Synthesis of fluorescent molecular probes specific for the receptor of blepharismone, a mating-inducing pheromone of the ciliate Blepharisma japonicum

Article abstract of DOI:10.1016/j.bmc.2006.12.021

Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molecular probes for t

Full text of DOI:10.1016/j.bmc.2006.12.021

Bioorganic & Medicinal Chemistry 15 (2007) 1622–1627
Synthesis of fluorescent molecular probes specific for the receptor
of blepharismone, a mating-inducing pheromone of
the ciliate Blepharisma japonicum
Yoshiyuki Uruma,a Mayumi Sugiura,b Terue Harumoto,b
Yoshinosuke Usukia and Hideo Iioa,*
aDepartment of Material Science, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan
bCellular and Animal Biology, Nara Women’s University, Nara 630-8506, Japan
Received 30 September 2006; revised 10 December 2006; accepted 12 December 2006
Available online 14 December 2006
Abstract—Blepharismone (gamone 2) is a mating-inducing pheromone of the ciliate Blepharisma japonicum. N-Pyrenylbutyryl-
blepharismone and N-biphenylacetyl-blepharismone, which are fluorescent derivatives of blepharismone, were synthesized as molec-
ular probes for the gamone 2 receptor. Further, we proved that they have inhibitory activities against the blepharismone-induced
monotypic pairing of B. japonicum.
Published by Elsevier Ltd.
1. Introduction
dimethyl-silanyloxy)-2-trimethylstannanyl-phenyl]-car-
bamic acid tert-butyl ester with an acid chloride derived
from (S)- and (R)-malic acid; this was a key reaction
(Scheme 1). Further, we showed that the mating-induc-
ing activity of synthetic (S)-blepharismone was as effec-
tive as that of its natural counterpart, while the
enantiomer (R)-blepharismone showed no mating-
inducing activity.7
Sexual reproduction in protozoan ciliates occurs by con-
jugation via specific interactions between cells of com-
plementary mating types.1 The ancestral ciliate
Blepharisma japonicum has two mating types I and II.
Substances that play the role of signaling molecules in
these extracellular interactions for conjugation are
termed gamones.2 Gamone 1 is a glycoprotein produced
by mating type I cells of B. japonicum.3 It is a key factor
required to trigger such interactions, and its complete
amino acid sequence was recently determined.4 We
showed that transcription of the gamone 1 gene is strictly
regulated by developmental and environmental factors.5
On the other hand, blepharismone (1) that was isolated
as gamone 2 from mating type II cells induces conjuga-
tion when present in a concentration of 1 ng/mL. The
structure of 1 was determined to be that of a calcium salt
of (S)-4-(2-formylamino-5-hydroxy-phenyl)-2-hydroxy-
4-oxo-butyric acid, which is a small molecule presum-
ably derived from tryptophan.6 Recently, we reported
the practical synthesis of both the enantiomers of 1 via
the Stille cross-coupling reaction of [4-(tert-butyl-
Miyake proposed that preconjugant interactions in
B. japonicum occur via the following mechanisms. Mat-
ing type I cells autonomously secrete gamone 1 (step 1).
Then, gamone 1 specifically acts on mating type II cells
(step 2) to transform them so that they can undergo con-
jugation (step 3) and it simultaneously induces or
enhances the production and excretion of gamone 2 by
these cells (step 4). Next, gamone 2 specifically acts on
mating type 1 cells (step 5) to transform them so that
they can undergo conjugation (step 6); it also enhances
the production and excretion of gamone 1 by these cells;
the transformed cells form pairs following contact (step
7). Thus, cells of the two mating types stimulate each
other through a positive feedback loop.2 This indicates
that the receptors for gamones are nonself recognized,
which is against the self-recognition hypothesis pro-
posed by Luporini and Miceli.8 To elucidate the molec-
ular mechanism of conjugation in B. japonicum, we
designed and synthesized fluorescent molecular probes
Keywords: Fluorescent probes; Cell–cell interaction; Ciliate phero-
mones; Inhibitory activity.
*
Corresponding author. Tel.: +81 6 6605 2562; fax: +81 6 6605
3152; e-mail: iio@sci.osaka-cu.ac.jp
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.12.021

Y. Uruma et al. / Bioorg. Med. Chem. 15 (2007) 1622–1627
1623
O
O
O
O
O
O
TBDMSO
SnMe3
NHBoc
+
TBDMSO
a
1
O
Cl
O
NHR
4: R = Boc
5: R=H
Scheme 1. Synthesis of blepharismone (1). Reagents and condition: (a) TFA, anisole, acetone, and rt.
specific for the gamone 2 receptor, which would be use-
ful in studying the localization of this receptor. In this
paper, we describe the syntheses of fluorescent probes
2 and 3, and their inhibitory activities against the bleph-
arismone-induced monotypic pairing of B. japonicum
type I cells (see Fig. 1).
group of the side chain appears to be essential, and
the formylamide group appears to be more important
than the 5-OH group on the benzene ring. The activity
of 2-deformylamino-blepharismone is approximately
1/1200 that of blepharismone; whereas 2-acetamide-
blepharismone has no activity. However at a concentration
of 10 lM, 2-acetamide-blepharismone demonstrates
inhibitory activity, that is, complete inhibition of
blepharismone-induced (concentration = 50 nM) mono-
typic pairing.10 Based on this information, we assumed
that chemical modification of the 2-amino group affor-
ded inhibition against gamone 2. Fluorescent probes
are widely used to track the localization of a receptor.11
Tryptophan and its congeners are fluorescent com-
pounds, but relatively common substrates for a cell.
Hence, we required fluorescent probes with higher spec-
ificity to unambiguously track the localization of the
receptor for blepharismone. Thus, we designed fluores-
cent molecular probes 2 and 3 in which the formylamide
group of blepharismone was modified by a fluorescent
pyrenyl group or a biphenyl group.
2. Results and discussion
2.1. Chemistry
Miyake reported that L-tryptophan and 5-hydroxy
L-tryptophan competitively inhibit blepharismone; the
L-isomers are stronger inhibitors than the D-isomers.9
Entzeroth and Jaenicke also showed that N-formyl-
5-hydroxy-L-kynurenine, N-formyl-L-kynurenine, and
5-hydroxy-L-kynurenine, which are similar to blepharis-
mone in structure, interfere with the activity of gamone
2 and desensitize the receptor system.10 None of these
inhibitors had gamone 2 activities. These results suggest
that gamone 2 acts on type I cells by binding reversibly
to the receptor on type I cells.1b According to the struc-
ture–activity relationship studies on 1, the 2-hydroxy
Treatment of the synthetic intermediate 4 formed during
the synthesis of blepharismone (1)7 with trifluoroacetic
O
OH
OH
O
OH
Ca
O
O
2
OH
OH
HO
HO
O
O
HO
O
NH
NH
NHCHO
O
O
Blepharismone (1)
3
2
Figure 1. Blepharismone and fluorescent probes.
O
O
O
CH3
R'O
O
O
NH
R
NH
R
O
O
A
B
6 R = A, R' = TBDMS
7 R = A, R' = H
10 R = A
8 R = B, R' = TBDMS
9 R = B, R' = H
11 R = B
Figure 2. Intermediates for the synthesis of fluorescent probes of blepharismone and their control samples.

1624
Y. Uruma et al. / Bioorg. Med. Chem. 15 (2007) 1622–1627
acid (TFA) in acetone in the presence of anisole12
achieved selective deprotection of the Boc group to
afford amine 5 in 67% yield. Condensation of 5 with
4-pyrene-1-yl-butyric acid by N,N0-dicyclohexyl-carbo-
diimide (DCC) was not successful. Consequently, we
examined amide formation by using 2-aminoacetophe-
none, a model compound. The reactivity of the amino
group is too low; this is presumably due to the reaction
of the vinylogous aromatic amide group with the car-
boxylic acid. Therefore, we converted the carboxylic
acid to its corresponding acid chloride11a to enhance
reactivity and construct an amide bond.
thus far the most effective known inhibitor against
blepharismone-induced
Table 1).1b
monotypic
pairing
(see
2.3. Fluorescence spectra of probes 2 and 3
Blepharismone (1) shows intrinsic relatively weak fluo-
rescence emission at kmax 484 nm. Emission from the
chromophore of the pyrenyl or biphenyl group is at kmax
400 and 310 nm, respectively, and when these chromoph-
ores attach to 1, the intensity of the fluorescence emission
of pyrenyl-blepharismone 2 and biphenyl-blepharismone
3 at kmax 484 nm, which is attributable to blepharismone
chromophore, is increased by a factor of 10 or 6, respec-
tively. This increase in fluorescence intensity at kmax
484 nm may be explained by FRET (fluorescence reso-
nance energy transfer)15 of excited pyrenyl or biphenyl
groups to the blepharismone chromophore.
Treatment of 4-pyren-1-yl-butyric acid or biphenyl-4-yl-
acetic acid with oxalyl chloride in dichloromethane in
the presence of a catalytic amount of DMF13 afforded
the corresponding acid chlorides. 2-Aminoacetophenone
was treated with the acid chlorides in dichloromethane
in the presence of pyridine14 to afford 10 and 11 in
83% and 88% yields, respectively. Similarly, amides 6
and 8 were prepared from amine 5 and acid chlorides
in 72% and 79% yields, respectively. Removal of the
TBDMS group of intermediates 6 and 8 by HFÆPy affor-
ded intermediates 7 and 9 in 71% and 58% yields, respec-
tively. Finally, the acetonide group was deprotected by
TFA in THF:H2O = 4:1 to afford 2 and 3 in 72% and
88% yields, respectively (see Fig. 2).
3. Conclusion
Fluorescent molecular probes 2 and 3 were successfully
synthesized from key intermediate 4 for the synthesis of
(S)-blepharismone, a mating-inducing pheromone of cil-
iate B. japonicum type II cells. These probes showed no
gamone activities; instead, they demonstrated marked
inhibitory activities against the blepharismone-induced
monotypic pairing of B. japonicum type I cells. These
probes may be useful for studying and tracking the
receptor sites of gamone 2 at the cellular level. Further
experiments are currently being performed.
2.2. Biological activity
The mating-inducing activity of compounds 2, 3, 10,
and 11, and their inhibitory activity against blepharis-
mone-induced monotypic pairing of B. japonicum were
then evaluated. With regard to mating-inducing activ-
ity, these compounds were inactive at concentrations
of up to 1.0 mM. Inhibitory activities against gamone
2 were examined in the presence of 74 nM of synthetic
blepharismone, which was 40-fold (40 U/mL activity)
the minimum concentration that induced one pairing
between cells. Fluorescent derivatives 2 and 3 of
blepharismone effectively induced complete inhibition
of blepharismone-induced monotypic pairing of B.
japonicum at concentrations of 4.0 and 4.8 lM, respec-
tively, while controls 10 and 11 showed no inhibitory
activities, as expected. This study did not provide a di-
rect comparison of the inhibitory activities of 2 and 3
with those of tryptophan, 5-hydroxy-tryptophan, and
others; however, the result reported by Jaenicke10c
revealed that the inhibitory activities of 2 and 3 were
effective; their inhibitory activities were more than
double that of 2-acetamide-blepharismone, which is
4. Experimental
4.1. General methods
1H and 13C NMR spectra were recorded at 400 and
100 MHz (JEOL JNM LA400) or 300 and 75 MHz
(JEOL JNM LA300), respectively. Chemical shifts
relative to Me4Si, CDCl3 with CHCl3 and DMSO-d6
with DMSO-d5 as the internal reference were reported
1
in ppm (7.26 ppm for H NMR and 77.0 ppm for 13C
NMR). Coupling constants were reported in hertz
(Hz) and determined directly from the 1H NMR spectra.
Spectral splitting patterns were designated as s (singlet),
d (doublet), t (triplet), m (multiplet), or br (broad). Mass
spectra were obtained using JEOL JMS-700T or JEOL
JMS-AX500 spectrometer. Infrared spectra were ob-
tained using a Jasco A-100 spectrometer or a Hitachi
270-30 spectrophotometer. Fluorescence spectra were
measured using a Hitachi F-4500 fluorophotometer. Ele-
mental analyses were carried out at the Analytical Cen-
ter of Osaka City University.
Table 1. Inhibitory activities of fluorescent probes 2, 3, 10, and 11
against the blepharismone-induced monotypic pairing of B. japonicum
Compound
Inhibitory activitya (lM)
All air- and moisture-sensitive reactions were performed
in a flame-dried, argon-flushed, two-necked flask sealed
with a rubber septum. Dry solvents and reagents were
introduced with a syringe. THF was freshly distilled from
sodium benzophenone ketyl. CH2Cl2 was distilled from
2
4.0
4.8
3
10 (control)
11 (control)
>1 · 103
>1 · 103
a Inhibitory activity indicates the inhibitory concentration at which
mating-inducing activity was completely inhibited in the presence of
74 nM of blepharismone.
˚
P2O5 and stored over 4-A molecular sieves. Pyridine was
˚
dried over KOH and stored over 4-A molecular sieves.

Y. Uruma et al. / Bioorg. Med. Chem. 15 (2007) 1622–1627
1625
TLC was performed on Merck precoated silica gel
(#5715) or RP-18 plates (#15685), and the TLC spots
were visualized under 254 nm UV light and/or by charring
after dropping the plate into vanillin solution in 5% sulfu-
ric acid/methanol. The products were purified via Merck
silica gel (#7734 and #9385) flash column chromatogra-
phy or ODS (Wakogelꢀ 50C18) column chromatography
to obtain compounds 2 and 3. Hexane and ethyl acetate
were distilled and used for column chromatography.
with brine (40 mL), dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography (hexane/EtOAc 1:1 v/v) to af-
ford 7 (127 mg, 71%) as a yellow solid. To a stirred solu-
tion of 7 (60 mg, 0.11 mmol) in THF (0.44 mL) were
added TFA (20 lL) and H2O (0.11 mL) at 0 ꢁC. After
being stirred for 16 h at 0 ꢁC, the reaction mixture was
concentrated in vacuo, and the residue was purified with
ODS column chromatography (CH3OH/H2O 1:1 v/v) to
afford 2 (225 mg, 72%) as a yellow solid.
4.2. (S)-{2-[2-Amino-5-(tert-butyl-dimethyl-silanyloxy)-
phenyl]-2-oxo-ethyl}-2,2-dimethyl-[1,3]dioxolan-4-one (5)
4.4. (S)-N-{4-(tert-Butyl-dimethyl-silanyloxy)-2-[2-(2,2-
dimethyl-5-oxo-[1,3]dioxolan-4-yl)-acetyl]-phenyl}-4-
pyren-1-yl-butyramide (6)
To a solution of (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 (4)7 (1.82 g,
3.79 mmol) in acetone (31 mL) were added anisole
(5.5 mL, 48.5 mmol) and TFA (18.1 mL, 235 mmol),
and the mixture was stirred for 9 h at room temperature.
To the reaction mixture was added aq NaHCO3 (excess)
and the mixture was extracted with EtOAc. The com-
bined organic layers were washed with water and brine,
dried over anhydrous Na2SO4, and solvent was removed
in vacuo. The residue was purified by flash column chro-
matography on silica gel (EtOAc/hexane 1:5 v/v) to give
5 (970 mg) in 67% yield. Oil; Rf = 0.30 (EtOAc/hexane
1:5 v/v); IR (neat) mmax 3470, 3345, 1770, 1735, 1640,
Yellow solid; Rf = 0.35 (EtOAc/hexane 1:3 v/v) mp 64–
;
1H
66 ꢁC; IR (nujol) mmax 3297, 1792, 1515 cmꢀ1
NMR (CDCl3, 400 MHz) d 0.21 (s, 6H), 0.99 (s, 9H),
1.55 (s, 6H), 2.28–2.35 (m, 2H), 2.55 (t, 2H,
J = 7.8 Hz), 3.37 (dd, 1H, J = 18.1, 7.1 Hz), 3.43 (t,
2H, J = 6.8 Hz), 3.56 (dd, 1H, J = 18.1, 2.7 Hz), 4.80
(dd, 1H, J = 7.1, 2.7 Hz), 7.06 (dd, 1H, J = 9.2,
2.8 Hz), 7.22 (d, 2H, J = 2.8 Hz), 7.90 (d, 1H,
J = 7.8 Hz), 7.98 (t, 1H, J = 8.1 Hz), 8.01 (AB q, 2H),
8.07–8.17 (m, 4H), 8.30 (d, 1H, J = 9.3 Hz), 8.63 (d,
1H, J = 9.2 Hz), 11.2 (s, 1H); 13C NMR (CDCl3,
100 MHz) d ꢀ4.4 (2C), 25.7 (3C), 25.8, 26.8, 27.2,
30.8, 32.6, 37.7, 41.2, 69.7, 111.1, 120.9, 121.9, 122.4,
123.3, 124.6, 124.8 (2C), 125.0, 125.8, 126.6, 127.33,
127.38, 127.43, 127.49, 128.8, 139.9, 130.8, 131.3,
133.4, 135.2, 135.7, 150.3, 171.8, 172.9, 198.4;
HRFABMS m/z 651.3013 (calcd for C39H45NO6Si,
651.3016 [M+H]+).
1370, 1170, 1145, 1110 cmꢀ1
;
1H NMR (CDCl3,
400 MHz) d 0.17 (s, 6H), 0.98 (s, 9H), 1.61 (s, 3H),
1.65 (s, 3H), 3.33 (dd, 1H, J = 17.7, 7.2 Hz), 3.55 (dd,
1H, J = 17.7, 2.8 Hz), 4.92 (dd, 1H, J = 7.2, 2.8 Hz),
5.98 (br s, 2H), 6.58 (d, 1H, J = 8.8 Hz), 6.88 (dd, 1H,
J = 8.8, 2.9 Hz), 7.08 (d, 1H, J = 2.9 Hz); 13C NMR
(CDCl3, 100 MHz) d ꢀ4.5 (2C), 18.1, 25.7 (3C), 25.9,
26.9, 40.6, 70.2, 111.1, 117.1, 118.5, 119.9, 128.6,
145.3, 145.6, 173.4, 195.6; HREIMS m/z 379.1833 (calcd
for C19H29NO5Si, 379.1815 [M]+).
4.5. (S)-N-{2-[2-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4-yl)-
acetyl]-4-hydroxy-phenyl}-4-pyren-1-yl-butyramide (7)
Yellow solid; Rf = 0.25 (EtOAc/hexane 1:1 v/v); mp
105–106 ꢁC; IR (neat) mmax 3296, 1789 cmꢀ1; FABMS
4.3. General procedure for the synthesis of fluorescent
probes
1
m/z 536 (M++H); H NMR (CDCl3, 400 MHz) d 1.48
(s, 6H), 2.28 (m, 2H), 2.52 (t, 2H, J = 7.3 Hz), 3.25
(dd, 1H, J = 18.3, 6.5 Hz), 3.41 (br t, 2H, J = 7.8 Hz),
3.43 (d, 1H, J = 18.3, 2.9 Hz), 4.66 (dd, 1H, J = 6.5,
2.9 Hz), 7.02 (dd, 1H, J = 9.0, 2.9 Hz), 7.13 (d, 1H,
J = 2.9 Hz), 7.72 (br s, 1H), 7.81 (d, 1H, J = 7.7 Hz),
7.90 (t, 1H, J = 7.7 Hz), 7.93 (AB q, 2H), 7.99 (d, 1H,
J = 9.3 Hz), 8.03 (d, 1H, J = 7.7 Hz), 8.07 (d, 2H,
J = 7.7 Hz), 8.21 (d, 1H, J = 9.3 Hz), 8.40 (d, 1H,
J = 9.0 Hz), 11.1 (s, 1H); 13C NMR (CDCl3,
100 MHz) d 25.6, 26.6, 27.1, 32.6, 37.9, 40.9, 69.8,
111.4, 116.5, 122.2, 122.6, 122.8, 123.2, 124.7, 124.8
(2C), 124.9, 125.0, 125.7, 126.6, 127.2, 127.3, 127.4,
128.6, 129.8, 130.7, 131.3, 133.2, 135.4, 151.7, 172.4,
173.3, 198.4; HRFABMS m/z 536.2067 (calcd for
C33H30NO6, 536.2073 [M+H]+).
To a stirred solution of 4-pyren-1-yl-butyric acid
(167 mg, 0.58 mmol) in CH2Cl2 (1.0 mL) were added a
drop of DMF and oxalyl chloride (60 lL, 0.64 mmol)
at 0 ꢁC. After being stirred for 45 min at room tempera-
ture, the reaction mixture was concentrated in vacuo to
give 4-pyren-1-yl-butyryl chloride. The resulting acid
chloride was dissolved in CH2Cl2 (1.0 mL) and added
to a solution of 5 (183 mg, 0.48 mmol) and pyridine
(90 lL) in CH2Cl2 (1.4 mL), and the mixture was stirred
at room temperature for 7 h. To the reaction mixture was
added H2O (2 mL), and the resulting mixture was
extracted with EtOAc (2· 20 mL). The combined organic
layers were washed with brine (5 mL), dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue
was purified by flash chromatography (hexane/EtOAc
5:1 v/v) to afford 6 (225 mg, 72%) as a yellow solid. To
a solution of 6 (225 mg, 0.35 mmol) in THF (1.75 mL)
was added HFÆPy (0.39 mL, 0.39 mmol) at 0 ꢁC. Stirring
was continued for 1 h at 0 ꢁC and for an additional 1 h at
room temperature. To the reaction mixture was added
water (3 mL), and the mixture was extracted with EtOAc
(2· 20 mL). The combined organic extracts were washed
4.6. (S)-2-Hydroxy-4-[5-hydroxy-2-(4-pyren-1-yl-buty-
rylamino)-phenyl]-4-oxo-butyric acid (2)
Yellow solid; Rf (ODS) = 0.5 (CH3OH/H2O 1:1 v/v); mp
148–149 ꢁC; IR (nujol) mmax 3000–3600 (br), 2953, 2924,
2854, 1461, 1376 cmꢀ1; 1H NMR (DMSO-d6, 400 MHz)

1626
Y. Uruma et al. / Bioorg. Med. Chem. 15 (2007) 1622–1627
d 2.10 (m, 2H), 2.46 (t, 2H, J = 7.1 Hz), 3.19 (dd, 1H,
J = 16.3, 7.6 Hz), 3.22 (dd, 1H, J = 16.3, 4.6 Hz), 3.36
(t, 2H, J = 7.6 Hz), 3.57 (br s, 1H), 4.43 (dd, 1H,
J = 7.6, 4.6 Hz), 6.96 (dd, 1H, J = 8.8, 2.7 Hz), 7.18
(d, 1H, J = 2.7 Hz), 7.77 (d, 1H, J = 8.8 Hz), 7.96 (d,
1H, J = 7.7 Hz), 8.04 (t, 1H, J = 7.7 Hz), 8.12 (AB q,
2H), 8.20–8.28 (m, 4H), 8.41 (d, 1H, J = 9.3 Hz), 9.63
(br s, 1H), 10.4 (s, 1H); 13C NMR (DMSO-d6,
100 MHz) d 27.2, 32.1, 36.2, 44.5, 66.9, 115.6, 120.1,
123.5, 124.0, 124.1, 124.2, 124.8, 124.9, 125.0, 126.1,
126.5, 127.3, 127.4, 127.6, 128.2, 129.1, 129.32, 129.35,
130.4, 130.9, 136.4, 153.3, 170.9, 174.8, 200.8;
HRFABMS m/z 496.1759 (calcd for C30H26NO6,
496.1760 [M+H]+).
J = 8.8 Hz), 9.43 (br s, 1H), 10.6 (s, 1H); 13C NMR
(DMSO-d6, 100 MHz) d 43.6, 44.8, 67.2, 116.0, 120.6,
124.5, 126.9 (2C), 127.1 (2C), 127.7, 129.3 (2C), 129.4,
129.6, 130.2 (2C), 134.8, 139.0, 140.2, 153.7, 169.7,
175.2, 201.2; HRFABMS m/z 420.1444 (calcd for
C24H22NO6, 420.1447 [M+H]+).
4.10. N-(2-Acetyl-phenyl)-4-pyren-2-yl-butyramide (10)
According to the general procedure, 2-aminoacetophe-
none (200 mg, 1.48 mmol) and 4-pyren-1-yl-butyryl
chloride, which was prepared from 4-pyren-1-yl-butyric
acid (640 mg, 2.22 mmol), were condensed. Purification
by flash column chromatography (hexane/EtOAc 5:1
v/v) afforded 10 (501 mg, 83%) as a yellow solid;
Rf = 0.30 (EtOAc/Hexane 1:3 v/v); IR (film) mmax 3489,
4.7. (S)-2-Biphenyl-4-yl-N-{4-(tert-butyl-dimethyl-sila-
nyloxy)-2-[2-(2,2-dimethyl-5-oxo-[1,3]dioxolan-4-yl)-ace-
tyl]-phenyl}-acetamide (8)
1654, 1605 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d
;
2.12 (m, 2H), 2.49 (s, 3H), 2.55 (t, 2H, J = 7.1 Hz),
3.38 (t, 2H, J = 8.1 Hz), 7.18 (t, 1H, J = 8.0 Hz), 7.57
(t, 1H, J = 8.0 Hz), 7.94 (d, 1H, J = 8.0 Hz), 7.96 (d,
1H, J = 7.7 Hz), 8.04 (t, 1H, J = 7.7 Hz), 8.12 (AB q,
2H), 8.18–8.31 (m, 5H), 8.41 (d, 1H, J = 8.0 Hz), 11.2
(s, 1H); 13C NMR (DMSO-d6, 100 MHz,) d 27.1, 28.7,
32.0, 36.8, 120.7, 122.9, 123.5, 124.1, 124.2 (2C), 124.6,
124.8, 124.9 (2), 126.1, 126.5, 127.2, 127.4, 127.5,
129.3, 130.4, 130.9, 131.3, 133.9, 136.2, 138.7, 171.3,
202.5; HRFABMS m/z 405.172 (calcd for C28H23NO2,
405.1729 [M]+).
Yellow solid; Rf = 0.20 (EtOAc/hexane 1:3 v/v); mp
176–177 ꢁC; IR (nujol) mmax 2924, 2854, 1461,
1376 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 0.20 (s,
;
6H), 0.98 (s, 9H), 1.54 (s, 3H), 1.57 (s, 3H), 3.36 (dd,
1H, J = 18.0, 6.8 Hz), 3.57 (dd, 1H, J = 18.0, 3.1 Hz),
3.76 (s, 2H), 4.82 (dd, 1H, J = 6.8, 3.1 Hz), 7.06 (dd,
1H, J = 9.1, 2.8 Hz), 7.22 (d, 1H, J = 2.8 Hz), 7.34 (br
t, 1H, J = 9.3 Hz), 7.43 (m, 4H), 7.59 (m, 4H), 8.64 (d,
1H, J = 9.1 Hz), 11.0 (s, 1H); 13C NMR (CDCl3,
100 MHz) d ꢀ4.4 (2C), 18.2, 25.6 (3C), 25.7, 26.7,
41.0, 45.3, 69.8, 111.1, 121.0, 122.5, 127.0, 127.2,
127.6, 128.7, 139.9 (2C), 133.4, 135.1, 140.1, 140.8,
150.5, 170.0, 172.8, 198.2; HRFABMS m/z 574.2637
(calcd for C33H40NO6Si, 574.2625 [M+H]+).
4.11. N-(2-Acetyl-phenyl)-2-biphenyl-4-yl-acetamide (11)
According to the general procedure, 2-aminoacetophe-
none (280 mg, 2.07 mmol) and biphenyl-4-yl-acetyl
chloride, which was prepared from biphenyl-4-yl-acetic
acid (526 mg, 2.48 mmol), were condensed. Purification
by flash column chromatography (hexane/EtOAc 5:1 v/v)
afforded 11 (602 mg, 88%) as a yellow solid; Rf = 0.30
(EtOAc/hexane 1:3 v/v) mp 128 ꢁC; IR (neat) mmax
4.8. (S)-2-Biphenyl-4-yl-N-{2-[2-(2,2-dimethyl-5-oxo-[1,3]-
dioxolan-4-yl)-acetyl]-4-hydroxy-phenyl}-acetamide (9)
Yellow solid; Rf = 0.25 (EtOAc/hexane 1:1 v/v); mp
1
2953, 1732, 1460, 1376 cmꢀ1
;
1H NMR (CDCl3,
154–155 ꢁC; IR (nujol) mmax 1647, 1459, 1376 cmꢀ1; H
400 MHz) d 2.62 (s, 3H), 3.79 (s, 2H), 7.09 (ddd, 1H,
J = 8.1, 7.2, 1.2 Hz), 7.33 (tt, 1H J = 7.1, 1.5 Hz),
7.44–7.49 (m, 4H), 7.53 (ddd, 1H, J = 8.5, 7.2,
1.5 Hz), 7.58–7.61 (m, 4H), 7.86 (dd, 1H, J = 8.1,
1.5 Hz), 8.76 (dd, 1H, J = 8.5, 1.2 Hz), 11.8 (s, 1H);
13C NMR (CDCl3, 100 MHz) d 28.5, 45.6, 120.7,
121.8, 122.4, 127.1 (2C), 127.2, 127.6 (2C), 128.7
(2C), 129.8 (2C), 131.5, 133.5, 135.1, 140.2, 140.9,
141.0, 170.5, 202.5; Anal. C22H19NO2: C, 80.22; H,
5.81; N, 4.25. Found: C, 80.19; H, 5.75; N, 4.24;
HRFABMS m/z 330.1486 (calcd for C22H20NO2,
330.1494 [M+H]+).
NMR (CDCl3, 400 MHz) d 1.54 (s, 3H), 1.56 (s, 3H),
3.37 (dd, 1H, J = 18.0, 7.3 Hz), 3.57 (dd, 1H, J = 18.0,
3.1 Hz), 3.77 (s, 2H), 4.79 (dd, 1H, J = 7.3, 3.1 Hz),
5.74 (s, 1H), 7.00 (dd, 1H, J = 9.2, 2.9 Hz), 7.24 (d,
1H, J = 2.9 Hz), 7.34 (br t, 1H, J = 9.3 Hz), 7.43 (m,
4H), 7.59 (m, 4H), 8.57 (d, 1H, J = 9.2 Hz), 11.1 (s,
1H); 13C NMR (CDCl3, 75 MHz) d 25.7, 26.7, 41.0,
45.3, 69.8, 111.4, 116.4, 122.7, 122.8, 127.1 (2C), 127.3,
127.6 (2C), 128.7 (2C), 130.0 (2C), 133.4, 135.1, 140.2,
140.5, 140.8, 150.8, 170.1, 172.8, 198.1; HRFABMS m/
z 460.1756, (calcd for C27H26NO6, 460.2412 [M+H]+).
4.9. (S)-4-[2-(2-Biphenyl-4-yl-acetylamino)-5-hydroxy-
phenyl]-2-hydroxy-4-oxo-butyric acid (3)
4.12. Cells and cell culture for biological assay
Blepharisma japonicum strain R1072 (mating type I)
was used to assay for mating-inducing and inhibitory
activities. Cells were cultured on Enterobacter aerogenes
in WGP (Wheat Grass Powder, Pines) medium,4 con-
centrated by low-speed centrifugation, washed with
physiological balanced solution SMB (synthetic medi-
um for Blepharisma),4 and suspended in SMB at a
density of 1500–2000 cells/mL. Cultures were main-
tained at 25 ꢁC.
Yellow solid; Rf (ODS) = 0.25 (CH3OH/H2O 1:1 v/v);
mp 184–185 ꢁC; IR (nujol) mmax 2924, 2854, 2340,
1462, 1376 cmꢀ1 1H NMR (DMSO-d6, 400 MHz) d
;
3.15 (dd, 1H, J = 16.3, 7.6 Hz), 3.22 (dd, 1H, J = 16.3,
4.9 Hz), 3.68 (s, 2H), 4.41 (dd, 1H, J = 7.6, 4.9 Hz),
6.95 (dd, 1H, J = 8.8, 2.9 Hz), 7.17 (d, 1H, J = 2.9 Hz),
7.34 (br t, 1H, J = 8.3 Hz), 7.39 (d, 2H, J = 8.3 Hz),
7.45 (t, 2H, J = 8.3 Hz), 7.62 (m, 4H), 7.81 (d, 1H,

Y. Uruma et al. / Bioorg. Med. Chem. 15 (2007) 1622–1627
1627
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Academic Press: New York, 1981; pp 125–198; (b)
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C., Eds.; Gustav Fischer Verlag: New York, 1996; pp 243–
290.
4.13. Preparation of a solution of blepharismone and
fluorescent compounds, and gamone activity assay
Synthetic blepharismone7 was dissolved in SMB (20 lg/mL)
and filtered. One percentage of DMSO in SMB solution
did not have any influence in inducing monotypic pair-
ing of B. japonicum type I cells. For the biological assay,
fluorescent compounds were dissolved in DMSO and
diluted using SMB.
2. (a) Miyake, A. Proc. Jpn. Acad. 1968, 44, 837–841; (b)
Miyake, A.; Beyer, J. Exp. Cell Res. 1973, 76, 15–24; (c)
Miyake, A. Cell interaction by gamones in Blepharisma. In
Sexual Interactions in Eukaryotic Microbes; O’Day, D. H.,
Horgen, P. A., Eds.; Academic Press: New York, 1981; pp
95–129.
3. (a) Miyake, A.; Beyer, J. Science 1974, 185, 621–623;
(b) Honda, H.; Miyake, A. Nature 1975, 257, 678–680;
(c) Braun, V.; Miyake, A. FEBS Lett. 1975, 53, 131–
134.
Biological gamone activity is represented in units (U). A
unit of activity was defined as the smallest amount of
gamone activity that could induce at least one face-
to-face pair in 750–1000 cells suspended in 1 mL SMB,
and the activity was measured by the method described
before.2c,16
4. Sugiura, M.; Harumoto, T. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 14446–14451.
5. Sugiura, M.; Kawahara, S.; Iio, H.; Harumoto, T. J. Cell
Sci. 2005, 118, 2735–2741.
6. Kubota, T.; Tokoroyama, T.; Tsukuda, Y.; Koyama, H.;
Miyake, A. Science 1973, 179, 400–402.
4.14. Assay for inhibitory activities against blepharismone-
induced monotypic pairing of B. japonicum
7. Takihiro, H.; Uruma, Y.; Usuki, Y.; Miyake, A.; Iio, H.
Tetrahedron: Asymmetry 2006, 17, 2339–2343.
8. Luporini, P.; Miceli, C. Mating pheromones. In The
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Academic Press: New York, 1986; pp 263–299.
9. (a) Miyake, A. Curr. Top. Microbiol. Immunol. 1974, 64,
49–77; (b) Miyake, A. Conjugation of ciliates in biochem-
istry of multicellular morphogenesis. In Biochemistry of
Differentiation and Morphogenesis; Janicke, L., Ed.;
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10. (a) Entzeroth, M.; Jaenicke, L. Z. Naturforsch. 1982, 37C,
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Gruyter: Berlin, 1984; pp 815–826.
The mating-inducing activity of gamone 2 with fluores-
cent samples 2, 3, 10, and 11 was represented by unit
and index of pair formation (0–5). The index of pair for-
mation was determined on the basis of the ratio of the
tester cell forming hemolytic pairs. Each well contained
500 mL of fluorescent sample and 40 ng of synthetic
blepharismone, and 500 mL of tester cells suspension
(1500–2000 cell/mL) was added to each well. The ratio
of pairs was judged after 3, 5, and 20 h. The index of
pair formation (0–5) indicates no pair (0), a few pairs
(1), approximately half the cells forming pairs (3), and
most cells forming pairs (5). Index (2) and index (4) were
between index (1) and (2), index (3) and (5), respectively.
The minimum concentration of fluorescent compounds
at index (0) is reported as inhibitory activity (lM).
11. (a) Barrantes, F. J.; Sakmann, B.; Bonner, R.; Eibl, H.;
Jovin, T. M. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 3097–
3101; (b) The Handbook—A Guide to Fluorescent Probes
and Labeling Technologies; 10th ed.; Web edition: http://
probes.invitrogen.com/handbook/.
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Jpn. 1980, 53, 464–468.
13. Wissner, A.; Grudzinskas, C. V. J. Org. Chem. 1978, 43,
3972–3974.
Acknowledgments
We thank Professor Akio Miyake, Camerino University,
Italy, and Professor Junji Teraoka, Osaka City Universi-
ty, Japan, for their helpful advice. This work was sup-
ported 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)
of the Ministry of Education, Science, Culture and
Sports of Japan.
14. Furstner, A.; Hupperts, A.; Seidel, G. Org. Synth. 1999,
¨
76, 142–150.
15. Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2003,
21, 1387–1395.
16. Kubota, T.; Miyake, A.; Tokoroyama, T. In Experimental
Methods on Natural Organic Compounds–Extraction and
Isolation of Physiologically Active Substances; Natori, S.,
Ikegawa, N., Suzuki, M., Eds.; Kodansha: Tokyo, 1997;
pp 485–498 (in Japanese).
References and notes
1. (a) Miyake, A. Physiology and biochemistry of
conjugation in ciliates. In Biochemistry and Physiology of