Tritylisation of pyrene, perylene and coronene: A new family of switchable fluorescent labels

Article abstract of DOI:10.1016/S0040-4039(00)00737-1

The synthesis of novel fluorescent labels based on pyrene, perylene and coronene is described. Due to the trityl-type structure, their fluorescence may be reversibly switched on and off by changing the pH. This property can be used to expand the palette of fluorophores available for multicolour DNA detection on DNA chips. Some FRET and surface chemistry applications are also demonstrated. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00737-1

Tetrahedron Letters 41 (2000) 4943±4948
Tritylisation of pyrene, perylene and coronene: a new family
of switchable ¯uorescent labels
Mikhail S. Shchepinov,a,* Vladimir A. Korshun,b Ryan D. Egelanda and
Edwin M. Southerna
aDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
bMRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK
Received 31 March 2000; accepted 4 May 2000
Abstract
The synthesis of novel ¯uorescent labels based on pyrene, perylene and coronene is described. Due to the
trityl-type structure, their ¯uorescence may be reversibly switched on and o€ by changing the pH. This
property can be used to expand the palette of ¯uorophores available for multicolour DNA detection on
DNA chips. Some FRET and surface chemistry applications are also demonstrated. # 2000 Elsevier
Science Ltd. All rights reserved.
Keywords: polycyclic aromatic compounds; Friedel±Crafts reactions; Grignard reactions; ¯uorescence; nucleic acids.
As part of a programme to optimise the manufacture and use of DNA chips we developed a
new family of ¯uorescent tags derived from polycyclic aromatic hydrocarbons (PAHs). Both single
¯uorophore1 and energy transfer2-based ¯uorescence detection methods ®nd wide applications in
the analysis of nucleic acids. Some PAHs have certain advantages over the ¯uorophores currently
used to label DNA, such as ¯uorescein: they are less prone to photobleaching and have high
molar absorbance and high quantum yields. Furthermore, molecules are available with a range of
excitation and emission maxima and large Stokes shifts. We chose pyrene,3 perylene4 and coronene5
as PAHs with useful ¯uorescent properties. The introduction of a carbinol±carbocation switchable
element would allow one to controllably turn the ¯uorescence on and o€ by changing the pH. The
conversion of these PAHs into trityl-type structures also seems advantageous due to the non-planar
conformation of triarylmethanols, which would prevent the p±p stacking interactions with a
resulting increase in the solubility.
Friedel±Crafts acylation of PAHs with 2,4-dimethoxybenzoyl chloride in presence of AlCl3
in DCM for pyrene or chlorobenzene6 for perylene and coronene gave 1-acylpyrene 2a, 3-acyl-
perylene 2b and acylcoronene 2c (Scheme 1). These ketones were converted into the corresponding
* Corresponding author. Tel: +44 1865 275226; e-mail: misha@bioch.ox.ac.uk
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00737-1

4944
R,S-oxazolyltritanols 3a±c using appropriate phenylmagnesium bromide as a nucleophile in
Grignard synthesis. Further conversion7 yielded N-hydroxysuccinimide (NHS)-derivatives 4a (as
pale yellow), 4b (as dark red) and 4c as yellow solids.8 NHS-esters 4a±c can be used for labelling
of biomolecules or for derivatisation of surfaces. The labelling procedure would involve reacting
4a±c with an amino group(s)-containing analyte; we therefore synthesised7b,c the corresponding
model butylamides 7±9 (Fig. 1) for ¯uorescence studies.
Scheme 1. Reagents and conditions: (i) 2,4-dimethoxybenzoylchloride/AlCl3 in DCM (a, 86%) or PhCl (b, 53%; c,
48%); (ii) 3-(4,4-dimethyl-1,3-oxazoline-2-yl)phenylmagnesium bromide/THF, re¯ux; (a, 73%; b, 64%; c, 49%); (iii)
80% AcOH, (or TFA, 72 h, for c), at 70ꢀC, 48 h, then re¯ux in 20% NaOH in EtOH/H2O, 3 h; a, 96%; b, 93%, c,
85%; (iv) DCC, NHS, dioxane/THF (a, 91%; b, 87%; c, 79%)
A modi®ed trityl group bearing a pyrenyl residue in place of one of the phenyls has ¯uorescent
properties similar to non-modi®ed pyrene.9 Triarylmethyl cation derived from tritanol by an acidic
treatment (Scheme 2) would have completely di€erent ¯uorescence properties, while remaining
covalently linked to a probe molecule if attached to it through a side-chain.7 These features are
combined in compounds 4a±c. The pH-threshold for the formation of trityl carbocations (like 6)
from corresponding tritanols at low pH can be controlled by electron withdrawing or donating
groups on the aromatic rings.10 Two methoxy groups and one carboxyl group give 7±9 an acidic
stability similar to that of a standard DMTr and MMTr groups.11 The UV spectra of 7±9 were of
the same shape but slightly red-shifted (3±10 nm) as compared to the starting PAHs.2,3
Multicolour detection (use of more than one ¯uorophore in one reaction) is a useful feature of
¯uorescent dyes; it enables di€erent sequences to be detected simultaneously and has been used
to good e€ect in DNA sequencing,1 ¯uorescence in situ hybridisation (FISH)12 and di€erential
gene expression analysis on DNA chips.13 The size of the palette is limited by the overlap of
the excitation and emission spectra; it has proved dicult to use more than four colours in FISH
and two colours are normal in expression analysis. An advantage of trityl-based ¯uorescent tags
is the potential to `switch' the spectra on and o€ by simply changing the pH. The magnitude of the
shifts is very large. For example, moving from neutral or alkaline to acidic pH shifts the excitation
maximum of the pyrene-based compound 7 from 346 to 711 nm (Fig. 1), the property earlier used
to generate triarylmethyl carbocations with a variety of di€erent colours.14

4945
Figure 1. A. Absorption spectra of butylamides 7 (solid line), 8 (dotted line) and 9 (dashed line) (10^5 M in DCM). B.
Fluorescence spectra of butylamides 7±9 (10^6 M in DCM); excitation wavelengths: 330 nm (7), 420 nm (8), 343 nm (9).
C. Absorption spectra of cations of butylamides 7±9 (10^5 M in 1% TFA/DCM)
Scheme 2.
Trityl carbocations do not ¯uoresce in the range detected for the corresponding tritanols. This
property can be used to improve the discrimination of labels: ®rst by increasing the accuracy of
intensity measurements; and, second, increasing the potential number of colours in the palette.
For example, targets can be labelled with two ¯uorophores having similar excitation and emission
spectra, but only one of which is switchable by pH change. After hybridisation, measurements are
taken at two pH values: under ambient conditions and after exposing the array to acidic vapour,
which is enough to switch the emission of ¯uorescent trityls o€ immediately, but reversibly. Using

4946
a single excitation source, both ¯uorophores emit at neutral pH but only one will emit in acid.
These two measurements alone would be enough to distinguish the two patterns of hybridisation.
But a third measurement, using a source which excites the second ¯uorophore in acid, can give
more analysis. In this way it may be possible to double the number of labels that can be used
together.
To evaluate the suitability of our ¯uorescent labels as components for ¯uorescence resonance
energy transfer (FRET), a model compound 10 was synthesized8 by the stepwise acylation of
4,7,10-trioxa-1,13-tridecanediamine with 4b followed by 4a in DCM. While the absorption spectra
for both non-ionised and bis-cationic forms essentially represent a superposition of 7 and 8 (Fig.
2A), 10 ¯uoresces only at 450, 480 and 515 nm when excited at the pyrene absorption maximum
of 330 nm (Fig. 2B), with no detectable ¯uorescence of pyrene (at 377, 388 or 396 nm). When
mixed in equimolar amounts, 7 and 8 retain their own ¯uorescence properties (data not shown).
This suggests a possibility of designing ¯uorescent labels having increased Stokes' shifts by
arranging the necessary ¯uorophores (perhaps even more than two) in the vicinity of each
other.15 Furthermore, additional control can be achieved by making some of these parts more
acid-labile than the others, so that some selected components of the chain may be reversibly
switched o€ by decreasing the pH. Mass-spectrum (LDI-TOF) of 10 (calculated exact mass for
C78H70N2O11: 1210.50) showed, apart from the molecular ion, two fragments, lacking one
(1193.46) and two (1176.47) hydroxyl groups. The fact that the latter ¯ies in mono-charged
(mono-cationic) and not in bis-cationic form7b,c (the signal for 1176.49/2=588.25 was not detected)
suggests some unusual interactions, perhaps FRET during the LDI-TOF process, initiated by
laser irradiation at 340 nm, which is almost a perfect match with the absorption maximum for 10.
Figure 2. A. Absorption spectra of 10 (10^5 M in DCM) in non-ionised (solid line) and bis-cationic (dotted line) forms
(in 1% TFA/ DCM). B. Fluorescence spectrum of bis-chromophore 10 (10^6 M in DCM); excitation wavelength:
330 nm
To demonstrate the reversibility of the ¯uorescence in real time, acid was generated using a
microelectrode technique. A series of 20 linear iridium microelectrodes 40 mm wide (80 mm
between centres) on a silicon dioxide wafer were manufactured using photolithography methods.
A glass microscope slide was silanized with 3-aminopropyltrimethoxysilane and treated overnight
with a 0.1 M solution of 4b in THF. This slide was then washed and placed atop of the micro-
electrode array, separated by ca. 3 mm layer of an electrolyte (hydroquinone in CH3CN). Confocal

4947
microscopy was used to examine the ¯uorescence emission at 520 nm with Ar/Kr laser excitation.
Application of voltage across adjacent electrodes generated protons electrochemically at the
anodes, resulting in a lower pH and a corresponding decrease in ¯uorescent emission at 520 nm
(Fig. 3). Upon switching o€ the microelectrodes, the ¯uorescent emission reverts to the starting,
voltage-o€ condition, demonstrating the reversibility of the acid-catalysed carbocation con-
formation. Aminated polypropylene can also be used instead of glass (data not shown).
Figure 3. Fluorescent response of an aminated glass surface coated with 4b (Scheme 2) to acid generated electro-
chemically above horizontal microelectrodes, observed using confocal microscopy. Left, no voltage applied. The signal
consists of ¯uorescence from 4b on the surface and re¯ection from iridium-covered electrodes A±D. Right, the voltage
(1.2 V between cathode B and anode C and 0.8 V between cathode D and anode C) is applied. The acid generated on
the anode C leads to conversion of 4b into cationic form with consequent disappearance of the signal
At present, we are preparing the corresponding phosphoramidite derivatives of 4a±c to facilitate
their incorporation into synthetic oligonucleotides.
Acknowledgements
The authors would like to express their gratitude to Drs. D. A. Stetsenko for helpful discussions,
A. A. Arzumanov for running the ¯uorescence spectra, R. Chalk for mass-spectra and R. Quarrell
for valuable suggestions and comments. M.S.S was supported by an MRC grant.
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