6.17; 0, 32.62 %). Iodoform

test on curvulinic acid

Curvuhnic acid (0.2 g) dissolved in dioxane (10 ml) was treated with aqueous sodium hydroxide (10%; 2.0 ml). A solution prepared from potassium iodide (20 mg) and iodine (10 mg) in water (80 ml) was added till absorption of iodine was complete and the mixture left on a water-bath (1 hr) until a distinct smell of iodoform was detectable. The filtrate was acidified with dil. HCI extracted with ether, dried and after removal of ether the iodoform, m.p. 119”, was undepressed by an authentic sample. Potassium

permanganate

oxidation

of O,O-dimethylcuruulin

O,O-Dimethylcurvulin (1.5 g) was dissolved in 1N aqueous potassium hydroxide (12 ml) by refluxing on a sand-bath. To the warm solution aqueous 5 ‘A potassium permanganate was added in portions with shaking until a permanent colour persisted (total vol. of KMnO, solution consumed: 40 ml). The filtrate was extracted with ethyl acetate, dried and after removal of the solvent the oil crystallized on standing; (0.17 g) and recrystallized from ethyl acetate in prisms m.p. 213” (effervesence) and was undepressed by an authentic sample of 2-carboxymethyl-4,6-dimethoxyphenylglyoxylic acid (m.p. 212”) prepared in accordance with the method of Musgrave.’ The acid gives UV absorption bands at A,,, 303, 275 and 230 rnp, identical with those of an authentic sample. The IR spectra are also identical. (Found C, 53.41; H, 444; 0, 42.03; C,,H,,O, requires: C, 53.73; H, 4.51; 0, 41.76%). Permanganate

oxidation

of the sulphuric acid reaction product

qf O,O-dimethylcurvulin

The product (from sulphuric acid reaction on O,O-dimethylcurvuhn) in alkali (0.3 g; in 10 ml water) was heated with mild reflux. Aqueous potassium permanganate (5 ‘A) was added drop-wise (30.0 ml) till a permanent colour persisted, then the excess permanganate was destroyed with methyl alcohol and the filtrate cooled and acidified whereby a solid (0.15 g) separated. This was washed with ice cold water, dried and recrystallized from ethyl acetate-light petroleum; m.p. 181”, undepressed by dimethyl-a-resorylic acid (m.p. 181”). (Found: C, 59.55; H, 5.63; 0, 34.73%. M.W. (Rast) 180; C$H,,O, (M.W. 182); requires: C, 59.33; H, 5.53; 0, 35.13%). Reduction of cur&in (a) With Na-borohydride.

To curvuhn (500 g) suspended in water (5.0 ml) cooled to 5” a saturated aqueous solution of sodium borohydride (1.0 g) was added and the contents left to stand (1 hr) and then acidified with sulphuric acid (50%). The solid product 105 mg, m.p. 199-204” was undepressed on admixture with curvulol m.p. 204”. Recrystallization from ethyl alcohol raised the m.p. 204”; extraction of the mother liquor gave an oil which could not be crystallized and was not investigated. (b) Reduction of curvulin (with LIAIH,). To lithium aluminium hydride (400 mg) dissolved in pure dry ether (100 ml) curvulin (200 mg) in dry ether (100 ml) was added drop by drop with ice cooling. After standing 1 hr, ice-cold water was added and the solution acidified with dil. H,SO,. The ethereal layer was separated and the aqueous phase twice extracted with ether. The crystalline residue on drying and removal of ether, was recrystallized from ethyl alcohol; 123 mg, m.p. 204”, undepressed on admixture with curvulol (m.p. 204”). (Found: C, 60.92; H, 6.44; 0,32.44; Calc. for C,,H,,O,; (M.W. 194): C, 61.21; H, 6.17; 0, 32.62%). The product gives a brownish-red colour with ferric chloride and a cherry-red colour with cone H,SO,. The UV absorption bands at A,, 275 rnp (log E 3.77); 285 rnp (log E 3.96) and Amin 255 rnp (log E 3.65); 280 m/l (log E 3.38) are identical with those of natural curvulol. of O-methylcurvulinic acid isomer M.P. 172” O-methylcurvuhnic acid isomer m.p. 172” (200 mg) in absolute ethyl alcohol (6 ml) containing a drop of cone HCI acid was refluxed (5 hr) and worked up. The product crystallized from ether-light petroleum (-0”) in tiny plates; m.p. 84”. identical with mono 0-methylcutvulin isomer m.p. 840.1 Esterification

thanks are due to Mr. A. Rauf Anwar,forcarryingout the IR determinations. All analyses were carried out by Dr. A. Bernhardt, Mikroanalytisches Laboratorium, 22a, Mulheim (Ruhr), West Germany.

Acknowled~ement-Our