EP1163372A1 - Colorants a base de cyanine rigidifies dans un plan et chimiquement reactifs, et derives de ces derniers - Google Patents

Colorants a base de cyanine rigidifies dans un plan et chimiquement reactifs, et derives de ces derniers

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Publication number
EP1163372A1
EP1163372A1 EP00919582A EP00919582A EP1163372A1 EP 1163372 A1 EP1163372 A1 EP 1163372A1 EP 00919582 A EP00919582 A EP 00919582A EP 00919582 A EP00919582 A EP 00919582A EP 1163372 A1 EP1163372 A1 EP 1163372A1
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EP
European Patent Office
Prior art keywords
dye
group
nucleic acid
biological molecule
molecule
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EP00919582A
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German (de)
English (en)
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EP1163372A4 (fr
Inventor
Derek Levison
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Princeton Separations Inc
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Princeton Separations Inc
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Publication of EP1163372A1 publication Critical patent/EP1163372A1/fr
Publication of EP1163372A4 publication Critical patent/EP1163372A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)

Definitions

  • the present invention relates to plane-rigidized cyanine near-infra red (NIR) fluorescent dyes, their methods of synthesis and use in the labeling of biological molecules
  • NIR near-infra red
  • Cyanine dyes are of increasing interest as near-infra-red absorbing and fluorescing chromophores Cyanine dyes have distinct advantages over most other fluorescent markers widely in use
  • One common problem encountered in biochemistry is the fact that most proteins, lipids and nucleic acids autofiuoresce in the same range as their fluorescent labels, thereby significantly reducing the sensitivity of diagnostic assay applications utilizing these dyes
  • the use of a chromophore emitting in a range with essentially no natural background autofluorescence would greatly increase detection sensitivity of the labeled molecule
  • Two of the most popular fluorescent dyes presently utilized as biomolecular labels are fluorescein and rhodamine Their respective fluorescent emissions are approximately 530 and 618 nm This fluorescent emission overlaps the natural fluorescent emission of most biological molecules Their use is therefore limited by this factor
  • NIR cyanine fluorophores typically occurs in a region with very low background fluorescence, making them useful for labeling of biological moleucles
  • NIR cyanine fluorophores have the additional benefit of absorbing in a region between 700-850 nm, which corresponds closely with the 785 nm wavelengths of the relatively cost-effective and commercially available GaAlAs diode lasers
  • a number of laser applications of these dyes in biotechnology have already been proposed Inclusive of these applications is automated DNA sequencing
  • NIR cyanine dyes in use today are not without their own disadvantages They do have both high extinction coefficients and high quantum yields, both desirable characteristics, however, possess only moderate chemical and photo stabilities
  • Most existing NIR cyanine fluorophores can be described as "non-rigidized" Due to the long inflexible central conjugated cyanine structure, these dyes have a tendency to twist and bend Upon absorption of light, these dyes tend to form radicals, which in turn can react with adjacent molecules resulting in a total loss of fluorophore Such phenomenon is referred to as photo bleaching
  • a further disadvantage is that these NIR fluorophores are for the most part large, bulky and very hydrophobic thus making them generally insoluble in aqueous solutions
  • This dye has incorporated a single 6-membered ring to stabilize the conjugated polyene system, as well as an isocyocyanate functional group for the attachment of this label onto primary amino groups of proteins or functionalized nucleotides, for example
  • this design has incorporated two sulfate groups to increase the water solubility of this otherwise rather insoluble molecule
  • NIR cyanine dyes each having a slightly different emission wavelength to allow for simultaneous tracking of more than one target molecule by providing each target molecule with its own unique dye label
  • the need for a new label for biomolecules can clearly be fulfilled by the creation of a class of rigidized NIR cyanine dyes with these properties and the low background fluorescence described in the foregoing discussion
  • the present invention seeks to improve upon existing NIR plane-rigidized cyanine dyes by providing for dyes with a central cyclic hydroanthraquinone framework inclusive of a tri-cyclic framework as well as frameworks containing 4, 5 and 6 stabilizing 6-membered rings in the conjugated polyene system Emission wavelengths will depend, at least in part, upon the number of stabilizing 6-membered rings within the hydroanthraquinone framework Moreover, various end groups are described for each of the 3, 4, 5 and 6 ring -containing dyes Such end groups flank the central planar network, allowing the formation of a large number of novel plane-rigidized cyanine dyes, each having a different emission wavelength It will then be possible to label different target molecules each with its unique plane-rigidized NIR cyanine dye and to perform a single assay simultaneously for the target molecules with these probes mixed in the same solution, as for example in automated fluorescent- based DNA sequencing with dye-labeled dideoxynucleotide chain terminators
  • the central planar structure that is of the hydroanthraquinone framework by addition of six-membered rings allows for an increase in the Stokes shift, offering a sharper absorption band with greater separation between the wavelengths of absorption and emission and longer wavelengths of absorption and emission, with less overlap with the natural fluorescent emission of most biological molecules The result is a greater signal to noise ratio
  • the dyes of the present invention provide highly stable and rigid structures to ensure low reactivity with neighboring molecules, a sharp absorption band and high quantum yields
  • Groups R b and R c which may be the same or different are conjugated to the central planar network. These groups allow the dye to fluoresce and also serve to alter the wavelength emission of the dye. Therefore, numerous dyes are possible depending both on which groups are placed at positions R b and R c as well as the number of rings present within the central hydroanthraquinone network.
  • a reactive substituent for bonding of the dye to a biological molecule may be placed at any one of positions R a , R b or R c .
  • groups R a , R and R c may contain a linker portion between their attachment to the dye and the reactive substituent in order to reduce steric hindrance between the dye and a biological molecule.
  • a linker When a linker is present, it may have a variety of structures inclusive of a linear or branched, cyclic or heterocyclic, saturated or unsaturated groups, wherein the length of the linker varies from between 1-20 carbons atoms.
  • the linker can also include one or more atoms inclusive of nitrogen, phosphorus, oxygen and sulfur atoms.
  • the linker may contain any combination of ether, thioether, amine, ester or amide bonds.
  • the linker may also contain one or more double or triple bonds and these bonds may include carbon-carbon, phosphorous-oxygen, phosphorous-sulfur, nitrogen-nitrogen, nitrogen-oxygen, as well as aromatic or heteroaromatic bonds.
  • the reactive substituent present on at least one of R , R b or R c for direct bonding of the dye to a biological molecule or indirect bonding of the dye to a biological molecule via a linker can be a number of compounds, classes of compounds or reactive groups.
  • the reactive substituent may also be selected from a number of classes of molecules inclusive of alcohols, aldehydes, thiols, reactive esters, acids, acid halides, sulfonyl halides, hydrazines, ketones, haloacetamides, amides, and azides Moreover, the reactive substituent may be a hydroxyl, amido, carboxylic, or amino group In reference to particular compounds, any one or more of the following may be used phosphoramidite, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, aziridine, hydroxsuccinimide ester, hydroxysulfosuccinimide ester and imido ester
  • the reactive substituent is most likely to form a bond with moieties largely present on biological molecules inclusive of, but not limited to, amino, hydroxy, thiol, carboxyl, aldehyde and ketone groups
  • the terminal amino groups of proteins as well as amino groups on amino acid residues such as lysine would be available for reactivity with a reactive substituent on the dye such as a carboxylic group for formation of an amide bond
  • hydroxyl groups present on sugar residues can react with an OR group on the dye, where R is H, for example
  • a phosphoramidite functionality on the dye can react with a 3' OH group on DNA or an oligonucleotide to form a covalent bond
  • a modified thymine phosphoramidite amino modifier dT
  • R a may be a group containing a reactive substituent for bonding of the dye to a biological molecule, it may also be a non-reactive grouping present on the dye such as H or a substituted or unsubstituted aliphatic or aromatic group
  • the substituted or unsubstituted group may be from the classes inclusive of but not limited to alkyl, alkenyl, alkynyl, aryl and combinations thereof
  • a polar moiety may be placed at one or more positions of R a , R b and R c in order to increase the solubility of the dye in aqueous solutions
  • Polar moieties which may be placed at these positions are inclusive of, but not limited to, hydroxy, nitro, sulfonate, sulfate, carboxylate, substituted amine, quaternary amine and nitrile groups
  • R b and R c which again may be the same or different, can specifically be O, OR, R R 2 and CR 3 , R 4 , wherein R is H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or a mono- or polysaccharide such as a sugar residue
  • R 1 , R 2 and R 4 are independently H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or contain a reactive substituent selected from reactive substituents including, but not limited to a number of classes of molecules inclusive of alcohols, aldehydes, thiols, reactive esters, acids, acid halides, sulfonyl halides, hydrazines, ketones, haloacetamides, amides, and azides
  • the reactive substituent may be a hydroxyl, amido, carboxylic, or amino group
  • any reactive substituent may be a hydroxyl,
  • ringed structures When such ringed structures are present, they may contain a reactive substituent selected from the same groups, classes of compounds or specific compounds aforementioned Moreover, the ringed structures may carry a polar moiety as a substituent to increase the solubility of the dye in aqueous solutions These polar moieties would be inclusive of, but not limited to, hydroxy, nitro, sulfonate, sulfate, carboxylate, substituted amine, quaternary
  • R , R and R 4 may independently contain a polar moiety inclusive of, but not limited to, the same groups
  • the present invention also describes the fluorescent complex which would be formed between a biological molecule which can be bound directly or indirectly to one or more di- molecules of the formula
  • the reactive functional groups present on either the dye or the biological molecule for formation of the complex are inclusive of, but not limited to, amines, amino group, hydroxy group, alcohols, thiols, acids, aldehydes and ketones
  • the biological molecule present in the fluorescent complex may be bound directly to the dyes via any combination of ether, thioether, amine, ester, amide, or a thiourea bonds Wherein the biological molecule is bound indirectly to the dye via a linking group, the biological molecule would be bound to the linker via these same types of bonds.
  • the attachment of the dye to a biological molecule, or for that matter to a linker can include a nitrogen-containing group, a sulfur-containing group or an oxygen-containing group
  • One preferred bond which may be formed between the dye and the biological molecule is from the reaction of an activated ester of N-hydroxysuccinimide and a carboxylic acid
  • the carboxylic acid is envisioned to be present on the biological molecule
  • the terminal carboxyl group of a protein may be conjugated to this activated ester functionality
  • the biological molecule suitable for conjugation with the dye of the present invention may include proteins, nucleic acid polymers, polysaccharides, cells, as well as lipids
  • the biological molecule is a protein
  • the protein may be an antibody Its presence within the fluorescent complex can allow for tracking of the antibody's reactivity with specific antigenic determinates using immuno assays as well as flow cytometric analysis for example
  • the biological molecule is a nucleic acid polymer, such polymers are inclusive of natural or synthetic oligon
  • a method of detecting a target biological molecule is also encompassed by the present invention. Such a method involves combining a sample that contains or is thought to contain a target molecule with a dye of the following formula:
  • the further step of detecting the signal and hence the target molecules can be performed.
  • the reaction conditions under which the target molecule may be directly conjugated with the dye compound may depend specifically on the nature of the biological molecule as well as on the solubility of the dye.
  • standard procedures for labeling biological molecules exist. Among these is the procedure of Motsenbocker, et al.
  • the pH of the labeling buffer should be sufficiently basic to allow the amino group to maintain its reactivity for conjugation with the dye.
  • solubility groups on the dye molecule allow it to remain in solution in such aqueous buffers.
  • the target molecules useful for this method are inclusive of nucleic acid polymers, nucleic acid bases, proteins, lipids, cells, tissue specimens, and polysaccharides
  • the target molecule may be a nucleotide base inclusive of adenine, thimine, cytosene, guanine and other nitrogen heterocyclic bases
  • the target molecule may also be a polysaccharide (sugar), a natural or synthetic oligonucleotide or oligonucleoside, DNA, RNA as well as a chromosome
  • the target molecule is nucleic acid polymer, such as DNA it may be labeled by PCR incorporation of a dye labeled nucleotide base such as adenine, thymine, cytosine, guanine or other nitrogen heterocyclic bases within the DNA molecule
  • DNA or RNA can be labeled at the 3' position by reaction of the 3' OH group with a reactive substituent on the dye such as phosphoram
  • the method just described may include the step of adding one or more reagents to the sample prior to or during the step of combining, where each additional reagent is capable of a response that is detectably different from the dye-target molecule complex
  • This further detection reagent may be an antibody, oligonucleoside or oligonucleotide, for example
  • the aforementioned method may further comprise the step of adding one or more reagents to the sample during the step of incubating where each additional reagent is capable of a response that is detectably different from the dye-target molecule complex
  • the further detection reagent may be inclusive of, but not limited to, antibodies, oligonucleosides, and oligonucleotides
  • the present invention also covers a method of detecting a target molecule wherein a probe is provided which is capable of recognizing or reacting in a specific way with the target molecule of interest
  • the probe is a fluorescent complex formed from a reaction of the probe with the dye molecule of the formula
  • a sample that contains or is thought to contain a target molecule is combined with the probe.
  • the combined sample is thereafter incubated for a time sufficient to allow the target molecule to bind with the probe to form one or more dye-probe-target molecule complexes that give a detectable signal followed by detection of the signal.
  • the target molecule may be qualitatively or quantitatively detected.
  • standards would be required for comparison.
  • the target molecule may again be selected from nucleic acid polymers, nucleic acid bases, proteins, lipids, cells, tissue specimens and polysaccharides.
  • the target molecule may specifically be DNA or RNN for example.
  • the target molecule may also be a protein located within a cell or an antigenic determinant present on a cell.
  • the probe may in this case be an antibody specific for such a protein or antigenic determinant which when bound thereto enables one to detect the molecules of interest.
  • the target molecule is DNN
  • the probe may be an oligonucleotide complimentary in sequence to the DNN as an example.
  • it may be necessary to modify the oligonucleotide probe by incorporation of an amino modified base or a 5' amino linker in order to enable the oligonucloetide to be conjugated to the dye or, alternatively, the 3' OH group on the oligonucleotide can be conjugated to the dye, depdening on the dye's reactive substituent.
  • the present invention provides for a method of characterizing a nucleic acid sample.
  • this method one or more fluorescent complexes are provided wherein the complexes are formed from a reaction of one or more biological molecules with at least one dye compound of the formula:
  • the sample is incubated with the complexes under reaction conditions which would allow for the fluorescent complexes to combine with or incorporate within the nucleic acid to form a staining profile having detectable fluorescent signals.
  • This staining profile would be characteristic of the sample.
  • Biological molecules suitable for combination with the dye compound would include natural, synthetic or modified nucleotides, nucleosides, oligonucleotides, oligonucleosides, DNN R ⁇ A or proteins. Most notably, this method may be applied to the sequencing of D ⁇ A wherein the fluorescent complexes incorporated within the D ⁇ A can be dideoxynucleotide triphosphate chain terminators bound to the dyes.
  • the nucleic acid may be characterized with regard to its sequence when a standard Sanger method of D ⁇ A sequencing is followed. Using PCR amplification techniques, a heat-stable D ⁇ A polymerase is used to make a copy of the D ⁇ A template. By choice of an appropriate primer the region of the nucleic acid that is copied can be predetermined. D ⁇ A synthesis occurs by incorporation of unlabeled deoxynucleotide triphosphates and random termination of the chain occurs when the dideoxynucleotide fluorescent complexes are incorporated.
  • the nucleic acid sample is a solution made up of nucleic acid polymers, in this case DNA fragments, separable by means of relative mobility. Characterization may be with respect to purity of the solution, composition of polymers in solution and composition of components present in solution.
  • the method of characterizing a nucleic acid sample may also involve adding one or more additional reagents to the sample wherein each additional reagent is capable of response that is detectably different from the fluorescent signal of the dye- nucleic acid complex.
  • the additional detection reagent may be an antibody, for example.
  • nucleic acid sample to be characterized can include not only DNA but RNA as well.
  • RNA is to be sequenced using the Sanger method, reverse transcriptase is used to make a DNA copy of an RNA template and the resulting DNA is sequenced by the same methods herein described.
  • one embodiment of the present invention encompasses a chemical complex of the formula:
  • D is of the formula:
  • the dye represented by D in the formula is bound either directly to a nucleotide or nucleoside base, as used in the foregoing DNA sequencing method, or indirectly to a nucleotide or nucleoside base via a linker
  • the linker (L) is a linear, branched, cyclic or heterocyclic, saturated or unsaturated group, each having between 1-20 carbon atoms
  • R-0-M s -B is a natural or modified nucleotide or nucleoside inclusive of DNA chain terminators wherein R can be H, P0 3 2" , P 2 O ⁇ 3" , P 3 0 9 4 ⁇ , alpha-thiophosphates, alpha-BH 3 -phosphates and derivatives thereof
  • O is an oxygen molecule
  • M s is a sugar residue, most notably ribose M s includes, but is not limited to, ribosyl, 2'-deoxyribosyl, 3'- deoxyribosyl,
  • many types of biological molecules may be chemically labeled with one or more of these dyes including, but not limited to, polysaccharides, nucleic acid polymers such as DNA, RNN oligonucleotides and oligonucleosides, nucleic acid bases, proteins, cells and lipids
  • buffers suitable for chemical labeling of the biological molecule may vary
  • the reactive moiety on the dye is an isothiocyanato moiety to form a thiourea linkage between the dye label and a primary amine of the biological molecule
  • a high pH buffer pH 9 2
  • the dye should contain polar moieties for increasing the solubility of the dye in aqueous solutions
  • the dye may be included
  • a DNA sequencing kit which includes a mixture containing dideoxynucleotide chain terminators for use in DNA sequencing methods based on the Sanger method In this mixture each of the 4 dideoxynucleotide chain terminators corresponding to ddATP, ddGTP, ddCTP and ddTTP would each be bound independently to a dye of the formula
  • each of the terminators would be bound to a dye which has an emission wavelength which is different from that of the other terminators in the mixture.
  • Another DNA sequencing kit is provided for by the present invention having additional components. These components include the same mixture just described containing dideoxynucleotide triphosphate chain terminators, wherein the terminators are each independently bound to a dye of the formula:
  • an oligonucleotide primer complimentary in sequence to a region of a vector template suitable for DNA sequencing, or to a region of the nucleic acid to be copied, a vector suitable for DNA sequencing, a heat-stable DNA polymerase such as Taq polymerase, a standard PCR buffer, and unlabeled deoxynucleotide triphosphates are also included.
  • each terminator provided in the mixture has bound to it a dye with an emission wavelength which is different from that of the other terminators in the mixture.
  • a DNA sequencing kit for wherein the components include an oligonucleotide primer bound to a dye of the formula:
  • kits include deoxynucleotide triphosphates as well as dideoxynucleotide triphosphate chain terminators, a PCR buffer, a heat-stable DNA polymerase such as Taq polymerase and a vector suitable for DNA sequencing.
  • the Sanger method of DNA sequencing provides for a display of a continuous set of DNA fragments that differs in length by only one nucleotide and relies on random termination of reactions using dideoxynucleotide triphosphate chain terminators which are, in this kit, unlabeled.
  • Reaction mixtures are set up to contain four deoxynucleotide triphosphates and a single dideoxynucleotide triphosphate.
  • reaction 1 using ddATP, containing all A terminations
  • reaction 2 using ddCTP, contains all C terminations
  • so on After incubation the reaction products are separated by electrophoresis on polyacrylamide gels and detected as a result of the dye bound to the extended primer via excitation of the flurophore and detection of the infrared emission.
  • the dye-bound oligonucleotide primer provided in the kit can be one which is complementary in sequence to a region of a vector suitable for DNA sequencing or to a region of the nucleic acid to be copied.
  • R is methyl or alkyl C ⁇ - 6 and R a optionally contains a reactive functional group for bonding to a biological molecule, x is 0 or 1 and z is 0 or 1; and N is 1-4.
  • the aryl alkali metal compound is lithium-2- lithio-5-methoxybenzoate or lithium 3-lithio-7-methoxy-2-naphthoate.
  • Other alkali metals are also useful.
  • the alkoxy aryl acid halide may be selected from a wide variety of materials. Included among these are ?-methoxybenzoylchloride, 6-methoxy-2-naphthoylchloride, isophthaloyldichloride and naphthalene-2,7-dicarbonyl chloride.
  • the keto-acid formed is desirably one of the following: 2-(-4-methoxybenzoyl)-3- methoxybenzoic acid, 2-(6-methoxy-2-naphthoyl)-3-methoxybenzoic acid, l,3-bw-(2- carboxy-4-methoxybenzoyl)-benzene or 2,7-bt.s-(2-carboxy-4-methoxybenzoyl)-naphthalene.
  • the quinone formed is desirably 2,7-dimethoxyanthraquinone, 2,9-dimethoxy-5,12- naphthacenedione, 2, 10-dimethoxy-6,13-pentacenedione, 2,10-dimethoxy-5,7,12,14- pentacenetetraone or 2,1 l-dimethoxy-5, 8,13, 16-hexacenetetraone.
  • the linear polyarene of step (iv) is desirably 2,7-dimethoxyanthracene, 2,9- dimethoxynaphthacene, 2,10-dimethoxypentacene or 2,11-dimethoxyhexacene.
  • the partially reduced linear polyarene compound is desirably 2,7-dimethoxy- 1,4,5,8,9, 10-hexahydroanthracene, 2,9-dimethoxy-l,4,5,6,7,10,l l,12-octahydronaphthacene, 2, 10-dimethoxy- 1,4,5,6,7,8,11,12,13,14-decahydropentacene, or 2, 11 -dimethoxy- 1,4,5,6,7,8,9, 12, 13, 14, 15, 16-dodecahydrohexacene.
  • the method further comprises the step of attaching the precursor dye to a biomolecule as previously described herein.
  • Birch Reduction Under an inert atmosphere were condensed 750 ml liquid ammonia at - 40°C. To this solution, 4 g lithium metal were added in small pieces. After the lithium was dissolved, 17.5 g of 2, 7-dimethoxy anthracene dissolved in 500 ml dry tetrahydrofuran was added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature was lowered to about -60°C and 75 ml dry ethanol followed by an additional 4 grams of lithium metal were added, being careful to maintain the temperature at -60°C. After stirring three additional hours, 75 gram of ammonium chloride was added to the reaction and the temperature was allowed to reach room temperature, during which time the ammonia evaporated.
  • the residue is purified by recrystallization from benzene-petroleum ether, from chloroform-hexane, or by column chromatography to yield 2-(6-methoxy-2-naphthoyl)-3-methoxybenzoic acid .
  • the solid extract After removal of the methanol, the solid extract is recrystallized from chloroform-hexane or purified using column filtration over alumina with chloroform as eluent to yield 2,9-dimethoxynaphthacene.
  • Birch Reduction Under an inert atmosphere are condensed 800 ml liquid ammonia at -40°C. To this solution, 5 g lithium metal are added in small pieces. After the lithium is dissolved, 17 g of 2,9-dimethoxynaphthacene dissolved in 500 ml dry tetrahydrofuran are added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature is lowered to about -60°C and 80 ml dry ethanol followed by an additional 5 grams of lithium metal are added, being careful to maintain the temperature at -60°C. After stirring three additional hours, 80 grams of ammonium chloride are added to the reaction and the temperature is allowed to reach room temperature, during which time the ammonia evaporates.
  • the alkaline extract is then acidified, extracted with ether, dried over sodium sulfate, filtered and concentrated The residue is purified by recrystallization from benzene-petroleum ether, from chloroform-hexane, or by column chromatography to yield l,3-b/5 , -(2-Carboxy-4-methoxybenzoyl)-benzene.
  • Birch Reduction Under an inert atmosphere are condensed 850 ml liquid ammonia at -40°C. To this solution, 6 g lithium metal are added in small pieces. After the lithium is dissolved, 17 g of 2,9-dimethoxynaphthacene dissolved in 500 ml dry tetrahydrofuran are added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature is lowered to about -60°C and 80 ml dry ethanol followed by an additional 6 grams of lithium metal are added, being careful to maintain the temperature at -60°C. After stirring three additional hours, 85 grams of ammonium chloride are added to the reaction and the temperature is allowed to reach room temperature, during which time the ammonia evaporates.
  • the alkaline extract is then acidified, extracted with ether, dried over sodium sulfate, filtered and concentrated.
  • the residue is purified by recrystallization from benzene-petroleum ether, from chloroform-hexane, or by column chromatography to yield 2,7-bts-(2-Carboxy-4-methoxybenzoyl)-naphthalene.
  • Birch Reduction Under an inert atmosphere are condensed 900 ml liquid ammonia at -40°C. To this solution, 8 g lithium metal are added in small pieces. After the lithium is dissolved, 17 g of 2,1 1-dimethoxyhexacene dissolved in 500 ml dry tetrahydrofuran are added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature is lowered to about -60°C and 90 ml dry ethanol followed by an additional 8 grams of lithium metal are added, being careful to maintain the temperature at -60°C. After stirring three additional hours, 90 grams of ammonium chloride are added to the reaction and the temperature is allowed to reach room temperature, during which time the ammonia evaporates.
  • the ether layers are combined, washed with water, and are extracted with 10% sodium hydroxide solution.
  • the alkaline extract is then acidified, extracted with ether, dried over sodium sulfate, filtered and concentrated.
  • the residue is purified by recrystallization from benzene-petroleum ether, from chloroform-hexane, or by column chromatography.
  • Birch Reduction Under an inert atmosphere are condensed 800 ml liquid ammonia at -40°C. To this solution, 5 g lithium metal are added in small pieces. After the lithium is dissolved, 70 mmol of 2,9-Dimethoxy-l 1-naphthacenecarboxylic acid dissolved in 500 ml dry tetrahydrofuran are added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature is lowered to about -60°C and 80 ml dry ethanol followed by an additional 5 grams of lithium metal are added, being careful to maintain the temperature at - 60°C.
  • the ether layers are combined, washed with water, and are extracted with 10% sodium hydroxide solution.
  • the alkaline extract is then acidified, extracted with ether, dried over sodium sulfate, filtered and concentrated.
  • the residue is purified by recrystallization from benzene-petroleum ether, from chloroform-hexane, or by column chromatography.
  • Birch Reduction Under an inert atmosphere are condensed 900 ml liquid ammonia at -40°C. To this solution, 8 g lithium metal are added in small pieces. After the lithium is dissolved, 70 mmol of 2,l l-Dimethoxy-14-hexacenecarboxylic acid_dissolved in 500 ml dry tetrahydrofuran are added dropwise over one hour. After an additional one hour of stirring at -40°C, the temperature is lowered to about -60°C and 90 ml dry ethanol followed by an additional 8 grams of lithium metal are added, being careful to maintain the temperature at - 60°C.
  • the solvent is removed by vacuum and the adduct is oxidized to the carboxyarene by addition of 15 ml water and 60 mmol bromine at 0°C.
  • the reaction is stirred at room temperature for 3 days.
  • the reaction is carefully poured over ice cold 5% hydrochloric acid and stirred for 30 minutes.
  • the layers are separated and the aqueous layer is extracted with ether.
  • the ether layers are combined, washed with water, and are extracted with 10% sodium hydroxide solution.
  • the alkaline extract is then acidified, extracted with ether, dried over sodium sulfate, filtered and concentrated.
  • the residue is purified by recrystallization from benzene- petroleum ether, from chloroform-hexane, or by column chromatography.
  • R H, alkyl, alkenyl, alkynyl, aryl or substituted versions thereof which may also contain a reactive but separate substituent selected from phosphoramidite, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono-or di-halogen substituted diazine, aziridine, sulfonyl halide, acid halide, glyoxal, reactive ester, aldehyde, alcohol, hydroxyl group, carboxylic group, amine, amide, thiol, amino group, sulfonyl halide, azides, acids, hydrazines, ketones, haloacetamides, hydroxysuccinimide ester, hydroxysulfosuccinimide ester and imido ester for covalent attachment to a biological molecule or which may also contain a polar moiety to increase solubility of
  • a modified thymine phosphoramidite (amino modifier dT) and a 5' amino linker (5' amino modifier C6) is obtained from Glen Research, Inc (Sterling, VA)
  • DNA oligomer is made on a PE Biosystems Nucleic Acid, Synthesizer Model No ABI 3948 synthesizer using standard B-cyanoethyl phosphoramidite chemistry and incorporating either a modified thymine base with a terminal amino linker arm at a position within the sequence or an amino modifier at the 5 terminus of the oligomer EXAMPLE 16
  • oligonucleotide, protein or aminosaccharide is dissolved in PBS buffer, pH 8, to a concentration of 10 nmol in 150 ⁇ l and the solution is vortexed
  • the Dye-Ester is dissolved in 50 ⁇ l amine-free DMF, using a five to 10 molar equivalent excess of dye to oligonucleotide (50 - 100 nmol ester)
  • the DMF/ester solution is added to the oligonucleotide solution and vortexed
  • the reaction is allowed to occur for 12 hours at room temperature Purification of the labeled biomolecule is done with an Amersham Pharmacia Biotech NAP- 10 column.

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  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention concerne de nouveaux colorants fluorescents proches de l'infrarouge (NIR), à base de cyanine et rigidifiés dans un plan, ainsi que des procédés de préparation et d'utilisation de ces derniers dans l'étiquetage non isotopique de molécules biologiques. Ces colorants contiennent une structure hydroanthraquinone centrale et cyclique qui est stable et rigide, et qui assure une faible réactivité par rapport aux molécules voisines, une bande d'absorption précise, un rendement quantique élevé et des émissions dans une région largement dépourvue d'auto-fluoescence de fond naturelle. Ces colorants comprennent en outre des groupes terminaux variables qui flanquent la structure cyclique centrale, permettant ainsi la formation de plusieurs colorants différents et rigidifiés sur un plan qui possèdent chacun une longueur d'onde d'émission légèrement différente des autres. Des groupes fonctionnel sont également prévus pour la fixation à des molécules biologiques cibles. Des groupes fonctionnels hydrophiles sont en outre prévus là où cela est nécessaire afin d'accroître sensiblement la solubilité dans l'eau des molécules de colorant.
EP00919582A 1999-03-24 2000-03-23 Colorants a base de cyanine rigidifies dans un plan et chimiquement reactifs, et derives de ces derniers Withdrawn EP1163372A4 (fr)

Applications Claiming Priority (3)

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US12583999P 1999-03-24 1999-03-24
US125839P 1999-03-24
PCT/US2000/007783 WO2000056933A1 (fr) 1999-03-24 2000-03-23 Colorants a base de cyanine rigidifies dans un plan et chimiquement reactifs, et derives de ces derniers

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EP1163372A1 true EP1163372A1 (fr) 2001-12-19
EP1163372A4 EP1163372A4 (fr) 2002-10-02

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EP (1) EP1163372A4 (fr)
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CA2401487C (fr) 2000-02-29 2011-06-21 Japan Science And Technology Agency Derives de polyacene et leur production
US6331632B1 (en) * 2000-11-09 2001-12-18 Beckman Coulter, Inc. Cyanine dye phosphoramidites
JP2005504811A (ja) 2001-09-27 2005-02-17 スリーエム イノベイティブ プロパティズ カンパニー 置換ペンタセン半導体
US20030097010A1 (en) 2001-09-27 2003-05-22 Vogel Dennis E. Process for preparing pentacene derivatives
WO2005080304A1 (fr) * 2004-02-25 2005-09-01 Asahi Kasei Kabushiki Kaisha Composé de polyacène et couche mince semi-conductrice organique
JP2006335719A (ja) * 2005-06-03 2006-12-14 Asahi Kasei Corp ポリアセン化合物及びその製造方法並びに有機半導体素子
JP7418323B2 (ja) 2017-08-24 2024-01-19 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ 遠赤および近ｉｒ範囲におけるシアニンフルオロフォアの配座の束縛

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US4600775A (en) * 1984-08-27 1986-07-15 Spyros Theodoropulos Isomaleimide and isophthalamide derivatives of chromophors
CA1340806C (fr) * 1986-07-02 1999-11-02 James Merrill Prober Methode, systeme et reactifs pour le sequencage de l'adn
US4783401A (en) * 1986-10-31 1988-11-08 Smithkline Beckman Corporation Viable cell labelling
US5187085A (en) * 1990-09-28 1993-02-16 Applied Biosystems, Inc. Nucleic acid sequence analysis with nucleoside-5'-o-(1-thiotriphosphates)
GB9024775D0 (en) * 1990-11-14 1991-01-02 Axis Research Chemical compounds
DE4326466A1 (de) * 1993-08-06 1995-02-09 Boehringer Mannheim Gmbh Infrarot-Farbstoff-markierte Nucleotide und ihre Verwendung in der Nucleinsäure-Detektion
US5453505A (en) * 1994-06-30 1995-09-26 Biometric Imaging, Inc. N-heteroaromatic ion and iminium ion substituted cyanine dyes for use as fluorescence labels
CA2194150A1 (fr) * 1994-06-30 1996-01-11 Linda G. Lee Colorants a base de cyanine substitues par des ions n-heteroaromatiques et par des ions iminium, utilises comme marques fluorescentes

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EP1163372A4 (fr) 2002-10-02
WO2000056933A1 (fr) 2000-09-28
AU4024500A (en) 2000-10-09

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