3.1. Structure Elucidation
Compounds
1 and
2 were obtained as a colorless mixture, and determination of the planar structures was realized without any further purification step.
1H NMR signal at
δ 1.26 ppm indicated that compounds
1 and
2 share a long alkyl chain (see
Table 1). The signal at
δ 81.3 (C, C-18) ppm on the
13C NMR spectrum, together with a thin doublet at
δ 3.07 (d,
J = 2.3 Hz, 1H, H-19) ppm on the
1H NMR spectrum, were attributed to an acetylenic proton. COSY correlations between this acetylenic proton and ethylenic protons at
δ 5.44 (ddd,
J = 10.9, 3.5, 1.4 Hz, 1H, H-17) and
δ 6.00 (dt,
J = 10.4, 7.3 Hz, 1H, H-16) ppm evidenced a
Z enyne terminus substituted by three spin-coupled methylenes at
δ 2.32 (q,
J = 7.6 Hz, 2H, H-15), 1.41 (m, 2H, H-14) and 1.26 (m, 2H, H-13) ppm. The
Z configuration of the C=C double bond was confirmed by the shielding of the the adjacent vinylic methylene at
δ 30.4 (CH
2, C-15) ppm. Downfield shifted signals (> 35 ppm) are usually observed for an
E configuration [
27].
Inspection of the
1H NMR spectrum also evidenced characteristic cyclopropane signals at
δ 0.78 (ddd,
J = 8.0, 6.5, 4.1 Hz, 1H, H-20a), 0.96 (dt,
J = 10.2, 7.1, 5.2Hz, 1H, H-20a) and 1.08 (ddd,
J = 8.5, 8.0, 4.6 Hz, 1H, H-20b) and 1.22 (m, 1H, H-20b) ppm. Indeed an important delocalisation of the
σ electrons in a cyclopropane can exert a considerable influence on the chemical shifts of neighbouring protons. The resulting
1H chemical shifts are lower than those observed for usual methylenes [
28].
Analysis of the HMBC spectrum gave supplementary information on the structure. HMBC correlations H-16/C-18, H-16/C-17 and H-16/C-15 confirmed the position of the enyne terminus. Another HMBC correlation was observed between the non equivalent protons of the cyclopropane ring H-20a and H-20b with the C-4 connecting the ring with the rest of the molecules. Although two carboxylic acid were identified on the HMBC spectrum, the absence at 2.2 ppm in the
1H NMR spectrum of the characteristic triplet signal of a methylene adjacent to a carbonyl, suggested that the cyclopropane ring was alpha connected to the carboxylic acid function. This conclusion was supported by the HMBC correlations H-20a/C-1 and H-20b/C-1. After anlysis of the HMBC and COSY spectra, two sets of correlations were identified for two distinct cyclopropane rings, but no correlations where observed to link them together. We came to the conclusion that two diastereoisomers of the cyclopropane ring (
cis and
trans) were present in the mixture. A comparison with
1H and
13C NMR data obtained by dAuria
et al. for cladocroic acid (
1) was consistent with this conclusion [
15]. Indeed, in the previously published paper, only two signals for the cyclopropane ring were reported at
δ 0.96 and 1.08 ppm while four signals were observed on the
1H NMR spectrum at
δ 0.78, 0.96, 1.08 and 1.22 ppm in our case.
To separate both compounds
1 and
2, a methyl esterification was realized in order to increase the hydrophobic interactions of the compounds with the reverse phase column [
29]. A mixture of the two methyl ester derivatives was obtained and subsequently purified using the same conditions than those previously described.
1H NMR spectra of collected peaks were recorded to confirm that both compounds were successfully separated (
Table 2).
Finally, the length of the methyl ester derivatives alkyl chain was reliably determined by GC-MS. A fairly similar fragmentation pattern and the same molecular ion at m/z 318 were observed for compounds 1a and 2b. Identification of the fragments led to the conclusion that the alkyl chain contained 12 methylenes (Figure S11).
Comparing NMR data and the signs of the optical rotation, we concluded that compound
2a was cladocroic acid previously isolated from
C. invurvata. Since the authors were unable to assign the absolute configuration [
15], compound
2 can be first described as (2
S*, 3
R*)-cladocroic acid while compound
1 can be identified as (2
S*,3
S*)-cladocroic acid. To determine the absolute configuration of both compounds, we then decided to use ECD, a nondestructive and very sensitive method that has been increasingly employed in the last few years in natural product chemistry [
16,
17,
18].
3.2. Determination of the Absolute Configuration
To take advantage of the presence of the ester function chromophore close to the chiral centers of the two compounds
1a and
2a, a study of the absolute configuration was performed by ECD. The double bond of the ester implied two possible electronic transitions, the allowed
π →
π* transition that gives a high
ε value, and the forbidden
n →
π* transition which has a far less important
ε value [
30]. Since the
π →
π* transition has a
λmax = 185 nm that falls below the measurement window (> 200 nm), the study was realized on the
n →
π* forbidden transition whereby the energy level is lower (
λmax = 205 nm). The linear methylenes induced a high degree of free rotation during the conformational analysis; the theoretical calculations were performed on a simplified analog (
i.e., the cyclopropane substituted by the ester and an ethyl chain). This approximation was considered to be realistic as the effects of the enyne terminus were considered negligible.
Only one Cotton effect (CE) was observed on the ECD spectrum at
λ = 210 nm, and the difference of 5 nm between the value and the experimental observation was explained by a slight bathochrome effect of the aliphatic chain (Woodward-Fieser rules). TD-DFT calculations using the integral equation formalism variant (IEFPCM) have been performed on the most stable conformer to simulate the ECD spectrum of both compounds
1a and
2a (
Figure 2).
A simple comparison of the sign of the CE at ca. 210 nm indicated that both compounds shared the same absolute configuration on C-2 and could be describe as (2S,3S)-epi-cladocroic methyl ester (1a) and (2S,3R)-cladocroic methyl ester (2a). Since the methyl esterification did not change the absolute configuration of 1 and 2, these results also provided information on the absolute configuration of the previously isolated cladocroic acid (2), which also had the same S configuration at C-2.