Centrifugal partition chromatography: an efficient tool to access highly polar and unstable synthetic compounds on a large scale

Abstract

Centrifugal partition chromatography (CPC) is an efficient method adaptable for the purification of synthetic compounds with chemical value, significantly simplifying the reaction work-up. From Aucuba japonica Thunb., an abundant natural terpenoid, aucubin, was isolated. Its reduced derivatives and corresponding unstable aglucons were purified by CPC. The latter chiral synthons were obtained under protecting group free conditions using eco-friendly, non-chlorinated solvents and without silica gel.

---

Introduction

Natural products play an important role in chemical ecology and have been a source of inspiration for organic chemists.¹ The perfect synthetic strategy complies with the concept of protecting group free chemistry² engaging renewable and readily available starting materials in a minimum number of simple, safe and quick steps with high overall yield.³ As the chirality of the natural products is one of the key elements responsible for the biological activities, the production of enantiomerically pure chemicals is of high value for pharmaceutical, food, agrochemical, fragrance and flavor industries.⁴

Classically, chiral pool is exploited in asymmetric synthesis as the source of starting materials, chiral auxiliaries or resolving reagents and in the preparation of enantioselective catalysts.⁵ In respect to green chemistry, the employment of other common and abundant renewable resources, not commercialized, could minimize the number of synthetic steps and/or enlarge the panel of chiral building blocks. In order to develop this concept, natural highly functionalized starting material, with low toxicity have to be eco-extracted⁶ and isolated in large scale. Those unprotected products can be further engaged in multi-step synthesis employing environmentally friendly reagents and solvents. In this context, the purification methods are in particular focus for the effective isolation of highly polar and unstable products in large amounts in line with saving of consumption of labor and time. In addition, this manipulation has to avoid the use of solid stationary chromatographic phases and chlorinated eluents.

Centrifugal partition chromatography uses two immiscible liquids as mobile and stationary phases avoiding solid support. The mobile phase percolates through the stationary phase, which is under the centrifugal field.⁷ This chromatography technique has become more and more frequently employed for the analytic and preparative separations and isolations of natural products, macromolecules, enantioseparations, fractionation of extracts and other miscellaneous uses.⁸ Although rotors of CPC apparatus vary from 100 mL to 25 L allowing various academic and industrial applications,⁷ this technique has been rarely applied for the purification of synthetic compounds.⁹ For example, the CPC was performed for the purification of glycosylated derivatives of squamocin, human surfactant protein SP-C, quinolines, analogues of ilomastat, salidroside and boronic acid derivatives of tyropeptin.¹⁰

Common iridoids and secoiridoids are widespread in nature and can be produced by numerous plants in abundant amounts.¹¹ We have used them as chiral renewable starting materials for the synthesis of new scaffolds and building blocks particularly useful for the construction of new heterocyclic systems and bioactive compounds.¹² A significant member of this monoterpenoid group is aucubin (1). Its polyfunctional aglycon part has been used for several decades by organic chemists as chiral source.^13–15 The purifications of 1 or of its unprotected derivatives are often challenging due to high polarity and instability of these products. As such, they serve as an excellent model for scaling-up of the engaged quantities and to probe the utilization of CPC for synthetic purposes.

Herein, we explored the scope of CPC for the isolation of highly polar natural product aucubin (1) and for the direct separation of compounds with similar polarity, linaride (2) and 6,10-dideoxyaucubin (3), from salts produced by Birch reduction. Furthermore, CPC was tested for the direct isolation of highly unstable aglucons 4 and 5 together with natural repellent, (R)-rotundial (6).

Results and discussion

Aucubin (1) was extracted by aqueous media in large amounts from the fresh aerial parts of Aucuba japonica Thunb.¹⁶ The simplicity of this process permits an easy scaling-up engaging more than 10 kg of plant material in academic laboratory. Classically to purify 1 from the extract, we have employed vacuum liquid column chromatography (VLC) on silica-gel using a gradient mixture of dichloromethane and methanol (CH₂Cl₂–MeOH (95/5) → CH₂Cl₂–MeOH (85/15)) as eluent. We improved this method by developing a quick and efficient CPC purification employing an environmentally friendly, low cost solvent system containing H₂O–n-PrOH–EtOAc (10/3/7)¹⁷ in isocratic mode. The used ternary system was superior over the tested Arizona¹⁸ systems (Hept–EtOAc–MeOH–H₂O) as these latter were not sufficiently polar. In order to ensure an efficient retention of the highly-polar iridoids, the upper phase was chosen as mobile phase whereas the lower, more polar phase was employed as stationary. Consequently, the separation was conducted in ascending mode. The 1 L rotor was fitted with maximum working load corresponding to 35 g of dry extract. The isolation of aucubin (1) with high purity (3%/fresh material, 23.9 g) was performed with a total of 12 L of upper phase with a 17 mL min⁻¹ flow at 1000 rpm. To purify 105 g of extract, this procedure was repeated 3 times using recycled upper phase. We compared this protocol with the VLC purification of same amount of the extract (105 g) adsorbed on silica-gel (100 g, 63–200 μm) using a column (ø 25 cm) of silica-gel (2.5 L, 40–63 μm) and 80 L of eluent affording pure 1 after crystallization (59 g: 2.6%/fresh material). The VLC procedure was performed in seven working days whereas CPC took 13 h per run.

Our interest was further expanded to the purification of highly polar compounds (Scheme 1). We tested the CPC method for the purification of the reduced aucubin derivatives 2 and 3 obtained under Birch conditions (Li/NH₃ (liq.)). This reduction method affords hardly available iridoids; linaride (2) and 6,10-dideoxyaucubin (3), in 9/1 ratio at −90 °C or selectively 3 at −30 °C.¹³ When 1 (12 g) was engaged in the reaction at −90 °C, CPC of the crude afforded 2 (9.6 g, 84%) and 3 (872 mg, 8%) using 6 L upper phase of the ternary system (H₂O–n-PrOH–AcOEt, 10/2/8), with a flow of 20 mL min⁻¹ at 1000 rpm, in isocratic ascending mode. Similarly, the same quantity of aucubin (1) was employed in the reduction at −30 °C, providing selectively 3 (8.7 g, 80%) under previously mentioned conditions (5 L of upper phase). In total, both purifications can be performed in 4–5 h. The attempts to isolate products 2 and 3 by silica-gel or charcoal column chromatographies at this scale were not successful due to the difficult salt elimination, easy degradation and similar polarity of formed products.¹³

Scheme 1 CPC purifications of aucubin (1) and derivatives 2–6.

Aglucons of iridoids obtained by enzymatic cleavage easily decompose and produce blue to black polymeric substances.¹⁴ Genines of aucubin (1) or of its deoxy-derivatives 2 and 3 are particularly unstable due to the equilibria with their dialdehyde forms. Their purifications by classical techniques with solid stationary phases were impossible in multigram scale.¹⁹ However using CPC, the reaction mixtures were easily purified. Compounds 1 or 2, at 10 g scale, were hydrolyzed by β-glucosidase (Scheme 1). The lyophilized reaction mixtures were dissolved in lower phase of binary system (H₂O–EtOAc (1/1)) and directly injected into CPC instrument containing lower phase as stationary. The purifications of genines 4 and 5 were performed in 3–4 h under previously mentioned conditions consuming 4 and 5 L of upper phase. Genines 4 and 5 were isolated as mixtures of anomers with a ratio α/β = 1/9 and 6/94 in 91% (4.8 g) and 87% (4.3 g) yields, respectively.

Finally, we employed CPC in the isolation of semisynthesized mosquito repellent (R)-rotundial (6). This natural product was first isolated by Nishimura in 1995 from Vitex rotundifolia. Out of three total synthesis of 6, two were asymmetric and were conducted in 6 and 10 steps with 27% and 25% overall yield, respectively.²⁰ We obtained (R)-rotundial (6) simply by the hydrolysis of 6,10-dideoxyaucubin (3) with phosphate buffer (pH = 2). After a extraction by EtOAc, the crude 6 was injected into the CPC apparatus (250 mL rotor, flow 7 mL min⁻¹, 1700 rpm) using H₂O–EtOAc (1/1) solvent system in ascending mode to give pure (R)-rotundial (6) in 55% yield.

Conclusions

In addition to its simplicity and utilization in natural product isolations, CPC is a practical alternative to classical purification methods used in preparative organic synthesis. By appropriate selection of solvent systems, this technique is easily adaptable to green conditions. As demonstrated, it is a valuable improvement for the purification of unstable and highly polar compounds, simplifying the work-up and saving time, labor, organic solvents and reagents. The chemistry of abundant natural product aucubin (1), coupled with CPC purifications have furnished a collection of rare iridoids, deoxyderivatives 2 and 3, as well as corresponding aglycons 4, 5 and the repellent (R)-rotundial (6) in multigram quantities. The access to the products in these amounts was not successful by employing classical techniques. Avoiding protecting groups, silica-gel and chlorinated solvents, all steps of isolation and synthesis from plant Aucuba japonica Thunb. were improved, thus opening a scalable route towards a large number of chiral building blocks with divers molecular architecture. The ongoing research has been directed towards the utilization of CPC for the isolation of other natural abundant iridoids and secoiridoids and their exploitation in green organic synthesis.

Experimental section

General CPC separation procedure

Selection of solvent systems and the determination of the K_d values. The two-phase solvent systems were selected according to an evaluation of the distribution coefficient (K_d) with purpose to eliminate the impurities. K_d is defined as a ratio of concentrations of the compound between the two immiscible solutions in equilibrium state. The K_d values were first evaluated using the shake-flask method²¹ followed by thin layer chromatography. A crude extract, a reaction mixture or a compound of interest was dissolved in a variety of two-phase systems (5 mg mL⁻¹) and the optimal partition was identified. Classically, optimal K_d values are comprised between 0.5 and 2. Nevertheless, we employed the systems with higher K_d's in order to obtain pure compounds in only one run.²² K_d values of the pure compounds were determined by HPLC according to the following equation: K_d = [solute]lower-phase/[solute]upper-phase. Pure compounds 1–6 (2 mg) were dissolved in two phase system (10 mL) and aliquots (1 mL) of upper and lower-phase were taken and evaporated. The ternary solvent systems containing EtOAc, n-PrOH and H₂O were used for the CPC purification of aucubin 1 (7/3/10: v/v), 2 (8/2/10: v/v), 3 (8/2/10: v/v), while binary systems of EtOAc and H₂O (5/5: v/v) were employed for the purification of genines 4 and 5 as well as (R)-rotundial (6). The residue was redissolved in water (1 mL) and filtered before injection on UptiDisc 0.45M nylon filters (Interchim, Montluc, France). 20 μl of each solution were injected and chromatograms were recorded at 226 nm. The analysis was carried out at room temperature, using Aglient ZORBAX SB-C18 (5 μm, 4.6 × 150 mm) column and a gradient elution with MeCN–H₂O as following method: 5/95 (0–5 min), 5/95 → 30/70 (5–25 min, linear gradient), 30/70 → 100/0 (25–25.1 min), 100/0 → 5/95 (25.1–30 min) and 5/95 (30–40 min) Table 1.

Table 1 Retention times, employed method and K_d values of compounds 1–6

Compound	Flow rate/[ml min^−1]	tR/min	Kd
1	1	4.4	17.6
2	1	7.3	5.1
3	1	13.3	2.2
4	0.3	5.9	4.0
5	0.5	7.8	1.3
6	0.5	5.6	15.0

---

Equilibration procedure. The rotors of CPC instrument (250 mL or 1 L) were fed with lower phases of the binary systems in descending mode (flow rate: 7 or 20 mL min⁻¹, rotation speed: 1600 or 1000 rpm). The upper phase was pumped through the stationary phase (ascending mode; flow rate: 6 to 20 mL min⁻¹; rotational speed 1600 or 1000 rpm, vide infra) and the equilibrium was assumed to be achieved after the mobile phase emerged from the CPC.

Sample injection. As in all cases the compounds of interest showed higher affinity for more polar lower phase, the latter was employed for solubilization preceding the injection into the loop. Crude samples were dissolved in a minimum volume of stationary phase and filtrated under reduced pressure. The injection was executed using 10 mL (250 mL rotor) or 50 mL injection loop (1 L rotor). The separations were conducted employing 250 mL or 1 L rotors with a flow rate of 7 to 20 mL min⁻¹ at 1600 or 1000 rpm in ascending mode, respectively. The fractions of 25 mL (250 mL rotor) or 250 mL (1 L rotor) were gathered.

Aucubin (1)

Aucubin (1) was extracted from 10.5 kg of leafs of Aucuba japonica Thunb., collected in botanical garden at the University Paris Descartes, Faculty of Pharmacy Paris in June 2013, using aqueous method of Bourquelot¹⁶ to give 470 g of dried extract.

CPC purification. The aliquot of extract (35.2 g) was dissolved by addition of lower phase of the system consisting of AcOEt–n-PrOH–H₂O (7/3/10). The centrifugal partition chromatography apparatus was fitted with lower phase and after the equilibration with upper phase, the filtrated extract was injected. The CPC was conducted using 1 L rotor, 1000 rpm and flow of 17 mL min⁻¹ in ascending mode. The fractions of 250 mL were collected and CPC was followed by TLC (MeOH–CH₂Cl₂ = 8/2). This procedure led to isolation of aucubin (1) (23.9 g, 3%/fresh material) as amorphous powder.

Vacuum liquid column chromatography purification. The extract (105.6 g) was dissolved in methanol and silica gel (100 g, 63–200 μm) was added. The solvent was evaporated and the residue was purified by flash chromatography using silica gel (2.5 L, 40–63 μm) and column (ø 25 cm). The chromatography was performed employing gradient mixture of dichloromethane and methanol in order MeOH–CH₂Cl₂ (5/95, 5 L), MeOH–CH₂Cl₂ (1/9, 10 L), MeOH–CH₂Cl₂ (15/85, 20 L), MeOH–CH₂Cl₂ (8/2, 40 L) under vacuum. Evaporated fractions containing high proportion of aucubin (70.0 g) were crystallized from MeOH to obtain pure 1 (62 g, 2.6%/fresh material). ¹H NMR (CD₃OD), 300 MHz δ (ppm): 6.33 (dd, 1H, ³J_3–4 = 6.5 Hz, ⁴J_3–5 = 2.0 Hz, H–C(3)), 5.77 (brs, 1H, H–C(7)), 5.11 (dd, 1H, ³J_3–4 = 6.5 Hz, ³J_4–5 = 4 Hz, H–C(4)), 4.97 (d, ³J_1–9 = 7.5 Hz, H–C(1)), 4.69 (d, 1H, ³J_1′–2′ = 8 Hz, 1H, H–C(1′)), 4.45 (m, 1H, H–C(6)), 4.37 (brd, 1H, ²J_10a–10b = 15 Hz, H–C(10a)), 4.18 (brd, 1H, ²J_10b–10a = 15 Hz, H–C(10b)), 3.87 (dd, 1H, ²J_{6′a–6′b} = 12.5 Hz, ³J_6′a–5′ = 1.5 Hz, H–C(6′a)), 3.66 (dd, 1H, ²J_{6′a–6′b} = 12.5 Hz, ³J_6′a–5′ = 5.0 Hz, H–C(6′b)), 3.45–3.15 (m, 4H, H–C(5′), H–C(4′), H–C(3′), H–C(2′)), 2.91 (brt 1H, ³J_9–1 = ³J_9–5 = 7.5 Hz, H–C(9)), 2.67 (m, 1H, H–C(5)). ¹³C NMR (CD₃OD), 100 MHz δ (ppm): 146.6 C(8), 140.2 C(3), 128.9 C(7), 104.3 C(4), 98.5 C(1′), 96.3 C(1), 81.5 C(6), 76.9 C(3′), 76.5 C(5′), 73.5 C(2′), 70.2 C(4′), 61.3 C(6′), 60.0 C(10), 46.5 C(9), 44.9 C(5). Elemental analysis of aucubin purified by chromatography: anal. calcd for C₁₅H₂₂O₉·H₂O: C, 49.45; H, 6.64; O, 43.91. Found: C, 49.35; H, 6.78; O 44.11. Elemental analysis for aucubin purified by CPC: anal. calcd for C₁₅H₂₂O₉·H₂O: C, 49.45; H, 6.64; O, 43.91. Found: C, 49.28; H, 6.82; O 44.21%.

Linaride (10-monodeoxyaucubin) (2)

In a flask equipped with cold finger, aucubin (1) (12 g, 34.68 mmol) was dissolved in absolute EtOH (40 mL) and stirred at −35 °C for 10 min. The liquid NH₃ (300 mL) was condensed and the temperature was decreased to −90 °C. Li powder (2.68 g) was added and the solution was stirred for additional 20 min. After the reaction mixture was diluted by EtOH (40 mL), the disappearance of the deep blue color was observed. The addition of the same amount of Li and EtOH was repeated and the reaction mixture was heated to room temperature overnight. The traces of NH₃ were evaporated under vacuum and the residue was dissolved by the addition of 30 mL of lower phase of system consisting of AcOEt–n-PrOH–H₂O (8/2/10) and filtered. The centrifugal partition chromatography apparatus was fitted with lower phase and the filtrate was injected. The CPC was conducted using 1 L rotor, 1000 rpm and flow of 20 mL min⁻¹ in ascending mode. The fractions of 250 mL were collected and CPC was followed by TLC (MeOH–CH₂Cl₂ = 8/2). This procedure led to the isolation of 6,10-dideoxyaucubin (3) (872 mg, 8%) and linaride (2) (9.62 g, 84%) as brownish amorphous powders.¹H NMR (CD₃OD), 300 MHz δ (ppm): 6.23 (dd, 1H, ³J_3–4 = 6.0 Hz, ⁴J_3–5 = 2.0 Hz, H–C(3)), 5.52 (m, 1H, H–C(7)), 5.19 (d, 1H, ³J_1–9 = 6.0 Hz, H–C(1)), 4.97 (dd, ³J_3–4 = 6.0 Hz, ³J_4–5 = 3.5 Hz, H–C(4)), 4.69 (d, 1H, ³J_1′–2′ = 8.0 Hz, 1H, H–C(1′)), 4.36 (dd, ³J_6–7 = 3.0 Hz, ³J_6–5 = 1.5 Hz, 1H, H–C(6)), 3.90 (dd, 1H, ²J_{6′a–6′b} = 12.0 Hz, ³J_6′a–5′ = 1.0 Hz, H–C(6_′a)), 3.68 (brdd, 1H, ²J_{6′a–6′b} = 12.0 Hz, ³J_6′a–5′ = 4.0 Hz, H–C(6′b)), 3.44–3.16 (m, 4H, H–C(5′), H–C(4′), H–C(3′), H–C(2′)), 2.92 (brt, 1H, H–C(9)), 2.66 (m, 1H, H–C(5)), 1.86 (brs, 3H, H–C(10)). NMR (CD₃OD), 100 MHz δ (ppm): 143.4 C(8), 139.7 C(3), 129.0 C(7), 104.6 C(4), 98.1 C(1′), 94.9 C(1), 81.3 C(6), 76.8 C(3′), 76.6 C(5′), 73.5 C(2′), 70.3 C(4′), 61.4 C(6′), 46.7 C(9), 43.4 C(5), 14.8 C(10). Anal. calcd for C₁₅H₂₂O₈·2H₂O: C, 49.15; H, 7.15. Found: C, 49.36; H, 7.07%.

6′,10-Dideoxyaucubin (3)

In a flask equipped with cold finger, aucubin (1) (12 g, 34.68 mmol) was dissolved in absolute EtOH (40 mL) and stirred at −30 °C for 10 min. The liquid NH₃ (300 mL) was condensed and subsequently Li powder (2.68 g) was added. The solution was stirred for additional 20 min. The disappearance of the deep blue color was observed after the reaction mixture was diluted by EtOH (40 mL). The addition of same amount of Li and EtOH was repeated and the reaction mixture was heated to room temperature overnight. The traces of NH₃ were evaporated under vacuum and the residue was dissolved by the addition of lower phase of system consisting of AcOEt–n-PrOH–H₂O (8/2/10) and filtered. The centrifugal partition chromatography apparatus was fitted with lower phase and the filtrate was injected. The CPC was conducted using 1 L rotor, 1000 rpm and flow of 20 mL min⁻¹ in ascending mode. The fractions of 250 mL were collected and CPC was followed by TLC (MeOH–CH₂Cl₂ = 8/2). This procedure led to the isolation of 6,10-dideoxyaucubin (3) (8.72 g, 80%) as brownish amorphous powder. ¹H NMR (CD₃OD), 300 MHz δ (ppm): 6.23 (dd, 1H, ³J_3–4 = 6.5 Hz, ⁴J_3–5 = 2.0 Hz, H–C(3)), 5.42 (m, 1H, H–C(7)), 5.28 (d, 1H, ³J_1–9 = 5.0 Hz, H–C(1)), 4.80 (dd, ³J_3–4 = 6.5 Hz, ³J_4–5 = 3.5 Hz, H–C(4)), 4.69 (d, 1H, ³J_1′–2′ = 8 Hz, 1H, H–C(1′)), 3.90 (dd, 1H, ²J_{6′a–6′b} = 12.0 Hz, ³J_6′a–5′ = 2.0 Hz, H–C(6′a)), 3.68 (dd, 1H, ²J_{6′a–6′b} = 12.0 Hz, ³J_6′a–5′ = 5.5 Hz, H–C(6′b)), 3.47–3.20 (m, 4H, H–C(5′), H–C(4′), H–C(3′), H–C(2′)), 2.91 (m, 1H, H–C(5)), 2.69 (brt, 1H, H–C(9)), 2.63–2.49 (m, 1H, H–C(6a)), 2.10–1.96 (m, 1H, H–C(6b)), 1.86 (brs, 3H, H–C(10)). ¹³C NMR (MeOD), 100 MHz δ (ppm): 139.1 (2C atoms) C(8), C(3), 125.5 C(7), 107.5 C(4), 98.2 C(1′), 94.0 C(1), 76.7 C(3′), 76.5 C(5′), 73.5 C(2′), 70.2 C(4′), 61.3 C(6′), 50.1 C(9), 38.4 C(5), 32.8 C(6), 14.6 C(10). Anal. calcd for C₁₅H₂₂O₇·H₂O: C, 54.21; H, 7.28. Found: C, 54.45; H, 7.25%.

Aucubigenin (4)

Aucubin (1) (10 g, 28.9 mmol) was dissolved in H₂O (200 mL) and β-glucosidase (Aldrich) (2 g) was added. The hydrolysis was followed by TLC (EtOAc–MeOH = 8/2) and after 4 h at 37 °C, 1 was completely transformed into a less polar compound. The reaction mixture was lyophilized and the residue was purified by means of CPC. The crude 4 was dissolved in lower phase of the system (AcOEt–H₂O = 1/1). The CPC apparatus was fitted with lower phase and equilibrated with upper phase. The filtrated reaction mixture was injected. The CPC was conducted using 1 L rotor, 1000 rpm and flow of 20 mL min⁻¹ in ascending mode. The fractions of 250 mL were collected and CPC was followed by TLC (MeOH–CH₂Cl₂ = 8/2). This procedure led to isolation of aucubigenin (4) with anomeric ratio α/β = 1/9 (4.8 g, 91%) as brownish oil. ¹H NMR (D₂O), 300 MHz δ (ppm) 4β: 6.23 (dd, 1H, ³J_3–4 = 6.0 Hz, ⁴J_3–5 = 1.5 Hz, H–C(3)), 5.69 (brs, 1H, H–C(7)), 5.03 (dd, 1H, ³J_4–3 = 6.0 Hz, ³J_4–5 = 3.5 Hz, H–C(4)), 4.71 (d, ³J_1–9 = 7 Hz, H–C(1)), 4.42 (m, 1H, H–C(6)), 4.30–4.15 (2d, 2H, ²J_10a–10b = 15.0 Hz, H–C(10)), 2.72 (dd, 1H, ³J_9–5 = 7.5 Hz, ³J_1–9 = 7.0 Hz, H–C(9)), 2.56 (m, 1H, H–C(5)). ¹³C NMR (100 MHz, D₂O) δ (ppm): 147.4 C(8), 141.3 C(3), 129.7 C(7), 105.3 C(4), 95.3 C(1), 82.0 C(6), 60.8 C(10), 48.7 C(9), 45.1 C(5). Anal. calcd for C₉H₁₃O₃·1/2H₂O: C, 55.95; H, 6.78. Found: C, 56.30; H, 7.01%.

10-Monodeoxyaucubigenin (5)

Laniride (2) (10 g, 28.9 mmol) was dissolved in H₂O (200 mL) and β-glucosidase (Aldrich) (2 g) was added. The hydrolysis was followed by TLC (EtOAc–MeOH = 4/1) and after 10 h at 37 °C, 1 was completely transformed into a less polar compound. The reaction mixture was extracted with EtOAc (200 mL × 4). The combined organic layers were dried over Na₂SO₄ and evaporated in vacuo at room temperature. The obtained residue (5.8 g) was purified by means of CPC. The crude 5 was dissolved in upper phase of the system (AcOEt–H₂O = 1/1). The CPC apparatus was fitted with lower phase and the filtrated reaction mixture was injected. The CPC was conducted using 1 L rotor, 1000 rpm and flow of 20 mL min⁻¹ in ascending mode. This procedure led to isolation of 6′-monodeoxyaucubigenin (5) (4.3 g, 87%) with anomeric ratio α/β = 6/94 as brownish oil. ¹H NMR (acetone-d₆), 300 MHz δ (ppm) 5β: 6.30 (dd, 1H, ³J_3–4 = 6.5 Hz, ⁴J_3–5 = 1.5 Hz, H–C(3)), 5.96 (d, 1H, ³J_1–9 = 6.5 Hz, H–C(1)), 5.69 (brs, 1H, H–C(7)), 5.04 (dd, 1H, ³J_4–5 = 6.0 Hz, ³J_4–3 = 6.5 Hz, H–C(4)), 4.63 (dd, ³J_1-C(1)OH = 6.5 Hz, ⁴J_C(1)–OH-9 = 2.5 Hz, HO–C(1)), 4.38 (brs, 1H, HO–C(6)), 3.98 (d, 1H, ³J_6-C(6)OH = 6.0 Hz, H–C(6)), 2.88 (m, 1H, H–C(9)), 2.55 (dd, 1H, ³J_4–5 = 6.0 Hz, ³J_6–5 = 3.0 Hz, H–C(5)), 1.85 (s, 3H, H–C(10)). ¹³C NMR (100 MHz, acetone-d₆) δ (ppm): 142.2 C(8), 141.0 C(3), 131.0 C(7), 104.0 C(4), 96.2 C(1), 82.2 C(6), 51.4 C(9), 46.4 C(5), 16.1 C(10). ¹H NMR (acetone-d₆), 300 MHz δ (ppm) 5α: 6.16 (dd, 1H, ³J_1–9 = 6.50 Hz, ⁴J_1–5 = 1.5 Hz, H–C(3)), 5.46 (brs, 1H, H–C(7)), 4.95 (dd, 1H, ³J_4–3 = 6.0 Hz, ³J_4–5 = 3.0 Hz, H–C(4)), 4.69 (dd, ³J_1-C(1)OH = 6.50 Hz, ⁴J_C(1)–OH-9 = 2.5 Hz, HO–C(1)), 4.51 (brs, 1H, HO–C(6)), 3.80 (d, 1H, ³J_6-C(6)OH = 6.0 Hz, H–C(6)), 2.85 (m, 1H, H–C(9)), 1.80 (s, 3H, H–C(10)). Anal. calcd for C₉H₁₂O₃·1/2H₂O: C, 61.00; H, 7.39. Found: C, 61.08; H, 7.09%.

(R)-Rotundial [(R)-2′-methyl-5′-(2′′-oxoethyl)cyclopent-1′-enecarbaldehyde] (6)

Acid hydrolysis of 6,10-dideoxyaucubin (3) (500 mg, 1.59 mmol) was dissolved in phosphate buffer (pH = 2, 15 mL) and stirred at 25 °C. The hydrolysis was followed by TLC (CH₂Cl₂–MeOH = 95/5) and after 2 h, 3 was completely transformed into a less polar compound. The solution was extracted with EtOAc (4 × 50 mL). Collected extracts were neutralized with NaHCO₃, dried over Na₂SO₄ and evaporated in vacuo. The obtained residue (350 mg) was purified by means of CPC. The crude 6 was dissolved in organic phase of the system (AcOEt–H₂O = 1/1, 0.5 mL). The CPC apparatus was fitted with aqueous phase and the filtrated reaction mixture was injected. The CPC was conducted using 250 mL rotor, 1600 rpm and flow of 7 mL min⁻¹ in ascending mode. This procedure led to isolation of (R)-rotundial (6) (133 mg, 55%) as brownish oil. This procedure was also repeated using 2 g (6.36 mmol) of 3. (R)-rotundial (6) was isolated in (540 mg, 55%). [α]²⁰_D = +108.8 (c 1.15, CHCl₃) {lit.⁶ [α]²²_D = +108 (c 1.00, CHCl₃, >99% ee)}. ¹H NMR (300 MHz, CDCl₃) δ: 9.97 (s, 1H, H–C(1)), 9.80 (t, ³J_{1′′–2′′} = 2.0 Hz, 1H, H–C(1′′)), 3.46 (m, 1H, H–C(5′)), 2.93 (brdd, 1H, ²J_{2′′a–2′′b} = 17 Hz, ³J_{5′–2′′a} = 4.0 Hz, H–C(2′′a)), 2.75–2.5 (m, 2H, H–C(3′)), 2.32 (ddd, 1H, ²J_{2′′a–2′′b} = 17 Hz, ³J_{2′′b–5′} = 9.0 Hz, ³J_{1′′–2′′} = 2 Hz, H–C(2′′b)), 2.21–2.12 (m, 1H, H–C(4′a)), 2.15 (s, 3H, H₃C–C(2′)), 1.56 (ddt, 1H, ²J_{4′a–4′b} = 14.0, ³J_{3′a–4′b} = 9 Hz, ³J_5′–4′b = ³J_{3′b–4′b} = 6 Hz, H–C(4′b)). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 202.0 C(1′′), 188.0 C(1), 164.3 C(2′), 139.0 C(1′), 47.8 C(2′′), 39.1 C(3′), 38.2 C(5′), 28.2 C(4′), 14.5 CH₃. HRMS ([M + H]⁺) calcd for C₉H₁₃O₂ 153.0910, found 153.0901. Anal calcd for C₉H₁₂O₂: C, 71.03; H, 7.95 found: C, 70.71; H, 8.21%.

Acknowledgements

The authors thank the Agence Nationale de la Recherche (ANR) for the financial support (ANR-09-CP2D-09-01).

Notes and references

1. (a) D. J. Newman and G. M. Cragg, J. Nat. Prod., 2007, 70, 461; (b) G. M. Cragg, P. G. Grothaus and D. J. Newman, J. Nat. Prod., 2014, 77, 703; (c) F. E. Koehn and G. T. Carter, Nat. Rev. Drug Discovery, 2005, 4, 206; (d) D. J. Newman and G. M. Cragg, J. Nat. Prod., 2012, 75, 311.
2. P. S. Baran, Nat. Chem., 2009, 1, 193.
3. P. A. Wender, Chem. Rev., 1996, 96, 1.
4. (a) S. C. Stinson, Chem. Eng. News, 2001, 7920, 45; (b) Chirality in Industry II: Developments in the Commercial Manufacture and Applications of Optically Active Compounds, ed. A. N. Collins, G. Sheldrake and J. Crosby, Wiley, Chichester, 1998.
5. (a) H. B. Kagan, Actual. Chim., 2003, 62; (b) S. Haleema, P. Vavan Sasi, I. Ibnusaud, O. L. Polavarapu and H. B. Kagan, RSC Adv., 2012, 2, 9257.
6. Eco-extraction du végétal - Procédés innovants et solvants alternatifs, ed. F. Chemat, Dunod, Paris, 2011.
7. (a) K. Ingkaninan, A. Hazekamp, A. C. Hoek, S. Balconi and R. Verpoorte, J. Liq. Chromatogr. Relat. Technol., 2000, 23, 2195; (b) L. Marchal, J. Legrand and A. Foucault, Chem. Rec., 2003, 3, 133; (c) I. A. Sutherland, J. Chromatogr. A, 2007, 1151, 6; (d) E. Hopmann, I. Goll and M. Monceva, Chem. Eng. Technol., 2012, 35, 72.
8. (a) K. D. Yoon, Y.-W. Chin and J. Kim, J. Liq. Chromatogr. Relat. Technol., 2010, 33, 1208; (b) A. P. Foucault, J. Chromatogr. A, 2001, 906, 365; (c) A. Toribio, J.-M. Nuzillard and J.-H. Renault, J. Chromatogr. A, 2007, 1170, 44; (d) N. Rubio and C. Minguillón, J. Chromatogr. A, 2010, 1217, 1183.
9. A. Berthod, M. J. Ruiz-Ángel and S. Carda-Broch, J. Chromatogr. A, 2009, 1216, 4206.
10. (a) E. F. Queiroz, F. Roblot, P. Duret, B. Figadère, A. Gouyette, O. Laprévote, L. Serani and R. Hocquemiller, J. Med. Chem., 2000, 43, 1604; (b) E. Otsubo, T. Takey and M. Nomura, Biol. Pharm. Bull., 2001, 24, 1362; (c) M. A. Fakhfakh, X. Franck, A. Fournet, R. Hocquemiller and B. Figadère, Tetrahedron Lett., 2001, 42, 3847; (d) T. Watanabe, I. Momose, M. Abe, H. Abe, R. Sawa and Y. Umezawa, Bioorg. Med. Chem., 2009, 19, 2343; (e) R. Delépée, S. Berteina- Raboin, M. Lafosse, C. Lamy, S. Darnault, I. Renimel, N. About and P. André, J. Liq. Chromatogr. Relat. Technol., 2007, 30, 2069; (f) G. Moroy, C. Denhez, H. El Mourabit, A. Toribio, A. Dassonville, M. Decarme, J.-H. Renault, C. Mirand, G. Bellon, J. Sapi, A. J. P. Alix, W. Hornebeck and E. Bourguet, Bioorg. Med. Chem., 2007, 15, 4753.
11. J. Brunetton, Pharmacognosy, Phytochemistry, Medicinal Plants, Tec & Doc, Paris, 2nd edn, 1999.
12. (a) X. Cachet, B. Deguin, M. Koch, K. Makhlouf and F. Tillequin, J. Nat. Prod., 1999, 62, 400; (b) C. Mouriès, B. Deguin, F. Lamari, M. J. Foglietti, F. Tillequin and M. Koch, Tetrahedron: Asymmetry, 2003, 14, 1083; (c) C. Mouriès, V. C. Rakotondramasy, F. Libot, M. Koch, F. Tillequin and B. Deguin, Chem. Biodiversity, 2005, 2, 695; (d) V. C. Rakotondramasy, R. Laschiazza, M. Lecso, M. Koch, F. Tillequin and B. Deguin, J. Nat. Prod., 2007, 70, 19; (e) V. C. Rakotondramasy, C. Mouriès, X. Cachet, A. Neghra, M. El Mourabet, F. Tillequin, M. Koch and B. Deguin, Eur. J. Med. Chem., 2010, 45, 2314; (f) C. Lemus, M. Poleschak, S. Gailly, M. Desage-El Murr, M. Koch and B. Deguin, Chem.–Eur. J., 2013, 19, 4686; (g) K. Vougogiannopoulou, C. Lemus, M. Halabalaki, C. Pergola, O. Werz, A. B. Smith III, S. Michel, L. Skaltsounis and B. Deguin, J. Nat. Prod., 2014, 77, 441; (h) C. Lemus, M. Koch, S. Michel and B. Deguin, Arkivoc, 2014, 3, 184; (i) H. Lemoine, D. Markovic and B. Deguin, J. Org. Chem., 2014, 79, 4358.
13. (a) A. J. Birch, J. Grimshaw and H. R. Juneja, J. Chem. Soc., 1961, 5194; (b) C. Iavarone, F. Mataloni and C. Trogolo, J. Org. Chem., 1988, 53, 2089.
14. A. Bianco, M. Guiso, C. Iavarone, P. Passacantilli and C. Trogolo, Tetrahedron, 1977, 33, 851.
15. (a) K. Weinges, U. Dietz, T. Oeser and H. Irngartinger, Angew. Chem., 1990, 102, 685; (b) H. Franzyk, Fortschritte der Chemie Organischer Naturstoffe, ed. W. Herz, H. Falk, G. W. Kirby and R. E. Moore, Springer-Verlag, Wien, 2000, vol. 79, pp. 1–114; (c) A. Bianco, L. Celleti, R. A. Mazzei and F. Umani, Eur. J. Org. Chem., 2001, 1331; (d) A. Bianco, D. Celona, S. Di Rita, M. Guiso, C. Melchioni and F. Umani, Eur. J. Org. Chem., 2001, 4061.
16. E. Bourquelot and H. Herissey, C. R. Acad. Sci., 1902, 134, 1441.
17. A. Hermans-Lokkerbol and R. Verpoorte, Planta Med., 1987, 53, 546.
18. (a) A. P. Foucault and L. Chevolot, J. Chromatogr. A, 1998, 808, 3; (b) J. B. Friesen and G. F. Pauli, J. Liq. Chromatogr. Relat. Technol., 2005, 28, 2777; (c) J. B. Friesen and G. F. Pauli, J. Chromatogr. A, 2009, 1216, 4225.
19. (a) J. M. Bobitt and K.-P. Segebarth, Cyclopentanoid terpene derivatives, ed. W. I. Taylor and A. R. Battersby, Marcel Dekker, New York, 1969, vol. 1, ch. 1, p. 1; (b) E. Davini, C. Iavarone, C. Trogolo, P. Aureli and B. Pasolini, Phytochemistry, 1986, 25, 2420.
20. (a) K. Watanabe, Y. Takada, N. Matsuo and H. Nishimura, Biosci., Biotechnol., Biochem., 1995, 59, 1979; (b) E. Marques-Lopez, R. P. Herrera, T. Marks, W. C. Jacobs, D. Koenning, R. M. De Figueiredo and M. Christmann, Org. Lett., 2009, 11, 4116; (c) H. Takikawa, Y. Yamazaki and K. Mori, Eur. J. Org. Chem., 1998, 229.
21. A. Berthod and S. Carda-Broch, J. Chromatogr. A, 2004, 1037, 3.
22. A. Ollivier, R. Grougnet, X. Cachet, D. Meriane, J. Ardisson, S. Boutefnouchet and B. Deguin, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2013, 926, 16.

---

Footnote

† Electronic supplementary information (ESI) available: Purification procedures, Kd determination, NMR spectra, microanalyses. See DOI: 10.1039/c4ra12141d

---