A Photometric Screening Method for Dimeric Naphthylisoquinoline

An efficient evaluation procedure for the chemical screen- ing and on-line structural elucidation of dimeric naph- thylisoquinoline alkaloids has been...
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Anal. Chem. 2001, 73, 2571-2577

A Photometric Screening Method for Dimeric Naphthylisoquinoline Alkaloids and Complete On-Line Structural Elucidation of a Dimer in Crude Plant Extracts, by the LC-MS/LC-NMR/LC-CD Triad Gerhard Bringmann,*,† Michael Wohlfarth,† Heiko Rischer,† Markus Heubes,† Wael Saeb,† Stefanie Diem,‡ Markus Herderich,‡ and Jan Schlauer†

Institute of Organic Chemistry, University of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany, and Institute of Pharmacy and Food Chemistry, University of Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany

An efficient evaluation procedure for the chemical screening and on-line structural elucidation of dimeric naphthylisoquinoline alkaloids has been developed. The method is based on the lead tetraacetate oxidation of the central binaphthalene core of the alkaloids. UV spectra of the extracts after addition of the oxidant show, in the presence of naphthylisoquinoline dimers, the appearance of a characteristic long-wavelength absorption indicative of dinaphthoquinones. The efficiency and relevance of the method has been demonstrated in the discovery of a constitutionally and configurationally new dimeric naphthylisoquinoline alkaloid, named ancistrogriffithine A (4), from the previously uninvestigated Asian liana Ancistrocladus griffithii. After verification of this screening result by LC-ESI-MS/MS, the constitution and the relative configuration of the compound were elucidated on line, by LC-NMR and LC-CD on the extract. Using an LCNMR-WET-ROESY experiment, it was possible for the first time to determine the relative axial configuration of a natural biaryl compound on line, by observing long-range ROE interactions. Finally, an oxidative degradation right on the extract delivered the absolute configuration of 4, without isolation of the alkaloid. Ancistrogriffithine A is the as yet only dimeric naphthylisoquinoline from an Asian Ancistrocladaceae plant. Dimeric naphthylisoquinoline alkaloids (see Figure 1) are pharmacologically interesting target compounds because some of their representatives, in particular michellamines A (1a) and B (1b), exhibit strong anti-HIV activities.2 More recently, a * Corresponding author: (tel) +49 931 8884323; (fax) +49 931 8884755; (email) [email protected]. † Institute of Organic Chemistry. ‡ Institute of Pharmacy and Food Chemistry. (1) This paper is part 145 of the series Acetogenic Isoquinoline Alkaloids; for part 144, see: Bringmann, G.; Wenzel, M.; Bringmann, H.; Ake´ Assi, L.; Haas, F.; Schlauer, J. Carniv. Plant Newslett. 2001, 30, 15-21. (2) Boyd, M. R.; Hallock, Y. F.; Cardellina, J. H., II; Manfredi, K. P.; Blunt, J. W.; McMahon, J. B.; Buckheit, R. W., Jr.; Bringmann, G.; Scha¨ffer, M.; Cragg, G. M.; Thomas, D. W.; Jato, J. G. J. Med. Chem. 1994, 37, 17401745. 10.1021/ac001503q CCC: $20.00 Published on Web 04/28/2001

© 2001 American Chemical Society

“mixed”, constitutionally unsymmetric bis(naphthylisoquinoline) was isolated: korundamine A (2), which has good antimalarial activity.3 Dimeric naphthylisoquinolines such as 1a, 1b, and 2 along with four other michellamines2,4,5 have so far been known only from Ancistrocladus korupensis (Ancistrocladaceae), a rare liana growing in high forests of Cameroon and bordering Nigeria. The rarity of this plant together with the promising activities of the contained compounds has prompted screening campaigns by several groups.6-10 However, these did not reveal any further natural sources of michellamines or other dimeric alkaloids. The analytical on-line techniques used so far in the screening for michellamines were based on chromatographic separation11 and sophisticated detection methods such as HPLC-ESI-MS/MS,12 pending development of a more simplified test that consumes less time and resources. The key observation for the improved method presented here arose from our total synthesis of michellamines. Recently, it was shown that dimeric naphthylisoquinolines are biosynthetically derived from their respective monomers by oxidative coupling catalyzed by phenoloxidases.13 Synthetically, this principle was (3) Hallock, Y. F.; Cardellina, J. H., II; Scha¨ffer, M.; Bringmann, G.; Franc¸ ois, G.; Boyd, M. R. Bioorg. Med. Chem. Lett. 1998, 8, 1729-1734. (4) Hallock, Y. F.; Manfredi, K. P.; Dai, J.-R.; Cardellina, J. H., II; Gulakowski, R. J.; McMahon, J. B.; Scha¨ffer, M.; Stahl, M.; Gulden, K.-P.; Bringmann, G.; Franc¸ ois, G.; Boyd, M. R. J. Nat. Prod. 1997, 60, 677-683. (5) Hallock, Y. F.; Cardellina, J. H., II; Scha¨ffer, M.; Bringmann, G.; Franc¸ ois, G. Bioorg. Med. Chem. Lett. 1998, 8, 1729-1734. (6) Manfredi, K. P.; Britton, M.; Vissieche, V.; Pannel, L. K. J. Nat. Prod. 1996, 59, 854-859. (7) Anh, N. H.; Porzel, A.; Ripperger, H.; Bringmann, G.; Scha¨ffer, M.; God, R.; Sung, T. V.; Adam, G. Phytochemistry 1997, 45, 1287-1291. (8) Bringmann, G.; Gu ¨ nther, C.; Busemann, S.; Scha¨ffer, M.; Olowokudejo, J. D.; Alo, B. Phytochemistry 1998, 47, 37-43. (9) Tang, C.-P.; Yang, Y.-P.; Zhong, Y.; Zhong, Q.-X.; Wu, H.-M.; Ye, Y. J. Nat. Prod. 2000, 63, 1384-1387. (10) Bringmann, G.; Gu ¨ nther, C.; Saeb, W.; Mies, J.; Wickramasinghe, A.; Mudogo, V.; Brun, R. J. Nat. Prod. 2000, 63, 1333-1337. (11) McCloud, T. G.; Britt, J. R.; Cartner, L. K.; Pearl, K. C.; Majadly, F. D.; Muschik, G. M.; Klueh, P. A.; Cragg, G. M.; Thomas, D. W.; Jato, J. G.; Simon, J. E. Phytochem. Anal. 1997, 8, 120-123. (12) Bringmann, G.; Ru ¨ ckert, M.; Schlauer, J.; Herderich, M. J. Chromatogr., A 1998, 810, 231-236.

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Figure 1. Michellamine A (1a) and B (1b), korundamine A (2), a quinoidic derivative 3 (with R, R′ ) protecting groups or H), and the novel dimeric naphthylisoquinoline alkaloid, ancistrogriffithine A (4).

used to build up the central biaryl axis of michellamines biomimetically by oxidative phenolic coupling, initially with oxidants such as Ag2O14,15 (which required, however, use of O- and N-protective groups). The product immediately formed was an intensely violet-blues“overoxidized”squinoid dimer 3 (e.g., for R ) R′ ) benzyl protective groups14) (see Figure 1), and only after mild reduction, was the colorless quateraryl (e.g., 1a or 1b) target obtained. The development of an optimized test for dimeric naphthylisoquinolines was initiated by the discovery that the use of lead tetraacetate [Pb(OAc)4] permits the smooth regioselective coupling of authentic, unprotected monomeric precursors to the corresponding dimers.16 The new test is based on the observation that even the biphenolic aromatic target dimers I themselves (e.g., 1a or 1b) can be reoxidized to the colored quinoid intermediates II by the same reagent (see Figure 2).16 As for similar (simpler) systems in the literature,17 the absorbance maximum of the dimer of type II was determined to be 525 nm, and fortunately, the naphthylisoquinoline-producing plants investigated here contain only very few plant pigments with absorbance maximums in this region. (13) Schlauer, J.; Ru ¨ ckert, M.; Wiesen, B.; Herderich, M.; Ake´ Assi, L.; Haller, R. D.; Ba¨r, S.; Fro ¨hlich, K.-U.; Bringmann, G. Arch. Biochem. Biophys. 1998, 350, 87-94. (14) Bringmann, G.; Go¨tz, R.; Harmsen, S.; Holenz, J.; Walter, R. Liebigs Ann. Chem. 1996, 2045-2058. (15) Hoye, T. R.; Chen, M.; Hoang, B.; Mi, L.; Priest, O. P. J. Org. Chem. 1999, 64, 7184-7201. (16) Bringmann, G.; Tasler, S. Tetrahedron 2001, 57, 331-343. (17) Laatsch, H. Liebigs Ann. Chem. 1991, 1085-1089.

2572 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

Figure 2. The key reaction and fundamental basis of the photometric analysis: formation of diquinoid intermediates II from binaphthols of type I, by oxidation with Pb(OAc)4.

With this new photometric assay, a number of Ancistrocladaceae and Dioncophyllaceae species were screened. These palaeotropical plant families are the, as yet exclusive, source of a broad variety of naphthylisoquinolines.18,19 The screening disclosed (18) Bringmann, G.; Pokorny, F. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: New York, 1995; Vol. 46, pp 127-171. (19) Bringmann, G.; Franc¸ ois, G.; Ake´ Assi, L.; Schlauer, J. Chimia 1998, 52, 18-28.

the presence of an as yet unknown dimeric naphthylisoquinoline alkaloid in Ancistrocladus griffithii, an Asian Ancistrocladaceae species not hitherto investigated phytochemically. Since only small amounts of cultivated plant material were available, crude extracts of twigs and leaves of A. griffithii were investigated by using the analytical triad LC-MS/MS, LC-NMR, and LC-CD/UV. In different combinations, these hyphenated techniques have already proven their value for the analysis of complex matrixes.20-24 The on-line elucidation of relative or absolute configurations of chiral molecules right from crude extracts, however, without the use of chiral chromatographic phases, is still a demanding task, which has been accomplished only rarely.25-29 In this paper, we present a screening procedure for this pharmacologically promising class of dimeric naphthylisoquinoline alkaloids. Crude plant extracts were investigated photometrically and then, if they gave positive test results, further examined in depth by LC-MS/MS, LC-NMR, and LC-CD. Using an on-line LC-ROESY experiment recently elaborated,26-29 applying the WET30 pulse sequence for solvent suppression, it has now become possible to determine the full structure of a dimeric naphthylisoquinoline directly in an extract, including the relative configuration at the stereogenic centers and at the chiral axis. A microscale oxidative degradation procedure developed by us earlier31 led to the final determination of the absolute configuration of the dimeric naphthylisoquinoline alkaloid ancistrogriffithine A (4). EXPERIMENTAL SECTION Plant Material. Leaves of the following species of Dioncophyllaceae and Ancistrocladaceae were analyzed: Triphyophyllum peltatum, Ancistrocladus barteri, and Ancistrocladus abbreviatus (all from Ivory Coast, supplied by Prof. Dr. L. Ake´ Assi); Ancistrocladus cf. pachyrrhachis (Sierra Leone, K. Dumbuya); Ancistrocladus cf. guinee¨nsis (Nigeria, Prof. B. Alo); Ancistrocladus korupensis (Cameroon, Prof. J. F. Ayafor); Ancistrocladus ealae¨nsis (Gabon, Dr. A. Louis); Ancistrocladus congolensis, A. ealae¨nsis, Ancistrocladus cf. letestui, and Ancistrocladus likoko (all from Democratic Republic of Congo, Prof. V. Mudogo); Ancistrocladus tanzanie¨nsis (Tanzania, F. Mbago); Ancistrocladus robertsoniorum (Kenya, Dr. R. Haller and Dr. S. Ba¨r); Ancistrocladus heyneanus (India, Dr. S. M. Ketkar); Ancistrocladus hamatus (Sri Lanka, Dr. A. Wickramasinghe); Ancistrocladus tectorius (Malaysia, Prof. H. Hadi); A. griffithii (20) Schneider, B.; Zhao, Y.; Blitzke, T.; Schmitt, B.; Nookandeh, A.; Sun, X.; Sto ¨ckigt, J. Phytochem. Anal. 1998, 9, 237-244. (21) Mistry, N.; Roberts, A. D.; Tranter, G. E.; Francis, P.; Barylski, I.; Ismail, I. M.; Nicholson, J. K.; Lindon, J. C. Anal. Chem. 1999, 71, 2838-2843. (22) Renukappa, T.; Roos, G.; Klaiber, I.; Vogler, B.; Kraus, W. J. Chromatogr., A 1999, 847, 109-116. (23) Albert, K. J. Chromatogr., A 1999, 856, 199-211. (24) Sandvoss, M.; Pham, L. H.; Levsen, K.; Preiss, A.; Mu ¨ gge, C.; Wu ¨ nsch, G. Eur. J. Org. Chem. 2000, 1253-1262. (25) Garo, E.; Wolfender, J.-L.; Hostettmann, K.; Hiller, W.; Antus, S.; Mavi, S. Helv. Chim. Acta 1998, 81, 754-763. (26) Bringmann, G.; Gu ¨ nther, C.; Schlauer, J.; Ru ¨ ckert, M. Anal. Chem. 1998, 70, 2805-2811. (27) Bringmann, G.; Messer, K.; Wohlfarth, M.; Kraus, J.; Dumbuya, K.; Ru ¨ ckert, M. Anal. Chem. 1999, 71, 2678-2686. (28) Bringmann, G.; Ru ¨ ckert, M.; Messer, K.; Schupp, O.; Louis, A. M. J. Chromatogr., A 1999, 837, 267-272. (29) Bringmann, G.; Ru ¨ ckert, M.; Saeb, W.; Mudogo, V. Magn. Reson. Chem. 1999, 37, 98-102. (30) Smallcombe, S. H.; Patt, S. L.; Keifer, P. A. J. Magn. Reson. A 1995, 117, 295-303. (31) Bringmann, G.; God, R.; Scha¨ffer, M. Phytochemistry 1996, 43, 1393-1403.

(originally from Thailand, ex cult., Botanical Garden of Wu¨rzburg, A. Kreiner); and A. tectorius (Indonesia, Prof. Dr. W. Smits). Herbarium specimens of these species are deposited at the Herbarium Bringmann, Institute of Organic Chemistry, University of Wu¨rzburg, Germany. Materials. Acetonitrile (MeCN, Pestanal quality) was from Riedel de Ha¨en (Deisenhofen, Germany) and deuterium oxide (D2O, 99.9%) from Promochem GmbH (Wesel, Germany). Water was demineralized with a Milli-Q water purification system from Millipore (Milford, CT). Trifluoroacetic acid (TFA, UV grade) was obtained from Merck (Darmstadt, Germany). Lead tetraacetate [Pb(OAc)4] was supplied by Sigma Aldrich Chemie GmbH (Deisenhofen, Germany). Pb(OAc)4 is classified as a highly toxic and potentially teratogenic substance. Any contamination or incubation should be strictly avoided. Sample Preparation for Dimer Assay. A 100-mg sample of air-dried leaves was ground to a fine powder in liquid nitrogen and suspended in 1 mL of acetic acid (glacial). The suspension was ultrasonified for 2 h at 25 °C. The extract was centrifuged for 15 min at 9000g. Fifty microliters of the supernatant was used for the photometric dimer assays. Photometric Assay. To 800 µL of acetic acid (glacial) in a microcuvette (1 mL total probe volume), 50 µL of crude extract was added, and the mixture was stirred until a homogeneous color was achieved. The blank absorbance of this solution at 525 nm was measured. Subsequently 15 µL of Pb(OAc)4 solution (10 mg/ mL in glacial acetic acid) was added, the solution was stirred, and the sample absorbance was again measured at 525 nm after 1 min. The difference between sample and blank measurements was taken as a measure of the total concentration of potentially oxidable naphthylisoquinoline dimers (including their respective monomers newly dimerized during the Pb(OAc)4 reaction) in the extracts. A value of ∆E525 ) 0.87, SD ) 0.022 was determined for 50 µL of a 1 mM michellamine B (1b) solution in the assay reaction (control samples gave a mean value of ∆E525 ) 0.02, SD ) 0.017). Estimated dimer concentrations calculated from this value are listed up in Table 1. Sample Preparation for Hyphenated Analysis. One gram of dried and ground leaves of A. griffithii was extracted with a mixture of water (pH 2 with TFA) and MeCN (8:2, v/v) in an ultrasonic bath at room temperature for 2 h. The extract solution was filtered and lyophilized to yield ∼30 mg of extract, of which 5 mg was redissolved in 500 µL of D2O/MeCN (8:2, v/v) and filtered through a 0.2-µm membrane filter. The resulting solution was directly used for hyphenated analyses. For twig extract analysis, 5 g of dried and ground twigs from A. griffithii was extracted and lyophilized as described above. The lyophilized extract (∼210 mg) was subsequently redissolved in methanol/dichloromethane (1:9, v/v, basified with ammonia) and filtered over a short silica gel column (deactivated with concentrated ammonia, 7.5, w/w) with methanol/dichloromethane (50 mL 9:1, v/v and 50 mL 8:2, v/v) as the eluent. The more polar crude fraction was dried in vacuo to yield ∼30 mg fractionated extract, of which 2 mg was redissolved in 150 µL of MeCN/D2O (8:2, v/v) and used for HPLC-NMR analysis. General HPLC Parameters. All chromatographic separations were performed using Symmetry C-18 columns from Waters (5 µm, 150 × 3.0 mm i.d. for HPLC NMR, 150 × 2.1 mm i.d. for Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

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Table 1. Results of the Photometric Screening for Dimeric Alkaloids in Plant Extracts taxon

∆E525

est dimer concn in extract (mM)

T. peltatum A. barteri A. abbreviatus A. cf. pachyrrhachis A. cf. guinee¨nsis A. korupensis A. ealae¨nsis (GA)a A. ealae¨nsis (CD)a A. cf. letestui A. likoko A. congolensis A. tanzanie¨nsis A. robertsoniorum A. heyneanus A. hamatus A. tectorius (MY)a A. tectorius (ID)a A. griffithii

0.00 0.01 0.06 0.00 0.21 1.48 0.09 0.16 0.10 0.32 0.40 0.02 0.02 0.11 0.00 0.04 0.00 0.73

0.00 0.01 0.05 0.00 0.18 1.28 0.08 0.14 0.09 0.28 0.35 0.02 0.02 0.10 0.00 0.03 0.00 0.63

a GA, Gabon; CD, Democratic Republic of Congo; MY, Malaysia; ID, India.

HPLC-MS, and 250 × 4.6 mm i.d. for HPLC-CD). The mobile phases used were (A) acetonitrile, (B) demineralized water, and (C) deuterium oxide. (B) and (C) were acidified to pH 3 with trifluoroacetic acid. A binary gradient (acetonitrile/water for HPLC-MS and HPLC-CD or acetonitrile/D2O for HPLC-NMR) was programmed as follows: 0 min 5% A, 15 min 25% A, 30 min 30% A, and 40 min 50% A. The flow rate was set to 0.5 mL/min for HPLC-NMR, 0.2 mL/min for HPLC-MS, and 1.2 mL/min for HPLC-CD. HPLC-NMR was performed on a Bruker DMX 600 NMR spectrometer operating at 600.13-MHz 1H frequency (Bruker, Rheinstetten, Germany) and controlled by the software system XWinNMR from Bruker. The outlet of the UV detector was connected to the flow probe by a PEEK capillary (0.25-mm i.d.) via a Bruker peak sampling unit (BPSU) interface (Bruker), controlling the experimental modes. The interface and the HPLC system were controlled by a PC using the software packages BPSU-12 and Chromstar from Bruker. The spectrometer was equipped with an inversely constructed 1H,13C flow probe with a 3-mm (60-µL) detection cell (Bruker). The chromatographic system consisted of a Bruker LC22 pump and a UV detector from Bischoff working at 254-nm absorption. The injection volume was 100 µL of leaf extract or 150 µL of twig extract. The one-dimensional 1H NMR spectra were obtained using the following parameters: Free induction decays (FIDs) were collected into 32K data points with a spectral width of 12 kHz. Ninetydegree pulses were used with an acquisition time of 1.36 s, and the relaxation time delay was set to 1 s. Typically 32 scans were accumulated. A WET solvent supression with 13C decoupling was performed on the signals of MeCN and HOD (from D2O) using standard Bruker pulse programs (lc1wetcd). Prior to Fourier transformation, an exponential apodization function (0.5 Hz) was applied. For calibration, the signal of the solvent acetonitrile was set to 2.0 ppm. TOCSY experiments were carried out using a standard Bruker pulse program (LCMLCWPS) with a binomial pulse solvent 2574

Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

supression. Typically the FIDs were collected into 2K data points with a spectral width of 12 kHz in both dimensions. Ninety-degree pulses were used with an acquisition time of 0.122 s, and the relaxation delay was set to 2 s. The MLEV spin-lock field was applied for 65 ms. The data were apodized with a squared π/2 shifted sinusoidal window function in both dimensions. For resolution enhancement in F1, a linear prediction to 1K data points was applied. Phase-sensitive ROESY spectra were acquired using a modified ROESY sequence with a WET pulse sequence30 and 13C decoupling for solvent supression. Typically the FIDs were collected into 2K data points with a spectral width of 12 kHz in both dimensions. Ninety-degree pulses were used with an acquisition time of 0.122 s, and the relaxation delay was set to 2 s. The mixing time was 600 ms. The same data manipulation was performed as for the TOCSY experiment described above. HPLC-ESI-MS/MS was performed with a triple-stage quadrupole TSQ 7000 mass spectrometer equipped with a pneumatically assisted electrospray interface (Finnigan MAT, Bremen, Germany), and a Personal DECstation 5000/33 (Digital Equipment, Unterfo¨hring, Germany) with ICIS 8.1 software (Finnigan MAT). Nitrogen served both as sheath and auxiliary gas, with argon as collision gas (1.8 mTorr). The spray capillary voltage was 3.5 kV, and the temperature of the heated inlet capillary was 230 °C. Five microliters of extract was injected. Positive ions were detected by scanning from 170 to 900 u with a total scan duration of 1 s for a single spectrum. MS/MS experiments were performed with a collision energy of 20 eV, scanning a mass range from 20 to 850 u with a total scan duration of 1 s. The mass spectra were measured in the profile mode and referenced to 100% intensitiy of the base peak. HPLC-CD spectra were measured on a J-714 CD spectrometer, equipped with a LC-980-025 Ternary Gradient Unit, a PU1580 pump, and the Borwin chromatographic software (all Jasco, GrossUmstadt, Germany). For CD measurement (190-400 nm), the flow was manually detached from the pump, using a Rheodyne 7010 injection valve, connected to a Besta Motorventil “H” (Besta, Wilhelmshafen, Germany). UV chromatograms were monitored at 254 nm. For analysis, 50 µL of the leaf extract was injected. RESULTS AND DISCUSSION Photometric Assay. In the photometric survey of a broad series of Dincophyllaceae and Ancistrocladaceae species for potential occurrence of naphthylisoquinoline dimers (see Table 1), significantly positive results were obtained only for very few extracts. As expected, A. korupensis gave a strong signal, the highest one of the test series. This is also the as yet only species in which dimeric naphthylisoquinoline alkaloids had been detected.2,4,5 Among the Asian species, A. griffithii showed a strongly positive reaction. This species was therefore investigated more thoroughly, with special emphasis on potential dimeric alkaloids (see below). A. congolensis, A. likoko, and A. cf. guinee¨nsis, three African species with phylogenetic affinities32 relatively close to A. korupensis, gave positive but much lower signals in the photometric test, supposedly due to low concentrations of michellamines (or their monomeric halves). In this context, it is noteworthy that the likewise related species A. letestui and A. ealae¨nsis, as also (32) Heubl, G.; Meimberg, H.; Schlauer, J.; Rischer, H.; Wohlfarth, M.; Bringmann, G., unpublished results.

Figure 3. LC-ESI-MS chromatogram of a leaf extract of A. griffithii: (a) reconstructed ion chromatogram of m/z 785.4; (b) reconstructed ion chromatogram of m/z 393.4; (c) reconstructed total ion chromatogram (TIC).

Figure 4. Product ion spectrum of precursor ion m/z 393.4 of peak A at tR ) 16.9 min.

the remaining species investigated (including T. peltatum), did not reveal significant dimer concentrations in the photometric test (see Table 1). This result is consistent with the fact that no dimers had ever been isolated from these plants. LC-ESI-MS/MS Analysis. Following the positive result for A. griffithii in the photometric assay, we analyzed a leaf extract by LC-MS/MS. One main compound (“peak A”) gave simultaneously signals at m/z 785.4 and 393.4, which is in accordance with the mono- and diprotonated molecular ions [M + H]+ and [M + 2H]2+ of a substance with a molecular formula C48H52N2O8 (see Figure 3). A subsequent MS/MS experiment on [M + 2H]2+ at m/z 393.4 delivered a mass spectrum with masses up to 742.4 u (see Figure 4). This mass corresponds to a monoprotonated fragment [M + H - C2H5N]+, arising from a retro-Diels-Alder reaction12 of one tetrahydroisoquinoline part of a 6,8-dioxygenated dimeric naphthylisoquinoline alkaloid. This was further confirmed by typical diprotonated mass fragments such as [M + 2H - NH3]2+ with m/z 384.8 and [M + 2H - C2H5N]2+ with m/z 371.7. LC-NMR and LC-CD Analysis. Since the mass could not be brought into accordance with any other known dimeric naphthylisoquinoline, the structure of the new alkaloid, now named ancistrogriffithine A, was examined more closely by using LC-NMR. A stopped-flow experiment carried out on peak A provided a 1H NMR spectrum typical of a naphthylisoquinoline alkaloid, although displaying only one set of 12 proton signals (see Figure 5a), not two, as expected from the “double” molecular mass. Therefore, different from michellamine B (1b), both “monomeric” halves of the compound had to be equivalent, as in the case of michellamine A (1a) (see Figure 1).

Figure 5. (a) Stopped-flow NMR spectrum of 4 (leaf extract, tR ) 19.7 min, 26.7% MeCN, 32 scans acquired); (b) 2D WET-ROESY of 4 (twig extract, tR ) 19.7 min, 72 scans, 359 increments in F1, 17 h 8 min total acquisition time).

By analysis of the coupling constants and from TOCSY data (see Table 2), some initial assignments could be made. In the aliphatic region of the spectrum, one spin system with signals at δ 1.41, 3.39, and 2.92 ppm was assigned to 3-CH3, H-3, and H-4 (axial and equatorial H-4’s overlapping). A doublet at δ 1.63 ppm (1-CH3) showed a coupling to the quartet at δ 4.63 ppm (H-1). Furthermore, H-4 exhibited a TOCSY correlation to the singlet at δ 6.63 ppm, which was therefore assigned to be H-5. From these data it was obvious that the tetrahydroisoquinoline moiety had to be linked in 7-position to the naphthalene part. The singlet at δ 7.27 ppm showed a typical downfield shift, which can be explained by a hydrogen atom positioned meta to an oxygen substituent in the naphthalene moiety, and was therefore assigned to H-7′. The positions of the two methoxy groups at δ 3.26 and 4.04 ppm could not be assigned from chemical shift data alone. For the investigation of spatial structures of molecules, the most informative NMR experiments are nuclear Overhauser measurements, such as NOESY33 or ROESY.34 Although delivering information similar to that from ROESY experiments, the NOESY sequence is critical with respect to the relative sign of the NOE (33) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. J. Chem. Phys. 1979, 71, 4546-4563. (34) Bax, A.; Davis, D. J. Magn. Reson. 1985, 63, 207-213.

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Table 2. HPLC 1H NMR On-Line Data of Ancistrogriffithine A (4)

a

δ (ppm)

H-atom

multiplicity

J (Hz)

1.41 1.63 2.33 2.92a 3.26 3.39 4.04 4.63 6.63 6.81 6.90 7.27

3-CH3 1-CH3 2′-CH3 Hax-4/Heq-4 8-OCH3 H-3 4′-OCH3 H-1 H-5 H-1′ H-3′ H-7′

d d s d s m s q s s s s

6.6 5.9 7.3

5.9

WET-ROESY correlation to H-...

TOCSY correlation to H-...

3,4 18-OCH3 1′,3′ 3,3-CH3, 5 1,1-CH3, 1′,7′ 1,3-CH3, 4 3′ 1-CH3, 3,8-OCH3 4 1-CH3, 2′-CH3, 8-OCH3 2′-CH3, 4′-OCH3 8-OCH3

3,4 1 3′ 3,3-CH3, 5 3-CH3, 4 1-CH3 4 2′-CH3

Overlapping signals.

effect, which can become very small or even zero. From our experience, the ROESY sequence is therefore distinctly preferable for LC-NMR,26-29 since it does not have this disadvantage.34 A major obstacle that complicates both NOE and ROE measurements is still the generally low analyte concentration after analytical HPLC in the probe flow cell,35 and the viscosity of the solvents used.36 Many approaches in recent years compensate for this problem. As an example, sensitivity is gained by the introduction of higher field strengths37 or by better solvent suppression techniques such as WET30 or excitation sculpting.38 Miniaturized experimental arrangements, such as microcoils in combination with capillary chromatography,39,40 can provide a gain of sensitivity by several orders of magnitude as well.23,41 However, in the latter case, special attention must be paid to the coordination of HPLC with NMR parameters.42 To optimize our experimental conditions, we used a 3-mm (i.d.) column for HPLC chromatography, in combination with a 60-µL probe head, instead of a 4.6-mm (i.d.) column and a 120-µL probe head as applied in earlier work.26-29 Additionally, we replaced in our LC-ROESY setup the original NOESY type double presaturation by the more powerful WET sequence. These modifications led to a significant improvement in sensitivity and spectra quality. Thus, by using the LC-WET-ROESY experiment (see Figure 5b), the structure of the novel dimeric naphthylisoquinoline, including the constitution, the coupling type, and the relative configuration, was determined, which confirmed the assignment derived from the previous 1H and TOCSY experiments (see Figure 6). The methoxy group at δ 3.26 ppm showed correlations to H-1 and 1-CH3 and was established to be located at C-8. Furthermore, a correlation sequence in the order δ 4.04 f 6.90 f 2.33 f 6.81 (35) Ndjoko, K.; Wolfender, J.-L.; Ro¨der, E.; Hostettmann, K. Planta Med. 1999, 65, 562-566. (36) Derome, A. D. Modern NMR Techniques for Chemistry Research; Pergamon Press: Oxford, U.K.,1987; Chapter 5. (37) Sidelmann, U. G.; Braumann, U.; Hofmann, M.; Spraul, M.; Lindon, J. C.; Nicholson, J. K.; Hansen, S. T. Anal. Chem. 1997, 69, 607-612. (38) Parella, T.; Adell, P.; Sanchez-Ferrando, F.; Virgili, A. Magn. Reson. Chem. 1998, 36, 245-249. (39) Behnke, B.; Schlotterbeck, G.; Tallarek, U.; Strohschein, S.; Tseng, L.-H.; Keller, T.; Albert, K.; Bayer, E. Anal. Chem. 1996, 68, 1110-1115. (40) Subramanian, R.; Kelley, W. P.; Floyd, P. D.; Tan, Z. J.; Webb, A. G.; Sweedler, J. V. Anal. Chem. 1999, 71, 5335-5339. (41) Lacey, M. E.; Subramian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J. V. Chem. Rev. 1999, 99, 3133-3152. (42) Griffiths, L. Anal. Chem. 1995, 67, 4091-4095.

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Figure 6. Selected WET-ROESY correlations of 4 observed online by LC-NMR, and resulting assignment of all of the relative configurations (absolute configuration here still arbitrarily depicted); interactions, establishing the relative configurations, are indicated by exclamation marks (!).

ppm was assigned to a spin system 4′-OCH3 f H-3′ f 2′-CH3 f H-1′. The position of the biaryl axis between the isoquinoline and the naphthalene portion was unambiguously deduced from ROE correlations from two naphthalene protons, H-1′ and H-7′, both to 8-OCH3. This is conclusive evidence of a 7,8′-coupling site in both monomeric halves. From this structural assignment it is obvious that the only position left for a linkage between the two monomers is 6′ of the identical naphthalene moieties. This established the structure of the novel naphthylisoquinoline dimer to be 7,8′-6′,6′′-8′′,7′′′-coupled (see Figures 6 and 1). Regarding the relative configurations at centers and axes, a very strong correlation between thesthus spatially closesatoms H-1 and H-3 indicated the presence of a cis array in the tetrahydroisoquinoline part. Even more interesting was a weak, but significant correlation observed between H-1′ and 1-CH3, as such long-range dipolar couplings are suitable for the determination of the axial configuration of naphthylisoquinoline alkaloids.43,44 This is a remarkable improvement since, in earlier online investigations, ROE correlations between the biaryl parts never had been observed,26-29 for the likewise 7,8′-coupled (albeit (43) Bringmann, G.; Koppler, D.; Scheutzow, D.; Porzel, A. Magn. Reson. Chem. 1997, 35, 297-301. (44) Bringmann, G.; Koppler, D.; Wiesen, B.; Franc¸ ois, G.; Sankara Narayanan, A. S.; Almeida, M. R.; Schneider, H.; Zimmermann, U. Phytochemistry 1996, 43, 1405-1410.

Figure 7. Experimental LC-CD spectrum of 4, i.e., peak A of the leaf extract of A. griffithii (tR ) 19.5 min, scan range 190-400 nm)

partially dehydrogenated) moiety of dimers such as korundamine A (2) (see Figure 1) not even off line.5 The fact that the two “outer” biaryl axes of 4 are configurationally stable was further confirmed by HPLC-CD experiments. The experimental on-line CD spectrum of 4 (see Figure 7) showed strong bands between 200 and 300 nm derived from π,π* transitions,27 as would be expected in the case of a stereochemically stable 7,8′ link in the monomers, even though the unprecedented structural type of 4 and its overall conformational flexibility (including its third, “inner” biaryl axis, which is configurationally unstable) did not permit an unambiguous attribution of the absolute configuration. Thus, at this point of the investigation, 4 had to be either 1S,3R,P or 1R,3S,M for both of its monomeric parts. Degradation Analysis of Leaf Extract. To determine the absolute configuration of 4, we thus subjected a portion of the leaf extract of A. griffithii to our ruthenium-catalyzed oxidative degradation procedure with subsequent GC-MSD analysis of the chiral degradation products.31 The exclusive (>99:1) formation of (S)-3-aminobutyric acid (data not shown) clearly revealed that only tetrahydroisoquinoline substructures with an S configuration at C-3 were present in the extract mixture, which is typical of Asian Ancistrocladaceae species.45,46 Thus, in combination with the fact that 4 is one of the main alkaloids of A. griffithii (see above), 4 can clearly be assigned the 3S configuration, too. From the cis configuration at C-1 versus C-3 and with the axial configuration relative to the centers likewise established by NMR (see above), the full stereostructure of 4 is clearly deduced as 1R,3S,5M in both molecular halves, as drawn in Figure 1. CONCLUSION The photometric test described in this paper has been used for the detection of naphthylisoquinoline dimers in a significant number of Ancistrocladaceae species (18 out of the ∼21 species known to date!47,48). Although false positive or false negative reactions in the photometric test cannot, in principle, be excluded with absolute (45) Bentley, K. W. Nat. Prod. Rep. 1999, 16, 367-388. (46) Bentley, K. W. Nat. Prod. Rep. 1998, 15, 341-362. (47) Gereau, R. E. Novon 1997, 7, 242-245.

certainty, the positive reactions obtained in only a limited set of species (as compared to the total number of plants tested) have facilitated further dimer screening considerably. The analytical potential and efficiency of the method is demonstrated by the confirmation of the presence of a naphthylisoquinoline dimer in crude extracts of a novel, “unexpected” source of dimers, the Southeast Asian plant species A. griffithii, by LC-MS and LC-NMR, ultimately leading to the discovery of an unprecedented dimeric naphthylisoquinoline alkaloid, ancistrogriffithine A. By long-range LC-NMR-ROESY measurements, it was possible for the first time to determine the relative configuration of the two stereochemically stable biaryl axes on line, right from the extract. Together with information from LC-CD and the microanalytical oxidative degradation method, the complete, absolute stereostructure of 4 has been established exclusively by trace analytical means. It is noteworthy that despite the relatively high concentration of 4 and the presence of a couple of monomeric naphthylisoquinolines in the extract according to LC-MS, neither the authentic monomeric “half” of 4 nor other dimers are detectable. Ancistrogriffithine A (4) is the first dimeric naphthylisoquinoline alkaloid ever found in an Asian Ancistrocladus species and, with its S configuration at C-3, the only one belonging to the “Ancistrocladaceae type”18 (i.e., with 3S and a oxygen function at C-6). Stereochemically and from its source, however, 4 is entirely unique.The knowledge of the complete stereostructure of 4 right from the plant extract makes the isolation of this novel quateraryl for pharmacological testing now most rewarding. This work is in progress. ACKNOWLEDGMENT We thank Prof. Dr. L. Ake´ Assi (Abidjan, Ivory Coast), K. Dumbuya (Freetown, Sierra Leone), Prof. B. Alo (Lagos, Nigeria), Prof. J. F. Ayafor (Yaounde´, Cameroon), Dr. A. Louis (Libreville, Gabon), Prof. V. Mudogo (Kinshasa, Democratic Republic of Congo), F. Mbago (Dar-es-salaam, Tanzania), Dr. R. Haller and Dr. S. Ba¨r (Mombasa, Kenya,), Dr. S. M. Ketkar (Pune, India,), Dr. A. Wickramasinghe (Peradenya, Sri Lanka), Prof. H. Hadi (Kuala Lumpur, Malaysia), A. Kreiner (Wu¨rzburg, Germany), Prof. W. Meijer, and Prof. Dr. W. Smits (Balikpapan, Indonesia) for the plant material, and M. Michel for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 251 and Graduiertenkolleg “NMR in vivo and in vitro” (fellowship for M.H.), by the Fonds der Chemischen Industrie (fellowship for M.W.), and the Max Buchner Stiftung (fellowship for M.W. and H.R.).

Received for review December 19, 2000. Accepted March 13, 2001. AC001503Q (48) Cheek, M.; Frimondt-Møller, C.; Hørlyck, V. Kew Bull. 2000, 55, 207212.

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