Effect of ph, buffer, and viscosity on the photolysis of formylmethylflavin: A kinetic study

Article abstract of DOI:10.1071/CH12457

The kinetics of the photolysis of formylmethylflavin, a major intermediate product in the aerobic and anaerobic photolysis of riboflavin, was studied in the pH range 2.0-11.0. Formylmethylflavin and its photoproducts, lumichrome and lumiflavin, were determined in degraded solutions using a specific multicomponent spectrophotometric method. The photolysis of formylmethylflavin in alkaline medium takes place by first-order kinetics and the rate constants (kobs) at pH 7.5-11.0 range from 0.27×10-4 to 3.88×10-4 and 0.36×10-4 to 5.63×10-4s-1 under aerobic and anaerobic conditions respectively. In acid medium, the photolysis involves a second-order mechanism and the rate constants at pH 2.0-7.0 range from 1.37 to 2.11 and 2.03 to 2.94M-1s-1 under aerobic and anaerobic conditions respectively. The rate-pH profiles for the photolysis reactions indicate the highest rate of formylmethylflavin degradation is at ～pH 4 and above pH 10. In the alkaline region, the increase in rate with pH is due to higher reactivity of the flavin triplet state. The photolysis of formylmethylflavin is catalyzed by phosphate ions and is affected by the solvent viscosity.

Full text of DOI:10.1071/CH12457

CSIRO PUBLISHING
Aust. J. Chem.
Full Paper
http://dx.doi.org/10.1071/CH12457
Effect of pH, Buffer, and Viscosity on the Photolysis
of Formylmethylflavin: A Kinetic Study
A
A
B
A
Iqbal Ahmad, Tania Mirza, Kefi Iqbal, Sofia Ahmed,
A
,
D
C
Muhammad Ali Sheraz,
and Faiyaz H. M. Vaid
A
Institute of Pharmaceutical Sciences, Baqai Medical University, Toll Plaza,
Super Highway, Gadap Road, Karachi-74600, Pakistan.
B
Department of Material Science, Baqai Dental College, Baqai Medical University,
Toll Plaza, Super Highway, Gadap Road, Karachi-74600, Pakistan.
C
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi,
Karachi-75270, Pakistan.
D
Corresponding author. Email: ali_sheraz80@hotmail.com
The kinetics of the photolysis of formylmethylflavin, a major intermediate product in the aerobic and anaerobic photolysis
of riboflavin, was studied in the pH range 2.0–11.0. Formylmethylflavin and its photoproducts, lumichrome and
lumiflavin, were determined in degraded solutions using a specific multicomponent spectrophotometric method. The
photolysis of formylmethylflavin in alkaline medium takes place by first-order kinetics and the rate constants (kobs) at
pH 7.5–11.0 range from 0.27 ꢀ 10ꢁ to 3.88 ꢀ 10 and 0.36 ꢀ 10 to 5.63 ꢀ 10
4
ꢁ4
ꢁ4
ꢁ4 ꢁ1
s under aerobic and anaerobic
conditions respectively. In acid medium, the photolysis involves a second-order mechanism and the rate constants at
ꢁ
1
ꢁ1
pH 2.0–7.0 range from 1.37 to 2.11 and 2.03 to 2.94 M
The rate–pH profiles for the photolysis reactions indicate the highest rate of formylmethylflavin degradation is at
pH 4 and above pH 10. In the alkaline region, the increase in rate with pH is due to higher reactivity of the flavin triplet
s
under aerobic and anaerobic conditions respectively.
,
state. The photolysis of formylmethylflavin is catalyzed by phosphate ions and is affected by the solvent viscosity.
Manuscript received: 7 October 2012.
Manuscript accepted: 29 January 2013.
Published online: 22 February 2013.
Introduction
Formylmethylflavin (2-(7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo
g]pteridin-10(2H)-yl)ethanone, FMF), was isolated by Smith
content, and light intensity. The present work involves a study of
the kinetics of FMF photolysis under aerobic and anaerobic
conditions over a wide range of pH and in organic solvents using
a low-intensity visible irradiation source. It would throw light on
the extent of the involvement of FMF in the kinetics of RF
degradation by different pathways and whether the reaction is
[
[
1]
and Metzler and identified as a major intermediate product in
the photolysis of riboflavin (RF, vitamin B ). It is degraded by
2
[
2,3]
[4]
thermolysis, and photolysis
[5–12]
hydrolysis,
chrome (LC), lumiflavin (LF), and carboxymethylflavin (CMF)
to lumi-
[17]
catalyzed by phosphate buffer, as in the case of RF,
and is
[11,27]
[
1–14]
in aqueous and organic solvents.
The kinetics of formation
affectedby solvent viscosity, in addition to solvent polarity.
The chemical structures of RF, FMF, and photoproducts (LC,
LF, and CMF) are shown in Fig. 1.
of FMF on the photodegradation of RF, followed by its disap-
pearance in aqueous solution, have been studied using a specific
[
9,15–21]
multicomponent spectrophotometric method.
The
mechanism of the photochemical formation of FMF from RF
and its degradation to further photoproducts has been discussed
Results and Discussion
[
2,22–26]
Characterization and Assay of FMF and Photoproducts
by several workers.
FMF is more sensitive to light than RF and its degradation is
FMF and its photoproducts (LC, LF, and CMF) were isolated
from an anaerobically photolyzed solution of RF using cellulose
column chromatography (Whatman CC 31) and solvent system
(b) described in the Experimental section under Thin-Layer
Chromatography. These compounds were characterized by
chromatographic (solvent systems (a) and (b)) and spectro-
[
5–8]
affected by pH in acid and alkaline media.
The photolysis of
FMF in aqueous solution is a controlling factor in the photo-
degradation sequence of RF and is important to understanding
the mode of RF degradation leading to the formation of the final
products (LC, LF, CMF).
RF is the prosthetic group of flavoenzymes involved in
biological redox reactions. It is an essential component of
vitamin preparations and its fate on photodegradation involves
the participation of FMF and ultimately its loss during the
reaction. This may be affected by factors such as pH, oxygen
[
28]
scopic techniques
as follows:
FMF: R 0.69 (a), 0.70 (b). lmax/nm (log e) (pH 7.0) 445
f
(4.023), 376 (3.996), 266 (4.450); fluorescence (pH 7.0) l /nm
460, lem/nm 528. nmax (KCl)/cm 3350 (–NH), 1705, 1660
(C¼O), 1580 (C¼C), 1545 (C¼N). HNMR (CF COOH): t, 7.20
ex
ꢁ
1
1
3
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

B
I. Ahmad et al.
5
Ј
2
4
CH OH
2
Table 1. Photolysis of 10 M formylmethylflavin (FMF) solution at
pH 9.0 and concentrations of FMF and photoproducts
Experimental conditions: wavelength, visible radiation; exposure time, 1 h;
temperature, 25 ꢃ 18C
4
Ј
H
H
H
C
OH
OH
OH
3
Ј
C
C
2Ј
1
Ј
CH2
N
Time [min]
FMF
LC
LF
Total
5
5
5
5
N
1
[M ꢀ 10 ]
[M ꢀ 10 ]
[M ꢀ 10 ]
[M ꢀ 10 ]
H C
O
3
9
1
0
8
7
2
3
0
10.00
8.55
7.40
6.34
5.25
3.78
–
–
10.00
9.98
6
5
N
NH
4
H C
3
10
0.23
0.41
0.61
0.74
0.93
1.20
2.29
3.12
3.99
5.26
O
2
3
0
0
10.10
10.07
9.98
Riboflavin
CHO
COOH
CH2
N
40
6
0
9.97
CH2
N
N
N
O
H C
O
H C
O
3
3
obtained during the reaction are in good agreement with the
initial concentration of FMF based on mole-to-mole conversion.
The accuracy of the analytical data is an important factor in the
evaluation of the kinetics of these reactions.
NH
NH
H C
3
N
H C
3
N
O
Formylmethylflavin
Carboxymethylflavin
CMF, a minor photoproduct, could not be accounted for in
the assay scheme. It was, therefore, separated by TLC of the
photolyzed solutions (R 0.38, solvent system (a)), extracted
with phosphate buffer (pH 7.0), concentrated under reduced
pressure and determined spectrophotometrically at 445 nm
CH3
H
N
N
O
O
H C
N
N
N
O
O
H C
f
3
3
NH
NH
H C
H C
3
3
N
ꢁ1
ꢁ1
using 10200 M cm as the value of molar absorptivity. At
ꢁ5
Lumiflavin
Lumichrome
50 % photolysis of FMF (i.e. 5 ꢀ 10 M), the concentration of
CMF in aerobically photolyzed solutions at pH 2–7 was found
to be ,0.1–0.2 ꢀ 10ꢁ M and at pH 8–10, ,0.2–0.4 ꢀ 10 M.
6
ꢁ6
Fig. 1. Chemical structures of riboflavin, formylmethylflavin and
photoproducts.
ꢁ6
The value was slightly higher, i.e. 0.5 ꢀ 10 M at pH 11.0,
indicating that the reaction is facilitated in alkaline medium.
(
[
s, CH ), 7.09 (s,CH ), 4.42(d, N–CH ), 1.97 (s, Ar–H). m/z 284
3 3 2
Spectral Characteristics of Photolyzed Solutions of FMF
þ
M ] (C H O N ).
4 12 3 4
1
FMF (in the protonated state) exhibits an absorption maximum
[9]
at 385 nm at pH 2.0 (aqueous layer), and the loss of absorption
LC: R 0.66 (a), 0.63 (b). lmax/nm (log e) (pH 7.0) 356
f
(3.929), 262 (4.435); fluorescence (pH 7.0) l /nm 368, lem/nm
ex
at this wavelength, with time, is an indication of the photo-
degradation of FMF in the reaction. The photoproducts (LC and
LF) were extracted in chloroform and do not interfere with the
absorption of FMF. The absorbance values of FMF at 385 nm
during photolysis at various pH values were used to determine
the rates of reaction.
ꢁ1
4
(
7
78. nmax (KCl)/cm 3380 (–NH), 1720, 1695 (C¼O), 1575
1
C¼C), 1560 (C¼N). HNMR (CF COOH): t, 7.31 (s, CH ),
3 3
þ
.26 (s,CH ), 1.80 (s, Ar–H). m/z 242 [M ] (C H O N ).
3 12 10 2 4
LF: R 0.53 (a), 0.41 (b). lmax/nm (log e) (pH 7.0) 445
f
(3.914), 370 (3.806), 263 (4.354); fluorescence (pH 7.0) l /nm
ex
ꢁ1
4
60, lem/nm 528. nmax (KCl)/cm 3350 (–NH), 1705, 1660
1
(
7
2
C¼O), 1575 (C¼C), 1560 (C¼N). HNMR (CF COOH): t,
3
Kinetics of Photolysis
.32 (s, CH ), 7.19 (s,CH ), 5.48 (d, NCH ), 1.80 (s, Ar–H). m/z
3 3 3
The kinetics of photolysis of FMF in acid and alkaline media
were studied. The rate constants reported were determined under
constant irradiation conditions to avoid any variation in the
results. The values of the rate constants are relative and may be
used for comparative purposes.
þ
56 [M ] (C H O N ).
1
3 12 2 4
CMF: R 0.38 (a), 0.20 (b). lmax/nm (log e) (pH 7.0) 445
f
(
4
4.008), 376 (3.981), 266 (4.435); fluorescence (pH 7.0) l /nm
ex
60, lem/nm 528.
FMF is photodegraded under aerobic and anaerobic condi-
The hydrolysis and photolysis of FMF in alkaline solution
tions to LC in acid medium and to LC and LF in alkaline medium
as detected by TLC. These compounds exhibit characteristic
fluorescence emission (FMF, LF, yellow-green; LC, sky blue)
[3]
follows apparent first-order kinetics and can be represented
by Eqn 1.
and were identified by comparison of their R values with those
f
k1
LC
LF
of the reference standards. Minor amounts of CMF (yellow-
green fluorescence), as an oxidation product of FMF under
aerobic conditions, were also detected in acid and alkaline
medium. LC, LF, and CMF have previously being reported as
FMF
ð1Þ
k2
[
6,9,11]
photoproducts of FMF.
FMF, LC, and LF were determined in degraded solutions
during photolysis by the multicomponent spectrophotometric
The differential equations for the reactant and the
products are:
[9]
ꢁ
d ½FMFꢂ
dt
method of Ahmad and Rapson. This method has been applied
of
¼
k1 ½FMFꢂ þ k2 ½FMFꢂ
ð2Þ
ð3Þ
[
3]
[4]
to study the hydrolysis, thermolysis, and photolysis
[8,9,11]
FMF in aqueous and organic solvents. The results of a typical
assayofFMFand photoproducts (LCandLF)duringthe reaction
at pH 9.0 are reported in Table 1. The values of the molar balance
d ½LCꢂ
dt
¼
k1 ½FMFꢂ

Photolysis of Formylmethylflavin
C
Table 2. Apparent first-order rate constants for aerobic photolysis of formylmethylflavin (FMF) (kobs), for the
formation of lumichrome (LC) (k ) and lumiflavin (LF) (k ) in alkaline solution and second-order rate constants for
the photolysis of FMF (k9) in acid solution
1
2
ꢁ4
Experimental conditions: initial concentration of FMF, 10 M; wavelength, visible radiation; exposure time, 1 h; temperature,
2
5 ꢃ 18C
obs ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k
ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k
ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k [M
0
ꢁ1
s
ꢁ1] ꢃ s.d.
A
pH
k
1
2
2
3
4
5
6
7
7
8
9
1
1
.0
–
–
–
1.37 ꢃ 0.056
.0
–
–
–
1.70 ꢃ 0.067
.0
–
–
–
2.11 ꢃ 0.066
.0
–
–
–
1.84 ꢃ 0.055
.0
–
–
–
1.73 ꢃ 0.047
.0
–
–
–
1.69 ꢃ 0.071
.5
0.27 ꢃ 0.017
0.36 ꢃ 0.022
1.13 ꢃ 0.038
2.72 ꢃ 0.091
3.88 ꢃ 0.108
0.23 ꢃ 0.014
0.32 ꢃ 0.018
0.95 ꢃ 0.033
2.01 ꢃ 0.081
2.43 ꢃ 0.087
0.03 ꢃ 0.001
0.05 ꢃ 0.002
0.17 ꢃ 0.010
0.70 ꢃ 0.026
1.45 ꢃ 0.058
–
–
–
–
–
.0
.0
0.0
1.0
A
n ¼ 3.
Table 3. Apparent first-order rate constants for anaerobic photolysis of formylmethylflavin (FMF) (kobs), for the
formation of lumichrome (LC) (k ) and lumiflavin (LF) (k ) in alkaline solution and second-order rate constants for
1
2
the photolysis of FMF (k9) in acid solution
Experimental conditions are as in Table 2
obs ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k
ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k
2
ꢀ 10 [sꢁ1] ꢃ s.d.
4
A
k [M
0
ꢁ1
s
ꢁ1] ꢃ s.d.
A
pH
k
1
2
3
4
5
6
7
7
8
9
1
1
.0
–
–
–
2.03 ꢃ 0.081
.0
–
–
–
2.41 ꢃ 0.075
.0
–
–
–
2.94 ꢃ 0.097
.0
–
–
–
2.61 ꢃ 0.071
.0
–
–
–
2.44 ꢃ 0.080
.0
–
–
–
2.39 ꢃ 0.079
.5
0.36 ꢃ 0.019
0.55 ꢃ 0.030
1.73 ꢃ 0.058
4.08 ꢃ 0.109
5.63 ꢃ 0.158
0.33 ꢃ 0.024
0.48 ꢃ 0.031
1.46 ꢃ 0.064
3.02 ꢃ 0.128
3.51 ꢃ 0.133
0.03 ꢃ 0.001
0.07 ꢃ 0.002
0.26 ꢃ 0.013
1.08 ꢃ 0.051
2.10 ꢃ 0.108
–
–
–
–
–
.0
.0
0.0
1.0
A
n ¼ 3.
d ½LFꢂ
dt
[11,25]
product.
The same mode of photolysis of FMF was
¼
k2 ½FMFꢂ
ð4Þ
observed in the present study and the rate constants for the
reactions carried out in acid and alkaline media under aerobic
and anaerobic conditions are reported in Tables 2 and 3
respectively. A difference in the rates of photolysis (kobs) was
observed under aerobic and anaerobic conditions. As reported in
the photolysis of RF, the rates under anaerobic conditions are
higher (,1 1/2 times) than those under aerobic conditions. This
may be due to the quenching of the flavin singlet state [ FL*]
The values of k and k for the parallel first-order reactions
1
2
leading to the formation of LC and LF respectively were
[
15]
determined according to the method of Ahmad et al.
In acid solution, FMF undergoes photolysis and thermolysis
[
29]
[
4,11]
by second-order kinetics
as follows:
1
0
hv; k
FMF ꢁꢁ ꢁꢁꢁꢁꢁc LC
3
[7]
2
ð5Þ
and the flavin triplet state [ FL*] under aerobic conditions.
This would lead to a decrease in the concentration of the flavin
3
triplet state [ FL*] and hence a lower rate of photolysis of FMF
The differential equations for the reactant and the product are:
compared with that under anaerobic conditions. The mechanism
of these reactions has been discussed by Heelis,
[25]
ꢁ
d ½FMFꢂ
Ahmad and
2
¼
k0 ½FMFꢂ
ð6Þ
ð7Þ
[
26]
[11]
dt
Vaid,
and Ahmad et al.
The degradation of FMF in the dark at pH 2.0–11.0 at
25 ꢃ 18C was also studied to determine its contribution in the
photolysis process. At the exposure times taken for 50 %
photolysis of FMF (t1/2), the percentage of FMF degraded during
the same period in the dark increased with pH and is given in
Table 4.
d ½LCꢂ
¼
k0 ½FMFꢂ
2
dt
The oxidation of flavin semiquinone radicals formed in this
reaction involves a bimolecular mechanism leading to the final

D
I. Ahmad et al.
2
4
Table 4. Degradation of 10 M formylmethylflavin (FMF) at 25 ± 18C in the dark during the exposure times (t1/2
)
required for 50 % photolysis of FMF (aerobic) at pH 2.0]11.0
First-order reactions at pH 8.0–11.0; second-order reactions at pH 2.0–7.0
pH
2.0
0.3
3.0
0.8
4.0
3.6
5.0
6.2
6.0
9.1
7.0
8.0
9.0
10.0
11.0
%
FMF degraded (in dark)
14.7
99
19.3
25.2
31.1
42.5
36.9
29.7
t1/2 [min] (in light)
122
98
79
91
96
315
102
pH could also be accounted for by the higher rate of hydrolysis
of ground-state FMF at 258C at high pH. The unprotonated form
Ϫ3.0
Ϫ3.4
Ϫ3.8
Ϫ4.2
Ϫ4.6
Ϫ5.0
3
of FL* (pK 6.5) is more reactive than the protonated form,
[7]
a
leading to a higher rate of FMF photolysis in the alkaline region.
It has also been suggested that the higher rate of photolysis of
flavins at high pH values results from both the high pK and
a
3
increased reactivity of FL*, which exists in a bent, diradical
[7]
form in this range.
The photolysis of FMF in the acid range results from a
bimolecular reaction and is affected by the protonation of FMF
[
31]
(
pK 3.5). The protonated form of FMF (97 %, pH 2.0) is non-
a
1
fluorescent owing to the quenching of FL* and is resistant to
photolysis. This is evident from the decrease in rate with a
decrease in pH below 4. In the pH range 5–6, the redox potentials
of RF are at their lowest, hence the lower rates of reaction in
[
this region.
same nucleus as that of RF and undergoes similar redox
6
7
8
9
10
11
pH
[
32]
Fig. 2. Log k–pH profiles for the photolysis of formylmethylflavin (FMF)
in alkaline solution under aerobic (J) and anaerobic (W) conditions.
16]
This also applies to FMF, which possesses the
reactions. This is supported by the fact that the pK of the
a
[33]
reduced form of FMF is 6.1, which results in a decrease in the
rate of photolysis in the pH range 5–6.
The ratios of k to k (Tables 2 and 3) increase with pH in the
3
3
2
2
1
1
0
.5
.0
.5
.0
.5
.0
.5
0
2
1
region 7.5–11.0, indicating that the formation of LF increases
with pH. LF formation results from the alkaline hydrolysis of the
side chain of a ground-state FMF molecule according to the
[
2]
mechanism suggested by Song et al. The formation of LC also
takes place in alkaline medium by cleavage of the side chain on
the addition of a water molecule, leading to the formation of an
isoalloxazine ring and glycolaldehyde. This is followed by the
rearrangement of the isoalloxazine ring to alloxazine, the
nucleus present in LC. However, the formation of LC in acid
medium results from a bimolecular mechanism as discussed
above in this section.
0
1
2
3
4
5
6
7
pH
0
Fig. 3. k –pH profiles for the photolysis of formylmethylflavin (FMF) in
acidic solution under aerobic (J) and anaerobic (W) conditions.
The photolytic degradation of FMF in both acid and alkaline
solutions is different from that of RF, which follows first-order
[
16]
kinetics in the entire pH range of 2.0–12.0.
leading to LC and LF takes place through the formation of FMF
RF photolysis
Effect of pH
[
1]
as an intermediate in this reaction. Therefore, the role of FMF
in the photodegradation sequence of RF is an important factor in
understanding the photolytic behaviour of RF in chemical,
biological, and pharmaceutical systems.
The photolysis of FMF is markedly affected by the pH of the
solution, which is a controlling factor in the kinetics of the
[
7,16]
photodegradation of RF.
The k versus pH profiles for
the photolysis of FMF under aerobic and anaerobic conditions in
alkaline and acidic solutions are shown in Figs 2 and 3 respec-
tively. As the photolysis of FMF in alkaline medium takes place
Effect of Phosphate Buffer
[
2,3]
by hydrolytic degradation,
an increase in the rate with pH
Phosphate buffer is known to catalyze the photolysis of RF. The
rate of photolysis of RF has been found to be proportional to the
was observed in the range 7.5–11.0. The reduced enhancement
in the rate constant above pH 10.0 may be due to the ionization
of the molecule to form an anion. The pK of RF, which pos-
[
34]
buffer concentration. Several studies have been conducted on
the effect of phosphate buffer on the photodegradation of RF and
a
ꢁ
2ꢁ
sesses the same isoalloxazine nucleus as that of FMF (N–3), is
[
the rate constants for H
tions have been determined.
2
PO
4
and HPO
4
ion-catalyzed reac-
In order to observe the
30]
[15,17,18]
1
0.2,
and as the ionized form of RF is less susceptible to
[16]
photolysis, the ionized form of FMF also appears to have low
susceptibility to photolysis above pH 10.0. A 15-fold increase in
the rate of photolysis on increasing the pH from 7.5 to 11.0
effect of phosphate species on the rate of photolysis of FMF,
reactions were carried out at pH 7.0. At this pH value, FMF
[
11]
undergoes second-order kinetics
and the rate constant is
(Tables 2 and 3) is probably due to a change in the reactivity of
3
FL*. The observed increase in rate constants with increasing
given in Table 5. It was found that the phosphate species cata-
lyze the photodegradation of FMF in aqueous solution. A graph

Photolysis of Formylmethylflavin
E
[
11]
Table 5. Second-order rate constant for the aerobic photolysis of
formylmethylflavin (FMF) (k9) at pH 7.0 in the presence of phosphate
buffer (0.2]1.0 M)
a particular solvent. A similar effect of viscosity on the rate of
[40]
[39]
photolysis of ascorbic acid
solvents has been observed.
and levofloxacin
in organic
Experimental conditions are as in Table 2
Mode of RF and FMF Photolysis
RF is photodegraded in aqueous solution by intramolecular
0
ꢁ1 ꢁ1
s
Buffer concentration [M]
k [M
] ꢃ s.d.
[
7,22,25,26]
photoreduction
as follows:
0
0
0
0
1
.00
.25
.50
.75
.00
2.39 ꢃ 0.079
5.50 ꢃ 0.175
9.70 ꢃ 0.272
12.48 ꢃ 0.312
15.28 ꢃ 0.367
hv
1
ꢄ
ox
RF ꢁꢁ ꢁꢁꢁꢁꢁc RF
ð8Þ
intersystem crossing
1
ꢄ
3
ꢄ
ox
RF ꢁꢁ ꢁꢁꢁꢁꢁꢁꢁꢁꢁꢁꢁꢁꢁc RF
ð9Þ
ox
Table 6. Second-order rate constants (kobs) for the anaerobic photoly-
sis of formylmethylflavin (FMF) in organic solvents
Experimental conditions are as in Table 2
3
ꢄ
RF þ RFox ꢁꢁ ꢁꢁꢁꢁꢁc RFHꢅ þ RFoxꢅ
ð10Þ
ox
2
RFHꢅ ꢁꢁ ꢁꢁꢁꢁꢁc RFox þ RFH2 ðcylic intermediateÞ ð11Þ
ꢁ
1
ꢁ1
0
ꢁ1
ꢁ1] ꢃ s.d.
Solvent
Viscosity [mPa s]
k [M
s
O2
RFH2 ꢁꢁ ꢁꢁꢁꢁꢁc FMF þ side chain products
ð12Þ
Water, pH 7.0
Acetonitrile
Methanol
1.000
2.898
1.838
0.931
0.514
0.393
1.193
2.39 ꢃ 0.079
1.50 ꢃ 0.073
1.25 ꢃ 0.055
1.16 ꢃ 0.051
1.02 ꢃ 0.036
0.98 ꢃ 0.039
0.95 ꢃ 0.042
In the above reaction scheme, RF is excited to the excited
1
singlet [ RF ] and excited triplet [ RF ] states. This is fol-
*
ox
3
*
ox
Ethanol
1
1
-Propanol
-Butanol
3
lowed by the conversion of [ RF ] to the semiquinone radical
*
ox
ꢅ
[RFH ]. The semiquinone radicals disproportionate to form an
oxidized RF and a reduced RFH cyclic intermediate, which is
Dichloroethane
2
oxidized to FMF and side chain products.
The role of FMF as an intermediate product in the photolysis
of RF is well established. It is photolyzed to LC in
acidic solutions and to LC and LF in alkaline solutions under
0
of second-order rate constants (k ) versus phosphate concen-
2
[1,5,7,9,16]
tration is linear (R ¼ 0.998), indicating that the phosphate
species exert a catalytic effect on the photolysis of FMF.
The effect of phosphate buffer on the reaction may also be
explained based on fluorescence studies. A decrease in the
fluorescence intensity of FMF solutions in the presence of
[2,3,6–8,10–13]
aerobic and anaerobic conditions directly
[1,5–7,9,13]
or
FMF may lead to the
through the photolysis of RF.
formation of LC in acidic solutions by a bimolecular photoredox
3
reaction involving an excited triplet state [ FMF*] and a ground
[11,25–27]
state [FMF] molecule as suggested earlier for flavins.
0
–1.0 M phosphate concentration (100–88% decrease) indicated
an interaction between FMF and the phosphate ions similar to
2
ꢁ
that observed in the case of the RF–HPO4 complex leading to
the formation of cyclodehydroriboflavin (CDRF) and the
3
FMF þ FMF ꢁꢁ ꢁꢁꢁꢁꢁc FMFꢅꢁ þ FMFꢅþ
ð13Þ
ð14Þ
ꢁ
[17].
RF–H PO complex forming LC
2
This complexation caused
þ
4
H
1
12 % quenching of the excited singlet state [ FMF]. As the
ꢁ
,
RF–HPO complex can only form CDRF by photoaddition,
FMFꢅ ꢁꢁ ꢁꢁꢁꢁꢁc FMFHꢅ
2
4
ꢁ
þ
which is not possible in the case of FMF, which lacks a ribose
side chain, the FMF–phosphate complex could form LC by
photoreduction as suggested previously for the photoreduction
pathway of RF. In view of the fact that phosphate ions exert a
catalytic effect on the photolysis of FMF, there is a strong
ꢁH
þ
FMFꢅ ꢁꢁ ꢁꢁꢁꢁꢁc FMFꢅ
ð15Þ
ð16Þ
ð17Þ
[
17]
2 FMFHꢅ ꢁꢁ ꢁꢁꢁꢁꢁc FMF þ FMFH2
–
4
2ꢁ
FMFꢅ þ FMF ꢁꢁ ꢁꢁꢁꢁꢁ FMFHꢅ þ LC
c
possibility that the H PO and/or HPO ions are involved in the
2
4
photolysis reaction.
3
The reaction of a triplet [ FMF*] with a ground state [FMF]
molecule leads to the formation of an anionic and a cationic
FMF radical. These radicals may accept or lose a proton to form
a reduced or an oxidized radical. The reduced FMF radicals may
lead to the formation of an oxidized and a reduced FMF
molecule. The oxidized FMF radical may react with a ground
state FMF molecule to give rise to a reduced FMF radical
and LC.
Effect of Solvent Viscosity
The solvent may exert a significant effect on the rate of a
[
35–37]
chemical reaction.
ysis of FMF on solvent dielectric constant has been reported.
The dependence of the rate of photol-
[
11]
In the present study, an attempt was made to correlate the rate of
photolysis with solvent viscosity (Table 6). A graph of rate
constants versus the inverse of solvent viscosity was found to be
2
FMF is photolyzed in alkaline solutions by hydrolytic degra-
linear (R ¼ 0.995). It appears that the rate of reaction increases
[
2,3]
dation.
reactivity and a change in the configuration of FMF.
The increase in rate with pH is due to the higher
3
linearly with the inverse of solvent viscosity. A similar obser-
[7]
[
27]
vation was made by Ahmad and Tollin for the dependence of
flavin triplet state quenching on solvent viscosity. The rate
constants for diffusion-controlled processes have been reported
ꢁ
OH
3
ꢁ
FMF ꢁꢁ ꢁꢁꢁꢁꢁc LF þ HCOO
ð18Þ
ð19Þ
[38]
as a function of solvent viscosity.
Thus, the rate of reaction
would be affected by the solute diffusion processes and, there-
fore, the degree of redox reactions undergone by the molecule in
þ
H
3
FMF þ H2O ꢁꢁ ꢁꢁꢁꢁꢁc LC þ OHCCH2OH

F
I. Ahmad et al.
Thus FMF on photolysis in aqueous solutions forms both LC
and LF by different mechanisms.
250-mm cellulose plates (Whatman CC 41). The following
solvent systems were used: (a) 1-butanol/acetic acid/water
(
40 : 10 : 50 v/v, organic phase); and (b) 1-butanol/propanol/
[
3]
Conclusion
acetic acid/water (50 : 30 : 2 : 18 v/v). FMF and its photo-
products were detected by their characteristic fluorescence
emission under UV (365 nm) excitation using a Uvitech lamp
The photolysis of FMF follows first-order kinetics in alkaline
medium and second-order kinetics in acid medium both under
aerobic and anaerobic conditions. The k–pH profiles for these
reactions indicate the maximum rate of reaction at ,pH 11 and
(
Cambridge, UK).
Spectral Determinations
4
. The rates are higher under anaerobic conditions owing to the
existence of a greater number of flavin singlet states and are
affected by the ionization of the molecule. The ionized species
of FMF, like those of RF, are less susceptible to photolysis both
in acid and alkaline media. The increase in rate in the pH range
All spectral determinations on FMF and its photolyzed solutions
were carried out on a Shimadzu UV-1601 recording spectro-
photometer using quartz cells of 10-mm path length.
7
.5–11.0 is due to greater reactivity of the flavin triplet state to
Light Intensity Measurement
base hydrolysis. The lower rate in the pH range 5–6 is probably
due to the molecule having its lowest redox potentials in this
region. FMF on photolysis undergoes a bimolecular reaction in
acid media to produce LC and a unimolecular reaction in alka-
line media to form LC and LF. The photodegradation of RF in
aqueous solution is dependent on the photolytic behaviour of
FMF as an intermediate product in this reaction. Phosphate ions
exert a catalytic effect on the photolysis of FMF in aqueous
solution. The rates of FMF photolysis are inversely proportional
to the solvent viscosity.
The intensity of the Phillips 25-W fluorescent lamp was deter-
[43]
mined using potassium ferrioxalate actinometry
16
and a value
ꢁ1
of 4.52 ꢃ 0.15 ꢀ 10 quanta s was obtained.
Spectrophotometric Assay
A multicomponent spectrophotometric assay method was used
for the determination of FMF and its major photoproducts, LC
[9]
and LF. The method involved pre-adjustment of the photo-
lyzed solution to pH 2.0 (HCl/KCl buffer) and extraction with
chloroform to remove LC and LF, followed by their determi-
nation at pH 4.5 (acetate buffer) by a two-component assay at
Experimental
4
determine FMF concentrations at 385 nm. It is stable in its
45 and 356 nm. The aqueous phase (pH 2.0) was used to
LC and LF were obtained from Sigma Chemical Co. FMF and
[
41]
CMF were synthesized by the method of Fall and Petering
and Fukumachi and Sakurai
[31]
[42]
protonated form (pK
chloroform.
a
3.5)
and is not extractable into
respectively. All reagents and
solvents were of the purest form available from BDH and
Merck. The following buffer systems were used throughout the
photolysis reactions: KCl/HCl, pH 2.0; citric acid/Na HPO ,
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