An unusual triterpene fatty acid conjugate from Dodonaea caespitosa

Triterpene fatty acid conjugates are highly lipophilic natural products, originating from the cuticle layer of plants,¹ which play an important role in strengthening the cuticle and preventing water loss at high ambient temperatures.² Although hundreds of triterpene fatty acid conjugates have been isolated,¹ only a single report describes conjugates incorporating a cutin monomer as a linking molecule.³ Inoue et al. showed a set of triterpene glycosides conjugated to 8/9-oxo-16-β-[(D-xylopyranosyl)oxy]hexadecanoic acids through polysaccharides, which likely played a role in the cytotoxicity of the plant.³ To date, non-glycosidic triterpene conjugates employing a cutin monomer are yet to be reported. 30-Hydroxybetulin has limited biological activity,^4,5 but its derivatives have, for example, a semisynthetic tyramine–betulin conjugate that has micromolar antiproliferative activity (IC₅₀ = 6 µM) against A-375 human melanoma cells.⁶ Herein, we report an unusual triterpene-fatty acid–cinnamate conjugate, along with 30-hydroxybetulin from Dodonaea caespitosa, a shrub endemic to Western Australia.⁷

Air-dried aerial parts of D. caespitosa, collected from Nowanup, WA,⁸ in 2021, were steeped in diethyl ether for 20 min to afford an oil accounting for 6.0% (w/w) of the dried plant after filtration and concentration. As the products could not be separated by normal phase flash chromatography due to poor solubility, a small portion of the extract was acetylated. The acetylated mixture was purified by normal phase flash chromatography to afford the known 30-hydroxybetulin triacetate 3 in 0.6% and the new lupane fatty acid conjugate 4 in 1% (Fig. 1). Both isolates were acetylated artefacts of the natural products 1 and 2. The triacetate 3 was unambiguously assigned by agreement of experimental NMR and specific rotation data published by Pramanick et al.⁹

Fig. 1.

Isolation of 1, 3, 4, 5 and 6 from D. caespitosa, yields of compounds are in weight/weight of dried plant and key HMBC correlations in red.

(+)-HRESIMS analysis of the conjugate 4 revealed multiple [M + H] ions, which made it unclear which formula was the molecular ion. Analysis of the ¹H and ¹³C NMR spectra of the conjugate 4 indicated that the key resonances assigned to the triacetate 3 were also present in the spectra of the conjugate 4; however, the conjugate was significantly more complex in structure, showing 61 distinct resonances in the ¹³C NMR spectrum. Further NMR analysis indicated the appearance of three ester carbonyl groups (δ_C 173.6), (δ_C 173.0) and (δ_C 169.7); an oxygenated sp² carbon (δ_C 149.2); two sp² methines (δ_H 7.20 dd, J = 10.8, 8.4 Hz; δ_C 129.4, C-5′) and (δ_H 6.99 d, J = 8.4 Hz; δ_C 129.4, C-6′); a quaternary sp² carbon (δ_C 138.3); an oxymethine (δ_H 4.85 pent., J = 6.2 Hz; δ_C 74.4); an oxymethylene (δ_H 4.05 t, J = 6.7 Hz; δ_C 74.4); three deshielded methylenes (δ_H 2.94 t, J = 7.8 Hz; δ_C 30.5, C-3′), (δ_H 2.61 t, J = 7.8 Hz; δ_C 36.0, C-2′) and (δ_H 2.34 t, J = 7.5 Hz; δ_C 34.5); and an acetate methyl group (δ_H 2.28 s), as well as 14 aliphatic methylene groups.

As a high number of overlapping resonances in the ¹H NMR spectrum were observed, 2-D-NMR data could not be used to elucidate the structure of the conjugate 4. Thus, a small portion of the unmodified crude extract was subjected to hydrolysis and separated by standard acid–base partitioning to afford the three known natural products 30-hydroxybetulin 1, para-hydroxydihydrocinnamic acid 5 and 8,16-dihydroxypalmitic acid 6 as the only products. 30-hydroxybetulin 1 and para-hydroxydihydrocinnamic acid 5 were unambiguously assigned based on agreement with literature reported spectroscopic data,^5,10,11 whereas 8,16-dihydroxypalmitic acid 6 was assigned by preparation of the corresponding methyl ester 7, which agreed with Siddiqui et al.’s reported NMR data.¹² Although NMR data for the palmitate methyl ester 7 have been previously reported,¹² the stereochemistry of the secondary alcohol (C-8) had never been assigned and required evaluation for the assignment of the betulin conjugate 4.

Owing to the high symmetry of 8,16-dihydroxypalmitic acid 6 and the lack of chromophores near the C-8 secondary alcohol, standard methods of stereochemical evaluation, such as Mosher’s esters and circular dichroism, were unsuitable. The stereochemistry at C-8 was therefore determined by conversion of the isolated 8,16-dihydroxypalmitic acid 6 to the known (S)-(+)-8-hydroxypalmitic acid 8 by a six-step synthesis employing a Barton–McCombie deoxygenation. The ¹H and ¹³C NMR spectra of the final reaction product were in agreement with literature reported data for (S)-(+)-8-hydroxypalmitic acid 8 confirming the connectivity of the reaction product.^13,14 The reported specific rotation values for (S)-(+)-8-hydroxypalmitic acid 8 varied from +0.15° to +1.23°^14–17; however, they agree with the observed sign and magnitude of the isolated product (+1.00° to +2.45°). As (S)-(+)-(8)-hydroxyplamitic acid 8 was obtained by semisynthesis from 8,16-dihydroxypalmitic acid 6, the C-8 stereochemistry of the starting material was also confirmed as (S)-configured (Fig. 2).

Fig. 2.

Semisynthesis of (S)-(+)-(8)-hydroxypalmitic acid 8 from (S)-(+)-8,16-dihydroxypalmitic acid 6 employing a Barton–McCombie deoxygenation.

After identifying all the hydrolysis products of the conjugate 4 as (S)-(+)-8,16-dihydroxypalmitic acid 6, para-hydroxydihydrocinnamic acid 5, and 30-hydroxybetulin 1, focus was returned to finalising the connectivity of the conjugate 4. Upon revisiting the ¹H NMR spectrum of the conjugate 4, the new oxymethine (δ_H 4.85 pent., J = 6.2 Hz; δ_C 74.4), oxymethylene (δ_H 4.05 t, J = 6.7 Hz; δ_C 74.4) and deshielded methylene group resonances (δ_H 2.34 t, J = 7.5 Hz; δ_C 34.5) were associated with (S)-(+)-8,16-dihydroxypalmitic acid 6. Moreover, the two new sp² methines (δ_H 7.20 dd, J = 10.8, 8.4 Hz; δ_C 129.4, C-5′), (δ_H 6.99 d, J = 8.4 Hz; δ_C 129.4, C-6′) and coupled deshielded methylenes (δ_H 2.94 t, J = 7.8 Hz; δ_C 30.5, C-3′), (δ_H 2.61 t, J = 7.8 Hz; δ_C 36.0, C-2′), were associated with p-hydroxydihydrocinnamic acid 5.

The ¹H–¹³C HMBC spectrum of the conjugate 4, when compared to the spectrum of the triacetate 3, confirmed that the acetates connected to the oxymethine (C-3) and oxymethylene (C-28) in the triacetate 3 were also present in the conjugate 4. Further analysis indicated that the allylic alcohol (C-30) no longer showed correlation to an acetate carbonyl in the conjugate 4 and instead correlated to the carbonyl group (δ_C 173.6) of the (S)-(+)-8,16-dihydroxypalmitic acid 6 portion. Further analysis of the HMBC spectrum indicated a correlation between the oxymethylene of (S)-(+)-8,16-dihydroxypalmitic acid 6 (δ_H 4.05 t, J = 6.7 Hz) and the carbonyl group (δ_C 173.0) of p-hydroxydihydrocinnamic 5. The connectivity of the conjugate 4 was therefore finalised as 30-(16-para-acetoxydihydrocinnimate-8-(S)-acetoxypalmitate)betulin diacetate 4. As the absolute configuration of the hydrolysed (S)-(+)-8,16-dihydroxypalmitic acid 6 and 30-hydroxybetulin 1 were assigned by specific rotation measurements, the absolute configuration of the conjugate 4 was determined as 3-(S), 5-(R), 8-(R), 9-(R), 10-(R), 13-(R), 14-(R), 17-(S), 18-(S), 19-(R), 8′-(S). Based on the high overlap of both proton and carbon resonances, most resonances associated with the (S)-(+)-8,16-dihydroxypalmitic acid portion could not be unambiguously assigned. In further proof of the assignment, returning to the (+)-HRESIMS spectrum after NMR analysis revealed an [M + H – AcOH] ion at m/z 985.6763, consistent with the molecular formula C₆₃H₉₆O₁₂. As the ¹H NMR spectrum of the initial extract lacked resonances characteristic of acetate functionalities, it was clear that the betulin conjugate 2 was the natural product present within the leaf resin and that the conjugate 4 was an acetylated artefact.

In summary, purification of the acetylated diethyl ether extract afforded the triacetate 3 and the novel betulin conjugate 4 in 0.6 and 1% respectively. The natural products present in the leaf resin were therefore 30-hydroxybetulin 1 and the betulin conjugate 2. As (S)-(+)-8,16-dihydroxypalmitic acid 6 is a known monomer of cutin,¹⁸ it is highly likely that (S)-(+)-8,16-dihydroxypalmitic acid 6 incorporated into the betulin conjugate 4 originated from the same source. The conjugate 4 marks the first isolation of a cutin monomer as a linking molecule directly conjugated to a triterpene. Although not completed within this study, exploration of the biological activity should be investigated to evaluate if conjugate 4 provides an ecological purpose independent of cuticle strengthening and plant moisture retention. Finally, a simple extraction, hydrolysis and partitioning procedure afforded 30-hydroxybetulin 3 in 2.9% (w/w) without chromatography. This method provides rapid access to a medicinally relevant molecule which could be used for semisyntheses, such as in the preparation of anti-cancer agents.

Supplementary material

Supplementary material is available online.