Synthetic GFP chromophore and control of excited-state proton transfer in DNA: An alternative concept for fluorescent DNA labels with large apparent stokes' shifts

Article abstract of DOI:10.1055/s-0030-1258400

Synthetic GFP-like chromophores bearing an ortho-phenol group instead of the para-phenol group of natural GFP can mimic the hydrogen bonding network in the protein by an intramolecular hydrogen bond to the imidazolone group. This hydrogen bond influences

Full text of DOI:10.1055/s-0030-1258400

502
PAPER
Synthetic GFP Chromophore and Control of Excited-State Proton Transfer
in DNA: An Alternative Concept for Fluorescent DNA Labels with Large
Apparent Stokes’ Shifts
Synthetic
G
FP Chr
l
omoph
r
ore ike Wenge, Hans-Achim Wagenknecht*1
University of Regensburg, Institute for Organic Chemistry, Universitätsstr. 31, 93053 Regensburg, Germany
Fax +49(941)9434617; E-mail: achim.wagenknecht@chemie.uni-regensburg.de
Received 22 September 2010; revised 17 November 2010
variants are the result of different types of interactions be-
Abstract: Synthetic GFP-like chromophores bearing an ortho-phe-
nol group instead of the para-phenol group of natural GFP can
mimic the hydrogen bonding network in the protein by an intramo-
lecular hydrogen bond to the imidazolone group. This hydrogen
tween the mature GFP chromophore and its immediate
protein neighborhood. The two parts of the p-conjugated
system, the phenol, and the imidazolone group, are inter-
bond influences the excited state of the model by proton transfer connected by a methine bridge, which is known from syn-
(ESPT). The corresponding GFP model chromophore 1 was synthe-
sized with an additional azide functionality that can be used to ligate
it to acetylene-modified biomolecules by Cu(I)-catalyzed cycload-
dition. The chromophore 1 was incorporated as a synthetic modifi-
cation into oligonucleotides using a postsynthetic methodology and
characterized within the DNA environment by optical spectrosco-
py. In order to elucidate the effect of DNA on ESPT a second GFP
chromophore 2 was synthesized carrying a methyl group that pre-
vents ESPT processes. It became evident that DNA is able to pro-
vide an artificial environment for the GFP chromophore that
controls the photophysical property in such a way that nearly solely
the ESPT-driven, red-shifted fluorescence is occurring. The appar-
ent Stokes’ shift is larger than 200 nm (9000 cm–1). Moreover, the
comparison with the methylated chromophore 2 in DNA elucidates
that DNA increases the fluorescence intensity of both GFP models
presumably by restricting the conformational flexibility. Although
the observable quantum yields are too low to consider the fluoro-
phore for any bioanalytical application, the combination of both ef-
fects, red-shifted ESPT-controlled fluorescence with large apparent
Stokes’ shifts and increase of intensity by restricting of internal con-
version, provides an important concept for the design of fluorescent
labels for DNA and RNA.
thetic fluorescent cyanine dyes. Due to the conformational
flexibility the fluorescence of the isolated GFP dye in so-
lution is quenched by radiationless internal conversion,
similar to other cyanine dyes, for instance thiazole orange.
The GFP chromophore inside the protein is buried deeply
in a rigid cylinder of b-sheets, which restricts the confor-
mational flexibility, and thus suppresses the fast depopu-
lation of the excited state by internal conversion.8 On the
other hand, the hydrogen bonding network of the GFP
chromophore in the protein environment promotes an ex-
cited state proton transfer (ESPT) resulting in a very ef-
fective and intense anion fluorescence.9 The two visible
absorption bands at 390–400 nm (A band) and 470–480
nm (B band) have been attributed to the protonated and
deprotonated state of this chromophore.4,9,10 Excitation of
the B band does not show unusual photophysical behav-
ior. However, excitation of the predominant A band
would normally emit blue light, but in fact emits green
light. Upon photoexcitation the pKa of the neutral chro-
mophore changes from a typical phenolic value to near
zero thus becoming a strong acid. Subsequent proton
transfer through a preorganized hydrogen-bonding net-
work rapidly generates the excited state anion with green
fluorescence. The GFP chromophore demonstrates clear-
ly that ESPT can be an extremely useful concept for the
development of synthetic bioanalytical tools and probes.
Hence, studies on synthetic GFP analogues are valuable
and highly important not only for the understanding of the
photophysical details of the natural chromophore inside
the protein but also for designing and developing new and
powerful synthetic fluorescent bioprobes.11–16
Key words: cyanine, fluorescence, green fluorescent protein, oli-
gonucleotide, proton transfer
Green fluorescent proteins (GFPs) have been widely ap-
plied as fluorescent labels for bioanalytical applications
and cell biology, especially live cell imaging.2–6 There is
continuous effort to further develop GFPs and GFP vari-
ants with enhanced brightness and with altered excitation
as well as emission wavelengths, respectively.5,6 GFP as a
fluorescent marker is particularly useful since the chro-
mophore is formed by a sequence of internal reactions that
do not require a cofactor. The GFP chromophore consists
of a p-hydroxybenzylidene-imidazol-5-one group and is
formed via a posttranslational cyclization of a Ser-Tyr-
Gly tripeptide followed by 1,2-dehydrogenation (autoox-
idation) of the Tyr moiety.7 The unique and remarkable
photophysical properties of the GFP chromophore and its
Synthetic GFP-like chromophores such as 1 bear an
ortho-phenol group instead of the para-phenol group of
natural GFP and thus can mimic the hydrogen bonding
network of the protein by an intramolecular hydrogen
bonding to the imidazolone group.14 As a result, it is re-
ported that a strongly enhanced fluorescence is observed
for this isolated synthetic chromophore. Other studies
have shown that encapsulation of the GFP chromophore17
or synthetic incorporation into linear alaninyl peptide nu-
cleic acids18 can enhance the fluorescence quite signifi-
SYNTHESIS 2011, No. 3, pp 0502–0508
x
x
.
x
x
.2
0
1
1
Advanced online publication: 10.01.2011
DOI: 10.1055/s-0030-1258400; Art ID: T19110SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthetic GFP Chromophore
503
cantly. These examples indicate clearly that ESPT-
controlled fluorescence becomes an increasingly impor-
tant concept for the design of synthetic fluorophores as
bioprobes with large apparent Stokes’ shifts. To our
knowledge, the concept of ESPT has not yet been applied
for the development of fluorescent labels for DNA or
RNA. Large Stokes’ shifts are important to prevent arti-
facts in the fluorescent readout that are caused by scatter-
ing of the excitation light. Recently, we demonstrated the
fluorescent labeling of DNA with so-called MegaStokes
dyes that combine to FRET pairs with apparent Stokes’
shifts of approximately 200 nm.19 Herein, we present the
principal achievement of large apparent Stokes’ shifts by
ESPT using a single fluorophore, the synthetic GFP mod-
el chromophore 1 (Scheme 1). In order to explore the po-
tential of ESPT-controlled emission in DNA we chose the
GFP chromophore due its well-characterized optical
properties and photophysical behavior. In principle, dou-
ble helical DNA should provide an artificial environment
with stacking interactions and a hydrogen-bond network
for the GFP chromophore. These structural features of
DNA could represent a basis to modulate the GFP fluores-
cence by controlling both the rapid internal conversion
due to restricted conformational flexibility and the ESPT
by hydrogen bonds. We present herein a synthetic route to
incorporate the GFP model chromophore 1 as a fluores-
cent modification into oligonucleotides using a postsyn-
thetic methodology and the optical characterization of the
GFP chromophore model within the DNA environment.
In order to elucidate the effect of DNA on ESPT the GFP
chromophore 2 was synthesized carrying a methyl group
that prevents ESPT processes.
O
O
MeO
MeCO2Na
OH
OMe
+
N
O
Ac2O
O
HN
4.5 h, 100 °C
O
3
4
5
60%
n-PrOH
H2NCH2CH2OH
4 h, reflux
MeO
MeO
Ph3P, CBr4
N
N
N
abs. CH2Cl2
7 h, 0 °C to r.t.
N
O
O
Br
OH
7
6
NaN3
DMF
4 h, 60 °C
46%
61%
MeO
N
1. BBr3
2. H2O
abs. CH2Cl2
N
O
14 h, 0 °C to r.t.
N3
2
40%
HO
HO
NaN3
N
N
N
Both chromophores are equipped synthetically with an
azide functionality. Among the various possible reactions
for postsynthetic modifications,20 the copper-catalyzed
1,3-dipolar cycloaddition between azides and terminal
alkynes,21 one of the so-called ‘click’-type ligations,22 en-
sures an easy access to covalent modifications of oligonu-
cleotides. This fast, high yielding and biocompatible
reaction does not interfere with other naturally occurring
reactive sites (bioorthogonality).23 This methodology is
particularly suitable for fluorophores and other modifica-
tion that would not be stable under the typical conditions
of DNA preparation and workup. Accordingly, we recent-
ly reported about the single labeling of oligonucleotides
with a coumarin dye and with MegaStokes dyes via the
‘click’-type chemistry.19,24 The dyes were ligated to a pro-
pargyl group that was attached to the 2¢-OH function of
the ribofuranoside part of uridine.25 With respect to the
acid sensitivity of 1 and 2 we applied this postsynthetic
methodology to modify covalently DNA with the GFP
chromophore.
DMF
6.5 h, 60 °C
N
O
O
Br
N3
1
8
66%
72%
Scheme 1 Synthesis of the azides 1 and 2
was attached to 5 via a one-pot ester cleavage and amide
formation with ethanolamine.27 Subsequently, the hy-
droxy group of the linker of 6 needs to be converted into
a good leaving group. The Appel reaction generated the
bromide 7,28 which was directly converted into the azide
2 by treatment with sodium azide in DMF.29 Alternative-
ly, the methylated hydroxy group of 7 can be deprotected
by a standard procedure using boron tribromide at low
temperature.14 The deprotected chromophore 8 can then
be converted into the azide 1, again by treatment with so-
dium azide in DMF (Scheme 1). It is important to note
that the azide formation must be the last step of the multi-
step synthesis, otherwise this functional group generates
substantial amounts of side products during the remaining
reactions. This was also the reason why the standard-type
activation of the hydroxy group of the short linker in 6 by
tosylation or mesylation29 failed.
The synthesis of the azides 1 and 2 started with the estab-
lished Erlenmeyer azlactone synthesis.26 The azlactone 5
was obtained from the starting materials 3 and 4 in good
yield of 60% due to the methylated hydroxy function of
the salicylic aldehyde 3 (Scheme 1). A free hydroxy group
led mainly to coumarin derivatives. A C2 linker function
Synthesis 2011, No. 3, 502–508 © Thieme Stuttgart · New York

504
U. Wenge, H.-A. Wagenknecht
PAPER
3'
5'
Before incorporating the GFP chromophores 1 and 2 into
DNA we characterized their optical properties in aqueous
buffer solution (Figure 1). At neutral pH, the chro-
mophore 1 shows a broad absorption band with a maxi-
mum at 376 nm that stands in agreement with the
published results.14 The fluorescence spectra of 1 exhibit
dual emission with peaks at 501 nm and at 596 nm. Ac-
cording to the literature, the latter band can be assigned to
the emission that originates from an intramolecular ESPT
state of the chromophore.14 Interestingly, the published
spectra of o-hydroxylated, synthetic GFP chromophores
in an organic solvent (cyclohexane) show pure ESPT-
driven fluorescence at 605 nm. The coexistence of both
emissions (501 and 596 nm) in buffer as the solvent can
be explained by incomplete intramolecular hydrogen
bonding in water probably due to alternative hydrogen
bonding possibilities with solvent molecules. If the ESPT
is blocked by methylation of the hydroxyl group – see
GFP chromophore 2 – the fluorescence is solely observ-
able at 478 nm. On the other hand, ESPT fluorescence can
be simulated by elevating the pH value of the aqueous so-
lution to 13. The deprotonated phenolic function of the
GFP chromophore 1 shifts the absorption to 448 nm and
the emission to 595 nm. The latter emission occurs at a
similar wavelength as the ESPT-driven emission of 1 at
pH 7, but with significantly stronger intensity (Table 1).
A
T
G
C
T
A
C
G
A
T
C
G
T
A
T
A
X
Y
T
A
T
A
C
G
T
A
G
C
A
T
C
G
G 5'
C 3'
DNA1Y: R = H DNA3Y: R = Me
3'
5'
A
T
G
C
T
A
C
G
A
T
C
G
T
A
G
C
X
Y
G
C
T
A
C
G
T
A
G
C
A
T
C
G
G 5'
C 3'
DNA2Y: R = H
O
X =
N
R
O
N
O
O
O
O
N
N
N
N
N
O
O
Figure 2 Sequences of DNA1Y and DNA2Y, modified with GFP
chromophore 1, and DNA3Y modified with the methylated GFP
chromophore 2 (each duplex set with Y = A, T, C, or G)
The melting temperatures (Tm) revealed that especially
purines opposite the GFP chromophore cause destabiliza-
tion by –1.8 °C to –5.2 °C (Table 1). Based on our expe-
rience with other chromophores,19,24 such values were
expected. Interesting is the observation that the presence
of T opposite to the chromophore 1 in DNA1T and
DNA2T yields a clear stabilizing effect of +4.6 and
+3.0 °C, respectively, although T forms a mismatch with
the GFP chromophore modified uridine. This effect is di-
minished by methylation of the chromophore hydroxy
group (in DNA3T). The measured Tm differences are in
the range of an additional hydrogen bond.30 Hence, we
propose that the chromophore arrangement in DNA1T
and DNA2T allows an additional hydrogen bond between
the phenolic hydroxy group of the chromophore and one
of the carbonyl groups of the opposite T. This special
structural scenario causes slightly changed optical proper-
ties as discussed below.
The following discussion of the optical properties is fo-
cused mainly on the two different structural effects how
DNA potentially affects the GFP chromophore fluores-
cence: (i) the control of ESPT by hydrogen bonding and
(ii) the control of the rapid internal conversion by less
conformational flexibility. Interactions of the GFP chro-
mophores, 1 and 2, with the DNA base stack can be clear-
ly seen by the bathochromic shift of both absorption and
emission of a few nm. The base opposite to the GFP chro-
mophore modification site has an additional influence on
the optical properties. Especially with T at the opposite
position (DNA1T and DNA2T) both absorption and fluo-
rescence are additionally red-shifted by up to 10 nm.
These observations go along with the enhanced thermal
stability of these two duplexes.
Figure 1 Normalized UV/Vis absorption and fluorescence of the
synthetic GFP chromophores 1 and 2 (100 mM) in Na-Pi (sodium
phosphate) buffer at pH 7 or pH 13, excitation at 400 nm
The ethynyl-modified oligonucleotides were presynthe-
sized according to the literature procedure,24,25 reacted
with the azides 1 or 2, respectively, according to our
‘click’-type ligation protocol, worked up, purified, and
identified according to the literature.24 The two represen-
tative double strands that were synthesized expose the
GFP chromophore 1 to two different variations in the se-
quential neighborhood: (i) The chromophore was placed
either in a T-A (DNA1Y) or G-C base pair environment
(DNA2Y). (ii) Within each duplex set, the base opposite
to the GFP chromophore site was varied (Y = A, T, C or
G). The methylated chromophore 2 was incorporated into
duplex set DNA3Y that has identical sequences as
DNA1Y to elucidate the ESPT effect (Figure 2).
Except for the small bathochromic shifts, the absorption
properties of DNA1Y and DNA2Y are very similar to the
absorption of the isolated chromophore 1 whereas the
fluorescence spectra display remarkable differences
(Figure 3). Both emission bands (ESPT-controlled and
Synthesis 2011, No. 3, 502–508 © Thieme Stuttgart · New York

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Synthetic GFP Chromophore
505
Table 1 Melting Temperatures, Optical Properties, and Quantum
Yields of 1, 2, and DNA1Y-3Y
Product Tm (°C)DTm (°C)alabs (nm) lem (nm) Dl (nm) FF (10–3)
1
–
–
376
448
370
393
396
403
397
393
396
400
397
382
386
389
389
501/596 125/220 0.19
1 (pH 13) –
–
595
478
609
611
617
609
610
607
613
607
460
458
467
467
147
108
216
215
214
212
217
211
213
210
78
0.67
0.14
4.3
2
–
–
DNA1A 58.3
DNA1G 53.0
DNA1T 57.8
DNA1C 52.5
DNA2A 66.0
DNA2G 62.0
DNA2T 63.3
DNA2C 60.5
DNA3A 57.3
DNA3G 51.7
DNA3T 53.5
DNA3C 50.5
–4.2
–2.0
+4.6
–0.3
–2.0
–1.8
+3.0
+2.2
–5.2
–3.3
+0.3
–2.3
b
–
b
–
b
–
1.9
Figure 3 Normalized UV/Vis absorption and fluorescence of
DNA1A, DNA1T, DNA2A, DNA2T, and DNA3A (2.5 mM) in Na-Pi
buffer at pH 7, excitation at 400 nm
b
–
b
–
b
–
DNA1A. Hence, the fluorescence enhancement cannot be
attributed to the ESPT but to the reduced conformational
flexibility and decreased radiationless decay by rapid in-
ternal conversion. Of course, we do not know if these ef-
fects are caused by intercalation of the GFP chromophore
(similar to ethidium bromide) or just by groove binding
(similar to cyanine dyes). Nevertheless, it is remarkable to
note that DNA is able (i) not only to control the ESPT for
increasing the apparent Stokes’ shift (of 1) but (ii) also to
increase the fluorescence intensity by restricting the con-
formational rotation freedom around the methine bridge
of synthetic GFP chromophores (in 1 and 2). The combi-
nation of both effects provides a promising concept to
achieve fluorescent labels for nucleic acids with large ap-
parent Stokes’ shifts.
2.7
b
72
–
b
78
–
b
78
–
a Compared to a completely unmodified DNA duplex.
b Not determined.
non-ESPT-controlled) of the isolated chromophore 1 at
pH 7 show almost equal intensities (as discussed above).
In contrast, the fluorescence spectra of all duplexes within
the set DNA1Y are dominated by the red-shifted, ESPT-
controlled band. The observable effect is even more pro-
nounced in the set DNA2Y; here the non-ESPT-con-
trolled band has nearly vanished. This is a remarkable
result, since it shows that DNA is able to provide an envi-
ronment that is similar to an organic solvent. That means
that DNA protects the chromophore from hydrogen bond-
ing with the surrounding water and enforces the presence
of the intramolecular hydrogen bond in the chromophore.
In conclusion, a synthetic route has been worked out to
prepare and equip the GFP model chromophores 1 and 2
with an azide group for ‘click’-type conjugation to bio-
molecules. The synthetic GFP-like chromophore 1 bears
an o-phenol group instead of the p-phenol group of natural
GFP and thus can mimic the hydrogen bond network in-
side DNA by an intramolecular hydrogen bond to the im-
idazolone group. At neutral pH, the spectra of 1 show dual
emission, the ‘normal’ and the ESPT-driven, red-shifted
fluorescence. Once incorporated into DNA as a 2¢-modi-
fication using a postsynthetic strategy, the fluorescence
spectra of all double strands within the set DNA1Y are
dominated by the red-shifted, ESPT-controlled band. Ob-
viously, DNA is similar to an organic solvent with respect
to providing an artificial environment for the GFP chro-
mophore that enforces the presence of the intramolecular
hydrogen bond in the chromophore and controls its photo-
physical property in such a way that nearly solely the
ESPT-driven, red-shifted fluorescence is occurring. The
apparent Stokes’ shift of ca. 210 nm (9000 cm–1) is re-
markably high for a single fluorophore and separates nice-
ly excitation from emission light, a desirable feature for
many imaging and bioanalytical applications. Moreover,
the comparison with the methylated chromophore 2 in
Measurements of the quantum yields of the isolated chro-
mophore are vey low (FF 0.19·10–3), but enhanced by fac-
tor of more than 20 inside the duplex DNA1A and 10 in
DNA2A. In order to avoid misunderstandings it is impor-
tant to mention here, that the quantum yields in DNA, al-
though enhanced, are still far too low to apply similar
GFP-modified oligonucleotides in any imaging or bio-
analytical application.
With respect to these results, the duplexes of set DNA3Y
represent important controls by two different means.
Firstly, the strongly enhanced red-shifted fluorescence at
~600 nm is completely absent due to the presence of the
methyl group as a blocker for ESPT. Secondly and more
importantly, the fluorescence of DNA3A exhibits a nearly
20-fold increase of the intensity compared to the isolated
chromophore 2, a similar enhancement as observed with
Synthesis 2011, No. 3, 502–508 © Thieme Stuttgart · New York

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U. Wenge, H.-A. Wagenknecht
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13C NMR (75 MHz, CD3OD): d = 15.8 (CH3C), 44.4 (CH2), 56.3
(CH3O), 60.7 (CH2), 111.9 (ArCH), 121.7 (ArCH), 122.3 (HC=C),
124.0 (ArCC), 133.3 (ArCH), 133.9 (ArCH), 139.0 (HC=C), 160.6
(C–O), 165.5 (C=N), 172.4 (C=O).
ESI-MS: m/z (%) = 261.0 (100, [M + H]+).
HRMS (EI-MS): m/z [M+·] calcd for C14H16N2O3: 260.1161; found:
DNA makes clear that the presence of DNA increases the
fluorescence intensity by restricting the conformational
flexibility in the GFP chromophore. The latter effect is
well known from various other cyanine dyes. The observ-
able quantum yields of the GFP chromophore in DNA are
too small to be relevant for applications. However, the
combination of both effects, red-shifted ESPT-controlled
fluorescence with large apparent Stokes’ shifts and in-
crease of intensity by restricting of internal conversion is
very attractive for the design of new and powerful fluores-
cent labels for DNA and RNA. Herein we evidenced these
principles for the well-known GFP chromophore attached
to DNA. Future work will show how this concept can be
transferred to other fluorophores with higher quantum
yields.
260.1161.
1-(2-Bromoethyl)-4-(2-methoxybenzylidene)-2-methyl-1H-imi-
dazol-5(4H)-one (7)
Compound 6 (600 mg, 2 mmol) was dissolved in anhyd CH2Cl2 (23
mL) under N2 atmosphere. First, Ph3P (670 mg, 3 mmol, 1.10 equiv)
was added at 0 °C, then, after 10 min, CBr4 (920 mg, 3 mmol, 1.20
equiv) was added. The mixture was stirred at r.t. for 7 h. Then addi-
tional Ph3P (670 mg, 3 mmol, 1.10 equiv) and CBr4 (920 mg, 3
mmol, 1.20 equiv) were added. After 1.5 h, the solvent was removed
under reduced pressure, and the residue was purified by flash chro-
matography with CH2Cl2–acetone (5:1); yield: 460 mg (61%); or-
ange solid; Rf = 0.86 (CH2Cl2–acetone, 10:1).
1H NMR (300 MHz, CDCl3): d = 2.41, (s, 3 H, CH3C), 3.56 (t,
J = 6.3 Hz, 2 H, CH2), 3.84 (s, 3 H, CH3O), 3.97 (t, J = 6.3 Hz, 2 H,
CH2), 6.85 (d, J = 8.4 Hz, 1 H, ArH), 6.99 (t, J = 7.7 Hz, 1 H, ArH),
7.28–7.34 (m, 1 H, ArH), 7.66 (s, 1 H, HC=C), 8.67–8.70 (m, 1 H,
ArH).
13C NMR (75 MHz, CDCl3): d = 16.0 (CH3C), 29.0 (CH2), 42.6
(CH2), 55.6 (CH3O), 110.6 (ArCH), 120.9 (ArCH), 122.2 (HC=C),
123.0 (ArCC), 132.0 (ArCH), 132.9 (ArCH), 137.4 (HC=C), 159.2
(C–O), 161.0 (C=N), 170.5 (C=O).
ESI-MS: m/z (%) = 324.9 (100, [M + H]+).
HRMS (EI-MS): m/z [M+·] calcd for C14H15BrN2O2: 322.0317;
TLC was carried out with silica gel 60 on aluminum foil (Merck).
1H and 13C spectra were recorded on Bruker Avance 300 and 400 in-
struments; chemical shifts (d) are referred to residual protonated
solvent. HRMS spectra were recorded on a Finnigan MAT 95 spec-
trometer. ESI-MS spectra were recorded on a ThermoQuest Finni-
gan TSQ 7000 spectrometer. CI-MS spectra were recorded on a
Finnigan MAT SSQ710 spectrometer. Absorption measurements
were carried out on Cary 100 (Varian) spectrometer at 20 °C. Fluo-
rescence measurements were performed on Fluoromax-3 (Horiba
Jobin Yvon) spectrometer at 20 °C. The quantum yields of the iso-
lated model compounds 1 and 2 were measured with perylene as
standard. The intensity of the excitation light had to be reduced to
0.1% by applying a neutral glass filter. In the case of modified
DNA, the quantum yields were measured with zinc(II)-5,10,15,20-
tetraphenyl-21H,23H-porphyrinate as standard, and the intensity of
the excitation light had to be reduced to 26.4%.
found: 322.0316.
1-(2-Azidoethyl)-4-(2-methoxybenzylidene)-2-methyl-1H-imi-
dazol-5(4H)-one (2)
4-(2-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (5)
o-Anisaldehyde (3; 14.0 mL, 15.8 g, 116 mmol), N-acetylglycine
(4; 14.0 g, 120 mmol, 1.03 equiv), and anhyd NaOAc (10.7 g, 130
mmol, 1.12 equiv) in Ac2O (47 mL, 51.0 g, 500 mmol) were stirred
at 100 °C for 4.5 h. The mixture was allowed to cool down to r.t. Ice
water (50 mL) was added. The resulting precipitate was collected by
filtration, washed with ice water, and dried in vacuum; yield: 15.2 g
(60%); yellow solid.
1H NMR (300 MHz, CDCl3): d = 2.35 (s, 3 H, CH3C), 3.85 (s, 3 H,
CH3O), 6.87 (d, J = 8.5 Hz, 1 H, ArH), 6.99 (t, J = 7.6 Hz, 1 H,
ArH), 7.32–7.38 (m, 1 H, ArH), 7.71 (s, 1 H, HC=C), 8.56–8.60 (m,
1 H, ArH).
Compound 7 (100 mg, 0.3 mmol) and NaN3 (200 mg, 3.1 mmol,
10.00 equiv) were dissolved in DMF (3 mL) and stirred for 3 h at
60 °C. The suspension was mixed with H2O (4 mL) and extracted
with CH2Cl2 (3 × 3 mL). The combined organic phases were washed
with brine (3 × 15 mL) and dried (Na2SO4). The solvent was evap-
orated and the residue was purified by flash chromatography using
CH2Cl2–acetone (400:1) as eluent; yield: 35 mg (40%); yellow sol-
id, Rf = 0.52 (CH2Cl2–acetone, 50:1).
1H NMR (300 MHz, CD3OD): d = 2.42 (s, 3 H, CH3C), 3.61 (t,
J = 5.5 Hz, 2 H, CH2), 3.74 (t, J = 5.5 Hz, 2 H, CH2), 3.88 (s, 3 H
CH3O), 6.89 (d, J = 8.3 Hz, 1 H, ArH), 7.03 (t, J = 7.6 Hz, 1 H,
ArH), 7.32–7.38 (m, 1 H, ArH), 7.69 (s, 1 H, HC=C), 8.71–8.74 (m,
1 H, ArH).
13C NMR (100 MHz, CDCl3): d = 15.6 (CH3C), 55.6 (CH3O), 110.7
(ArCH), 120.9 (ArCH), 122.2 (ArCC), 125.7 (HC=C), 131.8
(HC=C), 132.5 (ArCH), 132.8 (ArCH), 159.1 (C–O), 165.5 (C=N),
168.0 (C=O).
13C NMR (75 MHz, CD3OD): d = 14.8 (CH3C), 39.1 (CH2), 48.8
(CH2), 54.3 (CH3O), 109.6 (ArCH), 119.9 (ArCH), 121.0 (HC=C),
122.0 (ArCC), 130.9 (ArCH), 131.9 (ArCH), 136.5 (HC=C), 158.1
(C–O), 160.3 (C=N), 169.6 (C=O).
CI-MS: m/z (%) = 217.1 (29, [M+·]).
HRMS (EI-MS): m/z [M+·] calcd for C12H11NO3: 217.0739; found:
217.0740.
ESI-MS: m/z (%) = 286.0 (100, [M + H]+).
HRMS (EI-MS): m/z [M+.] calcd for C14H15N5O2: 285.1226; found:
285.1223.
1-(2-Hydroxyethyl)-4-(2-methoxybenzylidene)-2-methyl-1H-
imidazol-5(4H)-one (6)
1-(2-Bromoethyl)-4-(2-hydroxybenzylidene)-2-methyl-1H-imi-
dazol-5(4H)-one (8)
To a solution of 5 (5.00 g, 23 mmol) in anhyd n-propanol (23 mL),
ethanolamine (1.52 mL, 1.55 g, 25 mmol, 1.10 equiv) was added.
The reaction mixture was heated under reflux for 5 h. The solvent
was evaporated and the residue was purified by recrystallization
from pentanol–Et2O mixture, yield: 2.78 g (46%); yellow solid.
1H NMR (300 MHz, CD3OD): d = 2.44 (s, 3 H, CH3C), 3.73 (m, 4
H, CH2), 3.91 (s, 3 H, CH3O), 6.98–7.04 (m, 2 H, ArH), 7.36–7.42
(m, 1 H, ArH), 7.57 (s, 1 H, HC=C), 8.55–8.58 (m, 1 H, ArH).
Compound 7 (170 mg, 0.51 mmol) was dissolved in anhyd CH2Cl2
under N2 atmosphere and cooled to 0 °C. A 1.0 M solution of BBr3
in CH2Cl2 (0.54 mL, 1.06 equiv) was added dropwise over 15 min.
After stirring overnight, H2O (5 mL) was added and stirred for 0.5
h. The aqueous phase was extracted with CH2Cl2 (2 × 10 mL) The
combined organic phases were dried (Na2SO4) and the solvent was
Synthesis 2011, No. 3, 502–508 © Thieme Stuttgart · New York

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Synthetic GFP Chromophore
507
evaporated. The solid was purified by flash chromatography using
CH2Cl2–acetone (50:1) as eluent; yield: 113 mg (72%); yellow sol-
id; Rf = 0.68 (CH2Cl2–acetone, 50:1).
1H NMR (300 MHz, CDCl3): d = 2.44 (s, 3 H, CH3C), 3.60 (t,
J = 6.1 Hz, 2 H, CH2), 4.02 (t, J = 6.1 Hz, 2 H, CH2), 6.80–6.85 (m,
1 H, ArH), 6.96 (d, J = 8.2 Hz, 1 H, ArH), 7.15 (s, 1 H, HC=C),
7.24–7.35 (m, 2 H, ArH).
13C NMR (75 MHz, CDCl3): d = 15.6 (CH3C), 29.8 (CH2), 43.7
(CH2), 119.2 (ArCH), 120.7 (ArCH), 121.1 (ArCC), 129.2
(HC=C), 134.9 (ArCH), 135.0 (HC=C), 136.8 (ArCH), 159.6
(C=N), 160.9 (C–O), 170.1 (C=O).
2400 L/mol·cm for 1 and 3800 L/mol·cm for 2, both at 260 nm.
DNA duplexes are formed by heating in the presence of 1.2 equiv
of the unmodified counterstrands at 90 °C for 10 min followed by
slow cooling to r.t.
ESI-MS:
DNA1: m/z calcd for C181H225N61O107P16: 5460.0 Da; found: 1365.0
[M – 4 H]4–, 1820.3 [M – 3 H]3–
DNA2: m/z calcd for C181H223N67O105P16: 5510.0 Da; found: 1377.3
[M – 4 H]4–, 1837.2 [M – 3 H]3–
DNA3: m/z calcd for C182H227N61O107P16: 5474.0 Da; found: 1368.4
[M – 4 H]4–, 1825.0 [M – 3 H]3–
ESI-MS: m/z (%) = 308.8 (100, [M + H]+).
HRMS (EI-MS): m/z [M+·] calcd for C13H13BrN2O2: 308.0161;
found: 308.0163.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
1-(2-Azidoethyl)-4-(2-hydroxybenzylidene)-2-methyl-1H-imi-
dazol-5(4H)-one (1)
Acknowledgment
Compound 8 (40 mg, 0.14 mmol) and NaN3 (93 mg, 1.42 mmol,
10.0 equiv) were dissolved in DMF (1.4 mL) and stirred for 4 h at
60 °C. The suspension was mixed with H2O (2 mL) and extracted
with CH2Cl2 (3 × 2 mL). The combined organic phases were washed
with brine (2 × 10 mL) and dried (Na2SO4). The solvent was evap-
orated and the residue was purified by flash chromatography using
CH2Cl2–acetone (50:1) as eluent; yield: 25 mg (66%); light yellow
solid; Rf = 0.60 (CH2Cl2–acetone, 50:1).
1H NMR (300 MHz, CDCl3): d = 2.41, (s, 3 H, CH3C), 3.60–3.64
(m, 2 H, CH2), 3.72–3.77 (m, 2 H, CH2), 6.80–6.85 (m, 1 H, ArH),
6.92 (d, J = 8.0 Hz, 1 H, ArH), 7.15 (s, 1 H, HC=C), 7.24–7.35 (m,
2 H, ArH), 13.67 (s, 1 H, OH).
Financial support is acknowledged from the University of Regens-
burg and the Deutsche Forschungsgemeinschaft.
References
(1) New address: Karlsruhe Institute of Technology, Institute
for Organic Chemistry, Fritz-Haber-Weg 6, 76131
Karlsruhe, Germany; Fax: +49(721)60844825; e-mail:
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13C NMR (100 MHz, CDCl3): d = 15.4 (CH3C), 40.5 (CH2), 49.6
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(HC=C), 134.4 (ArCH), 136.5 (ArCH) 157.0 (C=N), 158.7 (C–O),
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271.1069
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Modified Oligonucleotides
Oligonucleotides were prepared on an Expedite 8909 Synthesizer
from Applied Biosystems (ABI) using standard phosphoramidite
chemistry. Reagents and controlled pore glass (CPG) (1 mmol) were
purchased from Proligo. 5¢-O-Dimethoxytrityl-N3-pivaloyloxy-
methyl-2¢-O-propargyluridine-3¢-O-(2-cyanoethyl-N,N-diisopropyl-
phosphoramidite was synthesized according to the literature
procedure.24 The concentration of the uridine building block was in-
creased to 0.1 M. After preparation, the trityl-off oligonucleotides
were cleaved from the resin and deprotected by treatment with con-
cd NH4OH at 50 °C for 15 h. The azide 1 or 2 (114 mL, 10 mM),
Cu(I) (17 mL, 100 mM), TBTA (34 mL, 100 mM), each in DMSO–
t-BuOH (3:1), and sodium ascorbate (25 mL, 400 mM) in H2O were
added to the oligonucleotide (1 mmol scale). The reaction mixture
was vortexed, shaken overnight at r.t., and then evaporated to dry-
ness using a speedvac. NaOAc (100 mL, 0.15 mmol) and EtOH (1
mL) were added, vortexed and frozen (–20 °C) overnight. The sus-
pension was centrifuged (13 000 rpm, 15 min), and the supernatant
removed. The pellet was dissolved in H2O (500 mL). Prior to purifi-
cation by HPLC, the DNA was desalted by NAP-5 column (GE
Healthcare). The modified oligonucleotides were purified by HPLC
on a semi-preparative RP-C18 column (300 Å, Supelco), using the
following conditions: A) NH4OAc buffer (50 mM, pH 6.5); B)
MeCN, gradient 0–20% B over 45 min, flow rate 2.5 mL/min, UV/
VIS detection at 260 nm and 378 nm. The modified oligonucleo-
tides were lyophilized, identified by ESI mass spectrometry, and
quantified by UV/Vis absorption using extinction coefficients of
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Synthesis 2011, No. 3, 502–508 © Thieme Stuttgart · New York