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EP-3262022-B1 - PHENYLETHYNYLNAPHTHALENE DYES AND METHODS FOR THEIR USE

EP3262022B1EP 3262022 B1EP3262022 B1EP 3262022B1EP-3262022-B1

Inventors

  • BATTRELL, C. FREDERICK
  • MATRAY, TRACY
  • SHERIF, Hesham
  • VANBRUNT, Michael

Dates

Publication Date
20260506
Application Date
20160226

Claims (16)

  1. A compound having the following structure (I): or a salt thereof, wherein: R 1a , R 1b , R 1c , R 1d , R 1e and R 1f are each independently absent, H, halo, nitro, C 1 -C 6 alkyl, -SO 3 - , -SO 3 alkyl, C 1 -C 6 alkoxy, aryl, aryloxy, C 1 -C 6 alkylcarbonyl, C 1 -C 6 alkylcarbonyloxy, arylcarbonyl, arylcarbonyloxy, amino, alkylamino, arylamino, aralklyamino, -L 1 -(R 3 ) z -L 2 -M or -L 1 -(R 3 ) z -L 2 -S-S-L 2 -(R 3 ) z -L 1 -I, where I represents, independently, a further compound of structure (I); or one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f joins with another one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f to form a heterocyclic ring and the remaining R 1a , R 1b , R 1c , R 1d , R 1e and R 1f are each independently absent, H, halo, nitro, C 1 -C 6 alkyl, -OSO 2 - , -OSO 2 alkyl, C 1 -C 6 alkoxy, aryl, aryloxy, C 1 -C 6 alkylcarbonyl, C 1 -C 6 alkylcarbonyloxy, arylcarbonyl, arylcarbonyloxy, amino, alkylamino, arylamino, aralklyamino, -L 1 -(R 3 ) z -L 2 -M or -L 1 -(R 3 ) z -L 2 -S-S-L 2 -(R 3 ) z -L 1 -I, where I represents, independently, a further compound of structure (I); R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i and R 2j are each independently H, halo, C 1 -C 6 alkoxy, aryloxy, amino, alkylamino, arylamino, aralklyamino, or heterocyclyl; R 3 is, at each occurrence, independently a mono or bivalent functional group selected from the group consisting of polyalkyether, polyalkylenether, hydroxylalkoxy, hydroxylalkyl, hydroxylalkylene, aminoalkylene, aminoalkoxy, hydroxylpolyalkyether, hydroxylpolyalkylenether, aminopolyalkyether, aminopolyalkylenether, phosphate, thiophosphate, phospho, thiophospho, phosphoalkyl, phosphoalkylene, phosphoalkylether, thiophosphoalkyl, thiophosphoalkylene, thiophosphoalkylether, and phosphoramidite; M is absent, H, sulfhydryl, disulfide, N-succinimide ester, imidoester, polyflourophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, maleimide, nucleic acid or a polymer thereof, amino acid or a polymer thereof, peptide, carbohydrate, lipid, enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, antigen, prion, or microparticle; and L 1 and L 2 are, at each occurrence, independently an optional linker; x and y are each independently an integer from 0 to 4, and the sum of x and y is 2 or greater; z is an integer from 1 to 10; and provided at least one of R 1a , R 1b , R 1c , R 1d , R 1e , or R 1f is -L 1 -(R 3 ) z -L 2 -M or -L 1 -(R 3 ) z -L 2 -S-S-L 2 -(R 3 ) z -L 1 -I, where I represents, independently, a further compound of structure (I).
  2. The compound of claim 1, wherein: A) the sum of x and y is 2; or B) x is 0 and y is 2.
  3. The compound of claim 1, wherein the compound has one of the following structures (la) or (Ia'):
  4. The compound of any one of claims 1-3, wherein: A) R 1a is -L 1 -(R 3 ) z -L 2 -M; or B) one of R 1d , R 1e or R 1f is -L 1 -(R 3 ) z -L 2 -M; or C) R 1a and R 1b are both -L 1 -(R 3 ) z -L 2 -M.
  5. The compound of any one of claims 1-2, wherein: A) one of R 1a or R 1b is -L 1 -(R 3 ) z -L 2 -M; or B) one of R 1a or R 1b is -L 1 -(R 3 ) z -L 2 -M and one of R 1d , R 1e or R 1f is -L 1 -(R 3 ) z -L 2 -M; or C) at least one of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i or R 2j is i) amino; ii) alkylamino; iii) arylamino; iv) aralkylamino; v) heterocyclyl; or D) R 2c or R 2h , or both, is i) amino; ii) alkylamino; iii) arylamino; iv) aralkylamino; v) heterocyclyl; or E) at least one of R 2a , R 2b , R 2c , R 2d , R 2e , R 2f , R 2g , R 2h , R 2i or R 2j is i) dimethylamino; ii) diethylamino; iii) diisopropylamino; iv) ethylisopropylamino; v) diphenylamino; vi) N-pyrrolidinyl; vii) N-pyrrolyl; or F) R 2c or R 2h , or both, is i) dimethylamino; ii) diethylamino; iii) diisopropylamino; iv) ethylisopropylamino; v) diphenylamino; vi) N-pyrrolidinyl; or vii) N-pyrrolyl.
  6. The compound of claim 1, wherein the compound has one of the following structures:
  7. The compound of any one of claims 1-6, wherein: A) at least one of R 2a , R 2b , R 2d , R 2e , R 2f , R 2g , R 2i or R 2j is halo; or B) at least one of R 2a , R 2b , R 2d , R 2e , R 2f , R 2g , R 2i or R 2j is alkoxy; or C) at least one of R 2a , R 2b , R 2d , R 2e , R 2f , R 2g , R 2i or R 2j is aryloxy; or D) at least one of R 2a , R 2b , R 2d , R 2e , R 2f , R 2g , R 2i or R 2j is H; or E) each of R 2a , R 2b , R 2d , R 2e , R 2f , R 2g , R 2i and R 2j is H.
  8. The compound of claim 1, wherein: A) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is C 1 -C 6 alkoxy, aryloxy or C 1 -C 6 alkylcarbonyloxy; or B) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is amino, alkylamino, arylamino or aralkylamino; or C) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is halo or nitro; or D) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is -SO 3 - or -SO 3 alkyl; or E) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is C 1 -C 6 alkyl or aryl; or F) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is alkylcarbonyl or arylcarbonyl; or G) at least one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f is methoxy, dimethylamino, methyl, or ethyl.
  9. The compound of any one of claims 1-7, wherein one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f joins with another one of R 1a , R 1b , R 1c , R 1d , R 1e or R 1f to form a carbocyclic or heterocyclic ring and the remaining R 1a , R 1b , R 1c , R 1d , R 1e and R 1f are each independently absent, H, halo, nitro, C 1 -C 6 alkyl, -OSO 2 - , -OSO 2 alkyl, C 1 -C 6 alkoxy, aryl, aryloxy, C 1 -C 6 alkylcarbonyl, C 1 -C 6 alkylcarbonyloxy, arylcarbonyl, arylcarbonyloxy, amino, alkylamino, arylamino, aralkylamino, -L 1 -(R 3 ) z -L 2 -M or -L 1 -(R 3 ) z -L 2 -S-S-L 2 -(R 3 ) z -L 1 -I, where I represents, independently, a further compound of structure (I).
  10. The compound of any one of claims 1-9, wherein the compound has one of the following structures:
  11. The compound of claim 1, wherein L 1 is C 1 -C 6 alkylene.
  12. The compound of any one of claims 1-11, wherein A) R 3 is selected from the group consisting of polyalkylenether, phosphate, phospho and phosphoalkylene; or B) R 3 is selected from the group consisting of: wherein n is an integer from 1 to 6 and m is an integer from 2 to 10.
  13. The compound of any one of claims 1-12, wherein -(R 3 ) z -L 2 -M has one of the following structures: or
  14. The compound of claim 1, wherein: A) R 3 is phosphoramidite and M is absent; or B) M is absent or H; or C) M is sulfhydryl, disulfide, N-succinimide ester, imidoester, polyfluorophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino or a maleimide; or D) M is a biomolecule, wherein the biomolecule is a nucleic acid, amino acid, peptide, carbohydrate, lipid, enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, antigen or prion.
  15. The compound of claim 1, wherein the compound has one of the following structures: or a salt thereof, wherein: A) M is -SH, disulfide, N-succinimide ester, imidoester, polyflourophenyl ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, maleimide, nucleic acid or a polymer thereof, amino acid or a polymer thereof, peptide, carbohydrate, lipid, enzyme, receptor, receptor ligand, antibody, glycoprotein, aptamer, antigen, prion, or microparticle.
  16. Use of a composition comprising the compound of any one of claims 1-15 and one or more biomolecules in an analytical method for detection of the one or more biomolecules.

Description

Field The present invention is directed to novel fluorescent or colored dyes comprising a phenylethynylnapthalene moiety and methods for their preparation and use in various analytical methods. Description of the Related Art There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microorganisms, ions, and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, peptides, e.g., antibodies and enzymes, and nucleic acids, particularly those implicated in disease states. The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity that characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable "label" which is attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding affinities of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed. A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations. However, such labels are expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Thus, there is wide interest in non-radioactive labels, particularly in labels that are observable by spectrophotometric, spin resonance, and luminescence techniques, and reactive materials, such as enzymes that produce such molecules. Labels that are detectable using fluorescence spectroscopy are of particular interest because of the large number of such labels that are known in the art. Moreover, the literature is replete with syntheses of fluorescent labels that are derivatized to allow their attachment to other molecules, and many such fluorescent labels are commercially available. Cyanine dyes have been widely used for labeling biomolecules including antibodies, DNA probes, avidin, streptavidin, lipids, biochemical analogs, peptides, and drugs, as well as for a variety of applications including DNA sequencing, DNA microarray, western blotting, flow cytometry analysis, and protein microarrays, to name a few. Scientists favor using cyanine dyes in biological applications because, among other reasons, cyanine dyes 1) are biocompatible; 2) have high molar absorptivity (c.a. 105 M-1 cm-1); 3) are readily modified to match a wide range of desired excitation and detection wavelengths (e.g., about 500 to about 900 nm); 4) are capable of incorporating water-soluble groups and linking groups; 5) and possess favorable fluorescence properties. In particular, Cy2 conjugates, with a maximum adsorption/excitation around 492 nm and emission around 510 nm, in the green region of the visible spectrum, are commonly used as an alternative to FITC due to reduced sensitivity to pH changes. However, the low fluorescence quantum yield, short fluorescence lifetime, propensity to photobleach, and poor chemical stability of Cy2 has limited its use in chemical and life sciences. WO2015/109136 relates to novel fluorescent or colored dyes and methods for their preparation and use in various analytical methods. US2009/299070A1 describes a semiconductor device comprising bis(substituted ethynyl) compounds with tri- or polycyclic aromatic hydrocarbon backbones with hetero-atoms, wherein the rings that constitute the backbone are annealed to each other via not more than two commonly shared ring atoms, respectively, and the number of the common atoms of the vicinal rings is twice the number of the common side areas (meaning that