Oxidation of ascorbic acid in the presence of phthalocyanine metal complexes. Chemical aspects of catalytic therapy of cancer 2. Catalysis by cobalt octacarboxyphthalocyanine. Reaction products

Article abstract of DOI:10.1007/s11172-005-0121-y

The products of ascorbic acid oxidation in the presence of cobalt octa-4,5-carboxy-phthalocyanine sodium salt (TPH) were identified. These include the ascorbate radical (A^?), hydroxyl radical (OH ^?), and hydrogen peroxide (H₂O₂). The kinetics of accumulation and consumption of the reaction products was studied. For the concentration ranges of ascorbic acid C_AH2 = 0-2.5·10^-3 mol L^-1 and the catalyst C _TPH = 0-3.5·10^-5 mol L^-1, the the highest possible concentration of the ascorbate radical is ～10^-7 mol L^-1, the concentration of H₂O₂ is 7·10^-4 (30% of the starting concentration of ascorbic acid) and the concentration of the hydroxyl radical is at most 10^-6 mol L^-1.

Full text of DOI:10.1007/s11172-005-0121-y

2322
Russian Chemical Bulletin, International Edition, Vol. 53, No. 10, pp. 2322—2326, October, 2004
Oxidation of ascorbic acid in the presence of phthalocyanine metal
complexes. Chemical aspects of catalytic therapy of cancer
2
.* Catalysis by cobalt octacarboxyphthalocyanine. Reaction products
E. G. Petrova,aꢀ S. A. Borisenkova, and O. L. Kaliya
b
aꢀ
aFederal State Unitary Enterprise State Research Center
"
Research Institute of Organic Intermediate Products and Dyes,"
building 4, 1 ul. B. Sadovaya, 101999 Moscow, Russian Federation.
Fax: +7 (095) 254 1200. Eꢀmail: jerrik@rol.ru
Department of Chemistry, M. V. Lomonosov Moscow State University,
Leninskie Gory, 119899 Moscow, Russian Federation.
b
Fax: +7 (095) 932 8846. Eꢀmail: borisenk@petrol.chem.msu.ru
The products of ascorbic acid oxidation in the presence of cobalt octaꢀ4,5ꢀcarboxyꢀ
•
–
phthalocyanine sodium salt (TPH) were identified. These include the ascorbate radical (A ),
•
hydroxyl radical (OH ), and hydrogen peroxide (H O ). The kinetics of accumulation and
2
2
consumption of the reaction products was studied. For the concentration ranges of ascorbic
acid CAH2 = 0—2.5•10–3 mol L and the catalyst CTPH = 0—3.5•10 mol L , the the
–1
–5
–1
–
7
–1
highest possible concentration of the ascorbate radical is ~10 mol L , the concentration of
–
4
H O is 7•10 (30% of the starting concentration of ascorbic acid) and the concentration of
2
2
–
6
–1
the hydroxyl radical is at most 10 mol L
.
Key words: cobalt phthalocyanine, ascorbic acid, oxidation, hydrogen peroxide, ascorbate
radical, hydroxyl radical, catalysis, cancer therapy.
Oxidation of ascorbic acid (AH ) is a highly important
mechanism. The aim of this research is to identify the
reaction products and to study the kinetics of product
accumulation and consumption.
2
process related to vital functions of living organisms. Sevꢀ
eral years ago this reaction has been used as the basis for
2
developing a new method of cancer therapy. The essence
of the method is in that a catalyst for AH oxidation is
2
Experimental
introduced into the tumor either systemically or regionꢀ
ally, and after a specified period, ascorbic acid is adminisꢀ
tered systemically into the body. The cytotoxic effect of
Sodium salt of cobalt octaꢀ4,5ꢀcarboxyphthalocyanine
CoРc(COONa)8 — Teraphthal (ТPH) was synthesized at the
Research Institute of Organic Intermediate Products and Dyes
and complies with the Enterprise Pharmacopeia Provision (FSP
No. 42ꢀ0047ꢀ1680) approved by the Russian Federation Pharꢀ
macopeia Committee. The electronic absorption spectrum of
Teraphthal in a 0.025 М aqueous phosphate buffer with рH 6.9
the catalyst—AH system is assumed to be due to the
2
presence of a free radical formed by the catalytic oxidaꢀ
tion of AH . However, no systematic study of the nature
2
of these radicals or quantitative characteristics of the proꢀ
cesses in which they are formed and consumed has been
carried out to date.
5
–1
–1
has maxima at λ 674 (ε = 1.2•10 L mol cm ) and 333 nm.
Ascorbic acid (AH ), pharmaceutical grade (Russia). The
2
Various phthalocyanine metal complexes (РcМ) exꢀ
hibiting enhanced tumorꢀseeking properties, were studied
as catalysts in the catalytic therapy of cancer.2,3 Among
these, the sodium salt of cobalt octaꢀ4,5ꢀcarboxyphthaloꢀ
cyanine (Teraphthal (ТPH)) proved to be the most effiꢀ
cient in experiments on cells and animals with transꢀ
planted tumors.
Previously, we studied the kinetics of oxidation of
ascorbic acid in the presence of TPH, obtained the kiꢀ
netic equation, and proposed a stepꢀbyꢀstep reaction
electronic absorption spectrum of a solution of the acid in a
0
2
.025 М phosphate buffer with рH 6.9 has a maximum at λ =
4
–1
–1
67 nm, ε = 1.4•10 L mol cm
.
General procedure for the catalytic oxidation of ascorbic acid
1
has been reported previously. The experimental conditions were
as follows: aqueous solutions (phosphate buffer with рH 6.9),
room temperature, air. The reaction products are ascorbate radiꢀ
cals, superoxide radical anions, hydroxyl radicals, hydrogen
peroxide.
1
Ascorbate radical. The ascorbate radical (A•–) was identiꢀ
fied and its concentration was determined by ESR spectroscopy.
The spectra were recorded on a Varian Eꢀ3 spectrometer
*
For Part 1, see Ref. 1.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 2224—2228, October, 2004.
066ꢀ5285/04/5310ꢀ2322 © 2004 Springer Science+Business Media, Inc.
1

Study of products of ascorbic acid oxidation
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 10, October, 2004
2323
4
–1
7
–1
САН2•10 /mol L
СА•–•10 /mol L
0
.2 G
1
1
2
0
8
6
4
2
1
3.5
1
´
3.0
2.5
2.0
1.5
1.0
2
2
´
Fig. 1. ESR spectrum of the ascorbate radical A•–. Solution in a
0.5
phosphate buffer, рH 6.9. Conditions of recording the spectra:
5
amplification 5•10 , power 12.5 mW; modulation 0.2 G; C
2
=
0
AH2
1
0
20
30
40
50
t/min
–
3
–1
–4
–1
.2•10 mol L , CTPH = 0.35•10 mol L .
Fig. 2. Typical kinetic curves for the oxidation of ascorbic
acid (1, 2) and the formation of the ascorbate radicals (1´, 2´)
5
(
power 6.3—10 mW, amplification 5•10 , modulation ampliꢀ
–4
–1
in the AH —ТPH system; C
CAH2, mol L : 1.06•10 (1); 6.5•10 (2); рH 6.9, air, room
= 0.35•10
mol L ,
2
–1
TPH
tude 1 G, magnetic field sweep 100 G). The resulting spectra
Fig. 1) were similar to those described in the literature; the
splitting between the main components in the spectrum (Fig. 1)
–3
–4
(
temperature.
4
was 1.8 G, which coincided with published data. The concenꢀ
•–
tration of A in the reaction mixture was determined from the
ESR signal intensity using the shape factor. The TEMPOL
nitroxide was used as the standard for determination of the shape
factor.
Ascorbate radical A•–
The concentrations of the radical anion A•– were meaꢀ
sured in two series of experiments in which the initial
concentration of either the substrate or the catalyst was
varied within the ranges indicated above, while the conꢀ
centration of the second component remained constant.
Figure 2 presents kinetic curves for the variation of the
Superoxide radical anion O •–. This species was determined
2
by ESR spectroscopy using spin traps. Tiron (sodium 4,5ꢀdiꢀ
hydroxyꢀ1,3ꢀbenzodisulfonate) was employed as the trap. The
spectra were recorded on an Xꢀrange Varian EPR Eꢀ4 spectromꢀ
3
eter (power 10 mW, amplification 5•10 , modulation ampliꢀ
tudes 1 and 0.25 G, magnetic field sweep 100 G).
•–
concentration of A during the experiment and the conꢀ
Hydroxyl radical. The OH•– radical was detected using a
sumption curves of the ascorbic acid. The pattern of the
variation of A• concentration indicates that this is a
reaction intermediate. Nevertheless, the fact that the
amount of the A• radical anion formed is three orders of
magnitude lower than the amount of the consumed acid is
less obvious. Analysis of the published data and the kiꢀ
5
method based on oxidation of 2ꢀdesoxyribose (DR). Prelimiꢀ
–
narily, it was shown that the addition of DR to the reacꢀ
tion mixture does not influence the oxidation kinetics of AH2
in the presence of TPH. This method permits determination
of the total amount of the OH• radicals formed by the time
instant t.
Hydrogen peroxide. 10ꢀAcetylꢀ3,7ꢀdihydroxyphenoxazine
reacts with hydrogen peroxide in 1 : 1 ratio in the presence of
peroxidase to give resorufin, which can be easily detected
based on the electronic absorption spectrum (λmax = 563 nm).6
It was shown by special experiments that this reaction is not
affected by the presence of ascorbic acid and that the product
–
1
netic results obtained previously suggests that the major
process giving rise to this radical anion over the given
range of parameters is the oxidation of the ascorbic acid
–
monoanion (AH ) with dioxygen coordinated to the coꢀ
balt atom of the Teraphthal
(
resorufin) does not undergo any transformations under these
HA– + ТPH•O → A + O2•– + H+ + ТPH,
•–
(1)
conditions.
2
while the consumption of the radical anion follows the
route
Results and Discussion
A•– + A•– + 2 H O → AH + A + 2 OH ,
–
(2a)
(2b)
1
2
2
According to the mechanism proposed previously for
the oxidation of ascorbic acid in the presence of TPH,
O + A → A + O2•–.
•–
2
•
this may yield the ascorbate radical (HA ), which exists
7
•–
in the dissociated form (рК 4.25), i.e., as the A radical
anion, in the рH range under study, the superoxide radiꢀ
cal anion O2•–, the hydroxyl radical OH , hydrogen perꢀ
oxide, and dehydroascorbic acid (A). The lastꢀmentioned
product was not studied in our work.
However, the rate constant for reaction (2b) is four
orders of magnitude lower than that for pathway (2a);
hence, the consumption of the ascorbate radical for the
reaction with oxygen can be excluded from consideration.
In this case, the kinetic equations for the accumulation
1
•

2324
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 10, October, 2004
Petrova et al.
and consumption of A•– at a fixed partial pressure of
dioxygen have the following form:
converted within the coordination sphere of the metal
atom without escape of free radicals, possible primary
intermediates, into the solution bulk.
Wform = kformСАН2СТPH
,
(3)
(4)
The reaction of A with O2 (reaction (6)) is known9
•–
•–
to proceed at a high rate even in solution. Therefore, it
can be reasonably assumed that the rate of the reaction
between these radicals should be even higher in the metal
coordination sphere.
Wcons = kconsC2A•–,
where kform, kcons are the rate constants for the formation
•–
and consumption of the A radical anion, respectively.
Within the framework of this assumptions, the rate of
variation of the concentration of the radical anion is equal
to the algebraic sum of the formation and consumption
rates
A•– + O2•– + H O → A + HO2– + OH–,
(6)
2
k = 2.6•10 L mol s–1
8
–1
.
•
Hydrogen peroxide and the HO radical
Wobs = Wform – Wcons
=
=
kformCAH2CТPH – kconsC2A•–.,
(5)
Hydrogen peroxide. The question of formation of
hydrogen peroxide upon the oxidation of ascorbic acid in
the presence of TPH is a key issue for the understanding
of the reaction mechanism and the therapeutic action of
this complex. Our experiments have shown that during
the process, hydrogen peroxide is accumulated in the reꢀ
action zone (subsequently it is slowly consumed) in conꢀ
centrations comparable, in the order of magnitude, with
the concentrations of ascorbic acid (Fig. 3).
and at the point of maximum, Wform = Wcons
Processing of the ascending and descending branches
of the kinetic curves and the points corresponding to the
position of the maximum given by Eq. (5) showed that the
curves for variation of the concentration of the ascorbate
radical during the oxidation of ascorbic acid are described
satisfactorily by Eq. (5). This made it possible to calculate
the rate constants for reactions (3) and (4): k
=
form
–1
3
–1 –1
0
.01 L mol–1
s
, kcons ≈ 5•10 L mol
s . The only rate
constant for the recombination of ascorbate radicals
AH + O → A + H O
2
(7)
2
2
2
7
(
7
reaction (2)) reported in the literature, equal to
4
–1
–1, differs from the value we found by
The position of the maximum in the kinetic curves for
H O formation coincides, most often, with the time of
•10 L mol
s
almost an order of magnitude. In our opinion, this differꢀ
ence is not essential and may be related to either different
experimental conditions (рH, temperature mode, and so
on) or to other reactions consuming the ascorbate radical.
The kform value proved to be three orders of magnitude
lower than the rate constant for the oxidation of ascorbic
2
2
complete consumption of ascorbic acid. At low concenꢀ
trations of TРH (at a constant CAH2 concentration, this
corresponds to a high substrate : catalyst ratio), the maxiꢀ
mum shifts toward residual concentrations of the acid
substantially differing from zero. This is due to the fact
that oxidation of ascorbic acid is accompanied by the
–
1
–1
acid (k
= 30 L mol
s ) calculated from kinetic
AH2
1
data at an atmospheric pressure of oxygen. This fact may
indicate that the major pathway of the catalytic reaction
we study is the oxidation of ascorbic acid to give dehyꢀ
droascorbic acid in the coordination sphere of the TPH
metal atom without escape of the intermediate radical
anion A•– into the solution bulk.
4
–1
САН2•10 /mol L
,
,
5
–1
7
–1
СTPН•10 /mol L
СА•–•10 /mol L
,
6
–1
4
–1
СOН•10 /mol L
СН O •10 /mol L
2
2
8
7
6
5
4
3
2
1
1
2
3
4
5
2
.0
Superoxide radical anion O •
–
2
1.5
The addition of a spin trap (Tiron) into a solution
containing ascorbic acid and a catalyst does not give rise
to a signal for the product of reaction between O2• and
the trap over the whole range of reactant concentrations.
1
0
.0
.5
–
8
This fact is consistent with the data according to which
0
•
–
O2 can be detected in "substituted Co phthalocyanine
including ТРH)—AH " systems only after the introducꢀ
20
40
60 80 100 120
t/min
(
2
Fig. 3. Kinetic curves for the accumulation and consumption of
tion of KCN whose anion replaces the superoxide from
the coordination sphere of cobalt.
This result also does not contradict the above hypoꢀ
thesis according to which the main bulk of ascorbic acid is
H O , oxidation of ascorbic acid, destruction of TPH, generaꢀ
2
2
•–
•
tion of the A
radical anion and the OH radical (integral
curve); CAH2 = 8.2•10–4 mol L , CТРH = 0.35•10–4 mol L
–1
–1
,
рH 6.9, air.

Study of products of ascorbic acid oxidation
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 10, October, 2004
2325
destruction of the catalyst. Full destruction at low conꢀ
centrations results in termination of the catalytic process
long before the substrate is exhausted.
Table 1. Rates of hydrogen peroxide decomposition (WH O )
2 2
and catalyst destruction (WТРH) in reaction mixtures of various
compositions
The concentration of H O at the point of maximum
increases with an increase in the initial concentration
2
2
Composition
of the mixture
WН2О2•107
WTPH•108
of AH ; however, it is always substantially lower than the
mol L–1 s–1
2
concentration of the converted acid, i.e., processes reꢀ
sulting in hydrogen peroxide consumption occur in parꢀ
allel with the formation of hydrogen peroxide in the
system.
1.3
2.7
3.3
2.0
—
2.7
2
TPH—H O2
A—H O2
TPH—A—H O2
2
2
Note. CТРH = 0.35•10–4 mol L–1, CA = 1.2•10–3 mol L–1
CH2O2 = 1.0•10–3 mol L–1
,
One process consuming H O is its catalytic decomꢀ
2
2
position in the presence of TPH. Special experiments
have shown that ТРH has a rather high catalytic activity
.
–
4
–1
in this reaction. For C
= (1.2—20)•10 mol L and
H2O2
–
4
–1
tions; about 40 products were found by HPLC. To our
knowledge, no other information has been reported.
Hydroxyl radical. The formation of the HO• radical
upon the oxidation of ascorbic acids in the presence of
ТРH was detected over the whole range of reactant conꢀ
centrations by the method described in the Experimental,
which is based on the use of 2ꢀdeoxyribose (see Fig. 3). It
can be assumed with sufficient grounds that the main bulk
of the radicals is formed upon decomposition of H2O2 in
the presence of ТРH. Therefore, its maximum possible
concentration by the end of the major reaction, taking
into account the residual content of H O in solution, its
CТРH = (0.1—1.6)•10 mol L , the initial reaction rate
is described by the kinetic equation
WH2O2 = kH2O2CH2O2CТPH
,
(8)
where kH2O2 = 2.3 L mol–1 s–1
.
For the reaction catalyzed by the Fe3+ ion, k =
.8•10–2 L mol
–1
–1, while for catalase, k = 3.0•107
s
8
(
рH 4.5—9).10
During decomposition of hydrogen peroxide, the cataꢀ
lyst also undergoes destruction. The kinetic equation in
the same concentration range has the form
2
2
consumption in the reaction with A, and catalytic deꢀ
–
4
–1
WТPH = kТРHCH2O2CТРH
,
(9)
composition, should be equal to ~3•10 mol L (for
init
–3
–1
C
= 1•10 mol L ). However, the С∑НО• value
AH2
where kТРH = 0.7 L mol–1 s–1
.
determined in our experiments did not exceed
–
5
–1
The ratio kH2O2 : kТРH = 3.3. This means that one
TPH molecule has time to perform, on average, only
three catalytic cycles before it decomposes.
The consumption of hydrogen peroxide still continues
when the catalyst concentration becomes negligibly small
10 mol L (see Fig. 3). This may be due to two reasons,
namely, the presence of a nonradical pathway for hydroꢀ
gen peroxide decomposition and competing consumption
•
of HO along pathways characterized by higher rate conꢀ
stants than the reaction with 2ꢀdesoxyribose (recombinaꢀ
tion, reaction with ascorbic acid, and so on). The rate
constants for these and other reactions involving the hyꢀ
(
see Fig. 3). We suggested and confirmed experimentally
for the H O —A system that this effect is due to the reacꢀ
2
2
9
10
–1 –1
tion of H O with dehydroascorbic acid. In the concenꢀ
droxyl radical are of the order of 10 —10 L mol
s .
2
2
–
3
–1
tration ranges C
0.2—2)•10– mol L , the reaction rate is described by
= (0.1—3.0)•10 mol L and C =
The results we obtained provide the conclusion that
the oxidation of ascorbic acid with dioxygen in the presꢀ
ence of TPH gives H O in a nearly equivalent amount
H2O2
A
3
–1
(
the equation
2
2
with respect to the consumed ascorbic acid. The intermeꢀ
WH2O2 = kH2O2CH2O2CA,
(10)
•–
•–
diate radical products (O2 and the major part of A
)
are subsequently converted within the coordination sphere
of the cobalt atom without leaving to the reaction bulk.
The resulting hydrogen peroxide is partially consumed in
where kH2O2 = 0.22 L mol–1 s–1
.
In the system containing all three components (A,
H O , and ТРH), the rate of catalyst destruction does not
the reactions with the AH oxidation product (dehydroꢀ
2
2
2
change and the rate of consumption of hydrogen peroxide
is the sum of the rates of its catalytic decomposition and
its reaction with A (Table 1). Thus, the products of ТРH
destruction do not exhibit catalytic activity in the decomꢀ
position of hydrogen peroxide. The products formed in
the reaction of dehydroascorbic acid with hydrogen perꢀ
oxide were beyond the scope of our study. According to
ascorbic acid) and in the catalytic decomposition. The
•
latter process is accompanied by the formation of HO
•
radicals; however, the amount of HO radicals detectable
in our experiments was much lower than the theoretically
possible value.
When considering the results from the standpoint of
chemical grounds of the catalytic therapy of cancer, one
can conclude that the hydroxyl radical and hydrogen
1
1
published data, this acid is unstable in aqueous soluꢀ

2326
Russ.Chem.Bull., Int.Ed., Vol. 53, No. 10, October, 2004
Petrova et al.
peroxide are among the key cytotoxic agents in the
Teraphthal—ascorbic acid system. The concentration of
hydrogen peroxide is comparable with that of ascorbic
acid, and its ability to destroy biological structures is wellꢀ
known.
and R. I. Yakubovskaya, Sposob Podavleniya Opukholevogo
Rosta, Pat. RF No. 2106146, 1997, US Patent 6,004,953.
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. A. B. Syrkin, O. S. Zhukova, B. S. Kikot´, and G. K.
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3
4
5
. G. P. Laroff, R. W. Fessenden, and R. H. Schuler, J. Am.
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The authors are grateful to A. Kh. Vorob´ev and V. S.
Gurman (Department of Chemistry, M. V. Lomonosov
Moscow State University) for assistance in recording and
discussing the ESR spectra and to V. I. Alekseeva and
L. E. Marinina (Prof. E. A. Luk´yants´ laboratory, State
Scientific Center NIOPIK) for providing an analytically
pure sample of 10ꢀacetylꢀ3,7ꢀdihydroxyphenoxazine.
This work was financially supported by the Moscow
Government within the bounds of the Moscow City Proꢀ
gram for Oncology.
6. J. G. Mohanty, J. S. Jaffe, E. S. Schulman, and D. G.
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in revised form July 2, 2004