# Patent

## Title
Phosphonylated fluorescent dyes and conjugates

## Abstract

Reagents are provided for the introduction of phosphonate groups into fluorescent dyes. Methods are also provided for preparing dye conjugates.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 11/202,635 which claims the benefit of U.S. Provisional application Ser. No. 60/601,599 filed Aug. 13, 2004, the contents of all is incorporated herein by reference.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK
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BACKGROUND OF THE INVENTION
The present invention provides a number of modified dyes, such as xanthene dyes, including rhodamines, rhodols and fluoresceins that are substituted with one to four phosphonate groups. The dyes of the invention, including chemically reactive dyes and dye-conjugates are useful as fluorescent probes, particularly in biological samples.
Fluorescent dyes are known to be particularly useful in biological applications where a highly sensitive detection reagent is required. Fluorescent dyes are used to label both visible color and fluorescence to other materials. The dyes of this invention are phosphonate derivatives of xanthene-based dyes that are typically fluorescein, rhodamine or rhodol derivatives




“Fluorescein” dyes include derivatives of 3H-xanthen-6-ol-3-one that are typically substituted at the 9-position by a 2-carboxyphenyl group. “Rhodamine” dyes include derivatives of 6-amino-3H-xanthen-3-imine that are typically substituted at the 9-position by a 2-carboxyphenyl group. “Rhodol” dyes include derivatives of 6-amino-3H-xanthen-3-one that are typically substituted at the 9-position by a 2-carboxyphenyl group. Fluoresceins, rhodamines and rhodols are typically substituted by a derivative capable of forming a 5- or 6-membered lactone or lactam ring. For example in the case of fluorescein the spirolactone form of the dye has the following structure:




Many commercial fluorescent dyes have a polycyclic aromatic nature and are hydrophobic. Those molecules are also prone to minimize exposure to any hydrophilic environment through interactions with nearby hydrophobic surfaces and residues. These interactions include dye-dye interaction and dye-biomolecule (e.g. proteins, lipids, oligonucleoties) interactions. Hydrophobic interactions can cause substantial quenching effect for fluorescent dyes (see for example Randolph, J. B.; Waggoner, A. S. Nucleic Acids Res. 1997, 25(14), 2923-2929 and references cited therein). One method to overcome this problem is to improve the hydrophilic character of the dye by, for example, introducing a sulfonate substituent into the dye molecule (sulfonated carbocyanine dyes are disclosed in U.S. Pat. No. 5,268,486 and sulfonated xanthene dyes are disclosed in U.S. Pat. No. 6,130,101).
The present invention describes dyes, including fluorescein, rhodol and rhodamine dyes that are substituted by at least one phosphonate moiety. The phosphonate-substituted dyes of the invention possess considerable advantages over their unsubstituted hydrophobic analogs. Being ionized, the phosphonate group brings additional hydrophilicity to the dye molecules, increasing their solubility in aqueous solutions. Importantly, the quantum yield of the new dyes is not decreased by introduction of a phosphonate moiety. In contrast to the sulfonated dyes, the dyes of the invention are compatible with the synthetic conditions used on commercial oligonucleotide synthesizers.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a fluorescent dye reagent having the formula:




wherein Fl is a fluorescent dye component; Lf is a linking group having an attached member selected from the group consisting of a protected or unprotected functional group, a reactive group, a polyfunctional linking moiety, a phosphoramidite moiety and a solid support; the subscript m is an integer of from 0 to 2; Pz is a zwitterionic phosphonate group having the formula (a) or a protected phosphonate group having formula (b) or (c):





wherein the wavy line indicates the direct attachment to a sp2 carbon of said fluorescent dye component; L is a linking group; Na is an ammonium ion group; each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group, such as C(O)CF3, FMOC, tButyl, tBOC, phthalimide and monomethoxytrityl; the subscript n is an integer of from 1 to 4, preferably 1 to 2; RC is H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a labile protecting group or an alkylene linking group having a distal hydroxy or protected hydroxy group, amino, protected amino, carboxy, protected carboxy, thio, or protected carboxy; and salts thereof. In some embodiments, RC is H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a labile protecting group or an alkylene linking group having amino, protected amino, carboxy, protected carboxy, thio, or protected carboxy group; and salts thereof. In one embodiment, the subscript n is an integer from 3-4.

In another aspect, the present invention provides fluorescent dye reagent having the formula:

Fl-(P1)n 

wherein Fl is a fluorescent dye component; the subscript n is an integer from 1 to 4; and P1 is functionalized phosphonate group having the formula:





wherein the wavy line indicates the direct attachment to a sp2 carbon of said fluorescent dye component; L1 is a linking group; R is a member selected from the group consisting of H, C1-C8 alkyl, aryl, arylC1-C4 alkyl, -La-Na, and -La-NRARB; wherein La is an alkylene linking group, Na is an ammonium ion group, and each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group; and Pa is a functional group component selected from the group consisting of a phosphoramidite moiety, a mono-, di- or tri-functional linking group having at least one terminal functional group or protected functional group, a solid support and a reactive group; and salts thereof. In some embodiments, the subscript n is an integer from 3 to 4. In another embodiment the subscript n is an integer from 2-4.

In other aspects, the present invention provides methods of preparing phosphonate fluorescent dye reagents as well as methods of using probes (e.g., oligonucleotide probes) having attached phosphonate fluorescent dyes as provided herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the structures of selected classes of benzoxanthene dyes of the present invention.
FIG. 2 illustrates the structures of selected classes of benzophenoxazine dyes of the present invention.
FIG. 3 illustrates an HPLC chromatogram of a MB-Q-oligonucleotide-PY (see Example 12).
FIG. 4 illustrates the titration of a HSV 1,2 MGB Eclipse probe assay wherein the FAM-labeled probe is multiplexed with a PY-labeled probe (Example 13).



DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions
Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
The term “alkyl” refers to a linear, branched, or cyclic saturated monovalent hydrocarbon radical or a combination of cyclic and linear or branched saturated monovalent hydrocarbon radicals having the number of carbon atoms indicated in the prefix. For example, (C1-C8)alkyl is meant to include methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl, cyclopentyl, cyclopropylmethyl and the like. For each of the definitions herein (e.g., alkyl, alkenyl, alkoxy, arylalkoxy), when a prefix is not included to indicate the number of main chain carbon atoms in an alkyl portion, the radical or portion thereof will have eight or fewer main chain carbon atoms.
The term “alkylene” means a linear saturated divalent hydrocarbon radical or a branched saturated divalent hydrocarbon radical having the number of carbon atoms indicated in the prefix. For example, (C1-C6)alkylene is meant to include methylene, ethylene, propylene, 2-methylpropylene, pentylene, and the like.
The term “alkenyl” refers to a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond. For example, (C2-C6)alkenyl is meant to include, ethenyl, propenyl, and the like.
The term “alkynyl” refers to a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond and having the number of carbon atoms indicated in the prefix. For example, (C2-C6)alkynyl is meant to include ethynyl, propynyl, and the like.
The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. Similarly, the term dialkylamino refers to an amino group having two attached alkyl groups that can be the same or different.
The term “aryl” means a monovalent monocyclic, bicyclic or polycyclic aromatic hydrocarbon radical of 5 to 10 ring atoms which is unsubstituted or substituted independently with one to four substituents, preferably one, two, or three substituents selected from alkyl, cycloalkyl, cycloalkyl-alkyl, halo, cyano, hydroxy, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl cut, phenyl or phenylalkyl, aryl or arylalkyl), —(CR′R′′)n—COOR (where n is an integer from 0 to 5, R′ and R′′ are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl cut, phenyl or phenylalkyl aryl or arylalkyl) or —(CR′R′′)n—CONRaRb (where n is an integer from 0 to 5, R′ and R′′ are independently hydrogen or alkyl, and Ra and Rb are, independently of each other, hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl, aryl or arylalkyl). More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl, and the substituted forms thereof. Similarly, the term “heteroaryl” refers to those aryl groups wherein one or more heteroatoms or heteroatom functional groups have replaced a ring carbon, while retaining aromatic properties, e.g., pyridyl, quinolinyl, quinazolinyl, thienyl, and the like. For brevity, the term aryl, when used in combination with other radicals (e.g., aryloxy, arylalkyl) is meant to include both aryl groups and heteroaryl groups as described above.
The term “arylalkyl” refers to a radical —RaRb where Ra is an alkylene group (having the indicated number of carbon atoms, or if unspecified having six or fewer main chain carbon atoms) and Rb is an aryl group as defined herein. Examples of arylalkyl groups include benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the like.
Similarly the term “arylalkenyl” means a radical —RaRb where Ra is an alkenylene group and Rb is an aryl group as defined herein, e.g., 3-phenyl-2-propenyl, and the like.
“Arylheteroalkyl” means a radical —RaRb where Ra is an heteroalkylene group (having the indicated number of carbon atoms) and Rb is an aryl group as defined herein, e.g., 2-hydroxy-2-phenyl-ethyl, 2-hydroxy-1-hydroxymethyl-2-phenyl-ethyl, and the like.
The term “aryloxy”, refers to a radical —OR where R is an aryl group, e.g., phenoxy, naphthyloxy and the like.
The prefix “halo” and the term “halogen” when used to describe a substituent, refer to —F, —Cl, —Br and —I.
The term “heteroalkyl” refers to an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, —ORa, —NRbRc, and —S(O)nRd (where n is an integer from 0 to 2), with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom of the heteroalkyl radical. Ra is hydrogen, alkyl, aryl, arylalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl. Rb is hydrogen, alkyl, aryl or arylalkyl. Rc is hydrogen, alkyl, aryl, arylalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl. Rd is hydrogen (provided that n is 0), alkyl, aryl, arylalkyl, amino, mono-alkylamino, di-alkylamino, or hydroxyalkyl. Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and 2-methylsulfonyl-ethyl. For each of the above, Ra, Rb, Rc, and Rd can be further substituted by NH2, fluorine, alkylamino, di-alkylamino, OH or alkoxy. Additionally, the prefix indicating the number of carbon atoms (e.g., C1-C10) refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, —ORa, —NRbRc, or —S(O)nRd portions.
The term “heterocyclic” refers to a saturated or unsaturated non-aromatic cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from O, NR (where R is independently hydrogen or alkyl) or S(O)n (where n is an integer from 0 to 2), the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group. The heterocyclic ring may be optionally substituted independently with one, two, or three substituents selected from alkyl, halo, cyano, hydroxy, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, —COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), —(CR′R′′)n—COOR (n is an integer from 0 to 5, R′ and R′′ are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), or —(CR′R′′)n—CONRaRb (where n is an integer from 0 to 5, R′ and R′′ are independently hydrogen or alkyl, and Ra and Rb are, independently of each other, hydrogen, alkyl, phenyl or phenylalkyl). More specifically the term heterocyclic includes, but is not limited to, tetrahydropyranyl, piperidino, N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl, 3-pyrrolidino, 2-pyrrolidon-1-yl, morpholino, thiomorpholino, thiomorpholino-1-oxide, thiomorpholino-1,1-dioxide, pyrrolidinyl, and the derivatives thereof. The prefix indicating the number of carbon atoms (e.g., C3-C10) refers to the total number of carbon atoms in the portion of the heterocyclic group exclusive of the number of heteroatoms.
The terms “heteroalkylene” means a linear saturated divalent hydrocarbon radical of one to six carbons or a branched saturated hydrocarbon radical of three to six carbon atoms with one, two or three substituents independently selected from —ORa, —NRbRc, and —S(O)nRd (where n is an integer from 0 to 2) where, Ra, Rb, Rc and Rd are as defined herein for a heteroalkyl radical. Examples include, 2-hydroxyethan-1,2-diyl, 2-hydroxypropan-1,3-diyl and the like.
Each of the above terms (e.g., “alkyl,” “heteroalkyl,” and “aryl”) are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, heterocycloalkyl, and heterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R′′, —SR′, -halogen, —SiR′R′′R′′′, —OC(O)R′, —C(O)R′, —CO2R′, —CONR′R′′, —OC(O)NR′R′′, —NR′′C(O)R′, —NR′—C(O)NR′′R′′′, —NR′′C(O)2R′, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R′′, —CN and —NO2 in a number ranging from zero to four, preferably, zero, one, two or three substituents. R′, R′′ and R′′′ each independently refer to hydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R′ and R′′ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R′′ is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” in its broadest sense is meant to include groups such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like). Preferably, the alkyl groups will have from 0-3 substituents, more preferably 0, 1, or 2 substituents, unless otherwise specified.
Similarly, substituents for the aryl groups are varied and are selected from: -halogen, —OR′, —OC(O)R′, —NR′R′′, —SR′, —R′, —CN, —NO2, —CO2R′, —CONR′R′′, —C(O)R′, —OC(O)NR′R′′, —NR′′C(O)R′, —NR′′C(O)2R′, —NR′—C(O)NR′′R′′′, —NH—C(NH2)═NH, —NR′C(NH2)═NH, —NH—C(NH2)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R′′, —N3, —CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R′′ and R′′′ are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.
Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)—(CH2)q—U—, wherein T and U are independently —NH—, —O—, —CH2— or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r—B—, wherein A and B are independently —CH2—, —O—, —NH—, —S—, —S(O)—, —S(O)2—, —S(O)2NR′— or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula —(CH2)s—X—(CH2)t—, where s and t are independently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)2—, or —S(O)2NR′—. The substituent R′ in —NR′— and —S(O)2NR′— is selected from hydrogen or unsubstituted (C1-C6)alkyl.
Certain compounds or oligonucleotides of the present invention may exist in a salt form. Such salts include base addition salts such as sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When the compounds or modified oligonucleotides of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, lactic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention. The methods for the determination of stereochemistry and the separation of isomers are well-known in the art (see discussion in Chapter 4 of ADVANCED ORGANIC CHEMISTRY, 4th edition J. March, John Wiley and Sons, New York, 1992).
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not (e.g, 2H), are intended to be encompassed within the scope of the present invention.
“Protecting group” or “protected form thereof” refers to a grouping of atoms that when attached to a reactive group in a molecule masks, reduces or prevents that reactivity. Examples of protecting groups can be found in T. W. Greene and P. G. Wuts, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, (Wiley, 2nd ed. 1991), Beaucage and Iyer, Tetrahedron 48:2223-2311 (1992), and Harrison and Harrison et al., COMPENDIUM OF SYNTHETIC ORGANIC METHODS, Vols. 1-8 (John Wiley and Sons. 1971-1996). Representative amino protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethyl silyl (TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl (NVOC) and the like (see also, Boyle, A. L. (Editor), CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY, John Wiley and Sons, New York, Volume 1, 2000). Representative hydroxy protecting groups include those where the hydroxy group is either acylated or alkylated such as benzyl and trityl ethers as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers. Additionally, hydroxy groups can be protected by photoremovable groups such as α-methyl-6-nitropiperonyloxycarbonyl (McGall, G. H. and Fidanza, J. A., Photolithographic synthesis of high-density oligonucleotide arrays, in DNA ARRAYS METHODS AND PROTOCOLS, Edited by Rampal J. B., METHODS IN MOLECULAR BIOLOGY, 170:71-101 (2001), Humana Press, Inc., NY; Boyle, Ann L. (Editor), Current Protocols in Nucleic Acid Chemistry, John Wiley and Sons, New York, Volume 1, 2000.)
The term “labile protecting group” refers to those protecting groups that are removeable under mild conditions that do not significantly impact the remainder of the molecule.
As used herein, the term “reactive group” refers to an electrophilic group or a nucleophilic group that can be used to form a covalent linkage with another component. Examples of of nucleophilic groups include —NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, —OH, —COOH, or —SH. The electrophilic groups can be activated esters, acrylamides, acyl azides, acyl halides, aldehyde or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boranates, carboxylic acids, carbodiimides, diazoalkanes, epoxides, haloacetamides, halotriazines, imidoesters, isocyanates, isothiocyanates, maleimides, phophoramidites, silyl halides, sulfonate ester and sulfonyl halides. Additionally, a spacer can include hetero atoms in linear or acyclic portions, cyclic portions, aromatic rings or combinations thereof. Within the above, an “activated ester group” refers to a carboxylic acid ester which is more reactive than an alkyl ester (e.g., methyl ester) in reactions in which the carbonyl moiety is an electrophilic center toward, for example, amide formation. Examples of activated esters include pentafluorophenyl (PFP) esters, N-hydroxysuccinimide esters, and the like.
A “polyfunctional linking moiety” is a linking group having two or more functional groups that can be used to attach or conjugate two or more components that can be the same or different. Polyfunctional linking moieties include, for example, trivalent linking groups and tetravalenet linking groups (see, for example, U.S. Pat. Nos. 5,512,677; 5,419,966; 5,585,481; 5,942,610 and 5,736,626).
A “phosphoramidite” is a term of art used to refer to a trivalent phosphorus group typically used in oligonucleotide synthesis. Detailed descriptions of the chemistry used to form oligonucleotides by the phosphoramidite method are provided in Caruthers et al., U.S. Pat. Nos. 4,458,066 and 4,415,732; Caruthers et al., Genetic Engineering, 4:1-17 (1982); Users Manual Model 392 and 394 Polynucleotide Synthesizers, pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991), each of which are incorporated by reference in their entirety. Labeled oligonucleotides can be synthesized enzymatically, e.g., using a DNA polymerase or ligase, e.g., Stryer, Biochemistry, Chapter 24, W. H. Freeman and Company (1981), or by chemical synthesis, e.g., by a phosphoramidite method, a phosphite-triester method, and the like, e.g., Gait, OLIGONUCLEOTIDE SYNTHESIS, IRL Press (1990). Labels can be introduced during enzymatic synthesis utilizing labeled nucleoside triphosphate monomers, or introduced during chemical synthesis using labeled non-nucleotide or nucleotide phosphoramidites, or may be introduced subsequent to synthesis. A typical phosphoramidite reagent used in oligonucleotide synthesis is represented by the structure below:




wherein the wavy line indicates the attachment to the remainder of the reagent and the substituents “a” and “b” are each independently isopropyl amino, diisopropylamino, 2-cyanoethyloxy, methoxy or morpholino; and “a” and “b” are not the same.

“Optional” or “optionally” in the above definitions means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “heterocyclo group optionally mono- or di-substituted with an alkyl group” means that the alkyl may but need not be present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted with the alkyl group.
The term “biological agent” refers to essentially any nucleoside, oligonucleotide, peptide, protein, aminocarbohydrate or ligand, as well as analogs thereof (e.g., oligonucleotides having modified or non-natural bases).
The term “conjugate” refers to a molecule formed by the covalent attachment of two or more components such as oligonucleotides, fluorophores, quenchers, minor groove binders, and the like.
“Oligonucleotide” and “polynucleotide” are used interchangeably and refers to a polymer of nucleotides, either natural or synthetic including, but not limited to those nucleotides having modified bases, sugar analogs, and the like. As noted above, an oligonucleotide conjugate will refer to an oligonucleotide as defined, having at least one covalently attached fluorophore, quencher, minor groove binder (MGB or MB) or other useful fragments, as well as combinations of the recited components.
The term “solid support” refers to essentially any solid or semisolid matrix that is useful for, and compatible with, automated oligonucleotide techniques and includes, glass, polystyrene, nylon, plastic, combinations and the like. Examples of useful solid supports have been described in, for example, U.S. Pat. Nos. 5,262,530, 5,419,966, 5,512,667 and 5,589,586.
As used herein, the term “phosphonylated dye” and “phosphonate dye” both refer to a dye having attached thereto a functionalized phosphonate group.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques in organic chemistry, biochemistry, oligonucleotide synthesis and modification, bioconjugate chemistry, nucleic acid hybridization, molecular biology, microbiology, genetics, recombinant. DNA, and related fields as are within the skill of the art. These techniques are fully explained in the literature. See, for example, Maniatis, Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (1982); Sambrook, Fritsch & Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press (1989); Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996); Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH, IRL Press (1984); Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, IRL Press (1991).
General
The present invention resides in the discovery that a wide variety of fluorescent dyes (or fluorophores) can be prepared having a zwitterionic phosphonate group (or a protected form thereof), providing reagents that are shelf-stable and that can be used to label essentially any biological agent (e.g., oligonucleotides, peptides, proteins, probes, and the like). Accordingly, the invention provides new “phosphonylated dyes” as well as methods of labeling biological agents using these “phosphonylated dyes”. The invention further provides reagents such as phosphoramidite-derivatized dyes that can be prepared from the phosphonate-substituted dyes described herein. Additionally, support-bound dyes, similarly prepared from the phosphonate dyes are also described. Additionally, reactive phosphonylated dyes for labeling biological agents are also disclosed.
The “phosphonate or phosphonylated dyes” (e.g., dyes having a zwitterionic phosphonate group or a protected form thereof), as well as reagents incorporating those dyes (e.g., support-bound dyes and phosphoramidites) have been found to be compatible with, for example, coumarin dyes, benzocoumarin dyes, fluorescein dyes, rhodol dyes, phenoxazine dyes, benzophenoxazine dyes, xanthene dyes, benzoxanthene dyes, and cyanine dyes.
Examples of these and other suitable dye classes can be found in Haugland, et al., HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, SIXTH ED., Molecular Probes, Eugene, Oreg. 1996; U.S. Pat. Nos. 3,194,805; 3,128,179; 5,187,288; 5,188,934; 5,227,487, 5,248,782; 5,304,645; 5,433,896; 5,442,045; 5,556,959; 5,583,236; 5,808,044; 5,852,191; 5,986,086; 6,020,481; 6,162,931; 6,180,295; and 6,221,604; EP 1408366; Smith, et al., J. Chem. Soc. Perkin Trans. 2, 1993, 1195-1204; Whitaker, et al., Anal. Biochem. 207:267-279 (1992); Krasoviskii and Bolotin, ORGANIC LUMINESCENT MATERIALS, VCH Publishers, NY., 1988; Zolliger, COLOR CHEMISTRY, 2nd Edition, VCH Publishers, NY., 1991; Hirschberg, et al., Biochemistry 37:10381-10385 (1998); Fieser and Fieser, REAGENTS FOR ORGANIC SYNTHESIS, Volumes 1 to 17, Wiley, US, 1995. Geiger, et al., Nature 359:859-861 (1992). Still other dyes are provided via online sites such as http://www.zeiss.com.
Embodiments of the Invention
In one aspect, the present invention provides fluorescent dye reagents having the general formula (A):




wherein Fl is a fluorescent dye component; Lf is a linking group having an attached member selected from the group consisting of a protected or unprotected functional group, a reactive group, a polyfunctional linking moiety, a phosphoramidite moiety and a solid support; the subscript m is an integer of from 0 to 1; Pz is a zwitterionic phosphonate group having the formula (a) or a protected phosphonate group having formula (b) or (c):





wherein the wavy line indicates the direct attachment to a sp2 carbon of said fluorescent dye component; L is a linking group; Na is an ammonium ion group; each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group, such as C(O)CF3, FMOC, tButyl, tBOC, phthalimide and monomethoxytrityl; the subscript n is an integer of from 1 to 4, preferably 1 to 2; RC is H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a labile protecting group or an alkylene linking group having a distal hydroxy or protected hydroxy group, amino, protected amino, carboxy, protected carboxy, thio, or protected carboxy; and salts thereof. In some embodiment, RC is H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a labile protecting group or an alkylene linking group having, amino, protected amino, carboxy, protected carboxy, thio, or protected carboxy group; and salts thereof.

In the context of the present invention, essentially any fluorescent dye can be modified to include either of Lf and Pz. Typically, Pz is attached to an sp2-hybridized carbon atom on an aromatic ring of the fluorescent dye using synthetic methods as described below. Similarly, Lf can be attached to an existing functional group on the dye or can be a vestige of, for example, a lactone dye (see co-pending application Ser. No. 10/026,374).
Suitable dyes can be selected from, for example, coumarins, benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, cyanines, acridines, dipyrrometheneboron difluorides, phenoxazines, benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines. Still other types of useful dyes are ruthenium bipyridyl dyes, energy transfer dyes, thiazole orange dyes, N-aryl-1,8-naphthalimide dyes, polymethines and other dyes shown in the present application.
In one group of embodiments, the fluorescent dye reagents are those in which the subscript m is 1 and the subscript n is 1. Within this group of embodiments, Pz can have formula (a) or (b). Preferably, as a reagent that will be subject to additional manipulations or synthetic methods, the phosphonate ester groups are protected (e.g., as in formula (b)). When the reagent is incorporated into a probe (see embodiments discussed below) and used in assays, the phosphonate is in a zwitterionic form (e.g., as in formula (a)).
In another group of embodiments, the fluorescent dye reagents are those in which the subscript m is 0 and the subscript n is 1. Within this group of embodiments, Pz can have formula (a), (b) or (c). Preferably, as a reagent that will be subject to additional manipulations or synthetic methods, the phosphonate ester groups are protected as in formula (b) and (c).
In still another group of embodiments, the fluorescent dye reagents are those in which the subscript m is 0 and the subscript n is 2. Within this group of embodiments, each of the Pz groups can be the same or different and can have formula (a), (b) or (c). As above, when the reagent is to be subjected to additional manipulations or synthetic methods, the phosphonate ester groups are preferably protected (e.g., as in formula (b) and (c)).
In a number of embodiments, L is C2-C8 alkylene. In other embodiments of the general formula above, Na is an ammonium ion group having the formula —N+(RD)3 wherein each RD is independently selected from H, C1-C8 alkyl, aryl and arylC1-C4 alkyl. Still more preferably, each RD is independently selected from H or C1-C8 alkyl.
Returning to the general formula above, Lf can be a variety of linking groups known to those skilled in the art. Many linking groups are available from commercial sources and can be utilized in the reagents above by coupling one end of the linker to the fluorescent dye and the other end of the linker to a protecting group. In one group of embodiments, Lf is a (C2-C20)alkylene group, terminating in a functional group such as hydroxy, protected hydroxy, amino, protected amino, carboxy, carboxylate ester, carboxamide, urea, and the like. In other embodiments, Lf is an alkylene group having an attached phosphoramidite moiety, preferably 2-cyanoethyl-N,N-diisopropylphosphoramidite. Similarly, the linking group L can be selected from a variety of linking groups having from 2 to 50 main chain atoms. Examples of linking group include, but are not limited to, alkylene linking groups, heteroalkylene linking groups, polyether linking groups, linking groups containing a combination of acyclic and cyclic groups (e.g., a alkylene group and a heterocyclic group, or a heteroalkylene group and a arylene group), and the like. In some embodiment, the linking group has from 2 to 50 main chain atoms and is a combination of acyclic and cyclic groups.
In sections below, a number of dyes and dye groups are discussed specifically and represent embodiments of particular interest. One of skill in the art, however, will appreciate that the invention is not limited to those embodiments.
Xanthene Dyes
In one group of embodiments, Fl is selected from the family of xanthene dyes, including benzoxanthenes, and more specifically, fluorescein dyes, rhodamine dyes and rhodol dyes.
In a particular group of embodiments, the fluorescent dye reagent has formula (I):




In certain aspects of this embodiment, the Ar ring, represented by the symbol,




in formula I is selected from the group consisting of:





In Ar1, A1 represents O, N—Z, or N+(Z)2. The bond, , in formula I, represents a single or double bond. In Ar2, A1 represents OR, or N(Z)2. At each occurrence, Z is independently hydrogen, (C1-C8)alkyl, aryl-(C1-C8)-alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, C1-C4alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with R2 or R4 to form a fused 5- to 7-membered ring, wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz. In some embodiments, the Z group is combined with the R2 or R4 group to form 7-membered ring, wherein the 7-membered ring is optionally fused to an optionally substituted aryl ring. In another embodiment, Z is independently aryl-(C1-C8)-alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, C1-C4alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with R2 or R4 to form a fused 5- to 7-membered ring, wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz. The substituent R, in Ar2, is selected from H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, protecting group and Lf. In one embodiment the substituent R in Ar2 is a protecting group

In certain aspects of this embodiment, A2 represents OR or N(Z)2 in which each Z is independently hydrogen, (C1-C8)alkyl, aryl(C1-C8)alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with with R1 or R1′′ to form a fused 5- to 7-membered ring wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz. In one embodiment, the Z group is optionally combined with R1 or R1′′ to form a 7-membered ring. In another embodiment, Z is independently aryl-(C1-C8)-alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with R2 or R4 to form a fused 5- to 7-membered ring, wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C1-C4)alkyl, Lf for Pz. The substituent R, in Ar2, is selected from H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a protecting group and Lf. In one embodiment, the group R in Ar2 is a protecting group.
In certain aspects of this embodiment, R1′, R1′′, R1, R2, R3 and R4 are each independently selected from the group consisting of H, halogen, cyano, sulfo, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz, wherein said aryl or heteroaryl group is optionally substituted with Pz; or optionally any two of the R1′, R1′′, R2 and R3 substituents that are attached to adjacent ring atoms are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic, and is optionally substituted with Pz. The alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkyl(C1-C6)amino, (C1-C6)alkoxy, cyano, haloacetyl or hydroxy. The aryl or heteroaryl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz.
R0 is halogen, cyano, CF3, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, substituted or unsubstituted heteroaryl or aryl having the formula:




wherein X1, X2, X3, X4 and X5 are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl, heteroaryl, SO3H, PO3H2, CO2H, Lf and Pz and optionally, any two adjacent X1 through X5 are combined to form an aromatic or heteroaromatic ring. As above, any aryl or heteroaryl portions of R0 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylaamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz. The substituent R0′ in formula I is absent or is (C1-C6)alkoxy, hydroxy, (C1-C6)alkylamino, or, di(C1-C6)alkylamino. Optionally, the R0 and R0′ groups are combined to form a 5- to 6-membered heterocyclic ring. Within the above formula, there will be from 0 to 1 Lf groups and from 1 to 4 Pz groups, preferably 1 to 2 Pz groups.

In related embodiments, the fluorescent dye reagents utilize a fluorescent dye which is a protected fluorescent dye. Examples of such protected fluorescent dyes are well known to those of skill in the art, and will be apparent from the description of particular embodiments below.
In some embodiments, the fluorescent dye is a protected xanthene dye having formula (Ia) or (Ib):




wherein A1 and A2 are each independently OR or N(Z)2. In one embodiment, A1 and A2 are each independently OR or N(Z)2, wherein R is selected from (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, Lf; and Z is independently selected from (C1-C8)alkyl, aryl(C1-C8)alkyl and aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl or Lf or Pz. R1′, R1′′, R1, R2, R3 and R4 are each independently selected from H, halogen, cyano, aryl, heteroaryl, sulfo, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, Lf and Pz, or optionally two adjacent members of R1′, R1′′, R2 and R3 are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic and which is optionally substituted with Pz, wherein the alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms. Within this embodiment, the symbols X1, X2, X3 and X4 are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl, heteroaryl, SO3H, PO3H2, CO2H, Lf and Pz and optionally, any two adjacent X1 through X4 are combined to form an aromatic or heteroaromatic ring that is optionally further substituted with from one to four substituents selected from halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio and (C1-C6)alkoxy. Within the above formulae (Ia and Ib), there will be from 0 to 1 Lf groups and from 1 to 2 Pz groups. Preferred groups for A1 and A2 are —O—C(O)-tButyl and —O—C(O)-isopropyl, di(C1-C8)alkylamino, N—(C1-C8)alkyl-N—C(O)(C1-C8)alkyl-amino.

In one embodiment of the invention, the fluorescent dye component (Fl) is a rhodamine dye having the formula Ic.




In formula Ic, the group Z′ or Z′′ at each occurrence is independently hydrogen, (C1-C8)alkyl, aryl-(C1-C8)alkyl or aryl, wherein the aliphatic or aryl portions of the Z′ or Z′′ groups are optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; and optionally the Z′ group at each occurrence, is independently combined with R2 or R4 to form a fused 5- to 7-membered ring, and optionally, the Z′′ group, at each occurrence, is independently combined with R1 or R1′′ to form a fused 5- to 7-membered ring. If present, said fused 5- to 7-membered ring is optionally fused to an aryl ring and is substituted with halogen, (C1-C4)alkyl, Lf or Pz. In one embodiment, the group Z′ or Z′′ at each occurrence is independently aryl-(C1-C8)alkyl or aryl, wherein the aliphatic or aryl portions of the Z′ or Z′′ groups are optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; and optionally the Z′ group at each occurrence, is independently combined with R2 or R4 to form a fused 5- to 7-membered ring, and optionally, the Z′′ group, at each occurrence, is independently combined with R1 or R1′′ to form a fused 5- to 7-membered ring. The R1′, R1′′, R1, R2, R3, R4 and R0 groups in formula Ic are the same as described above for formula I.
In certain aspects of this embodiment, the rhodamine dye component (Fl) has the formulae Ic1, Ic2, Ic3, Ic4, Ic5, or Ic6, as shown below.







In formulae Ic1-Ic6, Ra′ at each occurrence is independently selected from the group consisting of hydrogen, (C1-C6)alkyl and aryl, wherein the aryl group is optionally substituted with Pz or Lf. The substituents Rb′, Rb′′, Rc′, Rd′, Rg′, Rh′, Ri′, Rj′, Rk′, at each occurrence, are independently selected from the group consisting of hydrogen, (C1-C6)alkyl. Or optionally, any two substituents, i.e., any two of Rb′, Rb′′, Rc′, Rd′, Rg′, Rh′, Ri′, Rj′, or Rk′, located on adjacent ring atoms may be combined to form a fused 6-membered aryl ring, which is optionally substituted with Pz or Lf. In one embodiment, Ra′ is aryl which is optionally substituted with Pz or Lf. In formulae Ic1-Ic6, the subscript k and k′ are each independently an integer from 0-2; and the substituents X1—X4 are the same as set forth above for formula I.
Both “symmetrical” and “unsymmetrical” rhodamine dyes are useful in the compounds of the invention. Generally described here, symmetrical rhodamines are readily synthesized following the synthetic route shown below in Scheme A (below) starting from phthalic anhydride and a 3-aminophenol derivative following the synthetic procedures as described in (Color Index, 3rd Edition, Vol. 4: 420 (1971)). Unsymmetrical rhodamines can be synthesized following a similar synthetic route to the one described in Reaction Scheme 4 by substituting 2-(2,4-dihydroxy-3-chlorophenyl)propionic acid methyl ester in Reaction Scheme 4 with 3-Diethylamino-phenol, an example of a “3-aminophenol”.




3-Aminophenol analogs are well known in the art. Many are commercially available, or are readily accessible by following the synthetic methods reported in the literature or by methods generally known to a skilled artisan. In one embodiment, the 3-aminophenols that are useful in the the synthesis of rhodamine dyes components of the invention include, but are not limited to, those set forth in Table IA.








TABLE IA







1.
3-[methyl(phenyl)amino]phenol, 5-methyl-5,6-



dihydrophenanthridin-3-ol


2.
5-methyl-5,6-dihydrophenanthridin-1-ol


3.
1-phenylindolin-4-ol, 1-phenylindolin-6-ol


4.
1-phenyl-1,2,3,4-tetrahydroquinolin-7-ol


5.
1-phenyl-1,2,3,4-tetrahydroquinolin-5-ol


6.
5-phenyl-5,6-dihydrophenanthridin-3-ol


7.
5-phenyl-5,6-dihydrophenanthridin-1-ol


8.
2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-ol


9.
8H,13H-12b-Aza-dibenzo[a,de]anthracen-7-ol


10.
8H,9H-8a-Aza-benzo[fg]naphthacen-1-ol


11.
10-methyl-9,10-dihydroacridin-3-ol


12.
10-methyl-9,10-dihydroacridin-1-ol, 2,3-dihydro-1H


13.
7H-pyrido[3,2,10-de]acridin-4-ol


14.
2,3-Dihydro-1H,7H-pyrido[3,2,1-de]acridin-6-ol


15.
9-methyl-9H-carbazol-2-ol and 9-methyl-9H-carbazol-4-ol.









It will be appreciated by those skilled in the art that additional 3-aminophenol derivatives can be prepared from the 3-aminophenols compounds set forth above, that will also be useful for the synthesis of the rhodamine dye components (Fl) the invention. For example, other derivatives, (e.g., halogenated derivatives, and derivatives with other substituents, linkers or linking groups having the appropriate reactive groups), can be prepared from the 3-aminophenol compounds set forth in Table IA by methods known in the art and will also be useful synthetic precursors for preparing rhodamine dyes components (Fl). In one illustrative example, the halogenated derivatives of the compounds in Table IA can be prepared by, using known procedures, e.g., nitration, followed by reduction of the nitro compound to the amine, which can be converted to the halogenated analog using the Sandmeyer reaction (See, Reaction Scheme 5 which describes a similar halogenation reaction sequence on compound 28.). A specific group of halogenated 3-aminophenol derivatives that are useful for the synthesis of rhodamine dyes (Fl) are set forth in Table 1B.







TABLE IB













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 











X = halogen






It will be appreciated by those skilled in the art that the halogenated 3-aminophenols compounds shown in Table IB can be further reacted with, for example, phthalic anhydride, to form a halogenated rhodamine dye component, which can further be reacted with a compound (such as, for example, compound 3 shown in Reaction Scheme 1) using the Palladium-mediated synthetic methods described herein, to form rhodamine dye components (Fl) having formula Ic. Additionally, it will be appreciated that additional halogenated 3-aminophenols having a different halogen substitution patterns as well as additional non-halogenated derivatives can be prepared by similar methods.
Additional details concerning the syntheses of compounds having a rhodamine dye component (Fl) is provided in the Examples section of the application.
In one embodiment, the fluorescent dye (Fl) components are symmetrical rhodamine dyes, and in another embodiment, the fluorescent dye (Fl) components are unsymmetrical rhodamine dyes. In one embodiment, the symmetrical or unsymmetrical rhodamine dye components are prepared using the 3-aminophenol compounds set forth in Table IB.
In still other embodiments, the present invention provides compounds wherein Fl is a benzoxanthene dye such as, for example, a benzo[a]xanthene, a benzo[b]xanthene or a benzo[c]xanthene (see formulae II, IIa, III, IIIa, IV and IVa in FIG. 1 wherein the substituents have the meanings provided above, and in addition, subscript n in each of the formulae, is an integer of from 0 to 3 and each X0 is selected from the group consisting of halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl, heteroaryl, SO3H, PO3H2, CO2H, Lf and Pz and any aryl or heteroaryl portions of X0 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz).
Coumarin Dyes
In yet another group of embodiments, the fluorescent dye reagent has the formula:




wherein R1, R1′, R1′′, R2 and R3 are each independently selected from the group consisting of H, halogen, cyano, sulfo, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz; wherein the alkyl portions of any of R1′, R1′′ and R1 to R3 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl and heteroaryl portions of any of R1′, R1′′ and R1 through R3 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz; and A1 represents OR or N(Z)2, in which each Z is independently hydrogen, (C1-C8)alkyl, aryl-(C1-C8)alkyl, aryl or a protecting group, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz, said fused 5- to 7-membered ring is optionally fused with an aryl or heteroaryl ring which is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz. In one embodiment, the Z group is aryl-(C1-C8)alkyl, aryl or a protecting group, wherein the aliphatic or aryl protions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz. The R group of the A1 substituent in formula V is H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, protecting group or Lf. In one embodiment, the R group of the A1 substituent in formula V is a protecting group.

The benzocoumarin series (VI) of dyes are those of formula V in which R2 and R3 are combined to form a fused benzene ring, optionally substituted with one to four substituents selected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio and (C1-C6)alkoxy.
Cyanine Dyes
In still other embodiments, the present invention provides cyanine dye reagents having the general formula above wherein Fl is a cyanine dye component.
A variety of cyanine dyes are useful in the present invention and generally have the formula as provided below.




In formula VII, each of Q1 and Q2 are independently selected from O, S, N and CRaRb or —C(═CH2)— wherein Ra and Rb are independently H, methyl, ethyl or a linking group (e.g., Lf). The rings, Ax and Ay, each independently represent a condensed substituted or unsubstituted aryl or heteroaryl rings (see, for examples as represented in Table 1C below), wherein the aryl or heteroaryl rings are optionally substituted with halogen, CN, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C1-C8)alkylamino, di(C1-C8)alkylamino, an aryl or heteroaryl group that is optionally substituted with Lf or Pz. In one embodiment, the aryl or heteroaryl rings are optionally substituted with (C1-C8)alkylamino, di(C1-C8)alkylamino, an aryl or heteroaryl group that is substituted with Lf or Pz. The letter Q represents a conjugated linking system. Suitable conjugated linking systems are those below (wherein Rc, Rd, Re, Rf, Rg, Rh, Ri, Rj and Rk are each independently selected from hydrogen, halogen, —CN, —CF3, (C1-C6)alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein said aryl and heteroaryl groups, substituted or not, are optionally substituted with Lf or Pz; and Rn and Rp are each independently hydrogen, oxygen, halogen, —CN, —CF3, (C1-C6)alkyl, —NRm′, —ORm, —SRm, or —NRmRm, wherein at each occurrence, Rm is independently hydrogen, (C1-C6)alkyl, aryl, substituted aryl, heteraryl, substituted heteroaryl or Lf, wherein said aryl or heteroaryl group, substituted or not, is optionally substituted with Lf or Pz; and the subscript o is the integer 0 or 1) as well as the conjugated systems provided in the table of cyanine dyes. In one embodiment, the subscript o is the integer 0; Rn and Rp are each independently oxygen, halogen, —CN, —CF3, —NRm, —ORm, —SRm, —NRmRm; and Re, Rf, Rg, Rh, Ri and Rk, when present, are each independently selected from hydrogen, halogen, —CN, —CF3, (C1-C6)alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein said aryl and heteroaryl groups, substituted or not, are optionally substituted with Lf or Pz. In another embodiment, the subscript o is the integer 1; Rn and Rp are each independently hydrogen, oxygen, halogen, —CN, —CF3, (C3-C6)alkyl, —NRm, —ORm, —SRm, or —NRmRm; and Rc, Rd, Re, Rf, Rg, Rh, Ri, Rj and Rk are each independently selected from hydrogen, halogen, —CN, —CF3, (C1-C6)alkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, wherein said aryl and heteroaryl groups, substituted or not, are optionally substituted with Lf or Pz.




Returning to the cyanine dye formula above, the symbols RG and RG′ are independently selected from H, (C1-C8)alkyl, heteroalkyl, alkylene sulfonic acid, alkylene phosphoric acid, alkylene phosphonic acid, alkylene aryl, substituted alkylene aryl, alkylene carboxylic acid, a linking group (e.g., Lf), wherein said aryl group in RG or RG′ is optionally substituted with Pz, and wherein a compound of formula VII contains at least from about one to about two Pz groups, and from about 0 to about 2 linker, Lf, groups and wherein a linker, if present, in Ra, Rb, R, AX or AY contains a functional group such as a N, O or S nucleophile, or contains a functionality capable of reacting with N, O or S nucleophiles. In one embodiment, RG and RG′ is selected from the group consisting of alkylene phosphoric acid, alkylene, phosphonic acid, alkylene aryl and substituted alkylene aryl, wherein said aryl group in RG or RG′ is optionally substituted with Pz.
While the above formula is intended to convey the scope of cyanine dyes that can be modified according to the methods described herein, the term “cyanine dyes” or “cyanines” refers to any of the cyanine dyes that are well known in the art. Synthetic approaches have been disclosed in EP 1,065,250; WO 05/014723; WO 99/31181; U.S. Pat. Nos. 5,268,486; 5,658,751; 5,808,044; 5,981,747; 5,658,751; 4,937,198; 4,937,198; 6,080,868; 6,110,630; 6,225,050; 6,238,838; 6,716,994 and 6,207,464, as well as U.S Publication No. 2003/0113755. Representative examples of known cyanine dyes are shown in Table 1C. It will be appreciated by those of skill in the art that the synthesis of dyes of Table 1C can be modified to yield halogenated dyes which can be converted to the zwitterionic phosphonate dyes using the general methods described herein. In addition, substituents, linkers or linking groups with appropriate reactive functional groups can be incorporated in these dyes or dye analogs by other standard methods known in the art.







TABLE 1C





Known Cyanine Dyes













 








 








 








 








 








 








 








 








 








 








 








 








 


US 2004/0260093


 








 








 


WO 05118839


 








 


WO 05033245


 








 


US 6,238,838


 








 


Mishra et al, Chem. Rev., 100: 1973–2011 (2000)


 








 








 








 








 








 


WO 05118839


 








 


EP 1,408,366


 








 


US 5,658,751


 








 


Mishra et al, Chem. Rev., 100: 1973–2011 (2000)


 








 


Mishra et al, Chem. Rev., 100: 1973–2011 (2000)


 








 


Mishra et al, Chem. Rev., 100: 1973–2011 (2000)


 








 


Mishra et al, Chem. Rev., 100: 1973–2011 (2000)









Dipyrrometheneboron Difluoride Dyes (Bodipy Dyes)
Phosphonate dipyrrometheneboron difluoride dyes are provided as shown in the formula:




wherein X1, X2, X3, and X4, are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, SO3H, PO3H2, CO2H, Lf and Pz and optionally, any two adjacent X1 through X4 are combined to form a non-aromatic, aromatic or heteroaromatic five or six-membered ring that is optionally further substituted with from one to four substituents selected from halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio and (C1-C6)alkoxy. Within the above formulae (VIII), there are from 0 to 1 Lf groups and from 1 to 2 Pz groups.

Phenoxazine Dyes
In still another group of embodiments, the fluorescent dye reagents have the formula:




wherein A1 represents O, N+(Z)2 or N—Z in which Z is H, (C1-C8)alkyl, aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally each Z group is independently combined with R2 or R4 to form a fused 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused with an aryl ring, and is optionally further substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; A represents OR or N(Z)2 in which each Z is independently H, (C1-C8)alkyl, aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally each Z group is independently combined with R1 or R1′′ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused with an aryl ring, and is optionally further substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; R is H, a protecting group, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl and Lf, or is optionally or is combined with R1 or R1′′ to form a fused 5- to 7-membered ring. In one embodiment, A1 represents O, N+(Z)2 or N—Z in which Z is aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally each Z group is independently combined with R2 or R4 to form a fused 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused with an aryl ring, and is optionally further substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; A2 represents OR or N(Z)2 in which each Z is independently aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally each Z group is independently combined with R1 or R1′′ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused with an aryl ring, and is optionally further substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz.

In formula IX, the symbols R1′, R1′′, R1, R2, R3 and R4 are each independently selected from H, halogen, cyano, CF3, sulfo, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz; wherein the alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz, such that within the above formula there will be from 0 to 1 Lf groups and from 1 to 4 Pz groups, preferably 1 to 2 Pz groups.
In still other embodiments of the invention, Fl is a phenoxazine dye such as, for example, a benzo[a]phenoxazine dye, a benzo[b]phenoxazine dye, or a benzo[c]phenoxazine dye (see formula X, XI and XII in FIG. 2 wherein the substituents have the meanings provided above, and in addition the subscript n in each of the formulae is an integer of from 0 to 3 and each X0 is selected from the group consisting of halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl, heteroaryl, SO3H, PO3H2, CO2H, Lf and Pz and any aryl or heteroaryl portions of X0 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz).
Transition Metal Complexes
Phosphonate metal complexes such as 2,2′-dipyridyl-based ruthenium (II) complexed dyes are provided as shown in the formula:




wherein each of X1, X2, X3 and X4 is independently selected from H, OH, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz; wherein the alkyl portions of any of X1, X2, X3 and X4 are optionally substituted with halogen, carboxy, carboxy esters, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of X1, X2, X3 and X4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz; or optionally, any adjacent combination of two X1-4 form saturated or unsaturated substituted or unsubstituted 5- or 6-member rings which contain 0, 1 or more heterocyclic ring atoms selected from N or S. M is a metal ion selected from a lanthanide metal and a transition metal. Within the above formula (XIII), there will be from 0 to 1 Lf groups and from 1 to 2 Pz groups.

In another related embodiment one of X1-4 is Lf or Pz and the remainder of X1-4 are hydrogen.
In a still another related embodiment, M is selected from ruthenium, rhenium or osmium.
In another exemplary embodiment, the metal ion complex is stable to nucleic acid synthesis conditions, as well as nucleic acid deprotection and cleavage reagents.
Other Phosphonate-Substituted Dyes
A variety of other dyes can be modified into the dye reagents of the present invention, employing the methods described herein. Still other synthetic methods useful in the transformations are known to one skilled in the art. In particular, the dye can be prepared by, a) halogenation to allow introduction of the phosphonate group and b) be made reactive to various functional groups on biological agents or material proteins, peptides, oligonucleotides, substrates and the like (see Table 2). Known fluorescent dye structures are shown in Table 2 in the left column. An example of a halogenated dye precursor based on the dye structure in the left column is shown in the right column of Table 2. Those skilled in the art will appreciate that the phosphonate group can be introduced into any of the halogenated rings or where halogenation is possible. In addition, groups to attach the phosphonate dyes to biological materials can be introduced in various positions in dyes of Table 2. It will further be appreciated that the proposed dyes of Table 2 not only contain one or more phosphonate group, a group for the attachment of the dye to a biological or other substrate, but can optionally contain one or more substituents on any of the rings to modulate the fluorescence emission properties.







TABLE 2







Structure and Physical Properties of Known Dyes


and the Structure of Halogenated Dyes








Known Dye
Halogenated Dye Precursor















 


Acridine Orange
U.S. Pat. No. 4,060,527


λex 400 λem 500 (ethanol)


 












 


λex 420 λem 490 (ethanol)
Zeitschrift fuer Angewandte Physik, 14: 43-8 (1962)


λex 409 λem 558


 












 


(DMSO; MP)
U.S. Pat. No. 3,257,203


 












 


λex 358 λem 517 (H2O; MP)
Tetrahedron Letters 1321–1324 (1976)


 









 



J. Combinatorial Chemistry 7:463–473 (2005)


 









 



X = O or S



WO 04014366


 












 


λex 370 λem 570 (methanol; MP)
n = 8 or 2



Gaodeng Xueaiao Huaxue Xuebao 13:



1251-4 (1992) OR CAN 119:8716


 












 


λex 395 λem 601 (methanol; MP)
J de Chimie Phys. et de Physico-Chimie Biol., 75:961-8 (1978)


 












 


λex 420 λem 620 (methanol)
Materials Res. Soc. Symp.Proceed. (2001), 677


 












 


λex 400 λem 590 (benzene)
Helvitica Chim. Acta, 23: 292–302 (1940)


 









 



Helvitica Chim. Acta, 23: 292–302 (1940)


 












 


λex 485 λem 530 (toluene)


 












 


λex 410 λem 520 (Ethanol)
http://www.interchim.com


 












 


λex 380 λem 540 (water)
J. Org. Chem., 42:2426 (1977)


 












 


λex 350 λem 410, 470 (hexane)
U.S. Pat. No. 3,843,632


 









 



U.S. Pat. No. 3,843,632


 












 


λex 410 λem 430, 460, 490 (cyclohexane)
Ber., 73B: 1187-92 (1940)


 









 



Monalsh. 52: 1–6 (1929) (CAN 23:36005)


 












 


λex 350 λem 500 (water)
Internat. J. of Radiat. Biol., 66: 517-21 (1994)


 












 


λex 336 λem 450 (cyclohexane)
Tetrahedron Letters, 915–920 (1963)


 









 



Zhurnal Obshchei Khimii, 49:167–1670 (1979)


 









 



EP 1408366


 












 


λex 530 λem 480 (ethanol)
EP 1408366


 









 



EP 1408366


 









 



EP 1408366









    
    
        Unless otherwise indicated, the excitation and emission wavelengths were taken from the chemical spectra from the PhotochemCAD package by Jonathan Lindsey. (http://omlc.ogi.edu/spectra/PhotochemCAD/html/). The emission wavelengths were estimated from the emission spectra.
    
    


The halogenated dye intermediates shown in Table 2 can be converted to the phosphonate dyes by the methods of the invention. It will be apparent to those skilled in the art that fluorescent dyes reported in the art can be converted to the corresponding halogenated dye by a variety of methods. One such method is to start with a halogenated dye intermediate or in some instances it will be possible to introduce a halogen substituent into one of its aromatic rings. It will be further apparent that a linker moiety can be introduced into the dye molecule by methods known in the art or those taught by this invention. Halogenation of aromatic rings with chlorine and bromine is well known in the art, for a review see Breandlin and McBee, in Olah, “FRIEDEL-CRAFTS AND RELATED REACTIONS” Vol 3, pp 1517-1593, Interscience Publishers, Inc. NY, 1964 and Berliner, J. Chem. Educ. 43:124-132 (1966)). Alternatively the halide group can be introduced into a aromatic ring via diazotization of an amino group using the Sandmeyer reaction (Hodgson, Chem. Rev., 40:251-277(1947)). It will further be apparent to those skilled in the art that aryl and heteroaryl moieties present in the dyes of Table 2 can be substituted. Substituents can be introduced in the dyes by methods known in the art. As a result, the structure analogs (e.g., those optionally incorporating Lf and Pz) of the dyes of Table 2 are also contemplated as specific embodiments of the present invention.
Energy Transfer Dyes
Phosphonate-substituted energy transfer dyes are another example of the dye reagents of the present invention. One example of such energy transfer dyes is the electron-donating and electron-accepting xanthene dye couple shown below. One of skill in the art will appreciate that other dye combinations are also part of the present invention.




More particularly, the electron transfer dyes employing xanthene moieties can include essentially any of the xanthene dyes (e.g. those provided above in Formulae I, Ia and Ib) or the benzoxanthene dyes. The linker portion can be a variety of linkers, generally having from about 3 to 50 main atoms selected from C, N, O, P and S which is either cyclic, acyclic, aromatic or a combination thereof, and having additional hydrogen atoms to fill available valences. Examples of energy transfer dyes have been disclosed in U.S. Pat. Nos. 5,800,996; 5,863,727 and 5,565,554; and in European Patent No. 0 747 700. Table 4 illustrates a few examples of energy transfer dyes that can be modified according to the methods described herein to attach a phosphonate moiety (and optionally a further linking group as provided in the general formula above).







TABLE 4







Examples of Dimeric Fluorescence Energy Transfer Dyes


Example











 








 








 








 








 








 








 








 















Synthetic procedures for the dimeric energy transfer dyes shown in Table 4 have been described in, for example, U.S. Pat. Nos. 5,800,996, 5,863,727 and 5,565,554, and in European Patent No. 0 747 700. Fluorescent energy transfer tags that contain more than two fluorescent moieties are described in U.S. Pat. No. 6,627,748. The dyes in Table 4 contain at least one xanthene moiety or at least one cyanine moiety and the introduction of a phosphonate group into the xanthene- or cyanine-dyes can be readily accomplished by the methods described herein.
Thiazole Orange Analogs
In still other embodiments, phosphonate-substitued thiazole orange analogs are provided as shown in the formula




wherein X2, X3, X4, X5, X6, X7, X8, X9, X10 and X11 are each independently selected from H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz; or optionally two adjacent members of X2 through X11 are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic and with is optionally substituted with Pz; and X1 and X12 are each independently H, (C1-C6)alkyl, aryl and substituted aryl; wherein the alkyl portions of any of X1 through X12 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of X1 through X12 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz.

N-Aryl-1,8-naphthalimides
Phosphonate-Aryl-1,8-naphthalimides analogs are provided as shown in the formula XV




wherein X1, X2, X3, X4, X5, X6 are each independently selected from H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz; or optionally two adjacent members of X1 through X6 are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic and with is optionally substituted with Pz; and X7 is aryl or heteroaryl; wherein the alkyl portions of any of X1 through X6 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of X1 through X7 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz.

Polymethine-Phosphonate Dyes
In still other embodiment, polymethine-based phosphonate dyes of Formula (XVIa-XVIc) are provided. Polymethine-based phosphonate dyes and their synthesis are described in US Publication No. 2004/0260093, incorporated herein in its entirety for all purposes.




In formulae XVIa-XVIc, A represents OR or N(Z)2 in which each Z is independently hydrogen, (C1-C8)alkyl, aryl-C1-C8 alkyl, a protecting group or aryl; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with Rcc or Rdd to form a fused 5-, 6- or 7-membered ring, wherein the resultant fused 5- to 7-membered ring is optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz, and is optionally fused with an aryl ring; the R group is hydrogen, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a protecting group or Lf. The substituent Ra′ at each occurrence is independently selected from the group consisting of hydrogen, (C1-C6)alkyl and aryl, wherein the aryl group is optionally substituted with Pz or Lf. The substituents Raa, Rbb, Rcc, Rdd, Ree, Rff, Rgg, Rhh and Rii are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, (C1-C8)alkanoic acid, (C1-C8)alkylsulfonic acid, aryl, heteroaryl, Lf and Pz; optionally any two of the Raa, Rbb, Rcc, Rdd, Ree, Rff, Rgg, Rhh, Rii and Ra′ substituents are combined to form a five or six-membered fused ring that is aromatic, non-aromatic or heteroaromatic and is optionally substituted with Pz. The alkyl portions of any of Raa, Rbb, Rcc, Rdd, Ree, Rff, Rgg, Rhh, and Ra′ are optionally substituted with halogen, carboxy, sulfo, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkoxy, cyano, haloacetyl or hydroxy. The aryl or heteroaryl portions of any of Raa, Rbb, Rcc, Rdd, Ree, Rff, Rgg, Rhh, Rii and Ra′ are optionally substituted with from one to four substituents selected from the group consisting of Pz, halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz. The group, -D-E-, is selected from the group consisting of —C(O)—O—, —O—C(O)—, —C(O)—NRa′′ or —NRa′—C(O)—, wherein Ra′′ is hydrogen, (C1-C6)alkyl or aryl, wherein the aryl group is optionally substituted with Pz or Lf. The substituent Rn and Rn′ is independently selected from the group consisting of hydrogen, (C1-C6)alkyl and aryl, wherein the aryl group is optionally substituted with Pz or Lf. The subscript nn is an integer from 1 to 3, and the subscript k in formulae XVIa-XVIc is an integer from 1 to 2. There is the proviso that any compound having formulae XVIa-XVIc contain at least one Pz.
In a related aspect, the present invention provides fluorescent dye reagents having the general formula (B):

Fl-(P1)n  (B)

wherein Fl is a fluorescent dye component; the subscript n is an integer from 1 to 4; and P1 is functionalized phosphonate group having the formula:





wherein the wavy line indicates the direct attachment to a sp2 carbon of the fluorescent dye component; L1 is a linking group; R is selected from H, (C1-C8) alkyl, aryl, aryl(C1-C4)alkyl, -La-Na, and -La-NRARB; wherein La is an alkylene linking group, Na is an ammonium ion group, and each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group, such as C(O)CF3, FMOC, C(O)t-Butyl, phthalimide and monomethoxytrityl; and Pa is a functional group component selected from the group consisting of a phosphoramidite moiety, a mono-, di- or tri-functional linking group having at least one terminal functional group or protected functional group, a solid support and a reactive group (e.g., an electrophilic or nucleophilic group as described below); and salts thereof. For those embodiments in which R is alkyl, preferred alkyl groups are those that can be removed in the presence of other functional groups in the molecule (e.g., tert-butyl, tert-amyl, methyl). In one embodiment the subscript n in formula B is an integer from 2 to 4.

As with the fluorescent dye reagents described initially, this aspect of the invention includes a variety of fluorescent dye components (Fl) that are typically selected from coumarins, benzocoumarins, xanthenes, benzo[a]xanthenes, benzo[b]xanthenes, benzo[c]xanthenes, cyanines, acridines, dipyrrometheneboron difluorides, phenoxazines, benzo[a]phenoxazines, benzo[b]phenoxazines and benzo[c]phenoxazines. In some embodiments, the fluorescent dye component is selected from coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, thiazole orange dyes, N-aryl-1,8-naphthalimide dyes, polymethine-phosphonate dyes, merocyanines, stilbenes, styryl-based dyes and dipyrrins. In still other embodiments, the fluorescent dye component is selected from fluorescein dyes, rhodamine dyes and rhodol dyes. More particularly, the fluorescent dye can be any of the dyes provides in formulae I, Ia, Ib, II, IIa, III, IIIa, IV, IVa, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI or the dyes of Tables 1C or 2, wherein P1 is attached to an sp2-hybridized carbon atom at a position as described above for Pz.
The phosphonate moiety can be attached to the dye component using methods described herein (e.g., via phosphonylation of a halogen-dye component intermediate) followed by suitable synthesis steps in which the phosphonate is provided with a linkage to a terminal group (e.g., Pa).
General Synthesis of Phosphonylated Dyes
In still another aspect, the present invention provides a method of preparing a phosphonylated-fluorescent dye derivative, the method comprising contacting a halo-fluorescent dye substrate having at least one halogen atom attached to an aromatic ring carbon atom, with a phosphite reagent under conditions sufficient to remove the halogen atom and covalently attach a phosphonate group to the aromatic ring carbon atom to form the phosphonylated-fluorescent dye derivative.
In a number of embodiments, the halo-fluorescent dye substrate is selected from halo coumarins, halo benzocoumarins, halo xanthenes, halo benzo[a]xanthenes, halo benzo[b]xanthenes, halo benzo[c]xanthenes, halo cyanines, halo acridines, halo dipyrrometheneboron difluorides, halo phenoxazines, halo benzo[a]phenoxazines, halo benzo[b]phenoxazines and halo benzo[c]phenoxazines.
The phosphite reagents of the invention can be symmetric or unsymmetric. Symmetric phosphite reagents are those having the formula: HP(═O)(OR)2, wherein each of the phosphite ester R groups is -La-NRARB; wherein La is an alkylene linking group, and each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group; and at least one of RA and RB is a protecting group. Examples of such protecting groups are C(O)CF3, phthalimide, C(O)Ot-Bu, C(O)OBn, monomethoxytrityl, FMOC, and the like.
Unsymmetric phosphite reagents are those having the formula: HP(═O)(OR)(OR′), wherein the phosphite ester R′ group is a protecting group that can be removed in the presence of R or is -La-NRARB; and the R group is -L1-Pa or -La-NRARB; wherein L1 is an alkylene linking group, and Pa is a protected or unprotected functional group. For each of R and R′, La is an alkylene linking group, and each of RA and RB is independently selected from the group consisting of H, (C1-C8)alkyl and a labile protecting group; and at least one of RA and RB is a protecting group. Examples of useful R′ ester groups are t-butyl, benzyl, phenyl, and the like. For those embodiments in which R is also -La-NRARB; wherein each of RA and RB is independently H, (C1-C8)alkyl or a labile protecting group; and at least one of RA and RB is a protecting group, examples of such protecting groups are C(O)CF3, phthalimide, C(O)Ot-Bu, C(O)OBn, monomethoxytrityl, Fmoc and the like. One of skill in the art will appreciate that for those embodiments in which RA or RB is a protecting group, and the R′ group is an ester (or protecting group) and the RA or RB groups are orthogonal protecting groups (i.e., protecting groups that can be selectively removed in the presence of the other protecting group).
In view of the above, the present invention provides a compound having the formula:




wherein L is a member selected from the group consisting of a (C1-C8)alkylene and (C1-C8)heteroalkylene linking group; X is a member selected from the group consisting of a functional group and a protected functional group wherein said functional group is hydroxyl, carboxy, thio, sulfonic acid or an amino having the formula NRARB; R is a member selected from the group consisting of a labile protecting group and -L-NRARB; and each of RA and RB is independently selected from H, (C1-C8)alkyl and a labile protecting group and at least one of RA and RB is other than H. In some embodiments, R is tertiary butyl. In other embodiments, R is -L-NRARB, and X is —NRARB. More preferably, each RA is H and each RB is a protecting group, preferably selected from —C(O)CF3, —C(O)OtBu, FMOC, phthalimide and —C(O)O-benzyl. In other embodiments, X is —O-dimethoxytrityl (DMT). In the most preferred embodiments, the compound is selected from:





While the above structures are provided in a single tautomeric form, one of skill in the art will appreciate the recitation is further meant to include all forms, including the commonly used trivalent phosphite form.

More specific schemes for the preparation of selected phosphite reagents and for their use in preparing fluorescent dye reagents of the present invention are provided in the schemes below.
Reagents for Phosphonylated Dye Synthesis and Preparation of Phosphonylated Dyes
Reaction Scheme 1 illustrates the synthesis of a symmetric phosphite reagent bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite (3).




2,2,2-Trifluoro-N-(4-hydroxybutyl)acetamide (1) was reacted with diphenyl phosphite (2) in pyridine to yield phosphite 3. This reagent was used to prepare aromatic bis-trifluoroacetamidophosphonate dyes of the invention. Deprotection of the diester in concentrated NH4OH generates the zwitter ionic 4-aminobutylphosphonate group.







Reaction Scheme 2 illustrates the synthesis of a phosphonylated pentachlorofluorescein phosphoramidite 11. The methyl ester 4 was reacted with 4-bromobenzene-1,3-diol in the presence of methylsulfonic acid to yield the intermediate 5 which was converted with trifluoroacetic acid/trifluoroacetic acid anhydride to the lactone 6. Lactone 6 was then reacted with 6-aminohexanol and dimethoxytrityl to form the DMT-intermediate 7 which was then reacted with the phosphite reagent 3 to yield the phosphonate ester 8. The ester 8 was treated with trimethylacetic anhydride to protect the hydroxyl groups and after SiO2 purification yielded the fully protected dye analog 9. Removal of the DMT-protecting group with TFA treatment yielded the alcohol 10 which on treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite (Aldrich, St. Louis, Mo.) resulted in the desired phosphoramidite 11.










Reaction Scheme 3 illustrates the synthesis of a fully protected phosphonylated benzofluorescein phosphoramidite 19. 4-Bromophthalic anhydride and methyl-3-(3-chloro-2,4-dihydroxyphenyl)propionate were combined in the presence of AlCl3 yielded the benzophenone 12 which was reacted to with 1,3-dihydroxynaphthalene to form two isomers of the fluorescein analog 13. Intermediate 13 was converted with trifluoroacetic acid/trifluoroacetic acid anhydride to the lactone 14. The lactone was then reacted with O-dimethoxytrityl-6-aminohexanol (Tetrahedron Letters, 2004, 45(2):317-320) to form the DMT-intermediate isomers which were separated on silica gel to yield the pure major 6-isomer 15. The 6-isomer 15 was then reacted with trimethyl acetic anhydride to yield the fully protected analog 16 which was reacted with the phosphite reagent 3 to yield the phosphonate ester 17. Removal of the DMT-protecting group with TFA treatment yielded the hydroxyl derivative 18 which on treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite resulted in the desired phosphoramidite 19.
Reaction Scheme 4 illustrates the synthesis of a fully protected 6-phosphonyl rhodol 27.










The reaction of 4-bromophthalic anhydride and 8-hydroxyjulolidine yielded the benzophenone 20 which was reacted with methyl 3-(3-chloro-2,4-dihydroxyphenyl)propionate to form the rhodol analog 21 which was converted with trifluoroacetic acid/trifluoroacetic acid anhydride to the lactone 22. The lactone was then reacted with O-dimethoxytrityl-6-aminohexanol to form the DMT-intermdiate 23 which was then reacted with trimethylacetic anhydride to yield the fully protected analog 24. Isomers were separated on silica gel and the 6-isomer was carried on. This analog was reacted with the phosphite reagent 3 to yield the phosphonate ester 25. Removal of the DMT-protecting group with TFA treatment yielded the alcohol 26 which on treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite resulted in the desired phosphoramidite 27.
Reaction Scheme 4b Illustrates the Synthesis of a 3-aminophenol Compound.




The synthesis of 1-(4-iodophenyl)indolin-4-ol (92) is provided in Reaction Scheme 4b. The reaction of 1-fluoro-4-nitrobenzene and 4-methoxy-1H-indole in the presence of Cs2CO3 gave 4-methoxy-1-(4-nitrophenyl)-1H-indole (88), which was reduced with H2/Pd to yield 4-(4-methoxy-1H-indol-1-yl)benzenamine (89). The amino group of 89 was converted via the Sandmeyer reaction to 1-(4-iodophenyl)-4-methoxy-1H-indole (90) which on reduction with sodium borocyanohyride gave 1-(4-iodophenyl)-4-methoxyindoline (91). The indoline 91 was demethylated with BBr3 to provide the desired 92.
Reaction Scheme 5 illustrates the synthesis of 4-(4-Iodophenyl)resorcinol (31).




2,4-Dimethoxyphenylboronic acid and 1-bromo-4-nitrobenzene in the presence of tetrakis(triphenylphosphine)palladium yielded 4-(4-nitrophenyl)-2,4-methoxybenzene (28). Hydrogenation of 28 in the presence of a palladium catalyst yielded the crude amine (29) which without purification was diazotized and treated with KI solution to yield the desired 4-(4-iodophenyl)-2,4-dimethoxybenzene (30). The dimethoxy analog 30 was treated with boron tribromide to yield the desired 4-(4-iodophenyl)resorcinol (31).










Reaction Scheme 6 illustrates the synthesis of phosphoramidite 39. The reaction of 3-chlorophthalic anhydride and methyl 3-(3-chloro-2,4-dihydroxyphenyl)propionate in the presence of AlCl3 to form the benzophenone analog, which on crystallization yielded the pure 6-chloroisomer 32. This isomer was reacted with 31 to yield the 7′-(4-iodophenyl) substituted fluorescein analog 33 which was treated with trimethyl acetic anhydride and N-methylimidazole to give the lactone 34. The lactone was then reacted with O-dimethoxytrityl-6-aminohexanol to form the DMT-intermediate 35 which was then reacted with trimethylacetic anhydride to yield the fully protected dye 36. This dye intermediate 36 was reacted with the phosphite reagent 3 to yield the phosphonate ester 37. Removal of the DMT-protecting group with TFA treatment yielded the alcohol 38 which on treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite resulted in the desired phosphoramidite 39.
Synthesis of a useful unsymmetric phosphite reagent 41 is shown in Reaction Scheme 7. Reagent 41 allows introduction of reactive linking groups such as phosphoramidite through the phosphonate moiety.




In an example, phosphite 41 was used for the preparation of a phosphonate-substituted fluorescein phosphoramidite 46 as shown in Reaction Scheme 8.







Reaction Scheme 8 illustrates the synthesis of phosphoramidite 46. The reaction of 4-bromophthalic anhydride and resorcinol in the presence of methanesulfonic acid yielded a mixture of 5- and 6-isomers of bromofluorescein 42 which was treated with trimethylacetic anhydride and N-methylimidazole to give after silica gel chromatography, the pure 6-isomer of the blocked diester 43. Intermediate 43 was reacted with intermediate 41 to yield the phosphonate 44 which was then treated trifluoroacetic acid to yield the alcohol 45. The alcohol on treatment with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite resulted in the desired phosphoramidite 46.
Synthesis of Phosphonate-Substituted Cyanine Dye Reagents
Reaction Scheme 9 illustrates the synthesis of a phosphonate-substituted cyanine phosphoramidite 53.







The reaction of 2,3,3-trimethylindolenine and 1,4-butane sultone (5.4 g, 40 mmol) yielded the desired indolinum salt 47 which reacted with N,N′-diphenylformamidine to afford formamidine 48. Reaction of 48 with 1-(sulfobutyl)-2,3,3-trimethyl-5-bromoindolinum, inner salt 49 (see, J. Heterocycle Chem., 39:263-269 (2002)) afforded the cyanine intermediate 50. Intermediate 50 was reacted with the phosphite 41 to yield the phosphonate 51 which was then treated with trifluoroacetic acid to yield the alcohol 52. The alcohol on treatment with 2-cyanoethyl N,N,diisopropylchlorophosphoramidite resulted in the desired phosphoramidite 53.
Reaction Scheme 10 shows the preparation of a solid support-bound dye reagent 59 of the present invention.










Reaction Scheme 10 illustrates the synthesis of the cyanine solid support-bound dye reagent 59. The reaction of 5-iodo-2,3,3-trimethyl-3H-indole with 1,3-propanesultone provided the desired indolinum salt 54, which reacted with N,N′-diphenylformamidine to afford cyanine intermediate 55. Intermediate 55 was reacted with the phosphite 41 to yield the dimethoxytrityl-phosphonate 56 which was then treated with trifluoroacetic acid to yield the monomethoxytritylphosphonate intermediate 57. Intermediate 57 was first reacted with succinic anhydride and then with PFP-TFA to yield the PFP-active ester 58. The PFP ester was then reacted with aminoalkyl control pore glass (NH2—CPG) to give the desired solid support 59.
Reaction Scheme 11 shows the preparation of an activated ester-phosphonate-substituted cyanine dye reagent of the present invention.




In Reaction Scheme 11, 5-iodo-2,3,3-trimethyl-3H-indolenine was reacted with either tert-butyl 6-iodohexanoate or with ethyl iodide to give indolinum salts 60 and 61, respectively. Compound 61 was then converted to intermediate 62 by reaction with malonaldehyde bis(phenylimino)monohydrochloride in the presence of acetyl chloride and acetic anhydride. Condensation of 61 and 62 resulted in dye 63 which was then phosphonylated by reaction with phosphite 3 in the presence of tetrakis(triphenylphosphine)palladium and N-ethylmorpholine to give phosphonate dye 64. To remove the protective, tert-butyl group, compound 64 was treated with trifluoroacetic acid. The resultant acid 65 was then converted into PFP ester 66 by reaction with pentafluorophenyl trifluoroacetate.
The synthetic route described in Reaction Scheme 11 can be used to prepare compounds 67 to 70 set forth below in Table 3.







TABLE 3





Preferred Phosphonate-Substituted Cyanine Phosphoramidites

















67













 








 









68













 








 









69













 








 









70













 















Reaction Scheme 12a shows the preparation of the phosphonylated cyanine dye reagent (80) in a form of an activated ester.







In reaction scheme 12a, the indolinium salt 54 was reacted with 3,4-dibutoxy-3-cyclobutene-1,2-dione and the resultant crude product was hydrolyzed to yield the mono-substituted intermediate 77, which was in turn was reacted with the indolinium salt 60 to give the unsymmetrical dye 78. The phosphonylated dye 79 was obtained by reaction with the phosphite 3. The carboxylate ester group of dye 79 was deprotected by treatment with trifluoroacetic acid and then converted into the PFP (pentafluorophenyl) ester to yield the desired activated dye derivative 80.
Reaction Scheme 12b illustrates the synthesis of intermediates 85 and 86.







Intermediates 85 and 86 can be used as starting materials for the synthesis of activated cyanine dye ester 87 by following the synthetic route outlined in Reaction Scheme 11b and substituting compounds 54 and 60 with compounds 85 and 86, respectively. Palladium catalyzed reaction of compound 81 (see, WO 02/085854) with 1-bromo-4-nitrobenzene afforded nitrophenylindolenine 82. Following transformations included hydrogenation and diazotation-iodination reactions and provided common intermediate 84 which was further alkylated either by 1,4 butanesulton or tert-butyl 6-iodohexanoate to give compounds 85 and 86, respectively.

Reaction Scheme 13 illustrates the deprotection of the protected phosphonylated dyes that is attached to a oligonucleotide.




The protected oligonucleotide-dye-phosphonate conjugate (71 and 74) can be synthesized with any of the phosphoramidites of the invention. Ammonium hydroxide deprotection yields the oligonucleotide phophonate-dye conjugates (73 and 76).
The novel bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite (3) reagent can be used in the synthesis of other dyes of the present invention.
In the sections below, Schemes are provided for exemplary syntheses of various dyes. One of skill in the art will appreciate that the invention is not limited to these methods. In addition, those skilled in the art will appreciate that the proposed reaction schemes may have to be modified to achieve the desired products in Reaction Schemes 14 to 19.




6-Substituted phosphonate coumarin dyes can be synthesized starting from the protected 6-bromo-7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (1) ((for synthesis see Baker, et al., Chem. Soc. 170-173 (1970)) as shown in Reaction Scheme 14. Compound 1 is reacted with 5-(bis(4-methoxyphenyl)(phenyl)methoxy)pentan-1-amine to yield DMT-amide analog 2. Reaction of 2 with bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite (3) provides the protected phophonate-substituted dye 4. In the next step the dimethoxytrityl protecting group is removed to yield the alcohol 5 which can be converted to the protected phosphoramidite 6.
The synthesis of 8-substituted halogen coumarin analogs was disclosed in WO03023357. The 8-bromocoumarin lactone 7 can be converted in a similar fashion described in Reaction Scheme 14 to yield the equivalent phosphoramidite 8 shown in Reaction Scheme 14a.







The synthesis of halogenated resorufin lactones 9 was disclosed in WO 03/023357. Briefly described here, the reaction of nitrosobenzene-1,3-diol and methyl 3-(3-bromo-2,4-dihydroxyphenyl)propanoate in the presence of acidic MnO2 yields the resorufin-N-oxide (9) which can be reduced to the bromo-resorufinpropionic acid 10 (Reaction Scheme 15). This compound was lactonized to provide compound 11 and can be converted to the phosphonate lactone analog 12 similarly as described in Reaction Scheme 15. The bromoacid (10) can also be converted to the phosphonate resorufin phosphoramidite 13.




The dipyrrometheneboron difluoride parent heterocyclic compounds with a reactive group for attachment to biological materials were first disclosed in U.S. Pat. No. 4,774,339. Dipyrrometheneboron difluoride products were reported to be modifiable in a subsequent reaction by chemical techniques known to one skilled in the art including but not limited to sulfonation, nitration, alkylation, acylation, and halogenation. It was further reported that the substituents can in some cases be further modified to introduce chemically reactive functional groups. Briefly these substituted dipyrrometheneboron difluorides are prepared as shown in Reaction Scheme 16. A substituted pyrrole-5-carboxaldehyde was reacted with a substituted pyrrole in the presence of hydrobromic acid to yield the substituted dipyromethene intermediate 14 which was converted to substituted dipyrrometheneboron difluoride dye 15. The symbols X1, X2, X3, and X4 have the same definition as described previously for this class of dyes.




Dipyrometheneboron difluoride phosphonate dyes of interest can be synthesized as shown in Reaction Scheme 17. The reaction starting from 4-bromopyrrole-2-carboxaldehye and 3-(2,4-dimethyl-1H-pyrrol-3-yl)propanoic acid yields the bromo-intermediate 14c, which with BF3 base yields the bromodipyrrometheneboron difluoride 15c. Dye 15c can further be converted via a phosphonate intermediate to the phosphonate dipyrometheneboron difluoride intermediate 16 where R* can be a solid support or a phosphoramidite.
In another embodiment carboxylic group in intermediate 15c can be converted in to a reactive moiety such as a pentafluorophenyl- or a succinimidyl esters.
Pyrrole-2-carboxaldehydes needed for the synthesis of pyrrolomethenes are either commercially available or readily prepared from the corresponding pyrroles by the Vilsmeyer Haak formylation (R. M. Silverstein, E. E. Ryskiewicz and C. Willard. ORG. SYNTH. COLL VOL IV, page 831).
Appropriately substituted pyrrole derivatives (Reaction Scheme 17) are either commercially available or may be synthesized by methods known in the art, for example as described (R. Elderfield, HETEROCYCLIC COMPOUNDS, vol. 1, ch. 6, THE CHEMISTRY OF PYRROLE AND ITS DERIVATIVES, by Corwin (1950); Silverstein, Ryskiewicz and Willard. ORG. SYNTH. COLL VOL IV, page 831 and Korostova et al., Russ. J. Org. Chem. 34, 1691 (1998)). Pyrrole fatty acids are most conveniently prepared by the Wittig reaction (see, Wittig & Schollkopf, Ber. 87: 1318 (1954)) of pyrrole aldehydes with carboxy or protected-carboxy terminated phosphonium or phosphonate derivatives.
Ruthenium (II) Complexed Phosphonate Dyes
The synthesis of mixed ligand ruthenium (II) complexed phosphoramidite dyes have previously been reported starting from commercially available cis-Bis(2,2′-bipyridyl)ruthenium(II)chloride dehydrate (Khan et al, Inorg. Chem. 38:3922-3925 (1999)). Starting with cis-Bis(2,2′-bipyridyl)ruthenium(II)chloride dehydrate (18a) and (2,2′-bipyridine)-5-carboxylic acid, 5′-bromo-methyl ester (17; Haino et al, Chem. Comm., 402-403 (2002)). The Khan et al procedure allows the synthesis of a ruthenium bipyrydyl phosphoramidite 19 as shown in Reaction Scheme 18.




Briefly the standard procedure converts the methyl ester 17 to the blocked phosphonate bipyridyl alcohol 18b, which reacts with 18a followed by the introduction of the phosphoramidite as taught by Khan et al, can provide phosphonate bipyridyl ruthenium phosphoramidite 19.
In another embodiment carboxylic group in compound 19 can be converted in to a reactive moiety such as a pentafluorophenyl- or a succinimidyl esters instead of the phosphoramidite group.
Polymethine-Based Phosphonate-Substituted Dyes
The phosphonate polymethine-based dyes can be synthesized by the basic method disclosed in US 2004/0260093. The approach is briefly outlined in Reaction Scheme 19.




The reaction of 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde (20a) with an active —CH compound 20b in the presence of Ac2O provides a bromo-intermediate 21 which on reaction with phosphate reagent 3 provides blocked phosphonate intermediate 22. This intermediate can be converted to the phosphoramidite reagent 23 in the usual fashion.
In a related embodiment carboxylic group in intermediate 23 can be converted to a reactive moiety such as a pentafluorophenyl- or a succinimidyl esters instead of the phosphoramidite group.
In yet another embodiment, in fluorescent dye reagents having formulae XVIa-XVIc, the phosphonate group (Pz) on compound 23 is alternatively is located on the 2-oxo-2H-chromene group in the compound. Compounds with this substitution of Pz can be prepared by starting with a halogen substituted 2-oxo-2H-chromene-3-carbaldehyde reagent. The halogen group can be substituted with the phosphonate group in any one of the sp2-hybridized carbon positions.
Additional analogs of 7-(amino)-2-oxo-2H-chromene-3-carbaldehyde (20a) and CH-active heteroaryl analogs 20b are available either from a commercial vendor or can be synthesized by methods known in the art. Appropriate halogenated analogs of 20a and 20b can be converted to the phosphonate derivatives using the methods described herein. CH-active pyridine and 3H-indole analogs are either commercially available or can be synthesized by methods known in the art. Certain 7-(amino)-2-oxo-2H-chromene-3-carbaldehyes that are useful for the synthesis of compounds of formulae XVIa-XVIc are shown in Table 5.







TABLE 5













Sonawane, N. et al., J. Heterocyclic Chem., 39: 303–308 (2002)


 








 


JP 2004323394


 








 


Kirpichenok, M. et al., Khimiya


Geterotsiklicheskikh Soedinenii, 1480-7 (1991)


 








 


Lim, N. et al., Inorg. Chem., 44:2018–2030 (2005).


 








 


Bakulev, V et al., Khimiya


Geterotsiklicheskikh Soedinenii, 338-48 (1993)


 








 


DE 2413281









Oligonucleotide Probes and Other Labeled Biological Agents
In another aspect, the present invention provides oligonucleotide probes and other biological agents in which one or more of the fluorescent dyes above have been attached to a nucleic acid, polynucleotide, oligonucleotide and the like. As noted above, the present invention finds broad application in labeling of nucleic acids (including nucleotides, nucleosides, DNA, RNA, PNA, locked nucleic acids, oligonucleotides and the like), peptides or proteins, oligosaccharides, glycosylated proteins, and other biological agents. Additionally, the nucleic acids can include modified bases (e.g., 5-substituted pyrimidines, 3-substituted purines, substituted deazapurines, substituted pyrazolo[3,4-d]pyrimidines, and the like). See, for example, co-pending application Ser. Nos. 09/724,988 and 09/447,936. The invention also finds utility in labeling of oligonucleotides and modified oligonucleotides having attached groups such as minor groove binders, quenching agents or quenchers, intercalators, crosslinking groups, and the like.
In one embodiment of the invention, the phosphonate dyes contain at least one group -L-RX where RX is the reactive group that is attached to the fluorophore by a covalent linkage L. In certain embodiments, the covalent linkage attaching the phosphonate dye to RX contains multiple intervening atoms that serve as a spacer. The dyes with a reactive RX group fluorescently label a wide variety of organic or inorganic substances that contain or are modified to contain functional groups with suitable reactivity, resulting in chemical attachment of the conjugated substance (RY), represented by -L-RY. The reactive group and functional group are typically an electrophile and a nucleophile that can generate a covalent linkage. Typically, the conjugation reaction between the reactive dye and the substance to be conjugated results in one or more atoms of the reactive group RX to be incorporated into a new linkage L attaching the phosphonate dye to the conjugated substance RY.
Selected examples of functional groups involved to form linkages where the reaction of an electrophilic group and a nucleophilic group yields a covalent linkage are shown below.
Examples of nucleophilic groups include —NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, —OH, —COOH, or —SH. The electrophilic groups are activated esters, acrylamides, acyl azides, acyl halides, aldehyde or ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boranates, carboxylic acids, carbodiimides, diazoalkanes, epoxides, haloacetamides, halotriazines, imidoesters, isocyanates, isothiocyanates, maleimides, phophoramidites, silyl halides, sulfonate ester and sulfonyl halides. Additionally, a spacer can include hetero atoms in linear or acyclic portions, cyclic portions, aromatic rings or combinations thereof.
Conjugated substances include nucleic acids, oligonucleotides, oligonucleotide conjugates, proteins, peptides, drugs, inmmunoglobulins, receptors, toxins, organic small molecule ligands, enzyme substrates, vitamins, carbohydrates, biotin, streptavidin, solid substrate, and a solid support for oligonucleotide synthesis) described and used herein.
In one embodiment, conjugates of biological polymers such as peptides, proteins, oligonucleotides, nucleic acid polymers are also labeled with a second fluorescent or non-fluorescent dye, including an additional dye of the present invention, to form an energy-transfer pair.
In yet another embodiment, conjugates of biological polymers such as peptides, proteins, oligonucleotides, nucleic acid polymers are also labeled with a second fluorescent or non-fluorescent dye, in addition to the dye of the present invention, to form an energy-transfer pair where the fluorescence of the latter is quenched. Binding of these conjugated to their natural receptor or complement result in conformational change or cleavage of bond with an increase in fluorescence. Preferred selected examples of dual labeled oligonucleotide probes are 5′-(MB)ny-FlA-oligonucleotide-FlB-3′, 5′-FlA-oligonucleotide-FlB-MB-3′ where FlA and FlB are either a fluorophore or a quencher with the proviso that a probe can contain only one quencher and one fluorophore, MB is a minor groove binder and ny is 0 or 1. In one embodiment the quencher is non-fluorescent.
In a related embodiment the preferred conjugate probes are used in amplification methods to detect nucleic acid targets, nucleic acid polymorphisms and gene expression analysis. These methods are disclosed in U.S. Pat. No. 6,312,894, WO 2004/018626, Livak, K J and Schmittgen, T D. Methods 25: 402-408 (2001).)
Examples of minor groove binders are disclosed U.S. Pat. No. 5,801,155 and quenchers in U.S. Pat. No. 6,699,975 and WO02099141 all which are included by reference in their entireties.
In another embodiment, the conjugated substance is a nucleic acid base, nucleoside, nucleotide or a nucleic acid polymer, including those that were modified to possess an additional linker or spacer for attachment of the dyes of the invention, such as an alkynyl linkage (U.S. Pat. No. 5,047,519; U.S. RE 38,416), an aminoallyl linkage (U.S. Pat. No. 4,711,955) or other linkage.
In one embodiment a xanthenephosphonate (Xp) is attached to a solid support through a cleavable linker. The linker molecule also contains a hydroxyl group protected with DMTr (or like) blocking group. After removal of the DMTr group, an oligonucleotide is synthesized on an automated oligonucleotide synthesizer by step-wise attachment of nucleotide units to the hydroxyl group. A quencher is introduced at the 5′-end with the appropriate phosphoramidite, or post-synthetically with a quencher containing a reactive group, to yield an oligodeoxynucleotide (ODN) having an attached xanthenephosphonate moiety (XP) and a quencher (Q). A solid support compatible with oligonucleotide synthesis includes controlled pore glass, polystyrene, plastic, nylon, gold and the like.
In one embodiment a quencher is attached to a solid support through a cleavable linker. The linker molecule also contains a hydroxyl group protected with DMTr (4,4′-dimethoxytrityl) (or like) blocking group. After removal of the DMTr group, an oligonucleotide is synthesized on an automated oligonucleotide synthesizer by step-wise attachment of nucleotide units to the hydroxyl group. A XP fluorophore is introduced at the 3′-end with the appropriate phosphoramidite, or post-synthetically with a XP fluorophore containing a reactive group, to yield an ODN having an attached quencher (Q) and XP moiety. Alternatively, in addition to the XP and a quencher (Q) a MB is introduced to yield a MB-Q-ODN-L-Fl conjugate. In this connection it is noted that the synthesis of MBs and their attachment to ODNs is well known (see for example U.S. Pat. No. 5,801,155, Ser. Nos. 09/539,097 and 09/141,764; all of which are expressly incorporated herein by reference).
EXAMPLES
General Experimental
All air and water sensitive reactions were carried out under a slight positive pressure of argon. Anhydrous solvents were obtained from Aldrich (Milwaukee, Wis.). Flash chromatography was performed on 230-400 mesh silica gel. Melting points were determined on a Mel-Temp melting point, apparatus in open capillaRY and are uncorrected. Elemental analysis was performed by Quantitative Technologies Inc. (Boundbrook, N.J.). UV-visible absorption spectra were recorded in the 200-400-nm range on a UV-2100 (Shimadzu) or a Lambda 2 (Perkin Elmer) spectrophotometers. 1H NMR spectra were run at 20° C. on a Varian 300 spectrophotometer; chemical shifts are reported in ppm downfield from Me4Si. Thin-layer chromatography was run on silica gel 60 F-254 (EM Reagents) aluminum-backed plates.
In the examples below, compound numbering refers to those numbers provided in Reaction Schemes 1-13.
Example 1
Bis(4-(2,2,2-trifluoroacetamido)butyl)phosphite (3)
This example demonstrates the synthesis of the novel phosphonylation reagent 3 as shown in Reaction Scheme 1.
A 250 mL flask was charged with magnetic stirring bar, compound 1 (see, J. Org. Chem. 62(20): 6712-6713 (1997)) (9.455 g, 51 mmol), pyridine (100 mL), and diphenylphosphite (2) (7.035 g, 25.5 mmol). The mixture was stirred for 1 h at room temperature, then concentrated on a rotary evaporator, diluted with ethyl acetate (200 mL) and washed with 10% citric acid (2×150 mL). Aqueous phase was re-extracted with ethyl acetate (100 mL) and organic phases were combined, washed with brine (100 mL), dried over Na2SO4 and concentrated in vacuo. Residue was chromatographed on silica eluting, first, with ethyl acetate to separate phenol and, second, with acetone to elute the desired phosphite 3. Concentration of the pure product fractions gave 8.0 g, (75%) of the phosphite 3 as viscous liquid. 1H NMR (dmso-d6): 9.44 (brs, 2H), 6.82 (d, J=695 Hz; 1H), 4.0 (m, 4H), 3.21 (q, J=6.2 Hz; 4H), 1.70-1.50 (m, 8H). 31P NMR (dmso-d6): 9.02 (d, J=695 Hz). 19F NMR: 0.86 (s).
Example 2
This example illustrates the synthesis of a protected 4′,4,5,6,7-pentachloro-7′-phosphono-fluorescein analog 11 as shown in Reaction Scheme 2.
Compound 5
A 0.5 L round bottom flask equipped with magnetic stirring bar was charged with 4-bromoresorcinol (3.62 g, 19.2 mmol), compound 4 (8.264 g, 16.0 mmol, prepared as described in WO 03/023357) and trifluoroacetic acid (50 mL). Methanesulfonic acid (50 mL) was added and resultant mixture was stirred at +80° C. for 3 h. Resultant red solution was cooled and poured onto ice ( ̃400 g). Obtained solid was collected by filtration on sintered glass funnel, washed with water (3×40 mL) and dissolved in a mixture of water (300 mL) and triethylamine (10 mL). The solution was heated to boiling point with stirring, then cooled and acidified to pH ̃2 by adding aqueous hydrochloric acid. The resultant fine suspension was extracted with ethyl acetate, washed twice with brine, dried over MgSO4. Concentration afforded crude product 5, which was contaminated with its debrominated analog formed during the condensation reaction. The mixture was separated by silica gel chromatography eluting with a gradient of methanol (0 to 15%) in 3% triethylamine in dichloromethane. Fractions containing pure compound 5 (triethylammonium salt) were collected and concentrated. Residue was suspended in 1N hydrochloric acid and extracted with ethyl acetate. The organic solution was separated, washed twice with brine, dried over MgSO4, filtered, concentrated and dried in vacuo to give pure desired product 5 (5.37 g, 51%).
Compound 7
A 0.5 L round bottom flask was charged with compound 5 (2.1 g, 2.29 mmol), trifluoroacetic acid (10 mL), CH2Cl2 (10 mL), and trifluoroacetic anhydride (10 mL). After being kept at room temperature for 10 min, the solution was concentrated. Residual TFA was removed by co-evaporation with dichloromethane. Drying under vacuum gave lactone 6 as an off-white solid. A solution of 6-aminohexanol (0.75 g, 6.44 mmol) and triethylamine (2.1 mL, 1.53 g, 15.0 mmol) in DMF (30 mL) was added to the lactone and resultant mixture was stirred for 20 min. Triethylamine (4.2 mL, 3.03 g, 30.0 mmol) was added followed by dimethoxytrityl chloride (13.47 g, 9.625 mmol. Reaction mixture was stirred overnight, diluted with ethyl acetate (300 mL) and washed with 10% citric acid (100 mL) and water (4×30 mL). The organic phase was separated, dried over Na2SO4, filtered and concentrated in vacuo. Residue was chromatographed on silica eluting with ethyl acetate to afford 2.2 g (91%) of desired product 7 as a red solid.
Compound 9
A 25 mL flask was charged with magnetic stirring bar, compound 7 (2.10 g, 1.99 mmol), DMF (5.0 mL), triethylamine (0.50 mL, 0.362 g, 3.58 mmol) and phosphite 3 (0.873 g, 3.15 mmol). Argon was bubbled through solution during 2 min and tetrakis(triphenylphosphine)palladium(0) (0.30 g, 0.26 mmol) was added. Reaction mixture was stirred at 80° C. for 3 h. Another portion of each triethylamine (0.25 mL, 0.181 g, 1.8 mmol) and phosphite 3 (0.426 g, 1.57 mmol) and Pd(PPh3)4 (0.15 g, 0.13 mmol) was added to complete the reaction to yield 8. After being stirred at 80° C. for additional 1 h, the reaction was cooled and treated with triethylamine (2.23 mL, 1.618 g, 16.0 mmol), 1-methylimidazole (0.1 mL, 0.104 g, 1.26 mmol) and trimethylacetic anhydride (1.622 mL, 1.49 g, 4.0 mmol). Reaction was allowed to proceed at room temperature overnight and then concentrated. The oil obtained was dissolved in ethyl acetate (200 mL), washed with saturated sodium bicarbonate (50 mL), brine (2×30 mL), dried over Na2SO4, filtered from drying agent and concentrated. Residue was chromatographed on silica (eluent 30% DCM in ethyl acetate) to give pure desired product 9 (1.27 g, 0.81 mol, yield=41%) as an amorphous solid.
Compound 10
A solution of compound 9 (1.20 g, 0.83 mmol) in methanol (50 mL) was treated with trifluoroacetic acid (0.5 mL, 0.74 g, 6.5 mmol). After being kept for 1 h at room temperature, the solution was neutralized by adding triethylamine (1.0 mL, 0.726, 7.2 mol), then concentrated in vacuo and re-dissolved in ethyl acetate (100 mL). The solution was washed with 10% citric acid (30 mL), brine (2×30 mL), dried over Na2SO4, filtered from drying agent and concentrated. Chromatography on silica gel eluting with ethyl acetate followed by concentration of the pure product fractions afforded 0.37 g (38%) of compound 10 as an amorphous solid.
Compound 11
To a solution of compound 10 (0.35 g, 0.28 mmol), diisopropylammonium tetrazolide (53 mg, 0.31 mmol) in anhydrous CH2Cl2 (10 mL) was added 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.169 g, 0.56 mmol). The reaction was stirred for 2 h and diluted with CH2Cl2 (80 mL). The solution was washed with saturated aqueous sodium bicarbonate (30 mL), brine (10 mL) and dried over Na2SO4. The extract was filtered and concentrated. The crude product was chromatographed on silica gel, which had been pre-washed with CH2Cl2/Et3N followed by CH2Cl2, using gradient (50-100%) of ethyl acetate in hexane to afford desired phosphoramidite 11 (0.22 g, 54%) as a white, amorphous solid.
Example 3
Protected 4-chloro-6-phosphono-benzofluorescein Analog (19)
This example illustrates the synthesis of a protected 4′-chloro-6-phosphonate benzofluorescein analog 19 as outlined in Reaction Scheme 3.
Compound 12
To a suspension of 4-bromophthalic anhydride (2.27 g, 10 mmol) in 30 mL of anhydrous 1,2-dichloroethane was added 3.3 g (25 mmol) of AlCl3 followed by methyl 3-(3-chloro-2,4-dihydroxyphenyl)propionate (WO03023357)(2.3 g, 10 mmol). The mixture was stirred for several minutes to give a tan solution. The reaction was refluxed for 30 min, then cooled and concentrated. The resultant gelatinous material was partitioned between cold 1N hydrochloric acid (70 mL) and ethyl acetate (100 mL). The organic phase was washed with saturated NaCl, dried over Na2SO4 and concentrated. The resultant oil was suspended in dichloromethane and cooled to initiate crystallization. The crystals were collected by filtration, washed with cold dichloromethane and dried to afford 2.3 g (51%) of isomeric mixture of benzophenone 12.
Compound 13
1,3-Dihydroxynaphthalene (4.0 g, 25 mmol), benzophenone 12 (7.5 g, 16.3 mmol) and trifluoroacetic acid (20 mL) were combined and stirred until a solution was formed. To this solution was added methanesulfonic acid (20 mL) and the reaction was heated at 80° C. for 7 h. The reaction was cooled and poured into a mixture of ice and water. Precipitated solid was collected by filtration and washed with water. Drying in vacuo afforded 9.0 g (97%) of dye 13 (mixture of two isomers).
Compound 14
To a solution of compound 13 (2.75 g, 4.84 mmol) of trifluoroacetic acid (20 mL) was added 20 mL of trifluoroacetic anhydride. After being kept at room temperature for 1 h, the reaction was evaporated and the residue dried by co-evaporation with ethyl acetate. The solid residue was suspended in ethyl acetate (25 mL) and filtered. Washing with a small amount of ethyl acetate/hexane mixture (50%/50%) and drying under vacuum afforded 2.35 g (75%) of the desired lactone 14 as an off-white solid.
Compound 16
To a solution of 6-(O-DMT)-hexylamine (3.07 g, 7.25 mmol) (Tetrahedron Letters (2004), 45(2), 317-320) and triethylamine (1.5 mL, 10.8 mmol) in 25 mL of anhydrous DMF was added 2.3 g (3.6 mmol) of solid lactone 14. After stirring for 1 h, N-methylimidazole (4 mL, 50 mmol) and trimethylacetic anhydride (4 mL, 19.7 mmol) were added. The reaction was allowed to proceed overnight. DMF was evaporated on a rotary evaporator and the resultant oil was partitioned between water and ethyl acetate. The organic phase was washed with brine and dried over Na2SO4. The material obtained after solvent evaporation was chromatographed on silica eluting with a gradient of ethyl acetate (30 to 50%) in hexane to remove excess DMT by-products (fastest moving components) and separate isomers. Minor, 5-isomer was eluted first followed by the major, 6-isomer. Fractions containing the 6-isomer were combined and concentrated. Drying under vacuum afforded 1.77 g (43%) of compound 16 as a pale yellow, amorphous solid.
Compound 17
Compound 16 (2.5 g, 2.2 mmol), phosphite 3 (1.63 g, 3.9 mmol) and triethylamine (0.82 mL, 5.9 mmol) were dissolved in a mixture of DMF (1.5 mL) and toluene (1.5 mL). To this solution was added 0.163 g (0.14 mmol) of tetrakis(triphenylphosphine)palladium (0). The mixture was warmed to  ̃50° C. to dissolve the catalyst and placed into an oil bath at 80° C. Heating with stirring was continued for 50 min. The reaction was cooled and diluted with ethyl acetate (30 mL). This mixture was loaded onto a silica gel column and eluted with ethyl acetate. Concentration of the pure product fractions and drying under vacuum afforded 2.2 g (68%) of the desired phosphonate 17 as a pale yellow, amorphous solid.
Compound 18
To a solution of compound 17 (2.1 g, 1.43 mmol) in a mixture of MeOH (30 mL) and CH2Cl2 (60 mL) was added 0.6 mL of trifluoroacetic acid. After being kept at room temperature for 30 min, the reaction was neutralized with triethylamine (0.9 mL) and concentrated. The resultant oil was chromatographed on silica eluting, first, with ethyl acetate and, second, with 5% MeOH in ethyl acetate. The pure product fractions were concentrated and dried to afford 1.5 g (90%) of compound 18 as a pale yellow, amorphous solid.
Compound 19
2-Cyanoethyl tetraisopropylphosphorodiamidite (0.57 g, 1.9 mmol) was added dropwise to a solution of compound 18 (1.4 g, 1.2 mmol) and diisopropylammonium tetrazolide (0.27 g, 1.56 mmol) in 20 mL of anhydrous CH2Cl2. After being kept at room temperature overnight, the reaction was diluted with ethyl acetate and washed with saturated NaHCO3. The organic phase was washed with brine, dried over Na2SO4 and concentrated to oil. This material was chromatographed on silica, which had been pre-washed with a mixture of triethylamine and ethyl acetate followed by ethyl acetate, eluting with ethyl acetate. Concentration of the product containing fractions and drying under vacuum afforded 1.2 g (73%) of the desired phosphoramidite 19 as a pale yellow, amorphous solid.
Example 4
This example illustrates the synthesis of a rotected 6-phosphonate rhodol analog 27 as outlined in Reaction Scheme 4.
Compound 23
4-Bromophthalic anhydride (6.47 g, 28 mmol) was dissolved in 7 mL of anhydrous DMF. To this solution was added 19 mL of toluene followed by 3.58 (18.9 mmol) of 8-hydroxyjulolidine. The reaction was heated with stirring at 110° C. for 30 min, then cooled to room temperature and concentrated. Obtained material was partitioned between water and CH2Cl2 (200 mL). The organic phase was dried over Na2SO4 and concentrated to give a dark oily material. It was chromatographed on silica eluting with CH2Cl2 followed by 5% MeOH in CH2Cl2. The product containing fractions were combined and concentrated. Drying under vacuum afforded 7.5 g (95%) of desired benzophenone 20 (mixture of isomers) as yellow, amorphous solid.
Compound 21
Compounds 20 (4.16 g, 10 mmol) and methyl 3-(3-chloro-2,4-dihydroxyphenyl)propionate (2.31 g, 10 mmol) were suspended in 21 mL of methanesulfonic acid and heated slightly to dissolve the solids. When a solution was obtained it was placed into an oil bath and heated at 80° C. for 1 h. The reaction was cooled, mixed with ice (300 mL) and treated with 13.5 g of NaOH. The resultant mixture was heated to 65° C. and allowed to slowly cool to room temperature. Concentrated hydrochloric acid (2 mL) was added to acidify the solution to pH of 2. Precipitated material was collected by filtration and washed with water. Drying under vacuum at 50° C. over P2O5 afforded 5.3 g (89%) of the desired dye 21 (mixture of isomers) as a dark-red solid.
Compound 24
To a solution of compound 21 (3.0 g, 5 mmol) in 30 mL of trifluoroacetic acid was added 30 mL of trifluoroacetic anhydride. After being kept at room temperature for 30 min, the reaction was concentrated and the residue was dried by co-evaporation with CH2Cl2 (2×100 mL) to give lactone 22 as a viscous syrup. It was dissolved in 40 mL of CH2Cl2 and treated with a solution of O-DMT-6-aminohexanol (2.5 g, 5.9 mmol) and triethylamine (10 mL) in 10 mL of CH2Cl2. After being stirred at room temperature for 2 h, to the reaction was added triethylamine (3 mL), 1-methylimidazole (3 mL) and 6 mL of trimethylacetic anhydride. In order to expedite the reaction, the mixture was concentrated to an oil form and heated at 45° C. for 8 h. Water and ethyl acetate were added to partition components of the reaction. Organic phase was washed with 10% citric acid, saturated NaHCO3, brine and dried over Na2SO4. The material obtained after solvent evaporation was chromatographed on silica eluting with 50% ethyl acetate in hexane to remove excess DMT by-products (fastest moving components) and separate isomers. Minor, 5-isomer was eluted first followed by the major, 6-isomer. Pure 6-isomer containing fractions were combined and concentrated. Drying under vacuum afforded 1.1 g (20%) of compound 24 as a pale-pink, amorphous solid.
Compound 25
Compounds 24 (0.8 g, 0.72 mmol), 7 (0.6 g, 1.7 mmol) and triethylamine (0.3 mL, 2.1 mmol) were dissolved in a mixture of DMF (0.7 mL) and toluene (0.7 mL). To this solution was added 70 mg (0.06 mmol) of tetrakis(triphenylphosphine)palladium (0). The mixture was slightly warmed ( ̃50° C.) to dissolve the catalyst and placed into an oil bath at 80° C. Heating with stirring was continued for 30 min. The reaction was cooled and diluted with ethyl acetate (10 mL). This mixture was loaded onto a silica gel column and eluted, first, with ethyl acetate to remove by-products followed by 5% MeOH in CH2Cl2 to elute the product. Concentration of the pure product fractions and drying under vacuum afforded 0.9 g (88%) of the desired phosphonate 25 as a light-pink, viscous syrup.
Compound 26
To a solution of compound 25 (0.9 g, 0.63 mmol) in a mixture of MeOH (15 mL) and CH2Cl2 (30 mL) was added 0.3 mL of trifluoroacetic acid. After being kept at room temperature for 30 min, the reaction was neutralized with triethylamine (0.45 mL) and concentrated. Obtained red residue was chromatographed on silica eluting with a gradient of MeOH (5-15%) in dichloromethane. The pure product fractions were concentrated and dried to afford 0.5 g (71%) of compound 26 as a pale yellow, amorphous solid.
Compound 27
2-Cyanoethyl tetraisopropylphosphorodiamidite (0.21 g, 0.7 mmol) was added dropwise to a solution of compound 26 (0.45 g, 0.45 mmol) and diisopropylammonium tetrazolide (90 mg, 1.56 mmol) in 10 mL of anhydrous CH2Cl2. After being kept at room temperature for 3 h, the reaction was diluted with ethyl acetate and washed with saturated NaHCO3. The organic phase was washed with brine, dried over Na2SO4 and concentrated to oil. This material was precipitated from small amount of ethyl acetate into anhydrous pentane to initially give an oily precipitate, which eventually solidified upon trituration with a fresh portion of pentane. The solid was collected by filtration and dried in vacuo to afford 0.4 g (74%) of phosphoramidite 27 as a light-pink, amorphous solid.
Example 5
This example illustrates the preparation of rescorcinol 31 and dye reagent 39 as provided in Reaction Schemes 5 and 6.
4-(4-Nitrophenyl)-2,4-methoxybenzene (28). To a degassed solution of 4-bromonitrobenzene (24.37 g, 120.63 mmol) and 2,4-dimethoxyphenylboronic acid (20.95 g, 115.07 mmol) in 600 mL of 1,4-dioxane was added tetrakis(triphenylphosphine)palladium (0) (8.0 g, 7 mmol) followed by a solution of potassium carbonate (17.06 g, 124.3 mmol) in 120 mL of water. The reaction was stirred with heating at 80° C. for 24 h, cooled, filtered and concentrated. The resultant material was partitioned between ethyl acetate and water. The organic layer was washed with saturated NaHCO3, brine, dried over MgSO4 and concentrated. The resultant solid material was re-crystallized from hexane-ethyl acetate to afford 23.56 g of the desired product 28 as yellow crystals. 1H NMR (dmso-d6): δ 8.22 (d, J=8.8 Hz, 2H), 7.73 (d, J=8.8 Hz, 2H), 7.34 (d, J=8.5 Hz, 1H), 6.71 (d, J=2.2 Hz, 1H), 6.67 (dd, J=8.5, 2.2 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H).
4-(4-Aminophenyl)-2,4-methoxybenzene (29). A solution of 28 (23.2 g, 89.5 mmol) in 300 mL of THF was hydrogenated at 50 psi in the presence of 2.0 g of 10% Pd/C for 8 h. The catalyst was removed by filtration through Celite. The filtrate was concentrated to afford crude amine 29 (20.8 g) which was used in the next reaction without additional purification. 1H NMR (dmso-d6): δ 7.10 (d, J=8.3 Hz, 3H), 6.60-6.50 (m, 4H), 5.04 (bs, 2H), 3.77 (s, 3H), 3.72 (s, 3H).
4-(4-Iodophenyl)-2,4-methoxybenzene (30). To a solution of crude amine 29 (20.8 g) in 80 mL of 1,4-dioxane was added a mixture of conc. HCl (347 mL) and ice (172 g). The reaction was placed in an ice/water bath and allowed to cool to 0-3° C. A solution of NaNO2 in 420 mL of water was added with stirring over 20 min and stirring was continued for another 1 h. A solution of potassium iodide (150.5 g) was added with stirring over 10 min. The reaction was allowed to warm up to room temperature ( ̃2 h) and extracted with ethyl acetate (1.2 L). The organic layer was washed with saturated NaHCO3, Na2SO3 and brine and dried over Na2SO4. The crude product was chromatographed on silica eluting with 20% ethyl acetate in hexane. Concentration of the pure product fractions and drying under vacuum afforded 15.83 g (51.3%) of 30. 1H NMR (dmso-d6): δ 7.72 (d, J=8.3 Hz, 2H), 7.24 (d, J=8.3 Hz, 2H), 7.20 (d, J=8.5 Hz, 1H), 6.66 (d, J=2.2 Hz, 1H), 6.61 (dd, J=8.5, 2.2 Hz, 1H), 3.80 (s, 3H), 3.75 (s, 3H).
4-(4-Iodophenyl)resorcinol (31). To a solution of 30 (15.83 g, 46.54 mmol) in 150 mL of anhydrous dichloromethane at −70° C. was added dropwise 13.63 mL (144.3 mmol) of boron tribromide. After 40 min, the solution was allowed to warm to room temperature and slowly quenched by pouring into 500 g of ice. The mixture was diluted with 1 N ( ̃390 mL) to pH of  ̃10 and extracted with ethyl acetate (300 mL). The organic layer was washed with brine and dried over Na2SO4. Evaporation of the solvent and drying under vacuum afforded pure 31 (14.4 g, 99%) as a pale pink solid. 1H NMR (dmso-d6): δ 9.48 (s, 1H), 9.39 (s, 1H), 7.68 (d, J=8.3 Hz, 2H), 7.29 (d, J=8.3 Hz, 2H), 7.04 (d, J=8.5 Hz, 1H), 6.40 (d, J=2.2 Hz, 1H), 6.29 (dd, J=8.5, 2.2 Hz, 1H).
6-Chloro-2-({3-chloro-2,4-dihydroxy-5-[2-(methoxycarbonyl)ethyl]phenyl}carbonyl)benzoic acid (32). To a solution of 3-chlorophthalic anhydride (3.7 g, 20 mmol) ((J. Org. Chem., 1987, 52, 129-134) in 60 mL of anhydrous 1,2-dichloroethane was added 6.6 g (50 mmol) of AlCl3 followed by 4.6 g (20 mmol) of methyl 3-(3-chloro-2,4-dihydroxyphenyl)propanoate (WO 03/023357). The reaction was heated at reflux for 30 min, cooled and concentrated. The resultant gelatinous material was partitioned between cold 2 N HCl (100 mL) and ethyl acetate (100 mL). The organic layer was washed with 1 N HCl, brine, dried over Na2SO4 and concentrated to afford an oily material (mixture of 6- and 2-chloroisomers). The oil was dissolved in dichloromethane ( ̃40 mL) and briefly sonicated (ultrasound bath) to initiate crystallization. The mixture was cooled (ice/water bath) and allowed to crystallize for 30 min (extended crystallization time will result in co-crystallization of undesired 2-chloro isomer). The crystals were collected by filtration washed with cold dichloromethane. Drying under vacuum afforded 4.01 g of desired 6-chloro isomer 32. 1H NMR (DMSO-d6) δ 12.47 (br s, 1H), 10.78 (br s, 1H), 8.06 (d, J=7.7 Hz, 1H), 7.89 (d, J=7.9 Hz, 1H), 7.71 (t, J=7.9 Hz, 1H), 6.76 (s, 1H), 3.46 (s, 3H), 2.27 (m, 2H), 2.41 (t, J=6.6 Hz, 2H).
3-(7,13-Dichloro-16-(4-iodophenyl)-12,15-dihydroxy-1-oxospiro[3-hydroisobenzofuran-3,9′-xanthene]-11-yl)propanoic acid (33). A suspension of 32 (2.45 g, 7.8 mmol) and 31 (2.48 g, 6.0 mmol) in a mixture of TFA (12 mL) and methanesulfonic acid (12 mL) was heated at 80° C. with stirring for 3 h. The reaction was cooled and diluted with water (200 mL). The resultant solid was collected by filtration, washed with water and dried. The crude material was crystallized from ethyl acetate/hexane to afford 3.2 g (78%) of the desired dye 33 as an orange solid. 1H NMR (dmso-d6): δ 12.07 (br s, 1H), 10.64 (s, 1H), 10.06 (s, 1H), 8.03 (d, J=7.2 Hz, 1H), 7.84 (d, J=7.6 Hz, 1H), 7.76 (t, J=7.6 Hz, 1H), 7.68 (d, J=8.2 Hz, 2H), 7.13 (d, J=8.2 Hz, 2H), 6.93 (s, 1H), 6.63 (s, 1H), 6.62 (s, 1H), 2.68 (m, 2H), 2.35 (t, J=7 Hz, 2H).
Compound 34
To a solution of the dye acid 33 (3.4 g, 5 mmol) in 30 mL of anhydrous DMF was added 3 mL of trimethylacetic anhydride followed by 3 mL of N-methylimidazole. After 1 h the reaction was concentrated and re-dissolved in ethyl acetate. The solution was washed with 10% citric acid, brine and dried over Na2SO4. Evaporation of the solvent afforded the crude product which was further purified by silica gel column chromatography eluting with 50% ethyl acetate in hexane. Concentration of the pure product fractions afforded 1.6 g (43%) of the desired lactone 34 as an off-white solid. 1H NMR (dmso-d6): δ 8.07 (d, J=7.2 Hz, 1H), 7.87 (d, J=7.7 Hz, 1H), 7.79 (t, J=7.7 Hz, 1H), 7.73 (d, J=8.2 Hz, 2H), 7.55 (s, 1H), 7.03 (s, 1H), 7.02 (d, J=8.2 Hz, 2H), 7.00 (s, 1H), 2.97 (m, 2H), 2.83 (t, J=7 Hz, 2H), 1.13 (s, 9H).
Compound 36
A solution of the lactone 34 (1.6 g, 2.16 mmol) in 15 mL of anhydrous DMF was added to a cold (0° C., ice/water bath) solution of 6-(O-DMT)-hexylamine (1.1 g, 2.6 mmol) (Tetrahedron Letters (2004), 45(2), 317-320) and triethylamine (0.3 mL) in 20 mL of anhydrous DMF. After being stirred at 0° C. for 2 h, the reaction was warmed to room temperature and treated with 1 mL of trimethylacetic anhydride and 0.44 mL N-methylimidazole. The reaction was allowed to proceed for 2 h and then concentrated. The residue was dissolved in ethyl acetate ( ̃75 mL), washed with saturated NaHCO3, brine and dried over Na2SO4. The solvent was evaporated and the resultant material chromatographed on silica eluting with 2:1 ethyl acetate:hexane. Concentration of the pure product fractions afforded 2.3 g (85%) of 36 as an amorphous, white solid. 1H NMR (dmso-d6): δ 8.06 (d, J=7.4 Hz, 1H), 7.86 (d, J=7.7 Hz, 1H), 7.78 (t, J=7.7 Hz, 1H), 7.73 (d, J=8.2 Hz, 2H), 7.65 (br t, 1H), 7.53 (s, 1H), 7.4-7.2 (m, 9H), 7.01 (d, J=8.2 Hz, 2H), 6.99 (s, 1H), 6.91 (s, 1H), 6.87 (d, J=8.8 Hz, 4H), 3.72 (s, 6H), 2.92 (t, J=6.3 Hz, 2H), 2.86 (m, 2H), 2.58 (m, 2H), 2.15 (m, 2H), 1.15 (m, 2H), 1.38 (s, 9H), 1.25 (m, 4H), 1.51 (m, 2H), 1.12 (s, 9H).
Compound 37
To a solution of 36 (2.3 g, 1.85 mmol), bis-(4-trifluoroacetamidobutyl)phosphite (1.15 g, 1.5 eq.), triethylamine (0.75 mL) in 4 mL of toluene was added tetrakis(triphenylphosphine)palladium (0) (130 mg, 0.11 mmol). The reaction was stirred at 80° C. for 1 h and cooled to room temperature. The mixture was chromatographed on silica eluting with a gradient of acetone (40 to 60%) in hexane. The pure product fractions were concentrated and dried under vacuum to afford 2.66 g (94%) of the desired phosphonate 37 as a white, amorphous solid. 1H NMR (dmso-d6): δ 9.42 (br t, 2H), 8.05 (d, J=7.4 Hz, 1H), 7.86 (d, J=7.8 Hz, 1H), 7.78 (t, J=7.9 Hz, 1H), 7.67 (m, 3H), 7.57 (s, 1H), 7.4-7.2 (m, 11H), 7.10 (s, 1H), 6.91 (s, 1H), 6.87 (d, J=8.8 Hz, 4H), 3.93 (m, 4H), 3.72 (s, 6H), 3.18 (m, 4H), 2.92 (t, J=6.3 Hz, 2H), 2.86 (m, 2H), 2.58 (m, 2H), 2.15 (m, 2H), 1.55 (m, 10H), 1.42 (s, 9H), 1.27 (m, 4H), 1.15 (m, 2H), 1.07 (s, 9H). 31P NMR (dmso-d6) δ 18.14 (s).
Compound 38
To a solution of 37 (2.4 g, 1.56 mmol) in a mixture of dichroromethane (50 mL) and methanol (50 mL) was added 0.6 mL of TFA. The solution was kept at room temperature for 30 min and neutralized by adding 0.9 mL of triethylamine. The reaction was concentrated and chromatographed on silica eluting with a gradient of methanol (0 to 5%) in ethyl acetate. Concentration of the pure product fractions afforded 1.86 g (97%) of compound 38 as a white, amorphous solid. 1H NMR (dmso-d6): δ 9.41 (br t, 2H), 8.08 (d, J=7.4 Hz, 1H), 7.87 (d, J=7.8 Hz, 1H), 7.80 (t, J=7.9 Hz, 1H), 7.67 (m, 3H), 7.58 (s, 1H), 7.38 (dd, J=13 Hz, 6 Hz, 2H), 7.10 (s, 1H), 6.91 (s, 1H), 4.33 (t, J=5 Hz, 1H), 4.11 (m, 4H), 3.37 (t, J=6 Hz, 2H), 3.18 (m, 4H), 2.88 (m, 2H), 2.58 (m, 2H), 2.16 (m, 2H), 1.55 (m, 10H), 1.40 (s, 9H), 1.34-1.16 (m, 6H), 1.03 (s, 9H). 31P NMR (dmso-d6) δ 18.05 (s).
Compound 39
Diisopropylammonium tetrazolide (0.26 g, 1.5 mmol) was added to a solution of 38 (1.79 g, 1.46 mmol) in 20 mL of anhydrous dichloromethane. The reaction was stirred for 5 min to dissolve the tetrazolide. To the resultant solution was added 0.7 g (2.3 mmol) of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite. After being stirred for 5 h, the reaction was diluted with ethyl acetate (100 mL), washed with saturated NaHCO3, brine and dried over Na2SO4. The extract was concentrated and re-dissolved in a small amount of ethyl acetate ( ̃2 mL). Ether (10 mL) was added to precipitate the product. The mixture was further diluted with anhydrous heptane (200 mL) and the resultant emulsion was allowed to settle ( ̃10 min). The residue, obtained after decanting the liquid phase, was dried under vacuum to afford 1.69 g (81%) of phosphoramidite 39 as an off white, amorphous solid. 1H NMR (dmso-d6): δ 9.41 (br t, 2H), 8.08 (d, J=7.4 Hz, 1H), 7.87 (d, J=7.8 Hz, 1H), 7.80 (t, J=7.9 Hz, 1H), 7.67 (m, 3H), 7.58 (s, 1H), 7.38 (dd, J=13 Hz, 6 Hz, 2H), 7.10 (s, 1H), 6.91) s, 1H), 3.93 (m, 4H), 3.72 (m, 2H), 3.54 (m, 4H), 3.18 (m, 4H), 2.88 (m, 2H), 2.92 (t, J=6.3 Hz, 2H), 2.58 (m, 2H), 2.16 (m, 2H), 1.55 (m, 10H), 1.40 (s, 9H), 1.34-1.16 (m, 6H), 1.12 (t, J=6 Hz, 12H), 1.03 (s, 9H). 31P NMR (dmso-d6) δ 146.73 (s), 18.05 (s).
Example 6
This example illustrates the preparation of an asymmetric mono-DMT-mono-trifluoroacetamidobutyl phosphite reagent 41 as an example of alternative phosphonylation reagent. A schematic representation of the reactions is provided in Reaction Scheme 7.
Compound 41
To a solution of mono-DMT-1,6-hexanediol (40) (Nucleic Acids Research (1993), 21(1), 145-150) (4.0 g, 9.5 mmol) and compound 5 (1.76 g, 9.5 mmol) in 40 mL of anhydrous pyridine was added 2.5 g ( ̃10 mmol, contained 10-15% phenol) of diphenylphosphite. After being kept at room temperature for 1 h the reaction was concentrated and dissolved in ethyl acetate (100 mL). The solution was washed with 10% citric acid, saturated NaCl and dried over Na2SO4. Material, which was obtained after concentration, was chromatographed on silica eluting with a gradient of ethyl acetate (60-100%) in hexane. Bis-DMT by-product was eluted first followed by desired mono-DMT-mono-trifluoroacetamido derivative 41. Appropriate fractions were combined, concentrated and dried in vacuo to afford 2.0 g (32%) of phosphite 41 as a colorless, viscous liquid. 1H NMR (dmso-d6) δ 9.44 (br t, 1H), 7.4-7.1 (m, 9H), 6.88 (d, J=8 Hz, 4H), 6.79 (d, J=694 Hz, 1H), 3.96 (m, 4H), 3.73 (s, 6H), 3.21 (m, 2H), 2.94 (t, J=6 Hz, 2H), 1.57 (m, 8H), 1.30 (m, 4H). 31P NMR (dmso-d6) δ 9.01 (d, J=694 Hz).
Example 7
This example illustrates the preparation of a hydroxyhexyl-6-phosphonofluorescein and its phosphoramidite 36 according to methods outlined in Reaction Scheme 8.
Compound 42
A suspension of 4-bromophthalic anhydride (6.8 g, 30 mmol) and resorcinol (8.8 g, 80 mmol) in 50 mL of methanesulfonic acid was heated with stirring at 80° C. for 5 h. The reaction was cooled and poured into a mixture of ice and water (300 mL). Solid precipitate was collected by filtration and washed with water. Drying under vacuum gave 12.1 g (98%) of bromofluorescein 42 (mixture of 5- and 6-isomers) as an orange solid.
Compound 43
A solution of compound 42 (4.1 g, 10 mmol), 1-methylimidazole (1 mL) and trimethylacetic anhydride (8 mL, 40 mmol) was heated at 50° C. for 2 h. TLC analysis (20% EtOAc in hexane) showed complete conversion of starting material into two new products (5- and 6-isomers). MeOH (5 mL) was added to quench excess anhydride. After being kept at room temperature for 1 h, the reaction was concentrated and re-dissolved in ethyl acetate. The solution was washed with 10% citric acid, saturated NaHCO3, saturated NaCl and dried over Na2SO4. After solvent evaporation, the mixture was chromatographed on silica eluting with 20% ethyl acetate in hexane. Three groups of fractions were collected. First one contained the faster eluting, 5-isomer. Second group was a mixture of 5- and 6-isomers, and, the third group of fractions contained pure 6-isomer, slower eluting product (compound 43). Evaporation of the solvent and drying under vacuum afforded 1.6 g of 5-isomer, 1.6 g of isomeric mixture and 1.9 g (33%) of the desired, 6-isomer (compound 43). 1H NMR (dmso-d6) δ 7.99 (m, 2H), 7.78 (s, 1H), 7.26 (d, 1.8 Hz, 2H), 6.95 (m, 4H), 1.31 (s, 18H).
Compound 44
A solution of compound 43 (1.16 g, 2.0 mmol), compound 41 (1.9 g, 2.9 mmol), triethylamine (0.8 mL, 5.75 mmol) and tetrakis(triphenylphosphine)palladium(0) (140 mg, 0.12 mmol) in 3 mL of toluene was heated at 80° C. for 1 h. The reaction was cooled and diluted with 20 mL of 66% ethyl acetate in hexane. Precipitated triethylammonium bromide was filtered off. The filtrate was loaded onto a silica gel column. Elution with a gradient (50 to 80%) of ethyl acetate in hexane followed by concentration and drying in vacuo afforded 1.95 g (85%) of phosphonate 44 as an amorphous, white solid material. 1H NMR (dmso-d6) δ 9.34 (t, 1H), 8.20 (dd, J1=8 Hz, J2=3 Hz, 1H), 8.04 (dd, J1=12.3 Hz, J2=8 Hz, 1H), 7.66 (d, J=12.9 Hz, 1H), 7.4-71 (m, 11H), 6.8 (m, 8H), 3.92 (m, 4H), 3.71 (s, 6H), 3.07 (q, J=6 Hz, 2H), 2.89 (t, 6 Hz, 2H), 1.46 (m, 8H), 1.28 (s, 18H), 1.28-1.05 (m, 4H). 31P NMR (dmso-d6) δ 15.35 (s).
Compound 45
To a solution of compound 44 (1.85 g, 1.6 mmol) in a mixture of MeOH (30 mL) and CH2Cl2 (60 mL) was added 0.6 mL of trifluoroacetic acid. After being kept at room temperature for 30 min, the reaction was neutralized with triethylamine (1 mL) and concentrated. Obtained red residue was chromatographed on silica eluting with a gradient of MeOH (0-5%) in ethyl acetate. The pure product fractions were concentrated and dried to afford 1.05 g (77%) of compound 45 as an amorphous, white solid. 1H NMR (dmso-d6) δ 9.35 (br t, 1H), 8.22 (dd, J1=8 Hz, J2=3 Hz, 1H), 8.06 (dd, J1=12.6 Hz, J2=8 Hz, 1H), 7.67 (d, J=12.9 Hz, 1H), 7.27 (s, 2H), 6.94 (m, 4H), 4.31 (t, J=5.4 Hz, 1H), 3.95 (m, 4H), 3.31 (t, 6 Hz, 2H), 3.07 (q, J=6 Hz, 2H), 1.46 (m, 8H), 1.31 (s, 18H), 1.18 (m, 4H). 31P NMR (dmso-d6) δ 15.27 (s).
Compound 46
2-Cyanoethyl N,N,N′N′-tetraisopropylphosphorodiamidite (0.57 g, 1.9 mmol) was added dropwise to a solution of compound 45 (1.0 g, 1.18 mmol) and diisopropylammonium tetrazolide (0.2 g, 1.17 mmol) in 20 mL of anhydrous CH2Cl2. After being kept at room temperature overnight, the reaction was diluted with ethyl acetate and washed with saturated NaHCO3. The organic phase was washed with brine, dried over Na2SO4 and concentrated. The residue was chromatographed on silica, which had been pre-washed with a mixture of triethylamine and ethyl acetate followed by ethyl acetate, eluting with ethyl acetate. Concentration of the product containing fractions and drying under vacuum afforded 0.72 g (58%) of the desired phosphoramidite 46 as a white, amorphous solid.
Example 8
This example illustrates the synthesis of phosphonate cyanine phosphoramidite (53) using methods outlined in Reaction Scheme 9.
1-(Sulfobutyl)-2,3,3-trimethylindolinum, inner salt (47). A mixture of 2,3,3-trimethylindolenine (6.3 g, 40 mmol) and 1,4-butane sultone (5.4 g, 40 mmol) was heated at 100° C. for 5 h. The resultant dark semi-solid was suspended in dichloromethane and cooled to inforce product crystallization. The crystals were collected by filtration, washed with cold dichloromethane and ether. Drying under vacuum afforded 9.9 g (83%) of the desired indolinum salt 47 as a pale pink, hydroscopic solid.
1-(Sulfobutyl)-2,3,3-trimethyl-5-bromoindolinum, inner salt (49) was prepared as described above for non-brominated analog starting from 5-bromo-2,3,3-trimethylindolenine (J. Heterocycle Chem. 2002, 39(2), 263-269).
Compound 48
A suspension of 47 (6.0 g, 20.3 mmol) and N,N′-diphenylformamidine (4.0 g, 20.4 mmol) in 25 mL of acetic anhydride was heated at 110° C. with stirring for 1.5 h. The reasultant solution was cooled to room temperature and left for product crystallization overnight. The crystals were collected by filtration washed with small amount of dichloromethane and plenty of ether. Drying under vacuum afforded 6.66 g (71%) of compound 48 as an orange-red solid.
Compound 50
A suspension of 49 (3.7 g, 9.9 mmol) and 48 (4.0 g, 10 mmol) in 7 mL of acetic anhydride was heated at 110° C. with stirring for 1 h and cooled to room temperature. The insoluble material was collected by filtration and washed with small amount of acetic anhydride and plenty of ether. Drying under vacuum afforded 4.0 g (61%) of compound 50. The crude material was utilized in the next reaction without additional purification.
Compound 51
A mixture of 50 (0.68 g, 1.0 mmol), triethylamine (0.5 mL, 3.75 mmol), phosphite 41 (0.96 g, 1.46 mmol) in 1.5 mL of DMF was degassed under vacuum. To this suspension was added 70 mg (0.06 mmol) of tetrakis(triphenylphosphine)palladium (0). The reaction was heated at 80° C. with stirring for 1 h and then cooled to room temperature. The mixture was chromatographed on silica eluting with a gradient of MeOH (0-15%) in 5% triethylamine in dichloromethane. Concentration of the product containing fractions and dryind under vacuum afforded 1.2 g (89%) of the desired phosphonate 51 as an amorphous, red solid.
Compound 52
To a solution of 51 (1.1 g, 0.81 mmol) in a mixture of dichloromethane (50 mL) and MeOH (50 mL) was added 0.1 mL of TFA. After being kept at room temperature for 1 h, the reaction was neutralized with 0.2 mL of triethylaamine and concentrated. The residue was then chromatographed on silica eluting with a gradient of MeOH (0-15%) in dichloromethane (plus 10% triethylamine). The pure product fractions were concentrated and dried to give 0.62 g (77%) of compound 52.
Compound 53
To a solution of 52 (0.6 g, 0.6 mmol) and diisopropylethylamine (1 mL) in 10 mL of anhydrous dichloromethane was added dropwise with stirring 0.2 mL (0.9 mmol) of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. After being kept at room temperature, the reaction was diluted with ether (100 mL). The liquid phase was decanted and the residue washed with more ether. The residual syrupy material was foamed up to afford 0.7 g of the unstable phosphoramidite 53 as a purple, amorphous solid. Due to the instability the phosphoramidite had to be utilized for DNA synthesis within one day after preparation. From a practical consideration, the phosphoramidte (53) was synthesized daily from intermediate 52 in quantities sufficient for oligonucleotide synthesis requirement for the day.
Example 9
This example illustrates the preparation of a phosphonate-substituted Cy5 CPG reagent 59 as outlined in Reaction Scheme 10.
Compound 54
A mixture of 5-iodo-2,3,3-trimethyl-3H-indolenine (Eur. J. Med. Chem. Chim. Ther. 1974, 9(3), 274-280) (4.74 g, 16.6 mmol) and propanesultone (2.0 g, 16.4 mmol) was heated at +100° C. for 30 min. The reaction mixture was cooled and triturated with ether. Solid material was filtered off, washed with ether, dichloromethane and dried in vacuum to give desired product (5.5 g, 82%) as off-white solid.
Compound 55
To a solution of 54 (2.3 g, 5.6 mmol) and malonaldehyde bis(phenylimino)monohydrochloride (0.72 g, 2.8 mmol) in 20 ml of pyridine was added acetic anhydride (3.4 g, 33.6 mmol). The reaction was stirred at room temperature for 4 h and concentrated. The resultant material was chromatographed on silica eluting with a gradient of methanol (0-10%) in dichloromethane (plus 5% pyridine). Yield of the desired dye 55 was 56% (purple solid).
Compound 56
A mixture of 55 (0.46 g, 0.49 mmol), phosphite 41 (1.0 g, 1.53 mmol), N-ethylmorpholine (0.3 ml) and DMF (1 ml) was degassed under vacuum for 1 min. Tetrakis(triphenylphosphine)palladium (0) (0.1 g, 0.086 mmol) was added, and the mixture was stirred at 80° C. for 1 h. The reaction mixture was cooled and chromatographed on silica eluting with a gradient of methanol (0-10%) in dichloromethane (plus 5% pyridine). Concentration of the pure product fractions and drying under vacuum afforded 0.8 g (82%) of the desired phosphonate dye 56.
Compound 57
To a solution of 56 (0.7 g, 0.35 mmol) in 20 ml of dichloromethane was added methanol (0.0115 g, 0.35 mmol) followed by TFA (0.05 ml). The reaction was allowed to proceed for 10 min and neutralized with triethylamine (0.1 ml). Solvent was evaporated and the resultant material was chromatographed on silica eluting with a gradient of methanol (0-20%) in dichloromethane (plus 5% pyridine). Concentration of the pure product fractions and drying under vacuum afforded 0.12 g (20%) of the desired product 57.
Compound 58
A solution of 57 (0.11 g, (0.065 mmol), N-methylimidazole (5 mg, 0.065 mmol), triethylamine (80 mg, 0.78 mmol) and succinic anhydride (40 mg, 0.39 mmol) in 1.5 ml of anhydrous DMF was stirred at room temperature for 20 h. Pentafluorophenyl trifluoroacetate (0.22 g, 0.78 mmol) was added. After being kept at room temperature for 1 h, the reaction was concentrated and triturated with ether followed by ethyl acetate. Drying under vacuum afforded crude (contaminated with TFA salts) product 58 which was used in the next reaction without additional purification.
Preparation of Controlled Pore Size Support (CPG) 59
Long chain aminoalkyl CPG, 500A (1.5 g, 105 μmol/g) was added to a solution of 58 (90 mg,  ̃45 μmol) and triethylamine (50 mg, 0.49 mmol) in 8 ml of anhydrous DMF. The suspension was stirred using an orbiter shaker for 3 h and then treated with pyridine (1 ml) and acetic anhydride (1 ml) to cap unreacted aminogroups. After being stirred for another 1 h, the CPG was collected by filtration, washed with DMF, ethyl acetate and dried in vacuo. DMT loading: 18 μmol/g.
Example 10
This example illustrates the preparation of a phosphonate-substituted Cy5 PFP ester reagent 66 as outlined in Reaction Scheme 11.
Compound 60
A mixture of 5-iodo-2,3,3-trimethyl-3H-indolenine (Eur. J. Med. Chem. Chim. Ther. 1974, 9(3), 274-280) (1.0 g, 3.5 mmol) and tert-butyl 6-iodohexanoate (1.382 g, 4.6 mmol) was heated at +90° C. for 9 h. The reaction mixture was cooled and triturated with ether. Solid material was filtered off, washed with ether and dried in vacuum to give desired product 60 (1.2 g, 59%) as pale pink solid. 1H NMR (dmso-d6): δ 8.31 (s, 1H), 8.00 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 4.42 (t, J=7.4 Hz, 2H), 3.91 (bs, 2H), 2.81 (s, 3H), 2.19 (t, J=7.2 Hz, 2H), 1.80 (qn, J=7.0 Hz, 2H), 1.53 (s, 6H), 1.50 (qn, J=7.2 Hz, 2H), 1.36 (s, 9H).
Compound 61
A mixture of 5-iodo-2,3,3-trimethyl-3H-indolenine (2.0 g, 7.0 mmol) and ethyl iodide (1.76 ml, 3.42 g, 22 mmol) was heated at +90° C. for 3 h. The reaction mixture was cooled and triturated with ether. Solid material was filtered off, washed with ether and dried in vacuum to give desired product 61 (2.45 g, 79%) as pink solid. 1H NMR (dmso-d6): δ 8.31 (s, 1H), 8.00 (d, J=8.4 Hz, 1H), 7.80 (d, J=8.4 Hz, 1H), 4.46 (q, J=7.4 Hz, 2H), 2.80 (s, 3H), 1.52 (s 6H), 1.41 (t, J=7.2 Hz, 3H).
Compound 62
A mixture of 61 (1.5 g, 3.4 mmol), malonaldehyde bis(phenylimino)monohydrochloride (1.76 g, 6.8 mmol), acetyl chloride (2.5 ml), and acetic anhydride (25 ml) was heated at +120° C. for 1.5 h. The reaction mixture was cooled and added dropwise to stirred ether. Solid material was filtered off, washed with ether, EtOAc and dried in vacuum to give crude product (1.87 g, 90%) as brown solid, which was used further without purification.
Compound 63
A mixture of 62 (1.25 g, 2.04 mmol), 60 (1.162 g, 2.0 mmol), acetic anhydride (2 ml), and pyridine (20 ml) was magnetically stirred at room temperature for 1.5 h. The reaction mixture was concentrated in vacuum, residue was dissolved DCM/MeOH (9:1) mixture, and solution was added dropwise to stirred ether. The precipitate was filtered off, washed with ether, and dried in vacuum to give crude product, which was purified on silica column (10% MeOH/DCM) to give desired product (0.94 g, 50%) as purple solid. 1H NMR (dmso-d6): δ 8.34 (t, J=13.0 Hz, 2H), 8.06 (s, 2H), 7.75-7.71 (m, 2H), 7.24 (d, J=8.5, 1H), 7.23 (d, J=8.5, 1H), 6.57 (t, J=12.2 Hz, 1H), 6.30 (d, J=13.3 Hz, 2H), 4.10 (m, 4H), 2.15 (t, J=7.2 Hz, 2H), 1.67 (s, 12H), 1.60 (m, 2H), 1.50 (m, 2H), 1.33 (s, 9H), 1.30 (m, 2H), 1.23 (t, J=7.2 Hz, 3H).
Compound 64
A 25 ml flask was charged with compound 63 (0.84 g, 0.9 mmol), phosphite 3 (2.24 g, 5.4 mmol), DMF (2 ml), toluene (2 ml), and ethylmorpholine (0.859 ml, 0.777 g, 6.75 mmol). The flask was flushed with argon and argon was bubbled through solution during 2 min. Tetrakis(triphenylphosphine)palladium (0) (208 mg, 0.18 mmol) was added and reaction mixture was magnetically stirred at +80° C. for 2 h. The reaction mixture was cooled to room temperature and added dropwise to stirred methyl t-butyl ether. The solution was decanted and residual oily material was rinsed with ether, dissolved in MeOH/DCM (9:1) mixture and separated on silica column (10-15% MeOH in DCM) to give desired product (250 mg, 19%) as a purple solid. 1H NMR (dmso-d6): δ 9.42 (t, J=4.8 Hz, 4H), 8.46 (t, J=13.0 Hz, 2H), 7.95 (d, J=12.6 Hz, 2H), 7.80-7.70 (m, 2H), 7.54-7.52 (m, 2H), 6.67 (t, J=12.4 Hz, 1H), 6.41 (d, J=13.5 Hz, 2H), 4.15 (m, 4H), 3.95 (m, 8H), 3.17 (qr, J=6.1 Hz, 8H), 2.18 (t, J=7.2 Hz, 2H), 1.71 (s, 12H), 1.65-1.45 (m, 20H), 1.33 (s, 9H), 1.30-1.20 (m, 5H). 31P NMR (dmso-d6): δ 18.72.
Compound 65
A mixture of 64 (0.25 g, 0.16 mmol), dichloromethane (5 ml), and trifluoroacetic acid (5 ml) was magnetically stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuum and co-evaporated with DMF. The residue was dissolved in acetone and diluted with ether. The resultant solution was decanted, and the remaining oily residue was rinsed with ether and dried in high vacuum to give desired product (0.20 g, 88%) as purple solid. 1H NMR (dmso-d6): δ 9.42 (t, J=4.7 Hz, 4H), 8.45 (t, J=13.0 Hz, 2H), 7.95 (d, J=12.6 Hz, 2H), 7.78-7.70 (m, 2H), 7.53 (d, J=7.7 Hz, 2H), 6.68 (t, J=12.4 Hz, 1H), 6.42 (d, J=13.5, 1H), 6.41 (d, J=13.5, 1H), 4.15 (m, 4H), 3.98 (m, 8H), 3.55 (bs, 1H), 3.17 (qr, J=6.1 Hz, 8H), 2.20 (t, J=7.2 Hz, 2H), 1.71 (s, 12H), 1.65-1.45 (m, 20H), 1.40 (m, 2H), 1.27 (t, J=7.2 Hz, 3H). 31P NMR (dmso-d6): δ 18.72.
Compound 66
Pentafluorophenyl trifluoroacetate (0.18 g, 0.64 mmol) was added to a mixture of 65 (0.20 g, 0.14 mmol), DMF (3 ml), and N,N-diisopropylethylamine (0.082 g, 0.64 mmol). The resultant mixture was magnetically stirred at room temperature for 10 min. The reaction mixture was then concentrated under reduced pressure and the residue was dissolved in acetonitrile and diluted with ether. The resultant solution was decanted from the oily residue. The operation was repeated, and the oily residue was then rinsed with ether and dried under high vacuum to give desired product (0.15 g, 56%) as purple solid. 1H NMR (dmso-d6): δ 9.43 (bs, 4H), 8.45 (t, J=13.0 Hz, 2H), 7.96 (d, J=12.4 Hz, 2H), 7.78-7.69 (m, 2H), 7.54 (d, J=6.2 Hz, 2H), 6.65 (t, J=12.3 Hz, 1H), 6.40 (d, J=12.9 Hz, 2H), 4.15 (m, 4H), 3.98 (m, 8H), 3.17 (qr, J=5.9 Hz, 8H), 2.80 (t, J=6.9 Hz, 2H), 1.8-1.7 (m, 2H), 1.71 (s, 12H), 1.65-1.40 (m, 18H), 1.3-1.2 (m, 5H). 31P NMR (dmso-d6): δ 18.72.
Example 11
Comparison of Reverse Phase HPLC Retention Times for Phosphonate and Non-Phosphonate Dye-Octathymidylates
This example demonstrates a) the oligonucleotide synthesis with a protected xanthene phosphonate phosphoramidite b) the deblocking of the oligonucleotide conjugate and c) the increased polarity of the xanthene phosphonate oligonucleotide conjugate.
Oligonucleotide synthesis was performed on a AB 3900 DNA Synthesizer (Applied Biosystems, Foster City, Calif.). The protected oligonucleotide conjugates was treated under standard conditions (10% ethanol in concentrated NH4OH for 2 hours at 70° C.) to yield the pure unprotected oligonucleotide conjugates.
The purified oligonucleotide conjugates in Table 6 were analyzed by C18 reverse phase chromatography. HPLC conditions were: C18 reverse phase, Luna, 4.6×100 mm column, gradient of acetonitrile (0-45%) in 30 min, 0.1 M triethylammonium acetate, pH 7.5 with a flow/rate of 1 mL/min.
As shown in Table 6, the retention times of the phosphonate xanthenes is much faster than the non-phosphonate xanthenes confirming their more polar nature.







TABLE 6







Comparison of reverse phase HPLC retention times, absorption and fluorescence


properties for phosphonate and non-phosphonate dye-octathymidylates













Abs
Fluor




Retention
max
max
Relative



time (min)
(nm)
(nm)
brightness




















R = H R = PO(OH)(O(CH2)4NH2)
17.6 11.6
520.5 523
550 554
1.0 1.1


 






R = H R = PO(OH)(O(CH2)4NH2)
20.7 14.6
552 554
576 577
1.0 1.0


 






R = H R = PO(OH)(O(CH2)4NH2)
17.5 15.7

540 548


 










12.8
495
516
1.0


 







11.4
494
515
1.0


 















Example 12
The Oligonucleotide Synthesis of a MB-Q-Oligonucleotide-PY Conjugate
This example demonstrates a) the synthesis of a MB-Q-oligonucleotide-PY conjugate, b) the removal of protecting groups and c) the purification of the conjugate.
FIG. 3 shows a example of oligonucleotide synthesis of 5′-minor groove binder-quencher DNA probes labeled at the 3′ end with a phosphonate xanthene dye using phosphoramidite reagent 18b. This example demonstrates the successful and reproducible phosphoramidite coupling chemistry and efficient removal of protecting groups during the deprotection. The oligonucleotide was synthesized as described in Kutyavin et al, Nucl. Acids Res., 28:655-661 (2000). The conjugate was purified on a Wave System, Transgenomic, Inc., Omaha, Nebr. The retention time of the conjugate corresponded to a position observed for similar fully deprotected conjugates.
Example 13
This example demonstrates the use of a phosphonate-lableled MGB Eclipse probe in a multiplexed hybridization-based detection assay, where the HSV 1,2 probe is labeled with FAM and the internal control is labeled with a phosphonate dye (14).
Real-Time PCR Using MGB Eclipse PY-Labeled Probe
Real-time PCR was conducted on either an ABI Prism® 7900 Sequence Detection System (SDS) (Applied Biosystems, Foster City, Calif.), or on a MJ Research PTC-200 Peltier Thermal Cycle (Waltham, Mass.) (Afonina, et al., J. Clin. Ligand Assay, Vol. 25, Vol. 23, pp. 268). On both instruments, 50 cycles of a three step PCR (95° C. for 5 s, 58° C. for 20 s and 76° C. for 30 s) profile was run, after an initial 2 min at 95° C. If necessary, fluorescent data were collected at 58° C. with an ABI 7900 SDS. Commercially available 2× Jump StartTM Taq Ready MixTM for Quantitative PCR with 2 mM final Mg++ concentration (Sigma #D 74403) supplemented with JumpStart Taq Polymerase (Sigma Catalog #90 4184) to a final amount of 0.37 U/μl was used. Final concentration of both probes was 0.2 μM; concentration of limiting primer was 0.1 μM and excess primer was 2 μM. Each 5 μl reaction contained 10 ng of genomic DNA lyophilized in 96 or 384 well plates with a speed vac prior to reaction set up. A Biomek® 2000 Laboratory Automation Station (Beckman Coulter, USA) was used to setup PCR reactions.
HSV 1,2 Detection
Successful multiplexing requires that the amplification of both the target and internal control are close to 100% efficient (Livak, K J and Schmittgen, T D. 2001. Methods 25: 402-408). When such PCR efficient reactions are multiplexed in a single reaction the real-time data from a titration curve of Ct against concentration should give a slope of −3.333 with R2 value of greater than 0.99. In the case where the HSV 1,2 FAM-labeled probe and the phosphonate-labeled internal control were multiplexed, the slope is −3.14 with a R2 of 0.996, an acceptable value to proceed with the detection of unknown samples.
FIG. 4 illustrates the titration of a HSV 1,2 MGB Eclipse probe assay wherein the FAM-labeled probe is multiplexed with either an internal control (IC) probe labeled with PY. The titration real-time data for the multiplexed FAM and PY probes is shown in a). The titration is performed with cloned HSV 1,2 plasmid target with 10 fold concentrations from 10o to 106 copies. 1000 copies of IC were used in all HSV dilutions, while the IC primer concentrations were 0.3 μM in all HSV dilutions. The sequences for HSV 1,2 assay are MGB-Q-CCCAGGTTZ*TCCTCGCT-FAM (probe), MGB-Q-GCAAAGTCCCATCGTT-PY(14) (internal control probe), CCGTCCAGTCGTTTATCTTC (limiting primer) and CGAACGCAGCCCCGCTGGA (excess primer). MGB ligand is DPI3; Q is the Eclipse Dark Quencher and Z is a universal base.
Example 14
This example illustrates the synthesis of a cyanine phosphonate-substituted reagent 80 of the present invention as described in Reaction Scheme 12a.
Compound 78
A mixture of compound 54(1.0 g, 2.46 mmol), 3,4-dibutoxy-3-cyclobutene-1,2-dione (0.556 g, 0.531 ml, 2.46 mmol), ethanol (25 ml), and triethylamine (2.48 g, 3.42 ml, 24.6 mmol) was refluxed for 5 h. Aqueous 1 M NaOH (5.41 ml, 5.41 mmol) was added and resultant mixture was further refluxed for 30 min. The reaction mixture was cooled to room temperature and 1 M HCl (5.66 ml, 5.66 mmol) was added. Evaporation of the solvent afforded crude intermediate 77, which was then dissolved in toluenelbutanol mixture (1:1, 50 ml) and compound 60 (1.363 g, 2.34 mmol) was added. The resultant mixture was refluxed with Dean-Stark trap for 3 h, cooled and concentrated. The residue was separated on a silica gel column (20% MeOH in DCM) to give desired product 78 (0.45 g, 0.48 mmol, yield=20%) as deep blue solid. 1H NMR (DMSO-d6): δ 7.90 (s, 2H), 7.67 (m, 2H), 7.28 (d, J=8.3 Hz, 1H), 7.17 (d, J=8.4 Hz, 1H), 5.80 (s, 1H), 5.77 (s, 1H), 4.18 (bt, J=6.7 Hz, 2H), 4.07 (bt, J=6.7 Hz, 2H), 2.53 (bt, J=7.2 Hz, 2H), 2.16 (t, J=7.2 Hz, 2H), 1.90 (m, 2H), 170 (m, 2H), 1.66 (s, 12H), 1.55 (m, 2H), 1.34 (m, 2H), 1.32 (s, 9H).
Compound 79
A mixture of compound 78 (0.39 g, 0.42 mmol), phosphite 3 (1.40 g, 3.36 mmol), DMF (4 ml), toluene (4 ml), and ethylmorpholine (0.535 ml, 0.484 g, 4.2 mmol) was degassed prior to addition of Pd(PPh3)4 (97 mg, 0.084 mmol). The resultant mixture was magnetically stirred at +80° C. for 2 h, cooled to room temperature, concentrated in vacuo and chromatographed on silica (20% MeOH in DCM) to give desired product 79 (0.43 g, 0.28 mmol, yield=67%) as blue solid. 1H NMR (DMSO-d6): δ 9.43 (s, 4H), 7.81 (s, 1H), 7.77 (s, 1H), 7.73-7.64 (m, 2H), 7.59 (m, 1H), 7.47 (m, 1H), 5.92 (s, 1H), 5.90 (s, 1H), 4.25 (m, 2H), 4.10 (m, 2H), 3.97 (m, 8H), 3.60 (m, 2H), 3.17 (q, J=7.4 Hz, 8H), 2.53 (bt, J=7.2 Hz, 2H), 2.18 (t, J=7.2 Hz, 2H), 2.03 (m, 2H), 1.75 (m, 2H), 1.70 (s, 12H), 1.68-1.48 (m, 16H), 1.36 (m, 2H), 1.32 (s, 9H).
Compound 80
Trifluoroacetic acid (5 ml) was added to solution of compound 79 (0.36 g, 0.24 m) in DCM (5 ml) and resultant mixture was stirred for 1 h. The reaction mixture was concentrated, co-evaporated with DCM and dried in vacuo. The residue was dissolved in acetone and added slowly to stirred ether. The resultant precipitate was filtered, washed with ether and dried under high vacuum to give deprotected intermediate (0.31 g, 0.21 mmol, yield=88%). Pentafluorophenyl trifluoroacetate (0.060 ml, 0.098 g, 0.35 m) was added to solution of obtained intermediate (0.22 g, 0.15 mmol) in DMF (3 ml) and diisopropylethylamine (0.061 ml, 0.045 g, 0.35 mmol). The reaction mixture was concentrated in vacuum after 10 min period. The residue was rinsed with ether, dissolved in acetonitrile and poured into stirred ether. The precipitate was filtered out, washed with ether, and dried in vacuo to give desired PFP ester 80 (210 mg, 0.13 mmol, yield=77% for two steps) as a dark blue solid. 1H NMR (DMSO-d6): δ 9.43 (s, 4H), 7.81 (s, 1H), 7.77 (s, 1H), 7.73-7.58 (m, 3H), 7.48 (m, 1H), 5.91 (s, 2H), 4.25 (m, 2H), 4.12 (m, 2H), 3.98 (m, 8H), 3.17 (q, J=7.4 Hz, 8H), 2.81 (t, J=6.5 Hz, 2H), 2.53 (bt, J=7.0 Hz, 2H), 2.0 (m, 2H), 1.75 (m, 2H), 1.70 (s, 12H), 1.65-1.45 (m, 18H), 1.25 (m, 2H).
Example 15
The following example illustrates the synthesis of various intermediates that can be used in a synthesis of a phosphonate-substituted cyanine reagent 87 as described in Reaction Scheme 12b.
Compound 82
A mixture of compound 81 (5.0 g, 17.5 mmol), 1-bromo-4-nitrobenzene (5.30 g, 26.25 mmol), potassium carbonate (6.772 g, 49.0 mmol in water 24.5 ml), and dimethoxyethane (75 ml) was degassed prior to Pd(PPh3)4 (2.022 mg, 1.75 mmol) addition. The mixture was magnetically stirred +83° C. for 3 h, then cooled and diluted with EtOAc. The organic phase was washed with brine and water, dried over MgSO4, and concentrated in vacuo. The residue was separated on silica gel column (10% acetone in DCM) to affored desired product 82 (4.04 g, 14.4 mmol, yield=82%) as yellow semi-solid. 1H NMR (DMSO-d6): δ 8.30 (d, J=8.8 Hz, 2H), 7.99 (d, J=8.8 Hz, 2H), 7.91 (s, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.55 (d, J=8.0 Hz, 1H), 2.25 (s, 3H), 1.31 (s, 6H).
Compound 83
Palladium on charcoal (10%, 0.5 g) was added to solution of compound 82 (4.0 g, 14.2 mmol) in methanol (200 ml), and resultant mixture was hydrogenated on Parr apparatus at 30 psi for 2 h. The reaction mixture was filtered through Celite, concentrated and dried in vacuo to give desired product 83 (3.5 g, 14.0 mmol, yield=99%). 1H NMR (DMSO-d6): δ 7.58 (s, 1H), 7.41-7.34 (m, 4H), 6.63 (d, J=8.2 Hz, 2H), 5.20 (s, 2H), 2.20 (s, 3H), 1.27 (s, 6H).
Compound 84
Cold hydrochloric acid (6.6 M, 150 ml) was added to solution of compound 83 (3.5 g, 14.0 mmol) in dioxane (20 ml). The resultant mixture was cooled to 0° C. using ice-water bath and solution of sodium nitrite (1.213 g, 17.57 mmol) in water (100 ml) was added drop wise over 10 min. The reaction mixture was magnetically stirred for 1 h at +0° C., and solution of potassium iodide (23.241 g, 140.0 mmol) in water (120 ml) was added over 15 min with vigorous stirring. The reaction mixture was stirred for 1 h at room temperature, and then diluted with EtOAc (300 ml). The organic solution was separated, washed with saturated aqueous sodium thiosulfate, brine, dried over MgSO4, and concentrated in vacuo. The crude product was chromatographed on silica eluting with 20% EtOAc in DCM to give desired product 84 (2.73 g, 7.6 mmol, yield=54%) as yellow solid. 1H NMR (DMSO-d6): δ 7.80 (d, J=8.3 Hz, 2H), 7.45 (d, J=1.5 Hz, 1H), 7.56 (dd, J=8.0, 1.5 Hz, 1H), 7.50 (d, J=8.4 Hz, 2H), 7.48 (d, J=7.8 Hz, 1H), 2.23 (s, 3H), 1.29 (s, 6H).
Compound 85
A mixture of compound 84 (1.0 g, 2.77 mmol), 1,4-butanesulton (0.446 g, 3.65 mmol), and dimethylacetamide (5 ml) was heated at +100° C. for 16 h. The resultant mixture was cooled and diluted with ether. The solution was decanted and residue was dissolved in methanol/DCM (1:9) mixture. The solution was slowly added to stirred ether. The solid material was filtered, washed with ether and dried in vacuo to give compound 85 (1.13 g, 2.34 mmol, yield=84%) as grey solid. 1H NMR (DMSO-d6): δ 8.18 (s, 1H), 8.11 (d, J=8.3 Hz, 1H), 7.92 (d, J=8.5 Hz, 1H), 7.87 (d, J=8.3 Hz, 2H), 7.60 (d, J=8.2 Hz, 2H), 4.68 (t, J=7.2 Hz, 2H), 2.85 (s, 3H), 2.64 (t, J=6.2 Hz, 2H), 2.17 (t, J=6.4 Hz, 2H), 1.58 (s, 6H).
One of ordinary skill in the art will recognize from the provided description, figures, and examples, that modifications and changes can be made to the various embodiments of the invention without departing from the scope of the invention defined by the following claims and their equivalents. Additionally, all references, patents, patent publications and the like are expressly incorporated herein by reference.

## Claims

1. A fluorescent dye reagent having the formula:




wherein
Fl is a fluorescent dye component;
Lf is a linking group having an attached member selected from the group consisting of a protected or unprotected functional group, a reactive group, a polyfunctional linking moiety, a phosphoramidite moiety and a solid support;
the subscript m is an integer of from 0 to 1;
Pz is a zwitterionic phosphonate group having the formula (a) or a protected phosphonate group having formula (b) or (c):




wherein the wavy line indicates the direct attachment to a sp2 carbon of said fluorescent dye component; L is a linking group; Na is an ammonium ion group; each of RA and RB is independently selected from the group consisting of H and a labile protecting group; RC is selected from the group consisting of H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a labile protecting group or an alkylene linking group having a distal hydroxy or protected hydroxy group;
the subscript n is an integer of from 1 to 2;
and salts thereof.

2. A fluorescent dye reagent of claim 1, wherein said fluorescent dye component is selected from the group consisting of coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, thiazole orange dyes, polymethines and N-aryl-1,8-naphthalimide dyes.

3. A fluorescent dye reagent of claim 1, having formula I:




wherein,
the symbol , represents a single or double bond;
the Ar ring, represented by the symbol





is selected from the group consisting of:



A1 in Ar1 represents O, N—Z, or N+(Z)2, and A1 in Ar2 represents OR, or N(Z)2, wherein at each occurrence Z is independently hydrogen, (C1-C8)alkyl, aryl-(C1-C8)alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with R2 or R4 to form a fused 5- to 7-membered ring, and the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; R, is selected from H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, protecting group and Lf;
A2 represents OR or N(Z)2, wherein each Z is independently hydrogen, (C1-C8)alkyl, aryl(C1-C8)alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; or optionally the Z group, at each occurrence, independently is combined with R1 or R1′′ to form a fused 5- to 7-membered ring wherein the resultant fused 5- to 7-membered ring is optionally fused to an aryl ring, and is optionally substituted with halogen, C1-C4 alkyl, aryl, Lf or Pz; and the substituent R is selected from H, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a protecting group and Lf;
R1′, R1′′, R1, R2, R3 and R4 are each independently selected from the group consisting of H, halogen, cyano, CF3, sulfo, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz, wherein said aryl or heteroaryl group is optionally substituted with Pz; or optionally any two of the R1′, R1′′, R1 and R3 substituents that are attached to adjacent ring atoms are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic, and is optionally substituted with Pz; and the alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; the aryl or heteroaryl portions of any R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz;
R0 is halogen, cyano, CF3, (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, substituted or unsubstituted heteroaryl or aryl having the formula:




wherein X1, X2, X3, X4 and X5 are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, aryl, heteroaryl, SO3H, PO3Fl2, CO2H, Lf and Pz and optionally, any two adjacent substituents of X1 to X5 are combined to form an aromatic or heteroaromatic ring; wherein the aryl or heteroaryl portions of R0 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz;
R0′ in formula I is absent or is (C1-C6)alkoxy, hydroxy, (C1-C6)alkylamino, or, di(C1-C6)alkylamino, or optionally, the R0 and R0′ groups are combined to form a 5- to 6-membered heterocyclic ring;
and wherein in formula I, there will be from 0 to 1 Lf groups and from 1 to 4 Pz groups, preferably 1 to 2 Pz groups.

4. A fluorescent dye reagent of claim 3, having formula I(a) or I(b):




wherein
each A1 and A2 are each independently OR or N(Z)2, wherein R and Z are as set forth in claim 3;
R1′, R1′′, R1, R2, R3 and R4 are each independently selected from H, halogen, cyano, sulfo, aryl, heteroaryl, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, Lf and Pz, or optionally two adjacent members of R1′, R1′′, R2 and R3 are combined to form a five or six membered fused ring that is aromatic, non-aromatic or heteroaromatic and with is optionally substituted with Pz, wherein the alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms and the aryl or heteroaryl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz;
X1, X2, X3 and X4 are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C1-C8)alkenyl, (C1-C8)alkynyl, SO3H, PO3H, CO2H, Lf and Pz and optionally, any two adjacent X1 through X4 are combined to form an aromatic or heteroaromatic ring that is optionally further substituted with from one to four substituents selected from halogen cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz.

5. A fluorescent dye reagent of claim 3, having formula Ic:




wherein the group Z′ or Z′′, at each occurrence, is independently hydrogen, (C1-C8)alkyl, aryl-C1-C8 alkyl or aryl, wherein the aliphatic or aryl portions of the Z′ or Z′′ groups are optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; optionally the Z′ group, at each occurrence is independently combined with R2 or R4 to form a fused 5- or 6-membered ring, and optionally, the Z′′ group, at each occurrence is independently combined with R1 or R1′′ to form a fused 5- or 6-membered ring; wherein if present, said fused 5- or 6-membered ring is optionally fuse to an aryl ring and is substituted with halogen, C1-C4alkyl, Lf or Pz; and the R1′, R1′′, R1, R2, R3, R4 and R0 groups in formula Ic are as described in claim 3.

6. A fluorescent dye reagent of claim 5, selected from the group consisting of







wherein in formulae Ic1-Ic6 
Ra′ at each occurrence is independently selected from hydrogen, C1-C6alkyl, aryl, wherein the aryl group is optionally substituted with Pz or Lf;
Rb′, Rb′′, Rc′, Rd′, Rg′, Rh′, Ri′, Rj′, Rk′, at each occurrence, is independently selected from the group consisting of hydrogen and (C1-C6)alkyl, or optionally, any two substituents of Rb′, Rb′′, Rc′, Rd′, Rg′, Rh′, Ri′, Rj′, or Rk′, that are attached to adjacent ring atoms are combined to form a fused 6-membered aryl ring, said fused ring is optionally substituted with Pz or Lf;
the subscript k and k′ are each independently an integer from 0-2; and
the substituents X1-X4 are as set forth in claim 3.

7. A fluorescent dye reagent of claim 1, having formula:




wherein
A1 represents O, N—Z or N+(Z)2 in which Z is independently H, (C1-C8)alkyl, aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; wherein the Z group, at each occurrence, is optionally combined with R2 or R4 to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused to an aryl ring and is optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz;
A2 represents OR or N(Z)2 in which Z is independently H, (C1-C8)alkyl, aryl-(C1-C8)-alkyl, a protecting group or aryl, wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, aryl Lf or Pz; wherein the Z group, at each occurrence, is optionally combined with R1 or R1′ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally fused to an aryl ring and is optionally substituted with halogen, (C1-C4)alkyl, aryl, Lf or Pz; and R is selected from the group consisting of H, a protecting group, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl and Lf, or is optionally combined with R1 or R1′′ to form a fused 5- to 7-membered ring;
R1′, R1′′, R1, R2, R3 and R4 are each independently selected from H, halogen, cyano, CF3, sulfo, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz;
wherein the alkyl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz.

8. A fluorescent dye reagent of claim 1, having formula:




wherein
R1, R1′, R1′′, R2 and R3 are each independently selected from the group consisting of H, halogen, cyano, sulfo, CF3, (C1-C8)alkyl, (C1-C8)alkylthio, (C1-C8)alkoxy, aryl, heteroaryl, Lf and Pz;
wherein the alkyl portions of any of R1′, R1′′ and R1 through R3 are optionally substituted with halogen, carboxy, sulfo, amino, mono- or dialkylamino, alkoxy, cyano, haloacetyl or hydroxy, and the alkyl portions of the substituents have from 1 to 6 carbon atoms; and the aryl or heteroaryl portions of any of R1′, R1′′ and R1 through R4 are optionally substituted with from one to four substituents selected from the group consisting of halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and P2; and
A1 represents OR, N(Z)2, in which Z is independently hydrogen, (C1-C8)alkyl, aryl-(C1-C8)alkyl, aryl or a protecting group; wherein the aliphatic or aryl portions of the Z group are optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; or optionally the Z group at teach occurrence is independently combined with R1 or R1′′ to form a 5- to 7-membered ring, wherein the resultant 5- to 7-membered ring is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz, and is optionally fused with an aryl or heteroaryl ring wherein the aryl or heteroaryl ring is optionally substituted with halogen, (C1-C4)alkyl, Lf or Pz; and the R substituent in A1 is hydrogen, (C1-C8)alkyl, aryl, aryl(C1-C4)alkyl, a protecting group or Lf.

9. A fluorescent dye reagent of claim 1, having the formula:




wherein
Q1 and Q2 are independently selected from O, S, N and CRaRb or —C(═CH2)— wherein Ra and Rb are independently selected from the group consisting of H, methyl, ethyl and Lf;
the rings, Ax and Ay, each independently represents a condensed substituted or unsubstituted aryl or heteroaryl rings wherein said rings are optionally substitutued with halogen, CN, CF3, (C1-C8)alkoxy, (C1-C8)alkylthio, (C1-C8)alkylamino, di(C1-C8)alkylamino, an aryl or heteroaryl that is optionally substituted with Lf or Pz;
Q represents a conjugated linking system;
RF and RG′ are independently selected from the group consisting of H, C1-C8 alkyl, heteroalkyl, alkylene sulfonic acid, alkylene phosphoric acid, alkylene phosphoric acid, alkylene aryl, substituted alkylene aryl, alkylene carboxylic acid, Lf and P2, wherein said aryl group in RG or RG′ is optionally substituted with from about one to two Pz groups and from about 0 to about 2 Lf groups.

10. A fluorescent dye reagent of claim 9, wherein A is a conjugated linking system selected from the group consisting of:




wherein
Rc, Rd, Re, Rf, Rg, Rh, Ri, Rj and Rk are each independently selected from the group consisting of hydrogen, halogen, —CN, —CF3, C1-C6 alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;
Rn and Rp are each independently selected from the group consisting of hydrogen, oxygen, halogen, —CN, —CF3, —(C1-C6)alkyl, —NRm, —ORm, —SRm, —NRmRm, wherein at each occurrence, Rm is independently selected from the group consisting of hydrogen, (C1-C6)alkyl, aryl, substituted aryl, heteraryl, substituted hetcroaryl or Lf, wherein said aryl or heteroaryl group is optionally substituted with Lf or Pz; and
the subscript o is the integer 0 or 1.

11. A fluorescent dye reagent of claim 1, having the formula:




wherein X1, X2, X3 and X4 are each independently selected from the group consisting of H, halogen, cyano, CF3, (C1-C8)alkyl, (C1-C8)alkoxy, (C1-C8)alkylthio, (C2-C8)alkenyl, (C2-C8)alkynyl, SO3H, PO3H, CO2H, Lf and Pz; and optionally, any two adjacent X1 through X4 are combined to form a non-aromatic, aromatic or heteroaromatic five or six-membered ring that is optionally further substituted with from one to four substituents selected from halogen, cyano, carboxy, sulfo, hydroxy, amino, mono- or di(C1-C6)alkylamino, (C1-C6)alkyl, (C1-C6)alkylthio, (C1-C6)alkoxy, Lf and Pz; and wherein there is present from 0 to 1 Lf groups and from 1 to 2 Pz groups.

12. A fluorescent dye reagent of claim 1, selected from the compounds consisting of:

13. A fluorescent dye reagent having the formula:


Fl-P1 


wherein
Fl is a fluorescent dye component; and
P1 is functionalized phosphonate group having the formula:




wherein the wavy line indicates the direct attachment to a sp2 carbon of said fluorescent dye component;
L1 is a linking group;
R is a member selected from the group consisting of H, C1-C8 alkyl, aryl, arylC1-C4 alkyl, -La-Na, and -La-NRARB;
wherein La is an alkylene linking group, Na is an ammonium ion group, and each of RA and RB is independently selected from the group consisting of H and a labile protecting group;

and
Pa is a functional group component selected from the group consisting of a phosphoramidite moiety, a mono-, di- or tri-functional linking group having at least one terminal functional group or protected functional group, a solid support and a reactive group;
and salts thereof wherein R═CF3CONH(CH2)4—.

14. A fluorescent dye reagent of claim 13, wherein said fluorescent dye component is selected from the group consisting of coumarin dyes, resorufin dyes, dipyrrometheneboron difluoride dyes, ruthenium bipyridyl dyes, energy transfer dyes, thiazole orange dyes, polymethines and N-aryl-1,8-naphthalimide dyes.

15. A fluorescent dye reagent of claim 13, selected from the group consisting of compound