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S. Weis et al. / Journal of Photochemistry and Photobiology A: Chemistry 241 (2012) 52–57
1H NMR (300 MHz, CDCl3) ı 7.71 (d, 2H, J = 7.5 Hz), 7.56 (d, 2H,
Asp(DEACE) resp.) are specified in Table 1 in the supporting infor-
mation. The coupling solution was filtered out and the resin was
washed five times with DMF. The resin was split in two parts after
coupling of Fmoc-d-Phe-OH, followed by the coupling of Fmoc-
Asp(DEACM)-OH 6 or Fmoc-Asp(DEACE)-OH 7. Cleavage of the
protected linear peptides from the resin was performed by treating
the resin three times with 3 ml of a solution of trifluorethanol/acetic
acid/DCM (1/3/6) for 30 min each. Toluene was added to the fil-
trate and the solvents were removed in vacuum. The crude peptides
were purified by HPLC using acetonitrile and water containing 0.1%
TFA as eluent to yield 5.1 moles (yield 30%) of H-Asp(DEACM)-d-
Phe-Lys(Boc)-Gly-OH obtained from 17.1 moles of TCP-resin and
10.9 moles (yield 62%) of H-Asp(DEACE)-d-Phe-Lys(Boc)-Gly-OH
obtained from 17.5 moles of TCP-resin. The peptides were char-
acterized by ESI-MS.
J = 7.2 Hz), 7.37–7.19 (m, 5H), 6.53 (dd, 1H, J = 9.3 Hz, 2.1 Hz), 6.46
(d, 1H, J = 2.1 Hz), 6.14 (s, 1H, CH), 6.02 (d, 1H, J = 8.1), 5.23 (s, 2H),
4.79–4.73 (m, 1H), 4.41–4.31 (m, 2H), 4.18 (t, 1H, J = 6.9 Hz), 3.35
(q, 4H, J = 6.9 Hz), 3.19 (dd, 1H, J = 17.3 Hz, 4.2 Hz), 3.05 (dd, 1H,
J = 17.0 Hz, 4.5 Hz), 1.15 (t, 6H, J = 6.9 Hz).
13C NMR (75 MHz, CDCl3) ı 173.9, 170.5, 162.8, 156.4, 151.0,
149.6, 143.9, 141.5, 127.9, 127.3, 125.4, 124.5, 120.2, 109.2, 106.4,
106.2, 98.1, 67.7, 62.1, 50.6, 47.3, 45.0, 36.8, 12.6.
MS (ESI, m/z) calcd for C33H32N2NaO8: 607.21; found: 607.19.
Anal. calcd for C33H32N2O8: C, 67.80; H, 5.52; N, 4.79. Found: C,
67.80; H, 5.86; N, 4.65%.
2.2.5. N-Fmoc-aspartic acid
1-(7-(N,N-diethylamino)-coumarin-4-yl)-1-ethyl ester (7)
N-␣-Fmoc-l-aspartic acid ␣-t-butyl ester (95 mg, 0.23 mmol,
MS (ESI, m/z) of H-Asp(DEACM)-d-Phe-Lys(Boc)-Gly-OH calcd
for C40H54N6O11: 795.39; found: 795.39.
1.2 equiv.),
hydrochloride
1-ethyl-3-(3-dimethylaminopropyl)carbodiimid
(60 mg, 0.32 mmol, 1.65 equiv.) and 4-
MS (ESI, m/z) of H-Asp(DEACE)-d-Phe-Lys(Boc)-Gly-OH calcd
C41H57N6O11 for: 809.92; found: 809.42.
(dimethylamino)-pyridine (2.3 mg, 0.02 mmol, 0.1 equiv.) were
dissolved in absolute dichloromethane (4 ml). A solution of 3
(50 mg, 0.19 mmol, 1 equiv.) in absolute dichloromethane (4 ml)
was added and the reaction mixture was stirred at r.t. in the dark
under an argon atmosphere for 2.5 h. A solution of 340 mg sodium
bicarbonate in 85 ml water was added and the reaction mixture
was extracted three times with ethyl acetate. The combined
organic layers were washed with water and dried over MgSO4.
The solvent was removed in vacuum and the crude material was
purified via column chromatography (silica gel, 2:1 hexane/ethyl
acetate) to obtain 118 mg (0.18 mmol, 94%) of green solid,
7-N,N-diethylamino-4-(1ꢀ-hydroxyeth-1ꢀ-yl)coumarin-methyl-
N-␣-Fmoc-l-aspartic acid ␣-t-butyl ester (5). Product 5 (118 mg,
0.18 mmol, 1 equiv.) was dissolved in dry dichloromethane (2.6 ml)
and trifluoroacetic acid (2.6 ml, 0.035 mmol, 195 equiv.) was added.
The mixture was stirred at room temperature in the dark for 2 h.
The solvents were evaporated and the product was dried in
vacuum. Dichloromethane (20 ml) was added and the solution
was washed twice with water. The combined organic layers were
dried over MgSO4 and the solvent was removed in vacuum to give
the product as a yellow-brown solid (82 mg, 0.14 mmol, 76%). The
product was further purified by RP-HPLC using water/acetonitrile
containing 0.1% TFA as eluent. The pure fractions were collected
and freeze dried to give a yellow powder.
2.3. Photochemical experiments
3 ml of 0.05 mM solutions of 6 and 7 in acetonitrile/water = 95:5
were irradiated at ꢀ = 360 nm. The UV spectra of the solutions were
recorded after selected exposure times. In a similar way, a 0.16 mM
solution of Fmoc-Asp(DEACM)-OH 6 and a 0.11 mM solution of
Fmoc-Asp(DEACE)-OH 7 in acetonitrile/water = 95:5 were irradi-
ated and the fluorescence emission spectra were recorded using an
excitation wavelength range of 350–450 nm.
The chemical yield of the photolysis reaction was determined by
quantitative analytical HPLC. 3.3 ml of 0.06 mM solutions of 6 and
7 in acetonitrile/water = 95:5 were irradiated at 360 nm for 4 h. The
solvents of the irradiated solutions were removed under reduced
pressure and the residue was redissolved in 100 l of acetoni-
trile/water = 95:5. Aliquots of 20 l were injected into an analytical
HPLC set-up using water/acetonitrile containing 0.1% TFA as elu-
ent. The concentration of the uncaged product was evaluated by
comparing the peak areas with a reference solution with known
concentration.
2.4. Determination of the quantum yield
Ferrioxalate actinometry was used to determine the photon flow
of the LUMOS 43 lamp at 360 nm. A value of 2.77 × 10−9 E/s was
obtained. A solution of the caged amino acids 6 or 7 in acetoni-
trile/water (95:5) with an absorbance >2 at 360 nm was irradiated
with increasing times. Aliquots (50 l) of the irradiated solution
were taken after exposure and 20 l were injected into analytical
HPLC to determine the amount of remaining caged product by peak
area quantification using the non-irradiated solution as a reference.
The ratio of the number of molecules that reacted to the num-
ber of photons absorbed gave the quantum yield of the photolytic
reaction. Data were obtained for conversions <10%.
mp: 117–123 ◦C (1 ◦C/min).
1H NMR (250 MHz, CDCl3) ı 7.71 (d, 2H, J = 7.3 Hz), 7.55 (d, 2H,
J = 7.0 Hz), 7.38–7.23 (m, 5H), 6.53 (dd, 1H, J = 8.9 Hz, 2.3 Hz), 6.45
(s, 1H), 6.16 (d, 1H, J = 4.5 Hz), 6.11–5.94 (m, 2H, CH, 4.79–4.70 (m,
1H), 4.41–4.27 (m, 2H), 4.17 (t, 1H, J = 7.0 Hz), 3.34 (q, 4H, J = 7.0 Hz),
3.20–2.98 (m, 2H), 1.55 (d, 3H, J = 6.5 Hz), 1.15 (t, 6H, J = 7.0 Hz).
13C NMR (75 MHz, CDCl3) ı 173.8, 169.8, 163.3, 156.6, 156.3,
155.8, 150.8, 143.9, 141.5, 127.9, 127.3, 125.4, 124.8, 120.1, 109.3,
105.9, 104.7, 98.3, 68.6, 67.6, 50.5, 47.3, 45.0, 36.9, 21.1, 12.6.
HRMS (ESI) calcd for C34H34N2O8Na 621.2213, found 621.2195.
2.2.6. General procedure for solid phase peptide synthesis (SPPS)
The linear peptides were synthesized by SPPS following the
Fmoc-strategy. The TCP-resin was first reacted with glycine and
diisopropylethylamine (DIPEA) in absolute dichloromethane to
yield 0.035 mmol of Gly-loaded resin. Deprotection of the Fmoc-
group was performed by treating the resin twice for 10 min with
20% piperidine in DMF under vigorous shaking. The resin was
washed five times with DMF. To couple the next amino acid,
a solution of the Fmoc-amino acid, HOBt, HBTU, and DIPEA in
DMF (10–15 ml/g of resin) was added to the reaction vessel and
left to react under vigorous shaking. The coupling conditions for
each of the Fmoc-amino acids (Lys(Boc), d-Phe, Asp(DEACM) and
2.5. Stability tests
The stability of compounds 6 and 7 in DMF/PBS (7:5) solu-
tion during 52.5 h in the dark was tested using 5.9 mM solutions
of (Fmoc-Asp(DEACM)-OH) and 3.9 mM (Fmoc-Asp(DEACE)-OH).
20 l of the solutions were injected in the analytical HPLC using
water/acetonitrile containing 0.1% TFA as eluent for quantification.
The stability of compounds 6 and 7 (0.01 M) in 20% piperi-
dine/DMF solution was tested by taking aliquots of 200 l after
different times between 5 and 90 min, removing the solvent in
high vacuum, redissolving the residue in 1 ml water/acetonitrile
(1:4) (7) or 0.8 ml water/acetonitrile (1:3) (6) and injecting 20 l