Synthesis and photochemical properties of a light-activated fluorophore to label His-tagged proteins

Article abstract of DOI:10.1039/b716486f

Rapid and efficient light-induced fluorescence enhancement is demonstrated on a DMNPB-"caged" coumarin derivative carrying a His-tag recognition motif. The Royal Society of Chemistry.

Full text of DOI:10.1039/b716486f

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Synthesis and photochemical properties of a light-activated fluorophore
to label His-tagged proteinswz
ab
Clelia Orange, Alexandre Specht,*a David Puliti,a Elias Sakr,a Toshiaki Furuta,c
´
Barbara Winsorb and Maurice Goeldnera
Received (in Cambridge, UK) 26th October 2007, Accepted 13th December 2007
First published as an Advance Article on the web 11th January 2008
DOI: 10.1039/b716486f
Rapid and efficient light-induced fluorescence enhancement is
demonstrated on a DMNPB-‘‘caged’’ coumarin derivative
carrying a His-tag recognition motif.
weakly so. Photoactivation removes the protecting group and
abruptly switches on the fluorescence of the parental dye.
Desirable properties for ‘‘caged’’ fluorophores include fast
release of the fluorophore and a large enhancement of the
fluorescence in response to brief irradiation with reasonable
photostability of the released dye to resist photobleaching.
‘‘Caged’’ fluorophores initially described on fluorescein8a and
rhodamine8b derivatives have been reviewed8a and prompted
innovative cell biology work.8c Subsequently, 7-hydroxycou-
marin derivatives were caged through Williamson coupling
reactions as o-nitrobenzyl ether derivatives.9 These molecules
allowed a 200-fold fluorescence enhancement after UV photo-
lysis and displayed an increased uncaging cross section over
previously reported ‘‘caged’’ fluorophores.9 Recently, novel
ortho-nitrobenzyl fluorescein derivatives (‘‘caged’’ Tokyo-
Greens) displaying a large fluorescence enhancement were
described.10
Studies of protein localization and movement in living cells are
most commonly achieved by expressing the protein as a fusion
to a fluorescent protein (FP).1 This genetic targeting allows
high sensitivity of detection with no background except in-
tracellular autofluorescence. However, FP’s are full size pro-
teins of at least 220 aa, which may severely perturb folding,
trafficking and functioning of the proteins studied.2 This
limitation can be overcome by using small genetically encoded
peptide tags (20 aa) and complementary small organic fluor-
escent probes3 with sufficient specificity and affinity to be used
in living cells. Since the pioneering work reported by Tsien and
co-workers, describing a tetracysteine motif (-Cys-Cys-X-X-
Cys-Cys-)/bisarsenical ligand (FlAsH) pair and its application
to bio-imaging experiments in living cells;4 many peptide tag/
probe pairs have been developed. For example, the oligo-
aspartate sequence (D4 tag) and the corresponding multi-
nuclear Zn(II) complexes (Zn(II)-DpaTyr) have been used for
the labelling and fluorescent imaging of a membrane-bound
receptor protein.5 The conventional His tag -(His)n-/Ni(II)-
NTA pair was used for fluorescent labelling of a protein on a
cell surface.6 Light-activated fluorescent molecules (also called
‘‘caged’’ fluorophores), including photoactivatable GFP’s,7
are important research tools for tracking the spatiotemporal
dynamics of molecular movements in biological systems.8a The
general strategy in masking fluorescence is to perturb the
electronic structure by attachment of the photoremovable
group to make the molecule either non-fluorescent or very
We report here a new class of ‘‘caged’’ 7-hydroxycoumarin
fluorophores using a DMNPB ether as a photoremovable
protecting group which was initially described for the caging
of carboxylic acids.11 This probe displayed high uncaging
cross section at 365 nm and showed a rapid enhancement of
fluorescence after irradiation. A Mitsunobu coupling reaction
was used for an efficient incorporation of the caging group as
an ether derivative and convenient chemical modifications
allowed the incorporation of the Ni-nitrilotriacetic (Ni2+
-
NTA) His-tag recognition motifs. Since the penetration assays
of this molecule into HeLa cells remained inconclusive, we
designed and synthesized a new cell-permeable and non-toxic
coumarin derivative (7-acetylcoumarin-Ni-NDA, 8a-Ni2+
)
able to penetrate into HeLa cells and regenerate the coumar-
in-Ni-NTA.
The synthesis of 7-DMNPB-coumarin-NTA (9), coumarin-
NTA (10), 7-acetylcoumarin-NTA (8a) and 7-acetylcoumarin-
NDA (8b) are outlined in Fig. 1. Starting from 2,4-dihydroxy-
5-chlorobenzaldehyde (1)12 we prepared 6-chloro-7-hydroxy-
coumarin 3-carboxylate (2) in one step. Reaction using stan-
dard DCC/NHS coupling conditions between 2 and (tert-
butoxy)-N,N-bis(tert-butoxycarbonylmethyl)-L-lysine (4a)13
or methyl-N,N-bis(tert-butoxycarbonylmethyl)-L-lysine (4b),
obtained in two steps from appropriately protected L-lysine
derivatives (3a,b), gave the tert-butyl ester-protected NTA and
NDA coumarins (5a,b). Coupling under Mitsunobu condi-
tion14 of the tert-butyl ester-protected NTA-coumarin (5a)
and the threo diastereoisomers of 3-(3,4-dimethoxyphenyl)bu-
tan-2-ol (DMNPB) (11),11 a photoremoval protecting group,
a
´
Universtte Louis Pasteur/CNRS UMR 7175-Laboratoire de chimie
bioorganique, Faculte de pharmacie, 74 route du rhin, 67400 Illkirch,
France. E-mail: specht@bioorga.u-strasbg.fr; Fax: +33 390244306;
Tel: +33 390244166
´
b
´
Universtte Louis Pasteur/CNRS UMR7156 Genetique moleculaire,
Genomique et Microbiologie, Dept. Genetique moleculaire et
´
´
´
´
´
´
´
´
´
cellulaire, IPCB, 21 rue Rene Descartes, 67084 Strasbourg, France
c Toho University, 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan
w Abbreviations: DMNPB, [3-(4,5-dimethoxy-2-nitrophenyl)but-2-yl];
DpaTyrs, (2,20-dipicolylamine) complex based on the L-tyrosine scaf-
fold; FCS, fluorescent correlation spectroscopy; FlAsH, fluorescein
arsenical helix binder; NDA, nitrilodiacetic acid; NPE, 2-(nitrophenyl)-
ethyl; NTA, nitrilotriacetic acid.
z Electronic supplementary information (ESI) available: chemical
synthesis and characterization, one and two photon photolysis, quan-
tum yield determination, laser flash photolysis, Cell membrane perme-
ability studies and FCS studies. See DOI: 10.1039/b716486f
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1217–1219 | 1217

View Article Online
Fig. 1 Synthesis of 7-DMNPB-coumarin-NTA, 7-acetylcoumarin-NTA and 7-acetylcoumarin-NDA.
plus a final deprotection of the tert-butyl protecting groups,
gave our new ‘‘caged’’ fluorophore: 7-DMNPB-coumarin-
NTA (9) with good yield. It is noteworthy that our coupling
reaction avoids synthetic problems due to the use of strong
basic conditions which do not accommodate the halogenated
o-nitro benzyl derivatives under Williamson reaction.15 To test
cell permeability to alkylated coumarin-NTA-Ni2+ probes, 7-
acetylcoumarin-NTA (8a) and 7-acetylcoumarin-NDA (8b)
were synthesized by acylation followed by tert-butyl deprotec-
tion of (5a) and (5b), respectively. The coumarin NTA dye (10)
was obtained after removal of the tert-butyl protecting groups
of 5a.
An almost quantitative ( Z 95%) formation of 10 after full
conversion of 9 was demonstrated by HPLC. The quantum
yield for coumarin-NTA (10) release from 9 was determined
by competition with the 2-(nitrophenyl)ethyl-ATP16 reference
molecule. A disappearance quantum yield of 0.49 could be
determined from this experiment by HPLC analysis. Our new
‘‘caged’’-coumarin derivative displayed high quantum yields
for uncaging, which conferred a high photolytic efficiency to
this molecule in the near-UV, generating robust change in
fluorescence quantum yield and conferring a high contrast
optical signal with minimum background after irradiation.
This dye absorbs maximally at 408 nm with an extinction
coefficient of 25 000, and a fluorescent emission at 450 nm with
a quantum yield of 0.95 measured in an aqueous buffer
(0.1 M phosphate buffer pH 7.4).
Table 1 Summary of the photochemical properties of 9 and 10 in
0.1 M phosphate buffer pH 7.4 (ff and fu fluorescence and uncaging
quantum yield, du two-photon uncaging action cross section, k first-
order uncaging rate constant, GM, Goeppert–Mayer)
The photochemical properties of 9 and 10 are summarized
in Table 1. The photolytic release of 10 from precursor 9 was
analyzed by UV and HPLC (see ESI,w Fig. S1) after irradia-
tion at 315, 365 or 403 nm in neutral buffered media. The UV
analysis showed a decrease in absorbance at 355 nm and an
increase at 406 nm, corresponding to the disappearance of 9
and the formation of 10. The presence of an isosbestic point at
375 nm was indicative of a homogeneous photolytic reaction.
Compound
9
10
lmax (excitation)/nm
355
15 000
450
408
25 000
450
0.95
—
e(lmax)/Mꢁ1 cmꢁ1
lmax (emission)/nm
ff
fu
du (740 nm)/GM
k/sꢁ1
o0.009
0.49
0.21
—
—
1.7 ꢂ 105
ꢀc
This journal is The Royal Society of Chemistry 2008
1218 | Chem. Commun., 2008, 1217–1219

View Article Online
His-tagged protein and changed the physical properties of
diffusion of the fluorescent probe.
In conclusion, we have described here the synthesis and the
characterization of a new ‘‘caged’’-coumarin bearing NTA,
the His tag recognition motif. Our new ‘‘caged’’ fluorophore
showed a rapid and large fluorescence enhancement after near-
UV activation (315–406 nm). For biological application we
also developed a strategy to obtain cell membrane permeable
Ni2+-NTA dyes.
This work was supported by the Universite Louis Pasteur,
´
the CNRS (ACI-DRAB03/135); and ANR (PCV07-188521)
and a MNERT (C.O.) fellowship. The authors thank Prof.
Jakob Wirz for the laser flash photolysis experiments, Pascal
Kessler and the imaging center technology platform of the
IGBMC at Strasbourg for the bio-imaging experiments,
Dr Pascal Didier for FCS experiments, Pascale Buisine for
mass analysis and HPLC purification, Adeline Martz and
Betty Heller for cell cultures and Prof. Carsten Schultz for
helpful discussions.
Fig. 2 Fluorescence imaging of HeLa cells loaded with cell permeable
coumarins 6a and 8b-Ni2+ (lemission: 410–510 nm).
We checked the two-photon sensitivity of 9 by irradiating
the sample with a femtosecond-pulsed mode-locked Ti-sap-
phire laser using a previously described method,17 and a cross
section of 0.21 GM was measured at 740 nm. Surprisingly, this
value remained lower than the value described for NPE
‘‘caged’’ coumarin9 despite a perfect overlap of the absor-
bances of the coumarin and caging group. Fragmentation
kinetics were determined after laser photolysis at 350 nm by
analysis of coumarin 10 formation at 410 nm. A monoexpo-
nential absorbance increase at 410 nm in the microsecond time
range (t1/2 = 4 ms) was observed, showing very fast release of
10 and corresponding presumably to the rate-limiting step of
o-quinonoid aci-nitro decay.18
Notes and References
1 B. N. Giepmans, S. R. Adams, M. H. Ellisman and R. Y. Tsien,
Science, 2006, 312, 217–224.
2 O. Agbulut, A. Huet, N. Niederlander, M. Puceat, P. Menasche
and C. Coirault, J. Biol. Chem., 2007, 282, 10465–10471.
3 B. A. Griffin, S. R. Adams and R. Y. Tsien, Science, 1998, 281,
269–272.
4 G. Gaietta, T. J. Deerinck, S. R. Adams, J. Bouwer, O. Tour, D.
W. Laird, G. E. Sosinsky, R. Y. Tsien and M. H. Ellisman,
Science, 2002, 296, 503–507.
5 A. Ojida, K. Honda, D. Shinmi, S. Kiyonaka, Y. Mori and I.
Hamachi, J. Am. Chem. Soc., 2006, 128, 10452–10459.
6 (a) E. G. Guignet, R. Hovius and H. Vogel, Nat. Biotechnol., 2004,
22, 440–444; (b) E. G. Guignet, J. M. Segura, R. Hovius and H.
Vogel, ChemPhysChem, 2007, 8, 1221–1227.
To test the cell-membrane permeability of these derivatives
for further imaging applications in living cells, we selected the
7-acylated-coumarins as model compounds given their rapid
cellular hydrolysis into the corresponding fluorescent 7-hydroxy-
coumarin derivatives. Effectively, after 30 min incubation of
1 mM of the tris-tert-butoxyesters 6a on a HeLa cell culture, a
strong coumarin fluorescent signal was observed inside the
cytoplasm of cells with 405 nm excitation, confirming that 6a
entered into the cell and that intracellular hydrolysis released
the fluorescent dye 5a (Fig. 2). A similar experiment using the
8a-Ni2+ complex, did not reveal any cell permeation since no
cytoplasmic fluorescent signal was observed. Because this
NTA-Ni2+ complex carries a negative charge, we synthesized
the NDA-series, a neutral precursor complex of the NTA-
Ni2+ motif by masking one carboxylic acid function of NTA
as a methyl ester group. Following hydrolysis of the methyl
ester by cellular esterases, the His-tag recognition NTA-Ni2+
motif would be regenerated in situ. Indeed, after 30 min
incubation with 20 mM of the 8b-Ni2+ complexes on a HeLa
cell culture, a coumarin fluorescent signal was observed with
405 nm excitation (Fig. 2). This fluorescent signal, although
weaker than that obtained with 6a, was stable over time.
Fluorescence measured after a long UV excitation time (1 h),
demonstrated the non-toxicity and the fluorescence stability of
our probe (data not shown). Finally a fluorescence correlation
spectroscopy (FCS) experiment was performed to determine
whether the Ni2+-NTA-coumarin (10) was able to bind to a
His-tagged protein, Bzz1p. For the Ni2+-NTA-coumarin
alone, the measured time of diffusion was 0.08 ms and when
a His-tagged protein was added, this time increased to 0.156
ms. This indicates that the Ni2+-NTA-coumarin bound to the
7 G. H. Patterson and J. Lippincott-Schwartz, Science, 2002, 297,
1873–1877.
8 (a) T. J. Mitchison, K. E. Sawin, J. A. Theriot, K. Gee and A.
Mallavarapu, Methods Enzymol., 1998, 291, 63–78; (b) J. Ottl, D.
Gabriel and G. Marriott, Bioconjugate Chem., 1998, 9, 143–151; (c)
J. A. Theriot and T. J. Mitchison, Nature, 1991, 352, 126–131.
9 Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez and W. H.
Li, J. Am. Chem. Soc., 2004, 126, 4653–4663.
10 T. Kobayashi, Y. Urano, M. Kamiya, T. Ueno, H. Kojima and T.
Nagano, J. Am. Chem. Soc., 2007, 129, 6696–6697.
11 A. Specht, J. S. Thomann, K. Alarcon, W. Wittayanan, D. Ogden,
T. Furuta, Y. Kurakawa and M. Goeldner, ChemBioChem, 2006,
7, 1690–1695.
12 E. Bui, J. P. Bayle, F. Perez, L. Liebert and J. Courtieu, Liq. Cryst.,
1990, 8, 513–526.
13 S. Lata, A. Reichel, R. Brock, R. Tampe and J. Piehler, J. Am.
Chem. Soc., 2005, 127, 10205–10215.
14 O. Mitsunobu, Synthesis, 1981, 1, 1–28.
15 (a) S. Watanabe, T. Sueyoshi, M. Ichihara, C. Uehara and M.
Iwamura, Org. Lett., 2001, 3, 255–257; (b) L. Peng and M.
Goeldner, J. Org. Chem., 1996, 61, 185–191.
16 J. W. Walker, G. P. Reid, J. A. McCray and D. R. Trentham, J.
Am. Chem. Soc., 1988, 110, 7170–7177.
17 T. Furuta, S. S. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee, E.
M. Callaway, W. Denk and R. Y. Tsien, Proc. Natl. Acad. Sci.
USA, 1999, 96, 1193–1200.
18 S. Walbert, W. Pfleiderer and U. E. Steiner, Helv. Chim. Acta,
2001, 84, 1601–1611.
ꢀc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 1217–1219 | 1219