β-Cyclodextrin as a mimetic of the natural GFP-chromophore environment

Article abstract of DOI:10.1016/j.tetlet.2011.12.053

The green fluorescent protein (GFP) chromophore has been anchored to β-cyclodextrin (βCD) via a copper(I)-catalyzed azide-alkyne cycloaddition. The photophysical properties of this new GFP-CD derivative have been evaluated, showing the formation of a self

Full text of DOI:10.1016/j.tetlet.2011.12.053

Tetrahedron Letters 53 (2012) 973–976
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Tetrahedron Letters
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b-Cyclodextrin as a mimetic of the natural GFP-chromophore environment
Martina Cacciarini a, , Mogens Brøndsted Nielsen b, Elisa Martinez de Castro b, Lavinia Marinescu b,
⇑
Mikael Bols b
a Department of Chemistry, University of Florence, Via della Lastruccia 3-13, I-50019 Sesto F.no (FI), Italy
b Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
The green fluorescent protein (GFP) chromophore has been anchored to b-cyclodextrin (bCD) via a cop-
per(I)-catalyzed azide-alkyne cycloaddition. The photophysical properties of this new GFP-CD derivative
have been evaluated, showing the formation of a self-inclusion complex and enhancement of fluores-
cence of the GFP-chromophore covalently bound to the bCD. This enhancement of fluorescence by encap-
sulation and hence fixation of the chromophore in a binding pocket mimics the effect exerted by the
natural protein environment.
Received 16 November 2011
Revised 8 December 2011
Accepted 12 December 2011
Available online 19 December 2011
Keywords:
Green fluorescent protein (GFP)
Chromophore
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorescence
Cyclodextrins
Click reaction
Inclusion complexes
In the last decade, green fluorescent protein (GFP) has changed
from an almost unknown protein to a widely used tool as biological
marker in molecular biology and medicine, because of the special
fluorescent properties of its chromophore.1–5 The chromophore of
the wild type GFP (Fig. 1) shows peculiar spectroscopic properties:
two absorption peaks at 479 nm and at 397 nm, usually ascribed,
respectively, to a deprotonated chromophore (phenolate) and to
a protonated chromophore (neutral phenol), and one emission
peak at 508 nm. It is well documented that the photophysical
properties of GFP are the results of interactions between the chro-
mophore itself and its immediate protein environment, in fact the
denatured GFP and the isolated chromophore loose totally their
emissive properties.6–8
Previous gas-phase absorption studies of the isolated GFP phe-
nolate chromophore revealed a similar absorption maximum
(479 nm) to that of the chromophore inside the protein,9 while
the absorption is red-shifted relative to the chromophore in alco-
holic solution. This observation can be explained in two ways;
either the protein binding pocket does not exert any absorption
tuning or it exerts as many red-shifting interactions as blue-
shifting ones. To take the study a step further and evaluate the
minimal environment required for the GFP-chromophore to show
fluorescence and to display the photophysical properties shown
This macrocycle has a hydrophobic cavity that should allow inclu-
sion of the GFP-chromophore. The hydrophobic nature of this cav-
ity presents an environment that is not likely to interfere with the
absorption properties of the chromophore, hence resembling the
natural environment, but may influence the fluorescence proper-
ties by shielding the chromophore from solvent molecules.
Macrocyclic hosts of different nature, shape, and characteristics
from crown ethers, to cryptands, from cyclophanes, to calixarenes
and cucurbiturils have received great attention as new supramolec-
ular systems and materials.10–14 Among them, cyclodextrins are the
most important and promising macrocyclic hosts because of their
many advantages: water solubility, low cost, commercial availabil-
ity, and easy functionalization.15,16 Besides, chromophore-modified
CDs have been studied for a long time by many researchers and sev-
eral sensors bearing different type of chromophores have been
reported,17–19 such as azobenzene and stilbene photoswitches.20,21
Herein we present the synthesis and characterization of a
GFP-chromophore derivative that is covalently attached to b-cyclo-
dextrin through a triazole linker, easily affordable using a ‘click reac-
tion’.22 We show that there is an enhancement of fluorescence when
the GFP-chromophore is covalently bound to the cyclodextrin
enforcing encapsulation in its cavity.
The GFP-chromophore appended b-CD has been obtained
through a procedure that identifies a copper(I)-catalyzed azide-al-
kyne cycloaddition (CuAAC)23–25 as the key step. The synthetic
pathway to obtain the GFP-chromophore bearing an alkyne moiety
is depicted in Scheme 1. The synthesis starts from 4-[(Z)-4-acet-
oxybenzylidene]-2-methyloxazol-5(4H)-one 1, which was pre-
pared from N-acetylglycine and 4-hydroxybenzaldehyde.25
in natural environment, we developed
a system where the
GFP-chromophore is anchored to a b-cyclodextrin (bCD, Fig. 1).
⇑
Corresponding author. Tel.: +39 055 4573544; fax: +39 055 4573531.
E-mail addresses: martina.cacciarini@gmail.com, martina.cacciarini@unifi.it
(M. Cacciarini).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.053

974
M. Cacciarini et al. / Tetrahedron Letters 53 (2012) 973–976
allene moiety, respectively, were detected, and a resonance at
205.8 ppm corresponding to the central sp carbon supported and
confirmed the structure of 3b. Numerous attempts to change the
reaction conditions to avoid the formation of the allene 3b were
not successful.
The
per-O-acetylated-6-azido-6-deoxy-b-cyclodextrin
4
(Scheme 2) was prepared following a three-step procedure previ-
ously reported28 starting from commercial b-cyclodextrin. The
copper-catalyzed cycloaddition was performed between the azide
4 and the mixture 3a–b29 (enriched in 3a, 2:1 ratio) in the presence
of copper iodide and diisopropylamine in acetonitrile at room tem-
perature for 65 h to give 5 (78% yield),30 which was in turn depro-
tected under basic conditions to furnish the final GFP-CD 6.31
Furthermore the synthesis of compound 7 (Scheme 3),32 bear-
ing the GFP-chromophore linked to a triazole ring but missing
the CD, has been accomplished for direct comparison of the fluo-
rescence properties of 6.
The absorption and emission spectra of 6 (0.03 mM) were re-
corded in different buffer solutions. The absorption spectra
(Fig. 2) at pH 4 and 7 are characterized by a maximum absorption
at 371 and 372 nm, respectively, and the one at pH 10 by a kmax at
432 nm. The redshift is determined by the deprotonation of the
phenol group on the chromophore of 6 and this is confirmed by
the absorption spectrum of 7 (Fig. 3) that presents an analogous
redshift at pH 10. Since 7 is soluble in water only at pH 10, absorp-
tion spectra were measured in the three selected buffered solu-
tions with the addition of commercial bCD. Indeed, bCD can
include in its cavity the aromatic moiety of 7 forming a complex,
hence bringing partially 7 in solution. This property has been evi-
denced at first by proton NMR spectra (see Supplementary data).
In fact, addition of solid 7 to a 5 mM solution of bCD allowed us
to register a spectrum that contains the signals of the model com-
pound 7, moreover a shift of the inner protons (H-3 and H-5) of
bCD indicates the presence of a guest in the hydrophobic cavity
Figure 1. GFP-chromophore and bCD structures.
Compound 1 was treated with an excess of propargylamine
(5 equiv) to give the ring-opened propargyl amide 2 in a 68% yield
(Scheme 1). As expected,26 under the described conditions deacet-
ylation occurred to afford directly 2 with the unprotected phenol
group. On the contrary use of minor amounts of propargylamine
led to a mixture of 2 and its direct precursor, acetylated on the phe-
nolic moiety. Formation of the imidazolone ring on 3 was accom-
plished by treatment of 2 with potassium carbonate in refluxing
ethanol in the presence of molecular sieves (4 Å). Unfortunately,
heating under basic conditions provoked a well-known27 base-cat-
alyzed rearrangement of the acetylene through a prototropic prop-
argylic rearrangement and led to a hardly separable mixture of
alkyne 3a and allene 3b. The allene structure was assigned by
means of the proton and the carbon NMR spectra, which reflect
typical signals in terms of resonances and multiplicity (see Supple-
mentary data). In fact, a doublet at 5.51 ppm and a triplet at
6.73 ppm corresponding to the terminal CH2 and the CH–N of the
Scheme 1. Synthesis of the GFP-chromophore alkyne 3a.
Scheme 2. Synthesis of GFP-CD 6.

M. Cacciarini et al. / Tetrahedron Letters 53 (2012) 973–976
975
of the macrocycle. Besides, Figure 3 shows that addition of com-
mercial bCD does not have any influence on the absorption spec-
trum of 7 at pH 10.
The emission spectra of 6 (0.03 mM) are reported in Figure 4
and show a maximum emission value at 466, 462, and 497 nm,
respectively, at pH 4, 7 and 10. In the same figure the emission
spectra of 7 (0.03 mM) in the absence and presence of bCD (1/1 ra-
tio) at pH 10 have been reported for direct comparison. The absorp-
tion maximum of 6 and 7 at different pH were used as excitation
wavelength (kex), that is, 371, 372, and 432 nm, respectively, at
pH 4, 7, and 10 for 6, and 428 nm for 7. The emission spectra mea-
sured at the same concentration (0.03 mM) revealed that the fluo-
rescence of 6 at pH 10 is more than two times higher than that of 7
(Fig. 4, thick curves). Moreover addition of commercial bCD to 7 in
an equimolar amount does not affect the emission properties at pH
10. The covalent attachment of the GFP-chromophore to the CD en-
hances the proportion of encapsulated chromophore (when of
intramolecular origin), hereby preventing fluorescence quenching
by surrounding water molecules. Moreover, it should limit out-
of-plane deformations and translational motion of the chromo-
phore inside the cavity. Thus, the conjugate 6 mimics the natural
protein environment in regard to its ability to enhance the fluores-
cence of the chromophore.
To obtain some evidence regarding the inclusion ability of 6, the
variation of absorption and emission intensities of 6 has been reg-
istered upon addition of 1-adamantanol, a guest well recognized
by bCD cavity (Figs. 5 and 6).33 The absorption intensity increases
slightly while, importantly, the emission decreases in the presence
of guest. As reported by Ueno and coworkers,34 usually the fluores-
cence intensity is enhanced when the fluorophore locates in the
hydrophobic environment inside the CD cavity, while it is de-
pressed when it locates in the aqueous environment. Hence the
guest-induced fluorescence decrease of 6 suggests that the GFP
moiety is excluded by 1-adamantanol from the hydrophobic CD
cavity to the bulk water environment and therefore in the absence
of the guest, the GFP-CD 6 forms a self-inclusion complex.35
In summary, the results reported herein show that the GFP-
chromophore group of GFP-CD 6 resides in the bCD cavity, forming
a self-inclusion complex that can be disrupted upon the addition of
1-adamantanol expelling the GFP-chromophore into the water
environment. Transfer of the chromophore group from inside of
the cavity to outside leads to a decrease of the fluorescence inten-
sity and to a slight increase of the absorption intensity. Moreover
the self-inclusion complex GFP-CD 6 showed an effective fluores-
cence enhancement in comparison to the chromophore itself with
no CD appended. Thus, shielding the GFP chromophore against
water molecules by encapsulation and fixation in a covalently
Scheme 3. Synthesis of the model compound 7.
0.60
0.40
0.20
0.00
GFP-CD 6 - pH 4
GFP-CD 6 - pH 7
GFP-CD 6 - pH 10
300
350
400
450
500
550
Wavelength / nm
Figure 2. UV–vis absorption spectra (pH 4, 7 and 10 ꢀ 0.03 mM) of 6.
compound 7 + CD - pH 4
compound 7 + CD - pH 7
compound 7 + CD - pH 10
compound 7 - pH 10
0.60
0.40
0.20
0.00
300
350
400
450
500
550
Wavelength / nm
Figure 3. UV–vis absorption spectra of 7 at pH 10 (0.03 mM) and of 7 in presence of
bCD at pH 4, 7 and 10.
1.00
GFP-CD 6
1.60E+06
GFP-CD 6 - pH 10
GFP-CD 6 -pH 4
1.40E+06
+ 1-adamantanol (sat.sol.)
0.80
GFP-CD 6 - pH 7
compound 7 - pH 10
1.20E+06
0.60
0.40
0.20
0.00
compound 7 + CD - pH 10
1.00E+06
8.00E+05
6.00E+05
4.00E+05
2.00E+05
0.00E+00
400
450
500
550
600
650
300
350
400
450
500
550
Wavelength / nm
Wavelength / nm
Figure 4. Emission spectra (0.03 mM) of 6 at different pH levels and of 7 in the
absence and presence of equimolar amount of bCD at pH 10.
Figure 5. UV–vis absorption spectra (pH 10 ꢀ 0.03 mM) of compound 6 and 6 upon
addition of 1-adamantanol.

976
M. Cacciarini et al. / Tetrahedron Letters 53 (2012) 973–976
17. Dsouza, R. N.; Pischel, U.; Nau, W. M. Chem. Rev. 2011. ASAP.
1.60E+06
1.40E+06
1.20E+06
1.00E+06
8.00E+05
6.00E+05
4.00E+05
2.00E+05
0.00E+00
18. (a) Ueno, A.; Kuwabara, T.; Nakamura, A.; Toda, F. Nature 1992, 356, 136–137;
(b) Ueno, A. Supramol. Sci. 1996, 3, 31–36.
19. Ogoshi, T.; Harada, A. Sensors 2008, 8, 4961–4982.
20. Tamesue, S.; Takashima, Y.; Yamaguchi, H.; Shinkai, S.; Harada, A. Angew.
Chem., Int. Ed. 2010, 49, 7461–7464.
21. Wang, Z.; Takashima, Y.; Yamaguchi, H.; Harada, A. Org. Lett. 2011, 13, 4356–
4359.
22. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021.
23. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064.
24. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596–2599.
25. For a recent paper applying click chemistry to CD, see: Menuel, S.; Azaroual, N.;
Landy, D.; Six, N.; Hapiot, F.; Monflier, E. Chem. -Eur. J. 2011, 3949–3955.
26. Petersen, M. Å.; Riber, P.; Andersen, L. H.; Nielsen, M. B. Synthesis 2007, 3635–
3638.
GFP-CD 6
GFP-CD 6 + 1-adamantanol
(sol.sat.)
400
450
500
550
600
650
Wavelength / nm
27. Favorsky, A. J. Prakt. Chem. 1888, 37, 382–395.
28. Sallas, F.; Marsura, A.; Petot, V.; Pinter, I.; Kovacs, J.; Jicsinszky, L. Helv. Chim.
Acta 1998, 81, 632–645.
Figure 6. Emission spectra (pH 10 ꢀ 0.03 mM ꢀ kex: 432 nm) of compound 6 and 6
upon addition of 1-adamantanol.
29. 3a–b have been separated for analytical purposes but used as a 2:1 mixture in
the cycloaddition reaction. Although an allene can in principle react with azide
(see: Wedegaertner, D. W.; Kattak, R. K.; Harrison, I.; Cristie, S. K. J. Org. Chem.
1991, 56, 4463–4467), no traces of cycloaddition product were found. Data for
3a: yellow powder; mp 187–190 °C; 1H NMR (300 MHz, CDCl3): 8.00–7.98 (m,
2H, Ar), 7.12 (s, 1H, CH olef.) 6.86–6.83 (m, 2H, Ar), 4.40 (d, J = 2.5 Hz, 2H,
CH2N), 2.48 (s, 3H, CH3), 2.30 (t, J = 2.5, 1H, CH) ppm. 13C NMR (75 MHz, CDCl3):
169.5, 160.1, 158.6, 135.5, 134.5, 129.2, 126.4, 116.0, 77.1, 72.7, 29.4, 15.6 ppm.
HRMS-ESI: m/z [M+H]+ calcd for C14H13N2O2+: 241.0977; found 241.098.
linked bCD cavity enhances its fluorescence; an effect which
resembles the natural protein environment. The absorption prop-
erties of the chromophore on 6 were not altered significantly by
inclusion in the CD cavity, which likely reflects the fact that the
phenol end of the chromophore is exposed to the water solution.
30. Data for 5: yellow solid; mp 151–154 °C. ½a D20
ꢁ
: +90.75 (c = 1, CHCl3). 1H NMR
(500 MHz, 11 mM in CDCl3): 8.06 (d, J = 8.8 Hz, 2H, Ar), 7.69 (s, 1H), 7.06 (s,
1H), 6.87 (d, J = 8.8 Hz, 2H, Ar), 5.61 (d, J = 3.9 Hz, 1H, H-1A), 5.38–5.19 (m, 9H),
5.18 (d, J = 4.2 Hz, 1H, H-1B), 5.12 (d, J = 4.2 Hz, 1H, H-1C), 5.11 (d, J = 3.9 Hz,
1H, H-1D), 5.09 (d, J = 3.9 Hz, 1H, H-1E), 5.07 (d, J = 3.9 Hz, 1H, H-1F), 5.04 (d,
J = 3.7 Hz, 1H, H-1G), 4.99–4.96 (m, 1H), 4.94 (dd, J = 8.5, 3.9 Hz, 1H, H-2A),
4.89–4.54 (m, 12H), 4.48–4.46 (m, 1H, H-6C), 4.33–4.09 (m, 14 H), 3.77–3.70
(m, 5H), 3.65 (t, J = 8.5 Hz, 1H, H-3C), 3.58 (t, J = 9.0 Hz, 1H, H-3G), 2.52 (s, 3H,
Ac), 2.154 (s, 3H, Ac), 2.149 (s, 3H, Ac), 2.141 (s, 3H, Ac), 2.130 (s, 3H, Ac), 2.127
(s, 3H, Ac), 2.107 (s, 6H, 2Ac), 2.099 (s, 6H, 2Ac), 2.096 (s, 6H, 2Ac), 2.092 (s, 3H,
Ac), 2.066 (s, 3H, Ac), 2.062 (s, 6H, 2Ac), 2.054 (s, 3H, Ac), 2.051 (s, 3H, Ac), 2.04
(s, 3H, Ac), 2.03 (s, 3H, Ac), 2.02 (s, 3H, Ac) ppm. 13C NMR (125 MHz, 11 mM in
CDCl3): 171.08, 171.02, 170.98, 170.95, 170.90, 170.87, 170.86, 170.80, 170.75,
170.69, 170.67, 170.61, 170.56, 169.88, 169.73, 169.71, 169.66, 169.61, 169.51,
165.01, 161.07, 158.35, 142.91, 136.50, 134.63, 128.16, 127.06, 126.10, 116.13,
97.40, 97.23, 97.11, 96.96, 96.88, 96.85, 96.67, 71.73, 71.46, 71.19, 71.07, 70.90,
70.68, 70.37, 70.11, 69.90, 69.76, 69.59, 63.01, 62.82, 62.68, 62.51, 49.54, 35.84,
21.28, 21.09, 21.07, 21.04, 21.01, 21.00, 20.96, 20.94, 20.90, 20.85, 16.19 ppm.
Acknowledgments
We acknowledge Mr. Martin Rosenberg for assistance with the
spectroscopic investigations, Miss Brunella Innocenti for HPLC
analysis and Professor Luca Calamai for helpful discussions on
mass spectra. The Danish Research Council for Independent Re-
search | Natural Sciences (#272-08-0540) is acknowledged for
financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.12.053.
MALDI-TOF (ditranol): calcd for
C96H121N5O56: 2239.677 (93%), 2240.681
(100%); found 2239.261, 2240.219 and calcd for C96H120N5O56Na: 2261.659
(93%), 2262.663 (100%); found 2261.237, 2262.244.
References and notes
31. Data for 6: yellow solid; mp dec >150 °C. ½a D20
ꢁ
: +89.75 (c = 0.1, H2O). 1H NMR
(500 MHz, D2O): 7.94–7.77 (m, 3H), 6.95–6.80 (m, 3H), 5.11–4.85 (m, 6H),
4.68–4.51 (m, 5H), 3.96–3.29 (m, 39H), 3.18 (d, J = 10.0 Hz, 1H), 2.82 (d,
J = 10.0 Hz, 1H), 2.38 (s, 3H) ppm. 13C NMR (125 MHz, D2O): 170.93, 142.17,
134.78, 133.68, 131.69, 125.12, 122.78, 117.79, 114.93, 102.05, 101.90, 101.81,
101.45, 82.89, 81.07, 81.02, 80.93, 80.77, 80.60, 73.05, 72.94, 72.68, 72.12,
72.05, 71.99, 71.91, 71.77, 70.54, 60.16, 59.22, 50.80, 35.29, 15.26 ppm. MALDI-
TOF (DHB): calcd for C56H81N5O36Na: 1422.46 (100%), 1423.46 (63%); found
1423.66, 1424.66.
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