Note: Descriptions are shown in the official language in which they were submitted.
212933
- 2 -
This invention relates to substituted
aminostyrylpyridinium salts and their use as viscosity
probes for measurement of the viscosity of a liquid.
The invention also relates to certain novel
substituted aminostyrylpyridinium salts.
The viscosity of a solution, for example, an
aqueous solution, is a function of the size,
concentration, and nature of the molecules,
particularly macromolecules, dissolved therein.
Normally, this parameter is deduced from the rate of
passage of a volume, typically several to tens of
milliliters, of the liquid through a small hole or
capillary tube. However, this technique is not
appropriate for measuring specific values or vari-
ations of viscosity on a very small scale such as are
known, or suspected, to accompany many important
biological processes and some pathological conditions;
motion of mucus along respiratory passages,
notoriously inefficient in people with cystic
fibrosis, partly because of admixture with extremely
viscous DNA; or motion, for example contraction in
smooth muscle, extrusion of pseudopods in leukocytes,
or tonicity or other changes, for example mitosis,
within individual cells. pne existing microscale
technique is to microscopically observe and quantify
the movement of tiny magnetic particles in a steady or
oscillating magnetic field (M. King and P. Macklem, J.
Appl. Physiol. 42, 797 (1977)).
A better method would involve the addiaion
of a fluorescent dye whose emission, wavelength,
intensity and/or polarization, is strongly and
specifically sensitive to the viscosity of the local
microenvironment; such a strategy, making use of
3 - 2129933
existing hardware such as that for determination of
intracellular calcium ion by fluorescence microscopy,
using the Fura-2,"molecular probe", could give xeal-
time measurements of viscosities in small volumes,
such as 'different parts within a living cell as it
actually functions and responds to stimuli.
V.W. Burns,.Biochem. Biophys. Res. Comm.
. 1008 (1969) and Experim. Cell. Res. 64, 35 (1971),
describe the use of fluorescein to determine average
viscosities within cells suspended in culture, of
yeast (10-15 cP) and Euglena (6 cP). The technique.
makes use of polarization spectroscopy; light emitted
from a fluorescein molecule irradiated with polarized
light is depolarized according to the rate of tumbling
of the entire molecule, which itself depends on the
viscosity of the medium. The technique was shown
useful only to measure the average viscosity of all
cells in a culture, rather than within individual
cells (non-"imaging"); it was also sensitive to
scattering, and interference from extracellular
fluorescein even with washed cells due to continuous
leakage of fluorescein back out of cells even at room
temperature.
M.S.A. Abdel-Mottlieb, Laser Chem. 4, 305
(1984), observed an increase in the quantum yield of
fluorescence of 2-(4-dimethylaminostyryl)-1-methyl
pyridinium iodide with increasing viscosity of its
solution, and suggested that this molecule . can
therefore 'be used as a fluorescent probe to study
microscopic structural changes in surrounding media
occurring on the picosecond time scale; however,
without 'provision for an internal standard, this
method cannot be used to measure the viscosity of a
liquid in which any of the intensity of exciting
35~ radiation, the concentration of the molecule or the
thickness of the sample are unknown. M. L. Viriot et
212933
- 4 -
al, Photobiochem. Photobiophys. y1984 v5 p293,
measured viscosity by comparing ("ratioing") two kinds
of fluorescence from dipyrenyl alkanes, which is a
technique that depends neither on the intensity of
excitation, the concentration of the fluorescent
molecule nor the geometry of the sample; but these
molecules prefer the hydrophobic environment of a
cell's membrane over its cytoplasm, and are similarly
not useful to measure viscosities of aqueous solutions
because of their insolubility therein; moreover the
viscosity-dependance of their fluorescence involves
intramolecular excimer formation, a different
mechanism than Twisted Intramolecular Charge Transfer
(TICT; W. Rettig, Angew. Chem. Int. Ed. Engl. 25, 971
(1986)).
Syrups or other edible liquids, petroleum
mixtures, lubricating liquids, polymerization
reactions, sewage, pulp effluent, and other aqueous or
non-aqueous liquids also vary widely in viscosity
depending on the composition of the mixture and its
processing, and viscosity probes would also have
application in the determination of the viscosity of
such mixtures at discrete times or on a continuing
basis.
The present invention seeks to provide a
family of substituted aminostyrylpyridinium salts
which are fluorescent dyes which can be employed with
advantage as viscosity probes.
In accordance with the invention there is
provided a fluorescent probe for measurement of
viscosity of a liquid comprising a substituted
aminostyrylpyridinium salt of formula (I)
- 5 -
2129933
Y1
Y2
(I)
OZ
J
Y
wherein one of Y and Y is ~R1
1 2 -N\ and the other is CH,
R2
Rl and R2, which may be the same or different are
straight or branched chain lower alkyl of 1 to 6
carbons or R1 and R2 together with the N atom to which
they are attached form a cyclic ring of 5 or 6 ring
atoms; one of Z and Y is >N+-R X- and the other is CH;
R is selected from: ,(a) -(CH2)nR3 in which n is an
integer of 0 to 15 and R3 is aryl of 6 to 14 carbon
atoms,. alkyl of 1 to 10 carbon atoms, amino,
dialkylamino in which each alkyl is straight or
branched chain of 1 to 6 carbon atoms or cyclic of 5
or 6 carbon atoms, or acyl R6-CO- in which R6 is alkyl
of 1 to 16 carbon atoms, aryl of 6 to 14 carbon atoms
or aralkyl in which the aryl moiety has 6 to l4 carbon
atoms and the alkyl moiety has 1 to 16 carbon atoms,
(b) -(CH2)mC00R4 in which m is an integer of 1 to 10
and R4 is a straight or branched chain alkyl of 1 to
18 carbon atoms, aryl of 6 to 14 carbon atoms, aralkyl
in which the alkylene moiety has 1 to 18 carbon atoms
and the aryl moiety has 6 to ~.4 carbon atoms, said.
aryl and aryl moiety being' unsubstituted or
substituted one or more times by one or more of vitro,
fluoro, chloro, bromo, iodo and carboxylate; (c)
- 6 - 2129933
-(CH2)pArC00- in which p is 0 to 15, Ar is arylene of
6 to 14 carbon atoms unsubstituted or substituted one
or more times by one or more of nitro, fluoro, chloro,
bromo, iodo, carboxylate or -COOR4 in which R4 is as
defined above, (d) -(CH2)pArC00R4 in which p, Ar and
R4 are as defined above, and X- is selected from
chloride, bromide, iodide, toslylate, mesylate and
brosylate.
In another aspect of the invention~there is
provided a method of measuring viscosity of a liquid
in which fluorescence of a fluorescent dye in the
liquid is measured and the viscosity is determined
from the measured fluorescence, wherein the
fluorescent dye is a substituted aminostyrylpyridinium
salt of the invention.
In still another aspect of the invention
there is provided a method of measuring viscosity of a
liquid comprising contacting the liquid with a
fluorescent probe of the invention, allowing the
fluorescent probe to fluoresce and evaluating the
fluorescence as a measure of viscosity of the liquid
The invention also provides novel salts of
formula (I).
The novel salts are in particular salts in
which R is selected from a) -(CH2)mC00R4 in which m is
an integer of l~to 10 and R4 is a straight or branched
chain alkyl of, 3 to 1$ carbon atoms, aryl of 6 to 14
carbon atoms,'aralkyl in which the alkylene moiety has
1 to 18 carbon atoms and the aryl moiety has 6 to 14
carbon atoms, the aryl and aryl moiety being
unsubstituted or substituted one or more times by one
or more of nitro fluoro, chloro, bromo, iodo and
carboxylate, (c). -(CH2)pArC00- in which p is 0 to 15,
Ar is arylene of 6 to 14 carbon atoms, unsubstituted
or substituted one or more times by one or .more of
nitro, fluoro, chloro, bromo, iodo, carboxylate or -
%~1~9933
COOR4 in which R4 is as defined above, (d) -
(CH2)pArC00R4 in which p, Ar and R4 are as defined
above.
Rspecially preferred salts ( I ) are those in
R
which Y1 is -N 1, Y2 is CH, Z is CH and Y. is >N+-R X'
R2
and in which (a) R is -CH2CH2N(CH3)2 and X' is
chloride; (b) R is benzyl and X- is chloride; (c) R
is phenylethyl and X' is bromide; and (d) R is
CH2COOBu, in which Bu is butyl, especially n-butyl,
and X- is chloride.
The invention also provides a process for
preparing the novel salts.
The new viscosity probes can be produced by
a simple synthesis and include "caged" dyes capable of
irreversible permeation into cells. They display
increased sensitivity and selectivity of fluorescence
to viscosity by a mechanism involving rapid
intramolecular charge transfer with formation of a
TICT state, and emission and excitation spectra of
shape, as well as size, dependent on viscosity,
permitting conclusions on viscosity which are
independent of actual concentration of dye loaded into
cells; they permit ready real-time images of viscosity
throughout a live cell; human muscle cells loaded with
the dye remain viable; by varying the temperature of
the sample, whether by pulse or slow drift, activation
energy (Ea) can be quantified for characterization of
the nature of the solutes, for example DNA vs
polypeptides vs carbohydrates.
In the case of aryl radicals or moieties of
6 to 14 carbon atoms these are, in particular, phenyl,
naphthyl and anthracyl.
- ~ X129933
A particularly preferred group of salts
within formula (I) is the class of
dialkylaminostyrylpyridinium salts of formula (II):
R~ R2
0
-I~ X-
R
(IT)
wherein Rl and R2, which may be the same or different
are straight or branched chain lower alkyl of 1 to 6
carbons; R is selected from: (a) -(CH2)nR3 in which n
is an integer of 1 to 15 and R3 is phenyl, alkyl of 1
to ZO carbon atoms or dialkylamino, in which each
alkyl is straight or branched chain of 1 to 6 carbon
atoms provided that when R3 is alkyl, the total carbon
atom content of R is not more than 16, and (b)
-(CH2)mC00R4 in which m is an integer of 1 to 10 and
R4 is a straight or branched chain alkyl of 1 to 6
carbon atoms; and X- is a halide ion selected from
chloride, bromide and iodide.
In one preferred embodiment the liquid is an
aqueous liquid, and in particular a very small volume
of aqueous liquid.
In another embodiment the liquid is
lipophilic, for example, a petroleum mixture. In this
embodiment the substituents R, Rl and R2 are
preferably large, hydrophobic radicals soluble in oil
- 9 - 2129933
phase. Tn this embodiment the viscosity may be
measured in a microvolume or a macrovolume.
In the embodiment in which the ,liquid is an
aqueous liquid the viscosity may be measured in a
microvolume, for example, within biological cells or
in a macrovolume.
The viscosity may be determined for
individual cells or as an average viscosity of a
plurality of the cells in suspension or as a film on a
ZO support layer or strip.
The viscosity measurement may employ single
excitation from a single emission, double excitation
from a single emission or double emission from a
single excitation.
The colour change or fluorescence which is
produced may be one detectable to the naked eye or one
which is detected by instruments, such as a
spectrofluorimeter or a fluorescence microscope with
attached image analyzer.
In vitro studies show that fluorescence of
these probes is independent of pH, ionic strength, and
other factors than viscosity, except insofar as these
affect viscosity, though high concentrations of some
anions, apart from chloride, have an effect.
Fluorescence of these molecules is also inversely
proportional to temperature, though where temperature
is known or kept constant, it can be accounted for.
fluorescence of these molecules is also affected by
polarity, but in a manner different and independent of
viscosity, by shifts in peak wavelengths
(solvatofluochromism) rather than changes in
intensities. Calibration. against standard viscometric
techniques yields similar calibration curves for.
different solutes; changes in the emission and
excitation spectra with viscosity lend themselves
- 1 a - 2129933
readily to ratioing techniques for viscosity
determination.
Applications of these dyes to measuring slow
changes in viscosity during cell division or rapid
changes during pseudopodal motion of leukocytes,
particularly, with contraction or relaxation of smooth
muscle cells to provide a means of directly observing
contractile state of smooth muscle cells in culture;
this technique may displace current measurements of
"second messenger" levels, such as calcium ions, to
evaluate cell response vs stimuli, for example for
drug screening,. tissue typing or diagnosis of medical
conditions.
In particular, the fluorescent dye viscosity
probes of the invention are intramolecularly-relaxing
compounds exhibiting dual fluorescence spectra in
polar solvents. Such dual fluorescence can be
explained by the presence of twa kinds of excited
states arising from twisted intramolecular charge
transfer, which makes the compounds very sensitive to
the microenvironment surrounding the fluophore.
Observed excitation wavelength dependence and strong
red edge effect in more viscous solvents support a
model of twisted intramolecular charge transfer tTICT)
accompanied by viscosity-dependent torsional motion
during irradiation of these compounds.
The manufacture of the substituted
aminostyrylpyridinium salts of the invention is illus-
trated below and specific compounds RX are identified
for tour preferred salts of the invention.
- ~ 1 - 2129533
NMe2 NMez
NMr.I NMe2
~ .
a
. HCONMe2 CHO
1 X'R
CH3
a
rr.- ~ ~*.x
X-R = C1-CH2CI-IzNMez (1)
= CI-CHZPh (2)
~ - $c-C~-lzCHzPh (3)
- Cl-CFTZCOOBu (4)
Salt (1) is found to be impermeable to cell
membranes and can be used as a viscosity probe for
determination of the viscosity of only extracellular
fluids. Salts (2) and (3) readily permeate into
smooth muscles at 37°C and remain within the muscles
upon cooling to room temperature; salt (4) permeates
readily but is then transformed by cellular enzymes to
a membrane-impermeable form that remains in .the cell
indefinitely even at 37°C. In this regard salt (4) is
an example of caged dyes, the ester being converted to
the free acid after passage into the environment whose
viscosity is to be determined.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a viscosity calibration curve for
a compound (I) of the invention, employing single
excitation-single emission (quantum yield, non
ratioing) mode;
- 1 a - 2129933
FIGS. 2A and . 2B illustrate graphically
changes in the emission spectrum from a single
excitation far a compound (I) of the invention, with
change in viscosity;
FTG. 3, which appears on the same sheet as
FIG. 1, is a calibration curve for the compound (I) of
the invention, employing single excitation-double
emission (ratioing) mode; and
FTG. 4 is a photograph showing a map of
viscosities by fluorescence in smooth muscle cells in
culture, employing double excitation-single emission
(also ratioing) mode.
BXAMPLSS
The invention is further illustrated by the
following Examples.
- 13 _ X129933
EXAMPLE 1(a)
Syrn~hesis o~ Carboxymeth'~I ~ esters of
Dimeth,~laminos~yrylp~~ridinium salts
Dirreefih~~laniinosfi.~~yrlp, r~ idi,ne
Reaction of 4-picoline with benzoyl chloride at 0°C to~ yield the
1-
benzoyl salt of 4-picoline. This was then reacted with p-
dimethylamino benzaldehyde (DMAB) to obtain the 4-p-
dimethylaminostyrylpyridine.
Materials:
4-picoline: 20m1
benzoyl chloride: 5ml
DMAB; 10.16g
Exaerimental:
To picoline (5ml) was added dropwise benzoyl chloride in an oven-
dried 3-necked 100m1 ~ round bottom flask with strong magnetic
stirring and cooling in an ice bath. To this mass was added the
solution of DMAB in l5ml picoline. The reaction mixture became red
and upon. heating. The mixture was heated to reflux 0150 C) for
5hrs. (reflux condensor was topped with a drying tube).
The reaction mixture was cooled and decomposed with ~30m1 of .
conc 12N HCI. After stirring the solution for about 20 min, the
solution was alkalized by the addition of ~50m1 5N KOH. The red-
brown precipitate was collected on fluted filter paper .and washed a
few times 'with hot water. The crystals were placed in a
recrystallizing dish in the fumehood overnight.
The solid was dissolved in ~300m1 0.5N HCI and heated to the
boiling point. 10 large scoops of active carbon was added and the
solution was boiled ~1/2 an hour. The carbon was filtered away and
the product was precipitated with ~lml 6N, and 15m1.3N ammonium
hydroxide. The brownish-red product was collected and then
dissolved in ~600m1 95% boiling ethanol. The solution was cooled,
crystals formed. 3N ammpnia was added dropwise. The muddy-
yellow precipitate was collected , washed with cold ethanol and
recrystallized once more from 95% ethanol, then dried in a vacuum
oven at 80 C overnight. ,
Wt of crystals: 3.11 g 32.2% yield , ,
mp: 245-246 °C
2129933
- 14 -
Quaternization ofi dimeth~~laminosty~pyrridine with
chloroacetate esters.
~'N~
Cl
o DMr loo°c
0
o~~
. C,_
N O
Reaction of chloroacetate esters with dimethylaminostyrylpyridine
(DSP) in Dimethylformamide (DMF)
Materials
0.988 DSP (4.37mmol)
0.98g Butyl chloroacetate (6.53mmol)
DMF l5mls (Reagent grade , BD1-I)
Diethyl ether, 150m1 (BDH)
Experimental
DSP (0.988, ~.37mmol) was dissolved in l0ml DMF in a flame dried
2-neck RBF at 100°C, in a nitrogen atmosphere.
Butylchloroacetate in 5ml DMF was added slowly dropwise to the hot
solution which turned deep red.
The solution was stirred 5hrs at 100°C under nitrogen. The reaction
was followed by TLC (dichloromethane as eluent ).
The mixture was cooled to 50°C and poured in 150m1 diethylether
whereupon a fed precipitate formed.
The precipitate was filtered and washed with ethanol then dried in
vacuo overnight.
Yield 1.87g , 3.65mmol, 83.6%
Melting point 197.5-199.2 °C
- 15 - 2129933
_,
EXAMPLE 1(b)
Following the procedure of Example 1(a) but
employing dimethylaminoethylene chloride there was
produced salt (1) in the form of an oil having the
following characteristics H-NMR 8.05 (d, aryl H), 7.43
(d, aryl H), 7.15 (d, vinyl H), 6.43 (d, vinyl H),
3.48 (s, aryl N-CH3), 3.08 (s, alkyl N-CH3), 2.48 (m,
-CH2CH2-).
EXAMPLE 1(c) '
Following the procedure of Example 1(a) but
employing benzyl chloride there was produced salt (2)
characterized by a melting point of 259-261°C.
EXAMPLE 1(d)
Following the procedure of Example 1(a) but
employing phenethyl bromide there was produced salt
(3) characterized by a melting point of 253-255°C.
EXAMPLE 2
Use of the Dye
a) Cuvette system (ex. for calibration with
solutions of known viscosity, ex. sucrose/water, as
measured with a capillary viscometer).
The dye was added to liquid to a
concentration of 3.8E-5M; 3 mL of this was placed in a
cuvette and inserted in. a PTI Deltascan 4000
spectrofluorimeter for analysis of fluorescence
through the bulk of the sample.
b) Imaging of individual living cells.
Cultured cells (ex. smooth muscle) adhering
to slides were incubated at 37°C. in Hank's buffer
with penetrable dye (see below) at concentrations, of
3.8E-5M for 10 minutes, then washed 3X with Hank's
buffer, then placed under a Nikkon microscope
connected to a PTI Deltascan 4000 spectrofluorimeter
for real-time visual observation, and 'quantitative
measurement of flurorescence at selected points, or
over the whole image. Examination of the cells while
- 16 - 2129933
changing focus established the dye to be distributed
throughout the cytoplasm inside the cell, and not in
either the nucleus or the membrane. In the case of
the salts (2) in which R1 and R2 were both methyl and
R was -CH2Ph or -CH2CH2Ph, the dye slowly leaked out
again at 37 °C, but not if the temperature was lowered
to 25°C during washing and observation (cells
subjected to this treatment and returned to the
incubator were still alive 24 hours later). The salts
in which Rl and R2 were methyl and R was -CH2CH2NMe2
or -CH2C00- did not penetrate the cell membrane; the
salt in which R1 and R2 were methyl and R was
CH2COOBu initially penetrated but became non
penetrating as enzymes digested it to -CH2C00- once
inside the cell.
There are three modes of measuring viscosity
by fluorescence of probes of the invention (with
calibration vs other viscometry 'techniques possible in
any of the modes) of which b) and c)'below (made
possible by the phenomenon of "dual fluorescence" in
these 'molecules) have the advantage of involving
ratioing, thus being independent. of excitation
intensity, dye concentration or sample geometry:
a) Single excitation-single emission: upon
excitation at a single wavelength (ex. at 469 nm),
absolute intensity of one emission peak (ex. at 603
nm) depends on viscosity of solution; numerical
quantum yield could be obtained by comparison with a
standard (rhodamine B/ethanol) in a separate cuvette.
However, besides viscosity°dependent quantum yield of
fluorescence, intensity of emission is also a function
of intensity of excitation, dye concentration and path
length through sample (i.e., dimensions or.geometry of
sample). Figure 1 is a calibration curve generated in
this way.
- 1 ~ - 2129933
b) Single excitation-double emission.
Following a single excitation (ex. at 369 nm), by
observing and comparing the intensities of emission at
two wavelengths, at one of which emission is very
viscosity-dependent (ex. 592 nm) and at the other
less or not (ex. 443 nm), viscosity can be deduced
without having to know excitation intensity, dye
concentration or sample geometry. . The change in
relative sizes of two peaks with viscosity is
illustrated by FIGS. 2a and 2b. FIG. 3 is a
calibration curve that makes use of this excitation-
intensity/concentration/geometry-independent ratio,
and so illustrates a different concept than FIG. 1.
c) Double excitation-single emission. In this
second ratioing technique, it is the changing shape of
the excitation spectrum for a given emission that is
made use of. The intensity of the same emission peak
(ex. 600 nm) when excited at one wavelength at which
it is sensitive to viscosity (ex. 469 nm), is compared
to its intensity when excited at another wavelength
(ex. 360 nm) at which it is rather less viscosity-
sensitive. Viscosity measurements of living cells
(FIG. 4) were obtained in this way.
The human eye may. judge the intensity of
fluorescence involved in mode a), and the colour
(blend of hues) involved in mode b), but is less
useful by itself for mode c).
Data for FIGS. 1-3 came vfrom experiments on
ca. 2 mL volumes.
FIG. l: Quantum yield of emission ~at 603 nm, ;upon
excitation at 469 nm (single excitation-single
emission), of 4-(4-dimethylamino)styryl)-1-phenethyl
pyridinium bromide in water + glycerol of increasing
viscosities (0 g/L and 0.95 cP, to 560 g/L and 4-5
cP).
- 1$ 2129933
FIG. 2a: Emission spectrum, upon excitation at 369
nm, from 4-(4-dimethylamino)styryl)-1-phenethylpyri-
dinium bromide in water (viscosity = 0.95 cP).
FIG. 2b: Emission spectrum, upon excitation at 369
nm, from 4-(4-dimethylamino)styryl)-1-phenethylpyri
dinium bromide in 60~ glycerol/H20 solution (viscosity
- 5 cP).
FIG. 3: Ratio of emission intensities at 592 nm and
443 nm, upon excitation at 360 nm (single exaitation
double emission), from 4-(4-dimethylamino)styryl)-1
phenethylpyridinium bromide, vs viscosity/temperature
in sucrose/water solutions of different concent-
rations, from 0 (0.95 cP) to 280 g/L.
FIG. 4: (Black-and-white reproduction of) colour
figure showing map of viscosities in smooth muscle
cells in culture. Cells were treated with 4-(4
dimethylamino)styryl)-1-(1-butoxy)carbonylmethyl-pyri
dinium chloride in water for 20 minutes then washed
with fresh buffer. Black shows where dye was not
present outside cells and within cell nuclei.
Remaining shades show regions of viscosity ranges of
1.36-1.76, 1.76-2.25, 2.25-2.88 and 2.88-3.73 cP, as
measured by comparison of emissions at 600 nm from
excitations at 469 and 360 nm (double excitation
single emission).
