Redox reaction between m-thiocresol and riboflavin glycosides with 2:1 complex formation; regulation by the steric effect of sugar in the side chain

Article abstract of DOI:10.1080/10610278.2010.521840

We investigated the reduction of riboflavin-2′, 3′, 4′, 5′-tetra-acetate (AcB₂), riboflavin-1″-glucoside- 2″, 3″, 4″, 6″, 2′, 3′, 4′, 5′-hepta-acetate (AcB₂gl) and lumiflavin using m-thiocresol (mTc) in the presence of tetrabutylammonium hydroxide. The series of rate constants for AcB₂ and AcB₂gl reductions indicated that modified Lineweaver-Burk plots were best fit by assuming a 1:2 complex formation. The complex formation in the reaction was supported by the 2-D nuclear Overhauser enhancement spectroscopy and circular dichroism spectra. The modified Michaelis-Menten constants (K_m) for AcB₂ and AcB₂gl with mTc were 1.32 and 0.86 × 10^-3M ², respectively, and the maximum rate constant k₂ were 4.45 and 4.35 × 10^-2M^-1s^-1, respectively. The E_1/2 values of AcB₂ and AcB₂gl were - 331 and - 341mV, respectively, which indicated that their reduction activities were almost the same. It was established that the redox function depended on the formation of the complex and was regulated by the steric effect of the sugar in the side chain.

Full text of DOI:10.1080/10610278.2010.521840

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Supramolecular Chemistry
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Redox reaction between m-thiocresol and riboflavin
glycosides with 2:1 complex formation; regulation by
the steric effect of sugar in the side chain
Keiko Takahashi a , Hiroshi Odajima a , Saori Nuiya a & Yasushi Hasebe b
a Department of Life Science and Sustainable Chemistry, Faculty of Engineering , The
Center for Nano Science and Technology, Tokyo Polytechnic University , 1583 Iiyama, Atsugi,
Kanagawa, 243-0297, Japan
b Department of Life Science and Green Chemistry, Faculty of Engineering , Saitama
Institute of Technology , Fukaya, Saitama, 369-0293, Japan
Published online: 13 Apr 2011.
To cite this article: Keiko Takahashi , Hiroshi Odajima , Saori Nuiya & Yasushi Hasebe (2011) Redox reaction between m-
thiocresol and riboflavin glycosides with 2:1 complex formation; regulation by the steric effect of sugar in the side chain,
Supramolecular Chemistry, 23:03-04, 252-255, DOI: 10.1080/10610278.2010.521840
To link to this article: http://dx.doi.org/10.1080/10610278.2010.521840
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Supramolecular Chemistry
Vol. 23, Nos. 3–4, March–April 2011, 252–255
Redox reaction between m-thiocresol and riboflavin glycosides with 2:1 complex formation;
regulation by the steric effect of sugar in the side chain
Keiko Takahashia*, Hiroshi Odajimaa, Saori Nuiyaa and Yasushi Hasebeb
aDepartment of Life Science and Sustainable Chemistry, Faculty of Engineering, The Center for Nano Science and Technology, Tokyo
Polytechnic University, 1583 Iiyama, Atsugi, Kanagawa 243-0297, Japan; bDepartment of Life Science and Green Chemistry, Faculty of
Engineering, Saitama Institute of Technology, Fukaya, Saitama 369-0293, Japan
(Received 9 July 2010; final version received 24 August 2010)
We investigated the reduction of riboflavin-20, 30, 40, 50-tetra-acetate (AcB2), riboflavin-10-glucoside-200, 300, 400, 600, 20, 30, 40,
50-hepta-acetate (AcB2gl) and lumiflavin using m-thiocresol (mTc) in the presence of tetrabutylammonium hydroxide.
The series of rate constants for AcB2 and AcB2gl reductions indicated that modified Lineweaver–Burk plots were best fit by
assuming a 1:2 complex formation. The complex formation in the reaction was supported by the 2-D nuclear Overhauser
enhancement spectroscopy and circular dichroism spectra. The modified Michaelis–Menten constants (Km) for AcB2 and
AcB2gl with mTc were 1.32 and 0.86 £ 1023 M2, respectively, and the maximum rate constant k2 were 4.45 and
4.35 £ 1022 M21 s21, respectively. The E1/2 values of AcB2 and AcB2gl were 2 331 and 2 341 mV, respectively, which
indicated that their reduction activities were almost the same. It was established that the redox function depended on the
formation of the complex and was regulated by the steric effect of the sugar in the side chain.
Keywords: riboflavin glycoside; m-thiocresol; Lineweaver–Burk plot; steric effect; host–guest complex
Flavoproteins play a key role in oxidation–reduction
systems catalysing the electron transfer (1). The functions
of flavoproteins may be modified artificially so that they
associate reversibly by providing an appropriate self-
organising system for flavin and the other electron
acceptors. Riboflavin (B2), 7,8-dimethyl-10-((2R,3R,4S)-
2,3,4,5-tetrahydroxyphenyl)benzo-[g ]pteridine-2,4(3H,
10H)-dione, which is known as vitamin B2, is the central
component of flavoproteins. B2 is generally stable during
heating, but is sensitive to light, oxygen and alkaline
solution. The derivatives of 7,8-dimethyl-iso-alloxazine
that possess different functional groups in the side chain
at position 10 are defined as flavins. In biochemical
reactions, flavins undergo reversible redox conversion
atoms N(5) and N(1). Many model systems for flavin
catalyst reactions have been reported (2).
revealed (3). B2gl exhibits high water solubility, but it is
still sensitive to light and oxygen. By photo-irradiation,
B2gl is resolved into B2 and glucose, and then B2 is
resolved to lumiflavin (LF) and lumichrome. Acetylated
B2gl is stable during photo-irradiation. To elucidate the
redox-reaction mechanism between riboflavin derivatives,
kinetic studies were carried out using acetylated flavin and
m-thiocresol (mTc) in acetonitrile with tetrabutylammo-
nium hydroxide (TBAH), by means of UV spectra. In this
paper, we report the preparation of acetylated B2
derivatives, and discuss the kinetic parameters of their
reduction using mTc which induces a 1:2 complex
formation.
Riboflavin-10-glucoside (B2gl) was prepared using
riboflavin and maltose with an enzyme prepared from
Aspergillus oryzae extract in a citrate buffer (pH 3.5) at
308C for 48 h, as shown in Figure 1 (4). The separation
and purification of B2gl was performed using HP-20. B2gl
was obtained in yields of 48% and Rf value of 0.21
(ethylacetate/pyridine/aq.:18/6/1). Riboflavin-20,30,40,50-
tetra-acetate (AcB2) was prepared by the reaction of
riboflavin with acetic anhydride in pyridine according
to a previous report (5). After removing pyridine, crude
AcB2 was recrystallised from a mixture of ethanol and
chloroform (12.7% yield). The identification of AcB2 was
Glycosides are molecules in which a sugar is bound to
a non-carbohydrate group; they play an important role in
living organisms. Many plants store chemical molecules
in the form of inactive glycosides, which can be activated
by hydrolysis using enzymes. The bioavailability of
glycoside usually depends on the type of glycosidic bond
and glycone. It has also been known that riboflavin
a-glycoside (B2gl) is a metabolite; however, its metabolic
pathway and the role of the glycoside have not yet been
*Corresponding author. Email: takahasi@chem.t-kougei.ac.jp
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2011 Taylor & Francis
DOI: 10.1080/10610278.2010.521840
http://www.informaworld.com

Supramolecular Chemistry
253
Figure 1. Preparation of riboflavin glycoside and acetylation.
Figure 2. Redox reaction between flavin and mTc.
carried out by TLC, NMR and ESI-MS spectroscopy.
Rf value: 0.21 (hexane/ethylacetate ¼ 1:3), ESI-MS m/z
at lmax 442 nm and the fluorescence emission intensity at
l
max 505 nm. The products, FH2 and di-(m-methyl)phenyl-
1
calcd for C25H28N4O10: 567.1698; found 567.1654. H
disulphide were identified by comparing the 1H NMR
spectra with the reported spectrum of FH2 and di-(m-
methyl) phenyl-disulphide (5b). Good pseudo-first-order
rate data were obtained. The value of the observed rate
constant kobs for the reduction of AcB2 (1.4 £ 1024 M)
with mTc (4.2 £ 1022 M) in the presence of TBAH
(3.3 £ 1023 M) is comparable to a previously reported
value (5b). The kobs of acetylated B2 flavins indicates three
times greater than that of LF. kobs decreases at high
temperatures (Table 1). The reduction reaction was
analysed according to the standard and modified
Michaelis–Menten scheme (Equations (1a), (1b) and
NMR (500 MHz, CD3CN, 608C, ppm): d 1.73, 2.04, 2.18,
2.24 (s 3H acetyl methyl), d 2.42 (s 3H methyl 8a), d 2.54
(s 3H methyl 7a), d 4.20 (dd 1H ribityl 40), d 4.37(dd 1H
ribityl 30), d 4.90 (brd 1H ribityl 50a), d 5.10 (brd 1H ribityl
50b), d 5.29 (m 1H ribityl 20), d 5.40 (m 1H ribityl 10b), d
5.65 (m 1H ribityl 10a), d 7.53 (s 1H phenyl 6), d 8.00 (s 1H
phenyl 9), d 8.34 (s 1H imide 3). Riboflavin-10-glucoside-
200, 300, 400, 600, 20, 30, 40, 50-hepta-acetate (AcB2gl) was also
obtained from riboflavin-10-glucoside by applying the
procedure described above. Rf value: 0.16 (hexane/ethyl-
acetate ¼ 1:3), ESI-MS m/z calcd for C37H43N4O18:
1
855.2542; found 855.2567. H NMR (500 MHz, CD3CN,
608C, ppm): d 1.65, 1.91, 1.95, 2.00, 2.15, 2.19 (s 3H
acetyl methyl), d 2.37 (s 3H methyl 8a), d 2.49 (s 3H
methyl 7a), d 3.73 (dd 1H ribityl 40), d 3.82 (dd 1H ribityl
30), d 3.92 (qq 1H glucose 500), d 4.04 (dd 1H glucose 600b),
d 4.14 (dd 1H glucose 600a), d 4.77 (dd 1H glucose 200), d
4.86 (brd 2H ribityl 50ab), d 4.95 (t 1H glucose 400), d 5.06
(d 1H glucose 100), d 5.25 (m 1H ribityl 20), d 5.35 (t 1H
glucose 300), d 5.39 (m 1H ribityl 10b), d 5.57 (m 1H ribityl
10a), d 7.38 (s 1H phenyl 6), d 7.95 (s 1H phenyl 9), d 8.25
(s 1H imide 3).
Table 1. Observed pseudo-first-order rate constants for the
reduction in acetylated B2 derivatives with m-thiocresol.
k
obs(102 s21
323 K
)
Flavin
293 K
343 K
0.55
Lumiflavin (LF)
0.9
0.8a
0.62
AcB2
2.67
2.30a
2.59b
2.97
3.16a
1.77
1.92
1.58
1.60
We studied the reduction of riboflavin to 1,5-
dihydroriboflavin in acetonitrile at 293 K (Figure 2). The
reaction was nearly quantitative, as determined by
spectroscopy when TBAH and an excess of mTc (300-
fold) was used. The rate of reduction was measured
by following the characteristic absorption of flavin (F)
AcB2gl
a Measured by the fluorescence emission intensity at lmax 505 nm.
b 298 K, ref. (5b).

254
K. Takahashi et al.
1
1
Km
k2
Table 2. Kinetic parameters, Michaelis–Menten constants
(dissociation constants) and rate constants for the reduction
obtained by modified Lineweaver–Burk plots (Equation (2c)).
2
¼
þ
½Sꢀ0 :
ð2cÞ
kobs k2
The correlation coefficients of the plots according to (1c)
for AcB2 and AcB2gl are 0.9514 and 0.958, respectively.
The coefficients according to (2c) for AcB2 and AcB2gl
are 0.9912 and 0.9976, respectively. The redox reactions
proceed through a complex formation of one acetylated B2
derivative and two mTc molecules. The rate constants k2
and Km for the reaction of totally complexed FS2 were
evaluated from (2c); the resultant kinetic parameters are
given in Table 2. The maximum rate constant k2 for
AcB2gl is almost the same as that for AcB2. Cyclic
voltammetry is generally used to study the electrochemical
properties in solution (6).This result is consistent with that
obtained by cyclic voltammetry (Table 3). The Km value
for AcB2gl is considerably smaller than that for AcB2. This
indicates that in AcB2gl, the mTc is embedded strongly in
the cleft surrounded by the acetyl glucose group and
alloxazine group, resulting in enhanced binding, whereas
the cleft in AcB2 is not deep enough to form a stable
complex. AcB2 and AcB2gl showed induced circular
dichroism (CD) spectra peaking at 440, 340 and 255 nm.
The result indicated that the sugar groups of AcB2 (ribityl
group) and AcB2gl (ribityl and glucose groups) stay close
to the 7,8-dimethyl-iso-alloxazine group. A direct
evidence for the relative orientation of the mTc and flavin
has been obtained by the 2-D NMR NOESY method
(Figure 3). The cross peaks between the 50ab protons and
the protons of mTc and between the 50ab protons and 6
proton were observed. These situations lead to an apparent
overall increase in the reduction rate k2/Km for AcB2gl;
this rate is approximately 1.5 times greater than that for
AcB2. The phenomenon ‘supramolecular regulation’, a
redox reaction, is regulated only by the steric effect of
sugar. Only methyl group is substituted at position 10 in
LF; a straight line in both of the plots according to (1c) and
(2c) could not be observed. No induced CD was observed.
The result is an example of how a catalytically inactive
group can regulate the catalytic activity. Sugar groups
Flavin
k2 (102 s21
)
Km(103 M2)
k2/Km
AcB2
AcB2gl
4.46
4.33
1.32
0.86
3.37
5.00
Table 3. E1/2 values of riboflavin derivatives.
E1/2(mV)
Flavin
293 K
323 K
343 K
AcB2
AcB2gl
B2gl
2331
2314
2446
2341
2338
2464
n.d.
2392
2480
(2a), (2b), respectively).
F þ S Y F·S ! P;
where S is the electron receptor and P is the product.
ð1aÞ
1
1
ðk21 þ k2Þ
¼
þ
½Sꢀ0;
ð1bÞ
kobs k2
k1k2
F þ 2S Y F·S ! P;
ð2aÞ
ð2bÞ
1
1
ðk21 þ k2Þ
2
¼
þ
½Sꢀ0;
kobs k2
k1k2
ðk21 þ k2Þ
Km
¼
;
ð3Þ
k1
1
1
Km
k2
¼
þ
½Sꢀ0;
ð1cÞ
kobs k2
mTC phenyl
9 6
mTC phenyl
(b)
(a)
3
9 6
3
0
9
8
7
6
0
9
8
7
6
5’2b
5’2b
Figure 3. NOESY spectra of AcB2 (a) and AcB2gl (b) in CD3CN at 293 K.

Supramolecular Chemistry
255
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