Color regulation and stabilization of chromophore by Cys69 in photoactive yellow protein active center

Article abstract of DOI:10.1039/b905835d

Model compounds of PYP chromophore were synthesized and characterized to investigate the role of the Cys69 residue in the active center, which has the intramolecular NH...OC hydrogen bond to the conjugated carbonyl oxygen and thioester linkage of the chro

Full text of DOI:10.1039/b905835d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Color regulation and stabilization of chromophore by Cys69 in photoactive
yellow protein active center
Kentaro Okamoto,a Norio Hamada,a Taka-aki Okamura,a Norikazu Ueyamaa and Hitoshi Yamamoto*a,b
Received 24th March 2009, Accepted 4th June 2009
First published as an Advance Article on the web 15th July 2009
DOI: 10.1039/b905835d
Model compounds of PYP chromophore were synthesized and characterized to investigate the role of
the Cys69 residue in the active center, which has the intramolecular NH ◊ ◊ ◊ OC hydrogen bond to the
conjugated carbonyl oxygen and thioester linkage of the chromophore. The results of UV-vis and 13
C
NMR spectroscopy of the model compounds indicated that they delocalized the negative charge of the
chromophore and increased the contribution of the quinoid-like resonance structure in the phenolate
anion state. Thus, the Cys69 residue plays an important role in the regulation of the color and the
stabilization of the chromophore anion in the active center.
Introduction
Photoactive Yellow Protein (PYP) isolated from the purple sulfur
bacterium Halorhodospira halophila by Meyer in 19851 is one of the
photoreceptor proteins. PYP has a yellow color (lmax = 446 nm),
and it is considered to function as a blue-light photosensor
implicated in the negative phototaxis of the bacterium.2 Typical
photoreceptor proteins such as rhodopsin and bacteriorhodopsin
are membrane proteins; however, PYP is water soluble, small
(14 kDa), and thermally stable.3 Therefore, PYP is well studied
and its three-dimensional structure has been reported, with its
resolution higher than any other photoreceptor proteins. PYP is
reported by some groups to be composed of 125 amino acids and
one chromophore (4-hydroxycinnamic acid thioester).4–6 Crystal
structure of PYP reported by Borgstahl et al. shows a sphere-like
structure with six b strands that are in anti-parallel arrangements
in the middle, which is sandwiched by five a helices on both sides.7
The PYP chromophore is covalently bound to Cys69 of
p-loop (residues 63–78) via a thiol ester linkage and exists as a
phenolate anion state having E configuration in the hydrophobic
core of the protein.4–8 The chemical structure and the hydrogen
bond network around amino acids (Tyr42, Glu46, and Cys69)
and the chromophore are summarized in Fig. 1a.7,9 Although
Glu46 (pKa = 4.3), an acidic residue, is located near the hydroxy
group of the chromophore (4-hydroxycinnamic acid thioester,
pKa = 8.8),10 it is proposed that the chromophore exists as a
phenolate anion state in the active center of PYP.5,8 Considering
the differences in the pKa values between phenols and carboxylic
acids, this correlation between the deprotonated chromophore and
the protonated Glu46 is considered to be distinctive of the active
center of PYP. This diagnostic chemical mechanism regulated by
the active center of PYP attracted considerable attention.
Fig. 1 (a) Schematic diagram around the chromophore at the ground
state of Photoactive Yellow Protein and (b) E formed model compounds
of the chromophore (X = S, O, NH).
regulate the electronic properties of the chromophore, the color of
PYP, and its stability.11,12 However, the role of the hydrogen bond
between the amide NH in Cys69 and the carbonyl oxygen in the
chromophore has been noticed in previous research,13 but not fully
investigated. It was also reported that a point mutation at Cys69
(C69S) had no pigment and could not be reconstituted.14 In order
for PYP to function as a blue-light photosensor, the process of the
chromophore binding to Cys69 via thioester linkage is considered
to be important.
To investigate the role of the hydrogen bond between Cys69
and the chromophore, we designed novel model compounds
(shown in Fig. 1b) containing an intramolecular hydrogen bond
to the carbonyl oxygen in the form of the seven-membered ring
like native PYP. In previous work,15 we synthesized the amide
Point mutation studies of Tyr42 and Glu46 have suggested that
hydrogen bonds to a phenolic hydroxy oxygen of the chromophore
aDepartment of Macromolecular Science, Graduate School of Science, Osaka
University, Machikaneyama-cho 1-1, Toyonaka, Osaka 560-0043, Japan;
Fax: +81-6-6850-5474; Tel: +81-6-6850-5451
bDepartment for the Administration of Safety and Hygiene, Osaka Uni-
versity, Yamadaoka 1-1, Suita, Osaka 565-0871, Japan. E-mail: jin@
chem.sci.osaka-u.ac.jp; Fax: +81-6-6879-4024; Tel: +81-6-6879-4023
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linked model compound (X = NH) and confirmed that the
intramolecular NH ◊ ◊ ◊ OC hydrogen bond is formed between the
pivaloyl amide NH and the carbonyl C O of the chromophore.
incorporated into micellar cavity, in which the phenolate anion of
the model compound is protected from the nucleophilic attack,
and it was observed as stable without decomposition of the
linkage for about one day. For various measurements of the
model compounds, we employed poly(ethylene glycol) lauryl ether
(C12E9) as the detergent since it is nonionic and has relatively little
effect on 1H NMR and UV-vis spectroscopy.
=
Then, we could investigate the effect of the hydrogen bond to
the carbonyl oxygen on the electronic state of the chromophore by
using our model compound. In UV-vis spectroscopy, it is observed
that the intramolecular NH ◊ ◊ ◊ OC hydrogen bond contributes
to the extension of p-electron delocalization in the part of the
chromophore. In this work, we investigate the effects of the
sulfur atom of the thioester group, in addition to the hydrogen
bond to the carbonyl oxygen, on the chemical properties of the
chromophore using the thioester or ester linked model compounds
(X = S, O). Through the characterization of these more detailed
model compounds, we can elucidate the role of the NH ◊ ◊ ◊ OC
hydrogen bond and sulfur atom (i.e. the Cys69 residue) in the
active center of PYP.
Formation of intramolecular NH ◊ ◊ ◊ OC hydrogen bond in solution
In our previous work,15 the results of X-ray crystal structure
1
analysis and H NMR spectroscopy of the amide linked model
compound (X = NH) confirmed the formation of an intramolecu-
lar NH ◊ ◊ ◊ OC hydrogen bond between the pivaloyl amide NH and
=
carbonyl C O in the 4-hydroxycinnamic acid moiety. In the first
step, we confirmed the formation of the intramolecular NH ◊ ◊ ◊ OC
hydrogen bond in (E)-S1-OH and (E)-O1-OH by using 1H NMR
and IR spectroscopy. The information regarding chemical shifts
and temperature coefficients of the amide NH signals in 1H NMR
spectra and the amide NH stretching vibrations in IR spectra
of (E)-S1-OH and (E)-O1-OH are summarized in Table 1. The
temperature coefficients of the amide NH signals of (E)-S1-OH
and (E)-O1-OH were -1.70 and -1.75 ppb/K in chloroform-d1,
respectively. These values were smaller than that for the reference
compound C6H5-NHCOtBu (-2.38 ppb/K in chloroform-d1),
which has no intramolecular hydrogen bond. These results suggest
the formation of an intramolecular NH ◊ ◊ ◊ OC hydrogen bond to
Results and discussion
Synthesis of model compound
Syntheses of model compounds are shown in Scheme 1. The
hydroxy group of 4-hydroxycinnamic acid was protected by
treating with tert-butyldimethylsilyl triflate. The thioester and ester
groups were synthesized by the reaction with a thiol or a phenol via
an acyl chloride, respectively. Subsequently, the protecting group
was removed by hydrogen fluoride.
Deprotonation of the hydroxy group was performed by neutral-
ization with sodium hydroxide in a micellar solution. Unlike the
amide linked model compounds (X = NH) reported in previous
work,15 the phenolate anion state of the compounds possessing the
thioester or ester group could not be isolated in organic solvents. In
the neutralization of the hydroxy group with hydroxide or alkoxide
in organic solvents, the thiolate or phenolate anion (i.e. the
fragment of model compound) was obtained from the reaction
solution. This result suggests that the thioester and ester linkages
were decomposed by nucleophilic attack of the base in the organic
solvent. However, in a micellar solution, the model compound is
=
the carbonyl C O of the chromophore in solution.
However, the coefficients of (E)-O1-OH, an ester model com-
pound, increased toward changes in the solvent polarity, whereas
those of (E)-S1-OH, a thioester model compound, showed little
change in both acetonitrile-d3 and 10% C12E9 micellar solution
(10% D2O, 80% H2O). Additionally, the amide NH stretching
vibration of (E)-S1-OH (3405 cm-1) shifts to a lower wavenumber
compared with those of (E)-O1-OH (3459 cm-1) and C6H5-
NHCOtBu (3455 cm-1) by IR spectroscopy in chloroform solution.
These results suggest that a more stable intramolecular NH ◊ ◊ ◊ OC
hydrogen bond is formed in the thioester model (E)-S1-OH than
Scheme 1 Synthesis of the model compound. (i) TBDMS(OTf), Et3N; CH2Cl2; rt; (ii) K2CO3; MeOH/THF/H2O; rt; (iii) SOCl2; CH2Cl2; reflux;
(iv) Ar-SH or Ar-OH, Et3N; CH2Cl2; rt; (v) HF·pyridine; THF; rt; (vi) NaOH aq; 10% C12E9 micellar solution.
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Table 1 Chemical shifts (ppm), temperature coefficients (ppb/K), and stretching vibrations (cm-1) of the model compounds
Chemical shift (ppm)/Temperature coefficient (ppb/K)
Stretching vibration (cm-1)
in CHCl3
in CDCl3
in CD3CN
in 10% micellar solution (10% D2O, 80% H2O)
(E)-S1-OH
(E)-O1-OH
8.17/-1.70
7.59/-1.75
8.11/-1.02
7.72/-2.02
8.31/-2.43
8.21/-6.98
3405
3459
in the ester model (E)-O1-OH. The downfield shifts of amide
NH signal in 1H NMR spectroscopy support this suggestion.
In addition, it is considered that the intramolecular NH ◊ ◊ ◊ OC
hydrogen bond in (E)-O1-OH is not formed or might be weak in
polar solutions.
In light of these findings, it is proposed that the difference
between bridging atoms (sulfur or oxygen) induces changes in
the formation of the intramolecular NH ◊ ◊ ◊ OC hydrogen bond.
Thus, we investigated the conformation of each model compound.
Superposition of the optimized structures of model compounds
obtained by ab initio calculation (B3LYP/6–31++G(d,p))16–19 is
shown in Fig. 2. Superposition of (E)-S1-OH and (E)-O1-OH
shows that the bond distance between the amide proton and
carbonyl oxygen is shorter in the thioester model compound
˚
(E)-S1-OH (1.940 A) than the ester model compound (E)-O1-OH
˚
(1.972 A). This conformational difference within a part of the
seven-membered ring is due to the change in C(carbonyl)-X-C(Ar)
bond angle (106.99 in (E)-S1-OH and 122.96 in (E)-O1-OH).
These optimized structures support the results obtained from 1H
NMR and IR spectroscopy. It is suggested that the thioester model
compound (E)-S1-OH has a conformation that is easy to form an
intramolecular hydrogen bond between the amide group and the
4-hydroxycinnamic acid moiety.
Fig. 3 1H NMR spectra of (a) (E)-S1-OH, (b) (E)-S1-O-, (c) (E)-O1-OH,
and (d) (E)-O1-O- in a 10% C12E9 micellar solution (10% D2O, 80% H2O)
at 303 K.
suggested that the deprotonation has a significant effect on the
p-electron conjugation system of the chromophore.
We investigated the detail of this change in the p-electron
conjugation system by using UV-vis spectroscopy. The absorption
spectra of model compounds obtained in a 10% C12E9 micellar
solution at 303 K are shown in Fig. 4, and these spectral data
assigned to the p–p* transition of chromophore using the MOPAC
program (INDO/S method)20 are summarized in Table 2. The
p–p* transition of the model compounds is located around
Fig. 2 Superposition of the part of the chromophore of the optimized
structure (black: (E)-S1-OH, gray: (E)-O1-OH).
The effects of NH ◊ ◊ ◊ OC hydrogen bond and sulfur atom on
p-electron conjugation system of PYP chromophore
1H NMR spectra of model compounds (E)-S1-OH, (E)-S1-O-,
(E)-O1-OH, and (E)-O1-O- in a 10% C12E9 micellar solution
(10% D2O, 80% H2O) at 303 K are shown in Fig. 3. The chemical
shifts of signals (Ha~Hd) derived from the 4-hydroxycinnamic acid
moiety show upfield shifts with deprotonation of the hydroxy
group. It is noteworthy that the signal of Hd, which is located
far away from the hydroxy group, shows an upfield shift. It is
Fig. 4 UV-vis spectra of the model compounds in a 10% C12E9 micellar
solution at 303 K. (gray: phenol state, black: phenolate anion state).
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Table 2 Absorption maxima of p–p* transition of model compounds in
the ester model compounds. It is observed that the extension of
p-electron delocalization is induced by the introduction of sulfur
into the bridging atom as well.
10% C12E9 micellar solution at 303K
-OH
-O-
From the results of UV-vis spectroscopy of the model com-
pound, it is indicated that the intramolecular NH ◊ ◊ ◊ OC hydrogen
bond to the conjugated carbonyl oxygen spatially interacts with
the p-electron conjugation system through a different mechanism
from substituent effects. Thus, we investigated the changes in the
electronic state of the chromophore in more detail using ab initio
calculation and 13C NMR spectroscopy. The bond lengths of the
4-hydroxycinnamic acid moiety and the Mulliken charges of the
hydroxy and the carbonyl oxygen are summarized in Tables 3
and 4. The numbering of the 4-hydroxycinnamic acid moiety are
shown in Scheme 2. The bonds of the phenyl ring moiety
(C1-C2, C2-C3, C3-C4) have aromatic characteristics in the phenol
state. However, the bonds of C1-C2 and C3-C4 are lengthened
and those of C2-C3 are shortened in the phenolate anion state,
suggesting the contribution of the quinoid-like resonance structure
(Scheme 3). It is considered that the shifts between phenol and
phenolate anion in 1H NMR and UV-vis spectroscopy are derived
from the contribution of this quinoid-like structure. Additionally,
the differences between (E)-X1-O- and (E)-X2-O- (X = S, O)
show that such contribution is increased by the formation of the
(E)-S1
(E)-S2
(E)-O1
(E)-O2
348
342
325
323
425
413
386
382
330 nm in the phenol state and around 400 nm in the phenolate
anion state. It is well known that the extension of p-electron
delocalization induces a red shift of the absorption maximum.
The absorption maximum is significantly red-shifted according to
the deprotonation of chromophore, indicating that the negative
charge delocalizes along the skeleton of the chromophore in the
phenolate anion state.
It is observed in both the phenol and phenolate anion states that
the absorption maximum of the p–p* transition is red-shifted by
the formation of the intramolecular NH ◊ ◊ ◊ OC hydrogen bond.
The absorption maximum of the thioester model compound
(E)-S1 is red-shifted by +6 nm in the phenol state and +12 nm in the
phenolate anion state compared with those of (E)-S2. These results
indicate that the formation of the intramolecular NH ◊ ◊ ◊ OC
hydrogen bond contributes significantly to the extension of
p-electron delocalization along the skeleton of the chromophore,
especially in the phenolate anion state. On the other hand, the
absorption maximum of the ester model compound (E)-O1 is red-
shifted by +2 nm in the phenol state and +4 nm in the phenolate
anion state compared with those of (E)-O2, indicating that the
contributions of the intramolecular NH ◊ ◊ ◊ OC hydrogen bond are
smaller in the ester model compounds than in the thioester model
compounds. These results reflect the differences in the formation
of the intramolecular hydrogen bond between thioester and ester
model compounds, as shown in Fig. 2.
Scheme 2 The numbering of the 4-hydroxycinnamic acid moiety.
Scheme 3 Resonance structure in the phenolate anion state.
The absorption maxima of the thioester model compounds
are red-shifted by about +20 nm in the phenol state and about
+30~40 nm in the phenolate anion state compared with those of
˚
Table 3 Bond length (A) of the 4-hydroxycinnamic acid moiety of the optimized structure
(E)-X1-OH
X = S
(E)-X2-OH
X = S
(E)-X1-O-
X = S
(E)-X2-O-
X = S
X = O
X = O
X = O
X = O
O1-C1
C1-C2a
C2-C3a
C3-C4a
C4-C5
C5-C6
C6-C7
C7-O2
1.364
1.402
1.389
1.411
1.456
1.354
1.468
1.227
1.364
1.402
1.389
1.410
1.457
1.351
1.466
1.223
1.365
1.401
1.390
1.410
1.459
1.352
1.474
1.215
1.366
1.401
1.390
1.410
1.459
1.350
1.471
1.214
1.255
1.460
1.369
1.435
1.410
1.392
1.419
1.243
1.257
1.459
1.371
1.433
1.414
1.387
1.422
1.239
1.258
1.458
1.372
1.432
1.417
1.385
1.430
1.222
1.259
1.458
1.373
1.431
1.419
1.382
1.431
1.225
a The average C-C bond lengths in the phenyl ring.
Table 4 Mulliken oxygen atomic charge value of the optimized structure
(E)-X1-OH
X = S
(E)-X2-OH
X = S
(E)-X1-O-
X = S
(E)-X2-O-
X = S
X = O
X = O
X = O
X = O
O1(O-R)
O2(C O)
-0.4835
-0.3762
-0.4835
-0.4751
-0.4894
-0.3112
-0.4888
-0.4443
-0.5872
-0.4396
-0.5962
-0.5495
-0.6030
-0.3742
-0.6081
-0.5152
=
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Table 5 13C chemical shift (ppm) of C1 in the model compound (5 mM,
change from oxygen to sulfur in the bridging atom (-0.4 units),
suggesting that the phenolate anion of the chromophore is
stabilized by their contributions.
10% C12E9 micellar solution (10% D2O, 80% H2O), 303K
-OH
-O-
Dd(O--OH)
In order to investigate in detail the chemical mechanism of
the deprotonation, we evaluated the relative difference in acidity
(i.e. stability of anion state) between the model compounds and
carboxylic acid under the influence of a hydrophobic environment.
The previous study of the quantum chemical calculation of the
chromophore model has reported that the chromophore exhibits
the solvatochromism,21 suggesting that the hydrophobic core of
the PYP active center plays an important role in the regulation of
the electronic state of the chromophore.
The tetra-n-butylammonium acetate (1.0 eq) was added to a
THF solution of the phenol model compound (1.0 eq), and the
color of the reaction solution changed into yellow. The absorption
spectra of the reaction solutions are shown in Fig. 5. These results
show a decrease of the absorption band around 310 nm, which is
derived from the p–p* transition of the phenol compound, and an
increase of a new band around 400 nm. As seen in the results of
the absorption spectra in the micellar solution (Fig. 4), this new
band is derived from the p–p* transition of the phenolate anion,
indicating that the equilibria between phenol and phenolate anion
of the model compound are observed in the reaction solution. As
mentioned above, the model compounds possessing the thioester
or ester group are decomposed in organic solvents by the
nucleophilic attack of the base. However, the absorption spectra
shown in Fig. 5 had no change for several hours, suggesting that
the phenolate anion is stable in the acid-dissociation equilibrium
between the model compound and the acetate.
Considering their pKa values of model compounds, it is sug-
gested that this equilibrium observed in THF solution is different
from those in aqueous solution, and it is a specific reaction in
the low-polar solvent. It is proposed that the relation of their pKa
values, which represents the stabilities of the anion state,22–24 be-
tween the model compounds and the acetate are largely altered in
THF solution compared with when they are in aqueous solution.
The chromophore has a tendency to delocalize the negative charge
overall along the p-electron conjugation system and stabilize its
electronic state. We propose that the delocalization of the negative
charge along the extended conjugation system of the chromophore
is more stable in low-polar solvents such as THF than the
localization at the short conjugation system of the acetate anion.
Populations of the residual phenol, which are calculated by
spectral changes between before and after the reaction, are shown
in Fig. 5. The obtained values show that this exchange reaction
between the chromophore phenol and acetate anion is promoted
and this equilibrium is controlled by the contributions of the
intramolecular NH ◊ ◊ ◊ OC hydrogen bond and the bridging sulfur
atom (Scheme 4). From the results of pH titrations and exchange
reactions with the acetate anion of the model compounds, it is
concluded that the Cys69 residue, which affects both the hydrogen
bond to the carbonyl group and the bridging sulfur atom, is
important in the deprotonation of the chromophore in the native
PYP active center.
(E)-S1
(E)-S2
(E)-O1
(E)-O2
163.5
162.7
163.1
162.9
174.0
171.5
171.6
171.3
+10.5
+8.8
+8.5
+8.4
intramolecular NH ◊ ◊ ◊ OC hydrogen bond and the introduction
of a sulfur atom into the bridging atom. It is also considered
that the intramolecular hydrogen bond increases the negative
charge on the conjugated carbonyl oxygen, and influences the
p-electron conjugation system of the chromophore through the
change in the characteristics of the carbonyl group. For example,
the intramolecular hydrogen bond stabilizes the negative charge
of the quinoid-like canonical form, as depicted in Scheme 3,
and consequently increases the contribution of the quinoid-like
structure. The changes in Mulliken atomic charge (shown in
Table 4) support this mechanism. The Mulliken oxygen atomic
charge values of the carbonyl group are increased by the formation
of the intramolecular NH ◊ ◊ ◊ OC hydrogen bond by about 0.06 and
0.03 in the thioester and ester model compounds, respectively.
This change in the electronic state of the phenolate anion was
also observed in 13C NMR spectroscopy. The chemical shift of C1,
which is directly bound to the phenolic hydroxy oxygen, should
undergo a downfield shift by the contribution of the quinoid-like
resonance structure since the hybrid orbital between this carbon
and the phenolic oxygen changes from sp3 to sp2. The 13C chemical
shift of the C1 observed in a 10% micellar solution (10% D2O,
80% H2O) at 303 K is shown in Table 5. The detection and
the assignment of 13C signal were performed by heteronuclear
correlation spectroscopy (HMBC). The obtained 13C chemical
shifts are shifted downfield by the formation of the intramolecular
NH ◊ ◊ ◊ OC hydrogen bond and the introduction of a sulfur atom
into the bridging atom, supporting the notion that their effects
increase the contribution of the quinoid-like resonance structure
shown in Scheme 3.
From these results, the hydrogen bond network from Cys69
NH to the conjugated carbonyl group is considered to have a
significant effect on the electronic state of the chromophore. In
the native PYP active center, the Cys69 residue plays a crucial role
in the color regulation for PYP to function as a blue-light sensor.2
Stabilization of phenolate anion state by NH ◊ ◊ ◊ OC hydrogen
bond and sulfur atom
In order to investigate the effects of the NH ◊ ◊ ◊ OC hydrogen
bond to the conjugated carbonyl oxygen and the sulfur atom
of the thioester group on the acidity of the chromophore in the
native PYP active center, we performed pH titrations of the model
compounds in a micellar solution and determined their pKa values.
The pKa values of (E)-S1-OH, (E)-S2-OH, (E)-O1-OH, and
(E)-O2-OH at 303 K are 8.2, 8.4, 8.6, and 8.8, respectively. These
pKa values are comparatively small in the phenolic compounds.
This is considered to be due to the contribution of the quinoid-like
The point mutant PYPs Y42F and Y42A were previously
reported by other groups.11,12 In these studies, the effect of
OH ◊ ◊ ◊ O hydrogen bond from Tyr42 to the hydroxy group of the
chromophore was removed. The absorption spectra of both Y42F
and Y42A are different from the native PYP and have two bands
resonance structure proposed from the results of UV-vis and 13
NMR spectroscopy. The pKa values decreased by the formation of
the intramolecular NH ◊ ◊ ◊ OC hydrogen bond (-0.2 units) and the
C
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Fig. 5 UV-vis spectra of (a) (E)-S1-OH, (b) (E)-S1-O-, (c) (E)-O1-OH, and (d) (E)-O1-O- in both of the phenol of the model compound (dotted line)
and the reaction solution with tetra-n-butylammonium acetate (solid line) in THF solution at 303 K.
Scheme 4 Proposed mechanism of deprotonation of the chromophore.
at around 450 and 380 nm, which are derived from the phenol and
phenolate anion state of the chromophore, respectively. Studies of
the model compounds suggest that the chromophore is in equilib-
rium with the phenol and the phenolate anion in the active center
of Y42F and Y42A. In our previous studies, we found that the
hydrogen bond to the phenolic hydroxy group lowers the pKa value
of phenol and stabilizes the phenolate anion state.24,25 Thus, it is
consideredthat thehydrogen bond from Tyr42 to the chromophore
is also important in the deprotonation of the chromophore.
The formation of the intramolecular NH ◊ ◊ ◊ OC hydrogen bond
to the conjugated carbonyl oxygen has significant effects on the
p-electron conjugation system of the chromophore. In the phe-
nolate anion state, the formation of this hydrogen bond increases
the contribution of the quinoid-like resonance structure, which
changes the chemical properties of the chromophore (e.g. the
red-shift of color and anion stabilization). Moreover, it was
observed that the relation of the acidity, representing the stabilities
of phenolate anion state, between the chromophore model and
the acetate anion were largely altered under a hydrophobic
environment as compared with in water or in a micellar solution.
This result suggests that the chromophore exists stably as a
phenolate anion under the influence of incorporation into the
hydrophobic active center of PYP and the stabilization of the
phenolate anion state by the Cys69 residue (i.e. the NH ◊ ◊ ◊ OC
hydrogen bond and sulfur atom).
Conclusion
We synthesized thioester and ester model compounds containing
an intramolecular NH ◊ ◊ ◊ OC hydrogen bond to the conjugated
carbonyl oxygen and investigated the role of Cys69 residue in the
native PYP active center. The difference in the bridging atom has
an effect on the seven-membered skeleton of the intramolecular
hydrogen bond. In the case of the chromophore with the thioester
group, a more stable NH ◊ ◊ ◊ OC hydrogen bond is formed.
In conclusion, the Cys69 residue in the active center of PYP is
considered to be an essential factor for 4-hydroxycinnamic acid,
which is incorporated as its prosthetic group, to function as a
chromophore.
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Preparation of (E)-S-2-(pivalamido)phenyl
3-(4-tert-butyldimethylsiloxy-phenyl)prop-2-enethioate
Experimental
Materials
4-tert-Butyldimethylsiloxycinnamic acid (792.8 mg, 2.848 mmol)
was refluxed with thionyl chloride (2.50 mL, 34.3 mmol) in
10.0 mL of CH2Cl2 for 5 hours. The reaction solution was
cooled to room temperature, and concentrated to give a yellow
oily solution. CH2Cl2 was added to it and the solution was
concentrated again to remove trace of thionyl chloride. This
oil was dissolved in 10.0 mL of CH2Cl2 and added to N-(2-
mercaptophenyl)pivalamide (594.2 mg, 2.839 mmol) in 10.0 mL
of CH2Cl2. The resulting solution was stirred at room temperature
and then Et3N (3.0 mL, 21.5 mmol) was added to it. The
mixed solution was stirred overnight. The resulting solution was
diluted with diethyl ether and successively washed with 10% HCl
aqueous solution, H2O, and saturated NaCl aqueous solution,
and this organic layer was dried with Na2SO4. The solution was
concentrated under reduced pressure to obtain a white oil. This oil
was dissolved in hexane and cooled in a refrigerator to give a white
powder. This powder was washed with hexane and dried under
All operations were performed under an argon atmosphere.
All solvents were dried and distilled under an argon at-
mosphere before use. 4-Hydroxycinnamic acid was purchased
from Tokyo-Kasei Co. tert-Butyldimethylsilyl trifluoromethane-
sulfonate and hydrogen fluoride pyridine were purchased
from Aldrich Chemical Company, Inc. Phenol and thiophenol
were purchased from Nacalai Tesque, Inc. The syntheses of
N-(2-hydroxyphenyl)pivalamide and 2,2¢-dithiobis(N-phenyl-2,2-
dimethylpropanamide) were carried out using the same previously
reported procedure.25,26
Preparation of 4-tert-butyldimethylsiloxycinnamic acid
tert-Butyldimethylsilyl trifluoromethanesulfonate (8.60 mL,
37.4 mmol) was added to a suspension of 4-hydroxycinnamic acid
(2.03 g, 12.4 mmol) in 40.0 mL of CH2Cl2 on an ice bath and
then Et3N (8.00 mL, 57.0 mmol) was added to it. The mixed
solution became a homogeneous and the color was changed into
light yellow. The solution was stirred for 21 hours. The reaction
solution was washed successively with 10% HCl aqueous solution,
H2O, and NaCl aqueous solution, and organic layer was dried with
MgSO4. The solution was concentrated under reduced pressure
to give bright yellow oil. This oil was dissolved in 1:2 MeOH-
THF (12.0 mL) in a round-bottom flask and stirred at room
temperature while K2CO3 (5.20 g, 37.6 mmol) in 3.0 mL of
H2O was added slowly. The mixture was stirred for 3 hours. The
resulting solution was diluted with diethyl ether and successively
washed with 10% HCl aqueous solution, H2O, and saturated NaCl
aqueous solution, and this organic layer was dried with Na2SO4.
The solution was concentrated under reduced pressure to a light
yellow powder. The powder was washed with hexane and dried
over P2O5 under reduced pressure. (Yield: 2.42 g, 68%). Mp 127–
129 ◦C. Anal. Calcd for C15H22O3Si: C, 64.71; H, 7.96. Found:
C, 64.82; H, 7.87. 1H NMR (500 MHz, 30 ◦C, in chloroform-d1,
TMS): d 7.66 (1H, d), 7.38 (2H, d), 6.78 (2H, d), 6.24 (1H, d), 0.92
(9H, s), 0.16 (6H, s). 13C-NMR (125 MHz, 30 ◦C, in chloroform-
d1, TMS): d 172.31, 158.20, 146.68, 129.97, 127.34, 120.53, 114.82,
25.69, 18.33, -4.27. ESI-MS: m/z-; 277.2 (M-H, calcd; 277.12).
◦
reduced pressure. (Yield: 760.0 mg, 57%). Mp 98–100 C. Anal.
Calcd for C26H35NO3SSi: C, 66.48; H, 7.51; N, 2.98. Found: C,
66.70; H, 7.59; N, 2.97. 1H-NMR (500 MHz, 30 ◦C, in chloroform-
d1, TMS): d 8.27 (dd, 1H), 8.10 (br, 1H), 7.61 (d, 1H, 15.57 Hz),
7.41 (d, 2H), 7.39 (td, 1H), 7.37 (dd, 1H), 7.07 (td, 1H), 6.80
(d, 2H), 6.63 (d, 1H, 15.57 Hz), 1.20 (s, 9H), 0.92 (s, 9H), 0.17
(s, 6H). 13C-NMR (125 MHz, 30 ◦C, in chloroform-d1, TMS):
d 186.02, 175.56, 157.88, 141.89, 139.27, 134.87, 130.45, 129.50,
125.89, 123.42, 121.44, 119.92, 119.92, 116.50, 38.96, 26.52, 26.50,
24.59, -5.37. ESI-MS: m/z; 492.1 (M + Na, calcd; 492.20).
Preparation of (E)-S-2-(pivalamido)phenyl 3-(4-hydroxyphenyl)-
prop-2-enethioate ((E)-S1-OH)
To a solution of (E)-S-(2-pivalamidophenyl)-3-(4-tert-butyldi-
methylsiloxy-phenyl)prop-2-enethioate (758.0 mg, 1.61 mmol) in
5.0 mL of THF was added 5.0 mL of 2 M HF solution, stirred at
room temperature for 5 hours. The resulting solution was diluted
with CH2Cl2 and successively washed with saturated NaHCO3
aqueous solution, 2% HCl aqueous solution, H2O, and saturated
NaCl aqueous solution, and this organic layer was dried with
Na2SO4. The solution was concentrated under reduced pressure
to give a yellow oil. This oil was dissolved in AcOEt and added
hexane to give a white powder, and this powder was dried over
P2O5 under reduced pressure. (Yield: 359 mg, 63%). Mp. 162–
168 ◦C. Anal. Calcd for C20H21NO3S: C, 67.58; H, 5.95; N, 3.94.
Preparation of N-(2-mercaptophenyl)pivalamide
2,2¢-Dithiobis(N-phenyl-2,2-dimethylpropanamide) (1.06 g,
2.544 mmol) was dissolved in 10.0 mL of MeOH and then a
large excess of NaBH4 was slowly added until the yellow color
changed to pale yellow. Then acetic acid and water were added
to pH 4~5. After removal of MeOH under reduced pressure, the
desired product was extracted with hexane and diethyl ether. The
organic layer was washed with saturated NaCl aqueous solution,
and this organic layer was dried with Na2SO4. The solution was
concentrated under reduced pressure to obtain a white powder.
This powder was washed with hexane and dried under reduced
pressure. (Yield: 820.2 mg, 77%). Anal. Calcd for C11H15NOS:
C, 63.12; H, 7.22; N, 6.69. Found: C, 63.43; H, 6.77; N, 6.72.
Found: C, 67.32; H, 6.00; N, 3.86. H-NMR (500 MHz, 30 ◦C,
1
in chloroform-d1, TMS): d 8.33 (dd, 1H), 8.17 (br, 1H), 7.67 (d,
1H, 15.57 Hz), 7.50 (d, 2H), 7.49 (td, 1H), 7.46 (dd, 1H), 7.15
(td, 1H), 6.87 (d, 2H), 6.70 (d, 1H, 15.57 Hz), 5.26 (br, 1H), 1.27
(s, 9H). 13C-NMR (125 MHz, 30 ◦C, in chloroform-d1, TMS):
d 187.08, 176.70, 158.48, 142.75, 140.23, 135.89, 131.49, 130.81,
126.60, 124.54, 122.57, 120.84, 117.61, 116.19, 39.99, 27.54. ESI-
MS: m/z-; 354.1 (M-H, calcd; 354.12).
Preparation of (E)-S-phenyl 3-(4-tert-butyldimethylsiloxy-
phenyl)prop-2-enethioate
◦
1H-NMR (500 MHz, 30 C, in chloroform-d1, TMS): d 8.34 (br,
1H), 8.23 (d, 1H), 7.43 (d, 1H), 7.23 (t, 1H), 6.93 (t, 1H), 3.00 (s,
4-tert-Butyldimethylsiloxycinnamic acid (524.5 mg, 1.884 mmol)
1H), 1.29 (s, 9H).
was refluxed with thionyl chloride (2.00 mL, 27.4 mmol) in 10.0 mL
3788 | Org. Biomol. Chem., 2009, 7, 3782–3791
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of CH2Cl2 for 5 hours. The reaction solution was cooled to room
temperature, and concentrated to give a yellow oily solution.
CH2Cl2 was added to it and the solution was concentrated again to
remove trace of thionyl chloride. This oil was dissolved in 5.0 mL of
CH2Cl2 and added to thiophenol (0.185 mL, 1.81 mmol) in 5.0 mL
of CH2Cl2. The resulting solution was stirred at room temperature
and then Et3N (2.80 mL, 20.0 mmol) was added to it. The
mixed solution was stirred overnight. The resulting solution was
diluted with diethyl ether and successively washed with 2% HCl
aqueous solution, H2O, and saturated NaCl aqueous solution,
and this organic layer was dried with Na2SO4. The solution was
concentrated under reduced pressure to obtain a yellow oil. This
oil was dissolved in methanol and cooled in a refrigerator to give
a crude yellow powder. This crude product was used for the next
reaction without further purification. 1H-NMR (500 MHz, 30 ◦C,
in chloroform-d1, TMS): d 7.56 (d, 1H, 15.75 Hz), 7.42 (m, 1H),
7.39 (d, 2H), 7.36 (m, 2H), 7.35 (m, 1H), 6.78 (d, 2H), 6.63 (d,
1H, 15.75 Hz), 0.92 (s, 9H), 0.16 (s, 6H).13C-NMR (125 MHz,
30 ◦C, in chloroform-d1, TMS): d 186.69, 157.19, 140.24,
133.48, 129.05, 128.15, 127.97, 126.75, 126.11, 119.49, 114.88,
24.58, 17.22, -5.37. ESI-MS: m/z; 763.1 (M ¥ 2 + Na, calcd;
763.27).
resulting solution was diluted with diethyl ether and successively
washed with 10% HCl aqueous solution, H2O, and saturated
NaCl aqueous solution, and this organic layer was dried with
Na2SO4. The solution was concentrated under reduced pressure
to obtain a yellow oil. This oil was chromatographed on silica gel
(3:1:2/CH2Cl2:THF:hexane) to obtain a white oil. (Yield: 1.21 g,
73%). H-NMR (500 MHz, 30 ◦C, in chloroform-d1, TMS): d
1
8.13 (dd, 1H), 7.82 (d, 1H, 15.87 Hz), 7.52 (br, 1H), 7.43 (d,
2H), 7.18 (td, 1H), 7.12 (dd, 1H), 7.07 (td, 1H), 6.82 (d, 2H),
6.44 (d, 1H, 15.87 Hz), 1◦.20 (s, 9H), 0.93 (s, 9H), 0.17 (s, 6H).
13C-NMR (125 MHz, 30 C, in chloroform-d1, TMS): d 175.44,
163.91, 157.75, 146.64, 140.07, 129.28, 129.20, 126.10, 125.43,
123.56, 121.95, 120.94, 119.72, 119.68, 38.81, 26.57, 24.60, 17.26,
-5.37. ESI-MS: m/z; 929.1 (M ¥ 2 + Na, calcd; 929.5).
Preparation of (E)-2-(pivalamido)phenyl 3-(4-hydroxyphenyl)-
acrylate ((E)-O1-OH)
To
a solution of (E)-(2-pivalamidophenyl)-3-(4-tert-butyldi-
methylsiloxy-phenyl)acrylate (1.21 g, 2.67 mmol) in 10 mL of
THF was added 10 mL of 2 M HF solution, stirred at room
temperature for 5 h. The resulting solution was diluted with
CH2Cl2 and successively washed with saturated NaHCO3 aqueous
solution, 2% HCl aqueous solution, H2O, and saturated NaCl
aqueous solution, and this organic layer was dried with Na2SO4.
The solution was concentrated under reduced pressure and added
hexane to give a white powder, and this powder was dried over
P2O5 under reduced pressure. (Yield: 734 mg, 81%). Mp. 214–
218 ◦C. Anal. Calcd for C20H21NO4: C, 70.78; H, 6.24; N, 4.13.
Preparation of (E)-S-phenyl 3-(4-hydroxyphenyl)prop-2-enethioate
((E)-S2-OH)
To
a solution of (E)-S-phenyl-3-(4-tert-butyldimethylsiloxy-
phenyl)prop-2-enethioate (490.1 mg, 1.323 mmol) in 10.0 mL of
THF was added 7.0 mL of 2 M HF solution, stirred at room
temperature for 5 hours. The resulting solution was diluted with
diethyl ether and successively washed with saturated NaHCO3
aqueous solution, 2% HCl aqueous solution, H2O, and saturated
NaCl aqueous solution, and this organic layer was dried with
Na2SO4. The solution was concentrated under reduced pressure
to give a yellow oil. This oil was dissolved in diethyl ether and
added hexane to give a light yellow powder, and this powder
was dried over P2O◦5 under reduced pressure. (Yield: 98.7 mg,
29%). Mp. 117–122 C. Anal. Calcd for C15H12O2S: C, 70.29; H,
Found: C, 70.24; H, 6.24; N, 4.13. H-NMR (500 MHz, 30 ◦C,
1
in chloroform-d1, TMS): d 8.18 (dd, 1H), 7.88 (d, 1H, 15.57 Hz),
7.59 (br, 1H), 7.52 (d, 2H), 7.26 (td, 1H), 7.19 (dd, 1H), 7.15
(td, 1H), 6.89 (d, 2H), 6.51 (d, 1H, 15.57 Hz), 5.19 (br, 1H), 1.27
(s, 9H). 13C-NMR (125 MHz, 30 ◦C, in chloroform-d1, TMS):
d 176.57, 164.93, 158.34, 147.49, 141.20, 130.51, 130.25, 126.85,
126.49, 124.72, 123.13, 121.99, 116.15, 113.43, 39.83, 27.59. ESI-
MS: m/z-; 338.0 (M–H, calcd; 338.14).
4.72. Found: C, 69.70; H, 4.72. H-NMR (500 MHz, 30 ◦C, in
1
Preparation of (E)-phenyl 3-(4-tert-butyldimethylsiloxy-
phenyl)acrylate
chloroform-d1, TMS): d 7.62 (d, 1H, 15.75 Hz), 7.49 (m, 2H), 7.47
(d, 2H), 7.42 (m, 2H), 7.42 (m, 1H), 6.85 (d, 2◦H), 6.60 (d, 1H, 15.75
Hz), 4.99 (br, 1H). 13C-NMR (125 MHz, 30 C, in chloroform-d1,
TMS): d 187.91, 157.91, 141.18, 134.67, 130.51, 129.36, 129.17,
127.91, 127.07, 122.01, 116.14. ESI-MS: m/z-; 255.0 (M-H, calcd;
255.05).
4-tert-Butyldimethylsiloxycinnamic acid (524.5 mg, 1.884 mmol)
was refluxed with thionyl chloride (2.00 mL, 27.4 mmol) in 10.0 mL
of CH2Cl2 for 5h. The reaction solution was cooled to room
temperature, and concentrated to give a yellow oily solution.
CH2Cl2 was added to it and the solution was concentrated again
to remove trace of thionyl chloride. This oil was dissolved in
5.0 mL of CH2Cl2 and added to phenol (170.0 mg, 1.81 mmol)
in 5.0 mL of CH2Cl2. The resulting solution was stirred at room
temperature and then Et3N (2.80 mL, 20.0 mmol) was added to it.
The mixed solution was stirred overnight. The resulting solution
was diluted with CH2Cl2 and successively washed with 2% HCl
aqueous solution, H2O, and saturated NaCl aqueous solution,
and this organic layer was dried with Na2SO4. The solution was
concentrated under reduced pressure to obtain a yellow oil. This oil
was dissolved in hexane and cooled in a refrigerator to give a white
powder. This powder was washed with hexane and dried under
reduced pressure. (Yield: 397.0 mg, 59%). Mp. 67–70 ◦C. Anal.
Calcd for C21H26O2Si·H2O: C, 67.71; H, 7.58. Found: C, 67.33;
Preparation of (E)-2-(pivalamido)phenyl
3-(4-tert-butyldimethylsiloxy-phenyl)acrylate
4-tert-Butyldimethylsiloxycinnamic acid (1.013 g, 3.638 mmol)
was refluxed with thionyl chloride (2.50 mL, 34.3 mmol) in 15.0 mL
of CH2Cl2 for 4 hours. The reaction solution was cooled to room
temperature, and concentrated to give a yellow oil. CH2Cl2 was
added to it and the solution was concentrated again to remove
trace of thionyl chloride. This oil was dissolved in 15.0 mL of
CH2Cl2 and added to N-(2-hydroxyphenyl)pivalamide (731.6 mg,
3.786 mmol) in 15.0 mL of CH2Cl2. The resulting solution was
stirred at room temperature and then Et3N (6.0 mL, 43.0 mmol)
was added to it. The mixed solution was stirred overnight. The
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◦
1
H, 7.43. H-NMR (500 MHz, 30 C, in chloroform-d1, TMS): d
7.75 (d, 1H, 15.75 Hz), 7.41 (d, 2H), 7.33 (td, 2H), 7.17 (tt, 1H),
7.10 (d, 2H), 6.80 (d, 2H), 6.42 (d, 1H, 15.75 Hz), 0.93 (s, 9H),
0.17 (s, 6H). 13C-NMR (125 MHz, 30 ◦C, in chloroform-d1, TMS):
d 164.45, 157.05, 149.74, 145.08, 128.77, 128.20, 126.35, 124.47,
120.51, 119.44, 113.76, 24.58, 17.22, -5.37. ESI-MS: m/z; 731.0
(M ¥ 2 + Na, calcd; 731.32).
pH Titration
The pH of 1 mM solution of each phenol model compound
was determined using a Metrohm 716 DMS titrino, which is
combined with Metrohm 728 stirrer and a saturated calomel
LL micro pH glass electrode. The saturated calomel micro glass
electrode was calibrated with the 0.05 M KHC6H4(COO)2 buffer
(pH = 4.01) and the 0.025 M KH2PO4-NaHPO4 buffer (pH = 6.86)
at 303 K. The micellar solution was titrated with 0.01 M NaOH
aqueous solution at 303 K. The pKa values was estimated by
the following equation: pKa = pH - log[Na+] + log{[phenol]0 -
[Na+]}.
Preparation of (E)-phenyl 3-(4-hydroxyphenyl)acrylate
((E)-O2-OH)
To
a
solution of (E)-phenyl-3-(4-tert-butyldimethylsiloxy-
phenyl)acrylate (389.1 mg, 1.098 mmol) in 5.0 mL of THF was
added 5.0 mL of 2 M HF solution, stirred at room temperature for
5 hours. The resulting solution was diluted with diethyl ether and
successively washed with saturated NaHCO3 aqueous solution,
2% HCl aqueous solution, H2O, and saturated NaCl aqueous
solution, and this organic layer was dried with Na2SO4. The
solution was concentrated under reduced pressure to give a white
powder. This powder was washed with hexane and dried over
P2O5 under reduced pressure. (Yield: 222 mg, 84%). Mp. 140–
Computational details
The Gaussian03 program27 was used for geometry optimization
and to obtain relative energies of the optimized structures. Final
geometry optimization was done at the B3LYP/6–31++G(d,p)
level.16–19 The MOPAC program (WinMOPAC3.9, Fujitsu.)20
was used for UV-vis absorption spectral analysis (INDO/S) of
geometrical optimized model compound.
◦
144 C. Anal. Calcd for C15H12O3: C, 74.99; H, 5.03. Found: C,
74.53; H, 4.96. H-NMR (500 MHz, 30 ◦C, in chloroform-d1,
1
Acknowledgements
TMS): d 7.81 (d, 1H, 15.75 Hz), 7.50 (d, 2H), 7.40 (tt, 2H), 7.24
(tt, 1H), 7.17 (dd, 2H), 6.87 (d, 2H), 6.49 (d, 1H, 15.94 Hz),
4.96 (br, 1H). 13C-NMR (125 MHz, 30 ◦C, in chloroform-d1,
TMS): d 165.69, 157.80, 150.95, 146.09, 130.26, 129.42, 127.32,
125.70, 121.69, 115.98, 114.96. ESI-MS: m/z-; 239.0 (M–H, calcd;
239.1).
This research was supported by “Supramolecules in Stress and
Cooperation” from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
References
1 T. E. Meyer, Biochim Biophys Acta., 1985, 806, 175–183.
2 W. W. Sprenger, W. D. Hoff, J. P. Armitage and K. J. Hellingwerf, J
Bacteriol., 1993, 175, 3096–3104.
3 T. E. Meyer, E. Yakali, M. A. Cusanovich and G. Tollin, Biochemistry.,
1987, 26, 418–423.
4 J. J. Van Beeumen, B. V. Devreese, S. M. Van Bun, W. D. Hoff, K. J.
Hellingwerf, T. E. Meyer, D. E. McRee and M. A. Cusanovich, Protein
Sci., 1993, 2, 1114–1125.
5 M. Baca, G. E. Borgstahl, M. Boissinot, P. M. Burke, D. R. Williams,
K. A. Slater and E. D. Getzoff, Biochemistry., 1994, 33, 14369–14377.
6 W. D. Hoff, P. Dux, K. Hard, B. Devreese, I. M. Nugteren-Roodzant,
W. Crielaard, R. Boelens, R. Kaptein, J. van Beeumen and K. J.
Hellingwerf, Biochemistry., 1994, 33, 13959–13962.
7 G. E. Borgstahl, D. R. Williams and E. D. Getzoff, Biochemistry., 1995,
34, 6278–6287.
Deprotonation of phenol model compound in 10% GL micellar
solution
The phenol model compound was dissolved in a small amount of
THF and to the solution was added poly(ethylene glycol) lauryl
ether (C12E9). After removal of THF under reduced pressure, the
obtained residue was diluted by degassed water to give a micellar
solution. Final concentration is 10% C12E9 aqueous solution. To
a micellar solution was added NaOH aqueous solution to give a
phenolate anion model compound.
Physical measurements
8 M. Kim, R. A. Mathies, W. D. Hoff and K. J. Hellingwerf, Biochemistry.,
1995, 34, 12669–12672.
500 MHz 1H-NMR and 125 MHz 13C-NMR spectra were recorded
on a JEOL Lambda spectrometer in 5 mM chloroform-d1,
acetonitrile-d3, and D2O (10%) micellar solution. Variable-
temperature NMR measurements were made in the range 228
K-303 K, and then the temperature was decreased in each 15 K
intervals. HMQC and HMBC spectra were recorded on a Varian
UNITY plus 600 MHz spectrometer at 303 K. Tetramethylsi-
lane or sodium 2,2-dimethyl-2-silapentane-5-sulfonate was used
as a standard (0 ppm). ESI-mass spectrometric analyses were
performed on a Finniganmat LCQ-MS instrument in methanol
solution. IR spectra were recorded on a Jasco FT-IR 8300
spectrometer in 5 mM and 1 mM chloroform solution. UV-vis
spectra were obtained on a Shimazu UV-3100PC spectrometer
in 0.5 mM micellar solution at 303 K. Elemental analysis was
performed at the Elemental Analysis Center, Faculty of Scienece,
Osaka University. Melting point was measured on a micro melting
point apparatus of YANAGIMOTO Co.
9 E. D. Getzoff, K. N. Gutwin and U. K. Genick, Nat Struct Biol., 2003,
10, 663–668.
10 A. R. Kroon, W. D. Hoff, H. P. Fennema, J. Gijzen, G. J. Koomen, J. W.
Verhoeven, W. Crielaard and K. J. Hellingwerf, J Biol Chem., 1996,
271, 31949–31956.
11 R. Brudler, T. E. Meyer, U. K. Genick, S. Devanathan, T. T. Woo, D. P.
Millar, K. Gerwert, M. A. Cusanovich, G. Tollin and E. D. Getzoff,
Biochemistry., 2000, 39, 13478–13486.
12 Y. Imamoto, H. Koshimizu, K. Mihara, O. Hisatomi, T. Mizukami,
K. Tsujimoto, M. Kataoka and F. Tokunaga, Biochemistry., 2001, 40,
4679–4685.
13 M. A. van der Horst, J. C. Arents, R. Kort and K. J. Hellingwerf,
Photochem. & Photobiol. Sci., 2007, 6, 571–579.
14 K. Mihara, O. Hisatomi, Y. Imamoto, M. Kataoka and F. Tokunaga,
J Biochem., 1997, 121, 876–880.
15 K. Okamoto, N. Hamada, T. Sumi, T. Okamura, N. Ueyama and H.
Yamamoto, Chem. Lett., 2009, 38, 456–457.
16 C. Lee, W. Yang and R. G. Parr, Phys. Rev. B, 1988, 37, 785–789.
17 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.
18 T. Clark, J. Chandrasekhar, G. W. Spitznagel and P. v. R. Schleyer,
J. Comput. Chem., 1983, 4, 294–301.
3790 | Org. Biomol. Chem., 2009, 7, 3782–3791
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The Royal Society of Chemistry 2009
©

View Article Online
19 M. J. Frisch, J. A. Pople and J. S. Binkley, J. Chem. Phys., 1984, 80,
3265–3269.
27 Gaussian 03, Revision B.05, M. J. Frisch, G. W. Trucks, H. B. Schlegel,
G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery Jr,
T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega,
G. A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, M. Klene, X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross,
V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, P. Y.
Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J. Dannenberg, V. G.
Zakrzewski, S. Dapprich, A. D. Daniels, M. C. Strain, O. Farkas,
D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. V.
Ortiz, Q. Cui, A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov,
G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, Al-M. A. Laham, C. Y. Peng, A. Nanayakkara, M.
Challacombe, P. M. W. Gill, B. Johnson, W. Chen, M. W. Wong,
C. Gonzalez, and J. A. Pople, Gaussian, Inc., Wallingford CT,
2004.
20 WinMOPAC ver.3.9.0: A General Molecular Orbital Package developed
by J. J. P. Stewart, and Fujitsu limited, Tokyo, Japan. All Rights
ꢀ
C
Reserved, Copyright FUJITSU LIMITED.
21 M. Yoda, H. Houjou, Y. Inoue and M. Sakurai, J. Phys. Chem. B, 2001,
105, 9887–9895.
22 N. Ueyama, M. Inohara, A. Onoda, T. Ueno, T. Okamura and A.
Nakamura, Inorg. Chem., 1999, 38, 4028–4031.
23 A. Onoda, Y. Yamada, J. Takeda, Y. Nakayama, T. Okamura, M. Doi,
H. Yamamoto and N. Ueyama, Bull. Chem. Soc. Jpn., 2004, 77, 321–
329.
24 D. Kanamori, Y. Yamada, A. Onoda, T. Okamura, S. Adachi, H.
Yamamoto and N. Ueyama, Inorg. Chim. Acta, 2005, 358, 331–338.
25 D. Kanamori, A. Furukawa, T. Okamura, H. Yamamoto and N.
Ueyama, Org Biomol Chem., 2005, 3, 1453–1459.
26 N. Ueyama, T. Okamura and A. Nakamura, J. Am. Chem. Soc., 1992,
114, 8129–8137.
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