N-Acyl-N-carboxymethyl-2-nitroaniline and its analogues: A new class of water-soluble photolabile precursor of carboxylic acids

Article abstract of DOI:10.1039/c2pp05322e

The synthesis and photochemistry of a new class of water-soluble, photolabile protecting group, the N-carboxymethyl-2-nitroanilinyl (CNA) group, are described. All three CNA-caging compounds synthesized underwent clean photochemical reaction in aqueous bu

Full text of DOI:10.1039/c2pp05322e

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Cite this: Photochem. Photobiol. Sci., 2012, 11, 493
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N-Acyl-N-carboxymethyl-2-nitroaniline and its analogues: a new class of
water-soluble photolabile precursor of carboxylic acids†‡
Takuya Honda, Atsuya Momotake and Tatsuo Arai*
Received 30th September 2011, Accepted 4th December 2011
DOI: 10.1039/c2pp05322e
The synthesis and photochemistry of a new class of water-
soluble, photolabile protecting group, the N-carboxymethyl-
2-nitroanilinyl (CNA) group, are described. All three CNA-
caging compounds synthesized underwent clean photochemi-
cal reaction in aqueous buffer solution to produce the
intended product of acetic acid, along with the corresponding
nitroso aromatic.
The photochemical release of bioactive molecules from their
photolabile precursors (otherwise known as caged compounds)
is a useful technique for the controlled initiation of biological
processes by selective stimulation at target sites.1–6 To date,
Fig. 1 Chemical structures of MNI-Glu, MNDQ-Glu and CNAn-Ac.
various bioactive molecules, including neurotransmitters such as
glutamate or γ-aminobutyric acid (GABA),7–10 peptides and pro-
teins,11 and RNAs,12 as well as calcium ions,13 have been
solubility. This molecular structure also allows the introduction
subject to caging. Caged neurotransmitters are one of the most
extensively researched and frequently utilized caged compounds,
because the photo-release of neurotransmitters satisfies the
requirements of recently developed functional imaging tech-
niques for rapid rises in species concentration coupled with a
significant temporal and spatial control in order to stimulate
synapses at the single cell level. The widely utilized MNI (4-
methoxy-7-nitroindolinyl) group (Fig. 1),8 used for γ-carboxyl
caging, possesses the essential property of stability in the
absence of light, due to the presence of an amide bond between
the chromophore and the glutamate moiety. We have previously
reported the development of caging chromophores such as the 5-
methoxy-8-nitro-1,2-dihydroquinolinyl group (MNDQ) (Fig. 1)
which has the capacity to bind to glutamate via an amide linkage
and has demonstrated both one- and two-photon uncaging in
hippocampal slices.14
of various substituents onto the phenyl ring without the loss of
water solubility.
Since little is known at present about the photochemistry of
CNA-caging chromophores, preliminary photolysis experiments
with model compounds were required using some simple var-
iants of the chromophore group. To this end, we designed three
model chromophores, CNAn-Ac (Fig. 1) that we expected to
undergo photolysis at the amide linkage with subsequent release
of acetic acid. In this chromophore, the 2-nitro group is essential
for the uncaging reaction, since the nitro compound
9
(Scheme 1) is not photoreactive. CNA1-Ac can be regarded as
the simplest possible model of this protecting group since it has
no substituents at the 3 to 6 positions. CNA2-Ac has a hydrophi-
lic and electron-donating carboxymethoxy group at the 5-pos-
ition. CNA3-Ac was designed with the expectation of high
quantum yields on photocleavage (Φp), based on a previous
study.8d
The synthetic pathways leading to these new caged com-
pounds are depicted in Scheme 1. The synthesis of CNA1-Ac
started with the N-acetylation of the known compound tert-
butyl-2-(phenylamino)acetate.15 Subsequently, nitration was
carried out under mild conditions to afford 2 without decompo-
sition of the tert-butyl ester. Deprotection of 2 by treatment with
trifluoroacetic acid gave the final product CNA1-Ac. The syn-
thesis of CNA2-Ac was accomplished by a somewhat circuitous
process. Since treatment of 3-aminophenol with excess tert-butyl
bromoacetate gives a complex mixture of mono-, di-, and tri-
alkylated products and the purification of each product is proble-
matic, the mono-N-alkylated product 3 was first prepared by the
use of a lesser amount of the bromoacetate. Compound 3 was
In this paper we report a new class of chromophore for the
caging of carboxylates; the N-carboxymethyl-2-nitroanilinyl
(CNA) protecting group (Fig. 1). CNA caging groups are charac-
terized by the presence of an anilino nitrogen which allows the
formation of an amide bond with a carboxylic acid as well as the
addition of a carboxymethyl group, resulting in improved water
Graduate School of Pure and Applied Sciences, University of Tsukuba,
Tsukuba, Ibaraki 305-8577, Japan. E-mail: arai@chem.tsukuba.ac.jp;
Fax: +81-298-53-6503; Tel: +81-298-53-4315
†This paper is part of a themed issue on photoremovable protecting
groups: development and applications.
‡Electronic supplementary information (ESI) available: Experimental
procedures and analytical and spectroscopic data. Synthetic scheme. Flu-
orescence decay plots. See DOI: 10.1039/c2pp05322e
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Scheme 1
di-acetylated by reaction with an excess of acetic anhydride to
give 4. The following four reactions proceeded readily, resulting
in CNA2-Ac. The methoxy-substituted analogue CNA3-Ac was
synthesized in a similar manner except for the second nitration
reaction where the activated zeolite Hβ was used.16 All three
compounds exhibited good solubility in water (>1 mM) without
the use of any organic co-solvents, and were stable in phosphate
buffer solution at pH 7.4, with no hydrolysis after 24 h in the
dark at room temperature, as confirmed by HPLC analysis.
The UV-Vis absorption bands of the CNA-caging groups
varied depending on the phenyl ring substituents. Fig. 2 shows
the comparative absorption spectra of CNAn-Ac variants in
phosphate buffer at pH 7.4. The main absorption peak of CNA1-
Ac appears at 255 nm with an extinction coefficient (ε255) of
4700 cm−1 M−1. When a carboxymethoxy group is introduced at
the 5-position, as in CNA2-Ac, the absorption maximum is red-
shifted to 312 nm (ε312 = 7800 M−1 cm−1). CNA3-Ac, with a
second nitro group, exhibited an absorption spectrum similar to
that of CNA2-Ac, but with an additional absorption peak at
270 nm. At 350 nm the extinction coefficients were 210, 3800,
and 3200 M−1 cm−1 for CNA1-Ac, CNA2-Ac, and CNA3-Ac,
respectively, suggesting that the addition of either methoxy or
carboxymethoxy substituents is advantageous with regard to the
uncaging of these molecules under biological conditions.
Fig. 3 Change in the absorption spectra of CNA1-Ac (a) and CNA2-
Ac (b) upon irradiation at 365 nm in aqueous phosphate buffer at pH 7.4.
the photoproduct is evidently photo-stable, since subsequent
photoreaction, as normally evident by a loss of the isosbestic
point, was not observed. The absorption spectrum after
irradiation of CNA1-Ac indicates that the photoproduct is a
nitroso aromatic which exhibits an absorption band at a longer
wavelength. The other photoproduct should be the intended
acetic acid. Changes in the absorption spectra of CNA2-Ac upon
irradiation also indicate a clean reaction with the development of
a pronounced band at 360 nm (Fig. 3b). This band is similar to
those observed in the photolysis of the NMI and MNDQ caging
groups, likely due to the production of nitroso aromatics.
Changes in the 1H NMR spectrum of CNA2-Ac during
irradiation are shown in Fig. S1 (see ESI‡). It was observed that
new peaks, increasing in intensity with irradiation time, devel-
oped at 2.09 ppm in D2O due to the release of acetic acid. New
peaks were also seen in the aromatic region due to the photopro-
duct of the caging group, again increasing with irradiation time.
Scheme 2 shows a plausible mechanism for the photolysis of
CNAn-Ac, based on our observations of clean reactions result-
ing in acetic acid and 2-((5-(carboxymethoxy)-2-nitrosophenyl)
Fig. 2 Absorption spectra of CNA1-Ac (thin line), CNA2-Ac (dotted
line), and CNA3-Ac (thick line) in aqueous phosphate buffer at pH 7.4.
Photoirradiation was performed in phosphate buffer at pH 7.4
without the exclusion of oxygen. When CNA1-Ac was subjected
to photoirradiation, the absorption spectra changed appreciably
with an isosbestic point at 276 nm (Fig. 3a), suggesting that this
photochemical reaction proceeds cleanly. The absorption bands
obtained during and after irradiation are likely due to a simple
mixture of CNA1-Ac and its single photoproduct. In addition,
Scheme 2
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imino)acetic acid. Due to the presence of both electron-donating
and accepting groups, the first step of photolysis is likely to be a
charge transfer from the anilino nitrogen to the neighboring nitro
group. Cleavage of the N–C bond then takes place to form acetic
acid and the nitroso product. The quantum yields of this photoly-
sis (Φp) in aqueous buffer were determined to be 0.06, 0.07, and
0.07, for CNA1-Ac, CNA2-Ac, and CNA3-Ac, respectively,
and these values are equivalent to the quantum yield for the pro-
duction of acetic acid due to the clean reaction. The photochemi-
cal efficiency of caged compounds has often been defined as the
product of Φp and the molar extinction coefficient ε (ε·Φp). At
the 350 nm wavelength the ε350·Φp values were calculated to be
13, 266, and 224 for CNA1-Ac, CNA2-Ac, and CNA3-Ac,
respectively. A suitably large ε·Φp value is important in a caged
compound in order to reduce the likelihood of cell phototoxicity.
Interestingly, the uncaging quantum yield of the three CNA-
caging chromophores was only slightly affected by their substitu-
ent functional groups, even though the absorption spectra of the
chromophores varied substantially with the addition of the differ-
ent substituents, indicating that the electronic structures of all
three compounds are different. A previous study of substituent
effects on the photocleavage of 1-acyl-7-nitroindolines, includ-
ing MNI-Glu, showed that both the introduction of an alkoxyl
group at the 4-position and a nitro group at the 5-position dra-
matically enhanced the photochemical efficiency of the uncaging
reaction.8 In addition, the quantum yield for the classical o-nitro-
benzyl caging group has been shown to be significantly affected
by the addition of substituents.7 In contrast to such well-estab-
lished caging groups as these, substituent effects on the photo-
chemical reactions of CNA-caging groups are still not clearly
understood. However, the present findings that CNAn-Ac
exhibit clean photoreactions with moderate efficiency and
demonstrate both stability in solution and water-solubility, even
in the case of the dinitro derivative CNA3-Ac, indicate that
CNA-caging groups show promise with regard to the rapid
photorelease of bioactive molecules. The photochemistry of
these chromophores is substituent-independent, with the for-
mation of acetic acid and nitroso aromatics as the sole bypro-
ducts in aqueous solution. Future detailed photochemical studies
will define the time scale of the photorelease more precisely. We
are also in the process of investigating additional CNA-caged
amino acid derivatives.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific
Research in a Priority Area “New Frontiers in Photochromism”
(No. 471) and a Grant-in-Aid for Scientific Research (B) (No.
23350075) and for young scientists (B) (No. 18750141 to AM),
from the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT), Japan.
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