1-Acyl-5-methoxy-8-nitro-1,2-dihydroquinoline: a biologically useful photolabile precursor of carboxylic acids

Article abstract of DOI:10.1016/j.tetlet.2009.12.081

The synthesis, photochemistry, and biological application of 1-acyl-5-methoxy-8-nitro-1,2-dihydroquinoline (MNDQ-caged carboxylic acid) are described. Optimization experiments were carried out on three acetyl derivatives (3a-c), and the most appropriate analogue for application to the caging of glutamate was determined to be 3c. Thus, a MNDQ-caged glutamate (MNDQ-Glu) was synthesized, and the photochemical release of glutamate by uncaging of MNDQ-Glu was confirmed by NMR, MS, and HPLC analysis. When MNDQ-Glu was tested with pyramidal neurons in hippocampal slices, whole-field UV illumination resulted in a large inward current due to the release of l-glutamate. A short two-photon uncaging of MNDQ-Glu at single dendritic spines induced a transient current that exhibited similar kinetic properties to miniature excitatory postsynaptic currents (mEPSC).

Full text of DOI:10.1016/j.tetlet.2009.12.081

Tetrahedron Letters 51 (2010) 1642–1647
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
1-Acyl-5-methoxy-8-nitro-1,2-dihydroquinoline: a biologically useful
photolabile precursor of carboxylic acids
Naoko Obi a, Atsuya Momotake a, Yuya Kanemoto b, Masanori Matsuzaki b,c, Haruo Kasai b, Tatsuo Arai a,
*
a Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan
b Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
c PRESTO, Japan Science and Technology Agency, Saitama, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, photochemistry, and biological application of 1-acyl-5-methoxy-8-nitro-1,2-dihydroquin-
oline (MNDQ-caged carboxylic acid) are described. Optimization experiments were carried out on three
acetyl derivatives (3a–c), and the most appropriate analogue for application to the caging of glutamate
was determined to be 3c. Thus, a MNDQ-caged glutamate (MNDQ-Glu) was synthesized, and the photo-
chemical release of glutamate by uncaging of MNDQ-Glu was confirmed by NMR, MS, and HPLC analysis.
When MNDQ-Glu was tested with pyramidal neurons in hippocampal slices, whole-field UV illumination
Received 15 October 2009
Revised 14 December 2009
Accepted 15 December 2009
Available online 21 December 2009
resulted in a large inward current due to the release of L-glutamate. A short two-photon uncaging of
MNDQ-Glu at single dendritic spines induced a transient current that exhibited similar kinetic properties
to miniature excitatory postsynaptic currents (mEPSC).
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tween the chromophore and the glutamate. Some improvements of
the MNI caging group have been carried out; for example, the
The photochemically triggered release (uncaging) of bioactive
molecules from their photolabile precursors (caged compounds)
is a valuable technique for selective stimulation at target sites in
or around cells and switching on of biological processes.1 In many
caged compounds, covalent bond formation with a photoremov-
able-protecting group (caging group) masks an important feature
for biological recognition. Before irradiation, the caged compound
is biologically inert; uncaging causes rapid jumps in the concentra-
tion of active molecules. To be useful biologically, the caging group
must undergo rapid and efficient photolysis upon photoexcitation
at wavelengths that are not harmful to cells. In addition, a caging
group with enhanced hydrophilicity enables the caged compound
to be soluble in aqueous buffer at higher concentrations without
the use of organic solvents. In previous studies, various molecules,
quantum yield for photolysis may be dramatically increased by
the introduction of an additional nitro group,8 or by conjugation
with a triplet sensitizer.9 The development of new varieties of cag-
ing chromophore will contribute further to the advancement of
caged technology.
Here, we propose a new caging chromophore, 5-methoxy-8-ni-
tro-1,2-dihydroquinolinyl (MNDQ), for the caging of carboxylates
(Fig. 1). Similarly to the MNI caging group, MNDQ contains an
including neurotransmitters such as glutamate or c-aminobutyric
acid (GABA),2 peptides and proteins,3 cyclic nucleotides,4 RNAs5
and calcium,6 have been subjected to caging. Among neurotrans-
mitters, caged glutamate has emerged as the most well-researched
caged compound. In the preparation of caged glutamates, a c-car-
boxyl group or amino group is generally the object to be caged. The
widely used 4-methoxy-7-nitroindolinyl (MNI) group,7 used for
c-carboxyl caging, displays essential properties such as light
absorption at preferable wavelengths, a suitable rate of uncaging,
and stability in the dark due to the presence of an amido bond be-
* Corresponding author.
E-mail address: arai@chem.tsukuba.ac.jp (T. Arai).
Figure 1. MNI- and MNDQ-caging group.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.081

N. Obi et al. / Tetrahedron Letters 51 (2010) 1642–1647
1643
Scheme 1. Synthesis of model compounds 3a–c.
Figure 3. UV–vis absorption spectra of 3a–c in MeOH/H2O (1:1).
anilino nitrogen for amide bond formation with a carboxylic acid,
providing high chemical stability under physiological conditions
in the caged state. In contrast with the well-known MNI group,
MNDQ has an additional C@C double bond in the neighboring
six-membered ring. The distance between the nitro oxygen and
the carbonyl carbon, a key reacting point, was expected to be closer
than that between MNI-carboxylates. However, since the photo-
chemistry of the MNDQ group was unknown, a preliminary
photochemical study was required before the preparation of
caged compounds. With this in mind, optimization experiments
were carried out with three acetyl derivatives of MNDQ 3a–c
(Scheme 1), and the most appropriate analogue (3c) was chosen
for application to the caging of glutamate. In this way, a new
MNDQ-caged glutamate (MNDQ-Glu) was synthesized. The photo-
chemical release of glutamate by uncaging of MNDQ-Glu was con-
firmed by NMR, MS, and HPLC analysis. Finally, MNDQ-Glu was
tested on pyramidal neurons for its ability to activate glutamate
ion channels upon one- and two-photon photolysis.
not stable in air and were therefore immediately treated with acet-
yl chloride to give 3a–c. The structures of 3a–c were confirmed by
1H and 13C NMR and elemental analysis. In the NMR spectra of 3a–
c, all the proton and carbon signals were split at room temperature,
indicating the presence of rotational isomers. In view of the fact
2. Results and discussion
The first goal was to study the effects of various substituent
groups on the photochemistry of the new caging group. Thus, three
test compounds 3a–c were prepared, starting from reduction of
8-nitroquinolines 1a–c with NaBH4 in AcOH (Scheme 1). The
resulting 1,2-dihydro-8-nitroquinolines 2a10, 2b11, and 2c were
Figure 4. Change in the absorption spectrum of 3c upon photoirradiation at
365 nm in 1:1 MeOH/H2O.
Figure 2. X-ray crystal structures of compounds 3b (left) and 3c (right).

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N. Obi et al. / Tetrahedron Letters 51 (2010) 1642–1647
Figure 5. Comparison of 1H NMR spectra of 3c before (a) and after (b) irradiation in CD3OD for 35 min. Small peaks (4) represent the rotational isomer of 3c. New peaks
appearing after photoirradiation are due to 5-methoxy-8-nitrosoquinoline 4 (d) and acetic acid (s), respectively.
was red-shifted to 345 nm, but the extinction coefficient at that
peak (e
345 = 1620 cmꢀ1 Mꢀ1) was similar to that of 3a. An increase
in the extinction coefficient as well as spectral red-shift was ob-
served for 3c, which has a 5-methoxy group (kmax = 345 nm,
e
345 = 4400 cmꢀ1 Mꢀ1). At the same wavelength (365 nm), the
extinction coefficients of 3a, 3b, and 3c were 900, 1500, and
3700 cmꢀ1 Mꢀ1, respectively. This suggests that the protecting
group in 3c is advantageous from the point of view of its use as a
caging group because near-UV light is generally used for uncaging
in biological conditions.
Scheme 2. Photolysis of 3c.
that MNI-acetate does not have a rotational isomer,7a the distance
between the nitro group and the carbonyl group in 3a–c was con-
cluded to be closer than that in MNI-based caged compounds. The
structures of 3b and 3c were determined unequivocally by X-ray
crystallography, and their ORTEP representations are shown in Fig-
ure 2.
The absorption spectra of compounds 3a–c varied depending on
the position of the methoxy substituent. Figure 3 shows the com-
parative absorption spectra of 3a–c in 1:1 MeOH/H2O. The longest
wavelength absorption peak of 3a appears at 325 nm, with an
extinction coefficient at 325 nm (e325 of 1490 cmꢀ1 Mꢀ1. When a
methoxy group was substituted at the 6-position (3b), the band
Table 1
Photochemical properties of 3a–c in MeOH/H2O (1:1) and MNDQ-Glu in buffer at pH
7.2
ekmax (Mꢀ1 cmꢀ1
(nm)
)
e365
Up
e365 Up
ꢁ
(Mꢀ1 cmꢀ1
)
(Mꢀ1 cmꢀ1
)
3a
3b
3c
1490 (325)
1650 (345)
4200 (345)
4200 (350)
4300 (350)
900
0.01
0.006
0.05
0.04
0.085
9
9
1500
3500
4000
175
160, 168 (
MNDQ-Glu
e350ꢁUp
)
MNI-Glu8a
366 (
e
350ꢁUp
)
Scheme 3. Plausible mechanism for photolysis of 3c.

N. Obi et al. / Tetrahedron Letters 51 (2010) 1642–1647
1645
Photolysis in 1:1 MeOH/H2O (without exclusion of O2) also gave
different results for the three test compounds. When 3a and 3b
were subjected to photoirradiation at 365 nm, the absorption spec-
tra changed without showing an isosbestic point. Irradiation of 3a
in an NMR tube (solvent: CD3OD) revealed that acetate was re-
leased, but the photoreaction of the protecting group led to compli-
cated spectra, probably due to the occurrence of side reactions or
immediate decomposition of the photoproduct. In contrast, clean
photolysis of 3c was confirmed by the observation of isosbestic
points at 257 and 340 nm and the development of a band at
384 nm (Fig. 4). This band, which is similar to those observed in
the photolysis of the MNI caging group,7a is probably due to the
production of nitroso aromatics. Figure 5 shows the change in
the 1H NMR spectrum during irradiation of 3c, in which new peaks
due to released acetate and the photoproducts of the caging group
appear around 6.5–8.5 ppm, increasing with irradiation time. This
set of signals in the aromatic region was equivalent to five protons.
High-resolution ESI-MS analysis after irradiation of 3c strongly
indicated that the photoproduct was not 1,2-dihydro-5-methoxy-
8-nitrosoquinoline but 5-methoxy-8-nitrosoquinoline 4 (Scheme
2), produced by oxidation of 1,2-dihydroquinoline during
photolysis.
Scheme 4. Synthesis of MNDQ-Glu.
We proposed a reaction mechanism for 3c based on the obser-
vation of a clean reaction forming acetate and 5-methoxy-8-nitros-
oquinoline (Scheme 3). The first step of photolysis is likely to be
intramolecular charge transfer from the anilino nitrogen to the 8-
nitro group. The reaction starts with N–C bond cleavage from the
excited singlet state, followed by formation of ground-state nitron-
ic anhydride, which undergoes unimolecular cleavage of the N–O
bond to give acetate and 4. The quantum yield for photolysis
(
Up) of 3c in 1:1 MeOH/H2O was 0.05, which, due to the clean reac-
tion, is equivalent to the quantum yield for production of acetate.
The photochemical efficiency of caged compounds is defined as
the product of Up and the molar extinction coefficient
For compound 3c this was calculated as 175, which is much greater
e
(e Up).
ꢁ
than the values calculated for 3a and 3b (Table 1). Since light can
be phototoxic to cells, a large
e
Up value is essential.6e,8a
ꢁ
Figure 7. 1H NMR spectra in D2O of the amino acid portion of MNDQ-Glu before (a)
and after (b) photolysis and authentic sample of -glutamate (c). On irradiation for
80 min. (b), the caging chromophore (d) decreases, -glutamate (N) is released, and
5-methoxy-8-nitroso quinoline (j) is simultaneously produced.
Figure 6. HPLC chromatogram of 3c before irradiation (a), 3c after irradiation for
5 min (b), MNDQ-Glu before irradiation (c), and MNDQ-Glu after irradiation for
5 min. (d).
L
L

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N. Obi et al. / Tetrahedron Letters 51 (2010) 1642–1647
Figure 8. (a) Whole-field UV illumination of 0.8 mM MNDQ-Glu induced a large inward current in a CA1 pyramidal neuron in an acutely isolated hippocampal brain slice. The
illumination time was 0.2 s (black bar) and the laser intensity was 14 mW/mm2. MNDQ-Glu was locally perfused onto the recording neuron through an application pipette.
(b) Two-photon uncaging of 10 mM MNDQ-Glu at a dendritic spine (arrowhead) induced a small inward current similar to a postsynaptic current. The illumination time was
1 ms (black bar) and the laser intensity was 9.8 mW.
Based on these experiments, we concluded that the substituent
pattern in 3c was the most appropriate among 3a–c for efficient
and clean photolysis, and therefore we chose the 1,2-dihydro-5-
methoxy-8-nitroquinolinyl group (MNDQ) as a caging group for
glutamic acid. The new caged glutamate MNDQ-Glu was synthe-
sized as shown in Scheme 4. A key intermediate in this synthesis
was the acid chloride of protected glutamate 5. Indeed, acylation
amplitude 0.68 0.06, mean S.E.M., n = 6 spines in 3 cells) than
that of the current elicited by MNI-Glu in the same spines. Thus,
the two-photon action cross section of MNDQ-Glu may be slightly
smaller than that of MNI-Glu.
In order to establish the full potential of MNDQ-Glu, further
physiological examinations are required. However, its high extinc-
tion coefficient (around 350 nm), moderate quantum yield for pho-
tolysis, high stability in the dark, biological inertness before
uncaging, and clean photolysis may extend the utility of the new
MNDQ-caging platform.
of 2c using
a-tert-butyl N-Boc glutamate with a peptide-coupling
reagent such as DCC or DMT-MM did not proceed under various
conditions. Finally, treatment with neat TFA removed all tert-bu-
tyl-protecting groups in precursor 6 to leave the water-soluble
product MNDQ-Glu. After reverse-phase HPLC purification,
MNDQ-Glu was identified by 1H-NMR and UV spectroscopy.
MNDQ-Glu showed good solubility in water (>10 mM) without
the use of any organic solvents, and stability in D2O and HEPES buf-
fer solution at pH 7.2, with no hydrolysis after 2 days in the dark at
room temperature.
3. Conclusion
A new photolabile-protecting group for carboxylic acids is de-
scribed. The 5-methoxy compound 3c was shown to release ace-
tate by clean and efficient photolysis. Caged glutamate MNDQ-
Glu was prepared using this compound as a caging group, and
The photochemical properties of MNDQ-Glu are shown in Table
1. Irradiation of MNDQ-Glu in buffer solution at pH 7.2 resulted in
the release of glutamate accompanied by quantitative rearrange-
ment of the MNDQ moiety to 5-methoxy-8-nitrosoquinoline 4.
This was confirmed based on the change in the UV absorption
spectrum and HPLC analysis (Fig. 6). The absorption change upon
irradiation of MNDQ-Glu in buffer solution at pH 7.2 (see SI) was
quite similar to that of 3c shown in Figure 3. Furthermore, NMR
analysis of the photolysis solution revealed that glutamate was re-
leased essentially quantitatively from MNDQ-Glu (Fig. 7). The
quantum yield for glutamate release from MNDQ-Glu was deter-
mined to be 0.04 in buffer solution at pH 7.2, which is a similar va-
lue to that for MNI-glu.8a
was shown to release L-glutamate with a quantum yield of 0.04
on irradiation with light at 365 nm. When MNDQ-Glu was tested
with pyramidal neurons, whole-field UV illumination resulted in
a large inward current due to the release of L-glutamate. A short
two-photon-uncaging reaction of MNDQ-Glu at single dendritic
spines induced a transient current that exhibited similar kinetic
properties to those induced by MNI-Glu. Further investigation of
the excited state and ground state reaction dynamics of this com-
pound as well as studies on the modification of quantum yield are
in progress.
Acknowledgements
MNDQ-Glu was tested on CA1 pyramidal neurons in acutely
isolated hippocampal brain slices for its ability to activate gluta-
mate ion channels upon photolysis. The current response of the cell
evoked by light flashes was recorded in whole cell voltage-clamp
mode, as shown in Figure 8. On whole-field UV illumination, a
large inward current was measured (Fig. 8a). This response was
This work was supported by Grants-in-Aid for Scientific Re-
search in a Priority Area ‘New Frontiers in Photochromism (No.
471 to T.A.)’, for young scientists (B) (No. 18750141 to A.M.), for
specially promoted research (No. 16002021 to H.K.), for scientific
research on priority areas (Elucidation of neural network function
in the brain) (No. 20021008 to M.M.), and for young scientists (A)
(No. 19680020 to M.M.) from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT), Japan.
clearly the result of light-induced release of L-glutamate, because
no response was evoked in slices containing no caged glutamate.
The rapid kinetics of the currents produced by photolysis of
MNDQ-Glu was comparable with those previously reported for
MNI-Glu. This result suggests that MNDQ-Glu does not act as a
partial antagonist for AMPA receptors. In addition, the fact that
the holding current did not change before UV irradiation of
MNDQ-Glu indicates that the compound remains caged under
physiological conditions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.12.081.
References and notes
Two-photon uncaging of MNDQ-Glu at a dendritic spine was
also carried out using a femtosecond laser with a wavelength of
720 nm. Based on the previously developed protocol for MNI-Glu,
glutamate-induced currents were recorded upon two-photon
uncaging of either MNDQ-Glu (Fig. 8b) or MNI-Glu (data not
shown). Uncaging of MNDQ-Glu-induced currents with similar ki-
netic properties to those induced by MNI-Glu; however, the ampli-
tude of the current elicited by MNDQ-Glu was smaller (relative
1. (a) Kaplan, J. H.; Forbush, B., III; Hoffman, J. F. Biochemistry 1978, 17, 1929–
1935; (b) Lester, H. A.; Nerbonne, J. M. Ann. Rev. Biophys. Bioeng. 1982, 11, 151–
175; (c) Adams, S. R.; Tsien, R. Y. Annu. Rev. Physiol. 1993, 55, 755–784; (d)
Marriott, G.. In Methods in Enzymology; Academic Press: San Diego, 1998; Vol.
;
(e) Goeldner, M.; Givens, R. Dynamic Studies in Biology; Wiley-VCH:
Weinheim, 2005; (f) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45,
4900–4921.
2. (a) Rossi, F. M.; Margulis, M.; Tang, C.-M.; Kao, J. P. Y. J. Biol. Chem. 1997, 272,
32933–32939; (b) Givens, R. S.; Jung, A.; Park, C.-H.; Weber, J.; Wenzel Bartlett,

N. Obi et al. / Tetrahedron Letters 51 (2010) 1642–1647
1647
W. J. Am. Chem. Soc. 1997, 119, 8369–8370; (c) Furuta, T.; Wang, S. S.-H.;
Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.; Denk, W.; Tsien, R. Y.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1193–1200; (d) Fedoryak, O. D.; Dore, T. M.
Org. Lett. 2002, 4, 3419–3422; (e) Shembekar, V. R.; Chen, Y.; Carpenter, B. K.;
Hess, G. P. Biochemistry 2005, 44, 7107–7114; (f) Senda, N.; Momotake, A.; Arai,
T. Bull. Chem. Soc. Jpn. 2007, 80, 2384–2388.
Soc. 1988, 110, 3212–3220; (c) Adams, S. R.; Lev-Ram, V.; Tsien, R. Y. Chem. Biol.
1997, 4, 867–868; (d) Ellis-Davies, G. C. R.; Kaplan, J. H. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 187–191; (e) Momotake, A.; Lindegger, N.; Niggli, E.; Barsotti, R.
J.; Ellis-Davies, G. C. R. Nat. Methods 2006, 3, 35–40; (f) Ellis-Davies, G. C. R.
Chem. Rev. 2008, 108, 1603–1613.
7. (a) Papageorgiou, G.; Corrie, J. E. T. Tetrahedron 2000, 56, 8197–8205; (b)
Matsuzaki, M.; Ellis-Davies, G. C.; Nemoto, T.; Miyashita, Y.; Iino, M.; Kasai, H.
Nat. Neurosci. 2001, 4, 1086–1092; (c) Canepari, M.; Ogden, D. J. Neurosci. 2003,
23, 4066–4071; (d) Smith, M. A.; Ellis-Davies, G. C. R.; Magee, J. C. J. Physiol.
2003, 548, 245–258; (e) Canepari, M.; Auger, C.; Ogden, D. J. Neurosci. 2004, 24,
3563–3573; (f) Bernardinelli, Y.; Haeberli, C.; Chatton, J.-Y. Cell Calcium 2005,
37, 565–572; (g) Maier, W.; Corrie, J. E. T.; Papageorgiou, G.; Laube, B.; Grewer,
C. J. Neurosci. Methods 2005, 142, 1–9; (h) Noguchi, J.; Matsuzaki, M.; Ellis-
Davies Graham, C. R.; Kasai, H. Neuron 2005, 46, 609–622; (i) Araya, R.;
Eisenthal Kenneth, B.; Yuste, R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18799–
18804; (j) Nikolenko, V.; Poskanzer, K. E.; Yuste, R. Nat. Methods 2007, 4, 943–
950; (k) Mim, C.; Tao, Z.; Grewer, C. Biochemistry 2007, 46, 9007–9018.
8. (a) Fedoryak, O. D.; Sul, J.-Y.; Haydonb, P. G.; Ellis-Davies, G. C. R. Chem.
Commun. 2005, 3664–3666; (b) Ellis-Davies, G. C. R.; Matsuzaki, M.; Paukert,
M.; Kasai, H.; Bergles, D. E. J. Neurosci. 2007, 27, 6601–6604.
9. (a) Papageorgiou, G.; Lukeman, M.; Wan, P.; Corrie, J. E. T. Photochem. Photobiol.
Sci. 2004, 3, 366–373; (b) Papageorgiou, G.; Ogden, D.; Corrie, J. E. T. J. Org.
Chem. 2004, 69, 7228–7233; (c) Papageorgiou, G.; Corrie, J. E. T. Tetrahedron
2005, 61, 609–616; (d) Papageorgiou, G.; Ogden, D.; Corrie, J. E. T. Photochem.
Photobiol. Sci. 2008, 7, 423–432.
10. Rao, K. V.; Jackman, D. J. Heterocycl. Chem. 1973, 10, 213–215.
11. Carroll, F. I.; Blackwell, J. T.; Philip, A.; Twine, C. E. J. Med. Chem. 1976, 19, 1111–
1119.
3. (a) Petersson, E. J.; Brandt, G. S.; Zacharias, N. M.; Dougherty, D. A.; Lester, H. A.
Methods in Enzymology, 360: Biophotonics; Academic Press: New York, 2003. pp
258–273; (b) Marriott, G.; Roy, P.; Jacobson, K. Methods in Enzymology, 360:
Biophotonics; Academic Press: New York, 2003. pp 274–289; (c) Lawrence, D. S.
Curr. Opin. Chem. Biol. 2005, 9, 570–575; (d) Li, H.; Hah, J.-M.; Lawrence, D. S. J.
Am. Chem. Soc. 2008, 130, 10474–10475.
4. (a) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M. J. Org. Chem. 1995, 60,
3953–3956; (b) Furuta, T.; Momotake, A.; Sugimoto, M.; Hatayama, M.; Torigai,
H.; Iwamura, M. Biochem. Biophys. Res. Commun. 1996, 228, 193–198; (c)
Schade, B.; Hagen, V.; Schmidt, R.; Herbrich, R.; Krause, E.; Eckardt, T.; Bendig, J.
J. Org. Chem. 1999, 64, 9109–9117; (d) Hagen, V.; Bendig, J.; Frings, S.; Eckardt,
T.; Helm, S.; Reuter, D.; Kaupp, U. B. Angew. Chem., Int. Ed. 2001, 40, 1046–1048;
(e) Hagen, V.; Frings, S.; Bendig, J.; Lorenz, D.; Wiesner, B.; Kaupp, U. B. Angew.
Chem., Int. Ed. 2002, 41, 3625–3628; (f) Hagen, V.; Dekowski, B.; Nache, V.;
Schmidt, R.; Geißler, D.; Lorenz, D.; Eichhorst, J.; Keller, S.; Kaneko, H.;
Benndorf, K.; Wiesner, B. Angew. Chem., Int. Ed. 2005, 44, 7887–7891.
5. (a) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet. 2001, 28, 317–325;
(b) Wenter, P.; Fürtig, B.; Hainard, A.; Schwalbe, H.; Pitsch, S. Angew. Chem., Int.
Ed. 2005, 44, 2600–2603; (c) Hobartner, C.; Silverman, S. K. Angew. Chem., Int.
Ed. 2005, 44, 7305–7309.
6. (a) Ellis-Davies, G. C. R.; Kaplan, J. H. J. Org. Chem. 1988, 53, 1966–1969; (b)
Adams, S. R.; Kao, J. P. Y.; Grynkiewicz, G.; Minta, A. V.; Tsien, R. Y. J. Am. Chem.