Rational design, synthesis, and characterization of highly fluorescent optical switches for high-contrast optical lock-in detection (OLID) imaging microscopy in living cells

Article abstract of DOI:10.1016/j.bmc.2010.07.015

A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-r

Full text of DOI:10.1016/j.bmc.2010.07.015

Bioorganic & Medicinal Chemistry 19 (2011) 1030–1040
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rational design, synthesis, and characterization of highly fluorescent
optical switches for high-contrast optical lock-in detection (OLID) imaging
microscopy in living cells
Chutima Petchprayoon a, Yuling Yan b,c, Shu Mao d, Gerard Marriott a,
*
a Department of Bioengineering, University of California, Berkeley, CA 94720, USA
b Department of Electrical Engineering, Santa Clara University, Santa Clara, CA 95053, USA
c Department of Otolaryngology, Stanford University, 801 Welch Road, Stanford, CA 94305, USA
d Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal
control of cellular processes. These studies require microscope imaging techniques and associated optical
probes that provide high-contrast and high-resolution images of specific proteins and their complexes.
Auto-fluorescence however, can severely compromise image contrast and represents a fundamental lim-
itation for imaging proteins within living cells. We have previously shown that optical switch probes and
optical lock-in detection (OLID) image microscopy improve image contrast in high background environ-
ments. Here, we present the design, synthesis, and characterization of amino-reactive and cell permeable
optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and
spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR–NISO undergoes
rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored
merocyanine (MC)-state in response to irradiation with 365 and >530 nm light. In the MC-state, the
TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET)
to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence
is maximal. Irradiation of TMR–NISO with a defined sequence of 365 and 546 nm manipulates the levels
of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High
fidelity optical switching of TMR fluorescence is shown for TMR–NISO probes in vitro and for membrane
permeable TMR–NISO within living cells.
Received 5 May 2010
Revised 25 June 2010
Accepted 7 July 2010
Available online 30 July 2010
Keywords:
Optical switch
FRET
Spironaphthoxazine
NISO
Tetramethylrhodamine
Fluorescence
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
cay back to the MC-ground state with the emission of red fluores-
cence, albeit with low quantum yield.3,5 In general, all known
Optical lock-in detection (OLID) microscopy allows for high-con-
trast imaging of a specific class of optically switchable fluorescent
probe even in the presence of time-varying background signals such
as cellular auto-fluorescence.1,2 Optical switch probes undergo rapid
and reversible, optically-drive transitions between two distinct
states that differ in their structural, physical, and spectroscopic
properties.3,4 For example, spironaphthoxazine (NISO; Fig. 1) exists
either as a colored merocyanine (MC)-state, or as the colorless and
non-fluorescent spiro (SP)-state.1,3,4 Transitions between SP- and
synthetic optical switches including NitroBIPS, NISO and diaryleth-
enes6 and genetically-encoded optical switches7 are better as optical
switches than they are as fluorophores.
Here, we present herein the rational design of optical switch
probes having greatly improved quantum yields for both fluores-
cence emission and optical switching. This property is achieved
by incorporating tetramethylrhodamine (TMR), a highly fluores-
cent probe for fluorescent imaging, and NISO, a highly efficient
optical switch,8–10 in the same molecule.11,12 Related probes based
on a fluorescent donor such as fluorescein and GFP and a switch-
able acceptor based on NitroBIPS were described by our group as
part of the OLID-FRET imaging technique.1 The highly overlapping
emission spectra of validated single molecule fluorophores such as
TMR, Cy3, and ATTO dyes with the MC-absorption of NitroBIPS
places a severe limitation on the ability to achieve orthogonal
modulation of the donor probe.1,13 In the probes detailed herein,
this limitation is overcome by using a MC-state whose absorption
MC-states occur via rapid (<ls), intramolecular, excited states reac-
tions.2 In particular, irradiation of SP with near ultraviolet (365 nm)
generates the MC-state, while irradiation of MC with visible
(>500 nm) light generates the SP-state. Interestingly, in the case of
nitrospirobenzopyran (NitroBIPS), the excited MC-state can also de-
* Corresponding author. Tel.: +1 510 664 4339.
E-mail address: marriott1@berkeley.edu (G. Marriott).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.015

C. Petchprayoon et al. / Bioorg. Med. Chem. 19 (2011) 1030–1040
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Figure 1. Optical control of TMR fluorescence intensity in TMR–NISO probes. Optical switching between SP- and MC-states of NISO generates the modulation of TMR
fluorescence intensity by FRET.
spectrum is much further red shifted compared to NitroBIPS,
allowing for orthogonal optical control of the TMR fluorescence.3
Thus the absorption spectrum of MC in TMR–NISO exhibits a high
spectral overlap with the emission spectrum of TMR and, given
their close proximity (<1 nm), FRET efficiency is close to perfect
in the MC-state of TMR–NISO and, concomitantly, TMR fluores-
cence is effectively extinguished. On the other hand, the absorption
spectrum of SP in TMR–NISO does not overlap with TMR emission
and so the FRET efficiency is zero and TMR fluorescence is maximal
(Fig. 1). Thus, by irradiating a TMR–NISO probe with cycles of a de-
fined sequence of near ultraviolet and visible light, one changes the
population of the SP- and MC-states with concomitant modulation
of the TMR fluorescence intensity. This property is useful in OLID
imaging microscopy and is useful feature for probes employed in
the stochastic optical reconstruction microscopy (STORM) tech-
nique.1,2 We present herein the design, synthesis, spectroscopic,
photochemical, and photophysical properties of various TMR–NISO
probes. Most of the studies that characterize the properties of the
amino-reactive and cell permeable TMR–NISO probes are per-
formed in vitro with select examples showing they perform as high
fidelity optical switches within living cells. More detailed applica-
tions of these probes within living cells will be presented
elsewhere.
and C-5 linked TMR–NISO probes (Fig. 2; 8a–c, 14, and 20) were
shown to undergo successful cycles of optical switching between
the SP- and MC-states with concomitant modulation of FRET effi-
ciency and TMR fluorescence (see characterization of optical
switching in the following section). The reactions employed for
the preparation of the TMR–NISO probes (8a–c, 14, and 20) are
shown in Schemes 1–3. The amino-substituted NISO (7a–c and
13) and N-hydroxysuccinimide ester of NISO (19) were synthesized
using standard methods and used for coupling to commercially
available fluorophores, namely 5-carboxytetramethylrhodamine,
succinimidyl ester (5-TAMRA-SE) or tetramethylrhodamine-5-car-
boxamide lysine (5-TAMRA-Lysine).
The reactions employed for the synthesis of several 4-amino
substituted NISO derivatives are shown in Scheme 1. The conden-
sation of indoline 3 with the corresponding 1-nitroso-2-naphthol
derivatives was used to prepare several of the TMR–NISO probes
(6a–c) with yields ranging from 20% to 40%. The amino-NISO deriv-
atives (7a–c) were obtained in good yield via the reaction of the
azido-NISO derivatives (6a–c) with triphenylphosphine (PPh3) in
a mixture of tetrahydrofuran (THF) and water. The reactions used
for the synthesis of the 5-amino substituted NISO derivative (13)
are shown in Scheme 2. A similar scheme was used to prepare
the 5-amino substituted NISO. The 4- and 5-amino-NISO deriva-
tives (7a–c and 13) were allowed to react with 5-TAMRA-SE,
affording the corresponding TMR–NISO probes (8a–c and 14) in
20–40% yield. The 5-TMR-Lys NISO (20) was synthesized as shown
in Scheme 3. The indoleninium salt (17) was prepared in three
steps from commercially available 4-(4-aminophenyl)butanoic
acid. Condensation of intermediate salt 17 with 1-nitroso-2-naph-
thol in ethanol in the presence of NEt3 led to the NISO (18) with a
yield of 32%. Treatment of NISO (18) with N-hydroxysuccinimide
(NHS) in the presence of N,N0-dicyclohexylcarbodiimide (DCC) gave
the N-hydroxysuccinimide ester derivative of NISO 19, which was
further used for coupling with 5-TAMRA-Lysine to give 5-TMR-
Lys NISO (20) in 42% yield.
2. Results and discussion
2.1. Design of highly fluorescent optical switches
The low fluorescence quantum yield of the MC-state of NitroB-
IPS is a result of the competing loss of the MC-excited state via the
MC to SP transition.3,4,14 We therefore rationalized that the best
design for highly fluorescent optical switches would involve
uncoupling the fluorescence and optical switching processes. In
particular, we designed a class of optical switch probes that con-
tain TMR, a highly fluorescent and validated probe for single mol-
ecule imaging15–17 and NISO, a highly efficient and robust optical
switch.4,8–10 The quantum yield of TMR fluorescence in TMR–NISO
was reasoned to be at a maximum in the colorless SP-state while
close to zero in the colored MC-state (kmax ꢀ620 nm) as a result
of FRET.
The next step in the design of fluorescent optical switches was
to introduce an amino-reactive functionality into the TMR–NISO
probes (Fig. 2; 28a–b) for coupling to proteins and other biomole-
cules. The reactions used to synthesize the amino-reactive TMR–
NISO probes are shown in Scheme 4. First, bi-functional NISO
derivatives were prepared by reaction of indoleninium halide 17
and 23 with nitrosonaphthol 22 in ethanol in the present of NEt3
to give compounds 24a and 24b, respectively. These NISO deriva-
tives (24a and 24b) were converted to the amino derivatives
(26a and 26b, respectively), in two steps. The amino-NISO deriva-
tives 26a and 26b were coupled to 5-TAMRA-SE following by a
reaction with NHS in the presence of DCC to generate the N-
hydroxysuccinimide esters of the TMR–NISO probes 28a and 28b
2.2. Syntheses
The design features outlined above were realized through the
synthesis of several TMR–NISO probes in which TMR was cova-
lently linked to NISO through the C-4 or C-5 carbon atoms on NISO.
These probes were used to investigate whether the coupling of
NISO to TMR affected its optical switching properties. Both C-4

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Figure 2. Structures of TMR–NISO probes.
Scheme 1. Synthesis of 4-TMR NISO derivatives.
in 63 and 53% yield, respectively. These NHS ester probes are reac-
tive with lysine residues in proteins and can be used to generate
protein conjugates of TMR–NISO. The characterization and applica-
tion of these conjugates will be presented elsewhere.
All TMR–NISO probes were confirmed from measurements of
their exact molecular mass obtained from ESI mass spectra.
2.3. Spectroscopic and photophysical properties of TMR–NISO
probes
Key spectroscopic and photophysical properties of TMR–NISO
probes were measured in solution and compared to the same prop-
erties found for free TMR and free NISO. The SP-state of TMR–NISO

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Scheme 2. Synthesis of 5-TMR NISO derivative.
Scheme 3. Synthesis of 5-TMR-Lys NISO derivative.
proved to be the most thermodynamically-favored form at room
temperature for all of the solvents used in this study (Fig. 3A).
The absorption spectra of TMR–NISO probes (8a–c, 14, 20, and
28a–b) in their SP- and MC-states in glycerol are shown in Fig-
ure 3A and B, respectively. The TMR moiety in TMR–NISO exhibits
a maximum (S0–S1) absorption at 556–557 nm. The SP-absorption
of NISO within the TMR–NISO molecule is buried within the S0–S2
transition of TMR making it difficult to analyze. Irradiation of TMR–
NISO with 365 nm generates the corresponding MC-state having
an absorption maximum of 612–625 nm (Fig. 3B). The intensity
of the MC-absorption in TMR–NISO decreased with time as a result
of a thermally-driven, ground state transition that formed the col-
orless SP-state.4 The time constant for this thermally-driven reac-
tion was measured for several TMR–NISO probes in glycerol at
room temperature (Table 1). These results show that the time con-
stant for the MC to SP transition is dependent on the nature of sub-
stituent groups on NISO.18–21 The C-5 alkylated-NISO stabilizes the
MC-state compared to alkylation at the 4-position (14 and 8a,
respectively). Substituent groups at the 90-position also stabilize
the MC-state (8a–c), as does the presence of longer side chains
(8b–c, 14, and 20).
free TMR. This results suggests that the high quantum efficiency
of TMR in TMR–NISO is unaffected by the presence of the closely
situated SP-state. On the other hand, the quantum yield for TMR
fluorescence is very low in the MC-state of TMR–NISO because of
the high efficiency of FRET between the TMR and MC groups. Thus,
irradiation of the SP-state of TMR–NISO (8b) with 365 nm light in
glycerol results in a significant decrease in the fluorescence inten-
sity of TMR that is caused by formation of the MC-state and FRET
(Fig. 3C). The recovery of the fluorescence signal from TMR follow-
ing this UV-pulse is primarily due to the thermally-driven MC to SP
transition (Fig. 3C). Optical manipulation of the SP- and MC-states
of TMR–NISO over several cycles of optical switching is shown to
modulate the fluorescence intensity of TMR and can be repeated
over many cycles with little evidence of bleaching or secondary
photochemical reactions (Fig. 3C).3 Accurate determination of the
quantum yield of TMR fluorescence in the MC-state of TMR–NISO
is quite difficult using cuvette measurements such as those shown
in Figure 3C owing to the large volume but it is far easier when
using a fast imaging OLID microscope as is shown later.
2.4. Optical switching of TMR fluorescence in TMR–NISO in
living cells
The quantum yield for TMR fluorescence in the SP-state of
TMR–NISO was measured for several TMR–NISO probes in glycerol,
methanol, and water using rhodamine 101 in ethanol as a refer-
ence22 (Table 1). The quantum yield for TMR fluorescence mea-
sured in these TMR–NISO probes is similar to that measured for
The TMR–NISO probes described in this study are readily taken
up by living cells where they localize to vesicle-like structures that
may include mitochondria, as shown in Figure 4A. Optical manip-

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Scheme 4. Synthesis of N-hydroxysuccinimide ester derivatives of TMR–NISO.
ulation of TMR fluorescence in cells loaded TMR–NISO was per-
formed using a home-built OLID microscope as detailed in the
methods section. The field of view was subject to several cycles
of defined excitation of the field with 546 and 365 nm light. In par-
ticular, we employed continuous irradiation of the field with
546 nm light while recording the TMR fluorescence and irradiated
the same field every 2 s with a 100 ms pulse of 365 nm light. The
365 nm pulse was shown to convert almost all of the SP-state of
TMR–NISO to MC in the field of view, as longer irradiation times
did not appreciably change the lowest measured value of TMR
fluorescence. A trace of the TMR fluorescence intensity over eight
cycles of optical switching within a region of interest in a cell-
loaded preparation is shown in Figure 4B. Immediately following
the 365 nm pulse, the TMR intensity decreases as a result of FRET
to a value that is about 65–70% below the SP level. The recovery of
TMR fluorescence, seen inFigure 4B, is due to both direct excitation
of the MC-state with 546 nm light (the MC has a non-zero absorp-
tion value at 546 nm) and the thermally-driven MC to SP transi-
tion. Given the presence of an auto-fluorescence background and
the relatively long UV-pulse, it is difficult to arrive at the time zero
value of TMR fluorescence from data shown in the trace (Fig. 4B).
However, extrapolation of the intensity trace of TMR fluorescence
following irradiation with 365 nm light (Fig. 4C) allows us to esti-
mate the TMR signal is close to zero at the end of the UV-pulse.
Other important properties deduced from this live cell study in-
clude; first, optical switching is robust and proceeds with high
fidelity over the eight cycles; second, the intensities of TMR fluo-
rescence are similar immediately before and immediately after
the UV-pulse; third, the recovery of the TMR fluorescence intensity
for each cycle of the study is best fit with a single exponential hav-
ing a time constant of ꢀ200 ms. These data strongly suggest that
optical manipulation of the SP- and MC-states of TMR–NISO in liv-
ing cells occurs with high fidelity and little evidence of fatigue or
secondary photochemistry. The rate of recovery of TMR–NISO fluo-
rescence during 546 nm excitation of the MC-state with 546 nm
light was shown to depend on the square of the 546 nm light inten-
sity. Thus the result shown in Figure 5A and B strongly suggests
that energy used to excite the MC group in TMR–NISO results is
sub-saturating. Thus the time constant measured for recovery of
TMR fluorescence intensity for each cycle of optical switching
shown in Figure 5B is primarily determined by the quantum yield
for the MC to SP transition and the number of molecules of MC in
the exited state. This time constant can be shortened by increasing
the number of MC molecules in the excited state, for example, by
increasing the energy of the 546 nm light, or by irradiating the
MC group closer to its maximum absorption wavelength, that is,
632 nm. In the limit, that is, under a condition of saturating excita-
tion, it should be possible to obtain a square wave modulation of
TMR intensity.
The excited-state driven reactions between the MC- and SP-
states are faster than 2 l
s,2 the modulation of TMR intensity is fas-
ter than any of the optical switch probes used for super-resolution
imaging, including photoactivatable proteins and Cy3/Cy5
probes.17 Perhaps more significant for studies in living cells, optical
switching of TMR fluorescence in TMR–NISO occurs under normal
conditions of cell culture, that is, in medium and without the need

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Figure 4. Optical switching of TMR–NISO (8a) in living cells. (A) Single image frame
from a movie reflecting the distribution of 8a within living NIH 3T3 cells. (B) A trace
of TMR fluorescence intensity over eight cycles of optical switching of the 8a within
a region of interest (red square) within the image field (A). (C) The rate of return of
the TMR fluorescence signal for one of the cycles shown in (B) following a 365 nm
pulse was best fit by a single exponential that having a 200 ms time constant.
Figure 3. Absorption spectra and fluorescence intensity profile of TMR–NISO
probes. Absorption spectra of TMR–NISO probes 8a–c, 14, 20, and 28a–b in SP- (A)
and MC-states (B) in glycerol (inset: expand region of MC-NISO absorption between
580 and 660 nm). (C) TMR fluorescence intensity profile of 8b over six cycles of
optical switching in glycerol. A typical optical switching cycle was composed of 30 s
of 365 nm light and 60 s of 546 nm light.
would be especially useful for super-resolution imaging and OLID
imaging of proteins in living cells where diffusion of the probe dur-
ing the acquisition period is a serious problem.
3. Conclusion
We have synthesized several members of a new class of highly
fluorescent optical switch that incorporates the highly fluorescent
TMR probe and NISO, a highly efficient optical switch. The NISO
of high concentrations (molar) mercaptans or other reducing
agents or anoxia.17 The rapid switching of TMR fluorescence in
TMR–NISO and the high quantum yield of TMR in the SP-state
Table 1
Kinetic and quantum yield of TMR–NISO derivatives in glycerol, methanol, and water at room temperature
Compound
Quantum yield
Thermal decay ratea
(MC-NISO, ꢁ10ꢂ2 sꢂ1
)
Glycerol
MeOH
Water
5-TAMRA, SE
0.76
0.89
0.91
0.98
0.58
0.63
0.65
0.73
0.71
0.77
0.51
0.53
0.43
0.38
0.29
0.20
0.25
0.21
—
8a
8b
8c
14
20
4.00
3.14
2.36
2.22
1.60
a
In glycerol.

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Figure 5. TMR fluorescence intensity recovery of TMR–NISO by OLID-FRET microscopy. (A) TMR fluorescence intensity recovery after 365 nm pulse using different
illumination energy: 100% (}), 50% (h), and 12.5% (4). (B) Half life of TMR fluorescence depends on illumination intensity.
group in TMR–NISO undergoes orthogonal, optically driven transi-
tions between a colorless SP-state, and a colorful MC-state that
serves as an acceptor probe in FRET with TMR. In particular, the
close proximity of the TMR and MC groups in TMR–NISO results
in almost perfect FRET and decreases the quantum yield of TMR
fluorescence to almost zero whereas the quantum yield for TMR
fluorescence in the SP-state is at a maximum. Optical switching be-
tween the SP- and MC-states was shown to result in robust modu-
lation of the intensity of TMR fluorescence over many cycles of
optical switching. The TMR–NISO probe is useful for imaging of
structures in living cells and to our knowledge, optical switching
of TMR fluorescence is faster than any genetically-encoded or syn-
thetic optical switch. Moreover the TMR–NISO probes detailed
herein undergo reversible optical switching without the need to
exogenous reactants and do so under conditions compatible with
healthy and aerobically grown cells.
carboxylate (DIAD, 1.5 equiv) was added dropwise over 1 min to
the reaction mixture. The reaction mixture was allowed to warm
to room temperature and stirred for 4 h. The reaction mixture
was concentrated and purified by a silica gel flash column using
appropriate eluent to give the product.
4.2.2. General procedure for synthesis indoline or indoleninium
salt
Method A: A mixture of indolenine and alkyl halide (8 equiv) in
dichloromethane (CH2Cl2) was heated under reflux for 12 h. The
reaction mixture was cooled to room temperature and then evap-
orated to give a residue. The residue was dissolved in an aqueous
sodium hydroxide (NaOH, 0.5 M) and stirred at room temperature
for 30 min and then extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4 and evaporated to give a crude ex-
tract that was further used for next reaction without purification.
Method B: A mixture of indolenine and alkyl halide (2–8 equiv) in
CH2Cl2 or 1,2-dichlorobenzene was heated under reflux for 12 h.
The reaction mixture was cooled to room temperature and concen-
trated. The precipitate was filtered and washed with hexane sev-
eral times to give a residue that was used for next reaction
without purification.
4. Experimental
4.1. General procedures
Starting materials were purchased from Sigma–Aldrich, Fisher
Scientific, TCI America, Invitrogen, and AnaSpec. 4-(Hydroxy-
methyl)-2,3,3-trimethyl-3H-indole (1) was prepared as previously
reported.3 1H NMR spectra were recorded at 300 MHz on a Bruker
AC-300 spectrometer or a Varian MercuryPlus 300. Mass spectra
were recorded on a Micromass LCT for ESI or a Micromass Auto-
4.2.3. General procedure for nitrosation
To a stirred solution of 2-hydroxynaphthalene derivatives
(1 equiv) in a 5:1 mixture of glacial acetic acid and water on an
ice bath, an aqueous sodium nitrite (NaNO2, 1 equiv) was added
dropwise. After 10 min, an additional aqueous NaNO2 (0.05 equiv)
was added and the reaction mixture was further stirred at 0 °C for
2 h. The precipitate was collected by filtration, washed with dilute
acetic acid and water, and dried in vacuo.
Spec for EI. UV spectra were determined with
a Shimadzu
1601PC instrument. Fluorescence spectroscopy was performed on
an SLM-AB2 instrument (Thermoelectron, Madison, WI).
4.2. Syntheses
4.2.4. General procedure for synthesis NISO
Method A: To a solution of 1-nitroso-2-naphthol derivatives
(1 equiv) in ethanol (EtOH), an equimolar solution of indoline
derivatives in EtOH was added. The reaction mixture was heated
under reflux for 2 h. After cooling to room temperature, the mix-
ture was evaporated to give a residue. The residue was purified
by a Sephadex LH-20 column (methanol; MeOH) and a silica gel
column (hexane/ethyl acetate; EtOAc) to give a product. Method
B: To a solution of 1-nitroso-2-naphthol derivatives (1 equiv) in
EtOH, indoleninium salt derivatives (1 equiv) in EtOH and NEt3
(1 equiv) were added. The reaction mixture was heated under re-
flux for 5 h. After cooling to room temperature, the mixture was
4.2.1. General procedure for Mitsunobu reaction
A paste of sodium azide (260 mg, 4 mmol) in water (260
was stirred on an ice bath, and benzene (1.6 mL) was added. Con-
centrated sulfuric acid (H2SO4, 106 L, 2 mmol) was carefully
lL)
l
added dropwise to the reaction mixture. After stirred for 10 min,
the organic layer containing hydrazoic acid was separated and
dried over anhydrous magnesium sulfate (MgSO4). To a mixture
of indolenine (1 equiv) and triphenylphosphine (PPh3, 1.5 equiv)
in dry tetrahydrofuran (THF) stirred on an ice bath under a nitro-
gen atmosphere, the solution of hydrazoic acid in benzene
(1.5 equiv) was added. After stirred for 10 min, diisopropyl azodi-

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1037
evaporated to give a residue that was further separated by a Sepha-
4.2.13. 1,3,3-Trimethyl-4-(azidomethyl)spiro[indoline-2,30-[3H
dex LH-20 column (MeOH) and a silica gel column (hexane/EtOAc)
to give the product.
]naphtho[2,1-b][1,4]oxazine] (6a)
This compound was synthesized according to general procedure
for synthesis NISO method A. Yield 6 mg, 41%. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 3H), 1.47 (s, 3H), 2.75 (s, 3H), 4.38 (d,
J = 13.6 Hz, 1H), 4.44 (d, J = 13.6 Hz, 1H), 6.58 (dd, J = 0.8, 7.8 Hz,
1H), 6.82 (dd, J = 0.8, 7.8 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 7.24 (t,
J = 7.8 Hz, 1H), 7.39 (ddd, J = 1.3, 8.1, 8.2 Hz, 1H), 7.58 (ddd,
J = 1.4, 8.2, 8.5 Hz, 1H), 7.67 (br d, J = 9.0 Hz, 1H), 7.742 (s, 1H),
7.745 (br d, J = 8.1 Hz, 1H), 8.56 (br d, J = 8.5 Hz, 1H) ppm; EIMS:
m/z 383.1741 (M)+, calcd for C23H21N5O, 383.1741.
4.2.5. General procedure for synthesis azido derivatives from
chloro derivatives
To a solution of 90-chloropropoxy derivatives of NISO in dry N,N-
dimethylformamide (DMF), sodium azide (2 equiv) was added.
After stirring for 24 h at 80 °C, water was added to the reaction
mixture and then extracted with CH2Cl2. The organic layer was
dried over anhydrous MgSO4 and evaporated to give the product.
4.2.6. General procedure for reduction azide to amine
To a stirred solution of azido-NISO derivatives in a 10:1 mixture
of THF and water, PPh3 (2 equiv) was added. The reaction mixture
was stirred at room temperature for 24 h. The solvent was re-
moved to give a residue that was further purified by a silica gel
TLC (CH2Cl2/MeOH) to give the product.
4.2.14. 1,3,3-Trimethyl-4-(azidomethyl)-90-hydroxyspiro[indo
line-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (6b)
This compound was synthesized according to general procedure
for synthesis NISO method A. Yield 4 mg, 20%. 1H NMR (CDCl3,
300 MHz) d 1.455 (s, 3H), 1.46 (s, 3H), 2.75 (s, 3H), 4.38 (d,
J = 13.7 Hz, 1H), 4.44 (d, J = 13.7 Hz, 1H), 6.59 (dd, J = 0.7, 7.8 Hz,
1H), 6.83 (dd, J = 0.7, 7.8 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 7.02 (dd,
J = 2.6, 8.6 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.59 (br d, J = 8.8 Hz, 1H),
7.66 (br d, J = 8.6 Hz, 1H), 7.70 (s, 1H), 7.86 (br d, J = 2.6 Hz, 1H)
ppm; ESIMS: m/z 400.1768 (M+H)+, calcd for C23H22N5O2, 400.1769.
4.2.7. General procedure for synthesis N-hydroxysuccinimide
ester derivatives
To a solution of N-hydroxysuccinimide (NHS, 1.5 equiv) and
N,N0-dicyclohexylcarbodiimide (DCC, 1.5 equiv) in dry THF (1 mL),
carboxyl derivative of NISO (or TMR–NISO, 1 equiv) in dry THF
(or a 1:1 mixture of dry THF and dry DMF) was added. The reaction
mixture was stirred at room temperature for 12 h. The precipitate
was filtered out and the filtrate was evaporated and purified by sil-
ica gel TLC (CH2Cl2/MeOH) to give the product.
4.2.15. 1,3,3-Trimethyl-4-(azidomethyl)-90-methoxyspiro[ind
oline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (6c)
This compound was synthesized according to general procedure
for synthesis NISO method A. Yield 5 mg, 22%. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 3H), 1.47 (s, 3H), 2.76 (s, 3H), 4.01 (s, 3H),
4.38 (d, J = 13.6 Hz, 1H), 4.44 (d, J = 13.6 Hz, 1H), 6.59 (br d,
J = 7.3 Hz, 1H), 6.82 (dd, J = 0.7, 7.3 Hz, 1H), 6.85 (d, J = 8.6 Hz,
1H), 7.04 (dd, J = 2.6, 9.0 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.58 (br
d, J = 8.6 Hz, 1H), 7.63 (br d, J = 9.0 Hz, 1H), 7.72 (s, 1H), 7.87 (br
d, J = 2.6 Hz, 1H) ppm; EIMS: m/z 413.1851 (M)+, calcd for
4.2.8. General procedure for synthesis TMR–NISO derivatives
A solution of 5-carboxytetramethylrhodamine, succinimidyl es-
ter (5-TAMRA-SE) or tetramethylrhodamine-5-carboxamide lysine
(5-TAMRA-Lys, 1 equiv) in dry DMF (100 lL) and triethylamine
(NEt3, 1.5 equiv) were added to a solution of amino-NISO deriva-
tives or NHS–NISO derivative (1.5 equiv), respectively, in dry
CH2Cl2. The reaction mixture was stirred at room temperature
overnight and then evaporated to give a residue. The residue was
purified by a silica gel TLC (CH2Cl2/MeOH; 7:3) to give product.
C24H23N5O2, 413.1847.
4.2.16. 1,3,3-Trimethyl-4-(aminomethyl)spiro[indoline-2,30-[3H
]naphtho[2,1-b][1,4]oxazine] (4-NH2 NISO, 7a)
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 3.4 mg, 91%. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 3H), 1.50 (s, 3H), 2.74 (s, 3H), 3.95 (m, 2H),
6.51 (br d, J = 7.9 Hz, 1H), 6.92 (br d, J = 7.9 Hz, 1H), 7.02 (d,
J = 8.7 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.40 (ddd, J = 1.3, 7.7,
8.0 Hz, 1H), 7.58 (ddd, J = 1.3, 8.0, 8.5 Hz, 1H), 7.67 (br d,
J = 8.7 Hz, 1H), 7.75 (s, 1H), 7.76 (br d, J = 7.7 Hz, 1H), 8.56 (br d,
J = 8.5 Hz, 1H) ppm; ESIMS: m/z 358.1929 (M+H)+, calcd for
4.2.9. 4-(Azidomethyl)-2,3,3-trimethyl-3H-indole (2)
This compound was synthesized according to general procedure
for Mitsunobu reaction. Yield 193 mg, 90%. 1H NMR (CDCl3,
300 MHz) d 1.41 (s, 6H), 2.28 (s, 3H), 4.49 (s, 2H), 7.15 (dd,
J = 0.9, 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.55 (dd, J = 0.9, 7.8 Hz,
1H) ppm; ESIMS: m/z 215.1285 (M+H)+, calcd for C12H15N4,
215.1291.
C23H24N3O, 358.1914.
4.2.17. 1,3,3-Trimethyl-4-(aminomethyl)-90-hydroxyspiro[indo
line-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (4-NH2, 90-OH NISO,
7b)
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 2.5 mg, 89%. 1H NMR (CDCl3,
300 MHz) d 1.44 (s, 3H), 1.46 (s, 3H), 2.74 (s, 3H), 3.91 (d,
J = 14.7 Hz, 1H), 3.99 (d, J = 14.7 Hz, 1H), 6.51 (br d, J = 7.8 Hz, 1H),
6.82 (d, J = 8.9 Hz, 1H), 6.90 (br d, J = 7.8 Hz, 1H), 7.00 (dd, J = 2.7,
9.5 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.57 (br d, J = 8.9 Hz, 1H), 7.64
(br d, J = 9.5 Hz, 1H), 7.66 (s, 1H), 7.82 (br d, J = 2.7 Hz, 1H) ppm;
ESIMS: m/z 374.1861 (M+H)+, calcd for C23H24N3O2, 374.1864.
4.2.10. 4-(Azidomethyl)-1,3,3-trimethyl-2-methyleneindoline
(3)
This compound was synthesized according to general procedure
for synthesis indoline or indoleninium salt method A.
4.2.11. 1-Nitroso-2,7-dihydroxynaphthalene (4)
This compound was synthesized according to general procedure
for nitrosation. Yield 116 mg, 88%. 1H NMR (DMSO-d6, 300 MHz) d
6.19 (br s, 1H), 6.90 (dd, J = 2.3, 7.9 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H),
7.62 (br s, 1H), 8.41 (br s, 1H) ppm; EIMS: m/z 189.0428 (M)+, calcd
for C10H7NO3, 189.0421.
4.2.18. 1,3,3-Trimethyl-4-(aminomethyl)-90-methoxyspiro[indo
line-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (4-NH2, 90-OMe NISO,
7c)
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 4.6 mg, 93%. 1H NMR (CDCl3,
300 MHz) d 1.46 (s, 3H), 1.50 (s, 3H), 2.74 (s, 3H), 3.99 (d,
J = 14.7 Hz, 1H), 4.01 (s, 3H), 4.01 (d, J = 14.7 Hz, 1H), 6.50 (br d,
4.2.12. 7-Methoxy-1-nitroso-2-naphthol (5)
This compound was synthesized according to general procedure
for nitrosation. Yield 199 mg, 98%. 1H NMR (DMSO-d6, 300 MHz) d
3.83 (s, 3H), 6.26 (br d, J = 9.3 Hz, 1H), 7.11 (dd, J = 2.7, 8.5 Hz, 1H),
7.55 (d, J = 8.5 Hz, 1H), 7.69 (br d, J = 9.3 Hz, 1H), 8.49 (br s, 1H)
ppm; ESIMS: m/z 204.0663 (M+H)+, calcd for C11H10NO3, 204.0656.

1038
C. Petchprayoon et al. / Bioorg. Med. Chem. 19 (2011) 1030–1040
J = 7.4 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 6.90 (br d, J = 7.4 Hz, 1H),
7.03 (dd, J = 2.6, 8.8 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 7.57 (br d,
J = 8.8 Hz, 1H), 7.64 (br d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 7.87 (br d,
J = 2.6 Hz, 1H) ppm; ESIMS: m/z 388.2020 (M+H)+, calcd for
300 MHz) d 1.34 (s, 3H), 1.35 (s, 3H), 2.74 (s, 3H), 2.87 (t,
J = 7.2 Hz, 2H), 3.49 (t, J = 7.2 Hz, 2H), 6.51 (d, J = 7.9 Hz, 1H), 6.93
(d, J = 1.6 Hz, 1H), 7.01 (d, J = 8.9 Hz, 1H), 7.05 (dd, J = 1.6, 7.9 Hz,
1H), 7.39 (ddd, J = 1.2, 7.8, 8.1 Hz, 1H), 7.57 (ddd, J = 1.0, 8.1,
8.5 Hz, 1H), 7.66 (br d, J = 8.9 Hz, 1H), 7.74 (dd, J = 1.0, 7.8 Hz,
1H), 7.744 (s, 1H), 8.56 (dd, J = 1.2, 8.5 Hz, 1H) ppm; EIMS: m/z
397.1910 (M)+, calcd for C24H23N5O, 397.1898.
C24H26N3O2, 388.2020.
4.2.19. 5-Carboxytetramethylrhodamine derivative of 1,3,3-tri
methyl-4-(aminomethyl)spiro[indoline-2,30-[3H]naphtho[2,1-
b][1,4]oxazine] (4-TMR NISO, 8a)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 0.56 mg, 38%. ESIMS: m/
z 770.3315 (M+H)+, calcd for C48H44N5O5, 770.3337.
4.2.26. 1,3,3-Trimethyl-5-(aminoethyl)spiro[indoline-2,30-[3H
]naphtho[2,1-b][1,4]oxazine] (5-NH2 NISO, 13)
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 2.0 mg, 91%. 1H NMR (CDCl3,
300 MHz) d 1.34 (s, 3H), 1.35 (s, 3H), 2.74 (s, 3H), 2.74 (t,
J = 6.7 Hz, 2H), 2.97 (m, 2H), 6.51 (d, J = 7.8 Hz, 1H), 6.92 (d,
J = 1.5 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 7.04 (dd, J = 1.5, 7.8 Hz,
1H), 7.39 (ddd, J = 0.8, 8.0, 8.1 Hz, 1H), 7.57 (ddd, J = 1.3, 8.0,
8.3 Hz, 1H), 7.66 (br d, J = 8.9 Hz, 1H), 7.74 (br d, J = 8.1 Hz, 1H),
7.74 (s, 1H), 8.56 (dd, J = 8.3 Hz, 1H) ppm; ESIMS: m/z 372.2067
(M+H)+, calcd for C24H26N3O, 372.2071.
4.2.20. 5-Carboxytetramethylrhodamine derivative of 1,3,3-tri
methyl-4-(aminomethyl)-90-hydroxyspiro[indoline-2,30-[3H
]naphtho[2,1-b][1,4]oxazine] (4-TMR, 90-OH NISO, 8b)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 0.37 mg, 25%. ESIMS: m/
z 786.3281 (M+H)+, calcd for C48H44N5O6, 786.3287.
4.2.21. 5-Carboxytetramethylrhodamine derivative of 1,3,3-tri
methyl-4-(aminomethyl)-90-methoxyspiro[indoline-2,30-[3H
]naphtho[2,1-b][1,4]oxazine] (4-TMR, 90-OMe NISO, 8c)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 0.33 mg, 22%. ESIMS: m/
z 800.3412 (M+H)+, calcd for C49H46N5O6, 800.3443.
4.2.27. 5-Carboxytetramethylrhodamine derivative of 1,3,3-
trimethyl-5-(aminoethyl)spiro[indoline-2,30-[3H]naphtho[2,1-
b][1,4]oxazine] (5-TMR NISO, 14)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 0.31 mg, 21%. ESIMS: m/
z 784.3458 (M+H)+, calcd for C49H46N5O5, 784.3494.
4.2.22. 5-(Hydroxyethyl)-2,3,3-trimethyl-3H-indole (9)
4.2.28. 2,3,3-Trimethyl-5-(ethyloxycarbonylpropyl)-3H-indole
Toastirredsolutionof4-aminophenethylalcohol(1 g, 7.29 mmol)
ina1:1mixtureofconcentratedHClandwater(4 mL)at0 °C, anaque-
oussolution(1.5 mL)of NaNO2 (504 mg, 7.30 mmol)wasaddeddrop-
wise. After 1 h, a solution of stannous chloride dihydrate (SnCl2ꢃ2H2O,
4.93 g, 21.87 mmol)inconcentratedHCl(1.5 mL)wasaddedtoareac-
tionmixture. The reaction mixture was stirred for additional 1 h, then
neutralized with an aqueous NaOH, and filtered to give a milk white
solid. The solid was dissolved in MeOH and filtered to remove an
insoluble solid. The filtrate was evaporated to afford 4-hydrazino-
phenethylalcohol, which was used fornextreaction withoutpurifica-
tion. The obtained 4-hydrazinophenethyl alcohol was dissolved in
EtOH (8 mL) and refluxed with 3-methyl-2-butanone (1.49 mL,
(15)
To
(100 mg, 0.56 mmol) in a 1:1 mixture of concentrated HCl) and
water (1.4 mL) at 0 °C, an aqueous solution (500 L) of NaNO2
a stirred solution of 4-(4-aminophenyl)butanoic acid
l
(42 mg, 0.61 mmol) was added dropwise. After 1 h, a solution of
stannous chloride dihydrate (SnCl2ꢃ2H2O, 377 mg, 1.67 mmol) in
concentrated HCl (500 lL) was added to a reaction mixture. The
reaction mixture was stirred for additional 1 h and filtered to give
a milk white solid. The obtained solid was dissolved in EtOH (3 mL)
and refluxed with 3-methyl-2-butanone (729
concentrated sulfuric acid (H2SO4, 56 L, 0.56 mmol) for 12 h. After
concentration, the residue was neutralized with a saturated aque-
ous sodium carbonate (Na2CO3) and extracted with diethyl ether
(Et2O). The organic layer was dried over anhydrous MgSO4 and
then evaporated to give 15 (117 mg, 76% based on 4-(4-aminophe-
nyl)butanoic acid). 1H NMR (CDCl3, 300 MHz) d 1.26 (t, J = 7.3 Hz,
3H), 1.29 (s, 6H), 1.97 (quin, J = 7.7 Hz, 2H), 2.26 (s, 3H), 2.34 (t,
J = 7.7 Hz, 2H), 2.69 (t like, J = 7.7 Hz, 2H), 4.13 (q, J = 7.3 Hz, 2H),
7.09 (s, 1H), 7.10 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H) ppm;
ESIMS: m/z 274.1815 (M+H)+, calcd for C17H24NO2, 274.1802.
lL, 1.13 mmol) and
l
14.59 mmol) and concentrated H2SO4 (729 lL) for12 h. Afterconcen-
tration, the reaction mixture was washed with diethyl ether, neutral-
ized with a saturated aqueous sodium carbonate (Na2CO3), and
extracted with CH2Cl2. The organic layer was dried over anhydrous
MgSO4 and evaporated to give 9 (450 mg, 30% based on 4-amino-
phenethyl alcohol). 1H NMR (CDCl3, 300 MHz) d 1.27 (s, 6H), 2.24 (s,
3H), 2.90 (t, J = 6.8 Hz, 2H), 3.86 (t, J = 6.8 Hz, 2H), 7.130 (d,
J = 1.9 Hz, 1H), 7.134 (d, J = 7.9 Hz, 1H), 7.41 (br d, J = 7.9 Hz, 1H)
ppm; ESIMS: m/z 204.1392 (M+H)+, calcd for C13H18NO, 204.1383.
4.2.29. 2,3,3-Trimethyl-5-(carboxypropyl)-3H-indole (16)
4.2.23. 5-(Azidoethyl)-2,3,3-trimethyl-3H-indole (10)
To a solution of 15 (68 mg, 0.25 mmol) in methanol (MeOH,
1 mL), an aqueous NaOH (1 M, 1 mL) was added and stirred at
room temperature for 1 h. The reaction mixture was acidified with
an aqueous HCl (1 M) and extracted with EtOAc. The organic layer
was dried over anhydrous MgSO4 and evaporated to give 16
(32 mg, 52%). 1H NMR (CDCl3,300 MHz) d 1.32 (s, 6H), 1.99 (quin,
J = 7.6 Hz, 2H), 2.36 (s, 3H), 2.39 (t, J = 7.6 Hz, 2H), 2.73 (t like,
J = 7.6 Hz, 2H), 7.13 (s, 1H), 7.14 (d, J = 7.9 Hz, 1H), 7.52 (d,
J = 7.9 Hz, 1H) ppm; ESIMS: m/z 246.1479 (M+H)+, calcd for
This compound was synthesized according to general procedure
for Mitsunobu reaction. Yield 33 mg, 74%. 1H NMR (CDCl3, 300 MHz)
d 1.30 (s, 6H), 2.27 (s, 3H), 2.93 (t, J = 7.4 Hz, 2H), 3.51 (t, J = 7.4 Hz,
2H), 7.13 (s, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.47 (br d, J = 8.6 Hz, 1H)
ppm; ESIMS: m/z 229.1452 (M+H)+, calcd for C13H17N4, 229.1448.
4.2.24. 5-(Azidoethyl)-1,3,3-trimethyl-2-methyleneindoline
(11)
This compound was synthesized according to general procedure
for synthesis indoline or indoleninium salt method A.
C15H20NO2, 246.1489.
4.2.25. 1,3,3-Trimethyl-5-(azidoethyl)spiro[indoline-2,30-[3H
]naphtho[2,1-b][1,4]oxazine] (12)
4.2.30. 1,2,3,3-Tetramethyl-5-(carboxypropyl)-3H-indoleninium
iodide (17)
This compound was synthesized according to general procedure
This compound was synthesized according to general procedure
for synthesis indoline or indoleninium salt method B.
for synthesis NISO method A. Yield 8 mg, 23%. 1H NMR (CDCl3,

C. Petchprayoon et al. / Bioorg. Med. Chem. 19 (2011) 1030–1040
1039
4.2.31. 1,3,3-Trimethyl-5-(carboxypropyl)spiro[indoline-2,30-
[3H]naphtho[2,1-b][1,4]oxazine] (5-CO NISO, 18)
300 MHz) d 1.33 (s, 3H), 1.35 (s, 3H), 1.97 (quin, J = 7.6 Hz, 2H),
2.31 (quin, J = 6.1 Hz, 2H), 2.41 (t, J = 7.6 Hz, 2H), 2.64 (t,
J = 7.6 Hz, 2H), 2.73 (s, 3H), 3.78 (t, J = 6.1 Hz, 2H), 4.32 (t,
J = 6.1 Hz, 2H), 6.48 (d, J = 7.7 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H),
6.90 (d, J = 1.3 Hz, 1H), 7.013 (dd, J = 1.3, 7.7 Hz, 1H), 7.014 (dd,
J = 2.4, 9.2 Hz, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.62 (d, J = 9.2 Hz, 1H),
7.73 (s, 1H), 7.87 (d, J = 2.4 Hz, 1H) ppm; ESIMS: m/z 507.2043
(M+H)+, calcd for C29H32ClN2O4, 507.2045.
This compound was synthesized according to general procedure
for synthesis NISO method B. Yield 15 mg, 32%. 1H NMR (CDCl3,
300 MHz) d 1.32 (s, 3H), 1.34 (s, 3H), 1.89 (quin, J = 7.7 Hz, 2H),
2.23 (t, J = 7.7 Hz, 2H), 2.60 (t like, J = 7.7 Hz, 2H), 2.70 (s, 3H), 6.50
(d, J = 8.0 Hz, 1H), 6.96 (d, J = 1.6 Hz, 1H), 7.01 (dd, J = 1.6, 8.0 Hz,
1H), 7.02 (d, J = 8.7 Hz, 1H), 7.38 (ddd, J = 1.1, 8.1, 8.2 Hz, 1H), 7.55
(ddd, J = 1.5, 8.1, 8.5 Hz, 1H), 7.72 (br d, J = 8.7 Hz, 1H), 7.77 (br d,
J = 8.2 Hz, 1H), 7.79 (s, 1H), 8.51 (dd, J = 1.1, 8.5 Hz, 1H) ppm; ESIMS:
m/z 437.1837 (M+Na)+, calcd for C26H26N2O3Na, 437.1836.
4.2.38. N-Carboxypentyl-3,3-dimethyl-90-chloropropoxyspi
ro[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (1-CO, 90-Cl
NISO, 24b)
4.2.32. 1,3,3-Trimethyl-5-(N-succinimidyloxycarbonylpropyl)
spiro[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-NHS–
NISO, 19)
This compound was synthesized according to general procedure
for synthesis NISO method B. Yield 287 mg, 55%. 1H NMR (CDCl3,
300 MHz) d 1.33 (s, 3H), 1.34 (s, 3H), 1.42–1.70 (6H), 2.26–2.32
(4H), 3.19 (t, J = 7.4 Hz, 2H), 3.78 (t, J = 5.9 Hz, 2H), 4.32 (t,
J = 5.9 Hz, 2H), 6.57 (d, J = 7.6 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H),
6.86 (t, J = 7.6 Hz, 1H), 7.02 (dd, J = 2.3, 9.2 Hz, 1H), 7.06 (d,
J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.61
(d, J = 9.2 Hz, 1H), 7.75 (s, 1H), 7.85 (d, J = 2.3 Hz, 1H) ppm; ESIMS:
m/z 521.2195 (M+H)+, calcd for C30H34ClN2O4, 521.2202.
This compound was synthesized according to general procedure
for synthesis N-hydroxysuccinimide ester derivatives. Yield 15 mg,
80%. 1H NMR (CDCl3, 300 MHz) d 1.34 (s, 3H), 1.36 (s, 3H), 2.07
(quin, J = 7.6 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 2.71 (t, J = 7.6 Hz,
2H), 2.74 (s, 3H), 2.85 (br s, 4H), 6.50 (d, J = 7.9 Hz, 1H), 6.92 (s,
1H), 7.034 (d, J = 8.9 Hz, 1H), 7.03 (d, J = 7.9 Hz, 1H), 7.39 (dt,
J = 1.3, 7.9 Hz, 1H), 7.57 (dt, J = 1.3, 7.9 Hz, 1H), 7.66 (br d,
J = 8.9 Hz, 1H), 7.741 (s, 1H), 7.745 (br d, J = 7.9 Hz, 1H), 8.56 (d,
J = 7.9 Hz, 1H) ppm; ESIMS: m/z 534.2003 (M+Na)+, calcd for
4.2.39. 1,3,3-Trimethyl-5-(carboxypropyl)-90-azidopropoxyspi
ro[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-CO, 90-N3
NISO, 25a)
C30H29N3O5Na, 534.2000.
This compound was synthesized according to general procedure
for synthesis azido derivatives from chloro derivatives. Yield 30 mg,
77%. 1H NMR (CDCl3, 300 MHz) d 1.30 (s, 6H), 1.94 (m, 2H), 2.12
(quin, J = 6.3 Hz, 2H), 2.38 (m, 2H), 2.60 (m, 2H), 2.69 (s, 3H), 3.55
(t, J = 6.3 Hz, 2H), 4.25 (t, J = 6.3 Hz, 2H), 6.44 (d, J = 7.6 Hz, 1H),
6.81 (d, J = 8.9 Hz, 1H), 6.88 (br s, 1H), 6.98 (br d, J = 7.6 Hz, 1H),
7.01 (dd, J = 2.2, 8.9 Hz, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.60 (d,
J = 8.9 Hz, 1H), 7.69 (s, 1H), 7.85 (d, J = 2.2 Hz, 1H) ppm; ESIMS: m/z
514.2443 (M+H)+, calcd for C29H32N5O4, 514.2449.
4.2.33. Tetramethylrhodamine-5-carboxamide lysine derivative
of 1,3,3-trimethyl-5-(N-succinimidyloxycarbonylpropyl)spiro
[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-TMR-Lys
NISO, 20)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 0.73 mg, 42%. ESIMS: m/
z 955.4377 (M+H)+, calcd for C57H59N6O6, 955.4389.
4.2.34. 7-(3-Chloropropoxy)-2-naphthol (21)
To a solution of 2,7-dihydroxynaphthalene (1.6 g, 9.99 mmol) in
dry acetone (50 mL), anhydrous potassium carbonate (8.3 g,
60.05 mmol) and 1-chloro-3-iodopropane (1.07 mL, 9.97 mmol)
were added. The suspension was stirred at 80 °C for 20 h and then
cooled down to room temperature. The solid was filtered off and
the filtrate was evaporated to give a residue. The residue was further
chromatographed on a silica gel flash column (hexane/EtOAc; 7:3) to
give 21 (793 mg, 34%). 1H NMR (CDCl3, 300 MHz) d 2.22 (quin,
J = 6.2 Hz, 2H), 3.73 (t, J = 6.2 Hz, 2H), 4.11 (t, J = 6.2 Hz, 2H), 5.80 (br
s, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 2.4, 8.9 Hz, 1H), 6.96 (dd,
J = 2.4, 8.9 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H)
ppm; ESIMS: m/z 237.0677 (M+H)+, calcd for C13H14ClO2, 237.0677.
4.2.40. N-Carboxypentyl-3,3-dimethyl-90-azidopropoxyspiro
[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (1-CO, 90-N3
NISO, 25b)
This compound was synthesized according to general procedure
for synthesis azido derivatives from chloro derivatives. Yield
114 mg, 88%. 1H NMR (CDCl3, 300 MHz) d 1.32 (s, 3H), 1.34 (s, 3H),
1.38–1.46 (m, 2H), 1.55–1.71 (m, 4H), 2.13 (quin, J = 6.4 Hz, 2H),
2.29 (t, J = 7.6 Hz, 2H), 3.19 (t, J = 7.2 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H),
4.27 (t, J = 6.4 Hz, 2H), 6.57 (d, J = 7.2 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H),
6.87 (t, J = 7.2 Hz, 1H), 7.02 (dd, J = 2.6, 8.8 Hz, 1H), 7.06 (dd, J = 1.8,
7.2 Hz, 1H), 7.19 (dt, J = 1.8, 7.2 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 7.62
(d, J = 8.8 Hz, 1H), 7.72 (s, 1H), 7.84 (d, J = 2.6 Hz, 1H) ppm; ESIMS:
m/z 528.2602 (M+H)+, calcd for C30H34N5O2, 528.2605.
4.2.35. 7-(3-Chloropropoxy)-1-nitroso-2-naphthol (22)
This compound was synthesized according to general procedure
for nitrosation. Yield 845 mg, 95%. 1H NMR (DMSO-d6, 300 MHz) d
1.75 (quin, J = 6.2 Hz, 2H), 3.36 (t, J = 6.2 Hz, 2H), 3.72 (t, J = 6.2 Hz,
2H), 5.82 (br d, J = 8.6 Hz, 1H), 6.69 (dd, J = 2.7, 8.7 Hz, 1H), 7.11 (d,
J = 8.7 Hz, 1H), 7.24 (br d, J = 8.6 Hz, 1H) ppm; ESIMS: m/z 266.0578
(M+H)+, calcd for C13H13ClNO3, 266.0579.
4.2.41. 1,3,3-Trimethyl-5-(carboxypropyl)-90-aminopropoxyspi
ro[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-CO, 90-NH2
NISO, 26a)
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 12 mg, 43%. 1H NMR (MeOD-d4,
300 MHz) d 1.28 (s, 3H), 1.30 (s, 3H), 1.88 (quin, J = 7.7 Hz, 2H),
2.07 (quin, J = 6.3 Hz, 2H), 2.21 (t, J = 7.7 Hz, 2H), 2.59 (t, J = 7.7 Hz,
2H), 2.67 (s, 3H), 2.97 (t, J = 6.3 Hz, 2H), 4.24 (t, J = 6.3 Hz, 2H), 6.47
(d, J = 7.2 Hz, 1H), 6.81 (d, J = 8.9 Hz, 1H), 6.94 (s, 1H), 6.99 (d,
J = 7.2 Hz, 1H), 7.02 (dd, J = 2.4, 9.3 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H),
7.65 (d, J = 9.3 Hz, 1H), 7.73 (s, 1H), 7.88 (d, J = 2.4 Hz, 1H) ppm;
ESIMS: m/z 488.2537 (M+H)+, calcd for C29H34N3O4, 488.2544.
4.2.36. N-Carboxypentyl-2,3,3-tetramethyl-3H-indoleninium
bromide (23)
This compound was synthesized according to general procedure
for synthesis indoline or indoleninium salt method B.
4.2.42. N-Carboxypentyl-3,3-dimethyl-90-aminopropoxyspiro
[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (1-CO, 90-NH2
NISO, 26b)
4.2.37. 1,3,3-Trimethyl-5-(carboxypropyl)-90-chloropropoxyspi
ro[indoline-2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-CO, 90-Cl
NISO, 24a)
This compound was synthesized according to general procedure
This compound was synthesized according to general procedure
for reduction azide to amine. Yield 21 mg, 45%. 1H NMR (MeOD-d4,
for synthesis NISO method B. Yield 58 mg, 22%. 1H NMR (CDCl3,

1040
C. Petchprayoon et al. / Bioorg. Med. Chem. 19 (2011) 1030–1040
300 MHz) d 1.32 (s, 3H), 1.34 (s, 3H), 1.36–1.72 (m, 6H), 2.10 (m,
2H), 2.18 (t, J = 7.1 Hz, 2H), 2.86 (dd, J = 7.1, 7.4 Hz, 2H), 3.09 (t,
J = 7.6 Hz, 2H), 4.29 (m, 2H), 6.57 (d, J = 7.5 Hz, 1H), 6.81 (d,
J = 8.8 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 7.02 (dd, J = 2.6, 9.0 Hz,
1H), 7.06 (dd, J = 1.1, 7.5 Hz, 1H), 7.13 (dt, J = 1.1, 7.5 Hz, 1H),
7.61 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H), 7.90
(d, J = 2.6 Hz, 1H) ppm; ESIMS: m/z 502.2697 (M+H)+, calcd for
images of the donor probe were collected on an Olympus Fluo-
View500 confocal microscope using a 60ꢁ, NA = 1.45 oil immer-
sion objective. UV-light pulses from a 100 W Hg-arc lamp filtered
through a 365 25 bandpass filter were controlled with a time
varying shutter (Vincent Associates) to drive the SP to MC transi-
tion as described in Marriott et al.2 In the second system, a
100 W Hg-arc lamp was used to continually excite the donor probe
at 546 nm while a second 100 W Hg-arc aligned orthogonal was
used to deliver 365 nm pulses whose duration was controlled by
a mechanical shutter (Melles-Griot). Typically, the 100 ms pulses
were used to convert SP- to MC-state. The UV-light was directed
into the optical path using a dichroic mirror from a GFP filter set,
which allowed for pulsed UV-irradiation of the sample while con-
tinuously illuminating the sample with 546 nm light. The donor
probe images were collected using a Hamamatsu EM-CCD camera
at ꢀ30 Hz (512 ꢁ 512) or at 300 Hz using a smaller region of
interest.
C30H36N3O4, 502.2700.
4.2.43. 5-Carboxytetramethylrhodamine derivative of 1,3,3-
trimethyl-5-(carboxypropyl)-90-aminopropoxyspiro[indoline-
2,30-[3H]naphtho[2,1-b][1,4]oxazine] (5-CO, 90-TMR NISO, 27a)
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 1.47 mg, 43%. ESIMS: m/
z 900.3962 (M+H)+, calcd for C54H54N5O8, 900.3962.
4.2.44. 5-Carboxytetramethylrhodamine derivative of N-
carboxypentyl-3,3-dimethyl-90-aminopropoxyspiro[indoline-
2,30-[3H]naphtho[2,1-b][1,4]oxazine] (1-CO, 90-TMR NISO,
27b)
Acknowledgments
This compound was synthesized according to general procedure
for synthesis TMR–NISO derivatives. Yield 1.55 mg, 45%. ESIMS: m/
z 914.4117 (M+H)+, calcd for C55H56N5O8, 914.4123.
This work was financially supported by grants awarded to G.M.
(NIH R01 EB005217-04 and 5R01GM086233-04 and the NDC for
Optical Control of Biological Function (PN2EY018241).
4.2.45. N-Hydroxysuccinimide ester derivative of 5-CO, 90-TMR
NISO (5-NHS, 90-TMR NISO, 28a)
Supplementary data
This compound was synthesized according to general procedure
for synthesis N-hydroxysuccinimide ester derivatives. Yield 1.03 mg,
63%. ESIMS: m/z 997.4117 (M+H)+, calcd for C58H57N6O10, 997.4131.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.015. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
4.2.46. N-Hydroxysuccinimide ester derivative of 1-CO, 90-TMR
NISO (1-NHS, 90-TMR NISO, 28b)
References and notes
This compound was synthesized according to general procedure
for synthesis N-hydroxysuccinimide ester derivatives. Yield
0.91 mg, 53%. ESIMS: m/z 1011.4313 (M+H)+, calcd for C59H59N6O10
,
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4.3. Spectroscopic and optical switching properties of TMR–
NISO derivatives in vitro
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Absorption spectra of each TMR–NISO solution were recorded
using a Shimadzu 1601PC spectrophotometer in solutions of glyc-
erol, methanol, or water. The MC-state was generated from the SP-
state following irradiation of the latter for 1 min with 365 nm light
delivered from a hand-held lamp. The rate for the thermally-driven
MC to SP transition was determined by measuring the decrease
absorption intensity in glycerol at room temperature as a function
of time. Fluorescence emission spectra were recorded using an
SLM-AB2 instrument.3 The SP- and MC-states of each TMR–NISO
derivative were excited at 555 nm and the emission spectra re-
corded between 560 and 750 nm. The fluorescence quantum yield
(U
) for TMR in the SP-state of each TMR–NISO derivative was deter-
mined using rhodamine 101 in ethanol as a reference ( = 1 in eth-
anol).22 A typical optical switching cycle was demonstrated using a
120 L sample solution of TMR–NISO 8b in glycerol. Each cycle of
U
l
optical perturbations involved a 30 s irradiation with 365 nm light
followed by excitation of the MC-solution with 550 nm laser light
for 60 s. The fluorescence intensity of TMR was monitored at
580 nm. This process was repeated for six cycles. All spectra were re-
corded at room temperature.
4.4. Spectroscopic and optical switching properties of TMR–
NISO derivatives in vivo
Two different microscope systems were used to image and
manipulate TMR–NISO probes. In the first system, fluorescence
22. Karstens, T.; Kobs, K. J. Phys. Chem. 1980, 84, 1871.