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Chemistry Letters Vol.38, No.5 (2009)
Investigation of the Effect of the NHÁÁÁOC Hydrogen Bond from Cys69
to PYP Chromophore Using Novel Active-center Model Compound
Kentaro Okamoto,1 Norio Hamada,1 Toshiaki Sumi,1 Taka-aki Okamura,1 Norikazu Ueyama,1 and Hitoshi Yamamotoꢀ1;2
1Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043
2Department for the Administration of Safety and Hygiene, Osaka University, Suita, Osaka 565-0871
(Received December 16, 2008; CL-081177; E-mail: jin@chem.sci.osaka-u.ac.jp)
A PYP active-center model compound containing a hydrogen
pound, which leads to the elucidation of the effect of the hydro-
gen bond from the amide NH in Cys69 to the conjugated carbon-
yl group on the chromophore.
The amide groups of (E)-N1–OH and (E)-N2–OH were syn-
thesized by using DCC. The deprotonation of the hydroxy
groups was performed by neutralization with sodium ethoxide,
and the counter cation was exchanged with tetraethylammonium
cation to increase the solubility in organic solvents.
bond to the conjugated carbonyl oxygen was synthesized and its
effect on the electronic properties of the chromophore were inves-
tigated. The intramolecular hydrogen bond induces significant
change in the absorption maximum of the ꢀ–ꢀꢀ transition in the
chromophore.
Photoactive yellow protein (PYP) isolated from the purple
sulfur bacterium Halorhodospira halophila1 is considered the
blue-light photosensor protein that is implicated in the negative
phototaxis of the bacterium.2,3 By other groups,4–6 it has been re-
ported that PYP is composed of 125 amino acids and one chro-
mophore (4-hydroxycinnamic acid), and the chromophore is
covalently bound to Cys69 of the ꢀ-loop (residues 63–78) via
a thiol ester linkage and exists as a phenolate anion in E config-
uration in the hydrophobic core of the protein. The chemical
structure and the hydrogen bond network between the chromo-
phore and the amino acid residues (Tyr42, Glu46, and Cys69)
are summarized in Figure 1a.7 Point mutation studies on Tyr42
and Glu46 have been revealed that hydrogen bonds to the hy-
droxy oxygen of the chromophore regulate the electronic proper-
ties of the chromophore, and have important roles in exerting the
biological function.8–10 However, the role of the hydrogen bond
from the amide NH in Cys69 to the carbonyl oxygen in the chro-
mophore is unknown.
To investigate the role of the hydrogen bond between Cys69
and the chromophore, we designed novel model compounds
(shown in Figure 1b) containing an intramolecular hydrogen
bond to the carbonyl oxygen in the form of a seven-membered
ring like native PYP. In order to form an intramolecular hydro-
gen bond stably, we chose a rigid 1,2-phenylenediamide skele-
ton as the linker between the hydrogen bond donor and acceptor
instead of the alkyl chain. Thus, we could investigate in detail
the effect of the hydrogen bond to the carbonyl oxygen on the
electronic state of the chromophore by using this model com-
In Figure 2, the crystal structure of (E)-N1–OH is shown.
The geometry of model compound (E)-N1–OH has twisted con-
formation. The 1,2-phenylenediamide skeleton was twisted be-
tween each amide plane and the phenylene ring, and the dihedral
angles (C9–N10–C11–C12 and C11–C12–N11–C17) were
54.9(4) and 133.2(4)ꢁ, respectively. In contrast, the dihedral an-
gle (C9–N10–C11–C12) of (E)-N2–OH without the intramolec-
ular hydrogen bond was ꢂ169:3ð1Þꢁ in the crystal structure de-
scribed in Figure S1.11 In the twisted conformation of (E)-N1–
OH, the amide group (–NHCOt-Bu), which is the hydrogen-
bond donor, is located in the vicinity of the carbonyl group
contained in the plane of the extended ꢀ-electron conjugation
system. Consequently, the distance between the amide nitrogen
˚
N11 and the carbonyl oxygen O2 is short (2.72(3) A). The orien-
tation between the amide and carbonyl groups and the short
N11–O2 distance strongly suggest the presence of an intramo-
lecular NHꢃꢃꢃOC hydrogen bond between amide proton H11
and carbonyl oxygen O2 in the crystal structure of model com-
pound (E)-N1–OH. Intermolecular hydrogen bonds were also
observed in the crystal structures of (E)-N1–OH and (E)-N2–
OH. In (E)-N1–OH, two types of the intermolecular hydrogen
bond are observed between the hydroxy group and the carbonyl
oxygen O11, the bridging amide NH and the cyclic ether oxygen
of tetrahydrofuran. In (E)-N2–OH, two intermolecular hydrogen
bonds are observed between the hydroxy group and the bridging
amide NH.11 Two types (intra- and inter-) of the hydrogen bond
observed in the crystal structure of (E)-N1–OH were also con-
firmed by IR spectroscopy. In the spectrum, two different bands
were observed in the region of the amide NH stretching vibration
(3231, 3260 cmꢂ1, Figure S211).
Figure 1. (a) Hydrogen-bond network of the active center in photoactive
yellow protein. (b) E formed model compounds.
Figure 2. Crystal structure of the model compound (E)-N1–OH.
Copyright Ó 2009 The Chemical Society of Japan