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S1
Supporting Information
A Novel Sperm-Activating and Attracting Factor (SAAF)
from the Ascidian Ascidia sydneiensis
Nobuaki Matsumori,*,†, ∆
Yuki Hiradate, ‡,§,&,∆
Hajime Shibata,† Tohru Oishi,
║ Shuichi Shimma,
┴, @ Michisato
Toyoda, ┴,^
Fumiaki Hayashi,# Manabu Yoshida,
*,§ Michio Murata,
*,† and Masaaki Morisawa
*,‡, %
†Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
‡Department of Biology, Faculty of Science, Yamagata University, Kojirakawa, Yamagata 990-8560, Japan.
§Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, Miura, Kanagawa 238-0225,
Japan.
║Department of Chemistry, Graduate School of Sciences, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
┴Department of Physics, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
#RIKEN Systems and Structural Biology Center, RIKEN Yokohama Institute, Yokohama, Kanagawa, 230-0045, Japan.
*Corresponding authors: [email protected]; [email protected];
[email protected]; [email protected].
∆These authors contributed equally to this work.
Present Addresses
&Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai,
Miyagi 981-8555, Japan.
@National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan.
^Project Research Center for Fundamental Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka
560-0043, Japan.
%Tokyo Kasei Gakuin University, Machida, Tokyo 194-0292, Japan.
S2
Table of Contents
Page
Experimental Section S3-S4
600 MHz 1H NMR spectrum of SAAF 2 in D2O S5
800 MHz DQF COSY spectrum of SAAF 2 in D2O S6
800 MHz TOCSY spectrum of SAAF 2 in D2O S8
600 MHz ROESY spectrum of SAAF 2 in D2O S10
600 MHz HSQC spectrum of SAAF 2 in D2O S12
600 MHz HMBC spectra of SAAF 2 in D2O S14
Spectral simulation of the olefin (H-22 and H-23) signals S15
S3
Experimental Section
Materials. The ascidians Ascidia sydneinesis and Ciona intestinalis were collected from Aburatsubo
Bay (Kanagawa prefecture), Onagawa Bay (Miyagi prefecture), and Otsuchi Bay (Iwate prefecture) and
maintained in circulating seawater aquaria under constant light for the accumulation of gametes without
spontaneous spawning. Eggs and semen were obtained by dissection. Semen was stored at 4°C, and eggs
were washed with artificial seawater (ASW, 462 mM NaCl, 9 mM KCl, 10 mM CaCl2, 48 mM MgCl2,
10 mM Hepes, pH 8.2) and maintained in ASW until further use.
Purification of Ascidia-SAAF 2. The eggs were washed with ASW, suspended in 16 volumes of ASW,
and incubated at 4 °C for 14–20 h. The egg suspension was centrifuged at 1,600 g for 15 min, and the
supernatant was centrifuged further at 22,000 g for 30 min at 4 °C. The clear supernatant was designated
as egg seawater (ESW), fractioned on an oasis HLB column, and the adsorbed substances were eluted
with 0 (deionized water) to 100% stepwise methanol gradient. Each fraction was dried in a centrifugal
vaporizer (CVE-200D; Tokyo Rikagaku, Tokyo), and dissolved in 1 ml of deionized water. An aliquot
S4
of each fraction was used for the assay of sperm-activating and attracting activities, which were
performed as reported previously.1 Fractions exhibiting these activities were obtained in 50% and 75%
MeOH elutions, loaded onto a TSK gel ODS-120T HPLC column (4.6 mm × 25 cm; Tosoh, Tokyo)
eluted with a linear gradient of 10–40% acetonitrile at 1 ml/min. The fractions (1 ml each) were
collected, dried in the centrifugal vaporizer, and dissolved in 20 μl of deionized water. All the HPLC
procedures were performed on an LC-10 HPLC system (Shimadzu).
Structure Analysis of Ascidia-SAAF 2. High-resolution MS measurement of Ascidia-SAAF 2 was
performed in negative-ion mode with a LTQ-Orbitrap XL (Thermo Scientific) mass spectrometer. The
NMR spectra were recorded in 150 μl of a D2O (Shigemi tube) using either an Agilent Inova-600,
Agilent Inova-600 equipped with a 5-mm cold probe (Agilent 5 mm HCN probe), or Agilent Inova-800
equipped with a 5-mm cold probe (Agilent 5 mm HCN probe). MALDI-TOF-TOF MS experiments
were carried out in negative-ion mode by using a JMS-S3000 SpiralTOF (JEOL) equipped with a
TOF-TOF attachment (JEOL). Collision-induced dissociation was induced by introducing helium.
REFERENCE
(1) Yoshida, M.; Murata, M.; Inaba, K.; Morisawa, M. Proc. Natl. Acad. Sci. USA 2002, 99, 14831–
14836.
1.0
2
.0
3.0
4
.0
5.0
6
.0
Fig
ure S
1. 6
00 M
Hz 1H
NM
R sp
ectrum
of S
AA
F 2
in D
2 O. C
ontam
inated
meth
anol sig
nal (*
) was u
sed as a referen
ce peak
(3.3
4 p
pm
). T
he co
ncen
tration o
f SA
AF
2 w
as determ
ined
by co
mparin
g sig
nal in
tensity
of resid
ual D
MF
peak
s (**) w
ith
tho
se of a D
MF
-D2 O
solu
tion w
ith k
now
n co
ncen
tration.
ppm
*
** **
S5
24/2
3
4/3
2/3
26/2
6
26/2
6
6/7
6/7
7/6
23/2
4
22/2
0
7/6
Fig
rure S
2. 8
00 M
Hz D
QF
CO
SY
spectru
m o
f SA
AF
2 in
D2 O
.
S6
3/4
26/2
5
21/2
0
17
/20
12/1
2
11/1
2
6/6
27/2
5
25/2
7
20/2
1
12/1
2
6/5
4/5
4/5
4/4
4/4
1/2
1/2
1/2
1/2
2/2
2/2
15/1
6
16/1
6
16/1
5
15/1
5
15/1
5
1/1
24/2
4
24/2
4
24/2
5
12/1
1
2/1
2/1
5/4
12/1
1
5/4
16/1
7
17/1
6
15/1
4
15/1
4
14/1
5
14/1
5
1/1
9/1
1
11/1
2
9/1
1
2/1
2/1
6/6
5/6
17/1
6
Fig
ure S
3. 8
00 M
Hz D
QF
CO
SY
spectru
m o
f SA
AF
2 in
D2 O
. The co
nnectiv
ity fro
m H
-14 to
H-2
0 is n
ot clear p
robab
ly d
ue to
a
poten
tial flexib
ility o
f the D
ring. H
ow
ever, th
e connectiv
ity w
as clearly assig
ned
in th
e TO
CS
Y sp
ectrum
(Fig
. S5).
S7
S8
Fig
ure S
4. 8
00 M
Hz T
OC
SY
spectru
m o
f SA
AF
2 in
D2 O
.
21/1
7
21/1
6
21/1
6
15/1
7
15/1
7
21/2
0
17/2
1
16/2
1
16/2
1
20/2
1
14/1
7
27/2
4
27/2
4
27/2
5
25/2
7
24/2
7
24/2
7
16/1
7
16/1
7
15/1
6
15/1
6
14/1
6
16/1
6
16/1
6
15/1
6
15/1
6
14/1
6
12/1
2
11
/12
9/1
2
12/9
12/1
2
12/1
1
6/6
6/6
16/1
5
16/1
5
16/1
4
16/1
5
Fig
ure S
5. 8
00 M
Hz T
OC
SY
spectru
m o
f SA
AF
2 in
D2 O
. The co
nnectiv
ity fro
m H
-14 to
H-2
0 can
be assig
ned
in th
is spectru
m.
S9
7/6
7/6
7/1
5
Fig
ure S
6. 6
00 M
Hz R
OE
SY
spectru
m o
f SA
AF
2 in
D2 O
. An R
OE
betw
een H
-7 an
d H
-15
is ob
served
.
S10
5/9
14/9
9/5
9/1
4
20/1
9
Fig
ure S
7. 6
00 M
Hz R
OE
SY
spectru
m o
f SA
AF
2 in
D2 O
. Cro
ss peak
s show
n b
y arro
ws su
ggest
trans fu
sion o
f B/C
and C
/D rin
gs. A
n R
OE
of H
-20/H
-19 in
dicates th
e R co
nfig
uratio
n at C
17.
S11
22
23
3 2
6
17
7
14
9
12
12 2
0
24
Fig
ure S
8. 6
00 M
Hz H
SQ
C sp
ectrum
of S
AA
F 2
in D
2 O. A
cross p
eak fro
m co
ntam
inated
meth
anol
(*) w
as used
as reference as 3
.34 p
pm
and 4
9.5
ppm
for 1H
and 1
3C, resp
ectively. *
F1 (p
pm
)
F2
(pp
m)
S12
26
7
17
14
9
12
12 2
0
24
26
24
25 1
1
11
5
6
6
16
16
2
21
4
15
15
19
18
27
3
Fig
ure S
9. 6
00 M
Hz H
SQ
C sp
ectrum
of S
AA
F 2
in D
2 O.
S13
1.0
2.0
14
0
12
0
10
0
80
6
0
40
H-2
1/C
22
H-2
4/C
22
H-2
4/C
23
H-2
7/C
26
H-1
8/C
17
H-2
1/C
17
H-1
8/C
14
H-1
9/C
9
H-1
8/C
13 H
-18/C
12
H-2
1/C
20
H-1
4/C
13
H-1
9/C
10
H-2
7/C
24 H-1
9/C
5
H-1
9/C
1
H-2
7/C
25
H-9
/C10
Fig
ure S
10
. 600 M
Hz H
MB
C sp
ectra of S
AA
F 2
in D
2 O. C
hem
ical shifts o
f quatern
ary carb
ons, C
10 an
d C
13, w
ere assigned
from
HM
BC
.
F1 (p
pm
)
F2 (p
pm
)
F2 (p
pm
)
F1 (p
pm
)
S14
4.5
Hz
AB
system (6
00
MH
z) A
: 5.3
81
pp
m
B: 5
.40
3 p
pm
J: 1
7 H
z Lin
e bro
aden
ing: 5
Hz
Fig
ure S
11
. Spectral sim
ulatio
n o
f the o
lefin (H
-22 an
d H
-23) sig
nals. S
imulatio
n w
as perfo
rmed
usin
g rN
MR
softw
are.
The o
bserv
ed sig
nal (u
pp
er pan
el) was rep
rodu
ced b
y J =
17 H
z (low
er pan
el), suggestin
g th
e E co
nfig
uratio
n o
f the o
lefin.
S15
in D
2 O
