Uncharged pyrenyloxy sulfonamide dyes for conjugation with biomolecules

Abstract

The invention relates to novel fluorescent dyes based on the following pyrenyloxy sulfonamide structure: ##STR00001##
wherein R.sup.1 is a leash joined to the pyrenyloxy group via an ether link containing generally a reactive functional group such as, activated carbonate, activated ester, amino group, azide or alkyne for conjugation with biomolecules; R.sup.2 and R.sup.3 are hydrogen atoms, or short alkyl chains, or cyclic rings with or without heteroatoms such as nitrogen, oxygen, sulfur, phosphorus. The spectral properties of the fluorescent dyes are sufficiently different in wave-lengths and intensity from fluorescein as to permit simultaneous use of fluorescein and/or more other fluorescent dyes with minimum interference and to avoid interference from endogenous green fluorescent protein in biological system. The dyes are non-ionic to facilitate their entry into cells for intracellular detection. The non-ionic structure also precludes undesired electrostatic reactions with ionic sites on biological components and structures. The dyes have bigger Stokes' shifts than other dyes with similar spectral properties allowing use of simpler, more efficient detection equipment, are not sensitive to pH, and have good solubility in aqueous solution.

Claims

1. A fluorescent dye having the structure: ##STR00006## wherein, (a) R.sup.1 is a leash that contains a chemically reactive functional group to form a conjugate having a biologically active ligand and a biomolecule, wherein the chemically reactive functional group is activated ester, activated carbonate, acyl azides, acid halides, or other reactive groups that include acrylamides, alkyl and arylazides, alkynes and constrained cyclic alkynes, anhydrides, halides, sulfonate esters, amines, alcohols, haloacetamides, isothiocyanates and isocyanates; and (b) R.sup.2 and R.sup.3 in sulfonamide are selected from an independent alkyl group which is methyl, ethyl, propyl, and cyclic rings with or without heteroatoms which are nitrogen, oxygen, sulfur, or phosphorus.

2. A fluorescent dye as recited in claim 1, wherein R.sup.1 is a leash containing an activated carbonate, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00007##

3. A fluorescent dye as recited in claim 1 wherein R.sup.1 is a leash containing an activated ester, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00008##

4. A fluorescent dye as recited in claim 1 wherein R.sup.1 is a leash containing an amine, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00009##

5. A fluorescent dye as recited in claim 1 wherein R.sup.1 is a leash containing an azide, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00010##

6. A fluorescent dye as recited in claim 1 wherein R.sup.1 is a leash containing an alkyne, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00011##

7. A fluorescent dye as recited in claim 1 wherein R.sup.1 is a leash containing a constrained cycloalkyne, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00012##

8. A fluorescent dye as recited in claim 1 wherein R.sup.1 is an elongated leash containing an amine, a sulfonamide moiety is N-sulfonylmorpholine, and the dye has the structure: ##STR00013##

9. A fluorescent dye as recited in claim 1 wherein the dye is conjugated with a biologically active ligand which is a cell, tissue, protein, antibody, enzyme, or a biomolecule which is a drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecules which are natural or synthetic macromolecules or polymers.

10. A composition of claim 9, wherein a nucleotide is a Morpholino oligo.

11. A composition of claim 10, wherein a conjugate of the Morpholino oligo having the fluorescent dye has the structure: ##STR00014##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Excitation and emission spectrum of pyrenyloxy sulfonamide (Compound 4a)

(2) FIG. 2. Excitation and emission spectrum of Cascade Blue

(3) FIG. 3. Excitation and emission spectrum of fluorescein

(4) FIG. 4. Excitation and emission spectrum of lissamine

(5) FIG. 5. Synthesis of activated carbonate of pyrenyloxy sulfonamide

(6) FIG. 6. Synthesis of pyrenyloxy sulfonamide derivative (Compound 6)

(7) FIG. 7. Photostability test of Compound 6

(8) FIG. 8. Synthesis of a Morpholino conjugate with a pyrenyloxy sulfonamide dye

DETAILED DESCRIPTION OF THE INVENTION

(9) The core structure of the improved, reactive, hydrophilic, uncharged fluorophores is a new class of pyrenyloxy sulfonamide compounds. This invention describes methods for the synthesis of novel derivatives with substantially improved properties, as well as demonstration that the materials can be chemically bonded to the functional groups present in many biomolecules to form fluorescent conjugate. The novelty of this invention involves the substantial improvement of the properties of Cascade Blue, a currently available blue-fluorescent dye. The high polarity of anionic sulfonate of Cascade Blue results in the membrane-impermeability which can only be used extracellularly, a major disadvantage for many desirable applications. The uncharged fluorophores of this invention do not impede cell entry. And the high aqueous solubility of the pyrenyloxy sulfonamide is useful for biological and physiological application, allowing staining the cell surface which is negatively charged, and even more useful to enter the cell for intracellular detection. In addition, the Stokes shift of Cascade Blue is undesirably small (ca. 20 nm, FIG. 2). It is highly desirable to increase the Stokes Shift so that the signal to noise ratio can be substantially improved. The Stokes shift of the compounds of this invention is a much more practical 44 nm (FIG. 1), a significant improvement in its utility as a tracer for biological applications. The molar extinction coefficient of the structure of the invention is slightly higher (3.110.sup.4 cm.sup.1 M.sup.1 at 423 nm) than that of Cascade Blue (2.910.sup.4 cm.sup.1 M.sup.1 at 410 nm), an additional advantage for increased sensitivity. Photostability of the dye (compound 6 in FIG. 6) was tested under the light exposure. The scans were overlaid up to 60 mins with 15 min interval. FIG. 7 shows the robust stability of the core structure of the dye of this invention. This resistance to photobleaching greatly enhances the utility of the dye for quantitative measurements and permits extended illumination time.

(10) Pyrenyloxy sulfonamide compounds, namely the phenol intermediates (analogous to Compound 3 in FIG. 5) were reported in literature (Finkler B. et al: Photochem. Photobiol. Sci. 2014, 13, 548-562). However, those compounds were used as end products for fluorescence spectroscopic studies. The phenolic moiety or other sites of the molecule were not further derivatized to install a leash containing a reactive functional group, lacking the function for covalent conjugation with biomolecules. Two filed patents [(Singaram B. and Wessling R. A. U.S. Pat. No. 7,470,420 and U.S. Pat. No. 8,394,357)] did not describe the installation of the reactive functional groups for conjugation with biomolecules, making them only useful for staining as dyes, not a tracer for the conjugate of the biomolecule of the interest. Reactive functional groups were installed in this invention which are useful to form covalent linkage with many of the functional groups found in biomolecules. Potentially reactive functional groups that are intrinsically present or that can be introduced into the pyrenyloxy sulfonamide compounds which can conjugate with biomolecules and macromolecules include but are not limited to activated carbonate, amines, thiols, alcohols, carboxylic acids, activated esters, aldehydes, ketones, azides, alkynes or constrained cycloalkynes. Chemically reactive fluorescent reagents developed in this invention are generally very mild for conjugation with biomolecules, and thus will be useful not only for modification of the synthetic macromolecules, but also for the biologically active ligands such as a cell, tissue, protein, antibody, enzyme, or a biomolecule such as a drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecules such as natural macromolecules or polymers. None of the reagents previously described in the chemical or biochemical literature are recognized as possessing the appropriate combination of chemical reactivity, spectral properties, high water solubility, non-ionic structural feature, fluorescence yield and lack of pH sensitivity to make them optimum for use in the presence of background green fluorescent protein, or for simultaneous use with one or more than one fluorescent dyes.

(11) The new pyrenyloxy sulfonamide derivatives that are the subject of this invention have the general structure below.

(12) ##STR00002##

(13) In this structure, R.sup.2 and R.sup.3 are hydrogen atoms, or short alkyl chains, or cyclic rings with or without heteratoms such as nitrogen, oxygen, sulfur, phosphorus to confer the sulfonamide with uncharged feature under physiological conditions and appropriate water solubility.

(14) ##STR00003##
R.sup.2, R.sup.3, R.sup.4H, CH.sub.3, C.sub.2H.sub.5, CH.sub.2CH.sub.2CH.sub.3, CH.sub.2CH.sub.2OR.sup.4, CH.sub.2CH.sub.2NHCOR.sup.4

(15) Furthermore, R.sup.1 is a leash that can be further modified to provide a chemically reactive functional group. The subsequent modifications include but are not limited to chemically reactive derivatives such as activated carbonate, and activated carboxylic acids such as p-nitrophenyl esters, pentafluorophenyl esters, succinimidyl esters, acyl azides, and acid halides, or other reactive groups that include acrylamides, alkyl and arylazides, alkynes and constrained cyclic alkynes, anhydrides, halides, sulfonate esters, amine and hydrazine derivatives, alcohols, haloacetamides, isothiocyanates and isocyanates.

(16) Several representative examples of derivatives that have the chemical structure and properties claimed by this invention and the precursors that are used in their synthesis are illustrated below. It should be clearly recognized that the leash length and structural architecture can vary as long as the reactive functional groups are constructed which can be used for conjugation with macromolecules and biomolecules. The listing is not meant to be inclusive of chemical reactivity since with the appropriate choice of solvent, temperature and catalysts, other functional groups can be made to react and the listed functional groups can be made to react with other reactive moieties.

(17) ##STR00004##

(18) Chemically reactive derivatives of fluorophores have wide utility as tracers. This invention describes methods for preparation of pyrenyloxy sulfonamide dyes that incorporate activated carbonate as shown in FIG. 5. Acetylation of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid protects the phenolic hydroxyl to give the corresponding acetate 1. Trisulfonyl chloride 2 was converted from trisulfonate sodium salt with thionyl chloride. Trisulfonamide 3 formation by treatment with a secondary amine resulted in concomitant generation of phenolic alcohol providing the substrate for phenolic alkylation. The alkyl leash with a terminal hydroxyl group 4, after exposed to bis(4-nitrophenyl) carbonate, furnishes an activated carbonate moiety 5, useful for conjugation with biomolecules. An example was shown by using the activated carbonate of pyrenyloxy sulfonamide dye of this invention to conjugate a Morpholino oligo (FIG. 8). The conjugation was carried out while the oligo was still on the synthesis resin. The conjugate was obtained by the global ammonolytic deprotection of the protecting groups of the Morpholino subunits and the cleavage from the synthesis resin. The process provides a practical means for efficient streamlined production of conjugate containing the pyrenyloxy sulfonamide dye of this invention and a biomolecule such as a Morpholino oligo.

(19) Other reactive functional groups include, but not limited to carboxylic acids and esters, amines, hydrazides, halides, alcohols and aldehydes and their subsequent modification to give chemically reactive reagents that can be coupled to other molecules for use as fluorescent tracers.

(20) The excitation and emission spectrum of compound 4a is shown in FIG. 1. Obvious is the spectra region shorter than 500 nm which is particularly valuable to get away from interference where green fluorescent protein is present or, to carry out the applications requiring multiple dyes such as flow cytometry, DNA sequencing and multiparameter microscopy. More remarkable is the significantly greater Stokes' shift (ca. 44 nm) of the pyrenyloxy sulfonamide dye (peak of excitation: 421 nm, peak of emission: 465 nm). This special feature increases the sensitivity of the fluorescence techniques where the emission signal can be maximized against the low background.

EXAMPLES

(21) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

(22) The following synthetic examples illustrate how one of ordinary skill in the art could synthesize a variety of chemically reactive derivatives containing the desired fluorophore that are the subject of this invention. The methods outlined are intended to be illustrative and not to define or limit the possible methods of dye synthesis. It is also to be recognized that the specific compounds described herein are included for the purpose of clearly demonstrating the nature of the invention and do not exhaust the structural and chemical variations which are also considered to fall within the scope of this invention. It is to be understood that certain changes and modifications which are apparent to one skilled in the art are included in the purview and intent of this invention. Inclusion of spectral and other characterization of some of the synthetic products and intermediates is intended to facilitate comparison and confirmation of products by one skilled in interpretation of spectral techniques and not to specifically define limitations or absolute values for physical properties of the materials.

(23) A general scheme for synthesis of several pyrenyloxy sulfonamide dyes that are or can be modified to have the desired chemical reactivity or chemical substituents falling within the scope of this invention is illustrated below. The general method consists of condensation of an alkylating reagent such as an alkyl halide or appropriately substituted alkyl halide, in the presence of a base with a substituted pyrene sulfonamide 3 having a hydroxyl on the 8-position to give an alkoxy intermediate such as compound 4 in FIG. 5.

(24) Frequently, there are a number of alternative routes whose choice depends primarily on the availability (or ease of synthesis) of the reactants, solvents and equipment. Suitable bases for the initial alkylation step include but are not limited to bicarbonate, carbonate, diisopropylethylamine, and triethylamine. Suitable catalysts for the initial alkylation step include but are not limited to potassium iodide, tetrabutylammonium iodide. Suitable substituents on the alkylating reagent include but are not limited to substituted or unsubstituted alkyl, cycloalkyl, arylalkyl and aryl derivatives. Pyrenyloxy sulfonamide products may be modified in subsequent reactions by known chemical techniques including but not limited to esterification, sulfonation, nitration, alkylation, acylation, and halogenation. Furthermore, the substituents can in some cases be further modified to introduce chemically reactive functional groups, or biologically active groups that are understood to fall within the scope of this patent. Examples of methods that are suitable for preparation of selected members of this new class of reactive dyes are given in the examples outlined below. It is recognized that variations in the synthetic methods and reactants are possible that would fall within the scope and intent of this invention.

Example 1

Trisodium 8-acetoxypyrene-1,3,6-trisulfonic acid (1)

(25) Trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid (4.56 g, 8.70 mmol) and sodium acetate (71.4 mg, 0.88 mmol) were suspended in acetic anhydride (50 ml) and refluxed for 35 hours (Finider, B. et al. Photochemical & Photobiological Sciences 13, 548-562 (2014)). After the suspension was cooled down to room temperature, it was diluted with THF and filtered off. The residue was washed with acetone and dried under vacuum yielding a grey powder (5.52 g, 8.0 mmol, 92%).

Example 2

8-Acetoxypyrene-1,3,6-trisulfonyl chloride (2)

(26) The above trisulfonic acid 1 (1.09 g, 1.93 mmol) was suspended in thionyl chloride (5 ml). After addition of dimethylformamide (30 l), the mixture was heated to reflux for 5 hours. The solution was cooled down to room temperature and poured on ice. After precipitation, the sulfonyl chloride was filtered off and was obtained as an orange powder after drying in vacuo (1.04 g, 1.88 mmol, 97%).

Example 3

8-Hydroxypyren-tris(morpholino)-1,3,6-trisulfonamide (3a)

(27) The above trisulfonyl chloride 2 (1.04 g, 1.88 mmol) was added to morpholine (10 ml) cooled in an ice bath. The mixture was kept at room temperature for 16 hours. Most reagents were removed by evaporation. The residue was dissolved in dichloromethane and washed with aqueous hydrochloric acid (1N). The organic layer was separated and dried over sodium sulfate. After removal of the solvent, the residue was obtained as orange powder (1.20 g, 1.80 mmol, 95%). R.sub.f=0.11 (ethyl acetate/hexanes: 1/1).

8-Hydroxypyren-tris(pyrrolidino)-1,3,6-trisulfonamide (3b)

(28) Compound 3b was prepared following the general procedure as for 3a. The product was obtained as orange powder (98%). R.sub.f=0.26 (ethyl acetate/hexanes: 1/1).

8-Hydroxypyren-tris(piperidino)-1,3,6-trisulfonamide (3c)

(29) Compound 3c was prepared following the general procedure as for 3a. The product was obtained as orange powder (97%). R.sub.f=0.45 (ethyl acetate/hexanes: 1/1).

8-Hydroxypyren-N,N,N-trimethoxy-N,N,N-trimethyl-1,3,6-trisulfonamide (3d)

(30) Compound 3d was prepared following the general procedure as for 3a. N,O-Dimethylhydroxylamine hydrochloride was deprotonated with triethylamine and reacted with the trisulfonyl chloride 2. The product was obtained as orange powder (90%). R.sub.f=0.38 (ethyl acetate/hexanes: 1/1).

Example 4

8-(3-hydroxypropyloxy)pyren-tris(morpholino)-1,3,6-trisulfonamide (4a)

(31) The above 8-hydroxypyrene derivative 3a (1.20 g, 1.80 mmol) was dissolved in acetonitrile (20 ml). 3-Bromopropanol (278 mg, 2 mmol), potassium carbonate (690 mg, 5 mmol) and potassium iodide (10 mg, 0.06 mmol) were added to the mixture. The mixture was kept at 80 C. for 16 hours. After removal of the solvent, the residue was dissolved in dichloromethane and washed with water. The organic layer was dried over sodium sulfate and evaporated to give a solid as yellow powder (1.07 g, 1.48 mmol, 82%). R.sub.f=0.25 (ethyl acetate/hexanes: 3/1).

8-(3-hydroxypropyloxy)pyren-tris(pyrrolidino)-1,3,6-trisulfonamide (4b)

(32) Compound 4b was prepared following the general procedure as for 4a. The product was obtained as yellow powder (80%). R.sub.f=0.08 (ethyl acetate/hexanes: 1/1).

8-(3-hydroxypropyloxy)pyren-tris(piperidino)-1,3,6-trisulfonamide (4c)

(33) Compound 4c was prepared following the general procedure as for 4a. The product was obtained as yellow powder (77%). R.sub.f=0.20 (ethyl acetate/hexanes: 1/1).

8-(3-hydroxypropyloxy)pyren-N,N,N-trimethoxy-N,N,N-trimethyl-1,3,6-trisulfonamide (4d)

(34) Compound 4d was prepared following the general procedure as for 4a. The product was obtained as yellow powder (83%). R.sub.f=0.22 (ethyl acetate/hexanes: 1/1).

Example 5

8-[3-(4-nitrophenoxycarbonyloxypropyloxy)]pyren-tris(morpholinyl)-1,3,6-trisulfonamide (5a)

(35) The above 3-hydroxypropyloxypyrene derivative 4a (1.07 g, 1.48 mmol) was dissolved in acetone (20 ml). Bis(4-nitrophenyl) carbonate (912 mg, 3 mmol) and triethylamine (1 ml) were added to the solution. The mixture was kept at room temperature for 16 hours. After removal of the volatile materials, the residue was dissolved in dichloromethane and loaded on a silica gel column. The product was isolated by chromatography to give a yellow solid (1.20 g, 1.35 mmol, 91%). R.sub.f=0.73 (ethyl acetate/hexanes: 3/1).

(36) .sup.1H-NMR (400 MHz, CDCl.sub.3): =9.33 (d, 1H, J=9.94), 9.30 (d, 1H, J=9.68), 9.27 (s, 1H), 9.16 (d, 1H, J=9.94), 8.92 (d, 1H, J=9.58), 8.38 (s, 1H), 8.28 (d, 2H, J=9.24), 7.40 (d, 2H, J=9.21), 4.72 (t, 2H, J=6.20), 4.67 (t, 2H, J=5.87), 3.75 (m, 12H), 3.26 (m, 12H), 2.60 (m, 2H).

8-[3-(4-nitrophenoxycarbonyloxypropyloxy)]pyren-tris(pyrrolidino)-1,3,6-trisulfonamide (5b)

(37) Compound 5b was prepared following the general procedure as for 5a. The product was obtained as yellow solid (88%). R.sub.f=0.39 (ethyl acetate/hexanes: 1/1).

(38) .sup.1H-NMR (400 MHz, CDCl.sub.3): =9.36 (d, 1H, J=9.98), 9.31 (d, 1H, J=9.68), 9.21 (s, 1H), 9.18 (d, 1H, J=9.74), 8.87 (d, 1H, J=9.66), 8.43 (s, 1H), 8.28 (d, 2H, J=9.16), 7.41 (d, 2H, J=9.15), 4.72 (t, 2H, J=6.13), 4.65 (t, 2H, J=5.93), 3.50 (m, 4H), 3.48 (m, 4H), 3.44 (m, 4H), 2.58 (m, 2H), 1.94 (m, 8H), 1.86 (m, 4H).

8-[3-(4-nitrophenoxycarbonyloxypropyloxy)]pyren-tris(piperidino)-1,3,6-trisulfonamide (5c)

(39) Compound 5c was prepared following the general procedure as for 5a. The product was obtained as yellow solid (96%). R.sub.f=0.54 (ethyl acetate/hexanes: 1/1).

(40) .sup.1H-NMR (400 MHz, CDCl.sub.3): =9.28 (d, 1H, J=9.95), 9.27 (d, 1H, J=9.64), 9.24 (s, 1H), 9.13 (d, 1H, J=10.29), 8.87 (d, 1H, J=9.58), 8.39 (s, 1H), 8.29 (d, 2H, J=9.15), 7.41 (d, 2H, J=9.19), 4.72 (t, 2H, J=5.88), 4.64 (t, 2H, J=6.05), 3.30 (m, 4H), 3.29 (m, 4H), 3.24 (m, 4H), 2.58 (m, 2H), 1.64 (m, 12H), 1.48 (m, 6H).

8-[3-(4-(4-nitrophenoxycarbonyloxypropyloxy)]pyren-N,N,N-trimethoxy-N,N,N-trimethyl-1,3,6-trisulfonamide (5d)

(41) Compound 5d was prepared following the general procedure as for 5a. The product was obtained as yellow solid (93%). R.sub.f=0.41 (ethyl acetate/hexanes: 1/1).

(42) .sup.1H-NMR (400 MHz, CDCl.sub.3): =9.52 (d, 1H, J=9.48), 9.47 (d, 1H, J=10.02), 9.35 (d, 1H, J=10.05), 9.35 (s, 1H), 8.95 (d, 1H, J=9.82), 8.41 (s, 1H), 8.28 (d, 2H, J=9.17), 7.40 (d, 2H, J=9.21), 4.72 (t, 2H, J=6.24), 4.66 (t, 2H, J=5.90), 3.80 (s, 3H), 3.78 (s, 3H), 3.73 (s, 3H), 2.98 (s, 3H), 2.97 (s, 3H), 2.59 (m, 2H).

Example 6

(43) Determination of the Chemical Reactivity of the Dye and Conjugation with a Morpholino Oligo

(44) The chemical reactivity of the dyes that are the subject of this invention was determined by incubation of the reactive derivatives 5a in dimethylsulfoxide solution with a model Morpholino Oligo where the oligo is still on the synthesis resin. After incubation at 50 C. for 4 hours, the conjugate is then treated with concentrated ammonia to remove the protecting groups on the oligo subunits and to cleave the conjugate from the synthesis resin. It was demonstrated that the reactive dye derivative can be efficiently coupled with the Morpholino oligo while still on the synthesis resin, making the process in a practical manner where the excessive reactive dye can be removed by washing with the solvent, and the conjugate (structure shown below) can be obtained by deprotection of protecting groups of the oligo subunits and cleavage of the oligo conjugate from the synthesis resin.

(45) ##STR00005##