A Heartwood Pigment in Dalbergia Cell Cultures

Phytochemistry 57 (2001) 1013–1022 www.elsevier.com/locate/phytochem

A heartwood pigment in Dalbergia cell cultures

Miha´ ly Czako´ *, La´ szlo´ Ma´ rton Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC 29208, USA

Received 18 October 1999; received in revised form 11 January 2000

Abstract In an extensive survey of the genera Baphia, Caesalpinia, Dalbergia, Haematoxylon, and Pterocarpus, we have identified a number of species whose cell cultures accumulated pigments similar to those in heartwood. Thirteen rosewood (Dalbergia) species produced a purple quinonemethide pigment in the callus that was apparently identical between the species. The pigment was first purified from D. retusa cell culture and its structure was elucidated by mass, infrared, and detailed 1H and 13C NMR and NOE spectroscopic studies including 2D experiments (COSY, NOESY, HMQC, and HMBC). Retusapurpurin A (1a)isaC30 isoflavan of novel skeleton whose formation can be rationalized to occur via regioselective oxidative coupling of an isoflavan to 4,40-dihydroxy-20- methoxychalcone. Retusapurpurin A was also isolated from D. parviflora heartwood and cell culture indicating that stress metabolism pathways that are shared with heartwood-type secondary metabolism subpathways are initiated in Dalbergia cell cultures. Therefore, Dalbergia cell cultures afford a good model system for studying heartwood-type metabolic differentiation. # 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Baphia; Caesalpinia; Dalbergia; Haematoxylon; Pterocarpus; Leguminosae; Cell culture; Heartwood pigment; Isoflavan; Quinonemethide

1. Introduction for hardwoods) and intercalating parenchyma cells for food storage. Terminal differentiation of the fibers and Despite the importance of wood as the depository of a vessels quickly ends with programmed cell death while large fraction of the fixed carbon stored in the global there remain live parenchyma cells, sometimes deep in ecosystem and as a raw material for major global the no-longer-conducting sapwood (Allen and Hiatt, industries, we know little of the genes involved in the 1994); parenchyma cells can live for over a hundred formation of wood. While there has been great progress years (Shigo, 1994) in certain trees. in the understanding of the biology of xylem/sapwood Programmed cell death occurring during the terminal formation (Fukuda, 1996; Whetten et al., 1998; Gang et differentiation of ray and axial parenchyma is a develop- al., 1999; Lewis, 1999), information on gene expression mental process probably under strict control since it occurs during heartwood formation is not available. only within no-longer-conducting sapwood, spreading Wood (xylem), a tissue unique to higher plants, is a from the tree’s center. Ray parenchyma’s final function is highly ordered arrangement of living, dying, and dead the synthesis of secondary metabolites that impregnate the cells. Meristem cells of the vascular cambium differentiate wood, and this wood is called heartwood (Higuchi, 1997). into water-conducting and supportive fiber (and vessels Ray parenchyma cells in the intermediate (or transition) wood, the sapwood adjacent to heartwood, undergo * Corresponding author. Tel.: +1-803-777-8928; fax: +1-803-777- physiological differentiation and alter their metabolism. 4002. E-mail address: [email protected] (M. Czako´ ). The similarity of the spectrum of substances found in Abbreviations: APCI, atmospheric pressure chemical ionization; heartwood to those in pathogen- or pest-challenged or ESI, electrospray ionization; COSY, correlated spectroscopy; DQF- stressed plants (Harborne, 1994) suggests that some COSY, double-quantum COSY; DMSO, dimethylsulfoxide; HMBC heartwood- and active defense-related secondary meta- 1 13 n ( H, C-COSY- JCH=18 Hz, n=2 and 3), heteronuclear shift corre- bolism (phytoalexin) pathways may be common. lations via multiple bond connectivities (gradient enhanced); HMQC 1 13 1 Only a few of the enzymes of secondary metabolism ( H, C-COSY- JCH) heteronuclear multiple quantum coherence; MS, mass spectroscopy; NMR, nuclear magnetic resonance; NOESY, have been characterized in wood. Phenylalanine ammo- nuclear Overhauser and exchange spectroscopy nia-lyase (PAL) is active across the sapwood, while

0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00111-X 1014 M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 chalcone synthase is active exclusively in the vicinity of except for C. pulcherrima (Rahman et al., 1993), P. the heartwood boundary (Magel et al., 1991, 1995; indicus (Lee and Rao, 1980), D. latifolia, and D. sissoo Magel and Huebner, 1997). PAL is involved both in the (Mandal et al., 1994). formation of lignin and flavonoid biosynthesis; an inhi- Quinonemethide pigments were detected on the basis bitor of the enzyme fed to the sapwood prevented the of their intensive color and electrophoretic mobility formation of phenylpropanoid metabolites (Ohashi et towards the cathode on paper after conversion into al., 1991). The flavonoids in the heartwood thus are pyrylium ions by protonation in an acidic buffer. Spe- formed in situ; they are not transported to the heart- cies of Caesalpinia and Haematoxylon are well known wood via the phloem and the ray cells (Magel et al., for the intense colored brazilein and hematein heart- 1991, 1995). wood pigments (Robinson, 1985) which, however, were The characterization of novel genes involved in the not detectable in tissue cultures of 12 Caesalpinia and 2 biosynthesis of heartwood-specific secondary metabo- Haematoxylon species despite the dark pigmentation in lites in intermediate wood should lead to a better certain species. Similarly, tissue cultures of Baphia nitida understanding of mechanisms controlling this important and seven Pterocarpus species did not contain the san- differentiation pathway. Heartwood does not lend itself tarubins and/or santalins characteristic for the heart- to biosynthetic studies or mRNA extraction because it wood (Arnone et al., 1975, 1977a,b, 1981). Heartwood does not contain living cells, and access to the transition extracts were used as references where available. zone or intermediate wood is limited. To overcome this Since a quinonemethide pigment was also reported difficulty, we sought a system of lower complexity where from a Dalbergia species (Hamburger et al., 1988), we some aspect of heartwood-type metabolic differentiation tested callus cultures of numerous Dalbergia species for is recreated in cell cultures. Cell cultures of gymnosperm pigment production (Table 1). Several species of Dal- trees (Campbell and Ellis, 1992; Eberhardt et al., 1993; bergia proved especially prone to pigment production in Nose et al., 1995; Yamaguchi et al., 1997; Gang et al., vitro. Pigmentation in most species was localized and 1999) and two angiosperm trees (Pare´ et al., 1994; most intense in the cells that were in contact with the Davin et al., 1997; Zhentian et al., 1999) have been uti- medium and in adjacent cells, and the callus left a colored lized to study biochemical processes related to the for- ‘footprint’ in the medium. The emergent portion of the mation of wood, but for heartwood formation the callus usually was free of red, orange, brown, and pur- optimal indicator metabolite would be colored, easy to ple pigments, with the exception of certain species where assay, not far off the charted biosynthetic pathways, yet systemic coloration developed (Table 1). specialized enough not to occur outside heartwood. Besides the apparently water insoluble immobile pig- These criteria narrowed the choices to relatively very few ments in the ‘footprint’ and basal cell layers, diffusible candidates including the quinonemethide pigments in pigments were also produced in certain species. Paper angiosperm plants derived from precursors originating electrophoresis revealed a common major component in from the well studied isoflavonoid and flavonoid path- the pigment extracts of thirteen Dalbergia species ways in leguminous plants (Harborne, 1994; Dixon et (Table 1). It was a barely diffusible pigment located in the al., 1998). In an extensive survey of leguminous woody basal cell layers and the ‘footprint’. It appeared orange- plants, we have identified several species of Dalbergia yellow in the acidic buffer, and turned purple with that produce brilliantly colored pigments in cell cultures ammonia fumes. It showed strong fluorescence under under controlled conditions. We describe herein the UV. This component was easily extractable with etha- isolation and structural elucidation of a novel isoflavan- nol. On silica thin layer chromatograms, it was the lar- derived quinonemethide pigment, retusapurpurin A, from gest spot common to all the above thirteen species. It cell cultures and heartwood. The structure has been was conveniently detected by its yellow/orange fluores- established by spectral analysis, mainly based on detailed cence under UV, and by its pink fluorescence in ammonia 1H and 13C NMR and NOE studies including 2D fumes. The major component was accompanied by sev- experiments (COSY, NOESY, HMQC, and HMBC). eral similar fluorescent, as well as non-fluorescent, purple pigments. Since we first discovered pigment production in D. retusa, the major pigment was named retusapurpurin 2. Results and discussion A. Pigmentation in cell cultures of other Dalbergia species was either not observed under the conditions tested, or As part of our ongoing research on heartwood devel- the above purple pigment was not present and the color opment, cell cultures of a great number of leguminous and solubility of the pigments were different (Table 1). plants were established in order to find species whose Rapid pigment accumulation can be induced by cell cultures undergo metabolic differentiation to pro- transferring the emergent portion of D. retusa callus on duce heartwood secondary metabolites. This is the first DM-2, and in D. parviflora callus upon transfer to DM- report of in vitro culture of all the species listed under 10 medium. Coloration started within 24 h and pigment ’seeds and live plants’ in the experimental procedures accumulation continued thereafter. An orange to deep M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 1015

Table 1 Culture conditions and pigment accumulation in Dalbergia cell culturesa

Culture mediumb for

Dalbergia sp. Callus initiation Maintenance Type of colorationc Retusapurpurin A present

D. arbutifolia DM-1 DM-1 L-Or, S-lBr N D. assamica DM-1 DM-8 L-Or, S-dBr, L-RBr N D. balansae DM-1 DM-7 L-RBr Y D. brownei DM-3 DM-3 L-RBr N D. calycina DM-1 DM-3 L-Re, L-Bl, S-Bl, D-Br Y D. candenatensis DM-3 DM-3 L-Re, L-RBr N D. cochinchinensis DM-1 DM-3 L-PRe N D. cultrata DM-1 DM-3 L-Pu, S-Br Y D. densa DM-1 DM-1 L-lBr Y D. ecastaphyllum DM-1 DM-6 L-bRe Y D. glabra DM-3 DM-3 L-ROr, S-dBr, D-Or N D. hainanensis DM-1 DM-1 L-Br N D. hupeana DM-1 DM-3 DM-1: L-dBr Y D. latifolia DM-1 DM-3 L-Re, D-Or N D. martinii DM-1 DM-1 S-Gy N D. melanocardium DM-5 DM-6 L-dBr N D. melanoxylon DM-1 DM-3 L-Pu, S-Pu N D. miscolobium DM-1 DM-3 L-RBr, S-Bl Y D. nigra DM-4 DM-8 L-dBr, S-dBr, D-dBr Y D. nigrescens DM-1 DM-1 L-RBr Y D. obovata DM-1 DM-6 L-dBr N D. oliveri DM-1 DM-6 DM-3: L-Or N D. parviﬂora DM-1 DM-9 DM-7: L-Bl, L-bRe Y D. retusa DM-1 DM-1 DM-2: L-Bl, L-bRe, S-Bl, D-Or Y D. sericea DM-1 DM-3 L-PBr, S-PBr N D. sissoo DM-1 DM-3 L-Or Y D. trichocarpa DM-1 DM-3 DM-1: L-PRe Y D. tucurensis DM-1 DM-6 L-dBr, S-lOr N

a Normal callus color is a light to dark, chlorophyllous green. b Composition of media is given in the text. c Coloration on maintenance medium unless indicated otherwise. Explanation of color codes: L: Localized to the tissue in contact with the medium and some of it is secreted into the medium but not very diffusible (‘footprint’); S: systemic, expressed in nearly all cells; D: diffusible in the medium; b: bright; d: dark; l: light; Bl: black; Br: Brown; Gy: grey; Or: orange; P: purplish; Pu: purple; R: reddish; Re: red. blood red stain, occasionally intensifying to black, was dent; unsealed solutions gradually turned the same color deposited at the foot and in the core of the compact as can be obtained by addition of traces of water. Retu- callus. sapurpurin A also displayed phase transition induced Retusapurpurin A and the accompanying pigments chromism in DMSOsolution: it turned red upon freez- were not soluble in water but were readily extractable by ing. The solution in acidified DMSOwas dark orange polar organic solvents. Retusapurpurin A (1a) was recov- that turned red on freezing. ered from a methanol eluate of preparative TLC sepa- The characteristic fluorescence, color, and electro- rated band as an amorphous deep reddish purple powder. phoretic mobility of retusapurpurin A facilitated identi- It exhibited interesting physicochemical properties. The fication of the cell culture-produced pigment in heartwood color was pH sensitive. The UV/vis spectrum of a fresh extracts. We searched for retusapurpurin A in heartwood ethanol solution (red) retusapurpurin A dried in vacuo containing samples of 50 Dalbergia species from both the was close to what could be measured under pH controlled Old and the New World tropics. D. parviflora was identi- conditions in ethanol (pH 5.0). A bathochromic shift on fied as the only species whose heartwood appeared to addition of NaHCO3 (purple red) suggested the presence contain retusapurpurin A accompanied by similar UV of free phenolic hydroxyl groups. Identical UV spectra fluorescent purple pigments in smaller amounts. Retusa- were obtained with addition of AlCl3 and HCl or with purpurin A was also found in a sample of Dalbergia HCl alone, indicating that the hypsochromic shift (orange wood which was reported to be D. candenatensis solution) was solely caused by the acidity of AlCl3. Retu- (Hamburger et al., 1988; courtesy of the authors) and sapurpurin A also showed a pronounced solvatochro- from which the quinonemethide pigment candenatone mism: it dissolved in dimethylsulfoxide (DMSO) with a (3) was isolated. Comparison of the pigment profile of deep blue color. Color in DMSOwas moisture depen- authentic D. parviflora wood (supported by a herbarium 1016 M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 voucher) and that of ‘D. candenatensis’ (Hamburger et retusapurpurin A in wood or in cell cultures under the al., 1988), as well as a wood of identical appearance conditions tested so far, while D. parviflora callus does from a commercial source, suggested that these samples produce it (Table 1). represent the same species, while a specimen of authen- Retusapurpurin A was most conveniently purified in a tic D. candenatensis shoot containing traumatic wood quantity sufficient for structure elucidation from D. near a split (also supported by a herbarium voucher) retusa callus because of the high concentration and rela- was very different in color and pigment composition. D. tive purity. The structure of retusapurpurin A (1a, Fig. 1) parviflora wood is dark reddish brown to blood red was determined on the basis of MS and IR spectroscopy while the wood of D. candenatensis is blackish violet. D. as well as detailed 1H and 13C NMR and NOE studies candenatensis was not one of the species that produced including 2D experiments (COSY, NOESY, HMQC, and

Fig. 1. Retusapurpurin A (1a), three of its possible tautomers (1b–1d), their protonated form (2), the partial structure (4), and the related pigment, candenatone (3). M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 1017

HMBC) leading to complete assignment of the proton (d) and 6.43 (dd)). The signal at 6.43 was assigned to and carbon chemical shifts. Its molecular formula H-6 since it was a double doublet and showed a three- C32H26O7 was deduced from the pseudo-molecular ion bond interaction with C-4a and C-8, the latter also pre- [M+H]+ peak at m/z 523 in the ESI and APCI MS senting an interaction with C-4a. These observations, spectra. In the upfield region of the 1H NMR spectrum of also supported by the NOE between H-5 (peri position) 1a two pairs of methylene protons were observed (Table 2, and the methylene on C-4 NOE (Fig. 2 all NOEs were at 2.71 and 2.89 and 4.02 and 4.19, respectively). The positive), confirmed (Hamburger et al., 1988) that 1a COSY spectrum revealed a methine proton hidden under had an isoflavan A/C-ring partial structure. C-7 showed the HDOpeak at 3.43. The coupling patterns of the five correlation to a methoxy group ( 3.70) (HMBC), and aliphatic protons were typical for the C-ring of iso- so did H-6 and H-8 (NOESY) (Fig. 2). From these flavans (Kurosawa et al., 1978; Hamburger et al., 1988). observations it was concluded that 1a has a 7-methoxy- This partial structure was further supported by the substituted A-ring, similar to that of neovestitol (7- COSY cross-peaks (between H-2/H-3 and H-3/H-4), methoxy-20,4-dihydroxyisoflavan; Ingham, 1979). 13C NMR (Table 2), and 2D NMR spectral data, which Three singlet signals were observed downfield in the provided an overdetermined system of correlations. 1H spectrum of 1a and they appeared to belong to a Partially overlapping signals for 13 protons belonging fifth ring or moiety because all other aromatic protons to three aromatic rings were also observed in the 1H could be assigned to two additional aromatic rings. The NMR spectrum, in addition to two aromatic methoxy proton signals at 6.38, 6.42, and 7.82 (Table 2) signals (Table 2). H-5 (d, 6.93) showed three-bond suggested a 1,2,4-tri-substituted ring. Their coupling HMBC correlations with C-4 and C-8a (Fig. 2), in was also confirmed by DQF-COSY. These chemical addition to COSY correlations with two protons ( 6.31 shifts were in good agreement with those obtained for

Table 2 1H NMR and 13C NMR assignmentsa of Retusapurpurin A

Chemical shifts for 1a, TMS (ppm) in DMSO-d6 Chemical shifts for 2, TMS (ppm) in DMSO-d6 /DCl

1 13 1 13 No. d H (number, multiplicity, JHH in Hz) d C d H (number, multiplicity, JHH in Hz) d C 2 3 2 3 2 4.02 (1H, dd, JH2eq,H2ax10.0, JH2,H37.0) 69.03 4.25 (1H, dd, JH2eq,H2ax10.9, JH2,H35.4) 68.46 2 3 2 3 4.19 (1H, dd, JH2eq,H2ax10.0, JH2,H33.0) 4.30 (1H, dd, JH2eq,H2ax10.9, JH2,H32.8) 3 3.43 (1H, m, coupled to H2ax, H2eq, H4ax, H4eq) 31.64 3.61 (1H, m, coupled to H2ax, H2eq, H4ax, H4eq) 31.58 2 3 2 3 4 Hax 2.71 (1H dd, JH4eq,H4ax16.0, JH3,H4ax7.5) 28.98 Hax 3.11 (1H, dd, JH4eq,H4ax16.2, JH3,H45.7) 28.91 2 3 2 3 Heq 2.89 (1H, dd, JH4ax,H4eq16.0, JH3,H4eq5.5) Heq 2.87 (1H, dd, JH4ax,H4eq16.2, JH3,H45.5) 4a 114.26 113.67 3 3 5 6.93 (1H, d, JH6,H58.4) 130.71 6.98 (1H, d, JH6,H58.5) 130.99 3 4 3 4 6 6.43 (1H, dd, JH5,H68.4, JH8,H62.6) 107.32 6.48 (1H, dd, JH5,H68.5, JH8,H62.4) 107.94 7 159.03 159.33 4 4 8 6.31 (1H, d, JH6,H82.6]) 101.56 6.32 (1H, d, JH6,H82.0) 101.80 8a 155.21 155.20 20 158.03 166.80 30 7.27 (1H, s) 108.37 8.20 (1H, s) 115.65 40 152.07 162.81 40a 112.57 115.30 50 7.28 (1H, s) 125.02 7.81 (1H, s) 127.98 60 139.26 133.90b 70 180.09 157.70 80 6.33 (1H, s) 103.35 7.73 (1H, s) 103.10 80a 159.31 165.52 100 107.93 109.79 200 161.31 163.70 00 4 4 3 6.38 (1H, d, poorly res. JH500,H300 2.1) 100.85 6.74 (1H, d, JH500,H3002.0) 100.68 400 167.63 167.54 00 3 4 3 4 5 6.42 (1H, dd, unres. JH600,H5008.9, JH300,H5002.6) 111.35 6.80 (1H, dd, JH600,H5009.0, JH300,H5002.0) 111.30 00 3 3 b 6 7.82 (1H, d, JH500,H600 8.9) 130.66 8.23 (1H, d, JH500,H6009.0) 133.81 1000 126.78 125.42 000 00 3 3 2 /6 ’ 7.25 (2H, d, JH3000,H200 08.6) 131.30 7.43 (2H, d, JH3000,H20008.5) 133.05 000 000 3 3 3 /5 6.87 (2H, d, JH2000,H300 08.6) 116.30 7.00 (2H, d, JH2000,H300’8.5) 117.33 4000 160.08 162.45 7OMe 3.70 (3H) 55.54 3.69 (3H) 55.78 200OMe 3.83 (3H) 56.16 3.97 (3H) 57.21

a Assignments were conﬁrmed by HMBC and HMQC. b Interchangeable. 1018 M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022

Fig. 2. Diagnostic HMBC ( — ) and NOE (- - - -) interactions in compounds 1a and 2. the 2-methoxy-4-hydroxy substituted phenyl ring of complete structure was deduced from further HMBC candenatone (3), an isoflavan-chalcone dimer isolated correlations. C-40a displayed a cross peak with 7.27 to from Dalbergia wood (Hamburger et al., 1988). The which four other cross-peaks belonged, e.g. the carbon ortho position of the methoxy group ( 3.83) was con- at 152.7 to C-40, which also showed a three-bond cor- firmed by HMBC. The doublets (showing the char- relation with the doublet at 7.25 belonging to a para- acteristic second order effects typical for an AA’XX’ substituted ring, and to the carbon at 158.03, which system) at 6.87 and 7.25 were assigned to a para sub- showed a three-bond correlation to a doublet at 7.82 stituted ring. Their coupling was confirmed in a DQF- belonging to the ortho position of the 1,2,4-trisubstituted COSY experiment. The signals for protons belonging to aromatic ring. The NOE between this latter proton and hydroxyl groups predicted by the molecular formula the singlet assigned to C-80 allowed the deduction of the were apparently missing. connectivity of the two aromatic rings to the chromo- Considerations of possible biogenesis, the identical phore. The NOE cross-peak between signals at 7.23 molecular formula and chemical and physicochemical and 3.83 represents interaction between the proton at H- similarity of retusapurpurin A to candenatone (3), the 30 and methyl protons confirming that the methoxy necessity to assign three singlets in the 1H NMR spec- group is in the ortho position in the 1,2,4-trisubstituted tra, and 2D NMR spectroscopic data obtained for aromatic ring whose connection to the central ring retusapurpurin A led to the partial structure 4. system is thus defined. NOEs were also expected The carbon at 139.26 was assigned to C-60, which is between H-50 and H-2000/6000, H-30 and H-2000/6000, and linked to the isoflavan moiety, because it showed three- between H-2 or H-4 and H-2000/H-6000 but were not bond correlations in the HMBC spectrum with the ali- detected due to spectral overlap (Table 2, Fig. 2). NOE phatic protons H-2 and H-4, and one of the two partially was expected also between H-30 and H-6’’ but was not overlapping singlet signals at 7.27. The latter displayed detected. NOEs with all aliphatic protons indicating that there is The assignment of a methoxy group to ring A of the free rotation around the bond connecting the C-ring of isoflavan unit was corroborated by the positive ion ESI the isoflavan A/C-ring system to the rest of the molecule. MS spectrum which exhibited a peak at m/z 387 which One of these two singlet signals was assigned to C-50 resulted from RDA cleavage of 1a at the isoflavan C-ring. because of its three-bond correlations to two oxygen- The NMR and MS data established that retusa- bearing carbons at 180.09 and 159.31. The singlet sig- purpurin A is a quinonemethide represented by the nal at 6.33 was assigned to C-80 because of its two-bond structural formula 1a (Fig. 1). The stability and simplicity correlation to the carbon at 159.31, assignable to C-80a, of the 1H NMR spectrum indicated that, of the at least and based on the three-bond HMBC correlations to C-60 four tautomeric structures of retusapurpurin A, (1a) is by and a carbon at 112.57 assignable to C-40a. The carbonyl far the most stable in DMSOsolution at ambient tem- carbon signal at 180.09 (supported by the carbonyl peratures. Addition of deuterium chloride (DCl) caused absorption at 1635 cmÀ1 in the IR spectrum) was left to the color to change (hypsochromic shift) from blue to be assigned to C-70 thus suggesting a quinonemethide orange, consistent with the conversion of the quinone- structure for the chromophore. The chromophore’s methide into a pyrylium chromophore. Furthermore, the M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 1019 addition of DCl shifted nearly all the signals downfield authentic samples of D. parviflora collected in Java and in the 1H NMR spectrum. While most overlapping sig- Borneo and D. candenatensis collected in Singapore, nals in the downfield region were resolved, some of the each supported with herbarium vouchers. Botanical lit- signals in the upfield region became congested. Com- erature also supports our determination of the red wood parison of the 1a spectra and that of the protonated which is the source of both candenatone and retusa- form (2), however, made assignment of the signals pos- purpurin A. D. parviflora is a giant liana that develops a sible. The isoflavan partial structure was corroborated by dark red heartwood; records of its use as a medicinal 2D NMR spectral data obtained for the protonated form plant and for incense date back into the middle ages (2). All NOEs were positive except for the one between (Prain, 1904; Burkill, 1935). D. candenatensis is a climb- the protons at 4.30 and 3.61, which, we assume, was ing shrub of no economic importance (Chayamarit, evidence of a three-spin effect between methine and 1986; Niyomdham et al., 1997). There exists a possible methylene protons. NOE between the singlet at 8.20 source of confusion about these two species which might and a 2H doublet (H-2000 and 6000) corroborated the con- have passed down in botanical literature since a mono- nectivity of the para-substituted aromatic ring to the rest graph of Indian Dalbergia (Thothathri, 1987) apparently of the molecule. NOE between the singlet at 8.20 and misquoted an earlier work (Kurz, 1877) attributing the the proton signal at 8.23 belonging to the 1,2,4-trisub- red wood D. parviflora to D. candenatensis. Identification stituted ring was expected but could not be detected of the botanical sources of candenatone and retusa- because of spectral overlap. The signals for protons purpurin A as same means that candenatone and simple belonging to hydroxyl groups predicted by the molecular isoflavonoids hitherto attributed to D. candenatensis formula were apparently missing. 13C NMR as well as 2D (Hamburger et al., 1987, 1988) should be referred to D. NMR spectroscopic data provided an overdetermined parviflora. system of correlations from which the unambiguous A rigorous comparison of the tissue culture- and structure 2 could be deduced. The 2,4-diarylbenzopyr- heartwood-derived pigment has been carried out to ylium structure of 2 is in complete agreement with 2 being support the premise that stress metabolism pathways the protonated form of the quinonemethide 1a. that are shared with heartwood-type secondary meta- Retusapurpurin A (1a) is the first example of a new bolism subpathways are active in Dalbergia cell cultures. type of C30 isoflavan whose formation can be rationa- Retusapurpurin A purified from callus tissue of D. lized to occur via regioselective oxidative coupling of the retusa and D. parviflora, respectively, and from D. par- isoflavan neovestitol (Ingham, 1979) to 4,40-dihydroxy- viflora wood had identical color reactions, fluorescence, 20-methoxychalcone (Carlson and Dolphin, 1982). UV/VIS spectrum, solvatochromic, and thermochromic Retusapurpurin A belongs to a relatively small group behavior, molecular weight determined by ESI MS, and of natural products: isoflavonoids condensed with a they comigrated in HPLC and several TLC solvent sys- chalcone unit. The closest relative is candenatone (3), in tems (see Experimental). which the A-ring of an isoflavan (Hamburger et al., The subpathway to 4,40-dihydroxy-20-methoxychalcone 1988) is coupled to the chalcone unit (6!b linking). The (Carlson and Dolphin, 1982) is already known at the level orange pigments of sandalwood (Pterocarpus santalinus, of genes in alfalfa, and most of the genes for the isoflavan P. soyauxii, and P. osun; Arnone et al., 1977a,b, 1981) pathway are known (Akashi et al., 1998; Dixon et al., and camwood (Baphia nitida; Arnone et al., 1975) have 1998). Retusapurpurin A represents an extension of the an entirely different fused ring system formally derived known isoflavonoid pathway in the direction of heart- from two-point linking of an isoflavonoid to a chalcone. wood-type metabolism, and the biosynthetic gene(s) must The structures of the other fluorescent and non-fluor- be responsive to both stress and developmental cues. The escent purple pigments of D. parviflora cell cultures have next challenge is to identify the substrates undergoing not been elucidated thus far. Based on chromatographic coupling, and to determine whether the enzymatic cou- behavior, UV spectral data (not shown) and preliminary pling is as regioselective in vitro as in vivo. Identification NMR spectroscopic data, at least some of these fluor- of the enzyme(s) will afford access to genes involved in escent pigments appear to have the same skeleton as stress metabolism common to several species and possibly retusapurpurin A (1a). The differences may be in ring support research into the yet uncharted heartwood-type substitution, and some of the minor pigments may be metabolic and cellular differentiation. similar to candenatone (3) or represent other bimole- cular coupling modes. D. parviflora wood was reported to contain arylben- 3. Experimental zofurans, neoflavonoids, and a benzophenone (Muang- noicharoen and Frahm, 1981, 1982). The wood from 3.1. General experimental procedures which candenatone (3) was isolated as the major heartwood pigment (Hamburger et al., 1988) was identified in For structural assignments, NMR spectra were our laboratory as D. parviflora by direct comparison to recorded on solutions (0.8 ml) of 1a and 2 in DMSO-d6 1020 M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022

(Cambridge Isotope Laboratories, Andover, MA) with obovata E. Meyer (c), D. obtusa Lecomte (h), D. oliveri a Varian INOVA 500 spectrometer (1H NMR, 500.163 Gamble (h,c), D. ovata Graham (h), D. palo-escrito MHz, 13C NMR, 125.781 MHz) at 25C. The reso- Rzedowski & L. I. Guridi-Gomez (h), D. parviflora nances were measured relative to internal DMSO-d6 and Roxb. (h,c), D. pterocarpifolia Baker (h), D. retusa the chemical shifts are expressed in parts per million (). Hemsley (h,c), D. sericea G. Don (c), D. sissoo Roxb. Standard Varian supplied pulse sequences were used for (h,c), D. spruceana Benth. (h), D. stevensonii Standley gradient enhanced absolute value COSY, phase-sensi- (h), D. trichocarpa Baker (c), D. tucurensis J. D. Smith tive double quantum filtered DQF-COSY, phase sensi- (h,c), Haematoxylon braziletto Karst. (h,c), H. campe- tive NOESY, and gradient enhanced HMQC and chianum L. (h,c), Pterocarpus angolensis DC. (h,c), P. HMBC experiments. Mass spectra were recorded on a antunesii (Taub.) Harms (c), P. erinaceus Poir. (c), P. VG Trio 3 triple quadrupole instrument (Beverly, MA); indicus Willd. (h,c), P. macrocarpus Kurz (h,c), P. the ESI and APCI sources were designed by Analytica rotundifolius Druce (h,c), P. santalinus L.f. (h), P. (Branford, CT). IR spectrum of 1a was recorded on a soyauxii Taub. (h), P. tinctorius Welw. (h,c). Perkin Elmer FTIR 1600 Series instrument (16 scans, D. candenatensis stem and live plants were collected in resolution: 4 cmÀ1). Pulau Semakau (Singapore) and identified by Dr. Hugh T.W. Tan (School of Biological Sciences, The National 3.2. Plant materials University of Singapore, herbarium vouchers for our samples are SINU Nos. SM DC 27 Sep 1996, and SM The comparative phytochemical analysis included DC 2). Sample labeled as ’D. candenatensis’ was a gift heartwood samples (h) and cell cultures (c) of the spe- from Dr. N. Ruangrungsi (Faculty of Pharmaceutical cies listed below. Callus cultures were initiated from in Sciences, Chulalongkorn Univ., Bangkok). The sources vitro germinated seedlings or from explants of plants of D. parviflora wood samples were as follows. Sample cultivated in the greenhouse. RTIw7917 (for abbreviations of institutional wood col- Baphia nitida Lodd. (h,c), Caesalpinia bonducella (L.) lections see Stern, 1988), collected in Indonesia (cut off Fleming (h,c), C. cacalaco Bonpl. (c), C. coriaria from a larger sample supported by a voucher: Herbar- (Jacq.) Willd. (c), C. decapetala (Roth) Alston (c), ium Bogoriense #8326) and sample without number, C. ferrea Mart. (c), C. gilliesii (Wall. ex Hook.) Borneo, were from Dr. Iep Wiselius (I.W.R.A. Con- Dietr. (c), C. pulcherrima (L.) Swartz (c), C. sepiaria sultancy, Woodsection, Westzaan, The Netherlands). Roxb. (c), C. spinosa (Mol.) O. Kuntze (c), C. tinc- Commercial samples identical to D. parviflora were toria Dombey ex DC. (c), C. vesicaria L. (c), C. violacea from the Kamwo Trading Co., New York, NY (Herbal (Mill.) Standl. (h,c), Dalbergia arbutifolia Baker (c), Catalog #HA108). Seeds were from Dr. Ir. Suhirman, D. assamica Benth. (c), D. balansae Prain (a), D. bar- Bogor Botanic Gardens, Indonesia. D. retusa seeds were iensis Pierre (h), D. baroni Baker (h), D. boehmii from C. Elevich (AgroForester Tropical Seeds, Holua- Taubert (h), D. brownei (Jacq.) Urban (h,c), D. caly- loa, Hawaii), and the wood samples were RBHw 4807, cina Benth. (h,c), D. cambodiana Pierre (h), D. cande- RBHw 16229, and MADw 42824. Additional informa- natensis (Dennst.) Prain (h,c), D. cearensis Ducke tion on the sources of plant materials can be obtained (h), D. chermezonii R. Viguier (h), D. chlorocarpa R. from the authors. Viguier (h), D. cochinchinensis Pierre ex Laness. Seeds and leaves were surface sterilized with mercuric (h,c), D. congestiflora Pittier (h), D. cubilquitzensis (J. chloride (0.1% v/w, 15 min). The seeds were incubated D. Smith) Pittier (h,c), D. cultrata Graham (h,c), D. in sterile moist cotton. Rescue of living tissue by exci- cuscatlanica (Standley) Standley (h), D. decipularis sion from the testa was necessary with old seeds (D. Rizz. & A. Mattos (h), D. densa Benth. var. australis calycina, D. miscolobium, D. parviflora) in which large (c), D. ecastaphyllum (L.) Taubert (h,c), D. floribunda portions of the cotyledon or the embryo were dead. The Craib (h), D. foliolosa Benth. (h), D. frutescens (Vell. tissues rescued from seeds, cotyledons or leaves cut Conc.) Britton (h), D. funera Standley (h), D. fusca from germinated seeds, and the surface sterilized leaves Pierre (h), D. glabra (Miller) Standley (h,c), D. were placed on callus induction medium (26–28 oC; granadillo Pittier (h), D. greveana Baillon (h), D. hai- continuous illumination, 30–50 mmol mÀ2 sÀ1; mixture nanensis Merr. & Chun (c), D. hupeana Hance (h,c), of incandescent and cool white fluorescent tubes: Syl- D. hypoleuca Pittier (h), D. latifolia Roxb. (h,c), D. vania and Power-Twist Vita-Lite 40 W). lineata Pittier (h), D. louvelii R. Viguier (h), D. All tissue culture media were supplemented with 30 g madagascariensis Vatke (h), D. maritima R. Viguier lÀ1 sucrose, solidified with Bacto agar 7 g lÀ1, and ster- (h), D. mammosa Pierre (h), D. martinii F. White (c), ilized in a pressure cooker (109 oC, 35 kPa, 25 min); the D. melanocardium Pittier (c), D. melanoxylon Guille- pH was adjusted to 5.8 before autoclaving. DM-1 med- min & Perrottet (h,c), D. miscolobium Benth. (h,c), ium contained MS (Murashige and Skoog, 1975) basal D. nigra (Vell.) Allema‹ oexBenth.(h,c), D. nigres- salts (Sigma Fine Chemicals) 4.3 g; KH2PO4 (6% [w/v]), cens Kurz (h,c), D. nitidula Welw. ex Baker (h), D. 3 ml; myo-inositol, 100 mg; vitamin B1, 1 mg; vitamin M. Czako´,L.Ma´rton / Phytochemistry 57 (2001) 1013–1022 1021

B6, 0.1 mg; glycine, 0.2 mg, nicotinic acid, 0.1 mg, folic precipitate in H2O(1 g per 200 ml H 2O) with stirring. The acid, 0.05 mg, biotin, 0.1 mg, and supplemented with resulting brownish red solid was collected by centrifuga- the plant growth regulators adenine hemisulfate, 10 mg; tion and fractionation was repeated thrice with fresh H2O picloram, 0.12 mg; indole-3-butyric acid, 1 mg; 2,4- (200 ml). The water insoluble pellet was further fractio- dichlorophenoxyacetic acid, 0.5 mg; isopentenyladenine, nated by suspending in benzene (1 g per 200 ml) with 0.5 mg; and trans-zeatin, 0.5 mg per liter. DM-2 med- stirring and incubation at 50C. Benzene extraction was ium was DM-1 supplemented with 0.1 M mannitol. repeated thrice. The insoluble pigment was collected by DM-3 medium differs only in the plant growth reg- centrifugation, and redissolved in ethanol to give a dark ulators: adenine hemisulfate, 10 mg; 2,4-dichlor- brownish red solution. Preparative scale separation of the ophenoxyacetic acid, 0.2 mg per liter; thidiazuron 0.022 ethanol extract by TLC on Whatman PK5 preparative mg. DM-4 contained Anderson’s (Anderson, 1984) silica gel plates (1 mm on glass, pore size: 25–150 A˚ ) was rhododendron basal salt mixture (Sigma Fine Chemi- carried out first in solvent system II. There were at least cals) 1.89 g, vitamins of DM-1, and supplemented with three high mobility UV fluorescent (pink) and several UV the plant growth regulators; 2,4,5-trichlorophenoxy absorbent purple/red components of lower mobility. The acetic acid, 0.04 mg per liter; thidiazuron 0.022 mg. main component, retusapurpurin A (third band from top, DM-5 contained MS salts 4.3 g and 0.022 mg thidia- Rf. 0.63, color in HCl fumes is orange yellow, HCl+UV: zuron. DM-6 was MS salts, 4.3 g; vitamins of DM-1; orange yellow, in NH3 red purple, NH3+UV: mauve adenine hemisulfate, 10 mg; 1-naphthaleneacetic acid, pink, intermediate colors under neutral conditions), was 0.2 mg lÀ1; thidiazuron 0.022 mg. DM-7 was the same subjected to rechromatography first in solvent system I À1 as DM-6 except that it contained 0.2 mg l picloram (Rf 0.71) to separate it from traces of contaminants, then instead of 1-naphthaleneacetic acid. DM-8 was the same twice in solvent system II. Yield was approximately as DM-1 except that it contained also 3 mg thidiazuron, 0.01% of dry weight. and as much as 60 mg adenine hemisulfate per liter. Retusapurpurin A was identified in the ethanol extract DM-9 contained CLC/Ipomoea (CP) Basal salt mixture of D. parviflora heartwood as the main fluorescent pink (Che´ e et al., 1992; Sigma Fine Chemicals) 6.7 g, vita- band. It was isolated from the heartwood and callus by mins of DM-1, and supplemented with the plant growth the procedure applied to D. retusa callus (yield was regulators; 2,4,5-trichlorophenoxyacetic acid, 0.04 mg about 0.03 and 0.01% of dry weight, respectively). per liter; thidiazuron 0.022 mg. DM-10 contained C2d vitis basal salt mixture (Che´ e and Pool, 1987, Sigma 3.4. Retusapurpurin A (1a) Fine Chemicals) 4.47 g, vitamins of DM-1, 2,4,5-trichlorophenoxyacetic acid, 0.04 mg, and thidiazuron, Deep red amorphous powder. UV/VIS (EtOH–0.1 M 0.022 mg. Na-citrate (pH 5.0) 2:1 (v/v)): lmax nm: 533 sh, 510, 392, All Baphia, Caesalpinia, Haematoxylon,andPterocarpus 320 sh, 279. +NaHCO3: 567, 533 sh, 419, 401, 323, 300, as well as most Dalbergia species developed callus on 288 sh, 271. +AlCl3 +HCl or +HCl alone: 490, 420 sh, DM-1 medium; the exceptions are listed in Table 1. In 284. lmax (DMSO): 610, 571 sh, 526 sh, 433, 388, 338, À1 D. melanocardium, primary callus formed at the base of 280. IR max (KBr) cm : 1635. MS (ESI, positive ion shoot tips excised from in vitro germinated seedlings mode): 523 [M+H]+ and m/z 413 (7), 399 (8), 387 (48), and inserted into DM-5 medium, and this callus was 371 (7), and 233 (10). MS (APCI, high capture voltage): maintained on DM-6 medium. [M+H]+ 523. NMR: Table 2. Retusapurpurin A isolated from D. retusa callus 3.3. Pigment survey and isolation appeared homogeneous and comigrated on TLC with samples isolated from D. parviflora callus and wood in Pigments extracted in ethanol were separated by paper systems I (pink/red, UV: pink; Rf 0.71), II (Rf 0.63), III electrophoresis (in 50% acetic acid, 25 V/cm on What- (toluene-methanol-water 140:77:3, Rf 0.60), IV (chloro- man 3MM paper spanned between the buffer chambers form-tetrahydrofuran-water 40:70:2, Rf 0.33), V (ethyla- connected to platinum electrodes) and TLC on silica gel cetate–tetrahydrofuran–water 40:23:2, Rf 0.57), and in (250 mm on polyester backed flexible plates, PE SIL G, HPLC: 4.92 min (normal phase silica, 5 mm 10 cm, 100 ˚ Whatman) in solvent systems I (ethylacetate–methanol– A (Microsorb-MV) in MeOH-CHCl3 (1:19), isocratic water 100:16:13) and II (chloroform–methanol–water separation on a Beckman Model 332 gradient liquid 80:20:2) (Hamburger et al., 1988). chromatograph using a Model 165 variable wavelength For purification on a preparative scale, Dalbergia detector). retusa callus pieces showing red coloration were repeat- edly extracted with ethanol at 66 oC to give rise to a deep Acknowledgements orange red solution. 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