Synthesis and light triggered release of catecholamines from pyrenylmethyl carbamate cages

Article abstract of DOI:10.1039/c3nj00247k

A series of catecholamines (dopamine, norepinephrine, tyramine and octopamine) and their corresponding amino acid precursors in the biosynthetic pathway, l-phenylalanine, l-tyrosine and l-3,4-dihydroxyphenylalanine (DOPA), were reacted with 1-hydroxymethylpyrene in the presence of N,N′- carbonyldiimidazole (CDI). Photolysis studies on the corresponding carbamate conjugates were carried out under irradiation at different wavelengths (250, 300, and 350 nm), followed by HPLC/UV and ¹H NMR, with the aim of evaluating the applicability of the N-pyrenylmethoxycarbonyl group as a photolabile protecting group for the amino function of neuroactive amines and amino acids. It was found that the carbamate bond between the catecholamine and the pyrenylmethoxycarbonyl unit cleaved readily with the results obtained by irradiation at 350 nm being promising for practical applications.

Full text of DOI:10.1039/c3nj00247k

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Synthesis and light triggered release of catecholamines
from pyrenylmethyl carbamate cages
Cite this: NewJ.Chem., 2013,
37, 2369
´
Maria Jose G. Fernandes, Susana P. G. Costa and M. Sameiro T. Gonçalves*
A
series of catecholamines (dopamine, norepinephrine, tyramine and octopamine) and their
corresponding amino acid precursors in the biosynthetic pathway, L-phenylalanine, L-tyrosine and
L-3,4-dihydroxyphenylalanine (DOPA), were reacted with 1-hydroxymethylpyrene in the presence of
N,N0-carbonyldiimidazole (CDI). Photolysis studies on the corresponding carbamate conjugates were
carried out under irradiation at different wavelengths (250, 300, and 350 nm), followed by HPLC/UV
and 1H NMR, with the aim of evaluating the applicability of the N-pyrenylmethoxycarbonyl group as a
photolabile protecting group for the amino function of neuroactive amines and amino acids. It
was found that the carbamate bond between the catecholamine and the pyrenylmethoxycarbonyl
unit cleaved readily with the results obtained by irradiation at 350 nm being promising for practical
applications.
Received (in Montpellier, France)
6th March 2013,
Accepted 24th April 2013
DOI: 10.1039/c3nj00247k
www.rsc.org/njc
for alcohols,12 carboxylic acids,13 phosphates14 and amines.15
Pyrene has been used as an environment-sensitive fluorophore13
Introduction
Biogenic amines are involved in many functions at the central
nervous system such as the control of motor function, endocrine
secretion, appetite, mechanisms associated with cognition, emotion,
mood, various psychiatric and neurological disorders, including
depression, attention deficit hyperactivity disorder, schizophrenia,
anxiety, and drug addiction.1–8 Dopamine, norepinephrine
(noradrenaline), and epinephrine (adrenaline) are examples
of catecholamines, produced endogenously from a specific
amino acid, tyrosine. In the biosynthetic pathway of epinephrine,
tyramine and octopamine are intermediate amines.
In order to study fast neurophysiological processes, the use
of light triggering to photorelease caged molecules offers
several advantages, such as temporal and spatial resolution,
control of chemical identity (upon photolysis, the active molecule
is rapidly released in situ, not being subject to degradation, as for
example catecholamines which are very sensitive to oxidation by
air) and biological specificity.9 There have been reports on several
caged derivatives of epinephrine that were photolyzed in the
microsecond to the millisecond time region.10
and due to its strong absorption at wavelengths above 300 nm,
where irradiation is less damaging to biological entities, it has
also been applied in the photoinduced DNA cleavage activity and
photocytotoxicity by incorporation into pyrenyl–terpyridine
lanthanide complexes,16 in addition to its use in organic synthetic
methodologies.
Considering the above facts, our current research interests
include the design of new oxygen and nitrogen heterocycles, as
well as polycyclic aromatics, and their application as photocleavable
protecting groups for the amino and carboxylic functions.17–24 Now
we investigate the use of a N-pyrenylmethoxycarbonyl group in the
derivatisation of the amino function of relevant catecholamines
(dopamine, norepinephrine, tyramine and octopamine) and
their amino acid precursors (phenylalanine, tyrosine and
DOPA) in the biosynthetic pathway, cleaved under photolytic
conditions. Although this protecting group was previously
applied to the amino function of certain amino acids,15 this
is the first time that it is used with relevant catecholamines and
related hydroxylated amino acids.
Therefore, the search for new and more efficient photolabile
protecting groups has grown in recent years and found numerous
applications.11 Within the reported examples is the use of poly-
cyclic aromatics as photochemically removable protecting groups
Thus the release of the active amines was achieved by
irradiation at different wavelengths in a methanol–HEPES
buffer solution (80 : 20). Furthermore, in addition to being a
photocleavable protecting group and considering the fluores-
cence properties of the pyrene moiety, this group could also act
as a temporary fluorescent reporter. The linkage of neuroactive
molecules to a fluorescent moiety like pyrene is advantageous
as it allows easier visualisation and isolation of non-fluorescent
Centre of Chemistry, University of Minho, Campus of Gualtar, 4710-057 Braga,
Portugal. E-mail: msameiro@quimica.uminho.pt; Fax: +351-253604382;
Tel: +351-253604372
c
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Table 1 Yields and UV/visible data for caged catecholamines 3a–d, their amino
acid precursors 5a–c and 6a,b in absolute ethanol and methanol–HEPES buffer
(80 : 20) solution
systems whenever necessary during the course of organic reac-
tions or in photorelease applications.
MeOH–HEPES
(80 : 20)
Results and discussion
EtOH
Synthesis
labs (nm) log e
l
abs (nm) log e
Compound
Yield (%)
19
1-Hydroxymethylpyrene 1 was reacted with the amino function of
several catecholamines 2a–d, namely dopamine, norepinephrine,
tyramine and octopamine, in the presence of N,N0-carbonyldiimi-
dazole (CDI) as the carbonylating agent in DMF at room tempera-
ture. The corresponding N-pyrenylmethoxycarbonyl caged amines
3a–d with a carbamate bond between the fluorophore and the
active molecule were obtained in fair yields and fully characterised
by the usual spectroscopic techniques (Scheme 1, Table 1). The
N-pyrenylmethoxycarbonyl group will be designated in this report
by a four letter code (Pmoc) for simplicity of naming the various
compounds.
For the corresponding amino acid precursors in the biosyn-
thetic pathway, L-phenylalanine, L-tyrosine and L-3,4-dihydroxy-
phenylalanine (DOPA) 4a–c, the same coupling reaction was
performed to yield the corresponding amino acid carbamate
cages 5a–c. The ester group in compounds 5a,b was removed by
basic hydrolysis yielding the C-deprotected tagged amino acids
6a,b (Scheme 2, Table 1).
3a Pmoc-dopamine
3b Pmoc-norepinephrine 23
341
341
341
341
341
341
341
341
341
4.61 340
4.63 340
4.53 340
4.59 341
4.37 341
4.89 341
4.64 340
4.64 341
4.63 341
4.47
4.45
4.45
4.56
4.52
4.19
4.47
4.47
4.51
3c Pmoc-tyramine
3d Pmoc-octopamine
5a Pmoc-Phe-OMe
5b Pmoc-Tyr-OMe
5c Pmoc-DOPA-OMe
6a Pmoc-Phe-OH
6b Pmoc-Tyr-OH
32
36
15
20
40
74
90
The IR spectra of compounds 3a–d, 5a–c and 6a,b showed
bands due to stretching vibrations of the carbamate carbonyl
group between 1673–1693 cmꢀ1 and of the NH between 3296–
3360 cmꢀ1. H NMR spectra showed signals for the carbamate
1
NH at d 7.22–7.82 ppm and the pyrenyl methylene group
between d 5.62 and 5.87 ppm, as well as the a-CH (d 3.79 to
4.76 ppm) and the b-CH2 (d 2.66 to 3.19 ppm) for the amino
acid residues in compounds 5a–c and 6a,b. The confirmation of
the presence of the newly formed carbamate bond was also
Scheme 1 Synthesis of carbamate cages of catecholamines 3a–d.
c
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Scheme 2 Synthesis of carbamate cages of catecholamine amino acid precursors 5a–c and 6a,b.
supported by 13C NMR spectra signals of the carbonyl group, Absorption spectra showed a strong band centred at 341 nm and the
which were found between d 155.02–156.28 ppm. characteristic pyrene fine vibronic structure. The structure of the
Pyrene, widely known for its fluorescence, is a fluorogenic amines/amino acids did not influence the position of the wave-
reagent when used for the derivatisation of non-fluorescent length of maximum absorption and emission or the shape of
molecules, so the characterisation of the absorption and emis- the absorption and emission bands of compounds 3a–d, 5a–c
sion properties of the synthesised cages 3a–d, 5a–c and 6a,b and 6a,b. The wavelength of maximum emission was at about
was carried out in absolute ethanol and methanol–HEPES 375 nm for all compounds.
buffer (80 : 20) solution. The electronic absorption and emis-
sion spectra of 10ꢀ6 M solutions of the compounds were
Photolysis studies of catecholamines 3a–d, amino acids 5a–c
and 6a,b
measured: absorption (labs) and emission (lem) maxima, molar
absorption coefficients (e) and fluorescence quantum yields The purpose of this work was to assess the applicability of the
(FF) are reported in Tables 1 and 2. The FF was calculated N-pyrenylmethoxycarbonyl group as a photolabile protecting
using 9,10-diphenylanthracene as standard (FF = 0.95 in group for the amino function of neuroactive amines and amino
ethanol).25 The maximum absorption wavelength and the shape acids. As a result, the photostability of the carbamate bond
of the absorption curve were similar for all compounds, which between the pyrenyl moiety and the amines/amino acids was
was due to the presence of the pyrene moiety in the structures. studied at different wavelengths having in mind the development
c
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Table 2 Fluorescence data for caged catecholamines 3a–d, their amino acid precursors 5a–c and 6a,b in absolute ethanol and methanol–HEPES buffer (80 : 20)
solution
EtOH
MeOH–HEPES buffer (80 : 20)
lem (nm)
FF
Stokes’ shift (nm)
lem (nm)
FF
Stokes’ shift (nm)
Compound
3a Pmoc-dopamine
3b Pmoc-norepinephrine
3c Pmoc-tyramine
3d Pmoc-octopamine
5a Pmoc-Phe-OMe
5b Pmoc-Tyr-OMe
5c Pmoc-DOPA-OMe
6a Pmoc-Phe-OH
375
375
375
374
375
375
375
376
376
0.06
0.07
0.13
0.08
0.10
0.11
0.06
0.13
0.13
34
34
34
33
34
34
34
35
35
375
375
375
376
375
375
375
376
376
0.06
0.07
0.11
0.17
0.15
0.12
0.10
0.13
0.10
34
34
34
35
34
34
34
35
35
6b Pmoc-Tyr-OH
of photocleavable protecting groups that could be useful for that the presence of a second hydroxyl substituent at the phenyl
biological studies. Photolysis experiments were carried out by ring had a positive effect as the cleavage of the carbamate bond
irradiating 1 ꢁ 10ꢀ4 M solutions of carbamate cages 3a–d, 5a–c occurred more efficiently for compounds 3a (dopamine) and 3b
and 6a,b in methanol–HEPES buffer solution (80 : 20) using a (norepinephrine) when compared to compounds 3c (tyramine)
Rayonet RPR-100 reactor at 254, 300, and 350 nm. The solvent and 3d (octopamine), respectively. The same observation could
system was chosen based on previous studies regarding different be made for amino acids 5a–c (the DOPA derivative 5c was the
organic solvents with varying aqueous content, which revealed fastest to cleave), and 6a,b (the tyrosine derivative 6b had a
that photocleavage was more efficient in a mixture of methanol shorter irradiation time) at all wavelengths of irradiation.
and HEPES buffer solution.17 The course of the photocleavage
The effect of a hydroxyl group at the acyclic chain at the
reaction was followed by reverse phase HPLC with UV detection carbon next to the phenyl ring was less marked as for com-
and 1H NMR. The determined irradiation time represents the pounds 3a and 3b the irradiation times were very similar for all
time necessary for the consumption of the starting materials wavelengths of irradiation. For compounds 3c and 3d, the
until less than 5% of the initial peak area (A) was detected difference was larger as the latter compound displayed longer
(Table 3). Based on HPLC data, the plot of ln A versus irradiation irradiation times at 300 and 350 nm (at about 2.6 times longer).
time showed a linear correlation for the disappearance of
For biological applications, it is desirable the use of longer
the starting material, obtained using the linear least squares wavelengths of irradiation to minimise side reactions for the
methodology for a straight line, with high correlation coeffi- remaining functionalities of the active molecule and of the
cients. The photochemical quantum yields (FPhot) were calcu- biological media. From the data in Table 3 it could be seen that
lated based on half-lives (t1/2), molar absorption coefficients (e) for all compounds (except for 6a,b) the release of the active
and the incident photon flux (I0), which was determined using molecule required less time by irradiation at 300 nm, when
potassium ferrioxalate actinometry.26 The photocleavage process compared to the values obtained at 254 nm. At 350 nm,
was not as efficient as desirable (Table 3) (FPhot o 0.1), possibly the irradiation times for all compounds were longer (between
by deactivation via fluorescence pathways that compete with the 17 and 278 min) but still practicable with the exception of
photochemical reaction. Nevertheless, our results are generally compound 6a (more than 800 min). The obtained results for the
in accordance with the low photochemical quantum yields release of amines/amino acids from N-pyrenylmethoxycarbonyl
(o0.005) that have been reported for the pyrenylmethyl group.11 derivatives at 350 nm are feasible for organic synthesis and,
Taking into consideration the influence of the structure of depending on the biomolecule, might be also interesting for
the active molecule on the photocleavage rates, it was found biological applications.
Table 3 Irradiation times (tirr in min), rate constants (k ꢁ 10ꢀ2 minꢀ1) and photochemical quantum yields (FPhot) for the photolysis of caged catecholamines 3a–d,
their amino acid precursors 5a–c and 6a,b at different wavelengths in methanol–HEPES buffer (80 : 20) solution
254 nm
300 nm
350 nm
tirr
ka
FPhot
tirr
ka
FPhot
tirr
ka
FPhot
Compound
3a
3b
3c
3d
5a
5b
5c
6a
6b
Pmoc-dopamine
Pmoc-norepinephrine
Pmoc-tyramine
Pmoc-octopamine
Pmoc-Phe-OMe
Pmoc-Tyr-OMe
Pmoc-DOPA-OMe
Pmoc-Phe-OH
Pmoc-Tyr-OH
8
12
32
34
39
41
11
37
12
0.39
0.25
0.10
0.09
0.08
0.07
0.28
0.08
0.25
0.060
0.025
0.001
0.018
0.009
0.003
0.025
0.006
0.016
5
5
13
34
23
7
5
103
18
0.69
0.61
0.24
0.09
0.14
0.43
0.01
0.03
0.16
0.058
0.060
0.003
0.020
0.019
0.009
0.058
0.003
0.008
34
33
106
278
230
115
17
0.09
0.09
0.03
0.01
0.01
0.03
0.19
0.01
0.02
0.029
0.080
0.001
0.003
0.003
0.001
0.090
0.001
0.003
838
184
a
Initial values.
c
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of N,N0-carbonyldiimidazole to yield the corresponding N-pyrenyl-
methoxycarbonyl caged compounds. The assessment of the
suitability of the pyrenylmethoxycarbonyl group as a photolabile
protecting group for the amino function of neuroactive amines
and amino acids was carried out by photolysis studies under
irradiation at different wavelengths (250, 300 and 350 nm),
followed by HPLC/UV and 1H NMR. It was found that the
carbamate bond between the active amine and the pyrenyl-
methyl unit cleaved readily with the results obtained by irradia-
tion at 300 and 350 nm being the most promising.
Overall, the N-pyrenylmethoxycarbonyl group can be considered
a suitable photoactive group for the release of active molecules
bearing an amino group, for organic synthesis and eventually in
caging applications.
Experimental
Materials
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm
thick precoated silica plates (Merck Fertigplatten Kieselgel
60F254) and spots were visualised under UV light. Chromato-
graphy on silica gel was carried out on Merck Kieselgel
(230–240 mesh). IR spectra were determined on a BOMEM
MB 104 spectrophotometer using KBr discs. Absorption spectra
(200–700 nm) were obtained using a Shimadzu UV/2501PC
spectrophotometer. NMR spectra were obtained on a Bruker
Avance III 400 at an operating frequency of 400 MHz for 1H and
100.6 MHz for 13C using the solvent peak as internal reference
at 25 1C. All chemical shifts are given in ppm using dH Me4Si =
0 ppm as reference and J values are given in Hz. Assignments
were made by comparison of chemical shifts, peak multipli-
cities and J values and were supported by spin decoupling-
double resonance and bidimensional heteronuclear correlation
techniques. Mass spectrometry analyses were performed at
the ‘‘C.A.C.T.I. – Unidad de Espectrometria de Masas’’, at
University of Vigo, Spain. Fluorescence spectra were collected
using a FluoroMax-4 spectrofluorometer. Photolyses were car-
ried out using a Rayonet RPR-100 chamber reactor equipped
with 10 lamps of 254 (35 W), 300 (21 W) and 350 (24 W) nm.
HPLC analyses were performed using a Licrospher 100 RP18
(5 mm) column in a HPLC system composed of a Jasco PU-980
pump, a UV/Vis Shimadzu SPD-GAV detector and a Shimadzu
C-RGA Chromatopac register. All reagents were used as
received. The pyrene ring will be designated by a two letter
code (Py) in the NMR data.
Fig. 1 1H NMR spectra in methanol-d4–D2O (80 : 20) of the photolysis at
300 nm of caged norepinephrine 3b: (a) before irradiation; (b) after irradiation
for 60 min; (c) after irradiation for 120 min; (d) sample of free norepinephrine.
Additionally to monitoring the photolysis process by HPLC/
UV detection, the release of the neuroactive amine was also
followed by 1H NMR in a methanol-d4–D2O (80 : 20) solution.
Upon irradiation at 300 nm of a solution of norepinephrine
cage 3b, the signals due to the Pmoc CH2 group, visible at
about d 5.80 ppm, and the CH2–CH of norepinephrine in the
conjugate form at about d 3.30 and 4.60 ppm, respectively,
gradually decreased with time, and a close set of signals
appeared corresponding to norepinephrine in its free form
at about d 2.90 and 4.70 ppm, respectively. Signals due to
by-products related to the Pmoc group were also visible in the
spectra, namely the two singlets at d 5.20 and 5.40 ppm, related
to nucleophilic attack of solvent molecules at the intermediate
methylene carbocation. NMR monitoring was carried out with a
1 ꢁ 10ꢀ2 M solution, which led to an expected increase in the
photolysis time for the complete release of the molecule, when
compared to the irradiation times in Table 3 obtained with
dilute solutions (Fig. 1).
General procedure for the synthesis of carbamate cages 3a–d
and 5a–c
Conclusions
A series of carbamate derivatives of relevant catecholamines To a solution of N,N0-carbonyldiimidazole (CDI) (1 equiv.) in
(dopamine, norepinephrine, tyramine and octopamine) and dry DMF (1 mL) at 0 1C, 1-hydroxymethylpyrene 1 (1 equiv.)
their corresponding amino acid precursors in the biosynthetic dissolved in dry DMF (3 mL) was slowly added and the reaction
pathway, L-phenylalanine, L-tyrosine and L-3,4-dihydroxyphenyl- mixture was stirred at room temperature for 1 hour. After
alanine (DOPA), were obtained in fair yields by reaction of the addition of the corresponding catecholamines 2a–d or their
amines/amino acids with 1-hydroxymethylpyrene in the presence amino acid precursors 4a–c (1 equiv.) the mixture was kept stirring
c
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for 12 hours. The solvent was evaporated and purification was 238 (4.56). lmax (EtOH)/nm 341 (loge 4.53), 325 (4.40), 275 (4.56),
done by column chromatography, using chloroform as eluent. 264 (4.36) and 240 (4.76). nmax/cmꢀ1 3331, 3042, 2924, 1693, 1614,
N-[(Pyren-1-yl)methoxycarbonyl]-2-(30,40-dihydroxy-10-phenyl)- 1596, 1515, 1446, 1417, 1363, 1245, 1185, 1173, 1137, 1107, 1051.
ethylamine, 3a. Starting from 1-hydroxymethylpyrene 1 (0.122 g, dH (400 MHz; DMSO-d6) 2.61 (2H, t, J 7.2 Hz, CH2 a), 3.15–3.20
0.53 mmol) and 2-(30,40-dihydroxy-10-phenyl)ethylamine (dopamine) (2H, m, CH2 b), 5.75 (2H, s, CH2 Pmoc), 6.66 (2H, d, J 8.4 Hz, 30-H
2a (0.100 g, 0.53 mmol), compound 3a was obtained as a dark and 50-H), 6.98 (2H, d, J 8.4 Hz, H-20 and 60-H), 7.34 (1H, t, J 5.6
yellow solid (0.041 g, 19%), mp 114.5–115.8 1C, lmax (MeOH–HEPES Hz, NH), 8.07–8.11 (2H, m, Py-H), 8.16–8.21 (2H, m, 2H, Py-H),
80 : 20)/nm 340 (log e 4.47), 325 (4.37), 273 (4.41), 264 (4.31), 238 8.26–8.34 (5H, m, Py-H), 9.16 (1H, s, OH). dC (400 MHz; DMSO-d6)
(4.61) and 204 (4.41). lmax (EtOH)/nm 341 (log e 4.61), 325 (4.46), 274 34.58 (CH2 a), 42.28 (CH2 b), 63.54 (CH2 Pmoc), 115.05 (30-C and
(4.98) and 240 (4.93). nmax/cmꢀ1 3360, 3043, 1688, 1651, 1604, 1548, 50-C), 123.15 (Py-C), 123.76 (Py-C), 123.90 (Py-C), 124.65 (Py-C),
1519, 1444, 1359, 1283, 1256, 1194, 1113, 1066, 979, 838, 819, 754, 125.39 (Py-C), 125.47 (Py-C), 126.34 (Py-C), 127.31 (2 ꢁ Py-C),
712. dH (400 MHz; DMSO-d6) 2.54 (2H, t, J 7.2 Hz, CH2 a), 3.13–3.19 127.49 (Py-C), 127.85 (Py-C), 128.60 (Py-C), 129.27 (10-C), 129.47
(2H, m, CH2 b), 5.75 (2H, s, CH2 Pmoc), 6.43 (1H, dd, J 8.4 and 2.0 (20-C and 60-C), 130.20 (Py-C), 130.48 (Py-C), 130.70 (Py-C), 130.79
Hz, 60-H), 6.59 (1H, d, J 2.0 Hz, 20-H), 6.62 (1H, d, J 7.6 Hz, 50-H), 7.33 (Py-C), 155.58 (40-C), 156.05 (CQO). m/z 418 ([M+ + Na], 14%), 215
(1H, t, J 5.6 Hz, NH), 8.07–8.11 (2H, m, 2 ꢁ Py-H), 8.15–8.20 (2H, m, (100). HRMS calc. for C26H21NNaO3 [M+ + Na]: 418.14136; found:
2 ꢁ Py-H), 8.26–8.34 (5H, m, 5 ꢁ Py-H), 8.64 (1H, s, OH), 8.74 (1H, s, 418.14161.
OH). dC (400 MHz; DMSO-d6) 34.93 (CH2 a), 42.35 (CH2 b), 63.57
N-[(Pyren-1-yl)methoxycarbonyl]-2-(40-hydroxy-10-phenyl)-2-
(CH2 Pmoc), 115.45 (50-C), 115.98 (20-C), 119.23 (60-C), 123.15 (Py-C), hydroxyethylamine, 3d. Starting from 1-hydroxymethylpyrene
123.78 (Py-C), 123.91 (Py-C), 124.68 (Py-C), 125.41 (Py-C), 125.48 1 (0.061 g, 0.26 mmol) and 2-(40-hydroxy-10-phenyl)-2-hydroxy-
(Py-C), 126.35 (Py-C), 127.31 (Py-C), 127.33 (Py-C), 127.50 (Py-C), ethylamine (octopamine) 2d (0.050 g, 0.26 mmol), compound 3d
127.88 (Py-C), 128.60 (Py-C), 130.00 (10-C), 130.22 (Py-C), 130.49 was obtained as a brown solid (0.039 g, 36%). mp 155.3–157.2 1C.
(Py-C), 130.71 (Py-C), 130.79 (Py-C), 143.51 (40-C), 145.04 (30-C), lmax (MeOH–HEPES 80 : 20)/nm 341 (log e 4.56), 325 (4.40), 274
156.07 (CQO). m/z 434 ([M+ + Na], 45%), 359 (43), 288 (44), 227 (4.56), 264 (4.36) and 240 (4.78). lmax (EtOH)/nm 341 (log e 4.59),
(100), 215 (29), 201 (67). HRMS: calc. for C26H21NNaO4 [M+ + Na]: 325 (4.39), 312 (3.92), 275 (4.61), 264 (4.29) and 241 (4.79). nmax
434.13628; found: 434.13601.
cmꢀ1 3296, 3038, 1683, 1614, 1595, 1537, 1515, 1455, 1363, 1328,
/
N-[(Pyren-1-yl)methoxycarbonyl]-2-(30,40-dihydroxy-10-phenyl)- 1265, 1171, 1140, 1108, 1064, 1037, 972, 960, 897, 847. dH
2-hydroxyethylamine, 3b. Starting from 1-hydroxymethylpyrene (400 MHz; DMSO-d6) 3.08–3.19 (2H, m, CH2), 4.50–4.54 (1H, m,
1 (0.113 g, 0.49 mmol) and 2-(30,40-dihydroxy-10-phenyl)-2-hydro- CHOH), 5.20 (1H, d, J 4.4 Hz, CHOH), 5.70–5.78 (2H, m, CH2
xyethylamine (norepinephrine) 2b (0.100 g, 0.49 mmol), com- Pmoc), 6.69 (2H, d, J 8.4 Hz, 20-H and 60-H), 7.11 (2H, d, J 8.4 Hz,
pound 3b was obtained as a dark yellow solid (0.047 g, 23%). mp 2H, 30-H and 50-H), 7.23 (1H, t, J 6.0 Hz, NH), 8.07–8.09 (2H, m,
135.9–137.0 1C. lmax (MeOH–HEPES 80 : 20)/nm 340 (loge 4.45), Py-H), 8.16–8.21 (2H, m, Py-H), 8.26–8.34 (5H, m, Py-H), 9.26 (1H,
325 (4.35), 274 (4.39), 264 (4.29), 238 (4.59) and 204 (4.39). lmax s, OH). dC (400 MHz; DMSO-d6) 48.57 (CH2), 63.62 (CH2 Pmoc),
(EtOH)/nm 341 (loge 4.63), 325 (4.53), 275 (4.70), 264 (4.53) and 71.06 (CHOH), 114.74 (30-C and 50-C), 123.18 (Py-C), 123.79 (Py-C),
240 (4.88). nmax/cmꢀ1 3461, 3339, 3041, 1673, 1611, 1545, 1526, 123.91 (Py-C), 124.68 (Py-C), 125.42 (Py-C), 125.50 (Py-C), 126.37
1439, 1361, 1334, 1284, 1249, 1186, 1146, 1112, 1079, 1065, 973, (Py-C), 127.20 (20-C and 60-C), 127.26 (Py-C), 127.35 (Py-C), 127.51
846, 819, 755, 711. dH (400 MHz; DMSO-d6) 3.02–3.17 (2H, m, (Py-C), 127.88 (Py-C), 128.58 (Py-C), 130.23 (Py-C), 130.49 (Py-C),
CH2), 4.43–4.46 (1H, m, CHOH), 5.16 (1H, d, J 3.6 Hz, CHOH), 130.72 (Py-C), 133.85 (10-C), 156.24 (CQO), 156.38 (40-C). m/z 434
5.71–5.78 (2H, m, CH2 Pmoc), 6.55 (1H, dd, J 8.0 and 1.6 Hz, ([M+ + Na], 24%), 283 (17), 245 (19), 215 (100). HRMS calc. for
60-H), 6.65 (1H, d, J 8.0 Hz, 50-H), 6.74 (1H, d, J 1.6 Hz, 20-H), 7.22 C26H21NNaO4 [M+ + Na]: 434.13628; found: 434.13578.
(1H, t, J 6.0 Hz, NH), 8.07–8.11 (2H, m, Py-H), 8.15–8.20 (2H, m,
N-[(Pyren-1-yl)methoxycarbonyl]-L-phenylalanine methyl ester,
Py-H), 8.26–8.34 (5H, m, Py-H), 8.72 (1H, s, OH), 8.81 (1H, s, OH). 5a. Starting from 1-hydroxymethylpyrene 1 (0.108 g, 0.46 mmol)
dC (400 MHz; DMSO-d6) 48.72 (CH2), 63.66 (CH2 Pmoc), 71.22 and L-phenylalanine methyl ester 4a (0.100 g, 0.46 mmol), com-
(CHOH), 113.54 (20-C), 115.08 (50-C), 116.96 (60-C), 123.20 (Py-C), pound 5a was obtained as a yellow solid (0.029 g, 15%). mp
123.82 (Py-C), 123.93 (Py-C), 124.73 (Py-C), 125.45 (Py-C), 125.52 133.4–134.6 1C. lmax (MeOH–HEPES 80: 20)/nm 341 (loge 4.52),
(Py-C), 126.40 (Py-C), 127.27 (Py-C), 127.38 (Py-C), 127.53 (Py-C), 325 (4.39), 311 (4.01), 274 (4.50) and 264 (4.32). lmax (EtOH)/nm
127.92 (Py-C), 128.59 (Py-C), 130.26 (Py-C), 130.51 (Py-C), 130.75 341 (loge 4.37), 325 (4.05), 275 (4.26), 264 (3.20) and 241 (4.54).
(Py-C), 130.81 (Py-C), 134.66 (10-C), 144.29 (40-C), 144.91 (30-C),
n
max/cmꢀ1 3307, 3031, 2953, 1735, 1687, 1536, 1431, 1336, 1282,
156.28 (CQO). m/z 450 ([M+ + Na], 18%), 216 (24), 215 (100). 1261, 1249, 1201, 1011, 847, 840, 823, 713, 700. dH (400 MHz;
HMRS calc. for C26H21NNaO5 [M+ + Na]: 450.13119; found: CDCl3) 3.07–3.19 (2H, m, b-CH2), 3.73 (34, s, OCH3), 4.71–4.76
450.13183.
(1H, m, a-CH), 5.31 (1H, d, J 8.4 Hz, NH), 5.78–5.87 (2H, m, CH2
N-[(Pyren-1-yl)methoxycarbonyl]-2-(40-hydroxy-10-phenyl)ethyl- Pmoc), 7.07–7.09 (2H, m, 20-H-and 60-H), 7.20–7.22 (3H, m, 30-H,
amine, 3c. Starting from 1-hydroxymethylpyrene 1 (0.134 g, 40-H and 50-H), 8.01–8.07 (4H, m, Py-H), 8.13–8.16 (2H, m, Py-H),
0.58 mmol) and 2-(40-hydroxy-10-phenyl)ethylamine (tyramine) 8.20–8.29 (3H, m, Py-H). dC (400 MHz; CDCl3) 38.18 (b-CH2),
2c (0.100 g, 0.58 mmol), compound 3c was obtained as a brown 52.26 (OCH3), 54.85 (a-CH), 65.40 (CH2 Pmoc), 122.90 (Py-C),
solid (0.074 g, 32%). mp 144.8–146.1 1C. lmax (MeOH–HEPES 124.54 (Py-C), 124.57 (Py-C), 124.78 (Py-C), 125.35 (Py-C), 125.41
80 : 20)/nm 340 (log e 4.45), 325 (4.36), 273 (4.39), 264 (4.30) and (Py-C), 125.99 (Py-C), 127.04 (40-C), 127.29 (Py-C), 127.58 (Py-C),
c
2374 New J. Chem., 2013, 37, 2369--2376
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127.71 (Py-C), 128.09 (Py-C), 128.50 (30-C and 50-C), 129.04 (Py-C), The solution was stirred at room temperature for 8 hours and
129.18 (20-C and 60-C), 129.40 (Py-C), 130.64 (Py-C), 131.13 (Py-C), acidified to pH 2–3 with 1 M aqueous potassium hydrogen
131.65 (Py-C), 135.61 (10-C), 155.66 (CQO), 171.88 (CQO ester). sulphate. The reaction mixture was evaporated, dichloro-
m/z 460 ([M+ + Na], 100%), 215 (55). HRMS calc. for C28H23NNaO4 methane was added giving a precipitate that was filtered. The
[M+ + Na]: 460.15193; found: 460.15302.
N-[(Pyren-1-yl)methoxycarbonyl]-L-tyrosine methyl ester, 5b. obtained as a solid.
solvent was evaporated and the corresponding compound was
Starting from 1-hydroxymethylpyrene 1 (0.121 g, 0.52 mmol)
N-[(Pyren-1-yl)methoxycarbonyl]-L-phenylalanine, 6a. Start-
and L-tyrosine methyl ester 4b (0.124 g, 0.52 mmol), compound ing from compound 5a (0.023 g, 0.05 mmol) and 1 M sodium
5b was obtained as a yellow solid (0.047 g, 20%). mp 209.6– hydroxide (0.16 mL, 0.16 mmol) compound 6a was obtained as
210.7 1C. lmax (MeOH–HEPES 80 : 20)/nm 341 (log e 4.19), 325 a brown solid (0.016 g, 74%). mp 176.5–177.9 1C. lmax (MeOH–
(4.08), 274 (4.15), 264 (4.00) and 239 (4.37). lmax (EtOH)/nm 341 HEPES 80 : 20)/nm 341 (log e 4.47), 325 (4.33), 311 (3.95), 274
(log e 4.89), 325 (4.76), 274 (5.15), 264 (5.05), 240 (5.18) and 231 (4.45) and 264 (4.26). lmax (EtOH)/nm 341 (log e 4.64), 325
(5.08). nmax/cmꢀ1 3313, 3041, 2957, 2927, 1712, 1690, 1614, 1597, (4.48), 309 (4.24), 275 (4.76), 264 (4.52) and 241 (4.92). nmax
/
1540, 1517, 1448, 1432, 1341, 1292, 1256, 1226, 1107, 1052, 1014, cmꢀ1 3302, 3029, 2926, 1690, 1604, 1537, 1336, 1264, 1048,
846, 832. dH (400 MHz; DMSO-d6) 2.72–2.78 (1H, m, b-CH2), 1011, 842, 757, 700. dH (400 MHz; DMSO-d6) 2.81–2.87 (1H, m,
2.88–2.93 (1H, m, b-CH2), 3.61 (3H, s, OCH3), 4.19–4.24 (1H, m, b-CH2), 3.05–3.10 (1H, m, b-CH2), 4.18–4.24 (1H, m, a-CH),
a-CH), 5.69–5.77 (2H, m, CH2 Pmoc), 6.65 (2H, d, J 8.0 Hz, 30-H 5.67–5.75 (2H, m, CH2 Pmoc), 7.16–7.20 (2H, m, 20-H and 60-H),
and 50-H), 7.01 (2H, d, J 8.4 Hz, 20-H and 60-H), 7.82 (1H, d, J 8.0 7.22–7.26 (3H, m, 30-H, 40-H and 50-H), 7.62 (1H, d, J 8.0 Hz,
Hz, NH), 8.03–8.11 (2H, m, Py-H), 8.16–8.21 (2H, m, Py-H), 8.27– NH), 8.02–8.11 (2H, m, Py-H), 8.15–8.20 (2H, m, Py-H), 8.23–8.34
8.35 (5H, m, Py-H), 9.24 (1H, s, OH). dC (400 MHz; DMSO-d6) (5H, m, Py-H). dC (400 MHz; DMSO-d6) 36.50 (b-CH2), 55.70
35.75 (b-CH2), 51.86 (OCH3), 55.97 (a-CH), 63.87 (CH2 Pmoc), (a-CH), 63.73 (CH2 Pmoc), 123.08 (Py-C), 123.77 (Py-C), 123.88
115.02 (30-C and 50-C), 123.05 (Py-C), 123.76 (Py-C), 123.88 (Py-C), (Py-C), 124.65 (Py-C), 125.41 (Py-C), 125.49 (Py-C), 126.30 (Py-C),
124.67 (Py-C), 125.45 (Py-C), 125.53 (Py-C), 126.38 (Py-C), 127.15 126.35 (20-C and 60-C), 127.13 (Py-C), 127.33 (Py-C), 127.50
(Py-C), 127.33 (Py-C), 127.38 (10-C), 127.55 (Py-C), 127.90 (Py-C), (Py-C), 127.86 (Py-C), 128.10 (30-C and 50-C), 128.51 (Py-C),
128.52 (Py-C), 130.01 (20-C and 60-C), 130.12 (Py-C), 130.21 (Py-C), 129.09 (40-C), 130.21 (Py-C), 130.25 (Py-C), 130.71 (Py-C),
130.71 (Py-C), 130.82 (Py-C), 155.96 (CQO), 156.00 (40-C), 172.46 130.79 (Py-C), 138.01 (10-C), 155.99 (CQO), 173.31 (CQO acid).
(CQO ester). m/z 476 ([M+ + Na], 63%), 414 (21), 413 (58), 359 m/z 446 ([M+ + Na], 37%), 380 (22), 215 (100). HRMS calc. for
(39), 313 (61), 283 (34), 227 (100), 216 (21), 215 (80). HRMS calc.
for C28H23NNaO5 [M+ + Na]: 476.14684; found: 476.14751.
C
27H21NNaO4 [M+ + Na]: 446.13628; found: 446.13639.
N-[(Pyren-1-yl)methoxycarbonyl]-L-tyrosine, 6b. Starting from
N-[(Pyren-1-yl)methoxycarbonyl]-L-3,4-dihydroxyphenylalanine compound 5b (0.057 g, 0.13 mmol) and 1 M sodium hydroxide
methyl ester, 5c. Starting from 1-hydroxymethylpyrene 1 (0.061 g, (0.38 mL, 0.38 mmol) compound 6b was obtained as a yellow
0.26 mmol) and L-3,4-dihydroxyphenylalanine methyl ester 4c (0.065 solid (0.050 g, 90%). mp 186.9–188.2 1C. lmax (MeOH–HEPES
g, 0.26 mmol), compound 5c was obtained as a yellow solid (0.049 g, 80 : 20)/nm 341 (log e 4.51), 325 (4.39), 311 (4.01), 274 (4.50) and
40%). mp 101.0–102.2 1C. lmax (MeOH–HEPES 80 : 20)/nm 340 (log e 264 (4.33). lmax (EtOH)/nm 341 (log e 4.63), 325 (4.50), 311
4.47), 325 (4.38), 274 (4.42), 264 (4.32), 234 (4.61) and 204 (4.45). lmax (4.26), 275 (4.75), 264 (4.49), 241 (4.89) and 231 (4.77). nmax
/
(EtOH)/nm 341 (log e 4.64), 326 (4.51), 275 (4.66), 264 (4.45) and 240 cmꢀ1 3333, 3040, 1687, 1589, 1514, 1442, 1406, 1342, 1245,
(4.88). nmax/cmꢀ1 3417, 3043, 2954, 2927, 1717, 1604, 1505, 1439, 1138, 1064, 839, 757. dH (400 MHz; DMSO-d6) 2.77–2.82 (1H, m,
1365, 1346, 1260, 1131, 1066, 1032, 947, 846. dH (400 MHz; DMSO- b-CH2), 2.96–3.01 (1H, m, b-CH2), 3.79–3.83 (1H, m, a-CH),
d6) 2.66–2.72 (1H, m, b-CH2), 2.82–2.87 (1H, m, b-CH2), 3.61 (3H, s, 5.62–5.78 (2H, m, CH2 Pmoc), 6.31 (1H, d, J 6.8 Hz, NH), 6.54
OCH3), 4.18–4.23 (1H, m, a-CH), 5.69–5.78 (2H, m, CH2 Pmoc), 6.47 (2H, d, J 8.4 Hz, 30-H and 50-H), 6.90 (2H, d, J 8.4 Hz, 20-H and
(1H, dd, J 8.0 and 1.2 Hz, 60-H), 6.62 (1H, d, J 7.6 Hz, 50-H), 6.63 (1H, 60-H), 8.03–8.10 (2H, m, Py-H), 8.17–8.20 (2H, m, Py-H), 8.24–
d, J 2.0 Hz, 20-H), 7.79 (1H, d, J 8.0 Hz, NH), 8.03–8.11 (2H, m, Py-H), 8.31 (5H, m, Py-H), 9.29 (1H, s br, OH). dC (400 MHz; DMSO-d6)
8.15–8.20 (2H, m, Py-H), 8.27–8.34 (5H, m, Py-H), 8.76 (2H, s, OH). dC 36.61 (b-CH2), 57.08 (a-CH), 63.31 (CH2 Pmoc), 114.56 (30-C and
(400 MHz; DMSO-d6) 36.07 (b-CH2), 51.87 (OCH3), 56.01 (a-CH), 50-C), 123.14 (Py-C), 123.84 (Py-C), 123.93 (Py-C), 124.70 (Py-C),
63.91 (CH2 Pmoc), 115.38 (50-C), 116.41 (20-C), 119.82 (60-C), 123.06 125.42 (Py-C), 125.47 (Py-C), 126.37 (Py-C), 126.98 (Py-C), 127.39
(Py-C), 123.78 (Py-C), 123.90 (Py-C), 124.71 (Py-C), 125.47 (Py-C), (Py-C), 127.45 (Py-C), 127.87 (Py-C), 128.42 (Py-C), 129.25 (10-C),
125.53 (Py-C), 126.38 (Py-C), 127.13 (Py-C), 127.35 (Py-C), 127.54 130.28 (Py-C), 130.35 (20-C and 60-C), 130.72 (Py-C), 130.76 (Py-
(Py-C), 127.94 (Py-C), 128.07 (10-C), 128.53 (Py-C), 130.13 (Py-C), C), 130.80 (Py-C), 155.02 (CQO), 155.40 (40-C), 173.05 (CQO
130.23 (Py-C), 130.73 (Py-C), 130.83 (Py-C), 143.93 (40-C), 144.98 acid). m/z 462 ([M+ + Na], 100%), 396 (43), 348 (20), 215 (16), 203
(30-C), 156.03 (CQO), 172.51 (CQO ester). m/z 492 ([M+ + Na], (23). HRMS calc. for C27H21NNaO5 [M+ + Na]: 462.13119; found:
8%), 216 (24), 215 (100). HRMS calc. for C26H21NNaO5 [M+ + Na]: 462.13307.
492.14176; found: 492.14097.
General photolysis procedure
A 1 ꢁ 10ꢀ4 M methanol–HEPES buffer (80 : 20) solution of com-
General procedure for the synthesis of compounds 6a,b
To a suspension of compounds 5a,b (1 equiv.) in 1,4-dioxane pounds 3a–d, 5a–c and 6a,b (5 mL) was placed in a quartz tube and
(2.0 mL), 1 M aqueous sodium hydroxide (3 equiv.) was added. irradiated in a Rayonet RPR-100 reactor at the desired wavelength.
c
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NJC
11 P. Klan, T. Solomek, C. G. Bochet, A. Blanc, R. Givens,
M. Rubina, V. Popik, A. Kostikov and J. Wirz, Photoremo-
vable protecting groups in chemistry and biology: reaction
mechanisms and efficacy, Chem. Rev., 2013, 113, 119.
12 T. Furuta, Y. Hirayama and M. Iwamura, Anthraquinon-2-yl-
methoxycarbonyl (Aqmoc): a new photochemically removable
protecting group for alcohols, Org. Lett., 2001, 3, 1809.
13 A. Jana, S. Atta, S. K. Sarkar and N. D. P. Singh, 1-Acetylpyrene
with dual functions as an environment-sensitive fluorophore
and fluorescent photoremovable protecting group, Tetrahedron,
2010, 66, 9798.
14 T. Furuta, H. Torigai, T. Osawa and M. Iwamura, New
photochemically labile protecting group for phosphates,
Chem. Lett., 1993, 1179.
15 S. Okada, S. Yamashita, T. Furuta and M. Iwamura, (1-Pyrenyl)-
methyl carbamates for fluorescent ‘‘caged’’ amino acids and
peptides, Photochem. Photobiol., 1995, 61, 431.
16 A. Hussain, S. Gadadhar, T. K. Goswami, A. A. Karande and
A. R. Chakravarty, Photoactivated DNA cleavage and anti-
cancer activity of pyrenyl-terpyridine lanthanide complexes,
Eur. J. Med. Chem., 2012, 50, 319.
17 M. J. G. Fernandes, M. S. T. Gonçalves and S. P. G. Costa,
Photorelease of amino acid neurotransmitters from pyrenyl-
methyl ester conjugates, Tetrahedron, 2007, 63, 10133.
18 M. J. G. Fernandes, M. S. T. Gonçalves and S. P. G. Costa,
Neurotransmitter amino acid—oxobenzo[f]benzopyran con-
jugates: synthesis and photorelease studies, Tetrahedron,
2008, 64, 11175.
The lamps used for irradiation were of 254, 300 and 350 ꢂ 10 nm.
HEPES buffer solution was prepared in distilled water with HEPES
(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (10 mM),
sodium chloride (120 mM), potassium chloride (3 mM), calcium
chloride (1 mM) and magnesium chloride (1 mM) and the pH
adjusted to 7.2 with 1 M sodium hydroxide solution.
Aliquots of 100 mL were taken at regular intervals and
analysed using RP-HPLC. The eluent was acetonitrile–water,
3 : 1, at a flow rate of 0.6 (compounds 5c, 6a,b), 0.8 (compounds
3a–d, 5b) and 1.2 (compound 5a) mL minꢀ1, previously filtered
through a Millipore, type HN 0.45 mm filter and degassed by
ultra-sound for 30 min. The chromatograms were traced by
detecting UV absorption at the wavelength of maximum absorp-
tion for each compound (retention time: 3a, 5.8; 3b, 4.3; 3c, 7.6;
3d, 4.7; 5a, 8.8; 5b, 6.9; 5c, 7.3; 6a, 2.8; 6b, 2.8 min).
Acknowledgements
˜
ˆ
Thanks are due to the Fundaçao para a Ciencia e Tecnologia
(Portugal) for financial support through project PTDC/QUI/69607/
2006 (FCOMP-01-0124-FEDER-007449) and project FCT PEst-C/QUI/
UI0686/2011 (F-COMP-01-0124-FEDER-022716), FEDER-COMPETE.
A PhD grant to M.J.G.F. (SFRH/BD/36695/2007) is also acknowl-
edged. 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 Scientific Re-equipment, with funds from
POCI 2010 (FEDER) and FCT.
19 A. M. S. Soares, S. P. G. Costa and M. S. T. Gonçalves, 2-Oxo-
2H-benzo[h]benzopyran as a new light sensitive protecting
group for neurotransmitter amino acids, Amino Acids, 2010,
39, 121.
20 A. S. C. Fonseca, M. S. T. Gonçalves and S. P. G. Costa, Light-
induced cleavage of model phenylalanine conjugates based
on coumarins and quinolones, Amino Acids, 2010, 39, 699.
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