Blue Fluorescent Amino Acids
anhydrous toluene (10 mL). The mixture was stirred under nitrogen
at reflux temperature for 3 h. After being cooled, the solvent was
removed and the remaining white solid was dried in vacuo. The
crude product was used for the next reaction without further
purification. A mixture of ethyl acetate (25 mL) and concentrated
hydrochloric acid (10 mL) were added to the methylated salt. After
being stirred at room temperature for 18 h a concentrated aque-
ous solution of potassium hydroxide was added (pH 13) and the
mixture was extracted with dichloromethane (4ꢁ100 mL). Drying
with magnesium sulfate and evaporation of the solvent gave
duplex DNA and can be replicated by appropriate polymerases
with high efficiency and selectivity. This would lead to a greatly
expanded coding capacity of the genetic code.[47] These novel
noncanonical pairings could be based on sterical complemen-
tarity or hydrophobic interactions or on hydrogen bonding
through their tautomeric forms. For example, indole-like bases,
such as 7-azaindole or benzotriazole, could be good promising
substrates for incorporation into nucleic acids based on shape
complementarity.[48,49]
1
450 mg (90%) of an orange-colored oil. H NMR (400 MHz, CDCl3):
d=8.28 (d, J=1.8 Hz, 1H), 8.18 (d, J=7.7 Hz, 1H), 7.57 (d, J=
6.2 Hz, 1H), 6.94 (dd, J=7.7, 6.2 Hz, 1H), 6.44 (dd, J=1.8, 0.9 Hz,
1H), 4.15 (s, 3H) ppm.
Experimental Section
All chemicals were purchased from Sigma–Aldrich, unless other-
wise stated; 4AI and 7AI were purchased from Molekula and Bio-
synth. 1-Methyl-7-azaindole and 1-methyl-4-azaindole were synthe-
sized as described.[45,50] The protection of 4AI was performed as de-
scribed.[51] For the deprotection the literature procedure was
used.[52]
UV absorbance and fluorescence measurements: We acquired eM
of indole and azaindoles by UV spectra measurement with the UV/
Vis spectrometer lambda 19 (PerkinElmer and Life Sciences). Sam-
ples were measured in Tris-HCl buffer (100 mm, pH 7.5). Fluores-
cence spectra in a range of 300–500 nm were recorded with the lu-
minescence spectrometer LS50B (PerkinElmer and Life Sciences) at
208C at the determined maximum absorbance wavelengths as fol-
lows: W (278 nm), 4AI (288 nm), 1M4AI (289 nm), 4M4AI (285 nm),
1,4M4AI (290 nm), 7AI (288 nm), 1M7AI (289 nm), 1,7M7AI
(290 nm), 4AmI (270 nm), 4Am4AI (300 nm). Different excitation/
emission slits were used for the different substances measured be-
cause the fluorescence emission of some derivatives could not be
measured in a reasonable dilution at slit 2.5/2.5. Therefore, in case
of 4AI, 7AI, 4AmI and 4Am4AI the slits were set to 5/5. By co-
measuring with tryptophan as standard, measurements at different
slits and concentrations were later normalized with respect to each
other.
Syntheses
1-Methyl-7-azaindole (1M7AI): 7-Azaindole (472 mg, 4 mmol) and
sodium hydride (192 mg, 4.8 mmol; 60% in oil) were cooled to
08C under a nitrogen atmosphere. Then anhydrous dimethylforma-
mide (10 mL) was added and the mixture was stirred until the evo-
lution of hydrogen was complete. After the dropwise addition of
iodomethane (0.25 mL, 4 mmol) the reaction was allowed to warm
to room temperature. After being stirred at this temperature for
3 h, the solvent was removed and the residue was purified by flash
chromatography (PE:EE:DCM, 8:1.8:0.2) to give 494 mg (94%) of a
light yellow oil. 1H NMR (400 MHz, CDCl3): d=08.34 (dd, J=4.7,
1.5 Hz, 1H), 7.90 (dd, J=7.8, 1.5 Hz, 1H), 7.18 (d, J=3.5 Hz, 1H),
7.05 (dd, J=7.8, 4.7 Hz, 1H), 6.45 (d, J=3.5 Hz, 1H), 3.900 (s, 3H)
ppm. The NMR data are in agreement with those reported in the
literature.[50]
Excitation spectra were corrected automatically by the instrument
for artifacts originating from the xenon lamp (energy output is
wavelength and time dependent) and from the excitation mono-
chromator (efficiency is wavelength dependent). Emission spectra
were corrected with respect to artifacts originating from the emis-
sion monochromator (efficiency is wavelength dependent) and the
sample multiplier (sensitivity is wavelength dependent). Correction
files for emission spectra are yearly updated and provided by the
manufacturer of the instrument (PerkinElmer and Life Sciences). All
measurements were made with constant detector current.
1-Methyl-4-azaindole (1M4AI): 4-Azaindole (354 mg, 3 mmol) and
sodium hydride (144 mg, 3.6 mmol; 60% in oil) were cooled to
08C under a nitrogen atmosphere. Then dry dimethylformamide
(10 mL) was added and the mixture was stirred until the evolution
of hydrogen was complete. After the dropwise addition of iodome-
thane (0.19 mL, 3 mmol) the reaction was allowed to warm to
room temperature. After being stirred at this temperature for 3 h,
the solvent was removed and the residue was purified by flash
chromatography (PE:EE, 1:1) to give 330 mg (83%) of a light
brown oil. 1H NMR (400 MHz, CDCl3): d=8.46 (dd, J=4.6, 1.2 Hz,
1H), 7.62 (d, J=8.2 Hz, 1H), 7.32 (d, J=3.2 Hz, 1H), 7.12 (dd, J=
8.2, 4.6 Hz, 1H), 6.69 (dd, J=3.2, 0.8 Hz, 1H), 3.81 (s, 3H) ppm.
Acknowledgements
We thank Prof. Luis Moroder for critical reading of the manu-
script and Dr. Sandra Lepthien for the protein gels in Figure 1.
We are also grateful to Dr. Stephan Uebel from the Microchemis-
try Core Facility of the MPI for provided us with instrumentation
for spectroscopic measurements. This work was supported by the
BioFuture program of the Federal Ministry of Science and Educa-
tion (BMBF) of Germany.
tert-Butyl-4-azaindole-1-carboxylate:
N,N-Dimethylamino-pyridine
(49 mg, 0.4 mmol) and di-tert-butyl dicarbonate (1.05 g, 4.8 mmol)
were added to a solution of 7-azaindole (472 mg, 4 mmol) in anhy-
drous acetonitrile (10 mL). After being stirred under nitrogen at
room temperature for 3 h, the solvent was removed and the resi-
due was purified by flash chromatography (PE:EE, 1:1) to give
1
874 mg (quant.) of a white solid; m.p. 69–718C. H NMR (400 MHz,
Keywords: charge transfer · green fluorescence · proteins ·
tautomerism · tryptophan
CDCl3): d=8.34 (dd, J=4.7, 1.3 Hz, 1H), 7.90 (d, J=6.8 Hz, 1H),
7.83 (d, J=3.7 Hz, 1H), 7.05 (dd, J=8.3, 4.7 Hz, 1H), 6.78 (d, J=
3.7 Hz, 1H), 1.68 (s, 9H) ppm. 13C NMR (100 MHz, CDCl3): d=149.1,
148.8, 145.5, 128.9, 122.2, 118.7, 108.3, 84.5, 28.1 ppm. The NMR
data are in agreement to those reported in the literature.[53]
[1] I. M. Dobbie, N. F. Lowndes, K. F. Sullivan in Autofluorescent proteins,
Vol. 85 (Ed.: K. F. Sullivan), Academic Press, London, 2008, pp. 1–22.
4-Methyl-4-azaindole (4M4AI): Dimethylsulfate was added to a solu-
tion of tert-butyl-4-azaindole-1-carboxylate (775 mg, 3.55 mmol) in
ChemBioChem 2010, 11, 305 – 314
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313