Eur. J. Biochem. 241, 865-871 (1996) 0 FEBS 1996

Bacterial triterpenoids of the hopane series from the prochlorophyte Prochlorothrix hollandica and their intracellular localization Pascale SIMONIN', Uwe J. JURGENS' and Michel ROHMER3 ' Ecole Nationale Suptrieure de Chimie de Mulhouse/CNRS, Mulhouse, France

Albert-Ludwigs Universitat, Institut fur Biologie 11, Freiburg i. Br., Germany UniversitC Louis PastedCNRS, Institut Le Bel, Strasbourg, France

(Received 14 May/9 August 1996) - EJB 96 0709/5

35-0-/3-GaIacturonopyranosyl-, 35-0-~-3,S-anhydro-galacturonopyranosyl- and 35-0-a-altrurono- pyranosylbacteriohopanetetrol accompanied by their 2-methyl homologues have been isolated from Prochlorothrix hollandicu. We report here C,, triterpenoids of the hopane series in a prochlorophyte, a group of prokaryotic oxigenic phototrophs of the cyanobacterial lineage. Like many cyanobacteria, I? hollundicu contains a mixture of non-methylated as well as 2D-methylhopanoids. After side-chain cleavage by periodic acid oxidation followed by sodium borohydride reduction, these hopanoids could be localized in cell walls and thylakoids, in accordance with their role as membrane stabilizers.

Keywords: hopanoid; prochlorophyte ; Prochlorothrix; triterpenoid.

The prochlorophytes are a small polyphyletic group of pro- karyotic oxigenic phototrophs. They belong to the cyanobacter- ial lineage, but contain chlorophyll u and b like plants and green algae and no phycobiliproteins like cyanobacteria (Urbach et al., 1992; Bullerjahn and Post, 1993). Lipid analysis (fatty acids and hydrocarbons) was in accordance with their classification into the prokaryotic kingdom. Diploptene (1) (Fig. l), a triterpene of the hopane series which is usually a minor compound in most of the hopanoid-producing eubacteria, has already been detected in Prochlorothrix hollundicu (Volkman et al., 1988). In all hopa- noid-producing prokaryotes the major compounds are always the bacteriohopanepolyols resulting from a carbodcarbon linkage between the triterpenic hopane skeleton and a D-pentOSe deriva- tive (Rohmer et al., 1984, 1989; Flesch and Rohmer, 1988). As hopanoids are essential metabolites for the bacteria producing them, acting mainly as membrane stabilizers (Flesch and Rohmer, 1987; Rohmer et al., 1979; Sahm et al., 1993; refer- ences cited in the latter), an axenic strain of Z? hollundica was examined for these compounds. In this paper we report the very peculiar hopanoid composition of this prokaryote containing only bacteriohopanetetrol glycosides with three different hexo- suronic acid moieties and resembling the cyanobacterial hopa- noids by the presence of a 2D-methyl group, as well as data on their intracellular localization.

MATERIALS AND METHODS

Strain, culture conditions and subcellular fractions from R hollandica. Prochlorothrix hollundica [Culture Collection of Algae and Protozoa (CCAP) 1490/1] was obtained from T. Burger- Wiersma (Laboratorium voor Microbiologie Universtiteit van Amsterdam). The prochlorophyte was cultivated photoauto-

Correspondence to M. Rohmer, UniversitC Louis Pasteur, Institut Le

Fax: +33 03 88 41 61 01. Bel, 4 rue Blaise Pascal, F-67070 Strasbourg Cedex, France

trophically in BG 11 medium at pH 7.5 and 25 "C (Rippka et al., 1979). Mass cultures were prepared in a 10-1 Biostat E fermentor (Braun Diessel Biotech) without stirring. They were constantly gassed with a mixture of air and carbon dioxide at flow rates of 100 1 . h-' and 1 1 . h-' respectively and illuminated with white fluorescent light (500- 1000 Ix). Cells were harvested by cen- trifugation (12000 g, 30 min) after 28 days growth, washed once with distilled water and subjected to lyophilization. Cell wall and thylakoid preparations were obtained and characterized as described previously (Jiirgens, 1990).

Analytical methods and isolation of hopanoids. All analyt- ical procedures and spectroscopic identifications were as pre- viously described (Peiseler and Rohmer, 1992). Freeze-dried cells (200 mg), thylakoid membranes (4 mg) or cell wall prepa- rations (64 mg) were extracted with CHCI,/CH,OH (2: 1, by vol.) and treated by our H,IO,/NaBH, method, in order to deter- mine by GLC their hopanoid content (Rohmer et al., 1984).

In order to isolate intact hopanoids, the extract from freeze- dried cells (10 g) was acetylated and separated by TLC (cyclo- hexane/ethyl acetate, 1 : 1, by vol.), giving the tetra-acetates of bacteriohopanetetrols (2a) and (2b) (R, = 0.47, 1 .S mg) (Fig. I ) , the penta-acetates of (3a) and (3b) (R, = 0.4, 1.8 mg) (Fig. 1 ) and the hexa-acetates of (4a) and (4b), (5a) and (5b) on the base-line. The latter polar hopanoids were treated with a solution of diazomethane in diethyl ether, and further separation by TLC (cyclohexane/ethyl acetate, 6 : 4, by vol., two migrations) yielded the hexa-acetates of the methyl esters of (4a) and (4b) (R, = 0.54, 3.5 mg) (Fig. 1) and the hexa-acetates of the methyl esters (5a) and (5b) (R, = 0.64, 0.4 mg) (Fig. 1). The penta-acetates of (3a) and (3b) were further purified by reverse-phase HPLC on a Dupont Zorbax ODS column (2SOX4.6 mm) using CH,OH as eluent (1 ml/min) and a Spectra Physic 6040 differential refrac- tometer as detector.

Identification of compounds. All hopanoids were isolated as a mixture of the non-methylated hopanoid and the corre- sponding 2a-methylhopanoid in a 4:6 ratio. The 2Bmethyl-

866 Simonin et al. ( E m

19-29

R = H compounds labelled a R = CH, compounds labelled b

on OH

A,.. + 22 R

28 OH

(2a) and (2b)

OH

r

(3a) and (3b)

OH OH OH OH OH

OH

,COOH (4a) and (4b) (5a) and (Sb) 6,CooH

Fig. 1. Hopanoids isolated from Prochlorothrix hollandica.

hopanoid could not be separated from its non-methylated com- panion either by TLC or by reverse-phase HPLC. Consequently, only 'H- and 'T-NMR spectra and mass spectra of the mixtures are given. As the 'H and chemical shifts corresponding to the hopane skeleton were identical for all compounds, they were given only for the spectra of acetylated compounds (3a) and (3b). Assignments were made by comparison with reference spectra of pure non-methylated hopanoids and of 2P-methyl- hopanoids (Stampf, 1992).

Spectroscopic data labelled with an asterisk (*) correspond to the spectrum of a non-methylated hopanoid, and those la- belled with a degree symbol (") to the spectrum of its 2-methyl homologue. Those without superscript are common to both com- pounds. Numbering of non-methylated hopanoids includes an a after the number, and that of 2P-methylhopanoids a b (Fig. 1).

35-0-~-3,5-Anhydrogalacturonopyranosylbacteriohopanetet- rols (3a) and (3b). 'H-NMR (250MHz, CDCl,) of the penta- acetates of (3a) and (3b): &ppm = 0.685 (3H, s, 18a-CH,), "0.791 (3H, s, 4P-CH3), "0.814 (3H, s, 4a-CH3), "0.827 (3H, d,

IOP-CH,), "0.883 and "0.891 (6H, 2s, 4n- and lO/I-CH,), "0.896

CH,), "0.933 (6H, s, 8P- and 14a-CH,), *0.945 (6H, s, 8P- and 14a-CH,), 2.067 (3H, s, CH,COO-), 2.080 (3H, s, CH,COO-),

J = 6.5 Hz, 2P-CH,), "0.828 (3H, S, 4P-CH,), *0.845 (3H, S,

(3H, d, J = 6.5 Hz, 22-CH3), "0.920 (3H, d, J = 6.5 Hz, 22-

2.1 00 (6H, S, 2 CH,COO-), 2.1 72 (3H, S, CH,COO-), 3.70 (1 H, dd, J x ; , . , s , =11.4 Hz, J31,3se = 3.2 Hz, 35-H,), 3.82 (IH, dd, J v a , i T h = 11.4 Hz, J,,,s, = 7.4 Hz, 35-H,), 4.07 (1 H, dd, J1,,5, = 1.6 Hz, J.t,.s. = 1.3 Hz, 5'-H), 4.85 (lH, S, 1'-H), 4.87 (lH, dd,

(1H, t, J32.3, = J33.3, = 4.9 Hz, 33-H), 5.19 (lH, d, Jz,3, = J1..5, =1.6Hz, JT., = 4.8Hz, 3'-H), 4.98 (lH, m, 32-H), 5.13

4.8 Hz, 2'-H), 5.23 (IH, m, 34-H), 5.32 (lH, d, J4.,s = 1.3 Hz, 4'-H).

"C-NMR (65 MHz, CDCI,) of the penta-acetates (3a) and (3b): Glppm = "15.8 and *15.9 (C25 and C28), "16.0, "16.2 and

J . Biochem. 241)

"16.4 (C26, C27 and C28), "16.5 and *16.6 (C26 and C27), "18.7 (C2 and C6), 19.9 (C29), "20.0 (C6), 20.7 (2 CHI,COO-), 20.8 (CH,COO-), 20.9 (CH,COO-), 21.0 (CH,COO- and "Cll), *21.6 (C24), "21.8 (C25), "22.0 (Cll), 22.8 (C16), "23.2 (C2B), "24.0 (C12), "24.3 (C12), "24.8 (C2), "26.1 (C24), 26.3 (C31), 27.6 (C20), 30.9 (C30), "31.1 (C23), "32.5 (C4), "32.6 (C7), "33.3 (C4 and C7), "33.4 (C23), *33.74 (C15), "33.78 (C15), 36.1 (C22), *37.4 (CIO), "37.9 (CIO), "40.4 (CI), 41.6- 41.8 (C19, C14 and CS), *42.1 (C3), 44.4 (C18), "45.2 (Cl), 46.0 (C21), "49.3 (C13), "49.7 and "49.8 (C3, C9 and C13), "50.4 (C9), "51.0 (C5), 54.5 (C17), "56.2 (C5), 66.9 (C35), 69.4, 70.1, 70.8, 71.6, 72.1, 72.3, 77.4, 99.5 (Cl'), 169.0, 169.6, 169.8, 170.2, 170.4 and 171.4 (6 -CO-).

Mass spectrum (direct inlet): mlz ="928 (M+, 1 %), "914 (M+, 0.6%), "868 (M'-AcOH, 7%), 693 (ring C cleavage, 48%), "685 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, 1 %), "669 (cleavage of the glycosidic bond between C35 and oxygen atom at C35, 6%), "383 (M'-side- chain, 17%), "369 (M+-side chain, 17%), 243 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, 87%), "205 (ring C cleavage, loo%), *I91 (ring C cleavage, 10%).

35-O-~-Galacturonopyranosylbacteriohopanetetrols (4a) and (4b). 'H-NMR (250 MHz, CDCl,) of the acetylated methyl es- ters of (4a) and (4b): dlppm = "0.909 (3H, d, J = 6.5 Hz, 22- CH,), "0.920 (3H, d, J = 6.5 Hz, 22-CH,), 1.988 (3H, S,

CH,COO-), 2.051 (3H, S, CH,COO-), 2.062 (3H, S, CH,COO-), 2.066 (3H, S, CH,COO-), 2.071 (3H, S, CH,COO:), 2.1 10 (3H, S, CH,COO-), 3.76 (3H, S, CH,O-), 3.87 (lH, dd, J,sd.qsh = 12.4 Hz, J34,3Sa = 2.8 Hz, 35-H,), 4.03 (lH, dd, J,,,,, = 7 Hz, J,5s,3Sb 1 12.4 Hz, 35-H,), 4.30 (lH, d, 5q.y = 1.3 Hz, 5'-H), 4.53 (lH, d, J,,,,. = 7.7 Hz, 1'-H), 4.98 (IH, m, 32-H), 5.06 (lH, dd, JZ',,, = 10.4 Hz, J,.,4. = 3.4 Hz 3'-H), 5.13 (IH, t, J32,3, =

7.4Hz, 2'-H), 5.19 (IH, m, 34-H), 5.69 (lH, dd, 53,.4. = 3.4Hz, J4,.5, = 1.4 Hz, 4'-H).

"C-NMR (65 MHz, CDCl,) of the acetylated methyl esters of (4a) and (4b): G/ppm = 20.58 (CH,COO-), 20.60 (CH,COO-), 20.64 (CH,COO-), 20.78 (CH,COO-), 20.89 (CH,COO-), 21.0 (CH,COO- and "Cll) , 52.7 (CH,O-), 67.3 (C35), 68.2 (C2'), 68.3 (C4'), 70.3 (C34), 70.5 (C3'), 71.9 (C32), 72.1 (C33), 72.4 (CS'), 100.6 (Cl'), 166.3, 169.3, 169.6, 169.8, 170.0, 170.1 and

Mass spectrum (direct inlet): mlz ="I002 (M+, 0.5%), "988

"822 (M+-3AcOH, 0.5%), 767 (ring C cleavage, 6%), "685 (cleavage of the glycosidic bond between C1' and oxygen atom at C35,9%), *671 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, 2%), "669 (cleavage of the glycosidic bond between C35 and oxygen atom at C35, 9%), *655 (cleav- age of the glycosidic bond between C35 and oxygen atom at C35, 3%), "383 (M+-side-chain, 8%), "369 (M'-side-chain, 9%), 317 (cleavage of the glycosidic bond between C1' and oxy- gen atom at C35, loo%), 257 (317-AcOH, 28%), "205 (ring C cleavage, 43%), "191 (ring C cleavage, 19%).

35-0-a-Altruronopyranosylbacteriohopanetetrols (5a) and (5b). 'H-NMR (250 MHz, CDCl,) of the acetylated methyl esters of (5a) and (5b): G/ppm = "0.896 (3H, d, J = 6.5 Hz,

J33.3, = 5.3 Hz, 33-Hj, 5.19 (IH, dd, Jy.3. = 10.4 Hz, J, , , y =

170.5 (7 -CO-)

(M+, 0.1 %), "942 (M.'-AcOH, 1 %), "882 (M+-2AcOH, 0.5%),

22-CH,), "0.920 (3H, d, J = 6.5 Hz, 22-CH3), 2.019 (3H, S,

CH,COO-), 2.065 (3H, S, CH,COO-), 2.074 (3H, S, CH,COO-), 2.103 (3H, S, CH,COO-), 2.109 (3H, S, CH,COO-), 2.1 14 (3H, s, CH,COO-), 3.79 (lH, s, CH,O-), 3.80 (2H, m, 35-H, and 35- H,), 4.56 (IH, d, J4.,5f = 7.6 Hz, 5'-H), 4.87 (IH, d, J f , , y = 3.3 Hz, 1'-H), 5.02 (lH, m, 32-H), 5.02 (IH, dd, JT1. = 5.6 Hz,

3'-H), 5.20 (2H, m, 32-H and 34-H), 5.42 (lH, dd, J4.,5. = 7.5 Hz, J3.,4, = 3.3 Hz, 4'-H).

Ji:y = 3.3 Hz, 2'-H), 5.16 (lH, dd, J,,,4, = 3.3 Hz, J,,,,, = 5.4 Hz,

Simonin et al. (Eua J. Biochern. 241) 867

"C-NMR (65 MHz, CDCI,) of the acetylated methyl esters of (5a) and (5b): G/ppm = 20.58 (CH,COO-), 20.69 (CH,COO-), 20.79 (CH,COO-), 20.82 (CH,COO-), 20.95 (CH,COO- and "Cll), 52.8 (CH,O), 66.5 (C35), 66.8, 67.4, 68.4 ((257, 68.6, 70.2, 71.6, 71.9, 97.8 (Cl'), 168.4, 169.3, 169.6, 169.7, 169.9, 170.0 and 170.3 (7 -CO-).

Mass spectrum (direct inlet): m/z ="lo02 (M+, 0.5%), *988

"822 (M+-3AcOH, 0.5%), 767 (ring C cleavage, 6%), "685 (cleavage of the glycosidic bond between C1' and oxygen atom at C35,2%), *671 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, 1 %), "669 (cleavage of the glycosidic bond between C35 and oxygen atom at C35, 8%), "655 (cleav- age of the glycosidic bond between C35 and oxygen atom at C35, 3 %), "383 (M+-side-chain, 9%), *369 (M+-side-chain, lo%), 317 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, loo%), 257 (317-AcOH, 28%), "205 (ring C cleavage, 43%), "191 (ring C cleavage, 21 %).

Methanolysis of acetylated methyl esters of glycosides (4a) and (4b) and synthesis of galacturonic acid derivatives (6) and (7). The hexa-acetates, methyl esters of (4a) and (4b) (4 mg) were treated for 16 h at 110°C with a 10% solution of HCI in CH,OH (1 ml). The reaction mixture was taken to dryness, acetylated and separated by TLC (cyclohexanelethyl ac- etate, 7 : 3, by vol.) to yield the tetra-acetoxybacteriohopane (R, = 0.22, 2 mg) and the acetylated galacturonic acid methyl glycosides (6) and (7) on the base-line. These were further sepa- rated by TLC (CHCl,, three migrations) giving acetylated meth- yl p-glycoside (6) (R, = 0.67, 0.8 mg) and acetylated methyl a- glycoside (7) (R, = 0.73, 0.8 mg) which were shown to be iden- tical ('H-NMR, "C-NMR, GLC, GLC/MS) with acetylated methyl glycosides obtained by identical treatment of galacturo- nic acid.

Methyl ester of acetylated methyl a-galacturonoside (6). 'H- NMR (250 MHz, CDC1,): G/ppm = 1.993 (3H, s, CH,COO-),

(Mi , 0.1 %), "942 (M+-AcOH, 1 %), "882 (M+-2AcOH, 0.5%),

2.061 (3H, S, CH,COO), 2.116 (3H, S, CH,COO-), 3.57 (3H, S, CH,O-), 3.77 (3H, S, CH,OCO-), 4.30 (lH, d, 54.5 = 1.3 Hz, 5- H), 4.43 (lH, d, J l . 2 = 7.9 Hz, 1-H), 5.08 (lH, dd, 52.3 ~ 1 0 . 4 Hz, J,,4 = 3.4 Hz, 3-H), 5.25 (ZH, dd, J2,, = 10.5 Hz, J,,2 = 7.9 Hz,

"C-NMR (65 MHz, CDCI,): G/ppm = 20.6 and 20.7 (3

(C2), 70.6 (C3), 72.4 (C5), 102.0 (Cl), 166.5, 169.4, 169.9 and

Mass spectrum (GLCNS): m/z = 348 (M', O.l%), 289

2-H), 5.70 (lH, dd, J,,4 = 3.3 Hz, J4.5 = 1.2 Hz, 4-H).

CH,COO-), 52.8 (CH3OCO-), 57.3 (CH,O-), 68.4 (C4), 68.5

170.1 (4 -CO-).

(M+-C02CH,, 1 %), 257 (289-CzH20, 0.5%), 229 (269-AcOH, lo%), 215 (257-C2Hz0, 2%), 186 (15%), 169 (229-AcOH, 40%), 127 (169-C,H,O, loo%), 103 (diacetyl, 31%) (Biemann et al., 1963).

Methyl ester of acetylated methyl a-galacturonoside (7). 'H-NMR (250MHz, CDCI,): G/ppm = 1.988 (3H, s, CH,COO-),

s, CH,O-), 3.75 (3H, s, CH,OCO-), 4.60 (IH, dd, = 1.4 Hz

3.6 Hz, JZ , , = 10.7 Hz, 2-H), 5.39 (lH, dd, J2,, = 10.7 Hz, J3.4 =

IT-NMR (65 MHz, CDCI,): G/ppm = 20.5, 20.6 and 20.7

(C2), 68.3 (C5), 69.2 (C4), 97.6 (Cl), 167.4, 169.8, 169.9 and

Mass spectrum (GLCNS): m/z = 289 (M+-C02CH,, 2%),

2.080 (3H, S, CH,COO-), 2.099 (3H, S, CH,COO-), 3.44 (3H,

5-H), 5.13 (IH, d, J , , = 10.6 Hz, I-H), 5.20 (IH, dd, Jj.2 =

3.4 Hz, 3-H), 5.76 (lH, dd, J3.4 = 3.4 Hz, 54.5 = 1.6 Hz, 4-H).

(3 CH,COO-), 52.7 (CH,OCO-), 56.2 (CH,O-), 67.1 (C3), 67.6

170.2 (4 -CO-).

257 (289-CZH20, l%), 229 (269-AcOH, 13%), 215 (257- C2H20, 4%), 169 (229-AcOH, 40%), 127 (169-CzH20, loo%), 103 (diacetyl, 34%) (Biemann et al., 1963).

Reduction of the acetylated methyl esters of (5a) and (5b). Synthesis of altropyranose derivatives (9) and (10). The

hexa-acetates, methyl esters of (5a) and (5b) (1 mg) were treated with LiAlH, (10 mg) in tetrahydrofuran (1 ml) under reflux for 3 h. After addition of methanol and evaporation of reagents, the residue was acetylated and separated by TLC (CHCI,) giving the hepta-acetates of (Sa) and (Sb) (R, = 0.37, 0.8 mg).

The tetra-acetates of methyl altropyranose (9) and (10) were synthesized from altrose as described above and separated by TLC (CHCI,, three migrations) into the tetra-acetates (9) and (10) of methyl a-glycoside (R, = 0.76) and methyl 8-glycoside (R, = 0.82) in a 1 : l ratio.

Hepta-acetates of bncteriohopanetetrol altrosides (8a) and (8b). 'H-NMR (250 MHz, CDCI,): G/ppm = 2.020 (3H, s, CH,COO-), 2.037 (3H, S, CH3COO-), 2.079 (3H, S, CH,COO-), 2.102 (6H, S, 2 CH,COO-), 2.129 (3H, S, CH,COO-), 2.139 (3H, S, CH,COO-), 3.64 (ZH, dd, J,,,?,, = 3.8 Hz, J,s,,?,, = 10.2 Hz, 35-H,), 3.81 (3H, dd, j34,35b = 6 Hz, J,s:t.35h = 10.2 Hz, 35-H,), 4.21 (m, 5'-H, 6'-H, et 6'-H,), 4.72 (lH, broad s, 1'-H), 4.93 (ZH, dd, J,,,, = 1, J2,,,, = 3.6 Hz, 2'-H), 5.03 (IH, m, 32-

m, 33-H, 34-H et 3'-H). Mass spectrum (direct inlet): m/z =O669 (cleavage of the

glycosidic bond between C35 and oxygen atom at C35, 3%), *655 (cleavage of the glycosidic bond between C35 and oxygen atom at C35, 2%), "383 (M+-side-chain, 2%), *369 (M+-side- chain, 1 %), 331 (cleavage of the glycosidic bond between Cl' and oxygen atom at C35, 66%), 271 (331-AcOH, 16%), 211 (271-AcOH, 16%), "205 (ring C cleavage, 25%), *191 (ring C cleavage, 15%), 169 (331 -2AcOH-CH2C0, 100%) (Biemann et al., 1963).

Acetylated methyl a-altroside (9). 'H-NMR (250 MHz, CDCI,): G/ppm = 2.023 (3H, s, CH,COO-), 2.110 (3H, s,

3.41 (3H, s, CH,O-), 4.18 (lH, m, 6-H,), 4.32 (2H, m, 6-H, and

H), 5.13 (lH, dd, J,,,c = 3.6 Hz, JC,s, = 9.6 Hz, 4'-H), 5.24 (3H,

CH,COO-), 2.116 (3H, S, CH,COO-), 2.142 (3H, S, CH,COO-),

5-H), 4.65 (IH, t, J1.z = 1.4 Hz, J1.3 = 1 Hz, 1-H), 4.94 (lH, dd, J1.2 = 1.4 Hz, J2.3 = 3.6 Hz, 2-H), 5.19 (lH, dd, 53.4 =3.6 Hz, J4.5 = 9.6Hz, 4-H), 5.25 (lH, td, J, , j = 1 Hz, J, , = J3.4 = 3.6 Hz, 3-H).

Mass spectrum (GLCNS): m/z = 362 (M+, O.l%), 331 (M+-OCH,, 4%), 289 (M+-CHZCOOCH,, 3%), 243 (M+- ~AcOH, 4%), 229 (289-AcOH, 2%), 200 (242-CHZC0, 35%), 169 ( ~ ~ I - ~ A c O H - C H ~ C O , 43%), 157 (80%), 140 (~OO-ACOH, 44%), 115 (loo%), 109 (169-AcOH, 30%), 98 (242- (CH,CO),O, 68%).

Acetylated methyl P-altroside (10). 'H-NMR (250 MHz, CDCI,): Nppm = 2.037 (3H, s, CH,COO-), 2.095 (3H, s,

3.51 (3H, s, CH,O-), 4.06 (lH, rn, 5-H), 4.28 (2H, m, 6-H), 4.85 CH,COO-), 2.106 (3H, S, CH,COO-), 2.153 (3H, S, CH,COO-),

(IH, d, Jl.2 12.1 Hz, I-H), 5.12 (lH, dd, Jl.2 = 2.1 Hz, J y = 5.8 Hz, 2-H), 5.21 (lH, dd, J,,4 = 3.4 Hz, J , , = 7.8 Hz, 4-H), 5.41 (IH, dd, J2.3 = 5.8 Hz, J3,4 = 3.4 Hz, 3-H).

"C-NMR (65 MHz, CDCI,): G/ppni = 20.7 (2 CH,COO-),

66.5 (Is), 66.7 (Is), 68.2 (Is), 71.4 (Is), 98.8 (Cl), 169.2, 169.7, 169.8 and 170.7 (4 -CO-).

Mass spectrum (GLC/MS): mlz = 362 (M+, O.l%), 331 (M+-OCH,, 2%), 289 (M'-CHzCOOCH,, 8%), 243 (M+-

20.8 (CH3COO-), 20.9 (CH,COO-), 57.2 (CHXO-), 63.1 (C5),

~AcOH, 4%), 229 (289-AcOH, 2%), 200 (242-CHZC0, 30%), 169 (331 -2AcOH-CHZCO,39%), 157 (60%), 140 (~OO-ACOH, 40%), 109 (169-AcOH, 22%), 98 (242-(CH,C0)20, 72%), 81 (1 00 %).

Reduction of penta-acetates of (3a) and (3b). The penta- acetates of the glycosides of the bacteriohopanetetrols (3a) and (3b) (1.1 mg) were reduced and acetylated as described for the acetylated methyl esters of (Sa) and (5b) to give the hexa-ace- tates of compounds ( l la) and ( l lb) (CHCI,, R, = 0.38, 0.8 mg).

868 Simonin et al. (Eur: J. Biochem. 241)

Galactose series

Altrose series

OAc

CH,OAc

(9)

OAc

(7)

OH OH OH OH OH

J"... e 0 .,,? 2 ,~.oH

22R OH o;:oH OH

61'OH

(8a) and (8b) (1la)and ( I lb)

Fig. 2. Synthetic derivatives obtained from galacturonic acid [(6) (7) and (13)], altropyranose [(9) and (lo)] and from the penta-acetates of hopanoids (3a) and (3b) [(l la) and (l lb)] and hopanoids (5) and (5b) [@a) and (Sb)].

Heptci-acetates of bacteriohopanetetrol gulactosides (l la) and ( l lb) . 'H-NMR (250 MHz, CDCI,): rS/ppm = 1.978 (3H, s, CH,COO-), 2.051 (3H, S, CH,COO-), 2.055 (3H, S, CH,COO-), 2.068 (9H, S , 3CH,COO-), 2.148 (3H, S, CH,COO-), 3.8 to 4.2 (SH, m. 5'-H, 6'-H and 35-H;,, 35-H,,), 4.50 (lH, d, =7.8 Hz, 1'-H), 5.00 (lH, m, 32-H), 5.01 (lH, dd, JZ..?, =10.4 Hz, J,,,. = 3.4 Hz, 3'-H), 5.14 (lH, dd, J,.,. = 7.8 Hz, = 10.2 Hz, 2'- H), 5.17 (2H, m, 33-H, 34-H), 5.38 (lH, dd, J2.,1. = 3.4Hz,

Mass spectruni (direct inlet): mlz = "671 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, l%), "669 (cleavage of the glycosidic bond between C3S and oxygen atom at C35, 5 % ) , "655 (cleavage of the glycosidic bond be- tween C35 and oxygen atom at C35, 2 %), "383 (M ' -side-chain, 3 %), "369 (M -side-chain, 1 %), 331 (cleavage of the glycosidic bond between C1' and oxygen atom at C35, loo%), "205 (ring C cleavage, 25%) , "191 (ring C cleavage, 12%), 169 (331- 2AcOH-CH2C0, 47%) (Biemann et a/., 1963).

The acelylated methyl ester of methyl p-galacturonoside (6) ( 5 mg) was treated identically i n order to obtain galactose deriv- ative (12) (CHCI;, R, = 0.38, 4 mg)

Acetylaterl methyl /I-galnctoside (12). 'H-NMR (250 MHz, CDCI,): cS/ppm = 1.983 (3H, s, CH,COO-), 2.053 (3H, s, CH,COO-), 2.063 (3H, s, CH,COO-), 2.152 (3H, s, CH,COO-), 3.52 (3H, s, CH,O-), 3.91 (IH, td, J , , = 0.9, J,,<,, = 6.5 Hz,

J , , , , = 0.8 Hz, 4'-H).

5-H), 4.13 (lH, dd, Jq.6, = 6.7 Hz, Je,c.i,', = 1 1 . 1 Hz, 6-HJ, 4.19 (IH, dd, J,,,, = 6.7 Hz, J6J.i,h = 11.2 Hz, 6-H,,), 4.40 (IH, d, J I . 2 17.9 Hz, I-H), 5.01 (IH, dd, J z , i = 10.4 Hz, J3.4 = 3.4 Hz, 3-H), 5.20 (IH, dd, Jl.2 = 7.8 Hz, J , , = 10.4 Hz, 2-H), 5.39 ( lH, dd, J,, = 3.4 Hz, J,,5 = 0.9 Hz, 4-H).

RESULTS

After HJOJNaBH, treatment of the crude bacterial extract, GLC and GLC/MS indicated the presence of diploptene and of the monoacetates of (22R)-trinorbacteriohopan-32-01 and of its

Table 1. Hopanoid content (dry mass basis) of whole cells and sub- cellular fractions from Prochlorothrix hollandica.

~

Fraction Diploptene (1) Trinorbacterio- hopan-32-01 acetate

Whole cells 15 Cell wall 90 Thylakoids 300

1 50 1000 2350

higher homologue possessing an additional methyl group in whole cells and subcellular fractions such as cell wall and thyla- koids (Table 1). Indeed GLC/MS showed two fragments corre- sponding to ring C cleavage at m/z 191 as well as 205 and al- lowed the additional methyl group on ring A or B of the hopane skeleton to be located. The two compounds coeluted in our GLC conditions : this excluded the structure of a 3/hethylhopanoid as such compounds have retention times much longer than those of non-methylated hopanoids and strongly suggested the pres- ence of a 2p-methyl group, Structure elucidation of the bacterio- hopanepolyols had therefore in each case to be performed on a mixture of two compounds which could not be separated either by TLC or by reverse-phase HPLC. NMR spectroscopy always showed in the methyl region the superposition of the spectra of the non-methylated hopanoids and of its methylated homologue and allowed the localization of the additional methyl group at C-2p by comparison of the methyl region of the 'H-NMR spectrum as well as the signals from hopane skeleton of the 'Y-NMR spectrum with those of synthetic 2p-methylhopanoids obtained either by synthesis (Stampf, 1992), or isolated from Nostoc species (Bisseret et al., 1985 ; Simonin, 1993). Structure elucidation of the side-chain was however not hampered by the presence of such mixtures of methyiated and noii-methylated compounds as the presence of the 2P-methyl group did not mod- ify the 'H- or the "C-NMR signals from the side-chain atoms.

Bacteriohopanetetrols (2a) and (2b). The tetra-acetates of the bacteriohopanetetrol (2a) and (2b) were identified by 'H-NMR spectroscopy by comparison with the similar tetra-acetate niix- ture isolated from Nostoc tnuscoriim. The (32R, 33R, 34s) con- figuration of the side-chain was determined by comparison of the 'H-NMR spectrum with those of the eight synthetic bacterio- hopanetetrol side-chain diastereoniers (Bisseret and Rohmer, 1989).

35-O-~-Galacturonosylbacteriohopanetetrols (4a) and (4b). The acetylated methyl esters of (4a) and (4b) were readily ob- tained after treatment with diazomethane, indicating the pres- ence of a carboxylic group. These derivatives were much easier to purify than hexa-acetates of (4a) and (4b) with a free carbox- ylate group.

The 'H-NMR spectrum showed six signals between 1.988- 2.110 ppm corresponding to six acetoxy groups and one singlet at 3.76 ppm characteristic of a methyl ester. With regard to other bacteriohopanetetrol glycosides, the two most shielded protons at 3.87 and 4.03 ppm corresponded to the (23.5 methylene group bearing the glycosyl residue (Simonin et al., 1992; Llopiz et al., 1992). Selective 'H/'H decoupling experiments allowed assign- ments of all the signals and identification of the structure of the side-chain. The large coupling constant of the anomeric proton

= 7 Hz) was consistent with the presence of a /I-glycosidic bond in an hexopyranose ring i n a chair conformation and with an equatorial acetoxy group at C-2'. Similarly, the coupling con-

Simonin et al. (Eur: J. Biochern. 241) 869

stant ( J = 10 Hz) between the 2'-H and 3'-H protons corres- ponded to a trans diaxial coupling. The value of the coupling constant between the protons 3'-H and 4'-H ( J = 3.4 Hz) ex- cluded a trans diaxial coupling. The 4'-H proton had therefore to be in equatorial position. The lower value of the coupling constant between the protons 4'-H and 5'-H (1.4 Hz) indicated a probable axial position for the 5'-H proton. According to the 'H-NMR spectroscopy, the side-chain of the hopanoids (4a) and (4b) contains a galacturonic acid moiety linked via a glycosidic bond to the C35 atom of the tetrol side-chain. The mass spectrum was in agreement with the proposed structure, showing a molecular ion at mlz = 1002, a ring C cleavage fragment bear- ing the side-chain at miz = 767, as well as the typical fragmenta- tions around the glycosidic bond at mlz = 685 and 317 (Renoux and Rohmer, 1985).

Methanolysis of the glycosidic bond of (4a) and (4b) in CH,OH/HCI followed by acetylation allowed the triterpenic part and the carbohydrate moiety to be identified separately. The con- figuration of the tetra-acetoxybacteriohopane obtained was (32R, 33R, 34s) as determined by 'H-NMR spectroscopy (Bisseret and Rohmer 1989). The two methyl a- and methyl P-galacturono- sides obtained after methanolysis of the hopanoids were iden- tical (TLC, GLC, GLClMS, 'H-NMR) with the corresponding reference derivatives prepared from galacturonic acid. This extensive structure determination of the tetrol galacturonoside from f? hollandica allowed later the easy identification by com- parison of spectroscopic data of the same hopanoid isolated from the cyanobacterium Synechococcus PCC 6907, but available in much smaller amounts (Llopiz et al., 1996).

35 - 0 -/I- 3,5 - Anhydrogalacturonopyranosylbacteriohopane - tetrols (3a) and (3b). The 'H-NMR spectrum of the acetylated glycosides (3a) and (3b) showed five signals between 2.067- 2.172 pprn corresponding to methyls of five acetoxy groups, and the signals corresponding to the 32-H, 33-H, 34-H and 35-H protons of the bacteriohopanetetrol moiety. 'H/'H decoupling ex- periments did not allow the assignment of the signals. However, the signal of the anomeric proton was identified as a singlet at 4.85 ppm by 'H/"C selective decoupling : indeed, the signal of the anomeric carbon could be easily located at 99.5 ppm. The structure of this new hopanoid could not be elucidated by 'H- NMR spectroscopy alone. There was no observable coupling be- tween the 1'-H and 2'-H protons and it was impossible to local- ize the 2'-H proton. However, the low values of all coupling constants excluded trans diaxial couplings. On the mass spectrum, the molecular ion at mlz = 928 and the fragmentations at mlz = 685 and 243 corresponding to the glycosidic bond cleavage revealed the presence of an additional ring indicating most probably a lactonization between the carboxylate and an hydroxy group.

In order to determine the spacial relationships of the protons, nuclear Overhauser effects were determined on the mixture of acetylated hopanoids (3a) and (3b) (Fig. 3). They revealed that the most likely structure for these hopanoids was that of a bac- teriohopanetetrol galacturonoside with a lactone ring in the ga- lacturonic acid moiety. On the 'H-NMR spectrum, the 5'-H pro- ton appeared as a triplet at 4.71 pprn, being coupled with the 4'- H proton (3J = 1.3 Hz) as well as with the 3'-H proton (45 = 1.4 Hz) indicating a planar W system consistent with the pro- posed structure.

The structures of the hopanoids (3a) and (3b) could be fully elucidated after LiAlH, reduction into 35-O-~-galactopyranosyl- bacteriohopanetetrols (lla) and (llb). These reduced hopanoids were acetylated and identified after acetylation by 'H-NMR spectroscopy, mass spectrometry and comparison with ace-

"\\

NOE

R = becteriohopanetetrol moiety

Fig. 3. Nuclear Overhauser Effects (NOE) observed by 'H-NMR on the penta-acetates of hopanoids (3a) and (3b).

tylated methyl P-galactopyranoside (12) which was synthesized from galacturonic acid.

All these results were in agreement with a 35-0-P-3,5-anhy- dro-galactopyranosylbacteriohopanetetrol. The configuration of the side-chain of the bacteriohopanetetrol moiety remained how- ever undetermined. This would require acid solvolysis in order to obtain the free tetrol and was not performed as too small amounts of hopanoid (3a) and (3b) were available. It was also not possible to correlate the tetrol galactoside obtained after re- duction of hopanoid (3a) and (3b) derivatives with those ob- tained after similar reduction of the derivatives of tetrol glyco- sides (4a) and (4b). LiAlH, reduction of the acetylated methyl esters of hopanoids (4a) and (4b) yielded as major products bacteriohopanetetrol glycosides arising most probably from the expected reduction of the methyl ester into a primary alcohol accompanied by an unexpected easy elimination of the C-4' sub- stituent which is in axial position in galactose derivatives.

35-0-a-Altruronopyranosylacteriohopanetetrols (5a) and (5b). As for hopanoids (4a) and (4b), the bacteriohopane deriva- tives (5a) and (5b) were isolated and identified as methyl esters. The 'H-NMR spectrum revealed the presence of six methyl sin- glets between 2.019-2.1 14 ppm corresponding to six acetoxy groups and one singlet at 3.79 ppm characteristic of the ester methoxy group. The chemical shift of the two C35 protons (3.80 ppm) indicated that the glycosyl residue was linked to the C35 oxygen atom. The full structure of the side-chain was deter- mined by selective decoupling experiments. Heteronuclear 'Hi "C selective decoupling performed on the doublets at 4.56 ppm and 4.87 ppm permitted to localize the anomeric proton signal at 4.87ppm. Indeed, the anomeric carbon was the most des- hielded of all tertiary carbon atoms of the side-chain and could be readily identified at 97.8 ppm. Assignment of the doublet at 4.87 ppm to 1'-H allowed thus assignment of the 4.56-ppm doublet to the 5'-H proton. All other proton signals could be identified by 'HPH selective decoupling experiments. Assuming a chair conformation for the hexopyranose moiety, the coupling constant (7.6 Hz) observed between the 4'-H and 5'-H protons corresponded to a trans diaxial coupling. The lower values of the coupling constant between the 3'-H and 4'-H protons ex- cluded a trans-diaxial coupling and suggested an equatorial posi- tion for the 3'-H proton. Similarly, the values of the coupling constants of the 1'-H and 2'-H proton signals (J1..2. = 3.3 Hz and JT,3, = 5.6 Hz) indicated that 1'-H and 2'-H were probably equa- torial. This implied that the linkage between the tetrol and the altruronic acid moiety was an a glycosidic bond. From 'H-NMR spectroscopy, a side-chain structure containing a glycosyl moiety

870 Simonin et al. (Eu,: J. Biochem. 241)

derived from altruronic acid in a chair conformation was the most likely one. The molecular ion at mlz = 1002, as well as the fragmentations at mlz = 685 and 317 characteristic of the glycosidic bond cleavage, observed on the mass spectrum, were in full agreement with the proposed structure. Final confirmation was obtained after LiAlH, reduction of the acetylated methyl esters of hopanoids (5a) and (5b) which afforded the altropyra- nosylbacteriohopanetetrols (8a) and (8b). They were identified after acetylation by 'H-NMR spectroscopy, by mass spectrome- try and by comparison with the two acetylated methyl altropyra- nosides (9) and (10) obtained from altrose after MeOH/HCl treatment and acetylation.

All data confirmed clearly a 35-0-a-altruronopyranosylbac- teriohopanetetrol structure for hopanoids (5a) and (5b). The side-chain configuration of the tetrol moiety could in this case also not be determined as too small amounts were available for the isolation of free bacteriohopanetetrol after acidic solvolysis.

DISCUSSION

Diploptene (1) was the only hopanoid reported from f? hol- landica some years ago (Volkman et al., 1988). As this C,, triter- penic hydrocarbon is nearly always present in trace amounts in hopanoid-producing bacteria, it was interesting to look for the C,, bacteriohopane derivatives which are always the major com- pounds. This search was successful as free bacteriohopanetetrol and three new bacteriohopanetetrol glycosides were found. All these hopanoids were indeed a mixture of the non-methylated hopanoid and of its 2P-methylated higher homologue in a 4:6 ratio according to NMR spectroscopy. Except for 2P-methyl- diplopterol found as major compound in all Methylobacterium species (Renoux and Rohmer 1985 ; Knani et al., 1994) and in Beijerinckiu indicu (Vilchkze et al., 1994), and 2P-methyl- bacteriohopanetetrol present in trace amounts in Methylobacter- ium organophilum (Renoux and Rohmer, 1985), the 2P-methyl- hopanoids have to date only been recorded as major compounds in significant amounts from cyanobacteria as shown by their iso- lation from two Nostoc strains (Bisseret et al., 1985; Simonin, 1993), 'Atlacystis montarza' (Henmann et al., 1996). Synechn- coccus PCC 6760 (Llopiz et al., 1996) and Gloeobacter sp. (Herrmann and Rohmer, unpublished results). The presence of 2P-methylhopanoids in f? hollundica is in accordance with the conclusion drawn from ultrastructural studies (Jiirgens and Speth, 1991), cell wall composition (Jurgens, 1989 ; Jurgens and Burger-Wiersma, 1989) and investigations on phylogenetic rela- tionships based on 16s ribosomal RNA gene sequence analysis (Nelissen et al., 1994, and references cited therein). Although possessing a photosynthetic machinery closely related to that of green algae and plants, f? hollandica is a typical prokaryote re- lated to the cyanobacteria.

The major hopanoids from F! hollundica each containing a glycuronic acid moiety could not be readily detected and puri- fied as long as their carboxylate group was free. Isolation of pure compounds with satisfactory yields required the formation of the methyl ester derivative, as already observed for the isola- tion of the bacteriohopanetetrol glucuronoside from Rhodospiril- lum rubrutn (Llopiz et al., 1992). Many different carbohydrate- derived moieties linked to bacteriohopanetetrol either by a gly- cosidic or by an ether bond have been identified in bacterial hopanoids (Rohmer, 1993). There is no satisfactory explanation for such a structural variability. All bacterial hopanoids, how- ever, satisfy the basic requirements for membrane stabilizers, i.e. a quasi-planar rigid ring system, an amphiphilic skeleton with an hydrophobic triterpenic skeleton and an hydrophilic side- chain and finally a length corresponding to about half the thick-

ness of a phospholipid bilayer (Demel and De Kruyff, 1976; Rohmer et al., 1979; Ourisson et al., 1987). Provided these basic features are met, many kinds of modifications in the side-chain as well as in triterpenic skeleton (methylation, introduction of double bonds) are probably allowed. One has just to remember the extraordinary diversity found for sterols, the eukaryotic membrane stabilizers, in algae, plants and above all in marine invertebrates (Djerassi et al., 1977; Djerassi and Silva, 1991). Intracellular localization of hopanoids has been determined for a few gram-negative bacteria. It is i n accordance with such a role i n membranes: they have been found in the plasma mem- brane and in the outer membrane of Zymomonas mobilis (Tdhara et al., 1988) as well as in the cell wall and the thylakoids of the cyanobacterium Synechocystis PCC 6907 (Jiirgens et al., 1992; Simonin et al., 1992). The high hopanoid concentrations found in the thylakoid and the cell-wall preparations obtained from F! hollandica (Table 1) represent additional proofs for this role as membrane stabilizers.

The absence of sterols, which could not be detected in this microorganism, has finally to be pointed out. With the exception of a very few species, sterols are absent in prokaryotes [Bouvier, 1978 (and references cited therein); Rohmer et al., 19791. The sterols found in another prochlorophyte, a Prochloron sp. living in symbiosis with the ascidian Lissoclinum patella (Perry et al., 1978) and unambiguously identified, arise therefore most proba- bly from the tunicate host.

The skilful technical assistance of D. Le Nouen and R. Graff (NMR spectroscopy) and P. Wehmng and M. C. Schweigert (mass spectrome- try) is gratefully acknowledged. This work was supported by the Centre National de la Recherche Scienfifique (Unit6 de Recherche Associe'e 135) and by the European Community generic project Biotedmtrlogy of Extremophile (contract BI02-CT-93-0274).

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