U.S. patent application number 15/529700 was filed with the patent office on 2017-11-16 for hydrodabcyl.
The applicant listed for this patent is Universitat Bayreuth. Invention is credited to Elisa BOMBARDA, Karl KEMPF, Oxana KEMPF, Rainer SCHOBERT, G. Matthias ULLMANN.
Application Number | 20170327460 15/529700 |
Document ID | / |
Family ID | 54707786 |
Filed Date | 2017-11-16 |
United States Patent
Application |
20170327460 |
Kind Code |
A1 |
BOMBARDA; Elisa ; et
al. |
November 16, 2017 |
Hydrodabcyl
Abstract
A new azobenzene-based fluorescence quencher with excellent
solubility in aqueous solution is described here. This compound
represents an optimized alternative to dabcyl in a variety of
biomolecular applications, like fluorogenic protease substrates or
nucleic acids probes.
Inventors: |
BOMBARDA; Elisa;
(Eckersdorf, DE) ; KEMPF; Oxana; (Kulmbach,
DE) ; KEMPF; Karl; (Kulmbach, DE) ; SCHOBERT;
Rainer; (Bayreuth, DE) ; ULLMANN; G. Matthias;
(Eckersdorf, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universitat Bayreuth |
Bayreuth |
|
DE |
|
|
Family ID: |
54707786 |
Appl. No.: |
15/529700 |
Filed: |
November 27, 2015 |
PCT Filed: |
November 27, 2015 |
PCT NO: |
PCT/EP2015/077982 |
371 Date: |
May 25, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/64 20130101;
C09B 29/081 20130101; C07C 245/08 20130101 |
International
Class: |
C07C 245/08 20060101
C07C245/08; G01N 21/64 20060101 G01N021/64; C09B 29/08 20060101
C09B029/08 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 28, 2014 |
EP |
14195529.4 |
Dec 10, 2014 |
EP |
14197181.2 |
Claims
1. A compound having formula (I) ##STR00005##
2. Method of producing a compound having formula (I) ##STR00006##
wherein the method comprises the steps of a) producing
5-dimethylamino-resorcinol (5-(dimethylamino)benzene-1,3-diol)
##STR00007## by reacting phloroglucinol (benzene-1,3,5-triol) with
dimethylamine (HN(CH.sub.3).sub.2) to obtain
5-dimethylamino-resorcinol; and b) azo-coupling 4-diazo-salicylic
acid to 5-dimethylamino-resorcinol to obtain the compound having
formula (I).
3. Method of claim 2 further comprising step a1), wherein the
compound obtained in step a) of claim 2 is purified by (i)
concentrating the 5-dimethylamino-resorcinol; (ii) purifying the
residue obtained in step (i) by column chromatography; and (iii)
crystallizing the purified compound obtained in step (ii).
4. Method of claim 2, wherein 4-diazo-salicylic acid is obtained by
reacting 4-aminosalicylic acid with NaNO.sub.2 and HCl to obtain a
4-diazo-salicylic acid having the formula: ##STR00008##
5. Method of claim 2, wherein the method further comprises a step
b1), wherein the compound obtained in step b) of claim 2 is
purified by precipitation at pH<4 followed by
centrifugation.
6. (canceled)
7. A probe comprising a reporter and the compound of claim 1.
8. A method of measuring fluorescence comprising administering a
probe comprising a reporter and the compound of claim 1 to a sample
and measuring the fluorescence of the sample, wherein substantially
no fluorescence is observed from the compound of claim 1.
9. The method of claim 8, wherein the sample is a biological
system.
10. The method of claim 9, wherein the administering is in
vivo.
11. The method of claim 9, wherein the administering is in vitro.
Description
FIELD OF INVENTION
[0001] The present invention concerns the provision of a novel dark
quencher, called Hydrodabcyl, which is water-soluble and suitable
for assembling a fluorogenic probe for in vivo as well as in vitro
application. The present invention also concerns a method of
preparing Hydrodabcyl and the use of Hydrodabcyl as a dark quencher
in biological systems.
BACKGROUND
[0002] Biomolecular processes are extensively studied by employing
fluorescent dyes that either bind non-covalently to a target in the
system or, to gain specificity, are covalently linked to the
investigated biomolecule. Changes in fluorescence intensity or
wavelength indicate that a biochemical event has taken place. There
is a large choice of fluorescent dyes whose signal is affected by
several physico-chemical parameters, such as pH, hydrophobicity,
oxidation state or ionic strength. To improve the strategy of using
probes comprising a single label, quencher dyes have been developed
to provide dual-labeled probes, in which the quencher is paired
with the reporter dye to enhance the observable change in
fluorescence. Typically, these probes have a closed (i.e. quenched)
form in which the reporter and the quencher are close to each other
in space and an open form (i.e. fluorescent) in which the reporter
and the quencher are spatially separated.
[0003] The quencher can be a second fluorescent dye. In this case,
the fluorescence of the reporter can be monitored alone, or both
the increase in the fluorescence of the quencher and the decrease
in fluorescence of the reporter are observed. An overlap between
quencher and reporter fluorescence spectra may cause background
noise, which necessitates dedicated care in the instrumental set-up
and data analysis as well.
[0004] Dark quenchers (e.g. non-fluorescent dyes) offer a solution
to this problem because they do not occupy an emission bandwidth.
The dual-labeled probes including a reporter and a dark quencher
are also called fluorogenic or turn-on probes, since a
(bio-)chemical event causes their transition from a non-fluorescent
to a (typically highly) fluorescent form.
[0005] Dabcyl (4-(4'-dimethylamino-phenylazo)benzoic acid) is a
widely used dark quencher [1] in dual-labeled probes for a variety
of biomolecular applications, like enzymatic catalysis and nucleic
acid probes [2, 3].
[0006] Dabcyl is a molecule based on an azobenzene scaffold, which
consists of two phenyl rings linked by an azo group (N.dbd.N) in
which each nitrogen atom carries a non-bonding pair of
electrons:
##STR00001##
[0007] This aromatic system confers high hydrophobicity to dabcyl
making it insoluble in aqueous solution. Therefore, stock solutions
of dabcyl need to be prepared in DMSO.
[0008] The absorption band of dabcyl in the range of 400-550 nm
overlaps with the emission band of many common fluorescent dyes
such as EDANS (5-((2'-aminoethyl)amino)naphthalene-1-sulfonic acid;
.lamda..sub.em, Max=490 nm), monobromobimane (mBBr; .lamda..sub.em,
Max=480 nm), and many fluorescein, coumarin and rhodamine
derivatives, e.g. carboxyfluorescein (FAM; .lamda..sub.em=515 nm;
in water), coumarine 1 (.lamda..sub.em=448 nm; in ethanol),
rhodamine 123 (.lamda..sub.em=512 nm; in ethanol) to cite only a
few.
[0009] Although dabcyl is one of the most popular acceptors for
developing fluorescence resonance energy transfer-(FRET)-based
biological probes, the very poor solubility in water set limits to
its use in biological systems where the natural solvent is water.
Although this hydrophobicity can be compensated by the
hydrophilicity of the substrate to which dabcyl is linked (e.g.
long DNA segments or peptide chains), it represents a real problem
in case of comparatively small substrate (e.g. glutathione) in
which this compensation is more difficult.
[0010] Solubility problems have been observed for several
dabcyl-labeled substrates [4]. Incomplete dissolution leads to
incorrect estimation of the concentrations and consequently wrong
calculations of the stability and rate constants. Attempts to
overcome the problems deriving from the insolubility have been
done, e.g. by performing the enzymatic assays in mixture of water
and DMSO [2].
[0011] Decreasing the hydrophobicity of a compound is usually done
by adding either sulfonate or hydroxyl groups to the compound. The
modification, however, must not lead to a change of the desired
properties of the compound. In the case of dabcyl, it is imperative
that the fluorescence properties, namely the function as dark
quencher, are not changed. For example, the emitting properties of
the compound must not be significantly affected by the
modification. If the modification leads to a change of the emitting
properties, the compound might become fluorescent itself and
therefore is no longer suitable as a dark quencher. This effect is
known to occur when hydroxyl groups are added to a compound. For
example, the addition of one hydroxyl group transforms the weak
fluorescence of phenylalanine in the red-shifted stronger
fluorescence of tyrosine.
[0012] Furthermore, the modification must not lead to a significant
change in the electrostatic profile of the compound, as its
function is to interact with molecules in the context of biological
systems, wherein the molecular interactions are often driven by
electrostatics (e.g. enzymatic reactions). A change in the
electrostatic profile is a known effect of the addition of
sulfonate groups [5].
[0013] In addition, the modification must not lead to a significant
structural change of the compound, which affects the interaction
with molecules in the biological systems, where the compound is to
be used. For example, it has to be prevented that catechols are
formed by the modification, as the catechols strongly chelate
metals (e.g. Fe(III)). The chelation leads to unwanted reactions
that may interfere with the system under investigation. This aspect
is particularly important for the investigation of enzymatic
reactions in which metals are essential cofactors and for possible
applications in vivo.
[0014] It was the problem to be solved by the present invention to
provide a compound suitable as a dark quencher, which can be used
in aqueous systems, is superior in spectroscopic properties
compared to dark quenchers of the state of the art, and which is
easier to handle.
[0015] This problem was solved by providing the compound
4-((4'-(dimethylamino)2',6'-dihydroxyphenyl)azo)2-hydroxybenzoic
acid, which is herein also called Hydrodabcyl. Hydrodabcyl is
easier to handle compared to dabcyl as it is soluble in aqueous
solutions. It is superior to dabcyl as it has superior quenching
abilities due to a higher molar absorbance compared to dabcyl.
[0016] A further problem to be solved by the present invention was
to provide an improved method for synthesizing Hydrodabcyl. This
problem is solved by providing the method as described.
SUMMARY OF THE INVENTION
[0017] A compound is provided, which acts as a dark quencher, and
is water soluble. This compound is
4-((4'-(dimethylamino)2',6'-dihydroxyphenyl)azo)2-hydroxybenzoic
acid, which is herein also called Hydrodabcyl.
##STR00002##
[0018] Further provided is a method of producing the compound.
[0019] Details of the present invention are set forth in the
accompanying drawings and the description below. Other features,
objects, and advantages will be apparent from the description and
the drawings.
DESCRIPTION OF DRAWINGS
[0020] FIG. 1 shows the scheme of the synthesis of
4-((4'-(dimethylamino)2',6'-dihydroxyphenyl)azo)2-hydroxybenzoic
acid (Hydrodabcyl). 5-dimethylamino-resorcinol (1) is produced and
azo-coupled to a diazotized 4-aminosalicylic acid to synthesize
4-((4'-(dimethylamino)2',6'-dihydroxyphenyl)azo)2-hydroxybenzoic
acid (2).
[0021] FIG. 2 shows a comparison of the absorption spectra of
dabcyl (dash-dotted line, .lamda..sub.max=451 nm) and Hydrodabcyl
(solid line, .lamda..sub.max=470 nm) at a concentration of
2.times.10.sup.-5 M in DMSO at 20.degree. C. The bathochromic shift
(red-shift) of Hydrodabcyl can be observed.
[0022] FIG. 3 shows a comparison of the absorption spectra of
Hydrodabcyl (concentration: 2.times.10.sup.-5 M; temperature
20.degree. C.) in DMSO (solid line, .lamda..sub.max=470 nm,
.epsilon.=37000.+-.1000 Lmol.sup.-1cm.sup.-1) and in 50 mM sodium
phosphate buffer pH=8.0 (dotted line, .lamda..sub.max=445 nm,
c=43000.+-.1000 Lmol.sup.-1cm.sup.-1). The hypsochromic shift
(blue-shift) in the buffer can be observed.
[0023] FIG. 4 shows the molar absorbance of Hydrodabcyl in
DMSO.
[0024] FIG. 5 shows the molar absorbance of Hydrodabcyl in 50 mM
sodium phosphate buffer at pH=8.0.
[0025] FIG. 6 shows the result of the Electrophoretic Mobility
Shift Assay (EMSA) carried out with circular double-stranded DNA
pBR322. The DNA was incubated for 24 hours with various
concentrations (0, 5, 10, 25, 50 .mu.M) of (a) doxorubicin and (b)
Hydrodabcyl. FIGS. 6a and 6b show the ethidium bromide staining of
DNA separated by agarose gel electrophoresis.
[0026] FIG. 7 shows the result of the Electrophoretic Mobility
Shift Assay (EMSA) carried out with linear double-stranded DNA
pBR322. The DNA was incubated for 24 hours with various
concentrations (0, 5, 10, 25, 50 .mu.M) of (a) doxorubicin and (b)
Hydrodabcyl. FIGS. 7a and 7b show the ethidium bromide staining of
DNA separated by agarose gel electrophoresis.
[0027] FIG. 8 shows the results of the in vivo fluorescence
assay.
[0028] FIG. 8a shows an oocyte injected with buffer. No
fluorescence is detected.
[0029] FIG. 8b shows an oocyte injected with Substrate-Bim.
Fluorescence is detected.
[0030] FIG. 8c shows an oocyte injected with
Bim-Substrate-Hydrodabcyl. No fluorescence is detected, because
Hydrodabcyl quenches the fluorescence of Substrate-Bim.
[0031] FIG. 9 shows evidence of precipitation of dabcyl in water
(tentative concentration of about 7 .mu.M (on the left); and a
clear solution of Hydrodabcyl in water (7 .mu.M) on the right.
[0032] FIG. 10 shows the concentration dependence of the absorbance
at 445 nm of Hydrodabcyl in 50 mM sodium phosphate (NaP) buffer
pH=6.0 at T=20.degree. C.
[0033] FIG. 11 shows the concentration dependence of the absorbance
at 445 nm of Hydrodabcyl in 50 mM sodium phosphate (NaP) buffer
pH=4.3 (obtained from pH 7.0 with 5M HCl) at T=20.degree. C. At
pH<4.3 Hydrodabcyl precipitates.
[0034] FIG. 12 shows absorbance at 470 nm of Hydrodabcyl, 14 .mu.M
in DMSO T=20.degree. C., monitored over 2 h (7200 s).
[0035] FIG. 13 shows absorbance at 451 nm of dabcyl, 7 .mu.M in
DMSO T=20.degree. C., monitored over 2 h (7200 s).
[0036] FIG. 14 shows absorbance at 445 nm of Hydrodabcyl, 22 .mu.M
in buffered aqueous solution pH=8 T=20.degree. C., monitored over 2
h (7200 s).
[0037] FIG. 15 shows the absorbance of Hydrodabcyl 17.5 .mu.M in
DMSO at 20.degree. C. Spectrum recorded after the solution
preparation in the dark (continuous line) and spectrum recorded
after 5 min exposure to the light of a 60 W tungsten lamp (broken
line). The two curves are indistinguishable.
[0038] FIG. 16 shows the absorbance of dabcyl 17.5 .mu.M in DMSO at
20.degree. C. Spectrum recorded after the solution preparation in
the dark (continuous line), spectrum recorded after 5 min exposure
to the light of a 60 W tungsten lamp (broken line) and spectrum
recorded after 10 min in the dark at 20.degree. C. (crossed line).
The continuous and crossed lines are indistinguishable.
DETAILED DESCRIPTION OF INVENTION
[0039] A new compound is provided that is compatible with aqueous
systems and thereby overcomes the problem of insolubility in
aqueous solution and avoids the need for organic co-solvents, and
at the same time has spectroscopic properties like dabcyl. The
structure of the compound was designed and synthesized and it was
found that a dimethylamino phenyl azobenzoic acid that is
substituted with 3 hydroxyl groups at specific sites as indicated
in the formula above is water-soluble and is surprisingly a
superior dark quencher. The new compound--Hydrodabcyl or
(4-((4'-(Dimethylamino)-2',6'-dihydroxyphenyl)azo)-2-hydroxybenzoic
acid), thus, provides a very useful combination of properties.
##STR00003##
[0040] The modification of the ring system with hydroxyl groups has
the advantage that no charged groups are present, in contrast to
the presence of sulfonate groups. Consequently, Hydrodabcyl will
not significantly modify the electrostatic profile of the molecule
to which it is linked and thus the binding properties of the
labeled molecule are not altered. In addition, the position of the
hydroxyl groups prevents the formation of catechols. Moreover, the
choice of modifying the ring system has the practical advantage to
keep the carboxyl group available for the coupling to an amino
group in the substrate through a standard amide bond formation.
[0041] Furthermore, although the new compound carries three
hydroxyl groups in comparison to dabcyl, the emitting properties of
the compound compared to those of dabcyl are not adversely
affected. As shown in FIG. 2, the absorption spectra of dabcyl and
Hydrodabcyl are similar. Thus, Hydrodabcyl is not only a water
soluble alternative to dabcyl, but provides even better
properties.
[0042] It was surprisingly found that by addition of the three
hydroxyl groups at these specific locations, the molar absorbance
and the width at half the peak height of Hydrodabcyl is increased
compared to the molar absorbance and the width at half the peak
height of dabcyl (see FIG. 2 and Example 2). A slight extension of
the absorption band of Hydrodabcyl towards longer wavelength
(bathochromic shift) compared to dabcyl is observed as well. The
bathochromic shift together with its comparatively higher molar
absorbance extends the quenching power of this new dark quencher
compared to dabcyl. As it has been shown in FIG. 3, Hydrodabcyl has
a higher molar absorbance in aqueous solution compared to DMSO.
This property makes Hydrodabcyl an effective quencher for
wavelengths up to 530 nm.
[0043] The changed absorbance properties alone make Hydrodabcyl a
better dark quencher compared to dabcyl. The solubility in water
crucially improves the value of Hydrodabcyl and makes it a superior
dark quencher as it essentially improves the use of the compound in
biological systems.
[0044] The solubility of Hydrodabcyl was tested at different pH
values. At pH>7, solutions in the mM range can be directly
prepared. At acidic pH (pH 4.5-7), the solubility is lower, however
concentrations in the mM range can be nevertheless obtained by
gradual acidification of the solution. It was found that
Hydrodabcyl is soluble in aqueous solution in a mM concentration
range at pH>7. At a pH of about 4.5 to 7, the solubility in a mM
concentration range was confirmed by decreasing the pH-value of an
alkaline solution (see Example 7 and FIGS. 9-11).
[0045] Hydrodabcyl is also substantially easier to handle than
dabcyl. In fact due to the water solubility, the aqueous solutions
are easier to prepare, and glassware washing becomes easier.
Moreover, Hydrodabcyl shows higher stability to light exposure in
comparison to dabcyl (see Example 8 and FIGS. 15 and 16).
[0046] A further advantage of the compound of the present invention
is that the solubility of Hydrodabcyl is achieved without changing
the dimension of the chromophore appreciably. A small chromophore
has the advantage to minimize the sterical hindrances introduced in
the molecular system by the labelling process. Ideally, a labelling
chromophore should not affect the molecular system to investigate
at all; in practice, the influence of the labelling chromophore,
which is often unavoidable, has to be minimized. Therefore the
comparatively small dimension of Hydrodabcyl is a crucial feature
that, together with the absence of charged groups, makes it an
excellent chromophore, especially in the case of small
substrate.
[0047] Hydrodabcyl is also suitable to be used in biological
systems as it has been determined that Hydrodabcyl is neither
carcinogenic (see Example 3), nor cytotoxic (see Examples 3 and 4),
or teratoxic (see Example 6). It was further shown that Hydrodabcyl
is functional in vivo, as it effectively quenches the fluorescence
of a fluorescent substrate in vivo (see Example 5).
[0048] A compound suitable for use as a component of a fluorogenic
probe in biological systems has to be stable and is preferably in
purified form. Therefore, a further aspect of the present invention
is a method for producing Hydrodabcyl in stable and pure form. It
has been found that Hydrodabcyl can be synthesized and obtained as
non-fluorescent compound that is useful as dark quencher by using
the method as claimed in claim 2.
[0049] According to the method of the present invention, a compound
having formula (I)
##STR00004##
is produced wherein the method comprises the steps of [0050] a)
producing 5-dimethylamino-resorcinol
(5-(dimethylamino)benzene-1,3-diol) by reacting phloroglucinol
(benzene-1,3,5-triol) with dimethylamine (HN(CH.sub.3).sub.2) to
obtain 5-dimethylamino-resorcinol; [0051] b) azo-coupling
4-diazo-salicylic acid to 5-dimethylamino-resorcinol to obtain the
compound having formula (I). 4-diazo-salicylic acid can be obtained
by reacting 4-aminosalicylic acid with NaNO.sub.2 and HCl.
[0052] Thus, Hydrodabcyl can be synthesized in two steps (see also
FIG. 1 and Example 1). The first step in the chemical synthesis
consists in exchanging one --OH group of phloroglucinol with a
dimethylamino group and isolating the intermediate product (see
FIG. 1 and Example 1), in a second step 4-diazo salicylic acid and
5-dimethylamino-resorcinol are reacted to yield the compound of the
present invention.
[0053] It was found that the synthesis of
5-dimethylamino-resorcinol as disclosed by Petrzilka [6] did not
result in a useful product. The crystals of
5-dimethylamino-resorcinol appeared pink as reported in the work of
Petrzilka. If these crystals were used in the second step, the
final product, i.e. the compound obtained by reaction of
5-dimethylamino-resorcinol with diazotized 4-aminosalicylic acid to
obtain Hydrodabcyl, was fluorescent and, thus, could not be used as
dark quencher. Furthermore, it was observed that the solution of
5-dimethylamino-resorcinol prepared according to the method of [6]
became intensely colored after being stored for some days. A new
method of producing a purified 5-dimethylamino-resorcinol was
therefore needed.
[0054] It was surprisingly found that the fluorescence and the
coloring of the final product can be avoided by purifying the
intermediate product 5-dimethylamino-resorcinol before using it in
the second step. It is assumed that the undesirable fluorescence is
caused by an impurity.
[0055] It was found that by performing a purification step based on
column chromatography, preferably using silica gel, a stably
colorless solution can be obtained.
[0056] Therefore, the method of producing the compound having
formula (I) can comprise a further step a1) wherein the compound
obtained in step a) is purified by (i) concentrating the
5-dimethylamino-resorcinol; (ii) purifying the residue obtained in
step (i) by column chromatography; and (iii) crystallizing the
purified compound obtained in step (ii).
[0057] This improvement to the method of the prior art by adding
the above mentioned purification process leads to a non-fluorescent
final product, which can be used as dark quencher. The method of
the present invention provides Hydrodabcyl efficiently and in high
quality. An overall yield of about 60% or more can be obtained. A
process for the preparation of Hydrodabcyl is described in detail
in Example 1.
[0058] Briefly, the method of the present invention comprises the
following steps.
[0059] In a first step 5-dimethylamino-resorcinol is prepared by
reacting phloroglucinol and dimethylamine hydrochloride. In one
embodiment of the method, phloroglucinol is dissolved in a mixture
of dimethylformamide and water, preferably degassed, under argon.
Dimethylamine hydrochloride is added. Subsequently in the course of
the reaction, which can take some hours, for example about 4-6
hours, a base, such as NaOH is added to adjust the pH. The addition
of dimethylamine hydrochloride and base can be repeated several
times. The resulting dark solution comprises the desired
intermediate product 5-dimethylamino-resorcinol. The intermediate
product is isolated and purified to remove undesirable impurities.
In one embodiment the intermediate product is concentrated in vacuo
and the residue is purified by column chromatography on a silica
gel. Purified 5-dimethylamino-resorcinol can be crystallised from
dichloromethane as white crystals. These crystals can be used
directly for the next step without storage.
[0060] The second step consists of azo-coupling
5-dimethylamino-resorcinol and 4-diazo-salicylic acid to get the
final product
4-((4'-(dimethylamino)-2',6'-dihydroxyphenyl)azo)-2-hydroxybenzoic
acid. 4-diazo-salicylic acid can be prepared by adding a solution
of NaNO.sub.2 to a solution of 4-aminosalicylic acid. The reaction
can be carried out by heating. Finally the end product can be
isolated by dissolving the desired product in a solvent, for
example in methanol, filtering off impurities, and removing the
solvent, such as by evaporation.
[0061] The obtained product can be further purified, for example as
follows. The sediment can be diluted in a base, for example NaOH,
and then filtered. An acid, for example formic acid, for
acidification and a solvent, for example ethanol, are then added to
the filtrate and the mixture is cooled, for example, in a fridge.
After one or more centrifugation and resuspension steps, the
residue is dispersed in distillate water by ultrasonic bath and
then frozen, for example in liquid nitrogen and dried, for example
by lyophilisation.
[0062] The purification of the product can be further optimized
through its precipitation at pH<4 and centrifugation. Therefore,
the method of producing the compound having formula (I) can further
comprise a step b1), wherein the compound obtained in step b) is
purified by precipitation at pH<4 followed by
centrifugation.
[0063] In conclusion, Hydrodabcyl is a novel dark quencher, based
on an azobenzene-scaffold, with an optimal solubility in aqueous
solution. Its small dimension, the absence of charged groups and
its absorption range make Hydrodabcyl the dark quencher of choice
in tandem with many commercially available fluorescence donors. The
novel dark quencher Hydrodabcyl represents an improved alternative
to the very popular dabcyl in the design of fluorogenic probes.
EXAMPLES
[0064] Preferred embodiments of the invention are outlined in the
following examples which should not be interpreted as restricting
the scope or spirit of the invention.
[0065] The chemicals used were purchased from commercial sources
and used without further purification, unless indicated otherwise.
DMSO was obtained from Sigma. The typical aqueous solution
consisted in 50 mM sodium phosphate buffer pH=8.0.
[0066] The following apparatuses and methods were used.
[0067] The reaction progress was monitored by Thin Layer
Chromatography (TLC) on pre-coated silica plates (Merck TLC Silica
gel 60 F254) and the spots were visualized by UV light and stained
with ceric ammonium molybdate.
[0068] Chromatography was carried out using Macherey-Nagel 60
silica gel (230-400 mesh).
[0069] The .sup.1H and .sup.13C NMR spectra were taken on a Bruker
Avance 300 MHz spectrometer.
[0070] Chemical shifts are reported in parts per million (ppm)
referenced with respect to residual solvent (CDCl.sub.3=7.26 ppm,
D.sub.2O=4.49 ppm).
[0071] IR spectra were recorded with a FT-IR spectrometer
PerkinElmer S100 equipped with an Attenuated Total Reflection (ATR)
unit.
[0072] High Performance Liquid Chromatography (HPLC) was performed
on Phenomenex RP Kinetex 5 um C18 100 .ANG., 250.times.4.6 mm
(analytical) column. 0.1% HCOOH/H.sub.2O and MeOH were used as
eluents for HPLC experiments with flow rate of 0.7 ml/min.
[0073] Double beam Perkin Elmer Lambda 750 UV/Vis spectrophotometer
equipped with a thermostated cuvette holder was used to record the
absorption spectra over a wavelength range 200-800 nm at 20.degree.
C.
[0074] The quartz cuvettes with 1 cm light path used were from
Hellma.
[0075] Fluorescence was tested with a Cary Eclipse fluorimeter
equipped with a thermostated cuvette holder.
Example 1: Synthesis of Hydrodabcyl
[0076] The compound
4-((4'-(dimethylamino)-2',6'-dihydroxyphenyl)azo)-2-hydroxybenzoic
acid (Hydrodabcyl) was synthesized in two steps as outlined in the
scheme shown in FIG. 1.
[0077] The first step consists in the production of
5-dimethylamino-resorcinol (1), based on the method of (Petrzilka
[6]), which has been further developed and improved to allow to
obtain Hydrodabcal in the desired quality. The method comprises a
purification step over silica gel following the completion of the
reaction. The purification step yields colourless crystals, whereas
the crystals obtained by the method described previously yielded
pink crystals [6]. The reported pink color is most probably caused
by contaminations by a degradation product. This assumption is
supported by the observation that the product turns pink when it is
stored for several weeks in the refrigerator. This contamination
leads in the next step to an undesired fluorescent product (in
addition to the desired Hydrodabcyl), which cannot be separated
even by HPLC. In contrast thereto, the method of the present
invention avoids the formation of this by-product.
5-Dimethylamino-resorcinol (1)
[0078] phloroglucinol (9.23 g, 73.2 mmol) was dissolved in a
degassed mixture of dimethylformamide (128 ml) and water (95 ml)
under argon. Then dimethylamine hydrochloride (8 g, 91.5 mmol) was
added. Subsequently over a time period of 5 hours one pellet of
NaOH after another was added (whole amount 3.66 g, 91.5 mmol). The
mixture was stirred overnight at room temperature. During the next
48 hours dimethylamine hydrochloride and NaOH were added 3 times
(every time 10% of first addition). Then the dark solution was
concentrated in vacuo and the residue was purified by column
chromatography (silica gel 60; cyclohexane/ethyl acetate 1:1,
R.sub.f 0.38). The product was crystallised from dichloromethane
with yield: 8.2 g (73%) as white crystals of m.p. 151.degree. C.
and used directly for the next step without storage.
[0079] IR (ATR): 3276, 2972, 2884, 2512, 1604, 1512, 1462, 1435,
1377, 1347, 1312, 1276, 1247, 1128, 1042, 1005, 987, 853, 831, 809,
686, 634, 576 cm-1; .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. ppm
2.78 (s, 6H), 5.61 (s, 3H), 8.82 (s, 2H); .sup.13C NMR (75 MHz,
DMSO-d.sub.6) .delta. 158.8, 152.3, 92.0, 91.5, 40.1 ppm.
[0080] The second step consists of modified azo-coupling to get the
final product
4-((4'-(dimethylamino)2',6'-dihydroxyphenyl)azo)2-hydroxybenzoic
acid (2) with overall yield 58% after 2 steps [7]. The purification
of the product is optimized through its precipitation at pH<4
and centrifugation.
4-((4'-(Dimethylamino)-2',6'-dihydroxyphenyl)azo)-2-hydroxybenzoic
acid (2)
[0081] A cooled freshly prepared solution of 2.5 M NaNO.sub.2 (9
ml, 22.5 mmol) was added dropwise to a cooled solution of
4-aminosalicylic acid (3.46 g, 22.5 mmol) in a half concentrated
HCl (6 ml) at 0-5.degree. C. The solution was then stirred for
another 15 min and introduced dropwise at 0-5.degree. C. to
5-dimethylamino-resorcinol (3.45 g, 22.5 mmol) in 1M NaOH (23.5
ml). The mixture was heated at 70.degree. C. for 15 min and then
stirred for 1 h at room temperature. Methanol was added to the red
mixture and the mixture was then put in an ultrasonic bath for
several minutes.
[0082] The impurities were filtered and the filtrate was
evaporated. The sediment was diluted in 0.1N NaOH and then
filtered. Formic acid for acidification and ethanol were added to
the filtrate and the mixture was placed into the fridge for 15 h.
Then the mixture was centrifuged at -4.degree. C., and the pellet
was suspended in 0.1% formic acid and centrifuged again. This
procedure was repeated 3 times. Afterwards the residue was
resuspended in bi-distillate water and centrifuged twice. Finally,
the substance was dispersed in bi-distillate water by ultrasonic
bath and then frozen in liquid nitrogen and dried by
lyophilisation.
[0083] Pure final product (2) was obtained with yield: 5.7 g (80%)
as red powder of m.p. 253.degree. C. and stored in the darkness in
the fridge at 4-8.degree. C.
[0084] HPLC: T=16.5 min (MeOH--0.1% HCOOH/H.sub.2O 55-45,
.lamda..sub.max=455 nm); IR (ATR): 3352, 3083, 2908, 2737, 2475,
1874, 1656, 1620, 1502, 1473, 1426, 1387, 1337, 1291, 1226, 1191,
1133, 1087, 1018, 983, 965, 886, 847, 811, 776, 729, 675 cm-1;
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 3.07 (s, 6H), 5.73 (s,
2H), 7.12 (dd, J=8.2, 1.9 Hz, 1H), 7.29 (d, J=1.9 Hz, 2H), 7.78 (d,
J=8.5 Hz, 2H) ppm; .sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta.
172.1, 163.3, 158.2, 152.0, 131.7, 124.9, 113.6, 110.19, 105.6,
91.9, 40.4 ppm; ESI-MS: 316.0925 [M-H].sup.-, 317.0982[M] and
318.1001 [M+H].sup.+. HRMS calc. 317.1012 for
C.sub.15H.sub.15N.sub.3O.sub.5, found 317.0982.
Example 2: Comparison of Absorption Spectra of Dabcyl and
Hydrodabcyl
[0085] The absorption spectra of Hydrodabcyl and dabcyl were
compared (see FIG. 2). Dabcyl has a .lamda..sub.max=451 nm, whereas
Hydrodabcyl has a .lamda..sub.max=470 nm at a concentration of
2.times.10.sup.-5 M in DMSO at 20.degree. C. The molar absorbance
and the width at half the peak height of Hydrodabcyl is increased
compared to the molar absorbance and the width at half the peak
height of dabcyl. The bathochromic shift (red-shift) of Hydrodabcyl
can be observed.
[0086] The absorption spectrum of Hydrodabcyl in aqueous solution
(.lamda..sub.Max=445 nm, .epsilon..sub.445=43000 M.sup.-1
cm.sup.-1) shows a hypsochromic shift in comparison to its spectrum
in DMSO (.lamda..sub.Max=470 nm, .epsilon..sub.470=37000 M.sup.-1
cm.sup.-1) as shown in FIG. 3.
Example 3: Electrophoretic Mobility Shift Assay (EMSA)
[0087] In order to prove in vivo suitability of Hydrodabcyl, an
Electrophoretic Mobility Shift Assay (EMSA) was carried out.
[0088] Circular and linear double-stranded DNA was incubated with
various concentrations of Hydrodabcyl and doxorubicin (a known
intercalating substance). After 24 h of incubation time, agarose
gel electrophoresis was carried out. The results are shown in FIGS.
6 and 7.
[0089] The intercalation of doxorubicin into the DNA results in a
shift of the DNA bands with increasing doxorubicin concentration,
as can be seen in FIGS. 6a and 7a, where the results of the
incubation of the DNA with doxorubicin are shown. The shifting of
the bands shows the changes in mobility of the DNA in the agarose
gel due to the structural changes in the DNA due to intercalation
of doxorubicin into the DNA. The absence of the shift when
incubated with Hydrodabcyl shows that Hydrodabcyl does not
intercalate or bind to the DNA, presumably due to the decreased
lipophilicity of Hydrodabcyl. Since intercalation is an important
indication of toxicity (in particular carcinogenicity), this
finding is an indication that Hydrodabcyl is not toxic.
Example 4: Cytotoxicity
[0090] To further prove in vivo suitability of Hydrodabcyl, a
cytotoxicity assay was carried out as well.
[0091] Three different cell culture types were incubated with
various concentrations (from 5 to 100 .mu.M) of Hydrodabcyl.
[0092] Table 1 below shows the inhibitory concentration (IC.sub.50)
after incubation for 72 hours.
TABLE-US-00001 TABLE 1 Cell culture type IC.sub.50 [.mu.M] HT-29
>50 EaHy.926 >50 CHF >100
[0093] This test shows that Hydrodabcyl is not cytotoxic.
Example 5: In Vivo Fluorescence
[0094] Two sorts of labelled molecules were injected into oocytes
of Xenopus laevis: [0095] A molecule labelled with monobromobimane
(mBBr; a fluorescent dye, which emits light in the absorption
spectrum of Hydrodabcyl); the molecule is called Substrate-Bim
[0096] A molecule labelled with mBBr and Hydrodabcyl (a
fluorescence quencher); the molecule is called
Bim-Substrate-Hydrodabcyl
[0097] As negative control oocytes were injected with buffer.
[0098] FIG. 8 shows the results of the in vivo fluorescence
assay.
[0099] FIG. 8a shows an oocyte injected with buffer. No
fluorescence is detected.
[0100] FIG. 8b shows an oocyte injected with Substrate-Bim.
Fluorescence is detected.
[0101] FIG. 8c shows an oocyte injected with
Bim-Substrate-Hydrodabcyl. No fluorescence is detected, because
Hydrodabcyl quenches the fluorescence of Substrate-Bim. This assay
proves that Hydrodabcyl is an effective fluorescence quencher in
vivo.
Example 6: Influence of Hydrodabcyl on Embryogenesis
[0102] The effect on embryogenesis of the molecules injected into
the oocytes as described in Example 5 was observed. The results are
shown in Table 2 below.
TABLE-US-00002 TABLE 2 Injected molecule Living embryos after 24 h
(%) Only Buffer 65 Substrate-Bim 61 Bim-Substrate-Hydrodabcyl
71
[0103] The similarity between the percentages shows that
Hydrodabcyl does not influence embryogenesis.
Example 7: Quantitative Determination of the Solubility of
Hydrodabcyl (in Comparison to the Parent Molecule Dabcyl)
[0104] In order to prove the superiority of hydrodabcyl, the
solubilities of hydrodabcyl and dabcyl in pure water and buffered
aqueous solutions were measured and compared.
[0105] Hydrodabcyl is soluble in water, in contrast to dabcyl. A
limiting concentration of 5.71.times.10.sup.-4 M for Hydrodabcyl in
water was measured at 20.degree. C. It was not possible to obtain a
saturated solution of dabcyl due to precipitation. Evidence of
precipitation of dabcyl in water (tentative concentration of about
7 .mu.M) is shown in FIG. 9 on the left. On the right in FIG. 9, a
clear solution of Hydrodabcyl in water (7 .mu.M) can be seen.
[0106] In order to increase the solubility, the same tests were
performed in a buffered aqueous solution at pH 8. As expected, the
concentration of a saturated solution of Hydrodabcyl was much
higher (25 mM). In a buffered solution pH 8 dabcyl is also soluble,
although poorly; in fact a saturated solution of dabcyl has a
concentration of orders of magnitude lower than a saturated
solution of Hydrodabcyl, confirming the superiority of
Hydrodabcyl.
[0107] The excellent solubility of the bare chromophore in aqueous
solution already confers to Hydrodabcyl several practical
advantages, e.g., aqueous solutions of Hydrodabcyl are easier to
prepare and glassware can be more readily cleansed. Nevertheless,
in most of applications the chromophores are linked to the
molecules of interest. For example, to monitor protease activity
chromophores are usually linked to the peptidic substrate. Both
dabcyl and Hydrodabcyl can be easily coupled to an amino group
through a standard amide bond formation. This reaction, however,
eliminates the charge of the carboxylate that proved to contribute
to the solubility of the bare chromophores at basic pH, with the
risk to lead to insolubility. To investigate the behavior of dabcyl
and Hydrodabcyl coupled to an amino acid bearing an amino group,
dabcyl and Hydrodabcyl were linked to the amino group of a Lys side
chain, and the solubility of the Lys-dabcyl and Lys-Hydrodabcyl
moieties in a buffered aqueous solution at pH 8 were tested.
[0108] Interestingly, the concentration of the saturated solution
of Lys-dabcyl was only 7.6.times.10.sup.-6 M, whereas with
Lys-Hydrodabcyl a 6.6 mM solution could be prepared without
reaching saturation. This data indicates that the solubility of
Lys-Hydrodabcyl is much higher than 6.6 mM and closer to the
solubility of L-Lys which is reported to be 5.8 g per 1 kg of
water, corresponding to about 40 mM [8].
[0109] These results indicate that Hydrodabcyl has minimal effect
on the solubility of the peptidic substrate, whereas the
hydrophobicity of dabcyl drastically affects the solubility of
natural substrates in aqueous solution. A strongly reduced
solubility of the products of an enzymatic reaction may hinder
their release from the active site resulting in an inhibiting
effect, which distorts the catalytic mechanism, thus preventing its
understanding. These tests emphasize the superiority of Hydrodabcyl
in biochemical applications.
TABLE-US-00003 TABLE 3 Summary of the results of the solubility
tests (NaP buffer = sodium- phosphate buffer) Solvent Substance
Solubility Comments Water, 20.degree. C. Hydrodabcyl 5.71 .times.
10.sup.-4M pH = 4.5 dabcyl n.d. not determined due to precipitation
50 mM NaP Hydrodabcyl 2.54 .times. 10.sup.-2M buffer, pH 8.0 dabcyl
5.41 .times. 10.sup.-4M 50 mM NaP Lys-Hydrodabcyl >6.61 .times.
10.sup.-3M The solution was buffer, pH 8.0 not saturated Lys-dabcyl
7.62 .times. 10.sup.-6M
[0110] Hydrodabcyl is very well soluble in aqueous solution over
the whole biologically relevant pH range. From basic pH down to pH
6, solutions with concentrations in the millimolar range can be
prepared directly at the desired pH. Although at pH<6 the
solubility is lower, as the solubility test in pure water showed,
solutions with mM concentrations can still be prepared by gradual
acidification of an alkaline solution down to a pH value of
4.3.
[0111] Description of the Experiment:
[0112] Buffered solutions (50 mM sodium phosphate) have been
prepared at four different pH: 8.0, 7.0, 6.0 and 5.5, respectively.
The amount of Hydrodabcyl required to reach a concentration of 5 mM
was immersed in each buffer solution and kept for 1 h at 30.degree.
C. in an ultrasonic bath. Clear solutions of Hydrodabcyl have been
obtained at pH 8.0, 7.0 and at pH 6.0, whereas at pH 5.5 the
substance was not completely soluble. To confirm its solubility,
the absorbance of Hydrodabcyl was monitored as function of its
concentration. Since the stock solution was too concentrated to be
measured directly, three diluted solutions with different
concentrations (10 .mu.M, 20 .mu.M and 30 .mu.M) were prepared from
each stock solution at pH 8.0, pH 7.0 and pH 6.0. The aliquots have
been taken from different part of the volume of the stock solution
to test the homogeneity.
[0113] The linear increase of the absorbance with the concentration
of the solute according to the Lambert-Beer law indicates that the
stock solution is homogeneous. The result at pH 6 (lower limit for
the solubility in case of direct preparation) is shown in FIG. 10,
which shows the concentration dependence of the absorbance at 445
nm of Hydrodabcyl in 50 mM NaP buffer pH=6.0 at T=20.degree. C.
[0114] To reach complete solubility at pH<6.0, the pH of the
stock solution (5 mM range) at pH 7.0 was decreased at the desired
value with few drops of 5M HCl. Following gradual acidification of
the medium the mM solution of Hydrodabcyl remained clear till pH
4.3. With the same procedure described above, solubility of
Hydrodabcyl could be proven till pH 4.3 The result at pH 4.3 (lower
limit for the solubility obtained with gradual acidification of the
medium) is shown in FIG. 11 as an example, which shows
concentration dependence of the absorbance at 445 nm of Hydrodabcyl
in 50 mM NaP buffer pH=4.3 (obtained from pH 7.0 with 5M HCl) at
T=20.degree. C. At pH<4.3 Hydrodabcyl precipitates.
Example 8: Stability Under Light Exposure
[0115] The effect of exposure to the light beam of a commercial
spectrophotometer was tested to mimic the common experimental
condition. A 2-hour long continuous irradiation at a wavelength
corresponding to the maximum of absorption in DMSO of a solution of
Hydrodabcyl and one of dabcyl provided a constant signal,
indicating that the absorption properties under these conditions
are not affected for both compounds. These findings can be seen in
FIGS. 12 and 13 showing absorbance at 470 nm of Hydrodabcyl, 14
.mu.M in DMSO T=20.degree. C., monitored over 2 h (7200 s), and
absorbance at 451 nm of dabcyl, 7 .mu.M in DMSO T=20.degree. C.,
monitored over 2 h (7200 s).
[0116] Additionally, in the case of Hydrodabcyl, stability under a
2-hour long irradiation is observed also in buffered aqueous
solution pH8. This is shown in FIG. 14, showing absorbance at 445
nm of Hydrodabcyl, 22 .mu.M in buffered aqueous solution pH=8
T=20.degree. C., monitored over 2 h (7200 s).
[0117] However, Hydrodabcyl is much more stable under light
exposure than dabcyl. One solution of dabcyl and one of
Hydrodabcyl, both at 17.5 .mu.M in DMSO prepared in the dark, were
exposed for 5 minutes to the light of a common 60 W tungsten lamp.
This relatively short time of exposure was enough to strongly
modify the absorption spectrum of dabcyl, whereas it had no effect
on the absorption spectrum of Hydrodabcyl. The original spectrum of
dabcyl could be recovered only after 10 min in the dark (see FIGS.
15 and 16).
REFERENCES
[0118] [1] Gershkovich A. A. & Kholodovych V. V. Fluorogenic
substrates for proteases based on intramolecular fluorescence
energy transfer (IFETS). J. Biochem. Biophys. Methods 1996, 33,
135-162. [0119] [2] Mayatoshi E. D., Wang G. T., Kraft G. A. &
Erickson J. Novel fluorogenic substrates for assaying retroviral
proteases by resonance energy transfer. Science, 1990, 247,
954-958; [0120] [3] Tyagy S. & Kramer F. R. Molecolar Beacons:
probes that fluoresce upon hybridization. Nat. Biotech. 1996, 14,
303-308. [0121] [4] Holskin B P., Bukhtiyarova M., Dunn, B. M.,
Baur P., de Chastonay J. & Pennington M. W. A continuous
fluorescence-based assay of human cytomegalovirus protease using a
peptide substrate. Anal. Biochem. 1995, 226, 148-155. [0122] [5]
Loudwig S. & Bayley H. Protoisomerization of an individual
azobenzene molecule in water: an on-off switch triggered by light
at a fixed wavelength. J. Am. Chem. Soc. 2006, 126, 12404-12405
[0123] [6] Petrzilka T. & Lusuardi W. G. HELVETICA CHIMICA
ACTA, 1973, 56, 515. [0124] [7] Christie, R. M. Colour Chemistry.
Cambridge: Royal Society of Chemistry: Cambridge 2001. [0125] [8]
Handbook of Chemistry and Physics 85.sup.th Ed. 2004-2005, CRC
Press, David R. Lide Editor-in Chief
* * * * *