Synthesis of 9-CD3-9-cis-retinal cofactor of isorhodopsin

Article abstract of DOI:10.1016/j.tetlet.2018.11.034

We report the synthesis of 9-CD₃-9-cis-retinal via a six-step procedure from β-ionone. The steps involve an initial deuteration of the methyl ketone of β-ionone followed by two consecutive Horner-Wadsworth-Emmons (HWE) coupling reactions and th

Full text of DOI:10.1016/j.tetlet.2018.11.034

Accepted Manuscript
Synthesis of 9-CD3-9-cis-Retinal Cofactor of Isorhodopsin
Mozhgan Navidi, Shreya Yadav, Andrey V. Struts, Michael F. Brown, Nasri
Nesnas
PII:
S0040-4039(18)31362-5
https://doi.org/10.1016/j.tetlet.2018.11.034
TETL 50419
DOI:
Reference:
Tetrahedron Letters
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5 September 2018
7 November 2018
9 November 2018
Please cite this article as: Navidi, M., Yadav, S., Struts, A.V., Brown, M.F., Nesnas, N., Synthesis of 9-CD3-9-cis-
Retinal Cofactor of Isorhodopsin, Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.11.034
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Synthesis of 9-CD3-9-cis-Retinal Cofactor of Isorhodopsin
Mozhgan Navidia, Shreya Yadava, Andrey V. Strutsb, Michael F. Brownb, Nasri Nesnasa,*
aDepartment of Biomedical & Chemical Engineering & Sciences, Florida Institute of Technology, Melbourne, Florida 32901, USA
bDepartment of Chemistry, University of Arizona, Tucson, Arizona 85721, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report the synthesis of 9-CD3-9-cis-retinal via a six-step procedure from -ionone.
The steps involve an initial deuteration of the methyl ketone of -ionone followed by
two consecutive Horner-Wadsworth-Emmons (HWE) coupling reactions and their
corresponding DIBAL reductions. A final oxidation of the allylic alcohol of the
retinol leads to the target compound. This deuterium labeled retinoid is an important
cofactor for studying protein-retinoid interactions in isorhodopsin.
Available online
Keywords:
Retinoids
9-CD3-9-cis-retinal
isorhodopsin
labeled retinal
rhodopsin
Introduction
While 9-cis-retinal is now commercially available, at relatively
high prices, it is useful to present an efficient pathway for its
Retinoids are a group of natural and synthetic analogues of
vitamin A with critical roles in various biological processes
including vision, cell differentiation, immune responses,
reproduction, and brain functions.1–5 The chromophore for the
visual pigment, rhodopsin, is 11-cis-retinal. Isomerization of 11-
cis-retinal to all-trans-retinal initiates the visual signal
transduction pathway.6 During the last decades, many researchers
have aimed to uncover the details of the intricate steps of the
visual cycle through preparation of 11-cis-retinal and a wide
variety of its analogues.7 These analogues have been used to
assess the critical components of the chromophore and their
effect on spectral sensitivities and function of rhodopsin.5
Examples of some of these analogues include: demethylated and
synthesis to enable isotopic labeling and for others to design
pertinent analogues. Due to the similarities between rhodopsin
and isorhodopsin, and to further study the importance of this
methyl group, we have synthesized 9-CD3-9-cis-retinal. One goal
is to use solid-state NMR spectroscopy to investigate the
structure and dynamics of 9-cis-retinal in the isorhodopsin
binding pocket.23,24 Isotopically labeled retinoids are also useful
in studies employing neutron diffraction25–27 and vibrational
spectroscopy.28,29 To our knowledge, this is the first report on
the preparation of this labeled molecule.
methyl
switched
retinoids,
haloretinoids,
arotinoids,
conformationally locked retinoids, and retinoid isotopomers.8–14
An especially interesting analogue of the native chromophore
11-cis-retinal (1) is 9-cis-retinal (2), which can also bind to opsin
via the Schiff base with Lys296 forming isorhodopsin (Figure 1).
Here we present the previously unreported organic synthesis of
the deuterated analogue at C9, which is 9-CD3-9-cis-retinal.
Rhodopsin and isorhodopsin go through a similar bleaching
sequence;15 however, isorhodopsin undergoes a slower and less
efficient bleaching, and this is significant, as it would facilitate its
photophysical studies. Furthermore, 9-cis-retinal shows distinct
spectral properties in comparison to 11-cis-retinal, which makes
it an interesting candidate for the study of vision. As such,
having a facile synthesis for 9-cis-retinal becomes of great value.
Earlier reports of organic synthesis of 9-cis-retinal are scarce, and
the compound was only reported in passing as a by-product from
other pathways, or in a mixture of photoisomerized products of
Figure 1. Structures of 11-cis-retinal and 9-cis-retinal.
all-trans-retinal.
For example, Duhamel and coworkers
published two papers in 1987 on retinal preparation.16,17 They
mentioned 9-cis-retinal as one of the retinoids formed through
their synthesis. In 2003, Sashima et al. reported that irradiation
of all-trans-retinal could lead to several of its isomers, including
9-cis-retinal which was separated through HPLC.18 More recent
reports are mostly focused on 9-cis-retinoic acid and 9-cis-
retinoic ester, and occasionally 9-cis-retinal derivatives,
examples of which are shown in Figure 2 (compounds 3-6).8,19–22
Figure 2. Examples of reported retinoids.
-1-

Results and discussion
dimethyltetrahydropyrimidin-2(1H)-one (DMPU) in THF
(Scheme 2).31 Ester 15 formed through this method (83%) mostly
contained the desired E product (E/Z ratio of 17:1 based on
NMR). Separation of the stereoisomers was not feasible at that
step; therefore, reduction to the alcohol was performed using the
isomeric mixture in hand. Because alcohol 16 is not very stable,
it was obtained in sufficient purity simply after extraction of the
crude, and was used in the last step without further purification.
Oxidation of 16 was achieved using commercially activated
MnO2. The 9-CD3-9-cis-retinal, product 17, was obtained as a
yellow solid after purification by column chromatography (49%).
The ratio of the formed diastereomers was in favor of the desired
E isomer (E/Z: 12:1 based on isolated yields). Replacing the last
step with an alternative published oxidation condition using
MnO2 at room temperature32 afforded similar yield of the target
aldehyde. The structure of the labeled retinal was fully
characterized using IR, UV, 1H NMR, 13C NMR, and DART-MS
(see Supplementary Data). The electronic UV spectrum of 9-
CD3-9-cis-retinal (this work) is compared to authentic 9-cis-
retinal and all-trans-retinal UV spectra in Figure 4. For these
retinoids n-hexanes solutions with a concentration of 1.7  10-3
M were prepared and studied at room temperature. The
synthesized labeled retinal 17 and the commercially available 9-
cis-retinal had identical max at 366 nm, as expected. All-trans-
retinal showed a slight red shift and gave an observed max at 370
nm. The similarity between the max of the synthesized molecule
and the authentic commercial 9-cis-retinal added confidence to
our confirmed stereochemistry of the reported 9-CD3-9-cis-
retinal.
Our synthesis began with the commercially available -ionone
7 which was almost quantitatively converted to its deuterated
form, labeled -ionone 8, using previously reported methods with
greener modifications that avoided the use of pyridine.30
Compound 8 was then treated with diethyl (cyanomethyl)
phosphonate 9 to afford a mixture of a 2:1 E/Z isomers, as
determined by NMR, of 10 in 89% yield (Scheme 1). The E/Z
mixture cannot be separated at that stage, and was therefore
subjected to reduction as a mixture. One of the challenges in
isotope labeling is designing the reaction conditions in a way that
minimizes the possibility of H/D exchange. Our earlier attempts
to synthesize 10, using sodium hydride as base instead of n-BuLi,
resulted in some loss of the D-label as observed by NMR and
DART-MS. This problem was solved by replacing NaH with n-
BuLi. It is likely that the intimate ion pair in carbanion 13
(Figure 3), when M = Li, possesses lower basicity than when M =
Na, and is therefore not sufficiently basic to exchange the -
deuterium of 8. Attempts to increase Z selectivity for this step
via other phosphonates or bases were not successful. These
attempts either led to higher Z selectivity, with much lower
yields, or resulted in undesirable H/D exchange. A summary of
the optimization for this HWE step can be found in Table S1 (see
Supporting Information).
Scheme 1. Synthesis of the Z aldehyde 11.
Scheme 2. Synthesis of 9-CD3-9Z-retinal.
Figure 3. Carbanion formed in Horner–Wadsworth–Emmons
reaction.
Isotope labeling is a commonly used technique to track
changes as they occur in biological processes. A major
advantage of isotopic labeling is the fact that the labeled
molecules are sterically and electronically identical in properties
to their native analogues. There are many reports available on
using deuterium and carbon-13 enriched retinoids for
investigating different aspects of the visual cycle, with the help of
spectroscopic methods such as solid-state magic-angle-spinning
NMR, Raman, or FTIR.32–35 Studies have shown that the non-
bonding interactions of the methyl group on C9 of 11-cis-retinal
Reduction of 10 using diisobutylaluminum hydride (DIBAL)
in dichloromethane (DCM) solvent, resulted in the expected 2:1
ratio of E/Z isomers. The desired Z aldehyde 11 was separated
(25%) through column chromatography from the overall mixture
(76%) of the two diastereomers (Scheme 1). It should be noted
that aldehyde 12 is indeed a useful intermediate and precursor for
synthesis of the all-trans labeled retinal. It was then used for the
formation of C11=C12 bond according to the procedure reported
by Bennani, using phosphonate 14, n-butyllithium and 1,3-
-2-

with opsin is necessary for rhodopsin activation.11,36 Our
synthesis of the 9-CD3-9-cis-retinal gives a new route to testing
structure-function hypotheses for visual rhodopsin and other
retinal proteins. Research indicates the C9-methyl group of
retinal in the 11-cis (dark state of rhodopsin) and all-trans (active
metarhodopsin-II state) conformations is a dynamical hot spot of
the chromophore,37,38 due to weak nonbonded interactions with
surrounding amino acids of the binding pocket. As for intra-
retinal interactions of the C9-methyl group, they are reduced by
its approximately symmetric position with respect to the adjacent
polyene.37–40 For 11-cis-retintal the (1,6) interactions of the C9-
methyl hydrogens with the 7H and 11H atoms of the adjacent
C7=C8 and C11=C12 double bonds give a flattened potential
energy surface with a smaller activation barrier.37,39 In 9-cis-
retinal the (1,6) interaction involving the C11=C12 double bond
is replaced by a (1,5) interaction with the (e)H of the same double
Acknowledgments
This research was funded in part by the US NIH (R15
GM112119 to N.N., R01 EY026041 and R01 EY012049 to
M.F.B.). A.V.S. was supported by the Russian Foundation for
Basic Research (16-04-00494A).
A. Supplementary data
Supplementary
data
(experimental
procedures,
characterization data, and H and 13C NMR spectra) associated
with this article can be found in the online version, at
https://doi.org/…
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In summary, we report the synthesis of 9-CD3-9-cis-retinal.
This route is efficient in that the desired stereoisomers were the
major diastereomers formed in the reactions, except for the
aldehyde 11 where the minor Z isomer had to be isolated, and the
E isomer kept for other the synthesis of the all-trans labeled
retinoid. Furthermore, the isotopomer composition of the
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Highlights
 Deuterium labeled 9Z retinoid synthesized
in 6 steps
 2 Steps require no purification
 Efficient greener introduction of a
deuterium label in the first step
 Negligent loss of the deuterium label
-5-

Graphical Abstract:
Leave this area blank for abstract info.
Synthesis of 9-CD3-9-cis-Retinal Cofactor of Isorhodopsin
Mozhgan Navidi, Shreya Yadav, Andrey V. Struts, Michael F. Brown, Nasri Nesnas*