BIOCHEMICAL
Vol. 71, No. 4, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PRE-RESONANCE RAMAN SPECTRA OF ADRIAMYCIN* Kurt W. Hillig, II and Michael D. Morris Department of Chemistry, University of Michigan Ann Arbor, Michigan 48109 U.S.A. Received
June 25,1976
Raman spectra have been SUMMARY: Pre-resonance aqueous solutions of adriamycin. All bands are the anthracycline chromophore and are vibrations first (20,800 cm-l) electronic transition of the Both ring modes and vibrations of the carbonyl, methoxy substituents are observed. INTRODUCTION. currently tive
Adriamycin
available
of adriamycin
"unwinding" toxicity
of
There gross
with
to a lesser
extent,
DNA or metal
The most suitable actions *
with
to
ions. for
for is
from
normal
enzymatic
and skeletal
of the
to complexation
kinetics
ions.
There
of the
binding
design
activity
environment ions
the
The
by the drug
Such studies,
anti-tumor
DNA and metal
metal
effec-
toxic.
and related
details
information
of improved
studies
of adriamycin
drug to either
derivatives
cations
of the
very
Cardiotoxicity
metal
have been many previous
useful
extremely
DNA, inhibiting (2).
magnesium and other
of the molecular
very
also
While
have been shown to result
investigations
provide
is
(1).
are known to be due in part
mechanisms of binding
to DNA and,
it
the DNA molecule
of adriamycin
of calcium,
drugs
agent,
properties
intercalation
among the most promising
anti-cancer
as an anti-tumor
tumor-inhibiting
is
obtained for attributable to coupled to the molecule. hydroxy and
and
molecules are few of
however,
the can
of adriamycin
or lower
studying
(3).
toxicity.
adriamycin
an aqueous solution,
interwhere
Supported in part by Institutional Research Grant No. IN-40P to the University of Michigan from the American Cancer Society and in part by NIH Grant GM 22604.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in my form reserved.
1228
Vol. 71, No. 4, 1976
BIOCHEMICAL
Raman spectroscopy structural the
oxide
using
adriamycin
(doxorubicin
Wilmington,
were prepared
Del.)
probe
studies
into we
in water
and deuterium
tentative
assignments
in distilled
adjusted
water
Adria *
with
or deuterium
Solu-
oxide.
0.2 M_2-(N-morpholino)ethane-
to pH 6.15 with
were observed
or unbuffered
hydrochloride,
was used as received.
were buffered
acid,
No differences
between
potassium
spectra
hydroxide.
obtained
in buffered
solutions.
Raman spectra
were recorded
equipped
A Spectra-Physics
with
with
a cooled
adequate
provided
Generally,
offset
a Spex 1401 double
mono-
RCA C31034 photomultiplier.
126 He-Ne laser power.
60 mwatt
obtain
of these
and present
Adriamycin
Aqueous solutions
with
of
part
useful
bands.
Laboratories,
chromator,
first
He-Ne excitation
EXPERIMENTAL.
sulfonic
the only
almost
As the
Raman spectrum
of the major
tions
provides
details.
report
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
632.8
DC detection
of the high
nm excitation
was employed
fluorescence
to
backgrounds
encountered. Spectra
were obtained
were obtained
within
on samples
24 hours
0.02
All
- 0.04 2.
of sample preparation
spectra
to avoid
decomposition. RESULTS AND DISCUSSION. water
and deuterium
level
encountered
oxide
are
precluded
and depolarization
ratios,
Examination resonance of the *
The Raman spectra
spectra,
anthracycline
of
the
shown as Figure
obtaining data
to the
Adria
1.
meaningful
shows that
they
lowest
lying
First,
the
chromophore.
We thank Dr. Luigi Lenaz, sample of adriamycin.
adriamycin
Laboratories,
1229
in
The noise depolarization
is not presented
spectra
coupled
of
here. are pre* transition n-n
sample concentration for
a generous
BIOCHEMICAL
Vol.71,No.4,1976
Figure
range
(I
fairly
1.
intense
lower
spectra
which
visible
or absent
first
is only
electronic Extended
have confirmed
involves
primarily
cantly,
the
carbonyl
transition.
Thus,
Raman spectrum stretches and the
of adriamycin
that
the
first
the hydrocarbon pi-system we would
of the
C=O stretch
strong
band,
Finally,
the
is excita-
the
frequency of the -1 (20,800 cm , 480 nm).
calculations
on anthra-
electronic
pi-system
contributes
expect
no bands ring
anthraquinone
spectra. -1 below some 5000 cm
weak or absent
Although
saturated
a fairly
orbital
to give
Second,
in these
dominated
amino-sugar
the
normally
molecular
quinones
or the
Third, is
required
a He-Ne laser.
transition H&kel
normally
sugar moiety
are visible.
frequency
of 0.03 M adriamycin, He-Ne (a) H20 solution, pH 6.15 (b) D20 solution, unbuffered.
than
with
1625 cm-l),
barely tion
is
to the
chromophore (IR,
Raman spectra excitation. MES buffer.
0.04 M),
attributable
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transition Signifi-
(4-6). little
to this
to see a preresonance
by aromatic
ring
and vibrations
modes, with of
the
saturated
carbonyl ring
absent.
low frequency
bands do not
1230
change
in frequency
on
Vol. 71, No. 4, 1976
going
BIOCHEMICAL
from H20 to D20 solution,
unambiguously.
Gaziz
in the
anthraquinone
at
(8).
It
adriamycin coupled
contain
large
bending
the
modes.
bonds of the
assignment
hydroxy
same vibrabands of
from carbonyl
bending,
With
less
certainty,
to bending
we attri-
to the
side
carbon-oxygen
chains.
A similar
376 cm-'
band in the
-1 The weak 1083 cm band is attributahle
to either
spectrum
of
4-nitrocatechol
carbon-oxygen
stretch
the
Raman
(9).
or an aromatic
can not distinguish
434 cm-1
the
tetrahydroxy-
to the
and methoxy
has been made for
of
while
443 and 466 cm-'
contributions
352 cm-1 band primarily
the
single
spectrum
them
490 cm-1
the
(7),
4o K, has been assigned that
to assign
attribute bending
fluorescence
is probable
to ring
is difficult
to carbonyl
band observed
tion
it
and co-workers
band in anthraquinone
bute
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
between
these
carbon
a methoxy
hydrogen
possibilities
bend.
We
on the basis
of our data. Hydroxyanthraquinones in the
1200-1300
oxygen
stretching
10-12).
When D20
and a new strong
coupling
of
these
the phenolic
bands carhon-
(8,
adriamycin.
hands disappear the
phenolic
bending.
for
on the mass of
by deuterium Although
due to coupled
1213-1305
Since
appears.
depends strongly tution
at
H20, these
1335 cm-l
show two or three
and oxygen-hydrogen
These are observed replaces
typically
cm-1 region
band at
vibrations
hydrogen,
substi-
causes them to disappear.
assignment
probably
predominantly
hydrogen
bonding
increase
in the
of the
a carbon-oxygen
from phenolic nominal
oxygen
due to conjugation
formed
(4,5,8).
Thus,
new mode is not stretch.
to carbonyl
carbon-oxygen around
the
assignment
of
1231
certain,
is
Internal
groups
bond order
it
causes some of
chelate-type a hand in the
the phenolic
ring 1330 cm-1
Vol. 71, No. 4, 1976
region,
well
BIOCHEMICAL
removed
from the normal
The pair
of
the aromatic
commonly observed region
suggests
in the
(13,14).
that
-1 cm .
this
We do not
found
in the
quinone
about
observe
and most of
cm-1 band contains
its
1590 cm-'
its
is very
band in D20 spectra,
in
bands in D20
of the band at 1565
band aromatic of
We believe
C=C stretching
C=C stretch anthra-
that
the
component,
the hydroxy
1565
but must
or hydrogen
to 1543 cm-1 in D20.
shift
the C=O stretch
spectra.
these
Raman spectra
derivatives.
because of
modes are
correct.
and normal
(1637 cm-')
pre-resonance
is
of
to
of anthraquinones
the assignment
the
a large
As expected, carbonyl
spectra
to modes involving
bonded carbonyls
Ring stretching
infra-red
assignment
infra-red
be coupled
rings.
The invariance
We are uncertain
lower
C-O stretch
1418 and 1443 cm-1 are assignable
of bands at
C=C stretches
also
phenolic
1100 cm-1 , is reasonable.
around
this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the hydrogen-bonded
weak in our aqueous solution
We have been unable where it
is expected
to locate to appear
this at somewhat
frequency. Clearly,
actions
it
with
Although argon
ion
under
true
would
either
be useful
DNA or metal
the adriamycin laser
adriamycin
ions
adriamycin
at lower
inter-
concentrations.
480 nm band coincides
nicely
observation
adriamycin
488 nm line,
resonance
Raman conditions
of
is precluded
with
the
spectra
by intense
fluorescence.
In recent spectroscopic transitions measurements of adriamycin
to study
years
several
techniques
fluorescence-rejecting
based on time-resolution We are presently
have been proposed. of the and its
resonance
Raman spectra
biochemically
1232
significant
of
Raman or stimulated undertaking low concentrations complexes
by
BIOCHEMICAL
Vol. 71, No. 4,1976
coherent in a later
anti-Stokes
AND BIOPHYSICAL
Raman spectroscopy
RESEARCH COMMUNICATIONS
and will
report
these
publication.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Goldin, A. and Johnson, R.K. (1971) Cancer Chemother. Rep., Part 3, 5, 137-145. Kersten, H. and Kersten, W. (1974) Inhibitors of Nucleic Acid Synthesis, ~~-67-86, Springer-Verlag, New York. Young, D.M. (1975) Cancer Chemother. Rep., Part 3, 6, 159-175. Yoshida, 2. and Takabayashi, F. (1968) Tetrahedron, 24, 913-943. Issa, I-M., Issa, R-M., El-Ezaby, M.S. and Ahmed, Y-2. (1969) Z. Phys. Chem., 242, 169-176. Chem. Sot. Japan, 2, Kuboyama, A. and Wada, K. (1966) Bull, 1874-1877. Gazis, E., Heim, P., Meister, Ch. and Dorr, F. (1970) Spectrochim. Acta, 26A, 497-516. ..Gastilovich, E-A., Kryuchova, G.T. and Shigorin, D.N. (1975) 38, 500-505. opt. Spektrosk., _-Nothnagel, E.A. and Zitter, R.N. (1976) J. Phys. Chem., 80, 722-727. H. and Shephard, N. (1954) Trans. Farad. SOC., 2, Hadzi, 911-918. Z and Urbanski, T. (1969) Bull. Acad. Pal. Sci., Jaworska, Ser. Sci. Chem. 17, 579-585. Issa, R-M., Ahmed., Y.Z. and Zewail, A.H. (1972) Egypt- J. Chem., 15, l-10. J. (1971) Coil. Czech. Comm., Stepan, C.V. and Voderhall, 36, 3964-3979. Acta, e, 1785-1791. Walker, R.A. (1971) Spectrochim.
1233
Vol. 71, No. 4, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PRE-RESONANCE RAMAN SPECTRA OF ADRIAMYCIN* Kurt W. Hillig, II and Michael D. Morris Department of Chemistry, University of Michigan Ann Arbor, Michigan 48109 U.S.A. Received
June 25,1976
Raman spectra have been SUMMARY: Pre-resonance aqueous solutions of adriamycin. All bands are the anthracycline chromophore and are vibrations first (20,800 cm-l) electronic transition of the Both ring modes and vibrations of the carbonyl, methoxy substituents are observed. INTRODUCTION. currently tive
Adriamycin
available
of adriamycin
"unwinding" toxicity
of
There gross
with
to a lesser
extent,
DNA or metal
The most suitable actions *
with
to
ions. for
for is
from
normal
enzymatic
and skeletal
of the
to complexation
kinetics
ions.
There
of the
binding
design
activity
environment ions
the
The
by the drug
Such studies,
anti-tumor
DNA and metal
metal
effec-
toxic.
and related
details
information
of improved
studies
of adriamycin
drug to either
derivatives
cations
of the
very
Cardiotoxicity
metal
have been many previous
useful
extremely
DNA, inhibiting (2).
magnesium and other
of the molecular
very
also
While
have been shown to result
investigations
provide
is
(1).
are known to be due in part
mechanisms of binding
to DNA and,
it
the DNA molecule
of adriamycin
of calcium,
drugs
agent,
properties
intercalation
among the most promising
anti-cancer
as an anti-tumor
tumor-inhibiting
is
obtained for attributable to coupled to the molecule. hydroxy and
and
molecules are few of
however,
the can
of adriamycin
or lower
studying
(3).
toxicity.
adriamycin
an aqueous solution,
interwhere
Supported in part by Institutional Research Grant No. IN-40P to the University of Michigan from the American Cancer Society and in part by NIH Grant GM 22604.
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in my form reserved.
1228
Vol. 71, No. 4, 1976
BIOCHEMICAL
Raman spectroscopy structural the
oxide
using
adriamycin
(doxorubicin
Wilmington,
were prepared
Del.)
probe
studies
into we
in water
and deuterium
tentative
assignments
in distilled
adjusted
water
Adria *
with
or deuterium
Solu-
oxide.
0.2 M_2-(N-morpholino)ethane-
to pH 6.15 with
were observed
or unbuffered
hydrochloride,
was used as received.
were buffered
acid,
No differences
between
potassium
spectra
hydroxide.
obtained
in buffered
solutions.
Raman spectra
were recorded
equipped
A Spectra-Physics
with
with
a cooled
adequate
provided
Generally,
offset
a Spex 1401 double
mono-
RCA C31034 photomultiplier.
126 He-Ne laser power.
60 mwatt
obtain
of these
and present
Adriamycin
Aqueous solutions
with
of
part
useful
bands.
Laboratories,
chromator,
first
He-Ne excitation
EXPERIMENTAL.
sulfonic
the only
almost
As the
Raman spectrum
of the major
tions
provides
details.
report
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
632.8
DC detection
of the high
nm excitation
was employed
fluorescence
to
backgrounds
encountered. Spectra
were obtained
were obtained
within
on samples
24 hours
0.02
All
- 0.04 2.
of sample preparation
spectra
to avoid
decomposition. RESULTS AND DISCUSSION. water
and deuterium
level
encountered
oxide
are
precluded
and depolarization
ratios,
Examination resonance of the *
The Raman spectra
spectra,
anthracycline
of
the
shown as Figure
obtaining data
to the
Adria
1.
meaningful
shows that
they
lowest
lying
First,
the
chromophore.
We thank Dr. Luigi Lenaz, sample of adriamycin.
adriamycin
Laboratories,
1229
in
The noise depolarization
is not presented
spectra
coupled
of
here. are pre* transition n-n
sample concentration for
a generous
BIOCHEMICAL
Vol.71,No.4,1976
Figure
range
(I
fairly
1.
intense
lower
spectra
which
visible
or absent
first
is only
electronic Extended
have confirmed
involves
primarily
cantly,
the
carbonyl
transition.
Thus,
Raman spectrum stretches and the
of adriamycin
that
the
first
the hydrocarbon pi-system we would
of the
C=O stretch
strong
band,
Finally,
the
is excita-
the
frequency of the -1 (20,800 cm , 480 nm).
calculations
on anthra-
electronic
pi-system
contributes
expect
no bands ring
anthraquinone
spectra. -1 below some 5000 cm
weak or absent
Although
saturated
a fairly
orbital
to give
Second,
in these
dominated
amino-sugar
the
normally
molecular
quinones
or the
Third, is
required
a He-Ne laser.
transition H&kel
normally
sugar moiety
are visible.
frequency
of 0.03 M adriamycin, He-Ne (a) H20 solution, pH 6.15 (b) D20 solution, unbuffered.
than
with
1625 cm-l),
barely tion
is
to the
chromophore (IR,
Raman spectra excitation. MES buffer.
0.04 M),
attributable
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
transition Signifi-
(4-6). little
to this
to see a preresonance
by aromatic
ring
and vibrations
modes, with of
the
saturated
carbonyl ring
absent.
low frequency
bands do not
1230
change
in frequency
on
Vol. 71, No. 4, 1976
going
BIOCHEMICAL
from H20 to D20 solution,
unambiguously.
Gaziz
in the
anthraquinone
at
(8).
It
adriamycin coupled
contain
large
bending
the
modes.
bonds of the
assignment
hydroxy
same vibrabands of
from carbonyl
bending,
With
less
certainty,
to bending
we attri-
to the
side
carbon-oxygen
chains.
A similar
376 cm-'
band in the
-1 The weak 1083 cm band is attributahle
to either
spectrum
of
4-nitrocatechol
carbon-oxygen
stretch
the
Raman
(9).
or an aromatic
can not distinguish
434 cm-1
the
tetrahydroxy-
to the
and methoxy
has been made for
of
while
443 and 466 cm-'
contributions
352 cm-1 band primarily
the
single
spectrum
them
490 cm-1
the
(7),
4o K, has been assigned that
to assign
attribute bending
fluorescence
is probable
to ring
is difficult
to carbonyl
band observed
tion
it
and co-workers
band in anthraquinone
bute
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
between
these
carbon
a methoxy
hydrogen
possibilities
bend.
We
on the basis
of our data. Hydroxyanthraquinones in the
1200-1300
oxygen
stretching
10-12).
When D20
and a new strong
coupling
of
these
the phenolic
bands carhon-
(8,
adriamycin.
hands disappear the
phenolic
bending.
for
on the mass of
by deuterium Although
due to coupled
1213-1305
Since
appears.
depends strongly tution
at
H20, these
1335 cm-l
show two or three
and oxygen-hydrogen
These are observed replaces
typically
cm-1 region
band at
vibrations
hydrogen,
substi-
causes them to disappear.
assignment
probably
predominantly
hydrogen
bonding
increase
in the
of the
a carbon-oxygen
from phenolic nominal
oxygen
due to conjugation
formed
(4,5,8).
Thus,
new mode is not stretch.
to carbonyl
carbon-oxygen around
the
assignment
of
1231
certain,
is
Internal
groups
bond order
it
causes some of
chelate-type a hand in the
the phenolic
ring 1330 cm-1
Vol. 71, No. 4, 1976
region,
well
BIOCHEMICAL
removed
from the normal
The pair
of
the aromatic
commonly observed region
suggests
in the
(13,14).
that
-1 cm .
this
We do not
found
in the
quinone
about
observe
and most of
cm-1 band contains
its
1590 cm-'
its
is very
band in D20 spectra,
in
bands in D20
of the band at 1565
band aromatic of
We believe
C=C stretching
C=C stretch anthra-
that
the
component,
the hydroxy
1565
but must
or hydrogen
to 1543 cm-1 in D20.
shift
the C=O stretch
spectra.
these
Raman spectra
derivatives.
because of
modes are
correct.
and normal
(1637 cm-')
pre-resonance
is
of
to
of anthraquinones
the assignment
the
a large
As expected, carbonyl
spectra
to modes involving
bonded carbonyls
Ring stretching
infra-red
assignment
infra-red
be coupled
rings.
The invariance
We are uncertain
lower
C-O stretch
1418 and 1443 cm-1 are assignable
of bands at
C=C stretches
also
phenolic
1100 cm-1 , is reasonable.
around
this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the hydrogen-bonded
weak in our aqueous solution
We have been unable where it
is expected
to locate to appear
this at somewhat
frequency. Clearly,
actions
it
with
Although argon
ion
under
true
would
either
be useful
DNA or metal
the adriamycin laser
adriamycin
ions
adriamycin
at lower
inter-
concentrations.
480 nm band coincides
nicely
observation
adriamycin
488 nm line,
resonance
Raman conditions
of
is precluded
with
the
spectra
by intense
fluorescence.
In recent spectroscopic transitions measurements of adriamycin
to study
years
several
techniques
fluorescence-rejecting
based on time-resolution We are presently
have been proposed. of the and its
resonance
Raman spectra
biochemically
1232
significant
of
Raman or stimulated undertaking low concentrations complexes
by
BIOCHEMICAL
Vol. 71, No. 4,1976
coherent in a later
anti-Stokes
AND BIOPHYSICAL
Raman spectroscopy
RESEARCH COMMUNICATIONS
and will
report
these
publication.
REFERENCES 1. 2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Goldin, A. and Johnson, R.K. (1971) Cancer Chemother. Rep., Part 3, 5, 137-145. Kersten, H. and Kersten, W. (1974) Inhibitors of Nucleic Acid Synthesis, ~~-67-86, Springer-Verlag, New York. Young, D.M. (1975) Cancer Chemother. Rep., Part 3, 6, 159-175. Yoshida, 2. and Takabayashi, F. (1968) Tetrahedron, 24, 913-943. Issa, I-M., Issa, R-M., El-Ezaby, M.S. and Ahmed, Y-2. (1969) Z. Phys. Chem., 242, 169-176. Chem. Sot. Japan, 2, Kuboyama, A. and Wada, K. (1966) Bull, 1874-1877. Gazis, E., Heim, P., Meister, Ch. and Dorr, F. (1970) Spectrochim. Acta, 26A, 497-516. ..Gastilovich, E-A., Kryuchova, G.T. and Shigorin, D.N. (1975) 38, 500-505. opt. Spektrosk., _-Nothnagel, E.A. and Zitter, R.N. (1976) J. Phys. Chem., 80, 722-727. H. and Shephard, N. (1954) Trans. Farad. SOC., 2, Hadzi, 911-918. Z and Urbanski, T. (1969) Bull. Acad. Pal. Sci., Jaworska, Ser. Sci. Chem. 17, 579-585. Issa, R-M., Ahmed., Y.Z. and Zewail, A.H. (1972) Egypt- J. Chem., 15, l-10. J. (1971) Coil. Czech. Comm., Stepan, C.V. and Voderhall, 36, 3964-3979. Acta, e, 1785-1791. Walker, R.A. (1971) Spectrochim.
1233
