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Structure determination of a very unusual peroxide from solitary ascidians, Phallusia mammillata, ascidia ahodori, styela pricata and halocynthia roretzi.
TetrahedronLetters,Vo1.32.No.34,pp43554358.1991
0040-4039191 $3.00+.00 Pergmm Pressplc
Printed in Great Britain
STRUCTURE SOLITARY
DETERMINATION
ASCIDIANS, STYELA Akira
PHALLUSIA
PRICATA Nakamura*l,
Departments
OF A VERY UNUSUAL MAMMILLATA,
AND Tetsuya
of Chemistry1
ASCZDZA
HALOCYNTHIA
RORETZI.
Ashinol,
Yamamoto2
and Biology2,
Akita
PEROXIDE
Masahiko College
FROM
AHODORI,
of Education,
University,
Akita, Akita
JAPAN 010
Abstract: Cl-hydroperoxy diethyl peroxide was commonly extracted from the tunic of the titled four different ascidian soecies. The structure was determined by spectroscopic data, polarographic data and oxidation reactions.
Natural products carotenoids,lb)
found in ascidians
macrolideslc)
has began to attract many chemist’s
interests;
alkaloids,la)
and tunichromes. Id) The fact that the tunicates has the largest number in its
species among the marine animals directly observed
by rude eyes would be one of the reasons for the
variety of the natural products extracted from ascidians.2)
In the course of studying the oxygen carrier in
ascidian, we isolated a very unusual organic peroxides which are commonly found in the tunic of the titled ascidians.3) Taking phallusia mammillata as an instance, followings.
a typical extraction
procedure
is described
in the
About 100 ml of methanol distiled over CaH2 was used at one time against ca.3.5 g of wet
tunic which is freshly separated and cut into small pieces. The extraction was repeated three times at least. The collected methanolic
extract was concentrated
to about 50 ml on rotary evaporator.
To this solution,
50 ml of saturated aqueous NaCl was added and the solution was extracted with methylene chloride to be dried over sodium sulfate. After removal of the solvent, ca.O.15 g of slightly yellowish oily mixture which was positive against KI-starch test was obtained. column (hexane/benzene=2/1)
The oil (154 mg) was then separated through silica gel
to get a KI-starch positive fraction (57 mg, Rf=O.48).4) The spectroscopic
data of this fraction were summarized
in Table 1. As the fraction was explosive
analysis furnace, the values of C and H contents were not satisfactorily (CAUTION).!)
under the elemental
obtained even in several trials
The observed values are most closely related to a molecular formula of C4H1004. The UV
spectra of the fraction showed no maximum peak except for a gradually increasing
absorption line in the
region from 320 to 200 nm (E = 21 in hexane at 250 nm). In mass spectra of this fraction no parent peak was observed O[WA)n+Hl,
observed
(EI and FAB).
However,
in the FAB mass spectra (TEA/Ar),
O2[(TEA)~+Hl,03[(TEA)~+Hl,C2H40~[(TEA)n+Hl
fragment
peaks due to
and C2H403[UEA)n+HJ
where n is an integer from 1 to 3. These crude data strongly suggest the existence
group in the fraction.
were
of peroxy
According to the NMR data, three possible srmctures could be. tentatively nominated;
A, B and C (shown in Fig. 1). Anisotropic a particular conformation
character in vicinal methylene protons would be explained by
due to an intramolecular interaction in the solvent. The structure B is excluded by
4355
4356
comparisons
with the reported IR spectra.5)
Structure A can be differentiated
from C by a comparison of
their redox processes.
Table
1.
Spectroscopic
and
Element
Analytical
17.96(Cl),
Data
of
64.04(C3),
the
Ascidian
‘3C-NMR(SCDC13)
15.29(C4),
‘H-NMR(GCDCl3)
(assignment by COSY)
1.26(dd 7.0, 7.0; 3H; C4), 1.37(d 5.5; 3H; Cl) 3.65(dq 9.5, 7.0; 1H; C3), 3.88(dq 9.5, 7.0; IH; 5.03 (q 5.5; 1H; C2), 9.02(bs; 1H)
IR(neat/KBr) (cm-l) a)
3590sh,3350s(VOH),2975s(vCH),293Om,2890m,l635wbd, 1446m(6~CH~/6~~CHg),l380s(6~CH3),1345w,1324m,
Peroxide.
104.3O(C2) C3)
1174msh,l126vs(~CH-00H),1097vs(vCH-00Et), 1046s(~CH~-0),950m(~OH),880w(vOO),842m(~OO),798w Elemental Analysis (C/H/N) (averaged from seven samplings)
(found) C(39.71), H(8.80), N(O.0) _____ __ ___ ________ __ _ __ ___ __ _ _ ____ (calcd. for C4H1003, B, C) (calcd. for C4Hlc04, A) C(45.27), H(9.50) C(39.34), H(8.25)
a) Assignments of vibrational
mode
in the parenthesis
,CH,-CH, 3 4
;H~-C,H,/0---O”
CH,-C,” 1 2/O”
o-o
To determine peroxide,
1.
1C”,--C,”2,O-OH 3
Structures
hydroperoxide
transfered
for
Structure C the
Ascidian
in a reduction
(‘BuOOH) and di-rert-butyl
Peroxide.
process,
polarography
of ascidian peroxide.
reduction
were calculated from Ilkovic equation. these diffusion
coefficients
in
tBuOOBut did not show any
reduction wave in the sweeping region until the reduction wave of proton appeared. shows a single two-electron
of the ascidian
peroxide (tBuOOBur) was performed
acidic and aq.NaCl solutions with use of Ag/AgCl as a reference electrode.@ were observed in the polarograms
4
0-CH,-CH,
Structure B Possible
the number of electron
rert-butyl
,CH,-CH, 3 4
thoroughly.
o-o
Structure A Fig.
are not confirmed
Two reduction waves
In the media employed, it is known that rBuOOH
wave.7) The diffusion coefficients
(D) of tBuOOH in both media
Assuming the similarity in molecular weight and the applicability of
to the ascidian peroxide,
the numbers of electron transfer (n) of individual
reduction waves in acidic and aq.NaCl solutions were calculated.
The half-wave potentials as well as the
values of n thus evaluated are summarized in Table 2. In the polarogram of the ascidian peroxide in acidic media, two reduction
waves of one-electron
transfer process can be rationalized
shown in Scheme 1. In aq.NaCl media, on the other hand, two processes participated
in the reduction,
environments. hydroperoxy considered
revealing
the existence
of two peroxy
by a successive reaction
of two-electron groups
transfer were
of different
chemical
The fist reduction wave of the ascidian peroxide at -0.2OV is ascribed to the reduction of group by a similar mechanism
to be due to the two-electron
to that of ‘BuOOH.
The second reduction wave at - 1.8 1V is
reduction of dialkyl peroxy group. The lower reduction potential
4357
of dialkyl peroxy group comparing
with that of tBuOOBut could be explained
density in O-O bond which caused by a geminal hydroperoxy
Table
2.
Polarographic
Data
of Ascidian
Peroxide,
Ascidian Peroxide (1.98 mM; C) in 0.05 M H2SO4 Ascidian Peroxide (1.99 mM; C) in 0.1 M NaCl ‘BIJCCH (2.50 mM; C) in 0.05 M H2SO4 ‘BllCOH (2.85 mM; C) in 0.1 M NaCl
‘BuOOBu’ (2.08 mM; C) in 0.1 M NaCl
v V
1 .l 1 .l
-0.20 -1.81
v V
2.1 2.1
4.7 4.7
-0.21
v
2
4.0
D = 8.0 x 10s cm2/s -0.24 V
2
4.4
a) n=t,j/(607Cm2/3D1/2t116);
Scheme
1.
Possible
Reaction
1.5-2.0mV/s)
-0.09 -0.82
D = 9.8 x 10s cm2/s ca.-1.2 V (shoulder at H+ reduction) no wave until -1.9V
tBuOOBu’ (1.66 mM; C) in 0.05 M H2SO4
rate;
Diffusion Current Constant; kD b, 2.2 2.2
E1/2 (V vs Ag/AgCI)
Number of Electron Transfer;
n a)
_ -
__
_
__
- -
_- -
b) kD=id/Cm2/3t1/6=607nD112
Paths
of
Ascidian
Peroxide
in
Acidic
-OH
Me-CH
of electron
tB u 00 H and tB u 0 0 But.
(sweep Peroxide
by decreasing
group.
Media.
/“’ ,p”
/”
Me-CH \
\o_oiEt
0 -0
‘o-o
/Et
Et HO The existence of two kinds of peroxy group, e.g., hydroperoxy
/
and dialkyl peroxy, is also supported
by the oxidation reaction of thioanisole by the ascidian peroxide; a reaction of 1 to 1 molar ratio of ascidian peroxide to thioanisole
in chloroform
at room temperature
gave corresponding
sulfone quantitatively,
and
that of 1 to 2 molar ratio gave selectively sulfoxide in a quantitative yield. This fact shows the electrophilic character of the ascidian peroxide.
The structure A in Fig. 1, e.g., a-hydroperoxy
thus elucidated for the structure of ascidian peroxide.
diethyl peroxide,
was
4358
Finally,
the ascidian
peroxide
(200-350
nm).
The biological
of redox
reaction
in various
Acknowledgments: Tohoku University
was concluded
character aqueous
The authors
to be racemic
of this unusual
solutions
modified
gratefully
express
peroxide
from
the data of [a]405
is under investigation,
as well as CD
from the viewpoint
of sea water.
their thanks
to Prof.
M. Hirama
for their efforts in facilities of taking spectral data. In measuring
and Dr. Y. Sato at
the polarographic
data, Dr. N. Ogawa of Akita University was kindly offered his many efforts.
References,
Notes
and
la) J. Kobayashi, J. Cheng, M. R. Walchli, H. Nakamura, Org. Chem., (1988) a ISOOPS. J. Bloor and F. J. Schnitz, lb) T. Matsuno, Pure Appl. Chem., (1985) s 659; lc) T. Clardy and C. M. Ireland, J. Amer. Chem. Sot., (1990) 112 Nakamura, S. Nozoe, Y. Hirata, Y. Ohizumi and T. Sasaki, M. Tanaka, M. Nabae, M. Inoue, S. Kato, Y. Hamada and M. J. Smith, K. Kustin Id) E. M. Oltz, R. C. Bruening, (1988) 110 6162 2)
Norman
J. Berrill
in
“The
mammiflata 3) Phallusia sent alive to Akita by Prof. and ascidia ahodori were Tohoku University.
Origin
of
Vertebrates”,
CAUTION Y. Hirata, T. Sasaki and Y. Ohizumi, J. J. Amer. Chem. Sot., (1987) m 6134; M. Za riskie, M. P. Foster, T. 3. Stout, 3. 8080: 9 Kobayashi, J.Cheng, T,: ,Ohta, H. I. Org. Chem., (1988) Xj_ 6147y.T. Ishida, T. Shioiri, J. Org. Chem., (1988) ti 107; and K. Nakanishi, J. Amer. Chem. Sot.,
Clarendon
Press,
Oxford
(195.5)
and sryela pricata were collected in the Mediterranean Sea and F. Rafargue of University of P. and M. Curie; halocynthia roretzi collected in Asamushi Bay in Japan by Prof. T. Numakunai of
4) For the other ascidian species, a set of values (weight of wet tunic/weight name of ascidian) are listed; (12 g/O.17 g: uscidiu ahodori), (29 g/O.13 g: styela (40 g/O.13 g: halocynthia roretzi). Judging from the odor in a trapping device, of target compound is lost at a process of concentration. 5)‘$F. Welch, H. R. Williams and H. S. Mosher, J. Amer, Chem. Sot., Szymanski in “Interpreted Infrared Spectra”, vol 3, p.243, Plenum Press,
of crude oil: pricata) and small amount
(195.5) 21 557; N.Y. (1967)
H. A.
6) The ascidian peroxide is miscible with water in any ratio. As we want to determine the number of electron in a reduction process, a non-buffered media was employed, by which the second reduction wave was observed. In acetate buffer solution, on the other hand, only the first reduction wave of the peroxide was appeared. &v~ 7) R. D. Mair and R. T. Hall, im.533 ._,, an L. S. Silbert, Chapt. VII,p637 in “Organic --4 Peroxides” Ed. by D. Swem, vol 2, Wiley-Interscience, N.Y. (1971) !) This slightly viscous colorless liquid is stable to be stored at a temperature of -30 Co for several months. On heating the open end of a capillary tube which contained ca.10 ~1 in volume, however, the peroxide detonates with complete disappearance of the tube of 50 ~1. The explosion has enough power to blow off a frame of torch at a distance of 10 cm apart.
(Received in Japan 22 April
1991)
0040-4039191 $3.00+.00 Pergmm Pressplc
Printed in Great Britain
STRUCTURE SOLITARY
DETERMINATION
ASCIDIANS, STYELA Akira
PHALLUSIA
PRICATA Nakamura*l,
Departments
OF A VERY UNUSUAL MAMMILLATA,
AND Tetsuya
of Chemistry1
ASCZDZA
HALOCYNTHIA
RORETZI.
Ashinol,
Yamamoto2
and Biology2,
Akita
PEROXIDE
Masahiko College
FROM
AHODORI,
of Education,
University,
Akita, Akita
JAPAN 010
Abstract: Cl-hydroperoxy diethyl peroxide was commonly extracted from the tunic of the titled four different ascidian soecies. The structure was determined by spectroscopic data, polarographic data and oxidation reactions.
Natural products carotenoids,lb)
found in ascidians
macrolideslc)
has began to attract many chemist’s
interests;
alkaloids,la)
and tunichromes. Id) The fact that the tunicates has the largest number in its
species among the marine animals directly observed
by rude eyes would be one of the reasons for the
variety of the natural products extracted from ascidians.2)
In the course of studying the oxygen carrier in
ascidian, we isolated a very unusual organic peroxides which are commonly found in the tunic of the titled ascidians.3) Taking phallusia mammillata as an instance, followings.
a typical extraction
procedure
is described
in the
About 100 ml of methanol distiled over CaH2 was used at one time against ca.3.5 g of wet
tunic which is freshly separated and cut into small pieces. The extraction was repeated three times at least. The collected methanolic
extract was concentrated
to about 50 ml on rotary evaporator.
To this solution,
50 ml of saturated aqueous NaCl was added and the solution was extracted with methylene chloride to be dried over sodium sulfate. After removal of the solvent, ca.O.15 g of slightly yellowish oily mixture which was positive against KI-starch test was obtained. column (hexane/benzene=2/1)
The oil (154 mg) was then separated through silica gel
to get a KI-starch positive fraction (57 mg, Rf=O.48).4) The spectroscopic
data of this fraction were summarized
in Table 1. As the fraction was explosive
analysis furnace, the values of C and H contents were not satisfactorily (CAUTION).!)
under the elemental
obtained even in several trials
The observed values are most closely related to a molecular formula of C4H1004. The UV
spectra of the fraction showed no maximum peak except for a gradually increasing
absorption line in the
region from 320 to 200 nm (E = 21 in hexane at 250 nm). In mass spectra of this fraction no parent peak was observed O[WA)n+Hl,
observed
(EI and FAB).
However,
in the FAB mass spectra (TEA/Ar),
O2[(TEA)~+Hl,03[(TEA)~+Hl,C2H40~[(TEA)n+Hl
fragment
peaks due to
and C2H403[UEA)n+HJ
where n is an integer from 1 to 3. These crude data strongly suggest the existence
group in the fraction.
were
of peroxy
According to the NMR data, three possible srmctures could be. tentatively nominated;
A, B and C (shown in Fig. 1). Anisotropic a particular conformation
character in vicinal methylene protons would be explained by
due to an intramolecular interaction in the solvent. The structure B is excluded by
4355
4356
comparisons
with the reported IR spectra.5)
Structure A can be differentiated
from C by a comparison of
their redox processes.
Table
1.
Spectroscopic
and
Element
Analytical
17.96(Cl),
Data
of
64.04(C3),
the
Ascidian
‘3C-NMR(SCDC13)
15.29(C4),
‘H-NMR(GCDCl3)
(assignment by COSY)
1.26(dd 7.0, 7.0; 3H; C4), 1.37(d 5.5; 3H; Cl) 3.65(dq 9.5, 7.0; 1H; C3), 3.88(dq 9.5, 7.0; IH; 5.03 (q 5.5; 1H; C2), 9.02(bs; 1H)
IR(neat/KBr) (cm-l) a)
3590sh,3350s(VOH),2975s(vCH),293Om,2890m,l635wbd, 1446m(6~CH~/6~~CHg),l380s(6~CH3),1345w,1324m,
Peroxide.
104.3O(C2) C3)
1174msh,l126vs(~CH-00H),1097vs(vCH-00Et), 1046s(~CH~-0),950m(~OH),880w(vOO),842m(~OO),798w Elemental Analysis (C/H/N) (averaged from seven samplings)
(found) C(39.71), H(8.80), N(O.0) _____ __ ___ ________ __ _ __ ___ __ _ _ ____ (calcd. for C4H1003, B, C) (calcd. for C4Hlc04, A) C(45.27), H(9.50) C(39.34), H(8.25)
a) Assignments of vibrational
mode
in the parenthesis
,CH,-CH, 3 4
;H~-C,H,/0---O”
CH,-C,” 1 2/O”
o-o
To determine peroxide,
1.
1C”,--C,”2,O-OH 3
Structures
hydroperoxide
transfered
for
Structure C the
Ascidian
in a reduction
(‘BuOOH) and di-rert-butyl
Peroxide.
process,
polarography
of ascidian peroxide.
reduction
were calculated from Ilkovic equation. these diffusion
coefficients
in
tBuOOBut did not show any
reduction wave in the sweeping region until the reduction wave of proton appeared. shows a single two-electron
of the ascidian
peroxide (tBuOOBur) was performed
acidic and aq.NaCl solutions with use of Ag/AgCl as a reference electrode.@ were observed in the polarograms
4
0-CH,-CH,
Structure B Possible
the number of electron
rert-butyl
,CH,-CH, 3 4
thoroughly.
o-o
Structure A Fig.
are not confirmed
Two reduction waves
In the media employed, it is known that rBuOOH
wave.7) The diffusion coefficients
(D) of tBuOOH in both media
Assuming the similarity in molecular weight and the applicability of
to the ascidian peroxide,
the numbers of electron transfer (n) of individual
reduction waves in acidic and aq.NaCl solutions were calculated.
The half-wave potentials as well as the
values of n thus evaluated are summarized in Table 2. In the polarogram of the ascidian peroxide in acidic media, two reduction
waves of one-electron
transfer process can be rationalized
shown in Scheme 1. In aq.NaCl media, on the other hand, two processes participated
in the reduction,
environments. hydroperoxy considered
revealing
the existence
of two peroxy
by a successive reaction
of two-electron groups
transfer were
of different
chemical
The fist reduction wave of the ascidian peroxide at -0.2OV is ascribed to the reduction of group by a similar mechanism
to be due to the two-electron
to that of ‘BuOOH.
The second reduction wave at - 1.8 1V is
reduction of dialkyl peroxy group. The lower reduction potential
4357
of dialkyl peroxy group comparing
with that of tBuOOBut could be explained
density in O-O bond which caused by a geminal hydroperoxy
Table
2.
Polarographic
Data
of Ascidian
Peroxide,
Ascidian Peroxide (1.98 mM; C) in 0.05 M H2SO4 Ascidian Peroxide (1.99 mM; C) in 0.1 M NaCl ‘BIJCCH (2.50 mM; C) in 0.05 M H2SO4 ‘BllCOH (2.85 mM; C) in 0.1 M NaCl
‘BuOOBu’ (2.08 mM; C) in 0.1 M NaCl
v V
1 .l 1 .l
-0.20 -1.81
v V
2.1 2.1
4.7 4.7
-0.21
v
2
4.0
D = 8.0 x 10s cm2/s -0.24 V
2
4.4
a) n=t,j/(607Cm2/3D1/2t116);
Scheme
1.
Possible
Reaction
1.5-2.0mV/s)
-0.09 -0.82
D = 9.8 x 10s cm2/s ca.-1.2 V (shoulder at H+ reduction) no wave until -1.9V
tBuOOBu’ (1.66 mM; C) in 0.05 M H2SO4
rate;
Diffusion Current Constant; kD b, 2.2 2.2
E1/2 (V vs Ag/AgCI)
Number of Electron Transfer;
n a)
_ -
__
_
__
- -
_- -
b) kD=id/Cm2/3t1/6=607nD112
Paths
of
Ascidian
Peroxide
in
Acidic
-OH
Me-CH
of electron
tB u 00 H and tB u 0 0 But.
(sweep Peroxide
by decreasing
group.
Media.
/“’ ,p”
/”
Me-CH \
\o_oiEt
0 -0
‘o-o
/Et
Et HO The existence of two kinds of peroxy group, e.g., hydroperoxy
/
and dialkyl peroxy, is also supported
by the oxidation reaction of thioanisole by the ascidian peroxide; a reaction of 1 to 1 molar ratio of ascidian peroxide to thioanisole
in chloroform
at room temperature
gave corresponding
sulfone quantitatively,
and
that of 1 to 2 molar ratio gave selectively sulfoxide in a quantitative yield. This fact shows the electrophilic character of the ascidian peroxide.
The structure A in Fig. 1, e.g., a-hydroperoxy
thus elucidated for the structure of ascidian peroxide.
diethyl peroxide,
was
4358
Finally,
the ascidian
peroxide
(200-350
nm).
The biological
of redox
reaction
in various
Acknowledgments: Tohoku University
was concluded
character aqueous
The authors
to be racemic
of this unusual
solutions
modified
gratefully
express
peroxide
from
the data of [a]405
is under investigation,
as well as CD
from the viewpoint
of sea water.
their thanks
to Prof.
M. Hirama
for their efforts in facilities of taking spectral data. In measuring
and Dr. Y. Sato at
the polarographic
data, Dr. N. Ogawa of Akita University was kindly offered his many efforts.
References,
Notes
and
la) J. Kobayashi, J. Cheng, M. R. Walchli, H. Nakamura, Org. Chem., (1988) a ISOOPS. J. Bloor and F. J. Schnitz, lb) T. Matsuno, Pure Appl. Chem., (1985) s 659; lc) T. Clardy and C. M. Ireland, J. Amer. Chem. Sot., (1990) 112 Nakamura, S. Nozoe, Y. Hirata, Y. Ohizumi and T. Sasaki, M. Tanaka, M. Nabae, M. Inoue, S. Kato, Y. Hamada and M. J. Smith, K. Kustin Id) E. M. Oltz, R. C. Bruening, (1988) 110 6162 2)
Norman
J. Berrill
in
“The
mammiflata 3) Phallusia sent alive to Akita by Prof. and ascidia ahodori were Tohoku University.
Origin
of
Vertebrates”,
CAUTION Y. Hirata, T. Sasaki and Y. Ohizumi, J. J. Amer. Chem. Sot., (1987) m 6134; M. Za riskie, M. P. Foster, T. 3. Stout, 3. 8080: 9 Kobayashi, J.Cheng, T,: ,Ohta, H. I. Org. Chem., (1988) Xj_ 6147y.T. Ishida, T. Shioiri, J. Org. Chem., (1988) ti 107; and K. Nakanishi, J. Amer. Chem. Sot.,
Clarendon
Press,
Oxford
(195.5)
and sryela pricata were collected in the Mediterranean Sea and F. Rafargue of University of P. and M. Curie; halocynthia roretzi collected in Asamushi Bay in Japan by Prof. T. Numakunai of
4) For the other ascidian species, a set of values (weight of wet tunic/weight name of ascidian) are listed; (12 g/O.17 g: uscidiu ahodori), (29 g/O.13 g: styela (40 g/O.13 g: halocynthia roretzi). Judging from the odor in a trapping device, of target compound is lost at a process of concentration. 5)‘$F. Welch, H. R. Williams and H. S. Mosher, J. Amer, Chem. Sot., Szymanski in “Interpreted Infrared Spectra”, vol 3, p.243, Plenum Press,
of crude oil: pricata) and small amount
(195.5) 21 557; N.Y. (1967)
H. A.
6) The ascidian peroxide is miscible with water in any ratio. As we want to determine the number of electron in a reduction process, a non-buffered media was employed, by which the second reduction wave was observed. In acetate buffer solution, on the other hand, only the first reduction wave of the peroxide was appeared. &v~ 7) R. D. Mair and R. T. Hall, im.533 ._,, an L. S. Silbert, Chapt. VII,p637 in “Organic --4 Peroxides” Ed. by D. Swem, vol 2, Wiley-Interscience, N.Y. (1971) !) This slightly viscous colorless liquid is stable to be stored at a temperature of -30 Co for several months. On heating the open end of a capillary tube which contained ca.10 ~1 in volume, however, the peroxide detonates with complete disappearance of the tube of 50 ~1. The explosion has enough power to blow off a frame of torch at a distance of 10 cm apart.
(Received in Japan 22 April
1991)