Synthesis and photochemistry of pH-sensitive GFP chromophore analogs

Article abstract of DOI:10.1016/j.tetlet.2010.12.082

GFP chromophore analogs (7a-e, 8, and 10a,b) containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-methyl-2-pyridyl-groups were synthesized and their fluorescence spectra recorded in the pH range 1-7. NMR studies showed that protonation of 8 (2-thieny

Full text of DOI:10.1016/j.tetlet.2010.12.082

Tetrahedron Letters 52 (2011) 2224–2227
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Tetrahedron Letters
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Synthesis and photochemistry of pH-sensitive GFP chromophore analogs
Alan R. Katritzky a, , Megumi Yoshioka-Tarver a,b, Bahaa El-Dien M. El-Gendy a,c, C. Dennis Hall a
⇑
a Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611, USA
b Cotton Chemistry and Utilization, Southern Regional Research Center, USDA—ARS, New Orleans, Louisiana 70124, USA
c Department of Chemistry, Faculty of Science, Benha University, Benha, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 24 December 2010
GFP chromophore analogs (7a–e, 8, and 10a,b) containing 2-thienyl-, 5-methyl-2-furyl-, 2-pyrryl, and 6-
methyl-2-pyridyl-groups were synthesized and their fluorescence spectra recorded in the pH range 1–7.
NMR studies showed that protonation of 8 (2-thienyl system) inhibited photoisomerization (Z–E) about
the exocyclic double bond but that protonation of 7c (E + Z) (2-pyrryl system) gave only 7cE. Fluorescence
studies revealed enhancement of fluorescence intensity of 7c and 7b,e (furyl system) below pH 2.5 and
gave a similar result for 10a (pyridyl system) below pH 6. Quantum yields at pH 1 were low, probably due
to excited state proton transfer (ESPT).
Keywords:
GFP chromophores
Fluorescence
Photoisomerization
Hydrogen bonding
15N NMR
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Zelewsky and co-workers showed that the proton, the smallest
known cation, can act as a coordinating center and fix bipyridine
Fluorescent peptide labeling is useful for monitoring biological
activity since a fluorophore in a peptide or a protein enables li-
gands, inhibitors, and antigens to be detected at low concentra-
tion.1 Natural aromatic amino acids (Phe, His, Trp, and Tyr) play
key roles in the recognition of receptors and have frequently been
replaced by unnatural aromatic amino acids in bioactive peptides.2
Green fluorescent protein (GFP) chromophore 1, Scheme 1a, and
similar proteins (CFP or YFP) are well established as fluorescent
markers for monitoring biological activity because they have high
light emission (quantum yields up to Uf = 0.8) and work well both
in vitro and living mammalian cells.3 However, the large size (up to
238 amino acids) of GFP can cause misfolding or other structural
changes in target proteins. Unlike the chromophore of wild-type
GFP, which is surrounded by its protein sequence (1–64 and 68–
238) and stabilized as the Z-isomer,3c,4 the GFP model chromoph-
ores of type 2 show only low fluorescence at 20 °C due to Z–E
photoisomerization at the exo-methylene group (Scheme 1b).5 Arai
et al. demonstrated that hemi-indigo derivative 36 exists as the Z-
isomer stabilized by six-membered ring intramolecular hydrogen
bonding, thus preventing or minimizing photoisomerization
(Scheme 1c).The GFP chromophore analog 4 is also stabilized as
the Z-isomer by boron ligation and shows high fluorescent activity
ligands in a helical conformation.7b We reasoned, therefore, that
molecules of types 7, 8, and 10, might be stabilized as the Z-isomer
by protonation of the imidazolinone nitrogen (or pyridine N in the
case of 10a) and subsequent hydrogen bonding with the hetero-
atom of the adjacent heterocyclic ring. The objective of the work
was to test this hypothesis and monitor the effect of protonation
on fluorescence activity.
2. Results and discussion
2.1. Preparation of imidazolinone chromophores 7a–e and 8
Azalactones 6a–e were each synthesized by reaction of hippuric
acid 5a or 2-(2-naphthamido)acetic acid 5b with the appropriate
aldehyde in the presence of sodium acetate and acetic anhydride
(Scheme 2).8 Compounds 6a–e reacted under microwave condi-
tions with N,N-dimethylethylenediamine to give 7a–e in yields of
30–81% (Table 1).
Compound 8 was also synthesized from 6a and p-toluidine in
56% yield (Scheme 3a). Fluorophore 8 was isolated as the Z-isomer
as revealed by 1H NMR (Fig. S1a, see ESI) which showed an upfield
resonance at 6.8 ppm for the olefinic proton analogous to that
found in the boron complex of 4-Z and in contrast to the downfield
resonance of the olefinic proton of 4-E.7a After 1.5-5.5 h under UV
light (365 nm) a solution of 8 in DMSO-d6, revealed the formation
of increasing amounts of the E-isomer (Fig. S1b and c, see ESI). In
the presence of concd HCl, the NMR spectrum showed only the
Z-isomer even after 16 h under UV irradiation (Fig. S1d, see ESI)
(
Uf = 0.89) compared to low fluorescence of the boron ligated E-
isomer (Uf = 0.0007, Scheme 1d).7a
⇑
Corresponding author. Tel.: +1 352392 0554; fax: +1 352 392 9199.
E-mail address: katritzky@chem.ufl.edu (A.R. Katritzky).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.12.082

A. R. Katritzky et al. / Tetrahedron Letters 52 (2011) 2224–2227
2225
Scheme 3.
4'''
O
O
N
N
N
1'''
5
3'''
6
4
N
N
X
N
2'
1
3'
4'
5'
O
2'''
Scheme 1.
X
N
2
N
R
1'
X
3
1''
R
N
2''
R
6'
4''
3''
7b,c
7b,c
7b,c
- Z
- E
7b: R = CH3, X = O
7c: R = H, X = NH
Figure 1.
Scheme 2. (For designation of R1 and R2 see Table 1).
demonstrating stabilization of the Z-isomer by intramolecular
hydrogen bonding (Scheme 3b).
Scheme 4.
2.2. 1H, 13C, 15N NMR study of 7b and 7c
chemical shift of N-3 was identified by three bond correlation to
H6. The dimethylamino nitrogen (N-3000) chemical shift was re-
vealed by long range correlation with the protons of the two
methyl groups (H-4000) and the two methylene groups (H-1000 and
H-2000). The data for 7b and 7c are reported in Supplementary data
(Table S1). In TFA-d the 15N chemical shift of N-3 in 7b moves up-
field by 90.5 ppm consistent with protonation and formation of the
Z-isomer of 7b by intramolecular hydrogen bonding with furyl
1H and 13C chemical shifts were assigned based on the 1H–1H,
one-bond, and long-range 1H–13C couplings, of the gDQCOSY,
gHMQC, and gHMBC spectra. Protonation of 7b and 7c was studied
by 15N NMR in trifluoroacetic acid-d (TFA-d) using 1H–15N CIGAR-
gHMBC experiment (see Fig. 1 for numbering in 7b and 7c).
The 15N chemical shift of N-1 was identified by long range cor-
relation with the two methylene groups 1000 and 2000. The 15N NMR
Table 1
R1 and R2 designation for 6a–e and 7a–e
Entry
R1
R2
Compd (Yield)a
Compd (Yield)a
1
2
3
4
5
Ph
Ph
Ph
2-Thienyl
5-Methyl-2-furyl
2-Pyrryl
2-Thienyl
5-Methyl-2-furyl
6a (68%)
6b (59%)
6c (35%)
6d (50%)
6e (40%)
7a (33%)
7b (81%)
7c (55%)
7d (51%)
7e (30%)
Naphth-2-yl
Naphth-2-yl
a
Isolated yield.

2226
A. R. Katritzky et al. / Tetrahedron Letters 52 (2011) 2224–2227
pH1
1
0.5
0
pH1.3
pH1.6
pH2
pH2.5
pH3
pH4
Scheme 6.
pH5
pH7
nitrogens, sp3 nitrogens are known to be deshielded on
protonation.9
2.3. Fluorescence studies of 7a–e
200
300
400
500
Wavelength (nm)
The absorption and emission spectra of the fluorophores 7a-e at
10ꢀ5 M in Britton–Robinson Buffer solution were recorded over the
pH range 1–7. From pH 7 to pH 3 there is virtually no change in the
absorption spectra of 7a–e but a distinct decrease in the intensity
of the emission spectra by factors that range between 2 and 4
(Fig. 2 for 7b, S2-4 for 7a, 7c, and 7e, see ESI). The origin of the de-
crease in fluorescence intensity is clearly not associated with the
furyl, thienyl, or pyrryl units or to protonation of N-3 of the imidaz-
olinone ring (pKa ca. 1.8).10 Compounds 7a and 7d, both containing
the 2-thienyl group, gave virtually identical absorption and emis-
sion spectra (shown only for 7a, Fig. S2) that revealed a small
(ca. 20 nm) bathochromic shift of the absorption spectra below
pH 2.5 but no change in the emission spectra. The results are best
explained by protonation of N-3 of the imidazolinone ring but only
weak H-bonding with sulfur that may allow the thiophene ring to
twist out of planarity with the imidazolinone system (Scheme 5).
With compounds 7b and 7e containing the 5-methyl-2-furyl
unit, the absorption spectra again showed bathochromic shifts
(ca. 40 nm) below pH 2.5, but these were accompanied by signifi-
cant intensity increases of the emission spectra by factors of 7
(Fig. 2 for 7b) and 10 (Fig. S4 for 7e). Clearly, protonation of N-3
(Thousands)
pH1
pH1.3
pH1.6
pH2
pH2.5
pH3
pH4
pH5
400
300
200
100
pH6
pH7
0
400
500
600
700
Wavelength (nm)
Figure 2. Absorption (top) and emission (bottom) spectra of 7b at 10ꢀ5 M in
Britton–Robinson Buffer at pH 1-7. (Fluorescent spectra were subjected to Raman
peak correction).
oxygen. The coupling of 3.7 Hz between H-6 and C-5 confirms the
Z-isomer. Compound 7c exists in CDCl3 as a mixture of E- (75%) and
Z-isomers (25%), but in TFA-d only one isomer is found with the
15N chemical shift of N-3 shifted upfield by 95.9 ppm from the E-
isomer and 82.7 ppm from the Z-isomer. We suggest that in this
case, the E-isomer (7c2H+) predominates in the protonated system
due to hydrogen bonding between the NH proton of the pyrryl
group and the carbonyl oxygen (Scheme 4).
This hypothesis was confirmed by a coupling of 8.5 Hz between
H-6 and C-5. Furthermore, the 1H-15N CIGAR experiment failed to
detect 3JH6-N3 coupling which suggests a value < 3.0 Hz and further
supports the E-isomer assignment. The amide nitrogen of the imi-
dazolinone ring, however, shifted downfield by 9.2 ppm (7b),
9.2 ppm (7c from E-isomer), and by 4.9 ppm (7c from Z-isomer)
which provides evidence for protonation of this nitrogen. For the
dimethylamino nitrogen –N(CH3)2, prominent deshieding was ob-
served in protonated 7b2H+ (+17.5 ppm) and 7c2H+ (+16.0 ppm
from E-isomer and 16.2 ppm from Z-isomer). In contrast to sp2
pH1
1
0.5
0
pH1.3
pH1.6
pH2
pH2.5
pH3
pH4
pH5
pH6
pH7
200
300
400
500
pH1
Wavelength (nm)
(Millions)
4
pH1.3
pH1.6
pH2
pH2.5
pH3
pH4
pH5
3
2
1
pH6
pH7
0
400
450
500
550
600
650
Wavelength (nm)
Figure 3. Absorption (top) and emission (bottom) spectra of 10a at 10ꢀ5 M in
Britton–Robinson Buffer at pH 1–7. (Fluorescent spectra was subjected to Raman
peak correction).
Scheme 5.

A. R. Katritzky et al. / Tetrahedron Letters 52 (2011) 2224–2227
2227
3. Conclusions
GFP modified pH-sensitive chromophores were synthesized and
their fluorescence activity measured in Britton–Robinson Buffer
over the pH range 1–7. Chromophores containing five-membered
heterocyclic rings (7b,c,e, and 10b) showed an increase in fluores-
cence below pH 2.5 but the best quantum yield (observed with
10a) was only 2.9% of the value found for wild type GFP probably
due to excited state proton transfer. The results demonstrate that
high increase of fluorescence intensity requires a planar configura-
tion within the protonated conjugated molecular system. In the
case of furan-containing systems, NMR studies indicate that pro-
tonation of N-3 enforces planarity by H-bonding with furanyl oxy-
gen in the Z-configuration. However, protonation of N-3 in the
pyrrole system may also create planarity within the E-configura-
tion by H-bonding between pyrrole –NH and carbonyl oxygen.
Scheme 7.
Table 2
Quantum yields and excitation coefficients of 7a–e and 10a,b at pH 1
Entry
Compound
kabs
e
(Mꢀ1 cmꢀ1
)
kem
Uf
max
max
1
2
3
4
5
6
7
7a
7b
7c
7d
7e
409
444
462
410
451
405
449
24313
22376
45580
26913
28617
20343
20046
488
502
494
512
511
526
503
0.0008
0.0037
0.0017
0.0008
0.0060
0.0293
0.0070
Acknowledgments
10a
10b
We thank Professor Kirk Schanze, Professor Katsu Ogawa, and
Dr. Ion Ghiviriga for their kind help.
in 7b and H-bonding with furyl oxygen enforces planarity on the
cis configuration and the ensuing higher degree of conjugation en-
hances both the fluorescence wavelength and intensity (Scheme 5).
Finally, compound 7c containing the pyrrole system, shows
similar behavior to that of 7b and 7e with emission intensity
increasing five fold as the pH falls from 2.5 to 1 (Fig. S3, see ESI).
With 7c, however, protonation may lead to the Z or E isomers as
depicted in Scheme 4, either or both of which may account for
the increase in fluorescence intensity.
Supplementary data
Supplementary data (experimental procedures; photoisomer-
ization of 8; absorption and emission spectra of 7a,c,e, and 10b;
1H, 13C and 15N NMR chemical shift assignments of 6a, 7b, and
7c; characterization of 56, 6a–e, 7a–e, 8, and 10a,b) associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2010.12.082.
References and notes
2.4. Preparation and fluorescence of imidazolinone
chromophores 10a,b
1. (a) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006,
312, 217–224; (b) Suzuki, T.; Matsuzaki, T.; Hagiwara, H.; Aoki, T.; Takata, K.
Acta Histochem. Cytochem. 2007, 40, 131–137.
Compounds 10a and 10b were synthesized in yields of 18% and
29%, respectively, from compound 97a as shown in Scheme 6.
The absorption and emission spectra of 10a and 10b were re-
corded at 10ꢀ5 M in Britton–Robinson Buffer (Fig. 3, S5, see ESI).
Fluorescence of 10a increased below pH 6 initially due to proton-
ation of the pyridine nitrogen to 10aH+-Z followed by tautomerism
of the amide group to 10a0H+-Z (Scheme 7) as indicated by the red
shift in both absorption and emission spectra. However, fluores-
cence of 10b increased only below pH 2.5 thus exhibiting similar
behavior to that of 7b and 7e.
2. (a) Sisido, M.; Hohsaka, T. Appl. Microbiol. Biotechnol. 2001, 57, 274–281; (b)
Murakami, H.; Hohsaka, T.; Ashizuka, Y.; Hashimoto, K.; Sisido, M.
Biomacromology 2000, 1, 118–125; (c) De Filippis, V.; De Boni, S.; De Dea, E.;
Dalzoppo, D.; Grandi, C.; Fontana, A. Protein Sci. 2004, 13, 1489–1502; (d) Royo,
S.; Jiménez, A. I.; Cativiela, C. Tetrahedron: Asymmetry 2006, 17, 2393–2400.
3. (a) Brejc, K.; Sixma, T. K.; Kitts, P. A.; Kain, S. R.; Tsien, R. Y.; Ormö, M.;
Remington, S. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2306–2311; (b) Tsien, R. Y.
Annu. Rev. Biochem. 1998, 67, 509–544; (c) Malo, G. D.; Pouwels, L. J.; Wang, M.;
Weichsel, A.; Montfort, W. R.; Rizzo, M. A.; Piston, D. W.; Wachter, R. M.
Biochemistry 2007, 46, 9865–9873.
4. Wachter, R. M.; King, B. A.; Heim, R.; Kallio, K.; Tsien, R. Y.; Boxer, S. G.;
Remington, S. J. Biochemistry 1997, 36, 9759–9765.
5. He, X.; Bell, A. F.; Tonge, P. J. FEBS Lett. 2003, 549, 35–38; (b) Dong, J.; Solntsev,
K. M.; Poizat, O.; Tolbert, L. M. J. Am. Chem. Soc. 2007, 129, 10084–10085.
6. Arai, T.; Hozumi, Y. Chem. Lett. 1998, 1153–1154.
2.5. Quantum yields (Uf) measurements
7. (a) Wu, L.; Burgess, K. J. Am. Chem. Soc. 2008, 130, 4089–4096; (b) Düggeli, M.;
Christen, T.; Zelewsky, A. Chem. Eur. J. 2005, 11, 185–194.
The molar extinction coefficients (e
, Mꢀ1 cmꢀ1) and quantum
8. (a) Prüger, B.; Bach, T. Synthesis 2007, 7, 1103–1106; (b) Saravanan, V. S.;
Kumar, S. P. V.; De, B.; Gupta, J. K. Asian J. Chem. 2005, 17, 576–580.
9. Someswara, N. R.; Babu, G. R.; Murthy, B. N.; Das, M. M.; Prabhakar, T.; Lalitha,
M. Spectrochim. Acta, Part A: Mol. Biomol. Spec. 2002, 58A, 2737–2757.
10. Bell, A. F.; He, X.; Wachter, R. M.; Tonge, P. J. Biochemistry 2000, 39, 4423–4431.
11. White, A. Biochem. J. 1959, 71, 217–220.
yields (Uf) of 7a–e and 10a,b at pH 1 are shown in Table 2. The best
potential fluorescence marker, 10a, provides only 2.9% of the fluo-
rescence intensity of GFP and then only at pH 1. It seems likely that
the presence of protons necessary to enforce planarity and thus en-
hance conjugation, also provides a pathway for fluorescence decay
via the phenomenon of excited state proton transfer.11