Synthesis and biological evaluation of acylated oligorhamnoside derivatives structurally related to mezzettiaside-6 with cytotoxic activity

Article abstract of DOI:10.1039/c6ob00862c

Two partially acylated oligorhamnoside derivatives 1 and 2 structurally related to the natural product mezzettiaside-6 were synthesized via a '2 + 1 + 1' convergent strategy. The bioassay results showed that the introduction of the acetyl groups to the 2-position of the terminal l-rhamnose was helpful to improve in vitro cytotoxicity. Compound 1 showed both extensive in vitro cytotoxicity in tumor cell lines and potential antimultidrug resistance capability. Preliminary mechanistic studies demonstrated that compound 1 could inhibit cell growth by inducing apoptosis, arresting cell cycle progression at the S phase in K562 cells.

Full text of DOI:10.1039/c6ob00862c

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Synthesis and biological evaluation of acylated
oligorhamnoside derivatives structurally related
to mezzettiaside-6 with cytotoxic activity†
Cite this: DOI: 10.1039/c6ob00862c
Gaopeng Song,‡a,b Sumei Li,‡c Zhiwei Lei,d Yibin Li,a Junhua Li,a Yixian Liaoa and
Zi-Ning Cui*b
Two partially acylated oligorhamnoside derivatives 1 and 2 structurally related to the natural product
mezzettiaside-6 were synthesized via a ‘2 + 1 + 1’ convergent strategy. The bioassay results showed that
the introduction of the acetyl groups to the 2-position of the terminal L-rhamnose was helpful to improve
in vitro cytotoxicity. Compound 1 showed both extensive in vitro cytotoxicity in tumor cell lines
and potential antimultidrug resistance capability. Preliminary mechanistic studies demonstrated that com-
pound 1 could inhibit cell growth by inducing apoptosis, arresting cell cycle progression at the S phase in
K562 cells.
Received 21st April 2016,
Accepted 19th May 2016
DOI: 10.1039/c6ob00862c
www.rsc.org/obc
various cancer cells, and induce the apoptosis of certain
cancer cells.9,10
Introduction
Oligosaccharides, as an important family of natural products,
In a continuing search for novel plant-derived anticancer
play an important role in biological systems, such as cell–cell agents, two series of partially acetylated n-octyl tri-rhamnoside
interaction, cell adhesion, and immunogenic recognition.1,2 derivatives mezzettiaside-2–4, 8 and n-octyl two-rhamnoside
The diversity of structures, the challenges of isolation and derivatives mezzettiaside-9–11 (Fig. 1), have been obtained
synthesis, as well as the broad spectrum of biological and from Mezzettia leptopoda by the Kinghorn group.11 Among
pharmacological activities have driven more and more them several compounds have shown significant in vitro cyto-
chemists into the research field of oligosaccharides.3–5 toxic activity against three types of tumor cells. For example,
Rhamnose-containing oligosaccharides are widely distributed one of the oligorhamnoside derivatives, mezzettiaside-2
in natural products, such as K-antigens, triterpenoid glyco- initially isolated from M. leptopoda by G. Waterman et al. in
sides and a series of glycolipids, which have received consider- 199112 and subsequently characterized by the Kinghorn group
able attention due to the increased understanding of their in 1998,11 was found to possess the prominent in vitro cyto-
wide spectrum of biological and pharmacological applications, toxic activity against Co12 with IC50 of 4.3 μg mL−1
.
including antibacterial, anticancer, and antiviral activities.6–8
The ability of generating in vitro cytotoxicity by partially
Moreover, they have been shown to inhibit the growth of acetylated simple rhamnoside derivatives is interesting.
However, not all compounds in the series have cytotoxic activi-
ties as efficient as those mentioned above. Because the series
of mezzettiaside-2 analogs share the same oligorhamnoside
skeleton, the observed variety of cytotoxic activities indicates
that the sugar chain length seems to have an important influ-
ence on the cytotoxic activities and the acetyl groups might
play a key role in the underlying mechanism of action.
Previously, two tetra-α-L-rhamnosides (Fig. 2), mezzettiaside-6
aCollege of Materials and Energy, South China Agricultural University, Guangzhou,
510642, China
bState Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Integrative Microbiology Research Centre, Guangdong Province
Key Laboratory of Microbial Signals and Disease Control, South China Agricultural
University, 510642 Guangzhou, China. E-mail: ziningcui@scau.edu.cn;
Fax: +86 20 85288229; Tel: +86 20 85288229
(octyl
2-O-acetyl-α-L-rhamnopyranosyl-(l→3)-4-O-acetyl-α-L-
cDepartment of Human anatomy, School of Medicine, Jinan University, Guangzhou,
rhamnopyranosyl-(l→3)-2,4-di-O-acetyl-α-L-rhamnopyranosyl-
(l→3)-4-O-hexanoyl-α-L-rhamnopyranoside) and mezzettiaside-
510632, China
dGuizhou Tea Reasearch Institute, Guizhou Academy of Agricultural Science,
7
(octyl rhamnopyranosyl-(l→3)-4-O-acetyl-α-L-rhamnopyrano-
Guiyang, Guizhou 550006, China
†Electronic supplementary information (ESI) available: Spectra of all synthesized
compounds. See DOI: 10.1039/c6ob00862c
syl-(l→3)-2,4-di-O-acety-α-L-rhamnopyranosyl-(l→3)-4-O-hexanoyl-
α-L-rhamnopyranoside), were obtained from M. leptopoda,13
and synthesized in 2014 by the G. A. O’Doherty group via a
‡These authors contributed equally to this work.
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Fig. 1 Seven partially acetylated oligorhamnosides from Mezzettia leptopoda.
Fig. 2 Structures of mezzettiaside-6 and -7, and compounds 1 and 2.
de novo approach,14 and their preliminary cytotoxicity against conditions. Thus, compounds 1 and 2 were constructed via a
H460 and antibacterial activity in vitro was reported by O. Bajaj ‘2 + 1 + 1’ convergent strategy by glycosylation of disaccharide
et al. in 2014.15 As part of a program aimed at the design, syn- building block 3 with thioglycoside 4 and the trichloroacet-
thesis and biological investigation of anticancer oligorhamno- imidate 5 (Scheme 1). Building block 3 as a glycosyl acceptor
side derivatives,16 we became interested in the synthesis of could be derived from the glycosylation of monosaccharide
mezzettiaside derivatives. This interest came out of our syn- acceptor 6 with donor 7. The exclusive α-glycosidic linkage in
thesis and study of a related class of tri- and tetrasaccharide the glycosylation of rhamnopyranosyl donors that was either
natural products, the cleistriosides and cleistetrosides,1,16 glycosylated at C-2 or blocked at this position with a participat-
where it was found that the pattern of acetylation had a signifi- ing group could be obtained.
cant effect on the cytotoxic activity. Given that the acetyl group
First of all, building block 3 was synthesized by coupling of
at the 2-position of the third rhamnose unit was beneficial to glycosyl acceptor 6 with donor 7. As outlined in Scheme 2,
the cytotoxic activity,11,16 compounds 1 and 2 (Fig. 2) were the glycosylation with L-rhamnopyranosyl tetraacetate 817 and
designed with the incorporation of an Ac group on the 2′′′-OH n-capryl alcohol using SnCl4 as a catalyst was carried out
group of mezzettiaside-6 and -7 to further understand the cor- smoothly to give the pure α-isomer 9 in an 83% yield. The
relation between the sugar chain length and inhibitory activi- acetyl groups in 9 were removed with NaOMe in MeOH to get
ties, as well as to elucidate the contribution of the acetyl 10, followed by protecting the corresponding 2,3-positions
groups on cytotoxic activities. The results obtained provide a with an isopropylidene group affording 11. Treatment of 11
facile approach to other structural analogues and render new with HexCl in the presence of DMAP afforded 12, which was
clues on the understanding of the cytotoxic profile for these subjected to removal of the 2,3-O-isopropylidene group (80%
types of compounds.
HOAc, 80 °C) to produce 13. Compound 13 was refluxed with
Bu2SnO in toluene, followed by reaction with p-methoxybenzyl
chloride in the presence of TBAI in DMF (r.t.) to provide 14 in
a favorable yield (75%). Treatment of 14 with levulinic acid
(LevOH) in the presence of EDC·HCl and DMAP afforded 15, in
which the 3′-O-PMB group was removed to give the important
Results and discussion
Chemistry
The five or four acetyl groups in compound 1 or 2 require intermediate 6. The known compound 7 was synthesized
attention in the convergent synthesis due to their possible according to our previous procedures.8 With glycosyl acceptor
acetyl migration under some acidic or basic conditions. To 6 and the donor 7, the coupling reaction was then performed
address this issue, we chose the levulinoyl group (Lev) as the under NIS/AgOTf activation to afford 16, and the subsequent
hydroxy protecting group, which could be selectively removed deprotection of the PMB group with DDQ gave the building
without affecting acetyl and hexanoyl groups under mild block 3.
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Scheme 1 Retrosynthetic analysis of compounds 1 and 2.
Scheme 2 Reagents and conditions: (a) SnCl4, CH2Cl2; (b) CH3ONa, CH3OH–CH2Cl2; (c) 2,2-dimethoxylproprane, p-TsOH, DMF; (d) HexCl, DMAP,
pyridine; (e) 80% AcOH; (f) (i) Bu2SnO, toluene; (ii) p-methoxybenzyl chloride, TBAI, DMF; (g) levulinic acid, EDC·HCl, DMAP, CH2Cl2; (h) DDQ,
CH2Cl2–H2O; (i) NIS/AgOTf, CH2Cl2.
The next step was to elaborate monosaccharide acceptor 4 from thioglycoside 19 in two steps. Hydrolysis of 19 with
and donor 5. As shown in Scheme 3, acceptor 4 was prepared N-bromosuccinimide (NBS) in acetone–H2O, followed by treat-
from 17 in a simple one-pot method.17 Treatment of 188 ment with CCl3CN and 1,8-diazabicyclo[5.4.0]undec-7-ene
with triethylorthoacetate and a catalytic amount of (1S)- (DBU) led to trichloroacetimidate 5 in an 81% yield in two
(+)-camphor-10-sulfonic acid (CSA) formed the corresponding steps.
orthoester, and then the reaction mixture was diluted with di-
With an efficient synthetic access to the key intermediates 4
chloromethane and shaken with 1 M HCl solution to effect the and 5, we next turned our attention to a ‘one-pot sequential
orthoester rearrangement, which was directly reacted with glycosylation’ strategy for the efficient synthesis of tetra-
levulinic acid in the presence of EDC·HCl and DMAP to rhamnoside derivatives 1 and 2 utilizing the trichloroacet-
protect the remaining hydroxy group giving the expected com- imidates and thioglycosides as sequential glycosyl donors for
pound 19 in a 79% yield. Glycosyl donor 5 was synthesized two glycosidic linkages. As depicted in Scheme 4, coupling of
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Scheme 3 Reagents and conditions: (a) (i) MeC(OEt)3, CSA, DMF; (ii) 1 M HCl; (iii) levulinic acid, EDC·HCl, DMAP, CH2Cl2, 79% for three steps;
(b) (i) MeC(OEt)3, CSA, DMF; (ii) Ac2O, DMAP, TEA, pyridine; (iii) 1 M HCl, 83% for three steps; (c) (i) NBS, acetone–H2O; (ii) CCl3CN, DBU, CH2Cl2.
Scheme 4 Reagents and conditions: (a) (i) TMSOTf, CH2Cl2, −40 °C; (ii) NIS, CH2Cl2, 0 °C; (b) AcOH·NH2NH2, MeOH–CH2Cl2, r.t. 5 h;
(c) AcOH·NH2NH2, MeOH–CH2Cl2, 50 °C, 24 h.
thioglycoside 4 with imidate 5 was completed within 30 min Biology
using a catalytic amount of TMSOTf (0.1 equiv.) at −40 °C,
affording the desired disaccharide p-tol thioglycoside donor.
In vitro cytotoxicity against tumor cells. Preliminary screen-
Without purification, the reaction mixture was warmed ing of synthetic oligorhamnosides 1 and 2 employing the MTT
to 0 °C, then building block 3 as the acceptor was added, assay18 was performed to evaluate cytotoxic activity against a
followed by addition of N-iodosuccinimide (NIS) (2.0 equiv.) panel of selected human cancer cell lines namely: human oral
and AgOTf (0.3 equiv.), affording the fully protected tetra- epidermoid carcinoma KB cell line, human lung carcinoma
rhamnoside intermediate 20 in an 83% yield for two steps. cell line A549, human colon cancer Co12 cell line, human
Finally, deprotection of the Lev groups on the sugar residues myelogenous leukemia cell line K562, and human breast
with AcOH·NH2NH2 (20 eq.) in MeOH and CH2Cl2 at r.t. for cancer cell line MDA-MB-231. The results of cytotoxicity,
5 h afforded the expected compound 1 in a satisfactory yield. expressed as IC50 values in comparison with positive control
Interestingly, removal of the 2″″-O-Ac group with all the Lev adriamycin, are summarized in Table 1. Interestingly, both
groups was carried out simultaneously to furnish compound 2 oligorhamnosides 1 and 2 were selectively cytotoxic to cancer
when the dosage of AcOH·NH2NH2 was raised to 40 eq. cells with a weak effect on normal human embryonic kidney
at 50 °C for 24 h.
cells (HEK-293). Moreover, both compounds exhibited more
Table 1 IC50 values (μM) of two oligorhamnosides 1 and 2 against tumor cell linesa
Compd
KB
A549
Co12
K562
MDA-MB-231
1
2
5.03 0.73
6.22 0.61
1.12 0.36
0.95 0.20
1.72 0.33
0.90 0.15
2.15 0.12
3.68 0.63
2.03 0.56
0.70 0.06
1.82 0.43
1.55 0.73
3.82 0.65
5.46 0.78
10.20 1.2
Adriamycin
a Data derived from the mean of three independent assays (means SD).
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Table 2 IC50 cytotoxicity of compound 1 against two multidrug-resistant tumor cell lines
IC50(μM SD)
IC50(μM SD)
Compd
MCF-7
MCF-7/ADM
RFa
KB
KB/VCR
RFa
1
2.35 0.45
0.30 0.08
0.025 0.002
3.26 0.20
47.20 2.25
4.22 0.25
1.4
157.3
168.8
4.88 0.62
2.88 0.32
0.02 0.03
6.13 0.54
94.82 0.89
1.26 0.13
1.3
32.9
63.0
ADMb
VCRc
a Relative resistance index (RF) = IC50 (drug-resistant)/IC50 (drug-sensitive). b ADM means adriamycin. c VCR means vincristine.
pronounced activity against K562 cells compared to the other fragmented nuclei are the classic characteristics of apoptosis.
cell lines tested. It was considered of interest to investigate the apoptosis
Compound 1 showed significant cytotoxicity with IC50 inducing effect of these conjugates by the Hoechst staining
values in the micro to submicromolar (ranging from 0.7 to (H33258) method in the K562 cancer cell line. Thus, with
5.0 μM) against all the tested cancer cell lines, and exhibited adriamycin as positive control at 1 μM for 24 h, cells were
enhanced cytotoxic activity relative to adriamycin against K562 treated with 1 at 0.5 and 1 μM concentrations for 24 h. Manual
and MDA-MB-231 cells. The observation that compound 1 field quantification of apoptotic cells based on cytoplasmic
exhibited enhanced cytotoxic activity compared to compound condensation, the presence of apoptotic bodies, nuclear
2 indicates that the positions of acetyl groups are important, fragmentation and relative fluorescence of the test compounds
especially for the 2-position of the terminal L-rhamnose.
revealed significant increases in apoptotic cells as shown
In vitro antimultidrug resistance capability of compound 1. in Fig. 3.
P-glycoprotein (P-gp) overexpression, as a major multidrug
Effect on morphological character of mitochondrial
resistance (MDR) mechanism, is the significant impediment membrane potential. Mitochondria play an essential role in
to the success of cancer chemotherapy with naturally derived the propagation of apoptosis. It is well established that at
anticancer drugs. In order to test the antimultidrug resistance an early stage, apoptotic stimuli alter the mitochondrial trans-
capability of compound 1, P-gp over-expressed tumor cell lines membrane potential. This potential was monitored by the
MCF-7/ADM and KB/VCR and their normal counterparts were fluorescence of the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-
employed. High degrees of drug resistance were observed for benzimidazolcarbocyanine (JC-1).19 K562 cells were growth-
the reference compounds, adriamycin (RF = 157.3, Table 2) arrested for 24 h and stimulated with increasing concen-
and vincristine (RF = 63.0, Table 2). Compound 1 displayed trations from 0.5 to 2 μM of compound 1 for the next 48/72 h
equipotent cytotoxicity in MDR cells and their corresponding in DMEM (10% FBS). Then the cells were harvested and
parental cells, with the RF values of 1.4 and 1.3 respectively stained with JC-1 and imaged by fluorescence microscopy.
(Table 2). These findings indicated that compound 1 was not a As shown in Fig. 4, the increase of green emission (in the
substrate of the P-gp pump and had potential antimultidrug merged picture) corresponded with increased mitochondrial
resistance capability, although the cytotoxic activity was not at depolarization, which led to higher mitochondrial membrane
the same level as those of adriamycin and vincristine in the potential caused by cell apoptosis. The shift of mitochondrial
current evaluation.
orange-red fluorescence to green fluorescence among groups
Hoechst staining for morphological analysis. Potential lead indicated that the extent of apoptosis increased with increas-
compound 1 displayed a spectrum of antitumor activity. A pre- ing dose.
liminary anticancer mechanism on K562 cell lines was further
discussed.
Annexin V-FITC/propidium iodide analysis of apoptosis. To
characterize the mode of cell death induced by compound 1, a
Apoptosis is one of the major pathways to the process of biparametric cytofluorimetric analysis was performed using
cell death. It is well known that chromatin condensation and propidium iodide (PI), which stains DNA and enters only dead
Fig. 3 Hoechst staining of K562 cells treated with compound 1 (from left to right): A (1, 0 μM), B (adriamycin, 0.2 μM), C (1, 0.5 μM), D (1, 1.0 μM).
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Fig. 4 K562 cells were harvested, stained with JC-1 and imaged by fluorescence microscopy (from top left to bottom right): A (1, 0 μM),
B (1, 0.5 μM), C (1, 1 μM), D (1, 2 μM). From left to right: green, red and green-red merged.
cells, and fluorescent immunolabeling of the protein annexin-
Measurement of caspase-3 activity. The occurrence of the
V, which binds to phosphatidyl serine (PS) in a highly selective apoptotic process was confirmed by the presence of caspase-3,
manner.20 Dual staining for annexin-V and with PI permits dis- which plays a crucial role and is a vital element in the
crimination between live cells (annexin-V−/PI−), early apopto- execution phase of apoptosis.21 The enzyme activity of
tic cells (annexin-V+/PI−), late apoptotic cells (annexin-V+/PI+), caspase-3 was determined by spectrophotometry, as shown in
and necrotic cells (annexin-V−/PI+). In this study K562 cells Fig. 6. After incubation with compound 1 for 24 h, the activity
were treated with compound 1 for 24 h at 1 μM concentration of caspase-3 increased compared to the control. These results
to examine the apoptotic effect. Four quadrant images were clearly indicated that induction of cell apoptosis took place
observed by flow cytometric analysis: the R2 area represented when the cells were exposed to the drugs.
damaged cells appearing in the process of cell collection, the
Investigation of cell cycle distribution. To determine the
R3 region showed necrotic cells and late apoptotic cells, the R4 possible role of cell cycle arrest in oligorhamnoside-induced
area showed normal cells, and the early apoptotic cells were growth inhibition, K562 cells were treated with various concen-
located in the R5 area. As shown in Fig. 5, the late apoptotic trations of compound 1. Cell cycle distribution was observed
cells were significantly more populous than those in the by flow cytometric analysis after staining of the DNA with pro-
untreated group since the R3 region showed that compound 1 pidium iodide (PI). With increased concentration of com-
exhibited 14.0% of apoptosis, whereas 3.4% of apoptosis was pound 1, cells accumulated in the G0/G1 phase and G2/M
observed in the control (untreated cells). This experiment phase cells were gradually depleted, while S period cells
suggests that the cells reacting to compound 1 have the ability gradually increased (Fig. 7), which implied that compound 1
to significantly induce apoptosis in K562 cells.
arrested K562 cells in the S phase.
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Fig. 7 Investigation of cell cycle distribution. (A) Demonstration of
apoptosis by flow cytometric analysis. (1) Untreated K562 cells. (2)–(3)
Cells treated with increasing concentrations of compound 1 (1 and 2 μM)
for 24 h. (B) Dose-dependent induction of apoptosis by compound 1.
Fig. 5 Annexin V-EGFP/PI apoptosis detection assay. (A) Demonstration
of apoptosis or necrosis by flow cytometric analysis. (1) Untreated K562
cells. (2) Cells were treated with compound 1 at 1 μM for 24 h. (B) Induc-
tion of apoptosis or necrosis by compound 1.
ing the activity of caspase-3. These findings can serve as the
basis for further research on the chemical modifications,
structure–activity relationship, and bioactivity of oligorhamno-
side derivatives.
Experimental protocols
Chemistry
Solvents were purified in a conventional manner. Thin layer
chromatography (TLC) was performed on precoated E. Merck
silica gel 60 F254 plates. Flash column chromatography was
performed on silica gel (200–300 mesh, Qingdao, China). 1H
NMR and 13C NMR spectra were taken on a JEOL JNM-ECP 600
spectrometer with tetramethylsilane as an internal standard,
and chemical shifts were recorded in ppm values. Mass
spectra were recorded on a Q-TOF Global mass spectrometer.
1-O-n-Octyl-2,3,4-tri-O-acetyl-α-L-rhamnopyranoside (9). To a
solution of compound 8 (11.9 g, 36.0 mmol) in dry CH3CN
(100 mL) was added SnCl4 (3.8 mL, 32.4 mmol) at 0 °C and the
mixture was stirred for 30 min. To the cold solution, n-octanol
(3.6 g, 27.7 mmol) solution in CH3CN was added dropwise
Fig. 6 Effect of compound
activity.
1 or ADM (adriamycin) on caspase-3
Conclusion
Two acylated oligorhamnoside derivatives 1 and 2 structurally over a period of 30 min. The reaction was stirred for an
related to the natural product mezzettiaside-6 were synthesized additional 30 min while warming to room temperature. After
via a ‘2 + 1 + 1’ convergent strategy. Cytotoxic activities were the reaction was complete, as judged by TLC, the reaction
tested in vitro. The preliminary results showed that the intro- was quenched with water (30 mL) and extracted with CH2Cl2
duction of an acetyl group to the 2-position of the terminal (3 × 200 mL). The chloroform layer was washed with saturated
L-rhamnose could significantly improve cytotoxicity. Com- aqueous NaHCO3 (2 × 300 mL), and brine (2 × 300 mL), dried
pound 1 displayed extensive in vitro cytotoxicity against tumor over Na2SO4, and concentrated under reduced pressure. The
cell lines, and potential antimultidrug resistance capabilities. resulting crude oil was purified via silica gel column chromato-
Preliminary mechanistic studies demonstrated that compound graphy (8 : 1, petroleum ether–EtOAc) to provide 9 (9.2 g, 83%)
1 could inhibit cell growth by inducing apoptosis, arresting as a colorless oil. 1H NMR (CDCl3) δ 5.31 (dd, 1H, J = 10.1,
cell cycle progression in the S phase in K562 cells, and increas- 3.7 Hz, H-3′), 5.23 (dd, 1H, J = 3.7, 1.9 Hz, H-2′), 5.06 (t, 1H,
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J = 9.9 Hz, H-4′), 4.71 (d, 1H, J = 1.8 Hz, H-1′), 3.86–3.90 (m, 12 (2.5, 96%). 1H NMR (CDCl3): δ 4.99 (brs, 1H, H-1′), 4.87 (dd,
1H, H-5′), 3.62–3.68 (m, 1H, OCHH), 3.40–3.43 (m, 1H, OCHH), 1H, J = 10.1, 3.7 Hz, H-4′), 4.16 (dd, 1H, J = 7.7, 5.5 Hz, H-3′),
2.15 (s, 3H, COCH3), 2.05 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 4.13 (d, 1H, J = 5.4 Hz, H-2′), 3.71–3.75 (m, 1H, H-5′), 3.66–3.69
1.55–1.60 (m, 2H, βCH2), 1.30–1.33 (m, 2H, γCH2), 1.28 (brs, (m, 1H, OCHH), 3.41–3.45 (m, 1H, OCHH), 2.32–2.38 (m, 2H,
8H, 4 × CH2), 1.22 (d, 3H, J = 6.0 Hz, H-6′), 0.88 (t, 3H, J = OCOCH2), 1.30–1.66 (m, 18H, 9 × CH2), 1.15 (d, 3H, J = 6.6 Hz,
7.1 Hz, CH3); 13C NMR (CDCl3) δ 170.1, 169.9 (two), 97.4 H-6′), 0.90 (t, 3H, J = 7.1 Hz, CH3), 0.89 (t, 3H, J = 7.1 Hz, CH3);
(Rha-C-1), 71.2, 70.0, 69.2, 68.2, 66.1, 31.7, 29.3, 29.2, 29.1, 13C NMR (CDCl3): δ 172.9, 109.5, 96.8 (Rha-C-1), 76.2, 75.9,
26.0, 22.5, 20.8, 20.7, 20.6; HRMS(ESI-TOF): calcd for 74.3, 67.8, 63.9, 34.3, 31.8, 31.2, 29.4, 29.3, 29.2, 27.7, 26.4,
C20H34O8Na [M + Na]+ 425.2151; found 425.2145.
1-O-n-Octyl-α-L-rhamnopyranoside (10). To a stirred solution for C23H42O6Na [M + Na]+ 437.2879; found, 437.2860.
of 9 (2.5 g, 6.2 mmol) in dry CH2Cl2 (10 mL) and CH3OH 1-O-n-Octyl-4-O-hexanoyl-α-L-rhamnopyranoside (13). Com-
26.1, 24.6, 22.6, 22.3, 17.0, 14.0, 13.9; HRMS(ESI-TOF): calcd
(10 mL) was added MeONa (200 mg). The reaction mixture was pound 12 (4.0 g, 9.7 mmol) was dissolved in 80% HOAc
stirred at room temperature for 1 h, after which the reaction (40 mL) and stirred for 2 h at 80 °C, then the reaction mixture
mixture was neutralized with Dowex 50 × 8 (H+) resin until was concentrated in vacuo. The residue was purified by silica
pH 7, filtered and concentrated to give a crude product, which gel column chromatography (15 : 1, CHCl3–MeOH) to afford
1
was purified via silica gel column chromatography (15 : 1, compound 13 (3.2 g, 89%) as a colorless oil. H NMR (CDCl3):
trichloromethane–methanol) to yield 10 (1.6 g, 93%) as a δ 4.81 (d, 1H, J = 1.1 Hz, H-1′), 4.80 (t, 1H, J = 9.6 Hz, H-4′),
1
colorless oil. H NMR (CDCl3): δ 4.64 (d, 1H, J = 1.6 Hz, H-1′), 3.93–3.94 (m, 1H, H-2′), 3.86–3.89 (m, 1H, H-3′), 3.79–3.83 (m,
3.77 (dd, 1H, J = 3.4, 1.7 Hz, H-2′), 3.64–3.67 (m, 1H, OCHH), 1H, H-5′), 3.66 (dt, 1H, J = 9.9, 6.8 Hz, H-1-1), 3.66 (dt, 1H, J =
3.62 (dd, 1H, J = 9.5, 3.4 Hz, H-3′), 3.53–3.59 (m, 1H, H-5′), 9.9, 6.5 Hz, H-1-2), 3.08 (d, 1H, J = 6.6 Hz, OH), 2.74 (d, 1H, J =
3.34–3.41 (m, 1H, OCHH), 3.36 (t, 1H, J = 9.5 Hz, H-4′), 3.5 Hz, OH), 2.39 (td, 2H, J = 7.6, 3.7 Hz, COCH2), 1.57–1.67
1.50–1.60 (m, 2H, βCH2), 1.29–1.38 (m, 12H, 5 × CH2), 1.25 (d, (m, 2H, H-2), 1.29–1.35 (m, 10H, 5 × CH2), 1.22 (d, 3H, J =
3H, J = 6.2 Hz, H-6′), 0.89 (t, 3H, J = 7.2 Hz, CH3); 13C NMR 6.2 Hz, H-6′), 0.90 (t, 3H, J = 7.2 Hz, CH3); 13C NMR (CDCl3):
(CDCl3): δ 101.6 (Rha-C-1), 74.0, 72.5, 72.4, 69.7, 68.6, 33.0, δ 175.1, 99.2 (Rha-C-1), 75.5, 71.1, 70.4, 68.0, 65.5, 34.4, 31.8,
30.4, 27.3, 23.7, 18.0, 14.4; HRMS(ESI-TOF): calcd for 31.2, 29.4, 29.3, 29.2, 26.1, 24.6, 22.6, 22.3, 17.5, 14.1, 13.9;
C14H28O5Na [M + Na]+ 299.1834; found 299.1822.
HRMS(ESI-TOF): calcd for C20H38O6Na [M + Na]+ 397.2566;
1-O-n-Octyl-2,3-O-isopropylidene-α-L-rhamnopyranoside (11). found, 397.2554.
To a solution of compound 10 (3.5 g, 12.7 mmol) in dry DMF
1-O-n-Octyl-4-O-hexanoyl-3-O-(p-methoxybenzyl)-α-L-rhamno-
(30 mL) were added 2,2-dimethoxypropane (7.8 mL, pyranoside (14). A mixture of 13 (1.2 g, 3.2 mmol) and Bu2SnO
63.5 mmol) and p-toluene sulfonic acid (224 mg, 1.3 mmol) at ( 1.9 g, 4.8 mmol) in toluene (30 mL) was refluxed for 5 h.
r.t. After 10 h stirring, Et3N was added dropwise to attain Then the solution was concentrated under reduced pressure.
pH 7.0. The mixture was diluted with EtOAc (250 mL), washed The residue was suspended in DMF (30 mL), and Bu4NI (1.0 g,
with H2O (3 × 100 mL), and brine (2 × 100 mL), dried over 3.2 mmol) and PMBCl (0.72 mL, 5.3 mmol) were added. After
Na2SO4, and concentrated in vacuo. The resulting crude being stirred at r.t. for 24 h, the mixture was diluted with
product via silica gel column chromatography (8 : 1, petroleum CHCl3 (150 mL) and washed with H2O (3 × 50 mL). The separ-
ether–EtOAc) furnished 11 (3.6 g, 90%) as a white solid. ated water phase was extracted with CHCl3 (3 × 100 mL). The
1H NMR (CDCl3): δ 4.95 (s, 1H, H-1′), 4.15 (dd, 1H, J = 5.8, combined organic phase was washed with water (3 × 150 mL),
0.4 Hz, H-2′), 4.11 (t-like, 1H, J = 6.9, 5.8 Hz, H-3′), 3.67–3.72 and brine (2 × 150 mL), dried over Na2SO4, and then filtered
(m, 2H, H-5′, OCH2-1), 3.43 (dt, 1H, J = 9.6, 6.5 Hz, OCH2-2), through a short column of silica gel. The eluent was concen-
3.40–3.41 (m, 1H, H-4′), 1.57–1.61 (m, 2H, H-2), 1.54, 1.37, trated to a residue, which was purified by silica gel column
(each s, each 3H, each CH3), 1.27–1.35 (m, 10H, 5 × CH2), 1.31 chromatography (8 : 1, petroleum ether–EtOAc) to yield a white
(d, 3H, J = 6.4 Hz, H-6′), 0.90 (t, 3H, J = 7.0 Hz, CH3); 13C NMR solid 14 (1.2 g, 75%). 1H NMR (CDCl3): δ 7.23 (d, 2H, J =
(CDCl3): δ 109.4, 97.0 (Rha-C-1), 78.3, 75.9, 74.4, 67.7, 65.9, 8.6 Hz, Ar–H), 6.89 (d, 2H, J = 8.6 Hz, Ar–H), 5.06 (t, 1H, J =
31.8, 29.2, 26.1, 22.7, 17.6, 14.0; HRMS(ESI-TOF): calcd for 9.7 Hz, H-4′), 4.83 (d, 1H, J = 1.6 Hz, H-1′), 4.58 (d, 1H, J =
C17H32O5Na 339.2147 [M + Na]+; found 339.2136.
11.6 Hz, Ar–CH2-1), 4.50 (d, 1H, J = 11.6 Hz, Ar–CH2-2),
1-O-n-Octyl-2,3-O-isopropylidene-4-O-hexanoyl-α-L-rhamnopyrano- 3.86–4.00 (m, 1H, H-5′), 3.76 (dd, 1H, J = 9.5, 3.4 Hz, H-3′),
side (12). To a stirred solution of 11 (2.0 g, 6.3 mmol) in dry 3.66–3.69 (m, 1H, OCHH), 3.41–3.45 (m, 1H, OCHH), 2.32–2.38
pyridine (25 mL) were added hexanoyl chloride (1.3 g, (m, 2H, OCOCH2), 2.26–2.29 (m, 2H, OCOCH2), 1.30–1.66 (m,
9.6 mmol) and DMAP (77 mg, 0.6 mmol) at 0 °C, while 18H, 9 × CH2), 1.18 (d, 3H, J = 6.6 Hz, H-6′), 0.90 (t, 3H, J =
warmed to room temperature until TLC indicated that the reac- 7.1 Hz, CH3), 0.89 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3):
tion was complete. The reaction mixture was concentrated, δ 172.9, 159.4, 129.9, 129.3, 113.9, 98.8 (Rha-C-1), 72.3, 71.5,
and then diluted with EtOAc (100 mL), washed with 1 M HCl 68.5, 67.8, 66.1, 55.3, 34.4, 31.8, 31.3, 29.4, 29.3, 29.2, 26.1,
(2 × 50 mL), saturated aqueous NaHCO3 (2 × 50 mL), and brine 24.6, 22.6, 22.3; HRMS(ESI-TOF): calcd for C28H46O7Na: calcd
(2 × 50 mL), dried over Na2SO4, and concentrated in vacuo. The for [M + Na]+ 517.3141; found, 517.3133.
obtained residue was purified by silica gel column chromato-
1-O-n-Octyl-4-O-hexanoyl-3-O-(p-methoxybenzyl)-2-levulinoyl-
graphy (15 : 1, petroleum ether–EtOAc) to give a white solid α-L-rhamno-pyranoside (15). To a solution of compound 14
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(2.4 g, 4.9 mmol) in dry CH2Cl2 (30 mL), levulinic acid (0.72 g, and washed with aqueous Na2S2O3 (100 mL), saturated
6.2 mmol), EDC·HCl (1.4 g, 7.2 mmol), and DMAP (0.12 g, aqueous NaHCO3 (2 × 50 mL), and brine (2 × 50 mL), dried
0.96 mmol) were added under argon. The mixture was stirred over Na2SO4, and concentrated in vacuo. The residue was puri-
for 36 h, diluted with CH2Cl2 (100 mL), washed with H2O fied by silica gel column chromatography (5 : 1, petroleum
(50 mL), 1 M HCl (2 × 50 mL), satd aq. NaHCO3 (2 × 50 mL), ether–EtOAc) to give a white solid 16 (3.0 g, 86%); 1H NMR
and brine (2 × 50 mL), dried over Na2SO4, and concentrated (CDCl3): δ 7.19 (d, 2H, J = 8.6 Hz, Ar–H), 6.85 (d, 2H, J = 8.6 Hz,
in vacuo. The residue was purified by silica gel column chrom- Ar–H), 5.11–5.13 (m, 2H, H-2′, H-2″), 5.05 (t, 1H, J = 9.8 Hz,
atography (8 : 1, petroleum ether–EtOAc) to give 15 (2.6 g, H-4′), 4.96 (t, 1H, J = 9.8 Hz, H-4″), 4.85 (d, 1H, J = 1.1 Hz,
1
90%). H NMR (CDCl3): δ 7.19 (d, 2H, J = 8.6 Hz, Ar–H), 6.85 H-1″), 4.66 (brs, 1H, H-1″), 4.53 (d, 1H, J = 11.6 Hz, Ar–CH2-1),
(d, 2H, J = 8.6 Hz, Ar–H), 4.83 (dd, 1H, J = 3.4, 1.9 Hz, H-2′), 4.37 (d, 1H, J = 11.6 Hz, Ar–CH2-2), 4.07 (dd, 1H, J = 9.8, 3.2
4.99 (t, 1H, J = 9.7 Hz, H-4′), 4.73 (d, 1H, J = 1.7 Hz, H-1′), 4.55 Hz, H-3″), 3.79 (s, 3H, OCH3), 3.73–3.77 (m, 3H, H-3′, H-5′,
(d, 1H, J = 11.6 Hz, Ar–CH2-1), 4.33 (d, 1H, J = 11.6 Hz, Ar–CH2- H-5″), 3.61–3.65 (m, 1H, H-1-1), 3.37–3.40 (m, 1H, H-1-2), 2.71
2), 3.80 (dd, 1H, J = 9.5, 3.4 Hz, H-3′), 3.74–3.76 (m, 1H, H-5′), (t, 2H, J = 6.3 Hz, COCH2), 2.60–2.65 (m, 2H, COCH2), 2.36
3.62–3.66 (m, 1H, OCHH), 3.38–3.42 (m, 1H, OCHH), 2.65–2.76 (t, 2H, J = 6.6 Hz, COCH2), 2.18, 2.13, 2.02 (each s, each 3H,
(m, 4H, 2 × COCH2), 2.25–2.28 (m, 2H, COCH2), 2.17 (s, 3H, each CH3CO), 1.63–1.66 (m, 2H, CH2), 1.54–1.56 (m, 2H, CH2),
COCH3), 1.56–1.62 (m, 4H, 2 × CH2), 1.30–1.33 (m, 14H, 7 × 1.28–1.33 (m, 14H, 7 × CH2), 1.19 (d, 3H, J = 6.4 Hz, H-6′), 1.14
CH2), 1.18 (d, 3H, J = 6.3 Hz, H-6′), 0.90 (t, 3H, J = 7.1 Hz, CH3), (d, 3H, J = 6.3 Hz, H-6″), 0.90 (t, 3H, J = 7.1 Hz, CH3), 0.89
0.89 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3): δ 206.4, 172.7, (t, 3H, J = 7.2 Hz, CH3); 13C NMR (CDCl3): δ 205.9, 176.5, 173.0,
171.9, 159.2, 130.1, 129.3 (two), 113.5 (two), 97.6 (Rha-C-1), 171.9, 170.2, 159.1, 133.7, 130.1, 129.3 (two), 113.3 (two), 99.8,
74.4, 70.8, 68.0, 66.4, 55.2, 38.1, 34.4, 31.8, 31.3, 29.8, 29.4, 97.1, 75.2, 73.8, 72.3, 72.2, 72.0, 71.0, 68.8, 68.1, 67.3, 66.5,
29.3, 29.2, 28.3; 26.0, 24.5, 22.7, 22.3, 17.5, 14.1, 13.8; HRMS 55.2, 37.8, 34.1, 31.8, 31.3, 29.3 (two), 29.2, 28.5, 28.2, 26.1,
(ESI-TOF): calcd for C33H52O9Na [M + Na]+ 615.3509; found, 24.6, 22.6, 22.3, 20.9, 17.5, 17.3, 14.1, 13.9; HRMS(ESI-TOF):
615.3502.
calcd for C43H66O15Na [M + Na]+ 845.4299; found, 845.4275.
1-O-n-Octyl-4-O-hexanoyl-2-levulinoyl-α-L-rhamnopyranoside
1-O-n-Octyl-4-O-hexanoyl-2-levulinoyl-α-L-rhamnopyranosyl-
(6). To a stirred mixture of 15 (1.2 g, 2.0 mmol) in CH2Cl2 (1→3)-2,4-di-O-acetyl-α-L-rhamnopyranoside (3). Reaction of
(18 mL) and H2O (2 mL), was added DDQ (689 mg, 3.0 mmol) compound 16 (2.5 g, 3.0 mmol), and DDQ (0.90 g, 4.0 mmol)
at r.t. The reaction was stirred for 12 h until the reaction was in CH2Cl2 (60 mL) and H2O (4 mL) was essentially as described
complete as judged by TLC. The reaction mixture was poured for 6 and gave 3 (1.9 g, 89%) as a white solid; 1H NMR
into saturated aqueous NaHCO3 (20 mL) and extracted with (600 MHz, CDCl3): δ 5.12 (dd, 1H, J = 3.3, 1.8 Hz, H-2″), 5.05 (t,
CH2Cl2 (2 × 50 mL). The combined organic phase was washed 1H, J = 9.9 Hz, H-4′), 4.90 (d, 1H, J = 1.2 Hz, H-1″), 4.87 (dd,
with satd aq. NaHCO3 (2 × 50 mL), and brine (2 × 50 mL), 1H, J = 3.5, 1.6 Hz, H-2′), 4.83 (t, 1H, J = 9.8 Hz, H-4″), 4.66 (d,
dried over Na2SO4, and concentrated under reduced pressure. 1H, J = 1.6 Hz, H-1′), 4.07 (dd, 1H, J = 10.0, 3.4 Hz, H-3″),
The residue was purified by silica gel column chromatography 3.94–3.97 (m, 1H, H-3′), 3.74–3.82 (m, 2H, H-5′, H-5″), 3.63 (dt,
(5 : 1, petroleum ether–EtOAc) to give 6 (0.88 g, 92%) as a 1H, J = 9.5, 2.8 Hz, H-1-1), 3.39 (dt, 1H, J = 9.6, 6.6 Hz, H-1-2),
1
syrup. H NMR (CDCl3): δ 5.12 (dd, 1H, J = 3.5, 1.6 Hz, H-2′), 2.70–2.80 (m, 4H, 2 × COCH2), 2.46 (dt, 1H, J = 16.1, 7.5 Hz,
4.84 (t, 1H, J = 9.8 Hz, H-4′), 4.72 (d, 1H, J = 1.2 Hz, H-1′), 4.00 COCH2-1), 2.34 (dt, 1H, J = 16.0, 7.6 Hz, COCH2-2), 2.22, 2.14,
(d, 1H, J = 8.9 Hz, H-3′), 3.78–3.82 (m, 1H, H-5′), 3.65 (dt, 1H, 2.13 (each s, each 3H, each CH3CO), 1.63–1.66 (m, 2H, CH2),
J = 9.5, 6.8 Hz, H-1-1), 3.40 (dt, 1H, J = 9.6, 6.5 Hz, H-1-2), 2.81 1.54–1.59 (m, 2H, CH2), 1.26–1.34 (m, 14H, CH2), 1.18 (d, 3H,
(t, 2H, J = 6.4 Hz, COCH2CH2CO), 2.66 (t, 2H, J = 6.4 Hz, J = 6.4 Hz, H-6′), 1.17 (d, 3H, J = 6.3 Hz, H-6″), 0.90 (t, 3H, J =
COCH2CH2CO), 2.37 (td, 2H, J = 7.2, 3.7 Hz, COCH2), 2.21 (s, 7.2 Hz, CH3), 0.89 (t, 3H, J = 7.2 Hz, CH3); 13C NMR (CDCl3):
3H, CH3CO), 1.63–1.67 (m, 2H, CH2), 1.55–1.60 (m, 2H, H-2), δ 206.1, 173.1, 171.9, 171.5, 170.2, 99.1, 97.1, 74.9, 74.3, 72.9,
1.29–1.34 (m, 14H, 7 × CH2), 1.20 (d, 3H, J = 6.4 Hz, H-6′), 0.90 72.2, 71.9, 68.1, 67.8, 66.8, 66.6, 37.8, 34.0, 31.8, 31.3, 29.8,
(t, 3H, J = 7.0 Hz, CH3), 0.89 (t, 3H, J = 7.1 Hz, CH3); 13C NMR 29.3 (two), 29.2, 28.2, 26.1, 24.5, 22.6, 22.3, 21.0, 20.9,
(CDCl3): δ 207.1, 174.1, 172.2, 97.2 (Rha-C-1′), 74.4, 72.9, 68.7, 17.5, 17.2, 14.1, 13.9; HRMS(ESI-TOF): calcd for C35H58O14Na
68.1, 66.0, 38.2, 34.3, 31.8, 31.2, 29.4, 29.3, 29.2, 28.2, 26.0, [M + Na]+ 725.3724; found, 725.3732.
24.6, 22.6, 22.3, 17.4, 14.1, 13.9; HRMS(ESI-TOF): calcd for
C25H44O8Na [M + Na]+ 495.2934; found, 495.2923.
p-Tolyl 2,4-di-O-acetyl-1-thio-α-L-rhamnopyranoside (4). To a
solution of compound 17 (2.5 g, 9.4 mmol) in dry DMF
1-O-n-Octyl-4-O-hexanoyl-2-levulinoyl-α-L-rhamnopyranosyl- (30 mL), triethylorthoacetate (2.4 mL, 14.0 mmol) was added,
(1→3)-2,4-di-O-acetyl-3-O-(p-methoxybenzyl)-α-L-rhamnopyrano- followed by a catalytic amount of CSA (434 mg, 1.9 mmol). The
side (16). To a solution of compound 6 (2.0 g, 4.5 mmol), 7 mixture was stirred for 5 h. After complete conversion by TLC
(3.0 g, 6.3 mmol) and 4 Å molecular sieves in dry CH2Cl2 (1 : 1, petroleum ether–EtOAc), Et3N was added to neutralize
(50 mL) were added NIS (1.7 g, 7.3 mmol) and AgOTf (0.24 g, the solution. Ac2O (1.8 mL, 19.0 mmol), Et3N (4.0 mL,
0.90 mmol) at 0 °C under argon. The reaction mixture was 28.0 mmol), and DMAP (114 mg, 1.0 mmol) were added, and
allowed to stir for 2 h under these conditions, while warmed to the mixture was allowed to stir for 3 h at room temperature.
room temperature until TLC indicated that the reaction was When TLC (2 : 1, petroleum ether–EtOAc) showed complete
complete. Then the residue was diluted with CH2Cl2 (200 mL), conversion, MeOH (0.5 mL) was carefully added to destroy
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excess Ac2O, and the mixture was diluted with EtOAc (100 mL). concentrated under reduced pressure. The residue was purified
The organic layer was shaken with 1 M HCl (3 × 50 mL), fol- by silica gel column chromatography (3 : 2, petroleum ether–
lowed by washing with satd aq. NaHCO3 (3 × 50 mL) and brine EtOAc) to afford 5 (3.5 g, 81%) as a white solid. 1H NMR
(2 × 50 mL). Finally the organic layer was separated, dried over (CDCl3): δ 8.73 (s, 1H, NH), 6.21 (d, 1H, J = 1.7 Hz, H-1), 5.44
Na2SO4, and evaporated into a syrup. The crude product was (dd, 1H, J = 3.5, 1.8 Hz, H-2), 5.41 (dd, 1H, J = 10.1, 3.5 Hz,
purified by silica gel column chromatography (5 : 1, petroleum H-3), 5.14 (t, 1H, J = 10.1 Hz, H-4), 4.09–4.13 (m, 1H, H-5),
1
ether–EtOAc) to afford 4 (2.5 g, 74%) as a white solid; H NMR 2.79–2.85 (m, 3H, CH2CO), 2.60–2.68 (m, 4H, CH2CO),
(DMSO-d6): δ 7.35 (d, 2H, J = 8.0 Hz, Ph), 7.17 (d, 2H, J = 2.43–2.48 (m, 1H, CH2CO), 2.20, 2.19, 2.18 (each s, each 3H,
8.0 Hz, Ph), 5.50 (d, 1H, J = 5.9 Hz, 3-OH), 5.40 (d, 1H, J = each CH3CO), 1.30 (d, 3H, J = 6.2 Hz, H-6); 13C NMR (CDCl3):
1.6 Hz, H-1), 5.18 (dd, 1H, J = 3.4, 1.5 Hz, H-2), 4.79 (t, 1H, J = δ 206.3, 206.2, 172.0, 171.7, 169.8, 160.0, 94.7 (C-1′), 70.2, 69.4,
9.8 Hz, H-4), 4.09–4.12 (m, 1H, H-5), 3.85 (ddd, 1H, J = 9.6, 6.0, 68.8, 68.2, 37.8, 37.7, 29.7 (two), 27.9, 27.8, 20.8, 17.4; HRMS
3.8 Hz, H-3), 2.30 (s, 3H, PhCH3), 2.08 (s, 3H, COCH3), 2.07 (ESI-TOF): calcd for C20H26O10NCl3Na [M + Na]+ 568.0520;
(s, 3H, COCH3), 1.08 (d, 3H, J = 6.0 Hz, H-6).
found, 568.0532.
p-Tolyl 3,4-di-O-levulinoyl-2-O-acetyl-1-thio-α-L-rhamnopyra-
1-O-n-Octyl-4-O-hexanoyl-2-levulinoyl-α-L-rhamnopyranosyl-
noside (19). To a solution of compound 18 (4.0 g, 10.9 mmol) (1→3)-2,4-di-O-acetyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-
in dry DMF (60 mL), triethylorthoacetate (3.7 mL, 21.8 mmol) acetyl-α-L-rhamnopyranosyl-(1→3)-3,4-di-O-levulinoyl-2-O-acetyl-
was added, followed by a catalytic amount of CSA (517 mg, α-L-rhamnopyranoside (20). A solution of tol thioglycoside 4
2.2 mmol). The mixture was stirred for 4 h at r.t. When TLC (500 mg, 1.4 mmol) and compound 5 (0.95 g, 1.7 mmol) in dry
(1 : 1, petroleum ether–EtOAc) showed complete conversion, CH2Cl2 (20 mL) and 4 Å MS (300 mg) was stirred at room temp-
the mixture was diluted with EtOAc (200 mL). The organic erature under argon for 20 min, and then cooled to −40 °C. At
layer was shaken with 1 M HCl (3 × 100 mL), followed by this temperature, TMSOTf (25 μL) was injected, and the result-
washing with water (3 × 100 mL), and brine (2 × 100 mL), dried ing mixture was stirred for additional 30 min and then
over Na2SO4, and concentrated in vacuo. The residue was dis- warmed up to −10 °C. To the above-mentioned mixture was
solved in dry CH2Cl2 (60 mL), followed by levulinic acid (1.9 g, added a solution of acceptor 3 (470 mg, 1.0 mmol) in CH2Cl2
16.4 mmol), EDC·HCl (3.8 g, 19.6 mmol), and DMAP (0.14 g, (5 mL), followed by NIS (393 mg, 1.6 mmol). After stirring for
1.2 mmol) and stirred for 24 h at 30 °C. The mixture 3 h, the reaction was quenched with Et3N, and then filtered
was diluted with CH2Cl2 (200 mL), washed with 1 M HCl through a pad of Celite. The filtrate was concentrated under
(2 × 100 mL), satd aq. NaHCO3 (2 × 100 mL), and brine reduced pressure. The residue was purified by silica gel
(2 × 100 mL), dried over Na2SO4, and concentrated in vacuo. column chromatography (2 : 1, petroleum ether–EtOAc) to give
1
The residue was purified by silica gel column chromatography 20 (605 mg, 83%) as a white solid. H NMR (600 MHz, CDCl3):
(1 : 1, petroleum ether–EtOAc) to provide 19 (4.3 g, 79%). δ 5.15 (dd, 1H, J = 10.1, 3.4 Hz, H-3″″), 5.11 (dd, 1H, J = 3.2, 1.8
1H NMR (CDCl3): δ 7.35 (d, 2H, J = 8.0 Hz, Ar–H), 7.12 (d, 2H, Hz, H-2″″), 5.02–5.06 (m, 4H, H-4′, H-4″, H-4′′′, H-4″″),
J = 8.0 Hz, Ar–H), 5.46 (dd, 1H, J = 3.3, 1.8 Hz, H-2), 5.32 (s, 5.00–5.01 (m, 2H, H-2″, H-2′′′), 4.94 (d, 1H, J = 1.4 Hz, H-1′′′),
1H, H-1), 5.30 (dd, 1H, J = 10.0, 3.4 Hz, H-3), 5.14 (t, 1H, J = 4.93 (dd, 1H, J = 3.4, 1.7 Hz, H-2′), 4.09 (dd, 1H, J = 10.0, 3.4
10.0 Hz, H-4), 4.34–4.39 (m, 1H, H-5), 2.76–2.83 (m, 3H, Hz, H-3″), 4.04 (dd, 1H, J = 10.0, 3.4 Hz, H-3′), 3.92 (dd, 1H, J =
CH2CO), 2.58–2.66 (m, 4H, CH2CO), 2.43–2.48 (m, 1H, 10.0, 3.4 Hz, H-3′′′), 3.74–3.83 (m, 3H, H-5″, H-5′′′, H-5″″),
CH2CO), 2.33 (s, 3H, Ar–CH3), 2.19, 2.17, 2.13 (each s, each 3H, 3.66–3.69 (m, 1H, H-5′), 3.61–3.64 (m, 1H, H-1-a), 3.36–3.40
each CH3CO), 1.25 (d, 3H, J = 6.2 Hz, H-6); 13C NMR (CDCl3): (m, 1H, H-1-b), 2.58–2.79 (m, 12H, 6 × COCH2), 2.41–2.46 (m,
δ 206.3 (two), 172.0, 171.1, 170.0, 138.2, 132.5 (two), 129.9 1H, COCH2-1), 2.30–2.36 (m, 1H, COCH2-2), 2.22, 2.19, 2.17,
(two), 129.4, 92.1 (C-1′), 86.1, 71.4, 71.2, 69.5, 67.8, 37.8, 37.7, 2.16, 2.16, 2.15, 2.13, 2.12 (each s, each 3H, each CH3CO),
27.9, 27.8, 21.0, 20.8; HRMS(ESI-TOF): calcd for C25H32O9Na 1.54–1.63 (m, 4H, 2 × CH2), 1.26–1.32 (m, 14H, 7 × CH2), 1.19
[M + Na]+ 531.1665; found, 531.1676.
(d, 3H, J = 6.2 Hz, H-6″″), 1.18 (d, 3H, J = 6.2 Hz, H-6′′′), 1.16
3,4-Di-O-levulinoyl-2-O-acetyl-α-L-rhamnopyranosyl-trichloro- (d, 3H, J = 6.2 Hz, H-6′), 1.14 (d, 3H, J = 6.2 Hz, H-6″), 0.89 (t,
acetimidate (5). To a stirred solution of 19 (4.0 g, 7.9 mmol) in 3H, J = 7.1 Hz, CH3), 0.88 (t, 3H, J = 7.2 Hz, CH3); 13C NMR
acetone (36 mL) and H2O (4 mL) at −20 °C was added NBS (CDCl3): δ 206.4 (two), 206.0, 173.0, 171.9 (two), 171.4,
(2.1 g, 11.9 mmol). After stirring the mixture for 20 min, the 170.4, 170.3, 170.2 (two), 170.1, 99.4, 99.2, 98.7, 97.2, 75.4,
reaction was quenched with saturated aqueous NaHCO3 solu- 75.1, 74.7, 72.2, 72.0, 71.8 (two), 71.3, 70.8, 70.1, 68.5, 68.2,
tion and the resulting mixture was concentrated under 67.2 (two), 66.5, 37.8, 37.7 (two), 34.0, 31.8, 31.3, 29.7 (two),
reduced pressure. The residue was diluted with EtOAc 29.3 (two), 29.2, 28.2, 27.9, 27.8, 26.1, 24.5, 22.6, 22.3, 21.0
(150 mL) and washed with saturated aqueous NaHCO3 (two), 20.9, 20.8, 20.7, 17.5, 17.2, 17.1, 17.0, 14.1, 13.9;
(50 mL), brine (2 × 50 mL), dried over Na2SO4, and concen- HRESIMS calcd for C63H96O29Na [M + Na]+ 1339.5935; found
trated in vacuo. The residue was purified by silica gel column 1339.5940.
chromatography (1 : 1, petroleum ether–EtOAc) to afford a
white solid. To a stirred solution of the above white solid and O-acetyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-acetyl-α-L-rhamno-
DBU (0.62 mL, 4.0 mmol) in dry CH2Cl2 (50 mL) at 0 °C was pyranosyl-(1→3)-2-O-acetyl-α-L-rhamnopyranoside (1). To
added CCl3CN (4.8 mL, 47.4 mmol). After 1 h, the solution was stirred solution of 20 (0.40 g, 0.30 mmol) in CH2Cl2 (5 mL)
1-O-n-Octyl-4-O-hexanoyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-
a
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and CH3OH (5 mL) at r.t. was added AcOH·NH2NH2 (0.60 g, Biology
6.0 mmol). After stirring the mixture for 5 h, the reaction
mixture was concentrated under reduced pressure. The residue
MTT assay for cell viability/proliferation. The cytotoxicity
was purified by silica gel column chromatography (3 : 2, pet- of the derivatives was determined by the MTT assay.18 Cells
roleum ether–EtOAc) to afford a white solid 1 (0.26 g, 84%). 1H (1 × 104 per well) were plated in 100 μL of medium per well in
NMR (600 MHz, CDCl3): δ 5.05 (t, 1H, J = 9.9 Hz, H-4′), 5.04 (t, 96-well plates. After incubation overnight, the cells were
1H, J = 9.9 Hz, H-4″), 5.02 (t, 1H, J = 9.7 Hz, H-4′′′), 4.97 (dd, treated with different concentrations of drugs in RPMI 1640
1H, J = 3.0, 1.7 Hz, H-2″), 4.95 (dd, 1H, J = 3.0, 1.6 Hz, H-2′′′), with 10% FBS for 24 h. Finally, MTT was added, and then the
4.89 (dd, 1H, J = 3.0, 1.4 Hz, H-2″″), 4.87 (d, 1H, J = 1.4 Hz, H-1′ cells were incubated for another 4 h. The MTT–formazan
′′), 4.84 (s, 1H, H-1″″), 4.82 (d, 1H, J = 1.6 Hz, H-1″), 4.76 (d, formed by metabolically viable cells was dissolved in 150 mL
1H, J = 1.4 Hz, H-1′), 4.05 (dd, 1H, J = 9.9, 3.3 Hz, H-3″), of DMSO and shaken for 10 min. The absorbance was then
3.90–3.94 (m, 4H, H-5″, H-2′, H-3′, H-3″″), 3.74–3.78 (m, 2H, measured on an ELISA reader at a test wavelength of 490 nm.
H-3′′′, H-5′′′), 3.63–3.70 (m, 2H, H-5′, H-5″″), 3.54–3.59 (m, 1H, Each test was repeated at least three times. The concentration
H-1-a), 3.42 (t, 1H, J = 9.7 Hz, H-4″″), 3.39–3.41 (m, 1H, H-1-b), of the compound, which gives 50% growth inhibition value,
2.40–2.45 (m, 1H, COCH2-1), 2.31–2.36 (m, 1H, COCH2-2), corresponds to the IC50.
2.17, 2.16, 2.14, 2.14, 2.12 (each s, each 3H, each CH3CO),
Hoechst staining. Cells were seeded at a density of 1 × 105
1.56–1.63 (m, 4H, 2 × CH2), 1.26–1.32 (m, 17H, 7 × CH2, H-6″″), cells over 18 mm cover slips and incubated for 24 h. After incu-
1.20 (d, 3H, J = 6.2 Hz, H-6′′′), 1.17 (d, 3H, J = 6.2 Hz, H-6′), bation, they were treated with compound 1 at 0.5 and 1 μM for
1.15 (d, 3H, J = 6.2 Hz, H-6″), 0.89 (t, 3H, J = 7.1 Hz, CH3), 0.88 24 h, respectively. Hoechst 33258 (Sigma Aldrich) was added to
(t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3): δ 173.1, 170.7, 170.6, the cells at a concentration of 0.5 mg mL−1 and incubated for
170.5, 170.2, 170.1, 99.4, 99.3, 99.2, 99.1, 78.7, 75.6, 74.4, 73.1, 30 min at 37 °C. Later, the cells were washed with phosphate
72.4, 72.2, 71.9, 71.7 (two), 71.6, 71.0, 69.6, 69.0, 68.0, 67.4, buffered saline (PBS). The cells from each cover slip were cap-
67.3, 66.2, 34.0, 31.8, 31.3, 29.4, 29.3, 29.2, 26.1, 24.5, 22.6, tured from randomly selected fields under a fluorescence
22.3, 21.0, 20.9, 20.8, 20.7, 17.4, 17.2, 17.0, 14.1, 13.9; HRMS microscope (Leica, Germany) to qualitatively determine the
(ESI-TOF): calcd for C48H78O23Na [M + Na]+ 1045.4832; found proportion of viable and apoptotic cells based on their relative
1045.4869.
fluorescence and nuclear fragmentation.22
1-O-n-Octyl-4-O-hexanoyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-
Measurement of the mitochondrial membrane potential.
O-acetyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-acetyl-α-L-rhamno- The mitochondrial membrane potential was measured
pyranosyl-(1→3)-α-L-rhamnopyranoside (2). To a stirred solu- with the lipophilic cationic dye JC-1 (Molecular Probes), as
tion of 20 (0.32 g, 0.24 mmol) in CH2Cl2 (5 mL) and CH3OH described.19
(5 mL) at r.t. was added AcOH·NH2NH2 (0.95 g, 10.8 mmol).
Flow cytometric evaluation of apoptosis. K562 cells (1 × 106)
After stirring the mixture to at 50 °C for 24 h, the reaction were seeded in six-well plates and allowed to grow overnight.
mixture was concentrated under reduced pressure. The residue The medium was then replaced with complete medium
was purified by silica gel column chromatography (1 : 1, pet- containing 1 μM concentration of compound 1 for 48 h
roleum ether–EtOAc) to give 2 (0.17 g, 71%) as a white solid. along with vehicle alone (0.001% DMSO) as the control. After
1H NMR (600 MHz, CDCl3): δ 5.07 (t, 1H, J = 10.0 Hz, H-4′), 48 h of drug treatment, the cells from the supernatant and
5.05 (t, 1H, J = 10.0 Hz, H-4″), 4.98–5.01 (m, 3H, H-4′′′, H-2″, adherent monolayer cells were harvested by trypsinization, and
H-2′′′), 4.88 (d, 1H, J = 1.5 Hz, H-1′′′), 4.87 (d, 1H, J = 1.4 Hz, washed with PBS at 3000 rpm. Then the cells (1 × 106) were
H-1″″), 4.85 (d, 1H, J = 1.4 Hz, H-1″), 4.71 (d, 1H, J = 1.2 Hz, stained with annexin V-FITC and propidium iodide using
H-1′), 4.07 (dd, 1H, J = 9.9, 3.4 Hz, H-3″), 3.91–3.97 (m, 4H, the annexin-V-PI apoptosis detection kit (Invitrogen). Flow
H-2′, H-3′, H-3′′′, H-3″″), 3.81 (dd, 1H, J = 3.2, 1.4 Hz, H-2″″), cytometry was performed using a FACScan (Becton Dickinson)
3.76–3.79 (m, 1H, H-5″), 3.72–3.74 (m, 1H, H-5′′′), 3.64–3.68 equipped with a single 488 nm argon laser as described
(m, 2H, H-5″″, H-1-a), 3.55–3.58 (m, 1H, H-5′), 3.42 (t, 1H, J = earlier.23 Annexin V-FITC was analyzed using excitation
9.9 Hz, H-4″″), 3.40–3.42 (m, 1H, H-1-b), 2.40–2.46 (m, 1H, and emission settings of 488 nm and 535 nm (FL-1
COCH2-1), 2.31–2.37 (m, 1H, COCH2-2), 2.18, 2.16, 2.15, 2.09 channel); PI, 488 nm and 610 nm (FL-2 channel). Debris and
(each s, each 3H, each CH3CO), 1.57–1.64 (m, 4H, 2 × CH2), clumps were gated out using forward and orthogonal light
1.26–1.32 (m, 14H, 7 × CH2), 1.28 (d, 3H, J = 6.2 Hz, H-6″″), scatter.
1.21 (d, 3H, J = 6.2 Hz, H-6′′′), 1.19 (d, 3H, J = 6.2 Hz, H-6′),
Cell cycle assay. The cell cycle was analyzed by flow cyto-
1.16 (d, 3H, J = 6.2 Hz, H-6″), 0.91 (t, 3H, J = 7.1 Hz, CH3), 0.89 metry. Briefly, K562 cells were treated with different concen-
(t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3): δ 173.1, 170.7, 170.3, trations of compound 1 (1 or 2 μM) for 24 h. After incubation,
170.2, 170.1, 101.5, 99.2 (three), 78.7, 75.4, 74.2, 73.0, 72.5, a total of 1 × 108 cells were harvested from the treated and
72.0, 71.8 (two), 71.6, 71.4, 71.1, 70.9, 68.8, 68.0, 67.3 (two), normal samples. The cells were washed twice with PBS and
66.2, 34.1, 31.8, 31.3, 29.7 (two), 29.4, 29.3, 29.2, 26.1, 24.5, fixed in 75% ice-cold ethanol at least overnight. The sample
22.6, 22.3, 21.0, 20.9, 20.7, 17.4 (two), 17.2, 17.1, 14.1, 13.9; was concentrated by removing the ethanol and staining the
HRMS(ESI-TOF): calcd for C46H76O22Na [M + Na]+ 1003.4726; cellular DNA with a fluorescent solution (1% (v/v) Triton X-100,
found 1003.4720.
0.01% RNase, 0.05% PI) for 30 min at 4 °C in darkness. The
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Organic & Biomolecular Chemistry
cell cycle distribution was then detected by flow cytometry. All
experiments were performed three times.
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Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
Financial support was provided by the Special Fund for the
Research and Construction of Public Service Ability in Guang-
dong (2016A020217014, 2014A020210019), the National Key
Project for Basic Research (973 Project, 2015CB150600), the
National Natural Science Foundation of China (21202047,
31570122), the Pearl River S&T Nova Program of Guangzhou
(201506010029), and the Specialty and Innovation Project from
the Department of Education in Guangdong (2014KTSCX031).
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