Decarboxylative photorelease coupled with fluorescent up/down reporter function based on the aminophthalimide-serine system

Article abstract of DOI:10.1039/c001622e

The fluorescence of caged phthalimide-serine couples 2 and 4 is up/down modulated by decarboxylative photorelease with fluorescence decrease (2) vs. moderate fluorescence increase (4) serving as reporter function.

Full text of DOI:10.1039/c001622e

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Decarboxylative photorelease coupled with fluorescent up/down reporter
function based on the aminophthalimide–serine system
b
a
a
Alberto Soldevilla,*a Rau
l Perez-Ruiz, Yrene Dıaz Miara and Axel Griesbeck*
´ ´
´
Received 26th January 2010, Accepted 13th April 2010
First published as an Advance Article on the web 23rd April 2010
DOI: 10.1039/c001622e
The fluorescence of caged phthalimide–serine couples 2 and
4 is up/down modulated by decarboxylative photorelease with
fluorescence decrease (2) vs. moderate fluorescence increase (4)
serving as reporter function.
acetone/water (1 : 1) at pH = 3.5, which decreased to less
than 5% at pH > 6.8
In a first approach, we prepared and tested caged acetate 2.
When this photocage was irradiated at 350 nm, acetate release
took place and N-vinylphthalimide 3 was formed (Scheme 1).
This photochemical process is analogous to the previously
described photorelease of acetate in phthaloyl–serine derivatives,6
and it is related to the decarboxylation reaction of the glycine
derivative.8 In order to introduce a potential covalent peptide-
binding site into the caged compound, we also investigated the
4-aminophthalimide skeleton, a highly fluorescent chromophore.9
The corresponding caged 4 cleanly underwent the photo-
chemical decarboxylation/release process with the formation
of phthalimide 5.
Molecular fragments that are protected by covalent binding to
a light-sensitive group and released from an electronically
excited state are called photocaged compounds.1 Spatial as
well as timely control of the release process is crucial for the
use as bioanalytical tools,1,2 which in turn requires a fast and
efficient photoliberation. Furthermore, a physical reporting
function (emission) is desirable, which is able to map the
region of photorelease.3,4 Following a report on a new type
of photodecarboxylative caging-system,5 we have recently
described the use of phthalimide–serine/threonine couples
for photorelease of acetate with high diastereoselection in
the liberation process.6 These photocages, however, absorb
light in the UV-C region, which is not advantageous for
applications in biological environments, and there is no fluores-
cence observable, neither from the substrate nor from the
separated chromophore.
As in the case of photocage 2, the preparation of carbamate-
protected derivative 4 (Scheme 2) was accomplished by acyla-
tion of the phthalimide–serine couples (6 and 7, respectively).
The synthesis of these reagents started with the corresponding
anhydrides, 4,5-dimethoxyphthalic anhydride10 (8) and 4-iso-
cyanatophthalic anhydride11 (9, Scheme 3). They were con-
verted by methanolysis into the ring-opened phthalates 10 and
11. DCC-coupling with N-hydroxysuccinimide led to the
activated phthalates 12 and 13, which subsequently were
reacted with serine. The standard technique for the amino
acid phthaloylation with succinimido esters,12 using potassium
carbonate as base, did not give satisfactory results in our
hands when using serine. However, replacing carbonate with
the stronger base, triethylamine, we were able to carry out the
reaction in good yields.
From studies on PET reactions with 4,5-dimethoxyphthal-
imides, we became aware that this chromophore is highly
fluorescent and an excellent electron acceptor in the excited
state.7 Indeed, the corresponding glycine-derived 4,5-di-
methoxyphthalimide showed a 60 nm red-shift in absorption8
(like the serine derivative in Fig. 1) and a fluorescence emission
at lem = 503 nm with a fluorescence quantum yield of 0.6 in
Scheme 1 Decarboxylative photorelease of acetate from 2 and 4.
Fig. 1 Absorption spectra of donor-substituted phthalimide photo-
cages 2 and 4 in comparison with the parent compound 1.
a Department of Chemistry, Greinstr. 4, D-50939 Koln, Germany.
¨
E-mail: griesbeck@uni-koeln.de; Fax: 0049-221-4705057
b
´
Departamento de Quımica-Instituto de Tecnologıa Quımica
UPV-CSIC, Universidad Politecnica de Valencia,
´
´
Scheme 2 Preparation of caged acetates 2 and 4 by mono-acetylation
of phthalimide–serine couples 6 and 7.
´
Camino de Vera s/n, 46022, Valencia, Spain
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Scheme 3 Syntheses of caging reagents 6 and 7.
Fig. 3 Fluorescence emission spectra of 4 (lexc = 340 nm) at different
irradiation times, showing fluorescence increase and red-shift reporting
the photorelease of acetate.
involving a decarboxylation process but without liberation
of caged molecular fragments.13
Mechanistically, the photodecarboxylation process initiated
in the unsubstituted phthalimides most probably occurs from
an upper triplet state as has been shown by time-resolved
conductometry, quantum yield determination and oxygen
quenching experiments.7
In order to examine if this is also the case for the serine-
based photocages 4, laser flash photolysis (LFP) experiments
of the unsubstituted phthalimides (for the L- and D-enantiomers
of 1) were carried out in acetonitrile. Direct excitation of the
L- (or D-) enantiomer at 308 nm resulted in an intense band at
340 nm (Fig. 4) which was assigned to the triplet–triplet
transient absorption. This observation was in agreement with
previous experimental data for other phthalimide derivatives.14
The triplet lifetimes were found to be 1.25 ms for the L-enantiomer
and 1.32 ms for the D-enantiomer. When both enantiomers
were photolyzed by laser flash in pH 7.4 phosphate buffer (in
order to observe a reaction transient) no absorption lines were
Fig. 2 Fluorescence emission spectra of 2 (lexc = 340 nm) at
different irradiation times, showing fluorescence decrease and red-shift
reporting the photorelease of acetate.
The UV-absorption (Fig. 1) of 2 is red-shifted by 60 nm with
respect to the unsubstituted phthalimide 1 and 20 nm red-
shifted with respect to the carbamate 4. In the ‘‘armed’’ state
of 2 in aqueous media at pH = 7, strong fluorescence is
detected with lmax = 513 nm. Through irradiation, 2 is
transformed into 3 as the photoliberation of acetate takes
place (Scheme 1), and the fluorescence decreases with a 10 nm
red-shift of the emission maximum (Fig. 2). The fluorescence
decrease indicates a stronger electron transfer donor capability
of the enamide group in 3 in comparison with the carboxylate
in the starting material 2. The additional conjugation of the
vinyl group in the enamide 3 is represented by the batho-
chromic shift in absorption and emission. It is striking to see
that the fluorescence of 2 is more than 50 times stronger than
that of the glycine derivative under the same pH conditions.8
This indicates that the intramolecular fluorescence quenching
is strongly inhibited by the presence of the acetate group in 2.
Remarkably, this effect is inverted for compound 4: this
photocage shows a low fluorescence intensity at 458 nm
(compared to the dimethoxy compound 2) which is also red-
shifted during photolysis but subsequently increases by a
factor of 2 (Fig. 3). We have currently no explanation for this
reversal in fluorescence modulation. A similar fluorescence
increase has been observed in xanthone acetic acids, also
Fig. 4 Transient absorption spectra obtained upon LFP (lexc = 308 nm)
of L-enantiomer of 1 (Abs308 nm = 0.2) in acetonitrile, under argon.
Spectra recorded at 0.1 ms (—), 0.5 ms ( ), 1 ms ( ), 3 ms ( ) and 5 ms
(
) after the laser pulse. Inset: transient decay monitored at 340 nm
under argon.
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observed—neither triplet–triplet absorption nor reaction inter-
mediates. This suggests that the photodecarboxylation
process is faster than a possible formation of the unsubstituted
phthalimide excited triplet state under this condition and,
subsequently, the intermediate carbanion lifetime is too
short-lived to be detected in the nanosecond transient system.
In summary, an efficient and fast photoremovable protecting
group in two substituent-modified chromophores has been
described, which also includes a simple but effective fluores-
cent reporting function associated with a photorelease process.
Depending on the substitution pattern at the aromatic ring,
fluorescence up/down reporter function was observed. These
modifications on previously described photocaging systems
substantially improve phthalimide-photocages to a state of
usability in real applications. Moreover, these photocages
include a stereogenic center, readily available from a con-
venient source (serine), opening the gate for the study of
photoliberation of selected molecules in combination with
chiral recognition and enantiodifferentiation processes similar
to the phthalimide–urea couples we have reported recently.15
2 J. A. McCray and D. R. Trentham, Annu. Rev. Biophys. Biophys.
Chem., 1989, 18, 239.
3 (a) J. R. R. Majjigapu, A. N. Kurchan, R. Kottani,
T. P. Gustafson and A. G. Kutateladze, J. Am. Chem. Soc.,
2005, 127, 12458; (b) J.-P. Pellois, M. E. Hahn and T. W. Muir,
J. Am. Chem. Soc., 2004, 126, 7170; (c) W. F. Veldhuyzen,
Q. Nguyen, G. McMaster and D. S. Lawrence, J. Am. Chem.
Soc., 2003, 125, 13358.
4 Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez and
W.-H. Li, J. Am. Chem. Soc., 2004, 126, 4653.
5 (a) G. Cosa, M. Lukeman and J. C. Scaiano, Acc. Chem. Res.,
2009, 42, 599; (b) M. Lukeman and J. C. Scaiano, J. Am. Chem.
Soc., 2005, 127, 7698.
6 A. Soldevilla and A. G. Griesbeck, J. Am. Chem. Soc., 2006, 128,
16472.
7 K.-D. Warzecha, H. Gorner and A. G. Griesbeck, J. Phys. Chem.
A, 2006, 110, 3356.
¨
8 A. G. Griesbeck and S. Schieffer, Photochem. Photobiol. Sci., 2003,
2, 113.
9 (a) T. G. Kim, M. F. Wolford and M. R. Topp, Photochem.
Photobiol. Sci., 2003, 2, 576; (b) G. Saroja, B. Ramachandram,
S. Saha and A. Samanta, J. Phys. Chem. B, 1999, 103,
2906.
´ ´
10 J. Gawronski, K. Gawronska and M. Brzostowska, Tetrahedron
Lett., 1999, 40, 1191.
11 H. Ulrich and R. Richter, J. Org. Chem., 1973, 38, 2557.
12 J. R. Casimir, G. Guichard and J.-P. Briand, J. Org. Chem., 2002,
67, 3764.
Notes and references
13 J. A. Blake, E. Gagnon, M. Lukeman and J. C. Scaiano, Org. Lett.,
2006, 8, 1057.
1 (a) R. S. Givens, P. G. Conrad and A. L. Yousef, CRC Handbook
of Organic Photochemistry and Photobiology, ed. W. M. Horspool,
F. Lenci, CRC Press, Boca Raton, FL, 2nd edn, 2004, ch. 69;
(b) A. P. Pelliccioli and J. Wirz, Photochem. Photobiol. Sci., 2002,
1, 441.
14 H. Gorner, A. G. Griesbeck, T. Heinrich, W. Kramer and
¨
M. Oelgemoller, Chem.–Eur. J., 2001, 7, 1530.
¨
´
rez-Ruiz, Y. Diaz, B. Goldfuss, D. Hertel, K. Meerholz and
A. G. Griesbeck, Org. Biomol. Chem., 2009, 7, 3499.
15 R. Pe
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