Push-pull pyropheophorbides for nonlinear optical imaging

Article abstract of DOI:10.1039/C6OB02319C

Pyropheophorbide-a methyl ester (PPa-OMe) has been modified by attaching electron-donor and -acceptor groups to alter its linear and nonlinear optical properties. Regioselective bromination of the terminal vinyl position and Suzuki coupling were used to attach a 4-(N,N-diethylaminophenyl) electron-donor group. The electron-acceptor dicyanomethylene was attached at the cyclic ketone position through a Knoevenagel condensation. Four different derivatives of PPa-OMe, containing either electron-donor or electron-acceptor groups, or both, were converted to hydrophilic bis-TEG amides to generate a series of amphiphilic dyes. The absorption and emission properties of all the dyes were compared to a previously reported push-pull type porphyrin-based dye and a commercial push-pull styryl dye, FM4-64. Electrochemical measurements reveal that the electron donor group causes a greater decrease in HOMO-LUMO gap than the electron-acceptor. TD-DFT calculations on optimized geometries (DFT) of all four dyes show that the HOMO is mostly localized on the donor, 4-(N,N-diethylaminophenyl), while the LUMO is distributed around the chlorin ring and the electron-acceptor. Hyper-Rayleigh scattering experiments show that the first-order hyperpolarizabilities of the dyes increase on attaching either electron-donor or -acceptor groups, having the highest value when both the donor and acceptor groups are attached. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of the bis-TEG amide attached dyes in lipid monolayer-coated droplets of water-in-oil reveal that the TPEF and SHG involve transition dipole moments in different orientations.

Full text of DOI:10.1039/C6OB02319C

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Push-pull pyropheophorbides for nonlinear optical imaging
Anjul Khadria,a Yovan de Coene,b Przemyslaw Gawel,a Cécile Roche,a Koen Clays*b and Harry L.
Anderson*a
Received 00th January 20xx,
Accepted 00th January 20xx
Pyropheophorbide-a methyl ester (PPa-OMe) has been modified by attaching electron-donor and -acceptor groups to alter
its linear and nonlinear optical properties. Regioselective bromination of the terminal vinyl position and Suzuki coupling
were used to attach a 4-(N,N-diethylaminophenyl) electron-donor group. The electron-acceptor dicyanomethylene was
attached at the cyclic ketone position through a Knoevenagel condensation. Four different derivatives of PPa-OMe,
containing either electron-donor or electron-acceptor groups, or both, were converted to hydrophilic bis-TEG amides to
generate a series of amphiphilic dyes. The absorption and emission properties of all the dyes were compared to a
previously reported push-pull type porphyrin-based dye and a commercial push-pull styryl dye, FM4-64. Electrochemical
measurements reveal that the electron donor group causes a greater decrease in HOMO-LUMO gap than the electron-
acceptor. TD-DFT calculations on optimized geometries (DFT) of all four dyes show that the HOMO is mostly localized on
the donor, 4-(N,N-diethylaminophenyl), while the LUMO is distributed around the chlorin ring and the electron-acceptor.
Hyper-Rayleigh scattering experiments show that the first-order hyperpolarizabilities of the dyes increase on attaching
either electron-donor or -acceptor groups, having the highest value when both the donor and acceptor groups are
attached. Two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) images of the bis-TEG amide
attached dyes in lipid monolayer-coated droplets of water-in-oil reveal that the TPEF and SHG involve transition dipole
moments in different orientations.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Both TPA and SHG are enhanced when the electric field of the light
is polarized in the direction of the transition dipole moment (TDM)
Cellular imaging techniques based on nonlinear optical processes
such as two-photon excited fluorescence (TPEF) and second
harmonic generation (SHG) are gaining prominence because they
can probe deeper into the biological tissues, exhibit reduced out of
of the molecule.5,11 TPA and SHG have both been used to probe the
electric potentials across cellular membranes.12-17
Porphyrins and related porphyrinoid chromophores such as
focus fluorophore bleaching and generate less out of focus emitted chlorins and bacteriochlorins find applications in various fields such
and scattered light, compared to other optical microscopy as photodynamic therapy (PDT), fluorescence microscopy, chemical
techniques.1-4 Both TPEF and SHG involve simultaneous interaction sensing, and photovoltaics because they have excellent linear and
of two photons with the chromophore; hence, they require a high nonlinear optical properties due to their large ꢀ-system, which can
density of photons delivered by pulsed (typically sub-picosecond) be engineered by various structural modifications.4,18-23 TPA in
focused laser beams. While TPEF is detected in all directions around porphyrin-related compounds has already been applied in PDT.24,25
the sample, SHG is mostly detected in the forward direction of the Previously, we demonstrated that the TPA efficiency of porphyrin
incident light. TPEF signals from individual dyes placed in anti- dimers could be significantly increased by attaching electron-donor
parallel orientation add up, while the overall SHG signal from anti- and -acceptor groups.26 We found that apart from increased TPA
parallel dyes cancels. The intensity of an SHG signal is dependent on cross-section, donor-acceptor–based porphyrins give a high SHG
the second-order polar tensor, ꢀ2 induced when
a
non- response.2 One of our donor-acceptor free-base porphyrins, JR1, is
centrosymmetric molecule is placed in a non-centrosymmetric highly voltage-sensitive in comparison to commercial dyes such as
medium.5-10 SHG is an ideal technique to image interfaces while FM4-64, Di-4-ANEPPS and RH237 (Fig. 1), which makes it a good
avoiding background signals from isotropic regions. TPEF takes candidate to probe electrical potential differences across cellular
place via two-photon absorption (TPA) and requires population of membranes.2,14 We studied the effect of individual electron-
real excited states, while SHG is a scattering effect that generates donating and -accepting groups attached to porphyrin systems on
light of twice the incident frequency, only involving virtual states. SHG efficiency, and found that the free-base porphyrin core is
substantially electron-deficient, making the electron-withdrawing
group redundant.8 It has been previously suggested that porphyrins
substituted with phenyl rings at opposite meso-positions have
ambivalent nature, i.e. they can behave both as a donor and an
acceptor to create a push-pull effect when connected with either an
acceptor or a donor group at meso or β-positions.27,28 The
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Organic & Biomolecular Chemistry
1a and its derivatives have been extensively studied fVoirewinArvtiictlreoOnalninde
35-38
DOI: 10.1039/C6OB02319C
in vivo imaging, as well as photodynamic therapy.
The effects of
chemical modification of the ꢀ-system of 1a on its optical
properties have been explored in several studies.39-46 The electronic
absorption spectrum of 1a is characteristic of chlorins, and is
dominated by two absorption bands, one in the high-energy region
at the border of the UV (Soret band) and one in the far-red visible
region (Q band). Absorption in the far-red region is useful for one-
photon techniques because photo-damage is reduced at these
energies compared to shorter wavelengths. Although the linear
optical properties of 1a and its various derivatives have been widely
studied, there are few reports on its nonlinear optical
properties.47,48 The TPA cross-section, ꢀ of 1a at 800 nm in
methanol is only 3.5 GM, which is low compared to many other
dyes.4,24,48,49 It has recently been shown that aggregates of
pyropheophorbide-a (1b, the acid form of 1a) give an SHG
response.47 Cui et al. formed aggregates of 1b, organized non-
centrosymmetrically in liposomes and studied its optoelectronic
properties. They investigated the uptake of 1b-containing
liposomes by live cells and imaged the interior of the cells through
SHG and third harmonic generation microscopy.47
In order to explore the potential of 1a as a structural framework
for the linear and nonlinear optical imaging of membrane potentials
of excitable cells14,15,50,51 and for imaging biological structures
through TPEF and SHG-based microscopy,2-4,47 we describe the
design, synthesis, and properties of a new group of chromophores
based on 1a (Scheme 1). The parent structure of 1a was modified
by attaching electron-donating and -accepting groups to form
compounds 2a, 3a and 4a. We aimed to increase the non-
centrosymmetry and alter the electronic structure of the parent
molecule, to tune its linear and nonlinear optical properties. In
order to increase the amphiphilicity of the compound, we also
Fig. 1 Molecular structures of voltage-sensitive dyes.14
orientations of SHG-based probes within biological membranes play
crucial roles for an effective SHG response. Various studies
demonstrate different methods to determine the orientation of
molecular probes, such as TPEF-based microscopy, solid-state NMR,
fluorescence anisotropy, and SHG-based microscopy,29-33 while we
utilized the optical anisotropy of dyes in lipid-based water-in-oil
monolayers and bilayers.11 In this study, we modified the structure
of a chlorin molecule, pyropheophorbide-a methyl ester to enhance
its linear and nonlinear optical properties.
attach
a hydrophilic group. We investigate the first-order
hyperpolarizability β of the chromophores to study the effect of
individual donor and acceptor groups on their SHG efficacies. TD-
DFT calculations show that the HOMO is localized on the electron-
donor group while the LUMO is localized around the chlorin ring
and the acceptor group. While studying the orientation of the
compounds in lipid-based water-in-oil monolayer droplets, we
found that TPA and SHG in these set of molecules occur through
different TDMs.
Pyropheophorbide-a methyl ester (PPa-OMe, 1a, Fig. 2) is a
photosynthetic chlorin obtained by decarboxylation of methyl
pheophorbide-a, which is commonly obtained from Spirulina
maxima algae.34 Being a non-centrosymmetric ꢀ-system, 1a is
expected to give a SHG response when located in a non-
centrosymmetric environment. Since it consists of a conjugated
macrocycle with several positions available for chemical
modifications and creates singlet oxygen with high quantum yield,
Results and Discussion
Synthesis
We modified the structure of 1a (Scheme 1) by attaching electron-
donor
(4-(N,N-diethylaminophenyl))
and
-acceptor
(dicyanomethylene) groups to increase the polarization of its ꢀ-
system, with the objective of increasing its SHG intensity. We
attached a hydrophilic group, bis-triethyleneglycol (TEG) amide, at
the carboxylic positions of derivatives of 1a to make it more
amphiphilic and to favor incorporation into lipid membranes.
Fig. 2 Modification of pyropheophorbide-a methyl ester (PPa-OMe)
In order to attach the donor group, we developed a new
strategy to selectively brominate at the 32-vinyl position of 1a and
then perform Suzuki coupling. Functionalization at the 32-position
of 1a is often carried out by oxidation of the vinyl group to an
for enhanced nonlinear optical properties. Electron-donor,
-
acceptor and hydrophilic groups are attached at various positions of
PPa-OMe.
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aldehyde using OsO4.43,52 Lonin et al. achieved bromination at the 32 acids. Compounds 1b, 2b, 3b and 4b were reactedViwewitAhrticbleisO-TnlEinGe
55
DOI: 10.1039/C6OB02319C
vinyl position of 1a by reacting it with Br2 at –90 °C. The resulting amine in the presence of the coupling reagent COMU to form the
dibromoolefin was heated to eliminate HBr.53 In our attempts, this amphiphilic compounds 1c, 2c, 3c and 4c (step (v), Scheme 1),
respectively.
procedure was low yielding and gave inconsistent results. Vincente
et al. used two molar equivalents of N-bromosuccinimide (NBS) in
the presence of azobisisobutyronitrile (AIBN) on octaethylporphyrin
to form trans-(2-bromovinyl)-heptaethylporphyrin.54 Based on this
method, we used NBS in the absence of radical initiator to
brominate the 32-vinyl position of 1a (Scheme 1), and monitored
the reaction by reverse-phase HPLC (RP-HPLC, Fig. S1-A, ESI).
Bromination occurs selectively at the 32-vinyl position giving 5 in
78% yield. The reaction is stereoselective giving the E-product, as
Linear Optical and Electrochemical Properties
The electronic absorption and emission spectra of all the
compounds were measured in dichloromethane solution at 25 °C.
The absorption spectra are characteristic of chlorins with Soret
bands in the blue region and intense Q bands in the far-red region.
The absorption and emission spectra of 1a, 2a, 3a and 4a (Fig. 3)
show a clear effect of functionalization with electron-donating and -
accepting groups. The presence of the strongly electron-
withdrawing dicyanomethylene group in 3a causes a greater
1
evident from the HPLC trace (Figure S1) and H NMR spectrum (J =
14.0 Hz; Figure S2). Suzuki coupling was successfully applied (step
(ii) in Scheme 1) to attach 4-(N,N-diethylaminophenyl) to 5 to give
compound 2a.
bathochromic-shift
than
the
electron-donating
4-(N,N-
diethylaminophenyl) in 2a. On attaching either electron-donating or
accepting groups, the intensity of the Soret band decreases slightly,
and absorption in this region becomes structured into a complex
pattern of bands. The presence of various transitions within the
Soret region is supported by TD-DFT calculations (discussed below).
The multiple transition bands in the Soret region of 3a are of similar
intensities unlike the bands of 1a, 2a and 4a. We observe a nearly
Compounds 3a, 3b and 4a were synthesized by attaching
malononitrile at the 131-carbonyl positions of chlorins 1a, 1b and
2a, respectively, by Knoevenagel condensation.39,41 This method
affords derivatives functionalized with electron-acceptor groups in
good yields.
The polar TEG group was attached by first hydrolyzing the two-fold increase in intensity of the Q band on attachment of the
methyl esters 2a, 3a and 4a to carboxylic acids 2b, 3b and 4b (step only acceptor group in 3a. This effect is not observed on
(iv), Scheme 1), respectively. Compound 3b can be formed with attachment of the donor group (compound 2a), or of both the
higher yield by first performing the Knoevenagel reaction on 1a to donor and acceptor groups (compound 4a). Similarly, in the
form 3a and then hydrolyzing the ester rather than directly emission spectra, a greater bathochromic shift was observed for
attaching malononitrile on the carboxylic acid derivative 1b. The compound 3a (with electron-acceptor) than for compound 2a (with
higher isolated yield may be the result of easier purification of the electron-donor), compared to the parent compound 1a. The
methyl ester derivatives compared to the corresponding carboxylic emission maximum is further shifted towards a longer wavelength
Scheme 1 Synthesis of derivatives of pyropheophorbide-a methyl ester 1a. Reagents and conditions: (i) N-bromosuccinimide (NBS), 1,2-
dichloroethane, reflux; (ii) 4-N,N-diethylaminophenyl boronic acid, Pd(PPh3)4, Bu4NBr, toluene, NaOH (0.2 M aq.), 80 °C; (iii) CH2(CN)2,
Et3N, reflux; (iv) HCl (aq.), 20 °C; (v) HN(TEG)2, COMU, DMF, iPr2NEt, 0 °C (TEG: triethyleneglycol).
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Table 1 Linear optical properties of 1a–4a and other dyes
Organic & Biomolecular Chemistry
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DOI: 10.1039/C6OB02319C
Dye
1aa
2aa
3aa
λmaxabs (log ε) / nm
413 (5.0), 667 (4.7)
420 (4.9), 678 (4.7)
453 (4.9), 704 (5.0)
λmaxem/nm φF
Δṽ /eV
678
689
713
0.2239
0.030
0.18
0.24
0.029
0.022
4aa
458 (4.8), 716 (4.8)
448 (5.0), 723 (4.9)
726
803
761
0.003
0.13
0.35
0.023
1.708
5.690
JR12,b
FM4-646,b 564 (4.7)
a in CH2Cl2, b in CHCl3, Δṽ is the Stokes shift, 1a was used as
reference for calculating the fluorescence quantum yields φF of
all other compounds.39 ε is the molar absorption coefficient in
M–1 cm–1
when both the electron-donating and -accepting groups are
attached in compound 4a.
Fig. 3 Absorption and emission spectra of dyes, 1a–4a. Attaching
the electron donor and acceptor groups causes a red shift in the
absorption and emission bands, indicating a decrease in the HOMO-
LUMO band gap. Measurements were performed in CH2Cl2 at 25 °C.
For fluorescence measurements, the compounds were excited at
their respective Soret bands (414 nm for 1a, 420 nm for 2a, 453 nm
for 3a, and 459 nm for 4a). The fluorescence intensity is normalized
such that the areas of the peaks are proportional to their
fluorescence quantum yields. The fluorescence intensity for 4a is
multiplied by a factor of 50 for visible comparison
The attachment of electron-donor and -acceptor groups did not
substantially affect the Stokes shift, which is around 0.025 eV for all
compounds. In the PPa-OMe family of dyes (1a, 2a, 3a and 4a), the
fluorescence quantum yield decreases slightly while substituting
with the electron-donating group; however, it increases while
attaching the electron-accepting group (Table 1). In the push–pull
derivative 4a the quantum yield decreases by more than 99% (Table
1), which is beneficial for SHG imaging as there is less collateral
TPEF. Tamiaki and co-workers have studied the properties of
derivatives of 1a constructed by various functionalizations at its 31
and 32 positions and attaching malononitrile at its 132 position.39,43
None of those compounds exhibited quantum yields as low as 4a. It
appears that the 4-(N,N-diethylaminophenyl) moiety plays a critical
role in fluorescence quenching, probably via intramolecular charge
transfer.56-58 We also measured the absorption and emission
spectra of 1b, 2b, 3b and 4b to investigate the effect of the
hydrolysis of ester to carboxylic group on the optical properties.
This change does not alter the optical properties significantly (Fig.
S36).
We performed electrochemical measurements (square wave
voltammetry) on the chromophores to determine the energies of
their frontier orbitals (Table 2, Fig. S37–S40). As expected, the
1
electrochemical HOMO–LUMO gap (Eox − Ered1) decreases on
attaching electron-donor and -acceptor groups. However, in
contrast to the UV-Vis spectra, attachment of the donor group in 2a
decreases the HOMO-LUMO gap more than the acceptor group in
3a. On attaching the donor group, both the first oxidation and first
reduction potentials decrease, while on attaching the acceptor
group, they both increase. Tamiaki and co-workers found that on
attaching only electron-withdrawing groups (carbonyl and
dicyanomethene) at the 131 and 32 positions of 1a, the potentials of
oxidation and reduction increase, in agreement with our results.39
Table 2 Oxidation and reduction potentials of 1a–4aa
Dye
1a
E1ox (V)
E1red (V)
Eox1–Ered1 (V)
Electronic Structure Calculations
The ground-state geometries of 1a, 2a, 3a and 4a were optimized
by DFT at the B3LYP/6-31G(d) level of theory with solvation in
CH2Cl2 using the polarizable continuum model (PCM) implemented
in the Gaussian09 package (for details see the ESI).59 On these
geometries, vertical optical transitions were calculated by TD-DFT at
the B3LYP/LANL2DZ level of theory.60-62 In the case of 2a and 4a,
the lowest energy transition (Q band) is found with high precision
(difference of 0.06 eV and 0.07 eV, respectively, compared with
experiment; Fig. 4). On the contrary, the Q band transition energy
of 1a and 3a is underestimated by 0.31 eV and 0.30 eV,
0.43
–1.62
2.05
2a
0.17
–1.63
1.80
3a
4a
0.52
0.20
–1.38
–1.40
1.90
1.60
a All potentials were measured in THF with 0.1 M NBu4PF6,
relative to internal ferrocene, Fc/Fc+ at 0 V.
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Fig. 4 Overlay of the measured spectrum of 4a (CH2Cl2) and
calculated electronic transitions (TD-DFT B3LYP/LANL2DZ, PCM
solvation in CH2Cl2).
respectively. The splitting of the Soret band into multiple transitions
in the higher energy region was predicted with variable precision
and the general shape of the spectra could be reproduced (Fig. 4
and Section S7).
The calculated frontier molecular orbitals show a significant
difference in the localization of the HOMO and LUMO in 2a and 4a
(Fig. 5). This suggests that the HOMO-to-LUMO transitions in these
derivatives have intramolecular charge-transfer (ICT) character. In
the case of 1a and 3a, which do not contain the 4-(N,N-
diethylaminophenyl) group, both the HOMO and LUMO are located
on the central chlorin ring. The fact that the LUMO is localized on
the chlorin in 2a, while the HOMO is localized on the 4-(N,N-
diethylaminophenyl) group, implies that the chlorin ring can act as
an electron-acceptor. The calculated ground-state dipole moments
show a strong increase in the order: 1a (7.6 D), 2a (12.5 D), 3a (14.6
D) to 4a (20.7 D).
Fig. 5 Frontier molecular orbitals of 1a, 2a, 3a and 4a at the
B3LYP/6-31G(d) level of theory. 0.02 isovalue.
along the molecular-axis (βzzz) were measured for the series of PPa-
OMe derivatives 1a–4a in chloroform at 800 nm and 840 nm by
hyper-Rayleigh scattering (Table 3).63,64 The contribution to the β
values of both the donor and acceptor at 800 nm are similar. This
contrasts with our previously reported porphyrin-based dyes,
where an acceptor does not contribute to the β value.8 The
observed difference between the series of dyes may be due to the
nature of the conjugated core (porphyrin vs. chlorin). A free-base
porphyrin, when substituted with a strong donor group can itself
act as an acceptor, making the need of an additional acceptor
redundant. However, chlorins have higher electron density than
porphyrins, which makes them weaker electron acceptors. The β
values increase on attaching either the donor in 2a or the acceptor
in 3a, which means that the chlorin moiety can act as both the
acceptor (for compound 2a) and the donor (for compound 3a). The
βzzz value of 4a at 800 nm is about half that of the previously
studied porphyrin-based SHG voltage sensitive dye, JR1.2,14
However, it is more than the value that we measured in the same
conditions for the commercial SHG dye, FM4-64 (Table 3). The
donor and acceptor groups in JR1 are attached at meso-positions,
whereas, in 4c they are attached to the chlorin ring at the beta-
positions. This change of conjugation path undoubtedly contributes
towards the difference in HRS behavior.
Hyper-Rayleigh Scattering
The second-order polarizabilities (βHRS) and the component of β
Table 3 Nonlinear optical properties a
Dye
βzzz (λ)/10–30 esu (nm)
βHRS (λ)/10–30 esu (nm)
350 ± 50 (800),
1760 ± 340 (840)
140 ± 30 (800),
730 ± 140 (840)
1a
790 ± 150 (800),
2700 ± 500 (840)
330 ± 60 (800),
1100 ± 200 (840)
2a
3a
800 ± 150 (800),
3200 ± 560 (840)
330 ± 60 (800),
1300 ± 200 (840)
1330 ± 240 (800),
4700 ± 800 (840)
550 ± 100 (800),
2000 ± 340 (840)
4a
JR12
FM4-64
2300 ± 150 (800),
5800 ± 200 (840)
970 ± 50 (800),
2440 ± 70 (840)
Multi-Photon Microscopy
1150 ± 220 (800)
470 ± 90 (800)
We imaged the amphiphilic dyes, 1c–4c, in droplets of water in oil
a All measurements in chloroform
encapsulated by
microscope, to test their suitability for SHG and TPEF imaging, and
a lipid monolayer using a multi-photon
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Fig. 6 Two-photon excited fluorescence (TPEF) second harmonic generation (SHG) images of 3c and Di-8-ANEPPS. The cusp shape of TPEF
and SHG are in different directions for 3c, while they are in the same direction for Di-8-ANEPPS. The angular based intensity plots describe
the change of intensity as a function of the phase angle (angle around the droplet). The black dotted line shows the angle at which the
TPEF intensity for compound 3c is at maximum, but SHG is at minimum. The intensities maxima and minima of TPEF and SHG for Di-8-
ANEPPS are the same at the same angle (red dotted line). Droplet diameter: 70 µm (3c), 150 µm (Di-8-ANEPPS). ꢀext = 840 nm (3c), 850 nm
(Di-8-ANEPPS).
to study their average orientation (tilt angle) and probability active TDM at the excitation wavelength. Aiming to obtain average
distribution of the orientations. Droplets of phosphate buffer tilt angles and probability distribution of 1c, 2c, 3c and 4c, through
(aqueous phase) were mixed with lipids (DPhPC) and dye, and their TPEF and SHG images, we attempted to fit the images using
suspended in dodecane (oil phase) already dissolved with DPhPC our MATLAB-based image processing software.11 In 3c, we found
lipids. The lipid-based water-in-oil monolayer droplets are a simple that the intensity maxima (bright regions) and minima (dark
model for cell membranes, which we previously used to calculate regions) of SHG are shifted from their counterparts in TPEF. These
the average tilt angle and probability distribution of the tilt of SHG- results suggest that at the wavelength of excitation (840 nm), 3c
based dyes.11 The amphiphilic dyes locate at the water-oil interface has different TDMs active for TPA and SHG. We compared the
of the spherical droplets. All four compounds give clear SHG images, change of phase angles (angle around the droplet at which the
and the SHG signal is brightest for the push-pull dye 4c. Upon intensity is at maximum or minimum) for TPA and SHG of 3c with
imaging under the multi-photon microscope, two distinct dark that of Di-8-ANEPPS, an SHG-based dye, which we used previously
regions are observed around the edge of the droplets where the to demonstrate our model for measuring the probability
TDM of the dye is perpendicular to the direction of the polarization distribution of tilt angles (Fig. 6). Di-8-ANEPPS is a linear molecule
of the excitation light, and two bright regions where the TDM is with a single dominant TDM, which makes it a good candidate for
parallel to the polarization of the excitation light (Fig. 6). To comparison. No shift in the phase angles was observed in the
calculate the average tilt angle and their distribution in monolayers, droplets imaged with Di-8-ANEPPS. The phase angles of 1c, 2c and
we used the previously reported model and software.11 The model 4c in SHG and TPEF were also slightly shifted from each other when
is based on anisotropy data obtained from at least two of the processed in the MATLAB-based software. Hence, we presume that
following imaging techniques: one-photon fluorescence, TPEF and these chromophores also have different TDMs active for TPEF and
SHG (TPEF and SHG used here). Since the model determines the SHG. TD-DFT calculations predicted that 1c, 2c and 4c have several
probability distribution of the average tilt angle, it gives information TDMs (Section S7.2, ESI). This circumstance makes our model
about the preferred tilt (the angle which has the highest unsuitable to measure the probability distribution of tilt angles
probability). The model is based on photoselection rules, which because it is based on the assumption that both TPEF and SHG are
have the primary requirement that the molecule must have a single occurring through the same TDM and requires the use of both
6 | Org. Biomol. Chem., 2016, 00, 1-3
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acceptor groups (1c to 4c). The average tilt angle from SHG for all
View Article Online
DOI: 10.1039/C6OB02319C
the compounds vary between 30° to 40°, while that of TPEF varies
between 40° to 50° (Fig. 7). We also processed the TPEF and SHG
images of Di-8-ANEPPS as a control experiment, for which the
average tilt angles for both TPEF and SHG were found to be the
same (the difference of around 5% lies within experimental error).
In the PPa-OMe family dyes, SHG is most likely to be resonance
enhanced by the TDM lying in the donor-acceptor axis (long axis of
the molecule) as evident from the change of βzzz value on attaching
individual donor and acceptor groups. This means that the
information about the orientation of the whole molecule can be
predicted from the average tilt angle values of SHG for 1c, 2c, 3c
and 4c (Fig. 8).
Fig. 7 Comparison of average tilt angles obtained from TPEF and
SHG images. The average tilt angle obtained from the TPEF and SHG
images for 1c, 2c, 3c and 4c differ by more than 20%, while the
difference for Di-8-ANEPPS is around 5%. This means that there are
different TDMs active for TPA and SHG for 1c, 2c, 3c and 4c but
same TDM for Di-8-ANEPPS. The angles decrease on attaching the
electron-acceptor group, while they increase on attaching the
electron donor group. Error bars represent standard deviation (n ≥
4; nDi-8-ANEPPS = 3; n = number of samples).
Since these molecules contain a non-centrosymmetric core
macrocycle, they have TDMs pointing at various directions, as
shown by TD-DFT calculations (Section S7.2, ESI). To experimentally
confirm the existence of various TDM directions at different
wavelengths, we measured the polarized fluorescence excitation
spectra of 3c (Fig. S51). This experiment was performed in
glycerine-water mixture (9:1), to minimize diffusional reorientation
of molecules. The orientation of the polarization filter placed
between the sample and the light source was kept constant, while
the polarization filter (analyzer) placed between the sample and the
detector was rotated by 0° or 90° and the excitation spectrum was
measured. The difference in the intensity for these two spectra
varies across the UV and visible range, indicating that the direction
of TDMs varies at different wavelengths (Fig. S51). This observation
is supported by TD-DFT calculations, which show that the TDMs
within the Soret band of 3a are situated at approx. 90°. In contrast
to other derivatives, in 3a the three strong perpendicular
transitions dominate the Soret band (Fig. S58 and S59, ESI).
techniques. Nonetheless, the individual TPEF and SHG images can
be used to determine their individual average tilt angles (angle
between the active TDM and the normal to the membrane plane),
assuming that all the molecules in the droplet orient at the same tilt
angle.11 We processed the TPEF and SHG images of 1c, 2c, 3c and 4c
and fitted the model in order to calculate their respective average
tilt angles (Fig. 7). These average tilt angles obtained individually by
TPEF and SHG images differ by more than 20% for all the dyes. The
value of tilt does not change substantially on attaching the donor
group (1c to 2c) but decreases on attaching the acceptor group (1c
to 3c) and remains similar on attaching both the donor and
Conclusions
We have synthesized donor-acceptor functionalized
amphiphilic derivatives of pyropheophorbide-a methyl ester
and studied their linear and nonlinear optical properties in
organic solutions and at water-in-oil droplet interfaces. We
demonstrated a new approach to functionalize the 32-vinyl
position of pyropheophorbide-a methyl ester 1a via a
regioselective bromination. This approach gives high yields,
and could prove useful for the introduction of a wide range of
functional groups. The electronic absorption and emission
spectra of all the derivatives are characteristic for chlorins. The
push-pull substitution pattern results in a strong red-shift in
the absorption and fluorescence spectra, with a corresponding
reduction in the electrochemical HOMO-LUMO gap and an
increase in the hyperpolarizability β, measured by hyper-
Rayleigh scattering. The fluorescence quantum yield of the
push-pull derivative 4a is drastically reduced, possibly due to
photoinduced charge-separation. Analysis of the TPEF and SHG
images of lipid-monolayer coated droplets containing these
dyes shows that the TDMs dominating the SHG are sometimes
orthogonal to those responsible for two-photon absorption,
depending on the wavelength of excitation. The presence of
several almost perpendicular TDMs was confirmed by TD-DFT
calculations and by polarized fluorescence excitation spectra.
Since the SHG of a dye depends on the orientation of its
Fig. 8 Physical interpretation of orientation of 4c, based on the
assumption that the average tilt angle of the dye is the angle
derived from SHG image.
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Organic & Biomolecular Chemistry
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P. Theer, W. Denk, M. Sheves, A. Lewis and VPie.wB.ArDticeletwOinlleinre,
dominant TDM, control of the orientation of the TDMs is
crucial in the design of a SHG dye. Amphiphilic chromophores
based on 4c are promising prototypes for voltage-sensitive
dyes for monitoring membrane potential in excitable cells such
as neurons.
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Acknowledgements
We thank the Engineering and Physical Sciences Research
Council (EPSRC) for support, and the John Fell fund and the EP
Abraham Cephalosporin Fund, University of Oxford, for partly
funding a multi-photon microscope. A.K. acknowledges the
Systems Approaches to Biomedical Sciences and Systems
Biology Doctoral Training Center, the Clarendon Scholarships
Fund, Hilla Ginwala Scholarships, Radhakrishnan Memorial
Fund, and St. Catherine’s College Overseas Graduate
Scholarships at the University of Oxford. We thank Wojciech
Kaluza and Shifali Shishodia (both University of Oxford), and
17.
18.
19.
Carmen Bartic and Veerle Baekelandt (both University of 20.
Leuven) for valuable discussion. Y.C. thanks the Government of
21.
Flanders (Belgium) for an IWT Scholarship. P.G. acknowledges
the receipt of an Early Postdoc Mobility fellowship from the
Swiss National Science Foundation (SNF). The authors would
like to acknowledge the use of the University of Oxford
Advanced Research Computing (ARC) facility in carrying out the
computational work. http://dx.doi.org/10.5281/zenodo.22558.
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