U.S. patent application number 13/229259 was filed with the patent office on 2012-02-23 for labels, their production process and their uses.
This patent application is currently assigned to LABORATOIRES SYNTH-INNOVE. Invention is credited to Vincent Guyon, Marie-Noelle Rager, Francois SCHERNINSKI.
Application Number | 20120045851 13/229259 |
Document ID | / |
Family ID | 35985984 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120045851 |
Kind Code |
A1 |
SCHERNINSKI; Francois ; et
al. |
February 23, 2012 |
LABELS, THEIR PRODUCTION PROCESS AND THEIR USES
Abstract
Labels are disclosed capable of forming a covalent or
non-covalent bond with a target molecule, particularly a biological
molecule. The structure of these labels may consist of a dye
covalently bound by one or more carbons on its chemical structure
to one or more [FUNC] group(s), and optionally one or more [SOL]
group(s). The structure of these labels allow selection of dyes
from a wide variety of different excitation and emission
wavelengths and allow easy functionalization of the dye without
appreciably altering its spectral characteristics or its solubility
characteristics.
Inventors: |
SCHERNINSKI; Francois;
(Paris, FR) ; Guyon; Vincent; (Montigny Le
Bretonneux, FR) ; Rager; Marie-Noelle; (Paris,
FR) |
Assignee: |
LABORATOIRES SYNTH-INNOVE
Paris
FR
|
Family ID: |
35985984 |
Appl. No.: |
13/229259 |
Filed: |
September 9, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12063520 |
Mar 18, 2008 |
8034626 |
|
|
PCT/FR2006/001947 |
Aug 11, 2006 |
|
|
|
13229259 |
|
|
|
|
Current U.S.
Class: |
436/547 ;
204/461; 530/391.3; 540/123; 540/145 |
Current CPC
Class: |
C07D 403/06 20130101;
Y10T 436/25 20150115; C09B 23/086 20130101; G01N 33/583 20130101;
C07D 487/22 20130101; C07D 403/12 20130101; G01N 33/582 20130101;
C07D 403/14 20130101; C07D 209/10 20130101; C09B 11/08 20130101;
C07D 209/60 20130101; C07D 493/10 20130101; C09B 23/083
20130101 |
Class at
Publication: |
436/547 ;
540/145; 540/123; 530/391.3; 204/461 |
International
Class: |
C09B 62/84 20060101
C09B062/84; G01N 27/447 20060101 G01N027/447; C07K 1/13 20060101
C07K001/13; G01N 33/559 20060101 G01N033/559; C07K 16/00 20060101
C07K016/00; G01N 21/75 20060101 G01N021/75 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2005 |
FR |
05 08525 |
Claims
1.-23. (canceled)
24. A label consisting of a dye comprising, a carbon covalently
bonded to: one or more [FUNC] group(s), and optionally one or more
[SOL] group(s), said label having the following formulae (9) or
(10): ##STR00058## each of Q'.sub.1, Q'.sub.2, Q'.sub.4, Q'.sub.5,
Q'.sub.7, Q'.sub.8, Q'.sub.10 and Q'.sub.11 each independently from
each other represents hydrogen, hydroxyl, halogen, acetyl, amine,
substituted amine, quaternary ammonium, phosphate, nitro,
carboxylic acid and its salts, sulphonic acid and its salts,
alkylcarboxy with 2 to 30 carbon atoms, linear or branched alkyl
having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon
atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1
to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms,
alkylester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30
carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl
having 1 to 30 carbon atoms, sulphoalkyl having 1 to 30 carbon
atoms, aryl, aryloxy, arylalkyl, halogenoaryl, arylester, [SOL] or
[FUNC], Q'.sub.3, Q'.sub.6, Q'.sub.9 and Q'.sub.12 not being able
to represent aryl or aryloxy, providing that at least one of
Q'.sub.1 to Q'.sub.12 represents H or an aromatic ring, and
providing that at least one of Q'.sub.1, Q'.sub.2, Q'.sub.4,
Q'.sub.5, Q'.sub.7, Q'.sub.8, Q'.sub.10 and Q'.sub.11 represents
[FUNC]; ##STR00059## in which each of D'.sub.1 to D'.sub.16, each
independently from each other represents hydrogen, hydroxyl,
halogen, acetyl, amine, substituted amine, quaternary ammonium,
phosphate, nitro, carboxylic acid and its salts, sulphonic acid and
its salts, alkylcarboxy with 2 to 30 carbon atoms, linear or
branched alkyl having 1 to 30 carbon atoms, cycloalkyl having 3 to
14 carbon atoms, alkyloxy having 1 to 30 carbon atoms,
halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to
30 carbon atoms, alkylester having 2 to 40 carbon atoms, nitroalkyl
having 1 to 30 carbon atoms, carboxyalkyl having 2 to 30 carbon
atoms, aminoalkyl having 1 to 30 carbon atoms, sulphoalkyl having 1
to 30 carbon atoms, aryl, aryloxy, arylalkyl, halogenoaryl,
arylester, [SOL] or [FUNC], providing that at least one of D'.sub.1
to D'.sub.16 represents H, and providing that at least one of
D'.sub.1 to D'.sub.16 represents [FUNC]; [FUNC] each representing
independently an --X-A-Z group, in which: X is chosen from the
group consisting of an oxygen atom, a sulphur atom, an
NR.sub.1R.sub.2group, R.sub.1 and R.sub.2 each being independently
of each other a hydrogen atom or a linear or branched
C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18 and more
preferentially C.sub.1-C.sub.5 alkyl group; A is an alkylene group,
or an alkylene-arylene group; Z is a reactive chemical function
chosen from the group comprising the functions carboxylic acid and
its salts, sulfonic acid and its salts, acid anhydride, acid
chloride, esters such as alkyl ester, p-nitrophenyl ester,
succinimidyl ester, sulphosuccinimidyl ester, azido such as acyl
azide, azidonitrophenyl, hydrazide,
3-acyl-1,3-thiazolidine-2-thione, amine, substituted amine,
quaternary ammonium, isocyanate, isothiocyanate, hydrazine,
phthalimido, maleimide, haloacetamide, monochlorotriazine,
dichlorotriazine, mono- or dihalogenated pyridine, mono- or
dihalogenated diazine, aziridine, thiol, sulphonyl chloride,
vinylsulphone, disulphide, methanethiosulphonate, hydroxyle,
phosphoramidite, epoxy, aldehyde, carbonate, glyoxal, imidazolyl;
[SOL] each representing independently an --X'-A'-Z' group, in
which: X' is chosen from the group consisting of an oxygen atom, a
sulphur atom, an NR.sub.1R.sub.2 group, R.sub.1 and R.sub.2 each
being independently of each other a hydrogen atom or a linear or
branched C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18 and more
preferentially C.sub.1-C.sub.5 alkyl group; A' is an alkylene group
or alkylene-arylene group; Z' is either a polar group chosen from
the group comprising the groups carboxylic acid and its salts,
sulphonic acid and its salts, substituted amine, quaternary
ammonium, carbohydrate, glycol, hydroxyl, nitro, phosphate, or an
apolar group chosen from the group comprising the linear or
branched alkyl groups having 1 to 30 carbon atoms, cycloalkyl
having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms,
halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to
30 carbon atoms, alkylester having 2 to 40 carbon atoms, aryl,
aryloxy, substituted arylalkyl, arylalkyl, halogenoaryl.
25. The label according to claim 24, wherein the alkylene group is
a cyclic, linear or branched hydrocarbon chain having two free
bonds, comprising 1 to 30 carbon atoms and the arylene group is an
aromatic group, having two free bonds, comprising one or more
aromatic rings, optionally substituted.
26. The label according to claim 24, wherein [FUNC] is chosen from
--X--(CH.sub.2).sub.r--COOSu,
--X--(CH.sub.2).sub.r--COOSuSO.sub.3Na,
--X--(CH.sub.2).sub.r--COOH, --X--(CH.sub.2).sub.rSO.sub.3H,
--X--(CH.sub.2).sub.r--SO.sub.3Na,
--X--(CH.sub.2).sub.r--COO--C.sub.6H.sub.4--NO.sub.2,
--X--(C.sub.6H.sub.4)--(CH.sub.2).sub.r--COOSu,
--X--(CH.sub.2).sub.r--NHCOCH.sub.2I, --X--(CH.sub.2).sub.r--NCS,
--X--(CH.sub.2).sub.r--C.sub.6H.sub.4CH(CH.sub.3)--COOSu,
--N(CH.sub.3).sub.2--(CH.sub.2).sub.r--SO.sub.3H, and
--X--(CH.sub.2).sub.r--OP[N(iPr).sub.2][CH.sub.2CH.sub.2CN], X
being as defined in claim 24, r is an integer from 1 to 18, and Su
representing the succinimidyl group.
27. The label according to claim 24, wherein [SOL] is chosen from
--X--(CH.sub.2).sub.r--SO.sub.3Na,
--X--(CH.sub.2).sub.r--SO.sub.3H,
--X--(CH.sub.2).sub.r--C.sub.6H.sub.3(NO.sub.2).sub.2,
--X--(CH.sub.2).sub.r--CH.sub.3, and X being as defined in claim 24
and, r is an integer from 1 to 18.
28. The label according to claim 24, wherein said label is chosen
from the group consisting of the structures (6), (7), (11) or (12):
##STR00060## in which each of y and z, identical or different, is
an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z represents --COOH or
##STR00061## Y represents SO.sub.3.sup.- or SO.sub.3Na ##STR00062##
in which each of y and z, identical or different, is an integer
equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z represents --COOH or
##STR00063## Y represents SO.sub.3.sup.- or SO.sub.3Na.
29. A process for preparing labels as defined in claim 24,wherein
said process comprises a nucleophilic substitution reaction between
either: Z-A-L and [DYE']-Nu or: [DYE'']-L and Z-A-Nu Z and A being
as defined in claim 24; L representing a leaving group; Nu
representing a nucleophilic group chosen from the group comprising
--OH, --SH or --NR.sub.1R.sub.2, R.sub.1 and R.sub.2 each being
independently of each other a hydrogen atom or a linear or branched
C.sub.1-C.sub.30 alkyl group; [DYE'] and [DYE''] representing the
dye formula (4) or (5): ##STR00064## in which: Q.sub.1 to Q.sub.12
and D.sub.1 to D.sub.16, each independently represents hydrogen,
hydroxyl, halogen, acetyl, amine, substituted amine, quaternary
ammonium, phosphate, nitro, carboxylic acid and its salts,
sulphonic acid and its salts, alkylcarboxy with 2 to 30 carbon
atoms, linear or branched alkyl having 1 to 30 carbon atoms,
cycloalkyl having 3 to 14 carbon atoms, alkyloxy having 1 to 30
carbon atoms, halogenoalkyl having 1 to 30 carbon atoms,
hydroxyalkyl having 1 to 30 carbon atoms, alkylester having 2 to 40
carbon atoms, nitroalkyl having 1 to 30 carbon atoms, carboxyalkyl
having 2 to 30 carbon atoms, aminoalkyl having 1 to 30 carbon
atoms, sulphoalkyl having 1 to 30 carbon atoms, aryl, aryloxy,
arylalkyl, halogenoaryl, arylester, Q.sub.3, Q.sub.6, Q.sub.9 and
Q.sub.12 not being able to represent aryl or aryloxy, providing
that at least one of Q.sub.1 to Q.sub.12 represents H or an
aromatic ring, and, providing that at least one of D.sub.1 to
D.sub.16 represents H.
30. The process according to claim 29, wherein the nucleophilic
substitution reaction is a Williamson reaction in the presence of a
base.
31. The process according to claim 29, wherein the leaving group L
is chosen from the group consisting of a halogen, a
methanesulphonate, para-toluenesulphonate group and a diazonium
group.
32. The process according to claim 29, wherein [DYE']-Nu is
obtained by the following successive reactions: a) nitration of
[DYE']; b) reduction of the product obtained in step a); c)
optionally: either alkylation of the product obtained in step b),
or diazotization of the product obtained in step b) in order to
obtain a diazonium salt then nucleophilic substitution on this
diazonium function.
33. The process according to claim 29, wherein [DYE]-L is obtained
by the following successive reactions: a) nitration of [DYE'']; b)
reduction of the product obtained in step a); c) diazotization of
the product obtained in step b) in order to obtain a diazonium
salt; d) optionally: nucleophilic substitution on the diazonium
function of the product obtained in step c).
34. The process according to claim 29, wherein said process
comprises a nucleophilic substitution reaction between either:
Z'-A'-L and [DYE']-Nu or: [DYE'']-L and Z'-A'-Nu Z' and A' being as
defined in claim 24; L, Nu, [DYE'] and [DYE''] being as defined
previously in claim 29; [DYE']-Nu and [DYE'']-L being prepared
according to the process of claim 29.
35. A process of labelling a target molecule comprising the step of
coupling said target molecule with a label according to claim
24.
36. A method for labelling biological molecules comprising the step
of coupling said biological molecule with a label according to
claim 24.
37. The method for labelling biological molecules according to
claim 36, wherein the step of coupling is performed in the presence
of a peptide coupling agent, for example carbodiimide-type
reagents, IDDQ
(1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline),
phosphonium-type reagents, uronium-type reagents, Woodward reagents
or Curtius reagents.
38. The method for labelling biological molecules according to
claim 37, wherein the coupling is carried out in the presence of
dimethylformamide.
39. A method for detecting a biological or non-biological molecule
comprising the step of coupling a label according to claim 24 with
said biological or non-biological molecule, and the step of
detecting said molecule coupled with the label by absorption
spectrometry, fluorescence spectrometry, infrared spectrometry,
electrophoresis, near infra-red or infra-red medical imaging.
40. The label according to claim 24, wherein said label is selected
from the group consisting of: ##STR00065##
Description
[0001] A subject of the present invention is novel labels which can
be used for labelling target molecules. The invention also relates
to a process for obtaining these labels. Finally, it relates to the
use of these novel labels for the detection and/or quantification
of target molecules.
[0002] The labelling of a target molecule and in particular of
biological molecules by labels is of great interest in the field of
cell and molecular biology, in particular in cytology/histology,
flow cytometry, for the sequencing of nucleic acids. In the field
of medical imaging, .sub.the use of fluorescent labels makes it
possible inter alia to locate lesions or tumours.
[0003] Fluorescent labels are widely used in biology, biophysics,
or physiology not only in order to study the biological processes
at cell and molecular level or to quantify the physiological
substances to be analyzed but also in medicine in non-invasive
imaging techniques for surveillance or diagnosis.
[0004] A few specific examples of the use of fluorescent labels in
biology are: [0005] identification and separation of
sub-populations of cells in mixtures of cells by flow cytometry or
microscopy techniques, [0006] determination of the concentration of
a substance which binds to a second species such as an antibody to
an antigen in immunological test techniques, sequencing of the
nucleic acids, the demonstration of the pairing of nucleotide
sequences, [0007] location of substances such as DNA and proteins
in electrophoresis gels or other insoluble supports by fluorescent
staining techniques.
[0008] In numerous techniques, including fluorescence optical
microscopy, it is possible to simultaneously use several
fluorescent labels capable of emitting at different wavelengths.
This "multi-labelling" is carried out without any confusion arising
between the signals during detection.
[0009] The multi-labelling principle is also used in the particular
technique of FRET (Fluorescence Resonance Energy Transfer) which
exploits the transfer of energy from the excited state of a donor
fluorescent label towards a second acceptor fluorescent label, this
transfer of energy being possible only when the two labels are in
proximity. This technique makes it possible to measure the
interactions (association or dissociation) between two proteins,
one of which is coupled to a donor fluorescent label and the other
to an acceptor fluorescent label.
[0010] However, during these multi-labellings, account must be
taken of the problems of any partial overlaps of the emission
spectra of the labels used which falsify the reading of the
results. Consequently, during multi-labelling, it is of primary
importance to limit the spectra covering phenomenon, which makes it
necessary to have access to a large range of labels the spectral
characteristics of which are completely controlled.
[0011] The first subject of the present invention is novel labels
composed of "functionalized dyes" the resonance system of which is
not affected by the functionalization.
[0012] By "dye" is meant any coloured substance, natural,
artificial or synthetic, absorbing light in the ultra-violet,
visible and/or infrared range. Within the framework of the
invention, the dyes can be fluorescent. By "fluorescent dyes" is
meant dyes having the ability to be excited in a transient manner
by absorption of a luminous radiation then to return to their
initial state by emitting radiation the wavelength of which is
higher than that of the excitation radiation.
[0013] By "functionalized dyes", is meant dyes having at least one
reactive chemical function allowing their coupling to target
molecules and optionally at least one function allowing them to be
soluble under conditions of use.
[0014] The labels of the prior art can be classified in three
categories according to the way in which their reactive chemical
function is linked to the ring structure of the dye.
[0015] The labels of the first category are defined by the fact
that their reactive chemical function is linked directly to a
carbon atom of a ring of the dye's hydrocarbon skeleton. They have
been described in particular in U.S. Pat. No. 5,627,027,
US2002/077487, FR2764605, US4,614,723, JP03133629, JP03133628,
JP63277680, Masiero, S. et al. "G-quartets as a self-assembled
scaffold or circular porphyrin arrays" Chemical communications,
vol. 15, 2003 pages 1995-1996, Yu, Junhua et al. "Porphyrin capped
TiO.sub.2 nanoclusters, tyrosine methyl ester enhanced electron
transfer", FR2 748 027.
[0016] However, the major drawback of such labels comes from the
fact that the reactive chemical function is found in immediate
proximity to a ring of the molecule of the dye. This proximity
leads to a significant steric hindrance which strongly hinders
coupling to target molecules. On the other hand this proximity
affects the dye's absorption and fluorescence spectra. The choice
of a dye for the synthesis of a label is dictated by its spectral
properties. Consequently, obtaining a label the coupling ability of
which is hindered by a steric hindrance and the spectral properties
of which are different from those of the chosen dye is not
particularly useful.
[0017] In order to remedy the drawbacks mentioned previously, a
second category of labels has been developed. The labels of this
second category are defined by the fact that their reactive
chemical function is linked indirectly by means of an alkyl chain
to an atom which belongs to the molecule of the initial dye, this
atom not being a carbon atom. It is frequently a nitrogen atom
which, most often, belongs to the ring structure of this dye.
[0018] The production of these second category labels is limited to
the use of dyes most often constituted by one or more nitrogenous
heterocycles. Each nitrogen atom of the initial dye carries a chain
which strongly influences the solubility of the dye: these are
often sulphoalkyl chains if the dye must be solubilized in an
aqueous medium or alkyl chains if the dye must be solubilized in a
hydrophobic medium. The technique for producing labels of this
second category involves replacing one or more of these alkyl or
sulphoalkyl chains with a hydrocarbon chain carrying the reactive
chemical function. Nevertheless, this technique has the consequence
of reducing in particular the solubility of the label in relative
to the initial characteristics of the dye. Moreover, the production
of labels of this second category only gives access to a very
limited number of entities as it is applicable only to a restricted
number of dyes which comprise one or more heteroatoms.
[0019] For example, the patents WO2005/058370, WO2003/082988,
EP1209205 U.S. Pat. No. 6,027,709 and U.S. Pat. No. 6,224,644
disclose labels of this second category.
[0020] The patent U.S. Pat. No. 6,027,709 describes
carbocyanine-type dyes in which a sulphoalkyl chain has been
replaced by an alkyl chain carrying a carboxylic acid function
(reactive chemical function with an electrophilic character).
[0021] The patent U.S. Pat. No. 6,224,644 also describes
carbocyanines modified in a similar way and intended to be used as
labels of biological molecules. However, these labels differ from
those of the patent U.S. Pat. No. 6,027,709 in that the reactive
chemical function is not electrophilic but nucleophilic (hydroxyl,
amine and thiol function).
[0022] This category of labels is therefore of limited usefulness
as it applies only to a few carbocyanine-type dyes with a reduction
in solubility of the label relative to that of the initial dye
while it would have been desirable to increase this solubility in
order to facilitate coupling with a large number of target
molecules. In the case where the hydrosolubility of the label is
desired, the direct addition of groups such as NO.sub.2 and
SO.sub.3.sup.- to the aromatic rings remedies this problem but
affects the absorption and fluorescence spectra of the initial
dye.
[0023] A third category of labels, as described in EP 1 209 205,
WO02/32466, WO94/08631 and Lobnik, A. et al. "pH optical sensors
based on sol-gels. Chemical doping versus covalent immobilization"
Analytica Chimica Acta, vol. 367, no. 1-3, 1998, pages 159-165, is
defined by the fact that their reactive chemical function is linked
indirectly by means of an alkyl or amidoalkyl chain to a carbon
atom, and not a nitrogen atom, of the ring structure of the dye,
which avoids the suppression of one of the sulphoalkyl or alkyl
chains which conditions the solubility of the label.
[0024] Nevertheless, the production of labels of this third
category gives access to a number of entities even more limited
than those of the second category as they have the major drawback
of being difficult to synthesize. In fact, the synthesis precursors
of such labels are not commonly commercially available and their
synthesis requires the implementation of numerous stages with low
final yields.
[0025] At present, the synthesis of functionalized dyes serving as
labels for detecting and/or quantifying target molecules is known.
However, the labels of the prior art have at least one of the
following three fundamental drawbacks which considerably limit
their use: [0026] the reactive chemical function of these labels is
in immediate proximity to a ring of the dye, which results in a
steric hindrance and strongly hinders coupling to target molecules,
thus making them of little use. Moreover, their spectral properties
are different from those of the dyes from which they are obtained,
due to the electronic effects which are produced between the
reactive chemical function and the aromatic rings of the dye.
[0027] These labels are difficult to solubilize in the coupling
medium the nature of which is dictated by the identity of the
target molecule. [0028] These labels cannot be synthesized from
reagents which are easily commercially accessible and according to
simple and rapid synthesis processes; the available number of these
labels is therefore limited.
[0029] The present invention for its part proposes to prepare
according to a particular process, labels of a fourth category
having none of the drawbacks of the labels of the prior art.
[0030] Moreover, to the applicant's knowledge, there has never been
described, in the prior art, a process making it possible to easily
functionalize a very broad panel of dyes. In fact, the processes of
the prior art are applicable only to a restricted number of dyes
since a large number of dyes exists having specific spectral
characteristics useful for labelling target molecules.
[0031] Moreover, during multi-labelling processes, it is essential
to take into account any problems of partial overlaps of the
emission spectra of the labels used which falsify the reading of
the results. Within the framework of the present invention, this
problem of covering is minimized to the extent that the user has a
large panel of labels, optionally fluorescent having different
spectral properties, i.e. different excitation and emission
wavelengths covering a wide region of the detection spectrum.
[0032] The benefit of the process of the invention is that it
allows the selection of a dye as a function of its particular
spectral characteristics and being able to easily functionalize it
without appreciably altering its spectral characteristics or its
solubility characteristics.
[0033] In light of the prior art, it is apparent that a need exists
for a wide variety of labels which are easy to synthesize from dyes
the choice of which is mainly dictated by their absorption and
emission properties, said labels not only having substantially the
same spectral properties as the dyes from which they are obtained
but also a solubility in the coupling medium which is equivalent or
even improved relative to the initial dye.
[0034] The inventors, at the end of in-depth research, have found
that it is possible to obtain a large panel of novel labels
combining all the sought functional and spectral characteristics
from a large panel of dyes or intermediates commonly commercially
available.
[0035] The first subject of the present invention relates to novel
labels capable of forming a covalent or non-covalent bond with a
target molecule, consisting of a dye to which there is bonded in
covalent manner by one or more carbons of its chemical structure:
[0036] one or more [FUNC] group(s), and [0037] optionally one or
more [SOL] group(s), [0038] said label having the general
formula:
[0038] ##STR00001## [0039] [DYE] representing a molecule chosen
from the group comprising the phthaleins, carbocyanines,
merocyanines, porphyrins, phthalocyanines; [0040] [FUNC] each
representing independently an --X-A-Z group, in which: [0041] X is
chosen from the group consisting of an oxygen atom, a sulphur atom,
an NR.sub.1R.sub.2 group, R.sub.1 and R.sub.2 each being
independently of each other a hydrogen atom or a linear or branched
C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18 and more
preferentially C.sub.1-C.sub.5 alkyl group; [0042] A is chosen from
the group consisting of an alkylene group or an alkylene-arylene
group; [0043] Z is a reactive chemical function; [0044] [SOL] each
representing independently a [0045] X'-A'-Z' group, in which:
[0046] X' is chosen from the group consisting of an oxygen atom, a
sulphur atom, an NR.sub.1R.sub.2 group, R.sub.1 and R.sub.2 each
being independently of each other a hydrogen atom or a linear or
branched C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18 and more
preferentially C.sub.1-C.sub.5 alkyl group; [0047] A' is chosen
from the group consisting of an alkylene group, or an
alkylene-arylene group; [0048] Z' is a polar or apolar group.
[0049] In the present invention, by "phthaleins" is meant dyes
having the following structure (1):
##STR00002##
by "carbocyanines" is meant dyes having the following structure
(2):
##STR00003##
by "merocyanines" is meant dyes having the following structure
(3):
##STR00004##
by "porphyrins" is meant dyes having the following structure
(4):
##STR00005##
by "phthalocyanines" is meant dyes having the following structure
(5):
##STR00006##
in which: [0050] M.sub.1 to M.sub.4, M.sub.9 to M.sub.12, Q.sub.1
to Q.sub.12, D.sub.1 to D.sub.16, which are identical to or
different from each other, are chosen from the group comprising the
following radicals or groups: hydrogen, hydroxyl, halogen, acetyl,
amine, substituted amine, quaternary ammonium, phosphate, nitro,
carboxylic acid and its salts, sulphonic acid and its salts,
alkylcarboxy with 2 to 30 carbon atoms, linear or branched alkyl,
having 1 to 30 carbon atoms, cycloalkyl having 3 to 14 carbon
atoms, alkyloxy having 1 to 30 carbon atoms, halogenoalkyl having 1
to 30 carbon atoms, hydroxyalkyl having 1 to 30 carbon atoms,
alkylester having 2 to 40 carbon atoms, nitroalkyl having 1 to 30
carbon atoms, carboxyalkyl having 2 to 30 carbon atoms, aminoalkyl
having 1 to 30 carbon atoms, sulphoalkyl having 1 to 30 carbon
atoms, aryl, aryloxy, arylalkyl, halogenoaryl, arylester, [0051]
Q.sub.3, Q.sub.6, Q.sub.9 and Q.sub.12 not being able to represent
aryl or aryloxy, [0052] providing that at least one of Q.sub.1 to
Q.sub.12 represents H or an aromatic ring, [0053] providing that at
least one of D.sub.1 to D.sub.16 represents H; [0054] Z.sub.1 and
Z.sub.2 each represent independently of each other the atoms
necessary to complete an indole, benzindole or naphthindole
nucleus; [0055] Z.sub.3 represents O or S; [0056] V and W are each
independently of each other chosen from CR.sub.7R.sub.8, O, S, Se
and NR.sub.9, where R.sub.7, R.sub.8 and R.sub.9 are each
independently of each other chosen from hydrogen and a
(CH.sub.2).sub.mR.sub.10 group, or m is an integer from 1 to 18 and
R.sub.10 is selected from hydrogen, amine, substituted amine,
quaternary ammonium, aldehyde, halogen, cyano, aryl, heteroaryl,
hydroxyl, amide, sulphonic acid and its salts, carboxylic acid and
its salts; [0057] n is an integer from 1 to 10, preferably 1 to 7
and more preferentially 1 to 3; [0058] i is an integer from 1 to n;
[0059] R.sub.3 to R.sub.6 each represent independently of each
other a hydrogen atom, a linear or branched C.sub.1-C.sub.30,
preferably C.sub.1-C.sub.18, more preferentially C.sub.1-C.sub.5
alkyl group, cycloalkyl, aryl, aryloxy, nitroalkyl, alkylamine,
substituted alkylamine, quaternary alkylammonium, alkylphosphate,
alkylsulphonic acid and its salts; [0060] T.sub.1 to T.sub.8, each
represent independently of each other a hydrogen atom, a halogen
atom, a linear or branched C.sub.1-C.sub.30, preferably
C.sub.1-C.sub.18, more preferentially C.sub.1-C.sub.5 alkyl group,
sulphoalkyl, cycloalkyl, aryl, aryloxy, nitro, amine, substituted
amine, quaternary ammonium, phosphate, sulphonic acid and its
salts, OR.sub.11 with R.sub.11 chosen from hydrogen and a
C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18, more preferentially
C.sub.1-C.sub.5 alkyl group, COOR.sub.11 or CONHR.sub.11 with
R.sub.11 as defined previously; [0061] G.sub.11, G.sub.21 and
G.sub.3 and M.sub.5 to M.sub.8 each represent independently of each
other a hydrogen atom, a linear or branched C.sub.1-C.sub.30,
preferably C.sub.1-C.sub.18, more preferentially C.sub.1-C.sub.5,
cycloalkyl alkyl group, aryl; [0062] B.sub.11, B.sub.21, B.sub.3
each represent independently of each other a methine (.dbd.CH--)
group, mono- or di-unsaturated cycloalkyl with 4 to 8 carbon atoms,
mono- or di-unsaturated aryl-cycloalkyl with 4 to 8 carbon atoms,
or one of the following groups:
[0062] ##STR00007## [0063] each of these groups being either
unsubstituted, or substituted by one or more of the following
groups: linear or branched C.sub.1-C.sub.18, preferably
C.sub.1-C.sub.5 alkyl, halogen, sulphonate, sulphoalkyl, aryl,
sulphoaryl, aryloxy, hydroxy, hydroxylate, ketone, nitro, amine,
substituted amine, quaternary ammonium; [0064] Y represents a
counter-ion chosen from the following ions: halide,
p-toluenesulphonate, methanesulphonate,
trifluoromethane-sulphonate, perchlorate, acetate, sodium,
potassium, calcium, magnesium, lithium, ammonium and
trialkylammonium; `p is an integer from 0 to 8 necessary to the
neutrality of the molecule.
[0065] By the expression "reactive chemical function", is meant any
functional group capable of binding by a covalent or non-covalent
bond (electrostatic, hydrogen, coordinative, ionic or complex)
directly or after activation, to at least one of the functions
naturally present or artificially introduced onto a target
molecule. By way of non-limitative examples of reactive chemical
functions appropriate to the purposes of the invention, there can
be mentioned in particular the functions carboxylic acid and its
salts, sulphonic acid and its salts, acid anhydride, acid chloride,
ester (alkyl ester, p-nitrophenyl ester, succinimidyl ester,
sulphosuccinimidyl ester, etc.), azido (acyl azide,
azidonitrophenyl, etc.), hydrazide,
3-acyl-1,3-thiazolidine-2-thione, amine, substituted amine,
quaternary ammonium, isocyanate, isothiocyanate, hydrazine,
phthalimido, maleimide, haloacetamide, monochlorotriazine,
dichlorotriazine, mono- or dihalogenated pyridine, mono- or
dihalogenated diazine, aziridine, thiol, sulphonyl chloride,
vinylsulphone, disulphide, methanethiosulphonate, hydroxyl,
phosphoramidite, epoxy, aldehyde, carbonate, glyoxal,
imidazolyl.
[0066] By the expression "polar or apolar group", is meant any
functional group capable of facilitating the solubilization of a
chemical compound in polar or apolar solvents. By way of
non-limitative examples of polar groups appropriate to the purposes
of the invention, there can be mentioned in particular the groups
carboxylic acid and its salts, sulphonic acid and its salts, amine,
substituted amine, quaternary ammonium, carbohydrate, glycol,
hydroxyl, nitro, phosphate. By way of non-limitative examples of
apolar groups there can be mentioned in particular the linear or
branched alkyl groups having 1 to 30 carbon atoms, aryl, cycloalkyl
having 3 to 14 carbon atoms, alkyloxy having 1 to 30 carbon atoms,
halogenoalkyl having 1 to 30 carbon atoms, hydroxyalkyl having 1 to
30 carbon atoms, alkylester having 2 to 40 carbon atoms, aryloxy,
arylalkyl, substituted arylalkyl, halogenoaryl.
[0067] By "alkylene", is meant a cyclic, linear or branched
hydrocarbon chain, having two free bonds, containing 1 to 30 carbon
atoms, preferably 1 to 18 carbon atoms and still more
preferentially 1 to 5 carbon atoms and being able to be in
particular a methylene, ethylene, propylene, 2-methylpropylene,
n-butylene, i-pentylene, n-pentylene, hexylene, heptylene,
octylene, nonylene chain, or a cyclopentadiene group.
[0068] By "arylene", is meant an aromatic group, having two free
bonds, containing one or more optionally substituted aromatic
rings, including in particular one phenylene which can be
substituted or unsubstituted.
[0069] By "alkyl", and "C.sub.1-C.sub.30 alkyl" is meant a cyclic,
linear or branched hydrocarbon chain, having a free bond,
containing 1 to 30 carbon atoms, preferably 1 to 18 carbon atoms
and still more preferentially 1 to 5 carbon atoms and being able to
be in particular a methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, dry-butyl, tert-butyl, n-pentyl, 1-methylbutyl,
2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, isopentyl,
neopentyl, 2-pentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl,
heptyl, octyl, nonyl, decyl or dodecyl chain.
[0070] By "target molecule", is meant a biological or
non-biological molecule intended to be coupled to a label. This
expression includes but is not limited to organic molecules,
polymers, natural or synthetic silicate materials, lipid vesicles,
amino acids, nucleic acids, nucleotides, oligonucleotides,
peptides, proteins, carbohydrates, oligosaccharides,
polysaccharides, antibodies, antigens, cell receptors, haptenes,
pectines, cytokines, hormones, toxins, bacteria, viruses,
eukaryotic cells.
[0071] The label according to the invention is characterized by the
fact that each of the [FUNC] groups or [SOL] groups is attached to
a carbon of the chemical structure of the dye using its respective
X or X' group as defined previously. Therefore, the hydrophilic or
hydrophobic groups initially present on the dye and which can be
carried by the nitrogen atoms or by any other atom of the
structure, are not modified.
[0072] Particular but non-limitative examples of [FUNC] groups are
given hereafter (Su represents the succinimidyl group): [0073]
--X--(CH.sub.2).sub.r--COOSu,
--X--(CH.sub.2).sub.r--COOSuSO.sub.3Na,
--X--(CH.sub.2).sub.r--COOH, --X--(CH.sub.2).sub.r--SO.sub.3H,
--X--(CH.sub.2).sub.r--SO.sub.3Na,
--X--(CH.sub.2).sub.r--COO--C.sub.6H.sub.4--NO.sub.2,
--X--(C.sub.6-C.sub.4)--(CH.sub.2).sub.r--COOSu,
--X--(CH.sub.2).sub.r--NHCOCH.sub.2I, --X--(CH.sub.2).sub.r--NCS,
--X--(CH.sub.2).sub.r--C.sub.6H.sub.4CH (CH.sub.3)--COOSu,
--N(CH.sub.3).sub.2--(CH.sub.2).sub.r--SO.sub.3H,
--X--(CH.sub.2).sub.r--OP[N(iPr).sub.2][CH.sub.2CH.sub.2CN], X is
as defined previously, r is an integer from 1 to 18, preferably 2
to 10, and more preferentially 3 to 5.
[0074] Generally, the conditions for a coupling reaction between a
label and a target molecule are dictated by the nature of the
target molecule. In the case of the labelling of certain fragile
biological molecules, in particular proteins, it is preferable for
the coupling reaction to take place in aqueous solution in order to
avoid the denaturation of the latter. To this end, it is useful
that the label is provided with one or more Z' groups conferring
upon it a water-soluble character in the coupling medium which is
non-denaturing for the protein. On the other hand, for other target
molecules hydrophobic or non-polar groups are preferable. In this
case, the label must preferentially have at least one Z' group
conferring upon it a hydrophobic or non-polar character.
[0075] These hydrophilic or lipophilic (Z') groups can optionally
be introduced in the same way as the reactive chemical function Z.
Particular but non-limitative examples of a [SOL] group comprising
a hydrophilic or lipophilic Z' group, illustrating this structural
analogy are given hereafter: [0076]
--X--(CH.sub.2).sub.r--SO.sub.3Na,
--X--(CH.sub.2).sub.r--SO.sub.2H,
--X--(CH.sub.2).sub.r--C.sub.6H.sub.3(NO.sub.2).sub.2,
--X--(CH.sub.2).sub.r--CH.sub.3, X is as defined previously, r is
an integer from 1 to 18, preferably 2 to 10, and more
preferentially 3 to 5.
[0077] The labels of the invention can be fluorescent, when they
are made from fluorescent dyes, and have fluorescent spectral
characteristics which can range from the ultraviolet to the
infrared.
[0078] Thus, the invention relates to labels derived from dyes
chosen from the group comprising the following dyes: the
phthaleins, carbocyanines, merocyanines, porphyrins and
phthalocyanines, said dyes being as defined previously.
[0079] The label according to the invention obtained from a
phthalein, is a compound having the structure (6),
##STR00008##
in which: [0080] each of M.sub.5 to M.sub.g is as defined
previously; [0081] each of M'.sub.1 to M'.sub.4 and M'.sub.9 to
M'.sub.12, independently of each other, can have the definition
given previously for M.sub.1 to M.sub.4 and M.sub.9 to M.sub.12, or
can represent [SOL] or [FUNC], [SOL] and [FUNC] being as defined
previously, and providing that at least one of M'.sub.1 to M'.sub.4
and M'.sub.9 to M'.sub.12 represents [FUNC].
[0082] According to a particular embodiment of the label of
structure (6), at least one of M'.sub.1, M'.sub.2, M'.sub.3 or
M'.sub.4 represents [FUNC], and/or at least one of M'.sub.9,
M'.sub.10, M'.sub.11 or M'.sub.12 represents [FUNC].
[0083] The carbocyanine-type label according to the invention is a
compound having the following structure (7)
##STR00009##
in which [0084] Z.sub.1, Z.sub.2, R.sub.3, R.sub.4, B.sub.11,
B.sub.21, B.sub.3, G.sub.11, G.sub.21, G.sub.3, V, W, Y, i, n, and
p are as defined previously, [0085] each of T'.sub.1 to T'.sub.8,
independently of each other, can have the definition given
previously for T.sub.1 to T.sub.8, or can represent [SOL] or
[FUNC], [SOL] and [FUNC] being as defined previously, and providing
that at least one of T'.sub.1 to T'.sub.8 represents [FUNC].
[0086] According to a particular embodiment of the label of
structure (7), at least one of T'.sub.1 to T'.sub.4 and/or at least
one of T'.sub.5 to T'.sub.8 represents [FUNC].
[0087] The merocyanine-type label according to the invention is a
compound having the following structure (8):
##STR00010##
in which [0088] Z.sub.1, Z.sub.3, R.sub.3, R.sub.5, R.sub.6,
G.sub.11, G.sub.21 and G.sub.3, B.sub.11, B.sub.21, B.sub.3, V, Y,
i, n and p p are as defined previously, [0089] each of T'.sub.1 to
T'.sub.4, independently of each other, can have the definition
given previously for T.sub.1 to T.sub.4, or can represent [SOL] or
[FUNC], [SOL] and [FUNC] being as defined previously, and providing
that at least one of T'.sub.1 to T'.sub.4, represents [FUNC].
[0090] The porphyrin-type label according to the invention is a
compound having the following structure (9):
##STR00011##
in which [0091] Q.sub.3, Q.sub.6, Q.sub.9 and Q.sub.12 are as
defined previously, each of Q'.sub.1, Q'.sub.2, Q'.sub.4, Q'.sub.5,
Q'.sub.7, Q'.sub.8, Q'.sub.10 and Q'.sub.11 independently of each
other can have the definition given previously for Q.sub.1,
Q.sub.2, Q.sub.4, Q.sub.5, Q.sub.7, Q.sub.8, Q10 and Q.sub.11 or
can represent [SOL] or [FUNC], [SOL] and [FUNC] being as defined
previously, and providing that at least one of Q'.sub.1, Q'.sub.2,
Q'.sub.4, Q'.sub.5, Q'.sub.7, Q'.sub.8, Q'.sub.10 and Q' .sub.11
represents [FUNC].
[0092] The phthalocyanine-type label according to the invention is
a compound having the following structure (10):
##STR00012##
in which [0093] each of D.sub.1' to D.sub.16', independently of
each other, can have the definition given previously for D.sub.1 to
D.sub.16, or can represent [FUNC] or [SOL], [FUNC] and [SOL] being
as defined previously, and providing that at least one of D'.sub.1
to D'.sub.16 represents [FUNC].
[0094] According to an embodiment of the invention, the label is
water-soluble, preferably at a concentration greater than 0.5%
(m/v), more preferentially at a concentration greater than or equal
to 2% (m/v), and also still more preferentially greater than 10%
(m/v).
[0095] According to another embodiment of the invention, the label
is liposoluble, preferably at a concentration greater than 0.5%
(m/v), more preferentially at a concentration greater than or equal
to 2% (m/v), and still more preferentially greater than 10%
(m/v).
[0096] Specific examples of labels according to the invention are
the following compounds:
##STR00013## [0097] in which each of y, z, identical or different,
is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8, Z represents
--COOH or
[0097] ##STR00014## [0098] Y represents SO.sub.3.sup.- or
SO.sub.3Na
[0098] ##STR00015## [0099] in which each of y, z and n, identical
or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8, Z
represents --COOH or
[0099] ##STR00016## [0100] Y represents SO.sub.3.sup.- or
SO.sub.3Na in particular
[0100] ##STR00017## ##STR00018## [0101] in which each of y, z and
n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6,
7 or 8 [0102] Z reoresents --COOH or
[0102] ##STR00019## [0103] Y represents SO.sub.3.sup.- or
SO.sub.3Na in particular
[0103] ##STR00020## ##STR00021## [0104] in which each of y, z and
n, identical or different, is an integer equal to 1, 2, 3, 4, 5, 6,
7 or 8 Z represents --COOH or
[0104] ##STR00022## [0105] Y represents SO.sub.3.sup.- or
SO.sub.3Na in particular
[0105] ##STR00023## [0106] in which each of y, z and n, identical
or different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z
represents --COOH or
[0106] ##STR00024## [0107] Y represents SO.sub.3 or SO.sub.3Na in
particular
##STR00025##
[0107] in which each of y and z, identical or different, is an
integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 [0108] Z represents
--COOH or
[0108] ##STR00026## [0109] Y represents SO.sub.3.sup.- or
SO.sub.3Na in particular
[0109] ##STR00027## [0110] in which each of y and z, identical or
different, is an integer equal to 1, 2, 3, 4, 5, 6, 7 or 8 Z
represents --COOH or
[0110] ##STR00028## [0111] Y represents SO.sub.3 or SO.sub.3Na in
particular
##STR00029##
[0112] The process for the preparation of the labels of the
invention as described previously, and in particular the
introduction onto a carbon atom of the dye structure, of at least
one [FUNC] group, making it possible to conjugate said labels to a
target molecule in a given coupling medium, and optionally the
introduction of at least one [SOL] group, constitutes the second
subject of the invention.
[0113] The process according to the invention by means of which it
has been possible to synthesize labels according to the invention,
is characterized by the fact that it comprises a nucleophilic
substitution reaction between: either:
Z-A-L and [DYE']-Nu
or:
[DYE'']-L and Z-A-Nu [0114] Z and A being as defined previously;
[0115] L representing a leaving group; [0116] Nu representing a
nucleophilic group chosen from the group comprising --OH, --SH or
--NR.sub.1R.sub.2, R.sub.1 and R.sub.2 each being independently of
each other a hydrogen atom or a linear or branched
C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18, more preferentially
C.sub.1-C.sub.5 alkyl group; [0117] [DYE'] and [DYE''] representing
[DYE] or a precursor or synthesis intermediate of [DYE], [DYE]
being as defined previously.
[0118] By "leaving group", is meant a group which can be replaced
by another group during a substitution reaction.
[0119] Said leaving group can be in particular a halogen or a
methanesulphonate, para-toluenesulphonate group or a diazonium
group. This list is not limitative. The leaving group therefore
allows the binding of the chain carrying either the reactive
chemical function or the polar or apolar function, to a carbon atom
of the dye's ring structure or of its synthesis intermediate.
[0120] According to a particular embodiment, the nucleophilic
substitution reaction can be carried out by opening of a cyclic
molecule of formula:
##STR00030##
[0121] According to a particular embodiment, the nucleophilic
substitution reaction can be a Williamson-type reaction, carried
out between the molecule [DYE']-Nu and the molecule Z-A-L, in the
presence of a base.
[0122] Said base can be in particular sodium hydroxide, potassium
carbonate, potassium t-butylate, sodium hydride, sodium amide. This
list is not limitative.
[0123] The invention also relates to the processes for the
preparation of [DYE']-Nu and [DYE'']-L. [0124] [DYE']-Nu is
obtained by the following successive reactions: [0125] 1. nitration
of [DYE']; [0126] 2. reduction of the product obtained in stage 1;
[0127] 3. optionally: either alkylation of the product obtained in
stage 2, or diazotization of the product obtained in stage 2 in
order to obtain a diazonium salt then nucleophilic substitution on
this diazonium function. For its part [DYE'']-L is obtained by the
following successive reactions: [0128] 1'. nitration of [DYE''];
[0129] 2'. reduction of the product obtained in stage 1'; [0130]
3'. diazotization of the product obtained in stage 2' in order to
obtain a diazonium salt; [0131] 4'. optionally: nucleophilic
substitution on the diazonium function of the product obtained in
stage 3'.
[0132] According to a particular embodiment, [DYE']-Nu and
[DYE'']-L represents the dye itself, i.e. [DYE].
[0133] The nitration can be carried out for example by the action
of nitric acid, nitronium tetrafluoroborate, sodium nitrate or
sodium nitrite in acid medium. The reduction of the nitrated
derivative to amine can be carried out by catalytic hydrogenation,
by transfer of hydrogen in the presence of a catalyst or by the
action of stannous chloride in acid medium for example. This amine
function can itself be used as a nucleophilic group or can be
converted to another nucleophilic group. To the extent that it is
preferable to obtain another nucleophilic group, the latter can
easily be obtained by substitution of the corresponding diazonium
salt. In this case, the preceding amine function is converted to a
diazonium salt by the action of sodium nitrite in acid medium. In
order to obtain a hydroxy function, the diazonium salt is
hydrolyzed in diluted sulphuric medium for example. In order to
obtain a thiol function, the diazonium salt is reacted with a
sulphur-containing compound, for example a xanthate, then
cleaved.
[0134] The moment in the synthesis when the introduction of the
nucleophilic group Nu (or of the leaving group L) then the
substitution reaction with the molecule Z-A-L (or Z-A-Nu) are
carried out, i.e. the choice of the precursor [DYE'] (or [DYE'']),
of course depends on the nature of the dye used. A person skilled
in the art will be able of course to choose this moment in order to
limit the operations of protection and deprotection of sensitive
functional groups.
[0135] In the case where the synthesis of the functionalized dye
must start with one of its synthesis intermediates carrying the
nucleophilic group Nu, it is recommended to protect this group
beforehand in order to avoid undesired secondary reactions.
[0136] Said nucleophilic group can be protected by a protective
group, for example in the form of an ether (methoxy . . . ) for a
hydroxy group, a thioether (benzylthio . . . ) for a thiol group,
an amide or a urethane-type group for example a t-butyloxycarbonyl
group or a benzyloxycarbonyl group for an amine.
[0137] After obtaining the dye or one of its synthesis
intermediates, when the last stages of synthesis are no longer
likely to interfere with the reactive chemical function, the
nucleophilic group is deprotected under the action of an acid, a
base or by reduction or oxidation and is then reacted with the
molecule Z-A-L in order to introduce the reactive chemical function
Z.
[0138] According to a particular embodiment, the process of the
present invention can comprise the binding of one or more [SOL]
groups using one or more of the stages of previously defined
chemical reactions for the binding of the [FUNC] group. In this
case, the nucleophilic substitution reaction is carried out using a
molecule Z'-A'-L (or Z'-A'-Nu) in which: Z', A', Nu and L are as
defined previously. Thus, the process can comprise a nucleophilic
substitution reaction between: either:
Z'-A'-L and [DYE']-Nu
or:
[DYE'']-L and Z'-A'-Nu [0139] Z' and A', L, Nu, [DYE'] and [DYE'']
being as defined previously; [0140] [DYE']-Nu and [DYE'']-L being
prepared according to the process described previously.
[0141] The process of the invention therefore makes it possible to
obtain, from easily accessible dyes, new functionalized derivatives
the spectral characteristics and the solubility characteristics of
which are particularly useful for labelling target molecules.
[0142] Examples of reaction diagrams of the preparation of labels
from carbocyanines (Diagrams Ia and Ib), phthaleins (Diagram II)
and porphyrins (Diagram III) are given hereafter in a
non-limitative manner.
In these reaction diagrams, NHS represents N-hydroxysuccinimide and
DCC dicyclohexylcarbodiimide.
##STR00031##
##STR00032##
##STR00033##
[0143] The use of the labels of the invention, as described
previously, for the detection and/or quantification of target
molecules constitutes the third subject of the invention.
[0144] As indicated previously, the labels of the invention are
suitable as agents for labelling target molecules, the labelling
being carried out by coupling of said label by a covalent or
non-covalent bond with the target molecule to be assayed or
detected. This coupling can be carried out according to standard
coupling processes used in this field.
[0145] According to a particular embodiment, the target molecule is
reacted with 1 to 200 equivalents, more preferentially 3 to 20
equivalents of the label. When the target molecule is
water-soluble, the reaction takes place in an aqueous buffer
solution, preferably based on phosphate, carbonate or borate, the
pH of which is comprised between 7.0 and 11.0. When the target
molecule is liposoluble, the reaction takes place in an organic
solvent.
[0146] The coupling reaction can also be carried out in the
presence of a peptide coupling agent, for example carbodiimide-type
reagents such as DCC (dicyclohexylcarbodiimide),
carbonyldiimidazole, IDDQ
(1-isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline),
phosphonium-type reagents such as BOP
(benzotriazol-1-yloxytris-(dimethylamino)phosphonium
hexafluorophosphate), uronium-type reagents such as HBTu
(o-benzotriazolyl-tetramethyluronium hexafluorophosphate) and TBTu
(o-benzotriazolyltetramethyluronium tetrafluoroborate), Woodward
reagents such as N-ethyl-5-phenylisoxazolium-3'-sulphonate, Curtius
reagents (hydrazine and nitrite).
[0147] The coupling reaction is carried out if necessary in the
presence of an organic base and dimethylsulphoxide, or preferably
dimethylformamide.
[0148] The labelled target molecule is isolated from the reaction
medium and purified by separation on silica gel, by gel permeation
and/or by ultrafiltration.
[0149] The invention also relates to a method for detecting a
biological or non-biological molecule comprising the coupling of a
label according to the invention with said molecule, and the actual
detection of said molecule coupled with the label by absorption
spectrometry, fluorescence spectrometry, infrared spectrometry,
electrophoresis, infra-red or near infra-red medical imaging (NIRS,
Near Infra-Red Spectroscopy). This list is not limitative.
[0150] The invention also relates to synthesis precursors of the
labels of the invention, which are novel products consisting of
molecules of general formula:
##STR00034##
in which [0151] Z.sub.1, V, p, Y are as defined previously, [0152]
R'.sub.3 represents an electronic doublet or represents R.sub.3 as
defined previously; [0153] .mu. is an integer equal to 0 or 1;
[0154] R.sub.12 represents a linear or branched, saturated or
unsaturated C.sub.1-C.sub.30, preferably C.sub.1-C.sub.18, more
preferentially C.sub.1-C.sub.5 alkyl group, sulphoalkyl,
cycloalkyl, aryl, aryloxy, preferably a methyl group, each of
T'.sub.1 to T'.sub.4, independently of each other, can have the
definition given previously for T.sub.1 to T.sub.4 or can represent
[SOL] or [FUNC], [SOL] and [FUNC] being as defined previously, and
providing that at least one of T'.sub.1 to T'.sub.4 represents
[FUNC].
[0155] Non-limitative examples of precursors are given
hereafter:
##STR00035##
[0156] The invention will be described in more detail by means of
the following examples which are not limitative but relate to
advantageous embodiments.
EXAMPLES
Example 1
##STR00036##
[0157] 5-nitro-2,3,3-trimethyl-(3H)-indole (product A)
[0158] A solution of 3.4 g of sodium nitrate in 100 ml of sulphuric
acid is added to a mixture of 6.4 g of 2,3,3-trimethyl-(3H)-indole
and 50 ml of sulphuric acid cooled down to 0-5.degree. C., without
exceeding 5.degree. C. After stirring for one hour at 0-5.degree.
C., the reaction medium is diluted in 600 ml of water and
neutralized by the addition of solid sodium hydroxide. The
precipitate formed is filtered and solubilized in 200 ml of ethyl
acetate. After washing with water, drying over magnesium sulphate
and evaporation, 8 g of product A is obtained (Yield: 97.9%).
Example 2
##STR00037##
[0159] 5-amino-2,3,3-trimethyl-(3H)-indole (product B)
[0160] A mixture of 6.6 g of product A, 43.7 g of stannous chloride
dihydrate and 215 ml of hydrochloric acid is taken to reflux for
two hours. After cooling down to ambient temperature and
filtration, the solid collected is solubilized in 130 ml of water.
This solution is neutralized by the addition of 20% soda. The
precipitate formed is filtered then washed with water and dried
under vacuum in the presence of phosphoric anhydride. 5.4 g of
product B is obtained (Yield: 96%).
Example 3
##STR00038##
[0161] 5-hydroxy-2,3,3-trimethyl-(3H)-indole (product C)
[0162] A mixture of 3.5 g of product B, 5 ml of sulphuric acid and
25 ml of water is cooled down to 0.degree. C. A solution cooled
down to 0-5.degree. C. of 1.6 g of sodium nitrite in 4 ml of water
is added without exceeding 5.degree. C. After stirring at
0-5.degree. C. for 10 minutes, the reaction medium is added slowly
to a mixture of 15 ml of sulphuric acid and 20 ml of water taken to
90.degree. C. After stirring at this temperature for one hour then
cooling down to ambient temperature, the reaction medium is
neutralized by the addition of 20% soda. The precipitate formed is
filtered then washed with water and dried under vacuum in the
presence of phosphoric anhydride. 2.6 g of product C is obtained
(Yield: 74.3%).
Example 4
##STR00039##
[0163] 5-methoxy-2,3,3-trimethyl-benz(e)indole (product D)
[0164] A mixture of 35.4 g of 7-methoxy-2-naphthylhydrazine and 65
g of sodium bisulphate monohydrate in 180 ml of water is heated at
90.degree. C. for 15 minutes then 23 g of 3-methyl-2-butanone is
added. The reaction medium is maintained at 90.degree. C. for 7
hours, cooled down to ambient temperature then extracted with
dichloromethane. The organic phase is washed with water then
evaporated under vacuum. The residue is purified by silica gel
chromatography (dichloromethane/methanol:10/0.2). 28.9 g of product
D is obtained (Yield: 64.2%).
Example 5
##STR00040##
[0165] 5-hydroxy-2,3,3-trimethyl-benz(e)indole (product E)
[0166] 8.2 g of product D are introduced into a three-necked 250-ml
flask. 82 ml of HBr (33% in acetic acid) is added.
[0167] The mixture is maintained at 70.degree. C. for 7 hours then
cooled down to ambient temperature. 2.5 litres of water then 120 ml
of 5M soda are added to the reaction medium. The precipitate formed
is filtered then washed with water and dried under vacuum in the
presence of P.sub.2O.sub.5. 6.8 g of product E is obtained (Yield:
88.7%).
Example 6
##STR00041##
[0168] 5-[(5-carbethoxypentyl)oxy]-2,3,3-trimethyl-benz(e)indole
(product F)
[0169] A mixture of 20 g of product E, 21.7 g of ethyl
6-bromohexanoate and 13.5 g of potassium carbonate in 120 ml of
acetone is taken to reflux for 8 hours then cooled down to ambient
temperature. The reaction medium is filtered then evaporated under
vacuum. The residue is dissolved in 400 ml of ethyl ether and
washed three times with water. After evaporation and elimination of
the excess ethyl 6-bromohexanoate by distillation under vacuum,
32.5 g of product F is obtained (Yield: 100%) in the form of a
brown oil.
Example 7
##STR00042##
[0170] 5-[(5-carbethoxypentyl)oxy]-2,3,3-trimethyl-(3H)-indole
(product G)
[0171] A mixture of 23.1 g of product C, 32.4 g of ethyl
6-bromohexanoate and 20.1 g of potassium carbonate in 180 ml of
acetone is taken to reflux for 8 hours then cooled down to ambient
temperature. The reaction medium is filtered then evaporated under
vacuum. The residue is dissolved in 600 ml of ethyl ether and
washed three times with water. After evaporation and elimination of
the excess ethyl 6-bromohexanoate by distillation under vacuum,
41.8 g of product G is obtained (Yield: 100%) in the form of a
brown oil.
Example 8
##STR00043##
[0172] 5-[(5-carboxypentyl)oxy]-2,3,3-trimethyl-benz(e)indole
(product H)
[0173] 11.6 g of product F in 100 ml of 1M KOH are heated at
80.degree. C. for 30 minutes. After cooling down to ambient
temperature, the reaction medium is neutralized by the addition of
1M hydrochloric acid. The precipitate formed is filtered then
washed with water and dried under vacuum in the presence of
P.sub.2O.sub.5. 9 g of product H is obtained (Yield: 84.2%).
Example 9
##STR00044##
[0174] 5-[(5-carboxypentyl)oxy]-2,3,3-trimethyl-(3H)-indole
(product I)
[0175] 4.2 g of product G in 45 ml of 1M KOH are heated at
80.degree. C. for 30 minutes. After cooling down to ambient
temperature, the reaction medium is neutralized by the addition of
1M hydrochloric acid. The precipitate formed is filtered then
washed with water and dried under vacuum in the presence of
P.sub.2O.sub.5. 2.5 g of product I is obtained (Yield: 66.5%).
Example 10
##STR00045##
[0176] 5-[(4-sulphobutyl)oxy]-2,3,3-trimethyl-benz(e)indole, sodium
salt (product J)
[0177] A mixture of 1.3 g of product E, 0.9 g of 1,4-butane sultone
and 0.3 g of sodium hydroxide in 10 ml of ethanol is taken to
reflux for two hours. After cooling down to ambient temperature,
the reaction medium is added to 100 ml of acetone under stirring.
The precipitate formed is filtered then washed with acetone and
dried under vacuum. 1.8 g of very hygroscopic product J is obtained
(Yield: 81.2%).
Example 11
##STR00046##
[0178]
2-[7-[8-(5-carboxypentyloxy)-1,3-dihydro-1,1-dimethyl-3-(4-sulphobu-
tyl)-benz(e)indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulphob-
utyl)-1H-benz(e)indolium, internal salt, sodium salt (product
K)
[0179] A mixture of 10.2 g of product H and 32.7 g of 1,4-butane
sultone is heated at 115.degree. C. for 16 hours then cooled down
to ambient temperature. 120 ml of toluene is added, then the medium
is filtered in order to recover the insoluble fraction. The
precipitate is rinsed with acetone and dried under vacuum. 13.3 g
of this solid is reacted with 15.2 g of
2-(6-acetanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(4-sulphobutyl)-b-
enz(e)indolium, internal salt, in 90 ml of ethanol. 2.9 g of
triethylamine is added progressively and the mixture is taken to
reflux for 5 minutes then cooled down to ambient temperature. 3.8 g
of sodium acetate trihydrate is added and the mixture is stirred
for 10 minutes. The precipitate formed is filtered then washed with
acetone and dried under vacuum. 18.5 g of product K is obtained
(Yield: 72.8%).
Example 12
##STR00047##
[0180]
2-[5-[8-(5-carboxypentyloxy)-1,3-dihydro-1,1-dimethyl-3-(4-sulphobu-
tyl)-benz(e)indol-2-ylidene]-1,3-pentadienyl]-1,1-dimethyl-3-(4-sulphobuty-
l)-1H-benz(e)indolium internal salt, sodium salt (product L)
[0181] A mixture of 8.1 g of product H and 26.2 g of 1,4-butane
sultone is heated at 120.degree. C. for 12 hours then cooled down
to ambient temperature. 80 ml of toluene is added, then the medium
is filtered in order to recover the insoluble fraction. The
precipitate is rinsed with acetone and dried under vacuum. 10.6 g
of this solid is reacted with 11.5 g of
2-(4-acetanilido-1,3-butadienyl)-3,3-dimethyl-1-(4-sulphobutyl)-benz-
(e)indolium, internal salt, in 75 ml of ethanol. 2.2 g of
triethylamine is added progressively and the mixture is taken to
reflux for 5 minutes then cooled down to ambient temperature. 3 g
of sodium acetate trihydrate is added and the mixture is stirred
for 10 minutes. The precipitate formed is filtered then washed with
acetone and dried under vacuum. 15.2 g of product L is obtained
(Yield: 75.2%).
Example 13
##STR00048##
[0182]
2-[5-[6-(5-carboxypentyloxy)-1,3-dihydro-1,1-dimethyl-3-(4-sulphobu-
tyl)-indol-2-ylidene]-1,3-pentadienyl]-1,1-dimethyl-3-(4-sulphobutyl)-1H-b-
enz(e)indolium, internal salt, sodium salt (product M)
[0183] A mixture of 10.8 g of product I and 40.8 g of 1,4-butane
sultone is heated at 130.degree. C. for 8 hours then cooled down to
ambient temperature. 80 ml of toluene is added, then the medium is
filtered in order to recover the insoluble fraction. The
precipitate is rinsed with acetone and dried under vacuum. 13.4 g
of this solid is reacted with 16.3 g of the internal salt of
2-(4-acetanilido-1,3-butadienyl)-3,3-dimethyl-1-(4-sulphobutyl)-benz(e)in-
dolium in 175 ml of ethanol. 3.2 g of triethylamine is added
progressively and the mixture is taken to reflux for 5 minutes then
cooled down to ambient temperature. 4.3 g of sodium acetate
trihydrate is added and the mixture is stirred for 10 minutes. The
precipitate formed is filtered then washed with acetone and dried
under vacuum. 18.2 g of product M is obtained (Yield: 69.6%).
Example 14
##STR00049##
[0184]
2-[7-[8-(5-carboxypentyloxy)-1,3-dihydro-1,1-dimethyl-3-(4-sulphobu-
tyl)-benz(e)indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulphob-
utyl)-8-(4-sulphobutyloxy)-1H-benz(e)indolium, internal salt,
sodium salt (product N)
[0185] A mixture of 0.6 g of product J and 1.7 g of 1,4-butane
sultone is heated at 120.degree. C. for 16 hours then cooled down
to ambient temperature. 5 ml of toluene is added, then the medium
is filtered in order to recover the insoluble fraction. The
precipitate is rinsed with acetone and dried under vacuum. 0.7 g of
this solid is reacted with 0.9 g of
2-(6-acetanilido-1,3,5-hexatrienyl)-5-(5-carboxypentyloxy)-3,3-dimethy-
l-1-(4-sulphobutyl)-benz(e)indolium, internal salt, (obtained from
the internal salt of
5-(5-carboxypentyloxy)-1-(4-sulphobutyl)-2,3,3-trimethyl-benz(e)indolium,
the preparation of which is described in Examples 11 and 12, and
glutaconic aldehyde dianilide hydrochloride) in 10 ml of ethanol.
0.14 g of triethylamine is added progressively and the mixture is
taken to reflux for 5 minutes then cooled down to ambient
temperature. 0.18 g of sodium acetate trihydrate is added and the
mixture is stirred for 10 minutes. The precipitate formed is
filtered then washed with acetone and dried under vacuum. 1.1 g of
product N is obtained (Yield: 75.5%).
Example 15
##STR00050##
[0186]
2-[7-[8-(4-sulphobutyloxy)-1,3-dihydro-1,1-dimethyl-3-(4-sulphobuty-
l)-benz(e)indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulphobut-
yl)-8-(4-sulphobutyloxy)-1H-benz(e)indolium, internal salt,
trisodium salt (product O)
[0187] A mixture of 0.6 g of product J and 1.7 g of 1,4-butane
sultone is heated at 120.degree. C. for 16 hours then cooled down
to ambient temperature. 5 ml of toluene is added, then the medium
is filtered in order to recover the insoluble fraction. The
precipitate is rinsed with acetone and dried under vacuum. 0.7 g of
this solid is reacted with 0.97 g of
2-(6-acetanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(4-sulphobutyl)-5-
-(4-sulphobutyloxy)-benz(e)indolium, internal salt, sodium salt,
(obtained from
1-(4-sulphobutyl)-5-(4-sulphobutyloxy)-2,3,3-trimethyl-benz(e)indoli-
um, internal salt, sodium salt, the preparation of which is
described previously, and glutaconic aldehyde dianilide
hydrochloride) in 10 ml of ethanol. 0.14 g of triethylamine is
added progressively and the mixture is taken to reflux for 5
minutes then cooled down to ambient temperature. 0.18 g of sodium
acetate trihydrate is added and the mixture is stirred for 10
minutes. The precipitate formed is filtered then washed with
acetone and dried under vacuum. 1.2 g of product O is obtained
(Yield: 79.1%).
Example 16
##STR00051##
[0188] Sulphosuccinimidyl Ester of Product K (Product P)
[0189] 0.09 g of product K is solubilized in 5 ml of DMF. 0.11 g of
sodium 1-hydroxy-3-succinimide-sulphonate and 25 mg of
DiCyclohexylCarbodiimide are added. The reaction medium is
maintained under stirring at ambient temperature for 24 hours then
filtered. 100 ml of ethyl ether is added to the filtrate. The
precipitate formed is filtered and dried under vacuum. 105 mg of
product P is obtained (Yield: 95.4%).
Example 17
##STR00052##
[0190] Succinimidyl Ester of Product K (Product Q)
[0191] 1.08 g of product K is solubilized in 50 ml of DMF. 0.69 g
of N-hydroxysuccinimide and 0.31 g of DiCyclohexylCarbodiimide are
added. The reaction medium is maintained under stirring at ambient
temperature for 24 hours then filtered. 250 ml of ethyl ether is
added to the filtrate. The precipitate formed is filtered and dried
under vacuum. 1.1 g of product Q is obtained (Yield: 91.7%).
Example 18
##STR00053##
[0192] p-nitrophenyl Ester of Product K (Product R)
[0193] 90 mg of product K and 17 mg of p-nitrophenol are
solubilized in 2 ml of DMF. 25 mg of DiCyclohexylCarbodiimide is
added. The reaction medium is maintained under stirring at ambient
temperature for 24 hours then filtered. 100 ml of ethyl ether is
added to the filtrate. After filtration and drying under vacuum 101
mg of product R is obtained (Yield: 98%).
Example 19
##STR00054##
[0194] Succinimidyl Ester of Product L (Product S)
[0195] 0.24 g of product L and 0.12 g of TSTU
(N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate)
are solubilized in 5 ml of DMF. After stirring for 10 minutes, 64
mg of diisopropylethylamine is added. After stirring for 2 hours,
100 ml of ethyl ether is added. The precipitate formed is filtered,
washed with ethyl ether and dried under vacuum. 0.25 g of product S
is obtained (Yield: 92.6%).
Example 20
##STR00055##
[0196] Succinimidyl Ester of Product M (Product T)
[0197] 83 mg of product M and 40 mg of TSTU are solubilized in 5 ml
of DMF. After stirring for 10 minutes, 25 mg of
diisopropylethylamine is added. After stirring for 2 hours, 100 ml
of ethyl ether is added. The precipitate formed is filtered, washed
with ethyl ether and dried under vacuum. 90 mg of product T is
obtained (Yield: 96.8%).
Example 21
##STR00056##
[0198] Succinimidyl Ester of Product N (Product U)
[0199] 1.08 g of product N is solubilized in 50 ml of DMF. 0.57 g
of N-hydroxysuccinimide and 0.26 g of DiCyclohexylCarbodiimide are
added. The reaction medium is maintained under stirring at ambient
temperature for 24 hours then filtered. 250 ml of ethyl ether is
added to the filtrate. The precipitate formed is filtered and dried
under vacuum. 0.99 g of product U is obtained (Yield: 84.2%).
Example 22
##STR00057##
[0200] Phenethyl Amide of Product L (Product V)
[0201] 93 mg of product S and 39 mg of phenethylamine are
solubilized in 5 ml of DMF. After stirring for 45 minutes, 60 ml of
ethyl ether is added. The precipitate formed is filtered, washed
with ethyl ether and dried under vacuum. 57 mg of product V is
obtained (Yield: 61.3%).
Example 23
Labelling of Proteins with Product S
[0202] 1 ml of sodium carbonate-sodium bicarbonate buffer (0.1 M,
pH 9.3) containing 10% dimethylformamide is added to a solution of
1 mg of protein (molecular weight 150 kDa) in 0.5 ml of phosphate
buffer solution (0.1 M), then 25 .mu.l of a solution of 9.1 mg of
product S in 2 ml of dimethylformamide is added dropwise. The
mixture is stirred for 30 minutes at ambient temperature. 200 .mu.l
of this mixture is eluted in fractions of 0.5 ml with phosphate
buffer (0.1 M, pH 7.4, 10% of dimethylformamide) on a Sephadex
G25/PD-10 column (Amersham Biosciences) previously equilibrated
with 25 ml of the same buffer. The dye coupled with the protein is
separated from the uncoupled dye by gel permeation: the first
coloured band (fractions 7 to 9) corresponds to the coupled dye and
the second coloured band (fractions 14 to 17) corresponds to the
free dye. Reading the optical densities at 685 nm indicates an
average substitution level of 4.7 molecules of dye per molecule of
antibody. The conjugate is stored at 4.degree. C. away from the
light.
* * * * *