WO2010059880A2 - Agents d'atténuation du dommage photo-induit et préparation et procédés d'utilisation de ces agents - Google Patents

Agents d'atténuation du dommage photo-induit et préparation et procédés d'utilisation de ces agents Download PDF

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WO2010059880A2
WO2010059880A2 PCT/US2009/065222 US2009065222W WO2010059880A2 WO 2010059880 A2 WO2010059880 A2 WO 2010059880A2 US 2009065222 W US2009065222 W US 2009065222W WO 2010059880 A2 WO2010059880 A2 WO 2010059880A2
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compound
formula
photo
induced damage
reaction
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PCT/US2009/065222
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WO2010059880A3 (fr
Inventor
Xiangxu Kong
Gene Shen
Andrei Federov
John Lyle
Grace Lee
Lubomir Sebo
Duc Do
Robert Weber
Steve Dudek
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Pacific Biosciences Of California, Inc.
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Publication of WO2010059880A3 publication Critical patent/WO2010059880A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
    • C07C49/255—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing ether groups, groups, groups, or groups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Definitions

  • optically detectable labeling groups and particularly those groups having high quantum yields, e.g., fluorescent or chemiluminescent groups, is ubiquitous throughout the fields of analytical chemistry, biochemistry, and biology. In particular, by providing a highly visible signal associated with a given reaction, one can better monitor that reaction as well as any potential effectors of that reaction.
  • analyses are the basic tools of life science research in genomics, diagnostics, pharmaceutical research, and related fields.
  • Such analyses have generally been performed under conditions where the amounts of reactants are present far in excess of what is required for the reaction in question. The result of this excess is to provide ample detectability, as well as to compensate for any damage caused by the detection system and allow for signal detection with minimal impact on the reactants.
  • analyses based on fluorescent labeling groups generally require the use of an excitation radiation source directed at the reaction mixture to excite the fluorescent labeling group, which is then separately detectable.
  • an excitation radiation source directed at the reaction mixture to excite the fluorescent labeling group, which is then separately detectable.
  • optically detectable labeling groups is that prolonged exposure of chemical and biochemical reactants to such light sources, alone, or when in the presence of other components, e.g., the fluorescent groups, can damage such reactants.
  • the traditional solution to this drawback is to have the reactants present so far in excess that the number of undamaged reactant molecules far outnumbers the damaged reactant molecules, thus minimizing or negating the effects of the photo-induced damage.
  • the present invention is generally directed to compounds, compositions, methods, devices and systems for preventing, reducing, or limiting the effects of photo-induced damage during illuminated reactions, particularly reactions that employ fluorescent or fluorogenic reactants.
  • photo-induced damage refers generally to any direct or indirect impact of illumination on one or more reagents in a reaction resulting in a negative impact upon that reaction.
  • illumination reactions refers to reactions which are exposed to an optical energy source. Typically, such illumination is provided in order to observe the generation and/or consumption of reactants or products that possess a particular optical characteristic indicative of their presence, such as a shift in the absorbance spectrum and/or emission spectrum of the reaction mixture or its components.
  • compositions of the invention can comprise a first reactant, a second reactant, and a photo-induced damage mitigating agent, wherein interaction of the first reactant with the second reactant under excitation illumination causes photo-induced damage to the first reactant in the absence of the photo-induced damage mitigating agent.
  • the first reactant is confined, e.g., immobilized on a surface and/or in an optical confinement.
  • the first reactant is an enzyme, e.g., a polymerase.
  • the photo-induced damage mitigating agent is a triplet-state quencher and/or a free radical quencher, and in certain specific embodiments the photo-induced damage mitigating agent is a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxyric acid or a derivative thereof.
  • a photo-induced damage mitigating agent is a photo- induced damage mitigating agent admixture comprising multiple quinone derivatives.
  • the second reactant is a fluorogenic or fluorescent molecule.
  • the reaction mixture comprises first reactant, a second reactant, and a photo- induced damage mitigating agent, wherein the photo-induced damage mitigating agent reduces an amount of photo-induced damage to the first reactant that would otherwise result from interaction of the first reactant with the second reactant under excitation illumination in the absence of the photo-induced damage mitigating agent.
  • the method further comprises monitoring a reaction between the first and second reactants during illumination.
  • the reaction is a base extension reaction and/or the first reactant is a polymerase enzyme.
  • the second reactant is a fluorogenic or fluorescent molecule.
  • the photo-induced damage mitigating agent is a triplet-state quencher and/or a free radical quencher
  • the photo-induced damage mitigating agent is a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid or a derivative thereof.
  • a photo-induced damage mitigating agent is a photo-induced damage mitigating agent admixture comprising multiple quinone derivatives.
  • certain methods of the invention monitor a reaction mixture comprising at least one enzyme, a fluorescent or fluorogenic substrate for the enzyme, and a photo-induced damage mitigating agent, wherein interaction of the enzyme and the substrate under excitiation illumination result in altered activity of the enzyme.
  • Such methods can comprise directing an excitation radiation at a first observation region for a first period that is less than a photo-induced damage threshold period, which is lengthened by the presence of the photo-induced damage mitigating agent.
  • the photo-induced damage mitigating agent is a triplet-state quencher and/or a free radical quencher
  • the photo-induced damage mitigating agent is a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid or a derivative thereof.
  • a photo-induced damage mitigating agent is a photo-induced damage mitigating agent admixture comprising multiple quinone derivatives.
  • certain methods of the invention lengthen a photo-induced damage threshold period.
  • Such methods can comprise including in a reaction mixture a photo- induced damage mitigating agent, such as a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid or a derivative thereof.
  • a photo-induced damage mitigating agent is a photo-induced damage mitigating agent admixture comprising multiple quinone derivatives.
  • compounds of the invention include or comprise one or more quinone derivatives, e.g., one or more benzoquinone derivatives, one or more hydroquinone derivatives, one or more epoxy quinone derivatives, one or more further quinone derivatives provided herein, or a combination thereof.
  • compounds of the invention further include or comprise 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or a derivative thereof.
  • a compound of the invention comprises a linking group, e.g., linking a photo-induced damage mitigating agent to at least one of a nucleotide polyphosphate, a polymerase, a surface, another component of an illumination reaction, or a combination thereof.
  • a linking group e.g., linking a photo-induced damage mitigating agent to at least one of a nucleotide polyphosphate, a polymerase, a surface, another component of an illumination reaction, or a combination thereof.
  • devices of the invention can comprise a substrate having an observation region, a first reactant immobilized within the observation region, a second reactant disposed within the observation region, and at least one photo-induced damage mitigating agent disposed within the observation region, where interaction between the first and second reactants under excitation illumination causes photo-induced damage to the first reactant, and further wherein the photo-induced damage is reduced by the presence of the photo-induced damage mitigating agent.
  • the first reactant is an enzyme (e.g., a polymerase) and/or the observation region is within a zero mode waveguide.
  • the photo-induced damage mitigating agent is a triplet-state quencher and/or a free radical quencher, and in certain specific embodiments the photo-induced damage mitigating agent is a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or a derivative thereof.
  • the photo-induced damage mitigating agent comprises multiple quinone derivatives, e.g., in an admixture.
  • a system of the invention can serve to analyze an illuminated reaction that is susceptible to photo-induced damage when illuminated for a period longer than a photo-induced damage threshold period.
  • a system can comprise a substrate having reagents for the reaction disposed thereon, including at least one photo-damage mitigating agent of the invention; a mounting stage supported by the substrate and configured to receive the substrate; an optical train positioned to be in optical communication with at least a portion of the substrate to illuminate the portion of the substrate and detect signals emanating therefrom; and a translation system operably coupled to the mounting stage or the optical train for moving one of the optical train and the substrate relative to the other.
  • the illuminated reaction is a sequencing reaction, e.g., a nucleotide sequencing-by-synthesis reaction.
  • the substrate comprises at least one optical confinement, e.g., a zero mode waveguide.
  • a reaction mixture of the invention can comprise at least one fluorescent dye molecule and at least one photo-induced damage mitigating agent provided herein.
  • a photo-induced damage mitigating agent can be a triplet-state quencher and/or a free radical quencher, and in certain specific embodiments, the agent is a quinone derivative, e.g., a benzoquinone derivative, a hydroquinone derivative, an epoxy quinone derivative, or other quinone derivative described herein, e.g., compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • the photo-induced damage mitigating agent may further comprise 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid or a derivative thereof.
  • the photo-induced damage mitigating agent comprises multiple quinone derivatives.
  • one or more components of the reaction mixture are immobilized within an optical confinement, e.g., a zero mode waveguide.
  • the reaction mixture comprises an enzyme, such as a polymerase enzyme.
  • the reaction mixture is a sequencing-by-incorporation reaction mixture, e.g., comprises all components necessary for a sequencing-by-incorporation reaction.
  • photo-induced damage mitigating agent is chemically linked to at least one other of the reaction components.
  • photo-induced damage mitigating agent admixtures are provided.
  • such admixtures comprise at least about 2%, 5%, 10%, 15%, or 20% of a given component of the admixture, e.g. a quinone derivative provided herein.
  • such admixtures comprise no more than about 50%, 40%, 30%, 20%, or 10% of a given component of the admixture, e.g. a quinone derivative provided herein.
  • such admixtures comprise at least about 2%, 5%, 10%, 15%, or 20% of a first component of the admixture, and no more than about 50%, 40%, 30%, 20%, or 10% of a second component of the admixture.
  • various methods for preparing photo-induced damage mitigating agent admixtures are provided.
  • 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid is dissolved in methanol and the pH of the resulting solution is raised to at least about 11.
  • the resulting basic solution is incubated for at least about 15 hours.
  • the pH is typically readjusted after the incubation, e.g., to about 7-8.
  • the concentration of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid in the incubated solution is about 100 mM.
  • the incubation preferably is performed at room temperature (e.g., 18°C - 22°C).
  • the incubation is performed under ambient light, e.g., for about 15-20 hours. In more preferred embodiments, the incubation is performed in the absence of light (i.e., in the dark), e.g., for about 48-72 hours.
  • the invention also provides methods for preparing reaction mixtures comprising adding a photo- induced damage mitigating agent admixture so prepared.
  • compositions, systems, methods, and devices provided by the invention for mitigating photo-induced damage can be used alone or in combination.
  • kits that incorporate photodamage-induced mitigating agents, or admixtures thereof, optionally with additional useful reagents.
  • kits can include, e.g., a photodamage-induced mitigating agent of the invention packaged in a fashion to enable use of the agent with any of a variety of enzymes that participate in a reaction with one or more fluorescent or fluorogenic substrate.
  • kits of the invention optionally include, e.g., buffer solutions and/or salt solutions, divalent metal ions, i.e., Mg ++ , Mn ++ , Ca ++ , and/or Fe ++ , enzyme cofactors, substrates, standard solutions, e.g., dye standards for detector calibration, etc.
  • Kits can optionally include reagents and instructions for preparing photo-induced damage mitigating agent admixtures.
  • Such kits also typically include instructions for use of the compounds and other reagents in accordance with the desired application methods, e.g., nucleic acid sequencing and the like.
  • Figure 1 is a schematic illustration of a proposed mechanism of photo-induced damage to DNA polymerase in template-dependent synthesis using fluorescent nucleotide analogs while under excitation illumination.
  • Figure 2 provides certain exemplary embodiments of the photo-induced damage mitigating agents provided herein.
  • Figure 3 provides certain exemplary embodiments of the photo-induced damage mitigating agents provided herein.
  • Figure 4 provides certain exemplary embodiments of the photo-induced damage mitigating agents provided herein.
  • Figure 5 provides certain exemplary embodiments of the photo-induced damage mitigating agents provided herein.
  • Figure 6 provides a scheme showing the interconversion of certain photo-induced damage mitigating agents of the invention.
  • Figure 7 illustrates certain exemplary embodiments of compounds of formula III.
  • Figure 8 illustrates certain exemplary embodiments of compounds of formula IV.
  • Figure 9 provides fluorescence correlation spectroscopy (FCS) traces from experiments that measured the quenching activity of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid and derivatives thereof.
  • Figure 10 shows reverse phase HPLC traces for 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (A), photomodified 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid solutions (B, C), Fraction A (D), and Fraction B (E).
  • Figure 11 provides fluorescence correlation spectroscopy (FCS) traces from experiments that measured the quenching activity of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid and various solutions of photomodified 6-hydroxy-2,5,7,8-tetramethylchroman-
  • Figure 12 provides reverse phase HPLC traces showing photolysis of the major component of Fraction A to the major component of Fraction B.
  • Figure 13 provides results from an experiment comparing sequencing by incorporation readlengths in the presence of pure 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid and various mixtures of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and derivatives thereof.
  • Figure 14 provides a photodegradation pathway for 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid, as described further in Example 6.
  • Figure 15 provides a trace from a reverse phase HPLC performed on a mixture comprising photo-induced damage mitigating agents prepared by a method of the invention.
  • Figure 16 provides a trace from a reverse phase HPLC performed on a mixture comprising photo-induced damage mitigating agents prepared by a method of the invention.
  • Figure 17 provides data showing enzyme processivity in the presence of certain quinone derivatives of the invention.
  • Figure 18 provides a synthesis method for one of the compounds of formula II, as further described in Example 7.
  • Figure 19 provides a simplified scheme for the production of certain compounds of the invention.
  • Figure 20 provides illustrative examples of stereoisomers of certain compounds of formula IV (A, B) and formula III (C-F).
  • Figure 21 illustrates certain exemplary embodiments of compounds of formula V.
  • Figure 22 illustrates certain exemplary embodiments of compounds of formula
  • Figure 23 illustrates certain exemplary embodiments of compounds of formula
  • Figure 24 illustrates certain exemplary embodiments of compounds of formula
  • Figure 25 illustrates certain exemplary embodiments of compounds of formula
  • Figure 26 illustrates certain exemplary embodiments of compounds of formula V.
  • Figure 27 provides a crystal structure for a preferred embodiment of a compound of formula IV that corresponds to fraction 4 described in Example 7.
  • Figure 28 provides a crystal structure for a preferred embodiment of a compound of formula III that corresponds to fraction 5 described in Example 7.
  • Figure 29 provides a crystal structure for a preferred embodiment of a compound of formula III that corresponds to fraction 7 described in Example 7.
  • any concentration, percentage, or ratio values or ranges provided herein are generally given in terms of admixture values or ranges without regard to any conversion of the particular component of a mixture that occurs upon or following subsequent treatment of the mixture, e.g., addition of the mixture to an analytical reaction.
  • a mixture comprising 10% of component A may be added to an analytical reaction, and subsequent to that addition component A may be partially or completely converted to one or more additional components in the analytical reaction, e.g., by reaction with other components of the analytical reaction and/or by conditions present (e.g., electromagnetic radiation, optical energy, pH, temperature, etc.) present in the analytical reaction.
  • a mixture comprising essentially equivalent amounts of two or more different components may see the ratio of the amounts of these components change after addition to an analytical reaction.
  • the present invention is generally directed to compounds, compositions, methods, devices and systems for preventing, reducing, or limiting the effects of photo-induced damage during illuminated reactions, particularly reactions that employ fluorescent or fluorogenic reactants.
  • photo-induced damage refers generally to any direct or indirect impact of illumination on one or more reagents in a reaction resulting in a negative impact upon that reaction.
  • illumination reactions refers to reactions which are exposed to an optical energy source. Typically, such illumination is provided in order to observe the generation and/or consumption of reactants or products that possess a particular optical characteristic indicative of their presence, such as a shift in the absorbance spectrum and/or emission spectrum of the reaction mixture or its components.
  • the fluorescence detected in a fluorescence-based optical assay is the result of a three-stage process that occurs in the fluorophores or fluorescent dyes present in a reaction mixture.
  • the first stage is excitation in which a photon with quantized energy from an external light source having a specific wavelength (e.g., from a laser) is supplied and absorbed by a fluorophore creating an excited electronic singlet state (S 1 ').
  • the second stage is the excited-state lifetime in which the excited fluorophore undergoes several different changes to relax its energy to the lowest singlet state (Si). From the Si state several possible mechanisms can occur in the third stage, fluorescence, in which a photon of energy (Si - S 0 ) is emitted returning the fluorophore to its ground state.
  • ISC intersystem crossing
  • Ti the intersystem crossing
  • ISC intersystem crossing
  • the formation of the much longer lifetime triplet-state species greatly reduces the brightness of the fluorescence emission.
  • it exhibits a high degree of chemical reactivity in this state that often results in photobleaching and the production of damaging free radicals and reactive intermediates, e.g., radical ions, carbenes, carbocations, carbanions, etc.
  • the invention is directed to the performance of illuminated reaction analyses, where such analyses are illuminated for an amount of time that permits the effective performance of the analysis.
  • one or more photo-induced damage mitigating agents e.g., triplet-state quenchers and/or free radical quenchers
  • providing such an excess of the photo-induced damage mitigating agent can potentially interfere with the ability of a reaction to proceed to completion.
  • compounds of the invention function as triplet- state quenchers and/or free radical quenchers.
  • quenching slows the accumulation of damaging excited triplet-state forms of one or more reaction components.
  • quenching of a fluorescent dye in a reaction can slow the accumulation of the excited triplet state of the fluorophore, greatly improving the photophysical properties of the dye.
  • the invention provides methods and compositions for nucleic acid analysis in which a photo-induced damage mitigating agent is linked to another reaction component or to a reaction site to bring the photo-induced damage mitigating agent into close spatial proximity to the reactants susceptible to damage by the illumination.
  • the photo-induced damage mitigating agent may be linked to one or more of a reactant (e.g., a nucleic acid or fluorescent dye), an enzyme (e.g., a polymerase or nuclease), or to a surface at which the reaction will take place (e.g., a well, chip, fiber, bead, etc.).
  • a reactant e.g., a nucleic acid or fluorescent dye
  • an enzyme e.g., a polymerase or nuclease
  • a surface at which the reaction will take place e.g., a well, chip, fiber, bead, etc.
  • the invention provides methods and compositions for nucleic acid analysis in which a nucleoside polyphosphate is linked to a fluorescent dye, and wherein the compound further includes, integrated into its structure (e.g., in the linker itself), a photo- induced damage mitigating agent, which generally refers to any agent that can prevent and/or mitigate damages caused by illumination, for example, by triplet/radical quenching.
  • a photo- induced damage mitigating agent which generally refers to any agent that can prevent and/or mitigate damages caused by illumination, for example, by triplet/radical quenching.
  • the invention is generally applicable to any of a variety of optical assays that require substantial illumination and/or photoactivated conversion or excitation of chemical groups, e.g., fluorophores, and is particularly useful for assays that are impaired by the generation and/or accumulation of triplet-state forms or free radicals.
  • the compositions and methods provided herein may be used with fluorescence microscopy, optical traps and tweezers, spectrophotometry, fluorescence correlation spectroscopy, confocal microscopy, near-field optical methods, fluorescence resonance energy transfer (FRET), structured illumination microscopy, total internal reflection fluorescence microscopy (TIRF), etc.
  • the methods provided herein are particularly useful in analyses that utilize very limited concentrations of reactants that might be subject to photo-induced damage, such as single molecule detection/monitoring assays.
  • any degradation of a critical reagent will dramatically impact the analysis by further limiting the reagent, which not only can adversely effect the detectable signal, but may also directly impact the reaction being monitored, e.g., by changing its rate, duration, or product(s).
  • photo-induced damage can include a photoinduced change in a given reagent that reduces the reactivity of that reagent in the reaction, e.g., photobleaching of a fluorescent molecule, which diminishes or removes its ability to act as a signaling molecule.
  • photo-induced damage are other changes that reduce a reactant' s usefulness in a reaction, e.g., by making the reagent less specific in its activity in the reaction.
  • photo-induced damage includes undesired changes in a reagent that are caused by interaction of that reagent with a product of another photoinduced reaction, e.g., the generation of singlet oxygen during a fluorescence excitation event, which singlet oxygen may damage organic or other reagents, e.g., proteins.
  • damage to an enzyme that catalyzed a reaction being monitored may cause a reduction in the rate of the reaction, in some cases stopping it altogether, or may reduce the duration or fidelity of the reaction.
  • One particularly apt example of analyses that benefit from the invention are single-molecule biological analyses, including, inter alia, single molecule nucleic acid sequencing analyses, single molecule enzyme analyses, hybridization assays (e.g., antibody assays), nucleic acid hybridization assays, and the like, where the reagents of primary import are subjected to prolonged illumination with relatively concentrated light sources (e.g., lasers and other concentrated light sources, such as mercury, xenon, halogen, or other lamps) in an environment where photoconversion/excitation is occurring with its associated generation of products.
  • relatively concentrated light sources e.g., lasers and other concentrated light sources, such as mercury, xenon, halogen, or other lamps
  • the methods, compositions, and systems are used in nucleic acid sequencing processes that rely on detection of fluorescent or fluorogenic reagents.
  • SMRTTM nucleic acid sequencing (described in, e.g., U.S. Patent Nos. 6,399,335, 6,056,661, 7,052,847, 7,033,764, 7,056,676, 7,361,466, 7,416,844, and in Eid, et al. (2009) Science 323:133-138, the full disclosures of which are incorporated herein by reference in their entireties for all purposes), non-real time, or "one base at a time" sequencing methods available from, e.g., Illumina, Inc. (San Diego, CA), Helicos BioSciences (Cambridge, MA), Clonal Single Molecule ArrayTM, and SOLiDTM sequencing.
  • Such prolonged illumination can result in photo-induced damage to these reagents and diminish their effectiveness in the desired reaction.
  • illuminationted analysis and “illuminated reaction” are used interchangeably and generally refer to an analytical reaction that is occurring while being illuminated (e.g., with excitation radiation), so as to evaluate the production, consumption and/or conversion of luminescent (e.g., fluorescent) reactants and/or products.
  • luminescent e.g., fluorescent
  • reactant and “reagent” are used interchangeably.
  • the illuminated reaction is a sequencing reaction and the photo-induced damage results, directly or indirectly, from an excitation radiation source used to detect nucleotides as they are added to a nascent nucleic acid strand.
  • a photo-induced damage threshold period is assay-dependent, and is affected by various factors, including but not limited to characteristics of enzymes in the assay (e.g., susceptibility to photo-induced damage and the effect of such damage on enzyme activity/processivity), characteristics of the radiation source (e.g., wavelength, intensity), characteristics of the signal-generating molecule (e.g., type of emission, susceptibility to photo-induced damage, propensity to enter triplet state, and the effect of such damage on the brightness/duration of the signal), similar characteristics of other components of the assay.
  • characteristics of enzymes in the assay e.g., susceptibility to photo-induced damage and the effect of such damage on enzyme activity/processivity
  • characteristics of the radiation source e.g., wavelength, intensity
  • characteristics of the signal-generating molecule e.g., type of emission, susceptibility to photo-induced damage, propensity to enter triplet state, and the effect of such damage on the brightness/duration of the signal
  • the photo-induced damage threshold period is that period of illuminated analysis during which such photo-induced damage occurs so as to reduce the rate of the subject reaction by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over the same reaction in the absence of such illumination. It is an object of the invention to increase the photo-induced damage threshold period, thereby increasing the amount of time reactions can proceed toward completion with minimal damage to the reactants, thereby lengthening the time in which the detectable signal is an accurate measure of reaction progression.
  • a "photo-induced damaged" reaction may be subject to spurious activity, and thus be more active than desired.
  • the photo-induced damage threshold period of interest would be characterized by that period of illuminated analysis during which such spurious activity, e.g., as measured by an increase in reaction rate, or an increase in non-specific reaction rate, is no more than 10% over a non- illuminated reaction, no more than 20% over a non-illuminated reaction, no more than 50% over a non-illuminated reaction, and in some cases, no more than 90% over a non-illuminated reaction.
  • nucleic acid polymerase by virtue of a photodamaging event, begins to incorrectly incorporate nucleotides during template directed synthesis, such activity would impact the photo-induced damage threshold period as set forth above.
  • the compounds and methods of the invention would increase the photo- induced damage threshold period, thus increasing the amount of time the reaction could be illuminated before the above-described spurious activity occurred.
  • a fluorophore excited by exposure to electromagnetic radiation at an excitation wavelength can transition into a triplet state. This may occur directly, or as a result of multi-photon processes, where an excited fluorophore transitions to the triplet state upon contact with a photon of a wavelength that is shorter (or bluer) than the nominal excitation wavelength of the fluorophore. Subsequent relaxation of the triplet-state fluorophore can lead to generation of reactive intermediates, which can, in turn, damage one or both of the fluorophore or the enzyme processing the fluorophore, e.g., the polymerase.
  • photo-induced damage mitigating agents e.g., free radical and/or triplet-state quenching agents
  • agents can be included within the reaction mixtures or directly incorporated into compounds of the invention to alleviate and/or prevent the effects of reactive intermediates, as well as other species generated during illuminated reaction that can cause photo-induced damage.
  • the photo-induced damage sought to be prevented by the methods and compositions of the invention is not merely photo-induced damage to fluorescent reagents, e.g., photobleaching, but is also directed to prevention or reduction of the downstream effects of photoactivation of such fluorescent reagents to other reagents that are of limited quantity in a reaction mixture, and as such, their limited presence is more greatly impacted by even slight losses due to photo-induced damage, and particularly reactive proteins or enzymes, which, without being bound to a theory of operation, may include damage to the enzymes or reactive proteins or irreversible interactions between such enzymes or proteins and the photo- induced damaged reagents.
  • photo-induced damage generally refers to an alteration in a given reagent, reactant, or the like, that causes such reagent to have altered functionality in a desired reaction, e.g., reduced activity, reduced specificity, or a reduced ability to be acted upon, converted, or modified, by another molecule, that results from, either directly or indirectly, a photo-induced reaction, e.g., a photo-induced reaction creates a reactant that interacts with and causes damage to one or more other reactants.
  • a photo-induced reaction directly impacts either the reactant of interest, e.g., direct photo-induced damage, or impacts a reactant within one, two or three reactive steps of such reactant of interest.
  • photoreaction can directly impact the reaction of interest, e.g., causing a change in rate, duration, or fidelity of the reaction.
  • such limited quantity reagents or reactants may be present in solution, but at very limited concentrations, e.g., less than 200 nM, in some cases less than 10 nM and in still other cases, less than 10 pM.
  • such limited quantity reagents or reactants refer to reactants that are immobilized, or otherwise confined within a given area (e.g., a zero mode waveguide), so as to provide limited quantity of reagents in that given area, and in certain cases, provide small numbers of molecules of such reagents within that given area, e.g., from 1 to 1000 individual molecules, preferably between 1 and 10 molecules.
  • photo-induced damage of immobilized reactants in a given area will have a substantial impact on the reactivity of that area, as other, non-damaged reactants are not free to diffuse into and mask the effects of such damage.
  • the invention is directed to methods and compositions that reduce the amount of photo-induced damage to one or more reactants during an illuminated reaction, e.g., during excitation, the excited-state lifetime, or emission.
  • compositions are provided that yield a reduction in the level of photo-induced damage (or an increase in the photo-induced damage threshold period) as compared to such reactions in the absence of such compositions.
  • the components of such compositions that provide such effects are generally referred to as photo-induced damage mitigating agents.
  • photo-induced damage mitigating agents are provided in the context of the illuminated reaction to reduce the level of photo-induced damage (and/or increase the photo- induced damage threshold period), that would otherwise have occurred but for the presence of the photo-induced damage mitigating agent.
  • an agent as a photo-induced damage mitigating agent is generally reflective of the impact that such agent has on the actual photo-induced damage event or the downstream impacts of that damage.
  • the detrimental impact of the photo- induced damage event is generally referred to herein as photo-induced damage. Therefore, a photo-induced damage mitigating agent may prevent photo-induced damage of one or more reagents, or it may mitigate the impact that a photo-induced damaged reagent may have on a particular, limited reagent in the reaction of interest.
  • an agent that blocks a detrimental interaction between a photo-induced damaged fluorescent compound and a critical enzyme component e.g., by quenching the triplet state of the fluorescent compound
  • a photo-induced damage mitigating agent regardless of the fact that it did not prevent the initial photo-induced damage to the fluorescent reagent.
  • Measurements of reduction of photo-induced damage as a result of inclusion or treatment with one or more photo-induced damage mitigating agents may be characterized as providing a reduction in the level of photo-induced damage over an untreated reaction.
  • characterization of a reduction in photo-induced damage generally utilizes a comparison of reaction rates, durations, or fidelities, e.g., of enzyme activity, and/or a comparison of the photo- induced damage threshold period, between a treated reaction mixture and an untreated reaction mixture.
  • the inclusion of photo-induced damage mitigating agent(s) of the invention generally results in a reduction of photo-induced damage of one or more reactants in a given reaction, as measured in terms of "prevented loss of reactivity" in the system.
  • the amount of prevented loss of activity can at least 10%, preferably greater than 20%, 30%, or 40%, and more preferably at least 50% reduction in loss of reactivity, and in many cases greater than a 90% and up to and greater than 99% reduction in loss of reactivity.
  • the present invention is directed to the inclusion within the illuminated reaction volume of one or more photo-induced damage mitigating agents that function to block or otherwise minimize the pathways that lead to such photo-induced damage.
  • photo-induced damage mitigating agents include reducing and/or oxidizing agents or anti-fade agents that reduce the formation/lifetime of the triplet-state fluorophores (also referred to as triplet-state quenchers), in some cases by interacting/reacting with a triplet-state fluorophore, thereby preventing its interaction with (and resulting photo-induced damage to) other reaction components.
  • Such agents also include oxygen and/or radical scavenging/quenching agents that remove oxygen, reactive oxygen species, and other radicals from the reaction mixture, thus preventing downstream damage to enzymes within the system.
  • Such agents also include mixtures of agents having one or more reducing, oxidizion, anti-fade, triplet-state quenching, oxygen radical scavenging/quenching, or radical scavenging/quenching activities.
  • Certain examples of photo-induced damage mitigating agents are provided, e.g., in U.S. Published Patent Application No. 20070161017, previously incorporated herein by reference in its entirety for all purposes.
  • a photo-induced damage mitigating agent may be physically linked to one or more reaction components (e.g., a dye molecule, enzyme, nucleotide, etc.), or to a reaction site (e.g., on a substrate, in a well, in a zero mode waveguide, on a bead or optical fiber, etc.).
  • a photo-induced damage mitigating agent may be linked to multiple reaction components or to one or more reaction components and to a reaction site.
  • a tridendate structure may be formed connecting the photo- induced damage mitigating agent to two different reaction components, such as when the photo- induced damage mitigating agent is incorporated into a linker connecting two other components of the reaction.
  • such a tridendate structure may include a photo-induced damage mitigating agent, a dye, and a nucleotide, for example.
  • a photo-induced damage mitigating agent is a quinone derivative, as described below.
  • the present invention provides compounds of the formula:
  • each R 1 group is independently selected from the group consisting of hydrogen, halogen, alkyl, -CH 3 , — (CH 2 ) n R 2 , — (CH 2 ) n R 2 R 3 , -SO 3 H, -NO 2 , —OR 2 , —COR 2 , — COOR 2 , — (CH 2 ) «OR 2 , -CH(OH)R 2 , — S(O) m R 2 , -SO 3 R 2 , — C0NH m (R 2 ) 2 _ m , — SO 2 NH m (R 2 ) 2 m , NH m (R 2 ) 2 _ m , -CONHSO 3 H, — (CH 2 ) « R 5 , — CH(OR 2 )R 3 , — (CH 2 ) « R 2 R 5 , — R 5 , —OR 5 , —COR 5 , — COOR
  • R is selected from the group consisting of hydrogen, halogen, alkyl, — CH 3 , hydroxyl, — SO 3 H, -NO 2 , —OR 3 , —COR 3 ,— COOR 3 , — CH(OR 3 )R 4 , -C(OH)R 3 , -SO 3 R 3 , — R 5 , -OR 3 R 5 , —COR 5 , -COR 3 R 5 , -COR 3 R 4 R 5 , -CH(OH)R 5 , — S(O) m R 3 , -SO 3 R 3 , — C0NH m (R 3 ) 2 _ m , NH m (R 3 ) 2 _ m , — CONHSO 3 H,- SO 2 NH m (R 3 ) 2 ⁇ m , where m is an integer; R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alky
  • R 5 is a "linker" to physically link the compounds of the invention to other reactants or reaction sites so as to improve the ability of such compounds to mitigate photo-induced damage to materials normally susceptible to such damage.
  • a combination of at least one compound of formula I and at least one compound of formula II is a photo- induced damage mitigating agent.
  • a combination of 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (also a quinone derivative, and specifically a hydroquinone derivative) and at least one compound of formula I and/or at least one compound of formula II is a photo-induced damage mitigating agent.
  • the invention provides quinone derivatives of the formulas:
  • each R 1 group is independently selected from the groups provided above for R 1 of formulas I and II.
  • compounds of formula III are produced by base treatment of a compound of formula IV.
  • a scheme for the transformation of compounds of formula IV into compounds of formula III is described elsewhere herein and a reaction scheme is provided in Figure 6.
  • Such derivation can comprise a) hydrolytic opening of a ring by addition of H 2 O under acidic, basic, or neutral conditions, b) oxidation of a hydroquinone to quinone by loss of H 2 , c) further oxidation, e.g., epoxidation, by addition of O, and d) photolytic insertion of singlet oxygen.
  • the order of these three steps may vary. Equivalently, oxidation of hydroquinone to quinone may occur through addition of O 2 and release of H 2 O 2 , and/or the further oxidation step may occur through addition of H 2 O 2 and release of H 2 O.
  • a combination of at least two compounds of formulas III, IV, V, VI, VII, VIII, IX, and X is a photo-induced damage mitigating agent.
  • a combination of at least two compounds of formulas III is a photo-induced damage mitigating agent.
  • a combination of 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid and at least one compound of formulas III, IV, V, VI, VII, VIII, IX, and X is a photo-induced damage mitigating agent.
  • a combination of at least one compound of formula I and/or formula II and at least one compound of formula III, IV, V, VI, VII, VIII, IX, and X is a photo-induced damage mitigating agent.
  • linker encompasses any moiety that is useful to connect one or more compounds of the invention (e.g., a triplet-state or free radical quencher described herein) to a component of an illuminated reaction (e.g., a reporter molecule).
  • a component of an illuminated reaction e.g., a reporter molecule.
  • Linkers may also be branched to connect three or more components of a reaction mixture, e.g., in to a tridentate, tetradentate, or higher order structure.
  • a dye may be linked to one or more photo- induced damage mitigating agents, enzymes, or other reaction components.
  • a luminescently labeled reaction component e.g., fluorescent nucleotide
  • the linker is a member selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl.
  • the linker moiety is selected from straight- and branched carbon-chains, optionally including at least one heteroatom (e.g., at least one functional group, such as ether, thioether, amide, sulfonamide, carbonate, carbamate, urea and thiourea), and optionally including at least one aromatic, heteroaromatic or non-aromatic ring structure (e.g., cycloalkyl, phenyl).
  • at least one heteroatom e.g., at least one functional group, such as ether, thioether, amide, sulfonamide, carbonate, carbamate, urea and thiourea
  • aromatic, heteroaromatic or non-aromatic ring structure e.g., cycloalkyl, phenyl
  • molecules that have trifunctional linkage capability are used, including, but are not limited to, cynuric chloride, mealamine, diaminopropanoic acid, aspartic acid, cysteine, glutamic acid, pyroglutamic acid, S- acetylmercaptosuccinic anhydride, carbobenzoxylysine, histine, lysine, serine, homoserine, tyrosine, piperidinyl- 1,1 -amino carboxylic acid, diaminobenzoic acid, etc.
  • the linker as a whole may comprise a single covalent bond or a series of stable bonds.
  • a reporter molecule e.g., a fluorescent dye
  • a triplet- state or free radical quencher of the invention e.g., a quinone derivative
  • a linker that is a series of stable covalent bonds can incorporate non-carbon atoms, such as nitrogen, oxygen, sulfur and phosphorous, as well as other atoms and combinations of atoms, as is known in the art.
  • the attachment may comprise a combination of stable chemical bonds, including for example, single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds.
  • the dye is conjugated to the nucleoside triphosphate as an alkylated tetraphosphate analog.
  • linkers are derived from molecules which comprise at least two reactive functional groups (e.g., one on each terminus), and these reactive functional groups can react with complementary reactive functional groups on the various reaction components or used to immobilize one or more reaction components at the reaction site.
  • Reactive functional group refers to groups including, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho
  • Reactive functional groups also include those used to prepare bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and the like. Methods to prepare each of these functional groups are well known in the art and their application or modification for a particular purpose is within the ability of one of skill in the art (see, for example, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).
  • TetrafR ⁇ -l ⁇ -Benzoquinone and the compound of formula II may also be generically referred to as "2,3,5, ⁇ -TetrafR ⁇ -l ⁇ -Hydroquinone.”
  • one or more of the R groups may be chosen to bring additional properties to the compound, e.g., solubility, increased quenching abilities, or ability to physically link the compound to other reaction components (e.g., a dye molecule, enzyme, nucleotide, etc.) or to a reaction site (e.g., on a substrate, in a well, in a zero mode waveguide, on a bead or optical fiber, etc.), as described above.
  • the R 1 group comprises a Ci to Ce alkyl group, which may be branched or unbranched, and is substituted in certain preferred embodiments.
  • R 1 at C6 is (CH 2 ) 2 R 2 and R 2 is selected from the group consisting Of -COCH 3 (formula C), -COOH (formula D), and -C(OH)CH 3 (formula G).
  • R 1 is (CH 2 ) 3 R 2 and R 2 is — OH (formula E).
  • R 1 is selected from the group consisting of — (CH 2 ) 3 ⁇ S ⁇ 3 H (formula F), -(CHz) 2 CH(OSO 3 H)CH 3 (formula H), and — (CH 2 ) 2 COH— CH 3 COOH (formula B).
  • Formulas B-H are shown with only one substituted alkyl group, but as indicated by the R 1 groups at positions 2, 3, and 5, formulas having more than one substituted alkyl chain are contemplated and represent additional embodiments of the invention.
  • R 1 at C6 is (CH 2 ) 2 R 2 and R 2 is selected from the group consisting Of -COCH 3 (formula C), — COOH (formula D), and -C(OH)CH 3 (formula G).
  • R 1 is (CH 2 ) 3 R 2 and R 2 is — OH (formula E).
  • R 1 is selected from the group consisting of — (CH 2 ) 3 OSO 3 H (formula F),- (CH 2 ) 2 CH(OSO 3 H)CH 3 (formula H), and — (CH 2 ) 2 COH— CH 3 COOH (formula B).
  • formulas B-H in Figure 3 are shown with only one substituted alkyl group, but as indicated by the R 1 groups at positions 2, 3, and 5, formulas having more than one substituted alkyl chain are contemplated and represent additional embodiments of the invention.
  • Further preferred embodiments of formulas I and II are provided in Figure 4.
  • formulas shown at A, C, E, and G are embodiments of formula I
  • formulas shown at B, D, F, and H are embodiments of formula II.
  • Each of these formulas comprises at least two oxidizing or reducing groups on at least one substituted alkyl group extending from the ring.
  • formulas in Figure 4 are shown with only one substituted alkyl chain at C6, but as indicated by the R 1 groups at positions 2, 3, and 5, formulas having more than one substituted alkyl group are contemplated and represent additional embodiments of the invention.
  • the compound of formula I is compound A or
  • the compound of formula II is compound B or D in Figure 5, where A is -CH 2 -, -CO-, -CH(OH)-, or -CH(OSO 3 H)-; n represents an integer from one to eight; and R 6 is a hydrogen, alkyl, SO 3 H, acetate, or other group (see R'-R 5 above).
  • the compound of formula II is compound B or D in Figure 5, where A is -CH 2 -, -CO-, -CH(OH)-, or -CH(OSO 3 H)-; n represents an integer from one to eight; and R 6 is a hydrogen, alkyl, SO 3 H, acetate, or other group (see, e.g., R'-R 5 above).
  • FIG. 7 Certain exemplary embodiments of photo-induced damage mitigating agents of formulas III are shown in Figure 7.
  • the Z group can be essentially any bridging group, including but not limited to O, S, N- R 1 , (CH 2 ) n , CO, and combinations thereof, e.g., CONR, COO, CH 2 O, and the like.
  • the chemical name for formula H is 10-hydroxy-2,7,8,10-tetramethyl-6,9-dioxo-l-oxaspiro[4.5]dec-7-ene-2- carboxylic acid.
  • FIG. 8 Certain exemplary embodiments of photo-induced damage mitigating agents of formulas IV are shown in Figure 8.
  • the general structure of formula IV is shown (A), and further embodiments of formula IV are provided as formulas B, C, D, E, F, G, H, I, and J.
  • the Z group can be essentially any bridging group, including but not limited to O, S, N-R 1 , (CH 2 ) n , CO, and combinations thereof, e.g., CONR, COO, CH 2 O, and the like.
  • formula V For ease of reference, the general structure of formula V is shown (A), and further embodiments of formula V are provided as formulas B, C, D, and E.
  • the chemical name for formula E is 4-(l,6-dihydroxy-3,4,6-trimethyl-2,5-dioxocyclohex-3-enyl)-2-hydroxy-2- methylbutanoic acid.
  • formula X For ease of reference, the general structure of formula X is shown (D), and further embodiments of formula X are provided as formulas E, and F.
  • the chemical name for formula F is 4-hydroxy-l,4,5',6-tetramethyl-2,5-dioxodihydro-3'H-7-oxaspiro[bicyclo[4.1.0]heptane-3,2'- furan]-5'-carboxylic acid.
  • formula V For ease of reference, the general structure of formula V is shown (A), and further embodiments of formula V are provided as formulas B, C, D, and E.
  • the chemical name for formula E is 4-(4,5-dihydroxy-2,4,5-trimethyl-3,6-dioxocyclohex-l-enyl)-2-hydroxy-2- methylbutanoic acid.
  • the compounds provided may be used in combination with one another and/or with a variety of reducing agents, anti-fade agents, free radical quenchers/scavengers, singlet oxygen quenchers, and/or triplet-state quenchers (e.g., 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid), including, for example, those provided in U.S. Patent Publication No. 20070161017, previously incorporated by reference, which also provides methods of mitigating the impact of photo-induced damage on the results of a given analytical operation that may be used with the compounds and methods of the provided herein.
  • reducing agents e.g., 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid
  • a photo-induced damage mitigating agent is a mixture of at least two different quinone derivatives, e.g., at least about 2, 3, 4, 5, 6, or 7 quinone derivatives provided herein.
  • a mixture can comprise various ratios of any two of its constituent quinone derivatives, e.g., about 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 3:2, 1.5:1 1:1, 1:1.5, 2:3, 1:2, 1:4, 1:6, 1:8, 1:10, 1:20, etc.
  • there may be substantially equivalent amounts of multiple or each quinone derivative in the mixture and/or at least one quinone derivative may be at a significantly higher concentration than at least one other.
  • a mixture of at least two different quinone derivatives provided herein comprises various percentages of its constituent derivatives, e.g., less than about 95%, 85%, 75%, 65%, 55%, 45%, 35%, 25%, 15%, 10%, or 5%. Further, such mixtures may comprise various ranges of percentages for its constituent derivatives, such as, e.g., 5-10%, 10-15%, 10- 20%, 20-30%, and the like. In certain embodiments, at least one quinone derivative is below a threshold level, e.g., below 5%, 4%, 3%, 2%, or below 1%.
  • quinone derivatives of the invention fall within the ratios, percentages, and/or ranges provided herein.
  • percentages of quinone derivatives in a mixture are relative to the initial total concentration of a starting compound from which they are derived, but such percentages may alternatively or additionally be relative to the total concentration of all quinone derivatives in a mixture.
  • a solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is treated to produce a mixture of quinone derivatives and the resulting percentages of these derivatives is relative to the initial total concentration of the 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid prior to the treatment.
  • a photo-induced damage mitigating agent is a mixture comprising at least one benzoquinone derivative of formula I and at least one hydroquinone derivative of formula II.
  • a photo-induced damage mitigating agent is a mixture of at least two or more quinone derivatives of formulas III, IV, V, VI, VII, VIII, IX, and X, preferably at least one of formula III and another of formula IV.
  • at least one quinone derivative of each of formulas III and IV are present in a photo-induced damage mitigating agent, e.g., in a photo-induced damage mitigating agent admixture.
  • a photo-induced damage mitigating agent is a mixture of at least one photo-induced damage mitigating agent that is not a quinone derivative of formula I, II, III, IV, V, VI, VII, VIII, IX, and X and at least one quinone derivative of formula I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • a photo-induced damage mitigating agent is a mixture of at least one photo-induced damage mitigating agent that is not a quinone derivative of formula I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • a quinone derivative mixture of the invention comprises at least about 5%, 10%, 15%, 20%, 30%, or 50; or less than about 50%, 30%, 20%, 15%, 10%, or 5% of a single quinone derivative therein.
  • a quinone derivative mixture of the invention comprises at least 5%, 10%, 15%, or 20%; or less than about 30%, 20%, 15%, 10%, or 5% of multiple quinone derivatives therein.
  • a quinone derivative mixture of the invention comprises at least about 5-40% of at least one quinone derivative of formula III, e.g., of formula H in Figure 7.
  • a quinone derivative mixture of the invention comprises at least about 5-40% of at least one quinone derivative of formula IV, e.g., of formula J in Figure 8.
  • a quinone derivative mixture comprises at least about 1-10% of each of multiple quinone derivatives of formulas III, IV, V, VI, VII, VIII, IX, and X.
  • a quinone derivative mixture comprising at least about 2-15% of each of multiple quinone derivatives of formulas III, IV, V, VI, VII, VIII, IX, and X further comprises at least about 2-15% or less than about 10% of at least one quinone derivative of formula I and/or II.
  • quinone derivative mixtures also comprise 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid, e.g. less than about 20%, or 10% or 5%.
  • these percentages describing quinone derivative mixtures of the invention refer to percentages present in admixtures prior to addition to an analytical reaction of interest, regardless of changes in such percentages that may occur upon or subsequent to such addition, e.g., during the course of the analytical reaction.
  • the term "photo-induced damage mitigating agent admixture" indicates a mixture comprising one or more photo-induced damage mitigating agents to be added to a reaction mixture wherein such percentages may change.
  • a photo-induced damage mitigating agent admixture may further comprise additional components, e.g., buffers, salt, or other reagents necessary for initiation and/or progression of a reaction of interest, such as enzymes, substrates, cofactors, and the like.
  • additional components e.g., buffers, salt, or other reagents necessary for initiation and/or progression of a reaction of interest, such as enzymes, substrates, cofactors, and the like.
  • photo-induced damage mitigating agents may generally be provided as a component of the reaction mixture, either through addition as an additive, either liquid or solid, or through predisposition and/or immobilization of the photo-induced damage mitigating agents within the region where the reaction is taking place.
  • a photo-induced damage mitigating agent comprises cooperatively functioning components, e.g., dual enzyme systems, it may again be desirable to localize such components relative to each other, as well as to the reaction of interest.
  • the photo-induced damage mitigating agents may be immobilized upon the surfaces of the substrates or reactions wells, or may be provided in a configuration that permits them to freely interact with the aqueous system components by including such agents within or linked to structures (e.g., caging groups, tridentate structures, etc.) that render the agents suspended in aqueous systems and additionally available to interact with relevant portions of the reaction mixture, e.g., dissolved oxygen species.
  • structures e.g., caging groups, tridentate structures, etc.
  • a substrate may comprise any of a variety of formats, from planar substrates, e.g., glass slides or planar surfaces within a larger structure, e.g., a multi-well plates such as 96-well, 384-well, and 1536-well plates, or regularly spaced micro- or nano-porous substrates.
  • Such substrates may also comprise more irregular porous materials, such as membranes, aerogels, fibrous mats, or the like, or they may comprise particulate substrates, e.g., beads, spheres, metal or semiconductor nanoparticles, optical fibers, or the like.
  • the other reagents in a given reaction of interest may be provided in any of a variety of different configurations.
  • they may be provided free in solution, or complexed with other materials, e.g., other reagents and/or solid supports.
  • such reagents may be provided coupled to beads, particles, nanocrystals or other nanoparticles, or they may be tethered to larger solid supports, such as matrices or planar surfaces.
  • reagents may be further coupled or complexed together with other reagents, or as separate reagent populations or even as individual molecules, e.g., that are detectably resolvable from other molecules within the reaction space.
  • a particular reagent is confined by virtue of structural barriers to its free movement or is chemically tethered or immobilized to a surface of a substrate, it will be described as being "confined.”
  • one or more reagents in an assay system are confined within an optical confinement.
  • Such an optical confinement may be an internal reflection confinement (IRC) or an external reflection confinement (ERC), a zero mode waveguide, or an alternative optical structure, such as one comprising porous film with reflective index media or a confinement using index matching solids. More detailed descriptions of various types of optical confinements are provided, e.g., in International Application Publication No. WO/2006/083751, U.S. Patent No. 6,917,726, and U.S. Patent No. 7,170,050, the full disclosures of which are incorporated herein by reference in their entireties for all purposes.
  • the methods and compositions of the invention are useful in a broad range of illuminated analytical reactions, and particularly those using photoluminescent or fluorescent reactants, and particularly such reactions where the reagents that are susceptible to photo-induced damage are present at relatively low levels.
  • One exemplary application of the methods and compositions described herein is in single molecule analytical reactions, where the reaction of a single molecule (or very limited number of molecules) is observed in the analysis, such as observation of the action of a single enzyme molecule.
  • damage to any significant fraction of that population will have a substantial impact on the analysis being performed.
  • compositions and methods of the present invention can prevent or mitigate that impact by providing photo-induced damage mitigating agents in the reaction mixture.
  • the photo-induced damage of illuminated reactions sought to be prevented by the methods and compositions of the invention is not merely photo-induced damage to fluorescent reagents, e.g., photobleaching, but also includes the prevention or reduction of the downstream effects of photoactivation.
  • reagents with a limited presence are greatly impacted by even slight losses due to photo-induced damage, particularly reactive proteins or enzymes.
  • This damage may include damage to the enzymes or reactive proteins or irreversible interactions between such enzymes or proteins and the photo-induced damaged reagents.
  • the present invention is directed to illuminated reaction mixtures which include one or more agents that function to block or otherwise minimize the pathways that lead to damage due to the creation of reactive oxygen species during an illuminated reaction.
  • Photo-induced damage mitigating agents include quinone derivatives, as described herein, as well as reducing agents or anti-fade agents, such as those that prevent photo-induced damage resulting from the presence of triplet-state fluorophores (also referred to as triplet-state quenchers) that can form during the course of an illuminated reaction.
  • Photo-induced damage mitigating agents also include oxygen scavenging agents, which remove oxygen and reactive oxygen species from the reaction mixture. Such photo-induced damage mitigating agents are able to alleviate and/or prevent photo-induced damage by blocking the damage such species may cause to one or more reactants, particularly conjugates that include a dye. [00108] In general, the photo-induced damage mitigating agents described herein are present in the reaction mixture at levels sufficient to provide beneficial impact, e.g., reduced photo-induced damage and/or extension of the photo-induced damage threshold period, but are not present at such levels as to interfere with the reaction of interest, e.g., the sequencing reaction.
  • the photo-induced damage mitigating agents are present at 0.5-10.0 mM, or more preferably between about 0.5 mM and 5 mM, which represents the total concentration of a single or a combination of photo-induced damage mitigating agents presented herein.
  • concentrations are merely exemplary and may be change depending on various factors including, e.g., the particular photo-induced damage mitigating agent and/or mixture thereof, the type of reaction to which it is added, conditions under which such reaction is to be performed, and the like. Such adjustments are well within the abilities of the ordinary practitioner.
  • the photo-induced damage mitigating agents described herein are particularly suitable for mitigating photo-induced damage to reactants in small reaction volume concentrations, wherein such reactants may be present in solution, but at very limited concentrations, e.g., less than 200 nM, in some cases less than 10 nM, and in still other cases less than 10 pM.
  • such limited quantity reagents or reactants refer to reactants that are immobilized or otherwise confined within a given area, so as to provide a limited quantity of reagents in that given area, and in certain cases, provide small numbers of molecules of such reagents within that given area, e.g., from 1 to 1000 individual molecules, preferably between 1 and 10 molecules.
  • photo-induced damage of immobilized reactants in a given area will have a substantial impact on the reactivity of that area, as other, non-damaged reactants are not free to diffuse into the reaction volume and mask the damage effects.
  • the present invention is directed to illuminated reactions for single molecule analysis, including sequencing of nucleic acids by observing incorporation of nucleotides into a nascent nucleic acid sequence during template-directed polymerase-based synthesis.
  • sequencing-by-incorporation involve the observation of the addition of nucleotides or nucleotide analogs in a template-dependent fashion in order to determine the sequence of the template strand. See, e.g., U.S. Patent Nos. 6,780,591, 7,037,687, 7,344,865, 7,302,146, and Eid, et al.
  • Processes for performing this detection include the use of fluorescently labeled nucleotide analogs within a confined observation region, e.g., within a nanoscale well and/or tethered, either directly or indirectly to a surface.
  • excitation illumination i.e., illumination of an appropriate wavelength to excite the fluorescent label and induce a detectable signal
  • the fluorescently labeled bases can be detected as they are incorporated into the nascent strand, thus identifying the nature of the incorporated base, and as a result, the complementary base in the template strand.
  • One particularly preferred aspect of the invention is in conjunction with the sequencing by incorporation of nucleic acids within an optical confinement, such as a zero mode waveguide.
  • an optical confinement such as a zero mode waveguide.
  • Such reactions involve observation of an extremely small reaction volume in which one or only a few polymerase enzymes and their fluorescent substrates may be present.
  • Zero mode waveguides, and their use in sequencing applications are generally described in U.S. Patent Nos. 6,917,726 and 7,033,764, and preferred methods of sequencing by incorporation are generally described in Published U.S. Patent Application No. 2003-0044781, the full disclosures of which are incorporated herein by reference in their entireties for all purposes, and in particular for their teachings regarding such sequencing applications and methods.
  • arrays of zero mode waveguides may be employed as optical confinements for single molecule analytical reactions, e.g., for nucleic acid (e.g., DNA, RNA) sequence determination.
  • these ZMWs provide extremely small observation volumes at or near the transparent substrate surface, also termed the “base” of the ZMW.
  • a nucleic acid synthesis complex e.g., template sequence, polymerase, and primer, which is immobilized at the base of the ZMW, may then be specifically observed during synthesis to monitor incorporation of nucleotides in a template dependent fashion, and thus provide the identity and sequences of nucleotides in the template strand.
  • This identification is typically accomplished by providing detectable label groups, such as fluorescent labeling molecules, on the nucleotides.
  • the labeled nucleotides terminate primer extension, allowing a "one base at a time" interrogation of the complex. If, upon exposure to a given labeled base, a base is incorporated, its representative fluorescent signal may be detected at the base of the ZMW. If no signal is detected, then the base was not incorporated and the complex is interrogated with each of the other bases, in turn.
  • the labeling group is removed, e.g., through the use of a photocleavable linking group, and where the label was not the terminating group, a terminator, upon the 3' end of the incorporated nucleotide, may be removed prior to subsequent interrogation.
  • the above-described sequencing reaction may be carried out in the presence of one or more photo-induced damage mitigating agents (e.g., quinone derivatives, and conjugates and mixtures thereof) provided herein, either alone or in combination with other reaction mixture additives, such as reducing agents, antifade agents, free radical quenchers, triplet-state quenchers, singlet oxygen quenchers, or enzyme systems for depletion of oxygen species (e.g., comprising an oxidase).
  • the sequencing reactions may be carried out in the presence of at least one quinone derivative described herein.
  • a photo-induced damage mitigating agent may be a mixture comprising at least one hydroquinone derivative and at least one benzoquinone derivative, or at least two compounds of formulas I, II, III, IV, V, VI, VII, VIII, IX, and X.
  • a photo-induced damage mitigating agent may be a mixture comprising at least one compound of formula III and at least one compound of formula IV.
  • a photo-induced damage mitigating agent may be a mixture comprising one or more compounds of formulas III and IV and at least one compound of formula I.
  • a photo-induced damage mitigating agent may be a mixture comprising 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid and at least one quinone derivative provided herein.
  • the illuminated reaction mixture includes a nucleoside polyphosphate connected to a fluorescent dye by a linker.
  • the linker in such a reaction mixture itself may comprise one or more photo-induced damage mitigating agents, such as the quinone derivatives (or mixtures thereof) provided herein.
  • the present invention also provides alternative methods of mitigating the impact of photo-induced damage on a reaction.
  • Such alternative methods can be used in combination with the compositions and methods described above to further alleviate the effects of species that can be generated during an illuminated reaction.
  • One alternative method of mitigating the impact of photo-induced damage on the results of a given reaction is by only interrogating a reaction mixture, e.g., detecting fluorescent emission, during such portion of the illumination period before which excessive photo-induced damage has occurred.
  • This approach is particularly useful in the optical interrogation of reactions where components of the reaction that are susceptible to photo-induced damage are spatially confined on an assay plate or substrate, either through the presence of structural confinements and/or through immobilization of the components.
  • Examples of such confined reagents include surface immobilized or localized reagents, e.g., surface immobilized or associated enzymes, antibodies, etc. that are interrogated upon the surface, e.g., through fluorescence scanning microscopy or scanning confocal microscopy, total internal reflectance microscopy or fluorometry, surface imaging, or the like.
  • dissolved oxygen species may be flushed out of aqueous systems by providing the reaction system under different gas environments, such as by exposing an aqueous reaction to neutral gas environments, such as argon, nitrogen, helium, xenon, or the like, to prevent dissolution of excess oxygen in the reaction mixture.
  • neutral gas environments such as argon, nitrogen, helium, xenon, or the like.
  • the system is exposed to a xenon atmosphere.
  • xenon since xenon can be induced to form a dipole, it operates as a triplet-state quencher in addition to supplanting oxygen in the aqueous system. (See, e.g., Vierstra and Poff, Plant Physiol. 1981 May; 67(5): 996-998, which is incorporated herein by reference in its entirety for all purposes)
  • xenon would also be categorized as a quencher, as set forth above.
  • 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid is a vitamin E derivative that has been used to reduce radiation-induced damage, e.g., for fluorescent probes, and is generally commercially available as Trolox ® from Sigma-Aldrich (St. Louis, MO).
  • Trolox ® from Sigma-Aldrich (St. Louis, MO).
  • a solution of photomodified 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid was prepared by adding 0.2966 grams of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid to 1.278 mL of methanol and 9.58 mL of HPLC water. Drops of IM KOH were added to the mixture until it became a uniformly transparent solution. The solution was irradiated for one hour under a UV lamp, and was subsequently exposed to room-light irradiation at room temperature for 18 hours.
  • FCS Fluorescence correlation spectroscopy
  • FCS experiments using the fluorophore Alexa568 in low oxygen were used to monitor the amount of the dye triplet state upon excitation over time as a measure of triplet-state quenching by 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and derivatives thereof. Briefly, these experiments were performed on a confocal microscope setup (Olympus 1X71 with water-immersion objective UPlanSApo, 60x) using a single line laser excitation (532 nm) at 0.4- mW intensity and two single photon avalanche detectors (SPAD detectors; Perkin Elmer, Waltham, Massachusetts).
  • FCS measurements were done in a reaction buffer containing 50 mM ACES, pH 7.1; 75 mM potassium acetate; and 5 mM DTT.
  • a protocatechuic acid/protocatechuate-S ⁇ -dioxygenase (PCA/PCD) oxygen scavenging system (see, e.g., Aitken, et al. (2007) "An Oxygen Scavenging System for Improvement of Dye Stability in Single- Molecule Fluorescence Experiments," Biophys J. 94:1826-1835) was included, as well.
  • Correlation curves were generated and recorded by a FLEX digital photon correlator (Correlator.com; Bridgewater, NJ), and analyzed using Origin ⁇ .O (OriginLabs; Northampton, MA).
  • the aged solution was the second best performer with an autocorrelation amplitude of -0.07 at a lag time of 0.001 ms.
  • the freshly prepared solution not treated with UV was the least effective triplet- state quencher with an autocorrelation function of -0.2 at a lag time of 0.001 ms, and in general, increasing times of UV treatment increased effectiveness of triplet-state quenching by these solutions, although there were not large differences between the quenching ability of the solutions exposed for three, six, and 24 hours.
  • 2,5,7, 8-tetramethylchroman-2-carboxylic acid solutions A first, described above, involves exposing a freshly prepared solution to UV irradiation for one hour followed by 18 hours of room-light irradiation. In a second, a freshly prepared solution was irradiated for 36 hours under a UV lamp. Reverse phase HPLC was performed on a freshly prepared solution of 6-hydroxy- 2,5,7, S-tetramethylchroman ⁇ -carboxylic acid and photomodified solutions prepared by these two methods. The top trace in Figure 1OA shows that freshly prepared 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid ran as a single species on reverse phase HPLC with a migration time of 8.987 minutes.
  • the second trace (B) shows the peaks in the mixture produced by treating the fresh solution with UV light for one hour, followed by exposure to room-light radiation for 18 hours at room temperature. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and a first derivative termed "Fraction A" are clearly visible with migration times of 9.008 minutes and 9.791 minutes, respectively.
  • the third trace (C) shows the peaks in the mixture produced by treating the fresh solution with UV light for 36 hours. 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid (migration time 8.969 minutes) and two major derivatives are clearly visible.
  • Fraction A The derivative with a migration time of 9.756 minutes was identified as “Fraction A” and the derivative with a migration time of 14.8501 minutes was termed “Fraction B.”
  • Fraction B The derivative with a migration time of 9.756 minutes was identified as “Fraction A” and the derivative with a migration time of 14.8501 minutes was termed “Fraction B.”
  • Fraction B as triplet-state quenchers/radical scavengers was tested by fluorescence correlation spectroscopy (FCS) using Alexa568 in low oxygen, as described above in Example 1. Two different intensity of excitation settings, 0.2 mW and 0.4 mW, were used for power dependence.
  • Figure 1 IA provides an FCS autocorrelation curve at the 0.2 mW intensity of excitation setting for the aged solution, Fraction A, and Fraction B. At a lag time of 0.001 ms they had autocorrelation amplitudes of -0.09, -0.065, and -0.06, respectively. These data show that both Fraction A and Fraction B had better quenching activity than the aged solution.
  • Fraction A and Fraction B had autocorrelation amplitudes of -0.043 and -0.04, respectively, while the aged solution and the fresh solution exposed to one hour of UV light followed by 18 hours of room light had autocorrelation amplitudes of -0.073, and the fresh solution exposed to 36 hours of UV light had an autocorrelation amplitude of -0.065.
  • Fraction A was further converted to Fraction B by photolysis in a separate experiment. Specifically, purified Fraction A (reverse phase HPLC peak migration time of 9.791 minutes; Figure 12A) was subjected to UV irradiation in water for one hour to produce Fraction B (reverse phase HPLC peak migration time of 14.891 minutes; Figure 12B).
  • the HPLC traces in Figures 12A and 12B showed an 80% conversion from Fraction A to Fraction B, with a half- life of 26 minutes, and an apparently very clean conversion.
  • Figure 12C shows the conversion is essentially complete in about four hours under a portable UV lamp with the major component of Fraction B representing the majority of the resulting mixture with a peak migration time of 14.837.
  • the readlengths produced in sequencing-by-incorporation reactions in the presence of 0.5 mM freshly prepared 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid were compared to those produced in the presence of a) 0.5 mM of the mixture produced from the "one hour UV + 18 hours room temperature/light” method ("Photomodified mix”); b) 0.5 mM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and 0.05 mM purified Fraction A ("+0.05 mM FrA”); c) 0.5 mM 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid and 0.10 mM purified Fraction A ("+0.10 mM FrA”); and d) 0.5 mM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and 0.25 m
  • Figure 13 provides a graphical representation of the results from these experiments showing the relationship between the normalized counts versus the fraction of maximum readlength. Each point on each curve indicates a proportion of total sequencing reactions ("Normalized Counts") that achieved or exceeded a given sequencing readlength, where the readlength is measured as a fraction of the maximum readlength attained in any of the reactions.
  • Normalized Counts proportion of total sequencing reactions
  • the uppermost point on the graph is located at -0.6 (y-axis) and -0.1 (x-axis), indicating that 60% of the sequencing reactions in the presence of that solution attained a readlength of at least one-tenth that of the maximum readlength attained in any of the experiments.
  • This curve further indicates that only about 10% of sequencing reactions attained a readlength of at least one-fifth the maximum, and that very few attained a readlength of at least 30% of the maximum.
  • sequencing reactions performed in the presence of the "photomodified mix" of 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid and photo-induced derivatives ( ⁇ ) generally attained much longer readlengths, as did sequencing reactions in the presence of mixtures of 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid with (/) 0.05 mM purified Fraction A ( ⁇ ), (H) 0.10 mM purified Fraction A (o), or (Hi) 0.25 mM purified Fraction A (•).
  • the longer readlengths in these reactions were interpreted as being indicative of a decrease in photo-induced damage to the reaction components, and in particular are believed to be at least partially the result of increased processivity of the polymerase due to reduced photo -induced damage that would otherwise have reduced the activity and/or processivity of the enzyme earlier in the course of the reaction.
  • Fraction A is the compound 2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-l,4-dienyl)butanoic acid, which was previously identified as a derivative of 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Nonell, et al. (1995) "Solvent influence on the kinetics of the photodynamic degradation of Trolox, a water-soluble model compound for vitamin E," Photochem. Photobiol. 29:157-162).
  • Fraction B The major component of Fraction B is the decarboxylated product 2,3,5-trimethyl- 6-(3 — oxobutyl)-l,4-benzoquinone, herein termed "G-Lox.”
  • G-Lox The degradation pathway is shown in Figure 14.
  • LC/MS was carried out with a Thermo Scientific LCQ Fleet ion trap mass spectrometer.
  • For Fraction A the observed ion mass of 265.05 matches well with the expected M-I exact mass of 265.11.
  • 1 H NMR was carried out in deutero chloroform solvent with trimethylsilane as internal reference.
  • one method for preparing mixtures comprising certain quinone derivatives of the invention was by subjecting 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid to photodegradation by exposure to oxygen and irradiation under UV light (e.g., lamp) for one hour, followed by exposure to ambient light at room temperature for 18 hours to produce a mixture comprising quinone derivatives of the invention, including compounds of formula I such as 2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-l,4- dienyl)butanoic acid and 2,3,5-trimethyl-6-(3 — oxobutyl)-l,4-benzoquinone.
  • UV light e.g., lamp
  • 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid was subject 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid to 36 hours of UV irradiation in the presence of oxygen.
  • the product of both 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid photomodification methods is a mixture comprising, e.g., 2-hydroxy-2-methyl-4-(2,4,5- trimethyl-3,6-dioxocyclohexa-l,4-dienyl)butanoic acid and 2,3,5-trimethyl-6-(3 — oxobutyl)-l,4- benzoquinone.
  • 2,3,5-trimethyl-6-(3 — oxobutyl)-l,4-benzoquinone may also be prepared directly from 2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-l,4-dienyl)butanoic acid, as UV exposure in the presence of oxygen converts 2-hydroxy-2-methyl-4-(2,4,5- trimethyl-3,6-dioxocyclohexa-l,4-dienyl)butanoic acid into 2,3,5-trimethyl-6-(3 — oxobutyl)- 1 ,4-benzoquinone.
  • a further alternative method used for preparing a mixture comprising quinone derivatives of the invention was to expose 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid to a highly basic solution prior to UV irradiation.
  • 0.2503 grams of pure ⁇ -hydroxy ⁇ SJ ⁇ -tetramethylchroman ⁇ -carboxylic acid was added to 1.08 mL methanol, shaken to form a homogeneous solution, and subsequently diluted with 6.0 mL water. The dilution was followed by addition of 1 mL of a KOH solution, and mixing by vortex until a transparent solution was formed.
  • the dashed trace shows the HPLC peaks from a mixture of quinone derivatives prepared from a solution of 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid under neutral pH (7.5), and this mixture comprises both 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (at -8.1 minutes) and Fraction A (at -8.6 minutes).
  • the solid trace shows the peaks in a mixture produced by increasing the pH of a solution of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid prior to UV irradiation, as described above.
  • the type of UV and/or light exposure can also be modified.
  • 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid was dissolved in methanol and water was added to bring the volume up to 95% of the final total volume.
  • the pH was raised to 11.5-12.0 by addition of potassium hydroxide. Once a pH of 11.5-12.0 was reached, the volume was increased to the final total volume, which produced a 100 mM final concentration of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid in 10% methanol.
  • the solution was incubated in the dark at 18°C-22°C for 72 hours.
  • Figure 17 provides a graphical representation of the results from single-molecule, realtime sequencing reactions in the presence of purified fraction 5, purified fraction 7, no photo- induced damage mitigating agents ("no PIDMA"), 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (HTMCCA), Fraction A, and a mixture prepared by the method described in the previous paragraph (Mixture).
  • This graph shows the relationship between the normalized counts versus the fraction of maximum readlength.
  • Each point on each curve indicates a proportion of total sequencing reactions (“Normalized Counts") that achieved or exceeded a given sequencing readlength, where the readlength is measured as a fraction of the maximum readlength attained in any of the reactions.
  • the amount of time for UV irradiation and room light exposure can be adjusted, as can the concentration of the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid solution to be treated.
  • concentration of the 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid solution to be treated For example, one to twenty-four hours of UV irradiation have been employed, as have 18 to 36 hours room light exposure.
  • increasing UV exposures increase the ability of the mixture to effectively mitigate photo-induced damage, although there was no significant difference between the three, six, and 24 hour exposures ( Figure 9).
  • solutions of 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid subjected to photomodification were typically of a concentration of 95-100 mM, which is near the saturation point for the compound.
  • treatment of more or less concentrated solutions in contemplated and known chemical methods may be employed to increase the solubility of the compound prior to treatment.
  • this method of preparation of certain compounds of the inventions is exemplary and other methods for preparing this compound may be performed using chemistry methods well known to those of skill in the art, including direct synthesis methods, for example, starting with a quinone such as 1 ,4-benzoquinone or 1,4-hydroquinone.
  • FIG. 18 One method for preparing a compound of formula II is shown in Figure 18.
  • Friedel-Crafts acylation uses ethyl malonyl chloride as the agent and aluminum chloride (AICI 3 ) as a catalyst to add the ethyl malonyl group to 2,3,5-trimethylphenol at step A, which is then hydrolyzed to the corresponding acid at step B.
  • Reduction of the carbonyl group is achieved using either Wolff- Kishner reduction or Clemmensen reduction reaction at step C.
  • FIG. 19 provides a simplified scheme for the production of certain compounds of the invention from 6-hydroxy- 2,5,7, 8-tetramethylchroman-2-carboxylic acid solution. It is to be understood that this scheme for preparation of certain compounds of the inventions is exemplary and other methods for preparing this compound may be performed using chemistry methods well known to those of skill in the art.
  • Fractions 4, 5, and 7 in Figure 16 were purified by HPLC and concentrated to produce crystals. The crystal structure of these purified fractions was determined by X-ray crystallography and the stereoisomers so determined are provided in Figure 20.
  • Fraction 4 was determined to comprise a racemic mixture of compounds of formulas A and B.
  • Fraction 5 was determined to comprise a racemic mixture of compounds of formulas C and D.
  • Fraction 7 was determined to comprise a racemic mixture of compounds of formulas E and F.
  • formulas C and D are diastereomers of formulas E and F, respectively.
  • both fraction 5 and fraction 7 are formed when fraction 4 is subjected to the base (low pH) treatment used in the preparation of certain mixtures and admixtures of the invention.
  • This transformation is shown in Figure 6.
  • a five-carbon ring in the composition of fraction 4 interconverts with the six-carbon ring of the compositions of fractions 5 and 7, thereby creating an equilibrium of the three isomers.
  • the structures are stabilized by decreasing the pH, which inhibits the transformations between the three isomers and thereby stabilizes the amounts of the structures in an admixture useful for mitigation of photo-induced damage.
  • Figure 27 provides a three-dimensional crystal structure of a compound of fraction 4;
  • Figure 28 provides a three-dimensional crystal structure of a compound of fraction 5;
  • Figure 29 provides a three-dimensional crystal structure of a compound of fraction 7.
  • Crystals and crystal structures for the compounds of fractions 4, 5, and 7 were performed in a substantially similar way.
  • the data collection description for a crystal of fraction 4 is as follows: A colorless prism crystal of Ci 4H ⁇ gOg having approximate dimensions of 0.20 x 0.20 x 0.20 mm was mounted on a glass fiber. All measurements were made on a Rigaku RAXIS RAPID imaging plate area detector with graphite monochromated Cu-Ka radiation. Indexing was performed from 6 oscillations that were exposed for 120 seconds.
  • the crystal-to-detector distance was 127.40 mm.
  • any concentration values provided herein are generally given in terms of admixture values or percentages without regard to any conversion that occurs upon or following addition of the particular component of the mixture.
  • benzoquinone derivatives may be converted to hydroquinone derivatives by reducing agents (e.g., DTT) present in a reaction mixture.
  • reducing agents e.g., DTT

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Abstract

L'invention porte sur des compositions, des dispositifs, des systèmes et des procédés pour réduire et/ou prévenir un dommage photo-induit d'un ou plusieurs réactifs dans une réaction analytique éclairée en ajoutant au mélange réactionnel un ou plusieurs agents d'atténuation du dommage photo-induit et en amenant la réaction à se dérouler pendant une période de temps qui est inférieure à la période de seuil du dommage photo-induit.
PCT/US2009/065222 2008-11-19 2009-11-19 Agents d'atténuation du dommage photo-induit et préparation et procédés d'utilisation de ces agents WO2010059880A2 (fr)

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WO2010117420A2 (fr) 2009-03-30 2010-10-14 Pacific Biosciences Of California, Inc. Composés marqués par fret et leurs utilisations
US8834847B2 (en) 2010-08-12 2014-09-16 Pacific Biosciences Of California, Inc. Photodamage mitigation compounds and systems
US8956816B2 (en) 2012-06-05 2015-02-17 Pacific Biosciences Of California, Inc. Methods and compositions for performing analytical operations
US9637782B2 (en) 2012-09-28 2017-05-02 Pacific Biosciences Of California, Inc. Charged triplet-state quenchers for mitigation of photo-induced damage
EP3030683B1 (fr) 2013-08-05 2022-03-02 Pacific Biosciences of California, Inc. Composés réactifs fluorescents protégés
US10302972B2 (en) 2015-01-23 2019-05-28 Pacific Biosciences Of California, Inc. Waveguide transmission
WO2016126941A1 (fr) 2015-02-04 2016-08-11 Pacific Biosciences Of California, Inc. Réactifs fluorescents protégés multimères
CN108350487B (zh) 2015-11-19 2022-05-27 加利福尼亚太平洋生物科学股份有限公司 用于改善信号检测的化合物和系统
EP3376996B1 (fr) 2015-11-20 2022-09-07 Pacific Biosciences of California, Inc. Réactifs protégés marqués par un colorant
US10781483B2 (en) 2015-11-20 2020-09-22 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
US10676788B2 (en) 2015-11-20 2020-06-09 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
US10337050B2 (en) 2016-02-11 2019-07-02 Qiagen Sciences, Llc Polyphenolic additives in sequencing by synthesis
WO2020006421A1 (fr) 2018-06-29 2020-01-02 Pacific Biosciences Of California, Inc. Procédés et compositions pour l'administration de molécules et de complexes à des sites de réaction
CN114829369A (zh) 2019-11-27 2022-07-29 伊鲁米纳剑桥有限公司 含环辛四烯的染料和组合物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6068848A (en) * 1997-12-17 2000-05-30 Color Access, Inc. Antioxidant mixture comprising tocopherol
US20020037479A1 (en) * 1993-10-07 2002-03-28 Mallinckrodt Baker, Inc. Photoresist strippers containing reducing agents to reduce metal corrosion
US20030129642A1 (en) * 1997-04-09 2003-07-10 Biosite Diagnostics, Inc. Novel compositions and methods for inhibiting light-induced inactivation of biological reagents
US20070161017A1 (en) * 2005-12-02 2007-07-12 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2511428A (en) * 1947-05-27 1950-06-13 Nopco Chem Co Antioxidants
US4003919A (en) * 1973-11-19 1977-01-18 Hoffmann-La Roche Inc. Antioxidant chroman compounds
US3986980A (en) * 1974-04-29 1976-10-19 Hoffmann-La Roche Inc. Synergistic antioxidant composition comprising ascorbic acid and 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid
US4302569A (en) * 1979-06-21 1981-11-24 Argus Chemical Corporation 2-Chloro-2-alkyl substituted peroxyesters
US5242835A (en) * 1987-11-03 1993-09-07 Radiometer A/S Method and apparatus for determining the concentration of oxygen
US6881396B2 (en) * 2000-10-24 2005-04-19 Diatide, Inc. Stabilization of radiopharmaceutical compositions using hydrophilic 6-hydroxy-chromans
US20040219240A1 (en) * 2001-06-20 2004-11-04 Babish John G. Anti-inflammatory pharmaceutical compositions for reducing inflammation and the treatment or prevention of gastric toxicity
JP2003084002A (ja) * 2001-06-27 2003-03-19 Fuji Photo Film Co Ltd 蛍光検出におけるバックグラウンドの低減方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020037479A1 (en) * 1993-10-07 2002-03-28 Mallinckrodt Baker, Inc. Photoresist strippers containing reducing agents to reduce metal corrosion
US20030129642A1 (en) * 1997-04-09 2003-07-10 Biosite Diagnostics, Inc. Novel compositions and methods for inhibiting light-induced inactivation of biological reagents
US6068848A (en) * 1997-12-17 2000-05-30 Color Access, Inc. Antioxidant mixture comprising tocopherol
US20070161017A1 (en) * 2005-12-02 2007-07-12 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions

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