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

Article abstract of DOI:10.1039/c4ra12141d

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.

Full text of DOI:10.1039/c4ra12141d

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PAPER
Centrifugal partition chromatography: an efficient
tool to access highly polar and unstable synthetic
compounds on a large scale†
Cite this: RSC Adv., 2014, 4, 63254
Dean Markovic, Cedric Barboux, Yse Salle de Chou, Judith Bettach, Raphael Grougnet
´
´
´
¨
and Brigitte Deguin*
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.
Received 10th October 2014
Accepted 7th November 2014
DOI: 10.1039/c4ra12141d
www.rsc.org/advances
avoid the use of solid stationary chromatographic phases and
chlorinated eluents.
Introduction
Natural products play an important role in chemical ecology
Centrifugal partition chromatography uses two immiscible
1
and have been a source of inspiration for organic chemists. The liquids as mobile and stationary phases avoiding solid support.
perfect synthetic strategy complies with the concept of pro- The mobile phase percolates through the stationary phase,
2
7
tecting group free chemistry engaging renewable and readily which is under the centrifugal eld. This chromatography
available starting materials in a minimum number of simple, technique has become more and more frequently employed for
3
safe and quick steps with high overall yield. As the chirality of the analytic and preparative separations and isolations of
the natural products is one of the key elements responsible for natural products, macromolecules, enantioseparations, frac-
8
the biological activities, the production of enantiomerically tionation of extracts and other miscellaneous uses. Although
pure chemicals is of high value for pharmaceutical, food, rotors of CPC apparatus vary from 100 mL to 25 L allowing
4
7
agrochemical, fragrance and avor industries.
various academic and industrial applications, this technique
Classically, chiral pool is exploited in asymmetric has been rarely applied for the purication of synthetic
9
synthesis as the source of starting materials, chiral auxilia- compounds. For example, the CPC was performed for the
ries or resolving reagents and in the preparation of enantio- purication of glycosylated derivatives of squamocin, human
5
selective catalysts. In respect to green chemistry, the surfactant protein SP-C, quinolines, analogues of ilomastat,
10
employment of other common and abundant renewable salidroside and boronic acid derivatives of tyropeptin.
resources, not commercialized, could minimize the number
Common iridoids and secoiridoids are widespread in
of synthetic steps and/or enlarge the panel of chiral building nature and can be produced by numerous plants in abundant
1
1
blocks. In order to develop this concept, natural highly amounts. We have used them as chiral renewable starting
functionalized starting material, with low toxicity have to be materials for the synthesis of new scaffolds and building
6
eco-extracted and isolated in large scale. Those unprotected blocks particularly useful for the construction of new
1
2
products can be further engaged in multi-step synthesis heterocyclic systems and bioactive compounds. A signi-
employing environmentally friendly reagents and solvents. cant member of this monoterpenoid group is aucubin (1). Its
In this context, the purication methods are in particular polyfunctional aglycon part has been used for several
1
3–15
focus for the effective isolation of highly polar and unstable decades by organic chemists as chiral source.
The puri-
products in large amounts in line with saving of consump- cations of 1 or of its unprotected derivatives are oen
tion of labor and time. In addition, this manipulation has to 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 utili-
Universit ´e Paris Descartes, Facult ´e de Pharmacie de Paris, Sorbonne Paris Cit ´e , zation of CPC for synthetic purposes.
U.M.R./C.N.R.S. 8638, Laboratoire de Pharmacognosie, 4 Avenue de l'Observatoire,
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
F-75006 Paris, France. E-mail: brigitte.deguin@parisdescartes.fr; Fax: +33 140 46
9
6 58
†
Electronic supplementary information (ESI) available: Purication procedures,
6,10-dideoxyaucubin (3), from salts produced by Birch
Kd determination, NMR spectra, microanalyses. See DOI: 10.1039/c4ra12141d
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reduction. Furthermore, CPC was tested for the direct isolation
Our interest was further expanded to the purication of
of highly unstable aglucons 4 and 5 together with natural highly polar compounds (Scheme 1). We tested the CPC method
repellent, (R)-rotundial (6).
for the purication of the reduced aucubin derivatives 2 and 3
obtained under Birch conditions (Li/NH (liq.)). This reduction
3
method affords hardly available iridoids; linaride (2) and 6,10-
ꢁ
dideoxyaucubin (3), in 9/1 ratio at ꢀ90 C or selectively 3 at ꢀ30
Results and discussion
13
ꢁ
ꢁ
C. 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
Aucubin (1) was extracted by aqueous media in large amounts
1
6
upper phase of the ternary system (H O–n-PrOH–AcOEt, 10/2/8),
with a ow of 20 mL min at 1000 rpm, in isocratic ascending
mode. Similarly, the same quantity of aucubin (1) was employed
2
from the fresh aerial parts of Aucuba japonica Thunb. 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 (CH Cl –MeOH (95/5) / CH Cl –MeOH (85/15)) as
ꢀ1
ꢁ
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 purications 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
2
2
2
2
eluent. We improved this method by developing a quick and
efficient CPC purication employing an environmentally
13
polarity of formed products.
Aglucons of iridoids obtained by enzymatic cleavage easily
decompose and produce blue to black polymeric substances.
friendly, low cost solvent system containing H
2
O–n-PrOH–
1
7
EtOAc (10/3/7) in isocratic mode. The used ternary system
14
1
8
was superior over the tested Arizona systems (Hept–EtOAc–
Genines of aucubin (1) or of its deoxy-derivatives 2 and 3 are
particularly unstable due to the equilibria with their dialdehyde
forms. Their purications by classical techniques with solid
MeOH–H O) as these latter were not sufficiently polar. In
2
order to ensure an efficient retention of the highly-polar iri-
doids, 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 tted with maximum working load
corresponding to 35 g of dry extract. The isolation of aucubin
19
stationary phases were impossible in multigram scale.
However using CPC, the reaction mixtures were easily puried.
Compounds 1 or 2, at 10 g scale, were hydrolyzed by b-gluco-
sidase (Scheme 1). The lyophilized reaction mixtures were dis-
solved in lower phase of binary system (H O–EtOAc (1/1)) and
2
(
1) with high purity (3%/fresh material, 23.9 g) was per-
ꢀ1
directly injected into CPC instrument containing lower phase as
stationary. The purications 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 a/b ¼ 1/9 and 6/94 in 91% (4.8
g) and 87% (4.3 g) yields, respectively.
formed with a total of 12 L of upper phase with a 17 mL min

ow 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 purication of same
amount of the extract (105 g) adsorbed on silica-gel (100 g,
6
6
3–200 mm) using a column (ø 25 cm) of silica-gel (2.5 L, 40–
3 mm) and 80 L of eluent affording pure 1 aer crystalliza-
Finally, we employed CPC in the isolation of semi-
synthesized mosquito repellent (R)-rotundial (6). This natural
product was rst isolated by Nishimura in 1995 from Vitex
rotundifolia. Out of three total synthesis of 6, two were asym-
metric and were conducted in 6 and 10 steps with 27% and 25%
tion (59 g: 2.6%/fresh material). The VLC procedure was
performed in seven working days whereas CPC took 13 h per
run.
20
overall yield, respectively. We obtained (R)-rotundial (6) simply
by the hydrolysis of 6,10-dideoxyaucubin (3) with phosphate
buffer (pH ¼ 2). Aer a extraction by EtOAc, the crude 6 was
injected into the CPC apparatus (250 mL rotor, ow 7 mL
Table
d
1 Retention times, employed method and K values of
compounds 1–6
ꢀ1
Compound
Flow rate/[ml min
]
R
t /min
K
d
1
2
3
4
5
6
1
1
1
0.3
0.5
0.5
4.4
7.3
13.3
5.9
7.8
5.6
17.6
5.1
2.2
4.0
1.3
15.0
Scheme 1 CPC purifications of aucubin (1) and derivatives 2–6.
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ꢀ
1
min , 1700 rpm) using H
2
O–EtOAc (1/1) solvent system in elution with MeCN–H
2
O as following method: 5/95 (0–5 min), 5/
ascending mode to give pure (R)-rotundial (6) in 55% yield.
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.
Equilibration procedure. The rotors of CPC instrument (250
mL or 1 L) were fed with lower phases of the binary systems in
Conclusions
ꢀ1
In addition to its simplicity and utilization in natural product descending mode (ow rate: 7 or 20 mL min , rotation speed:
isolations, CPC is a practical alternative to classical purication 1600 or 1000 rpm). The upper phase was pumped through the
methods used in preparative organic synthesis. By appropriate stationary phase (ascending mode; ow rate: 6 to 20 mL min
ꢀ1
;
selection of solvent systems, this technique is easily adaptable rotational speed 1600 or 1000 rpm, vide infra) and the equilib-
to green conditions. As demonstrated, it is a valuable rium was assumed to be achieved aer the mobile phase
improvement for the purication of unstable and highly polar emerged from the CPC.
compounds, simplifying the work-up and saving time, labor,
Sample injection. As in all cases the compounds of interest
organic solvents and reagents. The chemistry of abundant showed higher affinity for more polar lower phase, the latter was
natural product aucubin (1), coupled with CPC purications employed for solubilization preceding the injection into the
have furnished a collection of rare iridoids, deoxyderivatives 2 loop. Crude samples were dissolved in a minimum volume of
and 3, as well as corresponding aglycons 4, 5 and the repellent stationary phase and ltrated under reduced pressure. The
(R)-rotundial (6) in multigram quantities. The access to the injection was executed using 10 mL (250 mL rotor) or 50 mL
products in these amounts was not successful by employing injection loop (1 L rotor). The separations were conducted
classical techniques. Avoiding protecting groups, silica-gel and employing 250 mL or 1 L rotors with a ow rate of 7 to 20 mL
ꢀ
1
chlorinated solvents, all steps of isolation and synthesis from min at 1600 or 1000 rpm in ascending mode, respectively. The
plant Aucuba japonica Thunb. were improved, thus opening a fractions of 25 mL (250 mL rotor) or 250 mL (1 L rotor) were
scalable route towards a large number of chiral building blocks gathered.
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
Aucubin (1)
exploitation in green organic synthesis.
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
Experimental section
General CPC separation procedure
16
aqueous method of Bourquelot to give 470 g of dried extract.
CPC purication. The aliquot of extract (35.2 g) was dis-
Selection of solvent systems and the determination of the Kd solved by addition of lower phase of the system consisting of
values. The two-phase solvent systems were selected according AcOEt–n-PrOH–H O (7/3/10). The centrifugal partition chro-
to an evaluation of the distribution coefficient (K ) with purpose matography apparatus was tted with lower phase and aer the
to eliminate the impurities. K is dened as a ratio of concen- equilibration with upper phase, the ltrated extract was injec-
trations of the compound between the two immiscible solutions ted. The CPC was conducted using 1 L rotor, 1000 rpm and ow
2
d
d
ꢀ
1
in equilibrium state. The K
d
values were rst evaluated using the of 17 mL min in ascending mode. The fractions of 250 mL
shake-ask method followed by thin layer chromatography. A were collected and CPC was followed by TLC (MeOH–CH Cl
crude extract, a reaction mixture or a compound of interest was 8/2). This procedure led to isolation of aucubin (1) (23.9 g, 3%/
21
2
2
¼
ꢀ1
dissolved in a variety of two-phase systems (5 mg mL ) and the fresh material) as amorphous powder.
optimal partition was identied. Classically, optimal K values Vacuum liquid column chromatography purication. The
are comprised between 0.5 and 2. Nevertheless, we employed extract (105.6 g) was dissolved in methanol and silica gel (100 g,
the systems with higher K 's in order to obtain pure compounds 63–200 mm) was added. The solvent was evaporated and the
values of the pure compounds were residue was puried by ash chromatography using silica gel
determined by HPLC according to the following equation: K (2.5 L, 40–63 mm) and column (ø 25 cm). The chromatography
solute]lower-phase/[solute]upper-phase. Pure compounds 1–6 was performed employing gradient mixture of dichloromethane
d
d
22
in only one run.
K
d
d
¼
[
(
2 mg) were dissolved in two phase system (10 mL) and aliquots and methanol in order MeOH–CH Cl (5/95, 5 L), MeOH–
2 2
(
1 mL) of upper and lower-phase were taken and evaporated. CH Cl (1/9, 10 L), MeOH–CH Cl (15/85, 20 L), MeOH–CH Cl
2 2 2 2 2 2
The ternary solvent systems containing EtOAc, n-PrOH and H
2
O
(8/2, 40 L) under vacuum. Evaporated fractions containing high
were used for the CPC purication of aucubin 1 (7/3/10: v/v), 2 proportion of aucubin (70.0 g) were crystallized from MeOH to
1
(
H
8/2/10: v/v), 3 (8/2/10: v/v), while binary systems of EtOAc and obtain pure 1 (62 g, 2.6%/fresh material). H NMR (CD
3
OD), 300
3
4
2
O (5/5: v/v) were employed for the purication of genines 4 MHz d (ppm): 6.33 (dd, 1H, J3–4 ¼ 6.5 Hz, J3–5 ¼ 2.0 Hz, H–C(3)),
3
3
and 5 as well as (R)-rotundial (6). The residue was redissolved in 5.77 (brs, 1H, H–C(7)), 5.11 (dd, 1H, J3–4 ¼ 6.5 Hz, J4–5 ¼ 4 Hz,
3
3
water (1 mL) and ltered before injection on UptiDisc 0.45M H–C(4)), 4.97 (d, J1–9 ¼ 7.5 Hz, H–C(1)), 4.69 (d, 1H, J
1
0
–2
0
¼ 8
0
2
nylon lters (Interchim, Montluc, France). 20 ml of each solution Hz, 1H, H–C(1 )), 4.45 (m, 1H, H–C(6)), 4.37 (brd, 1H, J
¼
1
0a–10b
2
were injected and chromatograms were recorded at 226 nm. 15 Hz, H–C(10a)), 4.18 (brd, 1H, J
The analysis was carried out at room temperature, using Aglient 3.87 (dd, 1H, J
ZORBAX SB-C18 (5 mm, 4.6 ꢂ 150 mm) column and a gradient (dd, 1H, J
¼ 15 Hz, H–C(10b)),
1
0b–10a
2
3
0
0
0
¼ 12.5 Hz, J
0
0
¼ 1.5 Hz, H–C(6 a)), 3.66
6
a–6
b
6
a–5
2
3
0
0
0
¼ 12.5 Hz, J
0
0
¼ 5.0 Hz, H–C(6 b)), 3.45–3.15
6
a–6
b
6
a–5
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0
0
0
0
3
(
m, 4H, H–C(5 ), H–C(4 ), H–C(3 ), H–C(2 )), 2.91 (brt 1H, J9–1
¼
to room temperature overnight. The traces of NH
3
were
3
13
J9–5 ¼ 7.5 Hz, H–C(9)), 2.67 (m, 1H, H–C(5)). C NMR (CD OD), evaporated under vacuum and the residue was dissolved by
3
1
9
7
00 MHz d (ppm): 146.6 C(8), 140.2 C(3), 128.9 C(7), 104.3 C(4), the addition of lower phase of system consisting of AcOEt–n-
0
0
0
0
8.5 C(1 ), 96.3 C(1), 81.5 C(6), 76.9 C(3 ), 76.5 C(5 ), 73.5 C(2 ), PrOH–H O (8/2/10) and ltered. The centrifugal partition
0.2 C(4 ), 61.3 C(6 ), 60.0 C(10), 46.5 C(9), 44.9 C(5). chromatography apparatus was tted with lower phase and
2
0
0
Elemental analysis of aucubin puried by chromatography: the ltrate was injected. The CPC was conducted using 1 L
ꢀ1
anal. calcd for C15
Found: C, 49.35; H, 6.78;
analysis for aucubin puried by CPC: anal. calcd for by TLC (MeOH–CH
C H O $H O: C, 49.45; H, 6.64; O, 43.91. Found: C, 49.28; H, isolation of 6,10-dideoxyaucubin (3) (8.72 g, 80%) as
H
22
O
9
$H
2
O: C, 49.45; H, 6.64; O, 43.91. rotor, 1000 rpm and ow of 20 mL min in ascending mode.
44.11. Elemental The fractions of 250 mL were collected and CPC was followed
Cl
¼ 8/2). This procedure led to the
O
2
2
1
5
22
9
2
1
6
.82; O 44.21%.
brownish amorphous powder. H NMR (CD OD), 300 MHz d
3
3
4
(
5
(
8
2
ppm): 6.23 (dd, 1H, J
¼ 6.5 Hz, J
¼ 2.0 Hz, H–C(3)),
3
–4
3–5
3
.42 (m, 1H, H–C(7)), 5.28 (d, 1H, J1–9 ¼ 5.0 Hz, H–C(1)), 4.80
Linaride (10-monodeoxyaucubin) (2)
3
3
3
dd, J3–4 ¼ 6.5 Hz, J4–5 ¼ 3.5 Hz, H–C(4)), 4.69 (d, 1H, J
1
0
–2
0
¼
¼
In a ask equipped with cold nger, aucubin (1) (12 g, 34.68
mmol) was dissolved in absolute EtOH (40 mL) and stirred at
0
2
3
Hz, 1H, H–C(1 )), 3.90 (dd, 1H, J
6
0
a–6
0
b
¼ 12.0 Hz, J
6
0
a–5
0
0
2
3
.0 Hz, H–C(6 a)), 3.68 (dd, 1H, J
6
0
a–6
0
b
¼ 12.0 Hz, J
6
0
a–5
0
¼ 5.5
ꢁ
ꢀ
35 C for 10 min. The liquid NH (300 mL) was condensed and
3
0
0
0
0
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–
.49 (m, 1H, H–C(6a)), 2.10–1.96 (m, 1H, H–C(6b)), 1.86 (brs,
H, H–C(10)). C NMR (MeOD), 100 MHz d (ppm): 139.1 (2C
ꢁ
the temperature was decreased to ꢀ90 C. Li powder (2.68 g) was
added and the solution was stirred for additional 20 min. Aer
the reaction mixture was diluted by EtOH (40 mL), the disap-
pearance 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
0
2
3
1
3
0
atoms) C(8), C(3), 125.5 C(7), 107.5 C(4), 98.2 C(1 ), 94.0 C(1),
0
0
0
0
0
7
6.7 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
$H O: C, 54.21; H, 7.28. Found: C, 54.45; H, 7.25%.
of NH were evaporated under vacuum and the residue was
dissolved by the addition of 30 mL of lower phase of system
3
C
15
H
22
O
7
2
consisting of AcOEt–n-PrOH–H O (8/2/10) and ltered. The
centrifugal partition chromatography apparatus was tted with
2
Aucubigenin (4)
2
lower phase and the ltrate was injected. The CPC was con- Aucubin (1) (10 g, 28.9 mmol) was dissolved in H O (200 mL)
ꢀ1
ducted using 1 L rotor, 1000 rpm and ow of 20 mL min in and b-glucosidase (Aldrich) (2 g) was added. The hydrolysis was
ꢁ
ascending mode. The fractions of 250 mL were collected and followed by TLC (EtOAc–MeOH ¼ 8/2) and aer 4 h at 37 C, 1
CPC was followed by TLC (MeOH–CH
2
Cl
2
¼ 8/2). This proce- was completely transformed into a less polar compound. The
dure led to the isolation of 6,10-dideoxyaucubin (3) (872 mg, reaction mixture was lyophilized and the residue was puried
%) and linaride (2) (9.62 g, 84%) as brownish amorphous by means of CPC. The crude 4 was dissolved in lower phase of
8
1
3
powders. H NMR (CD OD), 300 MHz d (ppm): 6.23 (dd, 1H, J
the system (AcOEt–H O ¼ 1/1). The CPC apparatus was tted
3
3–4
2
4
¼
6.0 Hz, J3–5 ¼ 2.0 Hz, H–C(3)), 5.52 (m, 1H, H–C(7)), 5.19 (d, with lower phase and equilibrated with upper phase. The l-
3
3
3
1
H, J1–9 ¼ 6.0 Hz, H–C(1)), 4.97 (dd, J3–4 ¼ 6.0 Hz, J4–5 ¼ 3.5 trated reaction mixture was injected. The CPC was conducted
3
0
ꢀ1
Hz, H–C(4)), 4.69 (d, 1H,
J
1
0
–2
0
¼ 8.0 Hz, 1H, H–C(1 )), 4.36 using 1 L rotor, 1000 rpm and ow of 20 mL min in ascending
3
3
(
dd, J6–7 ¼ 3.0 Hz, J6–5 ¼ 1.5 Hz, 1H, H–C(6)), 3.90 (dd, 1H, mode. The fractions of 250 mL were collected and CPC was
2
3
J
6
0
0
a–6
0
b
¼ 12.0 Hz, J
6
0
a–5
0
¼ 1.0 Hz, H–C(6
0
a)), 3.68 (brdd, 1H, followed by TLC (MeOH–CH
2
Cl
2
¼ 8/2). This procedure led to
2
3
0
J6
0
¼ 12.0 Hz, J
0
6 a–5
0
¼ 4.0 Hz, H–C(6 b)), 3.44–3.16 (m, 4H, isolation of aucubigenin (4) with anomeric ratio a/b ¼ 1/9 (4.8 g,
a–6 b
0
0
0
0
1
H–C(5 ), H–C(4 ), H–C(3 ), H–C(2 )), 2.92 (brt, 1H, H–C(9)), 91%) as brownish oil. H NMR (D O), 300 MHz d (ppm) 4b: 6.23
2
3
4
2
1
9
7
.66 (m, 1H, H–C(5)), 1.86 (brs, 3H, H–C(10)). NMR (CD OD), (dd, 1H, J ¼ 6.0 Hz, J ¼ 1.5 Hz, H–C(3)), 5.69 (brs, 1H, H–
3
3–4
3–5
3 3
00 MHz d (ppm): 143.4 C(8), 139.7 C(3), 129.0 C(7), 104.6 C(4), C(7)), 5.03 (dd, 1H, J4–3 ¼ 6.0 Hz, J4–5 ¼ 3.5 Hz, H–C(4)), 4.71
0
0
0
0
3
8.1 C(1 ), 94.9 C(1), 81.3 C(6), 76.8 C(3 ), 76.6 C(5 ), 73.5 C(2 ), (d, J1–9 ¼ 7 Hz, H–C(1)), 4.42 (m, 1H, H–C(6)), 4.30–4.15 (2d, 2H,
0
0
2
3
3
0.3 C(4 ), 61.4 C(6 ), 46.7 C(9), 43.4 C(5), 14.8 C(10). Anal.
J
10a–10b ¼ 15.0 Hz, H–C(10)), 2.72 (dd, 1H, J9–5 ¼ 7.5 Hz, J1–9
¼
1
3
calcd for C15
H, 7.07%.
H
22
O
8
$2H
2
O: C, 49.15; H, 7.15. Found: C, 49.36; 7.0 Hz, H–C(9)), 2.56 (m, 1H, H–C(5)). C NMR (100 MHz, D
2
O)
d (ppm): 147.4 C(8), 141.3 C(3), 129.7 C(7), 105.3 C(4), 95.3 C(1),
2.0 C(6), 60.8 C(10), 48.7 C(9), 45.1 C(5). Anal. calcd for
C H O $1/2H O: C, 55.95; H, 6.78. Found: C, 56.30; H, 7.01%.
8
0
6
,10-Dideoxyaucubin (3)
9
13
3
2
In a ask equipped with cold nger, aucubin (1) (12 g, 34.68
mmol) was dissolved in absolute EtOH (40 mL) and stirred at
10-Monodeoxyaucubigenin (5)
ꢁ
ꢀ
30 C for 10 min. The liquid NH
3
(300 mL) was condensed Laniride (2) (10 g, 28.9 mmol) was dissolved in H
2
O (200 mL)
and subsequently Li powder (2.68 g) was added. The solution and b-glucosidase (Aldrich) (2 g) was added. The hydrolysis was
ꢁ
was stirred for additional 20 min. The disappearance of the followed by TLC (EtOAc–MeOH ¼ 4/1) and aer 10 h at 37 C, 1
deep blue color was observed aer the reaction mixture was was completely transformed into a less polar compound. The
diluted by EtOH (40 mL). The addition of same amount of Li reaction mixture was extracted with EtOAc (200 mL ꢂ 4). The
and EtOH was repeated and the reaction mixture was heated combined organic layers were dried over Na
2
SO
4
and
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evaporated in vacuo at room temperature. The obtained residue
Acknowledgements
(5.8 g) was puried by means of CPC. The crude 5 was dissolved
in upper phase of the system (AcOEt–H O ¼ 1/1). The CPC The authors thank the Agence Nationale de la Recherche (ANR)
2
apparatus was tted with lower phase and the ltrated reaction for the nancial support (ANR-09-CP2D-09-01).
mixture was injected. The CPC was conducted using 1 L rotor,
ꢀ
1
1
000 rpm and ow of 20 mL min in ascending mode. This
Notes and references
0
procedure led to isolation of 6 -monodeoxyaucubigenin (5) (4.3
1
g, 87%) with anomeric ratio a/b ¼ 6/94 as brownish oil. H NMR
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.
3
(
acetone-d
6
), 300 MHz d (ppm) 5b: 6.30 (dd, 1H, J3–4 ¼ 6.5 Hz,
4
3
J3–5 ¼ 1.5 Hz, H–C(3)), 5.96 (d, 1H, J ¼ 6.5 Hz, H–C(1)), 5.69
1–9
3
3
(
brs, 1H, H–C(7)), 5.04 (dd, 1H, J ¼ 6.0 Hz, J ¼ 6.5 Hz, H–
4
4
–5
4–3
3
C(4)), 4.63 (dd, J
4
2
¼ 6.5 Hz, J
¼ 2.5 Hz, HO–C(1)),
1
-C(1)OH
C(1)–OH-9
3
.38 (brs, 1H, HO–C(6)), 3.98 (d, 1H, J6-C(6)OH ¼ 6.0 Hz, H–C(6)),
2 P. S. Baran, Nat. Chem., 2009, 1, 193.
3 P. A. Wender, Chem. Rev., 1996, 96, 1.
3
3
.88 (m, 1H, H–C(9)), 2.55 (dd, 1H, J4–5 ¼ 6.0 Hz, J6–5 ¼ 3.0 Hz,
1
3
H–C(5)), 1.85 (s, 3H, H–C(10)). C NMR (100 MHz, acetone-d
6
) d
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.
(
8
3
ppm): 142.2 C(8), 141.0 C(3), 131.0 C(7), 104.0 C(4), 96.2 C(1),
1
6
2.2 C(6), 51.4 C(9), 46.4 C(5), 16.1 C(10). H NMR (acetone-d ),
3
4
00 MHz d (ppm) 5a: 6.16 (dd, 1H, J ¼ 6.50 Hz, J ¼ 1.5 Hz,
1
–9
1–5
3
3
H–C(3)), 5.46 (brs, 1H, H–C(7)), 4.95 (dd, 1H, J ¼ 6.0 Hz, J
4
–3
4–5
3
4
¼
3.0 Hz, H–C(4)), 4.69 (dd, J
¼ 6.50 Hz, J
¼ 2.5
1
-C(1)OH
C(1)–OH-9
3
Hz, HO–C(1)), 4.51 (brs, 1H, HO–C(6)), 3.80 (d, 1H, J6-C(6)OH
.0 Hz, H–C(6)), 2.85 (m, 1H, H–C(9)), 1.80 (s, 3H, H–C(10)).
¼
6
Anal. calcd for C
1.08; H, 7.09%.
9
H
12
O
3
$1/2H
2
O: C, 61.00; H, 7.39. Found: C,
6 Eco-extraction du v ´e g ´e tal - Proc ´e d ´e s innovants et solvants
alternatifs, ed. F. Chemat, Dunod, Paris, 2011.
6
7
(a) K. Ingkaninan, A. Hazekamp, A. C. Hoek, S. Balconi and
R. Verpoorte, J. Liq. Chromatogr. Relat. Technol., 2000, 23,
0
0
00
0
(
R)-Rotundial [(R)-2 -methyl-5 -(2 -oxoethyl)cyclopent-1 -
2
195; (b) L. Marchal, J. Legrand and A. Foucault, Chem.
enecarbaldehyde] (6)
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. Minguill ´o n, J. Chromatogr. A, 2010, 1217,
1183.
Acid hydrolysis of 6,10-dideoxyaucubin (3) (500 mg, 1.59 mmol)
was dissolved in phosphate buffer (pH ¼ 2, 15 mL) and stirred
ꢁ
at 25 C. The hydrolysis was followed by TLC (CH Cl –MeOH ¼
2
2
9
5/5) and aer 2 h, 3 was completely transformed into a less
polar compound. The solution was extracted with EtOAc (4 ꢂ 50
mL). Collected extracts were neutralized with NaHCO , dried
over Na SO and evaporated in vacuo. The obtained residue (350
mg) was puried by means of CPC. The crude 6 was dissolved in
organic phase of the system (AcOEt–H
O ¼ 1/1, 0.5 mL). The
CPC apparatus was tted with aqueous phase and the ltrated
3
2
4
´
2
9 A. Berthod, M. J. Ruiz-Angel and S. Carda-Broch, J.
Chromatogr. A, 2009, 1216, 4206.
reaction mixture was injected. The CPC was conducted using 10 (a) E. F. Queiroz, F. Roblot, P. Duret, B. Figad `e re, A. Gouyette,
ꢀ
1
2
50 mL rotor, 1600 rpm and ow of 7 mL min in ascending
O. Lapr ´e vote, 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. Figad `e re, 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. Del ´e p ´e e, S. Berteina- Raboin,
M. Lafosse, C. Lamy, S. Darnault, I. Renimel, N. About and
P. Andr ´e , 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.
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
2
0
6
22
D
mg, 55%). [a]
1
D
¼ +108.8 (c 1.15, CHCl
3
) {lit. [a]
¼ +108 (c
1
.00, CHCl , >99% ee)}. H NMR (300 MHz, CDCl
3
3
) d: 9.97 (s, 1H,
3
00
3
H–C(1)), 9.80 (t, J 00 00 ¼ 2.0 Hz, 1H, H–C(1 )), 3.46 (m, 1H,
1
–2
0
2
H–C(5 )), 2.93 (brdd, 1H,
J
00
00 ¼ 17 Hz,
J
0
00 ¼ 4.0 Hz,
2
a–2 b
5 –2 a
00
0
2
H–C(2 a)), 2.75–2.5 (m, 2H, H–C(3 )), 2.32 (ddd, 1H, J
2
00a–200
b
¼
3
3
00
1
1
1
7 Hz, J
H, H–C(4 a)), 2.15 (s, 3H, H
4.0, J
2
00b–5
0
0
¼ 9.0 Hz, J
1
00–200 ¼ 2 Hz, H–C(2 b)), 2.21–2.12 (m,
0
2
0
0
¼
3
C–C(2 )), 1.56 (ddt, 1H, J
4
0
a–4
b
3
3
3
13
0
0
¼ 9 Hz, J
0
0
¼ J
0
0
¼ 6 Hz, H–C(4 b)).
C
3
a–4
b
5
–4
b
3
b–4
b
1
3
NMR (100 MHz, CDCl
(
4
(
3
) d (ppm): C NMR (100 MHz, CDCl
3
) d
0
00
0
ppm): 202.0 C(1 ), 188.0 C(1), 164.3 C(2 ), 139.0 C(1 ), 11 J. Brunetton, Pharmacognosy, Phytochemistry, Medicinal
00
0
0
0
7.8 C(2 ), 39.1 C(3 ), 38.2 C(5 ), 28.2 C(4 ), 14.5 CH . HRMS
[M + H] ) calcd for C H O 153.0910, found 153.0901. 12 (a) X. Cachet, B. Deguin, M. Koch, K. Makhlouf and
Plants, Tec & Doc, Paris, 2nd edn, 1999.
3
+
9
13 2
Anal calcd for C
H, 8.21%.
9
H
12
O
2
: C, 71.03; H, 7.95 found: C, 70.71;
F. Tillequin, J. Nat. Prod., 1999, 62, 400; (b) C. Mouri `e s,
B. Deguin, F. Lamari, M. J. Foglietti, F. Tillequin and
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(
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