- Email: [email protected]
Heterocyst glycolipids of the cyanobacterium Cyanospira rippkae
Phytockmistry, Vol.33, No. 2, pp. 393-395, 1393 Printed in GreatBritain.
GO3i-9422193 $6.00 + 0.00 @I 1993 F’crgan~oi~ Press Ltd
~ET~ROCYST GLYCOLIPI~S OF THE CYA~O~A~TERI~M CYANOSPIRA RIPPKAE* A~NUNZ~ATASORIENTE,AGATAGAM~ACO~TA,~ANTONIO~R~CON~~ CLAUDIA SIU,$ MASSIMOV~NCENZIN~~ and GUIDE SODANO Dipartimento di Fisk, Universitx4 di Salerno, 84081 Baronissi (SA), Italy; j-Istituto per la Chimica MIB, CNR, Via Toiano 6,80072 Arco Felice @A), Italy; fCentro di Studio dei Microrganismi Autotrofi, CNR, Pide delle Cascine 27, 50144 Fireme, Italy (Received in revised form 21 September 19902)
Key Word Index-Cyarwspira chemistry.
rippkae; Cyanobacteria;
heterocyst
glycolipids;
akinetes;
stereo-
Abstract-The heterocyst glycolipids of the cyanobacterium Cyanospira rippkae have been isolated and their structures established to be I~O-~-~-~ucopyranosyl)-3R,27~-~a~~n~iol and i-(~-~-D-glucopyranosyl~Z?-keto3R-~ta~sanol by spectroscopic and chemical means. The absolute conjuration at the two stereogenie centres in the aglywne moiety of the former compound has been established ia a single step by Masher’s method on the trio1 derivative. The akinetes of C. rippkae contain the same glycolipids.
INTRODUCXTON The ‘heterocyst glycolipids’ are constituents specific [l, 21 to the envelope of heterocysts, specialized cells of N,fixing cyanobacteria in which nitrogen fixation takes place. Glywlipids and polysaccharide layers seem to play a role in limiting oxygen diffusion into the heterocyst [3], thus prot~ting the oxygen-s~sitive ~trogen~ system. Earlier studies on Anabaena cytindrica r4, S] resulted in a partial characterization of the heterocyst glycolipids, while only recently heterocyst glycolipids have been isolated in a pure form from the marine ~yanoba~erium ~o~u~~ia ~~ueyana and their structures established to be l-(0-a-D-glucopyranosyi)-3R,25R-hexacosanediol (l), l-(O-a-D-glucopyranosyl)-3S,2SR-hexacosanediol(2) and l~~~-~~uwp~n~yl~3-keto-2SR-hex~~ol (3) [SJ. We report here the isolation and str~ture elucidation of the heterocyst glycolipids of the cyanobacterium Cyunospira rippkae f73. REsULm AND DISCUSSION Cyamspira rippkue filaments consist of vegetative cells and heterocysts, the latter representing 5.5-6.2% of the whole fitament. Since it is well d~umented that heterocyst glywlipids are specific to heterocysts and are absent in vegetative cells [2, 81, no attempts were made to separate heterocysts from vegetative cells. TLC examination of the chlorofo~-ho-propanol extract of whole C. rippkae freezedried filaments showed two bands in the R, range characteristic [S, 61 of heterocyst glycolipids
*Part 2 in the series ‘The Heterocyst Gly~li~~ Cyanobacteria’. For part 1 see ref. [6].
of
393
and the components were separated by a combination of chromatographic techniques. The major component (4) had ‘H and 13C NMR spectra identical to those of 1[6]. The FAB mass spectrum showed a quasi-EM] ’ at mJz 605 suggesting that 4 is a higher bomologue of 1 having two additional methylene units. Accordingly, the trio1 Ii obtained by acid hydrolysis of 4 showed ‘H and i3C NMR spectra su~rim~sabIe on those of the trio1 6, derived from 1,and CMH] + in the FAB mass spectrum 28 mu higher than that of 6. The absolute configuration at C3 in 4 should be R, since the proximity of the glucose moiety makes the ‘EI and l 3C chemical shift values of the relevant protons and carbons diagnostically different from those of the 3S-epimer [6]. Furthermore, the tris-pBr-benzoate, 7, exhibited typical [9] exciton split CD Cotton effects, A = - 14.9, which was pre~ously found also for the tris-p-Br-benzoate derivative, 8, [6). As far as the stereochemistry at C-25 is concerned, in the previous paper [6] we determined the stereochemistry at that centre in 1 and 2 by Masher’s method [lo] on the 1,3acetonides of the corresponding triols. In order to simplify the procedure, we checked the possibility of inferring both the absolute configurations at C-3 and at the w-l carbon by Mosher’s method. Thus, both the t&-(R)-(9) and t&(S)-MTPA (10) esters were prepared from the trio1 5 and analysed by ‘H NMR spectrometry. The C-27 methyl group exhibited a negative A&--0.08; 8,,,n,, - BtaS)MTPA) thus confirming the R absolute configuration at C-27. On the other hand, the C-2 protons exhibited a positive A&(f0.05) value which is consistent with the R absolute configuration at C-3 independently inferred by the NMR and CD data on 4 and 7, respectively. Thus, it seems that the simui~eo~ presence of MTPA ester groups at C-l and C-3 does not affect the expected Aa
394
A. SORIENTE
et al.
l:n-21 4:n=23
3
Br-ppG6H4CO-Z,
5:n-23 6:n-21
Q-COW.eBr :
:
E?r-pC6H4C0-0/
7:n-23 8:n=tl
MTPh-(S)-Ov
i
MTPA-(R)-O/\/2c-y\
n
MTPA-(S)-$I
MTPA-(R)-$l
y(R)-WPA 23
9
I)-(S)-MTPA 23
10
value of the C-2 protons. For confi~ation, we examined the MTPA derivatives of 3R- and 3S-1,3-butanediol and observed A6 values of the expected sign for both the C-2 and the methyl protons (see Experimental). Therefore, we propose that the above simp~fied procedure could be utilized for the determination of the absolute configuration of such polyols having a 1,3-diol moiety. The less polar compound (11) had a [MH]+ in the FAB mass spectrum at m/z 603, that is 2 mu iess than that of 4. The ‘HNMR spectrum of 11 was similar to that of 4; moreover, it lacked the C-27 proton signal and showed a methyl singlet at 62.14 and a methylene triplet at ~52.43which were considered to flank a carbonyl group replacing the carbynol moiety at C-27 in 4. Accordingly, in the 13CNMR spectrum a ketone carbonyl carbon at 6208.3 was present. Comparison of this spectrum with those of 4 and of 2-octanone allowed the assignment of all the resonances. Accordingly, compound 11 is l-(O-a-D-glu~pyranosyl)-27-keto-3R-~~cosanol. Confirmation for this structural assignment came from NaBH, reduction of 11 which afforded a product, maybe as a mixture of epimers at C-27, having TLC and ‘HNMR behaviour indistinguishable from that of 4. Akinetes, spore-like reproductive structures, of C. rippkae were also examined for their content of glycolipids and the extract revealed the presence of the same compounds, 4 and 11. This result was surprising because
akinetes possess mo~holo~~l and functional properties quite distinct from both heterocysts and vegetative cells and are characterized by a multilayered cell envelope thicker than that of vegetative cells. In this connection, it is worth mentioning that comparative chemical analyses [ill of vegetative cells, heterocysts and akinetes of A. cylindrica led to the suggestion that there was a close relationship between akinetes and heterocysts, the polysaceharide of the envelope of these differentiate cells being identical in repeating units and glycosidic bonds. The similar glycolipid composition shown by heterocysts and akinetes of C. rippkae seems to strengthen this suggestion. It should be noted that the heterocyst glycolipid composition of C. rippkue differs only slightly from that of N. harveyam In C: rippkae the major glycolipid fraction consists of only one epimer at C-3 and the chain is C-28 instead of C-26, while the minor one is again a hydroxyketone in which the keto function is at the w-l position of the side-chain instead of at C-3.
General. Mps: uncorr. “HNMR in pyridine-d, or CDCl, at 500 MHz with the downfield signal of pyridine (68.80) or CHCl, (67.26) as int. standards; 13C NMR in pyridine-d, at 62 MHz with the downfield signal of pyridine as int. standard (~150.0). Optical rotations in CHCl,-MeOH (2: 1). CD and IJV spectra in n-hexane.
Heterocyst glycolipids of Cyanospira rippkae
FAB-MS in glycerol matrix. CC: Sephadex LH-20 (70 x 3 cm; Pharmacia) and Lobar RP-8 (Merck). Prep. TLC: precoated silica gel 60 (0.5 mm; Merck). Organism and culture conditions.
Cyanospira
rippkae
Mag II 702 ATCC 43194=SAG 1.90, a heterocystous cyanobacterium [fl, was grown in batch mode under continuous light at 30” on the alkaline mineral medium previously described [12]. Trichomes were harvested by centrifugation when the culture entered the stationary phase of growth, washed twice with 0.2 M NaCl and freeze-dried. Akinetes were obtained from an old culture after several centrifugations at low speed, which reduced heterocysts and vegetative cells to less than 1% of the total cells, washed twice with sterile Hz0 and dried at 40”. Isolation of glycolipids. In a typical isolation procedure, 9.5 g of lyophilized cells were extracted with 0.41 (0.1 x 4) of CHCl,-iso-PrOH (1: 1) for 6 days and the soln evapd in oacuo. The extract (0.87 g) was dissolved in CHCl,-MeOH (2:l) and passed through a Sephadex LH-20 column eluting with MeOHCHCl, (9: 1). Frs containing glycolipids were pooled (0.55 g), dissolved in CHCl,-MeOH (2: 1) and passed through a RP-8 column eluting with MeOHCHCl, (9: 1). Frs containing glycolipids were dissolved in CHCl,-MeGH (2 : 1) and subjected to prep. TLC on five silica gel plates to afford 2.1 mg of 11 and 21 mg of 4. Analogously, from 0.95 g of lyophilized akinetes ca 0.2 mg of 11 and 4.4 mg of 4 were obtained. 1-(0-a-D-glucopyrunosyl)-3R,27R-Octacosunediol
(4).
Amorphous powder, mp 118-120”. [a], +44.3 (CHCl,-MeOH, 2: 1; c 0.4). FAB-MS m/z 605 [M + HI+, 443 (cleavage of the glucoside bond). ‘H NMR (pyridined,: 65.49 (d, J=3.7Hz, H-l’), 4.72 (t, J=9.1 Hz, H-3’), 4.59 (dd, J = 4.9 and 13.8 Hz, H&a), 4.48 (dd, J=4.9 and 13.8 Hz, H-5’ + H-B’b), 4.40 (dt, J = 6.2 and 9.8 Hz, H-la), 4.33(t,J=8.9Hz,H-4’),4.25(dd,J=3.8and9.6Hz,H-2’), 4.19 (m, H-3), 4.11 (m, H-lb+H-27), 2.07 (m, H-2), 1.45 (d, J = 6.2 Hz, Me-27), 1.42, 1.38,1.36 (methylene chain). i3C NMR pyridine-d,: 6100.7 (C-l’), 75.7 (C-3’), 74.5 (C-S), 74.0 (C-2’), 72.4 (C-4’), 68.6 (C-3), 67.2 (C-27), 66.2 (C-l), 63.0 (C-6’), 40.4 (C-26), 38.8 (C-4), 38.2 (C-2), 30.4, 30.2 (methylene chain), 26.6 and 26.5 (C-5 and C-25), 24.5 (G 28). Acid hydrolysis of compound 4. Compound 4 (5.6 mg) was dissolved in 1 M H,SO, (MeOH-H,O, 9: 1, 2 ml) and refluxed for 22 hr. After cooling, 2 ml of Hz0 was added, the MeOH removed in uucuo and the resulting suspension extracted with CHCl, (3 x 3 ml) to afford, after removal of solvent, 3.7 mg of $ pure by TLC. 1,3R,25R-Octucosanetriol(5). Amorphous powder. [a], + 8.1 (CHCl,-MeOH, 2: 1; ~0.37). FABMS, m/z 443 [M +H]+. ‘H NMR (pyridine-d,): 66.18 (m, OH-l), 5.98 (d, J =4.4 Hz, OH-3), 5.86 (d, J=4.3 Hz, OH-27), 4.32 (m, H-l +H-3), 4.11 (m, H-27), 2.14 (m, H-2), 1.45 (d, J=6.1 Hz, Me-27), 1.38 (methylene chain). 13C NMR (pyridine-d,): 669.8 (C-3), 67.2 (C-27), 60.5 (C-l), 41.3 (C-2), 40.4 (C-26X 38.9 (C-4), 30.3 (methylene chain), 26.6 and 26.5 (C-5 and C-25), 24.5 (C-28). Tris-(p-bromobenzoute) 7. To 2.5 mg of trio1 5 dissolved in dry pyridine (0.1 ml), 4-dimethylaminopyridine (ca
395
1 mg) and p-bromobenzoyl chloride (5 mg) were added and the mixt. heated at 50” in a capped vial for 16 hr. After cooling, MeGH (0.1 ml) was added and, after an additional 30 min, solvents were removed with a N, stream. The product was dissolved in CHCl, and chromatographed on a prep. silica gel TLC plate (n- hexaneEtOAc, 9: 1) and recovered from the plate with CHCl, to afford the tris-(p-bromobenzoate) 7 (1.8 mg). FAB-MS: no molecular ion. UV 1Kiy nm: 243.5 (~48000). CD 1Ey 252.1 run, As -9.4; A’&- 239.5 nm, AE + 5.5; A = - 14.9. ‘H NMR (CDCI,): 67.86 (m, 6H), 7.57 (m, 6H), 5.32 (m, lH), 5.14 (m, lH), 4.40 (m, 2H), 2.15 (m, 2H), 1.32 (d, J = 6.5 Hz, 3H), 1.24 (methylene chain). MTPA esters 9 and 10.To 1.5 mg of trio1 5, dissolved in dry pyridine (0.1 ml), were added 1 mg of rl-dimethylaminopyridine and an excess of S-(+)-a-methoxy-atrifluoromethylphenylacetyl chloride [S-( +)-MTPA-Cl; Fluka] and the mixt. heated for 24 hr at 50”. Spermine was added in order to destroy excess unreacted acyl chloride, pyridine removed with a N, stream and the residue dissolved in CHCI, and chromatographed on a silica gel TLC plate (n-hexane-EtOAc, 9: 1) to afford the R-MTPA triester 9. FAB-MS m/z 1091 [M+H]+, 623 [MH-ZMTPA]+. ‘HNMR (CDCI,): 67.50 (111,6H), 7.41 (m, 9H), 5.13 (m, H-3 + H-27), 4.35 and 4.24 (ms, H-la and H-lb), 3.55 (s, -Me), 3.54 (s, -GMe), 3.52 (s, -Me), 2.01 (m, H-2), 1.25 (methylene chain + 27-Me). Analogously, reaction of 1.5 mg of 5 with R-(-)-MTPA-Cl afforded the S-MTPA triester 10.FAB-MS m/z 1091 [M +H]+, 623 [MH-2MTPA]+. ‘HNMR G(CDC1,): 7.50 (m, 6H), 7.38 (m, 9H), 5.13 and 5.07 (ms, H-3 and H-27), 4.20 and 4.11 (ms, H-la and H-lb), 3.56 (s, -GMe), 3.54 (s, -GMe), 3.53 (s, -GMe), 1.96 (m, H-2), 1.33 (d, J=6.2 Hz, 27-Me), 1.25 (methylene chain). MTPA esters of (3R)- and (3S)-1,3-butanediol. The RMTPA and S-MTPA diesters of (3R)- and (3S)-1,3butanediol were prepd following the above procedure. The relevant ‘HNMR chemical shift values (CDCl,) were: (3R)-1,3-butanediol R-MTPA diester: 2.01 (H-2), 1.27 (Me-3); (3R)-1,3-butanediol S-MTPA diester: 1.97 (H-2), 1.35 (Me-3). (3S)-1,3-butanediol R-MTPA diesten 1.97 (H-2), 1.35 (Me-3k(3S)-1,3-butanediol S-MTPA diesten 2.0 (H-2), 1.26 (Me-3). l~Gu-~hrcopymnosy~)-n-1Yebo-3R-ocbacosal (11). Oil. FAB-MS m/z 603 [M +H]+, 441 (cleavage of glucoside bond). ‘HNMR 6 (pyridine-d,k 5.48 (d, J=3.7 Hz, H-l’), 4.71 (t, J =9.1 Hz, H-3’), 4.60 (dd, J=4.9 and 13.9 Hz, H&‘a), 4.49 (dd, J=4.9 and 13.9 Hz, H-5’+H6’b), 4.39 (dt, J=6.2 and 9.8 Hz, H-la), 4.32 (t, J=8.9 Hz, H-4’), 4.25 (dd, J = 3.8 and 9.6 Hz, H-2’), 4.19 (m, H-3), 4.11 (dt, J=6.8 and 9.8 Hz, H-lb), 2.43 (t, J=7.4H& H-26), 2.14 (s, Me-27), 2.07 (m, H-2), 1.38, 1.34, 1.31 (methylene chain). 13C NMR pyridine-d,: 6 208.3 (C-27), 100.7 (C-l’), 75.7 (C-3’), 74.5 (C-5’), 74.1 (C-2’), 72.3 (C-4’), 68.7 (C-3), 66.2 (C-l), 63.0 (C-6’), 43.6 (C-26), 38.8 (C-4), 38.2 (C-2), 30.1 (methylene chain), 29.8 and 29.5 (C-28 and C-24), 26.4 (C-5), 24.2 (C-25). 2-Octanone. 13C NMR pyridine -d,: 6207.8 (C-2), 43.7 (C-3), 32.1 (C-6), 29.7 (C-l), 29.1 (C-5), 24.2 (C-4), 22.9 (C7), 14.3 (C-8).
396
A. SRIENTE
NaBH, reduction of compound 11. To 1.8 mg of 11, dissolved in MeOH (0.5ml), an excess of NaBH, was added and the mixt. stirred at room temp. for 15 min. HOAc (5 ~1) was added and the mixt. chromatographed on a prep. TLC plate (CHCl,-MeOH, 4: 1) from which the product was recovered with CHCl,-MeOH (2: 1). The product obtained was indistinguishable from 4 by TLC and ‘H NMR. Acknowledgements-The 500 MHz NMR and mass spectra were obtained from the ICMIB-NMR Service and from the “Servizio di Spettrometria di Massa de1 CNR e dell’Universid di Napoli, respectively; the staff of both are gratefully acknowledged. This work was partially supported by Ministero dell’Universitl e della Ricerca Scientifica. REFERENCES
Nichols, B. W. and Wood, B. J. B. (1%8) Nature 217, 767. 2. Walsby, A. E. and Nichols, B. W. (1969) Nature 221, 673. 1.
et al.
3. Murry, M. A. and Wolk, C. P. (1989) Arch. Microbial. 151,469.
4. Bryce, T. A., Welti, D., Walsby, A. E. and Nichols, B. W. (1972) Phytochemistry 11,295. 5. Lambein, F. and Wolk, C. P. (1973) Biochemistry 12, 791. A. and 6. Soriente, A., Sodano, G., Gambacorta, Trincone A. (1992) Tetrahedron 48, 5375. 7. Florenzano, G., Sili, C., Pelosi, E. and Vincenzini, M. (1985) Arch. Microbial. 140,301. 8. Larch, S. K. and Wolk, C. P. (1974) J. Phycol. 10,352. 9. Harada, N., Saito, A., Ono, H., Gawronski, J., Gawronska, K., Sagioka, T., Uda, H. and Kurki, T. (1991) J. Am. Gem. Sot. 113,3842. 10. Dale, J. A. and Mosher, H. S. (1973) J. Am. Gem. Sot. 95, 512. 11. Cardemil, L. and Wolk, C. P. (1979) J. Biol. Chem. 254, 736. 12. Vincenzini, M., Sili, C., Tredici, M. R. and Materassi, R. (1989) in Nitrogen Fixation with Non-legumes (Skinner, F. A. et al., eds), p. 71. Kluwer Academic Publishers.
GO3i-9422193 $6.00 + 0.00 @I 1993 F’crgan~oi~ Press Ltd
~ET~ROCYST GLYCOLIPI~S OF THE CYA~O~A~TERI~M CYANOSPIRA RIPPKAE* A~NUNZ~ATASORIENTE,AGATAGAM~ACO~TA,~ANTONIO~R~CON~~ CLAUDIA SIU,$ MASSIMOV~NCENZIN~~ and GUIDE SODANO Dipartimento di Fisk, Universitx4 di Salerno, 84081 Baronissi (SA), Italy; j-Istituto per la Chimica MIB, CNR, Via Toiano 6,80072 Arco Felice @A), Italy; fCentro di Studio dei Microrganismi Autotrofi, CNR, Pide delle Cascine 27, 50144 Fireme, Italy (Received in revised form 21 September 19902)
Key Word Index-Cyarwspira chemistry.
rippkae; Cyanobacteria;
heterocyst
glycolipids;
akinetes;
stereo-
Abstract-The heterocyst glycolipids of the cyanobacterium Cyanospira rippkae have been isolated and their structures established to be I~O-~-~-~ucopyranosyl)-3R,27~-~a~~n~iol and i-(~-~-D-glucopyranosyl~Z?-keto3R-~ta~sanol by spectroscopic and chemical means. The absolute conjuration at the two stereogenie centres in the aglywne moiety of the former compound has been established ia a single step by Masher’s method on the trio1 derivative. The akinetes of C. rippkae contain the same glycolipids.
INTRODUCXTON The ‘heterocyst glycolipids’ are constituents specific [l, 21 to the envelope of heterocysts, specialized cells of N,fixing cyanobacteria in which nitrogen fixation takes place. Glywlipids and polysaccharide layers seem to play a role in limiting oxygen diffusion into the heterocyst [3], thus prot~ting the oxygen-s~sitive ~trogen~ system. Earlier studies on Anabaena cytindrica r4, S] resulted in a partial characterization of the heterocyst glycolipids, while only recently heterocyst glycolipids have been isolated in a pure form from the marine ~yanoba~erium ~o~u~~ia ~~ueyana and their structures established to be l-(0-a-D-glucopyranosyi)-3R,25R-hexacosanediol (l), l-(O-a-D-glucopyranosyl)-3S,2SR-hexacosanediol(2) and l~~~-~~uwp~n~yl~3-keto-2SR-hex~~ol (3) [SJ. We report here the isolation and str~ture elucidation of the heterocyst glycolipids of the cyanobacterium Cyunospira rippkae f73. REsULm AND DISCUSSION Cyamspira rippkue filaments consist of vegetative cells and heterocysts, the latter representing 5.5-6.2% of the whole fitament. Since it is well d~umented that heterocyst glywlipids are specific to heterocysts and are absent in vegetative cells [2, 81, no attempts were made to separate heterocysts from vegetative cells. TLC examination of the chlorofo~-ho-propanol extract of whole C. rippkae freezedried filaments showed two bands in the R, range characteristic [S, 61 of heterocyst glycolipids
*Part 2 in the series ‘The Heterocyst Gly~li~~ Cyanobacteria’. For part 1 see ref. [6].
of
393
and the components were separated by a combination of chromatographic techniques. The major component (4) had ‘H and 13C NMR spectra identical to those of 1[6]. The FAB mass spectrum showed a quasi-EM] ’ at mJz 605 suggesting that 4 is a higher bomologue of 1 having two additional methylene units. Accordingly, the trio1 Ii obtained by acid hydrolysis of 4 showed ‘H and i3C NMR spectra su~rim~sabIe on those of the trio1 6, derived from 1,and CMH] + in the FAB mass spectrum 28 mu higher than that of 6. The absolute configuration at C3 in 4 should be R, since the proximity of the glucose moiety makes the ‘EI and l 3C chemical shift values of the relevant protons and carbons diagnostically different from those of the 3S-epimer [6]. Furthermore, the tris-pBr-benzoate, 7, exhibited typical [9] exciton split CD Cotton effects, A = - 14.9, which was pre~ously found also for the tris-p-Br-benzoate derivative, 8, [6). As far as the stereochemistry at C-25 is concerned, in the previous paper [6] we determined the stereochemistry at that centre in 1 and 2 by Masher’s method [lo] on the 1,3acetonides of the corresponding triols. In order to simplify the procedure, we checked the possibility of inferring both the absolute configurations at C-3 and at the w-l carbon by Mosher’s method. Thus, both the t&-(R)-(9) and t&(S)-MTPA (10) esters were prepared from the trio1 5 and analysed by ‘H NMR spectrometry. The C-27 methyl group exhibited a negative A&--0.08; 8,,,n,, - BtaS)MTPA) thus confirming the R absolute configuration at C-27. On the other hand, the C-2 protons exhibited a positive A&(f0.05) value which is consistent with the R absolute configuration at C-3 independently inferred by the NMR and CD data on 4 and 7, respectively. Thus, it seems that the simui~eo~ presence of MTPA ester groups at C-l and C-3 does not affect the expected Aa
394
A. SORIENTE
et al.
l:n-21 4:n=23
3
Br-ppG6H4CO-Z,
5:n-23 6:n-21
Q-COW.eBr :
:
E?r-pC6H4C0-0/
7:n-23 8:n=tl
MTPh-(S)-Ov
i
MTPA-(R)-O/\/2c-y\
n
MTPA-(S)-$I
MTPA-(R)-$l
y(R)-WPA 23
9
I)-(S)-MTPA 23
10
value of the C-2 protons. For confi~ation, we examined the MTPA derivatives of 3R- and 3S-1,3-butanediol and observed A6 values of the expected sign for both the C-2 and the methyl protons (see Experimental). Therefore, we propose that the above simp~fied procedure could be utilized for the determination of the absolute configuration of such polyols having a 1,3-diol moiety. The less polar compound (11) had a [MH]+ in the FAB mass spectrum at m/z 603, that is 2 mu iess than that of 4. The ‘HNMR spectrum of 11 was similar to that of 4; moreover, it lacked the C-27 proton signal and showed a methyl singlet at 62.14 and a methylene triplet at ~52.43which were considered to flank a carbonyl group replacing the carbynol moiety at C-27 in 4. Accordingly, in the 13CNMR spectrum a ketone carbonyl carbon at 6208.3 was present. Comparison of this spectrum with those of 4 and of 2-octanone allowed the assignment of all the resonances. Accordingly, compound 11 is l-(O-a-D-glu~pyranosyl)-27-keto-3R-~~cosanol. Confirmation for this structural assignment came from NaBH, reduction of 11 which afforded a product, maybe as a mixture of epimers at C-27, having TLC and ‘HNMR behaviour indistinguishable from that of 4. Akinetes, spore-like reproductive structures, of C. rippkae were also examined for their content of glycolipids and the extract revealed the presence of the same compounds, 4 and 11. This result was surprising because
akinetes possess mo~holo~~l and functional properties quite distinct from both heterocysts and vegetative cells and are characterized by a multilayered cell envelope thicker than that of vegetative cells. In this connection, it is worth mentioning that comparative chemical analyses [ill of vegetative cells, heterocysts and akinetes of A. cylindrica led to the suggestion that there was a close relationship between akinetes and heterocysts, the polysaceharide of the envelope of these differentiate cells being identical in repeating units and glycosidic bonds. The similar glycolipid composition shown by heterocysts and akinetes of C. rippkae seems to strengthen this suggestion. It should be noted that the heterocyst glycolipid composition of C. rippkue differs only slightly from that of N. harveyam In C: rippkae the major glycolipid fraction consists of only one epimer at C-3 and the chain is C-28 instead of C-26, while the minor one is again a hydroxyketone in which the keto function is at the w-l position of the side-chain instead of at C-3.
General. Mps: uncorr. “HNMR in pyridine-d, or CDCl, at 500 MHz with the downfield signal of pyridine (68.80) or CHCl, (67.26) as int. standards; 13C NMR in pyridine-d, at 62 MHz with the downfield signal of pyridine as int. standard (~150.0). Optical rotations in CHCl,-MeOH (2: 1). CD and IJV spectra in n-hexane.
Heterocyst glycolipids of Cyanospira rippkae
FAB-MS in glycerol matrix. CC: Sephadex LH-20 (70 x 3 cm; Pharmacia) and Lobar RP-8 (Merck). Prep. TLC: precoated silica gel 60 (0.5 mm; Merck). Organism and culture conditions.
Cyanospira
rippkae
Mag II 702 ATCC 43194=SAG 1.90, a heterocystous cyanobacterium [fl, was grown in batch mode under continuous light at 30” on the alkaline mineral medium previously described [12]. Trichomes were harvested by centrifugation when the culture entered the stationary phase of growth, washed twice with 0.2 M NaCl and freeze-dried. Akinetes were obtained from an old culture after several centrifugations at low speed, which reduced heterocysts and vegetative cells to less than 1% of the total cells, washed twice with sterile Hz0 and dried at 40”. Isolation of glycolipids. In a typical isolation procedure, 9.5 g of lyophilized cells were extracted with 0.41 (0.1 x 4) of CHCl,-iso-PrOH (1: 1) for 6 days and the soln evapd in oacuo. The extract (0.87 g) was dissolved in CHCl,-MeOH (2:l) and passed through a Sephadex LH-20 column eluting with MeOHCHCl, (9: 1). Frs containing glycolipids were pooled (0.55 g), dissolved in CHCl,-MeOH (2: 1) and passed through a RP-8 column eluting with MeOHCHCl, (9: 1). Frs containing glycolipids were dissolved in CHCl,-MeGH (2 : 1) and subjected to prep. TLC on five silica gel plates to afford 2.1 mg of 11 and 21 mg of 4. Analogously, from 0.95 g of lyophilized akinetes ca 0.2 mg of 11 and 4.4 mg of 4 were obtained. 1-(0-a-D-glucopyrunosyl)-3R,27R-Octacosunediol
(4).
Amorphous powder, mp 118-120”. [a], +44.3 (CHCl,-MeOH, 2: 1; c 0.4). FAB-MS m/z 605 [M + HI+, 443 (cleavage of the glucoside bond). ‘H NMR (pyridined,: 65.49 (d, J=3.7Hz, H-l’), 4.72 (t, J=9.1 Hz, H-3’), 4.59 (dd, J = 4.9 and 13.8 Hz, H&a), 4.48 (dd, J=4.9 and 13.8 Hz, H-5’ + H-B’b), 4.40 (dt, J = 6.2 and 9.8 Hz, H-la), 4.33(t,J=8.9Hz,H-4’),4.25(dd,J=3.8and9.6Hz,H-2’), 4.19 (m, H-3), 4.11 (m, H-lb+H-27), 2.07 (m, H-2), 1.45 (d, J = 6.2 Hz, Me-27), 1.42, 1.38,1.36 (methylene chain). i3C NMR pyridine-d,: 6100.7 (C-l’), 75.7 (C-3’), 74.5 (C-S), 74.0 (C-2’), 72.4 (C-4’), 68.6 (C-3), 67.2 (C-27), 66.2 (C-l), 63.0 (C-6’), 40.4 (C-26), 38.8 (C-4), 38.2 (C-2), 30.4, 30.2 (methylene chain), 26.6 and 26.5 (C-5 and C-25), 24.5 (G 28). Acid hydrolysis of compound 4. Compound 4 (5.6 mg) was dissolved in 1 M H,SO, (MeOH-H,O, 9: 1, 2 ml) and refluxed for 22 hr. After cooling, 2 ml of Hz0 was added, the MeOH removed in uucuo and the resulting suspension extracted with CHCl, (3 x 3 ml) to afford, after removal of solvent, 3.7 mg of $ pure by TLC. 1,3R,25R-Octucosanetriol(5). Amorphous powder. [a], + 8.1 (CHCl,-MeOH, 2: 1; ~0.37). FABMS, m/z 443 [M +H]+. ‘H NMR (pyridine-d,): 66.18 (m, OH-l), 5.98 (d, J =4.4 Hz, OH-3), 5.86 (d, J=4.3 Hz, OH-27), 4.32 (m, H-l +H-3), 4.11 (m, H-27), 2.14 (m, H-2), 1.45 (d, J=6.1 Hz, Me-27), 1.38 (methylene chain). 13C NMR (pyridine-d,): 669.8 (C-3), 67.2 (C-27), 60.5 (C-l), 41.3 (C-2), 40.4 (C-26X 38.9 (C-4), 30.3 (methylene chain), 26.6 and 26.5 (C-5 and C-25), 24.5 (C-28). Tris-(p-bromobenzoute) 7. To 2.5 mg of trio1 5 dissolved in dry pyridine (0.1 ml), 4-dimethylaminopyridine (ca
395
1 mg) and p-bromobenzoyl chloride (5 mg) were added and the mixt. heated at 50” in a capped vial for 16 hr. After cooling, MeGH (0.1 ml) was added and, after an additional 30 min, solvents were removed with a N, stream. The product was dissolved in CHCl, and chromatographed on a prep. silica gel TLC plate (n- hexaneEtOAc, 9: 1) and recovered from the plate with CHCl, to afford the tris-(p-bromobenzoate) 7 (1.8 mg). FAB-MS: no molecular ion. UV 1Kiy nm: 243.5 (~48000). CD 1Ey 252.1 run, As -9.4; A’&- 239.5 nm, AE + 5.5; A = - 14.9. ‘H NMR (CDCI,): 67.86 (m, 6H), 7.57 (m, 6H), 5.32 (m, lH), 5.14 (m, lH), 4.40 (m, 2H), 2.15 (m, 2H), 1.32 (d, J = 6.5 Hz, 3H), 1.24 (methylene chain). MTPA esters 9 and 10.To 1.5 mg of trio1 5, dissolved in dry pyridine (0.1 ml), were added 1 mg of rl-dimethylaminopyridine and an excess of S-(+)-a-methoxy-atrifluoromethylphenylacetyl chloride [S-( +)-MTPA-Cl; Fluka] and the mixt. heated for 24 hr at 50”. Spermine was added in order to destroy excess unreacted acyl chloride, pyridine removed with a N, stream and the residue dissolved in CHCI, and chromatographed on a silica gel TLC plate (n-hexane-EtOAc, 9: 1) to afford the R-MTPA triester 9. FAB-MS m/z 1091 [M+H]+, 623 [MH-ZMTPA]+. ‘HNMR (CDCI,): 67.50 (111,6H), 7.41 (m, 9H), 5.13 (m, H-3 + H-27), 4.35 and 4.24 (ms, H-la and H-lb), 3.55 (s, -Me), 3.54 (s, -GMe), 3.52 (s, -Me), 2.01 (m, H-2), 1.25 (methylene chain + 27-Me). Analogously, reaction of 1.5 mg of 5 with R-(-)-MTPA-Cl afforded the S-MTPA triester 10.FAB-MS m/z 1091 [M +H]+, 623 [MH-2MTPA]+. ‘HNMR G(CDC1,): 7.50 (m, 6H), 7.38 (m, 9H), 5.13 and 5.07 (ms, H-3 and H-27), 4.20 and 4.11 (ms, H-la and H-lb), 3.56 (s, -GMe), 3.54 (s, -GMe), 3.53 (s, -GMe), 1.96 (m, H-2), 1.33 (d, J=6.2 Hz, 27-Me), 1.25 (methylene chain). MTPA esters of (3R)- and (3S)-1,3-butanediol. The RMTPA and S-MTPA diesters of (3R)- and (3S)-1,3butanediol were prepd following the above procedure. The relevant ‘HNMR chemical shift values (CDCl,) were: (3R)-1,3-butanediol R-MTPA diester: 2.01 (H-2), 1.27 (Me-3); (3R)-1,3-butanediol S-MTPA diester: 1.97 (H-2), 1.35 (Me-3). (3S)-1,3-butanediol R-MTPA diesten 1.97 (H-2), 1.35 (Me-3k(3S)-1,3-butanediol S-MTPA diesten 2.0 (H-2), 1.26 (Me-3). l~Gu-~hrcopymnosy~)-n-1Yebo-3R-ocbacosal (11). Oil. FAB-MS m/z 603 [M +H]+, 441 (cleavage of glucoside bond). ‘HNMR 6 (pyridine-d,k 5.48 (d, J=3.7 Hz, H-l’), 4.71 (t, J =9.1 Hz, H-3’), 4.60 (dd, J=4.9 and 13.9 Hz, H&‘a), 4.49 (dd, J=4.9 and 13.9 Hz, H-5’+H6’b), 4.39 (dt, J=6.2 and 9.8 Hz, H-la), 4.32 (t, J=8.9 Hz, H-4’), 4.25 (dd, J = 3.8 and 9.6 Hz, H-2’), 4.19 (m, H-3), 4.11 (dt, J=6.8 and 9.8 Hz, H-lb), 2.43 (t, J=7.4H& H-26), 2.14 (s, Me-27), 2.07 (m, H-2), 1.38, 1.34, 1.31 (methylene chain). 13C NMR pyridine-d,: 6 208.3 (C-27), 100.7 (C-l’), 75.7 (C-3’), 74.5 (C-5’), 74.1 (C-2’), 72.3 (C-4’), 68.7 (C-3), 66.2 (C-l), 63.0 (C-6’), 43.6 (C-26), 38.8 (C-4), 38.2 (C-2), 30.1 (methylene chain), 29.8 and 29.5 (C-28 and C-24), 26.4 (C-5), 24.2 (C-25). 2-Octanone. 13C NMR pyridine -d,: 6207.8 (C-2), 43.7 (C-3), 32.1 (C-6), 29.7 (C-l), 29.1 (C-5), 24.2 (C-4), 22.9 (C7), 14.3 (C-8).
396
A. SRIENTE
NaBH, reduction of compound 11. To 1.8 mg of 11, dissolved in MeOH (0.5ml), an excess of NaBH, was added and the mixt. stirred at room temp. for 15 min. HOAc (5 ~1) was added and the mixt. chromatographed on a prep. TLC plate (CHCl,-MeOH, 4: 1) from which the product was recovered with CHCl,-MeOH (2: 1). The product obtained was indistinguishable from 4 by TLC and ‘H NMR. Acknowledgements-The 500 MHz NMR and mass spectra were obtained from the ICMIB-NMR Service and from the “Servizio di Spettrometria di Massa de1 CNR e dell’Universid di Napoli, respectively; the staff of both are gratefully acknowledged. This work was partially supported by Ministero dell’Universitl e della Ricerca Scientifica. REFERENCES
Nichols, B. W. and Wood, B. J. B. (1%8) Nature 217, 767. 2. Walsby, A. E. and Nichols, B. W. (1969) Nature 221, 673. 1.
et al.
3. Murry, M. A. and Wolk, C. P. (1989) Arch. Microbial. 151,469.
4. Bryce, T. A., Welti, D., Walsby, A. E. and Nichols, B. W. (1972) Phytochemistry 11,295. 5. Lambein, F. and Wolk, C. P. (1973) Biochemistry 12, 791. A. and 6. Soriente, A., Sodano, G., Gambacorta, Trincone A. (1992) Tetrahedron 48, 5375. 7. Florenzano, G., Sili, C., Pelosi, E. and Vincenzini, M. (1985) Arch. Microbial. 140,301. 8. Larch, S. K. and Wolk, C. P. (1974) J. Phycol. 10,352. 9. Harada, N., Saito, A., Ono, H., Gawronski, J., Gawronska, K., Sagioka, T., Uda, H. and Kurki, T. (1991) J. Am. Gem. Sot. 113,3842. 10. Dale, J. A. and Mosher, H. S. (1973) J. Am. Gem. Sot. 95, 512. 11. Cardemil, L. and Wolk, C. P. (1979) J. Biol. Chem. 254, 736. 12. Vincenzini, M., Sili, C., Tredici, M. R. and Materassi, R. (1989) in Nitrogen Fixation with Non-legumes (Skinner, F. A. et al., eds), p. 71. Kluwer Academic Publishers.