5758
J. Am. Chem. Soc. 1997, 119, 5758-5759
Scheme 1
Chemoenzymatic Synthesis of 11-cis-Retinal
Photoaffinity Analog by Use of Squid Retinochrome
Babak Borhan, Roxanne Kunz, Andrew Y. Wang, and
Koji Nakanishi*
Department of Chemistry, Columbia UniVersity
New York, New York 10027
Nina Bojkova and Kazuo Yoshihara
Suntory Institute for Bioorganic Research
Osaka, Japan
ReceiVed March 26, 1997
We expect the photoactive 11-cis-3-diazo-4-oxoretinal (1) to
be crucial in clarifying the visual transduction process, but its
synthesis has not been successful. However, as described below,
the preparation has been achieved by incubation of trans isomer
2 with squid retinochrome which smoothly performed the critical
trans f 11-cis isomerization to yield 1.
The visual transduction process is initiated by isomerization
of the 11-cis-retinal chromophore in rhodopsin to all-trans,
cleavage of the chromophore/opsin bond and terminates in all-
trans-retinal and opsin.1 The cis f trans isomerization triggers
a chain of conformational changes in the opsin which induces
an enzymatic cascade leading to vision.1a,2 Scheme 1 depicts
the intermediates which have been identified based on low-
temperature spectroscopy.3 However, the movement of the
chromophore relative to the receptor opsin along the isomer-
ization pathway remains unknown.
Our objective is to clarify this crucial aspect on a molecular
structural basis using photoaffinity labeling as the main tool.
Photolysis of a pigment incorporating the nonisomerizable 11-
cis-locked retinal analog 3 (Scheme 1) resulted in clear-cut
cross-linking to Leu-266, thus revealing that the ionone C-3 is
in close contact with helix F of rhodopsin in the dark.4 On the
other hand, studies using a photoreactive 11-cis-retinal analog
in which the 11-ene is not locked showed that the C-3 region
cross-linked to both helices C and F;5 recent studies with spin
labels6 also showed that movements of these two helices were
involved in the light activation process. It is thus possible that
the cis f trans isomerization results in a “flip-over motion of
the ring” from the proximity of helix F to helix C as well as
movements of helices F and C and that this induces the
conformational changes responsible for the enzymatic cascade.
This scheme has been further corroborated by Sakmar and co-
workers who have demonstrated that the relative movements
of helices C and F is required for activation of the G-protein-
coupled receptor rhodopsin.7
We deemed it necessary to follow the isomerization pathway
in a temperature-resolved manner by using, in contrast to locked
analog 3, the rhodopsin analog incorporating unlocked retinal
1. After irradiation at 500 nm (-140 °C, “batho”), the
chromophore is cross-linked (254 nm) and the amino acid(s)
are sequenced. Similiar experiments will be performed at -40
°C (“lumi”), -15 °C (“meta-I”), etc. Since the photoaffinity
label should traverse the same path of 11-cis-retinal isomeriza-
tion, such sequential cross-linking experiments should allow one
to trace the relative movements of the chromophore and the
receptor opsin.8
Synthesis of 11-cis-retinal analogs, however, face problems
due to the chromophore instability. The problem is magnified
for analogs with functional groups which have to be introduced
after formation of the 11-cis-ene. Most schemes geared toward
introducing the 11-cis geometry close to the end of the synthesis
have either failed or resulted in low yields and formation of
complex isomeric mixtures.9 In contrast to 11-cis analogs, it
is known that synthesis of the more stable all-trans analogs are
more straightforward. It would therefore be advantageous to
synthesize the corresponding all-trans isomers and then intro-
duce the 11-cis geometry at the end of the synthesis. Herein,
we report a method for enzymatically introducing the 11-cis
moiety at the last stage of the synthesis from an all-trans retinal,
a reaction that could be performed with the necessary tritiated
analog and could be quite general.
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The visual cells of cephalopods contain in addition to
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performs the all-trans-retinal f 11-cis-retinal regeneration.10
In view of the efficiency of retinochrome in isomerization of
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