Absolute Configuration of Clemateol

The present study reports the determination of absolute stereochemistry of clemateol, an irregular monoterpene containing an epoxy group, which was isolated as the main component from the essential oil of Calea clematidea (Asteraceae). Its absolute stereochemistry was unambiguously established on the basis of detailed nuclear magnetic resonance (NMR) spectroscopic evidence (JH-H analysis, derivatization as Mosher’s esters and nuclear Overhauser effect (NOESY) spectrum) and also by resonance scattering effects in the single crystal X-ray diffraction (XRD) resolution of its (R)-mandelic acid ester derivative.


Introduction
Clemateol (1), an irregular monoterpene containing an epoxy group, has been isolated as main component (60-90%) from the essential oil of Calea clematidea (Asteraceae) and has been shown to possess moderate in vitro antifungal activity against some dermatophytes. 1 Structurally, clemateol contains two chiral centers (C-3 and C-4) and therefore it may give rise to four stereoisomers.In a previous work, 1 clemateol had its configuration determined as 3S, 4R by the Horeau method using enantioselective gas chromatography. 2At that time, reservations expressed by referees led us to reexamine this question by using another method to verify the absolute configuration of 1.The Horeau method, which relies on the kinetic resolution of the racemic 2-phenylbutiric anhydride by the chiral secondary alcohol, can fail due to the difficulty of unambiguously assigning the most "space-filling" substituent at the chiral center (R L and R S ).As in the case of R L and R S in clemateol, little stereo-differentiation between the large and small substituent may complicate the analysis and compromise the result (Figure 1). 3 In order to validate the method previously used or even to correct the absolute configuration proposed earlier, the absolute configuration of C-3 and C-4 of clemateol was unequivocally established on the basis of detailed nuclear magnetic resonance (NMR) spectroscopic data and resonance scattering effects in its single crystal X-ray diffraction analysis.Initially, the relative configuration of C-3 and C-4 was determined by analysis of the H-H coupling constant H-3 and H-4 in the 3,4-acetonide (4) obtained from its diol-derivative (3).Subsequently, the absolute configuration of C-4 was determined by the method of Mosher, 4 using the Dd RS values of the (R and S)-MPA-esters, of clemateol derivatives 5 and 6.[7][8][9]

General information
All the reactions were carried out under dry argon atmosphere employing oven dried glassware.Anhydrous methanol (MeOH) and ethanol (EtOH) were obtained by refluxing the solvent over clean Mg/I 2 and distilling from the resulting magnesium alkoxides, anhydrous dichloromethane was prepared by a 4 h reflux of the solvent over diphosphorus pentoxide (P 2 O 5 ) followed by atmospheric pressure distillation, and acetone was refluxed over calcium sulfate followed by fractional distillation.All other reagents were used as received.The hydrogen ( 1 H) NMR spectra were acquired at 400.1 MHz in deuterated chloroform (CDCl 3 ), except when noted otherwise, on a Bruker DPX-400 spectrometer.Chemical shifts are reported in parts per million (ppm) on the d scale and J values are given in hertz.The peak of the residual protonated solvent (chloroform (CHCl 3 ) in CDCl 3 , d 7.26) was used as the internal standard.The carbon-13 ( 13 C) NMR spectra were recorded at 100.6 MHz on a Bruker DPX-400 spectrometer.The solvent peak (CDCl 3 , d 77.0) was used as the internal standard.Distortionless enhancement by polarization transfer (DEPT) 135 and DEPT 90 experiments aided the interpretation and assignment of the fully decoupled 13 C NMR spectra.In special cases, two-dimensional (2D) NMR experiments (correlation spectroscopy (COSY), nuclear Overhauser effect (NOESY), heteronuclear multiple bond correlation (HMBC) and heteronuclear multiple quantum coherence (HMQC)) were also employed.

Single crystal X-ray diffraction study
A single crystal of 5 was glued to the end of a fine glass fiber and mounted in a Bruker D8 Venture dual source diffractometer equipped with a Photon 100 CMOS (complementary metal-oxide-semiconductor) area detector and a low temperature N 2 gas flow device operating at 100 K. Data were collected using Incoatec IμS microfocus copper anode source (λ = 1.541 78 Å) to a maximum 2θ of 144.84° with 2.0° j and ω scans using the APEX3 data collection suite. 10Data integration and scaling were done with SAINT 11 using the wide frame algorithm and SADABS 12 using a multi-scan adsorption correction, respectively.The space group chosen was P2 1 2 1 2 1 based on systemic absences and data statistics, and confirmed by successful solution and refinement.Structure solution and refinement were performed in Bruker XT 13 and XL, 14 respectively.Final R indices are R 1 = 0.0369 (I > 2σ(I)) and wR 2 = 0.0933 (all data); goodness of fit = 1.068.The Flack x = 0.04(9). 5,7Full structural data are available in the Supplementary Information (SI) section.

Plant material
Leaves of Calea clematidea were collected in November 2013 in Santana do Livramento, RS, Brazil.A voucher specimen (SMDB 7349) was identified by Prof R. Zachia, and was deposited in the herbarium of the Universidade Federal de Santa Maria, RS, Brazil.
For NMR data see Table 1.
For NMR data see Table 1.

Results and Discussion
Clemateol (1) was isolated as the main component of the essential oil of leaves of Calea clematidea.Despite its low molecular weight (212 Da), 1 is confirmed by three structurally relevant organic functional groups (alkene, ester and oxirane) behaving in addition two contiguous stereogenic centers.To determine clearly the absolute configuration of these stereogenic centers (C-3 and C-4), clemateol was initially subjected to alkaline hydrolysis (methanol/sodium) in order to obtain the secondary alcohol at C-4, affording the derivative 2. 15 Carbinol 2 was subjected to a regioselective opening of the epoxide function using FeCl 3 as catalyst and methanol as the nucleophile, 16 giving rise to compound 3.This oxirane ring opening mediated by anhydrous FeCl 3 is well precedented and occurs through the complexation of Fe +3 by the oxygen atom of epoxide as represented by structure 2a. 17,18This disposition allows the sole attainment of derivatives from the epoxide ring opening at the more substituted position, which in turn stabilizes better the incipient carbocationic character when compared with the contiguous less substituted position.Finally, a new C-OMe bond is formed rendering the tertiary methoxyether 3.Treatment of diol 3 with acetone in acidic medium gave the 3,4-acetonide derivative 4 (Scheme 1). 19ext, the relative stereostructure of 4 was determined by the comparison of the H-H-coupling constant between H-3 and H-4 in the 1 H NMR spectrum of 4, which showed 3 J 3,4 = 5.2 Hz and the absence of correlation between H-3 and H-4 (anti position) in the NOESY spectrum.These results determine the C-3 and C4-relative configurations of 4 (and therefore on 1) as being 3S*, 4R* (3,4-threo-form).
Applications of the Mosher's method to the MPA-ester derivative of 2 enabled the determination of the absolute configuration of C-4 of 1.For this, the ester derivatives were prepared by treatment of 2 with (−)-(R)-and (+)-(S)-MPA free acids and DCC-DMAP in dichloromethane affording 5 and 6, respectively.
The 1 H NMR spectra of the diastereoisomeric products were analyzed by the calculation of ∆d values, considering MPA-esters (∆d = d Rd S , shown in Table 1). 2 The groups attached to C-4 were called L 1 (H-5', H-5", H-6, H-7, H-7') and L 2 (H-1, H-1', H-3).All hydrogens in the L 1 group had positive values for ∆d RS , while the hydrogens bound to C-1 and C-1' in the L 2 group had negative values.Thereby, the groups L 1 and L 2 are arranged around the C-4 as can be seen in Figure 2. Thus, the absolute configuration at C-4 in 1 was determined to be R (Figure 2).
The complete absolute configuration of 1 was determined by an X-ray crystallographic study of single crystals of 2-(−)-R-MPA-derivative (5).The crystal structure is shown in Figure 3, and the result is consistent with the absolute configuration (3S, 4R), confirming the previous results obtained by Horeau's and Mosher's methods.
In continuing studies oriented to synthesize a nonnatural epimer of 2, we performed the oxidation of 2 with PCC in dichloromethane to give the ketone 7, 20 with diastereotopic faces close to a chiral center.In order to test the 1,2-asymmetric induction, ketone 7 was submitted to a diastereoselective reduction, using NaBH 4 in ethanol (Scheme 2). 21Alcohol 8 (4-epi-2) was formed with high diastereoselectivity (90%, GC, NMR), with almost complete inversion of C-4, which may be explained by the Felkin-Anh model shown in Scheme 3.
The ketone 7, represented by its Newman projection (A), behaves as the more stable conformers B and C, where the larger group -C(CH 3 ) 2 is as far away as possible from both the oxygen of the carbonyl and the R group of ketone.Consequently, no groups are eclipsed.As the nucleophiles   this case, the hydrogen in C-3 should exhibit the lowest interaction with borohydride than other groups.The reaction along this path leads to the product 8, represented by its Newman projection D.Here it can be observed that the obtained carbinol has the opposite configuration of C-4, related to the natural product 1.Thus, the ketone conformation explains the inversion of C-4, observed during the reduction of 7 with sodium borohydride.

Conclusions
In conclusion, the absolute configuration of the two stereogenic centers of clemateol were unequivocally determined to be 3S, 4R, by both the Mosher's method and resonance scattering effects in the X-ray diffraction experiment, confirming the previous determination by the Horeau method.

Figure 1 .
Figure 1.R L and R S substituents of clemateol.

Table 1 .
13 and13C NMR and Dd RS values data for 5 and 6 -methoxy-2-phenylacetyl, d were performed in CDCl 3 and expressed in ppm relative to tetramethylsilane (TMS).Multiplicities are expressed as: s, singlet; dd, doublet of doublets; m, multiplet. Copling constants are in hertz.