Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

Article abstract of DOI:10.1039/b711438a

Long wavelength voltage-sensitive dyes (VSDs) called Pittsburgh (PGH) dyes were recently synthesized by coupling various heterocyclic groups to a styryl-thiophene intermediate forming extended, partially rigid chromophores. Unlike most styryl VSDs, dyes with a sulfonic acid anchor directly attached to the chromophore showed no solvatochromic absorption shifts. The limited water solubility of many long wavelength VSDs requires the use of surfactants to transport the dye through physiological saline solutions and effectively label biological membranes. Here, we tested the chemical substitution of the sulfonic acid moiety with polyethyleneglycol (PEG) chains, ranging from MW 750 to 5000, to overcome the poor solubility of VSDs while retaining their properties as VSDs. The chemical synthesis of PGH dyes and their PEG derivatives are described. The PEG derivatives were soluble in aqueous solutions (>1 mM) and still reported membrane potential changes. In frog and mouse hearts, the voltage sensitivity (ΔF/F per action potential) and spectral properties of PEG dyes were the same as the sulfonated analogues. Thus, the solubility of VSDs can be considerably improved with small polyethyleneglycol chains and can provide an effective approach to improve staining of excitable tissues and optical recordings of membrane potential. The Royal Society of Chemistry.

Full text of DOI:10.1039/b711438a

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Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by
covalent attachment of polyethylene glycol†
Michael J. Patrick,*a Lauren A. Ernst,a Alan S. Waggoner,a Dung Thai,b Dean Taic and Guy Salamac
Received 26th July 2007, Accepted 31st August 2007
First published as an Advance Article on the web 14th September 2007
DOI: 10.1039/b711438a
Long wavelength voltage-sensitive dyes (VSDs) called Pittsburgh (PGH) dyes were recently synthesized
by coupling various heterocyclic groups to a styryl-thiophene intermediate forming extended, partially
rigid chromophores. Unlike most styryl VSDs, dyes with a sulfonic acid anchor directly attached to the
chromophore showed no solvatochromic absorption shifts. The limited water solubility of many long
wavelength VSDs requires the use of surfactants to transport the dye through physiological saline
solutions and effectively label biological membranes. Here, we tested the chemical substitution of the
sulfonic acid moiety with polyethyleneglycol (PEG) chains, ranging from MW 750 to 5000, to overcome
the poor solubility of VSDs while retaining their properties as VSDs. The chemical synthesis of PGH
dyes and their PEG derivatives are described. The PEG derivatives were soluble in aqueous solutions
(>1 mM) and still reported membrane potential changes. In frog and mouse hearts, the voltage
sensitivity (DF/F per action potential) and spectral properties of PEG dyes were the same as the
sulfonated analogues. Thus, the solubility of VSDs can be considerably improved with small
polyethyleneglycol chains and can provide an effective approach to improve staining of excitable tissues
and optical recordings of membrane potential.
chain. Many styryl dyes, including the series of RH and JPW
dyes, have been synthesised resulting in a wide range of excitation
Introduction
and emission wavelengths and a variety of voltage responses.4,5,11,12
VSDs that have high quantum yields and produce a large change in
fractional fluorescence (DF/F) are desirable because they generate
adequate signals at lower dye concentrations. Recently, Salama
et al. reported a series of long-wavelength VSDs designed to report
cardiac action potentials (APs) from deeper layers of the heart
by incorporating a thienyl group in the polymethine bridge to
maintain the rigidity of the styryl chromophore while extending
the fluorescence wavelengths.3
Staining of target tissues requires transport of the dye to the
plasma membrane and T-tubules in sufficient quantities for voltage
measurements. Most styryl VSDs require an additional reagent
such as DMSO, Pluronics or cyclodextrins for transport through
the aqueous medium to the tissues.8,13 Labelling conditions are
determined for each dye, which can be a time-consuming, trial and
error process.3 Attachment of an uncharged hydrophilic anchoring
group to the dye structure may eliminate the need for such agents.
Poly(ethylene glycol)s (PEGs) are commonly used to improve the
water solubility of small molecule pharmaceuticals and exhibit
water solubility, high mobility in solution, lack of toxicity, lack of
immunogenicity, and are readily cleared from the body.14 Covalent
linkage of PEGs to voltage dyes will greatly simplify protocols for
probe delivery to excitable tissue.12
Two- and three-dimensional mapping of electrical activity through
changes in voltage-sensitive dye (VSD) fluorescence has greatly
increased our understanding of the function of nerve networks and
of cardiac electrophysiology in normal and pathological states.1–5
Effective VSDs are amphiphilic molecules that contain a water
soluble moiety and a lipophilic region. To measure rapid changes
in cellular membrane potential, the sensor portions of these dye
molecules associate with the hydrophobic interior of the plasma
membrane where the electric field gradient is the highest. At the
same time, the dyes carry charged groups to facilitate transfer of
the dyes to the target tissues and to anchor the dyes at cell surfaces,
minimising diffusion of the VSDs to other membranes within the
cell.6 Effective imaging deep within tissues requires VSDs with
high molar extinction coefficients at red wavelengths and near-IR
emission with high quantum yields to reduce light scattering and
intrinsic tissue absorption and fluorescence.5,7,8
Styryl dyes are a class of fluorescent VSDs that are widely
used to measure membrane potentials because of their ability
to follow voltage changes on a millisecond timescale.2,5,9,10 Their
structures consist of an electron-rich aminophenyl group linked to
a quaternized nitrogen heterocycle by a conjugated polymethine
aMolecular Biosensor & Imaging Center, Carnegie Mellon University, 4400
Fifth Avenue, Pittsburgh, PA, 15213, USA. E-mail: mjp@andrew.cmu.edu;
Fax: +1-412-268-6571; Tel: +1-412-268-3462
In this report, we present the synthesis and spectral charac-
terization of a set of styryl dyes which fluoresce at near infrared
wavelengths. The series, PGH-1 through PGH-8, demonstrates
the effect of different heterocycles on the spectral properties of
the dyes. Analogues of two VSDs were synthesised and coupled to
PEG groups of varying chain lengths (MW 750–5000) to increase
their water solubility. The enhanced solubility of PEG derivatives
bAmgen Inc., South San Francisco, CA 94080. E-mail: dthai@amgen.com
cDepartment of Cell Biology and Physiology, University of Pittsburgh,
Pittsburgh, PA, 15261, USA. E-mail: gsalama@pitt.edu; Fax: +1-412-648-
8330; Tel: +1-412-648-9354
† Electronic supplementary information (ESI) available: Full synthesis and
characterization details. See DOI: 10.1039/b711438a
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facilitated delivery to target tissues while retaining their spectral
properties and voltage sensitivity (DF/F per action potential) in
cardiac tissues.
Results
Sulfonated dyes
VSDs were synthesised from heterocycles A or B quaternised
with sulfonic acid anchors or carboxylic acid groups (Table 1),
and a common styrene-thienyl aldehyde intermediate as shown
in Scheme 1. The structures of PGH-1 through PGH-10
are listed in Table 2. Briefly, styrene (1) was made from
dibutylamino-benzaldehyde via Wittig reaction and then coupled
with bromothiophene-carboxaldehyde via a Heck reaction to yield
the styryl-thiophene intermediate (2).15 Other syntheses of these
intermediates have been reported;16,17 however, the methods used
here require minimal purification of the styrene and fewer steps
to prepare the aldehyde. Wuskell et al. recently reported the
Table 1 Structures of heterocyclic intermediates used in dye syntheses.
Heterocycles (A) are lepidines (3, 10 and 11); or (B) are indolenines
(4, 6, 8 and 12); benzothiazole (5); benzoxazole (7); and quinaldine (9).
Intermediates 11 and 12 contain carboxylic acid functional groups
Scheme 1 Synthesis of voltage sensitive dyes described in this report.
synthesis of VSDs with similar chromophores utilizing quaternary
ammonium anchors.8,18 The sulfonic acid anchors used here in dyes
PGH-1 to PGH-8 produced zwitterionic dyes with the positive
charge of the chromophore delocalized between the nitrogen
atoms. The benzoxazole dye, PGH-5, was produced; however, the
dye rapidly decomposed after chromatographic purification upon
drying under mild conditions.
The spectral properties of these new dyes, PGH-1 to PGH-
8, were characterized in alcohols of different polarity to verify
the solvatochromic sensitivity of the various heterocycles. The
absorption maxima and extinction coefficients are shown in
Table 3, while the emission maxima and relative fluorescence
intensities are given in Table 4. The observed increased Stokes
shift with increasing solvent polarity is typical for styryl VSDs.19
For two of the dyes, PGH-4 and PGH-6, the increased Stokes
shift was asymmetrical; the absorption maxima did not change
with solvent polarity. These anomalous dyes have a sulfonic acid
Intermediate
Het.
X
Y
R
3
4
A
B
B
B
B
B
B
A
A
B
H
H
H
SO3K
H
SO3K
OMe
OMe
H
—
CMe2
S
CMe2
O
CMe2
CH CH
—
—
CMe2
C4H8SO3-
C4H8SO3-
C4H8SO3-
C4H8SO3-
C4H8SO3-
C2H5
C4H8SO3-
C4H8SO3-
C5H10CO2H
C5H10CO2H
5
6
7
8
=
9
10
11
12
SO3K
Table 2 Key to substitutions used in Scheme 1. Heterocycles A and B are shown in Table 1
Dye
Het.
X
Y
R
PGH-1
PGH-2
PGH-3
A
B
B
B
B
B
B
A
A
B
A
A
A
A
B
H
H
H
SO3K
H
SO3K
OMe
OMe
H
SO3K
H
—
CMe2
S
CMe2
O
CMe2
CH CH
—
—
CMe2
—
C4H8SO3-
C4H8SO3-
C4H8SO3-
C4H8SO3-
C4H8SO3-
C2H5
C4H8SO3-
C4H8SO3-
C5H10CO2H
C5H10CO2H
C5H10CONH[C2H4O]∼17Me
C5H10CONH[C2H4O]∼45Me
C5H10CONH[C2H4O]∼113Me
{C5H10CONH[C2H4O]∼37CH2}2
C5H10CONH[C2H4O]∼113Me
PGH-4
PGH-5a
PGH-6
PGH-7
PGH-8
PGH-9
=
PGH-10
PGH-9-PEG750
PGH-9-PEG2000
PGH-9-PEG5000
Bis-PGH-9-PEG3400b
PGH-10-PEG5000
H
H
H
SO3K
—
—
—
CMe2
a Decomposed after purification. b Contains two PGH-9 dyes with both R substitutions being either end of the same bis-functional PEG3400 linker.
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Table 3 Absorption properties of VSDs in various solvents
Methanol
max/nm
Ethanol
max/nm
2-Propanol
max/nm
n-Butanol
max/nm
n-Octanol
max/nm
Dye
A
e/M−1 cm−1
A
e/M−1 cm−1
A
e/M−1 cm−1
A
e/M−1 cm−1
A
e/M−1 cm−1
1
2
3
4
6
7
8
601
664
614
686
690
587
588
24900
32200
47400
53600
62500
42500
28400
608
673
620
690
690
594
598
24800
31500
46800
55200
67600
42100
34100
612
670
626
680
690
594
596
24300
33800
45750
53900
65800
43100
30300
619
678
630
688
696
604
602
24800
35200
47100
60600
52900
42000
32000
620
678
636
686
692
606
602
24700
33300
43000
55800
45500
43400
32000
Table 4 Emission properties of VSDs in various solvents. kEX = 610 nm
Methanol
Ethanol
2-Propanol
n-Butanol
n-Octanol
Dye
kmax/nm
Fl. rel. int.
kmax/nm
Fl. rel. int.
kmax/nm
Fl. rel. int.
kmax/nm
Fl. rel. int.
kmax/nm
Fl. rel. int.
1
2
3
4
6
7
8
1025
935
928
940
940
952
959
107
487
707
341
341
187
108
966
914
901
923
927
888
960
300
968
1489
832
707
496
351
934
887
890
888
892
870
940
714
1855
2621
1947
1397
1121
686
900
890
888
888
893
866
898
795
1882
2534
2046
486
1370
930
868
855
848
849
861
835
866
2859
4392
6626
2174
1607
4062
2283
anchor directly attached to the chromophore. This difference in
absorption behaviour is illustrated in Fig. 1. PGH-1 shows a
wavelength shift with similar extinction values, while PGH-6 gives
an absorbance difference with similar wavelength maxima.
A dye that exhibits a large DF/F response also must be soluble
in solvents or buffers that are compatible with physiological
conditions of excitable tissues. Great care must be taken to avoid
the precipitation of dye or the formation of dye particles in
the capillaries which would block flow and render the organ
ischemic. The solubilities of selected dyes were tested by diluting
a known concentration of dye in DMSO into various solvents. At
micro-molar concentrations, the dyes remained soluble in alcohol
solvents as well as aqueous buffers, and even buffered saline
solutions. However, effective labelling of tissues and intact organs
with a VSD is typically achieved by perfusing the tissue with a
bolus injection of dye at 1–2 mM resulting in a homogeneous
and intense VSD labelling on the membranes of the tissue. Fig. 2
shows the excitation and emission spectra of PGH-1 in cardiac
Fig. 2 Excitation–emission curve pair of PGH-1 in frog cardiac tissue.
Parameters: kEM = 875 nm for excitation scan, kEX = 600 nm for emission
scan.
Fig. 1 Absorption spectra of PGH-1 (A) and PGH-6 (B) in methanol (black lines) and octanol (gray lines).
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Table 6 Percentage aqueous solubility of voltage dyes and PEG
tissue. PGH dyes have desirable spectral properties in tissue but
in order to achieve sufficient loading, DMSO and Pluronic L-
64 are needed to keep the dyes from coming out of solution as
they course through the vascular bed.3 The search for alternative
methods to solubilise the dyes without extraneous surfactants led
to the development of PEG derivatives.
derivatives
Solubility of stock solutions
Dye
Water
PBS
PGH-1
40.7%
63.7%
77.5%
0%a
7.3%
69.0%
83.2%
0%a
PGH-9-PEG750
PGH-9-PEG5000
PGH-6
PEG derivatives
PGH-10-PEG5000
71.4%
78.9%
VSDs with functional groups (PGH-9 and PGH-10) were synthe-
sized as in Scheme 1. The highly water-soluble PEG derivatives
were prepared from different molecular weight amino-methoxy-
PEGs (mPEG-NH2) using standard methods and are listed in
Table 2.14,20 The combination of the hydrophilic polymer and
the hydrophobic styryl dye made purification of these PEG dyes
less straightforward than that of the sulfonated dyes. On acidic
alumina, the blue chromophore irreversibly disappeared for PGH-
9 derivatives of mPEG molecular weights 2000 to 5000. These
larger PEG-derivatives were successfully purified by C2-reversed
phase chromatography with water–methanol gradients containing
1% acetic acid. After purification, the PGH-9 derivatives had
an increased intensity of the short wavelength band (∼400 nm)
relative to long wavelength band (∼600 nm). Table 5 shows
a comparison of Ashort/Along of the PEG-dye derivatives, their
corresponding parent dyes (carboxylic acids PGH-9 and PGH-
10) and the sulfonate analogues (PGH-1 and PGH-6). When
compared with PGH-1 (containing no PEG group), the ratio of
a Solution was colourless.
m/z units apart. The products showed polydispersity similar to the
starting mPEG-amine reagents, but shifted by approximately the
mass of the dye.14 A marked change in the TLC migration of the
products from the starting materials, with no change in spectral
properties, gave further evidence of dye–PEG conjugation.
The PEG dye derivatives had much higher water solubility than
the sulfonated dyes. The aqueous solubilities of PGH-1, PGH-
6, PGH-9-PEG750, PGH-9-PEG5000 and PGH-10-PEG5000 are
shown in Table 6. With the aid of DMSO, PGH-1 showed limited
solubility in water and saline; however, PGH-6 was essentially
insoluble. Each of the PEG dye derivatives showed high water sol-
ubility without the need for DMSO; the relative aqueous solubility
of PGH-9-PEG5000 was approximately 20% higher than that of
PGH-9-PEG750 in both water and PBS. The fluorescence prop-
erties of PGH-10-PEG5000 and the four PGH-9 PEG-derivatives
were similar to those of PGH-6 and PGH-1, respectively.
PEG derivatives of PGH dyes had similar spectral, voltage-
sensitive responses and retention in cardiac tissues indicating that
these dyes provide similar voltage sensing and tissue retention as
their non-PEGylated analogues with the added advantage of being
highly water soluble. Fig. 3 illustrates action potentials recorded
from mouse hearts stained with PGH-1 and its PEG analogues.
Action potentials recorded from PGH-1 delivered with a pH 6
aqueous stock solution (A) were similar to those recorded with
PGH-1 delivered from a DMSO plus Pluronic stock solution
(B). Similar results were obtained with PGH-9-PEG750 derivative
delivered from Ringer’s stock solution in the absence of Pluronic
(C). PGH-9-PEG750 (D) shows the same inversion of the action
potential signal as observed with PGH-1 when the excitation
wavelength was shifted from 540 to 690 nm.3 PGH-9-PEG2000
(E) had similar spectral response characteristics and exhibited
action potential signals with similar signal to noise (S/N) ratio
compared to the PGH-9-PEG750 derivative. In contrast, the PEG-
5000 derivative yielded considerably lower S/N ratio compared
to the other dyes, most likely due to reduced insertion in the
membrane (F).
Ashort/Along increased as the molecular weight of PEG increased,
ranging from 1.4 to 2.7 for PEG750 and PEG5000 derivatives
respectively. This increased absorbance ratio could not be reversed.
Syntheses of two PGH-9 PEG-derivatives were repeated with
closer stoichiometric control. Product isolation by precipitation
from ether (PGH-9-PEG5000) or recovery by rotary evapora-
tion (PGH-9-PEG750) yielded sufficiently pure material (single
component by TLC), eliminating the need for chromatographic
purification. The Ashort/Along increase seen previously was not
observed with these resynthesized preparations and the ratio (0.6)
was the same as underivatized PGH-9. Mass spectral analyses
confirmed that the PGH-9 PEG-derivatives were intact. This
enhanced short wavelength absorption was not observed with
PGH-10-PEG5000 following chromatographic purification.
The mass profile of the PEG derivatives showed polydispersity
as evidenced by a distribution of mass spectral peaks spaced 44
Table 5 Absorbance ratios (short/long) were calculated for PGH-1,
PGH-6 and PEG derivatives of PGH-9 and PGH-10 in MeOH
Short kMax.
Long kMax.
Ashort/Along
Lepidine dyes:
PGH-1
392
398
396
390
390
390
398
398
601
606
596
590
588
588
610
608
0.71
0.56
1.39
1.99
1.44
2.71
0.58
0.60
PGH-9
Discussion
PGH-9-PEG750 (LC)
PGH-9-PEG2000 (LC)
PGH-9-PEG3400 (LC)
PGH-9-PEG5000 (LC)
PGH-9-PEG750 (No LC)
PGH-9-PEG5000 (No LC)
Indolenine dyes:
PGH-6
For a dye to function satisfactorily as a potentiometric probe, it
must exhibit the following essential properties: the dye must get to
target tissue and cells; must interact in the proper orientation to
sense membrane potentials across the plasma membrane; should
exhibit large and specific optical changes that vary only with
membrane potential changes; should induce minimal chemical-
and photo-toxicity to the cells and should be optically and
406
410
405
690
692
691
0.21
0.22
0.23
PGH-10
PGH-10-PEG5000
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are avoided and Rayleigh scattering is reduced at the longer
wavelengths. Far-red absorption was achieved with PGH-2, PGH-
4 and PGH-6 dyes.
The length of the alkyl chains on the styryl portion of the
dye can determine the depth of penetration into the lipid bilayer
and the rate of dye washout.13 Because of their effectiveness in
other dyes, butyl groups were selected to maximize tissue retention
without an excessive compromise on water solubility.12 The depth
of chromophore penetration into the lipid bilayer also increases
with the number of carbons between it and the covalently linked
anchor.13 The type of hydrophilic group is significant. Loew and
coworkers developed a family of styryl dyes which excite at 600–
650 nm and emit >750 nm, but have restricted application for
cardiac tissue studies.8,23 While these dyes have improved spectral
ranges, they exhibit limited photostability and low S/N ratio
signals. The hydrophilic anchor of these dyes is the quaternary
ammonium group, which can be drawn through the plasma
membranes of cells with large negative membrane potential and
thus is not effective for anchoring the dyes at the cell surface.3 We
have used sulfonic acid counter ions to anchor our dyes to the
membrane surface.
The choice of the heterocycle can impact the quantum yield
and voltage sensitivity of the dye within the membrane.5 In this
study, styryl dyes, PGH-1 through PGH-8, were synthesized from
lepidine, quinoline, indolenine, benzothiazole and benzoxazole
heterocycles. While most of the heterocycles gave absorption
maxima near 600 nm, the indolenine dyes (PGH-2, PGH-4
and PGH-6) showed maxima near 690 nm. Unlike most styryl
VSDs, the indolenine dyes with the sulfonic acid group attached
directly to the chromophore (PGH-4 and PGH-6) did not show
absorbance solvatochromism. The close proximity of the anchor
to the aromatic system may maintain a polar environment for
the chromophore. The sulfonate group will effectively orient the
hydroxyl groups of the solvent molecules near the chromophore,
even in octanol. While PGH-1 yielded larger DF/F signals in
heart, PGH-6 also was effective as a VSD, even though it shows
minimal absorption wavelength solvatochromism.3
Fig. 3 Comparison of mouse ventricular action potentials recorded with
PGH-1 and PGH-9 PEG derivatives. Excitation at 540 25 nm and
emission monitored >650 nm (except D). A: PGH-1 in Ringer’s solution
at pH 6. B: PGH-1 in a stock solution of 18% Pluronic L64 and DMSO. C:
PGH-9-PEG750 in Ringer’s at pH 7.4. D: PGH-9-PEG750 stained as C,
excited at 690 25 nm and the emission >750 nm. E: PGH-9-PEG2000 in
Ringer’s stock solution at pH 7.0. F: PGH-9-PEG5000 in Ringer’s stock
solution at pH 7.0.
The surfactant-like properties of PGH-9 PEG derivatives pre-
sented additional challenges for purification. Column purified
PGH-9 PEG derivatives showed an increased Ashort/Along ratio
while alternate synthesis of these derivatives, and the PGH-10
derivative, showed the same ratios as the parent dyes. As the
hydrophobic voltage dye core and the highly water soluble PEG
group became more dissimilar (as with increasing molecular
weight of PEG group), the Ashort/Along ratio increased. Interactions
between the PEG groups, the hydrophobic dye moiety and the
chromatographic stationary phase may have induced these spectral
changes. A shift in absorption maximum from 600 nm to 400 nm,
while maintaining an intact dye structure, can be caused by
a disruption of the p electron system. Encapsulation by the
lengthy PEG group could stabilize a folded conformation of the
dye. Johnsson et al. incubated other types of PEG derivatives
spatially stable for long-lasting experiments.4,6,9 The voltage sensor
component of the dye should lie in the membrane where the
electric field and charge movements are most significant during
electrical activity. Molecular design features include (1) extended
conjugation for long wavelength absorption while maintaining
photostability; (2) various nitrogen heterocycles contributing to
the long wavelength absorption and providing spectral sensitivity
to voltage changes; (3) hydrophobic groups for stable partitioning
into the lipid bilayer; and (4) a hydrophilic group making the
dye membrane impermeant and facilitating tissue staining.5,21
Through the structural modifications of the dyes, we have reached
near IR excitation wavelengths with increased brightness and
improved the delivery of the dyes to the tissues.
Increasing the absorption wavelength is typically done by
extending the conjugated system. Incorporation of a thienyl unit in
the conjugated system extended the wavelength and was effective
in expanding conjugation of donor–acceptor molecules provid-
ing increased photostability and greater oscillator strength.17,22
Extension of fluorescence to the near infra-red range improves
penetration of the excitation and transmission of emitted light
through thick muscle tissue. Intrinsic chromophores in tissue
◦
at 60–70 C for 3 h to obtain clear solutions.24 Heating had
no effect on the absorption spectra of our voltage dye-PEGs.
PEG lipids with molecular weight >1000 form spherical micelles
because of the steric hindrance of the larger molecular weight
PEG groups.24 Because of the duality inherent in their structure,
these new VSD PEG derivatives may behave like PEG lipids.
Practical considerations such as concentration may be necessary in
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applications where critical micellar concentration and formation
of spherical or lamellar aggregates are factors.24
concentrated to dryness by rotary evaporation. Dyes were purified
by either normal or reversed phase column chromatography. Refer
to ESI† for details.
Conjugation of PEG groups to VSDs significantly improved
water solubility and simplified delivery to excitable tissues.
Derivatives with a longer PEG group (MW 5000) allowed easier
isolation from reaction mixtures using precipitation with ether.
While derivatives with larger PEG groups are easier to synthesise,
the 2 mM stock concentrations of these materials approach 1%
solute concentration. At these concentrations, the viscosity of the
solution is high and can be problematic in applications where this is
a concern. Derivatives with a shorter PEG group (MW 750) could
not be isolated by precipitation, thus making product recovery
more arduous. However, the shorter PEG dyes labelled the tissues
better in mouse hearts than longer PEG dyes, and did not cause
ischemia. The difference in the relative aqueous solubilities of
PGH-9-PEG750 and PEG-9-PEG5000 (Table 5) shows that the
length of the PEG chain is also an important consideration.
The attachment of PEG groups to styryl VSDs, regardless of
their lengths, has made it possible to deliver these dyes to tissues
without the need for DMSO or Pluronics. The simplified delivery
method greatly improves the productivity of excitable membrane
experimentation and may prove to be particularly advantageous
in neurobiology and nerve networks.
Synthesis of PEG derivatives
Voltage dyes derivatised with PEG groups were synthesised
from PGH-9 and PGH-10 by conversion of carboxylic acid
functional groups to the highly reactive succinimidyl ester
using 1.5 molar equivalents of N,N,Nꢀ,Nꢀ-tetramethyl-O-(N-
succinimidyl)uronium tetrafluoroborate (TSTU) in DMF con-
taining 1% diisopropylethylamine. mPEG-amines were then added
to the reactive NHS esters in 1.1 molar excess and the progress of
the reaction monitored by TLC. PEG derivatives were isolated by
rotary evaporation or precipitation with ethyl ether. Additional
purification for some of the PEG derivatives was done by column
chromatography. Refer to ESI† for details.
Spectral analysis of dyes
Absorption spectra and extinction coefficients were determined
using a Hewlett Packard Spectrophotometer (8452A Diode Array)
or a Perkin Elmer Lambda 45 Spectrophotometer. Corrected
excitation and emission spectra were obtained using a PTI
QuantaMaster fluorescence system configured with double grating
excitation and emission monochromators and a Hamamatsu
R5108 photomultiplier tube cooled with dry ice and run at 1100 V
in photon-counting mode. Data acquisition and analysis was done
with Felix32 software. Fluorescence samples were prepared by 1 :
10 dilution of the dye solution used for absorption spectroscopy in
an appropriate solvent. Excitation wavelength was set 10–20 nm
below the absorption maximum of the dye (for lepidine-type
dyes at 600 nm; for indolenine-type dyes at 675 nm). Real-time
correction software was used. Extinction coefficients presented in
the synthesis methods (refer to ESI†) were calculated from samples
typically prepared by dissolving 1.00 mg of dye in 100.0 ml of
methanol or ethanol with the aid of sonication. Dye samples in
DMSO (1.00 mg ml−1) were diluted into methanol or ethanol to
produce an optical density of approximately 1.0 AU. The same
volume of DMSO stock solution was diluted into solvents for
measurement of absorption spectra. Further dilutions of these
solutions were made into corresponding solvents yielding optical
densities of approximately 0.1 AU for measurement of fluorescence
spectra.
Conclusion
The use of voltage sensitive dyes has been valuable for basic
research and medical physiology of excitable tissues like heart
and nerve networks. Improvements in the sensitivity of the probes
significantly enhance the data quality and range of possible
experiments. Furthermore, advantages can be obtained through
improved dye delivery and imaging of excitable cells from deeper
layers in heart and brain tissues. In developing new methods,
we have uncovered a better probe and enhanced membrane
staining using water-soluble VSDs. These developments provide
incremental improvements that may help us venture into new
paradigms that can produce major increments in dye performance.
Experimental
Chemical syntheses
Full synthetic and characterization details are available as elec-
tronic supplementary information (ESI†).
Synthesis of voltage dyes
Solubility of dyes and PEG derivatives
Briefly, fluorescent dyes (PGH-1 through PGH-10) were synthe-
sized by the following general method. Equi-molar quantities of
anilino-thiophene-carboxaldehyde (2) in ethanol (∼0.25 M) and
quaternized heterocyclic intermediate (3 through 12) dissolved in
methanol (∼0.25 M) were stirred at room temperature. Quinoline
and lepidine intermediates (3, 9, 10 and 11) required the addition
of 1% (v/v) piperidine to facilitate the reaction. At various times
a sample of the mixture was diluted in ethanol and measured
by UV/Vis spectroscopy. Reaction progress was monitored by
appearance of the product dye (Table 3) and decrease of the alde-
hyde (A444). If significant starting aldehyde remained unreacted,
additional heterocycle was added and the reaction continued.
When the Adye/A444 ratio remained constant, the reaction was
Dye solids were dissolved to make 2 mM solutions in water and
PBS. While PEG derivatives dissolved directly, PGH-1 and PGH-6
required initial dissolution in 10 ll of DMSO followed by dilution
into aqueous solvents (1000 ll). After brief vortex mixing and
1 h of sonication, samples were filtered through 0.22 lm filters
then diluted into methanol for concentration determination by
absorption spectroscopy.
Tissue labeling experiments
Frog hearts were isolated and perfused in a Langendorff apparatus
and stained with a bolus of concentrated dye solution (20 ll of
1 mg ml−1 dye in DMSO plus or minus Pluronic) until it was
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noticeably stained.3,6 The heart was placed in a 10 mm × 10 mm
cuvette and right-angle fluorescence measurements were taken
from re-emergent light through the tissue. Emission spectrum
parameters: excitation 600 nm, emission 620–1000 nm; excitation
spectrum parameters: emission 875 nm, excitation 500–865 nm,
Fluorolog 3 Jobin-Yvon, Hariba, Inc.
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Mouse ventricular action potential experiments
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◦
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emission with a long pass filter at 650 nm. All animal experiments
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Acknowledgements
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This work was supported by the National Heart, Lung and
Blood Institute with RO1 HL057929, HL70722 and HL69097
to Guy Salama. Thanks are due to Sujata Emani for synthesis
and purification of styryl intermediates, to Dr Frederick Lanni
and Patrick Byrne for spectral analysis of dyes in frog hearts;
to Ghassan Azour for preliminary evaluation of PEG dyes in
cardiac cells; to Jonathan Douds for spectral analysis method
development; to Eric Lanni for mass spectroscopy expertise; and
to Roberto Gil for help with NMR measurements. The NMR
spectrometers of the Department of Chemistry NMR Facility
at Carnegie Mellon University (USA) were purchased in part
with funds from NSF (CHE-0130903). The MALDI-TOF mass
spectrometer was funded by NSF (CHE-9808188).
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