Sulforhodamine phosphoramidite dyes
Inventors
Lund, Kevin P. • Sergueev, Dmitri • Qabar, Maher N. • Gall, Alexander
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Assignees
Member
CepheidCepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Cepheid is a global leader in molecular diagnostics, dedicated to improving healthcare by developing, manufacturing, and marketing automated, easy-to-use molecular systems and tests. Their mission is to provide rapid, accurate, and actionable genetic testing for a wide range of infectious diseases, oncology, and human genetics. Cepheid's flagship GeneXpert System delivers scalable, sample-to-answer PCR testing for institutions of any size, supporting both centralized and decentralized care. The company is committed to expanding access to high-quality diagnostics worldwide, supporting public health initiatives, driving innovation in molecular testing, and advancing sustainability and responsible business practices.
Publication Number
US-12071547-B2
Publication Date
2024-08-27
Expiration Date
Abstract
Automated oligonucleotide synthesis-compatible sulforhodamine dye phosphoramidite compounds and labeled polynucleotides incorporating these dyes are provided.
Core Innovation
Automated oligonucleotide synthesis-compatible sulforhodamine dye phosphoramidite compounds and labeled polynucleotides incorporating these dyes are provided. Provided herein are sulforhodamine dyes comprising one or more reactive groups, e.g., a phosphoramidite group, and sulforhodamine dye phosphoramidites that can be incorporated into oligonucleotides via standard automated oligonucleotide synthesis. The dyes disclosed herein can exist in open or closed form, wherein in their cyclic or “closed” form the sulforhodamine dyes are colorless and non-fluorescent and, upon protonation, the cyclic form can convert to the “open” form which is fluorescent.
The invention addresses limitations of previously known red labeling dyes by providing sulforhodamine dyes that are compatible with automated phosphoramidite oligonucleotide synthesis and that can be incorporated at any position in a polynucleotide. The background identifies drawbacks of post-synthetic coupling (including low conjugation yields and additional purification) and the lack of phosphoramidite derivatives for certain dyes such as Sulforhodamine 101 (Texas Red®), and sets forth the need for red fluorescent dyes that are compatible with automated synthesis, can be incorporated at any position, have emission and absorption maxima compatible with existing PCR instrumentation, and provide a high end-point fluorescence signal in PCR applications.
The disclosure further specifies structural embodiments of the dyes (e.g., compounds represented by Formula I, II, III, VI, IA, IIA, and IV) with defined substituents and linkers, and notes that the closed (sultam) form is non-polar and dissolves in organic solvents compatible with automated phosphoramidite synthesis and/or bioconjugation conditions, allowing ease of purification by conventional techniques such as silica gel column chromatography. The open form is described as having spectral properties comparable to or matching those of Sulforhodamine 101 dye or a TAMRA dye and, when conjugated to polynucleotides, polynucleotide conjugates of the dyes have fluorescence that is not pH-dependent in the pH range relevant for PCR applications and can be quenched with conventional quenchers such as BHQ-2 to produce high end-point fluorescence.
Claims Coverage
One independent inventive feature identified: a compound defined by a structural Formula I with specified substituents and optional stereoisomers, tautomers, or salts.
Compound represented by Formula I
A compound having a structure represented by Formula I or a stereoisomer, tautomer, or salt thereof, wherein: R1 is C1-C6 alkyl or L1-X; R2 is halogen or SO2NH2; R3 is H or halogen; R4, R7, R8, and R11 when taken alone are independently H, halogen, or optionally substituted C1-C6 alkyl; R5, R6, R9, and R10 when taken alone are independently H or optionally substituted C1-C6 alkyl; R4 and R5, R6 and R7, R8 and R9, and R10 and R11, when taken together, can be optionally substituted C2-C3 alkylene chains connecting the atoms to which they are attached; R5 and R6 and R9 and R10, when taken together with the nitrogen atom to which they are attached, can form a 5-membered or a 6-membered unsaturated ring; L1 is an optionally substituted C2-C10 alkylene or optionally substituted C2-C20 heteroalkylene; X is an activated ester, N3, propynyl, maleimido, or —O—P(OCH2CH2CN)NR12R13 or other defined reactive moieties; Q is a hydroxyl protecting group; Z is CH, N, NHC(O)N, or OC(O)N; and R12 and R13 are independently optionally substituted C1-C6 alkyl.
The independent claim covers sulforhodamine dye compounds defined by Formula I with specified substituents, linkers, reactive moieties, and optional stereoisomeric, tautomeric, or salt forms.
Stated Advantages
Compatibility with automated phosphoramidite oligonucleotide synthesis, allowing incorporation of the sulforhodamine dyes into oligonucleotides via standard automated synthesis.
Amenability to incorporation at any position in the oligonucleotide (not limited to 5′-end incorporation).
Spectral properties of the open form comparable to or matching Sulforhodamine 101 dye or a TAMRA dye, providing absorption and emission maxima compatible with existing PCR instrumentation.
Polynucleotide conjugates have fluorescence that is not pH-dependent in the pH range relevant for PCR applications (about 6.5 to about 8.5).
Can be quenched well with conventional quenchers such as BHQ-2 to produce high end-point fluorescence (EPF).
Closed (sultam) form is non-polar and dissolves in organic solvents compatible with automated phosphoramidite synthesis and/or bioconjugation conditions, facilitating purification by conventional techniques such as silica gel column chromatography.
Provides a viable red fluorophore alternative to Texas Red® dye by being compatible with automated synthesis and having spectral characteristics close to Texas Red®.
Documented Applications
Fluorescent labeling of oligonucleotides for applications ranging from PCR to sequencing.
Use as 5′-nuclease PCR probes and other fluorogenic probes such as probes used in 5′ nuclease assays and molecular beacons.
Preparation of labeled conjugates of ligands including polynucleotides, proteins, peptides, polysaccharides, polymers with an ethylenic backbone, and solid supports.
Incorporation into polynucleotides during automated phosphoramidite oligonucleotide synthesis to produce sulforhodamine dye-labeled polynucleotides (including internally labeled probes and 5′-labeled probes).
Multicolor labeling and staining applications enabled by longer wavelength emission maxima of rhodamine-type dyes.
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