Contrasting Dihydronaphthoquinone Patterns in Closely Related Drosera (Sundew) Species Enable Taxonomic Distinction and Identification

Dihydronaphthoquinones are described as constituents of sundews (Drosera), Venus flytraps (Dionaea), and dewy pines (Drosophyllum) for the first time. As in the corresponding naphthoquinones, these reduced derivatives may occur in two regio-isomeric series distinguished by the relative position of a methyl group (at position 2 or 7 in the naphthalene skeleton), depending on the taxon. Species producing plumbagin (2-methyljuglone, 1) do commonly contain the corresponding dihydroplumbagin (5), while species containing ramentaceone (7-methyljuglone, 2) also contain dihydroramentaceone (7-methyl-β-dihydrojuglone, 6). So far, only few species containing plumbagin (1) and dihydroplumbagin (5) additionally form dihydroramentaceone (6) but not ramentaceone (2). Thus, subtle but constant differences in the chemism of closely related and morphologically similar species reliably define and distinguish taxa within D. sect. Arachnopus, which is taken to exemplify their chemotaxonomic utility. The joint presence of quinones and hydroquinones allows observations and predictions on the chemical structures and the reactions of these intriguing natural products.

Upon closer inspection of the respective TLC chromatograms, all of the D. finlaysoniana samples stood out because of the consistent presence of an additional constituent that distinguishes this species even from its relatives (e.g., D. serpens) that share the same major Upon closer inspection of the respective TLC chromatograms, all of the D. finlaysoniana samples stood out because of the consistent presence of an additional constituent that distinguishes this species even from its relatives (e.g., D. serpens) that share the same major naphthoquinone ( Figure S11). In this paper, we identify this additional metabolite and evaluate its chemotaxonomic potential both within Drosera and among related genera and families.
GC-MS was used for the structural elucidation of the additional constituents coming directly from crude petroleum ether extracts from the fresh leaves of several representatives of Drosera sect. Arachnopus and of Dionaea muscipula Soland. ex J. Ellis (Droseraceae, Venus's flytrap) and Drosophyllum lusitanicum (L.) Link (Drosophyllaceae, dewy pine), which were chosen for outgroup comparison.

Results
Results are summarized in Table 1 (1). It is only after TMS-derivatization that all of the metabolites were separated clearly (Figure 1), and the respective mass spectra (Table S1 in Supplementary Materials) identify the additional metabolites as dihydronaphthoquinones dihydroplumbagin (5, R t = 15.90 min) and dihydroramentaceone (6, R t = 16.55 min), respectively. The extract of D. serpens only contained plumbagin (1) and dihydroplumbagin (3) but no dihydroramentaceone (6).
In addition to these naphthoquinone derivatives, different compounds (characterized by R t = 14.66 min, m/z 204; R t = 16.57 min, m/z 232; R t = 16.64 min, m/z 216; R t = 18.08 min, m/z 246, respectively) have been detected in the extracts from Drosophyllum but have not further identified. Possibly some of these represent derivatives of naphthoic acids that have been isolated from Drosophyllum and the related genus Ancistrocladus Wall. (Ancistrocladaceae) before [9,10].
Neither dihydronaphthoquinone has been isolated from Drosera or Drosophyllum before, but dihydroplumbagin (5) had been previously obtained and characterized from Juglans L. (Juglandaceae) species [11], and dihydroplumbagin (5) and dihydroramentaceone (6) were already known from Diospyros maritima Blume (Ebenaceae) [12]. Table 1. Naphthoquinone derivatives identified in petroleum ether extracts of the plant material investigated in this study.  In addition to these naphthoquinone derivatives, different compounds (characterized by Rt = 14.66 min, m/z 204; Rt = 16.57 min, m/z 232; Rt = 16.64 min, m/z 216; Rt = 18.08 min, m/z 246, respectively) have been detected in the extracts from Drosophyllum but have not further identified. Possibly some of these represent derivatives of naphthoic acids that have been isolated from Drosophyllum and the related genus Ancistrocladus Wall. (Ancistrocladaceae) before [9,10].

Discussion
The mass spectra of the TMS derivatives of both dihydronaphthoquinones indicate that the diketo-tautomers (assigned to structures 5a and 6a, characterized by m/z 247) are predominant in the petroleum ether extract. In contrast, the naphthotriol-tautomers (7 and 8) were not detected in underivatized samples but may be represented by their TMS derivatives (7c and 8c, characterized by m/z 406; 7a and 8a; m/z 318; 7b and 8b, m/z 319) that are less abundant (following the thermodynamic ratio of about 1:4) [13]. This corresponds well with the fact that natural dihydroplumbagin (5) isolated with chloroform from Diospyros maritima retains the same configuration (2R) as the corresponding position (3R) in isoshinanolone (3) from the same plant [12], so tautomerism and subsequent racemization may be limited in aprotic solvents. The hypothetical naphthotriol tautomers (7 and 8) are possibly able to form quinhydrones with the corresponding naphthoquinones. In the case of the 2-methyl derivative (7), this would have to occur in a fashion that conserves stereochemistry at C-2 upon the release of dihydroplumbagin (5) from the quinhydrone, even if the latter underwent an intermolecular proton transfer. Apparently, no mixed quinhydrones are formed from plumbagin (1) and dihydroramentaceone (6), or at least no intermolecular proton transfer occurs in such complexes. Otherwise, ramentaceone (2) would have been detected in D. finlaysoniana. The di-TMS derivatives with a phenolic hydroxyl group in peri-position to an O-TMS (7a and 8a) lose a methyl group from TMS together with a proton (assumedly from the phenol) in MS, while those with a more remote hydroxyl group (7b and 8b) only lose a methyl group. This may indicate that the resulting fragments from the former (7a and 8a) can form dioxydimethylsilane bridges between C-4 and C-5.
While dihydroplumbagin (5) can readily be construed as a metabolic precursor of plumbagin [6], the simultaneous presence of dihydroramentaceone (6) in D. finlaysoniana, that is otherwise devoid of any ramentaceone metabolites is outright surprising, especially if compared to D. serpens, which shares the plumbagin derivatives (1 and 3) but obviously lacks any trace of dihydroramentaceone (6) in all geographically diverse specimens that have been investigated to date.
A minor regio-isomer of a dihydronaphthoquinone derivative was observed before in D. intermedia Hayne that contains plumbagin (1) as the main constituent along with plumbaside-C (7d) and trace amounts of rossoliside (8d), while ramentaceone (2) was only detected after hydrolysis and oxidation [14]. The recurrent pattern of plumbagin (1) as the main constituent and minor amounts of reduced derivatives from only the 7-methyl series, e.g., dihydroramentaceone (6) or its 4-O-glucoside (8d), may represent a plesiomorphic character state in the genus Drosera because it is found in the related genus Drosophyllum and in species that are supposed to occupy the basal positions in their respective sections: D. finlaysoniana in D. sect. Arachnopus, and D. intermedia in D. sect. Drosera.
The joint presence of plumbagin (1) and ramentaceone (2) has repeatedly been found in species of known or assumed hybrid origin [4,15,16]. In species that display a similar "hybrid" quinone pattern but that lack quinone-heterogenous candidate parent species, e.g.,  [2,6,7], this condition may alternatively be derived from a predisposition similar to D. finlaysoniana or D. intermedia if the biosynthesis of the minor isomer is enhanced and its oxidation to the respective quinone is enabled or facilitated.
In contrast, D. serpens and its close relatives in which exclusively 2-methyl derivatives have been detected probably have a different genetic background, leading to higher regioselectivity.
The opposite extreme is observed in D. hartmeyerorum and D. aurantiaca, which exclusively contain derivatives of the 7-methyl series.

General Experimental Procedures
For GC-MS analyses of underivatized samples, a Gerstel MPS 2XL autosampler was used to inject (split ratio 20:1) the aliquots into an Agilent 7890B GC-system combined with an Agilent 5977B GC/MSD detector. The MS data at a mass range of 25-550 amu were collected and processed with Agilent MassHunter software. GC-MS runs were performed on an HP-5MS ultra inert silica capillary column (30 m, 0.250 mm ID, phase thickness 0.25 µm) using an oven temperature program from 50 • C to 260 • C at a rate of 10 • C min −1 . The injector, MS source, and MS quadrupole temperatures were 260, 230, and 130 • C, respectively.
For the GC-MS analyses of TMS derivatives, a Gerstel Maestro MPS 2 sampling system (Gerstel GmbH&Co. KG, Mühlheim am der Ruhr, Germany) was used to inject the aliquots into an Agilent 7890A GC combined with a 5975C mass selective detector, equipped with a DB-5 silica capillary column (30 m, 0.250 mm, 0.25 µm, Restek, Bellefonte, PA, USA), and the oven temperature program was from 80 • C (1 min) to 100 • C (5 • C min −1 ) and to 250 • C (10 • C min −1 ; 10 min). The split ratio was 10:1, and the data were collected at a mass range of 35-800 amu. The injector temperature was 250 • C, and those of the MS source and the MS quadrupole were 230 and 150 • C, respectively.
Thin-layer chromatography analysis was conducted on silica gel 60 F 254 plates (Macherey-Nagel, Düren, Germany) with toluene as the mobile phase. The spots were visualized using UV fluorescence (tetralones and dihydronaphthoquinones) and after exposure to NH 3 fumes from a saturated aq. soln. (naphthoquinones).

Plant Material
Fresh leaves from plants in cultivation (the annual representatives of D. sect. Arachnopus were grown from seed, Dionaea and Drosophyllum were obtained from horticultural trade) were used for extraction and analysis. Authentic specimens prepared at the sampling date (October 2020) were deposited in the first author's herbarium. Taxonomic identification of all samples was performed and confirmed by the authors. Their respective accession numbers are listed in Table 1.

Extraction and Isolation
Fresh leaf material (24-117 mg) was extracted with 500 µL petroleum ether (boiling range 60-80 • C, Emsure ® , Merck KGaA, Darmstadt, Germany) for 1.5 h at 20 • C. The crude extracts were analyzed directly by TLC for the screening of volatile constituents (a representative chromatogram is available as Supplementary Material) and by GC-MS for structure determination.
The extracts were concentrated, and 2 µL aliquots were analyzed using GC-MS. After direct runs, the samples were evaporated under nitrogen flow and were dissolved into dichloromethane and trimethylsilylated with MSTFA (N-Methyl-N-trimethylsilyltrifluoroacetamide, Pierce, Rockford, IL, USA) containing 0.1% trimethylchlorosilane (TMCS). The samples (2 µL aliquots) were analyzed through GC-MS (original spectra are available as Supplementary Material).