Water-soluble two-photon fluorophores based on paracyclophane

Article abstract of DOI:10.1080/15421400801925075

We report the synthesis, linear and nonlinear spectroscopy of the paracyclophane-based two-photon absorption (TPA) fluorophores. Two-photon induced fluorescence (TPIF) measurements in water show a substantial decrease in TPA cross section () and fluorescence quantum yield (), relative to toluene. We note that to obtain optimum two-photon action cross sections () in water, one needs to fine tune the intramolecular charge transfer (ICT). Microenvironment modulation using the micellar structure in water increases values similar to those obtained in toluene. The measured values in water are the highest among reported so far, suggesting important guidelines for designing of optimized TPIF molecular-labels.

Full text of DOI:10.1080/15421400801925075

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Water-Soluble Two-Photon
Fluorophores Based on
Paracyclophane
Han Young Woo a & Guillermo C. Bazan b
a Department of Nanomaterials Engineering (BK21) ,
Pusan National University , Miryang , South Korea
b Institute for Polymers and Organic Solids, UCSB ,
Santa Barbara , California , USA
Published online: 31 Aug 2012.
To cite this article: Han Young Woo & Guillermo C. Bazan (2008) Water-Soluble Two-
Photon Fluorophores Based on Paracyclophane, Molecular Crystals and Liquid Crystals,
485:1, 928-933, DOI: 10.1080/15421400801925075
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Mol. Cryst. Liq. Cryst., Vol. 485, pp. 180=[928]–185=[933], 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 1542-1406 print=1563-5287 online
DOI: 10.1080/15421400801925075
Water-Soluble Two-Photon Fluorophores Based
on Paracyclophane
Han Young Woo1 and Guillermo C. Bazan2
1Department of Nanomaterials Engineering (BK21), Pusan National
University, Miryang, South Korea
2Institute for Polymers and Organic Solids, UCSB, Santa Barbara,
California, USA
We report the synthesis, linear and nonlinear spectroscopy of the paracyclophane-
based two-photon absorption (TPA) fluorophores. Two-photon induced fluores-
cence (TPIF) measurements in water show a substantial decrease in TPA cross
section (d) and fluorescence quantum yield (g), relative to toluene. We note that
to obtain optimum two-photon action cross sections (gd) in water, one needs to fine
tune the intramolecular charge transfer (ICT). Microenvironment modulation
using the micellar structure in water increases gd values similar to those obtained
in toluene. The measured gd values in water are the highest among reported so far,
suggesting important guidelines for designing of optimized TPIF molecular-labels.
Keywords: bioimaging; paracyclophane; TPA molecular label; two-photon absorption
INTRODUCTION
Since the two-photon absorption (TPA) phenomenon was theoretically
¨
predicted by M. Goppert-Mayer in 1931 [1], two-photon induced fluor-
escence (TPIF) materials have received considerable attention [2] for
molecular labels in two-photon fluorescence microscopy (TPM) which
provides the best non-invasive means of biological imaging [3]. The
advantages of TPM relative to single-photon counterpart include
reduced photodamage, improved depth penetration, the ability to
This work was supported by the Korea Research Foundation Grant funded by the
Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-331-
D00139).
Address correspondence to Han Young Woo, Department of Nanomaterials Engineer-
ing (BK21), Pusan National University, Miryang 627-706, South Korea. E-mail: hywoo@
pusan.ac.kr
180=[928]

Water-Soluble Two-Photon Fluorophores
181=[929]
image turbid samples and reduced background cellular autofluores-
cence [4]. The application of TPM in understanding biological pro-
cesses is increasing exponentially and TPM-specific fluorophores for
biomolecular tags are an important consideration.
Best signal to noise is obtained with chromophores having a large
two-photon action cross section defined by gd, where g is the fluores-
cence quantum yield and d is the two-photon absorption (TPA) cross
section. Molecular design and TPA properties in organic solvents have
been extensively studied [5,6]. However, the effect of the medium, and
particularly water, on the magnitude of d remains less precisely
understood. There are conflicting theoretical expectations on the sol-
vent effects [7]. The currently used fluorescent reporters, which are
developed for single-photon microscopy, have gd values of 1–50 GM
in water (GM ¼ 1 ꢀ 10ꢁ50 ꢂ cm4 ꢂ s ꢂ photonꢁ1 ꢂ moleculeꢁ1). These values
are much lower than those (more than 1000 GM) in organic solvent.
There has been no systematic study on water-soluble TPA fluoro-
phores and TPIF properties in water.
In this contribution, we report the synthesis, linear and TPA spec-
troscopy of water-soluble TPA chromophores based on the [2.2]paracy-
clophane. We also discuss the molecular design strategies to get
efficient TPA fluorophores in water.
EXPERIMENT
All commercial chemicals were purchased from Aldrich and used as
received. All intermediates, including the final water-soluble fluoro-
phores, were characterized by common spectroscopic techniques such
1
1
as H-, 13C-NMR, UV=vis, and mass spectrometry. H- and 13C-NMR
spectra were collected on a Varian Unity 400 MHz (or 200 MHz) spec-
trometer. The UV=vis absorption spectra were recorded on a Shimadzu
UV-2401 PC diode array spectrometer. Photoluminescence spectra
were obtained on a PTI Quantum Master fluorometer equipped with
a Xenon lamp excitation source. Mass spectrometry analyses were
performed by UC, Santa Barbara, Mass Spectrometry Laboratory.
TPA spectra of the compounds were measured using two-photon
induced fluorescence spectroscopy [6]. Fluorescein (purchased from
Acros Inc.) was used as a reference material for TPA measurements.
RESULTS AND DISCUSSION
The molecular structures of two-photon fluorophores based on
[2.2]paracyclophane (pCp) are shown in Figure 1. Different substitu-
ents were chosen to modulate the intramolecular charge transfer

182=[930]
H. Y. Woo and G. C. Bazan
FIGURE 1 Molecular structures of neutral- and cationic paracyclophane
fluorophores.
(ICT) strength from the donor nitrogen groups to the pCp core and to
allow the molecules to be neutral (N series), and soluble in non-polar
organic solvents, or charged and water-soluble (C series). Four-fold
Horner-Emmons Wittig coupling reactions between the pCp core
(4,7,12,15-tetra(diethylphosphonatemethyl)-[2.2]paracyclophane) and
three donor structures with aldehyde functionality using potassium
tert-butoxide in THF at 0ꢃC give 1N, 2N and 3N, respectively, in yields
close to 60%. Successive quaternization with a large excess trimethy-
lamine provides 1C, 2C and 3C, respectively, in 85 ꢄ 90% yields.
Table 1 summarizes the absorption and photoluminescence (PL)
spectroscopy of the pCp fluorophores. A comparison of the linear spec-
tra of the C series in water with those of the N series in toluene reveals
TABLE 1 Spectroscopy Summary
Solvent
kabs (nm)
kPL (nm)
ga
kTPA (nm)
d (GM)b
gd (GM)
1N
1C
1C
2N
2C
3N
3C
3C
Toluene
Water
434
435
434
420
410
441
431
440
486
553
484
468
505
492
537
488
0.92
0.04
0.84
0.95
0.42
0.92
0.52
0.95
725
725
730
700
700
770
750
770
1290
370
1550
1690
700
2080
690
2020
1190
15
1300
1610
290
1910
360
1920
SDS (0.05 M)
Toluene
Water
Toluene
Water
SDS (0.05 M)
aMeasured relative to fluorescein at pH ¼ 11 in water.
bPeak TPA cross section at kTPA
.

Water-Soluble Two-Photon Fluorophores
183=[931]
the following trends. The kabs values are nearly the same (1N=1C) or
slightly blue shifted (2N=2C, 3N=3C). The emissions are broader, with
no vibronic structure, and larger Stokes shifts are observed. Most
importantly for the design of efficient TPM fluorophores, the g values
in water are inversely proportional to the donor strength of the ter-
minal groups (1N ꢄ 1C > 2N ꢄ 2C > 3N ꢄ 3C). A pronounced drop
in g is observed for 1C, while the g for 2C (0.42) and 3C (0.52) remain
high. The reduced g in water is caused by quenching related to strong
ICT in highly polar water. It is also noted that in a solvent of
intermediate polarity (DMSO), there are no measurable differences
in the linear spectroscopy of the N and C series. The charged
groups therefore are not interacting with the chromophores electronic
structures.
TPA spectra were obtained by using the two-photon induced fluor-
escence (TPIF) technique using a femtosecond pulsed laser source
[6]. The wavelength of TPA maximum, kTPA for 1N, 2N and 3N follow
the trend of kabs. Compounds 2N and 3N have higher d than 1N.
Higher d values may be attributed to additional electronic delocaliza-
tion within the extended p conjugation. Within the range of frequen-
cies accessible with our instrumentation (620 ꢄ 900 nm), the TPA
measurements in water show a substantial decrease in d, relative to
toluene (Table 1), with little change in the kTPA or general band shape.
We also measured a clear reduction in g in water owing to ICT related
quenching. Combining these results provides for gd values of 15 GM
for 1C, 294 GM for 2C and 359 GM for 3C, respectively, which are
much smaller than the values in toluene. The medium effect on d is
not clear at present stage although there have been several theoretical
approaches on the solvent effect. Interestingly, the larger gd values of
2C and 3C, relative to 1C, indicate that maximizing the electron den-
sity at the donor nitrogen atom is not necessarily a useful design para-
meter for TPM applications in water, due to ICT quenching. To
improve TPM imaging performance, it is recommended to make
the nonpolar microenvironment around the chromophore in water,
because typical TPA structures with strong ICT show larger gd in
nonpolar solvents.
Micelles are dynamic microheterogeneous structures containing
surfactant molecules and constitute an important research subject
[8]. It is possible within their internal environment to include some
compounds that are insoluble in water, to perturb the kinetics of many
photophysical processes and provide structural mimics of biological
membranes. Sodium dodecylsulfate (SDS) is a typical anionic surfac-
tant, displays a critical micelle concentration (cmc) of 8.1 mM at
25ꢃC and forms spherical micelles in aqueous solution.

184=[932]
H. Y. Woo and G. C. Bazan
Table 1 summarizes the PL and absorption spectra of 1C and 3C
([1C] or [3C] ¼ 10ꢁ5 M) in the presence of SDS at concentrations above
the cmc ([SDS] ¼ 0.05 M). Under these conditions the chromophores
are incorporated within the micellar core and the probability of finding
two chromophores within a single micelle is low. There is virtually no
change in the absorption maxima (kabs), however a significant increase
in the molar absorptivity of 3C is observed in micellar soultion. The PL
spectra are blue-shifted, have more pronounced vibronic structures
and larger g values are observed (0.84 for 1C and 0.95 for 3C), relative
to measurements in the absence of SDS. These PL characteristics are
similar to those obtained with 1N and 3N in toluene.
As shown in Figure 2, there is a significant increase in gd when one
compares the results in water (dmax ¼ 15 GM for 1C, 360 GM for 3C)
with those in the presence of SDS (1300 GM for 1C and 1920 GM for
3C). Indeed, the TPA action spectra under micellar conditions are
similar to the results in toluene (1190 GM for 1C, 1910 GM for 3C).
The combined maximum gd in SDS aqueous solution is 1300 GM for
1C and 1920 GM for 3C, respectively, which are more than 10 times
larger than those of the current fluorescent reporters in TPM. These
data show a large enhancement of gd in aqueous micellar solution
FIGURE 2 TPA action spectra of 1C and 3ꢁC5 in water (a, b) and in SDS
micellar solution (a0, b0). [1C] or [3C] ¼ 10
10ꢁ50 cm4 ꢂ s ꢂ photonꢁ1).
M, [SDS] ¼ 0.05 M. (GM ¼

Water-Soluble Two-Photon Fluorophores
185=[933]
by incorporation of the optically active units within hydrophobic
microenvironments.
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