- Email: [email protected]
A molecular weight revision for compounds of the oligomycin complex
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A MOLECULAR WEIGHTREVISION FOR COMPOUNDS OF THE OLIGOMYCINCOMPLEX* W. F. Prouty,
H. K. Schnoes, and F. M. Strong
Department of Biochemistry,
University
of Wisconsin,
Madison, Wisconsin 53706
ReceivedJanuary 17, 1969 SUIllIllCiX?y.Mass spectra and elementary analyses of oligomycins A and B and of several derivatives indicate that their molecular weights and formulas are C45H74011(M.W. 790) and C45H72012(M.W. 8041, respectively. Use of compoundsof the oligomycin complex for studying oxidative phosphorylation interest
(Lardy, et al.,
in the stoichiometry
1965, Racker, 1968) has stimulated of oligomycin binding with mitochondrial
components. Work done in this laboratory osmometry and integration
involving
vapor pressure
of the proton magnetic resonance spectrum of
oligomycin B indicated that present molecular weight data on the oligomycin compoundsare incorrect
(Foster,
1966).
Further studies have now shown
that the most probable values are approximately reported (Masamuni, et al.,
twice those previously
1956).
The current studies were initially
carried out on oligomycin
samples which were prepared in 1955 and had been stored meanwhile in a laboratory
cabinet at room temperature but protected
the newer results differed
so greatly
from the original
from light.
Since
ones, it seemed
possible that some change in the samples might have occurred, although in 1965 they still 1965).
exhibited
the sameenzyme-inhibitory
potency (Lardy,
New preparations of oligomycin A and B were therefore
-_ *Supported in part by NIH grant No. AI-04419.
511
isolated
Vol. 34, No. 4, 1969
from freshly
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
grown -Streptomyces -.-__--diastatochromogenes-Strain
Production and isolation described (Visser,
M-248*'".
of oligomycins were carried out as previously
et al.,
1960).
Identity
of the newly isolated
products with samples which had been stored for 13 years was established by comparisons of their behavior,
infrared
spectra,
massspectra,
chromatographic
and elementary composition.
The molecular weights were measuredby massspectroscopy.
The
molecular ions observed for oligomycins A, b, and C and for several of their
derivatives
are shown in Table I.
The data indicate
that
oligomycin B has a molecular weight of 804; the molecular ions of the corresponding acetate (M.W. 972) and TMSether (M.W. 1092) both agree exactly
with the values required for tetra-substituted
derivatives.
The molecular ions of dihydro and hexahydro-oligomycin thereof further
B and derivatives
confirm the value of 804 as the molecular weight of
oligomycin B. For oligomycin A the data in Table I indicate
a molecular weight
of 790. The next intense peak (greater than 5%of the base peak) at lower mass in the oligomycin A spectrum is at m/e 446, the molecular weight reported by Beechey et al. (1967) for this compound. The molecular weight of oligomycin A isolated from another organism was reported to be 772 (Forberg and Fricke, "molecule massspectrography"
1966).
This value was determined by
(Ardenne et al.,
1961).
Our data suggest
that this is probably an M-18 peak. The massspectral
data for oligomycin C (Table I) do not permit
an unequivocal measurementof the molecular weight. highest peak in the spectrum of the underivatized
Although the
compoundwas at
m/e 742, a peak was observed in the spectrum of the TMS-ether at m/e 1062. On the assumption that this also is a tetra-TMS derivative, a*We wish to thank Prof. E. McCoy of the Dept. of Bacteriology Univ. of Wisconsin for kindly supplying the culture.
512
the
of the
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Molecular Ions of Oligomycins and Derivativesa -..--~-
__
l__-___-----~_l.ll_
Underivatized ---
TMS-etherb
Acetate --
Oligomycin A
790
1078
958
Oligomcyin B
804
1092
972
806
1094
---
810
1098
978e
742f
----
---
Compound
dihydro-oligomycin hexahydro-oligomycin
BC Bd
Oligomycin C ~-I_
_--P--1-1__
_---_-.-
---
aAll massspectra were measured on an MS-902 massspectrometer at the Space Sciences Lab, Univ. of California, Berkeley, California with a source temperature not exceeding 22S" (as indicated by the source temperature meter), and an ionizing voltage of 70 EV. Wethank Dr. A. L. Burlingame for the use of the mass spectrometer and B. R. Simoneit for one of the massspectra. bThe trimethylsilyl (TMS) ethers were prepared by adding the compound to an excess of silylating reagent (pyridine, hexamethyldisilizane, chloro-trimethylsilane, 7~2~1, v/v), allowing the reaction to proceed off the resulting at room temperature for 30 minutes, filtering pyridine hydrochloride and removing solvents -in vacua. ?'repared by partial hydrogenation over 2% Pd on CaCO3in ethyl acetate (S. Masamuniand E. E. Van Tamelen, 1957). dprepared by hydrogenating in methanol for 24 hours at 3 atm. pressure over previously reduced Pt02. @Amorphousproduct obtained by acetylation with pyridine and excess acetic anhydride; homogeneouson thin layer chromatography. f Highest observed peak in the spectrum.
molecular weight of oligomycin C would have to be at least 774. The new values for the molecular weights of oligomycins A and B enable us to re-evaluate
the microanalytical
compounds. Table II indicates nearly fit
the available
the molecular formulas which most
evidence, G.,
andC 45H72012 for oligomycin B.
data compiled on these
C45H74011 for oligomycin A
The 14 massunit difference
513
between
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b z 3
514
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the molecular weights is attributed hydrogen
to one
oxygen and two less
more
in oligomycin B rather than to an extra methylene group
atoms
because the elementary compositions of the respective significantly
homologs differ
from the observed values given in Table II
(calcd.
(M.W. 804): C46H76011
C, 68.61; H, 9.52; 0, 21.87; calcd. for
C44H70012(M.W. 790):
C, 66.79; H, 8.92; 0, 24.28).
for
The molecular formula C28H4604proposed for oligomycin A by Beechey et al. analytical
(1967) is quite obviously
data (calculated
eliminated by the above
for C28H4604: C, 75.29; H, 10.38; 0, 14.33).
Likewise the formula C27H4404proposed by these authors for rutamycin does not agree with published analytical
data (Thompson, et al.,
Undoubtedly the peaks observed by Beechey, et al.
(1967) in the
1961). mass
spectra of these compoundsand considered to be the molecular ions corresponded instead to large fragments of the original It is obvious that the analytical
data in Table II do not fit
proposed formulas closely enough to be entirely no explanation
for the rather
discrepancies between the calculated
of oligomycin B. relatively
However, the agreement
for the acetates and for the dihydro derivative
Such discrepancies are not unknown in the case of
large molecular weight antibiotics
(Ceder, et al.,
We are convinced that our 13-year-old
preparations
oligomycin compoundsare unchanged and are identical isolated
samples. Wehave no satisfactory
explanation
large difference
in the presently
those originally
published (Masamuni, et al.,
values are approximately possibility
consistent
and observed values, particularly
for the carbon content of oligomycins A and B. is considerably better
our
unambiguous. Wehave
but disturbingly
small
molecules.
this quite definitely
seems
of the
with the newly for the very
proposed molecular weights from
double the original
that the original
1964).
1958).
Since the newer
ones, we considered the
preparations had undergone dimerization; not
to
be the case.
515
Vol. 34, No. 4, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Ardenne, V., Steinfelder, K., and T&nmler, F. Angew. Chemie73, 1936 (1961). Beechey, R. B., Williams, V., Holloway, C. T., Knight, I. G. , and Roberton, A. M. Biochem. Biophys. Res. Comm.23, 339 (1967). Ceder, O., Eriksson, G., Waisvisz, J. M., and Van Der Hoeven, M. G. Acta Chemica Scandinavica 18, 109 (1964). Dyer, J. Unpublished experiments, 1955. Foster, G. A. Ph.D. Thesis, University of Wisconsin (1966). Forberg, W., and Fricke, H. Personal communication (1966). Lardy, H. A. Univ. of Wisconsin, Madison, Wis., personal communication (1965). Lardy, H. A., Witonsky, P., and Johnson, D. Biochemistry 4, 552 (1965). Masamuni, S., Sehgal, J. M., Van Tamelen, E. E., Strong, F. M., and Peterson, W. H. J. Am. Chem. Sot. 80, 6092 (1956). Masamuni, S. and Van Tamelen, E. E. Unpub-rished experiments (1957). Racker, E. Sci. Am. 218, no. 2, 32 (1968). Thompson, R. Q., Hoehn, M. M. and Higgens, C. E. Antimicrobial Agents and Chemotherapy - 1961, American Association for Microbiology (1962), p. 474. Visser, J., Weinauer, D. E., Davis, R. C., Peterson, W. H., Nazarewicz, W., and Ordway, Ii. J. Biochem. Microbial. Technol. Eng. 2, 31 (1960).
516
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A MOLECULAR WEIGHTREVISION FOR COMPOUNDS OF THE OLIGOMYCINCOMPLEX* W. F. Prouty,
H. K. Schnoes, and F. M. Strong
Department of Biochemistry,
University
of Wisconsin,
Madison, Wisconsin 53706
ReceivedJanuary 17, 1969 SUIllIllCiX?y.Mass spectra and elementary analyses of oligomycins A and B and of several derivatives indicate that their molecular weights and formulas are C45H74011(M.W. 790) and C45H72012(M.W. 8041, respectively. Use of compoundsof the oligomycin complex for studying oxidative phosphorylation interest
(Lardy, et al.,
in the stoichiometry
1965, Racker, 1968) has stimulated of oligomycin binding with mitochondrial
components. Work done in this laboratory osmometry and integration
involving
vapor pressure
of the proton magnetic resonance spectrum of
oligomycin B indicated that present molecular weight data on the oligomycin compoundsare incorrect
(Foster,
1966).
Further studies have now shown
that the most probable values are approximately reported (Masamuni, et al.,
twice those previously
1956).
The current studies were initially
carried out on oligomycin
samples which were prepared in 1955 and had been stored meanwhile in a laboratory
cabinet at room temperature but protected
the newer results differed
so greatly
from the original
from light.
Since
ones, it seemed
possible that some change in the samples might have occurred, although in 1965 they still 1965).
exhibited
the sameenzyme-inhibitory
potency (Lardy,
New preparations of oligomycin A and B were therefore
-_ *Supported in part by NIH grant No. AI-04419.
511
isolated
Vol. 34, No. 4, 1969
from freshly
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
grown -Streptomyces -.-__--diastatochromogenes-Strain
Production and isolation described (Visser,
M-248*'".
of oligomycins were carried out as previously
et al.,
1960).
Identity
of the newly isolated
products with samples which had been stored for 13 years was established by comparisons of their behavior,
infrared
spectra,
massspectra,
chromatographic
and elementary composition.
The molecular weights were measuredby massspectroscopy.
The
molecular ions observed for oligomycins A, b, and C and for several of their
derivatives
are shown in Table I.
The data indicate
that
oligomycin B has a molecular weight of 804; the molecular ions of the corresponding acetate (M.W. 972) and TMSether (M.W. 1092) both agree exactly
with the values required for tetra-substituted
derivatives.
The molecular ions of dihydro and hexahydro-oligomycin thereof further
B and derivatives
confirm the value of 804 as the molecular weight of
oligomycin B. For oligomycin A the data in Table I indicate
a molecular weight
of 790. The next intense peak (greater than 5%of the base peak) at lower mass in the oligomycin A spectrum is at m/e 446, the molecular weight reported by Beechey et al. (1967) for this compound. The molecular weight of oligomycin A isolated from another organism was reported to be 772 (Forberg and Fricke, "molecule massspectrography"
1966).
This value was determined by
(Ardenne et al.,
1961).
Our data suggest
that this is probably an M-18 peak. The massspectral
data for oligomycin C (Table I) do not permit
an unequivocal measurementof the molecular weight. highest peak in the spectrum of the underivatized
Although the
compoundwas at
m/e 742, a peak was observed in the spectrum of the TMS-ether at m/e 1062. On the assumption that this also is a tetra-TMS derivative, a*We wish to thank Prof. E. McCoy of the Dept. of Bacteriology Univ. of Wisconsin for kindly supplying the culture.
512
the
of the
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE I Molecular Ions of Oligomycins and Derivativesa -..--~-
__
l__-___-----~_l.ll_
Underivatized ---
TMS-etherb
Acetate --
Oligomycin A
790
1078
958
Oligomcyin B
804
1092
972
806
1094
---
810
1098
978e
742f
----
---
Compound
dihydro-oligomycin hexahydro-oligomycin
BC Bd
Oligomycin C ~-I_
_--P--1-1__
_---_-.-
---
aAll massspectra were measured on an MS-902 massspectrometer at the Space Sciences Lab, Univ. of California, Berkeley, California with a source temperature not exceeding 22S" (as indicated by the source temperature meter), and an ionizing voltage of 70 EV. Wethank Dr. A. L. Burlingame for the use of the mass spectrometer and B. R. Simoneit for one of the massspectra. bThe trimethylsilyl (TMS) ethers were prepared by adding the compound to an excess of silylating reagent (pyridine, hexamethyldisilizane, chloro-trimethylsilane, 7~2~1, v/v), allowing the reaction to proceed off the resulting at room temperature for 30 minutes, filtering pyridine hydrochloride and removing solvents -in vacua. ?'repared by partial hydrogenation over 2% Pd on CaCO3in ethyl acetate (S. Masamuniand E. E. Van Tamelen, 1957). dprepared by hydrogenating in methanol for 24 hours at 3 atm. pressure over previously reduced Pt02. @Amorphousproduct obtained by acetylation with pyridine and excess acetic anhydride; homogeneouson thin layer chromatography. f Highest observed peak in the spectrum.
molecular weight of oligomycin C would have to be at least 774. The new values for the molecular weights of oligomycins A and B enable us to re-evaluate
the microanalytical
compounds. Table II indicates nearly fit
the available
the molecular formulas which most
evidence, G.,
andC 45H72012 for oligomycin B.
data compiled on these
C45H74011 for oligomycin A
The 14 massunit difference
513
between
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b z 3
514
Vol. 34, No. 4, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the molecular weights is attributed hydrogen
to one
oxygen and two less
more
in oligomycin B rather than to an extra methylene group
atoms
because the elementary compositions of the respective significantly
homologs differ
from the observed values given in Table II
(calcd.
(M.W. 804): C46H76011
C, 68.61; H, 9.52; 0, 21.87; calcd. for
C44H70012(M.W. 790):
C, 66.79; H, 8.92; 0, 24.28).
for
The molecular formula C28H4604proposed for oligomycin A by Beechey et al. analytical
(1967) is quite obviously
data (calculated
eliminated by the above
for C28H4604: C, 75.29; H, 10.38; 0, 14.33).
Likewise the formula C27H4404proposed by these authors for rutamycin does not agree with published analytical
data (Thompson, et al.,
Undoubtedly the peaks observed by Beechey, et al.
(1967) in the
1961). mass
spectra of these compoundsand considered to be the molecular ions corresponded instead to large fragments of the original It is obvious that the analytical
data in Table II do not fit
proposed formulas closely enough to be entirely no explanation
for the rather
discrepancies between the calculated
of oligomycin B. relatively
However, the agreement
for the acetates and for the dihydro derivative
Such discrepancies are not unknown in the case of
large molecular weight antibiotics
(Ceder, et al.,
We are convinced that our 13-year-old
preparations
oligomycin compoundsare unchanged and are identical isolated
samples. Wehave no satisfactory
explanation
large difference
in the presently
those originally
published (Masamuni, et al.,
values are approximately possibility
consistent
and observed values, particularly
for the carbon content of oligomycins A and B. is considerably better
our
unambiguous. Wehave
but disturbingly
small
molecules.
this quite definitely
seems
of the
with the newly for the very
proposed molecular weights from
double the original
that the original
1964).
1958).
Since the newer
ones, we considered the
preparations had undergone dimerization; not
to
be the case.
515
Vol. 34, No. 4, 1969
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Ardenne, V., Steinfelder, K., and T&nmler, F. Angew. Chemie73, 1936 (1961). Beechey, R. B., Williams, V., Holloway, C. T., Knight, I. G. , and Roberton, A. M. Biochem. Biophys. Res. Comm.23, 339 (1967). Ceder, O., Eriksson, G., Waisvisz, J. M., and Van Der Hoeven, M. G. Acta Chemica Scandinavica 18, 109 (1964). Dyer, J. Unpublished experiments, 1955. Foster, G. A. Ph.D. Thesis, University of Wisconsin (1966). Forberg, W., and Fricke, H. Personal communication (1966). Lardy, H. A. Univ. of Wisconsin, Madison, Wis., personal communication (1965). Lardy, H. A., Witonsky, P., and Johnson, D. Biochemistry 4, 552 (1965). Masamuni, S., Sehgal, J. M., Van Tamelen, E. E., Strong, F. M., and Peterson, W. H. J. Am. Chem. Sot. 80, 6092 (1956). Masamuni, S. and Van Tamelen, E. E. Unpub-rished experiments (1957). Racker, E. Sci. Am. 218, no. 2, 32 (1968). Thompson, R. Q., Hoehn, M. M. and Higgens, C. E. Antimicrobial Agents and Chemotherapy - 1961, American Association for Microbiology (1962), p. 474. Visser, J., Weinauer, D. E., Davis, R. C., Peterson, W. H., Nazarewicz, W., and Ordway, Ii. J. Biochem. Microbial. Technol. Eng. 2, 31 (1960).
516