Facile functionalization of a fully fluorescent perfluorophenyl BODIPY: Photostable thiol and amine conjugates

Article abstract of DOI:10.1039/c1cc13778f

Selective nucleophilic substitution on a perfluorophenyl unit comprising a BODIPY fluorophore using an alkanethiol or a primary amine offers a quantitative fluorophore labelling strategy, while retaining high photostability and emission quantum yields app

Full text of DOI:10.1039/c1cc13778f

View Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 10425–10427
www.rsc.org/chemcomm
COMMUNICATION
Facile functionalization of a fully fluorescent perfluorophenyl BODIPY:
photostable thiol and amine conjugatesw
a
a
a
a
a
´
Guillaume Vives, Carlo Giansante, Robin Bofinger, Guillaume Raffy, Andre Del Guerzo,*
Brice Kauffmann,b Pinar Batat,ac Gediminas Jonusauskasc and Nathan D. McClenaghan*a
Received 24th June 2011, Accepted 7th August 2011
DOI: 10.1039/c1cc13778f
Selective nucleophilic substitution on a perfluorophenyl unit
comprising a BODIPY fluorophore using an alkanethiol or a
primary amine offers a quantitative fluorophore labelling strategy,
while retaining high photostability and emission quantum yields
approaching unity.
Perfluorinated BODIPY 1 was obtained in a straight-
forward three step, one-pot reaction (see ESIw for detailed
procedure). Commercial perfluorobenzaldehyde was first reacted
with 2,4-dimethylpyrrole to afford a dipyrromethane inter-
mediate which was oxidized by p-chloranil to a dipyrromethene
intermediate. This was then reacted with BF3 to yield 1 in 33%
overall yield via a standard procedure.1a,c,4 Single crystals
suitable for X-ray diffraction were obtained from chloroform
(recently reported in acetonitrile).3a The crystallographic
structure shows the planar BODIPY moiety and the out-
of-plane perfluorinated phenyl substituent. Importantly for
high fluorescence, the phenyl ring plane presents a 90.01 angle
to the BODIPY plane in the solid state due to steric hindrance
induced partly by the methyl groups. On comparing with a
related hydrocarbon analogue, the fluorine atoms can also be
seen to be responsible for this behaviour. For example replacing
the pentafluorophenyl ring with a para-bromophenyl, such
that ortho protons replace fluorines resulted in a decreased angle
of 78.91 between planes; while that comprising a dimethylamino
group was 87.61.5
Perfluorinated rings are known to be susceptible to nucleophilic
substitution reactions, offering an effective reaction pathway
to C–C, C–S and C–N bond formation.6 This would thus offer
a high-yielding straightforward alternative to metal-catalysed
SN2 reactions on the BODIPY meso-phenyl ring.7 While the
Lewis acid boron centre is susceptible to such an attack from
aryl Grignards, acetylide anions or alkoxides,1a,8 it was anti-
cipated to be so less than the electron poor aromatic. Thus
under appropriately mild conditions selective F-displacement
reactions on the ring could be effected. Nucleophilic aromatic
substitution reactions on the perfluorinated ring were tested
with substrate 1 using thiol and amine compounds, as represented
in Scheme 1. 1 reacted smoothly at room temperature with
Photostable fluorescent dyes and labels are widely used in imaging
and various microscopies, different types of families being suited
to specific excitation wavelengths. Among these fluorophores,
dipyrrometheneboron difluoride dyes (commonly referred to
as BODIPY) have emerged as a popular choice of fluorophore
for different applications including implementation in biological
media due to high absorption cross-section, intense emission,
as well as specific membrane incorporation.1 While a large range
of chromophores exhibiting different photophysical properties
are known, the number of strategies for ad hoc introduction of
fluorescent labels on substrates is rather limited.2 Herein we
report the use of the specific chemistry of perfluorinated rings
to allow efficient labelling of versatile fluorescent synthon 1
(Scheme 1) with biologically relevant functional groups, in
both organic solvents and aqueous mixtures, whilst typically
retaining a quantum yield approaching unity, thus allowing
studies down to the single molecule level. It is noteworthy that
the methyl groups on the pyrrole rings provide an orthogonal
reaction site,3 as demonstrated in a structurally related fluorous
intermediate which should extend the scope of such synthons
in NIR emitter tag development. Indeed a range of chemical
reactions for colour tuning BODIPYs have been reported which
are a priori compatible with the current synthon. Harnessing
the current functionalisation approach with orthogonal reactions
holds promise for applications in materials development, e.g.
fluorescent focal points in dendrimer synthesis.
a Univ. Bordeaux, ISM, CNRS UMR 5255, F-33400 Talence, France.
E-mail: n.mc-clenaghan@ism.u-bordeaux1.fr,
a.del-guerzo@ism.u-bordeaux1.fr
b Univ. Bordeaux, IECB, CNRS UMS 3033, F-33607 Pessac, France
c Univ. Bordeaux, LOMA, CNRS UMR 5798, F-33400 Talence,
France
w Electronic supplementary information (ESI) available: Synthetic
procedures, HPLC traces for coupling reactions, steady-state absorp-
tion and emission spectra, Strickler–Berg treatment, time-resolved
emission and transient absorption spectra, photodegradation experi-
ments and confocal fluorescence microscopy setup. See DOI: 10.1039/
c1cc13778f
Scheme 1 Synthesis of 2–4 with quantitative conversion for 2 and 3.
Chem. Commun., 2011, 47, 10425–10427 10425
c
This journal is The Royal Society of Chemistry 2011

View Online
ethanethiol in the presence of K2CO3, HPLC analysis showing
full disappearance of 1 after 40 minutes concomitant with the
appearance of a new band with a longer retention time (6.3 cf.
7.7 min, eluting with 96 : 4, cyclohexane/ethyl acetate, v/v). 19F-NMR
analysis of the crude product showed a clean substitution of the
fluorine in the 4-position giving 2, as denoted by resonances of the
aromatic fluorine atoms at À134.1 and À143.6 ppm compared
with those of 1 at À161.9, À153.0 and À143.5 ppm (see ESIw),
while the quadruplet associated with boron-bound fluorines
remained similar at À146.7 ppm in both compounds.
Table 1 Spectroscopic data for 1–4 in air-equilibrated THF
Sample
l
max/nm e/MÀ1 cmÀ1
lem/nm Ff
t
calc/ns t/ns
1
2
3
4
516
516
514
515
78 600
77 000
78 700
77 200
527
527
525
523
0.98 5.8
0.90 5.4
0.93 6.1
0.96 6.0
6.1
5.9
5.7
5.7
and the maximum absorption wavelength is almost unchanging,
being almost coincident with popular argon ion laser, which
emits at 514 nm, and shows that remote substitution has a
negligible effect on absorption properties. Relatively few
fluorophores are perfectly adapted for this wavelength, one
example being expensive sulfonated rhodamine derivatives
(Alexa Fluor 514s). Importantly, the quantum yield which
is essentially unity for parent 1 remains high ( Z 90%) in all
cases although the substituted species are slightly lower than
the parent.9 The strongly allowed nature of the S1 ’ S0
transition, as denoted by the strong molar extinction coeffi-
cients allows estimation of the excited-state lifetime using a
Strickler–Berg analysis (see ESIw).10 Estimated fluorescence
lifetimes (tcalc, Table 1) on the nanosecond scale are in good
agreement with the measured values. This suggests that the
excited parent and derivatives are all structurally reminiscent
to that of the respective ground-state species and that solvent
interactions are unchanging.
Reaction with a primary amine was carried out in DMF at
room temperature using an 8-fold excess of butylamine. Only one
new HPLC signal for substitution product 3 could be observed as
the starting material was consumed, with 19F-NMR resonances
of the aromatic fluorines at À147.6 and À161.6 ppm confirming
that substitution in the 4-position had occurred. Secondary
amine diethylamine did not react with 1 at room temperature
despite using a large excess (40 eq.) of amine. 4 could be obtained
on heating the reaction mixture to 120 1C, however the formation
of side products was also observed.
Concerning relative reactivities, Fig. 1 shows the progres-
sion of the reaction of 1 with primary and secondary amines
and thiols, as judged by integrated peak areas in the HPLC
trace. The reaction with the thiol was found to be much more
rapid, in the presence of base, than the analogous reaction
with a primary amine. Simultaneously introducing thiol (5 eq.)
and primary amine (8 eq.) to a solution of 1 (24 mM) resulted
in the quasi-exclusive formation of 2. Indeed, integrated peak
areas corresponding to products 2 and 3 showed circa 99%
conversion to 2 after only 5 minutes. Thus the selectivity and
high reactivity for thiols with the perfluorophenyl moiety under
mild conditions is clearly demonstrated. In the absence of base
the reaction of 1 with ethanethiol is much more sluggish, with
only 54% conversion after 5 days. Importantly, in an aqueous
DMF mixture (1 : 1, v/v) the thiol reaction proved equally
efficient and a similar selectivity toward thiol vs. amine conjugate
formation was obtained (see ESIw). Indeed in this protic environ-
ment the amine proved unreactive under these mild conditions.
Parent BODIPY 1 and derivatives 2, 3 and 4 showed strong
absorption and intense emission, with a small Stokes shift as
shown by data regrouped in Table 1 (see spectra in Fig.
S12–S15 (ESIw)). Molar extinction coefficients for the lowest
energy visible absorption band are similarly high in all cases,
It is noteworthy that similarly high quantum yield values
were obtained for 1 and 2 in an H2O : DMF mixture (75 : 25, v/v)
at 91% (t = 6.6 ns) and 93% (t = 6.6 ns), respectively.
Meanwhile the amine-conjugate 3 exhibited a drastically
reduced value at 1%, presumably partly due to hydrogen-
bonding enhanced vibrational quenching. The photostability
of molecules 1–3 was evaluated on irradiation in air-equilibrated
solution. The quantum yields for photodegradation (Fbleach
)
were estimated to be 2.7 Â 10À5, 4.1 Â 10À5 and 2.7 Â 10À5
,
respectively, somewhat lower than the 7.5 Â 10À5 measured
for fluorescein isocyanate (which is on the same order of
magnitude as a literature value in deoxygenated EtOH).11
Picosecond transient absorption (Fig. S16, ESIw) spectro-
scopy was employed to follow the evolution of excited 1, a
representative member of the series, and notably to elucidate
the presence of any dark-states which may have been intro-
duced, could not be detected by fluorescence, and would
represent an unwanted alternative non-emissive deexcitation/
degradation pathway. A qualitatively similar excited-state
absorption spectrum to that of a multicomponent BODIPY
containing supermolecule, with positive absorption features at
350 nm and 420 nm and a band close to the ground state
absorption wavelength allows us to conclude that no significant
population of states other than the emissive S1 state occurs.12
High quantum yield, no apparent population of dark states
and relatively high photostability for 1 make this label potentially
suitable as a single molecule emitter. 1 was dispersed into a
PMMA matrix and evaluated using confocal fluorescence
microscopy under deoxygenated conditions (for more details,
see ref. 13 and ESIw). In preliminary studies, a linearly polarized
laser excitation at a standard wavelength of 488 nm with a
power of 1.5 kW cmÀ2 was used.14 Emission trajectories on single
molecules show the typical blinking of the fluorescence (Fig. 2a),
due to the reversible conversion between singlet ON states and
Fig. 1 Progression of nucleophilic substitution reaction of 1 with
different substrates at room temperature, based on HPLC analysis.
c
10426 Chem. Commun., 2011, 47, 10425–10427
This journal is The Royal Society of Chemistry 2011

View Online
Financial support from the European Research Council under
the European Community’s Seventh Framework Programme
(FP7/2008-2013) ERC grant agreement no. 208702; Region
Aquitaine; Ministere de la Recherche et de l’Enseignement
Superieur (P.B) is gratefully acknowledged.
´
´
Notes and references
1 (a) A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891;
(b) A. C. Benniston and G. Copley, Phys. Chem. Chem. Phys.,
2009, 11, 4124; (c) R. Ziessel, G. Ulrich and A. Harriman, New J.
Chem., 2007, 31, 496; (d) C. Nicoli, J. Baranski, S. Schlummer,
J. Palomo, M. Lumbierres-Burgues, M. Kahms, J. Kuhlmann,
S. Sanchez, E. Gratton, H. Waldmann and R. Winter, J. Am.
Chem. Soc., 2006, 128, 192; (e) F. Bestvater, E. Spiess,
G. Strobrawa, M. Hacker, T. Feurer, T. Porwol, U. Berchner-
Pfannschmidt, C. Wostlaw and H. Acker, J. Microsc., 2002,
208, 108; (f) C. Trieflinger, K. Rurack and J. Daub, Angew. Chem.,
Fig. 2 Single molecule emission of 1 in PMMA (lex = 488 nm, lem
>
500 nm): (a) emission trajectory of a single molecule (number of
photons detected per ms); (b–c) distribution for 82 single molecules
of: the total duration of emission before photobleaching (b); the decay
time (c); correlation between the decay time and the average number of
counts emitted before photobleaching (d).
Int. Ed., 2005, 44, 2288; (g) F. Bergstrom, I. Mikhalyov,
¨
P. Hagglof, R. Wortmann, T. Ny and L. B.-A. Johansson,
¨
J. Am. Chem. Soc., 2002, 124, 196.
¨
2 For examples of BODIPY–protein conjugates, see: (a) J. Lee,
S. Lee, D. Zhai, Y. Ahn, H. Yun Yeo, Y. Ling and T. Chang,
Chem. Commun., 2011, 47, 4508; (b) S. Niu, C. Massif, G. Ulrich,
R. Ziessel, P. Renard and A. Romieu, Org. Biomol. Chem., 2011,
9, 66; (c) D. Wang, J. Fan, X. Gao, B. Wang, S. Sun and X. Peng,
OFF states. For 50% of 82 molecules of 1 the peak emission
intensity reflecting the brightness reached at least 5.3 Â 103
photons sÀ1 detected, and up to 2.9 Â 104 photons sÀ1. These
values are in agreement with a maximum theoretical upper
limit of 2.9 Â 105 photons sÀ1 emitted,15,16 but lower due to
losses resulting from the random orientation of the molecules
relative to the excitation beam polarization and the instrumental
detection efficiency. From 50% of the molecules, we have
detected at least 2.1 Â 104 photons and in the best case up to
2.4 Â 105 photons before photobleaching (Fig. S19 in ESIw).
Considering the same limitations as above, this reflects the
measured 1/Fbleach = 3.7 Â 104 photons in solution and is in
agreement with known organic emitters.16 This emission occurs
within 11 seconds for 50% of the molecules and the duration
can reach 1 minute (Fig. 2b). The distribution of decay times
of 1 is represented in the bar chart in Fig. 2c, peaking around
4.9 ns and displaying some longer values around 6.8 ns. The
average decay time is 5.1 ns, with a standard deviation of 0.9 ns,
and is thus only slightly shorter than in THF solutions. This
could result from an enhanced non-radiative deactivation of
the excited states in the polymer environment.
J. Org. Chem., 2009, 74, 7675; (d) M. Isaksson, P. Hagglof,
¨
¨
P. Hakansson, T. Ny and L. Johansson, Phys. Chem. Chem. Phys.,
2007, 9, 3914.
3 (a) X. F. Zhou, Acta Crystallogr., Sect. E, 2010, 66, o757N;
(b) O. Galangau, C. Dumas-Verdes, R. Me
G. Clavier, Org. Biomol. Chem., 2010, 8, 4546.
4 (a) N. Basarıc, M. Baruah, W. Qin, B. Metten, M. Smet,
´
allet-Renault and
´
W. Dehaen and N. Boens, Org. Biomol. Chem., 2005, 3, 2755.
5 (a) D.-C. Wang, C. He, J.-L. Fan, W.-W. Huang and X.-J. Peng,
Acta Crystallogr., Sect. E: Struct. Rep. Online, 2007, 63, o2900;
(b) Y.-H. Yu, Z. Shen, H.-Y. Xu, Y.-W. Wang, T. Okujima,
N. Ono, Y.-Z. Li and X.-Z. You, J. Mol. Struct., 2007, 827, 130.
6 C. R. Becer, R. Hoogenboom and U. S. Schubert, Angew. Chem.,
Int. Ed., 2009, 48, 4900.
7 V. Leen, F. Schevenels, J. Cui, C. Xu, W. Yang, X. Tang, W. Liu,
W. Qin, W. M. De Borggraeve, N. Boens and W. Dehaen, Eur. J.
Org. Chem., 2009, 5920.
8 (a) C. Goze, G. Ulrich, L. J. Mallon, B. D. Allen, A. Harriman and
R. Ziessel, J. Am. Chem. Soc., 2006, 128, 10231; (b) G. Ulrich,
C. Goze, M. Guardigli, A. Roda and R. Ziessel, Angew. Chem., Int.
Ed., 2005, 44, 3694; (c) C. Tahtaoui, C. Thomas, F. Rohmer,
P. Klotz, G. Duportail, Y. Mely, D. Bonnet and M. Hibert, J. Org.
´
Chem., 2007, 72, 269; (d) Y. Gabe, T. Ueno, Y. Urano, H. Kojima
and T. Nagano, Anal. Bioanal. Chem., 2006, 386, 621;
(e) T. Rohand, M. Baruah, W. Qin, N. Boens and W. Dehaen,
Chem. Commun., 2006, 266.
Interestingly, a small population of molecules shows longer
decay times and emits a higher average total amount of photons
(Fig. 2d). In a previous study of single molecules in PMMA,17
two populations of emitters were also observed and the minor
one was attributed to molecules embedded in the more rigid
micro-environments of PMMA. 1 shows thus some sensitivity to
the environment while preserving suitable emissive properties.
In conclusion, perfluorophenyl rings can be readily and
selectively functionalised through C–N and C–S bond formation,
for example in a BODIPY chromophore derivative. These
conjugates are extremely photostable with high absorption
and emission in an organic solvent. In aqueous mixtures, high
selectivity for thiol vs. amine ( Z100-fold) conjugate formation
conspires with the 90-fold higher quantum yield to make the
thiol conjugate label particularly attractive. Applications in
labelling and staining are thus envisaged, which can in prin-
ciple similarly be applied to other fluorophores encompassing
the perfluorophenyl motif.
9 Fluorescein standard, Ff
= 0.95 in 0.1 M NaOH (aq);
J. H. Brannon and D. Magde, J. Phys. Chem., 1978, 82, 705.
10 (a) S. J. Strickler and R. A. Berg, J. Chem. Phys., 1962, 37,
814; (b) J. B. Birks, Photophysics of aromatic molecules, Wiley
Interscience, London, Date: 1970.
11 P. Meallier, S. Guittonneau, C. Emmelin and T. Konstantinova,
Dyes Pigm., 1999, 40, 95.
12 F. Puntoriero, F. Nastasi, S. Campagna, T. Bura and R. Ziessel,
Chem.–Eur. J., 2010, 16, 8832.
13 C. Giansante, G. Raffy, C. Schafer, H. Rahma, M.-T. Kao,
¨
A. G. L. Olive and A. Del Guerzo, J. Am. Chem. Soc., 2011, 133, 316.
=
14 Although the absorption and emission would be higher at lex
514 or 516 nm, this wavelength was not available on our micro-
scopy setup. 0.1 nM of 1 were dissolved in deoxygenated THF with
PMMA (10 mg mLÀ1) and spin-coated on a glass slide. Measure-
ments performed at 22 1C.
15 Assuming the same absorption cross-section and emission quantum
yield of 1 in PMMA and in solution.
16 W. E. Moerner and D. P. Fromm, Rev. Sci. Instrum., 2003,
74, 3597.
17 Y. Hou, A. M. Bardo, C. Martinez and D. A. Higgins, J. Phys.
Chem. B, 2000, 104, 212.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 10425–10427 10427