Tuning the Photophysical Properties of Spirolactam Rhodamine Photoswitches

Article abstract of DOI:10.1002/ijch.202000083

Spirolactam rhodamines are fluorescent photoswitches that are useful for single molecule localization microscopy, volumetric 3D digital light photoactivatable dye displays, and other applications. Measurement of the photophysical properties, particularly photoswitching kinetics and quantum yields, is challenging and a comprehensive understanding of how molecular structure affects these parameters remains incomplete. In this study, we have synthesized a series of N-aryl spirolactam rhodamine photoswitches with fluorescent emissions at 585 nm and 518 nm. Extinction coefficients and fluorescence quantum yields of the fluorescent form of the photoswitch have been measured using excess trifluoroacetic acid to drive the equilibrium to the open form. A method to determine photoswitching kinetics and quantum yields was developed by monitoring the kinetics to reach equilibrium between the on-state and off-state and fitting this data to a rate equation for a reaction in equilibrium. Trends based on the electronic and steric properties of the aryl substituents were evaluated. Using this information, a proof-of-principle demonstration of 3D voxel formation was accomplished using a green (518 nm) emitting photoswitch, setting the foundation for a multi-colour volumetric 3D display.

Full text of DOI:10.1002/ijch.202000083

Full Paper
DOI: 10.1002/ijch.202000083
Tuning the Photophysical Properties of Spirolactam
Rhodamine Photoswitches
[a]
[a]
[a]
[a]
[a, c]
[a, b]
Bo Li, Uroob Haris, Maha Aljowni, Andrew Nakatsuka, Shreya K. Patel,
and Alexander R. Lippert*
Abstract: Spirolactam rhodamines are fluorescent photo- excess trifluoroacetic acid to drive the equilibrium to the
switches that are useful for single molecule localization open form. A method to determine photoswitching kinetics
microscopy, volumetric 3D digital light photoactivatable dye and quantum yields was developed by monitoring the
displays, and other applications. Measurement of the photo- kinetics to reach equilibrium between the on-state and off-
physical properties, particularly photoswitching kinetics and state and fitting this data to a rate equation for a reaction in
quantum yields, is challenging and a comprehensive under- equilibrium. Trends based on the electronic and steric
standing of how molecular structure affects these parameters properties of the aryl substituents were evaluated. Using this
remains incomplete. In this study, we have synthesized a information, a proof-of-principle demonstration of 3D voxel
series of N-aryl spirolactam rhodamine photoswitches with formation was accomplished using a green (518 nm) emit-
fluorescent emissions at 585 nm and 518 nm. Extinction ting photoswitch, setting the foundation for a multi-colour
coefficients and fluorescence quantum yields of the fluores- volumetric 3D display.
cent form of the photoswitch have been measured using
Keywords: Photoswitches · spirolactam rhodamines · 3D displays · photophysical properties · kinetics
1
. Introduction
Spirolactam rhodamines are an important class of fluores-
cent photoswitches that have found widespread use in single
[24–27]
[22]
Photoswitches are molecules that undergo reversible light-
mediated isomerization reactions, with the reverse reaction
proceeding either thermally or by a second photolytic reaction,
often at a different wavelength than the forward reaction.
Many classes of photoswitches have been developed and
encompass a diverse set of molecular motifs, including
azobenzenes, diarylethenes, oxazines, acyl hydrazones,
spiropyrans,
indigoids,
molecule localization microscopy
and 3D displays.
Gleiter et al. first demonstrated that N-aryl spirolactam rhod-
amines adopt a colourless, non-fluorescent closed form (off-
state, Figure 1A), but upon illumination with ultraviolet light,
a photochemical reaction occurs which transforms the mole-
cule from the closed lactam form into an open fluorescent
form (on-state, Figure 1A) that persists for milliseconds to
[
1]
[2]
[3,4]
[5]
[6]
[7]
[8]
[28]
donor-acceptor
Stenhouse
adducts,
minutes depending on the solvent medium. Later, the same
[9]
[10]
[11]
spirolactam rhodamines,
dihydropyrenes,
group explored the kinetics of the colouring reaction and
thermal fading in mixed solvents of dioxane with water or
[12]
[13]
[14]
naphthopyrans, dihydroazulenes, and fulgides. In addi-
tion to being of fundamental scientific interest, photoswitches
have been used for numerous applications, including
[29]
alcohols.
Thermal isomerization rates and activation vol-
[15,16]
[17–19]
photopharmacology,
molecular machines,
and ad-
[20]
vanced materials. While these applications rely primarily on
shape-dependent features of molecular structures that change
upon light illumination, other applications depend on a change
in optical properties, including the use of photoswitches in
super-resolution
displays.
[a] B. Li, U. Haris, M. Aljowni, A. Nakatsuka, S. K. Patel,
A. R. Lippert
Department of Chemistry
Southern Methodist University
3215 Daniel Avenue, Dallas, TX 75206 USA
[21]
microscopy
and
volumetric
3D
E-mail: alippert@smu.edu
b] A. R. Lippert
[22,23]
Microscopy and optical display applications
[
depend on photoswitches that can be transformed from a non-
fluorescent off-state into a fluorescent on-state upon illumina-
tion with a specific wavelength of light. This fluorescent on-
state can then be excited to the emissive fluorescent excited
state by illumination with the same or a different wavelength
of light. Rapid relaxation to the non-fluorescent off-state is
usually a spontaneous thermal process for super-resolution
microscopy and 3D display applications, but this can also be
achieved by a photolytic process.
Center for Drug Discovery
Design and Delivery (CD4)
Southern Methodist University
Dallas, TX 75206 USA
c] S. K. Patel
Department of Chemistry and Biochemistry
University of California, Los Angeles
[
607 Charles E. Young Drive East
Los Angeles, CA 90095-1569 USA
Supporting information for this article is available on the WWW
under https://doi.org/10.1002/ijch.202000083
Isr. J. Chem. 2020, 60, 1–10
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generated with a visible light projector. Fluorescence only
occurs where the UV and visible light patterns intersect,
making it possible to produce free-floating volumetric 3D
images such as the resolution chart shown in Figure 1B and
the SMU Peruna Mustang shown in Figure 1C.
In order to better understand the design principles of
spirolactam rhodamine photoswitches and ultimately generate
photoswitches of different colours that would be suitable for
use in a 3D Light PAD, we synthesized and measured the
photophysical properties of a range of N-aryl spirolactam
rhodamine B (SRB) photoswitches with red (585 nm) emission
wavelengths and N-aryl spirolactam rhodamine derivatives
with fluorinated aminoalkyl groups (SRCF3) that have green
(518 nm) emission wavelengths. Herein, we report the syn-
thesis of these derivatives, including a new procedure to
prepare fluorinated spirolactam rhodamine derivatives, and
present a method to measure the kinetics and quantum yields
of the photoswitching process. This information is used to
provide a preliminary demonstration of 3D voxel formation
using a green emitting spirolactam rhodamine photoswitch in a
Figure 1. Photoswitching of N-aryl spirolactam rhodamines for use
in a volumetric 3D digital light photoactivatable dye display (3D
Light PAD). (A) Photoswitching of N-phenyl spirolactam rhodamine
B. (B) Resolution chart projected in a 3D Light PAD. (C) SMU
Peruna projected in a 3D Light PAD.
3D Light PAD display.
[30]
umes were also investigated by Sueishi and coworkers, and
the influence of rotameric isomers of the on-state were
postulated to explain the observed effects of solvent and
pressure. Bossi et al. further explored the kinetics of isomer-
ization and acidophotochromism in the presence of trifluoro-
acetic acid for N-phenyl spirolactam rhodamine and several
2. Experimental Section
2.1 Synthetic Procedures
2.1.1 General Materials and Methods
[31]
derivatives.
They proposed a four-state mechanism to
explain the acid-dependent photoswitching behaviour, and
used a mathematical model to extract kinetic parameters. The
acid-dependent properties of N-phenyl spirolactam rhodamine
and its derivatives have been explored independently by Scott
and Harbron, and these properties make molecules of this type
All reactions were performed in dried glassware under an
atmosphere of dry N . Silica gel P60 (SiliCycle) was used for
2
column chromatography and SiliCycle 60 F254 silica gel
(precoated sheets, 0.25 mm thick) was used for analytical thin
layer chromatography. Plates were visualized by fluorescence
quenching under UV light or by staining with iodine. Other
reagents were purchased from Sigma-Aldrich (St. Louis, MO),
Alfa Aesar (Ward Hill, MA), EMD Millipore (Billerica, MA),
or Oakwood Chemical (West Columbia, SC) and used without
[32–35]
useful as fluorometric pH probes.
The above studies have
been aided by advanced synthetic methodology for the
synthesis of a wide variety of spirolactam rhodamine
[10,24,32–35]
derivatives.
1
13
Despite progress in understanding the kinetics of photo-
switching for N-phenyl spirolactam rhodamine derivatives,
determining the photochemical parameters, particularly photo-
switching rate constants and quantum yields, remains difficult.
This difficulty has impeded understanding of molecular
structure-function relationships for spirolactam rhodamine B
photoswitches and full photophysical measurements have not
further purification. H NMR and C NMR spectra for
characterization of new compounds and monitoring reactions
were collected in CDCl (Cambridge Isotope Laboratories,
3
Cambridge, MA) on a JEOL 500 MHz spectrometer or Bruker
400 MHz spectrometer in the Department of Chemistry at
Southern Methodist University. All chemical shifts are
reported in the standard notation of parts per million using the
peak of residual proton signals of the deuterated solvent as an
internal reference. Coupling constant units are in Hertz (Hz).
Splitting patterns are indicated as follows: br, broad; s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of
doublets; dt, doublet of triplets. High resolution mass
spectroscopy was performed on a Shimadzu IT-TOF (ESI
source) at the Shimadzu Center for Advanced Analytical
Chemistry at the University of Texas, Arlington. Low
resolution mass spectrometry was performed using an Advion
[22]
been performed in dichloromethane. In our previous work,
we used N-phenyl spirolactam rhodamine B as a fluorescent
photoswitch to generate 3D images in a solution of dichloro-
methane in a 3D display that we call a volumetric 3D digital
light photoactivatable dye display (3D Light PAD, Figures 1B,
C). In this display, a solution of N-phenyl spirolactam rhod-
amine B in dichloromethane containing 7.2 μM triethylamine
is prepared and placed in an imaging chamber with glass or
quartz walls. An ultraviolet light projector is used to photo-
activate the spirolactam rhodamine photoswitch in a precise
pattern, which then intersects with a visible light pattern
L
Expression CMS (ESI source) at Southern Methodist Uni-
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versity. Complete synthetic details can be found in the
Supporting Information.
mixture was cooled to 0°C, then POCl (0.07 mL, 0.73 mmol,
3
1.2 equiv.) was added dropwise. The reaction mixture was
allowed to stir at 0°C for 15 min and then heated to 45°C for
20 h. The mixture was diluted with 10 mL dichloromethane
2.1.2 Representative Procedure for Fluorinated N-Aryl
and then washed with 1 M HCl (3×10 mL), 1 M NaOH (3×
10 mL), and brine. The organic layer was collected, dried over
Na SO , filtered, and concentrated under reduced pressure.
Spirolactam Rhodamine (SRCF3) Compounds
2
4
3
-oxo-3H-spiro[isobenzofuran-1,9’-xanthene]-3’,6’-diyl bis
The crude product was purified by silica gel chromatography
with ethyl acetate:hexane (1:4) as the eluent, affording the
product as a white solid (221.4 mg, 62% yield). The
compound can be additionally purified by recrystallization
[36]
(trifluoromethanesulfonate) (bis-triflate).
Fluorescein
(2.79g, 8.42mmol, 1.0 equiv.) and bis(trifluoromethanesulfon-
yl) aniline (7.52 g, 21.1 mmol, 2.5 equiv.) were dissolved in
dichloromethane (20 mL). Then, diisopropylethylamine
from ethyl acetate:hexanes (1:2) to provide a spectroscopi-
1
(4.31 mL, 25.2 mmol, 3.0 equiv.) was added into the mixture.
cally pure sample H NMR (500MHz, CDCl ) 8.01 (d, J=
3
The reaction mixture was allowed to stir at 25°C for 18 h. The
reaction was monitored by thin layer chromatography (TLC).
After the reaction was complete, the mixture was diluted with
7.1 Hz, 1H), 7.54–7.50 (m, 2H), 7.14–7.08 (m, 4H), 6.65 (d,
J=8.0 Hz, 4H), 6.30 (d, J=8.6Hz, 4H), 3.73 (q, J=6.6Hz,
1
3
4H). C NMR (125 MHz, CDCl ) 167.6, 152.7, 147.0, 135.9,
3
2
1
0 mL dichloromethane, washed with 1 M HCl (3×20 mL),
132.9, 131.3, 129.1, 127.3, 126.2, 123.7, 110.3, 99.3, 67.1,
+
M NaHCO (3×20 mL), and then washed with brine. The
45.9, 29.7. HRMS calculated for C H N O F [M+H]
3
30 21
3
2 6
organic layer was collected, dried over Na SO , filtered, and
570.1533, found 570.1531.
2
4
concentrated under reduced pressure. The crude product was
purified by silica gel chromatography with dichloromethane:
methanol (20:1) as the eluent, affording the product as a light
2.2 Determination of Photophysical Properties
1
yellow solid (3.56 g, 71% yield). H NMR (500MHz, CDCl )
3
8
(
.05 (d, J=7.0 Hz, 1H), 7.73–7.69 (m, 2H), 7.29 (s, 2H), 7.19
d, J=6.6 Hz, 1H), 7.01–6.97 (m, 4H).
’,6’-bis((2,2,2-trifluoroethyl)amino)-3H-spiro[isobenzo-
2.2.1 Extinction Coefficients of the On-State and Off-State,
ɛ556nm(on), ɛ495nm(on), ɛ254nm(off)
3
furan-1,9’-xanthen]-3-one (Rhodamine-CF ). The pure bis-
triflate (2.98 g, 5.04 mmol, 1 equiv.) was added into a 50 mL
Stock solutions (0.5 mM) of recrystallized N-aryl spirolactam
rhodamine B (SRB) derivatives 1–9 or N-aryl spirolactam
rhodamine derivatives with fluorinated aminoalkyl groups
(SRCF3) 10–17 were prepared in dichloromethane. A 25 mM
stock solution of trifluoroacetic acid in dichloromethane was
prepared for use with the SRB derivatives and a 250 mM stock
solution of trifluoroacetic acid was prepared for use with the
SRCF3 derivatives. For the SRB derivatives, the SRB stock
solutions and trifluoroacetic acid stock solutions were diluted
into 1 mL quartz cuvettes to prepare five solutions of the SRB
derivative with final concentrations between 1 μM and 30 μM,
each containing 50 equivalents of trifluoroacetic acid. The
capped cuvettes were allowed to sit for 40 minutes to ensure
complete reaction to the fluorescent on-form. A similar
procedure was used for the SRCF3 derivatives, except that
500 equivalents of trifluoroacetic acid were used. After
complete conversion, UV/Visible absorbance spectra were
acquired for each concentration and the extinction coefficient
was determined using the slope of the linear plot of
concentration versus absorbance at 556 nm for the SRB
derivatives and 495 nm for the SRCF3 derivatives according
to Beer’s law. A similar procedure was used to determine the
extinction coefficient at 254 nm of the off-state, but in the
absence of any trifluoroacetic acid. All values are the average
of n=3 independent experiments � standard deviation. More
details can be found in the supporting information.
3
pressure flask with Pd (dba) (462 mg, 0.504 mmol,
2
3
0.1 equiv.), XPhos (720 mg, 1.51 mmol, 0.3 equiv.) and
Cs CO3 (4.60 g, 14.1 mmol, 2.8 equiv.). The flask was
2
evacuated under high vacuum for 10 min and filled with N2.
The mixture was then dissolved in 20 mL dioxane and stirred
for 15 min under N . 2,2,2-trifluoroethylamine (0.92 mL,
2
12.9 mmol, 2.4 equiv.) was added into the mixture by
dropwise addition. The flask was capped and heated at 100°C
for 20 h. The mixture was then transferred into a round bottom
flask and the solvent was evaporated. The residue was
dissolved in ethyl acetate and poured into brine (50 mL), 1 M
HCl (10 mL) was added, and the mixture was extracted with
ethyl acetate (3×25 mL). The organic layer was collected,
dried over Na SO , filtered, and concentrated under reduced
2
4
pressure. The crude product was purified by silica gel
chromatography with dichloromethane:methanol (30:1) as the
eluent, affording the product as a red solid (1.59 g, 64%
1
yield). H NMR (500MHz, CDCl ) 7.99 (m, 1H), 7.65 (m,
3
1
6
H), 7.59 (m, 1H), 7.17 (d, 1H), 6.58 (s, 2H), 6.51 (m, 2H),
1
3
.36 (t, J=8.3Hz, 2H), 4.23 (br s, 2H), 3.78 (m, 4H).
C
NMR (125 MHz, CDCl ) 167.7, 154.8, 154.0, 133.3, 131.9,
3
1
1
30.6, 128.7, 123.9, 108.7, 98.1, 69.3, 44.6, 37.9, 30.0, 14.9,
+
2.6. HRMS calculated for C H N O F [M+H] 495.1121,
2
4
16
2
3 6
found 495.1119.
2
-phenyl-3’,6’-bis((2,2,2-trifluoroethyl)amino)spiro[isoin-
doline-1,9’-xanthen]-3-one (10). Rhodamine-CF3 (305 mg,
0
3
.607 mmol, 1.0 equiv.) and aniline (0.19 mL, 1.82 mmol,
.0 equiv.) were dissolved in dichloromethane (10 mL). The
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2
.2.2 Fluorescence Quantum Yield of the On-State, Φfl
12.0, with all parameters constrained to be positive. Values for
Imax were estimated by measuring the fluorescence emission of
1 or 10 upon addition of excess trifluoroacetic acid, and these
estimates were used to constrain the value of Imax. This
constraint was found to be critical to provide consistent values
of k . K was determined as k /k . All values are the
Fluorescence quantum yields were determined in dichloro-
methane solutions of SRB with the addition of 50 equivalents
of trifluoroacetic acid and SRCF3 with the addition of
5
00 equiv.alents of trifluoroacetic acid using rhodamine B as a
on
eq
on off
standard. Five solutions of the standard rhodamine B were
prepared in ethanol to have absorbance values between 0.01
and 0.1. The absorbance was measured at 550 nm and
integrated fluorescence emission was measured using an
excitation wavelength of 550 nm. A similar procedure was
used to prepare five solutions of the SRB and SRCF3
derivatives, measuring the absorbance and exciting the
fluorescence at 556 nm for the SRB derivatives and measuring
the absorbance and exciting the fluorescence at 495 nm for the
SRCF3 derivatives. The quantum yield was determined by
using equation 1, where G is the slope of the linear plot of
integrated fluorescence emission versus absorbance, Gstand is
the slope of the same plot for the standard, η is the index of
refraction for the sample solutions, approximated as the index
of refraction of dichloromethane (1.42) and ηstand is the index
of refraction of the standard solution, approximated as the
averages from fitting of n=3 independent experiments�
standard deviation, except for compounds 5 and 6, which were
the average values from fitting n=6 independent experiments.
Equation 2 is derived in the supporting information.
2.2.4 Determination of the Photochemical Quantum Yield for
Photoswitching to the On-State, Φpc
The photochemical quantum yields Φ were determined using
pc
[38]
equation 3, where k is the rate of conversion to the on-state
on
as determined by fitting equation 2, ɛ254nm is the extinction
coefficient of the off-state at 254 nm, V is the volume of the
index of refraction of ethanol (1.36). Φ is the fluorescence
fl
quantum yield of the sample and Φstand =0.5, the fluorescence
solution (0.3 mL), I is the intensity of the excitation light at
254 nm (50.8 μWcm , measured with a Digital Light Meter,
ThorLabs PM100D), and l is the path length of the cuvette
(1 cm). All values are the average of n=3 independent
experiments � standard deviation.
0
[37]
À 2
quantum yield of the standard, rhodamine B in ethanol. All
values are the average of n=3 independent experiments �
standard deviation.
2
.2.3 Determination of k , k , and K
off on eq
Kinetics were determined using fluorescence spectroscopy.
Samples of SRB and SRCF3 were prepared at 5 μM in
dichloromethane in a quartz cuvette and placed in a Hitachi F-
3. Results and Discussion
We became interested in spirolactam rhodamine photoswitches
due to photoswitching properties that make them ideal for use
in a volumetric 3D digital light photoactivatable dye display
(3D Light PAD): a non-fluorescent off-state, a fluorescent on-
state, fast UV photoactivated switching, and fast thermal
7000 fluorescence spectrophotometer. For the SRB derivatives,
the following parameters were used: λ =254 nm, λ =
ex
em
585 nm, excitation slits=5 nm, emission slits=2.5 nm. For
the SRCF3 derivatives, the following parameters were used:
[22]
λ =254 nm, λ =518 nm, excitation slits=5 nm, emission
relaxation. While these properties enabled the development
of a first generation monochrome 3D Light PAD, optimization
and expansion into multi-colour displays requires a thorough
understanding of the photoswitching design parameters and
how molecular structure affects these parameters. These
include the extinction coefficients of the off-state at the
ultraviolet photoactivation wavelength (ɛ254nm(off)) and of the
ex
em
slits=5 nm. Kinetic scans were performed with continuous
excitation at 254 nm until an equilibrium was reached, 100
seconds to 30 minutes, depending on the compound. The
254 nm illumination both initiates photochemical conversion
from the off-state to the on-state and excites fluorescence of
the on-state. The traces were then fit to an equation for a
reaction in equilibrium, equation 2, where I is the observed
on-state at the fluorescence excitation wavelength (ɛ556nm(on),
fl
fluorescence emission (proportional to the concentration of the
on-state), Imax is the theoretical maximum emission with
complete turn-on of the dye, kon is the rate constant for the
forward reaction, koff is the rate constant for the reverse
reaction, and B is a parameter to account for non-zero
background signal. Fitting was performed using Mathematica
ɛ495nm(on)), the fluorescence quantum yield of the on-state (Φfl),
the photochemical quantum yield for the photoactivation
reaction (Φ ), and the on and off kinetic rate constants for
pc
photoswitching (k ) and thermal relaxation (k ). Factors such
on
off
as solubility, stability, and toxicity are also important for
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Figure 2. Synthesis of SRB derivatives.
eventual commercialization of a 3D display of this type, but
these parameters are not considered in the current study.
Ideally, the on-rate should be fast and is dependent on the
extinction coefficient of the off-form of the photoswitch at the
photoactivation wavelength (ɛ254nm(off)), the excitation light
intensity (I ), and the photochemical quantum yield (Φ ), as
Figure 3. Synthesis of SRCF3 derivatives.
0
pc
[38]
described by equation 3. Additionally, the off-rate should be
fast to avoid diffusion of the on-form of the dye, which
generates blurry images. The brightness of the display depends
on the extinction coefficient at the visible excitation wave-
length of the on-form of the dye (ɛ556nm(on), ɛ495nm(on)) and the
recently developed Buchwald-Hartwig coupling conditions
[36]
(Figure 3). Starting from fluorescein, the bis-triflate can be
prepared in good yield using N-phenyl bis-triflimide as a
triflating reagent. The bis-triflate can then be coupled to
trifluoroethylamine under palladium catalysis using the XPhos
ligand to generate the fluorinated rhodamine derivative with
trifluorethylamino groups. The same phosphorus oxychloride
amide coupling reaction that was used to prepare the
spirolactam rhodamine B (SRB) derivatives can be used to
prepare the fluorinated spirolactam rhodamine (SRCF3)
derivatives. Here again, we prepared derivatives with electron-
rich aryl groups (Compounds 12–16), electron-poor aryl
groups (Compounds 11, 17), and sterically-hindered groups
(Compounds 12, 13, 16).
fluorescence quantum yield (Φ ), as well as the concentration
fl
of the on-form of the photoswitch at equilibrium (K ). Blue-
eq
shifting the emission wavelength of the on-form of the
[10]
photoswitch can be accomplished by fluorination, but the
effect that this has on the parameters described above and how
to modify the molecular structure to optimize these parameters
is unclear. In order to investigate these factors, we decided to
generate a library of derivatives by varying the N-aryl groups
and investigate if tuning this position could provide an avenue
towards new photoswitches for use in a volumetric 3D display.
We synthesized nine spirolactam rhodamine B (SRB)
derivates and eight fluorinated spirolactam rhodamine
With these compounds in hand, we first measured the
extinction coefficients (ɛ556nm(on), ɛ495nm(on)) and fluorescence
(SRCF3) derivatives. The SRB derivatives were prepared from
quantum yields (Φ ) of the on-form of the dyes. We capitalized
fl
rhodamine B using a phosphorus oxychloride mediated
on the light-independent, acid-mediated switching of these
[32–35]
[32–35]
coupling with a library of anilines (Figure 2).
Derivatives
compounds,
and determined that we could convert the
were chosen to explore electron-rich aryl groups (com-
pounds 2, 3, 7–9), electron-poor aryl groups (compounds 4–6),
and steric hindrance at the photoswitchable spirolactam motif
dyes completely to the on-form with the addition of 50 equiv-
alents of trifluoroacetic acid for the SRB photoswitch series
and 500 equivalents of trifluoroacetic acid for SRCF3 photo-
switch series, each measured after a 40 minute incubation time
(Figure S2). Using this method, we determined extinction
coefficients at 556 nm for the on-state of the SRB photoswitch
series (Table 1) and at 495 nm for the SRCF3 series (Table 2).
For the SRB series, extinction coefficients were found to be
between 84,700 and 132,100, while for the SRCF3 series,
extinction coefficients were found to be between 54,400 and
(compounds 7–9). We identified fluorinated spirolactam rhod-
amines containing trifluoroethylamino groups as a photoswitch
[24,25]
with green fluorescence emission at 518 nm.
We experi-
enced difficulty constructing the xanthene core using the
fluorinated aminophenol according to the literature
[24,25]
precedent,
so we decided to devise an alternative stream-
lined synthetic route to prepare these compounds using
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Table 1. Fluorescence quantum yields and extinction coefficients of
the on-state of N-aryl spirolactam rhodamine B (SRB) derivatives in
dichloromethane.
Table 2. Fluorescence quantum yields and extinction coefficients of
the on-state of fluorinated N-aryl spirolactam rhodamine (SRCF3)
derivatives in dichloromethane.
Compound
Φ /%
fl
ɛ495nm(on)
Compound
Φ /%
fl
ɛ556nm(on)
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
58
24
41
6
3
1
94,900
91,400
94,200
90,900
65,900
58,900
70,300
54,400
1
2
3
4
5
6
7
8
9
52
5
84,700
122,400
110,200
96,000
118,900
132,100
117,500
103,500
112,200
16
36
24
55
15
28
33
31
13
signal at the initial time point, likely due to light scattering and
a non-zero fluorescence of the off-state when excited at the
ultraviolet 254 nm wavelength. This background is accounted
for by adding a background parameter, B, into the mathemat-
ical fits.
9
4,900. The fluorescence quantum yields (Φ ) were also
fl
determined in the presence of excess trifluoracetic acid to
drive the equilibrium into the open form. Generally, quantum
yields were lower for derivatives with electron-rich aryl
groups (compounds 2, 3, 7, 13–15), likely due to photoinduced
The data in this experiment was fit to equation 3 and, in a
representative fit, the rate constants were determined as k =
on
[39]
electron transfer (PET) quenching. Interestingly, derivatives
that were electron-rich, but also had high steric hindrance at
the spirolactam motif (compounds 8, 9, 12, 16) showed
fluorescence quantum yields that were not as attenuated. We
hypothesize that this could be due to a deviation from planarity
reducing conjugation between the aryl group and amide, and
subsequently reducing PET quenching. While supported by a
trend of increased quantum yield as the steric hindrance
increases from compound 7 (2-methyl) to compound 8 (2,6-
dimethyl) to compound 9 (2,6-diisopropyl), further investiga-
tion would be needed to confirm this.
We next sought to determine the on-rates (k ) and
on
photochemical quantum yields (Φ ) of the photoswitching
pc
reactions. The on-rates and photochemical quantum yields are
related according to equation 3 and depend on the extinction
coefficients of the off-form (ɛ254nm(off)) and the excitation light
intensity (I ). Our approach was to measure the fluorescence
0
emission at the emission maximum of the on-form upon
continuous excitation at 254 nm and then fit this data to the
rate equation for a reaction at equilibrium, equation 2 (Fig-
ure 4). The 254 nm excitation light served to both mediate the
photochemical reaction from the off-state to the on-state and
excite the fluorescence of the on-state. We began by
formulating methods using N-phenyl spirolactam rhodamine
B, compound 1. We found that monitoring fluorescence
emission at 585 nm upon continuous excitation at 254 nm
provided a curve that reached an equilibrium value after
approximately 1000 seconds (Figure 4A). A digital light meter
Figure 4. Methodology to measure photoswitching rates. (A) Emis-
sion intensity at 585 nm of 5 μM 1 in CH Cl irradiated with
2
2
À 2
50.8 μWcm of 254 nm light (black trace) and the fit to equation 3
red trace). (B) Equation and parameters found for the trace shown
in (A). (C) Calibration curve generated from increasing concentra-
(
tions of 1 in CH Cl 40 min after addition of 50 equivalents of TFA.
2
2
Data are average values from n=6 independent experiments. Error
bars are � S.D. (D) Comparison of K determined from Method 1:
eq
k
off
and k found from equation 1 and Method 2: Determined by
on
evaluating the equilibrium concentration of the on-state using the
equilibrium fluorescence emission in (A) and the calibration curve in
was used to measure the light intensity, which was found to be
À 2
(C).
5
0.8 μWcm . We note that there is a non-zero fluorescence
Isr. J. Chem. 2020, 60, 1–10
© 2020 Wiley-VCH GmbH
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À 3 À 1
À 3 À 1
1
.53×10 s , k =1.14×10 s (Figure 4B). These values
Table 3. Kinetics and photochemical quantum yields of 5 μM N-aryl
spirolactam Rhodamine B (SRB) derivatives in dichloromethane.
off
allowed us to determine an equilibrium constant of K =1.4.
eq
It is important to note that the fitted parameters, particularly
kon, are sensitive to the constraints placed on the maximum
intensity (Imax). In order to confirm these fits and provide
reasonable constraints on the Imax, we compared the equili-
brium constant determined with this fit to the equilibrium
constant determined by estimating the maximum turn-on value
(
Imax) by addition of excess trifluoracetic acid to drive the
equilibrium to the on-state (Figure 4C). In Figure 4C, the
μM concentration data point on this calibration curve
[a]
[a]
Compound
1
ɛ254nm(off)
Keq
1.4
koff
kon
Φ /%
pc
40,300
1.2
�0.3
1.6
�0.4
6.0
�1.5
5
�
0.05
0.044
represents an estimate of the maximum fluorescence (Imax) if
the photoswitch is completely in the open fluorescent form.
The calibration curve in Figure 4C can be used to determine
the concentration of the fluorescent on-form once the photo-
chemical reaction reaches equilibrium by comparing the
equilibrium fluorescence emission in Figure 4A to the calibra-
tion curve in Figure 4C. The equilibrium constant determined
by these two methods is similar (Figure 4D), increasing
confidence in this approach. The addition of trifluoracetic acid
to estimate the maximum fluorescence emission of 1 for SRB
derivatives and 10 for SRCF3 derivatives was used in all
following measurements to provide reasonable constraints on
Imax and more reliable measures of kon.
2
3
4
42,600
41,800
40,400
47,000
42,300
41,300
34,500
38,900
6.1
�3.5
1.4
0.31
�0.26
0.25
1.1
�
0.023
�0.93
0.82
�0.45
4.1
0.17
�0.056
1.5
�0.44
0.72
�0.16
0.38
�0.24
0.57
�0.45
0.93
�0.56
2.7
�0.14
1.1
�
0.12
1.2
�0.2
0.49
�0.83
[
b]
5
6
7
8
9
1
1.6
�
0.15
1.9
�0.34
�1.1
3.1
[b]
0.97
�
0.62
1.6
�0.54
1.5
�1.8
6.7
�0.13
�1.0
�3.7
8.0
0.68
1.8
�
0.14
1.4
�1.1
0.60
�0.1
30
�1.0
0.84
�4.2
3.3
We fitted the fluorescence turn-on data and determined
photochemical quantum yields for the series of SRB photo-
�
0.46
�0.39
�1.5
5.5
�0.89
[c]
[d]
40,300
0.009
�0.007
1.7
switches (Table 3). Extinction coefficients (ɛ254nm(off)) were
�27
�0.28
determined at 254 nm for the off-form of the dyes in dichloro-
methane and were found to be between 34,500 and 47,000.
The off-rates, k , were determined to be in the range of 0.38–
6
[
a]
À 3 À 1 [b]
[c]
Values are 10
s
n=6 independent experiments in the
presence of 7.2 μM triethylamine ɛ254nm(off) of 1 in CH Cl2
[
d]
2
off
À 3 À 1
.1×10 s for the SRB photoswitch series. Generally, SRB
photoswitches with electron-poor aryl groups (2-fluoro, 3-
chloro, and 3-fluoro, compounds 4–6) and the very sterically
hindered 2,6-diisopropyl compound 9 had lower values for koff
between 0.38–0.72×10 s . On the other hand, electron-rich
aryl groups (4-methoxy, 3,4-dimethoxy, 2-methyl, and 2,6-
indicating that steric hindrance at the spirolactam unit can
increase the photoswitching on-rate and quantum yield of
photoactivation. However, the 2-fluoro, 3-chloro, and 3-fluoro
derivatives 4–6 all display kon and Φpc values slightly lower
than the parent phenyl derivative 1, indicating that the
À 3 À 1
dimethyl, compounds 2, 3, 7 and 8) had similar or higher
photochemical reaction rate k is less strictly dependent on the
on
values for k as the parent N-phenyl SRB 1 between 0.93–
electronic nature of the N-aryl substituent. We also performed
similar analyses of the N-phenyl spirolactam rhodamine B 1,
off
À 3 À 1
6
3
1
.1×10 s . The k values determined for the 4-methoxy and
off
,4-dimethoxy derivatives 2 and 3 were elevated to 1.4–6.1×
but in the presence of 7.2 μM triethylamine, as is used in the
À 3 À 1
[22]
0 s , consistent with these electron donating substituents
operation of the 3D Light PAD.
While other parameters
pushing the equilibrium towards the closed form of the
photoswitch. The koff values were plotted versus the Hammett
parameters of the substituents to show a reasonable trend with
a negative ρ value (Figure S6).
remained relatively constant, addition of triethylamine had a
dramatic effect on the k value, which increases from 1.2×
off
À 3 À 1
À 3 À 1
10 s in the absence of triethylamine to 30×10 s in the
presence of triethylamine. This 30-fold increase in the off-rate
is consistent with our preliminary evaluation
[
22]
We next evaluated the kon values and used these to
and helps
determine the equilibrium constant (K ) of the photostationary
state and the photochemical quantum yield for the photo-
explain the better image quality observed when triethylamine
is added to the solution.
eq
switching reaction (Φ ). While the 4-methoxy and 3,4-dimeth-
Next, we measured these parameters for the newly
synthesized SRCF3 photoswitch series. In general, we found
pc
oxy electron-rich derivatives 2 and 3 have lower k , Φ , and
on
pc
K , consistent with a preference for the closed form in these
eq
that the values for the off-rates, k , were much higher for
off
structures, the effect of electron withdrawing and sterically
hindered substituents on kon was less pronounced. Compared
to the electron-rich compounds 2 and 3, we generally do
observe higher kon and Φpc for the phenyl, 2-fluoro, 2-methyl,
these derivatives in comparison to the SRB derivatives, and
values as high as 180–360×10 s were measured for the
phenyl, 3,4-dimethoxy, and 4-methoxy compounds 10, 14, and
15 (Table 4). This is consistent with the fluorination of the
amino groups inductively shifting the equilibrium to prefer the
À 3 À 1
2,6-dimethyl, and 2,6-diisopropyl compounds 1, 4, 7, 8, and 9,
Isr. J. Chem. 2020, 60, 1–10
© 2020 Wiley-VCH GmbH
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Table 4. Kinetics and photochemical quantum yields of 5 μM
fluorinated N-aryl spirolactam rhodamine (SRCF3) derivatives in
dichloromethane.
[a]
[a]
Compound ɛ254nm(off) Keq
koff
kon
Φ /%
pc
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
41,300
37,600
36,500
40,400
36,300
41,100
36,800
43,400
0.016
�
180
�26
2.9
�0.29
11
�1
0.001
0.28
�
4.7
�1.4
2.8
1.3
�0.26
2.1
5.3
�1.0
0.051
0.75
�
8.8
0.038
�1.1
�0.79
�3.3
0.94
�0.002
11
0.094
3.2
0.25
�
0.064
0.0095
�1.2
290
�0.066
2.7
�
0.001
0.0094
0.002
0.78
�16
�0.47
�2.0
360
3.3
18
�
�88
1.9
�0.9
1.5
�5.0
6.1
�
0.10
0.069
�0.086
�0.14
�0.56
46
3.1
11
�
0.008
�19
�0.89
�3.1
[a]
À 3 À 1
Values are 10
s
Figure 5. Demonstration of 3D Voxel Formation. (A) Schematic and
B) photograph of the setup for 3D voxel formation using a 254 nm
TLC lamp, the blue LED of a LightCrafter 4500 projector, and a
(
closed conformation of the dye. This effect could be overcome
by introducing electron-withdrawing 2-fluoro and 3-chloro
groups (compound 11 and 17) or steric hinderance at the
spirolactam position (2,6-diisopropyl, 2-methyl, and 2,6-
dimethyl, compounds 12, 13, 16), which display slower koff
cuvette filled with 5 μM 11 and 7.2 μM triethylamine.
À 3 À 1
[22]
values between 1.9–4.7×10 s for compounds 11–13, 16
4500 UV projector (as used in our previous work), which is
À 3 À 1
and 46×10 s for the 3-chloro compound 17. On the other
equipped with a 385 nm LED.
hand, we found that the kon values and photochemical quantum
yields were slightly higher for SRCF3 derivatives versus SRB
derivatives, but showed less predictable trends across different
types of SRCF3 derivatives.
4. Conclusions
Finally, we used the information gathered from these
experiments to do a preliminary demonstration of 3D voxel
formation with green emission using the design principles of a
In summary, we have successfully synthesized a series of
fluorescent spirolactam rhodamine photoswitches with
fluorescence emission in the red (585 nm) or green (518 nm)
portion of the spectrum. We report measurements of extinction
coefficients, fluorescence and photochemical quantum yields,
and kinetics of photoswitching. The strengths of the study
include a useful new synthetic protocol to synthesize SRCF3
photoswitches using a Buchwald-Hartwig coupling with
trifluoroethylamine. The reported method to measure photo-
chemical quantum yields and photoswitching kinetics is useful
because it does not require ultrafast methods and can be
performed using fluorescence spectrophotometers that are
readily available. These parameters have been difficult to
measure and photoswitching parameters in dichloromethane
solvent were previously unavailable in the literature. This
study provides important insights into how molecular structure
of N-aryl spirolactam rhodamine derivatives affects the photo-
3D Light PAD. We used the 2-fluoro SRCF3 compound 11 in
dichloromethane and a 254 nm excitation source. The photo-
switch was dissolved at 5 μM in dichloromethane with 7.2 μM
triethylamine in a 2.5 cm×2.5 cm×5 cm cuvette. A mask was
constructed to place over a 254 nm TLC lamp so that a line of
UV light would project up from the TLC lamp into the cuvette
(Figure 5A). A square of blue light was projected from the
side using a LightCrafter 4500 projector. Using this setup, a
voxel of green light can be observed (Figure 5B), providing a
proof-of-principle demonstration for using green SRCF3
photoswitches in a 3D Light PAD. Unfortunately, photo-
activation was only efficient at 254 nm, so patterning could
not be achieved using the commercially available WinTech
Isr. J. Chem. 2020, 60, 1–10
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switching properties, with identification of steric hindrance at
the spirolactam motif and electronics of the N-aryl groups as
being key design principles for tuning photoswitch behaviour.
We do note some important limitations of the study. While
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FULL PAPER
B. Li, U. Haris, M. Aljowni, A.
Nakatsuka, S. K. Patel, A. R. Lippert*
1
– 10
Tuning the Photophysical Properties
of Spirolactam Rhodamine Photo-
switches