Long-wavelength photolysis of amino acid 6-(methoxy-2-oxo-2H-naphtho[1,2-b] pyran-4-yl)methyl esters

Article abstract of DOI:10.1002/ejoc.201100391

(6-Methoxy-2-oxo-2H-naphtho[1,2-b]pyran-4-yl)methyl esters of valine or phenylalanine as model bifunctional molecules were synthesised to assess its applicability as a photocleavable protecting group for solution phase organic synthesis and in caging applications at longer wavelengths. The behaviour of the corresponding derivatives towards photolysis was evaluated by irradiation at 350 and 419 nm in a mixture of HEPES buffer and acetonitrile or methanol in a photochemical reactor, followed by HPLC/UV monitoring. Time-resolved fluorescence measurements were used to elucidate the dynamics. Photocleavage studies of new valine orphenylalanine 2H-naphtho[1,2-b]pyran-4-yl)methyl esters revealed that the amino acid-heterocycle ester bond was efficiently photolysed, especially at 419 nm for all compounds in practical irradiation times. Copyright

Full text of DOI:10.1002/ejoc.201100391

FULL PAPER
DOI: 10.1002/ejoc.201100391
Long-Wavelength Photolysis of Amino Acid 6-(Methoxy-2-oxo-2H-
naphtho[1,2-b]pyran-4-yl)methyl Esters
Ana M. Piloto,[a] Ana M. S. Soares,[a] Graham Hungerford,[b] Susana P. G. Costa,[a] and
M. Sameiro T. Gonçalves*[a]
Keywords: Amino acids / Protecting groups / Photochemistry / Photolysis / Cage compounds / Oxygen heterocycles
(6-Methoxy-2-oxo-2H-naphtho[1,2-b]pyran-4-yl)methyl es-
ters of valine or phenylalanine as model bifunctional mole-
sis was evaluated by irradiation at 350 and 419 nm in a mix-
ture of HEPES buffer and acetonitrile or methanol in a photo-
cules were synthesised to assess its applicability as a photo- chemical reactor, followed by HPLC/UV monitoring. Time-
cleavable protecting group for solution phase organic syn-
thesis and in caging applications at longer wavelengths. The
behaviour of the corresponding derivatives towards photoly-
resolved fluorescence measurements were used to elucidate
the dynamics.
biology and biophysical studies with a range of chemical
functionalities.[3–6,13] As part of our research on the design
and evaluation of new polyaromatic and polyheteroarom-
atic systems, which include oxobenzopyrans and their fused
derivatives as photolabile protecting groups for biologically
relevant molecules,[14–19] we report the synthesis of new val-
ine and phenylalanine derivatives bearing a 2H-naphtho-
[1,2-b]pyran-2-one moiety as a light-sensitive group. In
order to evaluate the photorelease of the amino acids in N-
protected and free forms in practical times at longer wave-
lengths, photolysis studies were carried out in mixtures of
aqueous 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) buffer and an organic solvent at 350 and 419 nm.
Cleavage kinetic data obtained by HPLC/UV monitoring
was also collected and time-resolved fluorescence measure-
ments were used to elucidate the dynamics.
Introduction
The use of light of appropriate wavelength can liberate
synthetic and biochemically relevant molecules from their
corresponding nonreactive/inactive conjugate/caged precur-
sors possessing suitable light-sensitive moieties covalently
bonded to the functional groups of the molecule of interest.
In recent years this approach has been widely applied in
organic synthesis as an alternative to classical acid- and
base-labile protecting groups, as well as, in life sciences, to
investigate signal transduction mechanisms at the cellular
level and in drug delivery.[1] This broad range of applica-
tions supports the development of new photolabile groups
with enhanced properties that will allow fast cleavage and
release of various functionalities (such as alcohols, amines,
phosphates, aldehydes, ketones and carboxylic acids).[2–6]
Additionally, the use of longer wavelengths can minimise
side reactions and/or cell damage. Nevertheless, among the
considerable number of light-sensitive groups that have
been reported, only a few cleave in a practical time by irra-
diation at wavelengths longer than 400 nm.
Results and Discussion
In addition to the interest in natural or synthetic deriva-
tives of 2H-benzopyran-2-ones (coumarins) as antioxidants,
food components, stabilisers, immunomodulatory sub-
stances, fluorescent markers in analysis, laser dyes and in
clinical use,[7–12] they have also been recognised for their
application as photolabile protecting groups for the caging
of biomolecules. Oxobenzopyrans have been reported in cell
4-(Chloromethyl)-6-methoxy-2H-naphtho[1,2-b]pyran-2-
one[18] (1) was treated with N-(benzyloxycarbonyl)-l-valine
(2a) or N-(benzyloxycarbonyl)-l-phenylalanine (2b) in the
presence of potassium fluoride in DMF at room tempera-
ture[20] to give N-(benzyloxycarbonyl)-l-valine (6-methoxy-
2-oxo-2H-naphtho[1,2-b]pyran-4-yl)methyl ester (3a) and
N-(benzyloxycarbonyl)-l-phenylalanine (6-methoxy-2-oxo-
2H-naphtho[1,2-b]pyran-4-yl)methyl ester (3b), respectively,
in good yields (Scheme 1). In order to compare the release
of the model amino acids in their N-protected 2a and 2b
and free forms 5a and 5b under irradiation, the N-benzyl-
oxycarbonyl protecting group was removed by acidolysis
with hydrobromic acid in acetic acid to give 4a and 4b bear-
ing the amino acid and the photosensitive tag (Scheme 1).
[a] Centre of Chemistry, University of Minho, Campus of Gualtar,
4710-057 Braga, Portugal
Fax: +351-253604382
E-mail: msameiro@quimica.uminho.pt
[b] HORIBA Jobin Yvon IBH Ltd,
45 Finnieston Street, Glasgow G3 8JU, UK
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100391.
Eur. J. Org. Chem. 2011, 5447–5451
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5447

M. S. T. Gonçalves et al
FULL PAPER
(λabs) and emission maxima (λem), molar absorptivities (ε)
and relative fluorescence quantum yields (ΦF) are reported
(see Tables 1 and 2). Values of ΦF were calculated using
9,10-diphenylanthracene as standard (ΦF = 0.95 in eth-
anol).[21] For ΦF determination, the fluorescence standard
was excited at the wavelengths of maximum absorption
found for each of the compounds to be tested, and in all
fluorimetric measurements the absorbance of the solution
did not exceed 0.1.
By comparison of the absorption and emission maxima,
no significant differences were observed in these solvents,
with values in the range 371–376 and 471–481 nm, respec-
tively. The Stokes’ shift (Δλ) was between 97 and 106 nm,
which is an advantageous property in fluorescence tech-
niques as it minimizes self-quenching phenomena. It was
found that all compounds were highly emissive in mixtures
of HEPES buffer and acetonitrile or methanol (ΦF from
0.50 to 0.94) as well as in ethanol (ΦF from 0.49 to 0.65).
Absorption maxima at longer wavelengths indicates that
photolysis could be possible with shorter irradiation times.
Therefore considering that the present heterocycle is associ-
ated with a λabs at about 370 nm, which is 25 nm batho-
chromically shifted from the previously reported derivatives
6 and 7, possessing the same heterocycle with a different
ring fusion, namely 9-methoxy-3H-naphtho[2,1-b]pyran-3-
one (Bba) (Figure 1, Table 1), it was expected that the be-
haviour of 3 and 4 towards irradiation in mixtures of aceto-
nitrile and HEPES buffer (80:20) would be improved in the
visible region compared with results obtained with 6 and
7.[19]
Scheme 1. Synthesis of 3 and 4 and photorelease of 2 and 5.
The 2-oxonaphtho[1,2-b]pyran (Bbp) moiety will be des-
ignated in this report by a three-letter code for simplicity of
naming the various fluorescent derivatives, as indicated in
Tables 1–4.
Table 1. UV/Vis absorption and emission data (λ, in nm) for amino
acid derivatives 3, 4, 6 and 7 in mixtures of acetonitrile and HEPES
buffer (80:20).
λabs
logε
λem
ΦF
Δλ
3a
3b
4a
4b
6a
6b
7a
7b
Z-Val-OBbp
Z-Phe-OBbp
H-Val-OBbp
H-Phe-OBbp
Z-Val-OBba
Z-Phe-OBba
H-Val-OBba
H-Phe-OBba
373
373
371
373
346
346
345
347
3.82
3.92
3.68
3.78
4.07
4.06
4.02
4.00
479
476
475
478
481
478
476
480
0.80
0.50
0.84
0.58
0.54
0.59
0.63
0.77
106
103
104
105
135
132
129
135
All new compounds were fully characterised by HRMS
1
1
and IR, H and 13C NMR spectroscopy. H NMR spectra
showed signals of the amino acid residues, such as α-CH (δ
= 4.24–4.80 ppm), β-CH (δ = 2.00–2.40 ppm), β-CH2 (δ =
3.10–3.30 ppm) and γ-CH3 (δ = 0.95–1.05 ppm). The meth-
ylene group bound to the heterocycle was also visible for
all derivatives (δ = 5.30–5.90 ppm). The confirmation of the
Figure 1. Structure of 6 and 7.[19]
The evaluation of 3, 4, 6 and 7 towards UV/Vis irradia-
presence of the ester bond was also supported by 13C NMR tion was carried out by exposing solutions of the com-
signals at δ = 168.43 to 171.60 ppm.
pounds in mixtures of acetonitrile/HEPES buffer (80:20)
In order to obtain the parameters necessary for the mon- and methanol/HEPES buffer (80:20) with a Rayonet RPR-
itoring of the photolysis reaction, as well as the fluorescence 100 reactor at 350 and 419 nm (Scheme 1). The course of
properties, the UV/Vis characterization was carried out in the photocleavage reaction was followed by reverse phase
mixtures of different organic solvents (acetonitrile or meth- HPLC with UV detection. The plots of peak area (A) of
anol) with HEPES buffer. The absorption and emission the starting material vs. irradiation time (tirr) were obtained
spectra of 3 and 4 in degassed mixtures of acetonitrile and for each compound at the considered wavelengths. Peak
HEPES buffer (80:20), methanol and HEPES buffer areas were determined by HPLC, which revealed a gradual
(80:20), and absolute ethanol were measured; absorption decrease with time and were the average of three runs. The
Table 2. UV/Vis absorption and emission data (λ, in nm) for 3 and 4 in mixtures of methanol and HEPES buffer (80:20) and absolute
ethanol.
Methanol/HEPES (80:20)
Ethanol
λabs
λabs
logε
λem
ΦF
Δλ
logε
λem
ΦF
Δλ
3a
3b
4a
4b
376
375
375
374
3.70
3.91
3.75
3.71
481
481
474
471
0.51
0.72
0.70
0.94
105
106
99
376
374
375
375
3.66
3.63
3.65
3.69
474
474
476
472
0.65
0.49
0.51
0.58
98
100
101
97
97
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Photolysis of Amino Acid Ester Derivatives
determined tirr represents the time necessary for the con- of the processes involved a preliminary time-resolved fluo-
sumption of the starting materials until less than 5% of rescence study was performed using both acetonitrile/
the initial area was detected (Table 3). The photochemical HEPES and methanol/HEPES mixtures. The excitation
quantum yields (ΦP) were calculated based on half-lives wavelength of 375 nm (with λem = 474 nm) was chosen to
(t1/2), molar absorptivities (ε) and the incident photon flux monitor the coumarin species present. The expected cleav-
(I0), which was determined by potassium ferrioxalate acti- age alcohol product, Bbp-OH, was found to decay monoex-
nometry.[22]
ponentially [ΦF determined as 0.45 in both ethanol and
methanol/HEPES (80:20)].[18] This allowed the estimation
of the radiative and nonradiative rate constants (kr and knr)
of 5.2ϫ107 and 6.3ϫ107 s–1, respectively, in methanol/
HEPES and 4.6ϫ107 and 9.4ϫ107 s–1 in acetonitrile/
HEPES (ΦF = 0.45 and 0.33 for the two solvent systems,
respectively).[23]
Table 3. Irradiation times (tirr, in min) and photochemical quantum
yields (ΦP, ϫ10–3) for the photolysis of 3 and 4 at 350 and 419 nm
in mixtures of acetonitrile/HEPES buffer (80:20) and methanol/
HEPES buffer (80:20).
Acetonitrile/HEPES (80:20)
350 nm
Methanol/HEPES (80:20)
419 nm
350 nm
419 nm
With the exception of 3a, which decayed biexponentially,
the sum of three exponential components was required to
fit the decay data (see Supporting Information), indicating
the presence of different coumarin species. From the life-
times alone, it was not possible to clearly ascribe decay rates
to individual species, which necessitated the measurement
tirr
ΦP
tirr
ΦP
tirr
ΦP
tirr
ΦP
3a
3b
4a
4b
86
35
35
33
0.121
0.210
0.338
0.304
77
53
23
45
0.645
0.512
2.439
0.802
59
47
231
632
0.243
0.176
0.055 1253 0.047
0.023 1153 0.042
270 0.164
148 0.158
The photolysis irradiations times at 419 nm in a mixture of time-resolved emission spectra to obtain the decay asso-
of acetonitrile and HEPES buffer (80:20) obtained for 3 ciated spectra (see Supporting Information). Comparison
and 4 in comparison with those of 6 and 7, between 598 with the steady state spectrum (measured under the same
and 1155 min, confirmed the expectations related to the dif- experimental conditions) was in accordance in terms of
ference in the absorption maxima. Quantitative release of both spectrum and lifetime, and the presence of the alcohol
2a and 2b and the corresponding free amino acids 5a and product in the methanol/HEPES mixture was seen for 4a
5b from 3 and 4, bearing a naphtho[1,2-b]pyran group, was and 4b. The other assignments are an area of continued
achieved in less than 1.3 h, whereas cleavage from 6 and investigation, but assuming that the spectrum for the ester
7 required from 10 to 20 h. At 350 nm, differences in the is likely to be at longer wavelengths and its contribution
photolysis times were not significant enough to make a to the fluorescence significant (shown by pre-exponential
clear distinction in the behaviour of both systems. Regard- value), it is possible to estimate the rate constants for the
ing irradiation of 3 and 4 at 350 nm, it was found that irra- initial bond cleavage (kc), shown in Table 4. This generally
diation times were short, 33–86 min, and comparable to shows faster rates for 4, although these results are still pre-
those obtained at 419 nm (23–77 min). However, consider- liminary, there appears to be a correlation with the photo-
ing the benefits of the use of longer wavelengths for irradia-
tion in photolytic processes, the selected irradiation wave-
length for these conjugate systems would be 419 nm.
For both irradiation wavelengths, it was found that the
presence of the N-benzyloxycarbonyl protecting group in-
fluenced the cleavage times, as the release of 5a was faster
than the corresponding N-blocked form 2a. In the case of
phenylalanine, the cleavage times were similar. As reported
previously,[14] the N-benzyloxycarbonyl group was photo-
stable under the test conditions.
Table 4. Cleavage rate constants (kc in s–1) calculated from the fluo-
rescence lifetime data in mixtures of acetonitrile/HEPES buffer
(80:20) and methanol/HEPES buffer (80:20).
Acetonitrile/
Methanol/HEPES (80:20)
HEPES(80:20)
3a
3b
4a
4b
0.25ϫ108
0.58ϫ108
5.69ϫ108
9.35ϫ108
0.53ϫ108
1.11ϫ108
4.70ϫ108
6.60ϫ108
Photolysis of 3 and 4 was also carried out in a mixture
of HEPES buffer with a protic solvent, methanol, in the
same proportion as before, in order to compare the per-
formance of the (6-methoxy-2-oxo-2H-naphtho[1,2-b]pyr-
an-4-yl)methyl ester in different solvent systems. Overall,
the sensitivity of the ester linkage between the protecting
group and the amino acid was inferior in this solvent sys-
tem at both wavelengths tested. In addition, the release of
the free form of both amino acids required much longer
irradiation periods.
Very low photochemical quantum yields were obtained
in mixtures of acetonitrile and HEPES buffer (80:20) and
methanol and HEPES buffer (80:20), indicating strong
competition between the bond scission and radiative and
Figure 2. Plot of lnA vs. irradiation time for the photolysis of 3a
(ᮀ), 3b (Δ), 4a (᭺) and 4b (᭛) at 419 nm in mixtures of acetonitrile/
nonradiative relaxation. To help elucidate the photophysics HEPES buffer (80:20).
Eur. J. Org. Chem. 2011, 5447–5451
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5449

M. S. T. Gonçalves et al
FULL PAPER
lifetimes, generally fixed from the simple decay analysis, from which
decay associated spectra were then obtained.
chemical yield data presented in Table 3, and a full photo-
physical investigation is underway.
The disappearance of the starting material can also be
illustrated for each compound, and the plot of lnA vs. irra-
diation time based on HPLC data showed a linear corre-
lation for the disappearance of the starting material, which
suggests a first-order reaction, obtained by the linear least-
squares methodology for a straight line (Figure 2).
Synthetic Procedures for 3 and 4
N-(Benzyloxycarbonyl)-L-valine (6-Methoxy-2-oxo-2H-naphtho[1,2-
b]pyran-4-yl)methyl Ester, Z-Val-OBbp (3a): 4-(Chloromethyl)-6-
methoxy-2H-naphtho[1,2-b]pyran-2-one (Bbp-Cl, 1) (0.047 g,
1.7ϫ10–4 mol) was dissolved in dry DMF (2 mL) and potassium
fluoride (0.030 g, 5.1ϫ10–4 mol) and Z-Val-OH (2a) (0.047 g,
1.9ϫ10–4 mol) were added. The reaction mixture was stirred at
room temperature for 32 h and followed by TLC (ethyl acetate/n-
hexane, 3:7). The solvent was removed by rotary evaporation under
reduced pressure and the crude residue was purified by column
chromatography with silica gel using dichloromethane/methanol
mixtures with increasing polarity as eluent, to give 3a as a yellow
solid (0.061 g, 73%); m.p. 142.2–143.6 °C. TLC (ethyl acetate/n-
hexane, 3:7): 0.53. 1H NMR (400 MHz, CDCl3, 25 °C): δ = 0.95
(d, J = 6.8 Hz, 3 H, γ-CH3 Val), 1.04 (d, J = 6.8 Hz, 3 H, γ-CH3
Val), 2.22–2.36 (m, 1 H, β-CH Val), 4.04 (s, 3 H, OCH3), 4.40–4.50
Conclusions
The synthesis of new valine and phenylalanine oxo-
naphtho[1,2-b]pyran esters was achieved in good yields
through a standard procedure from the corresponding chlo-
romethylated oxonaphtho[1,2-b]pyran. In order to obtain
the parameters necessary for monitoring the photolysis re-
action, the UV/Vis characterization was carried out in mix- (m, 1 H, α-CH Val), 5.16 (s, 2 H, CH2 Z), 5.26 (d, J = 8.8 Hz, 1
H, α-NH Val), 5.35–5.50 (m, 2 H, CH2), 6.60 (s, 1 H, 3-H), 6.65
(s, 1 H, 5-H), 7.25–7.40 (m, 5 H, 5ϫ Ar-H Z), 7.60–7.74 (m, 2 H,
8-H and 9-H), 8.25–8.34 (m, 1 H, 7-H), 8.50–8.59 (m, 1 H, 10-H)
ppm. 13C NMR (100.6 MHz, CDCl3, 25 °C): δ = 17.58 (γ-CH3
Val), 19.15 (γ-CH3 Val), 31.04 (β-CH Val), 55.89 (OCH3), 59.33 (α-
CH Val), 62.38 (CH2), 67.29 (CH2 Z), 95.27 (C-5), 112.14 (C-4a),
113.20 (C-3), 122.29 (C-7), 122.44 (C-10), 124.01 (C10a), 127.43
(C-6a), 128.00 (C-9), 128.21 (2ϫ Ar-C), 128.28 (2ϫ Ar-C), 128.53
(Ar-C), 128.55 (C-8), 136.00 (C-1 Z), 145.82 (C-10b), 148.94 (C-4),
152.38 (C-6), 156.25 (CONH), 160.59 (C-2), 171.60 (CO2CH2)
tures of acetonitrile/HEPES buffer (80:20), methanol/
HEPES buffer (80:20) and absolute ethanol. Photocleavage
studies of the ester compounds, in a mixture of acetonitrile
and HEPES buffer (80:20), at 350 and 419 nm, revealed that
the amino acid–heterocycle ester bond was readily photo-
lysed, releasing the N-protected and free amino acids. The
most interesting results were obtained at 419 nm for all
compounds in practical irradiations times (23–77 min), con-
firming the suitability of oxonaphtho[1,2-b]pyran as a
photocleavable protecting group for release at longer wave- ppm. IR (KBr, 1%): ν = 3758, 3691, 3054, 2987, 2686, 2522, 2411,
˜
2306, 1726, 1603, 1551, 1508, 1422, 1386, 1265, 1160, 1114, 1029,
896, 738, 704 cm–1. HRMS (ESI): calcd. for C28H28NO7 [M+ +H]:
490.18603; found 490.18729.
lengths, which is an interesting improvement compared to
the previously reported 3-oxo-3H-naphtho[2,1-b]pyran with
a different ring fusion. The use of time-resolved fluores-
cence techniques can further elucidate the dynamics of the
photocleavage processes of these compounds.
N-(Benzyloxycarbonyl)-L-phenylalanine
(6-Methoxy-2-oxo-2H-
naphtho[1,2-b]pyran-4-yl)methyl Ester, Z-Phe-OBbp (3b): Starting
from Bbp-Cl (1) (0.060 g, 2.2ϫ10–4 mol), DMF (2 mL), potassium
fluoride (0.040 g, 6.9ϫ10–4 mol) and Z-Phe-OH (2b) (0.076 g,
2.5ϫ10–4 mol), following the same procedure described for 3a, 3b
was obtained as an orange solid (0.115 g, 81%); m.p. 134.6–
136.1 °C. TLC (ethyl acetate/n-hexane, 3:7): 0.58. 1H NMR
(400 MHz, CDCl3, 25 °C): δ = 3.16 (d, J = 6.4 Hz, 2 H, β-CH2
Phe), 3.98 (s, 3 H, OCH3), 4.73–4.80 (m, 1 H, α-CH Phe), 5.08–
5.14 (m, 2 H, CH2 Z), 5.30 (s, 2 H, CH2), 5.38 (d, J = 8.0 Hz, 1
H, α-NH Phe), 6.39 (s, 1 H, 3-H), 6.53 (s, 1 H, 5-H), 7.10–7.32 (m,
10 H, 5ϫ Ar-H Phe and 5ϫ Ar-H Z), 7.60–7.68 (m, 2 H, 8-H and
9-H), 8.20–8.26 (m, 1 H, 7-H), 8.38–8.50 (m, 1 H, 10-H) ppm. 13C
NMR (100.6 MHz, CDCl3, 25 °C): δ = 36.40 (β-CH2 Phe), 55.16
(α-CH Phe), 55.81 (OCH3), 62.35 (CH2), 67.15 (CH2 Z), 95.26 (C-
5), 112.05 (C-4a), 113.14 (C-3), 122.20 (C-7), 122.27 (C-10), 123.85
(C-10a), 127.28 (C-6a), 127.37 (Ar-C), 127.87 (Ar-C), 128.09 (2ϫ
Ar-C), 128.19 (2ϫ Ar-C), 128.39 (C-9), 128.46 (2ϫ Ar-C), 128.71
(2ϫ Ar-C), 129.01 (C-8), 135.13 (C-1 Phe), 135.95 (C-1 Z), 145.63
(C-10b), 148.53 (C-4), 152.20 (C-6), 160.41 (C-2), 162.50 (CONH),
Experimental Section
General: All melting points were measured with a Stuart SMP3
melting point apparatus. TLC analyses were carried out with
0.25 mm thick precoated silica plates (Merck Fertigplatten Kiesel-
gel 60F254) and spots were visualised under UV light. Chromatog-
raphy on silica gel was carried out on Merck Kieselgel (230–
240 mesh). IR spectra were determined with a BOMEM MB 104
spectrophotometer. UV/Vis absorption spectra (200–700 nm) were
obtained using a Shimadzu UV/2501PC spectrophotometer. NMR
spectra were obtained with a Bruker Avance III 400 at an operating
frequency of 400 MHz for 1H NMR and 100.6 MHz for 13C NMR
using the solvent peak as internal reference at 25 °C. All chemical
shifts are given in ppm using δ(Me4Si) = 0 ppm as reference and J
values are given in Hz. Assignments were made by comparison of
chemical shifts, peak multiplicities and J values and were supported
by spin decoupling–double resonance and bidimensional hetero-
nuclear correlation techniques. HRMS analyses were performed at
the C.A.C.T.I. – Unidad de Espectrometria de Masas, at University
of Vigo, Spain. Time-resolved fluorescence measurements were per-
formed using a HORIBA Scientific UltraFast-01 system equipped
with a DeltaDiode (DD-375L) excitation source running at 8 MHz,
171.15 (CO CH ) ppm. IR (KBr, 1%): ν = 3759, 3691, 3583, 3054,
˜
2
2
2987, 2686, 2522, 2411, 2306, 1725, 1677, 1602, 1551, 1507, 1422,
1386, 1265, 1163, 1113, 1086, 1029, 896, 740, 705 cm–1. HRMS
(ESI): calcd. for C32H28NO7 [M+ +H]: 538.18603; found
538.18639.
L-Valine (6-Methoxy-2-oxo-2H-naphtho[1,2-b]pyran-4-yl)methyl Es-
emitting at 374 nm and a microchannel plate detector. Data were ter Hydrobromide, HBr·H-Val-OBbp (4a): A 45% solution of hy-
analysed using DAS6 software, with the TRES (time-resolved emis-
sion spectra) fitting using a global analysis module linking common
drobromic acid in acetic acid (162 μL) and acetic acid (1 mL) were
added to 3a with stirring at room temperature. The reaction mix-
5450
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Eur. J. Org. Chem. 2011, 5447–5451

Photolysis of Amino Acid Ester Derivatives
ture was maintained in these conditions for 11 h, and the process
Supporting Information (see footnote on the first page of this arti-
was followed by TLC (ethyl acetate/n-hexane, 3:7). During this
cle): Time-resolved fluorescence measurements (decay curves and
time, additional amounts of 45% solution of hydrobromic acid in analysis) and rate-constant calculations.
acetic acid were added (total volume 972 μL). When the reaction
was completed, cold diethyl ether was added (0.5 mL), and the pre-
cipitate collected by filtration and washed with the same solvent to
Acknowledgments
give 4a as a light brown solid (0.021 g, 79%); m.p. 205.5–207.1 °C.
Thanks are due to the Portuguese Fundação para a Ciência e Tec-
TLC (ethyl acetate/methanol, 1:1): 0.53. 1H NMR (400 MHz, [D6]-
DMSO, 25 °C): δ = 0.98–1.05 (m, 6 H, 2ϫ γ-CH3 Val), 2.00–2.40
(m, 1 H, β-CH Val), 4.06 (s, 3 H, OCH3), 4.24 (br. s, 1 H, α-CH
Val), 5.60–5.90 (m, 2 H, CH2), 6.72 (s, 1 H, 3-H), 7.03 (s, 1 H, 5-
H), 7.70–7.80 (m, 2 H, 8-H and 9-H), 8.20–8.26 (m, 1 H, 7-H),
8.30–8.40 (m, 1 H, 10-H), 8.44 (br. s, 3 H, +NH3) ppm. 13C NMR
(100.6 MHz, [D6]DMSO, 25 °C): δ = 17.65 (γ-CH3 Val), 18.16 (γ-
CH3 Val), 29.52 (β-CH Val), 56.18 (OCH3), 57.33 (α-CH Val),
63.08 (CH2), 97.50 (C-5), 112.09 (C-3), 112.27 (C-4a), 121.66 (C-
10), 122.00 (C-7), 122.97 (C-10a), 126.48 (C-6a), 128.22 (C-8),
128.69 (C-9), 144.56 (C-10b), 150.08 (C-4), 151.26 (C-6), 159.63 (C-
nologia (FCT) for financial support through project PTDC/QUI/
69607/2006 (FCOMP-01-0124-FEDER-007449) and a Ph.D. grant
to A. M. P. (SFRH/BD/61459/2009). The NMR spectrometer
Bruker Avance III 400 is part of the National NMR Network and
was purchased in the framework of the National Program for Sci-
entific Re-equipment, under contract REDE/1517/RMN/2005,
with funds from the Fundo Europeu de Desenvolvimento Regional
(FEDER) (POCI 2010) and from FCT.
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2), 168.43 (CO CH ) ppm. IR (KBr, 1%): ν = 3450, 3072, 3009,
˜
2
2
2944, 2883, 1764, 1662, 1386, 1222, 1054, 1008, 823, 760 cm–1.
HRMS (ESI): calcd. for C20H22NO5 [M+ +H]: 356.14925; found
356.14906.
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L-Phenylalanine (6-Methoxy-2-oxo-2H-naphtho[1,2-b]pyran-4-yl)-
methyl Ester Hydrobromide, HBr·H-Phe-OBbp (4b): Starting from
3b (0.027 g, 5.0ϫ10–5 mol) and using 45% solution of hydrobromic
acid in acetic acid (1.2 mL), 4b was obtained as a brown solid
(0.017 g, 70%); m.p. 216.0–218.2 °C. TLC (ethyl acetate/methanol,
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1
1:1): 0.61. H NMR (400 MHz, [D6]DMSO, 25 °C): δ = 3.10–3.30
(m, 2 H, β-CH2 Phe), 4.05 (s, 3 H, OCH3), 4.62 (br. s, 1 H, α-CH
Phe), 5.50–5.70 (m, 2 H, CH2), 6.50 (s, 1 H, 3-H), 6.96 (s, 1 H, 5-
H), 7.10–7.30 (m, 5 H, 5ϫ Ar-H Phe), 7.70–7.90 (m, 2 H, 8-H and
9-H), 8.19–8.25 (m, 1 H, 7-H), 8.31–8.39 (m, 1 H, 10-H), 8.55 (br.
s, 3 H, +NH3) ppm. 13C NMR (100.6 MHz, [D6]DMSO, 25 °C): δ
= 36.07 (β-CH2 Phe), 53.25 (α-CH Phe), 56.20 (OCH3), 62.97
(CH2), 97.47 (C-5), 112.01 (C-4a), 112.21 (C-3), 121.63 (C-7),
121.99 (C-10), 122.96 (C-6a), 126.46 (C-10a), 127.37 (C-8 and Ar-
C Phe), 128.21 (C-9), 128.62 (2ϫ Ar-C Phe), 129.34 (2ϫ Ar-C
Phe), 134.38 (C-1 Phe), 144.50 (C-10b), 149.71 (C-4), 151.24 (C-6),
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159.54 (C-2), 168.48 (CO CH ) ppm. IR (KBr, 1%): ν = 3446,
˜
2
2
3073, 3010, 2970, 2882, 1765, 1663, 1383, 1222, 1031, 881, 823, 761
cm–1. HRMS (ESI): calcd. for C24H21NO5 [M+ +H]: 404.14925;
found 404.14882.
Photolysis: Photolysis was carried out using a Rayonet RPR-100
chamber reactor equipped with 10 lamps of 350 and 419Ϯ10 nm.
HPLC analyses were performed using a Licrospher 100 RP18
(5 μm) column with a JASCO HPLC system composed of a PU-
2080 pump and a UV-2070 detector with ChromNav software.
General Photolysis Procedure: A 1ϫ10–4 m solution of 3, 4, 6 and
7 in mixtures of acetonitrile/HEPES buffer (80:20, 5 mL), and 3
and 4 in methanol/HEPES buffer (80:20) was placed in a quartz
tube and irradiated in the reactor at the desired wavelength.
HEPES buffer solution was prepared in distilled water with HEPES
(10 mm), NaCl (120 mm), KCl (3 mm), CaCl2 (1 mm) and MgCl2
(1 mm) and pH adjusted to 7.2. Aliquots of 100 μL were taken at
regular intervals and analysed by RP-HPLC. The eluent was aceto-
nitrile/water (3:1) at a flow rate of 1 mL/min (retention time: 3a
7.6, 3b 8.3, 4a 10.9, 4b 8.2 min) or 0.8 mL/min (6a 7.1, 6b 7.4, 7a
and 7b 8.0 min), previously filtered through a Millipore, type HN
0.45 μm filter and degassed by ultrasound for 30 min. The chroma-
tograms were traced by detecting UV absorption at the wavelength
of maximum absorption for each conjugate.
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Received: March 21, 2011
Published Online: August 9, 2011
Eur. J. Org. Chem. 2011, 5447–5451
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5451