The antibacterial properties of 6-tuliposide B. Synthesis of 6-tuliposide B analogues and structure-activity relationship

Article abstract of DOI:10.1016/j.phytochem.2009.10.008

6-Tuliposide B is a secondary metabolite occurring specifically in tulip anthers. Recently, a potent antibacterial activity of 6-tuliposide B has been reported. However, its molecular target has not yet been established, nor its action mechanism. To shed light on such issues, 6-tuliposide B and tulipalin B analogues were synthesized and a structure-activity relationship (SAR) was examined using a broad panel of bacterial strains. As the results of SAR among a total of 25 compounds, only tulipalin B and the compounds having 3′,4′-dihydroxy-2′-methylenebutanoate (DHMB) moieties showed any significant antibacterial activity. Moreover, the 3′R analogues of these compounds displayed essentially the same activities as 6-tuliposide B and the structure of the 3′R-DMBA moiety was the same as that of the proposed active moiety of cnicin. These results suggest that 6-tuliposide B has the same action mechanism as proposed for cnicin and bacterial MurA is one of the major molecular targets of 6-tuliposide B.

Full text of DOI:10.1016/j.phytochem.2009.10.008

Phytochemistry 71 (2010) 312–324
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Phytochemistry
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The antibacterial properties of 6-tuliposide B. Synthesis of 6-tuliposide B
analogues and structure–activity relationship
Kengo Shigetomi a, Kazuaki Shoji b, Shinya Mitsuhashi a, Makoto Ubukata a,
*
a Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
b Agricultural Experiment Station, Toyama Agricultural Research Center, Toyama 939-8153, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2009
Received in revised form 9 September 2009
Available online 24 November 2009
6-Tuliposide B is a secondary metabolite occurring specifically in tulip anthers. Recently, a potent anti-
bacterial activity of 6-tuliposide B has been reported. However, its molecular target has not yet been
established, nor its action mechanism. To shed light on such issues, 6-tuliposide B and tulipalin B ana-
logues were synthesized and a structure–activity relationship (SAR) was examined using a broad panel
of bacterial strains. As the results of SAR among a total of 25 compounds, only tulipalin B and the com-
pounds having 30,40-dihydroxy-20-methylenebutanoate (DHMB) moieties showed any significant antibac-
terial activity. Moreover, the 30R analogues of these compounds displayed essentially the same activities
as 6-tuliposide B and the structure of the 30R-DMBA moiety was the same as that of the proposed active
moiety of cnicin. These results suggest that 6-tuliposide B has the same action mechanism as proposed
for cnicin and bacterial MurA is one of the major molecular targets of 6-tuliposide B.
Keywords:
Tuliposide
Tulipalin
SAR
MurA
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
6-tuliposide B and its 30R-epimer (epi-1) (Shigetomi et al., 2008).
Furthermore, we report a broad structure–activity relationship
Tuliposides are secondary metabolites occurring mainly in the
Liliaceae and Alstroemeriaceae (Slob, 1973; Slob et al., 1975; Chris-
tensen, 1995b). At present, tuliposides A, B, D, E and F have been
reported together with their chemical structures (Tschesche
et al., 1969; Christensen and Kristiansen, 1995a; Christensen,
(SAR) study to identify the structures responsible for antibacterial
activity, and thereafter discuss the target molecule of 6-tuliposide
B and its action mechanism.
2. Results and discussion
1995c, 1999), with all tuliposides composed of D
-glucose and 40-
hydroxy-20-methylenebutanoyl and/or (30S)-30,40-dihydroxy-20-
methylenebutanoyl side-chains. Both types of side-chains can
be released by either pH-dependent or enzymatic lactonization
to form tulipalin A (3) and tulipalin B (2) (Fig. 1), respectively
(Tschesche et al., 1968; Kato et al., 2009). Principally, these tulipa-
lins are known to be allergic and causative agents of ‘‘tulip fingers”
(Barbier and Benezra, 1982, 1986; Lahti, 1986; Marks, 1988; Gette
and Marks, 1990).
2.1. Synthesis of 6-tuliposide B and tulipalin B analogues
2.1.1. Synthesis of 40-deoxy and hexanoyl-type 6-tuliposide B
analogues (5, epi-5, 7 and epi-7)
The syntheses of 40-deoxy and hexanoyl-type 6-tuliposide Bs
were conducted on the basis of the total synthesis of 6-tuliposide
B (1). By employing acetaldehyde rather than (tert-butyldimethyl-
silyloxy)-acetaldehyde in the Baylis–Hillman reaction, both the 40-
deoxy 6-tuliposide B precursor and its 30-epimer were obtained via
chiral HPLC separation. Both diastereomers were deprotected by
TFA treatment to yield 40-deoxy 6-tuliposide B (5) and its 30-epimer
(epi-5), respectively (Scheme 1). Hexanoyl-type analogues (7 and
epi-7) were synthesized from 4-(tert-butyldimethylsilyloxy)-but-
anal (6) (Taillier et al., 2005) and sugar acrylate (4) in a similar way.
Recently, we found 6-tuliposide B (6-O-[(30S)-30,40-dihydroxy-
20-methylenebutanoyl]-
D-glucose) (1) to have potent antimicrobial
activity (Shoji et al., 2005), with its simple and low-molecular
weight structure displaying a broad antibacterial effect against
Gram-positive, Gram-negative, and certain fungicide-tolerant
strains of bacteria. However, neither its antibacterial molecular
target has been established, nor its action mechanism.
In this study, we describe the synthesis of 6-tuliposide B (1) and
tulipalin B (2) analogues based on our previous total synthesis of
2.1.2. Synthesis of hydrogenated 6-tuliposide Bs and hydrogenated
tulipalin Bs (8, epi-8, 9 and ent-9)
To survey the significance of the
a,b-unsaturated moiety,
hydrogenated 6-tuliposide B (8) and hydrogenated tulipalin B (9)
* Corresponding author. Tel./fax: +81 11 706 3638.
E-mail address: m-ub@for.agr.hokudai.ac.jp (M. Ubukata).
were synthesized by Pd/C catalytic hydrogenation of 6-tuliposide
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.10.008

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
313
1972; Kim et al., 1998; Rastelli et al., 2008) because of their
Michael acceptor properties. Based on the signal intensities of the
a
-H in the 1H NMR spectra, the diastereomeric syn/anti ratio of 8
and 9 were estimated to be 3:2 and 4:1, respectively. Diastereo-
meric mixtures were subjected to the antibacterial assays without
further separation. Similarly, hydrogenated products of non-natu-
ral type (epi-8 and ent-9) were prepared from epi-6-tuliposide B
(epi-1) and (+)-tulipalin B (ent-2) (see Scheme 2).
2.1.3. Synthesis of amide-type 6-tuliposide B and 1,2-dideoxy 6-
tuliposide Bs (19 and epi-19)
As mentioned above, 6-tuliposide B (1) can be converted into
tulipalin B (2) even at neutral conditions and is relatively stable
in mild acidic solutions below pH 5.0. To avoid this pH-dependent
lactonization in antibacterial assays, amide-linked 6-tuliposide B
analogues were synthesized. Amide-type analogues were con-
structed by a condensation of butanoic acid and amino sugar be-
cause aliphatic acrylamide has a low Baylis–Hillman reactivity
due to its low Michael acceptor properties (Guo et al., 2005).
The synthetic route of the butanoyl side chain is depicted in
Scheme 3. Baylis–Hillman reaction of (p-methoxybenzyloxy)-acet-
aldehyde (Smith and Fox, 2004) and methyl acrylate provided the
racemic adduct in good yield. The so-obtained racemate was then
separated by Katsuki-Sharpless kinetic resolution to afford 3S-
enantiomer (10, >99%ee) in 43% yield and the 3S-epoxide (11,
96%ee) in 40% yield, respectively. Similarly, the 3R-enantiomer
(epi-10) and 3R-epoxide (epi-11) were obtained by using (ꢀ)-diiso-
propyl tartrate in kinetic resolution. The pure 3S-enatiomer (10)
Fig. 1. Structures of 6-tuliposide B (1), epi-6-tuliposide (1a) (ꢀ)-tulipalin B (2) and
tulipalin A (3).
B (1) and (ꢀ)-tulipalin B (2), respectively. In general,
a,b-unsatu-
rated carbonyl compounds are known to be reactive and to have
various biological activities (Shibaoka et al., 1967; Iino et al.,
Scheme 1. Reaction and reagents: (a) 3-hydroxy quinuclidine (1.0 eq), CH3CN, 40 h, 31%, (b) TFA:CH2Cl2 = 2:1, 5:96%, epi-5:98%, (c) 3-hydroxy quinuclidine (1.0 eq), DMSO,
40 h, 40%, and (d) TFA:CH2Cl2 = 2:1, 7:95%, epi-7:95%.
Scheme 2. Reaction and reagents: (a) Pd/C, H2, MeOH, 72% and (b) Pd/C, H2, EtOAc, 65%.

314
K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
Scheme 3. Reaction and reagents: (a) 3-hydroxy quinuclidine (0.5 eq), DMSO, 15 h, 84%, (b) (+)-diisopropyl tartrate, Ti(OiPr)4, cumenehydroperoxide, MS 4A, CH2Cl2, 10; 43%
(>99%ee), 11; 40% (96%ee), (c) tert-butyldimethylsilyl trifuluoromethanesulfonate, Et3N, CH2Cl2, quant., and (d) LiOH (1.1 eq), MeCN:H2O = 1:1, 60 °C, 92%.
was protected by a TBDMS group and hydrolyzed to give butanoic
acid (13). The amino sugar (17) was synthesized from 1-O-(2-trim-
To study the role of the sugar moiety in 6-tuliposide B (1), 1,2-
dideoxy-6-tuliposide B (26) was synthesized (Scheme 6). The syn-
thesis was achieved by a condensation of butanoic acid with 1,2-
ethylsilylethyl)-b-
D-glucopyranoside
(14)
in
three
steps
(Scheme 4). The 6-hydroxyl group of (2-trimethylsilylethyl)- glu-
coside 14 was selectively tosylated and then substituted by azide,
which was reduced to the amine to give the amino sugar (17). Con-
densation of butanoic acid (13) with the amino sugar (17) afforded
an amide-type 6-tuliposide B analogue (19) after TFA treatment as
shown in Scheme 5. Likewise, the 30R-epimer (epi-19) was pre-
pared from the 3R Baylis–Hillman adduct (ent-13).
dideoxy-
from 3,4,6-tri-O-acetyl-
followed by methanolysis provided 1,2-dideoxy-
(21). Subsequently, the primary alcohol and 3,4-hydroxyl groups
were protected by pivaloylation and triethylsilylation, respectively.
D
-glucopyranose derivatives (24), which was prepared
-glucal (20) in five steps. Hydrogenation
-glucopyranose
D
D
Scheme 6. Reaction and reagents: (a) Pd/C, H2, EtOAc, (b) NaOMe, MeOH, 69% (two
steps), (c) PvCl, pyridine, 83%, and (d) triethylsilyl trifuluoromethanesulfonate2,6-
lutidine, CH2Cl2, 99%, (d) DIBAL, CH2Cl2, 79%.
Scheme 4. Reaction and reagents: (a) TsCl, pyridine, 0 °C, 86%, (b) NaN3, DMF,
90 °C, 92%, and (c) Pd/C, H2, EtOAc, quant.
Scheme 5. Reaction and reagents: (a) diisopropyl carbodiimide, 4-dimethylami-
nopyridine, CH2Cl2, 0 °C, 81% and (b) TFA:CH2Cl2 = 2:1, 90%.
Scheme 7. Reaction and reagents: (a) diisopropyl carbodiimide, 4-dimethylamin-
opryidine, CH2Cl2, 62% and (b) TFA:CH2Cl2 = 2:1, 70%.

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
315
tives (30 and epi-30) were also synthesized. The preparation of the
diastereomeric mixture of 1-O-methyl-b- -glucoside derivatives
D
was previously described (Shigetomi et al., 2006). To validate the
SAR analysis, the diastereomeric mixture was separated into its
natural-type 1-O-methyl-b-
mer (epi-27) by chiral HPLC. The 1-O-methyl-
D
-glucoside derivative (27) and its epi-
-glucoside deriv-
a-D
ative (30) and its 30R-epimer (epi-30) were individually
synthesized via condensation of acid (13, ent-13) with sugar (28)
as shown in Scheme 8 (see also Fig. 2).
Fig. 2. Structures of 1-O-methyl-b-6-tuliposide Bs (27 and epi-27).
2.1.5. Synthesis of methyl esters of tuliposide B side chain (31 and ent-
31) and their epoxides (32 and ent-32)
The Baylis–Hillman adduct (10) and the epoxide (11) were
deprotected by TFA treatment to give the methyl ester of 6-tulipo-
side B side chain (31) and its epoxide (32), respectively. Moreover,
the 3R-enantiomer of each product was prepared from the corre-
sponding starting materials. These products were also used in anti-
bacterial tests to probe the importance of the sugar moiety and the
double bond of the
Scheme 9).
a,b-unsaturated carbonyl moiety (see
2.2. Antibacterial activity evaluation
Antibacterial assays were performed against reported seven
strains (Shoji et al., 2005) that are Escherichia coli (NBRC 3972,
ATCC 8739), Salmonella enteritidis (NBRC 3313), Pseudomonas aeru-
ginosa (NBRC 13275, ATCC 9027), Burkholderia glumae (NBRC T
12119, ATCC 49703), Acidovorax avenae (NBRC T 9020), Staphylo-
coccus aureus (NBRC 13276, ATCC 6538) and Bacillus subtilis (NBRC
3007, ATCC 70385). A. avenae is a resistant strain against oxolinic
acid, and B. glumae is resistant to both oxolinic acid and kasugamy-
cin. To test the antibacterial generality of 6-tuliposide B (1) against
drug-resistant strains, methicillin-resistant S. aureus (ATCC
700699) was also subjected to assays.
Scheme 8. Reaction and reagents: (a) diisopropyl carbodiimide, 4-dimethylami-
nopyridine, CH2Cl2, 60% and (b) TFA:CH2Cl2 = 2:1, 80%.
The pivaloyl group was then removed by DIBAL reduction to give
24. Finally, 1,2-dideoxy-6-tuliposide B (26) was synthesized by
coupling of the sugar moiety (24) with butanoic acid (13) followed
by TFA treatment as shown in Scheme 7. The 30R-epimer of 26 (epi-
26) was also prepared in the same way.
2.1.4. Synthesis of methyl glucosides of 6-tuliposide B (27, epi-27, 30
and epi-30)
2.2.1. Antibacterial activities of 6-tuliposide B and its analogues
At first, 6-tuliposide B (1) and its analogues were evaluated for
their antibacterial activities. Tested compounds were namely
As sugar-modified analogues, the 1-O-methyl-b-
D-glucoside
derivatives (27 and epi-27) and 1-O-methyl- -glucoside deriva-
a
-D
Scheme 9. Reaction and reagents: (a) TFA:CH2Cl2 = 2:1, 80% and (b) TFA:CH2Cl2 = 2:1, 80%.
Table 1
MIC values of 6-tuliposide B (1) and its analogues against bacteria (mM).
Compounds
E. coli
S. enteritidis
P. aeruginosa
B. glumae
A. avenae
S. aureus
MRSA
B. subtilis
1 (epi-1)
0.2 (0.2)
0.3 (0.3)
0.2 (0.2)
0.3 (0.3)
0.2 (0.2)
0.2 (0.3)
0.2 (0.2)
0.3 (0.3)
0.2 (0.2)
0.2 (0.3)
0.2 (0.2)
0.3 (0.3)
0.3 (0.2)
0.3 (0.3)
0.3 (0.2)
0.3 (0.3)
0.1 (0.1)
0.1 (0.1)
0.1 (0.1)
0.1 (0.1)
0.2 (0.2)
0.2 (0.2)
0.2 (0.2)
0.3 (0.3)
0.3 (0.3)
0.2 (0.2)
0.3 (0.3)
0.3 (0.4)
0.5 (0.5)
0.5 (1.0)
0.5 (0.5)
0.5 (0.6)
26 (epi-26)
27 (epi-27)
30 (epi-30)
Compounds 5, 7, 8 and 19 and their epimers (epi-5, epi-7, epi-8 and epi-19) did not show any activity at 1.0 mM.

316
K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
6-tuliposide B (1), epi-6-tuliposide B (epi-1) and 14 synthetic ana-
logues (5, 7, 8, 19, 26, 27, 30 and their epimers).
higher activity than linear (not lactonized) esters such as 1, 26,
27, 30, 31 and their epimers. These results reflect that tulipalin B
formation plays a significant role for the antibacterial activity of
6-tuliposide B (1). Tulipalin A (3) showed a lower activity, which
is approximately a tenth to a twentieth part of tulipalin Bs (2
and ent-2). This indicated that presence of a hydroxyl group at b-
position is important, but its configuration is not significant for
antibacterial activities. Hydrogenated tulipalin Bs (9 and ent-9)
and epoxidized derivatives (32 and ent-32) exhibited no growth
inhibition at 1.0 mM and this is consistent with the results of 8
and epi-8.
The results are shown in Table 1. The antibacterial activities
were presented as minimal inhibitory concentration (MIC). All of
the analogues having 30,40-dihydroxy-2-methylenebutanoate
structures (26, 27, 30 and their epimers) retained antibacterial
activities irrespective of their sugar modification. Moreover, their
MIC values were essentially the same as 6-tuliposide B (1), which
suggests that the sugar moiety of 6-tuliposide B (1) is not indis-
pensable for inhibitory activities. Likewise, the absolute configura-
tions at the 30-position seemed to not affect activities. Active
compounds exhibited growth inhibition against MRSA as well as
the reported seven strains.
2.2.3. Discussion of the molecular target of 6-tuliposide B (1) and its
action mechanism
On the other hand, hydrogenated 6-tuliposide B (8 and epi-8)
did not show any activities at 1 mM. This indicates that the
a,b-
Recently, cnicin occurring in Cnicus benedictus has been shown
to inhibit bacterial MurA by an unusual 1,2-addition (Steinbach
et al., 2008). MurA is a bacterial cytoplasmic enzyme, which cata-
lyzes the first committed step of peptidoglycan biosynthesis that is
the coupling reaction of UDP-N-acetylglucosamine (UNAG) with
phosphoenolpyruvate (PEP) to yield a muramic acid precursor. Cni-
cin consists of a large sesquiterpenoid moiety and the same but-
anoate side chain as the synthetic 30R-epimer of 6-tuliposide B
(epi-1) (Fig. 3). Steinbach’s X-ray crystallographic analysis has
established that 3,4-dihydroxy-2-methylenebutanoate (DHMB)
moiety of cnicin reacts with UNAG to form an unusual adduct in
the active site of bacterial enzyme MurA by mimicking PEP (Fig. 3).
Results of the present SAR study proved that the 30,40-dihy-
droxy-20-methylenebutanoate structure is responsible for the
activities and both the 30S- and 30R-epimers show the same
activities, suggesting that 6-tuliposide B (1) has the same action
mechanism as cnicin. In general, MurA prefers high polar and low-
molecular weight substrates or inhibitors (e.g. PEP or fosfomycin);
therefore, it would be difficult especially for cnicin to interact with
MurA in its original form. 6-Tuliposide B (1) and cnicin could ex-
press their antibacterial activities by formation of (ꢀ)- and (+)-tuli-
palin B (2 and ent-2), respectively, and also released tulipalins
could be hydrolyzed into 3,4-dihydroxy-2-methylenebutanoic
acids to form UNAG-tulipalin B adducts as substrate-mimics.
Hydrolysis of tulipalin Bs (2 and ent-2) would proceed effectively
in the active site of MurA. The lower activity of tulipalin A (3)
can be attributed to its low polarity, because it has been considered
that the 3,4-dihydroxy moiety of the butanoic acid mimics phos-
unsaturated enone is essential for antibacterial activities. The 40-
deoxy (5 and epi-5), hexanoyl-type (7 and epi-7) and amide-type
analogues (19 and epi-19) were also inactive. These results might
stem from their inabilities to form tulipalin Bs (2 and ent-2), be-
cause 5, epi-5, 7 and epi-7 were structurally unable to form tulipal-
in Bs. Likewise, the amide-type analogues (19 and epi-19) would
be difficult to lactonize due to their strong amide bonds. In con-
trast, all of the antibacterial compounds tested (1, 26, 27, 30 and
their epimers) possess a 30,40-dihydroxy-20-methylenebutanoate
structure, which can be converted into tulipalin Bs. Although the
assay was carried out under weak acidic conditions, a part of these
compounds would be converted into tulipalin Bs at pH 6.0. There-
fore, SAR of tulipalin B (2) and its analogues should be studied to
establish the role of tulipalin B (2) in these activities.
2.2.2. SAR study of (ꢀ)-tulipalin B (2) and its analogues
Secondly, SAR of (ꢀ)-tulipalin B (2) and its analogues were stud-
ied. (ꢀ)-Tulipalin B (2), synthetic (+)-tulipalin B (ent-2) and other
six analogues described above (9, 31, 32 and their enantiomers)
were assayed. As a 3-deoxy analogue of tulipalin B (2), commer-
cially available tulipalin A (3) was also assayed.
As shown in Table 2, tulipalins (2, 3 and ent-2) and the methyl
esters of tuliposide B side chain (31 and ent-31) showed antibacte-
rial activities. The MIC values of the 3S- and 3R-enantiomers were
almost the same, this being analogous to the results of 6-tuliposide
B analogues. The methyl esters of the 6-tuliposide B side chain (31
and ent-31) showed equal activities to sugar esters, thus it was
confirmed that the sugar moiety was not necessary for growth
inhibition of bacteria. Tulipalin Bs (2 and ent-2) showed a little
phate group of PEP. The fact that the
a,b-unsaturated carbonyl
compounds, 5, epi-5, 7, and epi-7 do not show any antibacterial
Table 2
MIC values of (ꢀ)-tulipalin B (2) and its analogues against bacteria (mM).
Compounds
E. coli
S. enteritidis
P. aeruginosa
B. glumae
A. avenae
S. aureus
MRSA
B. subtilis
2 (ent-2)
3
0.2 (0.2)
3.4
0.1 (0.1)
1.8
0.1 (0.1)
2.0
0.2 (0.2)
2.8
0.1 (0.1)
1.2
0.2 (0.2)
3.2
0.2 (0.2)
4.8
0.3 (0.3)
>5.0
31 (ent-31)
0.3 (0.3)
0.3 (0.3)
0.3 (0.3)
0.3 (0.3)
0.1 (0.1)
0.3 (0.3)
0.4 (0.5)
0.8 (1.0)
Compounds 9 and 32 and their enantiomers (ent-9 and ent-32) did not show any activity at 1.0 mM.
Fig. 3. Structures of cnicin, PEP and the UNAG-DHMB adduct observed in the crystal structure (Steinbach et al., 2008).

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
317
activity at 1.0 mM indicates that a non-specific Michael addition of
biomolecule is not the major cause of antibacterial activity,
whereas a non-specific Michael addition might weaken the anti-
1-O-(2-trimethylsilylethyl)-b-
D
-glucopyranoside (0.22 mmol, 31%)
as a colorless syrup: IR (cmꢀ1) 3400, 1683, 1297, 1207, 1080 and
801, HR-FAB-MS m/z [MꢀH]+ calcd for C16H29O8Si, 377.1632;
found, 377.1608. The obtained mixture of diastereomers was fur-
ther separated by chiral HPLC (EtOH/hexane, 20:80; CHIRALPAKÒ
IA column (u 20 mm ꢁ 25 cm)).
bacterial activity of 6-tuliposide B. The
a,b-unsaturated structure
would be required for the unusual 1,2-addition of UNAG to the
double bond, although inhibition of the other PEP-related enzymes
could not be excluded completely.
4.1.1. First eluted (30R)- diastereomer
rt = 25.6 min, ½a 2D5
ꢂ
= ꢀ9.1 (c 1.77, MeOH), 1H NMR (500 MHz,
3. Conclusion
CDCl3) 0.03 (9H, s, CH2CH2SiMe3), 0.94–1.06 (2H, m,
CH2CH2SiMe3), 1.37 (1H, d, J = 6.5 Hz, CH3), 3.18 (1H, dd, J = 4.7,
4.4 Hz, H-2), 3.30–3.40 (2H, m, H-3 and H-4), 3.52 (1H, ddd,
J = 9.3, 6.6, 2.4 Hz, H-5), 3.63 (1H, m, CH2CH2SiMe3), 3.92 (1H,
ddd, J = 13.3, 7.1, 3.8 Hz, CH2CH2SiMe3), 4.27 (1H, m, H-6a), 4.33
(1H, d, J = 7.8 Hz, H-1), 4.52 (1H, dd, J = 6.4, 1.2 Hz, H-6b), 4.68
(1H, br q, J = 6.4 Hz, H-30), 5.93 (1H, dd, J = 1.4 Hz, @CH2, Ha),
6.24 (1H, s, @CH2, Hb), 13C NMR (125 MHz, CDCl3) ꢀ1.4
(CH2CH2SiMe3), 19.1 (CH2CH2SiMe3), 23.5 (CH3), 65.0 (C-6), 66.8
(CH2CH2SiMe3), 68.1 (C-30), 71.9 (C-4), 75.1 (C-5), 75.2 (C-2), 78.0
(C-3), 103.9 (C-1), 123.9 (@CH2), 146.7 (C-20), and 167.5 (C-10).
6-Tuliposide B (1) and (ꢀ)- tulipalin B (2) analogues were syn-
thesized and their structure–activity relationships studied against
a broad panel of bacteria. The results of the SAR study proved that
the 30,40-dihydroxy-20-methylenebutanoate structure is responsible
for antibacterial activities and the glucose moiety of 6-tuliposide B
did not directly relate to antibacterial activities. 6-Tuliposide B (1)
showed antibacterial activity against MRSA; however, no MIC
differences were observed between 30S- and 30R-epimers, and tuli-
palin B (2) showed a little higher activity than linear esters. There-
fore, formation of tulipalin B (2) is considered to be important for
bacterial growth inhibition of 6-tuliposide B (1). These results sug-
gest that the molecular target of 6-tuliposide B (1) is bacterial MurA
on which the (S)-30,40-dihydroxy-20-methylenebutanoic acid acts in
a similar fashion to cnicin. Although the sugar moiety is not impor-
tant for antibacterial activity in vitro, the existence of tuliposide-
converting enzymes in tulip tissues (Kato et al., 2009) might explain
the improved defense mechanism against both microbial and insect
attack. Given the present SAR study of tuliposide B (1) and its
analogues, the inhibitory activities against MurA and further studies
on action mechanism of these particular compounds will be
reported elsewhere.
4.1.2. Second eluted (30S)- diastereomer
rt = 28.5 min, ½a 2D5
ꢂ
= ꢀ23.6 (c 2.22, MeOH), 1H NMR (500 MHz,
CDCl3) 0.04 (9H, s, CH2CH2SiMe3), 0.95–1.04 (2H, m,
CH2CH2SiMe3), 1.34 (1H, d, J = 6.4 Hz, CH3), 3.19 (1H, dd,
J = 4.5 Hz, H-2), 3.36–3.40 (2H, m, H-3 and H-4), 3.52 (1H, ddd,
J = 12.9, 9.2, 2.4 Hz, H-5), 3.62 (1H, m, CH2CH2SiMe3), 3.91 (1H,
ddd, J = 13.3, 6.9, 3.7 Hz, CH2CH2SiMe3), 4.27 (1H, dd, J = 6.4,
3.3 Hz, H-6a), 4.33 (1H, d, J = 7.9 Hz, H-1), 4.57 (1H, dd, J = 6.4,
1.2 Hz, H-6b), 4.66 (1H, br q, J = 6.4 Hz, H-30), 5.93 (1H, dd,
J = 1.4 Hz, @CH2, Ha), 6.25 (1H, s, @CH2, Hb), 13C NMR (125 MHz,
CDCl3) ꢀ1.4 (CH2CH2SiMe3), 19.1 (CH2CH2SiMe3), 23.4 (CH3), 64.9
(C-6), 66.9 (CH2CH2SiMe3), 68.1 (C-30), 71.8 (C-4), 75.1 (C-5), 75.2
(C-2), 78.0 (C-3), 103.8 (C-1), 123.9 (@CH2), 146.7 (C-20), and
167.5 (C-10).
4. Experimental
The above (30S)-epimer (10.2 mg, 27.0
500 l of deprotection solution (CF3CO2H/CH2Cl2, 2:1, v/v) and stir-
lmol) was dissolved in
Unless otherwise stated, chemicals of the highest commercial
purity were used without further purification. Thin-layer and silica
gel column chromatographic steps were performed using Merck
SilicaGel 60 F254 and Kanto Chemicals Co. Silica Gel 60 N (spherical,
neutral), respectively. Chiral HPLC used a DAICEL CHIRALPAKÒ IA
column (u 20 mm ꢁ 25 cm) and a HITACHI L-7455 photodiode
array detector at 30 °C. IR spectra were recorded on a Digilab
FTS-50A. 1H, 13C, HH-COSY, HMBC and HMQC NMR spectra were
measured in CDCl3 or methanol-d4, with a Bruker AMX-500
(500 MHz) or a JEOL JNM-EX270 (270 MHz). Chemical shifts are
reported in d ppm using tetramethylsilane as internal standard.
Coupling constants (J) are given in Hertz. Mass spectra were acquired
using FD and FAB techniques using a JMS-SX102A. A part of the
NMR spectra and all of the mass spectra were measured at the
GC–MS and NMR Laboratory, Faculty of Agriculture, Hokkaido
University. Optical rotations were determined on a JASCO DIP-370
or a P-2200 polarimeter in u 3.4 mm ꢁ 5.0 cm cells at 25 °C. CH2Cl2
and CH3CN were distilled from phosphorous oxide, and pyridine
was distilled from calcium hydride.
l
red for 2 h at room temperature. Dilution of the reaction mixture
with toluene followed by evaporation was repeated until the TFA
was completely removed. The resulting mixture was then purified
by silica gel CC (H2O/CH3CN, 1:5). Subsequent evaporation of the
solvent provided 5 (7.20 mg, 96%) as a colorless syrup: IR (cmꢀ1
)
3412, 1684, 1289, 1205 and 1080, HR-FAB-MS m/z [M + Na]+ calcd
for C11H18O8Na, 301.0911; found, 301.0905. (30R)-Diastereomer
epi-5 was obtained from (R)-Baylis–Hillman adduct in 98% yield
by the same procedure.
4.1.3. 40-Deoxy-6-tuliposide B (5)
½
a 2D5 = +55.6 (c 1.30, MeOH), 1H NMR (270 MHz, CD3OD) 1.32
ꢂ
(3H, d, J = 6.3 Hz, CH3), 3.14 (dd, J = 7.6 Hz, H-2(b)), 3.31–3.56 (m,
H-2( ), H-3(b), H-4( ) and H-4(b)), 3.53 (m, H-5(b)), 3.69 (t,
J = 9.1 Hz, H-3( )), 4.03 (ddd, J = 9.1, 4.1, 1.9 Hz, H-5( )), 4.23–
4.32 (m, H-6a( ) and H-6a(b)), 4.45–4.55 (m, H-1(b), H-6b(
and H-6b(b)), 4.64 (br q, J = 6.3 Hz, H-30), 5.09 (d, J = 3.6 Hz, H-
1(
)), 5.91 (1H, s, @CH2, Ha), 6.23 (1H, s, @CH2, Hb), 13C NMR
(67.5 MHz, CD3OD) 23.3 (CH3) 65.0 (C-6), 66.9 (C-30), 70.7 (C-
5( )), 71.8 (C-4(b)), 72.0 (C-4( )), 73.8 (C-2( )), 74.8 (C-3( )),
)), 98.3 (C-
a
a
a
a
a
a)
a
4.1. 6-O-(30-hydroxy-20-methylenebutanoyl)-b-
epi-5)
D-glucopyranoside (5,
a
a
a
a
75.4 (C-5(b)), 76.3 (C-2(b)), 78.0 (C-3(b)), 94.0 (C-1(
a
To a solution of 4 (234.7 mg, 0.70 mmol) in dry CH3CN (7.0 ml),
acetaldehyde (88.3 mg, 2.10 mmol) and 3-hydroxy quinuclidine
(89.1 mg, 0.70 mmol) were added at room temperature. After
40 h, the reaction mixture was diluted with EtOAc (3.0 ml) and ex-
tracted with 3.0 ml of brine. The water layer was then washed with
EtOAc (3.0 ml ꢁ 2). The combined organic layer was dried over
anhydr. Na2SO4 and evaporated. Silica gel CC (MeOH–CHCl3, 1:9,
v/v) yielded 83.6 mg of 6-O-(30-hydroxy-20-methylenebutanoyl)-
1(b)), 123.8 and 126.9 (@CH2), 146.7 and 146.8 (C-20), 167.7 (C-10).
4.1.4. 40-Deoxy-epi-6-tuliposide B (epi-5)
½
a 2D5 = +34.7 (c 1.72, MeOH), 1H NMR (270 MHz, CD3OD) 1.29
ꢂ
(3H, d, J = 6.4 Hz, CH3), 3.12 (dd, J = 8.2 Hz, H-2(b)), 3.29–3.33 (m,
H-2( ), H-3(b), H-4( ) and H-4(b)), 3.51 (m, H-5(b)), 3.66 (t,
J = 9.2 Hz, H-3( )), 4.03 (m, H-5( )), 4.23–4.31 (m, H-6a( ) and
H-6a(b)), 4.39–4.49 (m, H-1(b), H-6b( ) and H-6b(b)), 4.56–4.65
a
a
a
a
a
a

318
K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
(m, H-30), 5.07 (d, J = 3.5 Hz, H-1(
a
)), 5.90 (1H, s, @CH2, Ha), 6.21
4.9, 2.0 Hz, H-5(
H-30 and H-1(b)), 5.08 (d, J = 3.6 Hz, H-1(
Ha), 6.25 (1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD) 29.9
and 34.3 (C-50 and C-60) 63.0 (C-6), 65.0 (C-30), 70.7 (C-5(
)), 71.8
(C-4(b)), 72.0 (C-4( )), 73.8 (C-2( )), 74.8 (C-3( )), 75.3 (C-5(b)),
)), 98.3 (C-1(b)), 124.7
a
)), 4.21–4.29 (1H, m, H-6a), 4.45–4.53 (m, H-6b,
(1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD) 23.3 (CH3) 65.0
a)), 5.89 (1H, s, @CH2,
and 65.1 (C-6(
70.7 (C-5( )), 71.8 (C-4(b)), 72.0 (C-4(
3(
a
) and C-6(b)), 66.8 and 66.9 (C-30 (
a
) and C-30(b)),
)), 74.7 (C-
)),
a
a
)), 73.8 (C-2(
a
a
a
)), 75.3 (C-5(b)), 76.2 (C-2(b)), 78.0 (C-3(b)), 94.0 (C-1(
a
a
a
a
98.3 (C-1(b)), 123.9 (@CH2), 146.6 and 146.7 (C-20), 167.6 and
76.2 (C-2(b)), 77.9 (C-3(b)), 94.0 (C-1(
a
167.7 (C-10).
(@CH2), 146.7 (C-20), 167.7 (C-10).
4.2. 6-O-(30,60-dihydroxy-20-methylenehexanoyl)-
D
-glucose (7, epi-7)
4.2.4. Hexanoyl-type epi-6-tuliposide B (epi-7)
= +25.7 (c 1.52, MeOH), 1H NMR (270 MHz, CD3OD) 1.45–
1.76 (4H, m, H-50 and H-60), 3.09 (t, J = 8.2 Hz, H-2(b)), 3.26–3.33
(m, H-40a, H-2( ) and H-4(b)), 3.50–3.55 (H-40b
), H-3(b), H-4(
and H-5(b)), 3.63 (1H, t, J = 9.0 Hz, H-5( )), 3.94–3.98 (1H, m, H-
5(
)), 4.23–4.30 (1H, m, H-6a), 4.34–4.56 (m, H-6b, H-30 and H-
1(b)), 5.04 (d, J = 3.3 Hz, H-1( )), 5.85 (1H, s, @CH2, Ha), 6.21 (1H,
s, @CH2, Hb), C NMR (67.5 MHz, CD3OD) 29.9 and 34.3 (C-50
and C-60) 63.0 (C-6), 65.0 and 65.1 (C-30), 70.7 (C-5(
)), 71.8 (C-
4(b)), 72.0 (C-4( )), 73.8 (C-2( )), 74.8 (C-3( )), 75.3 (C-5(b)),
)), 98.3 (C-1(b)), 124.7
½ ꢂ
a 2D5
Following the same procedure as 4.1, hexanoyl-type precursors
were synthesized from 4 (234.7 mg, 0.51 mmol), 4-(tert-butyldi-
methylsilyloxy)-butanal (6, 310.0 mg, 1.53 mmol) and 3-hydroxy
quinuclidine (65.0 mg, 0.51 mmol) in dry DMSO (5.1 ml). Silica
gel CC was conducted using MeOH/CHCl3 (5:95) then MeOH/CHCl3
(1:9) as developing solvent to yielded 114.9 mg of 6-O-[60-(tert-
butyldimethylsilyloxy)-30-hydroxy-20-methylenehexanoyl]-1-O-
a
a
a
a
a
13
a
(2-trimethylsilylethyl)-b-
D
-glucopyranoside (0.21 mmol, 42%) as a
a
a
a
colorless syrup: IR (cmꢀ1) 3410, 1715, 1250, 1161, 1079, 860 and
834, HR-FD-MS m/z [M + H]+ calcd for C24H49O9Si2, 537.2916;
found, 537.2921. The obtained mixture of diastereomers was fur-
ther separated by chiral HPLC (EtOH/hexane, 8:92; CHIRALPAKÒ
IA column (u 20 mm ꢁ 25 cm)).
76.2 (C-2(b)), 77.9 (C-3(b)), 94.0 (C-1(a
(@CH2), 145.6 and 145.7 (C-20), 167.7 (C-10).
4.3. Hydrogenation of 6-tuliposide Bs
To a solution of 6-tuliposide B (1) (8.70 mg, 29.5 lmol) in MeOH
(200 ll) was added Pd/C (2.1 mg). The mixture was allowed to stir
4.2.1. First eluted (30R)- diastereomer
rt = 97.8 min, ½a 2D5
ꢂ
= ꢀ14.7 (c 1.68, MeOH), 1H NMR (500 MHz,
under H2 atmosphere at room temperature overnight. Filtration
with a Celite pad and evaporation of solvent were followed by
purification via silica gel CC (H2O/MeCN, 1:5). Subsequent evapora-
tion provided 8 (6.35 mg, 72%) as a colorless syrup. The (30R)-dia-
stereomer epi-8 was obtained from epi-1 by the same procedure
in 68% yield.
CDCl3) 0.01–0.06 (15H, s, Me2Si and CH2CH2SiMe3), 0.89 (9H, a, t-
Bu), 0.94–1.03 (2H, m, CH2CH2SiMe3), 1.54–1.77 (4H, m, H-50 and
H-60), 3.16 (1H, t, J = 8.4 Hz, H-2), 3.29–3.37 (2H, m, H-4 and H-
40a), 3.49 (1H, ddd, J = 5.0, 3.5, 1.3 Hz, H-5), 3.60–3.66 (3H, m, H-
3, H-40b and CH2CH2SiMe3), 3.92 (1H, m, CH2CH2TMS), 4.24–4.28
(1H, m, H-6a), 4.27 (1H, d, J = 7.8 Hz, H-1), 4.48–4.51 (2H, m, H-30
and H-6b), 5.89 (1H, s, @CH2, Ha), 6.25 (1H, s, @CH2, Hb), 13C
NMR (125 MHz, CDCl3) ꢀ5.1 (Me2Si), ꢀ1.3 (CH2CH2SiMe3), 19.1
(CH2CH2SiMe3), 19.2 (Me3C), 26.5 (Me3C), 30.1 and 34.1 (C-50 and
C-60), 64.3 (C-6), 65.0 (C-40), 68.1 (CH2CH2SiMe3), 70.6 (C-4), 71.9
(C-30), 75.1 (C-5), 75.2 (C-2), 78.0 (C-3), 103.8 (C-1), 124.6
(@CH2), 145.6 (C-20), and 167.5 (C-10).
IR (cmꢀ1) 3401, 1769, 1182 and 1041, HR-FAB-MS m/z [M + Na]+
calcd for C11H20O9Na, 319.1006; found, 319.1003.
4.3.1. Hydrogenated 6-Tuliposide B (8)
1H NMR (270 MHz, CD3OD) 1.14–1.28 (3H, m, CH3), 2.42–2.77
(1H, m, H-20), 3.08–3.16 (m, H-2(b)), 3.25–3.90 (m, H-2(
a
), H-3,
)), 4.12–4.25 (m,
H-6a), 4.31–4.48 (m, H-6b and H-1(b)), 5.07–5.10 (m, H-1 ( )).
13C NMR (67.5 MHz, CD3OD) 11.9, 12.0, 13.7 and 13.8 (CH3),43.7,
44.3 and 44.3 (C-20), 64.7, 64.9, 65.0 and 65.1 (C-6), 70.7 (C-5(
)),
71.5, 71.8, 71.9 and 72.0 (C-4), 73.8 and 73.9 (C-2(
) and C-30),
74.7 (C-3(
)), 75.0 (C-40), 75.3 (C-5(b)), 76.2 (C-2(b)), 77.9 (C-
3(b)), 94.0 (C-1(
)), 98.3 (C-1(b)), 176.4 and 176.5 (C-10).
H-4, H-5(b), H-30 and H-40), 3.93–4.00 (m, H-5(
a
a
4.2.2. Second eluted (30S)- diastereomer
rt = 104.9 min, ½a D25
ꢂ
= ꢀ39.5 (c 2.01, MeOH), 1H NMR (500 MHz,
a
CDCl3) 0.01–0.06 (15H, s, Me2Si and CH2CH2SiMe3), 0.89 (9H, a, t-
Bu), 0.93–1.05 (2H, m, CH2CH2SiMe3), 1.53–1.78 (4H, m, H-50 and
H-60), 3.16 (1H, t, J = 8.3 Hz, H-2), 3.28–3.37 (2H, m, H-4 and H-
40a), 3.49 (1H, ddd, J = 5.0, 3.5, 1.3 Hz, H-5), 3.61–3.66 (3H, m, H-
3, H-40b and CH2CH2SiMe3), 3.92 (1H, ddd, J = 7.2, 3.8, 2.0 Hz,
CH2CH2SiMe3), 4.23 (1H, dd, J = 6.4, 3.3 Hz, H-6a), 4.27 (1H, d,
J = 7.8 Hz, H-1), 4.49 (1H, m, H-30), 4.52 (1H, dd, J = 6.4, 1.2 Hz, H-
6b), 5.89 (1H, s, @CH2, Ha), 6.25 (1H, s, @CH2, Hb), 13C NMR
(125 MHz, CDCl3) ꢀ5.1 (Me2Si), ꢀ1.3 (CH2CH2SiMe3), 19.1
(CH2CH2SiMe3), 19.2 (Me3C), 26.5 (Me3C), 30.2 and 34.2 (C-50 and
C-60), 64.3 (C-6), 65.0 (C-40), 68.1 (CH2CH2SiMe3), 70.7 (C-4), 71.8
(C-30), 75.1 (C-5), 75.2 (C-2), 78.0 (C-3), 103.8 (C-1), 127.7
(@CH2), 145.6 (C-20), and 167.5 (C-10).
a
a
a
4.3.2. Hydrogenated epi-6-tuliposide B (epi-8)
1H NMR (270 MHz, CD3OD) 1.13–1.19 (3H, m, CH3), 2.58–2.74
(1H, m, H-20), 3.09–3.15(m, H-2(b)), 3.25–3.69 (m, H-2(
a
), H-3,
H-4 and H-40), 3.73–3.81 (m, H-5(b) and H-30), 3.89 (t, J = 6.0 Hz,
H-30), 3.91–4.00 (m, H-5(
)), 4.16–4.27 (m, H-6a), 4.32–4.47 (m,
H-6b), 4.46 (d, J = 7.7 Hz, H-1(b)), 5.07 (d, J = 3.7 Hz, H-1 (
)), 13C
a
a
NMR (67.5 MHz, CD3OD) 11.8, 12.0, 13.6 and 13.7 (CH3),43.7,
43.7, 44.3 and 44.3 (C-20), 64.6, 64.8, 64.9 and 65.1 (C-6), 70.7 (C-
5(a
)), 71.6, 71.7, 71.9 and 72.0 (C-4), 73.8 and 73.9 (C-2(
a) and
Hexanoyl-type 6-tuliposide B (7) and its epimer (epi-7) were
obtained from the above 30R-diastereomer and 30S-diastereomer
in 95% yields, respectively, by using essentially the same deprotec-
tion procedures as mentioned in 4.1.2 except for reaction time
(1.5 h): IR (cmꢀ1) 3375, 1709, 1679, 1205, 1143 and 1057, HR-
FD-MS m/z [M + H]+ calcd for C13H23O9, 323.1342; found, 323.1370.
C-30), 74.7 (C-3(
a
)), 75.0 (C-40), 75.3 and 75.3 (C-5(b)), 76.2 (C-
2(b)), 77.9 (C-3(b)), 94.0 (C-1(a)), 98.3 (C-1(b)), 176.4 and 176.6
(C-10).
4.4. Methyl 3-hydroxy-4-(p-methoxybenzyloxy)-2-
methylenebutanoate (10/ent-10)
4.2.3. Hexanoyl-type 6-tuliposide B (7)
3-hydroxy quinuclidine (2.10 g, 16.5 mmol) was dissolved in
DMSO (10 ml) and methylacrylate (100 ml) was added. To the
½
a 2D5 = +37.6 (c 1.39, MeOH), 1H NMR (270 MHz, CD3OD) 1.48–
ꢂ
1.83 (4H, m, H-50 and H-60), 3.13 (t, J = 8.4 Hz, H-2(b)), 3.30–3.38
(m, H-40a, H-2( ) and H-4(b)), 3.54–3.58 (H-40b
), H-3(b), H-4(
and H-5(b)), 3.67 (1H, t, J = 9.3 Hz, H-5( )), 4.00 (1H, ddd, J = 9.7,
solution,
6.13 g
of
(p-methoxybenzyloxy)-acetaldehyde
a
a
(34.1 mmol) was added and stirred for 15 h at room temperature.
The reaction mixture was diluted with Et2O, washed with H2O
a

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
319
(50 ml), and subsequently with brine (50 ml). The resulting organic
layer was dried (anhydr. Na2SO4) and evaporated. Following
purification by silica gel CC (EtOAc/hexane, 1:4–1:2) afforded the
Baylis–Hillman adduct (10/ent-10, 7.61 g, 84%).
½
a 2D5
ꢂ
¼ þ17:3 (c 1.12 CHCl3), ent-12 ½a D25
ꢂ
¼ ꢀ18:4 (c 1.08, CHCl3),
IR (cmꢀ1) 1721, 1514, 1250, 1100, 1038 and 834, 1H NMR
(270 MHz, CDCl3) 0.03 and 0.09 (3H, s, SiMe2), 0.90 (9H, s, t-Bu),
3.37 (1H, dd, J = 10.1, 6.5 Hz, H-4a), 3.51 (1H, dd, J = 10.1, 3.3 Hz,
H-4b), 3.73 (3H, s, OMe), 3.80 (3H, s, PhOMe), 4.48 (2H, m, benzyl),
4.79–4.83 (1H, m, H-3), 6.01 (1H, dd, J = 1.3 Hz, @CH2, H-a), 6.31,
(1H, dd, J = 1.9, 0.9 Hz, @CH2, H-b), 6.86, (2H, dd, J = 8.8, 2.5 Hz, aro-
matic), 7.26, (2H, m, aromatic), 13C NMR (67.5 MHz, CDCl3) ꢀ5.0
and ꢀ4.8 (SiMe2), 18.2 (Me3C), 25.8 (Me3C), 51.8 (OMe), 55.2
(PhOMe), 70.4 (C-3), 72.8 (C-4), 74.7 (benzyl), 113.6 (aromatic),
126.1 (@CH2), 129.1 (aromatic), 130.6 (aromatic), 141.2 (C-2),
159.0 (aromatic), 166.5 (C-1), HR-FD-MS m/z [M]+ calcd for
C20H32O5Si, 380.2019; found, 380.2001.
IR (cmꢀ1) 3432, 1709, 1514, 1248, 1180, 1105, 1085 and 1033,
1H NMR (270 MHz, CDCl3), 3.39 (1H, dd, J = 9.6, 7.3 Hz, H-4a),
3.71 (1H, dd, J = 9.8, 3.6 Hz, H-4b), 3.76 (3H, s, OMe), 3.81 (3H, s,
PhOMe), 4.51 (2H, m, benzyl), 4.73 (1H, dd, J = 7.2, 3.3 Hz, H-3),
6.01 (1H, dd, J = 1.3 Hz, @CH2, H-a), 6.36, (1H, dd, J = 1.1 Hz,
@CH2, H-b), 6.89, (2H, dd, J = 8.8, 2.5 Hz, aromatic), 7.26, (2H, m,
aromatic), 13C NMR (67.5 MHz, CDCl3), 51.8 (OMe), 55.2 (PhOMe),
69.5 (C-3), 72.9 (C-4), 73.2 (benzyl), 113.8 (aromatic), 126.6
(@CH2), 129.4 (aromatic), 129.9 (aromatic), 139.1 (C-2), 159.3 (aro-
matic), 166.4 (C-1), HR-FD-MS m/z [M]+, calcd for C14H18O5,
266.1154; found, 266.1168.
4.4.1. Katsuki-Shrapless kinetic resolution of BH adduct (10/ent-10,
11/ent-11)
4.6. 3-(tert-Butyldimethylsilyloxy)-4-(p-methoxybenzyloxy)-2-
methylenebutanoic acid (13)
To a stirred suspension of MS 4A (1.50 g, activated by use of a
microwave) in CH2Cl2 (25 ml) was added Ti(OiPr)4 (0.50 g,
1.76 mmol) at room temperature. The mixture was then cooled
to ꢀ25 °C to which was added (+)-diisopropyl tartrate (0.70 g,
2.99 mmol) in CH2Cl2 (5.0 ml) dropwise. After stirring for 1 h, the
racemate of 10/ent-10 (1.00 g, 3.50 mmol) in CH2Cl2 (20 ml) was
added dropwise and left for an additional 1 h. Cumenehydroperox-
ide was added dropwise and the reaction mixture was allowed to
stir at ꢀ20 °C for 24 h. The reaction was quenched by addition of
H2O/acetone (15 ml: 4 ml) and stirred at room temperature. The
resultant emulsion was filtered through a Celite pad. The filtrate
was then diluted with Et2O and washed with 1 M HCl (80 ml),
NaHCO3 (80 ml) and brine (80 ml), respectively. The organic layer
was dried (anhydr.Na2SO4), evaporated, and then the resulting res-
idue was purified by silica gel CC (EtOAc/hexane, 1:2) to give the
allyl alcohol and the epoxide. Evaporation yielded 10 (430 mg)
(43%, >99%ee) and 11 (403 mg) (40%, 96%ee) as syrups. Kinetic res-
olution with (ꢀ)-diisopropyl tartrate gave (3R)-allyl alcohol (ent-
10) and (3R)-epoxide (ent-11), respectively, with enantiomeric ex-
cesses measured by chiral HPLC using a CHIRALPAK IA column. Al-
lyl alcohols were eluted with EtOH:hexane = 25:75, and epoxides
were eluted with EtOH:hexane = 60:40, S-allyl alcohol (10,
To a solution of 12 (1.28 g, 3.37 mmol) in 64 ml of H2O/CH3CN
(1:1, v/v), lithium hydroxide monohydrate (320 mg, 7.62 mmol)
was added and stirred at 55 °C. After 1 h, the reaction mixture
was acidified to pH 3 with dilute HCl and washed with EtOAc
(50 ml ꢁ 2). The combined organic layer was then washed with
brine (50 ml) and dried (anhydr. Na2SO4) followed by evaporation.
The crude product so obtained was further purified using silica gel
CC (EtOAc/hexane, 1:2) to give 1.13 g of 13 (3.10 mmol, 92%) as a
white solid. 13 ½a D25
ꢂ
¼ þ26:0 (c 1.20, CHCl3), ent-13 ½a D25
¼ ꢀ28:8
ꢂ
(c 1.03, CHCl3). IR (cmꢀ1) 3008, 1696, 1514, 1251 1104 and 833,
1H NMR (270 MHz, CDCl3) 0.05 and 0.09 (3H, s, SiMe2), 0.90 (9H,
s, t-Bu), 3.41 (1H, dd, J = 10.0, 6.4 Hz, H-4a), 3.53 (1H, dd, J = 10.1,
3.6 Hz, H-4b), 3.79 (3H, s, PhOMe), 4.48 (2H, m, benzyl), 4.76
(1H, br dd, J = 6.2 3.5 Hz, H-3), 6.03 (1H, s, @CH2, H-a), 6.43, (1H,
s, @CH2, H-b), 6.86, (2H, dd, J = 8.7, 2.4 Hz, aromatic), 7.23, (2H,
m, aromatic), 13C NMR (67.5 MHz, CDCl3) ꢀ5.0 and ꢀ4.8 (SiMe2),
18.2 (Me3C), 25.7 (Me3C), 55.2 (PhOMe), 70.8 (C-3), 72.9 (C-4),
74.4 (benzyl), 113.7 (aromatic), 128.5 (@CH2), 129.2 (aromatic),
130.2 (aromatic), 140.2 (C-20), 159.1 (aromatic), 169.9 (C-10), HR-
FAB-MS m/z [MꢀH]ꢀ calcd for C19H29O5Si, 365.1822; found,
365.1803.
rt = 26.9 min.) ½a D25
¼ þ5:3 (c 0.60, CHCl3), R-allyl alcohol (ent-10,
ꢂ
rt = 21.1 min.)
½
a 2D5
ꢂ
¼ ꢀ5:4 (c 0.52, CHCl3), S-epoxide (11,
¼ ꢀ10:7 (c 1.10, CHCl3), R-epoxide (ent-11,
¼ þ11:3 (c 1.73, CHCl3), 1H NMR (270 MHz,
rt = 18.1 min.) ½a D25
ꢂ
rt = 21.3 min.) ½a D25
ꢂ
4.7. 6-O-(p-Toluenesulfonyl)-1-O-(2-trimethylsilylethyl)-b-
D-
CDCl3) 3.05 (1H, d, J = 5.8 Hz, H2C-b), 3.12 (1H, d, J = 5.8 Hz, H2C-
glucopyranoside (15)
b), 3.66–3.80 (2H, m, H2C-c, 3.71 (3H, s, OMe), 3.80 (3H, s, PhOMe),
4.14 (1H, br t, J = 4.5 Hz, H-b), 4.48 (2H, s, benzyl), 6.87, (2H, d,
J = 8.8 Hz, aromatic), 7.23, (2H, d, J = 8.8 Hz, aromatic). 13C NMR
(67.5 MHz, CDCl3) 50.0 (oxyranyl-C-b), 52.6 (OMe), 55.2 (PhOMe),
56.3 (C-2), 69.2 (C-3), 70.4 (C-4), 73.1 (benzyl), 113.8 (aromatic),
129.4 (aromatic), 129.9 (aromatic), 159.3 (aromatic), 169.7 (C-1),
HR-FD-MS m/z [M]+ calcd for C14H18O6, 282.1103; found, 282.1106.
To a stirred solution of (2-trimethylsilylethyl)- glucoside (14,
1.22 g, 4.35 mmol) in pyridine (18 ml), p-toluenesulfonyl chloride
(860 mg, 4.51 mmol) solution in pyridine (2.0 ml) was added drop-
wise at 0 °C. After 15 h, the reaction was quenched by addition of
few drops of MeOH and diluted with toluene. The solvent was then
azeotropically removed by evaporation, with the addition and
evaporation of toluene repeated twice. The obtained crude product
was purified by silica gel CC (MeOH/CHCl3, 1:9) to give 1.58 g of 15
(3.63 mmol, 83%) as a white solid.
4.5. Methyl 3-(tert-butyldimethylsilyloxy)-4-(p-methoxybenzyloxy)-
2-methylenebutanoate (12, ent-12)
IR (cmꢀ1) 3392, 1364, 1248, 1190, 1178, 1096, 1040 and 836, 1H
NMR (270 MHz, CDCl3) 0.00 (9H, s, CH2CH2SiMe3), 0.88–1.08 (2H,
m, CH2CH2SiMe3), 2.43 (3H, s, PhCH3), 3.32 (1H, t, J = 8.4 Hz, H-
2), 3.39–3.61 (4H, m, H-3, 4, 5 and CH2CH2SiMe3), 3.82–3.92 (1H,
m, CH2CH2SiMe3), 4.22–4.33 (2H, m, H-1, H-6a and H-6b), 7.33
(2H, d, J = 8.2 Hz, aromatic), 7.80, (2H, d, J = 8.2 Hz, aromatic), 13C
NMR (67.5 MHz, CDCl3) ꢀ1.5 (CH2CH2SiMe3), 18.2 (CH2CH2SiMe3),
21.6 (PhCH3), 67.4 (C-6), 69.1(CH2CH2SiMe3), 69.6 (C-4), 73.2 (C-2),
73.3 (C-5), 76.1 (C-3), 101.8 (C-1), 128.0 (aromatic), 129.8(aro-
matic), 132.7 (aromatic), 144.8 (aromatic), HR-FAB-MS m/z
[M + Na]+ calcd for C18H30O8SiSNa, 457.1328; found, 457.1348.
To a solution of 10 (1.00 g, 3.50 mmol) in CH2Cl2 (20 ml), trieth-
ylamine (976 ll, 7.00 mmol) and tert-butyldimethylsilyl trifluoro-
methanesulfonate (1.02 g, 3.85 mmol) was added in series. The
reaction mixture was allowed to stir at room temperature for
1 h. After that, the reaction was quenched by satd. NH4Cl (20 ml)
and washed with Et2O (30 ml). The organic phase was dried (anhy-
dr. Na2SO4) and the solvent was removed by evaporation. The
resulting residue was subjected to short silica gel CC (EtOAc:hex-
ane, 1:4) to afford 1.33 g (12, quant.) as a colorless syrup. ent-12
was yielded from ent-10 by the same procedure as above. 12

320
K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
4.8. 6-Azido-6-deoxy-1-O-(2-trimethylsilylethyl)-b-
(16)
D
-glucopyranoside
3.77 (3H, s, OMe), 3.91–4.01 (1H, m, CH2CH2SiMe3), 4.24 (1H, d,
J = 7.7 Hz, H-1), 4.46, (2H, s, benzyl), 4.78 (1H, m, H-30), 5.64 (1H,
s, @CH2, Ha), 5.87 (1H, s, @CH2, Hb), 6.87 (2H, d, J = 8.5 Hz, aro-
matic), 7.21 (2H, d, J = 8.5 Hz, aromatic), 13C NMR (125 MHz,
CD3OD) ꢀ4.8 and ꢀ4.6 (SiMe2), ꢀ1.3 (CH2CH2SiMe3), 19.1
(CH2CH2SiMe3 and Me3C), 26.4 (Me3C), 41.5 (C-6), 55.7 (PhOMe),
68.2 (CH2CH2SiMe3), 73.0 (C-40), 73.5 (C-4), 73.8 (benzyl), 75.2
(C-30), 75.8 (C-5), 75.6 (C-2), 77.6 (C-3), 103.9 (C-1), 114.8 (aro-
matic), 121.7 (@CH2), 130.4 (aromatic), 131.4 (aromatic), 146.0 (),
160.8 (aromatic), 169.9 (C-10).
Tosylate 15 (1.58 g, 3.63 mmol) and NaN3 (484 mg, 7.44 mmol)
were dissolved in DMF (33 ml). The reaction mixture was allowed
to stir at 60 °C for 24 h. After that, the mixture was added satd.
NaHCO3 (30 ml) and washed with EtOAc (50 ml ꢁ 2). The com-
bined organic phase was then dried (anhydr. Na2SO4) and the sol-
vent was removed under reduced pressure. The resulting crude
product was next subjected to further purification by silica gel
CC (MeOH/CHCl3, 1:5–1:9) to give 1.02 g of 16 (3.34 mmol, 92%)
as a white solid.
4.10.2. Acrylamide epi-18 (30R)
IR (cmꢀ1) 3417, 2172, 2128, 2103, 1078, 1051 and 838, 1H NMR
(270 MHz, CDCl3) 0.00 (9H, s, CH2CH2SiMe3), 0.89–1.08 (2H, m,
CH2CH2SiMe3), 3.12–3.42 (6H, m, H-2, H-3, H-4, H-5, H-6a and
H-6b), 3.57–3.67 (1H, m, CH2CH2SiMe3), 3.92–4.02 (1H, m,
CH2CH2SiMe3), 4.28 (1H, d, J = 7.7 Hz, H-1), 13C NMR (67.5 MHz,
CDCl3) ꢀ1.5 (CH2CH2SiMe3), 19.4 (CH2CH2SiMe3), 52.8 (C-6),
68.0(CH2CH2SiMe3), 72.6 (C-4), 75.1 (C-2), 77.2 (C-5), 77.9 (C-3),
103.7 (C-1), HR-FAB-MS m/z [M + Na]+, calcd for C11H23O5N3SiNa,
328.1305; found, 328.1297.
½
a 2D5
ꢂ
¼ ꢀ94:8 (c 1.88, CHCl3), 1H NMR (270 MHz, CD3OD) 0.01
(9H, s, CH2CH2SiMe3), 0.07 and 0.11 (6H, s, SiMe2), 0.90 (9H, s, t-
Bu), 0.90–1.02 (2H, m, CH2CH2SiMe3), 3.13 (1H, t, J = 7.8 Hz, H-2),
3.19–3.26 (1H, m, H-5), 3.30–3.57 (2H, m, H-3, H-4, H-6a and
CH2CH2SiMe3), 3.66 (1H, dd, J = 14.0, 2.7 Hz, H-6b), 3.78 (3H, s,
OMe), 3.91 (1H, ddd, J = 13.3, 7.2, 3.8 Hz, CH2CH2SiMe3), 4.12
(1H, d, J = 7.8 Hz, H-1), 4.44, (2H, s, benzyl), 4.79 (1H, br dd,
J = 5.4, H-30), 5.64 (1H, s, @CH2, Ha), 5.89 (1H, s, @CH2, Hb), 6.87
(2H, d, J = 8.5 Hz, aromatic), 7.21 (2H, d, J = 8.5 Hz, aromatic), 13C
NMR (125 MHz, CD3OD) ꢀ4.8 and ꢀ4.6 (SiMe2), ꢀ1.3
(CH2CH2SiMe3), 19.1 (CH2CH2SiMe3 and Me3C), 26.4 (Me3C), 41.5
(C-6), 55.7 (PhOMe), 68.2 (CH2CH2SiMe3), 72.9 (C-40), 73.4 (C-4),
74.0 (benzyl), 75.2 (C-30), 75.4 (C-5), 75.6 (C-2), 77.6 (C-3), 104.0
(C-1), 114.7 (aromatic), 121.4 (@CH2), 130.5 (aromatic), 131.5 (aro-
matic), 146.2 (C-20), 160.8 (aromatic), 170.0 (C-10).
4.9. 6-Amino-6-deoxy-1-O-(2-trimethylsilylethyl)-b-
glucopyranoside (17)
D-
The azido sugar 16 (309 mg, 1.01 mmol) was dissolved in EtOAc
(3.0 ml) and Pd activated C (15 mg) was subsequently added, with
the suspension allowed to stir under H2 atmosphere overnight.
After that, the Pd/C was removed by filtration through a Celite
pad and the filtrate was evaporated to give the amino sugar 17
(280 mg quant., slightly impure).
4.11. 6-Deoxy-6-(30,40-dihydroxy-20-methylenebutanoamido)-b-
glucopyranoside (19, epi-19)
D-
IR (cmꢀ1) 3370, 1364, 1250, 1075, 1050, 862 and 837, 1H NMR
(270 MHz, CD3OD) 0.03 (9H, s, CH2CH2SiMe3), 1.00 (2H, m,
CH2CH2SiMe3), 2.72 (1H, dd, J = 13.4, 6.8 Hz, H-6a), 3.02 (1H, dd,
J = 13.3, 2.5 Hz, H-6b), 3.11–3.34 (4H, m, H-2, H-3, H-4 and H-5),
3.66 (1H, m, CH2CH2SiMe3), 3.97 (1H, m, CH2CH2SiMe3), 4.27,
(1H, d, J = 7.7 Hz, H-1), 13C NMR (67.5 MHz, CD3OD) ꢀ1.4
Following the procedure described in 4.2, amide product 19 and
epi-19 were obtained in 90% and 92% yield, respectively.
IR (cmꢀ1) 3402, 1687, 1206, 1141 and 1055, HR-FAB-MS m/z
[MꢀH]ꢀ calcd for C11H18NO8, 292.1033; found, 292.1050.
4.11.1. Amide-type 6-tuliposide B (19)
(CH2CH2SiMe3),
19.1
(CH2CH2SiMe3),
43.9
(C-6),
68.1
½
a 2D5
ꢂ
¼ þ24:7 (c 0.84, MeOH), 1H NMR (270 MHz, CD3OD) 3.08–
(CH2CH2SiMe3), 73.3 (C-4), 75.2 (C-2), 77.6 (C-5), 78.0 (C-3),
103.9 (C-1), HR-FAB-MS m/z [MꢀH]ꢀ, calcd for C11H24NO5Si,
278.1424; found, 278.1428.
3.19 (m, H-6a and H-2(b)), 3.30–3.70 (m, H-2(
a
), H-3(
a
), H-3(b), H-
)),
4.47 (d, J = 7.8 Hz, H-1(b)), 4.49–4.51 (m, H-30), 5.10 (d, J = 3.6 Hz,
H-1( )), 5.64 (1H, d, J = 3.5 Hz, @CH2, Ha), 5.87 (1H, s, @CH2, Hb),
13C NMR (67.5 MHz, CD3OD) 41.6 and 41.7 (C-6(
) and C-6(b)),
66.4 and 66.5 (C-40), 71.2 (C-5(
)), 73.0 (C-4(b)), 73.2 (C-4( )),
73.7 (C-30), 73.8 (C-2(
)), 74.4 (C-3( )), 75.9 (C-5(b)), 76.3 (C-
2(b)), 77.5 (C-3(b)), 94.0 (C-1( )), 98.2 (C-1(b)), 121.5 and 121.6
4(a a
), H-6b, H-40a, H-5(b), H-40b and H-4(b)), 3.81–3.88 (m, H-5(
a
4.10. 6-[30-(tert-Butyldimethylsilyloxy)-40-(p-methoxybenzyloxy)-20-
a
methylenebutanoyl]amino-6-deoxy-1-O-(2-trimethylsilylethyl)-b-
glucopyranoside (18, epi-18)
D-
a
a
a
a
a
To a solution of the amino sugar 17 (279 mg, 1.00 mmol) in
CH2Cl2 (3.0 ml), dimethylaminopyridine (61.0 mg, 0.5 mmol) and
carboxylic acid 13 (402 mg, 1.10 mmol) were added at ꢀ20 °C.
After that, diisopropylcabodiimide (126 mg, 1.00 mmol) was added
slowly dropwise and stirred at 0 °C overnight. The reaction mixture
was then diluted with EtOAc, whereupon it was washed with satd.
NH4Cl, brine and dried (anhydr. Na2SO4). Subsequent removal of
solvent by evaporation and purification by silica gel CC (MeOH/
CHCl3, 1:9) gave acrylamide 18 (508 mg, 0.81 mmol, 81%) as a syr-
up. The 30-epimer epi-18 was synthesized in a similar way in 70%
yield.
(@CH2), 145.4 (C-20), 170.7 and 170.8 (C-10).
4.11.2. Amide-type epi-6-tuliposide B (epi-19)
½
a 2D5
ꢂ
¼ þ11:9 (c 1.03, MeOH), 1H NMR (270 MHz, CD3OD) 3.08–
3.18 (m, H-6a and H-2(b)), 3.29–3.69 (m, H-2(
a
), H-3(
a
), H-3(b), H-
)),
4.46 (d, J = 7.7 Hz, H-1(b)), 4.48–4.52 (m, H-30), 5.08 (d, J = 3.7 Hz,
H-1( )), 5.62 (1H, d, J = 4.0 Hz, @CH2, Ha), 5.86 (1H, s, @CH2, Hb),
4(a a
), H-6b, H-40a, H-5(b), H-40b and H-4(b)), 3.80–3.87 (m, H-5(
a
13C NMR (67.5 MHz, CD3OD) 41.6 and 41.7 (C-6(
a
) and C-6(b)),
)), 73.0 (C-4(b)), 73.3 (C-4( )),
)), 74.4 (C-3( )), 75.9 (C-5(b)),
)), 98.3 (C-1(b)), 121.0 and
121.2 (@CH2), 145.7 and 145.8 (C-20), 170.6 and 170.6 (C-10).
66.5 and 66.6 (C-40), 71.2 (C-5(
73.6 and 73,7 (C-30), 73.9 (C-2(
a
a
a
a
IR (cmꢀ1) 3556, 3460, 3340, 1610, 1513, 1465, 1361, 1250,
1056, 861, 836 and 780, HR-FD-MS m/z [M]+ calcd for C30H53NO9-
Si2, 627.3259; found, 627.3249.
76.4 (C-2(b)), 77.5 (C-3(b)), 94.0 (C-1(a
4.12. 1,2-Dideoxy- -glucopyranose (21)
D
4.10.1. Acrylamide 18 (30S)
½
a 2D5
ꢂ
¼ ꢀ68:4 (c 2.22, CHCl3), 1H NMR (270 MHz, CD3OD) 0.01
To a solution of 3,4,6-tri-O-acetylglucal (5.11 g, 18.8 mmol) in
MeOH (50 ml) was added Pd/C (500 mg). The mixture was allowed
to stir under H2 atmosphere for 22 h at room temperature. After
that, the suspension was filtered through a Celite pad to remove
(9H, s, CH2CH2SiMe3), 0.06 and 0.09 (6H, s, SiMe2), 0.90 (9H, s, t-
Bu), 0.90–0.97 (2H, m, CH2CH2SiMe3), 3.15 (1H, t, J = 8.8 Hz, H-2),
3.30–3.63 (8H, m, H-3, H-4, H-5, H-6a, H-6b and CH2CH2SiMe3),

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
321
Pd/C and the filtrate was evaporated. The crude mixture was puri-
fied by silica gel CC (EtOAc:hexane = 1:2) to give 3,4,6-tri-O-acetyl-
1,2-dideoxy-glucopyranose (4.12 g, 15.0 mmol, 80%) as a viscous
syrup. IR (cmꢀ1) 1735, 1368, 1240 and 1049, 1H NMR (270 MHz,
CDCl3) 1.74–1.90 (1H, m, H-2a), 2.03–2.12 (1H, m, H-2b), 2.03,
2.04 and 2.09 (9H, s, Ac), 3.47–3.57 (2H, m, H-1a and H-5), 4.01–
4.26 (1H, m, H-1b), 4.06 (1H, dd, J = 5.0, 1.6 Hz, H-6a), 4.06 (1H,
dd, J = 5.0, 1.6 Hz, H-6a), 4.24 (1H, dd, J = 12.3, 5.0 Hz, H-6b),
4.92–5.03 (2H, m, H-3 and H-4), 13C NMR (67.5 MHz, CDCl3) 20.6,
20.6 and 20.8 (Me), 30.9 (C-2), 62.6 (C-1), 65.3 (C-6), 69.2 (C-4),
72.2 (C-3), 76.4 (C-5), 169.7, 170.3 and 170.6 (carbonyl), HR-FD-
MS m/z [M]+, calcd for C12H18O7, 274.1052; found, 274.1040.
6.0, 4.4 Hz, H-1b), 4.10 (1H, dd, J = 11.7, 5.5 Hz, H-6a), 4.46 (1H, dd,
J = 11.7, 2.5 Hz, H-6b), 13C NMR (67.5 MHz, CDCl3) 5.2–6.9
(SiCH2CH3), 27.2 (Me3C), 34.4 (C-2), 38.8 (Me3C), 63.5 (C-6), 64.4
(C-1), 73.1 (C-4), 74.3 (C-3), 79.0 (C-5), 178.3 (carbonyl), HR-FD-
MS m/z [MꢀEt]+ calcd for C21H43O5Si2, 431.2649; found, 431.2661.
4.15. 1,2-Dideoxy-3,4-di-O-triethylsilyl- -glucopyranose (24)
D
The pivaroyl ester 23 (3.60 g, 7.87 mmol) was dissolved in dry
CH2Cl2 (40 ml), and cooled to ꢀ78 °C. DIBAL (16 mmol) was added
dropwise and left for 2 h at ꢀ78 °C. The reaction was monitored by
TLC, and quenched by MeOH and then diluted with Et2O (100 ml)
when it was completed. After warming to room temperature, the
mixture was washed with 1 M HCl (100 ml), satd. NaHCO3
(100 ml) and brine (100 ml). The organic layer was purified by sil-
ica gel CC (EtOAc:hexane, 1:4). Evaporation yielded 24 (2.33 g,
6.18 mmol, 78%).
NaOMe (400 ll, 28% in MeOH) was added to a solution of above
3,4,6-tri-O-acetyl-1,2-dideoxy-glucopyranose (4.12 g, 15.0 mmol)
in MeOH (70 ml) at room temperature. Stirring was continued for
1 h, whereupon the reaction mixture was neutralized by adding
Amberlyst R-150 and filtered to give a solution. After evaporation
of the filtrate, the crude product was purified by short silica gel
CC (MeOH/CHCl3, 15:85) to give 21 (1.95 g, 87%) as a white solid.
IR (cmꢀ1) 1735, 1368, 1240 and 1049, 1H NMR (270 MHz, CDCl3)
1.74–1.90 (1H, m, H-2a), 2.03–2.12 (1H, m, H-2b), 2.03, 2.04 and
2.09 (9H, s, Ac), 3.47–3.57 (2H, m, H-1a and H-5), 4.01–4.26 (1H,
m, H-1b), 4.06 (1H, dd, J = 5.0, 1.6 Hz, H-6a), 4.06 (1H, dd, J = 5.0,
1.6 Hz, H-6a), 4.24 (1H, dd, J = 12.3, 5.0 Hz, H-6b), 4.92–5.03 (2H,
m, H-3 and H-4), 13C NMR (67.5 MHz, CDCl3) 20.6, 20.6 and 20.8
(Me), 30.9 (C-2), 62.6 (C-1), 65.3 (C-6), 69.2 (C-4), 72.2 (C-3),
76.4 (C-5), 169.7, 170.3 and 170.6 (carbonyl), HR-FD-MS m/z
[M]+, calcd for C12H18O7, 274.1052; found, 274.1040.
IR (cmꢀ1) 3478, 1460, 1415, 1239, 1128, 1104, 810 and 740, 1H
NMR (270 MHz, CDCl3) 0.59–0.70 (12H, m, SiCH2CH3), 0.94–1.01
(18H, m, SiCH2CH3), 1.52–1.67 (1H, m, H-2a), 1.88–1.97 (1H, m,
H-2b), 3.16 (1H, ddd, J = 8.8, 5.9, 2.9 Hz, H-5), 3.37 (1H, t,
J = 7.8 Hz, H-4), 3.37–3.47 (1H, m, H-1a), 3.58–3.77 (2H, m, H-3
and H-6a), 4.46 (1H, dd, J = 11.7, 2.5 Hz, H-1b), 13C NMR
(67.5 MHz, CDCl3) 5.2 and 5.4 (SiCH2CH3), 6.9 (SiCH2CH3), 34.9
(C-2), 62.7 (C-6), 65.1 (C-1), 73.6 (C-4), 74.5 (C-3), 80.9 (C-5), HR-
FD-MS m/z [MꢀEt]+ calcd for C16H35O4Si2, 347.2073; found,
347.2065.
4.16. 6-O-[30-(tert-Butyldimethylsilyloxy)-40-(p-methoxybenzyloxy)-
20-methylenebutanoyl]-1,2-dideoxy-3,4-di-O-triethylsilyl-
D-
4.13. 1,2-Dideoxy-6-O-pivaloyl-
D
-glucopyranose (22)
glucopyranose (25, epi-25)
Pivaroyl chloride solution (1.34 ml, 10.9 mmol)/30 ml of pyri-
dine was added dropwise to a solution of 21 (1.47 g, 9.94 mmol)
in pyridine (30 ml). The reaction was allowed to stir at room tem-
perature for 24 h. Pyridine was azeotropically removed with re-
peated addition of toluene, followed by evaporation which was
repeated twice. The residual mixture was purified by silica gel CC
(MeOH/CHCl3, 1:9) to give 22 (1.91 g, 83%) as a white solid.
IR (cmꢀ1) 3413, 1727 and 901, 1H NMR (270 MHz, CDCl3) 1.21
(9H, s, t-Bu), 1.66 (1H, dddd, J = 12.8, 11.5, 5.0 Hz, H-2a), 1.95
(1H, br dd, J = 13.0, 4.7 Hz, H-2b), 3.15 (1H, t, J = 9.0 Hz, H-4),
3.27 (1H, ddd, J = 9.6, 4.4, 2.1 Hz, H-5), 3.45 (1H, dd, J = 12.2,
1.9 Hz, H-3), 3.97 (1H, dd, J = 11.2, 4.9 Hz, H-1b), 4.28 (1H, dd,
J = 12.1, 2.1 Hz, H-6a), 4.42–4.46 (1H, m, H-6b), 13C NMR
(67.5 MHz, CDCl3) 27.1 (Me3C), 33.1 (C-2), 38.9 (Me3C), 63.7 (C-
6), 65.8 (C-1), 72.3 (C-4), 72.6 (C-3), 78.5 (C-5), 179.8 (carbonyl),
HR-FD-MS m/z [M]+ calcd for C11H20O5, 232.1311; found, 232.1307.
Following the procedure described in 4.10, 25 and epi-25 were
obtained from 24 and corresponding carboxylic acids (13 and ent-
13) in 62% and 71% yield, respectively. The reactions were con-
ducted at 0 °C. IR (cmꢀ1) 1717, 1514, 1463, 1415, 1380, 1303,
1249, 1129, 1109, 835, 809, 740 and 729, HR-FD-MS m/z [M]+ calcd
for C37H68O8Si3, 724.4222; found, 724.4200.
4.16.1. 1,2-Dideoxy-6-tuliposide B precursor (25)
½
a 2D5
ꢂ
¼ þ28:8 (c 1.55, CHCl3), 1H NMR (270 MHz, CDCl3) 0.03
and 0.08 (6H, s, SiMe), 0.58–0.68 (12H, m, SiCH2CH3), 0.90–1.00
(27H, m, t-Bu and SiCH2CH3), 1.54–1.69 (1H, m, H-2a), 1.89–1.95
(1H, m, H-2b), 3.28–3.47 (4H, m, H-3, H-4, H-5 and H-40a), 3.55
(1H, dd, J = 10.2, 2.6 Hz, H-40b), 3.64 (1H, ddd, J = 14.0, 6.4, 4.3 Hz,
H-1a), 3.79 (3H, s, OMe), 3.88 (1H, ddd, J = 12.5, 5.0, 2.6 Hz, H-
1b), 4.22 (1H, dd, J = 11.8, 4.3 Hz, H-6a), 4.41–4.48 (3H, m, H-6b
and benzyl), 4.84 (1H, br dd, H-30), 6.05 (1H, t, J = 1.6 Hz, @CH2,
Ha), 6.36 (1H, s, @CH2, Hb), 6.85 (2H, d, J = 8.8 Hz, aromatic), 7.24
(2H, d, J = 8.8 Hz, aromatic), 13C NMR (125 MHz, CDCl3) ꢀ5.0 and
ꢀ4.8 (Me2Si), 5.2–6.9 (Et), 18.2 (Me3C), 25.8 (Me3C), 34.6 (C-2),
55.2 (OMe), 64.0 (C-6), 64.7 (C-1), 70.5 (C-30), 72.7 (benzyl), 73.2
(C-40), 74.4 (C-4), 74.9 (C-3), 78.8 (C-5), 113.6 (C-1), 126.5 (aro-
matic), 129.0 (@CH2), 130.7 (aromatic), 140.9 (C-20), 158.9 (aro-
matic), 165.8 (C-10).
4.14. 1,2-Dideoxy-6-O-pivaloyl-3,4-di-O-triethylsilyl-D-glucopyranose
(23)
1,2-Dideoxy-D-glucopyranose 22 (1.85 g, 7.96 mmol) was dis-
solved in CH2Cl2 (40 ml) to which was added 2,6-lutidine (3.7 ml,
31.9 mmol) and triethylsilyl trifluoromethanesulfonate (4.0 ml,
17.9 mmol) in series. The reaction mixture was allowed to stir at
room temperature for 1 h. After that, the reaction was quenched
by 1 M HCl (40 ml) and washed with Et2O (50 ml). The organic
phase was washed with satd. NaHCO3 (40 ml), brine (40 ml) and
dried (anhyd. Na2SO4). After evaporation, the resulting residue
was subjected to short silica gel CC (EtOAc:hexane, 1:4) to afford
23 (3.63 g, 99%) as a colorless syrup.
4.16.2. 1,2-Dideoxy-epi-6-tuliposide B precursor (epi-25)
½
a 2D5
ꢂ
¼ ꢀ10:5 (c 1.68, CHCl3), 1H NMR (270 MHz, CDCl3) 0.04
and 0.08 (6H, s, SiMe), 0.60–0.68 (12H, m, SiCH2CH3), 0.89–1.01
(27H, m, t-Bu and SiCH2CH3), 1.55–1.71 (1H, m, H-2a), 1.89–2.00
(1H, m, H-2b), 3.31–3.47 (4H, m, H-3, H-4, H-5 and H-40a), 3.54
(1H, dd, J = 10.2, 2.9 Hz, H-40b), 3.63 (1H, ddd, J = 10.9, 5.6,
3.9 Hz,, H-1a), 3.79 (3H, s, OMe), 3.88 (1H, ddd, J = 10.9, 5.6,
3.9 Hz,, H-1b), 4.25 (1H, dd, J = 11.7, 5.5 Hz, H-6a), 4.41–4.48 (3H,
m, H-6b and benzyl), 4.84 (1H, br dd, J = 6.9, 2.7 Hz, H-30), 6.05
(1H, s, @CH2, Ha), 6.36 (1H, s, @CH2, Hb), 6.85 (2H, d, J = 8.7 Hz,
IR (cmꢀ1) 1733, 1481, 1461, 1284, 1240, 1152, 1131, 1108, 809
and 740, 1H NMR (270 MHz, CDCl3) 0.47–0.68 (12H, m, SiCH2CH3),
0.90–0.68 (18H, m, SiCH2CH3), 1.22 (9H, s, t-Bu), 1.52–1.67 (1H, m,
H-2a), 1.88–1.97 (1H, m, H-2b), 3.27–3.44 (3H, m, H-3, H-4 and H-
5), 3.63 (1H, ddd, J = 11.2, 9.7, 3.3 Hz, H-1a), 3.89 (1H, ddd, J = 11.7,

322
K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
aromatic), 7.24 (2H, d, J = 8.7 Hz, aromatic), 13C NMR (125 MHz,
CDCl3) ꢀ5.0 and ꢀ4.8 (Me2Si), 5.2–6.9 (Et), 18.2 (Me3C), 25.8
(Me3C), 34.5 (C-2), 55.2 (OMe), 64.1 (C-6), 64.6 (C-1), 70.4 (C-30),
72.7 (benzyl), 73.2 (C-40), 74.4 (C-4), 74.8 (C-3), 78.8 (C-5), 113.6
(aromatic), 126.4 (aromatic), 129.0 (@CH2), 130.7 (aromatic),
140.9 (C-20), 159.0 (aromatic), 165.8 (C-10).
6b), 4.56 (1H, br dd, J = 5.1 Hz, H-40), 5.97 (1H, dd, J = 1.5 Hz,
@CH2, Ha), 6.33 (1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD)
ꢀ5.2 and ꢀ5.1 (SiMe2), 19.2 (Me3C), 26.4 (Me3C), 57.2 (OMe),
65.0 (C-6), 68.0 (C-40), 71.8 (C-4), 72.0 (C-30), 75.0 (C-5), 75.3 (C-
2), 77.9 (C-3), 105.4 (C-1), 126.7 (C-b), 142.4 (C-a), 167.4
(carbonyl).
4.17. 1,2-Dideoxy-6-O-(30,40-dihydroxy-20-methylenebutanoyl)-
glucopyranose (26, epi-26)
D-
4.18.2. Second eluted (30S)-diastereomer
½
a 2D5
ꢂ
¼ ꢀ11:1 (c 1.31, CH3OH), 1H NMR (270 MHz, CD3OD) 0.05
and 0.06 (3H, s, MeSi), 0.89 (9H, s, t-Bu), 3.16 (1H, dd, J = 8.4,
8.4 Hz, H-2), 3.30–3.37 (2H, m, H-3 and H-4), 3.48 (3H, s, OMe)
3.48–3.51 (1H, m, H-5), 3.59 (1H, dd, J = 10.5, 6.2 Hz, H-40a), 3.79
(1H, dd, J = 10.4, 4.0 Hz, H-40b), 4.17 (1H, d, J = 7.8 Hz, H-1), 4.25
(1H, dd, J = 11.9, 6.0 Hz, H-6a), 4.50 (1H, dd, J = 11.9, 2.0 Hz, H-
6b), 4.56 (1H, br dd, J = 4.8 Hz, H-40), 5.97 (1H, s, @CH2, Ha), 6.35
(1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD), ꢀ5.2 and ꢀ5.1
(SiMe2), 19.2 (Me3C), 26.4 (Me3C), 57.2 (OMe), 65.0 (C-6), 68.0
(C-40), 71.8 (C-4), 72.0 (C-30), 75.0 (C-5), 75.3 (C-2), 77.9 (C-3),
The same deprotection procedures as mentioned in 4.1.2 was
applied for 25 and epi-25 to yield 26 (70%) and epi-26 (68%),
respectively. IR (cmꢀ1) 3407, 1709, 1277, 1204 and 1087, HR-
FAB-MS m/z [MꢀH]ꢀ calcd for C11H17O9, 261.0975; found,
261.0982.
4.17.1. 1,2-Dideoxy-6-tuliposide B (26)
½
a 2D5
ꢂ
¼ þ24:7 (c 0.84, MeOH), 1H NMR (270 MHz, CD3OD) 1.52–
1.67 (1H, m, H-2a), 1.87–1.94 (1H, m, H-2a), 3.20 (1H, t, J = 9.1 Hz,
H-4), 3.29–3.37 (1H, m, H-5), 3.41–3.57 (3H, m, H-40a, H-1a and H-
4), 3.71 (1H, dd, J = 11.3, 3.4 Hz, H-40b), 3.89 (1H, br dd, J = 11.6,
4.7 Hz, H-5), 4.24 (1H, dd, J = 11.9, 5.5 Hz, H-6a), 4.49 (1H, dd,
J = 11.9, 1.9 Hz, H-6b), 4.57 (1H, br m, H-30), 6.00 (1H, s, @CH2,
Ha), 6.35 (1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD) 35.0 (C-
2), 65.5 (C-6), 66.7 (C-1), 66.8 (C-40), 72.1 (C-30), 73.5 (C-4), 73.9
(C-3), 79.8 (C-5), 126.7 (@CH2), 142.2 (C-20), 167.5 (C-10).
105.4 (C-1), 126.9 (C-b), 142.4 (C-
The (30S)-Baylis–Hillman adduct (39.3 g, 93.0
solved in CH3CN (2.0 ml) and cooled to 0 °C. To the reaction mix-
ture, a stoichiometric amount of aqueous HF aqueous (10.0 l)
a), 167.4 (carbonyl).
l
mol) was dis-
l
was slowly added and stirring was continued for 1 h. Evaporation
of solvent and subsequent short silica gel CC (MeOH/CHCl3, 1:9)
produced 27 (25.8 g, 90%) as a colorless syrup. The 30R-epimer
(epi-27) was prepared following the same procedure in 93% yield.
IR (cmꢀ1) 3392, 1720, 1269, 1164 and 1083, HR-FAB-MS m/z
[MꢀH]ꢀ, calcd for C12H19O9, 307.1029; found, 307.1018.
4.17.2. 2. 1,2-Dideoxy-epi-6-tuliposide B (epi-26)
½
a 2D5
ꢂ
¼ þ11:9 (c 1.03, MeOH), 1H NMR (270 MHz, CD3OD) 1.52–
1.67 (1H, m, H-2a), 1.87–1.94 (1H, m, H-2a), 3.19 (1H, t, J = 9.1 Hz,
H-4), 3.29–3.37 (1H, m, H-5), 3.41–3.57 (3H, m, H-40a, H-1a and H-
4), 3.71 (1H, dd, J = 11.3, 3.6 Hz, H-40b), 3.90 (1H, ddd, J = 10.4, 4.6,
1.6 Hz, H-1b), 4.27 (1H, dd, J = 11.9, 5.7 Hz, H-6a), 4.45 (1H, dd,
J = 11.9, 2.1 Hz, H-6b), 4.57 (1H, br dd, J = 6.6, 3.3 Hz, H-30), 6.00
(1H, t, J = 1.6 Hz, @CH2, Ha), 6.35 (1H, dd, J = 1.4, 0.9 Hz, @CH2,
Hb), 13C NMR (67.5 MHz, CD3OD) 35.0 (C-2), 65.5 (C-6), 66.7 (C-
1), 66.8 (C-40), 72.1 (C-30), 73.5 (C-4), 73.9 (C-3), 79.8 (C-5), 126.7
(@CH2), 142.2 (C-20), 167.5 (C-10).
4.18.3. 1-O-Methyl-b-epi-6-tuliposide B (epi-27)
½
a 2D5
ꢂ
¼ ꢀ20:6 (c 1.67, CH3OH), 1H NMR (500 MHz, CD3OD) 3.17
(1H, dd, J = 7.8, H-2), 3.30–3.38 (2H, m, H-3, H-4), 3.44 (1H, dd,
J = 11.4, 6.7 Hz, H-40a), 3.49 (3H, s, OMe), 3.49–3.53 (1H, m, H-5),
3.71 (1H, dd, J = 11.3, 3.5 Hz, H-40b), 4.18 (1H, d, J = 7.8 Hz, H-1),
4.30 (1H, dd, J = 11.8, 6.1 Hz, H-6a), 4.48 (1H, dd, J = 11.9, 2.2 Hz,
H-6b), (1H, br dd, J = 6.8, 3.5 Hz, H-30), 6.00 (1H, t, J = 1.5 Hz,
@CH2, Ha), 6.36, (1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD)
57.3 (OMe), 65.0 (C-6), 66.7 (C-40), 71.8 (C-4), 72.1 (C-30), 75.0
(C-5), 75.2 (C-2), 77.9 (C-3), 105.4 (C-1), 126.8 (@CH2), 142.2 (C-
20), 167.3 (C-10).
4.18. 6-O-(30,40-Dihydroxy-20-methylenebutanoyl)-1-O-methyl-b-
glucopyranoside (27, epi-27)
D-
4.18.4. 1-O-Methyl-b-6-tuliposide B (27)
To a solution of 4 (54.3 mg, 162
(tert-butyldimethylsilyloxy)-acetaldehyde (84.9 mg, 487
and 3-hydroxy quinuclidine (20.6 mg, 162 mol) were added at
room temperature. After 40 h, the reaction mixture was diluted
with EtOAc (2.0 ml) and extracted with brine (2.0 ml). The water
layer was then washed with EtOAc (2.0 ml ꢁ 2). The combined or-
ganic layer was dried (anhydr.Na2SO4) and evaporated. Silica gel
CC (5% then 10% MeOH in CHCl3) yielded 6-O-[40-(tert-butyldi-
methylsilyloxy)-30-hydroxy-20-methylenebutanoyl]-1-O-methyl-
l
mol) in dry DMSO (1.62 ml), 2-
½
a 2D5
ꢂ
¼ ꢀ4:2 (c 1.14, CH3OH), 1H NMR (500 MHz, CD3OD) 3.15
l
mol)
(1H, dd, J = 7.8, H-2), 3.31–3.36 (2H, m, H-3, H-4), 3.43 (1H, dd,
J = 11.3, 6.8 Hz, H-40a), 3.47 (3H, s, OMe), 3.47–3.50 (1H, m, H-5),
3.71 (1H, dd, J = 11.3, 3.5 Hz, H-40b), 4.16 (1H, d, J = 7.8 Hz, H-1),
4.25 (1H, dd, J = 11.9, 5.9 Hz, H-6a), 4.52 (1H, dd, J = 11.9, 2.2 Hz,
H-6b), (1H, br dd, J = 6.6, 3.4 Hz, H-30), 6.00 (1H, t, J = 1.5 Hz,
@CH2, Ha), 6.34, (1H, s, @CH2, Hb), 13C NMR (67.5 MHz, CD3OD)
57.3 (OMe), 64.9 (C-6), 66.7 (C-40), 71.7 (C-4), 72.1 (C-30), 75.0
(C-5), 75.2 (C-2), 77.8 (C-3), 105.4 (C-1), 126.8 (@CH2), 142.2 (C-
20), 167.3 (C-10).
l
b-
D-glucopyranoside (32.4 mg, 64 lmol, 40%, 3% d.e.) as a colorless
syrup:
IR (cmꢀ1) 3421, 1718, 1257, 1053 and 838, HR-FD-MS m/z
[M + H]+, calcd for C18H35O9Si, 423.2050; found, 423.2023. The ob-
tained mixture of diastereomers was further separated by chiral
HPLC (EtOH/hexane, 8:92; CHIRALPAKÒ IA column (u
20 mm ꢁ 25 cm)).
4.19. 1-O-Methyl-6-O-(30,40-dihydroxy-20-methylenebutanoyl)-
a-D-
glucopyranoside (30, epi-30)
1-O-Methyl-6-O-[30-(tert-butyldimethylsilyloxy)-40-(p-
methoxybenzyloxy)-20-methylenebutanoyl]-2,3,4-tri-O-trimethyl-
silyl-a-D-glucopyranoside (29, epi-29) were prepared by the same
4.18.1. First eluted (30R)-diastereomer
procedure as described in 4.10. IR (cmꢀ1) 1718, 1515, 1473, 1362,
1303, 1251, 1163, 1099, 1080, 1043, 897, 871 and 841, HR-FD-MS
m/z [M]+, calcd for C35H66O10Si4, 758.3734; found, 758.3734.
½
a 2D5
ꢂ
¼ ꢀ8:6 (c 1.13, CH3OH), 1H NMR (270 MHz, CD3OD) 0.05
and 0.06 (3H, s, MeSi), 0.88 (9H, s, t-Bu), 3.16 (1H, dd, J = 9.0,
7.9 Hz, H-2), 3.28–3.37 (2H, m, H-3 and H-4), 3.48 (3H, s, OMe)
3.48–3.51 (1H, m, H-5), 3.60 (1H, dd, J = 10.4, 6.1 Hz, H-40a), 3.77
(1H, dd, J = 10.4, 4.1 Hz, H-40b), 4.17 (1H, d, J = 7.8 Hz, H-1), 4.27
(1H, dd, J = 11.8, 6.2 Hz, H-6a), 4.50 (1H, dd, J = 11.8, 2.2 Hz, H-
4.19.1. 30S product (29)
½
a 2D5
ꢂ
¼ þ49:8 (c 0.79, CHCl3), 1H NMR (270 MHz, CDCl3) 0.02
and 0.07 (3H, s, MeSi), 0.15, 0.15 and 0.16 (27H, s, TMS), 0.89

K. Shigetomi et al. / Phytochemistry 71 (2010) 312–324
323
(9H, s, t-Bu), 3.29–3.57 (4H, m, H-2, H-4, H-5 and H-40a), 3.34 (3H,
s, OMe), 3.73–3.85 (2H, m, H-3 and H-40b), 3.80 (3H, s, PhOMe),
4.12 (1H, dd, J = 11.8, 5.9 Hz, H-6a), 4.47 (1H, d, J = 6.8 Hz, benzyl),
4.49 (1H, dd, J = 11.7, 2.2 Hz, H-6b), 4.61 (1H, d, J = 3.6 Hz, H-1),
4.81–4.85 (1H, m, H-30), 6.07 (1H, dd, J = 1.7 Hz, @CH2, Ha), 6.37
(1H, dd, J = 1.3 Hz, @CH2, Hb), 6.85 (2H, d, J = 8.7 Hz, aromatic),
7.23 (2H, d, J = 8.7 Hz, aromatic), 13C NMR (67.5 MHz, CDCl3)
ꢀ5.0 and ꢀ4.9 (SiMe2), 0.5, 0.9 and 1.3 (TMS), 18.2 (Me3C), 25.8
(Me3C), 54.8 (OMe), 55.2 (PhOMe), 63.9 (C-6), 69.5 (C-5), 70.5 (C-
30), 72.6 (C-2), 72.8 (C-40), 73.8 (C-4), 74.8 (benzyl), 75.1 (C-3),
99.7 (C-1), 113.6 (aromatic), 126.6 (@CH2), 129.0 (aromatic),
130.6 (aromatic), 140.9 (C-20), 159.0 (aromatic), 165.7 (carbonyl).
S-diol (31)
½
a 2D5
ꢂ
¼ þ12:7 (c 1.80, MeOH), R-diol (ent-31)
½
a 2D5
ꢂ
¼ ꢀ12:1 (c 1.60, MeOH), IR (cmꢀ1) 3393, 1718, 1273, 1197
and 1085, 1H NMR (270 MHz, CD3OD) 3.42 (1H, dd, J = 11.3,
6.7 Hz, H-4a), 3.68 (1H, dd, J = 11.2, 3.5 Hz, H-4b), 3.74 (3H, s,
OMe), 4.55 (1H, br dd, J = 6.4, 3.4 Hz, H-3), 5.97 (1H, J = 1.5 Hz,
@CH2, Ha), 6.31 (1H, J = 1.4 Hz, 0.9 Hz, @CH2, Hb), 13C NMR
(67.5 MHz, CD3OD) 52.3 (OMe), 66.7 (C-4), 72.0 (C-3), 126. 5
(@CH2), 142.1 (C-2), 168.0 (C-1), HR-FAB-MS m/z [M + H]+ calcd
for C6H11O4, 147.0657; found, 147.0672.
4.21. Methyl 3,4-dihydroxy-2-oxyranyl-butanoate (32 ent-32)
The deprotection procedure shown in 4.1.2. was applied for 11
and ent-11 to yield 32 (80%) and ent-32 (80%), respectively.
4.19.2. 30R product (epi-29)
S-Oxide (32) ½a 2D5
¼ þ0:5 (c 1.01, MeOH), R-Oxide (ent-32)
ꢂ
½
a 2D5
ꢂ
¼ þ33:1 (c 2.66, CHCl3), 1H NMR (270 MHz, CDCl3) 0.02
½
a 2D5
ꢂ
¼ ꢀ2:2 (c 1.34, MeOH), IR (cmꢀ1) 3392, 1788, 1252, 1207,
and 0.07 (3H, s, MeSi), 0.15, 0.16 and 0.16 (27H, s, TMS), 0.89
(9H, s, t-Bu), 3.30–3.56 (4H, m, H-2, H-4, H-5 and H-40a), 3.33
(3H, s, OMe), 3.74–3.81 (2H, m, H-3 and H-40b), 3.80 (3H, s,
PhOMe), 4.14 (1H, dd, J = 11.8, 5.9 Hz, H-6a), 4.42–4.53 (1H, m,
H-6b and benzyl), 4.61 (1H, d, J = 3.6 Hz, H-1), 4.84 (1H, br dd,
J = 6.7, 2.7 Hz, H-30), 6.07 (1H, dd, J = 1.6 Hz, @CH2, Ha), 6.37 (1H,
s, @CH2, Hb), 6.85 (2H, d, J = 8.7 Hz, aromatic), 7.23 (2H, d,
J = 8.6 Hz, aromatic), 13C NMR (67.5 MHz, CDCl3) ꢀ5.0 and ꢀ4.9
(SiMe2), 0.5, 0.9 and 1.3 (TMS), 18.2 (Me3C), 25.8 (Me3C), 54.8
(OMe), 55.2 (PhOMe), 63.9 (C-6), 69.4 (C-5), 70.3 (C-30), 72.6 (C-
2), 72.8 (C-40), 73.7 (C-4), 74.7 (benzyl), 75.1 (C-3), 99.7 (C-1),
113.6 (aromatic), 126.6 (@CH2), 129.0 (aromatic), 130.6 (aromatic),
141.0 (C-20), 159.0 (aromatic), 165.7 (carbonyl).
1159, 1119 and 1001, 1H NMR (270 MHz, CD3OD) 3.03 (2H, m,
H2C-b), 3.62 (2H, m, H2C-
c
), 3.75 (3H, s, OMe), 4.17 (1H, m, H-b),
13C NMR (67.5 MHz, CD3OD) 50.0 (C-bOC-
a
), 53.0 (OMe), 58.3 (C-
bOC-a), 63.5 (C-4), 70.6 (C-3), 171.5 (C-1), HR-FD-MS m/z
[M + H]+ calcd for C6H11O5, 163.0606; found, 163.0615.
4.22. Assay for Antibacterial activity
Antibacterial activities were evaluated by a broth microdilution
method based on the National Committee for Clinical Laboratory
Standards (NCCLS) guideline. Bacteria were maintained with NA
medium (5.0 g/l meat extract, 10.0 g/l BactoTM Pepton, 5.0 g/l NaCl,
and 15.0 g/l agar) plates at 37ꢀC. Assay for antibacterial activity was
tested using Mueler-Hinton (MH) liquid medium containing 3.0 g/l
meat extract, 17.5 g/l BactoTM Pepton, and 1.5 g/l soluble starch.
MH liquid medium was acidified to pH 6.0 with aqueous HCl, be-
cause 6-tuliposide B (1) is rapidly converted into tulipalin B (2)
within 1–2 h even at neutral conditions. The tested compounds
were diluted with Milli Q-water except tulipalin A (3), which
was diluted with 5% DMSO. Tulipalin A (3) was tested at final con-
centrations of 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4,
3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8 and 5.0 mM and other compounds
were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mM. Minimum
inhibitory concentration (MIC) was defined as the lowest concen-
trations at which no growth was observed. Each bacterium was
pre-cultured for 6–10 h with MH liquid medium at 170 rpm, 35
ꢀC and then used for preparation of bacterial inoculum. The cell
density of obtained suspensions was adjusted to 1.0 ꢁ 107 col-
ony-forming unit (CFU)/ml and further diluted 19-fold with MH
IR (cmꢀ1) 3392, 1712, 1271, 1148 and 1056, HR-FAB-MS m/z
[MꢀH]ꢀ, calcd for C12H19O9, 307.1029; found, 307.1018.
4.19.3. 1-O-Methyl-a-6-tuliposide B (30)
Deprotection of 29 and epi-29 were carried out by procedure
described in 4.1.2.
½
a 2D5
ꢂ
¼ þ111:8 (c 0.72, CH3OH), 1H NMR (500 MHz, CD3OD)
3.28–3.30 (1H, m, H-4), 3.38–3.41 (4H, m, OMe and H-2), 3.43
(1H, dd, J = 11.3, 6.8 Hz, H-40a), 3.60 (1H, t, J = 9.3 Hz, H-3), 3.71
(1H, dd, J = 11.3, 3.5 Hz, H-40b), 3.72–3.76 (1H, m, H-5), 4.25 (1H,
dd, J = 11.8, 6.0, H-6a), 4.49 (1H, dd, J = 11.8, 2.1, H-6b), 4.57 (1H,
br dd, J = 6. 7, 3.4 Hz, H-30), 4.65 (1H, d, J = 3.8 Hz, H-1), 5.99 (1H,
t, J = 1.5 Hz, @CH2, Ha), 6.34, (1H, s, @CH2, Hb), 13C NMR
(67.5 MHz, CD3OD) 55.6 (OMe), 65.1 (C-6), 66.7 (C-40), 71.0 (C-5),
72.0 (C-30), 72.1 (C-2), 73.5 (C-4), 75.0 (C-3), 101.3 (C-1), 126.7
(@CH2), 142.3 (C-20), 167.3 (C-10).
medium. After that, 5 ll of test compound solutions and 95 ll of
4.19.4. 1-O-Methyl-a-epi-6-tuliposide B (epi-30)
bacterial inoculums were mixed to 5.0 ꢁ 105 CFU/ml in 96-well
microtiter plates. The plates were covered with sterile sealer and
incubated at 600 rpm, 35 °C for 16 h. Bacterial growth was evalu-
ated by measuring OD595 value (microplate spectrophotometer,
Sunrise Remote, TECAN). Assays were carried out in triplicate.
½
a 2D5
ꢂ
¼ þ144:0 (c 0.95, CH3OH), 1H NMR (500 MHz, CD3OD)
3.27–3.31 (1H, m, H-4), 3.37–3.40 (4H, m, OMe and H-2), 3.43
(1H, dd, J = 11.3, 6.8 Hz, H-40a), 3.61 (1H, t, J = 9.3 Hz, H-3), 3.70
(1H, dd, J = 11.3, 3.5 Hz, H-40b), 3.73–3.77 (1H, m, H-5), 4.27 (1H,
dd, J = 11.8, 6.2, H-6a), 4.45 (1H, dd, J = 11.8, 2.2, H-6b), 4.57 (1H,
br dd, J = 6.8, 3.5 Hz, H-30), 4.65 (1H, d, J = 3.8 Hz, H-1), 5.99 (1H,
t, J = 1.5 Hz, @CH2, Ha), 6.34, (1H, s, @CH2, Hb), 13C NMR
(67.5 MHz, CD3OD) 55.6 (OMe), 65.2 (C-6), 66.7 (C-40), 71.0 (C-5),
72.0 (C-30), 72.0 (C-2), 73.5 (C-4), 75.0 (C-3), 101.3 (C-1), 126.7
(@CH2), 142.3 (C-20), 167.4 (C-10).
Acknowledgement
This work was supported in part by Grant-in-Aid for Scientific
Research (A) provided by the Japan Society for the Promotion of
Science of Japan.
4.20. Methyl 3,4-dihydroxy-2-methylenebutanoate (31, ent-31)
To a stirred solution of PMB ether (10, 280 mg, 1.00 mmol) in
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