Photolysis of carboxymethylflavin in aqueous and organic solvent: A kinetic study

Article abstract of DOI:10.1039/c9ra02818h

This is the first study on the photolysis of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin (RF). CMF is photodegraded by removal of side-chain to lumichrome (LC) in acid solution and to LC and lumiflavin (LF) in alkaline solution. It also undergoes alkaline hydrolysis to 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxoquinoxaline (DQ) by cleavage of isoalloxazine ring. CMF degrades to LC in organic solvents. The formation of LC in acid solution and organic solvents takes place by second-order reaction and those of LC, LF, KA and DQ in alkaline solution by first-order reactions. The values of second-order rate constants for the photolysis of CMF at pH 2.0 to 7.0 are in the range of 1.13 to 2.45 M^-1 s^-1 and those of first-order rate constants (k_obs) at pH 8.0-12.0 from 1.53 to 4.18 × 10^-4 s^-1 and for the formation of photoproducts from 0.37 to 16.6 × 10^-5 s^-1. The photolysis of CMF is enhanced, with pH, in the alkaline region since the excited state is sensitive to alkaline hydrolysis. The photolysis and fluorescence quantum yields of CMF in aqueous and organic solvents have been reported. CMF and photoproducts have been assayed spectrofluorimetrically. The mode of CMF photolysis is discussed.

Full text of DOI:10.1039/c9ra02818h

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Photolysis of carboxymethylflavin in aqueous and
organic solvent: a kinetic study†
Cite this: RSC Adv., 2019, 9, 26559
a
b
a
*
Iqbal Ahmad,
Tania Mirza,a Syed Ghulam Musharraf,
Zubair Anwar,
Muhammad Ahsan Ejaza
a
a
Muhammad Ali Sheraz,
Sofia Ahmed,
and Adeela Khurshida
This is the first study on the photolysis of carboxymethylflavin (CMF), an intermediate in the photolysis of
riboflavin (RF). CMF is photodegraded by removal of side-chain to lumichrome (LC) in acid solution and
to LC and lumiflavin (LF) in alkaline solution. It also undergoes alkaline hydrolysis to 1,2-dihydro-1-
methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxoquinoxaline
(DQ) by cleavage of isoalloxazine ring. CMF degrades to LC in organic solvents. The formation of LC in
acid solution and organic solvents takes place by second-order reaction and those of LC, LF, KA and DQ
in alkaline solution by first-order reactions. The values of second-order rate constants for the photolysis
of CMF at pH 2.0 to 7.0 are in the range of 1.13 to 2.45 Mꢀ1 sꢀ1 and those of first-order rate constants
(kobs) at pH 8.0–12.0 from 1.53 to 4.18 ꢁ 10ꢀ4 sꢀ1 and for the formation of photoproducts from 0.37 to
Received 14th April 2019
Accepted 24th July 2019
16.6 ꢁ 10ꢀ5
s
ꢀ1. The photolysis of CMF is enhanced, with pH, in the alkaline region since the excited
state is sensitive to alkaline hydrolysis. The photolysis and fluorescence quantum yields of CMF in
aqueous and organic solvents have been reported. CMF and photoproducts have been assayed
spectrofluorimetrically. The mode of CMF photolysis is discussed.
DOI: 10.1039/c9ra02818h
rsc.li/rsc-advances
An important consideration in degradation of chemical and
pharmaceutical compounds is the effect of solvent on the rates of
reaction.30–35 Solvent polarity plays an important role in the
stabilization of drug formulations to prolong their shelf-lives.36–40
The effect of solvent on the photolysis of RF,5,16,41–44 FMF,20,21,45
LC45 and other avins46–48 has been studied. The photo-dynamics
aspects of RF have also been investigated.49 The photolysis of
CMF in organic solvents needs to be investigated to determine
the effect of solvent polarity on its degradation.
The present work involves a detailed kinetic study of
photolysis of CMF in aqueous and organic solvents, charac-
terize the photoproducts and evaluate the kinetics of its
degradation reactions. The rate–pH proles could provide
information about the regions of minimum and maximum
degradation of the compound. Attempts are made to correlate
the kinetic parameters with solvent polarity and viscosity. The
photochemical and uorescence quantum yields of CMF in
aqueous and organic solvents are determined. CMF and its
photoproducts are assayed by a recently developed multicom-
ponent spectrouorimetric method.29 The mode of photo-
degradation reactions of CMF is discussed.
Introduction
Riboavin (vitamin B2) RF (1) (Fig. 1) has long been studied
due to its biochemical importance, photosensitivity and
pharmaceutical applications.1–8 It is photodegraded to several
products in aqueous and organic solvents to yield for-
mylmethylavin (FMF) (2) as a major intermediate in the
reaction.9–16 FMF undergoes hydrolysis17–19 and photolysis to
form lumichrome (LC) (3) and lumiavin (LF) (4) in aqueous
solution20,21 and LC in organic solvents.20 It is photooxidized to
carboxymethylavin (CMF) (5) in aqueous and organic
solvents.12,13,16,21,22 CMF has also been suggested to act as an
intermediate in the formation of LC and LF during the
photolysis of RF in aqueous solution.23 It is an isoalloxazine
derivative like RF, FMF and LF1,24,25 and is susceptible to
hydrolytic degradation in alkaline solution by the cleavage of
the isoalloxazine ring to give 1,2-dihydro-1-methyl-2-keto-3-
quinoxaline carboxylic acid (KA) (6) and 1,2,3,4-tetrahydro-1-
methyl-2,3-dioxoquinoxaline (DQ) (7).26–29 So far no chemical
or photodegradation studies of CMF have been reported since
its isolation by Fukumachi and Sakurai in 1955.22
aBaqai Institute of Pharmaceutical Sciences, Baqai Medical University, Super Highway,
Gadap Road, Near Toll Plaza, Karachi, Pakistan. E-mail: zubair_ana@hotmail.com
bHEJ Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra02818h
Experimental section
Materials
LC and LF were procured from Sigma. CMF was prepared by
the method of Fukumachi and Sakurai.22 KA and DQ were
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Fig. 1 Chemical structures of riboflavin (RF) and photoproducts.
prepared from RF by the methods of Surrey and Nachod50 and solvent system.18 All the reagents and solvents of the highest
Miles et al.,51 respectively. The side-chain of KA and DQ was purity were procured from Merck & Co. The buffers used were
cleaved by periodic acid51 oxidation. The compounds were citric acid–Na2HPO4 (pH 2.5–8.0), H3BO3–KCl–NaOH (pH 9.0–
puried by cellulose column chromatography using 1- 10.0) and Na2HPO4–NaOH (pH 11.0–12.0). The ionic strength
butanol–1 propanol–acetic acid–water (50 : 30 : 2 : 18, v/v) as was 0.001 M in each case.
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period is determined and the quantum yield of LC is calculated
using the equation.
Precautions
The photolysis of CMF, thin-layer chromatography and uori-
NLC
metric assay have been carried out in a dark chamber.
FPL
¼
(1)
nph abs
Measurement of pH
where, NLC ¼ number density of generated LC
All pH measurements were carried out with a digital pH meter
(Model CP-501; sensitivity ꢂ0.01 units, Poland) using a combi-
nation pH electrode. The calibration of the electrode was per-
formed using buffer solutions of pH 4.0, 7.0 and 9.0.
Iexctexc
hvexc
Iexctexc
hvexc
1
nph abs
¼
aexc
¼
sexc ½CMFꢄ
(2)
nph abs [cmꢀ3] ¼ number density of CMF absorbed photons per
1 cm of sample length, Iexc [W cmꢀ2] ¼ excitation light intensity
in watt per cm2, texc [s] ¼ time duration of light exposure in
seconds, h [J s] ¼ Planck constant, nexc [sꢀ1] ¼ excitation light
frequency, hnexc [J] energy of an excitation photon, aexc [cmꢀ1] ¼
Measurement of UV-visible spectra
The spectral measurements were carried out with Thermo-
Scientic UV-visible spectrophotometer (Evolution 201) using
quartz glass cells of 10 mm path length.
absorption coefficient of CMF at excitation wavelength, sexc
¼
absorption cross-section of CMF at excitation wavelength.
HPLC-Tof-MS system. Samples were recorded on HPLC-Tof-
MS system (Agilent 6230). RP-C8 column was used for separa-
tion (1.0 ꢁ 50 mm, 3.5 mm, Agilent ZORBAX Eclipse XDB
columns – C18 column). The mass spectrometer with ESI source
was set in the positive ion mode. An isocratic mobile phase that
is 0.1% formic acid in both water (eluent A) and methanol
(eluent B) was used at a ow rate of 0.5 ml minꢀ1 for 20 min.
Injection volume was 2 mL.
Measurement of uorescence
The measurement of uorescence of CMF and photoproducts
ꢃ
was performed out at 25 ꢂ 1 C using Jasco FP-8300 spectro-
urimeter (Japan). The wavelengths used for the determination
of CMF and photoproducts are given in Table 1.
The uorescence intensity was measured in relative units.
Pure 0.01 mM solutions (pH 6.5) of these compounds were used
as standards.
Light intensity measurement
Photodegradation of CMF
The intensity of the radiation source, a Philips HPLN 125 W
high pressure mercury vapor lamp, was determined by ferriox-
alate actinometry52 and a value of 1.12 ꢂ 0.08 ꢁ 1017 quanta
per s was obtained.
Reaction description. The reactions involved in the photol-
ysis of CMF are described by eqn (12)–(16) in Scheme 2 (see
section on Mode of photolysis), followed by.
H
þ=OHꢀ
3
½CMFꢄ*
LC þ LF þ further photoproducts
(3)
ƒƒƒ!
photolysis
Determination of quantum yields of photolysis
The quantum yields (FPD) for the photochemical formation of
LC in acid solution and organic solvents by second-order reac-
Experimental procedure. The amount of photodegraded
tion and the photodegradation of CMF in alkaline solution by CMF and the amount of absorbed excitation light by CMF
rst-order reaction have been determined by the method of during a certain period is determined. The quantum yield of
Zirak et al.53,54 and Tyagi and Penzkofer55 as follows.
CMF is calculated using the equation.
NCMF
FPL
¼
(4)
Formation of LC
nph abs
Reaction description. The reactions involved in the forma-
tion of LC by CMF are described by eqn (8)–(11) and eqn (12)–
(21) in Scheme 2 (see section on Mode of photolysis).
Experimental procedure. The amount of formed LC and the
amount of absorbed excitation light by CMF during a certain
where, NCMF ¼ number density of degraded CMF, nph abs ¼ as
given in eqn (23) in Scheme 2
Fluorescence quantum yield. The uorescence quantum
yield (FF) is the ratio of photons emitted through uorescence
to photons absorbed.
number of photons emitted through fluorescence
FF ¼
number of photons absorbed
Table 1 Excitation and emission wavelengths for the determination of
CMF and photoproducts
It can be determined of a comparative method56 using
standard samples of a compound whose FF is known. The
standard and the test samples having identical absorbance at
the same excitation wavelength are assumed to absorb same
number of photons. Therefore, the ratio of integrated uores-
cence intensities of the two solutes determined under identical
conditions gives the ratio of values of the quantum yields. Since
Excitation wavelength
(lex) nm
Emission wavelength
(lem) nm
Compound
CMF
LC
LF
KA
DQ
445
356
445
364
332
530
478
530
443
420
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FF of the standard is known, the FF of test sample can be the same as those of its hydrolytic degradation (LC, LF, KA, DQ),
calculated. the method can be conveniently used for determination of these
The method involves the measurement of absorbance and compounds. It involves extraction of the photodegraded solu-
the integrated uorescence intensity (i.e. area of the uores- tions of CMF with chloroform at pH 2.0 to remove side-chain
cence spectra) of the sample of the standard and the test products, LC and LF, and their assay at 478 and 530 nm,
samples. The integrated uorescence intensity of the standard respectively. This is followed by adjustment of the pH of
and the test samples are plotted against the absorbance of each aqueous phase to 6.5 and assay of CMF and the isoalloxazine
sample. The gradients of the two plots are proportional to the ring cleavage products, KA and DQ, at 530, 443 and 420 nm,
quantum yields of the two compounds. The uorescence respectively.
quantum yield of the unknown is calculated from the product of
Organic solvents. A 1 ml aliquot of degraded solution of CMF
the quantum yield of the standard and the quotient of the two was evaporated to dryness under reduced pressure and the
gradients according to the following equation.
residue dissolved in 10 ml of pH 6.5 citro–phosphate buffer. The
solution was used for uorimetric assay of CMF and LC at 530
and 478 nm.
ꢀ
ꢁꢀ ꢁꢀ
ꢁ
2
As
Eu
hu
hs
FF ¼ Fs
Au
Es
ꢀ
ꢁꢀ
ꢁ
2
mu
hu
hs
¼ Fs
Results and discussion
Absorption characteristics of CMF
ms
where, Fs ¼ uorescence quantum yield of the standard, m ¼
gradient of the plot of integrated uorescence intensity against
absorbance, h ¼ refractive index of the solvent, A ¼ absorbance
of the solution at the uorescence excitation wavelength (445
nm), E ¼ integrated uorescence intensity, subscripts “s” and
“u” refer to the standard and unknown samples, respectively,
RF with a FF value of 0.24 (ref. 57) has been used as a standard
in this case.
The UV-visible and IR spectra of CMF are shown in Fig. 2a and
b, respectively. CMF exhibits absorption maxima at 223 nm
(molar decadic extinction coefficient, 3 ¼ 24 700 Mꢀ1 cmꢀ1),
266 nm (3 ¼ 27 800 Mꢀ1 cmꢀ1), 376 nm (3 ¼ 9000 Mꢀ1 cmꢀ1
)
and 445 nm (3 ¼ 10 200 Mꢀ1 cmꢀ1) at pH 7.0 (Fig. 2a). These
values are similar to those of RF and FMF having the same
nucleus and different side-chains.58 The FTIR spectrum of CMF
shows peaks at 3250 cmꢀ1 (NH), 1650 cmꢀ1 (C]O), 1580 cmꢀ1
Thin-layer chromatography (TLC)
CMF and its side-chain cleavage products (LC and LF) were
detected using 250 mm cellulose plates (Whatman CC41) and
the solvent systems: (a) 1-butanol–1-propanol–acetic acid–water
(50 : 30 : 2 : 18, v/v) and (b) 1-butanol–acetic acid–water
(40 : 10 : 50, v/v, organic phase).12
The isoalloxazine ring cleavage products of CMF, i.e. KA and DQ,
were detected using 250 mm silica gel G plates and the solvent
system (c) chloroform–acetic acid–pyridine (50 : 10 : 10, v/v).27 The
spots of all these products were located by their characteristic
uorescence on excitation at 366 nm.
Photodegradation of CMF
A CMF solution (5 ꢁ 10ꢀ5 M) was prepared at pH 2.0–12.0, using
appropriate buffer, in a 100 ml volumetric ask and placed in
a thermostat bath maintained at 25 ꢂ 1 ꢃC in the radiation
chamber. It was irradiated with the Philips HPLN 125 W high
pressure mercury vapor lamp (emission at 405 and 435 nm, the
later wavelength corresponding to the absorption maximum of
CMF at 445 nm),19,22,29 xed horizontally at a distance of 25 cm
from the center of the ask. The same procedure was used for
photolysis of CMF in organic solvents. Samples of degraded
solutions were subjected to TLC and assay at appropriate
intervals.
Assay of CMF and photoproducts
Aqueous solution. The assay of CMF and photoproducts has
been carried out by a recently developed and validated spec-
Fig. 2 (a) UV and visible absorption spectrum of CMF at pH 7.0. (b)
trouorimetric method.29 Since the photoproducts of CMF are FTIR spectrum of CMF.
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(C]C) and 1540 cmꢀ1 (C]N) (Fig. 2b). The broad absorption 10% and 2 to 6%, respectively. These results indicate that
band in the 3300–2800 cmꢀ1 region indicates hydrogen bonded the formation of these photoproducts is gradually increased
O–H stretching of the carboxylic group. This is further sup- with pH due to the hydrolytic degradation of CMF which is
ported by the peaks at 1700 cmꢀ1 and 1300 cmꢀ1
.
accelerated in the presence of light. The decrease in the
concentration of LC, with pH, is due to its formation by an
acid-catalyzed reaction.17
Nature of photoproducts
CMF was originally reported as a photoproduct in the photolysis
of RF in alkaline solution in the presence of H2O2, along with LC
and LF.22,23 It has also been detected in the photolysis of RF12,13,51
and FMF and may be formed directly from RF23 or by the
oxidation of FMF.21 LC and LF are formed on the photol-
ysis9,11,20,21 or hydrolysis of FMF17–19 and CMF29 in the alkaline
solution.
It has been found that the formation of LC and LF takes
place on the photolysis of CMF. Thus, a second route of the
formation of LC and LF, in addition to that of FMF, in the
photolysis of RF is through CMF. The other two products
formed by isoalloxazine ring cleavage of CMF in alkaline
solution are quinoxaline derivatives, KA and DQ. The
formation of both of these products is enhanced with an
increase in pH due to the hydrolysis of the isoalloxazine
ring,29 as has previously been observed in the case of the
hydrolysis of 9-methylisoalloxazine.27,28 The only product
detected on the photolysis of CMF in organic solvents is LC.
All these products have been identied on comparison of
their characteristics uorescence under UV light (CMF and
LF, yellow green; LC, sky blue; KA and DQ, blue) and
retention (Rf) values with those of the reference compounds
using the solvent systems reported in the experimental
section. The identication of photoproducts of CMF was
Spectral characteristics of photolyzed solutions
CMF has been found to undergo spectral changes during
photolysis in acid and alkaline solutions (Fig. 3). These
changes have been found to vary with pH and the loss of the
molecule on degradation. A typical set of absorption spectra
of CMF on degradation at pH 4.0 is presented in Fig. 3a. It
indicates the loss of peak at 445 nm and a shi towards
356 nm with concomitant increase in absorbance showing
the formation of LC in acid solution. Another set of
absorption spectra of CMF during photolysis at pH 11.0 is
shown in Fig. 3b. The loss of absorbance at 445 nm indicates
the degradation of CMF and a prominent shi of the 374 nm
peak towards 356 nm showing the formation of LC. The
absorption spectrum of LF is similar to that of CMF and,
therefore, it cannot be distinguished from CMF to indicate
any spectral change. The disappearance of 445 nm peak
indicates the cleavage of the isoalloxazine ring to form KA
and DQ absorbing in the 300–400 nm region.27,28,59 Thus, the
spectral changes on the photolysis of CMF at pH 11.0 indi-
cate that the molecule is being transformed into its side-
chain and ring cleavage products.
Assay of CMF and photoproducts
necessary to conrm their presence in degraded solutions The photoproducts of CMF in acid (LC) and alkaline solution
prior to the application of the spectrouorimetric method (LC, LF, KA and DQ) are same as those of the hydrolytic
for their assay during the photolysis reactions.
degradation reaction. However, the formation of LC and LF is
increases in presence of light. Therefore, a single method can be
applied to the determination of the products of hydrolytic and
photolytic degradation. Recently, a spectrouorimetric method
has been developed and validated for the determination of CMF
and its side-chain cleavage (LC and LF) and ring cleavage
products (KA and DQ).29 The accuracy of the method has been
reported to be within 1%.29 This is used for the determination of
CMF and photoproducts during photolysis. The results of the
assay of these compounds on the photolysis of CMF for a typical
reaction (pH 10) are given in Table 3. These indicate an almost
constant molar balance at various intervals showing the accu-
racy of the method. The assay data for the reactions carried out
at pH 2.0–12.0 have been used to evaluate the kinetics of
degradation of CMF.
Product composition
The photolysis of CMF leads to the formation of LC, LF, KA
and DQ in aqueous solution as described in the above
section. The composition of these products at 50% degra-
dation of CMF at pH 8.0–12.0 is given in Table 2. The
formation of LC in acid solution amounts to 50% and in the
alkaline solution from 20 to 35% while the formation of LF
in alkaline solution takes place to the extent of 8 to 13% of
the degraded CMF in the pH range studied. The ring
cleavage products, KA and DQ, occur to the extent of 5 to
Table 2 Product composition at 50% photolysis of 5.0 ꢁ 10ꢀ5 M CMF
solutions at pH 8.0–12.0
Kinetics of photolysis of CMF
CMF undergoes photolysis to form LC in acid solution and LC,
LF, KA and DQ in alkaline solution (Scheme 1).
pH
Time (min)
LC (%)
LF (%)
KA (%)
DQ (%)
8
9
10
11
12
75.3
36.5
30.8
28.7
27.6
34.8
30.8
26.2
23.2
20.2
8.4
10.4
11.0
12.4
13.0
4.8
6.0
7.8
9.2
10.4
2.0
3.0
4.8
6.0
6.4
The kinetics of these reactions has not so far been studied. A
recent study29 has shown that CMF is degraded by hydrolysis to
form the above mentioned compounds in aqueous solution.
Considering the photolysis of CMF in acid solution to form LC
by a second-order reaction (k1) and to form LC, LF, KA and DQ
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Fig. 3 Absorption spectra of CMF on photolysis at pH 4.0 (a), and at pH 11.0 (b). Times indicated are in min.
in alkaline solution by parallel rst-order reactions, the rate
constants, k2, k3, k4 and k5, can be calculated by the method of
Frost and Pearson60 an applied previously to the photolysis of
RF.61
Photolysis of CMF in acid solution
ꢀd½CMFꢄ
2
¼ k1½CMFꢄ
(5)
dt
Table 3 Photolysis of CMF at pH 10.0. Concentrations of CMF and photoproducts
Time (min)
CMF (M ꢁ 105)
LC (M ꢁ 105)
LF (M ꢁ 105)
KA (M ꢁ 105)
DQ (M ꢁ 105)
Total (M ꢁ 105)
0
5.00
2.38
1.22
0.59
0.31
—
—
—
—
5.00
5.03
5.05
4.98
5.02
15
30
45
60
0.91
1.31
1.43
1.52
0.85
1.15
1.28
1.39
0.46
0.85
1.10
1.18
0.42
0.51
0.56
0.62
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Scheme 1 Photolysis of CMF in acid and alkaline solution.
The rate constants, k1, for second-order photolysis reactions or
of CMF in acid solution determined from slopes of inverse
concentration (1/c) against time plots are given in Table 4.
Photolysis of CMF in alkaline solution. If A, U, V, W and X are
the corresponding concentrations of CMF, LC, LF, KA and DQ
during degradation and Ao is initial concentration of CMF, the
overall rate constant (kobs) can be written as:
À
À
Á
Á
U ¼ Uo þ ðk2Ao=kobsÞ 1 ꢀ eꢀkt
À
Á
V ¼ Vo þ ðk3Ao=kobsÞ 1 ꢀ eꢀkt
À
Á
(8)
W ¼ Wo þ ðk4Ao=kobsÞ 1 ꢀ eꢀkt
X ¼ Xo þ ðk5Ao=kobsÞ 1 ꢀ eꢀkt
If Uo ¼ Vo ¼ Wo ¼ Xo ¼ 0, the equations can be expressed as
ꢀdA
¼ k2A þ k3A þ k4A þ k5A ¼ ðk2 þ k3 þ k4 þ k5ÞA ¼ kobs
A
dt
V/U ¼ k3/k2
W/U ¼ k4/k2
X/U ¼ k5/k2
Therefore
kobs ¼ k2 + k3 + k4 + k5
(6)
or
and
or
ðAoÞ
U : V : W : X ¼ k2 : k3 : k4 : k5
(9)
ln
¼ kobst
A
A ¼ Ao eꢀkt
(7)
The concentrations of the photoproducts are in a constant
ratio to each other. These are independent of time and the
initial concentration of CMF and can be used to determine
the rate constants for the individual reactions. The values of
kobs for disappearance of CMF and k2, k3, k4 and k5 for
formation of LC, LF, KA and DQ, respectively, at pH 8–12 are
reported in Table 5. These rate constants vary with pH as
discussed in a later section. The sum of the individual rate
constants for the formation of the photoproducts is almost
equal to the values of the kobs for the photolysis of CMF at
a particular pH (Table 5).
Considering the degradation of A as a rst-order reaction
dU
¼ k2A ¼ k2Ao eꢀkt
dt
and
k2Ao
kobs
U ¼
eꢀkt þ constant
Table
4 Second-order rate constants (k1) and half-lives for the
Photolysis of CMF in organic solvents. The photolysis of
CMF in organic solvents has been found to follow second-order
kinetics to form LC as observed in the case of FMF.20
photolysis of CMF in acid solution
pH
k1 (Mꢀ1 sꢀ1) ꢂ SD
t1/2 (min)
Fa
hv;k6
ƒƒƒƒƒ!
2.0
3.0
3.5
4.0
4.5
5.0
6.0
7.0
1.13 ꢂ 0.01
4.20 ꢂ 0.05
8.05 ꢂ 0.09
10.85 ꢂ 0.11
6.04 ꢂ 0.07
2.75 ꢂ 0.03
1.99 ꢂ 0.02
2.45 ꢂ 0.02
295.0
79.3
41.4
30.7
55.2
121.2
167.5
136.1
0.16
0.20
0.23
0.29
0.06
0.05
0.05
0.12
CMF
LC
(10)
second-order
It is similar to that of CMF in acid solution as described
above. However, the rates of the reaction are lower than those
observed in the acid solution. This is due to a change in the
polarity of the medium. The second-order rate constants, k6, for
these reactions are reported in Table 6. The values increase on
increasing the polarity of solvent.
a
It represents the quantum yields for the formation of LC, the major
side-chain photoproduct.
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Table 5 Apparent first-order rate constants (kobs) for the photolysis of CMF at pH 8.0–12.0, and the rate constants for the formation of lumi-
chrome (LC) (k2), lumiflavin (LF) (k3), keto acid (KA) (k4) and dioxo-quinoxaline (DQ) (k5), second-order rate constants (k0) for OHꢀ ion catalyzed
reaction and quantum yields of photolysis (F)
pH
kobs ꢁ 104 (sꢀ1) ꢂSD
t1/2 (min)
k2 ꢁ 104 (sꢀ1
)
k3 ꢁ 104 (sꢀ1
)
k4 ꢁ 104 (sꢀ1
)
k5 ꢁ 104 (sꢀ1
)
k0 (Mꢀ1 sꢀ1
)
Fa
8
9
10
11
12
1.53 ꢂ 0.06
3.07 ꢂ 0.12
3.75 ꢂ 0.15
4.01 ꢂ 0.14
4.15 ꢂ 0.16
75.3
36.5
30.8
28.7
27.6
0.76
1.08
1.47
1.63
1.67
0.36
0.91
1.10
1.13
1.15
0.23
0.66
0.70
0.75
0.80
0.18
0.42
0.48
0.50
0.53
153.0
31.7
3.75
0.40
0.04
0.32
0.34
0.36
0.39
0.41
a
It represents the overall quantum yields for the photolysis of CMF.
alkaline hydrolysis.11 At pH 8 and above CMF undergoes isoal-
loxazine ring cleavage to form KA and DQ and, therefore, the
values of kobs are further increased with pH in this region as
observed in the case of FMF19 and 9-methylisoalloxazine.27,28 All
the products of alkaline photolysis of CMF (LC, LF, KA, DQ) are
formed by parallel rst-order kinetics and the rates of these
reactions are in the range of 0.18–1.67 ꢁ 10ꢀ4 sꢀ1 at pH 8.0–12.0
(Table 5). The rate–pH proles for formation of LC, LF, KA and
DQ in the alkaline region are similar to that of the degradation
of CMF (Fig. 6). However, the individual rates of formation of
these products are lower than that of the overall loss of CMF.
The ratio of the rate constants for the formation of side-chain
cleavage products, LC and LF (k2 and k3) and those of the
formation of ring cleavage products, KA and DQ (k4 and k5) on
the photolysis of CMF at pH 12 is 6.8 : 3.2.
pH effect
CMF is sensitive to pH in the presence or absence of light and
undergoes degradation to give LC in acid solution and LC, LF, KA
and DQ in alkaline solution. The kinetic results (Table 2) indicate
that CMF is more labile to side-chain cleavage than the ring
cleavage. The k–pH proles for photolysis of CMF in acid and
alkaline regions have been determined. A plot of k1 for the
photolysis of CMF in acid region versus pH is shown in Fig. 4. The
rate of the reaction is slow in the pH range 2 to 3 due to
protonation of the molecule (N-10) as observed in the case of RF
(pKa 1.7)13,62 and is then increased up to pH 4. Above this value
a rapid decrease in the rate up to pH 6 is observed. This is similar
to that of the photolysis of FMF (pKa 3.5)63 which also have the
highest rate around pH 4 in the acid region.21 The decrease in the
rate above pH 4 can be explained on the basis of redox potentials
of avins which have lowest values in the pH range of 5 to 6 (e.g.
RF ꢀ0.117 V at pH 5.0, ꢀ0.208 at pH 7.0).39 This would slow down
the photoreduction of CMF and lead to a decrease in rate in this
region. The values of k1 for the photolysis of CMF in acid solution
are in the range of 1.13 to 2.45 Mꢀ1 sꢀ1 at pH 2 to 7 (Table 4).
Flavins are known to degrade by photoreduction process.4,24,25
The k–pH prole for photolysis of CMF in alkaline region is
shown in Fig. 5. It represents a steep curve in the pH range 8–10
followed by a decline in rate upto pH 12 as a result of hydrolytic
degradation. The lowering of rate above pH 10 appears to be due
to the ionization of the N3–H group (pKa 10.2)62 of the isoal-
loxazine nucleus. This is in accordance with the photolysis
behavior of RF which is also less susceptible to degradation in
the pH range 10.0–12.0.13 The increase in the rate in alkaline
region is probably due to the fact that the avin triplet state
exists in a bent, diradical form which is more sensitive to
Hydroxyl ion catalysis
In alkaline solution the photolysis of CMF is catalyzed by OHꢀ
ions. In order to evaluate the effect of OHꢀ ions on the rate of
photolysis of CMF, the log kobs values were plotted against pH
which showed a biphasic linear relation (Fig. 7). The values of
second-order rate constants (k0) for the OHꢀ ion catalyzed
reactions at various pH values are reported in Table 5. The slight
deviation in rate constants at 10.5 to 12.0 is due to the
involvement of the N3-anion of CMF with a pKa value similar to
that of RF (10.2)62 and being less sensitive to photolysis.
Effect of solvent
Dielectric constant. The photolysis of CMF in organic solvents
follows second-order kinetics and the values of the rate constants
Table 6 Second-order rate constants (k6) for the photolysis of CMF in organic solvents and water
Inverse viscosity
(mPa s)ꢀ1 (25 ꢃC)
Solvent
Acceptor number
Dielectric constant (3)
k6 (Mꢀ1 sꢀ1
)
Ethyl acetate
1-Butanol
1-Propanol
Acetone
17.1
36.8
37.3
12.5
37.1
41.3
18.9
54.8
6.02
17.8
20.1
20.7
24.3
32.6
38.5
78.5
2.268
0.387
0.514
0.330
0.931
1.828
2.898
1.123
0.98
1.15
1.20
1.23
1.26
1.42
1.56
2.45
Ethanol
Methanol
Acetonitrile
Water
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Fig. 4 k1–pH profile for photolysis of CMF at pH 2.0–7.0.
Fig. 7 log kobs against pH plot for the photolysis of CMF in alkaline
solution.
photoreduction of avins in aqueous and organic solvents as
proposed by Ahmad and Tollin.48 The reactivity of this intermediate
depends on the extent of solvent interaction. This implies that if the
polarity of the intermediate state is greater than that of the reactant
it would lead to an increase in rate with an increase in relative
dielectric constant of the solvent as reported in the case of RF16 and
FMF.20
The effect of solvent on the rate of a reaction can also be
considered in terms of the acceptor number (AN) which deter-
mines the ability of a solvent to take shares in electron pairs
from suitable donors.64 When the ln (k6) values for the photol-
ysis of CMF are plotted against the AN of highly structured
solvents (i.e. above 25), a linear relation is observed (Fig. 9). The
AN is considered as a measure of the reactivity of a species in
different solvents.65 The solvent dielectric constant is consid-
ered as a good indicator for prediction of the extent of solute–
solvent interaction.
Fig. 5
kobs–pH profile for the photolysis of CMF at pH 8.0–12.0.
(k6) are given in Table 6. A plot of k6 versus relative dielectric constant
(3) of the solvents is shown in Fig. 8. The increase in rate with 3
indicates that the rate of reaction is controlled by the polarity of the
solvent. This is probably due to the existence of a dipolar species in
Viscosity. The rate of a reaction may be inuenced by the
viscosity of the solvent as observed in the case of RF16 and
Fig. 6 k–pH profiles for the formation of photoproducts of CMF, (-) LC, (:) LF, (ꢁ) KA, (*) DQ.
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Fig. 10 Plot of k6 for photolysis of CMF against inverse of viscosity:
(C) 1-butanol, (-) 1-propanol, (A) acetone, (:) ethanol, (*) meth-
anol, (ꢁ) acetonitrile.
Fig. 8 Plot of kobs for photolysis of RF against dielectric constant: (B)
ethyl acetate, (C) 1-butanol, (-) 1-propanol, (A) acetone, (:)
ethanol, (*) methanol, (ꢁ) acetonitrile, (>) water.
uorescence intensity of CMF was plotted against pH (Fig. 11).
The curve indicates a slow increase in uorescence intensity,
with pH, in acid region due to gradual deprotonation of CMF.
The maximum uorescence is exhibited in the pH range 7 to 8
followed by a decline due to anion formation of the molecule in
alkaline region. The ionized forms of avins are less susceptible
to photolysis in aqueous solution.13 The uorescence of CMF
has also been measured in organic solvents and the relative
values of uorescence intensity and quantum yields (0.09–0.27)
of CMF in aqueous and organic solvents are given in Table 7.
The deactivation of the excited singlet state (S1) is enhanced
with an increase in the polarity of the solvent and hence an
increase in k6 values with a loss in uorescence intensity of CMF
in the medium has been observed.
The quantum yield of uorescence (FF) of avins depends on
the solvent and is decreased with an increase in the polarity of
the solvent.41 This is evident from the values of FF and indicates
a higher photostability of avins in less polar solvents.67 The
uorescence quantum yields may be affected by the degree of
hydrogen bonding between the avin and the solvent.68
FMF.21 A plot of k6 versus inverse of solvent viscosity is shown in
Fig. 10. It follows a linear relation indicating that the rate of
reaction is slowed down by an increase in viscosity. This is in
accordance with the quenching of avin triplet state which is
inhibited with an increase in solvent viscosity.48 The rate of the
reaction is suppressed by the viscosity of the solvent probably
due to the involvement of a diffusion-controlled process.35 The
degradation of CMF in ethyl acetate does not comply with its
behaviour in other solvents. This may be due to a difference in
the orientation of the side-chain in that solvent42 that would
determine the rate of reaction.
Effect of uorescence
CMF possesses an isoalloxazine nucleus similar to that of other
avins (e.g. RF, FMF and LF) and emits a yellow green uores-
cence at 530 nm when excited at 445 nm.19,29 Flavins are dipolar
molecules and their uorescence is destroyed by acid or alkali
as a result of the cation or anion formation.66 To observe the
effect of pH on the ionization behavior of the molecule, the
Dissociation constants of CMF
CMF tends to ionize in acid and alkaline medium due to proton-
ation (N10+H) and anion formation (N10CH2COOꢀ and N3ꢀ) of the
molecule. The pKa values of these groups have not been reported.
Fig. 9 Relation between ln k6 for CMF photodegradation and solvent
acceptor number (AN) (symbols are as in Fig. 7).
Fig. 11 A plot of fluorescence intensity of CMF against pH.
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Table 7 Relative values of fluorescence intensity (% F) and quantum
yields (FF) in CMF (1.0 ꢁ 10ꢀ6 M) and organic solvents
negative ionization mode of MS. Extracted ion chromatograms
of all the mentioned compounds along with their high resolu-
tion masses (Fig. 1–5) are given in ESI.†
Solvent
% F
FF
Water (pH 7.0)
Acetonitrile
Methanol
48.5
0.13
0.18
0.23
0.27
0.21
0.10
0.20
0.09
Mode of photolysis
68.6
87.2
100.0
79.8
36.1
76.3
33.7
CMF undergoes photolysis to form LC in acid solution. The
kinetic data indicate that the transformation of CMF to LC takes
place by a second-order reaction. This is in accordance with the
photolysis of FMF which also yields LC in acid solution.21 The
formation of LC and LF in alkaline solution has been found to
take place by parallel rst-order reactions as observed in the
case of FMF.17–19,21 Therefore, an analogous scheme for the
photolysis of CMF is proposed (Scheme 2).
Ethanol
1-Propanol
1-Butanol
Acetone
Ethyl acetate
Since CMF possesses ꢀthe nucleus same as that present in RF, the
pKas of N10+H and N3 group would be similar to those of RF (1.7
and 10.2, respectively).62 However, since the uorescence of avins
is quenched by acid and alkali66 due to ionization, the pKa of the
N10CH2COOH group in the side-chain can be determined from the
plot of uorescence intensity versus pH (Fig. 11) according to the
Henderson–Hasselbalch equation:
In acid solution CMF is promoted to the excited singlet state,
1[CMF]*, by the absorption of a photon of light (eqn (12)) which may
be deactivated to the ground state, 1[CMF] (eqn (13)), or is converted
3
to triplet state, [CMF]* (eqn (14)) by intersystem crossing (ISC).
3[CMF]* may return to the ground state (eqn (15)) or on interaction
with a ground state molecule is photolyzed to LC and CH3COOH
(eqn (16)). Alternatively it may give rise to a cationic [CMFc+] and an
anionic [CMFcꢀ] radical (eqn (17)), which on losing or accepting
a proton form a neutral radical [CMFc] (eqn (18)) and a semiquinone
radical [CMFHc] (eqn (19)), respectively, as described previously in
the case of other avins.48 Two semiquinone radicals
½saltꢄ
pH ¼ pKa þ log
(11)
½acidꢄ
Considering the uorescence intensity–pH curve, there
appears to be an inection point around pH 4.1 (with a uo-
rescence intensity corresponding to 50% ionization of the
molecule). Therefore, a value of 4.1 can be considered as the pKa
of the N10CH2COOH group of CMF. N-heterocyclic carboxylic
acids have pKas in the range of 2.9 to 4.4.69,70 Fluorimetry has
been used for the determination of the pKa values of N-hetero-
cyclic bases such as 8-methylquinoline and acridine.71
Quantum yields of photolysis
The quantum yields (F) of the photolysis of CMF at pH 2.0–12.0
have been determined and are given in Tables 3 and 4. The
values of F in this pH range vary from 0.05 to 0.41 and are
affected by the pH and reactivity of the excited triplet state of
CMF. In the acid and neutral range (2.0–7.0), the F of formation
of LC are low. In alkaline range (8.0–12.0), the F gradually
increases since the triplet state has high reactivity in alkaline
solution.11 These values show a slight decrease in the pH range
10–12 due to anion formation of the molecule which has low
susceptibility to photolysis like those of RF13 and FMF.21 The
values of F of CMF are higher than those of RF photolysis in
presence of divalent anions.72
LC-ESI-MS analysis. Both, standard and CMF were analyzed
by LC-ESI-MS using Tof-MS spectrometry. Riboavin has shown
an [M + H]+ ion at m/z 377.1440 (calcd 377.1456) corresponding
to C17H20N4O6, CMF has shown [M + H]+ ion at m/z 301.0925
(calcd 301.0936) corresponding to C14H12N4O4. LC has shown
[M + H]+ ion at m/z 243.0872 (calcd 243.0882) corresponding to
C
12H10N4O2, similarly LF has shown [M + H]+ ion at m/z
257.1033 (calcd 257.1033) corresponding to C13H12N4O2. While
DQ has shown [M + H]+ ion at m/z 205.0971 (calcd 205.0972)
corresponding to C16H19N2O2. The peak for b-KA was not found
using positive ionization mode which could probably appear in
Scheme 2
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disproportionate73 to give a neutral molecule and a dihydro inter-
mediate product [CMFH2] (eqn (20)) similar to that predicted by
Berdicka74 in the photolysis of RF and suggested by Ahmad et al.
eqn (21) in the photolysis of FMF. The neutral radical reacts with
a neutral molecule and they are degraded to a semiquinone radical,
LC and CH3COOH (eqn (22)).
In the alkaline solution the photolysis of CMF takes place by
hydrolytic degradation as reported in the case of FMF.17,18,21 An
increase in rate at pH 8.0–10.0 could be attributed to congu-
rational changes and greater reactivity of the avin triplet state
to alkaline hydrolysis.11 This leads to the formation of LF and
formic acid (eqn (22)) and LC and acetic acid (eqn (23)). CMF is
also degraded by hydrolytic cleavage of isoalloxazine ring in
alkaline solution to yield KA (eqn (24)) and DQ (eqn (25)).29
3 P. F. Heelis, in Chemistry and Biochemistry of Flavoenzymes,
ed. F. Muller, CRC Press, Boca Raton, FL, 1991, pp. 171–193.
4 I. Ahmad and F. H. M. Vaid, in Flavins: Photochemistry and
Photobiology, ed. E. Silva and A. M. Edwards, The Royal
Society of Chemistry, Cambridge, 2006, pp. 13–40.
5 M. A. Sheraz, S. H. Kazi, S. Ahmed, Z. Anwar and I. Ahmad,
Beilstein J. Org. Chem., 2014, 10, 1999–2012.
6 Z. Anwar and I. Ahmad, in Advances in Medicine and Biology,
ed. L. V. Berhardt, Nova Science Publishers, USA, 2017, pp.
139–180.
7 British Pharmacopoeia, Her Majesty‘s Stationary Office,
London, UK, 2016.
8 United
States
Pharmacopeia
29,
United
States
Pharmacopoeial Convention, Rockville, MD, 2016.
9 E. C. Smith and D. E. Metzler, J. Am. Chem. Soc., 1963, 85,
3285–3288.
10 G. E. Treadwell, W. L. Cairns and D. E. Metzler, J.
Chromatogr., 1968, 35, 376–388.
Conclusions
CMF is an intermediate product in the photolysis of RF. On
visible irradiation the side-chain of CMF is removed to form LC 11 W. L. Cairns and D. E. Metzler, J. Am. Chem. Soc., 1971, 93,
in acid solution and LC and LF in alkaline solution. The isoal- 2772–2777.
loxazine ring of the molecule is also cleaved in alkaline solution 12 I. Ahmad and H. D. Rapson, J. Pharm. Biomed. Anal., 1990, 8,
to yield quinoxaline derivatives. In organic solvents CMF is 217–223.
photodegraded to LC only. CMF is degraded in acid solution 13 I. Ahmad, Q. Fasihullah, A. Noor, I. A. Ansari and Q. N. Ali,
and organic solvents by second-order kinetics and in alkaline Int. J. Pharm., 2004, 280, 199–208.
solution by simultaneous rst-order kinetics. The rate constants 14 I. Ahmad, S. Ahmed, M. A. Sheraz, F. H. M. Vaid and
for the formation of LC and LF in alkaline solution are higher
I. A. Ansari, Int. J. Pharm., 2010, 390, 174–182.
than those of the quinoxaline derivatives. CMF degradation is 15 I. Ahmad, Z. Anwar, K. Iqbal, S. A. Ali, T. Mirza,
increased in alkaline solutions due to the enhanced reactivity of
the excited triplet state. The photostability of CMF is greater in
Ad. Khurshid, Aq. Khurshid and A. Arsalan, AAPS
PharmSciTech, 2014, 15, 550–559.
organic solvents than in the aqueous solution due to a decrease 16 I. Ahmad, Z. Anwar, S. Ahmed, M. A. Sheraz, R. Bano and
polarity of the medium. The photoproducts of CMF have been A. Hafeez, AAPS PharmSciTech, 2015, 16, 1122–1128.
identied chromatographically and determined spectrouori- 17 P. S. Song, E. C. Smith and D. E. Metzler, J. Am. Chem. Soc.,
metrically in degraded solutions. The quantum yields of uo- 1965, 87, 4181–4184.
rescence and photolysis of CMF have been reported. A scheme 18 I. Ahmad, H. D. Rapson, P. F. Heelis and G. O. Phillips, J. Org.
for photodegradation reactions of CMF in aqueous and organic
solvent is presented.
Chem., 1980, 45, 731–733.
19 I. Ahmad, T. Mirza, Z. Anwar, M. A. Ejaz, M. A. Sheraz and
S. Ahmed, Spectrochim. Acta, Part A, 2018, 205, 540–550.
20 I. Ahmad, Q. Fasihullah and F. H. M. Vaid, Photochem.
Photobiol. Sci., 2006, 5, 680–685.
Conflicts of interest
There are no conicts to declare.
21 I. Ahmad, T. Mirza, K. Iqbal, S. Ahmed, M. A. Sheraz and
F. H. M. Vaid, Aust. J. Chem., 2013, 66, 579–585.
22 C. Fukamachi and Y. Sakurai, J. Vitaminol., 1955, 1, 217–220.
23 S. Svobodova-Leblova, J. V. Kostir and I. M. Hais, J.
Chromatogr., 1964, 14, 451–455.
Acknowledgements
The authors gratefully acknowledge Higher Education
Commission of Pakistan for nancial support to conduct this 24 P. F. Heelis, Chem. Soc. Rev., 1982, 11, 15–39.
study through a grant (Research Project # 20-3968) to Iqbal 25 P. Hemmerich, Fortschr. Chem. Org. Naturst., 1976, 33, 451–
Ahmad. They Thank Professor Alfons Penzkofer University of
527.
Regensburg, Germany and Professor Marek Sikorski, Adam 26 I. Ahmad, A. E. Beg and S. M. S. Zoha, Pak. J. Pharm. Sci.,
Mickiewicz University of Poznan, Poland for helpful
discussions.
1973, 2, 84–91.
27 D. A. Wadke and D. E. Guttman, J. Pharm. Sci., 1966, 55,
1088–1092.
28 D. A. Wadke and D. E. Guttman, J. Pharm. Sci., 1966, 55,
1363–1368.
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