Straightforward synthesis of the near-Infrared fluorescent voltagesensitive dye RH1691 and analogues thereof

Article abstract of DOI:10.1021/ol901846g

A highly straightforward synthesis of the near-infrared voltage-sensitive dye RH1691 is reported featuring two sequential anionic additions of C-nucleophilic heterocycles on a cyanine. This convergent approach led to the synthesis of four new probes, whic

Full text of DOI:10.1021/ol901846g

ORGANIC
LETTERS
2009
Vol. 11, No. 21
4822-4825
Straightforward Synthesis of the
Near-Infrared Fluorescent Voltage-
Sensitive Dye RH1691 and Analogues
Thereof
Raphae¨l Lebeuf, Isabelle Fe´re´zou,† Jean Rossier,† Stellios Arseniyadis,* and
Janine Cossy*
Laboratoire de Chimie Organique, ESPCI ParisTech, CNRS, 10 rue Vauquelin,
75231 Paris Cedex 05, France, and Fondation Pierre-Gilles de Gennes pour la
Recherche, 29 rue d’Ulm, 75005 Paris, France
stellios.arseniyadis@espci.fr; janine.cossy@espci.fr
Received August 8, 2009
ABSTRACT
A highly straightforward synthesis of the near-infrared voltage-sensitive dye RH1691 is reported featuring two sequential anionic additions of
C-nucleophilic heterocycles on a cyanine. This convergent approach led to the synthesis of four new probes, which also exhibit fluorescence
in the near-infrared region.
One of the most challenging objectives of modern neuro-
science is to understand the neuronal bases of behavior. It
is therefore crucial to perform functional studies of the
cerebral cortex as it is the most integrative structure of the
brain involved in many cognitive functions such as process-
ing of sensory information and generation of motor com-
mands. During the past two decades, massive efforts have
been made to record single cortical neurons in the intact brain
and to investigate how the properties of these neurons and
their intricate synaptic connections combine to form networks
allowing the processing of information. Recently, the re-
markable development of real time cortical imaging1 with
voltage-sensitive dyes2 has opened new avenues in this field
allowing the recording of cortical ensemble activity with a
temporal resolution reaching a millisecond and a spatial
resolution of a few tens of micrometers.2b,3-5 This experi-
mental approach is based on the use of dyes that bind to the
external surface of excitable membranes without interrupting
their normal function and act as transducers that transform
changes in membrane potential into optical signals. Various
molecules presenting such properties have been developed,6
(1) (a) Grinvald, A.; Anglister, L.; Freeman, J. A.; Hildesheim, R.;
Manker, A. Nature 1984, 308, 848–850. (b) Orbach, H. S.; Cohen, L. B.;
Grinvald, A. J. Neurosci. 1985, 5, 1886–1895. (c) Arieli, A.; Sterkin, A.;
Grinvald, A.; Aertsen, A. Science 1996, 273, 1868–1871. (d) Kleinfeld,
D.; Delaney, K. R. J. Comp. Neurol. 1996, 375, 89–108. (e) Tsodyks, M.;
Kenett, T.; Grinvald, A.; Arieli, A. Science 1999, 286, 1943–1946. (f) Spors,
H.; Grinvald, A. Neuron 2002, 34, 301–315. (g) Seidemann, E.; Arieli, A.;
Grinvald, A.; Slovin, H. Science 2002, 295, 862–865. (h) Slovin, H.; Arieli,
A.; Hildesheim, R.; Grinvald, A. J. Neurophysiol. 2002, 88, 3421–3438.
(i) Petersen, C. C. H.; Grinvald, A.; Sakmann, B. J. Neurosci. 2003, 23,
1298–1309. (j) Petersen, C. C. H.; Hahn, T. T. G.; Mehta, M.; Grinvald,
A.; Sakmann, B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13638–13643.
(k) Derdikman, D.; Hildesheim, R.; Ahissar, E.; Arieli, A.; Grinvald, A.
J. Neurosci. 2003, 23, 3100–3105.
(2) (a) Shoham, D.; Glaser, D. E.; Arieli, A.; Kenet, T.; Wijnbergen,
C.; Toledo, Y.; Hildesheim, R.; Grinvald, A. Neuron 1999, 24, 791–802.
(b) Grinvald, A.; Hildesheim, R. Nat. ReV. Neurosci. 2004, 5, 874–885.
(3) Kleinfeld, D.; Waters, J. Neuron 2007, 56, 760–762.
† Laboratoire de neurobiologie et diversite´ cellulaire, ESPCI ParisTech,
CNRS, 10 rue Vauquelin, F-75231 Paris Cedex 05, France.
10.1021/ol901846g CCC: $40.75
Published on Web 09/25/2009
 2009 American Chemical Society

however, most of them still suffer from limitations linked
to their intrinsic physicochemical properties. In this context,
the development of new high quality voltage probes which
allow real time imaging of cortical spatiotemporal dynamics
in vivo remains of great interest.
Scheme 2. Synthesis of the Two Heterocyclic Coupling Partners
Among all the dyes reported in the literature, we were
particularly interested in the “blue” oxonol dye RH16917
developed by Grinvald1h and co-workers which exhibits an
excitation wavelength that has minimal overlap with the
absorption of tissues thus conferring minimal pulsation
artifacts. Our interest for this dye was initially triggered by
the fact that, despite its extensive use, no synthesis had been
reported in the literature. In addition, we were particularly
motivated in developing an expedient and highly versatile
synthesis of this dye, which would offer an easy access to
other structurally related near-infrared voltage-sensitive
probes that may be more potent and less prone to photody-
namic damage or dye bleaching than RH1691. Herein, we
report the results of our endeavor.
Our strategy for the synthesis of RH1691 relied on two
main disconnections (Scheme 1). Hence, two sequential
anionic additions of C-nucleophilic heterocycles of type A
and C on a polyene precursor such as glutaconaldehyde8 (B)
were envisioned in order to access the entire carbon backbone
of RH1691 and analogues thereof.
was prepared from commercially available 4-chloro-3-
nitrobenzenesulfonyl chloride (3) (Scheme 2, eq 2). The
choice of the neopentylsulfonate ester protecting group12 for
the sulfonic acid moiety was prompted by its stability toward
a variety of reaction conditions12b and its ability to be easily
cleaved. Hence, 3 was first treated with neopentyl alcohol
in the presence of DMAP to afford the corresponding
sulfonic ester. The nitro group was then reduced using HCl
in combination with SnCl2, thus leading to the corresponding
aniline intermediate, which was then converted to the desired
hydrazine. The latter was finally condensed with a ꢀ-ketoester
in the presence of a catalytic amount of TsOH in refluxing
acetonitrile to produce compound 6. The synthesis of 6 was
thus achieved in five steps and 27% overall yield starting
Scheme 1
.
Retrosynthetic Analysis of RH1691 and Analogues
Thereof
(6) (a) Cohen, L. B.; Salzberg, B. M.; Davila, H. V.; Ross, W. N.;
Landowne, D.; Waggoner, A. S.; Wang, C. H. J. Membr. Biol. 1974, 19,
1–36. (b) Cohen, L. B.; Salzberg, B. M. ReV. Physiol. Biochem. Pharmacol.
1978, 83, 35–88. (c) Loew, L. M.; Bonneville, G. W.; Surow, J. Biochemistry
1978, 17, 4065–4071. (d) Loew, L. M.; Simpson, L. L. Biophys. J. 1981,
34, 353–365. (e) Fluhler, E.; Burnham, V. G.; Loew, L. M. Biochemistry
1985, 24, 5749–5755. (f) Wuskell, J. P.; Boudreau, D.; Wei, M.-D.; Jin,
L.; Engl, R.; Chebolu, R.; Bullen, A.; Hoffacker, K. D.; Kerimo, J.; Cohen,
L. B.; Zochowski, M. R.; Loewa, L. M. J. Neurosci. Methods 2006, 151,
200–215. (g) Grinvald, A.; Hildesheim, R.; Farber, I. C.; Anglister, L.
Biophys. J. 1982, 39, 301–308. (h) Grinvald, A.; Fine, A.; Farber, I. C.;
Hildesheim, R. Biophys. J. 1983, 42, 195–198. (i) Cacciatore, T. W.;
Brodfuehrer, P. D.; Gonzalez, J. E.; Jiang, T.; Adams, S. R.; Tsien, R. Y.;
Kristan, W. B.; Kleinfeld, D. Neuron 1999, 23, 449–459. (j) Gonzalez, J. E.;
Tsien, R. Y. Chem. Biol. 1997, 4, 269–277. (k) Schenk, O.; Fromherz, P.
Biochim. Biophys. Acta 1993, 1150, 111–122. (l) Fromherz, P.; Mu¨ller,
C. O. Biochim. Biophys. Acta 1994, 1191, 299–308. (m) Kuhn, B.;
Fromherz, P. J. Phys. Chem. B 2003, 107, 7903–7913. (n) Fromherz, P.;
Hu¨bener, G.; Kuhn, B.; Hinner, M. J. Eur. Biophys. J. 2008, 37, 509–514.
(7) May be purchased from Optical Imaging Ltd. (http://www.opt-imaging.
com).
The synthesis of 2 was achieved in three steps and 42%
yield starting from the corresponding aniline. Hence, 4-meth-
oxyaniline (1) was first converted into its corresponding
isothiocyanate following the procedure developed by Wong
and Dolman.9 The isothiocyanate was then treated with
thioglycolic acid and the resulting dithiocarbamate subse-
quently cyclized into the corresponding rhodanine by re-
fluxing in acetic anhydride (Scheme 2, eq 1).10,11
(8) For a review on the use of glutaconaldehyde potassium salt, see:
Becher, J. Synthesis 1980, 589–611.
(9) Wong, R.; Dolman, S. J. J. Org. Chem. 2007, 72, 3969–3971.
(10) Garraway, J. L. J. Chem. Soc. 1961, 3733–3735.
(11) This procedure was preferred to the condensation of bis(carboxy-
methyl)trithiocarbonate on anilines: Yarovenko, V. N.; Nikitina, A. S.;
Zavarzin, I. V.; Krayuskin, M. M.; Kovalenko, L. V. Synthesis 2006, 1246–
1248.
Compound 6, on the other hand, was obtained via a key
condensation between a ꢀ-ketoester and a hydrazine which
(12) Only a few examples of a protecting group have been reported for
sulfonic acid moieties: (a) Seeberger, S.; Griffin, R. J.; Hardcastle, I. R.;
Golding, B. T. Org. Biomol. Chem. 2007, 5, 132–138, and references cited
therein. (b) Roberts, J. C.; Gao, H.; Gopalsamy, A.; Kongsjahju, A.; Patch,
R. J. Tetrahedron Lett. 1997, 38, 355–358.
(4) Ferezou, I.; Bolea, S.; Petersen, C. C. H. Neuron 2006, 50, 617–
629.
(5) Ferezou, I.; Haiss, F.; Gentet, L. J.; Aronoff, R.; Weber, B.; Petersen,
C. C. H. Neuron 2007, 56, 907–923.
Org. Lett., Vol. 11, No. 21, 2009
4823

from 4-chloro-3-nitrobenzenesulfonyl chloride (3) and re-
quired only one purification by flash column chromatography
at the last stage. It is worth noting that the last step appeared
to be much more successful when using a ꢀ-ketoester
containing an aryloxy leaving group rather than an alkyloxy
group.
With the two heterocyclic building blocks in hand, we next
turned our attention toward the synthesis of the central core
of the molecule. As glutaconaldehyde potassium salt 7 is a
poor electrophile,8 the use of its ester derivative 8,13 the
enamine derivative 10 (Zincke aldehyde),14 or the related
cyanine 13 obtained by ring-opening of pyridine with the
corresponding aniline 1215 was envisaged (Scheme 3).16
[UV absorbance in DCM: λmax ) 542 nm, ε ) 7.5 × 104
L·mol-1·cm-1] similar to the one observed for compound 16
[UV absorbance in DCM: λmax ) 527 nm, ε ) 5.8 × 104
L·mol-1·cm-1].
The end-game sequence featuring the introduction of the
second heterocycle and the final deprotection of the sulfonic
acid is illustrated in Scheme 5. As we encountered difficulties
converting enol ester 14 into its corresponding aldehyde, we
decided to perform a direct nucleophilic displacement of the
ester moiety through a 1,8-addition/elimination sequence. To
validate this approach, a first try involving ethyl acetoacetate
as the nucleophile was performed on substrate 14. Hence,
by treating ethyl acetoacetate with NaH in DMF and adding
compound 14 to the resulting enolate, the desired product
17 was isolated in 24% yield. To our delight, when the
reaction was performed using 6 as the C-nucleophilic
heterocycle under otherwise identical conditions, the precur-
sor of RH1691 bearing a neopentylsulfonate ester protecting
group (18, not shown) was obtained in 19% yield. Interest-
ingly, higher yields were obtained when the reaction was
performed on compounds 16 bearing an enamine moiety
instead of the enol ester. Indeed, the desired coupled product
could now be isolated in a fair 40% yield.
Scheme 3. Synthesis of the Central Fragment of RH1691
Scheme 5. Condensation of All Three Fragments and
Completion of the Synthesis of RH1691
Both 8 and 10 reacted with rhodanine 2 in the presence
of TiCl4 to afford, respectively, the corresponding coupled
products 14 and 15 in excellent yields (Scheme 4).17
A
slightly lower yielding but overall more convenient approach
involved the use of cyanine 13 which could be condensed
with rhodanine 2 to afford the corresponding enamine 16 in
53% yield along with 8% of starting material, which were
difficult to separate. Fortunately, the mixture could be used
in the next step without further purification.
Scheme 4. Synthesis of the Western Fragment of RH1691
With these results in hand and in order to complete the
synthesis of RH1691, the final deprotection of the sulfonic
acid had to be performed. Among the various conditions
reported in the literature,12b,18 solvolysis appeared to be the
most practical as the free sulfonic acid would be released
without generating any undesirable or difficult to remove
side products during the process.
We found that heating a solution of the neopentylsulfonate
ester in trifluoroethanol at 120 °C under microwave irradia-
tion afforded the desired deprotected product RH1691
quantitatively and without the need of any purification. The
spectroscopic data of the synthetic material were identical
to those obtained from the purchased compound.
(13) (a) Becher, J.; O’Brien, J. P.; Teitel, S.; Saucy, G. Org. Synth. 1979,
59, 79-84. (b) Becher, J.; Frandsen, E. G. Tetrahedron 1977, 33, 341–
While both (E)- and (Z)-isomers of 14 gave pale yellow
solutions, compound 15 displayed a deep-violet coloration
343
.
(14) Zincke, T.; Wu¨rker, W. Ann. 1904, 107–140
.
4824
Org. Lett., Vol. 11, No. 21, 2009

to both the nature and the position of the substituents on the
aryl groups did not have a tremendous impact on the
fluorescence emission. However, the disparity in the substitu-
tion patterns may be of importance for cell-membrane
adsorption or penetration considering the differences in terms
of lipophilicity.
In summary, we have completed the synthesis of the near-
infrared voltage-sensitive dye RH1691 in seven steps and
11% overall yield via two sequential anionic additions of
C-nucleophilic heterocycles on a cyanine. In addition to
providing the title “blue” oxonol dye RH1691, the described
synthetic strategy is scalable and provides a straightforward
access to a variety of analogues for real time imaging of
cortical spatiotemporal dynamics in vivo. Such studies are
currently underway and will be reported in due course.
Acknowledgment. We thank the Fondation Pierre-Gilles
de Gennes pour la Recherche for financial support.
Supporting Information Available: Experimental details
and characterization data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL901846G
Figure 1. Structure of various RH1691 analogues. (a) Fluorescence
was recorded in a phosphate buffer. (b) Fluorescence was recorded
in a phosphate buffer/EtOH (3/1) solution.
(15) (a) Ko¨nig, W. J. Prakt. Chem. 1904, 69, 105. (b) Reichardt, C.;
Stein, J. Eur. J. Org. Chem. 1999, 2899–2908.
(16) Pyrilium perchlorate could also be envisaged; however, for safety
reasons, this approach was not investigated. For condensation of pyrilium
perchlorate with rhodanine, see: Tolmachev, A. I.; Sribnaya, V. P. Zh. Obsh.
Khim. 1965, 35 (2), 316–324 (Eng. Transl.). For its synthesis: Furber, M.;
Herbert, J. M.; Taylor, R. J. K. J. Chem. Soc., Perkin Trans. 1 1989, 683–
690.
Having validated the route to RH1691, we embarked in
the synthesis of various analogues in order to determine
qualitatively a structure-fluorescence relationship (Figure
1). Four new dyes were thus prepared in similar yields, and
the fluorescence spectra recorded in a phosphate buffer
solution (pH ) 7.2). As a general trend, the changes made
(17) Both stereoisomers of 14 could be separated; however, the exact
ratio could not be determined due to the poor solubility of the E-isomer
compared to the Z-isomer.
(18) (a) Roberts, D. D. J. Org. Chem. 1999, 64, 1341–1343. (b) Koh,
H. J.; Lee, H. W.; Lee, I. J. Chem. Soc., Perkin Trans. 2 1994, 253–257.
Org. Lett., Vol. 11, No. 21, 2009
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