Holzforschung, Vol. 59, pp. 413–417, 2005 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/HF.2005.067

Bioactive phenolic substances in industrially important treespecies. Part 4: Identification of two new 7-hydroxy divanillylbutyrolactol lignans in some spruce, fir, and pine species

Figure 1 Structures of 1 and 2 and of liovil.

Stefan Willfor1,*, Patrik Eklund2, RainerSjoholm2, Markku Reunanen1, Reijo Sillanpaa3,Sebastian von Schoultz4, Jarl Hemming1, LindaNisula1 and Bjarne Holmbom1

1 Abo Akademi University, Process Chemistry Centre,Laboratory of Wood and Paper Chemistry, Turku/Abo,Finland

2 Abo Akademi University, Process Chemistry Centre,Department of Organic Chemistry, Turku/Abo, Finland

3 University of Jyvaskyla, Department of Chemistry,Jyvaskyla, Finland

4 Firma von Schoultz, Turku/Abo, Finland

*Corresponding author.Abo Akademi University, Process Chemistry Centre, c/oLaboratory of Wood and Paper Chemistry, Porthansgatan 3,FIN-20500 Turku/Abo, Finland. Fax: q358-2-2154868E-mail: stefan.willfor@abo.fi

Abstract

A few lignans, that is, 7-hydroxymatairesinol, secoisola-riciresinol, lariciresinol, and nortrachelogenin, predomi-nate in a large proportion of the industrially importantsoftwood species used. Some other lignans, of whichsome still are unidentified, are also present in loweramounts. Softwood knots, i.e., the branch bases insidetree stems, commonly contain exceptionally largeamounts of free aglycone lignans, which has provided agreat opportunity to isolate sufficient amounts of soft-wood lignans for structural characterisation. Here wepresent the identification and characterisation of two new9-epimers of 7-hydroxy divanillyl butyrolactol lignans,(7S,8R,89R,99R)-4,49,7-trihydroxy-3,39-dimethoxylignano-99,99-lactol and (7S,8R,89R,99S)-4,49,7-trihydroxy-3,39-dimethoxylignano-99,99-lactol, in knotwood of Coloradospruce (Picea pungens), from tentative GC-MS analysisto final determination of the structure by NMR spectros-copy and X-ray analysis. Further analyses have verifiedthe occurrence of these lignans, which were earlier incor-rectly addressed by our group as isomers of liovil, in sev-eral spruce, pine, and fir species.

Keywords: Abies; fir; 7-hydroxy divanillyl butyrolactollignans; isoliovil; knots; knotwood; lignans; liovil; Picea;pine; Pinus; spruce; todolactol A.

Introduction

A few lignans, that is, 7-hydroxymatairesinol, secoisola-riciresinol, lariciresinol, and nortrachelogenin, predomi-nate in a large proportion of the industrially important

softwood species used (Holmbom et al. 2003). Someother softwood lignans, of which some still are uniden-tified, are also present in lower amounts. Softwoodknots, i.e., the branch bases inside tree stems, common-ly contain large amounts of free aglycone lignans (Willforet al. 2003a,b,c, 2004a). This has provided a great oppor-tunity to isolate sufficient amounts of softwood lignansfor structural characterisation. For example, we recentlyidentified the lignan nortrachelogenin in Scots pine (Pinussylvestris) knots (Ekman et al. 2002) and two diastereo-mers of the new lariciresinol-type butyrolactone lignan,isohydroxymatairesinol, in Norway spruce (Picea abies)knots (Eklund et al. 2004).

Lignans in Norway spruce have been extensively ana-lysed and most of the predominant lignans have beenknown for a long time (Freudenberg and Knof 1957;Weinges 1960; Andersson et al. 1975; Ekman 1976).Freudenberg and Knof (1957) reported the tetrahydrofu-ran lignan liovil (Figure 1) in Norway spruce stemwoodand the same lignan was later reported to occur in sev-eral tree species. Ekman (1976) showed mass spectra ofsilylated spruce lignans, including the then tentativelyidentified lignan liovil. Ekman’s work was the basis of ananalytical method for the analysis of spruce lignans inwood tissues (Ekman 1979) and in pulp and processwater samples (Ekman and Holmbom 1989a). The samemethod was later adopted for softwood lignans in gen-eral and up to three isomers of liovil were reported forsome spruce species (Willfor et al. 2003a,c, 2004a;

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414 S. Willfor et al.

Eklund et al. 2004). Nevertheless, none of these tenta-tively identified liovil isomers were isolated and com-pletely structurally characterised.

Here we report the identification and characterisationof two 7-hydroxy divanillyl butyrolactol lignans, com-pounds 1 and 2, in knotwood of Colorado spruce (Piceapungens), from tentative GC-MS analysis to final deter-mination of the structure by NMR spectroscopy andX-ray analysis. Further analyses also verified the occur-rence of these lignans in several spruce, pine, and firspecies.

Material and methods

Hydrophilic acetone extract, containing approximately 1.2 g ofpolyphenols, of which approximately 20% were the studiedcompounds 1 and 2, from knots of Picea pungens Engelm.reported earlier (Willfor et al. 2004a) were evaporated in a rotaryfilm evaporator under vacuum. The extract was fractionated onnormal-phase silica gel columns using a flash chromatographyapparatus, according to Ekman et al. (2002). Separation wasperformed with a stepwise gradient eluent composition ofdichloromethane/ethanol (3%, 5%, and 8% ethanol) at30 ml miny1. Fractions of 50 ml were collected and each fractionwas examined by GC on a short column (Orsa and Holmbom1994). The last fraction from the 8% elution was evaporated andweighed (53 mg), before it was re-dissolved in pure methanol.Pure compound 1 was crystallised from the solution; the crystalswere washed with methanol and dried (;20 mg). The remainingextract was fractionated on reversed-phase TLC plates (RP-8F254 S, Merck) with an eluent composition of water/methanol(60:40, v vy1). The zone containing the studied compounds (1and 2), was scraped off, extracted with acetone, and then theextract was dried and weighed (7.6 mg).

GC-MS analyses of the silylated lignans were performed on aHP 6890-5973 GC-MSD instrument with a HP-1 cross-linkedmethyl siloxane column (15 m=0.25 mm i.d., film thickness0.25 mm). The column oven temperature was programmed from80 to 2908C at a rate of 88C miny1. Helium was used as thecarrier gas at a constant flow of 0.9 ml miny1. The GC injectorwas set in split mode. The injector and MS interface tempera-tures were 260 and 2808C, respectively. Mass spectra wereobtained by electron impact (EI) ionisation energy of 70 eV. Ele-mental composition of the isolated lignan was determined witha Fisons ZAB-Spec high-resolution MS instrument. The samplewas applied through a direct insertion probe, and was ionisedby EI at 70 eV of energy. Perfluorokerosene (PFK) was used asthe standard for accurate mass determination at a resolution of10 000.

1H and 13C NMR spectra were recorded at 258C on a BrukerAV-600 spectrometer at 600.13 and 151.91 MHz, respectively.The sample was dissolved in acetone-d6 and tetramethylsilane(TMS) was used as an internal standard. The 1H and 13C NMRsignal assignments were based on chemical shifts and 1H-1Hand 1H-13C correlation spectroscopy wCOSY; heteronuclear sin-gle-quantum coherence (HSQC) and heteronuclear multiple-bond connectivity (HMBC)x.

Optical rotation was measured on a Perkin-Elmer 241 digitalpolarimeter, using a 1-dm, 1-ml cell.

X-ray data collection and processing

Crystallographic data were collected at 173 K on a Nonius Kap-pa CCD area-detector diffractometer using graphite monochro-matised MoKa radiation (ls0.71073 A). The data collection was

performed using w and v scans. The data were processed usingDENZO-SMN v0.93.0 (Otwinowski and Minor 1997).

The structure was solved by direct methods using theSHELXS-97 program and full-matrix least-squares refinementson F2 were performed using the same program (Sheldrick 1997).All heavy atoms were refined anisotropically. The OH hydrogenswere refined isotropically. The CH hydrogen atoms were includ-ed at fixed distances with fixed displacement parameters fromtheir host atoms. The figure was drawn with Ortep-3 for Win-dows (Farrugia 1997).

Crystallographic data (excluding structure factors) for thestructure reported in this paper have been deposited with theCambridge Crystallographic Data Centre as supplementarypublication CCDC 262610 for 1. Copies of the data can beobtained free of charge on application to CCDC (12 Union Road,Cambridge CB2 1EZ, UK; Fax: q44-1223-336-033, E-mail:deposit@ccdc.cam.ac.uk).

Results and discussion

Isolation by flash chromatography, crystallisation, andthin layer chromatography yielded pure crystals of 1 anda mixture of 1 (38%) and 2 (62%). Mass spectra of thesilylated derivatives of 1 and 2 showed a high intensityof the base peak at m/z 297 wAr-CHOTMSxq, suggestingthe presence of a free benzylic alcohol group, and amolecular ion at m/z 664, as reported by Ekman (1976)for liovil (Figure 2). However, Ekman did not fully explainin his work what data he based the tentatively identifiedstructure on.

High-resolution MS analysis showed the molecular ionof the underivatised compounds at m/z 376. The molec-ular ion intensity was 14% relative to the base peak atm/z 137. Accurate mass measurement gave a mass of376.1529 (theoretical mass 376.1522), and a molecularformula of C20H24O7. The optical rotation for 1 was alsodetermined (waxDsy61.58 in tetrahydrofuran, cs1). Itcan be assumed that 1 and 2 are optically pure accordingto the recent statement by Umezawa (2003) that buty-rolactone, butyrolactol, and other higher lignans haveoptical purity of )99% e.e. (enantiomeric excess).

The 1H and 13C NMR shifts of 1 and 2 (Table 1) werein conflict with the tentatively suggested liovil structure.However, the NMR signals of compounds 1 and 2 resem-bled those reported for the divanillyl butyrolactol lignanstodolactol A (Ozawa and Sasaya 1988; Omori et al. 1994)and isoliovil (Miller et al. 1982).

The 1H-NMR (in acetone-d6) spectra of compounds 1and 2 showed typical signals for the two guaiacyl (3-methoxy, 4-hydroxyphenyl) rings in the aromatic region.Compound 1 showed two doublets at 4.58 ppm(J7–8s7.5 Hz, H-7) and 5.07 ppm (J8–9s0.8 Hz, H-99),whereas compound 2 showed two doublets at 4.40 ppm(J7–8s7.3 Hz, H-7) and 5.09 ppm (J8–9s4.6 Hz, H-99),respectively. These doublets were correlated to the 13Csignals at 76.3 and 102.8 ppm (compound 1) and 76.2and 99.2 ppm (compound 2) in the HSQC spectrum, indi-cating the presence of a benzylic hydroxyl group and ahemiacetal function. In addition, each of these com-pounds showed signals of two diastereotopic methylenegroups and two methine groups, indicating the 7-hydroxydibenzylbutyrolactol structure.

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New butyrolactol lignans in softwood species 415

Figure 2 Mass spectra of the TMS derivatives of 1 and 2.

Table 1 1H and 13C chemical shifts (d) and spin-spin coupling constants (JH,H) of 1 and 2 in acetone-d6 at 258C.

Position 1 2

1H J 13C 1H J 13C(ppm) (Hz) (ppm) (ppm) (Hz) (ppm)

1 – – 136.8 – – 137.02 6.49 1.9 110.7 6.59 2.0 110.93 – – 148.3 – – 148.44 – – 146.4 – – 146.65 6.66 8.0 115.2 6.63 7.9 115.46 6.46 8.0, 1.9 119.9 6.52 7.9, 2.0 120.07 4.58 7.5 76.3 4.40 7.3 76.28 2.23 m 50.2 2.36 m 49.99a 4.04 7.1, 8.8 69.2 4.02 8.6 69.29b 3.94 8.3 – 3.92 8.6, 5.9 –Ome 3.74 s 56.1 3.76 s 56.219 – – 132.5 – – 133.829 6.85 – 113.1 – – 113.339 – – 148.3 – – 148.049 – – 145.6 – – 145.359 6.75 8.0 115.5 6.82 8.0 115.469 6.72 8.0, 1.7 122.0 6.83 8.0, 1.7 121.979a 2.42 8.7, 13.7 39.5 2.54 10.9, 13.4 43.279b 2.17 7.0, 13.7 – 1.93 4.2, 13.4 –89 2.12 3.7, 8.7 52.4 2.02 m 52.399 5.07 0.8 102.8 5.09 4.6 99.2OMe9 3.77 s 56.1 3.84 s 56.1

The structure of 1 was unambiguously determined byX-ray crystallography and the relative stereochemicalconfigurations of the four asymmetric centres were 99R*,8R*, 89R*, and 7S* (Figures 1 and 3). Spruce wood isknown to contain enantiopure lignans with the 8R,89Rconfiguration (Eklund et al. 2002). Therefore, we find itmost likely that compounds 1 and 2 have the 8R and 89Rabsolute configuration. The chemical shifts and the 1H-1H coupling constants (Table 1) of compound 1 showedsimilarities with those previously reported for the 7R-epi-mer, but were not in accordance with those reported forthe 7S-epimer of todolactol A (Ozawa and Sasaya 1988).Ozawa and Sasaya showed that todolactol A has the8R,89R,99R configuration. However, the configuration atposition 7 of the two 7-epimers was only tentativelyassigned. According to our results, the previously report-ed7R-epimer of todolactol A has in fact the 7S-configu-ration. However, like todolactol A, compound 1 has the8R,89R,99R configuration.

By comparison of the NMR data reported for the lactol-lignans todolactol A and isoliovil, it must be assumed thatisoliovil is the 99S-epimer of todolactol A. The couplingconstants (J H7-H8) for the acetylated derivative of 99R-todolactol A and acetylated isoliovil were previouslyreported to be 0 Hz (singlet) and 7 Hz, respectively. Thechemical shift and coupling constant (J H7-H8) of H-7 forisoliovil was in accordance with that of the previouslyincorrectly assigned 7S-epimer of todolactol A (Miller etal. 1982; Ozawa and Sasaya 1988; Omori et al. 1994).Therefore, the configuration of isoliovil seems to be7R,8R,89R,99S. Comparison of the coupling constants (JH7-H8 and J H89-H99) for compounds 1 and 2 (Table 1)indicate that these compounds have the same configu-ration at C-7 and an opposite configuration at C-99. Inconclusion, compound 1 is the 7S-epimer of todolactolA. Compound 2 is a 99-epimer of 7S-todolactol A and a

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416 S. Willfor et al.

Table 2 Tree species in which the divanillyl butyrolactol lignans 1, 2, todolactol A, and isoliovil have been identified in knots orstemwood and the most important references.

Compounds Tree species References

1, 2 Picea abies Ekman 19761, 19791;Ekman and Holmbom 1989a1,b1;Willfor et al. 2003a1,c1, 2004b1,2

1, 2 Picea sitchensis Holmbom et al. 20041;Lindberg et al. 20041;Willfor et al. 2004a1

1, 2 Picea glauca, P. mariana, P. pungens, Willfor et al. 2004a1

P. omorika, P. koraiensis1, 2 Pinus sylvestris Willfor et al. 2003b1

1, 2 Pinus contorta Willfor et al. 2004a1

1, 2 Abies sibirica, A. lasiocarpa, A. balsamea, Willfor et al. 2004c1

A. alba, A. amabilis, A. veitchii, A. concolor

1, 2 Abies sachalinensis Willfor et al. 2004c1

Todolactol A Abies sachalinensis Ozawa and Sasaya 1988;Sasaya and Ozawa 1991

Todolactol A Abies mariesii Omori et al. 1994Isoliovil Taxus wallichiana Miller et al. 19821 1 and 2 incorrectly reported as isomers of liovil.2 b-O-4-linked guaiacylglyceryl ethers of 1 and/or 2.

Figure 3 Molecular structure of 1. Only one of two similar mol-ecules in the asymmetric unit is shown. Thermal ellipsoids havebeen drawn at the 30% probability level.

7-epimer of isoliovil and has the 7S,8R,89R,99Sconfiguration.

This new evidence regarding the structure of thesoftwood lignans 1 w(7S,8R,89R,99R)-4,49,7-trihydroxy-3,39-dimethoxylignano-99,99-lactolx and 2 w(7S,8R,89R,99S)-4,49,7-trihydroxy-3,39-dimethoxylignano-99,99-lactolx,previously incorrectly identified as liovil, corrects earlierreports from our group concerning the lignan composi-tion in several softwood species. Consequently, we havenot identified liovil in the tree species studied shown inTable 2, but we have now instead identified the lignans1 and 2. This is especially important for Picea sitchensis,Picea pungens, and some fir species, where 1 and 2 arethe most abundant lignans in both stemwood and knots(Willfor et al. 2004a,c). Nevertheless, it is not ruled outthat liovil may also be present in the species studied asa minor component. Epimers of the lignans 1 and 2 werealso earlier identified in two Abies and one Taxus species(Miller et al. 1982; Ozawa and Sasaya 1988; Sasaya andOzawa 1991; Omori et al. 1994).

Concluding remarks

Two new 9-epimers of 7-hydroxy divanillyl butyrolactollignans, 1 w(7S,8R,89R,99R)-4,49,7-trihydroxy-3,39-dime-thoxylignano-99,99-lactolx and 2 w(7S,8R,89R,99S)-4,49,7-trihydroxy-3,39-dimethoxylignano-99,99-lactolx, were isol-ated from knotwood of Colorado spruce (Picea pungens).The lignans were identified and characterised, first bytentative GC-MS analysis and then by final determinationof the structure using NMR spectroscopy and X-ray anal-yses. The same lignans also occur in several spruce,pine, and fir species, including Norway spruce and Scotspine. We earlier incorrectly reported lignans 1 and 2 asisomers of the tetrahydrofuran lignan liovil. Nonetheless,it is not ruled out that liovil may also be present in thesespecies as a minor component.

Acknowledgements

This work is part of the activities at the Abo Akademi ProcessChemistry Centre within the Finnish Centre of Excellence Pro-gramme (2000–2005) by the Academy of Finland.

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Received December 2, 2004. Accepted February 10, 2005.

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