Synthesis and properties of the red chromophore of the green-to-red photoconvertible fluorescent protein Kaede and its analogs

Article abstract of DOI:10.1016/j.bioorg.2007.12.003

Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His-Tyr-Gly chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to the following tripeptides were obtained: His-Tyr-Gly, Trp-Tyr-Gly, Phe-Trp-Gly, Tyr-Trp-Gly, Asn-Tyr-Gly, Phe-Tyr-Gly, and Tyr-Tyr-Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds). The most red-shifted absorption peak at 595 nm was found for the chromophore Trp-Tyr-Gly in basic DMSO. Surprisingly, in basic DMF non-aromatic Asn-derived chromophore Asn-Tyr-Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus, Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.

Full text of DOI:10.1016/j.bioorg.2007.12.003

Available online at www.sciencedirect.com
Bioorganic Chemistry 36 (2008) 96–104
www.elsevier.com/locate/bioorg
Synthesis and properties of the red chromophore of the green-to-red
photoconvertible fluorescent protein Kaede and its analogs
Ilia V. Yampolsky, Alexander A. Kislukhin, Tynchtyk T. Amatov, Dmitry Shcherbo,
Victor K. Potapov, Sergey Lukyanov, Konstantin A. Lukyanov *
Institute of Bioorganic Chemistry, Lab of Molecular Techonologies, Miklukho-Maklaya 16/10, Moscow 117997, Russian Federation
Received 12 October 2007
Available online 11 February 2008
Abstract
Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocat-
alytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly
chromophore-forming triad. Here, we describe synthesis of Kaede red chromophore (2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxyben-
zylidene)-5-imidazolone) and its analogs that can be potentially formed by natural amino acid residues. Chromophores corresponding to
the following tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly, Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly, and
Tyr–Tyr–Gly. In basic conditions they fluoresced red with relatively high quantum yield (up to 0.017 for Trp-derived compounds).
The most red-shifted absorption peak at 595 nm was found for the chromophore Trp–Tyr–Gly in basic DMSO. Surprisingly, in basic
DMF non-aromatic Asn-derived chromophore Asn–Tyr–Gly demonstrated the most red-shifted emission maximum at 642 nm. Thus,
Asn residue may be a promising substituent, which can potentially diversify posttranslational chemistry in GFP-like proteins.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Green fluorescent protein; Fluorophore; Bathochromic shift; Fluorescent probes; Protein engineering
1. Introduction
phenolic ring connected by a methylene bridge (Scheme
1). In all known natural GFP-like proteins positions
corresponding to the chromophore-forming Tyr66 and
Gly67 are invariant, while the first chromophore-forming
residue 65 can vary (here and further the numbering is in
accordance to GFP). Studies of last several years clearly
demonstrated that 4-(p-hydroxybenzylidene)-5-imidazo-
lone chromophore core is common for all natural GFP-like
proteins. At the same time, red-shifted proteins can contain
a modification of the protein backbone at position 65 [1].
Two main types of red chromophores within GFP-like
proteins are known. One type was first described for
DsRed [3]. DsRed-type chromophore consists of GFP
chromophore core extended with an acylimine group
(Scheme 1) which is formed as a result of dehydrogenation
of CaAN bond of the first chromophore-forming residue
with molecular oxygen [3,4]. This oxidation is catalyzed
by nearby residues and occurs in the dark, although it
can be greatly facilitated by irradiation with light at about
In contrast to all other known natural protein-based
pigments, proteins of Green fluorescent protein (GFP)
family form their chromophores by autocatalytic modifica-
tion of internal amino acid residues [1]. This property
makes GFP-like proteins a useful fluorescent marker for
live cell labeling [2]. Mechanisms of chromophore forma-
tion within GFP-like proteins and properties of the chro-
mophores are of undoubted basic interest.
Chromophore in GFP is formed by cyclization and
further dehydration and oxidation of amino acids at
positions 65–67 (Ser–Tyr–Gly). These reactions result in
4-(p-hydroxybenzylidene)-5-imidazolone, which includes a
newly formed 5-membered heterocycle and Tyr66-derived
*
Corresponding author. Fax: +7 4953 30 7056.
E-mail address: kluk@ibch.ru (K.A. Lukyanov).
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.12.003

I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
97
OH
Tyr66
Xaa65
O
R
N
N
H2O2
Light (350-420nm)
O
HN
O
O2
O
O
Gly67
GFP
Tyr66
Tyr66
His65
N
Xaa65
O
R
N
N
N
N
N
N
H
O
O
H2N
Gly67
DsRed
Gly66
Kaede
Scheme 1. Origination of DsRed-type and Keade-type chromophores from GFP-type precursor in the course of posttranslational modifications of
fluorescent proteins.
400 nm [5]. In some proteins, DsRed-like chromophore
undergoes further chemical modifications of the acylimine
group [6–9].
ment by mutating adjacent amino acid positions. Another
explanation is a direct participation of His65 imidazole
ring in protonation–deprotonation events during photo-
conversion [11,14]. In this case, Phe, Tyr or Trp residues
cannot act as His. Anyhow, absence of corresponding
mutants of Kaede-like proteins precludes studying spectral
properties of Kaede-like chromophore variants.
Chemical synthesis of natural compounds often pro-
vides useful information on their structure and properties.
Synthetic imidazolones related to GFP chromophore have
been a subject of extensive research [22–26]. However, up
to date, a few compounds corresponding to natural chro-
mophores of red-shifted GFP-like proteins were synthe-
sized. Model compounds that are similar but not
identical to DsRed chromophore were described [27]. We
earlier synthesized a chromophore of a non-fluorescent
purple chromoprotein asFP595 [28]. Here, we describe a
general synthetic approach to the chromophores of Kaede
type. Synthesis of Kaede-like chromophores with different
groups corresponding to natural amino acids at positions
65 and 66 made it possible to evaluate their influence on
spectral properties of the chromophore and to find struc-
tures with the most red-shifted absorption and emission.
Second red chromophore type is characteristic for green-
to-red photoconvertible fluorescent proteins carrying His–
Tyr–Gly chromophore-forming residues. The first protein
of this type named Kaede was cloned from stony coral
Trachyphyllia geoffroyi in 2002 [10]. In the dark, Kaede is
a stable green fluorescent protein with a GFP-like chromo-
phore. However, irradiation with UV or violet light (at
approximately 350–420 nm) efficiently converts Kaede into
red fluorescent state. Kaede red chromophore is formed by
protein backbone break between N and Ca of His65 [11–
13]. As a result, GFP-like chromophore core is brought
into conjugation with His65 heterocycle (Scheme 1). This
reaction is non-oxidative and requires no molecular oxy-
gen. A number of Kaede-like proteins were characterized
and shown to be a useful tool to study movements of cells,
organelles and proteins [10,14–19], to perform ultra-high
resolution fluorescence imaging [20], and to visualize pro-
tein degradation in living cells [21].
Considering the role of His65 aromatic ring in Kaede-
type chromophore red shift, it is tempting to test other aro-
matic amino acids at this position. However, up to date all
attempts to create functional mutants of Kaede-like pro-
teins carrying Phe, Tyr or Trp as well as any other residues
instead of chromophore-forming histidine failed [11,14].
Such mutants are either non-fluorescent or have no ability
of green-to-red conversion. This failure might be a result of
improper orientation of the bigger (compared to His)
aromatic residues. It is known from crystal structure of
Kaede-like proteins that position and orientation of the
chromophore-forming His is almost unchanged during
green-to-red photoconversion [12,13]. Thus, possibly the
chromophore environment catalyses formation of the red
chromophore by stabilizing the product-like transition
state. If so, success in the histidine substitution might be
a matter of efforts to create a proper chromophore environ-
2. Materials and methods
2.1. General
NMR spectra were recorded on a Bruker DRX-500
instrument; chemical shifts are expressed in d scale down-
field from tetramethylsilane and are referenced to residual
protium in the NMR solvent (CHCl3, d 7.26; CHD2OD,
3.30; CD3S(O)CD2H, d 2.50). Analytical and preparative
TLC was performed on precoated Silufol UV 254 silica
or alumina plates impregnated with a fluorescent indicator
(254 nm). TLC plates were visualized by exposure to UV
light or treatment with saturated permanganate solution
followed by washing with excess water, or with phospho-

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I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
molybdic acid with subsequent heating, or by specific color
change of acidic compounds in ammonia vapors. Column
chromatography was performed on Merck Kieselgel 60
(70–230 mesh) silica or Aldrich CAMAG-A-1 (150 mesh,
basic, Brockman activity I) alumina. ESI mass-spectra
were obtained on Bruker Esquire6 Plus instrument.
of tetrabutylammonium fluoride trihydrate (438 mg,
1.39 mmol) in 5 mL THF was added. The mixture was stir-
red for 1 min and then neutralized with glacial acetic acid
(0.12 mL, 2 mmol). The volatiles were removed in
vacuoand the residue was redissolved in ethanol (5 mL).
The ethanolic solution (1 mL) was purified by preparative
TLC (15% EtOH:CHCl3). The combined target fractions
from 5 runs were chromatographed again in the same man-
ner, resulting in 10 mg (5%) of pure product as a red–
2.2. Phosphonium salts
1
Phosphonium salts were prepared from corresponding
bromides and triphenylphosphine by refluxing in toluene
until the bromide was consumed, except for (4-imidazo-
lyl)methyltriphenylphosphonium chloride (3a) [29]. N-Boc-
3-(bromomethyl)indole (3e) was prepared as described [30].
orange solid. Rf 0.30, silica, 15% EtOH:CHCl3; H NMR
(500 MHz, CD3OD) 8.10 (d, J = 8.6;Hz, 2H), 7.95 (d,
J = 15.6 Hz, 1H), 7.81 (s, 1H), 7.50 (s, 1H), 7,01 (s, 1H),
6.99 (d, J = 15.6 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 3.58
(s, 3H). MS (ESI-MS) calcd for C16H15N4O2 [M+H+]
+
295.12. Found: 295.27.
2.3. Aldehyde 2a
2.6. Chromophore YYG
A suspension of imidazolone 1a (2.98 g, 13.9 mmol) and
selenium dioxide (1.85 g, 16.7 mmol, 1.2 equiv) in anhy-
drous dioxane (140 mL) was stirred at reflux for 1 h and
carefully decanted from the red solid while hot. The solu-
tion was concentrated in vacuo, the crude product was dis-
solved in dichloromethane (50 mL), treated sequentially
with diisopropylethylamine (3.59 g, 27.8 mmol, 2.0 equiv)
and tert-butyldimethylsilyl chloride (4.19 g, 27.8 mmol,
2.0 equiv). After stirring for 2 h at room temperature, the
volatiles were removed in vacuo and the residue was purified
by column chromatography (silica, 2% EtOH:CHCl3) to
afford the aldehyde 2a as an orange–red solid (2.73 g,
57%). Rf 0.46, silica, 2% EtOH:CHCl3; 1H NMR
(500 MHz, CDCl3) d 9.68 (s, 1H), 8.28 (d, 2H, J = 8.4Hz),
7.48 (s, 1H), 7.00 (d, 2H, J = 8.4Hz), 3.32 (s, 3H), 0.96 (s,
9H), 0.27 (s, 6H).
To a suspension of aldehyde 2a (500 mg, 1.45 mmol)
and (4-(tert-butyldimethylsiloxy)benzyl)triphenylphospo-
nium bromide (3b) (817 mg, 1.45 mmol, 1.0 equiv) in
dichloromethane (60 mL) was added 2M NaOH (40 mL).
The orange organic layer turned instantly dark-red. The
resulting mixture was vigorously stirred for 30 min, and
the layers were separated. The aqueous layer was extracted
with dichloromethane (2 Â 30 mL). The combined organic
extracts were treated with tetrabutylammonium fluoride
trihydrate (960 mg, 3.04 mmol, 2.1 equiv) to produce a vio-
let suspension which was neutralized with 30% aqueous
CH3COOH. Solvents were removed in vacuo and the crude
product was purified by column chromatography (silica,
10% EtOH:CHCl3) and recrystallized from methanol to
give the pure product as red-brown crystals (108 mg,
23%). Rf 0.19, silica, 5% EtOH:CHCl3; 1H NMR
(500 MHz, DMSO-d6) d 10.13 (br s, 2H), 8.14 (d,
J = 8.7 Hz, 2H), 7.95 (d, J = 15.7 Hz, 1H), 7.71 (d,
J = 8.7 Hz, 2H), 6.99 (d, J = 15.7 Hz, 1H), 6.93 (s, 1H),
6.87 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 3.26 (s,
2.4. Aldehyde 2b
A suspension of imidazolone 1b (707 mg, 2.95 mmol)
and selenium dioxide (393 mg, 3.54 mmol, 1.2 equiv) in
anhydrous dioxane (50 mL) was stirred at reflux for 1 h
and carefully decanted from the red solid while hot. The
solvent was removed in vacuo and the residual red oil was
purified by column chromatography (basic alumina, 10%
EtOH in CHCl3) to afford the aldehyde 3b as a fine red
crystalline solid (549 mg, 73%). Rf 0.31, basic alumina,
10% EtOH:CHCl3; 1H NMR (500 MHz, DMSO-d6) d
12.51 (br s, 1H), 9.69 (s, 1H), 8.53 (s, 1H), 8.07 (s, 1H),
7.53 (d, J = 7.8 Hz), 7.32–7.24 (m, 3H), 3.35 (s, 3H).
+
3H); MS (ESI-MS) calcd for C19H17N2O3 [M+H+]
321.12. Found: 321.18.
2.7. Chromophore FYG
Procedure is essentially the same as for the chromophore
YWG; benzyltriphenylphosphonium bromide (3c) was
used. Yield 53%. Rf 0.35, silica, 5% EtOH:CHCl3; 1H
NMR (500 MHz, DMSO-d6) d 10.20 (br s, 1H), 8.20 (d,
J = 8.7 Hz, 2H), 7.99 (d, J = 15.7 Hz, 1H), 7.84 (d,
J = 7.5 Hz, 2H), 7.47–7.40 (m, 3H), 7.22 (d, J = 15.7 Hz,
1H), 6.99 (s, 1H), 6.89 (d, J = 8.7 Hz, 2H), 3.29 (s, 3H);
2.5. Chromophore HYG (Kaede chromophore)
MS (ESI-MS) calcd for C19H17N2O2 [M+H+] 305.13.
+
To a solution of 480 mg (1.39 mmol, 2.1 equiv) of alde-
hyde 2a in dichloromethane (10 mL) 2 M aqueous NaOH
(10 mL) was added, and then solid phosphonium salt 3a
(250 mg, 0.66 mmol) was added with vigorous stirring.
The stirring was continued for 30 min and the organic
phase was separated. Dichloromethane was evaporated,
the mixture was redissolved in 10 mL THF and a solution
Found: 305.25.
2.8. Chromophore NYG
Procedure is essentially the same as for the chromophore
YWG; (carbamoylmethyl)benzyltriphenylphosphonium

I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
99
bromide (3d) was used. Yield 49%. Rf 0.26, silica, 10%
EtOH:CHCl3; 1H NMR (500 MHz, DMSO-d6) d 10.29
(br s, 1H), 8.15 (d, J = 7.8 Hz, 2H), 7.93 (s, 1H), 7. 45 (s,
1H), 7.28 (d, J = 12.8 Hz, 1H), 7.22 (d, J = 12.8 Hz, 1H),
7.08 (s, 1H), 6.85 (d, J = 7.8 Hz, 2H), 3.20 (s, 3H); MS
(ESI-MS) calcd for C14H14N3O3+ [M+H+] 272.10. Found:
272.36.
J = 7.9 Hz, 1H), 7.91 (d, J = 15.6 Hz, 1H), 7.68 (d, J =
8.2 Hz, 2H), 7.49 (d, J = 7.9 Hz, 1H), 7.34 (s, 1H), 7.22
(t, J = 7.9 Hz, 1H), 7.20 (t, J = 7.9 Hz, 1H), 6.99 (d,
J = 15.6 Hz, 1H), 6.86 (d, J = 7.5 Hz, 2H), 3.18 (s, 3H);
MS (ESI-MS) calcd for C21H18N3O2 [M+H+] 344.14.
+
Found: 344.16.
2.11. Chromophore FWG
2.9. Chromophore WYG
To a stirred suspension of aldehyde 2b (50 mg,
0.20 mmol) and benzyltriphenylphosphonium bromide
(3c) (85 mg, 0.20 mmol, 1.0 equiv) in tert-butyl alcohol
(3 mL) was added a solution of potassium tert-butoxide
(33 mg, 0.30 mmol, 1.5 equiv) in tert-butyl alcohol
(1 mL). Instant color change from orange to dark-red
was observed. The reaction mixture was stirred for
30 min, the solvent was removed in vacuo and the residue
was purified by column chromatography on silica (5%
EtOH:CHCl3) to afford the product as fine red-brown crys-
talline solid (28 mg, 43%). Rf 0.38, silica, 5% EtOH–CHCl3;
1H NMR (500 MHz) d 12.06 (br s, 1H), 8.26 (s, 1H), 8.07
(d, J = 7.9 Hz, 1H), 7.94 (d, J = 7.8 Hz, 2H), 7.85 (d,
J = 7.9 Hz, 1H), 7.64–7.36 (m, 3H), 7.24–7.15 (m, 2H),
7.12 (d, J = 13.4 Hz, 1H), 7.08 (t, J = 7.9 Hz, 1H), 6.48
(d, J = 13.4 Hz, 1H), 3.18 (s, 3H); MS (ESI-MS) calcd
for C21H18N3O+ [M+H+] 328.14. Found: 328.17.
To a stirred suspension of aldehyde 2a (224 mg,
0.65 mmol) and ((N-Boc-3-indolyl)methyl)triphenylpho-
shonium bromide (3e) (372 mg, 0.65 mmol, 1.0 equiv) in
tert-butyl alcohol (9 mL) was added a solution of potas-
sium tert-butoxide (73 mg, 0.65 mmol, 1.0 equiv) in tert-
butyl alcohol (3 mL). After 1 h of stirring, more potassium
tert-butoxide (37 mg, 0.33 mmol, 0.5 equiv) was added and
stirring was continued for 30 min. Solvent was removed in
vacuo, the residue was dissolved in dichloromethane (5 mL)
and treated with tetrabutylammonium fluoride trihydrate
(215 mg, 0.68 mmol, 1.05 equiv) and stirred for 5 min. To
the resulting deep purple solution were added sequentially
p-cresol (70 mg, 1.0 equiv) and trifluoroacetic acid (1 mL)
and the mixture was refluxed for 30 min, cooled down
and neutralized with conc. ammonia (1.5 mL). The result-
ing precipitate was filtered and recrystallized from metha-
nol to yield the pure product as a red crystalline solid
1
(49 mg, 22%). Rf 0.21, silica, 5% EtOH:CHCl3; H NMR
2.12. Optical spectral measurement
(500 MHz, DMSO-d6) d 11.96 (br s, 1H), 10.16 (br s,
1H), 8.53 (s, 1H), 8.01 (d, J = 15.6 Hz, 1H), 7.99 (d,
J = 8.6 Hz, 2H), 7.97 (d, J = 7.8 Hz, 1H), 7.47 (d,
J = 7.8 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.16 (t,
J = 7.8 Hz, 1H), 6.75 (d, J = 15.6 Hz, 1H), 6.70 (s, 1H),
6.24 (d, J = 8.6 Hz, 2H), 3.25 (s, 3H). MS (ESI-MS) calcd
Absorption and excitation–emission spectra were
recorded at room temperature using Beckman DU520
UV/VIS spectrophotometer and Varian Cary Eclipse fluo-
rescence spectrophotometer, respectively. Emission spectra
were corrected for photomultiplier response. Fluorescence
quantum yields of chromophores in basic DMF were deter-
mined by using purified DsRed2 as a reference standard
(quantum yield 0.55) [31].
for C21H18N3O2 [M+H+] 344.14. Found: 344.17.
+
2.10. Chromophore YWG
To a stirred suspension of aldehyde 2b (76 mg,
0.30 mmol) and (4-(tert-butyldimethylsiloxy)benzyl)tri-
phenylphosphonium bromide (3b) (169 mg, 0.30 mmol) in
tert-butyl alcohol (4 mL) was added a solution of potas-
sium tert-butoxide (33 mg, 0.20 mmol) in tert-butyl alcohol
(2 mL). The reaction mixture was stirred for 30 min, the
solvent was removed in vacuo and the residue was purified
by column chromatography on alumina (CHCl3) to afford
the TBS-protected chromophore. Rf 0.32, silica, 5% EtOH–
CHCl3. TBS-protected chromophore was dissolved in
CH2Cl2 (5 mL) and treated with tetrabutylammonium fluo-
ride trihydrate (100 mg, 0.32 mmol, 1.05 equiv). The result-
ing purple solution was neutralized with acetic acid (25 lL,
0.44 mmol) and the reaction mixture was concentrated in
vacuo. The product crystallized on treatment with 80%
aqueous methanol (2 mL) to afford the title compound as
a red-purple crystalline solid (43 mg, 42%). Rf 0.23, silica,
5% EtOH:CHCl3; 1H NMR (500 MHz, DMSO-d6) d
11.93 (br s, 1H), 9.90 (br s, 1H), 8.54 (s, 1H), 8.22 (d,
3. Results and discussion
3.1. Synthesis
Kaede-like chromophores corresponding to the following
tripeptides were obtained: His–Tyr–Gly, Trp–Tyr–Gly,
Phe–Trp–Gly, Tyr–Trp–Gly, Asn–Tyr–Gly, Phe–Tyr–Gly,
and Tyr–Tyr–Gly. They were designated according to the
one-letter amino acid code as HYG, WYG, FWG, YWG,
NYG, FYG, and YYG, respectively, (Fig. 1).
Oxidation of readily available 2-methylimidazolones 1
[32] with selenium dioxide in refluxing dioxane provided
aldehydes 2 (Scheme 2). In case of imidazolone 1a, the
desired aldehyde underwent decomposition to unidentified
products on attempt of chromatographic purification. The
TBS-protected aldehyde 2a, synthesized by treatment of
the crude product with TBSCl and diisopropylethylamine,
was stable to chromatography and was isolated in 57%
combined yield. However, it decomposes significantly in 1

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I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
OH
O
OH
OH
OH
O
O
N
OH
N
N
N
N
N
N
N
N
N
H
NH2
O
O
FYG
NYG
YYG
HYG
Kaede chromophore
(red form)
OH
NH
NH
OH
N
N
N
N
NH
N
O
N
O
O
WYG
YWG
FWG
Fig. 1. Structures of Kaede and Kaede-like chromophores synthesized.
OTBS OH
OH
R
Chromophore
HYG
4-imidazolyl
3-indolyl
CONH2
1. SeO2
2. TBSCl, DIPEA
1. 3, base
WYG
2. deprotection
NYG
O
R
N
N
N
N
N
C6H5
FYG
(57%)
(see Table 1)
4-HOC6H4
YYG
N
O
O
O
1a
2a
NH
NH
NH
R
C6H5
4-HOC6H4
Chromophore
FWG
YWG
SeO2
1. 3, base
O
2. deprotection
R
N
N
N
N
N
(73%)
N
(see Table 1)
O
O
O
1b
2b
Scheme 2. Synthesis of Kaede-type chromophores.
Table 1
month even if stored at À20 °C. A number of other meth-
ods to bring about such oxidation failed. O-protected sub-
strates also did not produce the desired products with SeO2
or other oxidants. Indolic imidazolone 1b gave the corre-
sponding aldehyde 2b in 73% yield. Target chromophores
were obtained via Wittig reaction of aldehydes 2a and 2b
with appropriate phosphonium salts 3 (Table 1). In all
cases, the reaction proceeded stereoselectively to give
trans-product (E:Z > 15:1), with characteristic coupling
constant of about 15 Hz. Low yield of the Kaede chromo-
phore HYG is probably due to a competing deprotonation
of imidazole nitrogen in 3e and expulsion of PPh3 mole-
cule, producing the electrophilic diazafulvene intermediate
4 [29] rather than desired nucleophilic phosphorous ylide 5
(Scheme 3). If sterically hindered potassium tert-butoxide is
used as base, this pathway seems to become major and no
chromophore is formed. In this case, biphasic mixture of
aqueous NaOH and CH2Cl2 was employed. For other
Yields and conditions of Wittig reaction
Aldehyde
Phosphonium salt
Product (yield, %)
PPh3+Cl-
HYG (5)a
N
2a
(3a)
N
H
2a
2a
2a
4-(TBSO)C6H4CH2PPh3Br (3b)
BnPPh3Br (3c)
H2NCOCH2PPh3Br (3d)
YYG (23)a
FYG (53)b
NYG (49)b
PPh3+Br-
2a
(3e)
WYG (18)b,c
N
Boc
2b
2b
a
4-(TBSO)C6H4CH2PPh3Br (3b)
BnPPh3Br (3c)
YWG (42)b
FWG (44)b
2 M aqueous NaOH/CH2Cl2.
t-BuOK/t-BuOH.
After Boc cleavage (see Section 2).
b
c

I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
101
PPh3
H
PPh3
—PPh3
N
N
side products
B:
N
N
N
4
N
PPh3
H
3a
2a
N
HYG
5
N
H
Scheme 3. Reaction pathways for phosphonium salt 3a.
chromophores, the results obtained using either of these
two bases were comparable.
Other Kaede-like chromophores demonstrated similar
dependences on pH and solvent with some exceptions
(Table 2 and Fig. 3). As expected, chromophore FWG
showed no spectral changes at interval pH 5–10 since this
chromophore does not have acidic phenolic hydroxyl.
Instead, a spectral transition to a red-shifted form was
observed with pKa 11.6 probably associated with deproto-
nation of indole. At low pH Trp-containing chromophores
(WYG, FWG and YWG) demonstrated transition with
pKa ꢀ 2.5–3.5 into spectral forms characterized with con-
siderably higher extinction coefficient and red-shifted
absorption maxima compared to corresponding absorption
peaks at pH 4–7 (Table 3 and Fig. 3C). Most probably, this
transition can be attributed to appearance of positively
charged chromophore state due to protonation of N-3 of
the imidazolone ring, which is stabilized by indole. Other
Kaede-like chromophores also possessed similar spectral
transition but at considerably lower pH (pKa ꢀ 1.5) as
reported for GFP chromophore [23] and for HBMPI and
HBMPDI [27] chromophores. NYG chromophore had
the most red-shifted absorption in DMF, not in DMSO
as others.
3.2. Spectral properties
As expected, wild type Kaede chromophore HYG dem-
onstrated clear pH dependence of absorption spectrum. In
water solutions, acidic form absorbed at 430 nm (Table 2).
With an increase of pH, this peak transformed into a peak
at 490 nm with isosbestic point at 447 nm and pKa 7.7
(Fig. 2A). We attributed this transition to deprotonation
of phenolic oxygen that is a common process for chro-
mophores within other GFP-like proteins. No other spec-
tral transitions were observed in interval pH 3–14.
Nature of solvent was found to have a minor impact on
absorption maximum and extinction coefficient of the
acidic form of HYG (Fig. 2B). In contrast, in basic condi-
tions HYG chromophore possessed clearly different
absorption spectra in different solvents. Strong absorption
red shift was observed in the series water–ethanol–isopro-
panol–DMF–DMSO (Fig. 2C, Table 2). Compared to
HBMPI and HBMPDI [27], absorption maxima are at
longer wavelengths due to extension of conjugation to
another aromatic ring or electron-withdrawing substituent.
All Kaede-like chromophores in basic DMF and DMSO
demonstrated a well-detectable red fluorescence with a
Table 2
Spectral properties of Kaede-like chromophores
Chromo-phore Absorption max, nm (extinction coefficient,
mMÀ1 cmÀ1) in neutral solutionsa
pKa
Absorption max, nm (extinction coefficient,
Emission max,
nm (QY)c
(water) mMÀ1 cmÀ1) in basic solutionsb
Water
EtOH
iPrOH
DMF
DMSO
Water
EtOH
iPrOH
DMF
DMSO
HYG
WYG
FWG
YWG
NYG
FYG
YYG
a
430
(41)
460
(7)
455
(11)
462
(8)
424
(27)
425
(28)
436
(30)
443
(40)
462
(15)
456
(23)
466
(30)
433
(19)
437
(29)
459
(35)
444
(41)
460
(16)
460
(25)
468
(26)
433
(18)
451
(26)
483
(42)
446
(40)
460
(14)
456
(20)
464
(23)
431
(21)
437
(27)
445
(22)
448
(37)
465
(14)
462
(19)
468
(22)
435
(18)
438
(28)
447
(29)
7.7
8.1
490
(53)
494
(21)
498d
(13)
496
(27)
510
(35)
492
(43)
506
(49)
502
(79)
509
(24)
508
(23)
509
(34)
531
(36)
513
(45)
522
(61)
514
(80)
532
(27)
526
(26)
533
(40)
548
(30)
526
(44)
531
(60)
557
(77)
580
(27)
556
(22)
567
(38)
564
(34)
553
(49)
556
(59)
565
(80)
595
(27)
563
(24)
577
(44)
543
(25)
572
(50)
588
(59)
582
(0.005)
615
(0.017)
625
(0.016)
610
(0.017)
642
(0.005)
635
(0.009)
625
(0.016)
11.6
8.3
7.6
7.8
7.8
In water at pH 5 or in pure solvents.
b
c
In water at pH 10 or in solvents containg 50 mM NaOH.
In basic (50 mM NaOH) DMF.
At pH 13.
d

102
I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
A
A
490
0.5
1.0
WYG
FWG
430
0.4
0.3
0.8
0.6
YWG
3.5
0.2
0.1
0.0
7.2
7.7
8.5
0.4
0.2
10.1
0.0
350
400
450
500
550
600
400
450
500
550
600
650
Wavelength, nm
B
0.4
0.3
0.2
0.1
0.0
Water
EtOH
iPrOH
DMF
B
1.0
0.8
0.6
FYG
DMSO
YYG
NYG
0.4
0.2
0.0
350
400
450
iPrOH
500
550
C
DMSO
0.8
0.6
0.4
0.2
0.0
400
450
500
550
600
650
Wavelength, nm
EtOH
C
DMF
2.1
0.3
0.2
9.8
7.0
Water
500
400
450
550
600
650
0.1
0.0
Wavelength, nm
Fig. 2. Absorption spectra for HYG chromophore. (A) Absorption
spectra for buffered water solutions of HYG chromophore at different
pH as designated on the graphs. (B and C) Absorption spectra for
HYGchromophore in different solvents at pH 3.5 (B) or pH 10.1 (C).
300
400
500
600
Wavelength, nm
Fig. 3. Absorption spectra for Kaede-like chromophores. (A) Absorption
spectra for Trp-containing chromophores (WYG, FWG and YWG) in
basic DMF. (B) Absorption spectra for chromophores FYG, NYG and
YYG in basic DMF. (C) Absorption spectra for buffered water solutions
of WYG chromophore (15 lM) at pH 2.1 (dotted line), 7.0 (dashed line),
and 9.8 (solid line).
quantum yield well above that of HBMPI, HBMPDI [27],
and GFP and asFP595 chromophores [22,28] (Table 2 and
Fig. 4). The highest fluorescence quantum yield (about
0.017) was measured for Trp-derived chromophores
WYG and YWG (for comparison, we measured quantum
yield of GFP chromophore in basic DMF to be 0.00005).
Probably, bulky Trp side chain prevents to some extent fast
isomerization of the chromophore molecules and thus
decreases non-radiative decay of the excited state.
Emission spectra of HYG chromophore in DMF and
DMSO had maxima at 582 and 598 nm, respectively. Thus,
DMF appears to be close to the chromophore environment
within Kaede protein, which has emission maximum at

I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
103
Table 3
300
400
500
550
600
582
700
Absorption characteristics of indolic Kaede-like chromophores in acidic
solutions (in water at pH 2 or in solvents containg 50 mM HCl)
A
Chromo-
phore
pKa
(water)
Absorption max, nm (extinction
coefficient, mMÀ1 cmÀ1
)
HYG
625
Water
EtOH
iPrOH
DMF
DMSO
355
WYG
FWG
YWG
3.5
2.6
2.8
507
(23)
491
(24)
508
(32)
512
(27)
486
(21)
510
(32)
521
(30)
509
(27)
523
(40)
509
(25)
480
(25)
511
(26)
500
(23)
477
(21)
496
(22)
585
615
B
WYG
582 nm [10]. Earlier, the same similarity between spectra of
synthetic chromophore in basic DMF and native protein
was found for asFP595 [28]. In contrast to other Kaede-
like chromophores as well as GFP and asFP595 chromoph-
ores, HYG emission spectrum contained a shoulder at
620–640 nm which is separated from the main peak very
clearly (Fig. 4A). This well-defined long wavelength shoul-
der or even minor peak is characteristic for all Kaede-like
proteins [10,14,17,33]. Thus, His residue appears to deter-
mine this unusual shape of the emission curve.
385
580
625
635
642
C
YYG
All ‘‘non-natural” Kaede-like chromophores demon-
strated considerably red-shifted emission spectra compared
to HYG chromophore (Table 2 and Fig. 4). Surprisingly,
the most red-shifted emission was detected for NYG chro-
mophore. In basic DMF NYG possessed excitation and
emission maxima at 570 and 642 nm, respectively, demon-
strating a large Stokes shift (Fig. 4E). In case of successful
substitution H65N in a Kaede-like protein such mutant
could represent a new group of green-to-far-red photocon-
vertible fluorescent proteins.
387
568
D
FYG
308
The panel of Kaede-like chromophores allows for direct
comparison of some substituents (Tables 2 and 3). ‘‘Sym-
metrical” compounds WYG and YWG indeed possess
almost identical absorption maxima in all solvents in pos-
itively charged and neutral states and in almost all solvents
in negatively charged states. Only basic DMF and DMSO
provide red-shifted absorption for WYG. As expected, p-
hydroxyphenyl group gives red-shifted absorption in com-
parison with phenyl group (compare YYG and FYG).
FWG and FYG chromophores demonstrate that neutral
indole ensures stronger absorption red shift compared to
neutral p-hydroxyphenyl; however, in the negatively
charged states their spectral behavior is very similar.
Fluorescent proteins are chemically and optically com-
plex molecules. The availability of model compounds has
in the past proven useful for advancing our understanding
of their behavior [22–24,34,35]. In particular, the
chromophores described here might provide a useful refer-
ence for prediction of the optical properties of GFP-related
chromophores and proteins using quantum chemical
approaches.
570
E
NYG
350
300
400
500
600
700
Wavelength, nm
Fig. 4. Fluorescence spectra for Kaede-like chromophores. (A–E) Exci-
tation (dotted lines) and emission (solid lines) spectra for HYG, WYG,
YYG, FYG, and NYG solutions in basic DMF.
deprotonation of the indole group in FWG that is much
lower than of indole itself (pKa 17 in water). We found that
model compound corresponding to chromophore within
CFP (GFP-Y66W, structure 1b, Scheme 2) also shows
about 80-nm absorption red shift in a strong base (not
shown). Apparently, this state corresponds to deproto-
To the best of our knowledge, the present study
describes deprotonated indole-containing GFP-like chro-
mophore for the first time. We measured pKa 11.6 for

104
I.V. Yampolsky et al. / Bioorganic Chemistry 36 (2008) 96–104
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residues in the vicinity of chromophore-forming Trp66 into
ECFP (or similar mutants) could result in novel (and
potentially useful) fluorescent protein variant where Trp-
containing chromophore exists in anionic state.
One of the most interesting results of the present work is
that non-aromatic asparagine moiety provides a strong red
shift in NYG chromophore. In case of Asn65, Kaede-like
chromophore includes additional C@O group in the system
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We thank Dr. Andrey Formanovsky, Igor Prokhorenko
and Vadim Kublitsky for their help. This work was sup-
ported by grants from Molecular and Cell Biology Pro-
gram RAS, Howard Hughes Medical Institute Grant
HHMI 55005618, National Institutes of Health USA
(GM070358), Russian Foundation for Basic Research
grant 05-04-49316. K.A.L. is supported by Russian Science
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