Practical synthesis of blepharismone, a mating inducing pheromone of Blepharisma japonicum

Article abstract of DOI:10.1016/j.tetasy.2006.09.005

Both enantiomers of blepharismone, a mating inducing pheromone produced by type II cells of Blepharisma japonicum, were synthesized via the Stille cross-coupling reaction of [4-(tert-butyl-dimethyl-silanyloxy)-2-trimethylstannanyl-phenyl]-carbamic acid te

Full text of DOI:10.1016/j.tetasy.2006.09.005

Tetrahedron: Asymmetry 17 (2006) 2339–2343
Practical synthesis of blepharismone, a mating inducing
pheromone of Blepharisma japonicum
Hisashi Takihiro,a Yoshiyuki Uruma,a Yoshinosuke Usuki,a Akio Miyakeb and Hideo Iioa,*
aDepartment of Material Science, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan
bDepartment of Molecular Cellular and Animal Biology, University of Camerino, Via F. Camerini 2, 62032 Camerino (MC), Italy
Received 25 July 2006; accepted 4 September 2006
Abstract—Both enantiomers of blepharismone, a mating inducing pheromone produced by type II cells of Blepharisma japonicum, were
synthesized via the Stille cross-coupling reaction of [4-(tert-butyl-dimethyl-silanyloxy)-2-trimethylstannanyl-phenyl]-carbamic acid
tert-butyl ester with an acid chloride derived from (S)- and (R)-malic acid as a key reaction. The mating inducing activity of synthetic
(S)-blepharismone was as effective as that of the natural one. The enantiomer (R)-blepharismone showed no mating inducing activity.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
O
OH
HO
O Ca
2
In the ancestral ciliate Blepharisma japonicum, conjugation
occurs between mating type I and II cells under a nutrient-
deficient condition by secreting gamones (mating inducing
pheromones) in the medium.1 Blepharismoneꢀ 1 was iso-
lated as gamone 2 from mating type II cells, which induces
conjugation at 1 ng/ml. Further, the structure of 1 was
determined as a calcium salt of 4-(2-formylamino-5-hydroxy-
phenyl)-2(S)-hydroxy-4-oxo-butyric acid.2 On the other
hand, the amino acid sequence of blepharismone, a glyco-
protein isolated as gamone 1 from mating type I cells of B.
japonicum,3 was recently determined by Harumoto and
Sugiura;4 further, it was shown that the expression of
blepharismone was regulated developmentally and envi-
ronmentally.5 The syntheses of racemic 1 were reported
by Tokoroyama et al.6 and Jaenicke et al.,7 albeit in low
yield. The enantiomer of 1 has also been synthesized, and
it was reported that it had no mating inducing activity.8
In our effort to clarify the molecular mechanisms that reg-
ulate the conjugation of B. japonicum, we required molecu-
lar probes for elucidating the receptor of the gamones. In
this paper, we describe the practical syntheses of enantio-
merically pure 1 and its enantiomer using the Stille cross-
coupling reaction as a key reaction.
O
NHCHO
Blepharismone 1
2. Results and discussion
2.1. Synthesis of both enantiomers of blepharismone
Blepharismone 1 is a tri-substituted benzene derivative,
which was fully functionalized and presumably synthesized
biogenetically from tryptophan by hydroxylation at the C5
position of the indole ring, oxidative cleavage of the indole,
and replacement of the amino group by a hydroxyl group.
Compound 1 bears a structural resemblance to kynurenine,
which was successfully synthesized by Salituro et al.9 using
the Stille coupling method;10 their success has prompted us
to apply their method for the synthesis of 1. [4-(tert-Butyl-
dimethyl-silanyloxy)-phenyl]-carbamic acid tert-butyl ester
(2) was prepared from p-aminophenol according to the
procedure reported by Isobe et al.11 While the hydroxyl
group of p-aminophenol was initially protected by a
methoxy group, deprotection of derivative 6b could not
be achieved without a dehydration reaction of the side
chain (vide infra); therefore, the hydroxyl group was
protected by a tert-butyldimethylsilyl (TBDMS) group.
The directed metallation of 2 using tert-butyllithium12
proceeds regioselectively11 to afford 3 by the addition of
*
Corresponding author. E-mail: iio@sci.osaka-cu.ac.jp
ꢀ Although gamone 2 was originally called blepharismin, the name was
changed to blepharismone to avoid confusion since the red pigment of
Blepharisma, zoopurpurin, was renamed as blepharismin.1c
0957-4166/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2006.09.005

2340
H. Takihiro et al. / Tetrahedron: Asymmetry 17 (2006) 2339–2343
O
O
TBDMSO
R
R'O
O
O
O
Pd(CH3CN)2Cl2
69%
+
O
O
X
NHBoc
NHBoc
O
t
-BuLi
2 R = H
3 R = SnMe3
6a R' = TBDMS
6b R' = Me
4 X = OH
5 X = Cl
then
Me3SnCl
60%
O
OH
O
O
HO
O Ca
2
HO
O
HF-Py
79%
HCO2H, Ac2O
O
O
NHCHO
then CaCO3
52%
NHBoc
1
7
H6
O
OH
MeO
OMe
HCO2H, AcOH
then CH2N2
O
NHCHO
8
Scheme 1.
Table 1. 1H NMR chemical shifts of H6 in compounds (S)- and (R)-8
trimethyltin chloride. The crucial cross-coupling reaction
of 3 and acid chloride 5 derived from (S)-(2,2-dimethyl-5-
oxo-[1,3]dioxolan-4-yl)-acetic acid (4),13 which was pre-
pared from (S)-(+)-malic acid, was carried out using
10 mol % of PdCl2(CH3CN)2 in toluene to afford 6a in
63% yield. It should be mentioned that the use of acid chlo-
ride 5, which was free from acid and prepared by adding a
small amount of dimethylformamide in oxalyl chloride,14
was crucial. Otherwise, the coupling yield of 6a decreased
dramatically. The use of palladium catalyst Pd2(dba)3 in-
stead of PdCl2(CH3CN)2 did not improve the yield. The re-
moval of the TBDMS group by tetrabutylammonium
fluoride was unsuccessful15 because a dehydration reaction
occurred on the side chain of the b-alkoxy carbonyl func-
tionality to afford a 4-keto-2-butenoic acid derivative. In-
stead, removal was accomplished by the treatment of 6a
with HFÆpyridine16 to afford 7 in 79% yield. Removal of
the acetonide and N-Boc groups, and formylation of the
resulting amino group were carried out in one step by the
treatment of 7 with a 1:5 mixture of acetic anhydride and
formic acid. Triturating the resulting polar product in a
saturated aq calcium carbonate solution yielded crystalline
(S)-1 in 52% isolated yield (Scheme 1); this was identical to
natural blepharismone in all respects. For biological evalu-
ation, (R)-blepharismone ((R)-1), an enantiomer of natural
blepharismone, was also synthesized from (R)-malic acid
by using the same method described above.
Conditions
Chemical shift
(ppm) of H6
(S)- and (R)-8 without (+)-Eu(hfc)3
(S)-8 with 1 equiv of (+)-Eu(hfc)3
(R)-8 with 1 equiv of (+)-Eu(hfc)3
7.33
8.03
8.23
measurement of 8 in the presence of a chiral shift reagent
1
[(+)-Eu(hfc)3]. We discovered that the H NMR signals
of H6 on the aromatic ring of (S)- and (R)-8 in the presence
of 1 equiv of (+)-Eu(hfc)3 were separated at the baseline
level (Table 1); further, none of the enantiomers were
detected in each sample. Hence, it was concluded that the
synthetic (S)- and (R)-1 were enantiomeric pure.
The mating inducing activity of synthetic (S)-blepharis-
mone (1) was as effective as that of the natural one (1 ng/
l unit activity)ꢁ for the type I cell of B. japonicum. Synthetic
(R)-blepharismone, the enantiomer of 1, showed no mating
inducing activity at a concentration of up to 500 lg/mL.
3. Conclusion
We have synthesized enantiomerically pure (S)-blepharis-
mone (1), a mating inducing pheromone produced by type
II cell, of B. japonicum, via the Stille cross-coupling reaction
of [4-(tert-butyl-dimethyl-silanyloxy)-2-trimethylstannanyl-
phenyl]-carbamic acid tert-butyl ester (3) and acid chloride 5
derived from (S)-malic acid as a key reaction. Enantiomer
(R)-1 was also prepared by an identical method. The enan-
tiomeric purities of synthetic blepharismones were unam-
2.2. Enantiomeric purity and biological activity of synthetic
(S)- and (R)-blepharismones
In order to determine the enantiomeric purity of synthetic
(S)- and (R)-1, we tried to prepare a Mosher ester of 1;17
however, a dehydration reaction on the side chain oc-
curred, and we could not obtain the ester. Hence, dimethyl
derivative 8 was synthesized by treating with diazome-
thane, the crude intermediate after deprotection and
formylation of 7 with acetic anhydride in formic acid. We
ꢁ The mating inducing activity of blepharismone in the conjugation of
B. japonicum can be defined as follows: 1 ng/l unit activity denotes that a
concentration of 1 ng of blepharismone in 1 mL of the culture medium
containing 1000 cells of B. japonicum causes one conjugation pair in the
medium.2
1
could determine the enantiomeric purity using H NMR

H. Takihiro et al. / Tetrahedron: Asymmetry 17 (2006) 2339–2343
2341
biguously determined by 1H NMR analysis of the dimethyl
derivative of 1 in the presence of a chiral shift reagent. The
mating inducing activity of synthetic (S)-1 was found to be
as potent as that of natural 1, while enantiomer (R)-1 was
found to have no mating inducing activity at all. The inter-
mediates in our synthesis of 1 could be applied to the synthe-
sis of molecular probes for elucidating the gamone receptor
of B. japonicum, and the results will be reported in due course.
(60%) as a slightly yellow oil. 1H NMR (400 MHz, CDCl3)
d 0.18 (s, 6H), 0.31 (s, 9H), 0.98 (s, 9H), 1.49 (s, 9H), 6.11
(br s, 1H), 6.76 (dd, 1H, J = 8.2, 3.1 Hz), 6.85 (d, 1H,
J = 3.1 Hz), 7.25 (d, 1H, J = 8.2 Hz); 13C NMR
(100 MHz, CDCl3) d ꢀ8.7, ꢀ4.4, 18.2, 25.7, 26.3, 28.4,
80.0, 120.2, 120.6, 127.2, 136.3, 153.0, 154.4; IR (neat) mmax
3300, 1715, 1520, 1150 cmꢀ1; MS (EI) m/z 487 (M+, 0.8),
472 (11), 416 (100), 398 (36), 372 (17), 342 (6), 284 (5),
267 (21), 223 (25), 210 (31), 192 (13), 166 (64), 135 (6),
106 (5), 73 (41), 57 (46). HRDEIMS m/z; [MꢀH]+ calcd
for C20H36NO3SiSn, 486.1486. Found: 486.1491.
4. Experimental
4.1. General
4.3. (S)-{4-(tert-Butyl-dimethyl-silanyloxy)-2-[2-(2,2-
dimethyl-5-oxo-[1,3]dioxolan-4-yl)-acetyl]-phenyl}-carbamic
acid tert-butyl ester (S)-6a
1H and 13C NMR spectra were recorded, respectively, at
400 and 100 MHz (JEOL JNM LA400) or 300 and
75 MHz (JEOL JNM LA300). Chemical shifts are reported
in ppm relative to Me4Si and CDCl3 with CHCl3 as the
To a solution of (S)-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4-
yl)-acetic acid (S)-413 (1.19 g, 6.84 mmol) in CH2Cl2
(15 mL) in the presence of a catalytic amount of DMF
was added (COCl)2 (0.59 mL 16.9 mmol) at room tempera-
ture under an argon atmosphere, and the mixture was stir-
red for 30 min. The solvent was removed under reduce
pressure to afford acid chloride (S)-5. The acid chloride
was dissolved in anhydrous toluene (10 mL), and to a solu-
tion were added PdCl2(CH3CN)2 (187 mg, 0.720 mmol) and
a solution of 3 (3.25 g, 6.69 mmol) in anhydrous toluene
(30 mL) and the mixture was stirred for 4 h at room temper-
ature. Palladium was removed by filtration, and the filtrate
was concentrated under reduced pressure. The resulting
crude product was purified by silica gel chromatography
1
internal reference (7.26 ppm for H NMR and 77.0 ppm
for 13C NMR). Coupling constants are reported in hertz
(Hz) and determined directly from 1H NMR spectra. Spec-
tral splitting patterns were designated as s (singlet), d (dou-
blet), t (triplet), m (multiplet), and br (broad). Mass spectra
were obtained on JEOL JMS-700T or JEOL JMS-AX500
spectrometers. Infrared spectra were taken on a Jasco A-
100 spectrometer or a Hitachi 270-30 spectrophotometer.
Optical rotations were measured on a Jasco DIP-370
digital polarimeter. Elemental analyses were carried out
at Analytical Center of Osaka City University.
All air- and moisture-sensitive reactions were carried out in
flame-dried, argon-flushed, two-necked flask sealed with
rubber septum, and dry solvents and reagents were intro-
duced with a syringe. THF and Et2O were freshly distilled
from sodium benzophenone ketyl. CH2Cl2, CHCl3, and
(Et2O/hexane = 1:12–1:5) to give 2.21 g of (S)-6a (69%) as
28
a pale yellow oil. ½aꢁD ¼ þ4:61 (c 3.93, CH3OH). 1H
NMR (400 MHz, CDCl3) d 0.20 (s, 6H), 0.99 (s, 9H), 1.51
(s, 9H), 1.62 (s, 3H), 1.65 (s, 3H), 3.39 (dd, 1H, J = 18.0,
7.3 Hz), 3.61 (dd, 1H, J = 18.0, 3.1 Hz), 4.94 (dd, 1H,
J = 7.3, 3.1 Hz), 7.06 (dd, 1H, J = 9.2, 3.1 Hz), 7.24 (d,
1H, J = 3.1 Hz), 8.36 (d, 1H, J = 9.2 Hz), 10.44 (br s,
1H). 13C NMR (100 MHz, CDCl3) d ꢀ4.3, 18.2, 25.5,
25.6, 26.9, 28.3, 41.4, 70.0, 80.4, 111.3, 115.0, 120.9, 121.5,
127.5, 136.9, 149.3, 153.2, 173.0, 197.8. IR (Nujol): mmax
3220, 1785, 1715, 1655, 1515, 1180, 1150, 1120, 1045, 940,
840 cmꢀ1. MS (EI) m/z 479 (M+, 12), 423 (13), 379 (100),
348 (5), 321 (20), 293 (23), 276 (13), 264 (57), 250 (13),
220 (12), 192 (49), 166 (6), 146 (6), 129 (22), 106 (4), 85
(6), 73 (36), 57 (30). HRDEIMS m/z; [M]+ calcd for
C24H37NO7Si, 479.2339. Found: 479.2334.
˚
toluene were distilled from P2O5 and stored over 4-A
molecular sieves. Pyridine was dried over KOH and stored
˚
over 4-A molecular sieves. TLC was performed on Merck
precoated silica gel plates (#5715), and the TLC spots were
visualized under 254-nm UV light and/or by charring after
dropping the plate into vanillin solution in 5% sulfuric
acid/methanol. Purification of products was accomplished
via Merck silica gel (#7734 and #9385) flash chromatogra-
phy. Hexane and ethyl acetate were distilled and used in
column chromatography.
4.2. [4-(tert-Butyl-dimethyl-silanyloxy)-2-trimethylstannan-
yl-phenyl]-carbamic acid tert-butyl ester 3
4.4. (R)-{4-(tert-Butyl-dimethyl-silanyloxy)-2-[2-(2,2-
dimethyl-5-oxo-[1,3]dioxolan-4-yl)-acetyl]-phenyl}-carbamic
acid tert-butyl ester (R)-6a
To a solution of [4-(tert-butyl-dimethyl-silanyloxy)-phen-
yl]-carbamic acid tert-butyl ester 2 (3.62 g, 11.2 mmol) in
anhydrous THF (40 mL) was added t-BuLi (1.35 M pen-
tane solution, 20 mL, 27.0 mmol) at ꢀ78 °C under an
argon atmosphere. The mixture was stirred for 15 min at
ꢀ78 °C, and 2.5 h at ꢀ20 °C. After dropwise addition of
trimethyltin chloride (1.0 M THF solution, 11.2 mL,
11.2 mmol) at ꢀ20 °C, the mixture was stirred for an addi-
tional 20 min. The resulting mixture was treated with H2O
and extracted with AcOEt. The organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified by silica gel chro-
matography (Et2O/hexane, 1:14) to afford 3.25 g of 3
Using the same procedure as above, (R)-413 (398 mg,
2.29 mmol) and 3 (1.10 g, 2.27 mmol) gave 644 mg of (R)-
24
6a (59%) as a pale yellow oil. ½aꢁD ¼ ꢀ4:73 (c 4.14,
CH3OH). HRDEIMS m/z; [M]+ calcd for C24H37NO7Si,
479.2339. Found: 479.2320.
4.5. (S)-{2-[2-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4-yl)-acet-
yl]-4-hydroxy-phenyl}-carbamic acid tert-butyl ester (S)-7
To a solution of (S)-6a (962 mg, 2.01 mmol) in anhydrous
THF (10 mL) was added HFÆpyridine (2 mL) at 0 °C under

2342
H. Takihiro et al. / Tetrahedron: Asymmetry 17 (2006) 2339–2343
an argon atmosphere, and the mixture was stirred for 1 h.
The cooling bath was removed and stirred for additional
1 h. To the reaction mixture was added saturated aq NaH-
CO3 solution, and extracted with AcOEt. The organic layer
was washed with brine and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure, and the
resulting residue was purified by silica gel chromatography
(AcOEt/hexane = 1:3). The crystalline product was further
4.8. (R)-4-(2-Formylamino-5-methoxy-phenyl)-2-hydroxy-4-
oxo-butyric acid methyl ester (R)-8
Using the same procedure as above, (R)-7 (80.0 mg,
0.219 mmol) gave 46.2 mg of (R)-8 (75%) as a pale yellow
solid.
4.9. (S)-Blepharismone (S)-1
recrystallized from Et2O/hexane to give 580 mg of (S)-7
25
(79%) as pale yellow needles; mp 147–148 °C. ½aꢁD
¼
To a suspension of (S)-7 (166 mg, 0.455 mmol) in formic
acid (3 mL) was added acetic anhydride (0.6 mL) at room
temperature. The reaction mixture was stirred for 1.5 h,
and then concentrated in vacuo. To the residue were added
saturated aq CaCO3 (1 mL) and CaCO3 (21.2 mg, 0.21
mmol) to give a precipitate, which was collected by filtra-
1
þ6:55 (c 3.97, CH3OH). H NMR (400 MHz, CDCl3) d
1.51 (s, 9H), 1.62 (s, 3H), 1.65 (s, 3H), 3.39 (dd, 1H,
J = 18.0, 7.3 Hz), 3.61 (dd, 1H, J = 18.0, 3.1 Hz), 4.92
(dd, 1H, J = 7.3, 3.1 Hz), 5.70 (br s, 1H), 7.04 (dd, 1H,
J = 9.2, 2.4 Hz), 7.25 (d, 1H, J = 2.4 Hz), 8.30 (d, 1H,
J = 9.2 Hz), 10.36 (br s, 1H). 13C NMR (100 MHz, CDCl3)
d 25.9, 26.8, 28.3, 41.4, 70.0, 80.6, 111.6, 115.0, 121.3, 121.6,
123.1, 135.4, 149.7, 153.4, 173.4, 197.8. 1R (Nujol) mmax
3440, 3300, 1770, 1725, 1655, 1510, 1290, 1180, 1150,
1130, 830 cmꢀ1. MS (EI) m/z 365 (M+, 16), 354 (2), 309
(17), 291 (13), 265 (94), 234 (12), 220 (4), 207 (57), 179
(24), 162 (100), 151 (28), 136 (39), 108 (20), 91 (6), 85
(12), 71 (7), 57 (73). HRDEIMS m/z; [M]+ calcd for
C18H23NO7, 365.1475. Found: 365.1463. Elemental Anal.
Calcd: C, 59.17; H, 6.35; N, 3.83. Found: C, 59.10; H,
6.40; N, 3.95.
tion. Recrystallization from H2O gave 64 mg of (S)-1 (52%)
27
as a yellow solid. ½aꢁD ¼ ꢀ94:5 (c 1.07, DMSO). 1H
NMR (400 MHz, DMSO-d6) d 3.17 (br s, 1H), 3.34 (s,
2H), 4.31 (br s, 1H), 6.04 (br s, 1H), 6.92 (d, 1H, J = 8.8,
2.4 Hz), 7.49 (s, 1H), 8.07 (d, 1H, J = 8.8 Hz), 8.29 (d,
1H, J = 2.4 Hz), 10.64 (s, 1H). 13C NMR (100 MHz,
DMSO-d6) d 45.8, 69.3, 116.7, 120.1, 123.1, 127.5, 128.9,
153.4, 160.1, 177.6, 202.5. IR (Nujol) mmax 3600–3000
(br), 1680, 1650, 1600, 1440 cmꢀ1
.
4.10. (R)-Blepharismone (R)-1
Under the same procedure as above, (R)-7 (151 mg, 0.414
4.6. (R)-{2-[2-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4-yl)-acet-
yl]-4-hydroxy-phenyl}-carbamic acid tert-butyl ester (R)-7
26
mmol) gave 58 mg of (R)-1 (54%). ½aꢁD ¼ þ94:7 (c 1.08,
25
rt
DMSO), ½aꢁD ¼ þ17:9 (c 0.50, H2O), ðlit:: ½aꢁ578 nm
¼
þ24:3 (H2O)).8
Using the same procedure as above, (R)-6a (644 mg,
1.43 mmol) gave 432 mg of (R)-7 (83%) as pale yellow
26
needles; mp 147–149 °C. ½aꢁD ¼ ꢀ6:26 (c 3.92, CH3OH).
Acknowledgements
HRDEIMS m/z; [M]+ calcd for C18H23NO7, 365.1475.
Found: 365.1462. Elemental Anal. Calcd: C, 59.17; H,
6.35; N, 3.83. Found: C, 59.16; H, 6.38; N, 3.91.
This work was supported in part by a Grant-in-Aid for
Scientific Research (Grant No. 11680592) and Grants-in-
Aid for Priority Area Research Program (Grant No.
12045256) from the Ministry of Education, Science,
Culture and Sports of Japan.
4.7. (S)-4-(2-Formylamino-5-methoxy-phenyl)-2-hydroxy-4-
oxo-butyric acid methyl ester (S)-8
To a suspension of (S)-7 (80.3 mg, 0.22 mmol) in formic
acid (1.5 mL) was added acetic anhydride (0.30 mL) at
room temperature. The reaction mixture was stirred for
1.5 h and then concentrated in vacuo. The residue was
treated with an excess of diazomethane in Et2O (8 mL).
The reaction mixture was concentrated under reduced
pressure and the resulting crude product was purified
by preparative silica gel TLC (AcOEt/hexane = 1:1) to
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25
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.
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