Synthesis of alaninyl and N-(2-aminoethyl)glycinyl amino acid derivatives containing the green fluorescent protein chromophore in their side chains for incorporation into peptides and peptide nucleic acids

Article abstract of DOI:10.1002/ejoc.200600729

Artificial amino acids carrying either the chromophore of the Green Fluorescent Protein (GFP) or a modification as their side chains have been synthesized: Boc-protected alaninyl derivatives and Fmoc-protected N-(2-aminoethyl)glycine-functionalized amino

Full text of DOI:10.1002/ejoc.200600729

FULL PAPER
DOI: 10.1002/ejoc.200600729
Synthesis of Alaninyl and N-(2-Aminoethyl)glycinyl Amino Acid Derivatives
Containing the Green Fluorescent Protein Chromophore in Their Side Chains
for Incorporation into Peptides and Peptide Nucleic Acids
Thorsten Stafforst[a] and Ulf Diederichsen*[a]
Keywords: Fluorescence / Chromophores / Peptide nucleic acid / Green Fluorescent Protein (GFP) / Solid-phase synthesis
Artificial amino acids carrying either the chromophore of the
Green Fluorescent Protein (GFP) or a modification as their
side chains have been synthesized: Boc-protected alaninyl
derivatives and Fmoc-protected N-(2-aminoethyl)glycine-
functionalized amino acids were obtained and could be ap-
plied in solid-phase peptide synthesis. The incorporation of
the GFP chromophore into N-(2-aminoethyl)glycine-PNA
was achieved and fluorescence was studied as a function of
hybridization with complementary DNA.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
Introduction
Since 1992, when the Green Fluorescent Protein (GFP)
was cloned for the first time, it has become widely used as
a highly efficient tool in cell biology,[1] thanks to its interest-
ing photophysics, which allows monitoring of protein move-
ments and interactions inside the living cell by various tech-
niques such as Fluorescence Recovery After Photobleach-
ing (FRAP), Fluorescence Loss In Photobleaching (FLIP),
Fluorescence Localization After Photobleaching (FLAP),
and Fluorescence Resonance Energy Transfer (FRET).[2]
The photophysical properties of GFP and its homologues
are the products of interactions between the remarkable
small chromophore 1 and its immediate protein environ-
ment. Fluorescence of the isolated chromophore in solution
is quenched by radiationless internal conversion, but ap-
pears upon freezing.[3] Fast internal conversion is also re-
sponsible for photobleaching of the GFP fluorescence. A
rotation mechanism has been suggested (Figure 1): either
cis-trans isomerization, a rotation of the phenyl group, or a
combination of both phenomena results in a decay of the
chromophore’s excited state, so fluorescence can only be ob-
served if the fast internal conversion is suppressed by re-
striction of the chromophore flexibility, as is provided by
the rigid protein environment.[4] The role of the protonation
state of the chromophore 1 in internal conversion is still a
matter of debate, as is the role of stacking interactions vs.
the role of the hydrogen bond network for the restriction of
the chromophore flexibility.[5]
Figure 1. The proposed fluorescence quenching mechanism of the
GFP chromophore 1 and comparison with thiazole orange.
Thiazole orange is a chromophore with a similar mecha-
nism for fluorescence quenching through cis-trans isomer-
ization; it shows a strong increase in fluorescence emission
upon intercalation in DNA, a property that has been used
to test for single-point mutations in DNA.[6] This raises the
question of the minimal environment required for the small
GFP chromophore (GFPC) to show fluorescence and to
display the interesting photophysical properties we know
[a] Institut für Organische und Biomolekulare Chemie, Georg-Au-
gust-Universität Göttingen,
Tammannstrasse 2, 37077 Göttingen, Germany
Fax: +49-551-392944
E-mail: udieder@gwdg.de
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from the GFP. To answer this question, here we present the
synthesis of two GFP chromophores and their functionali-
zation as alaninyl and N-(2-aminoethyl)glycinyl amino ac-
ids for incorporation into regular peptides and into N-(2-
aminoethyl)glycine-PNA[7] by solid-phase peptide synthesis
(Figure 2). From the incorporation of the GFP chromo-
phore into peptides and nucleic acid analogues we might
expect to learn about the interactions between the GFP
chromophore and the protein environment, and one future
prospect might be the application of the fluorescence prop-
erties of the GFP chromophore 1 to test for DNA binding.
Figure 2. GFP chromophores 2 and 3 and their incorporation into
alanyl derivatives 4–6 and N-(2-aminoethyl)glycinyl amino acids 7
and 8.
Figure 3. Synthesis of the 3-methyl-GFP chromophores 2 and 3.
monomers 4–6 by thermal cyclization in DMF in the pres-
ence of catalytic amounts of DBU. The low temperature
and the presence of catalytic amounts of base reduced the
thermal degradation, giving 74–86% yields and complete
conversion. As a minor product, 22 (10–20% yield, charac-
Results and Discussion
Synthesis
The synthesis of the 3-methyl-GFP chromophores 2 and terized by NMR, EI-MS) was probably formed from the
3 was accomplished through preparation of the correspond- products 4–6 by intramolecular nucleophilic attack of an
ing azlactones 9 and 10 by literature procedures (Fig- adjacent carbonyl group with the 4-imidazolone anion as
ure 3).[8] The azlactones were then transformed into the cor- the leaving group. The enantiomeric excess was determined
responding amides 11 and 12 by treatment with methyl- to be Ͼ 95% by HPLC after derivatization with enantio-
amine, followed by thermal cyclization at 230 °C under vac- merically pure alanine. In addition, the phenol functionality
uum.[9] On application of the literature procedures for the of the (OH)GFPC building block 5 was protected with
synthesis of the GFP chromophores 2 and 3 in one step dichlorobenzyl bromide to yield 6 (63%) once more.[13]
starting from the azlactones 9 and 10, the major products
The synthesis of the GFPC-functionalized N-(2-amino-
turned out to be the acids 13 and 14.[10] The moderate ethyl)glycine (aeg) monomers 7 and 8 (Figure 5) required
yields of the thermal conversion of the amides into the GFP the three-step syntheses of GFPC acetic acids 23 and 24,
chromophores were the result of the low thermal stabilities starting from acids 13 and 17 by coupling with glycine
of the GFP chromophores, which did not allow complete methyl ester by the DCC/HOBt activation procedure, ther-
conversion.
mal cyclization to afford the GFPC acetic acid methyl esters
The synthesis of the alaninyl GFP chromophores 4–6 27 and 28, and basic saponification in the presence of LiOH
(Figure 4) started from the corresponding acids 13–15 (Fig- to give the corresponding GFPC acetic acids 23 and 24 in
ure 3), which were in turn synthesized from azlactones 9, 25% and 10% overall yields.
10, and 16 by mild hydrolysis in acetone/water. The hydroly-
The GFPC-functionalized Fmoc-aeg-monomers 7 and 8
sis of the acetyl group of 10 required further treatment with were synthesized by a recently developed strategy for the
potassium hydroxide after hydrolysis of the azlactone moi- synthesis of highly acid-labile building blocks for study of
ety in acetone/water. The acids 13–15 were then coupled charge transfer in nucleic acids.[14] In analogy with this pro-
with enantiomerically pure N-α-Boc--diaminopropionic cedure, the GFPC acetic acids 23 and 24 were coupled with
acid[11] 18 by use of the DCC/HOBt activation technique[12] Fmoc-aeg-OMe (29) by the HBTU/HOBt activation
in 73–90% yields (Figure 4), and the obtained amides 19– method and were saponified with retention of the Fmoc
21 were converted into the GFPC-functionalized alanine group in the presence of lithium iodide[15] to provide 7 and
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The (H)GFPC-functionalized Fmoc-aeg monomer 7 was
introduced into a PNA oligomer through solid-phase syn-
thesis by use of the Fmoc/Bhoc strategy, with a recently
developed method that allows for the synthesis of N-(2-ami-
noethyl)glycine-PNA on acid-labile Sieberamide resin for
this purpose.[14] The PNA oligomer Ac-t-t-t-t-(H)GFP-g-g-
c-g-g-c-Lys-Lys-Gly-NH2 (32) was synthesized on a 5 µ
scale and was obtained in 9% yield after HPLC purifica-
tion.
For solid-phase peptide synthesis by the Boc strategy one
has to allow for the fact that the GFP chromophore is not
stable to treatment with TFMSA, so a suitable linker and
side chain protecting group strategy was required. Alterna-
tively, it should also be possible to transform the Boc-pro-
tected alaninyl monomers 4–6 into the corresponding
Fmoc-protected monomers in order to use them with con-
ventional side chain protection methods. (The direct synthe-
sis of the Fmoc-protected monomers starting from Nα-
Fmoc-diaminopropionic acid is not possible because of the
low thermal stability of the Fmoc group.)
Fluorescence Properties
The synthesized GFP chromophores showed typical UV
spectra and fluorescence behavior, with almost no fluores-
cence being observed in methanol, water, or ethyl acetate.
Interestingly, though, fluorescence was observed in highly
Figure 4. Synthesis of Boc--alaninyl GFP chromophores 4–6.
8 in 51% and 12% overall yields. The remarkably low yield viscose solvents such as glycerin (Figure 6), in which the
for the coupling of (OH)GFPC acetic acid 24 with Fmoc- typical blue fluorescence of (H)GFPC (GFP mutant Y66F,
aeg-OMe (29) might be because of the requirement for prior λexc = 360 nm, λem = 442 nm) was observed for 30, with λexc
phenol protection.
= 350 nm, λem = 436 nm. On freezing of a solution of 30
Figure 5. Synthesis of N-(2-aminoethyl)glycine-functionalized GFP chromophores 7 and 8.
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Figure 6. Temperature dependences of the fluorescence emissions of Fmoc-aeg-H(GFPC)-OMe (30) [λexc = 350 nm, A350nm = 0.9, λem
=
436 nm, Φrel = 1 (80 °C) and λem = 413 nm, Φrel = 5 (–10 °C)] and of Fmoc-aeg-(OH)GFPC-OMe (31) [λexc = 430 nm, A430nm = 1.6, λem
= 499 nm, Φrel = 1 (80 °C) and λem = 492 nm, Φrel = 11 (–10 °C)] in glycerin (+ 0.1% NEt3 for 31) at 80 °C and in glycerin glass at
–10 °C. A = absorption; Φ = fluorescence quantum yield.
to –10 °C the fluorescence intensity increased by a factor of strongly increased by a factor of 11, with a slight blue shift
five with a strong blue shift of the fluorescence maximum of the fluorescence maximum of 7 nm.
of 23 nm (Figure 6).
The PNA oligomer 32, containing the (H)GFP chromo-
Analogous behavior was found for the (OH)GFP chro- phore, formed an antiparallel double strand with the com-
mophore 31. In glycerin with 0.1% NEt3, typical green plementary DNA 33 (5Ј-G-C2-G-C2-A5-3Ј). Adenine was
fluorescence with λexc = 430 nm, λem = 499 nm was ob- chosen as the counterbase for the (H)GFP chromophore.
served (wild-type GFP, λexc = 395/475 nm, λem = 509 nm), Temperature-dependent UV spectroscopy yielded a melting
while on freezing to –10 °C the fluorescence intensity temperature of 71 °C for the duplex 32+33, with a small
Figure 7. UV melting curve of oligomer 32 after hybridization with complementary DNA 33 (5Ј-G-C2-G-C2-A5-3Ј), each 3 µm in 10 mm
NaH2PO4 buffer, 0.1  NaCl, pH 6.85. The binding of Uronium-t-t-t-t-t-g-g-c-g-g-c-Lys-Lys-Gly-NH2 (34) with DNA 33 is also shown,
as a reference.[17]
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heating–cooling hysteresis of 3 °C (Figure 7). In compari-
The fluorescence spectrum of oligomer 32 upon exci-
son, the UV experiment with the reference oligomer 34+33, tation at 350 nm showed broad emission maxima at
with replacement of the (H)GFP chromophore by thy- 413 nm, 440 nm, and 510 nm (Figure 10). This fluorescence
mine – therefore giving the canonical A-T base pair – re- was sensitive to the addition of complementary DNA 33,
sulted in a melting temperature of 70 °C. Overall, this indi- which produced an increase in fluorescence intensity by a
cates a well stacked (H)GFP chromophore.[16]
factor of two and the upraising of a sharp emission band
CD spectroscopy with the DNA/PNA pairing complex at 417 nm with shoulders at 440 and 506 nm. In compari-
32+33 showed typical temperature-dependent bands at the son, the fluorescence of the free (H)GFP chromophore (27
optical transitions of the nucleobases (Figure 8, top) indi- as reference) showed a weak emission at 398 nm with 1/12
cating the formation of a B-DNA-like, right-handed pairing of the intensity of the double strand.
complex. At the optical transitions of the (H)GFP chromo-
The emission bands at 413 nm and 440 nm appeared
phore (Figure 8, bottom), centered around 293 nm close to the emission of the Y66F mutant of the GFP con-
(+3 mdeg) and 347 nm (–3 mdeg), significant CD bands taining the (H)GFP chromophore (442 nm) and are consis-
were found, their temperature dependency consistent with tent with the fluorescence behavior of the chromophore
the UV melting behavior (Figure 9). The appearance of CD monomer 30 observed in glycerin at 80 °C (440 nm) and
bands at the optical transitions of the (H)GFP chromo- –10 °C (413 nm). In comparison to the free chromophore
phore once more supported the assumption of a well 27 in methanol, with an emission maximum at 398 nm, the
stacked chromophore buried inside the DNA/PNA double typical blue fluorescence requires the incorporation of the
strand.
chromophore into the base stack. Interestingly, a more red-
Figure 8. Top: CD spectra of oligomer 32 after hybridization with complementary DNA 33, each 3 µm in 10 mm NaH2PO4 buffer, 0.1 
NaCl, pH 6.85. Bottom: Same pairing complex, but oligomer and DNA each 30 µm in the same buffer. The concentration needed to be
increased for monitoring of the CD effect at the optical transitions of the (H)GFP chromophore since its absorption coefficient (ε350nm
≈ 9·103 cm2·mol–1) is much smaller than that of the double strand (ε260nm ≈ 206·103 cm2·mol–1).
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Figure 9. Temperature dependency of the CD effect at the optical transitions of the (H)GFP chromophore at 293 nm and 347 nm.
Oligomer 32 + DNA 33 each 30 µm in 10 mm NaH2PO4 buffer, 0.1  NaCl, pH 6.85.
Figure 10. Fluorescence and UV spectra of oligomer 32 depending on hybridization; concentrations of oligomer 32 and DNA 33 each
30 µm in 10 mm NaH2PO4 buffer, 0.1  NaCl, pH 6.85. UV absorption: λmax = 356 nm; fluorescence emission: λexc = 350 nm, A350nm
=
0.27; single strand λem = 413, 440, 510 nm, Φrel = 6; double strand Φrel = 12; reference (H)GFPC-methyl acetate 27, λexc = 350 nm, A350nm
= 0.27 in methanol, λem = 398 nm, Φrel = 1; fluorescence excitation: 32+33, λem = 500 nm (fix), λmax = 356 nm (scanned). A = absorption,
Φ = fluorescence quantum yield.
shifted band, at 506–510 nm, was found both for duplex phore was clearly identified as the source of the red-shifted
32+33 and for single-strand 32, and might result from π- fluorescence by collection of a fluorescence excitation spec-
stacking inside the base stack as indicated by UV and CD trum at 500 nm, revealing a total overlap with the UV ab-
spectroscopy. On switching from single-strand 32 to duplex sorption spectra with λmax = 356 nm. Like the fluorescence
32+33 the fluorescence emission spectra changed not only emission, the UV ground state absorption was slightly red-
in fluorescence intensity but also in the emission band shifted (8 nm) in relation to that of the free chromophore
shape, which might be the result of the transition from a 27 with λexc = 348 nm. It is known from mutagenesis experi-
less ordered single strand with several conformations to the ments that stacking of the chromophore with an aromatic
well defined double helix structure. The (H)GFP chromo- amino acid as in T203F/H/Y mutations yields GFP variants
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ODS-H80, 250ϫ20 mm ID, 8–4 µm, 8 nm, C18 (YMC) column
with red-shifted (15–20 nm) fluorescence emission maxima.
X-ray structure analysis revealed coplanar stacking of the
aromatic amino acid in position 203 with the GFP chromo-
phore with a distance of 3.3–3.8 Å, comparable with the
raise of 3.4 Å in the DNA base stack.[18] Nevertheless, the
overall low quantum yield (Φ Ͻ 0.5%)[19] indicates that
blocking of the radiationless deactivation channels ad-
ditionally requires the incorporation of the chromophore
into the hydrogen bond network of the protein. It remains
an open question whether the hydrogen bond network in-
side the GFP protein barrel blocks the radiationless deacti-
vation through a protonation mechanism or simply by more
efficient restriction of the chromophore flexibility.[5,20]
(10 mLmin–1). DNA was purchased from Carl Roth GmbH.
Analytical Data: The NMR analysis of compounds 7, 8, 30, and
31 suffered from high rotation barriers caused by the tertiary amide
bond of the N-acyl-(2-aminoethyl)glycine, which produced broad-
1
ening and partly doubling of H and 13C NMR signals.[21] Mostly,
a complete set of NMR assignments was obtained separately for
each rotamer with the aid of H,H-COSY, HSQC, and HMBC ex-
periments. The 2-methyl group in the 4-imidazolone moiety was
found to be quite acidic, as is known from the literature.[22] H/D
exchange takes place within some hours and needs to be taken into
account for 2D NMR spectroscopy. The enantiomeric excesses
were determined by deprotection of monomer 4–6 with TFA fol-
lowed by coupling with enantiomerically pure - and -Ala-OSu
and analytical HPLC. In all cases the enantiomeric excess was
higher than 95%.
Synthesis of 3-Methyl-GFP Chromophores
Conclusions
3-Methyl-(H)GFPC (2): The GFPC-precursor 11 (218 mg,
1.00 mmol, 1.0 equiv.) was heated quickly to 230 °C under vacuum
in an oil bath and stirred for 5 min at 230 °C. After cooling, the
black residue was extracted with hexane (10ϫ5 mL) to yield an
orange solution, which was purified on silica gel (70 g, ethyl ace-
tate/hexane 3:1) to provide the chromophore 2 (89 mg, 45%) as
light red crystals. RF (ethyl acetate/hexane 3:1) = 0.49. 1H NMR
(200 MHz, CDCl3): δ = 2.34 (s, 3 H, CH3), 3.11 (s, 3 H, NCH3),
7.04 (s, 1 H, acryl-H), 7.34 (m, 3 H, phenyl-H), 8.06 (d, J = 8.1 Hz,
2 H, phenyl-H) ppm. 13C NMR (50 MHz, CDCl3): δ = 15.7 (CH3-
2), 26.6 (CH3-3), 127.2 (CH), 128.7 (CH), 130.0 (CH), 132.1 (CH),
134.1 (C-phenyl), 138.7 (C-5), 162.6 (C-2), 170.7 (CO-4) ppm. UV/
Vis (methanol): λmax (ε) = 236, 293, 350 nm. EI-MS: m/z (%): 200
(100) [M]+. HR-ESI: m/z [2M + H]+ = calcd. for C12H12N2O
401.1972; found: 401.1971.
The synthesis and characterization of two GFP chromo-
phores has been performed. In order to incorporate them
into alaninyl or N-(2-aminoethyl)glycinyl peptide nucleic
acids the chromophores were prepared as the side chains of
the corresponding amino acids. Incorporation into an N-(2-
aminoethyl)glycine-PNA oligomer was achieved by care-
fully optimized solid-phase peptide synthesis. The double
strand stability of an N-(2-aminoethyl)glycine-PNA func-
tionalized with the GFP chromophore with a complemen-
tary DNA was investigated by UV and CD spectroscopy,
while – since fluorescence of the GFP strongly depends on
nearest-neighbor interactions and shielding from solvent –
fluorescence within this model system was also investigated,
with the appearance of blue fluorescence with sensitivity to
the presence of the DNA counter-strand being observed.
Interestingly, an additional green fluorescence was found
and assumed to be caused by π-stacking interactions. Un-
fortunately, the overall quantum yield was very low, indicat-
3-Methyl-(OH)GFPC (3): Acrylamide 12 (298 mg, 1.27 mmol,
1.0 equiv.) was heated quickly to 230 °C under vacuum in an oil
bath and stirred at 230 °C for 5 min. After cooling, the residue was
extracted with hot (70 °C) ethyl acetate (8ϫ5 mL) to provide an
orange raw product (238 mg) that was purified on silica gel (80 g
ing insufficient blocking of the radiationless internal con- ethyl acetate/hexane 3:1) to give the chromophore 3 (124 mg, 45%)
1
as bright yellow crystals. RF (ethyl acetate/hexane 3:1) = 0.36. H
version by simple π-stacking, which might limit utility in
molecular biology diagnostics.
NMR (200 MHz, [D6]DMSO, 35 °C): δ = 2.32 (s, 3 H, CH3), 3.09
(s, 3 H, NCH3), 6.84 [d, JH,H = 8.4 Hz, 2 H, phenyl-H], 6.88 (s, 1
H, acryl-H), 8.07 (d, JH,H = 8.4 Hz, 2 H, phenyl-H), 9.84 (br. s, 1
3
3
H, phenyl-OH) ppm. 13C NMR (50 MHz, [D6]DMSO, 35 °C): δ =
15.6 (CH3), 26.9 (CH3), 116.4 (2ϫCH-phenyl), 125.9 (C), 126.8
(CH-methylene), 134.7 (2ϫCH-phenyl), 136.6 (C), 159.9 (C), 163.3
(CO), 170.9 (CO) ppm. UV/Vis (methanol): λmax (ε) = 248, 371 nm.
EI-MS: m/z (%): 216 (100) [M]+. HR-ESI: m/z [M + H]+ = calcd.
for C12H12N2O2 217.0972; found: 217.0971.
Experimental Section
General Methods: Infrared spectra were recorded on a Perkin–El-
mer FT-IR 1600 instrument. NMR spectra were recorded on a Var-
ian Unity 300 or a Varian Inova 600 spectrometer. UV absorption
spectra were recorded with a Jasco V-550 spectrometer; all extinc-
tion coefficients are given in cm2·mol–1. Fluorescence emission and
excitation spectra were collected with a Jasco FP 6200 instrument.
Fluorescence emission spectra were not corrected for the wave-
length dependency of the detector sensitivity, and fluorescence exci-
tation spectra were not corrected for the wavelength dependency of
the lamp emission intensity. ESI mass spectra were recorded with
a Finnigan LCQ iontrap spectrometer and high-resolution mass
2-Acetylamino-N-methylcinnamide (11): (H)Azlactone 9 (1.64 g,
8.76 mmol, 1.0 equiv.) was dissolved in ethanolic methylamine (8 ,
15 mL) and the mixture was stirred for 1 d. The product precipi-
tated in small crystals and was separated by filtration, and the title
compound 11 (1.02 g, 53%) was obtained as a colorless solid after
washing of the crystals with hexane and drying in vacuo. RF (ethyl
acetate/methanol 7:1) = 0.38. 1H NMR (200 MHz, [D6]DMSO,
3
spectra (HR-ESI) were recorded on
a
Bruker FTMS-7
35 °C): δ = 2.00 (s, 3 H, COCH3), 2.67 (d, JH,H = 5.2 Hz, 3 H,
APEX® IV 70e FT-ICR spectrometer. Reversed-phase HPLC was
performed with a Pharmacia Biotech Äkta Basic 900 system [el-
uents: A: deionized water (18 MΩ·cm–1) + 0.1% TFA, B: acetoni-
trile/water 8:2 + 0.1% TFA], analytical HPLC was carried out with
a Grom-Sil 80 ODS, 250ϫ4.6 mm, 4 µm, 80 Å, C-18 column
(0.5 mLmin–1), and preparative HPLC was done on a J’sphere
CH3), 7.05 (s, 1 H, acryl-H), 7.28–7.45 (m, 3 H, phenyl-H), 7.52
(d, JH,H = 8.0 Hz, 2 H, phenyl-H), 7.87 (br. s, 1 H, NHMe), 9.30
3
(s, 1 H, NHCO) ppm. 13C NMR (50 MHz, CDCl3): δ = 22.8 (Ac-
CH3), 26.1 (CH3), 127.4 (CH), 128.4 (2ϫCH-phenyl), 129.2
(2ϫCH-phenyl), 130.1 (CH-methylene), 134.2 (C-phenyl), 165.2
(CO), 169.2 (CO) ppm. UV/Vis (methanol): λmax (ε) = 216, 275 nm.
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EI-MS: m/z (%): 218 (40) [M]+. HR-ESI: m/z [M + H]+ = calcd.
for C12H14N2O2 219.1128; found: 219.1126.
aqueous NaOH (1 , ≈ 250 µL) was added to provide pH 11, and
2,5-dichlorobenzyl bromide (25 mg, 105 µmol, 1.2 equiv.) dissolved
in methanol (1000 µL) was then added. Each day, further 2,5-
dichlorobenzyl bromide (10 mg) and NaOH (1 , to keep the pH at
ca. 11) were added. After 3 d the reaction was terminated by ad-
dition of HCl (1 , ≈ 100 µL, final pH ≈ 5.5), the solvent was re-
moved in vacuo, and the crude product was purified on silica gel
(14 g, ethyl acetate/methanol 8:1 + 1.0% acetic acid) to provide the
title compound 6 (30 mg, 63%) as a bright yellow solid.
2-Acetamido-4-hydroxy-N-methylcinnamide (12): (OAc)Azlactone
10 (1.00 g, 4.01 mmol, 1.0 equiv.) was dissolved in aqueous methyl-
amine solution (40%, 10 mL) and the mixture was stirred for 5 h.
The solution was reduced in vacuo, water (10 mL) was added, and
the residue was dissolved with heating (100 °C). After the solution
had been cooled at 4 °C overnight, light yellow crystals (745 mg)
had precipitated and were separated by filtration. These crystals
were dissolved in water (25 mL, 100 °C) and precipitated by ad-
dition of acetic acid (10 drops) to provide the desired product 12
(710 mg, 74%) as colorless crystals. RF (ethyl acetate/methanol 7:1)
B) Boc--Ala-(OBzl)GFPC-precursor 21 (1.80 g, 3.18 mmol) was
dissolved in DMF (5 mL) and the system was flushed with nitrogen
for 10 min. DBU (5 drops) was added, the solution was heated
under reflux for 5 min, the solvent was removed under vacuum,
and the orange crude product was purified on silica gel (100 g,
ethyl acetate/methanol 7:1 + 0.5–2% acetic acid) to provide the title
compound 6 (1.51 g, 86%) as an orange solid. RF (ethyl acetate/
methanol 7:1 + 2% acetic acid) = 0.45. 1H NMR (300 MHz,
CD3OD): δ = 1.36 (s, 9 H, tBu), 2.42 (s, 3 H, CH3), 3.89 (dd, 2JH,H
1
= 0.30. H NMR (200 MHz, [D6]DMSO, 35 °C): δ = 2.00 (s, 3 H,
3
COCH3), 2.66 (d, JH,H = 6.0 Hz, 3 H, NCH3), 2.90–3.80 (br. s, 1
3
H, OH), 6.87 (d, JH,H = 8.6 Hz, 2 H, phenyl-H), 7.03 (s, 1 H,
3
3
acryl-H), 7.39 (d, JH,H = 8.6 Hz, 2 H, phenyl-H), 7.74 (dbr, JH,H
= 6.0 Hz, 1 H, NH), 9.16 (s, 1 H, NHCO) ppm. 13C NMR
(50 MHz, [D6]DMSO, 35 °C): δ = 23.2 (CH3), 26.6 (CH3), 115.9
(2ϫCH-phenyl), 125.3 (C), 126.6 (C), 129.8 (CH-methylene), 131.7
(2ϫCH-phenyl), 158.3 (C), 166.4 (CO), 170.6 (CO) ppm. UV/Vis
(methanol): λmax (ε) = 226, 299 nm. EI-MS: m/z (%): 234 (40) [M]+.
HR-ESI: m/z [M + H]+ = calcd. for C12H14N2O3 235.1077; found:
235.1076.
3
2
3
= 15 Hz, JH,H = 10 Hz, 1 H, Hβ), 4.09 (dd, JH,H = 15 Hz, JH,H
= 5.2 Hz, 1 H, HβЈ), 4.47 (m, 1 H, Hα), 5.35 (s, 2 H, benzyl-H),
3
7.03 (s, 1 H, acryl-H), 7.09 (d, JH,H = 7.6 Hz, 2 H, phenyl-H),
7.35–7.48 (m, 3 H, phenyl-H), 8.10 (d, 3JH,H = 7.6 Hz, 2 H, phenyl-
H) ppm. 13C NMR (75 MHz, CD3OD): δ = 18.0 (CH3, prone to
H/D exchange), 28.6 (3ϫBoc-CH3), 43.6 (β-CH2), 53.8 (α-CH),
66.3 (CH2), 80.8 (Boc-C), 116.0 (CH), 128.5 (C), 128.6 (CH), 129.7
(CH), 132.2 (CH), 133.1 (C), 135.3 (CH), 137.4 (C), 138.0 (C),
157.8 (C), 162.2 (C), 163.7 (C), 172.5 (C) ppm. UV/Vis (methanol):
λmax (ε) = 225, 248, 370 nm. ESI-MS: m/z (%): 548 (100) [M +
H]+. HR-ESI: m/z [M + H]+ = calcd. for C26H27Cl2N3O6 548.1350;
found: 548.1354.
Synthesis of Alaninyl GFP Chromophores
Boc-L-Ala-(H)GFPC-OH (4): Precursor 19 (800 mg, 2.04 mmol)
was dissolved in DMF (5 mL) and the solution was flushed with
nitrogen for 10 min. DBU (5 drops) was added and the solution
was heated under reflux for 5 min. The solvent was removed under
vacuum and the crude orange product was purified on silica gel
(100 g, ethyl acetate/methanol 7:1 + 0.5–2% acetic acid) to provide
amino acid 4 (562 mg, 74%) as light red crystals. RF (ethyl acetate/
methanol 7:1 + 2.0% acetic acid) = 0.35. 1H NMR (300 MHz,
CD3OD): δ = 1.33 (s, 9 H, tBu), 2.44 (s, 3 H, CH3; prone to H/D
exchange), 3.85 (m, 1 H, Hβ), 4.08 (m, 1 H, HβЈ), 4.42 (m, 1 H,
Hα), 7.02 (s, 1 H, acryl-H), 7.39 (m, 3 H, phenyl-H), 8.05 (d, 3JH,H
= 6.6 Hz, 2 H, phenyl-H) ppm. 13C NMR (75 MHz, CD3OD): δ =
18.0 (CH3-2, prone to H/D exchange), 28.6 (Boc-CH3), 44.1 (β-
CH2), 54.5 (α-CH), 80.7 (Boc-C), 128.2 (CH), 129.7 (CH), 131.3
(CH), 133.2 (CH), 135.4 (C), 139.5 (C), 157.5 (C), 165.4 (C), 172.5
(CO), 174.9 (CO) ppm. ESI-MS: m/z (%): 372 (60) [M – H]–, 396
2-Acetamido-4-hydroxycinnamic Acid (14): 4-Acetoxy-2-acet-
amidocinnamic acid (17, 2.10 g, 8.0 mmol, 1.0 equiv.) was sus-
pended in water (7 mL) and a solution of KOH (1.56 g, 2.5 equiv.)
in water (3 mL) was added slowly at 0 °C. The resulting orange
solution was stirred (5 h) at 0 °C and acidified with concentrated
HCl (37%) at 0 °C, and the precipitate was filtered off, washed with
cold HCl (0.1 ), and dried in vacuo. The title compound 14
(1.65 g, 7.44 mmol, 93%) was obtained as light pink crystals. RF
(ethyl acetate/methanol 7:1) = 0.15. 1H NMR (200 MHz, [D6]
DMSO, 35 °C): δ = 1.96 (s, 3 H, COCH3), 6.79 (d, 3JH,H = 8.1 Hz,
(100) [M + Na]+, 769 (40) [2M + Na]+. HR-ESI: m/z [M + H]+
=
3
2 H, phenyl-H), 7.18 (s, 1 H, acryl-H), 7.48 (d, JH,H = 8.1 Hz, 2
calcd. for C19H23N3O5 374.1711; found: 374.1713.
H, phenyl-H), 9.22 (s, 1 H, NH), 9.90 (br. s, 1 H, phenyl-OH) ppm.
13C NMR (50 MHz, [D6]DMSO, 35 °C): δ = 22.5 (CH3), 115.4
(2ϫCH-phenyl), 124.0 (C), 124.6 (C), 131.7 (2ϫCH-phenyl),
132.4 (C-methylene), 158.6 (C), 166.6 (CO), 169.1 (CO) ppm. UV/
Vis (methanol): λmax (ε) = 225, 301 nm. EI-MS: m/z (%): 221 (40)
[M]+. HR-ESI: m/z [M + H]+ = calcd. for C11H11NO4 222.0761;
found: 222.0760.
Boc-L-Ala-(OH)GFPC-OH (5): Precursor 20 (160 mg, 0.40 mmol,
1.0 equiv.) was dissolved in DMF (6 mL) and the system was
flushed with nitrogen for 15 min. The solution was heated at reflux
for 15 min, the solvent was removed in vacuo, and the residue was
cleaned on silica gel (50 g, ethyl acetate/methanol 7:1 + 0.5–2%
acetic acid) to provide amino acid 5 (142 mg, 75%) as a bright
yellow solid. RF (ethyl acetate/methanol 7:1 + 2% acetic acid) =
0.30. 1H NMR (300 MHz, CD3OD): δ = 1.31 (s, 9 H, tBu), 2.42
(s, 3 H, CH3), 3.83 (m, 1 H, Hβ), 4.06 (m, 1 H, HβЈ), 4.36 (m, 1
2-Acetamido-3-[4-(2,6-dichlorobenzyloxy)phenyl]cinnamic Acid (15):
Potassium acetate (2.5 g, 25 mmol, 0.8 equiv.) was dried in vacuo
with melting for 15 min. Aldehyde 35 (8.0 g, 28 mmol, 1.0 equiv.)
and N-acetylglycine (3.6 g, 31 mmol, 1.1 equiv.) were suspended in
acetic acid anhydride (15 mL) and the system was flushed with ni-
trogen (15 min). The resulting mixture was heated under reflux un-
til no aldehyde 35 was detectable by TLC (5 h), the solution was
kept in the fridge (4 °C) overnight, ice water (100 mL) was then
added, and the precipitated product was filtered off and washed
with aqueous K2CO3 (1 , 2ϫ100 mL). The brown residue was
dissolved in dichloromethane, silica gel (50 mL) was added, the sol-
vent was removed in vacuo, and the product was eluted with ethyl
3
H, Hα), 6.81 (d, JH,H = 8.1 Hz, 2 H, phenyl-H), 6.99 (s, 1 H,
3
acryl-H), 7.94 (d, JH,H = 8.1 Hz, 2 H, phenyl-H) ppm. 13C NMR
(50 MHz, CD3OD): δ = 17.8 (CH3), 28.7 (CH3), 44.7 (β-CH2), 55.7
(α-CH), 80.5 (Boc-C), 116.7 (2ϫCH-phenyl), 126.9 (C), 128.6
(CH), 129.5 (CH), 135.5 (2ϫCH-phenyl), 136.9 (C), 141.5 (CH),
157.5 (C), 161.5 (C), 163.4 (C), 172.8 (CO), 175.4 (CO) ppm. UV/
Vis (methanol): λmax (ε) = 249, 371 nm. ESI-MS: m/z (%): 390 (85)
[M + H]+, 412 (35) [M + Na]+. HR-ESI: m/z [M + H]+ = calcd.
for C19H23N3O6 390.1660; found: 390.1660.
Boc-L-Ala-(OBzl)GFPC-OH (6): A) Boc--Ala-(OH)GFP-OH (5, acetate/hexane 1:3 and dried in vacuo. This crude product 16 was
34 mg, 87 µmol, 1.0 equiv.) was dissolved in methanol (250 µL),
dissolved in acetone/water 3:1 with heating and heated at reflux for
906
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 899–911

GFP Chromophore in Peptides and Peptide Nucleic Acids
FULL PAPER
2 h. After the system had been stirred overnight at room tempera-
ture, the product precipitated, and was filtered off, washed with
ethyl acetate/hexane, and dried in vacuo to provide the title com-
pound 15 (4.6 g, 43%) as a light yellow solid. 1H NMR (300 MHz,
[D6]DMSO, 35 °C): δ = 1.99 (s, 3 H, CH3), 5.28 (s, 2 H, CH2), 7.10
and for 45 min at room temperature, and the precipitated urea was
filtered off and washed with DMF (6 mL). (S)-3-Amino-2-(tert-bu-
toxycarbonylamino)propionic acid (18, 332 mg, 1.62 mmol,
1.05 equiv.) and triethylamine (500 µL, 360 mg, 3.60 mmol,
2.5 equiv.) were added to the DMF solution, the resulting suspen-
sion was stirred until a clear solution was obtained (1 d), the sol-
vent was removed in vacuo, and the crude product was purified on
3
(d, JH,H = 8.0 Hz, 2 H, phenyl-H), 7.22 (s, 1 H, acryl-H), 7.40–
7.62 (m, 5 H, phenyl-H), 9.31 (s, 1 H, NH), 12.50 (br. s, 1 H,
OH) ppm. 13C NMR (75 MHz, [D6]DMSO, 35 °C): δ = 22.5 (CH3), silica gel (50 g, ethyl acetate/methanol 7:1, + 0.5–2% acetic acid)
62.8 (CH), 64.9 (CH2), 102.1 (CH), 114.6 (CH), 125.4 (C), 126.8 to provide the title compound 20 (446 mg, 73%) as a colorless so-
(C), 128.7 (CH), 131.3 (C), 131.4 (CH), 136.0 (C), 159.0 (C), 166.5 lid. RF (ethyl acetate/methanol 7:1 + 2% acetic acid) = 0.15. 1H
(C), 169.0 (C) ppm. EI-MS: m/z (%): 379.2 (50) [M]+. HR-ESI: m/z
NMR (200 MHz, CD3OD): δ = 1.42 (s, 9 H, tBu), 2.15 (s, 3 H,
[M + Na]+ = calcd. for C14H10Cl2O2 402.0270; found: 402.0269.
COCH3), 3.64 (m, 2 H, Hβ,βЈ), 4.20 (m, 1 H, Hα), 6.79 (d, JH,H
3
3
= 8.0 Hz, 2 H, phenyl-H), 7.19 (s, 1 H, acryl-H), 7.40 (d, JH,H
=
2-Acetamido-4-acetoxycinnamic Acid (17): (OAc)Azlactone 10
(11.4 g, 46.5 mmol, 1.0 equiv.) was heated under reflux in acetone/
water 3:1 (120 mL) for 4 h, the solution was kept at 4 °C overnight,
and the precipitate was filtered off with a Buchner funnel, washed
with cold water (30 mL), cold acetone (10 mL), and cold ethyl ace-
tate (20 mL), and dried in vacuo to provide the title compound 17
(11.5 g, 43.7 mmol, 94%) as light yellow crystals. 1H NMR
(300 MHz, [D6]DMSO, 35 °C): δ = 1.99 (s, 3 H, CH3), 2.27 (s, 3
8.0 Hz, 2 H, phenyl-H) ppm. 13C NMR (50 MHz, CD3OD): δ =
21.8 (CH3), 27.3 (CH3), 43.0 (β-CH2), 53.3 (α-CH), 77.2 (Boc-C),
114.5 (2ϫCH-phenyl), 123.9 (C), 125.8 (C), 128.1 (CH-methylene),
130.3 (2ϫCH-phenyl), 154.4 (C), 157.3 (C), 164.3 (C), 168.5 (C),
177.0 (CO) ppm. UV/Vis (methanol): λmax (ε) = 229, 300 nm. ESI-
MS: m/z (%): 406 (95) [M – H]–, 430 (95) [M + Na]+. HR-ESI: m/z
[M + H]+ = calcd. for C19H25N3O7 408.1765; found: 408.1766.
3
Boc-L-Ala-(OBzl)GFPC-Precursor 21: Acrylic acid 15 (2.50 g,
H, CH3), 7.17 (d, JH,H = 8.1 Hz, 2 H, phenyl-H), 7.23 (s, 1 H,
3
6.6 mmol, 1.05 equiv.) and HOBt (1.10 g, 8.1 mmol, 1.3 equiv.)
were dissolved in DMF (8 mL), DCC (1.29 g, 6.3 mmol, 1.0 equiv.)
dissolved in DMF (2 mL) was added at 0 °C, and the solution was
stirred for 30 min at 0 °C and for 60 min at room temperature. The
precipitated urea was filtered off and washed with DMF (25 mL)
until the urea was colorless, (S)-3-amino-2-(tert-butoxycarbonyla-
mino)propionic acid 18 (1.41 g, 6.9 mmol, 1.1 equiv.) and triethyl-
amine (1.5 mL, 1.33 mg, 13 mmol, 2.0 equiv.) were added to the
obtained yellow solution, and the resulting suspension was stirred
until a clear solution was obtained (1 d). The solvent was removed
in vacuo and the crude product was purified on silica gel (200 g,
ethyl acetate/methanol 7:1, + 0.5–1.5% acetic acid) to provide the
title compound 21 (3.21 g, 90%) as a colorless solid. 1H NMR
(300 MHz, [D6]DMSO, 35 °C): δ = 1.38 (s, 9 H, Boc), 2.05 (s, 3 H,
CH3), 3.31–3.42 (m, 2 H, β-H), 3.90 (m, 1 H, α-H), 5.21 (s, 2 H,
acryl-H), 7.65 (d, JH,H = 8.1 Hz, 2 H, phenyl-H), 9.42 (s, 1 H,
NH), 12.5 (br. s, 1 H, COOH) ppm. 13C NMR (125.7 MHz, [D6]
DMSO, 35 °C): δ = 20.8 (CH3), 22.5 (CH3), 121.9 (2ϫCH-phenyl),
127.3 (C), 124.6 (C), 130.2 (C-methylene), 130.9 (2ϫCH-phenyl),
132.4 (C), 150.8 (C), 166.3 (CO), 169.0 (CO), 169.2 (CO) ppm. UV/
Vis (methanol): λmax (ε) = 225, 301 nm. ESI-MS: m/z (%): 264.1
(20) [M + H]+, 286.1 (100) [M + Na]+, 549.2 (35) [2M + Na]+.
HR-ESI: m/z [M + H]+ = calcd. for C13H13NO5 264.0867; found:
264.0866.
Boc-L-Ala-GFPC-Precursor 19: 2-Acetamidocinnamic acid (13,
900 mg, 4.40 mmol, 1.05 equiv.) and HOBt (770 mg, 5.7 mmol,
1.3 equiv.) were dissolved in DMF (3 mL). DCC (850 mg,
4.11 mmol, 1.0 equiv.) dissolved in DMF (2 mL) was added at 0 °C
and the solution was stirred for 60 min at 0 °C and for 120 min at
room temperature. The precipitated urea was filtered off and
washed with DMF (18 mL) until the urea was colorless, (S)-3-
amino-2-(tert-butoxycarbonylamino)propionic acid (18, 1.00 g,
4.90 mmol, 1.1 equiv.), and triethylamine (1.2 mL, 890 mg,
8.8 mmol, 2.0 equiv.) were added to the obtained yellow solution,
and the resulted suspension was stirred until a clear solution was
obtained (1 d). The solvent was removed in vacuo and the crude
product was purified on silica gel (170 g, ethyl acetate/methanol
7:1, + 0.5–1.5% acetic acid) to provide the title compound 19
(1.42 g, 88%) as a colorless solid. RF (ethyl acetate/methanol 7:1 +
3
CH2), 6.63 (br. s, 1 H, NH), 7.05 (s, 1 H, acryl-H), 7.06 (d, JH,H
= 8.0 Hz, 2 H, phenyl-H), 7.40–7.60 (m, 5 H, phenyl-H), 8.12 (br.
s, 1 H, NH), 9.40 (s, 1 H, NH), 12.00 (br. s, 1 H, OH) ppm. 13C
NMR (50 MHz, [D6]DMSO, 35 °C): δ = 22.7 (CH3), 28.1 (3ϫBoc-
CH3), 41.8 (β-CH2), 54.1 (α-CH), 64.9 (CH2), 78.0 (Boc-C), 114.6
(CH), 127.2 (C), 128.0 (C), 128.3 (CH), 128.7 (CH), 131.0 (CH),
131.4 (C), 131.6 (CH), 136.0 (C), 155.3 (C), 158.6 (C), 165.0 (C),
169.3 (C), 172.4 (C) ppm. ESI-MS: m/z (%): 588.5 (100) [M +
Na]+. HR-ESI: m/z [M + Na]+ = calcd. for C26H29Cl2N3O7
588.1275; found: 588.1277.
1
1% acetic acid) = 0.18. H NMR (200 MHz, [D6]DMSO, 35 °C):
4-(2,6-Dichlorobenzyloxy)benzaldehyde (35): 4-Hydroxybenzalde-
hyde (10 g, 90 mmol, 1.1 equiv.) and 2,6-dichlorobenzyl bromide
(19.5 g, 80 mmol, 1.0 equiv.) were dissolved in dichloromethane
(60 mL), DBU (21 mL, 1.5 equiv.) was added, and the resulting
mixture was stirred for 17 h at room temperature. The reaction was
stopped by addition of acetic acid (3 mL, 0.6 equiv.), the solvent
was removed in vacuo, ethyl acetate (100 mL) was added, and the
mixture was stirred for 10 min. The resulting suspension was fil-
tered off and the organic layer was extracted with aqueous K2CO3
solution (0.1 , 2ϫ100 mL), washed with brine (100 mL), and
dried with Na2SO4 to provide the title compound 35 (14.4 g, 64%)
as a light brown solid. This product could be used without further
purification for the Erlenmeyer azlactone synthesis, but for NMR
analysis a small sample was recrystallized from ethyl acetate/hexane
1:6, 5 mL·g–1 to provide the title compound as long, colorless need-
les. RF (ethyl acetate/hexane 1:1) = 0.80. 1H NMR (200 MHz,
δ = 1.39 (s, 9 H, tBu), 2.00 (s, 3 H, COCH3), 3.30–3.48 (m, 2 H,
Hβ, HβЈ), 3.75 (m, 1 H, Hα), 6.44 (d, 3JH,H = 5.6 Hz, 1 H, NHBoc),
7.08 (s, 1 H, acryl-H), 7.28–7.44 (m, 3 H, phenyl-H), 7.54 (d, 3JH,H
= 8.3 Hz, 2 H, phenyl-H), 8.30 (br. s, 1 H, NHCH2), 9.56 (s, 1 H,
NHCO) ppm. 13C NMR (50 MHz, [D6]DMSO, 35 °C): δ = 22.6
(CH3), 28.1 (Boc-CH3), 42.8 (β-CH2), 54.2 (α-CH), 77.8 (Boc-C),
128.0 (CH), 128.4 (CH), 129.2 (CH), 129.8 (CH), 134.0 (CH), 155.2
(C), 164.4 (C), 169.5 (CO), 173.3 (CO) ppm. UV/Vis (methanol):
λmax (ε) = 217, 277 nm. ESI-MS: m/z (%): 391 (100) [M – H]–, 414
(100) [M + Na]+. HR-ESI: m/z [M + Na]+ = calcd. for C19H25N3O6
414.1636; found: 414.1638.
Boc-L-Ala-(OH)GFPC-Precursor 20: 2-Acetamido-4-hydroxycin-
namic acid (14, 370 mg, 1.67 mmol, 1.05 equiv.) and HOBt
(300 mg, 2.22 mmol, 1.3 equiv.) were dissolved in DMF (1 mL),
DCC (310 mg, 1.50 mmol, 1.0 equiv.) dissolved in DMF (1 mL)
was added at 0 °C, the resulting solution was stirred for 1 h at 0 °C
3
CDCl3): δ = 5.39 (s, 2 H, CH2), 7.12 (d, JH,H = 8.0 Hz, 2 H,
Eur. J. Org. Chem. 2007, 899–911
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
907

T. Stafforst, U. Diederichsen
FULL PAPER
3
phenyl-H), 7.20–7.42 (m, 3 H, phenyl-H), 7.88 (d, JH,H = 8.0 Hz,
(phenyl-C–CH), 125.9 (2ϫFmoc-CH), 120.0 (2ϫFmoc-CH),
115.7 (2ϫphenyl-CH), 65.4 (Fmoc-CH2), 47.8 (CH2COOH), 46.7
2 H, phenyl-H), 9.92 (s, 1 H, HCO) ppm. 13C NMR (50 MHz,
CDCl3): δ = 65.3 (CH2), 115.0 (CH), 128.5 (CH), 130.3 (C), 130.8 (Fmoc-CH), 46.6 (CH2–N), 41.1 (GFP-CH2), 37.9 (CH2–NH), 14.9
(CH), 131.2 (C), 132.0 (CH), 137.0 (C), 163.7 (C), 190.8 (CH3C) ppm. Second rotamer: 1H NMR (300 MHz, [D6]DMSO,
(CHO) ppm. EI-MS: m/z (%): 280.2 (10) [M]+, 159.1 (100) [benzyl 35 °C): δ = 8.08 (d, 3JH,H = 9.0 Hz, 2 H, phenyl-CH), 7.88 (d, 3JH,H
3
cation]. HR-ESI: m/z [M + Na]+ = calcd. for C14H10Cl2O2
302.9950; found: 302.9948.
= 7 Hz, 2 H, Fmoc-CH), 7.67 (d, JH,H = 7 Hz, 2 H, Fmoc-CH),
3
3
7.41 (t, JH,H = 7 Hz, 2 H, Fmoc-CH), 7.33 (t, JH,H = 7 Hz, 3 H,
Fmoc-CH, NH), 6.91 (s, 1 H, CH=C), 6.85 (d, JH,H = 9.0 Hz, 2
H, phenyl-CH), 4.60 (s, 2 H, GFP-CH2), 4.28 (d, JH,H = 6 Hz, 2
H, Fmoc-CH2), 4.24 (t, JH,H = 6 Hz, 1 H, Fmoc-CH), 4.17 (s, 2
H, CH2COOH), 3.36 (m, 2 H, CH2–N), 3.13 (m, 2 H, CH2–NH),
2.18 (s, 3 H, CCH3) ppm. 13C NMR (150 MHz, [D6]DMSO,
35 °C): δ = 170.9 (COOH), 169.5 (GFP-CO), 166.9 (GFP-CH2–
CO), 161.8 (C=N), 159.6 (phenyl-C–OH), 156.1 (Fmoc-CO), 143.8
(2ϫFmoc-C), 140.7 (2ϫFmoc-C), 136.0 (C=CH), 134.0
(2ϫphenyl-CH), 127.5 (2ϫFmoc-CH), 127.0 (2ϫFmoc-CH),
125.7 (CH=C), 125.2 (phenyl-C–CH), 125.9 (2ϫFmoc-CH), 120.0
(2ϫFmoc-CH), 115.7 (2ϫphenyl-CH), 65.4 (Fmoc-CH2), 47.1
(CH2COOH), 46.7 (Fmoc-CH), 46.6 (CH2–N), 40.6 (GFP-CH2),
3
Synthesis of (2-Aminoethyl)glycinyl GFPC Chromophores
3
3
Fmoc-aeg-(H)GFPC-OH (7): Fmoc-aeg-(H)GFPC-OMe (30,
463 mg, 0.83 mmol) was dissolved in ethyl acetate (20 mL), LiI
(480 mg, 3.6 mmol) was added, and the obtained solution was
heated under reflux for 25 h. After the mixture had cooled to room
temperature, ethyl acetate (200 mL) was added and the organic
layer was washed with NaHSO3/HCl (0.5 , pH 2, 3ϫ100 mL) and
saturated NaCl/HCl (pH 2, 2ϫ100 mL) and dried with Na2SO4.
After purification on RP-18 silica gel (methanol/water 7:3) title
compound 7 (0.30 g, 0.53 mmol, 64%) was obtained as a red solid.
M.p. 118 °C; NMR: two rotamers 1:1. 1H NMR (300 MHz, [D6]
DMSO, 35 °C): δ = 8.20 (d, 3JH,H = 8.1 Hz, 2 H, CH-phenyl), 7.88
37.9 (CH –NH), 15.0 (CH C) ppm. IR (KBr): ν = 3412, 1707,
˜
2
3
1646, 1599, 1516, 1447, 1252, 850, 746 cm–1. UV/Vis (methanol):
λmax (OD) = 370 (0.3), 300 (0.14), 223 (0.1) Ǟ same sample: UV/
Vis (+ 0.01% NEt3): λmax (OD) = 431 (0.22), 386 (0.22), 300 (0.15),
228 (3.0) nm. ESI-MS: m/z (%) = 583.2 (100) [M + H]+, 581.1 (90)
[M – H]–, 695.0 (100) [M + CF3COO–]–. HR-ESI-MS: m/z [M +
H]+ = calcd. for C21H30N4O7 583.2187; found: 583.2187.
3
3
(d, JH,H = 7.2 Hz, 2 H, CH-Fmoc), 7.68 (t, JH,H = 6.6 Hz, 2 H,
CH-Fmoc), 7.48–7.30 (m, 7 H, CH-Fmoc, CH-Fmoc, CH-phenyl,
CH-phenyl), 6.99 (s, 1 H, CH-methylene), 4.62 (s, 1 H, CH2-GFP),
4.46 (s, 1 H, CH2-GFP), 4.38–4.28 (m, 2 H, CH2-Fmoc), 4.28–4.21
3
(m, 1 H, CH-Fmoc), 3.97 (s, 2 H, CH2COOH), 3.49 (t, JH,H
=
6.9 Hz, 2 H, CH2–N), 3.16–3.12 (m, 2 H, CH2–NH), 2.20 (s, 1.5
H, CH3), 2.18 (s, 1.5 H, CH3) ppm. 13C NMR (150 MHz, [D6]-
DMSO, 35 °C): δ = 170.4 (COOH), 169.6 (CO-GFP), 167.0
(CH2CON), 164.3 (N=C–N), 156.4 (OCON), 143.9 (2ϫC-Fmoc),
140.7 (2ϫC-Fmoc), 138.8 (CH=C–N), 134.0 (C-phenyl), 131.9
(2ϫCH-phenyl), 130.0 (CH-phenyl), 128.7 (2ϫCH-phenyl), 127.6
(2ϫCH-Fmoc), 127.1 (2ϫCH-Fmoc), 125.1 (CH-methylene),
125.0 (2ϫCH-Fmoc), 120.1 (2ϫCH-Fmoc), 65.5 (CH2-Fmoc),
47.0 (CH-Fmoc), 46.7 (CH2–N, CH2COOH), 40.3 (CH2-GFP),
(H)GFPC-Acetic Acid (23): Aqueous LiOH (1.0 , 12 mL, 2 equiv.)
was slowly added (15 min) to a stirred solution of (H)GFPC-methyl
acetate (27, 1.50 g, 5.8 mmol, 1 equiv.) in methanol (60 mL). After
conversion was complete (TLC, 2 h), aqueous HCl (1.0 , 6 mL,
1 equiv.) was added and methanol was removed under reduced
pressure. The obtained aqueous suspension was acidified with sev-
eral drops HCl (12 ) and the precipitated product was filtered off
and washed with a small amount of cold aqueous HCl solution
(0.1 ) and dried in vacuo to provide product 27 (1.27 g, 5.2 mmol,
90%) as a yellow solid. RF (ethyl acetate/methanol 7:1) = 0.2 (prod-
uct), 0.86 (starting material); m.p. 79 °C (combustion). 1H NMR
39.4 (CH –NH), 15.2 (CH ) ppm. IR (KBr): ν = 3441, 1718, 1653,
˜
2
3
1458, 1253, 742 cm–1. UV/Vis (methanol): λmax = 347, 299, 289,
264, 210 nm. ESI-MS: m/z (%) = 565.2 (100) [M – H]–, 679.1 (90)
[M + TFA]–, 1131.2 (25) [2M – H]–, 1697.3 (10) [3M – H]–. HR-
ESI-MS: m/z [M + H]+ = calcd. for C32H30N4O6 567.2238; found:
567.2239.
3
(300 MHz, CD3OD): δ = 8.11 (dd, JH,H = 7.8 Hz, 2 H, CH-
phenyl), 7.46–7.39 (m, 3 H, CH-phenyl), 7.08 (s, 1 H, CH-methyl-
ene), 4.45 (s, 2 H, CH2), 2.35 (s, 3 H, CH3; prone to H/D ex-
change) ppm. 13C NMR (75.5 MHz, CD3OD): δ = 171.7 (CO),
171.0 (COOH), 164.8 (CCH3), 139.3 (CCH), 135.3 (C-phenyl),
133.3 (2ϫCH-phenyl), 131.5 (CH-phenyl), 129.7 (2ϫCH-phenyl),
128.6 (methylene-CH), 42.2 (CH2), 15.5 (CH3 prone to H/D ex-
Fmoc-aeg-(OH)GFPC-OH (8): Fmoc-aeg-(OH)GFPC-OMe (31,
394 mg, 0.66 mmol) was dissolved in ethyl acetate (17 mL), LiI
(408 mg, 3.1 mmol) was added, and the resulting solution was
heated under reflux for 25 h. After removal of the solvent under
reduced pressure, NaHSO3 (106 mg, 1.5 equiv.) in HCl (1.0 ,
4 equiv.) was added. Methanol (≈ 2 mL) was then added to the
suspension until a clear solution had been obtained, and this was
purified on RP-18 silica gel (methanol/water 7:3). The title com-
pound 8 (223 mg, 0.38 mmol, 58%) was obtained as a yellow solid.
M.p. 196 °C; NMR: two rotamers 1.4:1. First rotamer (major com-
change) ppm. IR (KBr): ν = 3500, 1715, 1645, 1413, 1260, 770,
˜
688, 608 cm–1. UV/Vis (methanol): λmax = 346, 291, 233, 205 nm.
ESI-MS: m/z (%) = 243.0 (100) [M – H]–, 487.1 (50) [2M – H]–,
489.2 (100) [2M + H]+. HR-ESI-MS: m/z [M + H]+ = calcd. for
C13H12N2O3 245.0921; found: 245.00920.
(OH)GFPC-Acetic Acid (24): (OAc)GFPC-Methyl acetate (28,
ponent): 1H NMR (300 MHz, [D6]DMSO, 35 °C): δ = 8.08 (d, 2.00 g, 6.33 mmol) was dissolved in methanol (100 mL) and aque-
3
3JH,H = 9.0 Hz, 2 H, phenyl-CH), 7.88 (d, JH,H = 7 Hz, 2 H,
ous LiOH (1.0 , 22.2 mL, 3.5 equiv.) was added slowly (1 h). After
the system had been stirred for 2 h at room temperature, aqueous
HCl (1.0 , 19 mL, 3.0 equiv.) was added and methanol was re-
moved under reduced pressure. To complete precipitation, further
aqueous HCl (1.0 , 9.5 mL, 1.5 equiv.) was added. The obtained
suspension was purified on RP-18 silica gel (methanol/water 2:3)
3
3
Fmoc-CH), 7.69 (d, JH,H = 7 Hz, 2 H, Fmoc-CH), 7.41 (t, JH,H
3
= 7 Hz, 3 H, Fmoc-CH, NH), 7.33 (t, JH,H = 7 Hz, 2 H, Fmoc-
CH), 6.91 (s, 1 H, CH=C), 6.85 (d, JH,H = 9.0 Hz, 2 H, phenyl-
CH), 4.44 (s, 2 H, GFP-CH2), 4.36 (d, JH,H = 6 Hz, 2 H, Fmoc-
CH2), 4.24 (t, JH,H = 6 Hz, 1 H, Fmoc-CH), 3.97 (s, 2 H,
3
3
3
CH2COOH), 3.49 (m, 2 H, CH2–N), 3.26 (m, 2 H, CH2–NH), 2.15 and the title compound 24 (860 mg, 3.3 mmol, 52%) was obtained
1
(s, 3 H, CCH3) ppm. 13C NMR (150 MHz, [D6]DMSO, 35 °C): δ
as an orange solid. M.p. 160 °C. H NMR (300 MHz, [D6]DMSO,
3
= 170.3 (COOH), 169.5 (GFP-CO), 167.4 (GFP-CH2–CO), 162.0 35 °C): δ = 8.07 (d, JH,H = 8.7 Hz, 2 H, CH-phenyl), 7.91(s, 1 H,
(C=N), 159.6 (phenyl-C–OH), 156.3 (Fmoc-CO), 143.8 (2ϫFmoc-
C), 140.6 (2ϫFmoc-C), 135.9 (C=CH), 134.0 (2ϫphenyl-CH),
127.5 (2ϫFmoc-CH), 127.0 (2ϫFmoc-CH), 125.8 (CH=C), 125.2
CH), 6.82 (d, 3JH,H = 8.7 Hz, 2 H, CH-phenyl), 4.35 (s, 2 H, CH2),
2.26 (s, 3 H, CH3) ppm. 13C NMR (75.5 MHz, [D6]DMSO, 35 °C):
δ = 169.5 (CO), 169.4 (CO), 161.2 (NC=N), 159.7 (phenyl-C–OH),
908
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 899–911

GFP Chromophore in Peptides and Peptide Nucleic Acids
FULL PAPER
135.6 (CH=C), 134.1 (2ϫphenyl-CH), 126.2 (CH=C), 125.1
(phenyl-C), 115.7 (2ϫphenyl-CH), 41.2 (CH2), 15.0 (CH3) ppm.
H]–. HR-ESI-MS: m/z [M + H]+ = calcd. for C16H18N2O6
335.1238; found: 335.1238.
IR (KBr): ν = 3426, 1595, 1383, 1251, 1165, 837 cm–1. UV/Vis
˜
(H)GFPC-Methyl Acetate (27): Under vacuum, (H)GFPC-methyl
acetate precursor 25 (2.80 g, 10.1 mmol) was quickly heated up to
220 °C in an oil bath and stirred at 220 °C for 10 min. The crude
product was dissolved in DCM and cleaned on silica gel (110 g,
ethyl acetate/pentane 1:2), and the title compound 27 was obtained
as a yellow solid (1.62 g, 6.3 mmol, 62%). RF (ethyl acetate/hexane
1:2) = 0.2 (product), 0.0 (starting material); m.p. 112 °C. 1H NMR
(300 MHz, CDCl3): δ = 8.11 (dd, 3JH,H = 8.1 Hz, 2 H, CH-phenyl),
7.43–7.32 (m, 3 H, CH-phenyl), 7.11 (s, 1 H, CH-methylene), 4.37
(s, 2 H, CH2), 2.93 (s, 3 H, OCH3), 2.31 (s, 3 H, CH3) ppm. 13C
NMR (75.5 MHz, CDCl3): δ = 170.0 (CO), 168.0 (COOMe), 161.3
(CCH3), 137.9 (CCH), 133.9 (C-phenyl), 132.2 (2ϫCH-phenyl),
130.3 (CH-phenyl), 128.7 (2ϫCH-phenyl), 128.2 (methylene-CH),
(methanol): λmax = 370, 246 nm. ESI-MS: m/z (%) = 259.1 (100)
[M – H]–, 518.8 (12) [2M – H]–. HR-ESI-MS: m/z [M + H]+
=
calcd. for C13H12N2O4 261.0870; found: 261.0870.
(H)GFPC-Methyl Acetate Precursor 25: α-Acetamidocinnamic acid
(13, 10 g, 48.7 mmol) was dissolved in DMF (100 mL), DCC
(10.6 g, 1.05 equiv.) was added, and the system was stirred for
30 min at 0 °C and for 1 h at room temperature. Glycine methyl
ester hydrochloride (7.96 g, 63.4 mmol 1.3 equiv.) and DIPEA
(33.4 mL, 4 equiv.) were added and the system was again stirred
overnight. The resulting suspension was filtered, the solid residue
was washed with DMF (50 mL), the combined organic layers were
reduced to dryness and taken up with ethyl acetate (300 mL), and
the organic layer was extracted with saturated KHCO3 solution
(3ϫ100 mL) and HCl solution (0.1 , 3ϫ100 mL). The title com-
pound 25 was obtained from the acidic water layer as a yellow solid
(2.80 g, 10.1 mmol, 21%) without further purification. The crude
product from the basic layer was cleaned by filtration through silica
gel (30 g silica gel, ethyl acetate/methanol 9:1 + 0.5% triethylamine,
1 L) to provide further title compound 25 as a yellow solid (3.92 g,
14.2 mmol, 29%). RF (ethyl acetate/methanol 9:1 + 0.5 triethyl-
amine) = 0.53; m.p. 117 °C. 1H NMR (300 MHz, CDCl3): δ = 7.91
(s, 1 H, NH), 7.58–7.48 (m, 1 H, NHCH2), 7.37–7.29 (m, 5 H, CH-
52.8 (OCH ), 41.2 (CH ); 15.5 (CH ) ppm. IR (KBr): ν = 3500,
˜
3
2
3
1743, 1645, 1561, 1406, 1363, 1232, 1144, 981, 904, 775, 700,
610 cm–1. UV/Vis (methanol): λmax = 348, 290, 234, 209 nm. ESI-
MS: m/z (%) = 257.1 (40) [M – H]–, 259.1 (60) [M + H]+, 281.0
(100) [M + Na]+, 538.8 (95) [2M + Na]+. HR-ESI-MS: m/z [M +
H]+ = calcd. for C14H14N2O3 259.1077; found: 259.1077.
(OAc)GFPC-Methyl Acetate (28): Under vacuum, (OAc)GFPC-
methyl acetate precursor 26 (5.3 g, 15.9 mmol) was quickly heated
up to 200 °C in an oil bath and stirred at 200 °C for 12 min. The
crude product was dissolved in DCM (5 mL) and cleaned on silica
gel (ethyl acetate/pentane 1:1), and the title compound 28 (2.2 g,
7.0 mmol, 44%) was obtained as a yellow solid. M.p. 75 °C. 1H
3
phenyl), 6.96 (s, 1 H, CH), 4.01 (d, JH,H = 5.7 Hz, 2 H, CH2),
3.68 (s, 3 H, OCH3), 2.01 (s, 3 H, CH3CONH) ppm. 13C NMR
(75.5 MHz, CDCl3): δ = 170.7 (CO), 170.5 (CO), 166.2 (CO), 133.4
(phenyl-C), 130.1–128.5 (CH=C–N, 5 ϫphenyl-CH), 52.3 (OCH3),
3
NMR (300 MHz, CDCl3): δ = 8.14 (d, JH,H = 8.7 Hz, 2 H, CH-
3
phenyl), 7.12 (d, JH,H = 8.7 Hz, 2 H, CH-phenyl), 7.07 (s, 1 H,
41.5 (CH ), 22.9 (NHCOCH ) ppm. IR (KBr): ν = 3445, 1635,
˜
2
3
1376, 1203, 573 cm–1. UV/Vis (methanol): λmax = 208, 281, 327 nm.
ESI-MS: m/z (%) = 299.1 (20) [M + Na]+, 574.8 (100) [2M +
Na]+, 275.1 (100) [M – H]–, 550.8 (30) [2M – H]–. HR-ESI-MS:
m/z [M + Na]+ = calcd. for C14H16N2O4 299.1002; found: 299.1003.
CH), 4.36 (s, 2 H, CH2), 3.75 (s, 3 H, OCH3), 2.30 (s, 3 H, CH3CO),
2.28 (s, 3 H, CH3) ppm. 13C NMR (75.5 MHz, CDCl3): δ = 169.9
(CO), 169.0 (CO), 167.9 (CO), 161.5 (NC=N), 151.9 (phenyl-C-
OAc), 137.9 (CH=C), 133.4 (2ϫphenyl-CH), 131.7 (phenyl-C),
126.9 (CH=C), 121.9 (2ϫphenyl-CH), 52.8 (OCH3), 41.2 (CH2),
(OAc)GFPC-Methyl Acetate Precursor 26: 2-Acetamido-4-acetoxy-
cinnamic acid (17, 10 g, 38 mmol) was dissolved in DMF (100 mL),
and DCC (7.84 g, 38 mmol) dissolved in DMF (10 mL) was added
slowly (5 min) at 0 °C with stirring in an ice bath. The resulting
solution was stirred at 0 °C for 30 min and for 1 h at room tempera-
ture, and the formed urea was filtered off and washed with DMF
(40 mL). Triethylamine (21.2 mL, 152 mmol, 4.0 equiv.) and glycine
methyl ester hydrochloride (6.18 g, 49 mmol, 1.3 equiv.) were
added, the DMF solution was stirred overnight, the solvent was
removed in vacuo, and the obtained solid was dissolved in HCl
(0.1 , 60 mL) and extracted several times with chloroform
(150 mL overall). Silica gel (30 g) was added to the organic layer,
which was reduced to dryness, and the product was eluted from the
silica gel (ethyl acetate/methanol 95:5 + 2% triethylamine), concen-
trated, and dried in vacuo. The title compound 26 (8.92 g,
27 mmol, 71%) was obtained as a yellow solid. RF (ethyl acetate/
21.1 (OCOCH ), 15.5 (CH ) ppm. IR (KBr): ν = 1747, 1646, 1367,
˜
3
3
1216, 980, 908, 609 cm–1. UV/Vis (methanol): λmax = 351, 292, 236,
203 nm. ESI-MS: m/z (%) = 339.1 (90) [M + Na]+, 654.9 (100)
[2M + Na]+. HR-ESI-MS: m/z [M + H]+ = calcd. for C16H16N2O5
317.1132; found: 317.1132.
Fmoc-aeg-(H)GFPC-OMe (30): (H)GFPC-Acetic acid (23, 0.61 g,
2.5 mmol, 1.3 equiv.), Fmoc-aeg-OMe·HCl[14] (29, 0.75 g,
1.9 mmol, 1.0 equiv.), HBTU (0.95 g, 2.5 mmol, 1.3 equiv.), and
HOBt (0.52 g, 3.8 mmol, 2.0 equiv.) were dissolved in DMF
(25 mL). DIPEA (1.65 mL, 5 equiv.) was added and the resulting
solution was stirred for 14 h at room temperature. The solvent was
removed in vacuo, and the obtained solid was dissolved in ethyl
acetate (100 mL), washed with NaHCO3 (0.5 , 100 mL), brine
(100 mL), saturated NH4Cl solution (100 mL), and brine (100 mL),
and dried with Na2SO4. After purification on silica gel (ethyl ace-
tate, 600 mL) title compound 30 was obtained as a yellow solid
methanol 95:5 + 2% triethylamine) = 0.29 (product); m.p. 94 °C.
3
1H NMR (300 MHz, CD3OD): δ = 7.55 (d, JH,H = 8.7 Hz, 2 H, (0.87 g, 1.5 mmol, 79%). M.p. 165 °C; NMR: two rotamers 2:1. 1H
3
3
CH-phenyl), 7.22 (s, 1 H, CH), 7.14 (d, JH,H = 8.7 Hz, 2 H, CH- NMR (300 MHz, CDCl3): δ = 8.10 (d, JH,H = 10.8 Hz, 2 H, CH-
3
3
phenyl), 4.02 (s, 2 H, CH2), 3.73 (s, 3 H, OCH3), 2.27 (s, 3 H,
phenyl), 7.72 (d, JH,H = 9.6 Hz, 2 H, CH-Fmoc), 7.57 (t, JH,H =
CH3CO), 2.11 (s, 3 H, CH3CONH) ppm. 13C NMR (75.5 MHz, 6.9 Hz, 2 H, CH-Fmoc), 7.43–7.24 (m, 7 H, CH-Fmoc, CH-Fmoc,
CD3OD): δ = 173.3 (CO), 171.8 (CO), 170.9 (CO), 168.3 (CO), CH-phenyl, CH-phenyl), 7.09 (s, 0.66 H, CH-methylene), 7.07 (s,
152.7 (phenyl-C-OAc), 132.8 (CH=C), 131.7 (2ϫphenyl-CH),
130.6 (CH), 129.9 (phenyl-C), 123.1 (2ϫphenyl-CH), 52.6 (OCH3),
0.33 H, CH-methylene), 5.85 (t, 3JH,H = 6.0 Hz, 0.66 H, NH), 5.30
3
3
(t, JH,H = 5.7 Hz, 0.33 H, NH), 4.50 (d, JH,H = 6.3 Hz, 1.3 H,
3
42.3 (CH ), 22.7 (NHCOCH ), 20.9 (OCOCH ) ppm. IR (KBr): ν
CH2-Fmoc), 4.36 (d, JH,H = 8.7 Hz, 2.7 H, CH2-Fmoc, CH2-
GFP), 4.19 (t, JH,H = 5.4 Hz, 1 H, CH-Fmoc), 3.99 (s, 2 H,
˜
2
3
3
= 3272, 1756, 1658, 1534, 1371, 1205, 1014, 910, 655 cm–1. UV/Vis
(methanol): λmax = 358, 281, 216 nm. ESI-MS: m/z (%) = 357.1
(15) [M + Na]+, 590.8 (100) [2M + Na]+, 1024.3 (20) [3M + Na]+,
333.1 (100) [M – H]–, 378.8 (70) [M + HCOO]–, 666.6 (30) [2M –
3
CH2COOMe), 3.75 (s, 2.0 H, OCH3), 3.72 (s, 1.0 H, OCH3), 3.53
(m, 2 H, CH2–N), 3.40–3.32 (m, 2 H, CH2–NH), 2.32 (s, 2.0 H,
CH3), 2.15 (s, 1.0 H, CH3) ppm. 13C NMR (150 MHz, CD3OD):
Eur. J. Org. Chem. 2007, 899–911
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
909

T. Stafforst, U. Diederichsen
FULL PAPER
first rotamer (major component): δ = 170.4 (COOMe), 169.8 (CO-
167.7 (GFP-CH2–CO), 160.4 (C=N), 158.7 (phenyl-C–OH), 156.8
GFP), 167.1 (CH2CON), 162.4 (N=C–N), 156.6 (OCON), 143.6 (Fmoc-CO), 143.8 (2ϫFmoc-C), 141.3 (2ϫFmoc-C), 135.5
(2ϫC-Fmoc), 141.3 (2ϫC-Fmoc), 138.2 (CH=C–N), 134.1 (C- (C=CH), 134.5 (2ϫphenyl-CH), 129.0 (CHC), 127.7 (2ϫFmoc-
phenyl), 132.2 (2ϫCH-phenyl), 130.1 (1 CH-phenyl), 128.6
(2ϫCH-phenyl), 127.7 (2ϫCH-Fmoc), 127.6 (CH-methylene),
127.0 (2ϫCH-Fmoc), 124.9 (2ϫCH-Fmoc), 120.0 (2ϫCH-
Fmoc), 66.6 (CH2-Fmoc), 52.6 (OCH3), 48.9 (CH2–N,
CH2COOMe), 47.3 (CH-Fmoc), 40.9 (CH2-GFP), 39.2 (CH2–NH),
15.4 (CH3) ppm; second rotamer: δ = 170.3 (COOMe), 169.8 (CO-
CH), 127.1 (2ϫFmoc-CH), 126.4 (phenyl-C–CH), 125.0
(2ϫFmoc-CH), 120.0 (2ϫFmoc-CH), 115.9 (2ϫphenyl-CH), 66.9
(Fmoc-CH2), 53.0 (OCH3), 50.0 (CH2COOMe), 49.0 (CH2–N),
47.2 (Fmoc-CH), 41.5 (GFP-CH2), 38.9 (CH2–NH), 15.2
(CH C) ppm. IR (KBr): ν = 3405, 1703, 1601, 1513, 1448, 1252,
˜
3
750 cm–1. UV/Vis (methanol): λmax (OD) = 370 (0.83), 300 (0.46),
GFP), 167.8 (NCO–CH2), 162.4 (N=C–N), 156.61 (OCON), 143.8 265 (0.8), 221 (1.0) Ǟ same sample: UV/Vis (+ 0.01% NEt3): λmax
(2ϫC-Fmoc), 141.3 (2ϫC-Fmoc), 138.2 (CH=C–N), 134.0 (C- (OD) = 431 (0.86), 300 (0.43), 264 (0.88), 227 (3.3). Fluorescence
phenyl), 132.2 (2ϫCH-phenyl), 130.2 (1ϫCH-phenyl), 128.6
(2ϫCH-phenyl), 127.7 (2ϫCH-Fmoc), 127.6 (CH-methylene),
127.0 (2ϫCH-Fmoc), 125.1 (2ϫCH-Fmoc), 119.9 (2ϫCH-
(glycerin + 0.01% NEt3): λexc. = 430 nm, λem. = 499 nm. ESI-MS:
m/z (%) = 597.3 (40) [M + H]+, 1193.4 (100) [2M + H]+. HR-
ESI-MS: m/z [M + H]+ = calcd. for C33H32N4O7 597.2344; found:
Fmoc), 66.8 (CH2-Fmoc), 53.0 (OCH3), 48.9 (CH2–N, 597.2346.
CH2COOMe), 47.1 (CH-Fmoc), 41.5 (CH2-GFP), 39.2 (CH2–NH),
PNA Oligomer Ac-t-t-t-t-(H)GFPC-g-g-c-g-g-c-Lys-Lys-Gly-NH2
15.6 (CH ) ppm. IR (KBr): ν = 3412, 2928, 1750, 1722, 1663, 1470,
˜
3
(32): The solid-phase peptide synthesis was performed on a 5 µ
scale in a plastic syringe by the Fmoc/Bhoc strategy on Sieberamide
resin as described elsewhere.[14] Commercially available amino acids
Fmoc-aeg-thymine-OH, Fmoc-aeg-guanine(Bhoc)-OH, Fmoc-aeg-
cytosine(Bhoc)-OH, Fmoc-Lys(Mtt)-OH, and Fmoc-Gly-OH on
Sieberamide resin (0.12 mmol·g–1) were used for the oligomer syn-
thesis with use of the HBTU/HATU activation technique. Double
coupling was performed in cases involving the coupling of guaninyl
amino acids following a thyminyl or guaninyl amino acid. Double
coupling was not required for introduction of Fmoc-aeg-
(H)GFPC-OH (7). Oligomer 32 was cleaved from the resin by con-
tinuous flow with 2% trifluoroacetic acid, 4% triethylsilane in
1,1,1,3,3,3-hexafluoropropan-2-ol/dichloromethane 1:2 and was
obtained (1.5 mg, 9% yield) after preparative HPLC purification.
HR-ESI-MS: [M]+: calcd. for C141H184N64O41 3429.4280; found:
3429.4306, [M + H]+: calcd. 3430.4329; found: 3430.4329, [M +
3H]3+: calcd. 1144.1500; found: 1144.1510, [M + 4H]4+: calcd.
858.3643; found: 858.3647; HPLC: analytical: 10% Ǟ 40% in
30 min (Grom25), tR = 26.00 min. UV: ε260nm = 96.9·103 [approxi-
mated by nearest neighbor approach with ε260nm = 9.0·103 for the
(H)GFP chromophore]. UV/Vis (pH 6.85, phosphate buffer): λmax
(ε/103) = 350 (9.0), 260 (97.0), 204 (204) nm. UV-melting: pairing
with complementary DNA 33 (5Ј-G-C-C-G-C-C-A-A-A-A-A-3Ј,
ε260nm = 110.5·103) each 3 µ in 10 mm phosphate buffer pH =
6.85, NaCl (0.10 ): H260nm = 10% at Tm = 71.0 °C, heating–cool-
ing hysteresis 3 °C; CD: pairing with complementary DNA 33 each
3 µm in phosphate buffer (10 mm) pH = 6.85, NaCl (0.10 ): tem-
perature-dependent band at 265 nm (10 mdeg at 20 °C), melting
temperature ca. 75 °C; pairing with complementary DNA 33 each
30 µm in 10 mm phosphate buffer pH = 6.85, NaCl (0.10 ): tem-
perature-dependent band at 293 nm (3 mdeg at 20 °C) and 347 nm
(–3 mdeg, 20 °C); fluorescence: oligomer 32 with and without com-
plementary DNA 33 (5Ј-G-C2–G–C2–A5-3Ј) each 30 µm in phos-
phate buffer (10 mm) pH 6.85, NaCl (0.10 ); excitation: 350 nm
(OD = 0.27), 20 nm bandwidth, emission: single strand 413, 441,
510 nm. pairing complex: 416, 440, 505 nm, quantum yield
Ͻ 0.5%.
1210, 1045, 745 cm–1. UV/Vis (methanol): λmax = 347, 299, 289,
264, 210 nm. Fluorescence (glycerin): λexc = 350 nm, λmax, em = 413,
436, 477 nm. ESI-MS: m/z (%) = 581.2 (25) [M + H]+, 1182.8 (100)
[2M + Na]+, 624.9 (100) [M + TFA]–, 1204.7 (70) [2M + TFA]–.
HR-ESI-MS: m/z [M + H]+ = calcd. for C33H32N4O6 581.2395;
found: 581.2396.
Fmoc-aeg-(OH)GFPC-OMe (31): (OH)GFPC-Acetic acid (24,
508 mg, 1.95 mmol, 1.1 equiv.), Fmoc-aeg-OMe·HCl[14] (29,
694 mg, 1.77 mmol, 1.0 equiv.), and HBTU (740 mg, 1.95 mmol,
1.1 equiv.) were dissolved in DMF (15 mL). DIPEA (1.22 mL,
920 mg, 7.1 mmol, 4 equiv.) was added, the resulting solution was
stirred for 2 d, the solvent was removed under reduced pressure,
and the residue was dissolved in ethyl acetate (100 mL), washed
(3ϫ100 mL 0.5  NH4Cl, 3ϫ100 mL 0.5  NaHCO3, 1ϫ100 mL
brine), and dried with Na2SO4. The crude product was purified on
silica gel (200 mL, ethyl acetate) and the title compound 31
(0.223 g, 0.37 mmol, 21%) was obtained as a yellow solid. M.p.
174 °C; NMR: two rotamers 2:1. First rotamer (major component):
3
1H NMR (300 MHz, CDCl3): δ = 7.89 (d, JH,H = 8.7 Hz, 2 H,
3
phenyl-CH), 7.70 (d, JH,H = 6.9 Hz, 2 H, Fmoc-CH), 7.57 (d,
3
3JH,H = 6.6 Hz, 2 H, Fmoc-CH), 7.35 (t, JH,H = 6.9 Hz, 2 H,
3
Fmoc-CH), 7.27 (t, JH,H = 6.6 Hz, 2 H, Fmoc-CH), 6.95 (s, 1 H,
3
3
CH=C), 6.76 (d, JH,H = 8.7 Hz, 2 H, phenyl-CH), 5.91 (t, JH,H
3
= 6.0 Hz, 1 H, NH), 4.48 (d, JH,H = 6.3 Hz, 2 H, Fmoc-CH2),
4.35 (s, 2 H, GFP-CH2), 4.19 (t, JH,H = 6.3 Hz, 1 H, Fmoc-CH),
3
3.98 (s, 2 H, CH2COOMe), 3.71 (s, 3 H, OCH3), 3.52–3.49 (m, 2
H, CH2–N), 3.41–3.39 (m,
2 H, CH2–NH), 2.11 (s, 3 H,
CCH3) ppm. 13C NMR (150 MHz, CD3OD): δ = 170.4 (GFP-CO),
169.7 (COOMe), 167.7 (GFP-CH2–CO), 160.4 (C=N), 158.7
(phenyl-C–OH), 156.8 (Fmoc-CO), 143.6 (2ϫFmoc-C), 141.3
(2ϫFmoc-C), 135.7 (C=CH), 134.5 (2ϫphenyl-CH), 128.7
(CHC), 127.8 (2ϫFmoc-CH), 127.1 (2ϫFmoc-CH), 126.4
(phenyl-C–CH), 124.9 (2ϫFmoc-CH), 120.0 (2ϫFmoc-CH),
115.9 (2ϫphenyl-CH), 66.7 (Fmoc-CH2), 52.6 (OCH3), 50.0
(CH2COOMe), 49.0 (CH2–N), 47.2 (Fmoc-CH), 41.0 (GFP-CH2),
39.2 (CH2–NH), 15.4 (CH3C) ppm. Second rotamer: 1H NMR
3
(300 MHz, CDCl3): δ = 7.89 (d, JH,H = 8.7 Hz, 2 H, phenyl-CH),
Abbreviations: aeg = N-(2-aminoethyl)glycine, Bhoc = benzhy-
dryloxycarbonyl, Boc = tert-butyloxycarbonyl, DAP = 2-amino-
propionic acid, DBU = 1,3-diazabicyclo[5.4.0]undecane, Fmoc =
3
3
7.72 (d, JH,H = 6.9 Hz, 2 H, Fmoc-CH), 7.55 (d, JH,H = 6.6 Hz,
3
2 H, Fmoc-CH), 7.35 (t, JH,H = 6.9 Hz, 2 H, Fmoc-CH), 7.27 (t,
3JH,H = 6.6 Hz, 2 H, Fmoc-CH), 6.95 (s, 1 H, CH=C), 6.76 (d, 9-fluorenylmethoxycarbonyl, GFPC = Green Fluorescent Protein
3
3JH,H = 8.7 Hz, 2 H, phenyl-CH), 5.39 (t, JH,H = 5.7 Hz, 1 H,
chromophore, HFIP = 1,1,1,3,3,3-hexafluoropropan-2-ol, HATU
NH), 4.35 (s, 2 H, GFP-CH2), 4.33 (d, 3JH,H = 6.3 Hz, 2 H, Fmoc- = O-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
3
CH2), 4.19 (t, JH,H = 6.3 Hz, 1 H, Fmoc-CH), 4.16 (s, 2 H, fluorophosphate, HOAt = 1-hydroxy-7-azabenzotriazole, Mtt =
CH2COOMe), 3.71 (s, 3 H, OCH3), 3.52–3.49 (m, 2 H, CH2–N), monomethyltrityl, PNA = peptide nucleic acid, OD = optical den-
3.37–3.31 (m, 2 H, CH2–NH), 2.27 (s, 3 H, CCH3) ppm. 13C NMR sity, TES = triethylsilane, TFA = trifluoroacetic acid, TFMSA =
(150.8 MHz, CD3OD): δ = 170.3 (GFP-CO), 169.7 (COOMe),
910
trifluoromethanesulfonic acid, Z = benzyloxycarbonyl.
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 899–911

GFP Chromophore in Peptides and Peptide Nucleic Acids
FULL PAPER
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fellowship from the Fonds der Chemischen Industrie for T. S.
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Received: August 17, 2006
Published Online: November 21, 2006
Eur. J. Org. Chem. 2007, 899–911
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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