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Cyanoglucosides from Osmaronia cerasiformis (Rosaceae) Article in Phytochemistry · November 1994 DOI: 10.1016/S0031-9422(00)89525-4 · Source: PubMed
4 authors, including: Adolf Nahrstedt
University of Münster
Helmholtz Centre for Infection Research
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Phytochemtitry. Vol. 37, No. 4, pp.,1039-1043, 1994 Copy&&t @TJ1994 Elswici Science Ltd Printed in Great Britain. All n&s rcsm’ed @331-9422,94 S7.00+0.00
FROM OSMARONIA CERASIFORMIS (ROSACEAE)*
MAITHIAS LECHTENBERG, ADOLF NAHRSTEDT,t VICTORWRAY$ and FRANK R. FRONCZEK~
Institut fiir Pharmazeutische Biologic und Phytochemie, Westf. Wilhelm+Universitlt,
HittorfstraBe 56, D-48149 Miinster, Germany; $.Gesellschaft fiir Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany; @epartment of Chemistry, Louisiana State University, Baton Rouge, LA 70803-1804, U.S.A.
(Receioed17 June 1994) IN HONOUR
Key Word Index-Osmaronia cerasijbmis; Rosaceae; Prunoideae; cyanogenesis; osmaronin; onin epoxide; sutherlandin; nitrile glucosides; X-ray analysis.
nitrile glucosides have been isolated from a methanolic extract of the leaves of Osmaronia (Torr. & Gray) Greene (Rosaceae, Prunoideae). One is the known sutherlandin (Z-4-b-Dglucopyranosyloxy-3-hydroxymethylbut-2-ene nitrile); the second is Z-4-/?-glucopyranosyloxy-3-methylbut-2-ene nitrile, which has recently been characterized but whose stereochemistry was not previously determined, the Z-isomer identified here was named osmaronin. The third is the new 4-b-D-glucopyranosyloxy-2R,3R-epoxy-Jmethylbutyronitrile (osmaronin epoxide). The nitrile glucosides occur in the leaves and flowers of the title plant; their aglycone is apparently derived from the aliphatic amino acid L-leucine.
Osmaronia cerasijbrmis (Torr. & Gray) Greene (Prunoideae; syn. Oemleria cerasijbrmis (Torr. & Gray) Landon; syn. Nuttallia cerasi$ormis Torr. & Gray) is a shrub or
small tree growing in western North America from British Columbia to California [l, 2, 31. It was reported to possess cyanogenic buds and leaves but non-cyanogenic seeds [4, 51; no benzaldehyde could be detected after treatment of the plant material with fl-glucosidase preparations , although members of the Prunoideae usually contain cyanogenic compounds such as prunasin or amygdalin which release benzaldehyde after degradation . We here report on the isolation and structure elucidation of three nitrile gluwsides, osmaronin (l), osmaronin epoxide (2) and sutherlandin (3), from the leaves of 0. cerasi$ormis. RESULTS AND DISCUSSION
The methanolic extract of the lyophilized leaves showed three weakly cyanogenic zones on TLC plates after detection with the sandwich-picrate test according to ref. . The extract was purified by chromatography on [email protected]
, silica gel and MCI gel. Final purification was achieved by MPLC (RP-18) yielding the chromatographically pure compounds 1, 2 and 3 (TLC, GC as
*Part of the projected Ph.D. thesis of Matthias Lechtenherp. fAuthor to whom correspondence should he addressed.
TMSi derivatives). Hydrolysis of 1, 2 and 3 using an unspecific enzyme preparation yielded glucose (TLC, GC). The coupling constants of the anomeric protons of 1, 2 and 3 in the ‘HNMR were 7.6, 7.8 and 7.7 Hz respectively indicating fl-D-glucosides [S]. The IR spectra clearly showed nitrile bands at v = 2220-2230 cm- ’ for 1, 2 and 3 which are usually weak or not present in glucosylated cyanohydrins [S]. Concerning 1 (M,: 259; D/CIMS: [M + 18]+ =277) the NMR spectroscopic data were very similar with those published for a 4-j&gluwpyranosyloxy-3-methyl-but-2ene nitrile with hitherto undetermined stereochemistry  isolated from the epidermal layer of barley leaves
M. LECHTENBERG et al.
Table 1. NMR data for 1 (osmaronin), 2 (osmaronin epoxide) and 3 (sutherlandin) rH NMR C 1
2 3 4
4 5 6
__ 5.46 (lH, m)
3.75 (lH, s) ._
H-4A 4.57 (lH, d, 13.2) H-4B 4.46 (lH, d, 13.2) 2.03 (3H, d, 1.5) 4.29 flH, d, 7.6)
3.89 (2H, s)
3.2-3.4 (4f-L m)
H-6A 3.88 (IH, dd) H-6B 3.70 (IH, dd)
1.48 (3H, s) 4.41 (lH, d, 7.8) 3.25 (lH, dd, 8.4) 3.43
(lH, dd, 8.8)
5.72 (lH, m)
H-4A 4.51 (lH, d, 13.2) H-4B 4.36 (lH, d, 13.2) 4.14 (3H, d, 1.8) 4.15 (IH, d, 7.7)
2.9-3.3 (4f-L m)
H-6A 3.67 (lH, ddf H-6B 3.45 (lH, dd)
‘%I NMR 2
117.1 91.5 163.0 70.7
117.3 47.4 62.7 72.0
116.5 94.0 164.9 66.4
71.5 78.0* 62.6
71.5 77.Y 62.8
69.7 76.4* 60.77
Compound 1 in MeOH-d,, 2 in acetone-d,, 3 in DMSO-d,; for numbering see formulae. *Assignments are interchangeable in vertical cohnnns. tAssignments are confirmed by lH/l~C-correlatlon (HETCOR).
oulgare L.). Proton NOE difference spectra of 1 indicated the stereochemistry of the olefinic carbons. Irradiation on H-5 caused a strong enhancement of H-2 and a weak enhan~ment of H-4, while irradiation on H-2 strongly enhanced H-5 only and irradiation of H-4 produced only a weak effect on H-5. Thus, H-2 is closer in space to the C-5 protons than to the C-4 protons. The c coupling constant of 1.5 Hz (J,,,, Table 1) is in agreement with a 4J long range coupling whereas 3J long range coupling (7-8 Hz, [lo]) could not be observed. Consequentty H-Z and the -CN grouping must be adjacent on the same olefinic carbon. Thus 1 is the Z-isomer of 4-pglucopyranosyloxy-3-methylbut-2-ene nitrile; as PourC13A mohseni et al.  were unable to fully assign the structure we due to lack of substance, we propose that the fully characterized Z-isomer from Osmaroniu cerasiforrnis be named osmaronin. The M, of underivatized 2 was 275 (D/CIMS: [M +18]+ = 293). After peracetylation 2 showed a M, of 443 by comparison with previous data [l l] (Table 1). The (D/CIMS: [M + 18-J’ =461) and, after trimethylremaining five aglycone signals were assigned to the three silylation of 563 (GC-CIMS: [M+ 181’ = 581). These groups mentioned above, to a -CN group and to a values indicate four derivatizable OH groups (all assigned to the glucose moiety). Together with the M, the ele- quaternary carbon (Table 1). A 13C-APT spectrum  mental analysis gave a molecular formula of facilitated these assignments. The large downfield shift of C,,H,,O,N. The ‘HNMR spectrum of underivatized 2 the quaternary carbon indicates a connection to an -OR showed in addition to the signals due to B-D-gh.iCOSe group. These data suggest an aglycone moiety with an oxirane grouping or a four-membered ring [ 131. Correlresonances of a -Me group (1.48 ppm, s), a - CH,-OR group (3.89 ppm, s) and a > CH-OR group (3.75 ppm, s). ations in the 2DHMBC spectrum (Table 2), indicating The t3C NMR exhibited 11 carbon resonances of which heteronuclear iong-range couplings, established the positions of all aglycone carbons and thus proved the prethe glucose resonances could be un~biguously assigned
Cyanoglucosides from Osmaronia cerasfirmis Table 2. 2D ‘H-detected multiple-bond t3C multiple-quantum coherence spectrum (HMBC) of 2: table of ‘cross-peaks’ involving the aglycone moiety ‘H
++ ++ ++
1% C-l c-2 c-3 c-4 C-5 C-l’
++ ++ * +
+ + Cross-peaks (strong intensity). +Cross-peaks (medium intensity). +Cross-peaks (weak intensity). sence of the suggested oxirane
structure. The relative stereochemistry at the oxirane carbons C-2 and C-3 was determined by ‘H NOE difference spectra which showed unambiguous enhancements for H-2 and H-4 upon irradiation of H-5, while irradiation of H-2 caused enhancement of H-S only. Irradiation of H-4 produced enhancements for H-5 and H-l’. Thus, H-2 is closer in space to the C-5 protons than to the C-4 protons and the configuration at C-2 and C-3 is either RR or SS with the -CN and -Me groupings trans to one another. The absolute stereochemistry of the oxirane carbons, as 2R,3R, was deduced from an X-ray crystallographic study of osmaronin epoxide peracetate. Two independent molecules of osmaronin epoxide peracetate exist in the crystal, with one water molecule of solvation. The two differ primarily in the conformation of the C(ring) -CH,-OAc portion of the glucoside. In one molecule, the G(ring)-C(ring)-C-O torsion angle is + 64.8 (4)“, while in the other it is - 63.6 (4)“. Smaller differences, of up to 33”, exist in the torsion angles about the C(ring)-O(acetate) bonds of the two molecules and within the epoxy-3methylbutyronitrile substituents. Compound 2 is thus 4-B-D-glucopyranosyloxy3R,3R-epoxy-3-methylbutyronitrile that was named osmaronin epoxide; it is one of the few cyanoglucoside epoxides, which are known from Sedum cepaea (Crassulaceae [ 143) and PassiJlora spp. (Passifloraceae [ 131). In the case of 3 (Z-4-/I-D-glucopyranosyloxy-3hydroxymethylbut-2-ene nitrile, sutherlandin) all spectroscopic data (‘H NMR, 13C NMR, NOE experiments, MS, IR) were consistent with the published data . Sutherlandin was found recently in leaves of Acacia sutherlandii (Mimosaceae)  and of Hordeum uulgare L. (Poaceae) . Compounds 1,2 and 3 do not belong to the cyanogenic glucosides as the cyanohydrin structure is lacking. Surprisingly the sandwich test detected all compounds by liberation of HCN; furthermore, leaves of 0. cerasiforrnis released detectable amounts of HCN (ca 5 mg 100 g-’ freeze-dried leaves, harvested in April 1994) when incubated in buffer in a semi-quantitative assay using the
*Heterogeneous; members of the tribe Sorbarieae.
Feigl/Anger test [16, 173. However, assuming that the entire amount of 1, 2 and 3 present in the leaves (see below) would have been converted to HCN it should account for ca 250 mg lOOg- ‘. The stability of 2 and 3 was investigated by measuring the liberation of HCN in an aqueous solution at pH 6. Pure 2 released 2-3 mol% HCN, while pure 3 did not show any detectable amount of HCN under these experimental conditions. The rate of HCN liberation from 2 or 3 was unaffected by addition of Heuea fi-glucosidase  or an unspecific enzyme preparation (see Experimental). Thus the weak cyanogenesis of the crushed material of 0. cerasijknis may be due to an as yet unknown degradation, concerning 2 probably via a vicinal diol arising from hydrolysis of its oxiran moiety. It should be noted, however, that during the purification of 3 by Swenson et al.  this compound was monitored using almond emulsin and the cyanide sensitive Feigl-Anger test [ 171. Within the Rosaceae several genera contain cyanogenie species [19-211. The well known phenylalaninederived cyanogenic glycosides prunasin and amygdalin are typical for the subfamilies Maloideae and Prunoideae, whereas the Spiraeoideae* are heterogeneous; members of the tribe Sorbarieae contain leucine-derived cyanogenics such as heterodendrin or derivatives of cardiospermin [22, 61. It is noteworthy that the carbon skeleton of 1, 2 and 3 suggest these are also biogenetically derived from L-leucine. For morphological reasons (seed orientation, innervation and placentation; number of carpels etc.) 0. cerasqormis differs from other Prunoideae and Mai has argued in favour of creating the separate subfamily Oemlerioideae [2, 231. Our results support a separation of Osmaronia from the Prunoideae. In all probability other members of the Rosaceae-Prunoideae also contain compounds such as 1,2 or 3; this is currently under investigation. EXPERIMENTAL
spectra of 2 were General. The ‘H and “CNMR recorded at 600.1 and 150.9 MHz, respectively (Bruker AM-600); those of 1 and 3 at 199.98 and 50.3 MHz, respectively (Varian Gemini 200). Chemical shifts are given in ppm relative to TMS, coupling constants in Hz. The ‘H-detected multiple-bond 13C multiplequantum coherence spectrum (HMBC) was recorded on a Bruker AM-600. The NOE experiments were performed on a Bruker AM-360 (2,3) and on a Varian Gemini 200 (l), all other NMR experiments (APT, HETCOR) on a Varian Gemini 200. X-ray experimental. A plate fragment of dimensions 0.05 x 0.18 x 0.90 mm was used for data collection on an Enraf-Nonius CAD4 diffractometer equipped with CuK, radiation (A= 1.54184 A); and a graphite monochromator. Crystal data are: C,,H,,NO,, x l/2 H,O, M,=452.4, triclinic space group Pl, a = 7.6868 (6), b=11.5713 (9), c=14.2154 (8) A, a=69.200 (5)0, B =79.635 (6)“, y=78.036 (7)“, I’=1148.5 (1) A3, Z=2, dc = 1.308 g cm- j, T = 24”. Intensity data were measured by
M. LECHTENBERC et al.
o-20 scans of variable rate. A hemisphere of data was collected within the limits 2 CO< 75”. Data reduction included corrections for background, Lorentz, polarization, decay (4.8%, linear), and absorption effects. Absorptions corrections (~=9.Ocm-“) were based on Y scans, with minimum relative transmission coefficient 91.4%. Of 4737 unique data, 4193 had I > 3a(Z) and were used in the refinement. The structure was solved by direct methods using program SHELXS  and refinded by full matrix least squares, treating nonhydrogen atoms anisotropically, using the Enraf-Nonius MolEN programs . Hydrogen atoms were placed in calcd positions, guided by different maps, and were not refined. H atoms of the water molecule were not located. Convergence was achieved with R= 0.04824, R,=0.05879, and GOF = 2.849 for 566 variables. Maximum residual electron density was 0.27 eA_“. The model with alternative absolute configuration was refined under identical circumstances, yielding slightly worse fit, R=0.04827, R,=0.0588.5, and GOF = 2.852. Coordinates, bond distances, and bond angles have been deposited with the Cambridge Crystallographic Data Centre. PIant material. Osmaronia cerasiformis was cultivated in the Experimental Garden of the Institute at Miinster. Young leaves were harvested in April 1994. The plants stem originally from the collection of Prof. R. Hegnauer, Leiden (No. 29160 and 29028). Vouchers are deposited under PBMS 88 in the Institute at Miinster. Isolation. Young leaves of 0. cerasiformis (85 g) were ground in liquid N,. After lyophilization the leaves (20.3 g) were exhaustively extracted with MeOH. The extract was taken to dryness, suspended in H,O and loaded on to an [email protected]
20 (Merck Darmstadt; No. 11737) column (2.5 x40cm). After elution with EtOEt (500 ml) and i-BuOH (500 ml) the l-BuOH fr. was taken to dryness and chromatographed on a silica gel (Merck, No. 7734) column (5 x 40 cm, 1.5 mlmin-‘) using EtOAcH,O-MeOH (79: 10: 11). Cyanogenesis was tested by TLC using the sandwich method according to ref. . Cyanogenic zones were detected within the 850-1500 ml (A) and 1500-2500 ml (B) frs. A (con~ning 1 and 2) was coned and chromatographed on a [email protected]
CHP 20P gel (Mitsubishi Kasei Corporation, Lot No. 91-01) column (2.5 x 25 cm, 2.2 ml min- ‘) with a H,O-MeOH gradient (H,O-MeOH 9: 1-*H,O-MeOH 1: 1). The cyanogenic frs (150-310ml) were coned. Final purification was achieved by MPLC (RP-18, see below). The cyanogenic frs (600-1000 ml [containing 21 and 110&1500 ml [containing 11) were chromatographically pure (TLC, GC) and yielded 55 mg of 1 and 350 mg of 2. For isolation of 3 the coned frs B were chromatographed on a [email protected]
CHP20P gel column (2.5 x25 cm, 1.55 mlmin-‘, gradient: MeOH-H,O, 1:9+MeOH-H,O, I : 1, elution vol.: 80-140 ml). Cyanogenic frs were monitored by TLC sandwich tests. Finally, chromatography on a RP-18 MPLC column (see below) yielded 75 mg of pure 3. Lyophilized 1,2 and 3 were used for structure elucidation. Hydrolysis. An unspecific enzyme prepn with ,&glucosidase, ~-glucuronida~ and esterase activity (Rohm EL I77) and @-glucosidase from Hevea [ 181 were used in
citrate-phosphate for 12 hr.
buffer (0.15 M, pH 6) at ambient temp.
~Zementa~ a~u~ys~sqf 2. The elemental analysis of 2 was performed ( x 2) with 5 mg each. Found 47.10% (C), 6.27% (H), 5.13% (N), 41.50 (0, diff.); talc.: 47.98% (C), 6.23% (H), 5.09% (N), 40.70% (0). TLC system. Precoated TLC plates (silica gel, Merck 5554) were used with EtOAc-Me,CO-CH,Cl,-H,OMeOH (20:15:6:4:5, system F,) and EtOAc-MeOHH,O (79: 11: 10, system F2) as mobile phases. Detection was with the sandwich-picrate test  or with R, of 1: O&/0.29; R, of 2: anisaldehyde-H,SO,. 0.47/0.35: R, of3: 0.34/0.21 (system F, or F, respectively). MPLC system. Biichi 688 Chromatography Pump; Europrep C-l 8 (Besta Tech& GmbH), 60 A, 20-45 pm, 26 x 450 mm, mobile phase: MeOH-H,O (2: 98) (elution vol.: 460-660 ml 3; 600-1000 ml 2; 1100- 1500 ml l), 5.5 ml min- ‘; 15-20 bar; UV detection at 210 nm with LKB 2151 Variable Wavelength Monitor. Derivatization. TMSi ether: The compounds were dissolved in pyridine- BSTFA-TMCS (I : 3: 1) and used for GC after 4 hr at ambient temp. Peracetates: the peracetates were prepd by dissolving 20 mg of the pure compounds in 1 ml of pyridine-Ac,O (1: 1) for 24 hr. After adding ice and water a white ppt. could be isolated. The peracetate of 2 crystallized in H,O-MeOH (1: 1) at ambient temp. GC system. Carlo Erba Instruments, HRGC 5160; DB5 capillary column, 30 m x 0.25 mm x 0.25 pm; He (1 mlmin-’ at 50”); 160-280”, 8” min- I, then isothermitally; injector 180”, FID and PND 270”; R, TMSi-2: 16.5 min, TMSi-1: 16.7, TMSi-3: 19.4min, TMSi-linamarin for comparison: 13.7min. Higher injector temperatures (Ti > 180’) affect epime~zation of TMSC2: We identified a second peak at 16.7 min with identical CIMS data and measured a linear increase of the peak area with increasing T, (160-+280”). Thus 2 is not a mixt. of 2 epimers as we postulated recently . MS systems. The GC-CIMS (with NH,) was performed on a Varian 3700 and a Varian MAT 445, HP-1 column, 50 m x 0.32 mm x 0.52 pm, 160-~300”,8” min-‘, then isothermically; R, TMSi-2: 12.5min, R, TMSi-1: 13.0min TMSi3: 15.1 mm. D/C1 (with NH,) mass spectra were recorded on a Varian MAT 44s. Measurement of HCN liberation. The liberation of HCN from plant material and isolated compounds was tested in the ‘Wissing Apparatus’ according to ref.  using citrate-phosphate buffer (pH 6, 0.15 M, 35”, N,: 30 ml min- ‘). HCN was trapped in 1 M NaOH solution (traps were changed after 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7 and 24 hr) and determined photometrically using the ABS method . In some preliminary experiments plant material and extracts were tested for cyanogenesis with the Feigl-Anger test in the same buffer solution [16, 171.
Acknowledgements-Thanks are due to Professor R. Hegnauer for providing the original plants, K. Busse (Inst. fur Org. Chemie Miinster) for performing the NOE experiments (1 and 3), Dr D. Bergenthal and M. Heim (Inst. fiir Pharm. Chemie Miinster) for recording several NMR
Cyanoglucosides from Osmaroniaceras~ormis(Rosaeeae)
spectra and the HETCOR of 3, H. Wennemer and T. Meiners (Inst. fur Pharm. Chemie Miinster) for running the MS and GC-MS, I. Simons (Inst. fur Org. Chemie Miinster) for performing the elemental analysis of 2, E. Schratz and B. Quandt for technical assistance and Dr A. Brinker for valuable linguistic advice.
13. Olafsdottir, E. E., Sorensen, A. M., Comett, C. and Jaroszewski, J. W. (1991) J. Org. Chem. 56, 2650. 14. Nahrstedt, A., Walther, A. and Wray, V. (1982) Phytochemistry 21, 107.
15. Swenson, W. K., Dunn, J. E. and Conn, E. E. (1987) Phytochemistry 26, 1835.
16. Brinker, A. M. and Seigler, D. S. (1989) Phytochemical REFERENCES
1. Hutchinson, J. (1964) 7’he Genera ofFlowering Plants (Angiospermae), Dicotyledones Vol. I, 187. Clarendon
Press, Oxford. Mai, D. H. (1984) Fe&es Repertorium 95, 299. Rydberg, P. A. (1918) North Amer. Flora 22, 481. Plouvier, V. (1948) Compt. Rend. 227, 1260. Plouvier, V. (1942) Compt. Rend. 214, 322. Fikenscher, L. H., Hegnauer, R. and Ruijgrok, H. W. L. (1981) Planta Med. 41, 313. 7. Brimer, L., Christensen, S. B., Molgaard, P. and Nartey, F. (1983) J. Agric. Food Chem. 31, 789. 8. Nahrstedt, A. (1981) in Cyanide in Biology (Vennesland, B., Conn, E. E., Knowles, C. J., Westley, J. and Wissing, F., eds), p. 145. Academic Press, London. 9. Pourmohseni, H., Ibenthal, W.-D., Machinek, R., Remberg, G. and Wray, V. (1993) Phytochemistry 33,
2. 3. 4. 5. 6.
O., Masuda, H. and Mihara, S. (1987)
J. Agric. Food Chem. 35, 338.
11. Hiibel, W., Nahrstedt, A. and Wray, V. (1981) Arch. Pharm. ( Weinheim) 314, 609.
12. Patt, S. L. and Shoolery, J. N. (1982) Journal of Magnetic Resonance 46, 535.
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Bulletin 21, 24.
17. Tantisewie, B., Ruijgrok, H. W. L. and Hegnauer, R. (1969) Pharm. Weekbl. 104, 1341. 18. Selmar, D., Lieberei, R., Biehi, B. and Voigt, J. (1987) Plant Physiol. 83, 557.
19. Hegnauer, R. (1973) Chemotaxonomie der Pflanzen 6, 84, 727. Birkhauser, Basel. 20. Hegnauer, R. (1986) Chemotaxonomie der Pflanzen 7, 345. Birkhauser, Basel. 21. Hegnauer, R. (1990) Chemotaxonomie der Pfanzen 9, 369. Birkhauser, Basel. 22. Nahrstedt, A. (1987) in Biologically Active Natural Products (Hostettmann, K. and Lea, P. J., eds), p. 213. Clarendon Press, Oxford. 23. Sterling, C. (1964) Am. J. Bot. 51, 354. 24. Sheldrick, G. M. (1990) Acta Cryst. A46, 467. 25. Fair, C. K. (1990) MolEN. An Interactive System for Crystal Structure Analysis. Enraf-Nonius, Delft, The Netherlands. 26. Lechtenberg, M. and Nahrstedt, A. (1993) Planta Med. 59, A616. 27. Wissing, F. (1981) in Cyanide in Biology (Vennesland,
B., Conn, E. E., Knowles, C. J., Westley, J. and Wissing F., eds), p. 473. Academic Press, London. 28. Nahrstedt, A. (1977) Dtsch. Apoth. Ztg. 117, 1357.