A method for the rapid discovery of naturally occurring products by proteins immobilized on magnetic beads and reverse affinity chromatography

Article abstract of DOI:10.1002/asia.200900357

A highly efficient screening method for naturally occurring products that bind to a specific target protein was demonstrated by using hVDR magnetic beads. The native ligand 1α, 25(OH)₂ VD₃ (1) was selectively bound by hVDR magnetic b

Full text of DOI:10.1002/asia.200900357

FULL PAPERS
DOI: 10.1002/asia.200900357
A Method for the Rapid Discovery of Naturally Occurring Products by
Proteins Immobilized on Magnetic Beads and Reverse Affinity
Chromatography
Midori A. Arai,*[a] Eiji Kobatake,[a] Takashi Koyano,[b] Thaworn Kowithayakorn,[c]
Shigeaki Kato,[d] and Masami Ishibashi*[a]
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Chem. Asian J. 2009, 4, 1802 – 1808

Abstract: A highly efficient screening
method for naturally occurring prod-
ucts that bind to a specific target pro-
tein was demonstrated by using hVDR
magnetic beads. The native ligand
1a,25(OH)2 VD3 (1) was selectively
bound by hVDR magnetic beads when
present in a mixture of natural com-
pounds. Furthermore, this method was
shown to be applicable to the identifi-
cation of natural products that interact
with a specific protein immobilized on
the beads from an extract of a natural
resource. Two new natural compounds
were isolated by this method. This ap-
proach will be helpful for the discovery
of novel, naturally occurring products
that bind to specific target proteins.
This method has the further advantages
that it can identify the HPLC peak cor-
responding to the target compound for
isolation, as well as provide important
UV, CD, or MS profile information.
Keywords: drug discovery · liquid
chromatography · magnetic beads ·
natural products · proteins
Introduction
from a compound mixture. Immobilized proteins might have
less mobility, which sometimes cause a less effective com-
pound binding. In addition, problems from false-positive re-
sults might occur as a result of alteration of the binding
character of a protein. However, technological innovations
and novel applications, such as protein microarrays[1] and
protein microplates,[2] have greatly advanced the field of im-
mobilized proteins. MS-based techniques with immobilized
proteins for lead compound discovery are also important.[3]
Therefore, there is still a huge advantage in using immobi-
lized proteins for finding new ligands. There are several re-
ports of the application of protein immobilized beads to nat-
ural extracts, which are a mixture of many compounds.[4] By
following the binding of the natural compound to the pro-
tein beads, HPLC analysis, in particular the retention time
on an HPLC column, can provide a large amount of infor-
mation concerning the nature of the natural compound. By
using a combination of protein immobilized beads followed
by HPLC analysis, the time for isolation of target com-
pounds can be greatly shortened. Herein, we describe a
method for the rapid discovery of naturally occurring prod-
ucts using an immobilized protein on magnetic beads and
reverse affinity chromatography (Figure 1).
The identification of novel, naturally occurring bioactive
products has played an important role in drug discovery.
Moreover, targeting of drugs to specific proteins, for the
treatment of specific diseases, is also of great clinical impor-
tance. There is a strong demand for “quick” discovery of
novel natural products that are potential new candidate
medicines. However, it can take enormous amounts of time
and effort to find such desirable natural products. The isola-
tion of natural products with a particular bioactivity is ach-
ieved by biological assay of the product during purification.
Often, repeated fractionations and assaying of bioactivity re-
sults in decreased bioactivity or loss of the desired bioactive
compound. We tested the possibility that protein immobi-
lized magnetic beads might be useful for rapidly obtaining
new natural products.
The use of protein-immobilized beads facilitates the isola-
tion of compounds that interact with a protein of interest
[a] Prof. Dr. M. A. Arai, E. Kobatake, Prof. Dr. M. Ishibashi
Graduate School of Pharmaceutical Sciences, Chiba University
Inage, Chiba 263-8522 (Japan)
Magnetic beads are particularly useful for immobilization
of proteins as bead-bound compounds can be easily separat-
ed from other soluble components by using a magnet; they
are thus compatible with high-throughput screening (HTS)
applications. Compared with the many centrifuge steps that
would be required for separation if sepharose beads were
used, the simple collection of magnetic beads on the wall of
the tube by the use of a magnet, followed by removal of the
supernatant, is a much easier and far less time-consuming
method (Figure 1B). Magnetic nanoparticles, synthesized
from cobalt, iron, nickel, and the oxides of these metals,
have been utilized widely in a range of fields, including biol-
ogy, chemistry, and molecular imaging.[5] The immobilization
Fax : +81432902913
E-mail: marai@p.chiba-u.ac.jp
[b] Dr. T. Koyano
Temko Corporation
4-27-4 Honcho, Nakano, Tokyo 164-0012 (Japan)
[c] Prof. Dr. T. Kowithayakorn
Faculty of Agriculture, Kohn Kaen University
Kohn Kaen 40002 (Thailand)
[d] Prof. Dr. S. Kato
Institute of Molecular and Cellular Biosciences, The University of
Tokyo
Yayoi, Tokyo 113-0032 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.200900357.
Chem. Asian J. 2009, 4, 1802 – 1808
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FULL PAPERS
beads and ligand 1 was gently
mixed at 48C for 2 h (Fig-
ure 1A), and then excess
ligand solution was easily re-
moved after the beads were
gathered on the wall of the
tube using
a magnet (Fig-
ure 1B). Because of the non-
specific binding to the magnet-
ic beads through hydrophobic
interactions, we carefully ex-
amined the washing method.
An
appropriate
washing
Figure 1. Protocol for screening of natural compounds by protein beads. A) Incubation of the extracts of natu-
ral resources (a mixture of compounds) with protein beads; B) washing of the beads by using a magnet and re-
lease of the compounds bound to the bead-bound protein; C) analysis of the released compounds by HPLC;
D) outline of the protein magnetic beads.
method was determined by
comparing washing methods,
types and volumes of buffer,
type and concentrations of de-
tergent, and washing times and
of proteins on magnetic beads has facilitated protein purifi-
cation,[6] detection of protein–protein interactions,[7] cancer-
cell detection (using immobilized antibody),[8] and isolation
of the target protein of small molecules.[9] Handa et al. have
described a method for the separation of FK506 from ex-
tracts by using FKBP (FK506 binding protein) immobilized
magnetic beads.[10] We report the use of protein-immobilized
magnetic beads for the isolation of new natural products.
temperature. Following two sequential washes with NET-N
buffer (50 mm Tris-HCl; pH 7.5, 150 mm NaCl, 5 mm EDTA;
pH 7.9) that includes 0.05% Nonidet P40, which is used in
the pull-down assay of VDR (0.5% Nonidet P40 was used
usually), 1 bound to hVDR on the beads was eluted with
EtOH. There are no leaching problems, and the protein-
beads seem to be stable in this assay. The dispersibility of
protein beads is good in buffer solution. An HPLC profile
of the eluted EtOH solution is shown in Figure 2A, and 1
bound to hVDR can be clearly detected. With 1 mg hVDR
immobilized magnetic beads, approximately 40 nmol of 1
Results and Discussion
Establishing the Method by Using hVDR Beads
To establish a method for the isolation of naturally occur-
ring small molecules from the complex mixtures that make
up extracts of natural resources (such as plants, marine or-
ganisms, actinomycetes, and myxomycetes), we first selected
hVDR, a member of the superfamily of nuclear receptors,
as the target protein. This receptor and its ligands have been
well studied. It is known that 1a,25-dihydroxyvitamin D3
[1a,25(OH)2 VD3] (1) is an endogenous cellular ligand of
hVDR, and that vitamin D3 (VD3) (2), which is the precur-
sor of 1, also binds but with much lower affinity than 1.
Compound 1 binding to hVDR transactivates the target
DNA and plays a vital role in calcium homeostasis,[11] osteo-
clastogenesis,[12] keratinocyte differentiation,[13] phosphate
homeostasis, the immune response, cellular proliferation,
differentiation, and apoptosis.[14] Therefore, 1 was selected
as a positive control for the bead-bound hVDR-binding
assay. Recombinant hVDR protein was prepared from
E. coli as a glutathione-S-transferase (GST) fused full-length
protein.[15] After cleavage of the GST moiety, hVDR was
immobilized on COOH-coated magnetic beads (Dynabeads
M-270 carboxylic acid; active chemical functionality,
150 mmolgꢀ1 beads; Invitrogen) that had been treated with
N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDC) (Figure 1D). GST
was similarly immobilized on magnetic beads as a negative
control. A suspension of hVDR- or GST-bound magnetic
Figure 2. HPLC profiles of compounds eluted from the protein magnetic
beads. A) 1a,25(OH)2 VD3 (1) eluted from hVDR beads. 3.0 nmol of 1
was detected; B) VD3 (2) eluted from hVDR beads; C) compound 1
eluted from GST beads; D) compound 2 eluted from GST beads; all
samples are the final EtOH-eluted solution obtained following incubation
of 1 or 2 with protein beads, washing of the beads with buffer, and re-
lease of the bound compound from the bead-bound protein by EtOH.
Assay conditions: Protein beads (1 mg) in NET-N buffer (225 mL) and
compounds (25 nmol) in EtOH 25 mL were incubated. HPLC conditions:
column: CAPCELL PAK C18 MGII (5 mm,
f
4.6ꢁ250 mm),
CH3CN+0.1% TFA/H2O=60:40 (for 1) or 100:0 (for 2) with isocratic
method, 1 mLminꢀ1, UV 254 nm detection.
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A Method for the Rapid Discovery of Naturally Occurring Products
can be detected. In the incubation step, the amount of
EtOH (10%) in buffer was important. Compound 1 binding
to negative-control GST-bound magnetic beads was similar-
ly examined, and a very small peak was observed for 1 (Fig-
ure 2C). When the binding of the weak compound 2 was as-
sayed, the peak of 2 bound to hVDR was very small (Fig-
ure 2B) and similar to the peak obtained following incuba-
tion with control GST magnetic beads (Figure 2D). The
binding affinity of VD3 (2) to VDR has been reported as a
relative competitive index value whereby 1a,25(OH)2VD3
(1) is 100.[16] The binding affinity of 2 was reported as
0.0001, which is below the detection limit of our system.
Therefore, the peaks shown in Figure 2B would result from
nonspecific binding. From the amounts of 1 (Figure 2C) and
2 (Figure 2D), the nonspecific binding of these types of
compounds to GST would be expected around 0.5 nmol.
Therefore, the lowest amount of 1 detectable by using VDR
magnetic beads would be approximately 0.5 nmol. As a fur-
ther check for nonspecific binding, we also assayed non-pro-
tein-linked magnetic beads and found that the peak was
negligible.
Isolation of New Natural Compounds by Using hVDR
Magnetic Beads
On the basis of this result, we then screened several plant
extracts. An extract of Limocharis flava (the aerial part)
yielded HPLC peaks following binding to and elution from
hVDR beads (Figure 4B). Since “peak b” was obtained fol-
Figure 4. Screening for natural compounds with hVDR magnetic beads.
A) The HPLC profile of a crude MeOH extract of L. flava. B) Com-
pounds eluted from hVDR beads that were incubated with a MeOH ex-
tract of L. flava. Assay conditions: hVDR beads (1 mg) in NET-N buffer
(225 mL) and compounds mixture in EtOH 25 mL (extract; 185 mg) were
incubated. HPLC conditions: column: CAPCELL PAK C18 MGII
(5 mm, f 4.6ꢁ250 mm), CH3CN+0.1% TFA/H2O=10:90–100:0 with gra-
dient method, 1 mLminꢀ1, UV 254 nm detection.
Application to Plant Extracts
We next attempted to apply this system to a mixture of a
plant extract and hVDR magnetic beads (Figure 3). The
plant extract used was a MeOH extract of Eleutherine pal-
mifolia. Ligand 1 (14 nmol) was mixed with extract solution
(185 mg in EtOH, 25 mL) prior to incubation with hVDR
magnetic beads (Figure 3A); this mixture was then added to
hVDR-bound magnetic beads (1.0 mg) in NET-N buffer
(225 mL). Even though the plant extract contained many
compounds, the hVDR magnetic beads selectively bound
ligand 1 (Figure 3B). There is no nonspecific binding (Fig-
ure 3C; GST beads).
lowing incubation with, and elution from, GST beads,
“peak a” from the hVDR beads was judged to be com-
pounds that specifically interacted with hVDR. We there-
fore proceeded to purify and identify “peak a” as follows.
The methanol extract of L. flava (2.2 g) was partitioned be-
tween H2O, and n-hexane and
ethyl acetate. Because HPLC
analysis of the n-hexane and
the ethyl acetate soluble frac-
tions both revealed the pres-
ence of “peak a”, we combined
these fractions and further pu-
rified them by consecutive
steps of silica gel column chro-
matography and reverse-phase
HPLC, which yielded two new
labdane type diterpenes (3 and
4) (Figure 5, Table 1). The mo-
lecular formulas of 3 and 4
were
determined
to
be
C25H40O5 on the basis of high-
resolution FAB mass-spectro-
scopic analysis. The 1H–1H
COSY spectrum, heteronu-
clear multiple bond correla-
tions (HMBCs) and other
spectral data of 3 revealed the
Figure 3. Application to a plant extract. A) HPLC profile of Eleutherine palmiforia MeOH extract mixed with
1; B) compounds eluted from hVDR beads that were incubated with a MeOH extract of E. palmiforia and 1.
1.14 nmol of 1 was detected; C) HPLC profile of eluent from GST beads that were incubated with a MeOH
extract of E. palmiforia and 1. Assay conditions: Protein beads (1 mg) in NET-N buffer (225 mL) and com-
pounds mixture in EtOH 25 mL (1; 13 nmol, extract; 185 mg) were incubated. HPLC profile of the mixture of
extract and 1. HPLC conditions: column: CAPCELL PAK C18 MGII (5 mm, f 4.6ꢁ250 mm), CH3CN+0.1%
TFA/H2O=10:90–100:0 with gradient method, 1 mLminꢀ1, UV 254 nm detection.
Chem. Asian J. 2009, 4, 1802 – 1808
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M. A. Arai, M. Ishibashi et al.
FULL PAPERS
Figure 5. Chemical structures of 3 and 4.
1
Figure 6. Key H–1H-COSY and HMBC data for 3.
Table 1. 1H and 13C NMR data for 3 and 4.
3 (CDCl3)
reochemistry of the aglycone
moiety of 3 was determined to
be ent-12(Z)-labda-8(17),12,14-
trien-18-ol.[22] The absolute ste-
reochemistry of the aglycone
moiety of 4 was determined by
the same methods to be ent-
12(E)-labda-8(17),12,14-trien-
18-ol[23] and the sugar moiety
to be l-arabinose.
4 (CDCl3)
position
dH[a] [ppm] (J [Hz])
dC[b] [ppm]
dH[a] [ppm] (J [Hz])
dC[b] [ppm]
1
1.76 m
38.7
1.76 m
1.05 td (12.4, 4.8)
1.57 m
38.7
1.05 td (12.8, 4.8)
1.56 m
1.35 m
2
3
4
5
6
18.6
36.4
37.2
49.0
24.1
18.6
36.4
37.2
49.0
24.0
1.35 m
1.35 m
1.56 m
1.35 m
1.57 m
1.35 m
2.36 m
1.35 m
7
2.38 m
37.8
37.7
An assay of the binding of
the isolated pure compounds
to hVDR beads revealed
stronger binding of 4 than 3
(Figure 7). We further exam-
ined the VDR-related tran-
scriptional activities of 3 and 4
by a cell-based luciferase re-
porter-gene assay.[15] Neither 3
nor 4 induced transcriptional
1.99 t (12.4)
1.99 t (12.8)
8
9
10
11
148.0
57.2
39.4
22.1
148.0
57.0
39.3
23.1
1.76 m
1.76 m
2.38 m
2.17 m
5.30 brt (6.4)
2.36 m
2.14 m
5.40 t (6.8)
12
13
14
15
131.6
131.6
133.8
113.2
133.4[c]
134.0[c]
141.6
6.79 ddd (17.2, 10.8, 0.8)
5.18 dd (17.2, 0.8)
5.09 dt (10.8, 1.6)
1.77 d (1.2)
4.83 d (1.2)
4.48 d (1.2)
3.32 d (9.6)
3.16 d (9.6)
0.78 s
0.74 s
6.30 dd (17.2, 10.8)
5.04 d (17.2)
4.88 d (10.8)
1.75 s
4.82 d (0.8)
4.46 d (0.8)
3.32 d (9.2)
3.17 d (9.2)
0.79 s
109.9
16
17
19.7
107.9
11.8
107.8
activation, although
a high
concentration of 4 slightly de-
creased the transcriptional ac-
tivity induced by 1 (see the
Supporting Information).
18
76.8
19
20
18.0
14.9
18.0
14.9
0.75 s
1’
2’
3’
4’
5’
4.96 s
4.03 brs
4.03 brs
4.18 q (2.0)
3.91 dd (11.6, 2.0)
3.85 dd (11.6, 2.0)
108.6
78.4[c]
78.2[c]
87.4
4.96 s
4.03 brs
4.03 brs
4.18 q (2.0)
3.92 dd (11.6, 2.0)
3.86 dd (11.6, 2.0)
108.6
78.4[c]
78.2[c]
87.4
Conclusions
In conclusion, we have estab-
lished a method of screening
for naturally occurring prod-
ucts that bind to a specific
target protein that is immobi-
62.1
62.2
[a] 400 MHz. [b] 100 MHz. [c] These values may be interchanged within the same column.
presence of 12(Z)-labda-8(17),12,14-trien-18-ol[17,18] and a-
lized on magnetic beads. We demonstrated the feasibility of
the method by using hVDR and its native ligand
1a,25(OH)2 VD3 (1). Compound 1 was selectively bound by
hVDR magnetic beads when present in a mixture of natural
compounds. Furthermore, this method was proven to be ap-
plicable to the identification of natural products, which in-
teract with a specific protein immobilized on the beads,
from an extract of a natural resource. Two new natural com-
pounds were isolated by this method. This method has the
further advantages that it can identify the HPLC peak cor-
arabinose[19] (Figure 6). The configuration of the C12 C13
ꢀ
double bond (Z) was determined by comparison of the
signal pattern in the 13C NMR spectra with those reported
for cis- and trans-methyl ozate.[20] Acid hydrolysis of 3 was
carried out to determine the absolute stereochemistry of the
aglycone and sugar moieties of the compound. The thiazoli-
dine derivative of the sugar moiety was analysed by HPLC
and determined to be l-arabinose.[21] On the basis of the
[a]D data of the purified aglycone moiety, the absolute ste-
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A Method for the Rapid Discovery of Naturally Occurring Products
and they were mixed for 15 min at
room temperature. The hVDR beads
were washed by Tris-HCl buffer
(200 mL, 50 mm, pH 7.5) four times,
then the beads were suspended in
NET-N buffer (225 mL).
Selective detection of 1 from Eleu-
therine palmifolia extract: The plant
extract used was a MeOH extract of
Figure 7. HPLC profiles of isolated pure compounds obtained by incubation with hVDR magnetic beads.
A) Profile of 3; B) profile of 4; all samples were the final EtOH eluate of hVDR magnetic beads incubated
with 3 or 4. Assay conditions: hVDR beads (1 mg) in NET-N buffer (225 mL) and compound (25 mL; 1 mm in
EtOH) were incubated. HPLC conditions: column: CAPCELL PAK C18 MGII (5 mm, f 4.6ꢁ250 mm),
CH3CN+0.1% TFA/H2O=10:90–100:0 with gradient method, 1 mLminꢀ1, UV 254 nm detection.
Eleutherine palmifolia. Ligand
1
(14 nmol) was mixed with this solu-
tion (17 mgmLꢀ1) prior to incubation
with hVDR magnetic beads. Even
though the plant extract contained
many compounds, the hVDR mag-
responding to the target compound for isolation, as well as
netic beads selectively bound ligand 1 (Figure 3B). There was no com-
provide important UV, CD, and MS profile information. Al-
though available recombinant proteins for this method
would be limited by protein purity, availability, and stability
of the functional form, this approach will be helpful and
give information for rapid isolation of natural products.
Along with phenotypic or cell-based screening, this protein-
based method is worth using for the discovery of natural
products.
pound bound to the GST beads (control beads), as shown in Figure 3C.
Extraction and isolation: The air-dried, powdered aerial part (13.7 g) of
Limnocharis flava was extracted with MeOH (300 mLꢁ3) at room tem-
perature. The MeOH extract (2.2 g) was subjected to Diaion HP-20
column chromatography (30ꢁ300 mm) using mixtures of MeOH/acetone
to yield the MeOH-soluble fractions. This MeOH-soluble part (1.7 g) was
partitioned with H2O (200 mL) between n-hexane (200 mLꢁ3), and the
aqueous phase was further extracted with EtOAc (200 mLꢁ3) and
nBuOH (200 mLꢁ3) to afford hexane extract (317.3 mg), EtOAc extract
(575.0 mg), nBuOH extract (276.8 mg), and H2O extract (454.3 mg).
Since the hexane and EtOAc extracts contained almost the same constit-
uents by TLC analysis, they were combined and used for silica gel
column chromatography (column A: 20ꢁ220 mm), eluted with gradient
mixtures of hexane/EtOAc (6/4 to 0/1) and EtOAc/MeOH (4/1 to 0/1) to
give seven fractions (fractions 2A–2G): 2A (315.5 mg), 2B (65.1 mg), 2C
(24.7 mg), 2D (200.6 mg), 2E (95.2 mg), 2F (20.8 mg), 2G (131.1 mg).
Fraction 2D (23.0 mg) of column A was separated by reverse-phase
HPLC (YMC-Pack ODA-AM, 10ꢁ250 mm; eluent: H2O/MeOH (15:85);
flow rate: 2.0 mLminꢀ1; detection: UV at 254 nm) to afford fraction 3A
(15.4 mg; mixture of 3:4=4:1, tR 37 min) and fraction 3B (7.1 mg). To
separate compounds 3 and 4, fraction 2D (18.7 mg) of column A was sep-
arated by reverse-phase HPLC (CAPCELL PACK C18 ACR 3 mm: 4.6ꢁ
250 mm; eluent: H2O/MeOH (15:85); flow rate: 0.6 mLminꢀ1; detection:
UV at 232 nm) to afford fractions 4A (4, 2.0 mg, tR 27.5 min), 4B (3,
5.7 mg), 4C (3, 3.3 mg, peak top of 3; tR 28.5 min), and 4D (8.2 mg). Frac-
tion 2D (13.2 mg) of column A was separated by reversed-phase HPLC
(CAPCELL PACK C18 ACR 5 mm; 6.0ꢁ250 mm; eluent: H2O/MeOH
(20:80); flow rate: 1.5 mLminꢀ1; detection: UV at 232 nm) to afford frac-
tions 5A (4, 2.0 mg), 5B (3, 5.3 mg), 5C (3, 3.8 mg), and 5D (7.9 mg). 3:
Experimental Section
A typical screening procedure was as follows: A MeOH extract of a nat-
ural resource (185 mg in EtOH, 25 mL) was added to hVDR-bound mag-
netic beads (1.0 mg) in NET-N buffer (225 mL) that includes 0.05% Noni-
det-P40 and the mixture was gently mixed for 2 h at 48C. The beads were
then gathered to the wall of the tube by using a magnet, the supernatant
solution was removed, and the beads were then washed twice with NET-
N buffer (0.3 mL) for 10 min at 48C. EtOH (0.15 mL) was then added to
the washed beads and the suspension was gently mixed for 10 min at
48C. The beads were then gathered on the wall of the tube using a
magnet and the supernatant was analyzed by HPLC.
Expression and purification of recombinant hVDR and GST: E. Coli
strain DH5a serves as a host for pGEXhVDR4-427. An overnight plateau
phase culture of DH5a was used to inoculate fresh LB medium (Invitro-
gen) containing 50 mgLꢀ1 ampicillin. Cells were grown at 378C to a den-
sity of OD600 of 0.6 and induced by 1 mm IPTG (isopropyl-1-thio-b-d-gal-
actopyranoside) followed by incubation for an additional 3 h at 188C
(378C for GST). The cells were harvested and were lysed by sonication.
The lysate was centrifuged at 6000 rpm for 5 min at 48C. To the resulting
supernatant was added triton X-100 (final concentration 1% (w/v)) and
the resulting solution was mixed at 48C for 30 min. The mixture was cen-
trifuged at 5000 rpm for 7 min at 48C. To the resulting supernatant was
added pre-washed glutathione sepharose 4B beads (GE Healthcare) and
gently mixed at 48C for 30 min. The beads obtained after centrifugation
(3200 rpm, 5 min, at 48C) were washed 5 times (2000 rpm, 1 min, at 48C)
with PBS-triton X-100 buffer (PBS plus 1% triton X-100). GST was
cleaved from recombinant protein (GST-hVDR4-427) by thrombin pro-
tease (GE Healthcare) for 15 h at 48C, then dialyzed against PBS buffer
using Slide-A-Lyzer Dialysis Cassette (Extra Strength, 10000 MWCO;
PIERCE). The recombinant proteins were estimated to be greater than
90% pure by SDS-PAGE.
white amorphous solid, FABMS m/z 443 [M+Na]+, 459 [M+K]+,
18
HRFABMS m/z: calcd for C25H40O5Na 443.2773, found 443.2733, ½aꢁD
=
ꢀ83.5 (c=0.4, MeOH), IR (ATR): n˜max =3351, 2925, 2867, 1062, 1033,
998, 899 cmꢀ1; 4: white amorphous solid, HRFABMS m/z: calcd for
C25H40O5Na 443.2773, found 443.2733, ½aꢁ1D8 =ꢀ62.2 (c=0.4, MeOH), IR
(ATR): n˜ =max 3375, 2928, 2867, 1092, 888 cmꢀ1
.
Determination of the Absolute Stereochemistry of Aglycones of 3 and 4
Hydrolysis of compounds: Fraction 3A (mixture of 3:4=4:1, 5.0 mg) was
stirred in 5% H2SO4 (3.0 mL) and 1,4-dioxane (4.5 mL) at 958C for 1 h.
H2O was added to the reaction mixture, and the mixture was extracted
with EtOAc. The organic layer was washed with saturated aqueous
NaHCO3 and H2O, and dried over Na2SO4 and concentrated. The residue
(7.3 mg) was purified by reverse-phase HPLC (CAPCELL PACK C18
ACR 5 mm; 6.0ꢁ250 mm; eluent: H2O/MeOH (15/85); flow rate:
Preparation of hVDR immobilized on COOH magnetic beads: Dyna-
beads M-270 carboxylic acid (1 mg, DYNAL) in MES buffer (33 mL) was
treated by N-hydroxysuccinimide (150 mL, 50 mgmLꢀ1, MES buffer solu-
tion) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC), (150 mL, 50 mgmLꢀ1, MES buffer solution) for 30 min at room
temperature. The beads were washed with MES buffer twice, then
hVDR in PBS (400 mL, 1 mgmLꢀ1) was added to the activated magnetic
beads and they were mixed at 408C for 30 min. The solution was re-
moved, Tris-HCl buffer (400 mL, 50 mm, pH 7.5) was added to the beads,
1.5 mLminꢀ1
; detection: UV at 232 nm) to give the main product
(1.8 mg), which was determined to be an aglycone of 3 from the NMR
peak pattern of the olefin part and the mass balance. The H2O-soluble
part was purified by Amberlite IRA 96 SB AG (10ꢁ210 mm; eluent,
H2O) to give the sugar part (3.5 mg). 1H NMR data of the aglycone of 3
(ent-12(Z)-labda-8(17),12,14-trien-18-ol) was the same as the reported
value.[18] ½aꢁD value of the purified aglicone part of 3: ½aꢁ1D8 =ꢀ35.0 (c=
0.4, CHCl3). Ref. ½aꢁ1D8 =ꢀ27.0 (c=1.6, CHCl3).[22]
Chem. Asian J. 2009, 4, 1802 – 1808
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1807

M. A. Arai, M. Ishibashi et al.
FULL PAPERS
The fractions 4 A and 5 A (4; 4.0 mg) were stirred in 5% H2SO4
(3.0 mL) and 1,4-dioxane (4.5 mL) at 958C for 1 h. H2O was added to the
reaction mixture, and it was extracted with EtOAc. The organic layer
was washed with saturated aqueous NaHCO3 and H2O, and dried over
Na2SO4 and concentrated. The residue (2.9 mg) was purified by reverse-
phase HPLC (CAPCELL PACK C18 ACR 5 mm; 6.0ꢁ250 mm; eluent:
H2O/MeOH (15:85); flow rate: 1.5 mLminꢀ1; detection: UV at 232 nm)
to give a mixture (2.9 mg) of aglycone of 4 and side product 4a (4/4a=
3:1), which is the more stable aglycone by isomerization of olefin part
(see the Supporting Information for the structure of 4a). The H2O-solu-
ble part was purified by Amberlite IRA 96 SB AG (10ꢁ210 mm; eluent:
H2O) to give the sugar part (1.9 mg). ½aꢁD value of the purified aglycone
part of 4 (ent-12(E)-labda-8(17),12,14-trien-18-ol); ½aꢁ1D8 =ꢀ24.0 (c=0.4,
CHCl3). Reference 12(E)-labda-8(17),12,14-trien-18-ol: ½aꢁ1D8 =+16.6 (c=
0.4, CHCl3).[23]
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Determination of the Absolute Stereochemistry of Sugars of 3 and 4
Thiazolidine derivatization of sugar parts:[21] To the sugar part (purified
by amberlite IRA 96 SB AG after hydrolysis) in pyridine (1.0 mL) was
added l-cysteine methyl ester hydrochloride (10.3 mg), and the mixture
was stirred at 608C for 1 h. After reaction completed, the reaction mix-
ture was concentrated in vacuo. As the control, l-arabinose and d-arabi-
nose were also converted into thiazolidine derivatives. Diastereomers
were detected as separated peaks: l-arabinose (27 min, 32 min) and d-
arabinose (25 min, 34 min). The samples of sugar parts of 3 and 4 were
analyzed by reverse-phase HPLC (CAPCELL PAL NH2 UG80 5 mm;
4.6ꢁ250 mm; eluent: H2O/CH3CN (3:97); flow rate: 0.7 mLminꢀ1; detec-
tion: UV at 210 nm). The retention times of the thiazolidine derivatives
of the sugar parts of 3 and 4 matched those of l-arabinose. The results
for 4 is shown in the Supporting Information.
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Acknowledgements
This work was supported by a Grant-in Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology,
Japan. M.A.A. also gratefully acknowledges financial support from the
Uehara Memorial Foundation, the Mitsubishi Chemical Corporation
Fund, and the Hamaguchi Foundation for the Advancement of Biochem-
istry.
1
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Received: August 9, 2009
Published online: November 18, 2009
1808
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ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2009, 4, 1802 – 1808