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WO2024013770A1 - Composés de 3-méthylbenzo[d]thiazol-3-ium substitués et leur utilisation - Google Patents

Composés de 3-méthylbenzo[d]thiazol-3-ium substitués et leur utilisation Download PDF

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Publication number
WO2024013770A1
WO2024013770A1 PCT/IN2023/050682 IN2023050682W WO2024013770A1 WO 2024013770 A1 WO2024013770 A1 WO 2024013770A1 IN 2023050682 W IN2023050682 W IN 2023050682W WO 2024013770 A1 WO2024013770 A1 WO 2024013770A1
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Prior art keywords
methyl
thiazol
compound
ium
ylidene
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Inventor
Atul Goel
Sajiya PARVEEN
Kundan Singh Rawat
Shradha GOENKA
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Biotech Desk Pvt Ltd
Council of Scientific and Industrial Research CSIR
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Biotech Desk Pvt Ltd
Council of Scientific and Industrial Research CSIR
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to substituted 3-methylbenzo[d]thiazol-3-ium compounds of the general formula I and their nucleic acid conjugates/complexes, salts which are potentially useful in chemical and biological sciences such as cell imaging applications, diagnostics, fluorescent tags, pharmaceuticals and other useful molecular biology applications, and a process of preparing said new compounds. More particularly, the present invention relates to 3-methyl-2- ((2-substituted- 1 -substituted/unsubstitued-phenylquinolin-4( 1 H)ylidene)methyl)benzo [d] thiazol- 3-ium compounds, processes for preparing the said compounds and their uses for detection of nucleic acids in fluorescence -based imaging and/or analysis. More specifically, the invention relates to the field of nucleic acid detection including genomic DNA, PCR products, plasmids, and RNA and visualization in gel electrophoresis and Real-Time Polymerase Chain Reaction (RT-PCR) processes.
  • RT-PCR Real-
  • step-II the precursor B (2-chloro-4-methyl- 1 -phenylquinolin- 1-ium) was synthesized by using DMF and POCI3 which was further reacted with 3-methyl-2-methylthiobenzothiazolium tosylate (Step-Ill) to afford the 2-((2-chloro-l-phenylquinolin-4(lH)-ylidene)methyl)-3-methylbenzo[d]thiazol-3- ium in situ as intermediate compound.
  • SYBR Safe In response to these concerns, safer alternatives such as SYBR Safe have been developed and adopted (US5436134A). SYBR Safe, being non-mutagenic, greatly reduces the health risks associated with DNA staining. Moreover, it offers high sensitivity, making it a valuable tool for visualizing nucleic acids in gel electrophoresis. However, this advancement is not without its shortcomings. Despite the lower toxicity, SYBR Safe and similar dyes still require special disposal procedures, contributing to the operational complexity and cost in laboratories. The environmental implications of these disposal procedures are significant, adding to the overall environmental footprint of research and diagnostic activities in molecular biology.
  • nucleic acid stain In light of these challenges, the need for a more versatile, safe, and efficient nucleic acid stain becomes evident.
  • This ideal stain would offer high sensitivity and safety, similar to SYBR Safe, while also being environmentally friendly by not necessitating any special disposal procedures.
  • this stain should offer broader utility, applicable not just to DNA staining in gel electrophoresis, but also to enhancing the efficiency and scope of RT-PCR, including the amplification of larger targets.
  • substituted 3-methylbenzo[d]thiazol-3-ium compounds are designed with these considerations in mind. It aims to address the limitations of existing dyes, offering a more versatile, safe, and environmentally friendly solution for nucleic acid visualization and RT- PCR enhancement.
  • the present invention provides substituted 3-methylbenzo[d]thiazol-3-ium compounds for nucleic acid staining and visualization in molecular biology and genetic diagnostic applications, addressing the limitations of current technologies. In addition to it the invention provides a new high yield process for the preparation of the said compounds.
  • the main object of the present invention is to provide substituted 3-methylbenzo[d]thiazol-3- ium compounds having general formula I for nucleic acid staining and visualization in molecular biology and genetic diagnostic applications, addressing the limitations of current technologies.
  • the invention provides a new high yield process for the preparation of 3 -methyl - 2-((2-substituted-l-phenylquinolin-4(lH)-ylidene)methyl)benzo[d]thiazol-3-ium compounds having general formula I.
  • Another key object of this invention is to ensure high sensitivity in nucleic acid detection, equivalent or superior to that of existing dyes like SYBR Safe. This ensures that even small quantities of nucleic acids can be effectively stained and visualized, enhancing the accuracy and reliability of subsequent analysis.
  • a further object of the invention is to enhance RT-PCR processes.
  • the compounds of present invention as DNA Gel Stain are formulated to not only serve as a nucleic acid stain but also to improve the sensitivity and scope of RT-PCR applications. This includes the amplification of larger targets, which remains a challenge with existing master mixes.
  • An additional object of this invention is to provide a safe solution for users and the environment.
  • Our DNA Gel Stain does not require special or hazardous waste disposal procedures, contrasting with many existing dyes. This aligns with global trends towards safer and more environmentally friendly laboratory practices, contributing to sustainable research and diagnostic activities.
  • the ultimate object of this invention is to provide a versatile, safe, and efficient tool for molecular biology and genetic diagnostics.
  • Our DNA Gel Stain aims to simplify laboratory procedures, enhance research capabilities, and facilitate accurate and efficient genetic analysis, all while prioritizing user safety and environmental sustainability.
  • the present invention relates to preparation of substituted 3 -methylbenzo [d]thiazol- 3-ium compounds as nucleic acid staining probes having general formula I.
  • the present invention provides a compound of general formula I, nucleic acid conjugates, complexes and salts thereof, wherein;
  • X, Z are independently selected from the group consisting of hydrogen, hydroxy, alkyl (Cl- C5), alkoxy (0C1-0C5), amine and halogen;
  • Y is independently selected from the group consisting of
  • the compounds are selected from the group consisting of: i. 3-methyl-2-((2-morpholino- 1 -phenylquinolin-4( lH)-ylidene)methyl)benzo[d]thiazol-3-ium; ii. 2-((2-((3-azidopropyl)amino)-l-phenylquinolin-4(lH)-ylidene)methyl)- 3- methylbenzo[d]thiazol-3-ium; iii.
  • X, Z are independently selected from the group consisting of hydrogen, hydroxy, alkyl (Cl- C5), alkoxy (0C1-0C5), amine and halogen;
  • Y is independently selected from the group consisting of
  • the organic solvent is selected from the group consisting of toluene, dichloromethane, dichloroethane, CH3CN, dimethylsulphoxide, dimethylformamide, water, methanol, ethyl acetate, hexane and tetrahydrofuran.
  • the present invention provides a compound of general formula I, useful for analyzing nucleic acids (DNA, RNA), and other biological substances of diagnostic importance; useful for development of diagnostic kit for detection of substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes and nucleic acids, particularly those implicated in disease states; and useful for the preparation of fluorescent probes, tags, markers, diagnostics, ion sensor, pharmaceuticals for detecting/trapping ions in fluorescence-based imaging and/or analysis of cells, biological fluids, chemical mixture and/or other useful applications.
  • nucleic acids DNA, RNA
  • diagnostic kit useful for detection of substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes and nucleic acids, particularly those implicated in disease states
  • fluorescent probes, tags, markers, diagnostics, ion sensor pharmaceuticals for detecting/trapping ions in fluorescence-based imaging and/or analysis of cells, biological fluids, chemical mixture and/or other useful applications.
  • the present invention provides a kit for staining nucleic acids, comprising the compound as claimed in claim 1, for detection of substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes and nucleic acids.
  • the present invention provides a kit for enhancing RT-PCR, comprising the compound as claimed in claim 1 and PCR Master mixes consisting of PCR buffer, dNTPs, TaqPolymerase, glycerol and water.
  • the present invention provides a nucleic acid gel stain, comprising the compound as claimed in claim 1 , which exhibits fluorescence in the presence of DNA/RNA.
  • the compound of general formula I is a nucleic acid stain exhibiting dual functionality in nucleic acid visualization and enhancing Real- Time Polymerase Chain Reaction (RT-PCR) processes, enhanced sensitivity in RT-PCR processes and amplification of larger targets in RT-PCR processes, thereby expanding the range of sequences that can be efficiently amplified and detected;
  • RT-PCR Real- Time Polymerase Chain Reaction
  • the compound exhibits affinity towards various types of nucleic acids, including but not limited to genomic DNA, PCR products, plasmids, and RNA; and the compound provides clear visualization of nucleic acids under exposure to blue light or UV excitation.
  • the stain demonstrates sensitivity comparable or superior to that of SYBR Safe, facilitating effective staining and visualization of small quantities of nucleic acids.
  • the stain contributes to enhanced sensitivity in RT- PCR processes by providing improved Relative Fluorescence Units (RFU) values compared to other dyes such as SYBR Green.
  • REU Relative Fluorescence Units
  • the stain facilitates the amplification of larger targets in RT-PCR processes, thereby expanding the range of sequences that can be efficiently amplified and detected.
  • the stain does not necessitate any special or hazardous waste disposal procedures as required for ethidium bromide, contributing to safer laboratory practices and environmental sustainability.
  • the present invention provides a method of staining nucleic acids using the compound of general formula I, comprising the steps of: (a) mixing a sample containing nucleic acids with the compounds of Claims 1 and 2; and (b) exposing said sample to blue light or UV excitation to visualize the nucleic acids.
  • the present invention provides a method for enhancing RT-PCR processes using the compound of general formula I, comprising the steps of: (a) incorporating the compounds of Claims 1 and 2 into an RT-PCR master mix; and (b) performing RT-PCR, resulting in enhanced sensitivity and the ability to amplify larger targets.
  • Fig.l Illustrates emission spectra of 3-methyl-2-((2-morpholino-l-phenylquinolin-4(lH)- ylidene)methyl)benzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig. 2 Illustrates emission spectra of 2-((2-((3-azidopropyl)amino)-l-phenylquinolin-4(lH)- ylidene)methyl)- 3-methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig. 3 Illustrates emission spectra of 2-((2-((3-(dimethylamino)propyl)amino)-l- phenylquinolin-4(lH)-ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1 Kb DNA.
  • Fig.4 Illustrates emission spectra of 2-((2-((3-azidopropyl)(methyl)amino)-l-phenylquinolin- 4(lH)-ylidene)methyl)-3- methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig. 5 Illustrates emission spectra of 3-methyl-2-((l-phenyl-2-(propylamino)quinolin-4(l- ylidene)methyl)benzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig.6 Illustrates emission spectra of 3-methyl-2-((2-(methyl(prop-2-yn-l-yl)amino)-l- phenylquinolin-4(lH)- ylidene)methyl)benzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig.7 Illustrates emission spectra of 3-methyl-2-((l-phenyl-2-(prop-2-yn-l-ylamino)quinolin- 4(lH)-ylidene)methyl)benzo[d]thiazol-3-ium with different concentration of 1Kb DNA.
  • Fig.8 Illustrates emission spectra of 2-((2-((l -(dimethyl (propyl)-14-azaneyl)propan-l-ylium-3- yl)amino)-l-phenylquinolin-4(lH)-ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig. 9 Illustrates emission spectra of 2-((2-amino-l-phenylquinolin-4(lH)-ylidene)methyl)-3- methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig.10 Illustrates emission spectra of 2-((2-(4-hydroxypiperidin-l-yl)-l-phenylquinolin-4(lH)- ylidene)methyl)-3-methylbenzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig.ll Illustrates emission spectra of 3-methyl-2-((l-phenyl-2-(piperidin-l-yl)quinolin-4(lH)- ylidene)methyl)benzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig.12 Illustrates emission spectra of 3-methyl-2-(( 1 -phen yl-2-((3-(4-phenyl-lH- 1,2, 3-triazol- l-yl)propyl)amino)quinolin-4(lH)-ylidene)methyl)benzo[d]thiazol-3-ium in PBS Buffer with different concentration of 1Kb DNA.
  • Fig. 13 Illustrate the absorption and emission spectra of the compound 1 in the presence of DNA
  • Fig.14 Illustrate the Cytotoxicity Study of the Compound 1.
  • Six bacterial plates are displayed, each treated with different concentrations of Compound 1 dissolved in DMSO: DMSO only (control), 10 pM Compound 1, 50 pM Compound 1, 0.1 mM Compound 1, 0.5 mM Compound 1 , and 1 mM Compound 1.
  • the absence of a clear zone in all plates indicates that Compound 1 , even at the highest concentration tested, does not inhibit bacterial growth or cause cell death, suggesting its non-cytotoxic nature.
  • Fig.15 Illustrate the Sensitivity Test of Compound 1. This figure showcases the sensitivity of Compound 1 as a nucleic acid stain. Different amounts of DNA (1 ng, 10 ng, 100 ng, 200 ng, and 500 ng) were loaded on an agarose gel and stained with Compound 1. The resulting image clearly shows that even as little as 10 ng of DNA can be effectively visualized using Compound 1 , highlighting its high sensitivity and effectiveness as a DNA stain. This exceptional sensitivity of Compound 1 makes it a powerful tool for detecting and quantifying even low amounts of DNA in research and diagnostic applications.
  • Fig. 16 Illustrate the Comparison Study of Compound 1 with Other Nucleic Acid Stains.
  • the figure presents a comparative study of the staining efficiency of Compound 1 DNA Gel Stain with two other commonly used nucleic acid stains. Three 1% agarose gels loaded with the same nucleic acid samples were stained differently: Gel 1 with Ethidium Bromide, Gel 2 with SYBR Safe dye from a third party, and Gel 3 with Compound 1 DNA Gel Stain.
  • the resulting images provide a visual comparison of the staining efficiency, clarity, and sensitivity of the three different dyes, underscoring the performance of Compound 1 DNA Gel Stain in nucleic acid detection and visualization.
  • Lane 1-5 represents the gel staining in different type of DNA (1) lOObp G2P ladder (Catalogue No. L15), (2) 1Kb G2P ladder (Catalogue No. L12); (3) PCR purified Product_ OmiS gene (849bp)-200ng, (4) Plasmid P01-200ng, (5) Genomic DNA-300ng
  • Fig. 17 Illustrate Real-Time PCR results recorded using the BioRAD CFX Opus 96 Dx Real- Time PCR System. Four images are presented together: Image A displays the amplification curve, and its corresponding melting curve is shown in Image B. Additionally, Image C compares the amplification curves of Compound 1 and SybrGreen from Vendor A, while Image D compares their respective melting curves. In the experiment, different DNA input concentrations (1 ng, 0.1 ng, and 0.01 ng) were tested with Compound 1 and SybrGreen from Vendor A. The results demonstrate the enhanced sensitivity of Compound 1, enabling the detection of low quantities of amplified nucleic acids.
  • the improved Relative Fluorescence Units (RFU) values obtained with Compound 1 highlight its higher sensitivity and accuracy compared to SybrGreen from Vendor A.
  • the corresponding melting curves in Image D provide further evidence of Compound 1's advantages.
  • the comparable shape and characteristics of the melting curves indicate the similar performance of Compound 1 and SybrGreen from Vendor A in differentiating and identifying specific DNA sequences.
  • Figure 18 highlights the enhanced sensitivity and accuracy of Compound 1 as a dye in real-time PCR, s featuring its advantages over existing dyes.
  • Fig. 18 illustrates the amplification of larger targets using Compound 1 and SybrGreen from Vendor an in real-time PCR.
  • A The amplification curves for both dyes are displayed . However, it is notable that the curve obtained for Vendor A's SybrGreen exhibits undesired amplification, likely originating from primer dimer formation, as confirmed by running the PCR product on an agarose gel. Compound 1, on the other hand, demonstrates successful amplification of the larger targets without the presence of primer dimer artifacts.
  • B Depicts the melting curve of the same experiment. This result highlights the advantage of Compound 1 in enabling the amplification of larger genetic sequences, overcoming the limitations experienced with Vendor A's SybrGreen. By effectively amplifying larger targets, Compound 1 expands the range of genetic material that can be efficiently detected and analyzed in real-time PCR experiments, contributing to the accuracy and reliability of the results.
  • Fig. 19 illustrates synthesis compound of formula (I)
  • Fig. 20(C) illustrates synthesis of compound (E)
  • Fig. 20(D) illustrates synthesis of compound (F)
  • Fig. 20(E) illustrates synthesis of compound (I)
  • the present invention relates to substituted 3-methylbenzo[d]thiazol-3-ium compounds of the general formula I and their ion complexes, salts which are potentially useful in chemical and biological sciences such as cell imaging applications, diagnostics, fluorescent tags, pharmaceuticals and other useful applications, and a process of preparing said new compounds. More particularly, the present invention relates to 3-methyl-2-((2-substituted-l-phenylquinolin- 4(lH)ylidene)methyl)benzo[d]thiazol-3-ium compounds, process for preparing the said compounds and their uses for detection of nucleic acids in fluorescence -based imaging and/or analysis.
  • the present invention more particularly relates to a compound of formula I: wherein X, Z are independently selected from the group consisting of hydrogen, hydroxy, alkyl (C1-C5), alkoxy (OC1-OC5), amine and halogen;
  • Y is independently selected from the group consisting of
  • Another embodiment of the present invention provides a process for the preparation of new 3- methyl-2-((2-substituted- l-phenylquinolin-4( lH)-ylidene)methyl)benzo[d]thiazol-3-ium having the general formula I as shown in scheme 1.
  • the compound 4-methyl-l-phenylquinolin-2(lH)-one (C) in 80% yield has been synthesized by using the 4-methylquinolin-2(lH)-one and diphenyliodonium trifluoromethanesulfonate in the presence of copper iodide and triethyl amine in toluene at reflux condition for 8h in step I.
  • step-II the 4-methyl-l-phenylquinolin-2(lH)-one (C) was reacted with 3-methyl-2- (methylthio)benzo[d]thiazol-3-ium iodide (D) (prepared by 2-methylthio-benzthiazole and methyl iodide) in the presence of di-isopropyl ethyl amine and trimethylsilyl trifluoro methane sulphonic acid in DCM at 50°C for 2h which gave the 4-((3-methylbenzo[d]thiazol-2(3H)- ylidene)methyl)-l-phenylquinolin-2(lH)-one (E) in 75% yield which was purified by washing with water and ethyl acetate and characterized by mass and NMR data.
  • D 3-methyl-2- (methylthio)benzo[d]thiazol-3-ium iodide
  • E 4-((3-methylbenzo[d]thiazol-2(
  • Step-1 Synthesis of 4-methyl-l-phenylquinolin-2(lH)-one
  • Step-2 Synthesis of 4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-l-phenylquinolin- 2(lH)-one
  • Step-3 Synthesis of 2-((2-chloro-l-phenylquinolin-4(lH)-ylidene)methyl)-3- methylbenzo[d]thiazol-3-ium
  • Step-4 Synthesis of 3-methyl-2-((2-substituted-l-phenylquinolin-4(lH)- ylidene)methyl)benzo[d]thiazol-3-ium
  • the compound 1 DNA Gel Stain exhibits unique fluorescence properties that are triggered upon binding with nucleic acids, making it a highly sensitive and responsive tool for molecular biology applications.
  • Compound 1 In its native state, dissolved in DMSO solution, Compound 1 is non-fluorescent. This property prevents any background fluorescence, facilitating a clean and clear readout when the dye is used for staining nucleic acids. Upon binding with nucleic acids such as DNA or RNA, Compound 1 undergoes a change that results in the emission of green fluorescence. This fluorescence change is indicative of the presence and quantity of nucleic acids, enabling accurate visualization and quantification.
  • the excitation maximum wavelength (X m ax) of Compound 1, when bound to nucleic acids, is 495 nm, and its emission maximum wavelength (X m ax) is 523 nm. These specific wavelengths fall within the green region of the visible light spectrum, and offering convenient and easily distinguishable visualization (Fig. 13).
  • Compound 1 makes it a highly effective tool for nucleic acid detection and quantification, whether in research or diagnostic applications. Its non- fluorescent nature in the unbound state and its green fluorescence upon binding with nucleic acids provide a high level of sensitivity and specificity in its applications.
  • a kit for staining nucleic acids is prepared by mixing Compound 1 as a nucleic acid gel stain soluble in DMSO for detection of substances, hormones, pathogenic microorganisms, viruses, antibodies, enzymes and nucleic acids.
  • a kit for enhancing RT-PCR efficiency is prepared by mixing Compound 1 and PCR Master mix consisting of PCR buffer, dNTPs, TaqPolymerase, glycerol and water.
  • Compound 1 DNA Gel Stain is supplied as a 10,000x concentrate in DMSO and is designed to be used in a similar manner to traditional nucleic acid stains like Ethidium bromide. For a standard mini gel, mix 5p I of Compound 1 stain into 50ml of agarose solution before pouring the gel into the casting tray. This ensures even distribution of the dye within the gel matrix.
  • Post-Electrophoresis Staining Alternatively, Compound 1 can be added to the lx TAE or TBE buffer used during electrophoresis for post-run visualization of DNA or RNA bands.
  • Compound 1 may also be incorporated directly into a PCR reaction, where it serves to enhance the efficiency and sensitivity of Real-Time PCR. Add the dye to the reaction at concentrations of less than 0.05x. This low concentration of the stain is sufficient to enable its function without inhibiting the PCR reaction.
  • the dye's unique properties non-fluorescent in its unbound state and green fluorescent upon binding to nucleic acids — facilitate clear, precise visualization of nucleic acids, offering an effective tool for nucleic acid detection and quantification in various experimental conditions.
  • Ethidium Bromide a widely used nucleic acid stain, is known for its mutagenic nature, which raises safety concerns in laboratory settings. In contrast, Compound 1 DNA Gel Stain is non-mutagenic, making it a safer alternative for routine use (Fig.14).
  • Compound 1 allows for easy and environmentally friendly disposal of gels and used buffers. This reduces the burden of special waste disposal procedures that are necessary with other, more hazardous dyes.
  • Fluorescent in Blue Light Compound 1 exhibits fluorescence under blue light illumination. This decreases the exposure risk to harmful UV light that is commonly associated with other nucleic acid stains. It's particularly handy during the elution process from DNA gels, as it allows for safe visualization and extraction of nucleic acids. 5. High Sensitivity: Fig. 15, showcases the sensitivity of Compound 1 as a nucleic acid stain.
  • Compound 1 exhibits high sensitivity as a dye in Real-Time PCR, allowing for the detection of even low quantities of amplified nucleic acids (Fig 17). Its ability to provide improved Relative Fluorescence Units (RFU) values compared to other dyes enhances the sensitivity and accuracy of real-time PCR results.
  • REU Relative Fluorescence Unit
  • Compound 1 offers the unique advantage of enabling the amplification of larger targets in Real-Time PCR. This addresses a significant limitation of existing master mixes, which often struggle to efficiently amplify larger genetic sequences. By facilitating the amplification of larger targets, Compound 1 broadens the range of genetic material that can be effectively detected and analyzed (Fig. 18). 3. Compatibility with Standard PCR Instruments: Compound 1 is compatible with standard realtime PCR instruments, making it a versatile and easily adoptable dye for routine laboratory use. It can be seamlessly incorporated into existing PCR workflows without requiring any specialized equipment or modifications.
  • Compound 1 offers the advantage of being non-mutagenic and safer to handle compared to traditional dyes such as Ethidium Bromide. It does not pose significant health risks to users and does not require special or hazardous waste disposal procedures. This aligns with the increasing emphasis on safety and environmental sustainability in laboratory practices.
  • Compound 1 provides a cost-effective alternative for real-time PCR applications. Compared to other commercial dyes, Compound 1 offers comparable or even superior performance at a potentially lower cost. This cost-effectiveness makes Compound 1 an attractive choice for laboratories seeking reliable and efficient nucleic acid detection in real-time PCR without compromising their budget.
  • Compound 1 demonstrates several advantages as a dye in real-time PCR, including enhanced sensitivity, the ability to amplify larger targets, compatibility with standard instruments, safety, environmental friendliness, and cost-effectiveness. These benefits contribute to improved performance and efficiency in real-time PCR applications, enabling accurate and reliable detection and analysis of nucleic acids in research and diagnostic settings.
  • agarose gel by directly incorporating Compound 1 DNA Gel Stain.
  • Compound 1 DNA Gel Stain is provided in a buffer form, so replace the buffer with the Compound 1 DNA Gel Stain when preparing the agarose gel.
  • the 10,000X Compound 1 concentrate dilute it 1: 10,000 in the agarose gel buffer (e.g., IX TBE or IX TAE).
  • the buffer-stain mixture to the powdered agarose. For instance, if you need 30 mL of molten agarose for your tray and are using TBE buffer, mix 3 pL of the 10,000X Compound 1 concentrate with 30 mL of IX TBE, and add it to the powdered agarose. Thoroughly mix the solution.
  • Gels containing Compound 1 DNA Gel Stain may exhibit slightly slower mobility of nucleic acid fragments compared to gels without stain when using precast gels with ethidium bromide.
  • a blue-light transilluminator such as the Safe ImagerTM 2.0 Blue-Light transilluminator (Cat. no. G6600), for optimal visualization of DNA stained with Compound 1.
  • a blue-light transilluminator such as the Safe ImagerTM 2.0 Blue-Light transilluminator (Cat. no. G6600)
  • UV light sources e.g., Safe ImagerTM 2.0 Blue-Light transilluminator
  • a blue-light source e.g., Safe ImagerTM 2.0 Blue-Light transilluminator
  • the combination of UV light sources with Compound 1 DNA Gel Stain may potentially reduce cloning efficiencies.
  • For photography purposes consider using PolaroidTM 667 black-and-white print film with a Compound 1 photographic filter (Cat. no. S37100), SYPROTM photographic filter (Cat. no. S6656), or a KodakTM Wratten #9 filter. These filters provide a similar detection sensitivity to ethidium bromide when working with Compound 1 DNA Gel Stain. Avoid using a standard ethidium bromide photographic filter with Compound 1 DNA Gel Stain.
  • gels stained with Compound 1 can be imaged using a CCD camera or a laser-based scanner.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
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Abstract

La présente invention concerne des composés de 3-méthylbenzo[d]thiazol-3-ium substitués, leurs conjugués d'acide nucléique, leurs complexes, leurs sels qui sont potentiellement utiles pour des applications d'imagerie cellulaire, des diagnostics, des marqueurs fluorescents, des produits pharmaceutiques et d'autres applications de biologie moléculaire utiles, et un procédé de préparation de ceux-ci. La présente invention concerne, plus particulièrement, des composés de 3-méthyl-2-((1-phénylquinolin-4(1H)ylidène-2-substitué)méthyl)-benzo[d]thiazol-3-ium, des procédés de préparation desdits composés et leurs utilisations en vue de la détection d'acides nucléiques dans l'imagerie et l'analyse par fluorescence. La présente invention concerne, plus précisément, un nouveau composé 1 de gel de coloration d'ADN double fonction, qui sert de colorant efficace pour les acides nucléiques, présentant une sensibilité et une affinité élevées vis-à-vis de divers acides nucléiques, y compris l'ADN génomique, les produits de PCR, les plasmides et l'ARN. De plus, le composé 1 améliore les processus de réaction en chaîne par polymérase en temps réel en fournissant des valeurs d'unités de fluorescence relative améliorées et en permettant l'amplification de cibles plus grandes. Le composé 1 a pour caractéristique de ne pas nécessiter de procédures particulières ou dangereuses d'élimination des déchets, ce qui en fait une option plus sûre et respectueuse de l'environnement pour les laboratoires.
PCT/IN2023/050682 2022-07-12 2023-07-12 Composés de 3-méthylbenzo[d]thiazol-3-ium substitués et leur utilisation Ceased WO2024013770A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
WO2025202488A1 (fr) * 2024-03-28 2025-10-02 Roche Diagnostics Gmbh Activateur d'amplification d'acide nucléique

Citations (1)

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US5863753A (en) * 1994-10-27 1999-01-26 Molecular Probes, Inc. Chemically reactive unsymmetrical cyanine dyes and their conjugates

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US5863753A (en) * 1994-10-27 1999-01-26 Molecular Probes, Inc. Chemically reactive unsymmetrical cyanine dyes and their conjugates

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SAARNIO VILLE K., ALARANTA JOHANNA M., LAHTINEN TANJA M.: "Systematic study of SYBR green chromophore reveals major improvement with one heteroatom difference", JOURNAL OF MATERIALS CHEMISTRY. B, ROYAL SOCIETY OF CHEMISTRY, GB, vol. 9, no. 16, 28 April 2021 (2021-04-28), GB , pages 3484 - 3488, XP093129987, ISSN: 2050-750X, DOI: 10.1039/D1TB00312G *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025202488A1 (fr) * 2024-03-28 2025-10-02 Roche Diagnostics Gmbh Activateur d'amplification d'acide nucléique

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