Catch and release microwave mediated synthesis of cyanine dyes

Article abstract of DOI:10.1039/b820719b

Unsymmetrical functionalised cyanine dyes, covering the whole colour range, were readily synthesised (in 100 mg amounts) by a combination of microwave and solid-phase methodologies.

Full text of DOI:10.1039/b820719b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Catch and release microwave mediated synthesis of cyanine dyes†
Maria Lopalco,a Eftychia N. Koini,b Jin Ku Choa and Mark Bradley*a
Received 19th November 2008, Accepted 12th December 2008
First published as an Advance Article on the web 22nd January 2009
DOI: 10.1039/b820719b
Unsymmetrical functionalised cyanine dyes, covering the
whole colour range, were readily synthesised (in 100 mg
amounts) by a combination of microwave and solid-phase
methodologies.
To avoid time-consuming purifications and allow enhanced
access to a broad range of cyanine dyes, solid-phase synthesis
methods17–19 have been reported. In particular, Mason et al.20
reported a highly versatile strategy based on the attack of a
heterocyclic carbon nucleophile onto a polyene-chain precursor
immobilised onto a solid support to generate unsymmetrical
trimethine and pentamethine dyes.
However, there is still a great demand for the development of
a facile synthetic method for the preparation of sulfonated and
non-sulfonated cyanine dyes, available in different colours (e.g.
far-red profiles for in vivo imaging) and amenable to biomolecule
conjugation (e.g. with an appendant carboxyl group or equivalent).
Herein, we report a common synthetic pathway for the prepa-
ration of a wide colour-range of hydrophobic and hydrophilic
cyanine dyes suitable for bioconjugation, using both microwave
and solid-phase methodologies, the combination of which offers
a practical approach to the rapid and practical preparation of
a variety of Cy-fluorophores. The general synthetic procedure
(Schemes 2 and 3) shows the parallel synthesis of the range of
cyanine dyes with the starting point being the N-alkylation of
indolenines (Scheme 1).
Fluorescent probes represent a versatile tool for visualising specific
molecular targets and events in vitro and increasingly in vivo.1–3
Multicolour fluorescence detection has proved to be useful for
multiplexed assays on a variety of microarray formats4,5 and for
the simultaneous investigation of multiple biological processes in
living cells.6 As a consequence, an increasingly large number of
fluorescent probes, available in different colours, with the ability
to be straightforwardly conjugated to biomolecules, are required.7
The cyanine dyes (Fig. 1), a class of highly fluorescent com-
pounds, have all the key requirements necessary for highly sensitive
multicolour detection, with wavelengths which are tunable, by
synthesis, across the visible spectrum. They also display excellent
photophysical properties, including high extinction coefficients
and quantum yields, while having fluorescence wavelengths remote
from the natural autofluorescence of biomolecules.8–10 They are
thus routinely used as fluorescent probes in a wide-range of
applications such as DNA sequencing,11 cellular analysis,12 flow
cytometry13 and in vivo imaging.14,15
1
2
1
-
=
Fig. 1 Structure of cyanine dye. R , R = H, -(CH CH)2-, R = SO3
,
R2 = H; n = 1, 2, 3.
Conventional synthetic methods for the preparation of un-
symmetrical cyanine dyes are based on the condensation of
quaternised indolenine derivatives with vinylogous homologues
of diphenyl formamidine and typically affording mixtures of
the desired compound along with unreacted hemicyanine and
undesired symmetrical analogues.16
Scheme 1 Microwave-mediated alkylation of indolenines. (i) methyl
iodide, acetonitrile, 150 ◦C, 30 min to 1 h, microwave; (ii) 6-bromohexanoic
acid, acetonitrile, 150 ◦C, 1 to 3 h, microwave.
The heterocyclic quaternary ammonium salts 2 and 3 are usually
obtained by heating the corresponding heteroaromatic base 1 with
an excess of alkylating agent in an aprotic solvent16,21 over several
days. In the first simplification of the synthesis it was possible
to reduce the reaction time by microwave heating the reaction
mixture at 150 ◦C in acetonitrile (Scheme 1) to give rapid access to
a broad range of desired heterocyclic quaternary ammonium salts
aSchool of Chemistry, The University of Edinburgh, West Mains Road,
Edinburgh, United Kingdom EH9 3JJ. E-mail: mark.bradley@ed.ac.uk
bNational Hellenic Research Foundation, 48 Vas. Constantinou Avenue,
11635 Athens, Greece
† Electronic supplementary information (ESI) available: Detailed exper-
imental procedures and characterisation data of the products. See DOI:
10.1039/b820719b
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Scheme 2 Solid phase synthesis of the cyanine dyes: (a) triethylorthoformate for 6a, malonaldehyde bis dimethyl-acetal for 6b, BF3·Et2O, DCM, 6 h;
glutaconaldehyde (2-pentenedial) dianilide hydrochloride, Ac2O, DIEA, and pyridine for 6c; (b) DMF, 120 ◦C, 15 min, microwave, for trimethine and
pentamethine dyes; Ac2O, DIEA, and pyridine, 1.5 h; (c) DCM, 1 h.
Table 1 Properties of dyes synthesised on solid phase
Dye
lmax, abs (nm)
lmax, em (nm)a
Yieldb
8a
8b
8c
8d
8e
8f
547
640
743
587
678
781
647
561
660
765
603
704
808
667
68%
84%
86%
49%
92%
51%
94%
8g
a Measured in MeOH (except for compound 8g which was dissolved in
H2O). b Yield of isolated product in the final step (based on the amount of
heterocycle used in the cleavage step).
it was observed that microwave heating in this step gave rise to
higher yields and purities of the final compound. Cleavage from
the solid support was then accomplished using the carboxylated
indolium salt (3a–c) to give functionalised cyanine dyes suitable
for conjugation with biomolecules (Scheme 3). The unsymmetrical
trimethine and pentamethine cyanine dyes were obtained in good
yield (Table 1) and purity (85–100% by ELSD detection) and in
preparative scale (up to 100 mg).
Scheme 3 Cleavage of the cyanine dyes from the resin. (i) Ac2O, (DIEA),
and pyridine, 2 h.
Further studies were carried out to extend this protocol to the
synthesis of heptamethine dyes, as they have attracted great interest
as fluorescent probes for in vivo imaging.15,22,23
in good yields (66–95%), including sulfonated (2c, 3c) and/or
carboxylated (3a–c) variants.
To synthesise cyanine dyes the alkylated indolenines (2a–c)
were reacted with the resin bound polymethine imines (6a–c)
(Scheme 2, route A) as originally reported by Mason et al.,20
although again using microwave irradiation (15 mins, 120 ◦C).
Though microwave heating accelerates the attack of a second
molecule of the indolenium salt 2 onto the polymer-bound imine
6, the symmetrical impurity obtained is cleaved from the resin and
In an initial approach the monofunctionalised heptamethine
dye 8c was obtained following the route A. Glutaconaldehyde
dianilide hydrochloride was reacted with the supported ani-
line 4, in the presence of Ac2O, to form the polymer-bound
polymethine 6c.
The reaction of the indolenium salt 2a with 6c produced the
desired supported hemicyanine 7c via loss of N-phenylacetamide,
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but resulted also in the release of the hemicyanine in solution
by cleavage from the resin, with a consequent decrease in the
yield of the hemicyanine 7c and of the final compound 8c (12%)
(Scheme 4).
hemicyanine 5a onto the resin in this manner was carried out
in DCM for 1 h, with an 86% yield as determined by nitrogen
elemental analysis. The desired dyes were then cleaved from the
resin by nucleophilic attack of 3a,b onto the intermediates 7c,f in
pyridine:Ac2O (10:1) to afford the unsymmetrical heptamethine
dyes 8c and 8f in good yield (Table 1) and purity (>90%), as
determined by HPLC analysis and 1H NMR (Fig. 2).
The catch-and-release protocol described above has proven to
be effective also for the synthesis of sulfoindocyanine dyes. The
presence of sulfonate groups on the ring systems improves water
solubility, prevents aggregation in water and reduces non-specific
binding to biomolecules and cellular constituents.8 However,
purification by chromatography of these dyes is difficult.
To overcome this limitation, Jiang et al.19 recently suggested the
use of poly(ethylene glycol) as a soluble support, which has its own
handling problems and low loading. In addition, Mason et al.20
described the synthesis of sulfonated cyanine dyes on polystyrene
resin; however they reported that the sulfonated heterocycles did
not react during the formation of the hemicyanine intermediate on
solid phase but they did react in the dye formation step allowing
the synthesis of mono-sulfonated cyanine dyes.
Scheme 4 Formation of hemicyanine 7c. (i) 2a, Ac2O, DIEA, pyridine,
1.5 h.
This led to the development of an alternative catch-and-release-
strategy (Scheme 2, route B) based on the formation of the
hemicyanine intermediates 5a,b in solution and the captive loading
onto the solid support (4) to form 7c,f. The loading of the
Microwave heating enabled the solid-phase synthesis of the
pentamethine sulfoindocyanine dye 8g from polystyrene resin
(route A), although in low yield (10%) and purity.
Fig. 2 1H NMR of the crude heptamethine dye 8c.
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The catch-and-release method (route B) proved to be far
more effective, resulting in the formation of 8g in excellent yield
and purity (94%), without the need for further purification by
chromatography (Fig. 3).
Notes and references
‡ Purification by column chromatography of the dyes obtained from the
supported imidates was carried out to provide their detailed characteri-
sation but may not be necessary for application of the dyes as labeling
reagents.
1 N. Johnsson and K. Johnsson, ACS Chem. Biol., 2007, 2, 31–38.
2 R. Weissleder, Science, 2006, 312, 1168–1171.
3 J. Zhang, R. E. Campbell, A. Y. Ting and R. Y. Tsien, Nat. Rev. Mol.
Cell Biol., 2002, 3, 906–918.
4 E. K. Lewis, W. C. Haaland, F. Nguyen, D. A. Heller, M. J. Allen, R. R.
MacGregor, C. S. Berger, B. Willingham, L. A. Burns, G. B. I. Scott,
C. Kittrell, B. R. Johnson, R. F. Curl and M. L. Metzker, Proc. Natl.
Acad. Sci. U. S. A., 2005, 102, 5346–5351.
5 J. Y. Ju, D. H. Kim, L. R. Bi, Q. L. Meng, X. P. Bai, Z. M. Li, X. X. Li,
M. S. Marma, S. Shi, J. Wu, J. R. Edwards, A. Romu and N. J. Turro,
Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 19635–19640.
6 J. K. Jaiswal, H. Mattoussi, J. M. Mauro and S. M. Simon, Nat.
Biotechnol., 2003, 21, 47–51.
7 A. Waggoner, Curr. Opin. Chem. Biol., 2006, 10, 62–66.
8 R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis and A. S.
Waggoner, Bioconjugate Chem., 1993, 4, 105–111.
9 C. Bouteiller, G. Clave, A. Bernardin, B. Chipon, M. Massonneau, P. Y.
Renard and A. Romieu, Bioconjugate Chem., 2007, 18, 1303–1317.
10 Y. H. Lin, R. Weissleder and C. H. Tung, Bioconjugate Chem., 2002,
13, 605–610.
11 R. S. Duthie, I. M. Kalve, S. B. Samols, S. Hamilton, I. Livshin, M.
Khot, S. Nampalli, S. Kumar and C. W. Fuller, Bioconjugate Chem.,
2002, 13, 699–706.
Fig. 3 HPLC chromatogram of the crude sulfonated dye 8g.
In conclusion, two practical approaches to the solid phase syn-
thesis of unsymmetrical cyanine dyes suitable for bioconjugation
have been reported. The method proposed is straightforward and
enables, in a few steps, the synthesis of cyanine dyes spanning the
whole colour range. Through a catch-and-release strategy we have
also been able to synthesise, on solid phase, heptamethine and
water soluble cyanine dyes in high yield and purity without the
need for chromatographic purification‡, thus providing an easy
route to the preparation of these invaluable fluorescent probes
in amounts sufficient to satisfy the most hungry of biological
appetites.
12 J. R. Carreon, K. M. Stewart, K. P. Mahon, S. Shin and S. O. Kelley,
Bioorg. Med. Chem. Lett., 2007, 17, 5182–5185.
13 S. C. De Rosa, J. M. Brenchley and M. Roederer, Nat. Med., 2003, 9,
112–117.
14 A. Becker, C. Hessenius, K. Licha, B. Ebert, U. Sukowski, W. Semmler,
B. Wiedenmann and C. Grotzinger, Nat. Biotechnol., 2001, 19, 327–331.
15 J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626–634.
16 M. E. Jung and W. J. Kim, Bioorg. Med. Chem., 2006, 14, 92–97.
17 S. J. Mason and S. Balasubramanian, Org. Lett., 2002, 4, 4261–4264.
18 J. Isacsson and G. Westman, Tetrahedron Lett., 2001, 42, 3207–3210.
19 L. L. Jiang, L. F. Dou and B. L. Li, Tetrahedron Lett., 2007, 48, 5825–
5829.
20 S. J. Mason, J. L. Hake, J. Nairne, W. J. Cummins and S. Balasubrama-
nian, J. Org. Chem., 2005, 70, 2939–2949.
21 T. Hirata, H. Kogiso, K. Morimoto, S. Miyamoto, H. Taue, S. Sano,
N. Muguruma, S. Ito and Y. Nagao, Bioorg. Med. Chem., 1998, 6,
2179–2184.
Acknowledgements
22 X. Y. Chen, P. S. Conti and R. A. Moats, Cancer Res., 2004, 64, 8009–
We thank the Basic Technology Programme of the RCUK. ENK
thanks EU for a Marie Curie Host Fellowship for the Transfer of
Knowledge.
8014.
23 C. H. Tung, Y. H. Lin, W. K. Moon and R. Weissleder, ChemBioChem,
2002, 3, 784–786.
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