structure elucidation and biosynthesis of 31-methylhopanoids from acetobacter europaeus : studies on...

Eur. J. Biochem. 225,765-771 (1994) 0 FEBS 1994

Structure elucidation and biosynthesis of 3Lmethylhopanoids from Acetobacter europaeus Studies on a new series of bacterial triterpenoids

Pascale SNONIN’, Brian TINDALL’ and Michel ROHMER’

’ Deutsche Sammlung von Mikroorganisrnen und Zellkulturen GmbH, Braunschweig, Germany

(Received May 20, 1994) - EJB 94 0724/5

Laboratoire de Chimie Microbienne, CNRS - URA 135, Ecole Nationale SupCrieure de Chimie de Mulhouse, Mulhouse, France

Apart from a nuxture of bacteriohopanetetrols already found in other Acetobacter species, four new 3P-methylhopanoids have been isolated from Acetobacter europaeus. All of them present an ether linkage between a bacteriohopanetetrol or a bacteriohopanepentol and a carbapseudopentose moiety often found in bacterial hopanoids. Three of these ethers were shown by comparison with synthetic reference hopanoids to posess a supplementary methyl group at C31. This novel series of methylhopanoids may be the precursor of yet unidentified molecular fossils found in sediments. [rnethyl-2H,]Methionine was efficiently incorporated into the 3 1 -methylhopanoids with retention of all three deuterium atoms in the transferred methyl group. This labelling pattern might be consistent with a rather rarely found methylation reaction of an enol.

Acetic acid bacteria are all good producers of hopanoids, a series of bacterial triterpenoids widely distributed among eubacteria [ l ] and acting as membrane stabilizers [2]. Aceto- bacter species present the most complicated pattern of struc- tural variations on the pentacyclic skeleton : double-bond at C6 and/or C11, additional methyl group at C3P and two ste- reoisomers at C22 [3-61. This paper describes the isolation, identification and biosynthesis from [methyl-’H,]methionine of a novel series of methylhopanoids with an additional methyl group at C31 from the recently described Acetobacter europaeus which, in contrast to other Acetobacter species, requires acetic acid for growth [7].

of the bacteriohopanetetrols (R, = 0.83) and acetylated tetrol ethers (RF = 0.09). Preparative reverse-phase HPLC on a Du- pont Zorbax ODS column (250X21.2 mm) using methanol as eluent (15 mYmin) and a Spectra Physic 6040 differential

MATERIALS AND METHODS Culture conditions and isolation of hopanoids

Acetobacter europaeus DSM 6160 (Deutsche Sammlung von Mikroorganismen, Braunschweig) was grown aerobi- cally at 30°C either in a 10-1 fermenter for 96 h (yield 0.2- 0.3 g/l, dry mass), or in 2-1 conical flasks on a rotatory shaker for 72 h (yield 0.2 g/l) using the medium described by Sie- vers et al. [7]. For labelling experiments, ~-[rnethyl-~H,]methionine (CEA, Saclay, France ; isotopic abundance 99%) was added to the culture medium at a 1 mM concentration. Most of the analytical procedures were as described previously [8, 91. The CHCl,/CH,OH (2: 1, by vol.) extract of a small sample of freeze-dried cells (0.1 g) was first treated by our HJOJNaBH, method in order to evaluate the hopanoid content [l]. Bacteriohopanepolyols were isolated from a larger sample (4 g) as acetylated derivatives by TLC (cyclohexane/ethyl acetate, 1 : 1, by vol.) giving the tetraacetates

Chimie de Mulhouse, 3 rue Alfred Werner, F-68093 Mulhouse CCdex, France

I*ll++*

OH HO @ (2) A1l ”*,,

(3)

HO

Correspondence to M . Rohmer, Ecole Nationale Superieure de (4)

Fig. 1. Bacteriohopanetetrol ethers from Acetobacter europaeus.

766

refractometer detector yielded the acetylated derivatives of 11 bacteriohopanetetrols and 4 bacteriohopanetetrol ethers (Fig. 1).

Identification of compounds (1-4) Structures of already known hopanoids were determined

by comparison of their 13C- and/or ‘H-NMR spectra and HPLC retention times with those of the corresponding hopanoids obtained from Acetobacter pasteurianus [9]. The acetylated novel hopanoids (1-4) were identified by ‘H- and ”C-NMR spectroscopy and direct inlet electron impact mass spectrometry. Definitive proof for the localization at C31 of the supplementary methyl group was obtained by comparison (‘H-NMR and GLC) of the aldehyde obtained after H,IO, treatment of hydrogenated hopanoid (2) with the two C31 diastereomers of 3/?,31-dirnethyltrinorhopan-32-a1 synthesized by methylation of 3P-methyltrinorhopan-32-a1 prepared from Acetocater aceti ssp xylinum hopanoids [6,9].

Bacteriohopanetetrol ether (1)

‘H-NMR (250 MHz, CDCl,) of the octaacetate of compound (1): G/ppm = 0.645 (3H, s, CH,), 0.738 (3H, s, CH,),

(3H, s, CH,), 0.863 (3H, s, CH,), 0.887 (3H, d, J = 6.5 Hz, 0.822 (3H, d, J = 6.5 Hz, 3P-CH3), 0.835 (6H, S, CH,), 0.849

22-CH3), 2.012 (3H, S, CH,COO-), 2.019 (3H, S, CH3COO-), 2.051 (3H, S , CH3COO-), 2.069 (3H, S, CH,COO-), 2.072 (3H, S, CH,COO-), 2.087 (3H, S, CH,COO-), 2.109 (3H, S, CH,COO-), 2.139 (3H, S , CH,COO-), 3.65 (lH, dd, J 34,3sa = 5.5 Hz, J 3 S a , 3 5 b = 10.5 Hz, 35-H,), 3.91 (lH, q, J1*,y= Jz,3’=J,:N,=7.5H~,2‘-H),3.98(lH,dd,J,,,,b=5.5H~, J 3Sa,35b = 10.5 HZ, 35-Hb), 4.09 (IH, d, J ga,6’b =11.5 HZ, 6’- Ha), 4.14 (lH, d, J 1 , , y = ~ H z , 1’-H), 4.24 (lH, d, Jga,gb=

5.16(1H,d,J,,,&= 5.5H~,4’-H),5.32(1H,dd,J,,,,= ~ H z , J 3334 = 6 Hz, 33-H), 5.32 (lH, dd, J 2.3’ =8 Hz, J y,c = 5.5 Hz, 3’-H), 5.46 (lH, dd, J31,3, = 3.5 Hz, J32.3, = 8 Hz, 32-H), 5.54 (lH, d, J = 10.5 Hz, 11-H), 5.64 (lH, d, J z10 .5 Hz, 12-H), 6.42 (lH, d, J y , N H = 7.5 Hz, -NH-).

0.1 %), 1011 (M’-AcOH, 7.5%), 952 (M+-AcOH-AcNH,,

11.5Hz, 6’-Hb), 5.01 (lH, m, 31-H), 5.07 (lH, m, 34-H),

Mass spectrum: d z = 1071 (M+, 0.2%), 1053 (M+-H,O,

2%), 805 (retro Diels-Alder after isomerization of A” into A” double-bond -AcOH, 5.5%), 725 (ether bond cleavage, 7%), 381 (M+-side-chain, 6.5%), 330 (ether bond cleavage, 6%), 232 (retro Diels-Alder reaction after isomerization of A” into A’-double-bond, 39%), 205 (ring C cleavage, 31%), 60 (100%).

Bacteriohopanepentol ether (2) ‘H-NMR (250 MHz, CDCl,) of the heptaacetate of com-

pound (2) : &ppm = 0.645 (3H, s, CH,), 0.755 (3H, s, CH,), 0.821 (3H, d, J = 6.5 Hz, 3P-CH3), 0.835 (3H, d, J = 6.5 Hz, 31-CH,), 0.835 (6H, S, CH,), 0.850 (3H, S , CH,), 0.864 (3H, S , CH,), 0.890 (3H, d, J = 6.5 Hz, 22-CH3), 1.994 (3H, S, CH,COO-), 2.059 (6H, S, 2 CH,COO-), 2.076 (3H, S, CH,COO-), 2.079 (3H, S, CH,COO-), 2.112 (3H, S, CH,COO-), 2.139 (3H, S, CH,COO-), 3.68 (lH, dd, J 34,3Sa = 7 Hz, J 35a.33, = 11 Hz, 35-Ha), 3.86 (lH, d, J 1 P . y = 6.5 Hz, I’-H), 4.05 (IH, dd, J 34.3-3 = 4 HZ, J 35=,35b = 11 HZ, 35-&), 4.08 (IH, dt, J 1:y = J y , y = 6.5 Hz, J , NH = 7.5 Hz, 2’-H), 4.10 (lH, d, Jgu,cb= 11.5 Hz, 6’-H,), 4.30 (lH, d, J g a , g b =

11.5 Hz, 6’-Hb), 5.03 (lH, dd, J3’ ,32 = 4.5 Hz, J 3 , 3 3 =7.5 Hz,

32-H), 5.10 (lH, dt, J 33.34 = J 34,35b = 3 Hz, J 34,3Sa =7 Hz, 34-H), 5.19 (lH, t, J y , y = J,:c= 6.5 Hz, 3’-H), 5.25 (lH, d, J y.4’ = 6 Hz, 4’-H), 5.32 (lH, dd, J 32,33 = 7.5 Hz, J 33.34 = 3 Hz, 33-H), 5.55 (lH, d, J = 10.5 Hz, 11-H), 5.63 (lH, d, J = 10.5 Hz, 12-H), 6.10 (lH, d, Jy, N H = 7.5 Hz, -NH-).

I3C-NMR (65 MHz, CDCl,) of the heptaacetate of compound (2): G/ppm = 15.9 (Is), 16.0 (Is), 16.6 (Is), 17.2 (Is), 17.27 (Is), 17.30 (Is), 17.34 (Is), 18.9 (Is), 19.5 (Is), 20.76 (Is), 20.85 (2s), 20.87 (Is), 20.93 (Is), 21.0 (Is), 23.4 (Is), 23.5 (Is), 27.3 (Is), 27.9 (Is), 28.9 (Is), 31.0 (Is), 31.1 (Is), 31.5 (Is), 34.0 (Is), 36.4 (ls,), 36.7 (Is), 37.1 (Is), 39.7 (Is), 40.6 (Is), 41.0 (Is), 41.1 (Is), 42.5 (Is), 43.5 (Is), 46.7 (Is), 53.3 (Is), 54.1 (Is), 54.5 (Is), 56.9 (Is), 57.9 (Is), 64.5(1s), 69.3(1s), 70.4(1s), 71.3(1s), 75.6(1s), 76.3(1s), 76.8(1s), 79.2(1s), 81.6(1s), 127.2(1s), 129.7(1s), 169.6, 170.3, 170.5, 170.8, 170.9 (7CH,CO-).

Mass spectrum: d z = 1027 (M+, 6%), 1009 (M+-H,O,

848 (908-AcOH, 3%), 806 (retro Diels-Alder reaction after isomerization of A” into 4” double-bond -Me, 3%) [6], 746 (806-AcOH, 3%), 330 (ether bond cleavage, 69%), 232 (retro Diels-Alder reaction after isomerization of A” into A9- double-bond, 33%) [6], 205 (ring C cleavage, 51%), 95 (100%).

2%), 967 (M’-AcOH, 8%), 908 (M’-AcOH-AcNH,, 7%),

Bacteriohopanetetrol ether (3) ‘H-NMR (250 MHz, CDCl,) of the heptaacetate of com-

pound (3): G/ppm = 0.633 (3H, s, CH,), 0.688 (3H, s, CH,), 0.781 (3H, S , CH,), 0.809 (3H, d, J = 6.5 Hz, 3P-CH,), 0.821 (3H, d, J = 6.5 Hz, 31-CH3), 0.861 (3H, S , CH,), 0.886 (3H, d, J = 6.5 Hz, 22-CH3), 0.939 (3H, S , CH,), 0.947 (3H, S, CH,), 1.996 (3H, S, CH,COO-), 2.061 (6H, S, 2 CH,COO-), 2.079 (6H, S, CH,COO-), 2.115 (3H, S, CH,COO-), 2.138 (3H, S, CH,COO-), 3.68 (lH, dd, J 34,3sa = 6 Hz, J 3sa,3sb = 11 Hz, 35-H,), 3.86 (lH, d, J 1 , , 2 , = 6.5 Hz, 1’-H), 4.05 (ZH, dd, J 3 4 , 3 5 b = 4 HZ, J3sa,35b = 11 HZ, 35-&), 4.08 (IH, dt, J i ’ , y = J z , , , = 6 . 5 H ~ , J,:N,=7.5Hz, 2’-H), 4.11 (lH, d, J6ta,6q,= 11.5 HZ, 6’-H,), 4.31 (1H, d, Jga,6q,= 11.5 Hz, 6’- Hb), 5.02 (IH, dd, J 11.32 = 4.5 Hz, J 32.33 = 7.5 Hz, 32-H), 5.10 (lH, dt, J 33,34 = J 34,35b = 3 Hz, J 34,3Sa = 7 Hz, 34-H), 5.19(1H,t,J,,,.=J,,,=6.5H~,3’-H),5.25(1H,d,J,,,,.= 6 Hz, 4’-H), 5.32 (lH, dd, J 32.33 = 7.5 Hz, J 3 3 . 3 = 3 Hz, 33- H), 6.09 (lH, d, J,: NH = 7.5 Hz, -NH-).

0.5%), 970 (M+-AcNH,, 4%), 969 (M+-AcOH, 7%), 910 Mass spectrum: m/z = 1029 (M+, l%), 1011 (M+-H,O,

(M’-AcOH-AcNH,, 2%), 850 (910-AcOH, l%), 794 (ring C cleavage, l%), 734 (794-AcOH, 3%), 683 (ether bond cleavage, 2%), 383 (M+-side-chain, 20%), 330 (ether bond cleavage, 18%), 270 (330-AcOH, 37%), 205 (ring C cleavage, 100%).


‘H-NMR (250 MHz, CDC1,) of the heptaacetate of compound (4): the methyl signals (0.6-1 ppm) are not resolved and could not be assigned. Between 1.8 -6ppm, the ‘H- NMR spectrum was identical with that of the heptaacetate of the bacteriohopanetetrol ether (2).

Mass spectrum (direct inlet):m/z = 1041 (M+, 5%), 981 (M’-AcOH, 5%), 922 (M+-AcOH-AcNH,, 5%) , 806 (retro Diels-Alder reaction after isomerization of A” into A’z double bond - Me, 2%), 794 (ring C cleavage, 2%), 695 (ether bond cleavage, 3%), 330 (ether bond cleavage, 75%),

767

r?

Fig. 2. Hopanoids identified from Acetobucter europueus. Compounds 6-9 correspond to the carbon skeleton of bacteriohopanepolyol derivatives obtained after H,IO,JNaBH, treatment.

246 (retro Diels-Alder reaction after isomerization of A" in A9 double-bond, 26%), 231 (246-Me, 31%), 219 (ring C cleavage, 41%), 95 (100%).

Synthesis of 3~,31-dimethyltrinorbacteriohopan-32-al (Fig. 3)

3P-Methylbacteriohopanetetrol(5 mg) was obtained from A. pasteurianus [9] and treated by periodic acid [l], leading after TLC separation (cyclohexane/ethyl acetate, 95 : 5, by vol.) to 3P-methyltrinorbacteriohopan-32-a1 (1 3) (RF = 0.5, 4mg). To a stirred suspension of potassium t-butoxide (30mg) in dimethoxyethane (1 ml), a solution of the aldehyde (13) (4 mg) in dimethoxyethane (2 ml) and methyl iodide (lOOi.1.1) were introduced under argon. The reaction mixture was stirred for 1.5 h. After addition of water and hexane extraction, the hexane extract was taken to dryness, and the residue separated by TLC (cyclohexane/ethyl acetate, 95 : 5, by vol.), giving the two isomers of the 3P,31-dimethyltrinorbacteriohopan-32-a1 (14) and (15) (RF = 0.56, 1.5 mg and R, = 0.64, 0.4 mg) and the 3P,31,31-trimethyltrinorbacteriohopan-32-a1 (16) (RF= 0.75, 2 mg).

Identification of compounds (14-16) NaBH, reduction of aldehydes (1 4 - 16) yielded the cor-

responding alcohols which were analyzed after acetylation by GLC/MS.

0.841 (3H, s, CH,), 0.942 (3H, d, J = 6.5 Hz, 22R-CH3), 0.953 (3H, s, 2CH,), 1.041 (3H, d, J = 6.9 Hz, 31-CH3), 2.5 (lH, m, 31-H), 9.61 (lH, d, J = 1.6 Hz, -CHO).

I3C-NMR spectrum (65 MHz): G/ppm = 12.9 (Is), 14.1 (Is), 15.8 (Is), 16.0 (Is), 16.4 (Is), 16.6 (Is), 18.8 (Is), 19.7 (Is), 20.9 (Is), 22.7 (Is), 24.0 (Is), 27.6 (Is), 28.0 (Is), 29.2 (Is), 31.6 (Is), 33.5 (Is), 33.8 (Is), 34.8 (Is), 36.3 (Is), 37.6 (Is), 40.4 (Is), 41.6 (Is), 42.4 (Is), 44.0 (Is), 44.3 (Is), 46.7 (Is), 49.4 (Is), 50.6 (Is), 54.6 (Is), 57.1 (Is), 205.5 (CHO).

Mass spectrum (GLCMS): d z = 482 (M+, 3%), 467 (M+-Me, 4%), 383 (M+-side-chain, 14%), 247 (ring C cleavage, 78%), 205 (ring C cleavage, 100%).

Melting point of crystals = 193 - 195 "C (obtained from Me0 H/CH,Cl,)

3P,31 -Dimethyltrinorbacteriohopan-32-a1 (15), minor diastereomer

'H-NMR spectrum (250 MHz): G/ppm = 0.631 (3H, s,

0.861 (3H, S, CH,), 0.935 (3H, S, CH,), 0.936 (3H, d, J =

7 Hz, CH,), 2.40 (lH, m, 31-H), 9.54 (lH, d, J = 3.1 Hz,

Mass spectrum (GLCMS): m/z=482 (M+, 3%), 467 (M+-Me, 4%), 383 (M'-side-chain, 13%), 247 (ring C cleavage, 75%), 205 (ring C cleavage, 100%).

CH,), 0.685 (3H, S , CH,), 0.808 (3H, d, J = 6.5 Hz, 3P-CH3),

6.5 Hz, 22R-CH3), 0.947 (3H, S, CH,), 1.071 (3H, d, J =

-CHO).

3b,31 -Dimethyltrinorbacteriohopan-32-a1 (14), major diastereomer

3P,31,31 -Trimethyltrinorbacteriohopan-32-al (1 6) 'H-NMR spectrum (250 MHz): G/ppm = 0.631 (3H, s,

CH,), 0.703 (3H, s, CH,), 0.780 (3H, s, CH,), 0.808 (3H, d, 'H-NMR spectrum (250 MHz): G/ppm = 0.635 (3H, s, CH,), 0.712 (3H, S, CH,), 0.810 (3H, d, J = 6.6 Hz, 3P-CH3), J = 6.5 Hz, 3P-CH3), 0.850 (3H, d, J = 6.5 Hz, 22-CH,),

768

A,, 31 CHO

~ l l r l ~ ~ o ____._) 2.CH31 fy (16)

1. t-BuOK

(14) and (15) '8, (13)

Fig. 3. Synthesis of the two diastereoisomers of 3/l,31-dimethyltrinorbacteriohopan-32-al.

0.860 (3H, s, CH,), 0.941 (6H, s, CH,), 1.025 (3H, s, CH,),

',C-NMR spectrum (65 MHz): G/ppm = 15.8 (Is), 16.0 (Is), 16.4 (2s), 16.6 (2s), 18.8 (Is), 20.5 (Is), 20.9 (Is), 22.1 (Is), 22.7 (Is), 23.0 (Is), 23.8 (Is), 24.0 (Is), 27.6 (Is), 28.6 (Is), 29.2 (Is), 31.6 (Is), 33.5 (Is), 33.8 (Is), 34.2 (Is), 36.4 (Is), 37.6 (Is), 40.4 (Is), 41.6 (Is), 42.4 (Is), 44.3 (Is), 44.8 (Is), 47.1 (Is), 49.5 (Is), 50.6 (Is), 54.5 (Is), 57.1 (Is), 206.9

Mass spectrum (GLCMS): m/z = 496 (M+, 3%), 481 (M+-Me, 4%), 383 (M'-side-chain, 17%), 261 (ring C cleavage, 66%), 205 (ring C cleavage, 100%).

1.040 (3H, S, CH,), 9.5 (lH, S, -CHO).

(IS, -CHO).

Monoacetate of 3p,31 -dimethyltrinorbacteriohopan-32-ol, major diastereomer

Mass spectrum (GLCMS): m/z = 526 (M+, l%), 511 (M+-Me, 3%), 383 (M'-side-chain, 14%), 291 (ring C cleavage, loo%), 231 (291-AcOH, 31%), 205 (ring C cleavage, 97 %)

Monoacetate of 3p,31 -dimethyltrinorbacteriohopan-32-01, minor diastereomer

Mass spectrum (GLCMS): d z = 526 (M+, l%), 511 (M'-Me, 4%), 383 (M+-side-chain, 17%), 291 (ring C cleavage, loo%), 231 (291-AcOH, 26%), 205 (ring C cleavage, 91 %)

Monoacetate of 3p,31,31 -trimethyltrinorbacteriohopan-32-ol

Mass spectrum (GLCMS): m/z = 540 (M+, l%), 526 (M+-Me, 2%), 466 (M+-Me-AcOH, 0.5%), 383 (M+-side- chain, 23%), 305 (ring C cleavage, 92%), 205 (ring C cleavage, 100%)

RESULTS H,IOflaBH, treatment of the crude extract yielded

diplopterol (120 pg/g) and a series of primary alcohols. Un- less indicated otherwise, all these alcohols are derived from carbon skeleton (7) (Fig. 2). The monoacetates of (22R)-3/3- methyltetranorbacteriohop-11-en-31-01 [skeleton (6), Fig. 21 and (22R)-3~-methyltrinorbacteriohop-11-en-32-ol (30%), (22S)-trinorbacteriohopan-32-01 [skeleton (S)] (12%), (22R)- 3P-methyltrinorbacteriohopan-32-01 and 3p,3 1 -dimethyl- trinorbacteriohop-l1-en-32-ol (42%), (22R)-trinorbacteriohop-6-en-32-01 (4%), (22R)-3/?-methyltrinorbacteriohop-6- en-32-01 (3%), (22R)-3~-methyltrinorbacteriohop-ll-en-32- 01 (3%), (22R)-3~-methyltrinorbacteriohopa-6,ll-dien-32-01 (1 %), (22R)-38,3 1-dimethyltrinorbacteriohopan-32-01 (4%) and 3 1 ,x,y-trimethyltrinorbacteriohop- 11 -en-32-01 [skeleton (9)] (1%) were identified by GLCMS (Fig. 2). These alcohols are typical for Acetobacter species, as they correspond

to a mixture of saturated, unsaturated and methylated bacteriohopane derivatives. GLCMS of their acetates permitted the identification of most of them by comparison with reference compounds prepared from the hopanoids of A. pasteurianus or A. aceti [9]. GLCMS revealed, however, the presence of new compounds with an additional methyl group. Indeed the two compounds with the longest retention times corresponded to the acetates of two C,, hopanic alcohols (a saturated and a &'-unsaturated alcohol) with two additional methyl groups, and molecular ions at respectively 526 and 524Da. One methyl group was located on ring A or B as shown by the fragment at m/z = 205 and the other one on the side-chain as shown by the fragments corresponding to the loss of the side-chain at m/z 383 or 381. In addition to these C,, alcohols, a C,, alcohol with a A" double bond and a methyl group on ring A was also among the major prod- ucts. All acetylated bacteriohopanetetrols (6 mg/g, freeze- dried cells) could be isolated by TLC followed by reverse- phase HPLC yielding the tetraacetates of (22R, 32R, 33R, 34S)-bacteriohopane-32,33,34,35-tetrol (10) (27 % of the tetrol fraction), (22R, 32R, 33R, 34S)-3P-methylbacteriohopane-32,33,34,35-tetrol [derived from skeleton (lo), Fig. 21 (17%), (22R, 32R, 33R, 34S)-bacteriohop-6-ene-32,33,34,35- tetrol [derived from (lo)] (14%), (22R, 32R, 33R,34S)-3P- methylbacteriohop-6-ene-32,33,34,35-tetrol [derived from (lo)] (5%), (22R, 32R, 33R, 34S)-bacteriohop-ll-ene- 32,33,34,35-tetrol [derived from (lo)] (11 %), (22R, 32R, 33R, 34S)-bacteriohopa-6,ll-diene-32,33,34,35-tetrol [derived from (lo)] (2%), (22R, 32R, 33R, 34S)-3p-methyl- bacteriohopa-6,11-diene-32,33,34,35-tetrol [derived from (lo)] and (22R, 32R, 33R, 34R)-3P-methylbacteriohop-6-ene- 32, 33,34,35-tetrol [derived from (12)] (6%), (22S, 32R, 33R, 34S)-bacteriohopane-32,33,34,35-tetrol [derived from (ll)] (11 %), (22R,32R,33R,34R)-3P-methylbacteriohopane-32,33, 34,35-tetrol [derived from (12)] (6%) and (22R, 32R, 33R,34R)-bacteriohop-ll-ene-32,33,34,35-tetrol [derived from (12)] (1 %) (Fig. 2). All these bacteriohopanetetrol derivatives were identified by comparison of their 13C- andor 'H- NMR spectra and their HPLC retention times with those of the acetylated tetrols obtained from A. pasteurianus and A. aceti ssp. xylinum [9]. The new acetylated bacteriohopanepolyols ethers (5 mg/g, freeze-dried cells) (1) (53% of the ether fraction), (2) (42%), (3) (4%) and (4) (1%) (Fig. 1) were identified by spectroscopic methods and comparison with reference material obtained by chemical synthesis.

Bacteriohopanepentol ether (1)

The structure of the acetylated bacteriohopanepentol ether (1) was determined by 'H-NMR spectroscopy by comparison with its saturated and non methylated homologue isolated from Azotobacter vinelandii [ 101. The 'H-NMR spectrum showed in the methyl region two doublets at 0.83 ppm corresponding to the C3p methyl group and at

769

+

d

Fig. 4. Fragmentations of hopanoids in mass spectrometry. Fragments d and e are only observed in the spectra of A"-hopanoids.

Table 1. Incorporation of [methyl-*H,]methionine into the hopanoids of Acetobucter europueus. Mass spectra of the 31-methylbacteriohopane derivatives: acetates of 3P,31-dimethyltrinorbac- teriohop-1 1 -en-32-01, 3P,31 -dimethyltrinorbacteriohopan-32-01 and 3l,x,y-trimethyltrinor-bacteriohop-ll-en-32-01. Structures of the fragments a-e are given in Fig. 4.

Hopanoid d z for acetate

molecular a b c d e ion

3p,3 1 -Dimethyltrinor-bacteriohop- 11 -en-32-01 non-deuterated 524 205 291 381 232 318 deuterated 527 208 294 384 235 321

530 3p,3 1 -Dimethyltrinor-bacteriohopan-32-ol

non-deuterated 526 205 291 383 deuterated 529 208 294 386

532

3 1 ,x,y-trimethyltrinor-bacteriohop- 11 -en-32-01 non-deuterated 538 219 291 395 246 318 deuterated 541 222 294 398 249 321

544 401 252

0.92 ppm characteristic of the C22 methyl group and six singlets corresponding to the other methyl groups of the hopane skeleton. Between 2.01 -2.40 ppm, eight singlets characteristic of acetoxy groups were observed. The presence of a A" double-bond was indicated by the two characteristic broad doublets at 5.54 and 5.65 ppm showing the 'J(,,.,,,,,, cou- pling constant. The presence of the C31 acetoxy group was revealed by the signal of the C32 proton: a doublet of doublet indicating the presence of a functional group at C31, and by the multiplet at 5.01 ppm corresponding to the C31 proton. The signals corresponding to the protons of the cyclitol moiety were identified by comparison with those of the spectrum of the bacteriohopanetetrol ether isolated from Methylobac- terium organophilum [l l] . The molecular ion at d z = 1071, the ring C fragmentation yielding an ion at d z = 205 and the two ions at d z = 232 and 805 were in accordance with the proposed structure.

Bacteriohopanetetrol ether (2) The 'H-NMR spectrum of the heptaacetate of tetrol ether

(2) showed in the methyl region three doublets and six singlets corresponding to nine methyl groups. The doublet at 0.89 ppm was characteristic of the C22 methyl group with a 22R configuration. The doublets at 0.81 and 0.83 ppm indicated the presence of two additional methyl groups located at C3p and the other in an unknown position. The presence

of nine methyl groups was confirmed by I3C-NMR spectroscopy. The existence of the cyclitol moiety could be deduced from the 'H-NMR spectrum by comparison with the bacteriohopanetetrol ether isolated from Methylobacterium organophilum [ l l ] . The two broad doublets at 5.54 and 5.65 ppm revealed the presence of a A" double-bond. The mass spectrum of the acetylated compound (2) showed a molecular ion at m/z = 1027 indicating the presence of two supplementary methyl groups, a ring C fragmentation at m/z = 205 and two ions at d z = 232 and 806 corresponding to a retro Diels-Alder reaction after migration of the A" double-bond to the A9 or A" position. These ions allowed the localization of the two additional methyl groups one on ring A or B (at C3p) and the other on ring C, D or E or on the side-chain. The ion at d z 381 resulting from the loss of the side-chain suggested the presence of one of these methyl groups in this side-chain. The supplementary methyl group could be located at C31 by 'H-NMR spectroscopy. Indeed, the signal of the C32 protons at 5.03 ppm is a doublet of doublet in accord with the presence of a substituent at C31. All spectroscopic data are in full agreement with a bacteriohopanetetrol ether containing an extra methyl group at C31. Confirmation of the structure was obtained after cleavage by HJO, of the hydrogenated hopanoid (2) giving a 3p,3 1 -dimethyltrinorbacteriohopan-32-a1 (14) which was identified by comparison with the corresponding aldehyde obtained by hemisynthesis. After synthesis of the two isomers of the 3p,31- dimethyltrinorbacteriohopan-32-a1 (14) and (15) (Fig. 3), the comparison of the physical data ('H, 13C-NMR spectra, GLC retention times, mass spectra, melting point and GLC/MS of the corresponding alcohols) of the aldehydes from the two origins indicated that the two compounds were identical. The 'H-NMR spectroscopy permitted the two isomers 31R and 31s of the methylated aldehyde to be distinguished, but it was not possible to determine by this method the C31 configuration of each isomer. The major synthetic aldehyde was identical with the compound resulting of the HJO, cleavage of the natural product.


The 'H-NMR spectrum of the acetylated tetrol ether (3) was identical with that of the heptaacetate of the bacteriohopanetetrol ether (2), except the two doublets at 5.54 and 5.65 ppm which were absent from the spectrum of the acetylated compound (3). This indicates that compound (3) is a saturated hopanoid. The molecular ion at d z = 1029, the ring C fragmentation at d z = 794 and 205 and the ether bond cleavage at m/z = 683 and 330 in the mass spectrum were compatible with the structure of a 3p,31-dimethyl- bacteriohopanetetrol ether.

770

- X

Fig. 5. Hypothetical biogenetic pathway for the biosynthesis of 31-methylhopanoids.


The compound (2) was accompanied by a minor hopanoid (4). Due to its low concentration (0.2 mg/g, freeze- dried cells), this bacteriohopanetetrol ether (4) was only analyzed by 'H-NMR spectroscopy and mass spectrometry. The 'H-NMR spectrum was similar to that of the compound (2), with the exception of the methyl region. The methyl signals appeared not resolved and could not be analysed in detail. The signals of the side-chain protons were identical with those of the acetylated hopanoid (2). As for as the identification of the hopanoid (2), the doublet of doublet representing the signal of the C32 proton indicated that one methyl group was located at C31. In the mass spectrum of the heptaacetate of the bacteriohopanetetrol ether (4), the molecular ion at m/z = 1041 revealed the presence of three additional methyl groups, by comparison with the mass spectrum obtained from the heptaacetate of the acetylated hopanoid (2). The ions at m/z = 219 corresponding to the ring C fragmentation, m/z = 246 and 806 characteristic of a retro Diels-Alder reaction after migration of the A'' double-bond to the A9 or A'* position, proved the existence of two methyl groups on the ring A or B and one methyl group on the ring C, D or E or on the side-chain, located by 'H-NMR at C31.

In this case, compound (4) was not completely identified by the spectroscopic data. This hopanoid is however a bacteriohopanetetrol ether containing most probably one methyl group at C31 and two methyl groups on rings A or B.

Biosynthesis of 31-methylhopanoids : incorporation of [methyl-2H,]methionine

After incorporation of [methyl-2H,]methionine, the labelled hopanoids obtained after treatment by HJOJNaBH, were analyzed by GLCMS and the characteristic ions (fig. 4) were checked for the presence of deuterium. All analyzed hopanoids were a mixture of a non-labelled triterpenoid aris- ing from non-labelled methionine synthesized from the nor- mal carbon source of the culture medium and of a labelled hopanoid synthesized from deuterated methionine. The deuterated hopanoids eluted slightly faster than the corresponding non-labelled hopanoids during GLCMS [12, 131. Best spectra were thus obtained on the ascending part of each GLC peaks. The fragments a, c, d corresponding respectively to the ring C fragmentation, to the side-chain loss and to a retro Diels-Alder reaction after migration of the A" double- bond in A9-position (Fig. 4) were shifted by 3 Da in the spectra of labelled hopanoids (Table l) , indicating that the 3P-methyl group arises from the methionine, most probably via S-adenosylmethionine, and confirming the results obtained with A. pasteurianus.

Concerning the origin of the 31-methyl group, the fragments b and e corresponding respectively to the ring C fragmentation and to a retro Diels-Alder reaction after migration of the A" double-bond in A" were shifted by 3 Da in the spectra of labelled hopanoids (Table l ) , showing clearly the methionine origin of the 3 1-methyl group. Furthermore, the three deuterium atoms of the transferred methyl group were retained in the triterpenoids. The molecular ions M++6 and M+ +3 observed on the mass spectra were in full accordance with the methionine as precursor for the the 3P- and 31- methyl groups. In the same way, the ions corresponding to the characteristic fragmentations a, b, c, d and e (Fig. 4) detected in the spectrum of the labelled acetylated bacteriohopane derivative 3 1 ,x,y-trimethyltrinorbacteriohop-11 -en-32- 01 [derived from skeleton (9)] revealed the origin by methionine of all three supplementary methyl groups (Table 1).

DISCUSSION According to the hopanoid composition, the recently de-

scribed A. europaeus is a typical Acetobacter as it contains, like all other Acetobacter species, the most complex mixture of unsaturated and methylated bacteriohopanepolyols found in all hopanoid-producing bacteria [3 -61. Furthermore, A. europaeus, like A. pasteurianus and A. aceti, possesses two bacteriohopane side-chains differing one from another by the stereochemistry at C34 [9]. This feature has been only found in Acetobacter species. However, hopanoids isolated from A. europaeus are the only ones possessing an extra methyl group at C31. To date only 2P-methylhopanoids were identified from Nostoc muscorum [14] or Methylobacterium organophilum [ 151, 2a-methylhopanoids from M. organophilum [ 161 and 3P-methylhopanoids from Acetobacter species [6], Methylococcus capsulatus [ 17 - 181, Methylococcus luteus [ 191 or Methylomonas methanica [ 181. The stereochemistry of the different bacteriohopanetetrols was determined by 'H-NMR spectroscopy by comparison with synthetic reference compounds [20]. The stereochemistry of the side-chain of the new bacteriohopanetetrol ether is still unknown. The configuration of the C31 asymmetric center could not be determined. This would require hemisynthesis of reference hopanoids of known C31 stereochemistry.

Concerning the formation of 2- and 3-methylhopanoids, it has been already shown by incorporation of deuterium- labelled methionine on the methyl group that this amino acid is the methyl donor. The substrate for the methylation could be a A'-hopanoid in the case of 2P-methylhopanoid biosynthesis and a A'-hopanoid or squalene in the case of formation of 3P-methylhopanoid [13]. The labelling of 3 1 -methylhopanoids obtained after incorporation of deuterated methio-

77 1

nine proved that this amino-acid was the precursor of the additional methyl group, most probably via S-adenosylmethionine as for instance for the side-chain methylation in phytosterols. All three deuterium atoms of the transferred methyl group were retained in the triterpenoids, excluding intermediates possessing a methylene group.

By comparison with other methylation reactions involv- ing S-adenosylmethionine, a hopanoid containing a A”- double-bond could be a suitable methylation substrate. An- other possibility could be the methylation of an enol, i.e. in an a-position of a carbonyl group (Fig. 5). Such a methylation reaction in a-position of a carbonyl group by S-adenosylmethionine has already been reported for the formation of indolmycin [21]. A suitable substrate for such a reaction is known in the hopane series. Indeed a 32-oxobacteriohopane- 33,34,35-triol glycoside has been already isolated as a minor compound from Zymomonas mobilis [22] (Fig. 5).

Owing to their poor biodegradability and their stable triterpenic polycyclic skeleton, hopanoids are widespread in the organic matter of sediments and are useful biological markers. In addition to 2 a-, 2 p-, 3 a- and 3 P-methylhopanoids, several unidentified methylhopanoids have been detected in sediments, some of them possessing an additional methyl group in the side-chain [23]. It has now to be determined whether 3 1 -methylbiohopanoids, such as those found in A. europaeus, have a significance in organic geochemistry and could be the precursors of molecular fossils.

We thank Dr D. Le Nouen for all NMR measurements and M. P. Wehrung and M. C. Schweigert for recording the direct inlet mass spectra. Financial support was provided by the Centre National de la Recherche Scientifique (Unite! de Recherche Associe‘e 135) and the European Community (contract BIO-CT93-02734) generic project ‘Biotechnology of Extremophiles’ .

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