Structure-activity relationship for FR901464: A versatile method for the conversion and preparation of biologically active biotinylated probes

Article abstract of DOI:10.1271/bbb.68.2178

The structure-activity relationship for FR901464, a potent cell-cycle inhibitor, was examined by synthesizing its analogs. A versatile method for converting FR901464 was devised. This method made it possible to synthesize biologically active FR901464-biotin conjugates which could be used to isolate the binding proteins.

Full text of DOI:10.1271/bbb.68.2178

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Structure-activity Relationship for FR901464: A
Versatile Method for the Conversion and Preparation
of Biologically Active Biotinylated Probes
Hajime MOTOYOSHIa, Masato HORIGOMEa, Ken ISHIGAMIa, Tatsuhiko YOSHIDAb, Sueharu
HORINOUCHIb, Minoru YOSHIDAbcd, Hidenori WATANABEa & Takeshi KITAHARAa
a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life
Sciences, The University of Tokyo
b Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The
University of Tokyo
c Chemical Genetics Laboratory, RIKEN
d CREST Research Project, Japan Science and Technology Agency
Published online: 22 May 2014.
To cite this article: Hajime MOTOYOSHI, Masato HORIGOME, Ken ISHIGAMI, Tatsuhiko YOSHIDA, Sueharu HORINOUCHI,
Minoru YOSHIDA, Hidenori WATANABE & Takeshi KITAHARA (2004) Structure-activity Relationship for FR901464: A Versatile
Method for the Conversion and Preparation of Biologically Active Biotinylated Probes, Bioscience, Biotechnology, and
Biochemistry, 68:10, 2178-2182, DOI: 10.1271/bbb.68.2178
To link to this article: http://dx.doi.org/10.1271/bbb.68.2178
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Biosci. Biotechnol. Biochem., 68 (10), 2178–2182, 2004
Structure-activity Relationship for FR901464: A Versatile Method for the
Conversion and Preparation of Biologically Active Biotinylated Probes
Hajime MOTOYOSHI,1 Masato HORIGOME,1 Ken ISHIGAMI,1 Tatsuhiko YOSHIDA,2
Sueharu HORINOUCHI,2 Minoru YOSHIDA,2;3;4 Hidenori WATANABE,1 and Takeshi KITAHARA
y
1;
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
4CREST Research Project, Japan Science and Technology Agency, Saitama 332-0012, Japan
Received June 8, 2004; Accepted July 14, 2004
The structure-activity relationship for FR901464, a
potent cell-cycle inhibitor, was examined by synthesiz-
ing its analogs. A versatile method for converting
FR901464 was devised. This method made it possible
to synthesize biologically active FR901464-biotin con-
jugates which could be used to isolate the binding
proteins.
Result and Discussion
We first obtained information on the structure-activity
relationship of 1 to construct derivatives which would
retain their biological activity. We had already com-
pleted the total synthesis of 1,5–8) so we tested several
intermediate compounds for their biological activity
(Fig. 2) on the basis of stimulating cytomegalovirus
(CMV) promoter-driven transcription (M. Yoshida,
unpublished results).
Key words: FR901464; cell cycle inhibitor; biotin; con-
version
Comparing compounds 2 and 3 showed that the
epoxide moiety on the right-hand pyran ring was
important for expression of the activity. More interest-
ingly, a methylated FR901464 derivative (2) exhibited
higher activity than that of natural 1, which was thought
to come from the greater stability of the methyl acetal in
contrast to the labile hemiacetal of 1. This indicated that
transforming the acetal moiety did not have a critical
effect on its biological activity. We therefore introduced
functionality into this acetal moiety by transacetaliza-
tion. However, this was difficult to achieve under the
usual acidic conditions, because 1 was extremely
susceptible to acid attack (e.g., acetic acid or PPTS)
and readily decomposed (Fig. 3). Irreversible cleavage
of the right-hand pyran ring accompanied by opening of
the epoxide occurred to give aldehyde A as a degrada-
tion product.
FR901464 (1), which has two highly functionalized
tetrahydropyran rings linked by a diene chain, has been
isolated from the culture broth of Pseudomonas sp.
No. 2663.1,2) This compound showed transcriptional
regulating activity and induced characteristic G1 and
G2/M phase arrest in the cell cycle. Related to these
activities, 1 also exhibited a potent antitumor effect.3,4)
These significant biological activities prompted us to
undertake the synthesis of biotinylated FR901464
derivatives, which could be useful for isolating the
binding proteins, and a mechanistic study on the
expression of the biological activities of 1. We describe
a method for converting FR901464 and the synthesis of
FR901464-biotin conjugate.
To overcome this problem, it was essential to select
suitable reaction conditions. After screening various
possibilities, Amberlyst 15, an ion-exchange resin,9) was
found to be an effective acid catalyst. Treatment of 1
with several alcohols as solvents in the presence of this
resin furnished the corresponding products (2, 6 and 7)
in moderate yields (Scheme 1).
All of these compounds exhibited biological activity,
Fig. 1.
y
To whom correspondence should be addressed. Tel: +81-3-5841-5119; Fax: +81-3-5841-8019; E-mail: atkita@mail.ecc.u-tokyo.ac.jp
Abbreviation: SE, succinimidyl ester

Conversion of FR901464
2179
Fig. 2. Results of the Biological Assay Testing the Structure-activity Relationship for FR901464.
þ/ꢂ shows the relative strength of activity determined by CMV promoter activation.
Fig. 3.
Scheme 1.
and our method was found to provide an efficient route
for preparing a biologically active probe of 1. We next
embarked on the synthesis of a biotin-labeled derivative
by using this method (Scheme 2). We decided to
connect 1 to the biotin portion through an amido
linkage. Therefore, 1 was treated with 2-azidoethanol to
introduce nitrogen functionality.10) The azide group of 8
was reduced with tri-n-butylphosphine and then reacted
with commercially available biotinylating agents to
provide desired FR901464-biotin conjugates such as 9
and 10. These products displayed sufficient activity for a
biological assay, even though they were weaker than
that of natural 1 (ꢀ1 ꢁ 10ꢂ2 compared to natural 1).
Following the example of the literature,10) it might be
possible to isolate binding proteins with probes having
weaker biological activity. We performed several
experiments with 9 and 10, and interestingly, several
binding proteins were positively isolated. Intensive
analyses are now underway.
In conclusion, we obtained information on the
structure-activity relationship of FR901464 and ex-
plored a route to produce derivatives of FR901464
which retained biological activity. We synthesized
FR901464-biotin conjugates which are helpful in com-
prehending the biological activities of FR901464. We
finally isolated the binding proteins. A comprehensive
report on the detailed results of the biological assay will
be presented in due course.

2180
H. MOTOYOSHI et al.
Scheme 2.
Experimental
20-H), 5.89 (1H, dd, J ¼ 7:7, 11.6 Hz, 30-H), 6.00 (1H, d,
J ¼ 9:3 Hz, –NH–), 6.26 (1H, m, 40-H), 6.41 (1H, d,
J ¼ 15:9 Hz, 7-H); HRMS (FAB) m=z: calcd. for
C28H44NO8, 522.3067 (Mþ þ H); found, 522.3091.
Dienal A: 1H-NMR (300 MHz, CDCl3) ꢂ (ppm): 1.05
(3H, d, J ¼ 7:2 Hz, 50-Me), 1.16 (3H, d, J ¼ 6:5 Hz, 20-
Me), 1.40 (3H, d, J ¼ 6:5 Hz, 5-H), 1.78–1.83 (1H, m,
50-H), 1.84 (3H, s, 300-H), 1.96 (1H, ddd, J ¼ 4:5, 4.5,
15.0 Hz, 40-Ha), 2.02 (1H, ddd, J ¼ 2:5, 2.5, 15.0 Hz, 40-
Hb), 2.05 (3H, s, 4-OAc), 2.32 (1H, ddd, J ¼ 6:0, 6.0,
16.0 Hz, 100-Ha), 2.48 (1H, ddd, J ¼ 7:0, 7.0, 16.0 Hz,
100-Hb), 3.61 (1H, ddd, J ¼ 4:0, 6.0, 7.0 Hz, 60-H), 3.69
(1H, dq, J ¼ 2:2, 6.5 Hz, 20-H), 3.94–4.00 (1H, m, 30-H),
5.72 (1H, d, J ¼ 12:0 Hz, 2-H), 5.90 (1H, dd, J ¼ 7:0,
12.0 Hz, 3-H), 6.02 (1H, brd, J ¼ 9:0 Hz, NH), 6.05 (1H,
dd, J ¼ 6:0, 7.0 Hz, 200-H), 6.12 (1H, dd, J ¼ 8:0,
15.5 Hz, 500-H), 6.24 (1H, dq, J ¼ 6:5, 7.0 Hz, 4-H), 7.14
(1H, d, J ¼ 15:5 Hz, 400-H), 9.58 (1H, d, J ¼ 8:0 Hz,
600-H).
IR spectra were measured with a Jasco FT/IR-230
1
spectrophotometer, H-NMR were recorded with a Jeol
JNM AL300, and mass spectra were recorded with a
Jeol JMS-700T. TLC was carried out on Merck glass
plates precoated with silica gel 60 F254 (0.5 mm). HPLC
was performed by a Showa Denko Shodex DS-4
instrument equipped with a Senshu Pak Pegasil ODS
(4:6ꢀ ꢁ 250 mm) or Senshu Pak Pegasil ODS (20ꢀ ꢁ
250 mm) column. The succinimidyl ester of 6-
{60-(biotinoylamino)hexanoylamino}hexanoic acid was
purchased from Molecular Probes, Inc.
FR901464 methyl acetal (2). To a cooled (ꢂ10 ꢃC)
solution of FR901464 (30.0 mg, 0.059 mmol) in meth-
anol (2 ml) was added Amberlyst 15 (13 mg), before the
mixture was stirred for 30 min at this temperature and
then for 3 h at room temperature. The reaction mixture
was diluted with ether and filtered through CeliteÒ. The
solvent was removed under reduced pressure. The
residue was purified by preparative TLC (1:3 hexane/
EtOAc) to provide 2 as a white powder (15.3 mg, 50%)
and unreacted starting material (4.3 mg), accompanied
by a small amount (ꢀ10%) of dienal A. The yield was
calculated to be 58% based on the recovered starting
material.
FR901464 ethyl acetal (6). A solution of FR901464
(10.0 mg, 0.020 mmol) in ethanol (2 ml) was cooled to
ꢂ10 ꢃC, treated with Amberlyst 15 (13 mg) and gradual-
ly warmed to room temperature during 6 h while stirring.
The reaction mixture was diluted with ether and filtered
through CeliteÒ, the solvent being removed under
reduced pressure. The residue was purified by prepara-
tive TLC (1:3 hexane/EtOAc) to provide 6 as a colorless
oil (2.6 mg, 25%).
IR (CHCl3 solution) ꢁmax cmꢂ1: 1732, 1670, 1505,
1372, 1250, 1056; 1H-NMR (300 MHz, CDCl3) ꢂ (ppm):
1.02 (3H, d, J ¼ 7:2 Hz, 20-H), 1.15 (3H, d, J ¼ 6:5 Hz,
16-H), 1.25 (3H, t, J ¼ 7:1 Hz, CH3 of –OEt), 1.39 (3H,
d, J ¼ 6:6 Hz, 50-H), 1.40 (3H, s, 17-H), 1.72 (1H, d,
J ¼ 10:7 Hz, –OH), 1.76 (1H, d, J ¼ 14:6 Hz, 2-Ha),
1.77–1.82 (1H, m, 12-H), 1.79 (3H, s, 19-H), 1.92–1.96
(2H, m, 13-H), 2.04 (3H, s, –OAc), 2.18–2.43 (2H, m,
10-H), 2.28 (1H, d, J ¼ 14:6 Hz, 2-Hb), 2.50 (1H, d,
J ¼ 4:5 Hz, 18-Ha), 2.98 (1H, d, J ¼ 4:5 Hz, 18-Hb),
3.52 (1H, m, 11-H), 3.53 (2H, q, J ¼ 7:1 Hz, CH2 of
–OEt), 3.60 (1H, dd, J ¼ 9:5, 10.7 Hz, 4-H), 3.67 (1H,
dq, J ¼ 2:0, 6.5 Hz, 15-H), 3.94 (1H, m, 14-H), 4.07
FR901464 methyl acetal (2): IR (KBr) ꢁmax cmꢂ1
:
3448, 2932, 1737, 1668, 1637, 1523; 1H-NMR
(300 MHz, CDCl3) ꢂ (ppm): 1.02 (3H, d, J ¼ 7:2 Hz,
20-H), 1.15 (3H, d, J ¼ 6:5 Hz, 16-H), 1.39 (3H, d,
J ¼ 6:3 Hz, 50-H), 1.39 (3H, s, 17-H), 1.71 (1H, d,
J ¼ 10:7 Hz, –OH), 1.74 (1H, d, J ¼ 14:7 Hz, 2-Ha),
1.78–1.82 (1H, m, 12-H), 1.80 (3H, s, 19-H), 1.92–1.98
(2H, m, 13-H), 2.04 (3H, s, –OAc), 2.18–2.45 (2H, m,
10-H), 2.31 (1H, d, J ¼ 14:7 Hz, 2-Hb), 2.50 (1H, d,
J ¼ 4:5 Hz, 18-Ha), 2.99 (1H, d, J ¼ 4:5 Hz, 18-Hb),
3.28 (3H, s, –OMe), 3.52 (1H, dt, J ¼ 2:6, 7.1 Hz, 11-
H), 3.61 (1H, dd, J ¼ 9:5, 10.7 Hz, 4-H), 3.67 (1H, dq,
J ¼ 2:4, 6.5 Hz, 15-H), 3.94 (1H, m, 14-H), 4.05 (1H,
dd, J ¼ 7:2, 9.5 Hz, 5-H), 5.51 (1H, t, J ¼ 6:9 Hz, 9-H),
5.65–5.75 (1H, m, 6-H), 5.71 (1H, dd, J ¼ 1:2, 11.6 Hz,

Conversion of FR901464
2181
(1H, dd, J ¼ 7:2, 9.5 Hz, 5-H), 5.51 (1H, t, J ¼ 6:9 Hz,
9-H), 5.65–5.75 (1H, m, 6-H), 5.71 (1H, dd, J ¼ 1:2,
11.5 Hz, 20-H), 5.89 (1H, dd, J ¼ 7:7, 11.5 Hz, 30-H),
5.99 (1H, d, J ¼ 8:7 Hz, –NH–), 6.26 (1H, m, 40-H),
6.38 (1H, d, J ¼ 15:6 Hz, 7-H); HRMS (FAB) m=z:
calcd. for C29H46NO8, 536.3223 (Mþ þ H); found,
536.3223.
(3H, d, J ¼ 6:6 Hz, 16-H), 1.2–2.1 (23H, m, 2-Ha, 12-H,
13-H, –OH, methylenes), 1.38 (3H, d, J ¼ 6:6 Hz, 50-H),
1.39 (3H, s, 17-H), 1.76 (3H, s, 19-H), 2.04 (3H, s,
–OAc), 2.1–2.45 (9H, m, 2-Hb, 10-H, three –C(O)CH2),
2.53 (1H, d, J ¼ 4:8 Hz, 18-Ha), 2.71 (1H, d, J ¼
12:5 Hz, biotin –S–CH–), 2.90 (1H, dd, J ¼ 4:8,
12.5 Hz, biotin –S–CH–), 3.07 (1H, d, J ¼ 4:8 Hz, 18-
Hb), 3.1–3.35 (5H, m, biotin –S–CH–, two –NCH2–),
3.45–3.75 (7H, m, 4-H, 11-H, 15-H, acetal –OCH2–,
–NCH2–), 3.92 (1H, m, 14-H), 4.07 (1H, dd, J ¼ 6:8,
9.5 Hz, 5-H), 4.31 (1H, m, biotin –N–CH–), 4.49 (1H,
m, biotin –N–CH–), 5.45–5.55 (2H, m, H-9, –NH–),
5.66 (1H, dd, J ¼ 6:8, 15.6 Hz, 6-H), 5.74 (1H, d,
J ¼ 11:7 Hz, 20-H), 5.89 (1H, dd, J ¼ 7:8, 11.7 Hz, 30-
H), 6.1–6.20 (2H, m, two –NH–), 6.27 (1H, m, 40-H),
6.3–6.37 (1H, m, –NH–), 6.35 (1H, d, J ¼ 15:6 Hz, 7-
H). 6.42 (1H, m, –NH–), 6.67 (1H, m, –NH–); LRMS
(FAB) m=z: calcd. for C51H83N6O12S, 1003.5790
(Mþ þ H); found, 1003.5788.
FR901464 2-azidoethyl acetal (8). A solution of
FR901464 (30.0 mg, 0.059 mmol) in 2-azidoethanol11)
(0.6 ml) was cooled to ꢂ10 ꢃC, treated with Amberlyst
15 (5 mg) and gradually warmed to room temperature
during 6 h while stirring. The reaction mixture was
diluted with ethyl acetate and filtered through CeliteÒ.
The resulting solution was successively washed with
water, a saturated NaHCO3 solution and brine, before
drying with Na2SO4. The solvent was removed, and the
residue was purified by preparative TLC (1:3 hexane/
EtOAc) to provide 8 as a colorless oil (24.9 mg, 73%)
and the unreacted starting material (3.3 mg). The yield
was calculated to be 82% based on the recovered
starting material.
Acknowledgments
IR (CHCl3 solution) ꢁmax cmꢂ1: 2112, 1732, 1670,
The authors sincerely thank Dr. H. Nakajima of
Fujisawa Pharmaceutical Co., Ltd., for a kindly present-
ing natural FR901464 and its spectral charts. This work
was supported by a Grant-in-Aid for Scientific Research
from the Japanese Ministry of Education, Culture,
Sports, Science and Technology. The authors also thank
Sankyo Foundation of Life Science and Suntory Institute
for Bioorganic Research for financial support.
1
1504, 1372, 1251, 1054; H-NMR (300 MHz, CDCl3) ꢂ
(ppm): 1.02 (3H, d, J ¼ 7:2 Hz, 20-H), 1.15 (3H, d,
J ¼ 6:5 Hz, 16-H), 1.39 (3H, d, J ¼ 6:6 Hz, 50-H), 1.43
(3H, s, 17-H), 1.75–1.80 (1H, m, 12-H), 1.76 (1H, d,
J ¼ 10:8 Hz, –OH), 1.79 (3H, s, 19-H), 1.80 (1H, d,
J ¼ 14:6 Hz, 2-Ha), 1.92–1.97 (2H, m, 13-H), 2.04 (3H,
s, –OAc), 2.18–2.45 (2H, m, 10-H), 2.31 (1H, d,
J ¼ 14:6 Hz, 2-Hb), 2.51 (1H, d, J ¼ 4:5 Hz, 18-Ha),
2.99 (1H, d, J ¼ 4:5 Hz, 18-Hb), 3.46 (2H, t, J ¼ 5:0 Hz,
–CH2N3), 3.52 (1H, dt, J ¼ 2:7, 7.2 Hz, 11-H), 3.58–
3.70 (2H, m, 4-H, 15-H), 3.63 (2H, t, J ¼ 5:0 Hz, acetal
–OCH2–), 3.94 (1H, m, 14-H), 4.15 (1H, dd, J ¼ 7:5,
9.0 Hz, 5-H), 5.51 (1H, t, J ¼ 7:2 Hz, 9-H), 5.65–5.74
(1H, m, 6-H), 5.70 (1H, dd, J ¼ 1:2, 11.6 Hz, 20-H),
5.89 (1H, dd, J ¼ 7:8, 11.6 Hz, 30-H), 6.01 (1H, d,
J ¼ 9:3 Hz, –NH–), 6.26 (1H, m, 40-H), 6.40 (1H, d,
J ¼ 15:6 Hz, 7-H); HRMS (FAB) m=z: calcd. for
C29H45N4O8, 577.3237 (Mþ þ H); found, 577.3241.
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