JP2002168867A - Rhodamine fluorophor useful as labelling reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescence-generating compound and composition for labelling amino acids, peptide, protein, nucleotide, oligonucleotide or nucleic acids stably without decreasing an efficiency and which can carry out the labelling inexpensively and conveniently. SOLUTION: A fluorescent pigment based on Rhodamine is induced, thereby forming a labelling combined body which generates fluorescence excited by a suitable wavelength light. A particularly preferred embodiment is a single isomer Rhodamine phosphoramidite. A synthesis efficiency for a Rhodamine labelled compound by a solid phase method is promoted by these Rhodamine phosphoramidites. The embodiment of the combined body includes a fully substituted amide bond induced from a 3-position carboxylic acid, and therefore prevents transformation to non-fluorescent lactam.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates generally to a method for labeling an organic compound for fluorescence detection. More particularly, the present invention relates to rhodamine-based fluorophores, which are useful for guidance by organic molecules, and may be applied to label labeled biological molecules such as synthetic oligonucleotides and proteins. Fluorophores are single isomers, are stable, reactive with standard phosphite chemistry, and leave ligation upon coupling.

[0002]

BACKGROUND OF THE INVENTION The use of fluorescent dyes as detection labels has found wide application in molecular biology, cell biology and molecular genetics. In particular, the use of fluorescently labeled oligonucleotides allows DNA sequencing, fluorescent in situ hybridization (FISH), nucleic acid arrays
("DNA chips"), including hybridization assays, probe capture assays, fluorescence polarity studies, and DNA amplification assays: polymerase chain reaction (PCR),
Isothermal amplification assay
(Strand displacement amplification (S
DA)), nucleic acid sequence-based amplification (nucleic acid sequence
based amplification) (NASBA), self-sustained sequence replication (3SR),
And fluorescent primer and / or probe detection
Progress has been seen in those involving the "Taqman" assay.

[0003]

Modern automated DNA sequencing methods use multiple fluorescent labels for simultaneous detection of base sequence per single gel lane or capillary.
The most commonly used fluorescent dyes for sequencing are synthesized as a mixture of isomers, including the rhodamine family. (As for Rhodamine dye, Association
Colou by Textile Chemists, 2nd Edition, 1971
Use the numbering scheme described in rIndex. 2.) Single isomer dye labels are preferred for high resolution techniques such as DNA sequencing and capillary electrophoresis. This is because the spectral properties present between the various isomers of the fluorophore are slightly different. In addition, due to differences in electrophoretic mobilities of 5- and 6-isomeric fluorophore-tagged primers (eg, 5- and 6-carboxytetramethylrhodamine), when using a mixture of isomers,
Bands can be broad (Hung et al., Analytical
Biochem, 238, 165-170, 1996). Therefore, the fluorochrome labeling reagent used for oligonucleotide labeling must be purified to a single isomer before being prepared for DNA sequencing.

[0004] Some fluorescent dye labels can be attached to the 5 'end of the oligonucleotide during primer synthesis.
(Eg, fluorescein using fluorescein phosphoramidite reagent). These dye phosphoramidites react under the conditions of appropriate phosphite chemistry. This is because protection of the two active oxygen groups of the fluorescein moiety can prevent possible side reactions between phosphoramidite and fluorescein. In addition, modification with a protecting group preserves the 3-carboxylic acid functionality in the ring-closed lactone form and prevents proton donation from the carboxylic acid to N, N-diisopropylamino phosphoramidite. Upon protonation, the diisopropylamino moiety is converted to a good leaving group and the reagent is decomposed. Some synthesized rhodamine phosphoramidites (e.g.,
US Patent 5,231,19 issued June 27, 1993
1, Inventor Woo et al.) Conclude that ring-closed (lactone) forms and ring-opened
It has a 3-carboxylic acid functional group for which an equilibrium exists between the (acid) form. When the reagent is used for oligonucleotide synthesis, "acid" conditions favor the formation of the carboxylic acid onium cation form. Proton transfer occurs from the carboxylic acid moiety to N, N-diisopropylamino phosphoramidite, making the reagent unstable and reducing the efficiency of oligonucleotide labeling.

[0005] Some fluorescent dye labels (eg, fluorescein and related derivatives) cleave labeled oligonucleotides from solid supports and remove protecting groups with concentrated aqueous ammonia (a standard method in recent practice). The fluorescein nature is maintained during the process. However, rhodamine family dyes are susceptible to chemical modification by ammonia modification which strongly reduces fluorescence. Therefore, after automated synthesis, cleavage and deprotection, the linker function (funct
Conjugation to the 5 'end of oligonucleotides modified with ionalities (eg, primary amines) is a common practice for rhodamine-type dyes. This dye labeling requires additional steps and manual work, resulting in significant costs and inconvenience throughout the synthesis of 5'-rhodamine dye labeled oligonucleotides.

[0006]

SUMMARY OF THE INVENTION In one aspect of the present invention, a fluorogenic compound and composition are provided based on rhodamine. Rhodamine has a 3-carboxylic acid. The fluorogenic compounds, or fluorophores, of the present invention have the 3-position carboxylic acid converted to a fully substituted amide. One substituent of the amide nitrogen is a group that effectively prevents lactam ring formation. Other substituents on the amide nitrogen are useful for making or containing the desired derivative. Useful derivatives of these fluorophores can be made for labeling organic compounds for fluorescence detection. Preferred organic compounds are biomolecules such as peptides, proteins, amino acids, nucleotides, oligonucleotides, or nucleic acid polymers. Such conjugation can occur via a phosphoramidite bond, preferably during the synthesis of the labeled oligonucleotide, and by various known protein conjugation chemistries during the synthesis or labeling of the labeled peptide.

Formula A below shows a fluorophore moiety based on the rhodamine of the present invention, wherein Ra and Ra 'are both non-hydrogen substituents on the amide nitrogen.

Embedded image

In the structure of formula A, R ₁ and R ₁₀ are independently hydrogen or halogen, and R ₂ , R ₅ , R ₆ and R ₉ are independently halogen, alkyl, carboxyalkyl, aminoalkyl , An alkyl ether, an alkyl thioether, a halogen or an alkoxy, R ₃ ,
R ₄ , R ₇ and R ₈ independently represent a halogen, an alkyl,
Carboxyalkyl, aminoalkyl, cycloalkyl, aryl, or alkyl, cycloalkyl or aryl substituted to have an additional functional group that binds, including but not limited to, alkoxy, sulfate, phosphate or nitrate. R ₂ and R ₃ are together an alkyl chain having 2 to 5 carbon atoms connecting the 2 ′ carbon atom to the nitrogen atom bonded to the 3 ′ carbon, and R ₉ and R ₈ are together, 6 2 to linking the 'nitrogen atom to 7 attached to a carbon atom' carbon atom is an alkyl chain having 5 carbon atoms each, R ₄
And R ₅ together are alkyl having 2-5 carbon atoms linking the 4 ′ carbon atom to the nitrogen atom bonded to the 3 ′ carbon atom, and R ₆ and R ₇ together , An alkyl having 2 to 5 carbon atoms each linking a 5 ′ carbon atom to a nitrogen atom bonded to a 6 ′ carbon atom, and R ₃ and R ₄ together form a carbon atom and a nitrogen atom or an oxygen atom Forming an alkyl or alkylene chain comprising up to 5 atoms in the main chain consisting of one or more heteroatoms from and having both ends of the main chain bonded to the nitrogen atom bonded to the 3 ′ carbon And R ₇ and R ₈ together form an alkyl or alkylene chain consisting of one or more heteroatoms from carbon and nitrogen or oxygen atoms and containing up to 5 atoms in the main chain And this Both terminal bonds of the main chain are bonded to the nitrogen atom bonded to the 6 ′ carbon, and R ₁₁ , R ₁₂ , R ₁₃ and R ₁₄ are each hydrogen or halogen.

One function of the _Ra substituent is to prevent the formation of a lactam ring, so that alkyl,
It can be selected from a variety of substituents such as carboxyalkyl, aminoalkyl, cycloalkyl, aryl or arylallyl. The size of the substituents that prevent the group from forming a lactam ring can vary considerably.

[0010] connection of the desired bound material forms via a R _{a 'substituent} or R _a' substituent derivatives. Typically, R _{a ′} can be selected which is alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, arylalkyl, but preferably, R _{a ′} is such that it comprises a further derivatizing chemically reactive functional group. Selected. Suitable functional groups include amines, alcohols, halogens, carboxylic acids, hydrazines,
Mercapto, sulfuric acid, phosphoric acid or nitric acid. R _{a 'substituent} definitive in simplest having chemically reactive functional groups are hydroxyl may be -CH ₂ CH ₂ OH which may be used in the preparation of the desired various derivatives or conjugates.

Since the compound of the present invention preferably has a functional group linked via a carboxyl group at the 3-position, the bond converts the 3-carboxylic acid to a non-acidic functional group (eg, amide). Derivatives such as phosphoramidites are more stable. Due to the advantage of performing chemistry via the 3-position carboxyl group, the dyes and labeled derivatives of the present invention are single isomers and require purification from a mixture of 5- and 6-position carboxy rhodamine before preparing the oligonucleotide labeling reagent. Is different from the above compound.

Because the compounds of the present invention have a fully substituted amide nitrogen atom, the dye prevents conversion to a non-fluorescent lactam form. The phosphoramidites and derivatized solid phase support matrix reagents of the present invention allow the efficient and fully automated synthesis of rhodamine-labeled oligonucleotides.

[0013]

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention is to provide a dye based on rhodamine that binds to organic molecules, more preferably biomolecules. Compounds previously described in the chemistry or biochemistry literature have functional groups that link to biomolecules such as oligonucleotides and proteins via the 3-position carboxyl group of rhodamine. Is thinking. Rhodamine-based dyes are more easily synthesized as single isomeric derivatives, which are important in labeling oligonucleotides for DNA sequencing. As a control, the conventionally used carboxy rhodamine requires the purification of the mixed isomer to a single isomer before the labeled derivative can be synthesized. Furthermore, the compounds based on rhodamines of the present invention are 3-
It has an amide bond derived from the carboxylic acid at the position and converts the carboxylate group to a non-acidic functional group. This improves the stability of the induced phosphoramidite. A fully substituted amide nitrogen atom prevents the dye from converting to a non-fluorescent lactam form.

FIGS. 1 and 2 show six embodiments of the present invention.
The use of a label for (compound 4-9) is illustrated and its preparation is described in Examples 4-10 below.

Briefly describing FIG. 1, rhodamine-labeled oligonucleotides were prepared using standard conditions for gel purification of oligonucleotides (Sambrook et al., Molecular Cl.
^{oning: A Laboratory Manual, 2 nd} Edition, Cold Sprin
g Harbor Laboratory Press, 1989) using 19% polyacrylamide, 10 M urea, 89 mM Tris-boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA) (T
Electrophoresis was performed on a (BE buffer) gel. The gel is irradiated in an ultraviolet light box, and a NucleoVision CCD camera system (NucleoT
(ech, Inc., Foster City, CA). Lane A contained a poly-dT9mer labeled at the 5 ′ end during synthesis using the rhodamine-based embodiment for which compound 5 was designed. Lane B contained a poly-dT10mer that used the rhodamine-based embodiment compound 7 and was labeled at the 3 ′ end during synthesis.
Lane C contained poly-dT11mer using compound 4 and labeled at the 5 ′ end during synthesis. Lane D
Contained poly-dT11mer, which used compound 6 and labeled the 5 ′ end during synthesis.

Briefly explaining FIG. 2, bovine serum albumin protein is ligated with the indicated labeling reagent and subjected to standard conditions (Sambrook et al., Molecular Cloning: A Laborator).
^{y Manual, 2 nd Edition, Cold} Spring Harbor Laborator
y Press, 1989), sodium dodecyl sulfate (SDS) -polyacrylamide gel (5% stacking gel, 25 m
M Tris, 250 mM glycine, 15% polyacrylamide with 0.1% SDS buffer). The gel is irradiated in an ultraviolet light box and a NucleoVision CCD equipped with a rhodamine filter
Camera system (NucleoTech, Inc., Foster City, CA)
Was used to obtain a fluorescent image. Lane A shows bovine serum albumin bound to compound 8. Lane B shows bovine serum albumin bound to compound 9.

As shown in FIG. 1, the 3′-end labeled oligonucleotide is automatically synthesized by the dye-derivatized solid support matrix of the present invention, and the 5′-end is synthesized by the dye phosphoramidite during the synthesis of the oligonucleotide. Is labeled. Further, the invention can be practiced such that a dye-labeled dU residue is added at any point within the sequence during automated oligo synthesis. The fluorescent excitation and emission properties of rhodamine used in the practice of the present invention are based on commercially available automated fluorescent DNA with improved spectral separation from commonly used fluorescein derivatives.
Similar to rhodamine used in sequencing and fluorescent assay detection instruments. The derivatives and conjugates of the present invention retain their fluorescence ability upon excitation with light of a determined wavelength. Typically, the derivatives and conjugates of the invention have a wavelength of 500
To 700 nm, and the fluorescence has a wavelength of 520 nm.
750 nm.

As shown in FIG. 2, proteins can be labeled with the rhodamine-based dyes described herein. The amino acid and peptide bonds of the present invention can be made using protein binding reagents known to those of skill in the art.
In addition, particles such as cells and viruses can be labeled using these protein binding reagents. This is because the link is formed with a protein on the cell surface membrane or the viral coat.

Derivatives and conjugates based on rhodamine, which are the subject of the present invention, are based on the general structure represented by the following formula A:

Embedded image

In the structure of formula A, R ₁ and R ₁₀ are independently hydrogen or halogen, and R ₂ , R ₅ , R ₆ and R ₉ are independently halogen, alkyl, carboxyalkyl, aminoalkyl , An alkyl ether, an alkyl thioether, a halogen or an alkoxy, R ₃ ,
R ₄ , R ₇ and R ₈ independently represent a halogen, an alkyl,
Carboxyalkyl, aminoalkyl, cycloalkyl, aryl, or alkyl, cycloalkyl or aryl substituted to have an additional functional group that binds, including but not limited to, alkoxy, sulfate, phosphate or nitrate. R ₂ and R ₃ are together an alkyl chain having 2 to 5 carbon atoms connecting the 2 ′ carbon atom to the nitrogen atom bonded to the 3 ′ carbon, and R ₉ and R ₈ are together, 6 2 to linking the 'nitrogen atom to 7 attached to a carbon atom' carbon atom is an alkyl chain having 5 carbon atoms each, R ₄
And R ₅ together are alkyl having 2-5 carbon atoms linking the 4 ′ carbon atom to the nitrogen atom bonded to the 3 ′ carbon atom, and R ₆ and R ₇ together , An alkyl having 2 to 5 carbon atoms each linking a 5 ′ carbon atom to a nitrogen atom bonded to a 6 ′ carbon atom, and R ₃ and R ₄ together form a carbon atom and a nitrogen atom or an oxygen atom Forming an alkyl or alkylene chain comprising up to 5 atoms in the main chain consisting of one or more heteroatoms from and having both ends of the main chain bonded to the nitrogen atom bonded to the 3 ′ carbon And R ₇ and R ₈ together form an alkyl or alkylene chain consisting of one or more heteroatoms from carbon and nitrogen or oxygen atoms and containing up to 5 atoms in the main chain And this Both terminal bonds of the main chain are bonded to the nitrogen atom bonded to the 6 ′ carbon, and R ₁₁ , R ₁₂ , R ₁₃ and R ₁₄ are each hydrogen or halogen.

To illustrate the amide nitrogen atom substituent of the structure of Formula A, R _a is alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, arylalkyl;
R _{a '} contains chemically reactive functional groups, which include amines, alcohols, halogens, carboxylic acids, hydrazines,
These include, but are not limited to, mercapto, sulfuric acid, phosphoric acid or nitric acid. The size of _Ra can vary considerably, but preferably, _Ra has one carbon atom. To be fully described and described below, and to select from the chemistry of derivatives and conjugates known to those of skill in the art,
A chemically reactive functional group contained in R _{a ′} is used. Suitable functional groups are amines, alcohols, halogens, carboxylic acids, hydrazine, mercapto, sulfuric acid, phosphoric acid or nitric acid. Typically, the chemically reactive functional group of R _{a '} is an organic molecule,
It is preferably selected to react with a reactive site of a biomolecule, or a molecule that binds to a solid support material, such as a conditioned pore glass or polystyrene resin.

When the intended biomolecule of the conjugate is a nucleotide, oligonucleotide or nucleic acid, the chemically reactive functional group contained in R _{a '} is preferably a phosphoramidite. Upon reacting with the hydroxyl function, the phosphoramidite forms a phosphite and then
Oxidizes to form phosphate esters. Phosphoramidite means a moiety having the structure of Formula B.

Embedded image

In formula B, L ₁ is cyanoethyl, alkyl, alkenyl, aryl, arylalkyl, or cycloalkyl, and L ₂ and L ₃ independently represent alkyl, arylalkyl, cycloalkyl, and cycloalkylaryl, respectively. In the formula, L ₁ and L ₂ are formed integrally with an alkylene chain containing up to 5 carbon atoms in the main chain and up to a total of 10 carbon atoms, and both ends of the main chain are bonded to a nitrogen atom. Combine or L ₂ and L ₃
Forms, together with the nitrogen atom to which they are attached, a saturated nitrogen atom heterocycle containing one or more heteroatoms from a group consisting of a nitrogen, oxygen or sulfur atom.

Therefore, the oligonucleotide is labeled by reacting a fluorophore having a phosphoramidite group with the 5 'hydroxyl of the oligonucleotide. Rhodamine 3 in the preparation of the fluorophore of formula A
Conversion to a non-acidic group (ie, an amide) by the -position carboxylate makes the phosphoramidite derivative more stable.

The prior art rhodamine phosphoramidites are prepared by reacting the amino alcohol (eg, ethanolamine, hexanolamine, etc.) and rhodamine in N, N-dimethylformamide (DMF) or similar aprotic polar solvent at room temperature. By reacting-or 6-N-hydroxysuccinimidyl ester, a rhodamine dye 5- or 6-alcoholamide can be synthesized,
It is then separated from the reaction mixture by standard methods. Then, the alcohol amide of the rhodamine dye was added to excess di-
React with (N, N-diisopropylamino) cyanoethylphosphine at room temperature in acetonitrile containing catalytic amounts of tetrazole and diisopropylamine to form rhodamine phosphoramidite and separate from the reaction mixture. As a control, rhodamine phosphoramidites of the invention are made by reacting the Formula A embodiment at a functional group linked through the 3-position carbon. For example, most simply, when an N-methylamino alcohol derivative of rhodamine is used, tetrazole-activated bis (N, N-diisopropylamino) β- in dry dichloromethane or acetonitrile at room temperature in a dry, inert atmosphere.
Cyanoethyl phosphite (BIS) can be reacted with hydroxyl. The resulting rhodamine phosphoramidite embodiment is produced from a reaction mixture for oligonucleotide synthesis.

The rhodamine phosphoramidites of the present invention are also useful for preparing novel rhodamine-inducible solid phase supports. Rhodamine-derivatized solid phase supports automatically synthesize 3 'rhodamine-labeled oligonucleotides.

As an example, the rhodamine-derivatized solid phase support of the present invention can be used to convert an N-methyl carboxylic acid derivative of rhodamine to a base labile ester bond, a branch point with an indicator (DMT or MMT) group. , And by reaction with terminal amine groups in dimethylformamide or other aprotic organic solvents. The rhodamine-derivatized solid phase support is filtered, washed and dried. The dye derivative component can be determined by a trityl cation assay. Suitable supports include:
Includes modified pore glass and polystyrene resins having functional groups capable of binding rhodamine-based dyes.

Detailed methods for solid phase synthesis by phosphite triester, phosphotriester and H-phosphonic acid chemistry are available (eg, US Pat. Nos. 4,401,796, 4,415,732 and 4,6
68,777; Oligonucleotide Synthesis: A Practica
l Approach, IRL Press, Washington, DC, 1984). Preferably, the practice of the present invention involves the synthesis of rhodamine-labeled oligonucleotides by the phosphite triester approach. Oligonucleotide synthesis is initiated by a nucleoside-derived solid support and is successfully added to the nucleotides of the growing strand by reacting the hydroxyls of the growing strand with nucleoside phosphoramidites. In particular, the oligonucleotide is labeled at the 5 'end by reacting a rhodamine phosphoramidite of the invention with the 5' hydroxyl of the binding oligonucleotide, or the oligonucleotide is a rhodamine derivatized solid support of the invention. Is reacted at the 3 'end by reacting a nucleoside phosphoramidite with the hydroxyl.

Usually, the cleavage of the rhodamine-labeled oligonucleotide from the solid phase support and the removal of the protecting group bound to the exocyclic amine are carried out at 4 to 55 ° C. for 15 minutes to 18 hours at a cleavage / deprotection reagent (0.1 N To 1.0 N sodium hydroxide), the rhodamine-labeled oligonucleotide bound to the solid phase support is preferably treated at 55 ° C. for 2 hours or Treated with 0.1 N sodium hydroxide at room temperature for 12-18 hours.
After cleavage and deprotection, the labeled or unlabeled oligonucleotide is purified by standard methods (Oligonucleotide Synth
thesis: A Practical Approach, IRL Press, Washingto
n, DC, 1984).

When R _{a '} contains a phosphoramidite group, the other substituents are alkyl, alkyl amides, alkyl ethers, polyethers or polyamides of up to 20 atoms, which are chemically inert chains [formula Wherein the atom is a carbon atom and is one or more heteroatoms from a group comprising a nitrogen atom or an oxygen atom and comprising a linking functionality capable of chemically linking the dye and the phosphoramidite moiety ], Preferably the inactive chain is 5-10
A chain containing atoms [the atoms are carbon atoms and are one or more heteroatoms from a group consisting of a nitrogen or oxygen atom]. Phosphoramidite itself,
Includes molecular structure with indicator protecting group (DMT or MMT) attached by reaction to hydroxyl function
[Wherein, DMT is a dimethoxytrityl group, and MMT
Is a monomethoxytrityl group].

For formula A, a number of functional groups other than phosphoramidites can be selected to be included in the R _{a '} substituent. This is because the selection depends on the biomolecule to be bound. Generally, the linkages of the present invention include Formula A fluorophores that bind to a biomolecule via an amide, ester, ether or thioether linkage.

Thus, if the biomolecule to be conjugated is, or contains, an amino acid, a peptide, or a protein, preferred derivatives of formula A fluorophores include functional groups that react with primary amines or mercapto groups. . As will be readily appreciated by those skilled in the art of protein conjugation, a variety of protein-linked derivatives are used for targeted amino acid reactions. Many protein binding reagents and techniques are available, for example, from Hermanson in Bioconjugate Tech.
niques, Academic Press, San Diego, California 1996
It is described in. Furthermore, because cells and viruses contain proteins on their surface membranes or virus coatings,
Cells or virus particles can be labeled by practicing the present invention. An example of a known protein binding reagent, ie, _a suitable functional group contained in the R _{a '} substituent, is a succinimidyl ester, which readily reacts with primary amines.

In summary, the fluorescent conjugate according to the present invention can be prepared to have the structure shown by Formula 1.

Embedded image [Wherein Z comprises a binding substance that is a peptide, protein, amino acid, nucleotide, oligonucleotide or nucleic acid]. That is, in Formula 1, the fluorophore is
The R _{a '} derivative reacts to include the binding substance, which can be as conventionally described by Formula A.

The following examples serve as illustrations and do not limit the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Reagent concentrations, temperatures, and various other parameter values are only illustrative of the invention and are not intended to be limiting. Synthetic methods and variants of reactants
It is within the scope of the present invention and may be considered an object of the present invention.

Table 1 shows the structures of the compounds prepared in Examples 1-9. Table 1.

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

[0036]

EXAMPLE 1 Tetramethylrhodamine N-methylethanolamine amide (Compound 1) Using N, N-dimethylaminophenol and phthalic anhydride as starting materials, according to Hung et al. (1996), tetramethylrhodamine Was synthesized.

Under a stream of nitrogen atoms, 2.00 g (4.73 mmol) of tetramethylrhodamine was added to 100 ml of dry dichloromethane.
After that, 2.3 g (5.20 mmol) of benzotriazol-1-yl-oxytris (dimethylamino) phosphonium hexafluorophosphate (BOP) and 0.76 ml (9.46 mmol) of N-methylethanolamine were added. The reaction mixture was stirred at room temperature and monitored by thin phase chromatography (TLC) using Kieselgel 60 F254 plates in 2: 1: 1 chloroform: methanol: acetone containing 2% (v / v) triethylamine. Subsequently, the reaction mixture was extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated to dryness. The product was purified by chromatography on silica gel with 2: 1: 1 chloroform: methanol: acetone containing 2% (v / v) triethylamine. Compound 1 was analyzed by MALDI-TOF mass spectrometry
Analyzed by V / visible spectrometry and analytical TLC. Absorption (excitation) maximum in methanol: 548 nm;
Fluorescence emission maximum: 543 nm.

Example 2 Preparation of Rhodamine B N-methylethanolamine amide (Compound 2) Rhodamine B (Aldrich Chemical Comp.
Any, Milwaukee, WI) was suspended in 25 ml of dry dichloromethane, and 0.34 ml (4.18 mmol) of N-methylethanolamine was added. The reaction mixture was stirred at room temperature and 2: 1: 1 chloroform: methanol:
Monitored by TLC using Kieselgel60 F254 plates in acetone. Subsequently, the reaction mixture was extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated to dryness. The product was purified by chromatography on silica gel with 2: 1: 1 chloroform: methanol: acetone. Compound 2 was analyzed by MALDI-TOF mass spectrometry with U
Analyzed by V / visible spectrometry and analytical TLC. Absorption (excitation) maximum in methanol: 560 nm;
Fluorescence emission maximum: 579 nm.

Example 3 Preparation of Rhodamine 101 N-methylethanolamine amide (Compound 3) Rhodamine 101 (Acros Organic, Pi
ttsburgh, PA) was suspended in 50 ml of dry dichloromethane, and then 0.49 g of BOP (1.11 mmol) was added.
2 mmol) and 0.162 ml of N-methylethanolamine
(2.02 mmol) was added. The reaction mixture is stirred at room temperature and 2:
Kies in 1: 1 chloroform: methanol: acetone
Monitored by TLC using elgel60 F254 plates. Successively, the reaction mixture was extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated to dryness. The product was used without further purification. Compound 3 was analyzed by MALDI-TOF mass spectrometry by UV / visible spectrometry and analytical TLC. In methanol, maximum absorption (excitation): 582 nm; maximum fluorescence emission: 602 nm.

Example 4 Preparation of tetramethylrhodamine phosphoramidite (compound 4) Preparation of activated bis (N, N-diisopropylamino) β-cyanoethyl phosphite (BIS) reagent. Tetrazole activated BIS was prepared by placing 8.5 mg (0.12 mmol) of tetrazole in a drying bottle and sealing with a rubber membrane. Dry acetonitrile (2.5 ml) was added by injecting through the membrane lid. BIS 0.0 was added to the resulting solution.
85 ml (0.24 mmol) were injected and then left at room temperature for 30 minutes.

Preparation of compound 4. In a stream of dry nitrogen, 1.0 ml of dry acetonitrile was added to 0.10 mg (0.02 mmol) of dry compound 1 in a bottle covered with a membrane. Activated BIS (0.17m
1, 0.017 mmol) was injected as a small aliquot through the membrane lid. The solution was left at room temperature for 15 minutes and a sample was removed for analysis by TLC (Kieselgel60 F254 in 2: 1: 1 chloroform: methanol: acetone). Addition of activated BIS and analysis of reaction sample
This was continued every 5 minutes until the reaction was judged complete. The resulting solution was used directly for the synthesis of the labeled oligonucleotide.

Example 5 Preparation of Rhodamine B phosphoramidite (Compound 5) Under a nitrogen atom stream, 1.20 g (2.24 mmol) of dry compound 2 was combined with 86.2 mg (1.23 mmol) of tetrazole and dissolved in 25 ml of dry dichloromethane. BIS (0.74 ml, 2.46 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. TLC
Sample analysis by (Kieselgel60 F254 in 10% methanolic chloroform containing 2% triethylamine) confirmed that the reaction was complete. The reaction was quenched and successively extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product is purified by chromatography on silica gel, 50:50 with 2% triethylamine.
Elution with dichloromethane: acetone. Purified compound 5
Was stored lyophilized until used for the synthesis of labeled oligonucleotides.

Example 6 Preparation of Rhodamine 101 Phosphoramidite (Compound 5) Under a nitrogen gas stream, 0.5 g (0.91 mmol) of the dry compound was combined with 35.2 mg (0.51 mmol) of tetrazole and dissolved in 20 ml of dry acetonitrile. BIS (0.30 ml, 1.01 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. TLC
Sample analysis by (Kieselgel60 F254 in 10% methanolic chloroform containing 2% triethylamine) confirmed that the reaction was complete. The reaction was quenched, extracted twice with an equal volume of petroleum ether and evaporated to dryness. The product is dissolved in chloroform containing 2% triethylamine and successively
(w / v) Extracted with sodium bicarbonate, then with saturated sodium chloride. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by chromatography on silica gel and eluted with 50:50 dichloromethane: acetone containing 2% triethylamine. Purified compound 6 was stored lyophilized until used for the synthesis of labeled oligonucleotides.

Example 7 Tetramethylrhodamine derivatized controlled pore glass (CPG)
Preparation of (Compound 7) Preparation of tetramethylrhodamine N-methylbutyryl ester. In a nitrogen stream, 663 mg of BOP were added to a mixture of 424 mg (1 mmol) of tetramethylrhodamine, 12 mg of dimethylaminopyridine and 251 mg of N-methylbutyryl-methylester-HCl in 20 ml of dimethylformamide. The solution was adjusted to pH 8.5 using diisopropylethylamine.
The reaction mixture was stirred at room temperature and monitored to completion using TLC (Kieselgel60 F254 in 15% methanolic chloroform). The reaction mixture was evaporated to a syrup and resuspended in 100 ml of dichloromethane. The solution was successively extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride.
The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The product was purified by silica gel chromatography using 7% methanolic chloroform to obtain 250 mg of a purified compound. Tetramethylrhodamine N-methylbutyryl ester was analyzed by MALDI-TOF mass spectrometry by UV / visible spectrometry and analytical TLC. Absorption (excitation) maximum value in methanol: 54
8 nm; fluorescence emission maximum: 572 nm.

Preparation of tetramethylrhodamine N-methylbutyrate Tetramethylrhodamine N-methylbutyryl ester (250 mg) was dissolved in 20 ml of methanol, and a 1N sodium hydroxide solution was added. The reaction mixture was stirred at room temperature and TLC (Kiese in 15% methanolic chloroform).
lgel60 F254). The reaction mixture was evaporated to an oil and dissolved in 200 ml of water. The aqueous phase was washed twice with an equal volume of dichloromethane. Aqueous layer with 6N hydrochloric acid
Adjusted to H3-4 and extracted with an equal volume of dichloromethane. Collect the organic layer, dry over anhydrous sodium sulfate,
Evaporated to dryness. The yield of the product is 254 mg
Met. N-methylbutyrate tetramethylrhodamine,
Used without further purification.

Preparation of tetramethylrhodamine N-methylbutyryl-CPG (compound 7). F-moc-3'-amino-modifier-CPG (Peninsula Laboratories, In
c., San Carlos, CA) with 20% piperidine-dichloromethane. The CPG was stirred at room temperature for 30 minutes, filtered and washed successively with acetone, methanol and ether. After drying under vacuum for 3 hours, CPG was washed with 250 mg of N-methylbutyrate tetramethylrhodamine, 0.5 ml of triethylamine, 0.39 N, N'-diisopropylcarbodiimide.
ml and a mixture of 20 ml of dichloromethane. The reaction mixture was stirred overnight. The CPG was filtered and washed as described above. The dye derivative component (“loading”) of the CPG solid support was determined to be 30-40 μmol / g by a trityl cation assay.

Example 8 Preparation of tetramethylrhodamine-((N-hydroxysuccinimidyl) (4-N-methylamino) (butanolate) amide (Compound 8) Tetramethylrhodamine- (methyl (4-N-methyl amino)
Preparation of (butanolate) amide. In a dry nitrogen stream, 1.0 g (2.58 mmol) of tetramethylrhodamine and 1.26 g of BOP (2.6 g
3 mmol), 4- (N-methylamino) butyric acid methyl hydrochloride 0.52 (3.12 mmol), triethylamine 0.87 ml (6.24 mmol)
And 100 ml of dry dichloromethane. The reaction was stirred overnight at room temperature. 2: 1: 1 dichloroform:
Sample analysis by TLC in methanol: acetone confirmed that the reaction was complete. The reaction mixture was successively extracted with an equal volume of 5% (w / v) sodium bicarbonate and then with saturated sodium chloride. The organic layer was collected, dried over anhydrous sodium sulfate and evaporated to dryness. The product was used without further purification.

Preparation of tetramethylrhodamine-((4-N-methylamino) butyric acid) amide. Compound tetramethylrhodamine- (methyl (4-N-methylamino)
1.6 g (3.19 mmol) of (butyric acid) amide were dissolved in 30 ml of methanol. Add 7.5 ml of 1N sodium hydroxide,
The reaction was stirred overnight at room temperature. Completion of the reaction was confirmed by TLC sample analysis in 2: 1: 1 chloroform: methanol: acetone. The reaction mixture was evaporated to dryness and diluted with 50 ml of water. The resulting mixture was extracted twice with an equal volume of dichloromethane. The aqueous phase was collected, acidified with 6N hydrochloric acid and extracted three times with an equal volume of dichloromethane. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated to dryness. The product was used without further purification.

Preparation of tetramethylrhodamine-((N-hydroxysuccinimidyl) (4-N-methylamino) butyric acid) amide (compound 8). In a stream of dry nitrogen, the compound tetramethylrhodamine-((4-N-methylamino) butyric acid) amide 0.2
3 g (0.46 mmol) was added to disuccinimidyl carbonate 0.16 g (0.69 mm
ol), 0.07 ml (0.923 mmol) of dry pyridine and 20 ml of dry dichloromethane. The reaction was stirred overnight at room temperature. Completion of the reaction was confirmed by TLC sample analysis in 2: 1: 1 chloroform: methanol: acetone. The reaction mixture is evaporated to dryness, dissolved in a minimum amount of dichloromethane and purified by chromatography on a silica column 50 ml, 2:
Elution was performed with 1: 1 chloroform: methanol: acetone. In methanol, maximum absorption (excitation): 559 nm; maximum fluorescence emission: 578 nm.

Example 9 Preparation of rhodamine B- (N-methylethanolamide) -succinate. 0.100 g (0.21 mmol) of compound 2 (rhodamine BN-methylethanolamine amide) was combined with 0.023 g (0.23 mmol) of succinic anhydride, 5.0 mg (0.04 mmol) of dimethylaminopyridine and 5 ml of dry acetonitrile. Was. The mixture was quenched with an equivalent of 5% sodium bicarbonate, then the acetonitrile was removed by evaporation. The resulting aqueous phase was collected, washed three times with an equivalent amount of saturated sodium chloride, dried over anhydrous sodium sulfate, and evaporated to dryness. The product was used without further purification.

Preparation of Rhodamine B- (N-methylethanolamine) -succinyl- (N-hydroxysuccinimide) (compound 9). 0.118 g (0.221 mmol) of the compound rhodamine B- (N-methylethanolamine) succinate was
N-hydroxysuccinimide 23.0 mg (0.23 mmo
l), 48.0 mg of dicyclohexylcarbodiimide (0.2
3 mmol) and 5 ml of dry dichloromethane. The reaction was stirred at room temperature and monitored by TLC in 2: 1: 1 chloroform: methanol: acetone. The reaction was extracted with an equivalent amount of saturated sodium chloride. The organic phase is collected, washed with an equivalent amount of saturated sodium chloride, dried over anhydrous sodium sulphate and evaporated to dryness. The product was used without further purification. In methanol, maximum absorption (excitation): 567 nm; maximum fluorescence emission: 586 nm.

Example 10 Solid Phase Synthesis of Rhodamine-Labeled Oligonucleotides and Cleavage and Deprotection with 0.1 N Sodium Hydroxide Oligonucleotide synthesis was performed according to the manufacturer's instructions.
The test was performed using a pendorf Ecosyn 300+ DNA synthesizer. Oligonucleotides were synthesized on a 1 μmol scale. Dye phosphoramidite in dry acetonitrile at a concentration of 100m
g / ml. Labeled oligonucleotide (5m
er to 10 mer) from the solid support,
Deprotection was achieved by treatment with 0.5 ml of 0.1N sodium hydroxide at 55 ° C. for 2 hours. The labeled oligonucleotide is purified and purified by TLC (Kieselgel 60 F254 in 55:10:35 isopropanol: water: ammonia) or by standard methods (Sambrook et al., Molecular Cloning: A Laborat
^{ory Manual, 2 nd Edition, Cold} Spring Harbor Laborat
ory Press, 1989) and analyzed by polyacrylamide gel electrophoresis.

Using compounds 5, 7, 4 and 6, the oligonucleotide is labeled. FIG. 1 shows data for a rhodamine-labeled oligonucleotide according to an embodiment of the present invention, wherein 19% polyacrylamide, 10 M urea, Tris-
Electrophoresis was performed on a boric acid (89 mM tris-boric acid, 2 mM EDTA) gel. Irradiate the gel in an ultraviolet light box, using a NucleoVision CCD camera system equipped with a rhodamine filter (NucleoTech, Inc., Foster City, CA)
Fluorescent images were obtained. The sample is shown in A) with compound 5
-DT9me labeled at the 5 'end during synthesis using
For r: B), compound 7 is a poly-dT10mer having a 3′-end labeled during the synthesis using tetramethylrhodamine derivatized controlled pore glass: For C), compound 4 is synthesized using tetramethylrhodamine phosphoramidite. The poly-dT11mer: D) labeled at the 5 ′ end during the 5 ′ synthesis during the synthesis using the rhodamine 101 phosphoramidite
End-labeled poly-dT11mer was loaded.

Table 2 below shows oligonucleotides labeled with three inventive rhodamine phosphoramidite embodiments and one oligonucleotide labeled with the present invention attached to a solid support (adjusted pore glass). 4 shows fluorescent excitation and emission. Table 2

[Table 1] All spectral measurements were performed on purified labeled oligonucleotides dissolved in phosphate buffered saline solution, pH 7.2 (JRH Biosciences) using a Shimadzu UV-1601 UV-visible spectrophotometer and a Shimadzu RF-5301PC spectrofluorophotometer. went.

The maximum excitation and emission of the labeled oligonucleotide is shifted to a longer wavelength than the maximum excitation and emission of the free dye reagent. The maximum excitation and emission red-shifts are also comparable to the prior art 5- or 6-TAMRA by conventional methods.
Seen in oligonucleotides labeled with.

Using a derivative such as DMT-5-dye-deoxyuridine phosphoramide (DMT-5-dye-dU-CEP), the dye-labeled deoxyuridine (dU) residue is added to the 5 ′ end of the oligonucleotide or It can be added at any position in the sequence between the 5 'and 3' ends during automated oligo synthesis. Usually, researchers replace the dye-dU in the sequence with thymidine (dT) so that hybridization base pairs are not affected.

Other derivatives used are dye-deoxynucleotide triphosphate (dye-dNTP), dye-dNTP
Ribonucleotide triphosphate (dye-NTP) and dye-dideoxynucleotide triphosphate (dye
-ddNTP) compound. These reagents are dye-bound d
Useful for labeling DNA or RNA by NTP or enzymatic incorporation of NTP.

Dye-labeled dideoxynucleotide triphosphate (ddNTP) was used for DNA sequencing applications by the Sanger dideoxy sequencing method (San
geret al., J. Mol. Biol., 143, pp. 161-178, 1980)
Can be enzymatically incorporated into DNA as a chain terminator. Prior art of these compounds exists. Dye-ddATP, dye-ddCTP, dye-ddGTP, dye-ddTTP analogs are also contemplated.

Example 11 FIG. 2 shows a protein labeled with a rhodamine succinimidyl ester binding reagent (compounds 8 and 9). Compounds 8 and 9 were treated with anhydrous dimethyl sulfoxide (DMS
O) at a concentration of about 10 mg / ml. The reagent concentration was calculated from the maximum absorption, and the molar extinction coefficient was 90,000 in methanol for rhodamine dye reagent. Bovine serum albumin (BSA) (molecular weight 66,200 daltons)
The phosphate buffered saline (PBS) solution (JRH Bioscienc
es, Lenexa, Kansas) at a concentration of 4.16 mg /
ml. 0.2 ml of BSA,
Reacted with rhodamine succinimidyl ester reagent in a reaction tube containing 20 μl sodium M bicarbonate pH 8.3 and a labeling reagent (compound 8 or 9) greater than 8 to 15 times in mole. The binding reaction is about 1
Performed on a tube shaker at room temperature for hours. A 6 μl volume of 6 M hydroxylamine pH 8.5 was added to stop the binding reaction. Unincorporated rhodamine reagent is applied to a BioGel P6 spin column (Bio-Rad Laboratories, Hercules, Califor
nia) by gel filtration chromatography. Labeled BSA was analyzed by UV / visible spectrophotometry, fluorescence spectrophotometry, and standard methods (Sambrook et al., Molecular Cloning: A Lab
^{oratory Manual, 2 nd Edition, Cold} Spring Harbor Lab
oratory Press, 1989) by SDS-polyacrylamide gel electrophoresis. For tetramethylrhodamine-coupled BSA in PBS solution, maximum absorption (excitation): 564 nm; maximum fluorescence emission: 581 nm. PBS
Absorption (excitation) for rhodamine B-bound BSA in solution
Maximum: 569 nm; maximum fluorescence emission: 585 nm.

Although the present invention has been described in connection with preferred specific embodiments, the description and examples are for the purpose of explanation and are set forth in the appended claims. Should not be limited to the range.

[Brief description of the drawings]

FIG. 1 is a photograph of a polyacrylamide gel after electrophoresis, in which the lane contains an oligonucleotide labeled according to the present invention (lane A is poly-
dT 9mer, lane B, poly-dT 10mer,
Lane C is poly-dT 11mer and lane D
Is a poly-dT 11mer, lanes A, C and D were labeled at the 5'-end during synthesis, while lane B was labeled at the 3'-end during synthesis) .

FIG. 2 shows a photograph of a polyacrylamide gel after electrophoresis of a fluorescent rhodamine-labeled protein according to the present invention.

────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] March 21, 2001 (2001.3.2)
1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0009

[Correction method] Change

[Correction contents]

One function of the _Ra substituent is to prevent the formation of a lactam ring, so that alkyl,
It can be selected from a variety of substituents such as carboxyalkyl, aminoalkyl, cycloalkyl, aryl or arylallyl. The size of the substituents that serve as protecting groups for lactam ring formation can vary considerably.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction contents]

[0013]

DETAILED DESCRIPTION OF THE INVENTION One aspect of the present invention is to provide a dye based on rhodamine that binds to organic molecules, more preferably biomolecules. We believe that compounds previously described in the chemistry or biochemistry literature do not have functional groups linked to biomolecules such as oligonucleotides and proteins via the 3-position carboxyl group of rhodamine. ing. Rhodamine-based dyes
It is more easily synthesized as a single isomeric derivative, which is important in labeling oligonucleotides for DNA sequencing. As a control, the conventionally used carboxy rhodamine requires the purification of the mixed isomer to a single isomer before the labeled derivative can be synthesized. Furthermore, the rhodamine-based compounds of the present invention have an amide bond derived from a carboxylic acid at the 3-position and convert a carboxylate group to a non-acidic functional group. This improves the stability of the induced phosphoramidite. A fully substituted amide nitrogen atom prevents the dye from converting to a non-fluorescent lactam form.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0015

[Correction method] Change

[Correction contents]

Briefly describing FIG. 1, rhodamine-labeled oligonucleotides were prepared using standard conditions for gel purification of oligonucleotides (Sambrook et al., Molecular Cl.
^{oning: A Laboratory Manual, 2 nd} Edition, Cold Sprin
g Harbor Laboratory Press, 1989) using 19% polyacrylamide, 10 M urea, 89 mM Tris-boric acid, 2 mM ethylenediaminetetraacetic acid (EDTA) (T
Electrophoresis was performed on a (BE buffer) gel. The gel is irradiated in an ultraviolet light box, and a NucleoVision CCD camera system (NucleoT
(ech, Inc., Foster City, CA). Lane A included a poly-dT9mer labeled at the 5 'end during synthesis using compound 5 designed in the rhodamine-based embodiment. Lane B included a 3′-end labeled poly-dT10mer during the synthesis using rhodamine-based embodiment compound 7. Lane C
Included a poly-dT11mer labeled at the 5 ′ end during synthesis using compound 4. Lane D shows poly-dT11 labeled at the 5 ′ end during synthesis using compound 6.
mer was included.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction contents]

As shown in FIG. 1, the 3′-end labeled oligonucleotide is automatically synthesized by the dye-derivatized solid support matrix of the present invention, and the 5′-end is synthesized by the dye phosphoramidite during the synthesis of the oligonucleotide. Is labeled. Further, the invention can be practiced such that a dye-labeled dU residue is added to the oligonucleotide at any point within the sequence during automated oligo synthesis. The fluorescent excitation and emission properties of rhodamine used in the practice of the present invention provide a commercially available automated fluorescent DNA sequencing and fluorescent assay detection instrument with improved spectral separation from commonly used fluorescein derivatives. Similar to the rhodamine used. The derivatives and conjugates of the present invention retain their fluorescence ability upon excitation with light of a determined wavelength. Typically, the derivatives and conjugates of the present invention are excited with light at a wavelength of 500-700 nm and excited with fluorescence at a wavelength of 520-750 nm.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction contents]

In the structure of formula A, R₁And R₁₀Is simply
R is hydrogen or halogen;₂, R₅, R₆You
And R₉Represents, independently, hydrogen, alkyl, carboxyal
Kill, aminoalkyl, alkyl ether, alkylthio
O ether, halogen or alkoxy, R₃,
R₄, R₇And R₈Is, by itself, halogen, alk
, Carboxyalkyl, aminoalkyl, cycloal
Kill, aryl, or alkoxy, sulfate, phosphate
Or binds nitric acid (including but not limited to)
Alkyl, cyclo substituted with an additional functional group
Alkyl or aryl;₂And R₃Is
Together, the nitrogen atom bonded to the 3 'carbon has a 2' carbon atom
Having 2 to 5 carbon atoms, respectively,
A killed chain and R₉And R₈Is 6 'together
Link a 7 'carbon atom to a nitrogen atom that bonds to a carbon atom
Alkyl chains each having 2 to 5 carbon atoms
R ₄And R₅Together form a 3 'carbon atom
2 to 5 linking the 4 'carbon atom to the bonding nitrogen atom
Alkyl having each carbon atom;₆and
R ₇Is a nitrogen source that, together, is attached to the 6 'carbon atom
2 to 5 carbon atoms connecting the 5 'carbon atom to the
Each having alkyl, R₃And R₄Is one
From the carbon and nitrogen or oxygen atoms
Consists of one or more heteroatoms and contains 5 atoms in the main chain
Form an alkyl or alkylene chain containing
The bonds at both ends of the main chain are bonded to the nitrogen atom bonded to the 3 'carbon.
And R ₇And R₈Is a carbon source
One or more of a nitrogen atom and a nitrogen atom or an oxygen atom
A chain consisting of the above heteroatoms and containing up to 5 atoms in the main chain
To form an alkyl or alkylene chain, both ends of the main chain
A joint is attached to the nitrogen atom attached to the 6 'carbon,
₁₁, R₁₂, R₁₃And R₁₄Are hydrogen or
Is halogen.

[Procedure amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction contents]

To illustrate the amide nitrogen atom substituent of the structure of formula A, _Ra is alkyl, carboxyalkyl, aminoalkyl, cycloalkylaryl, arylalkyl, _{Ra '} comprises a chemically reactive functional group, They include, but are not limited to, amines, alcohols, halogens, carboxylic acids, hydrazine, mercapto, sulfuric acid, phosphoric acid or nitric acid. The size of the _Ra substituents can vary considerably, but preferably, _Ra has one carbon atom. As described and explained fully below, R
_The chemically reactive functional group contained in _{a '} is used, and the derivative is selected from the chemistry of the derivative and conjugate known to those skilled in the art. Suitable functional groups include amines, alcohols, halogens, carboxylic acids, hydrazine, mercapto,
Sulfuric acid, phosphoric acid or nitric acid. Typically, the chemically reactive functional group of _{Ra '} is such that it reacts with a reactive site of an organic molecule, preferably a biomolecule, or a molecule that binds to a solid support material such as conditioned pore glass or polystyrene resin. Is selected.

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

In equation B, L₁Is cyanoethyl, alk
Alkenyl, aryl, arylalkyl, or
Cycloalkyl, L₂And L₃Are, separately,
Alkyl, arylalkyl, cycloalkyl and cyclo
L represents alkylalkylaryl, L₁And L
₂Contains up to 5 carbon atoms in the main chain and a total of 10
Form alkylene chain containing carbon atom at
And the bond at both ends of the main chain is L ₂And L₃Combine
Bonds to a nitrogen atom or₂And L₃Is that they
Nitrogen, oxygen or sulfur
One or more heteroatoms from a group consisting of yellow atoms
And a saturated nitrogen atom-containing heterocycle is integrally formed.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0030

[Correction method] Change

[Correction contents]

When _{Ra '} contains a phosphoramidite group, the other substituents are chemically inert chains of alkyl, alkyl amides, alkyl ethers, polyethers or polyamides of up to 20 atoms, of the formula Wherein the atom is a carbon atom and is one or more heteroatoms from a group comprising a nitrogen atom or an oxygen atom and comprising a linking functionality capable of chemically linking the dye and the phosphoramidite moiety ], Preferably the inactive chain is 5-10
A chain containing atoms [the atoms are carbon atoms and are one or more heteroatoms from a group consisting of a nitrogen or oxygen atom]. Phosphoramidite itself,
Contains a branched structure with an indicator protecting group (DMT or MMT) attached by reaction to the hydroxyl function
[Wherein, DMT is a dimethoxytrityl group, and MMT
Is a monomethoxytrityl group].

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Ronald H. Chiarello 9520 Hills Barrow Drive, Castro Valley, CA 94546, USA (72) Inventor Liu Win Cheon, Belmont, CA 94002, USA Monroe Avenue 2004 No. 72 (72) Inventor Cathy E. Yokobata United States 94086 Sunnyvale, California Astor Avenue 1013 F Term (Reference) 4B024 AA11 CA04 FA10 HA20 4B063 QA01 QQ08 QQ10 QR41 QR58 QS03 QS36 QX02

Claims (20)

[Claims] 1. A method for labeling an organic compound for fluorescence detection
A method of formula A[Wherein, R₁And R_TenIs, by itself, hydrogen or halogen
And R_Two, R_Five, R₆And R₉Is alone, haloge
, Alkyl, carboxyl, aminoalkyl, alkyl
Ether, alkylthioether, halogen or
Alkoxy and R_Three, R_Four, R₇And R₈Is, alone,
Halogen, unsubstituted or substituted alkyl
, Carboxylalkyl, aminoalkyl, cycloalkyl
Alkyl, aryl, R_TwoAnd R_ThreeIs one
Link the 2 'carbon atom to the nitrogen atom that is bonded to the 3' carbon
Alkyl chains each having 2 to 5 carbon atoms
R ₉And R₈Together form a 6 'carbon atom
2 to 5 carbon atoms linking the 7 'carbon atom to the merging nitrogen atom
An alkyl chain having an elemental atom,_FourAnd R_FiveIs one
As a body, the nitrogen atom bonded to the 3 'carbon atom has 4' carbon
A each having 2 to 5 carbon atoms connecting the atoms
It is R₆And R₇Is one, 6 'charcoal
2 linking a 5 'carbon atom to a nitrogen atom bonded to an elemental atom
Alkyl having from 5 to 5 carbon atoms,
_ThreeAnd R_FourAre, together, a carbon atom and a nitrogen atom
Or one or more hetero atoms from an oxygen atom
Alkyl or alkyl containing up to 5 atoms in the main chain
Form a kylene chain, and the both ends of the main chain are 3 'carbon
R is attached to the nitrogen atom₇And R₈Is one
Is it a carbon and nitrogen or oxygen atom
Consisting of one or more heteroatoms,
Form an alkyl or alkylene chain containing up to 5 atoms
And a nitrogen atom at which both ends of the main chain are bonded to the 6 ′ carbon
Child and R₁₁, R_{1 Two}, R₁₃And R₁₄Is it
Hydrogen or halogen, respectively, wherein R_aAnd R _a
'Is a non-hydrogen atom substituent, and R_aLactam ring formation by
Provided resistance to R_{a '}Is a group reactive with the derivative
And the labeled organic compound and fluorophore
A with the fluorophore R_{a '}And the resulting bond
Coalescence fluoresces upon excitation with light of the determined wavelength.
Emit. A fluorophore having a structure represented by
A method comprising providing. 2. The method of claim 1, wherein said linking comprises reacting an organic compound with a fluorophore under conditions that form a covalent bond. 3. The method according to claim 2, wherein the organic compound is a biomolecule.
The method described in. 4. The method according to claim 3, wherein the biomolecule is an amino acid, peptide, protein, nucleotide, oligonucleotide, or nucleic acid. 5. The method of claim 3, wherein the biomolecule is bound to a solid support. 6. The method of claim 3, wherein the biomolecule is an oligonucleotide and the fluorophore is attached via a phosphoramidite at the 5 ′ end of the attachment. 7. The method of claim 5, wherein the biomolecule is an oligonucleotide and the fluorophore is attached at the 3 'end of the linkage. 8. The method of claim 3, wherein the biomolecule is an amino acid, peptide, or protein, and the fluorophore is attached at an amine or mercapto group of the linkage. 9. The method of claim 3, wherein the biomolecule is part of a cell surface membrane or a viral coat. 10. The binding agent is an amino acid, peptide, protein, nucleotide, oligonucleotide or nucleic acid that binds to one or more fluorophores, and the fluorescent conjugate is of formula 1 Wherein R ₁ and R ₁₀ are independently hydrogen or halogen, and R ₂ , R ₅ , R ₆ and R ₉ are independently halogen, alkyl, carboxyalkyl, aminoalkyl,
R ₃ , R ₄ , R ₇ and R ₈ are independently halogen, unsubstituted or substituted alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, alkyl ether, alkylthioether, halogen or alkoxy. Aryl, R ₂ and R ₃ together are an alkyl chain having 2-5 carbon atoms connecting the 2 ′ carbon atom to the nitrogen atom bonded to the 3 ′ carbon, and R ₉ and R ₈ Is together an alkyl chain having 2-5 carbon atoms each connecting the 7 ′ carbon atom to the nitrogen atom bonded to the 6 ′ carbon atom, and R ₄ and R ₅ are together 3 2 to linking the carbon atoms' 4 nitrogen atoms bonded to a carbon atom 'is alkyl having 5 carbon atoms, respectively, R ₆ and R _7, together, 6' TansoHara 2 to linking the nitrogen atom at the 5 'carbon atom bonded to an alkyl having 5 carbon atoms, respectively, R ₃ and R _4, together, one of the carbon atoms and nitrogen atom or an oxygen atom or An alkyl or alkylene chain comprising up to 5 heteroatoms and containing up to 5 atoms in the main chain, wherein both ends of the main chain are bonded to a nitrogen atom bonded to the 3 ′ carbon, and R ₇ and R ₈ together comprise one or more heteroatoms from a carbon atom and a nitrogen or oxygen atom to form an alkyl or alkylene chain containing up to 5 atoms in the main chain; Are bonded to the nitrogen atom bonded to the 6 ′ carbon, and R ₁₁ , R ₁₂ , R ₁₃ and R ₁₄ are each hydrogen or halogen;
_a is an alkyl, carboxyalkyl, aminoalkyl, cycloalkyl, aryl, or arylalkyl having 1 to 10 carbon atoms, and Z includes a linking substance. The fluorescent conjugate comprising the binding substance and a fluorophore, having a structure represented by the following formula: 11. The conjugate of claim 10, wherein the conjugate is attached to the fluorophore via an amide, ester, ether, disulfide or thioether bond. 12. The conjugate according to claim 10, wherein the linkage between the fluorophore and the binding substance comprises a phosphate ester. 13. A method for binding a binding substance to a solid support.
The fluorescent conjugate according to claim 10. 14. The solid support is a controlled pore glass.
A fluorescent conjugate according to claim 13. 15. The solid support is a polymer support.
A fluorescent conjugate according to claim 13. 16. The fluorescent conjugate according to claim 10, wherein the binding substance is a part of a cell membrane. 17. The fluorescent conjugate according to claim 10, wherein the binding substance is part of a viral coat. 18. The fluorescent conjugate according to claim 10, wherein the fluorophore is derived from tetramethylrhodamine. 19. The fluorescent conjugate according to claim 10, wherein the fluorophore is derived from Rhodamine 101. 20. The fluorescent conjugate according to claim 10, wherein the fluorophore is derived from Rhodamine B.