Dual conjugate labels and methods of use

Abstract

Provided herein is a biomolecule comprising a fluorescent marker. The marker is Cy3B having formula (I), wherein Q 1 and Q 2 Independently a monomeric or oligomeric biomolecule. #imgabs0#

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 698,006, filed on July 13, 2018, which is hereby incorporated by reference in its entirety. Background Art

[0003] Labeled probes are widely used in methods for detecting biological analytes and analyzing biological processes. Some of these techniques involve real-time monitoring of biological reactions using luminescent labeled reaction components. The labeled molecules are illuminated with a light source to induce luminescence, and the luminescence is detected with a photodetector. These events can be recorded and analyzed to identify individual reaction components based on their corresponding luminescence properties. When identifying a specific type among multiple types of labeled molecules, it is critical that each type exhibits unique and easily distinguishable luminescence properties. However, the inherent sensitivity of complex biological processes requires careful consideration when designing labeled probes for use in these systems. Summary of the invention

[0004] Aspects of the present disclosure relate to labeled biomolecules comprising internally conjugated luminescent markers. In some aspects, the present disclosure provides labeled biomolecules comprising substrates configured for reactions. In some aspects, the present disclosure provides labeled biomolecules comprising nucleotides configured for polymerization reactions. In some aspects, the present disclosure provides methods for sequencing using labeled nucleotides described herein. In some aspects, the present disclosure provides dual conjugated luminescent markers and methods for preparing the same.

[0005] In some aspects, the present application provides a labeled biomolecule of formula (I):

[0006]

[0007] Where: Q 1 and Q 2 are independently monomeric or oligomeric biomolecules; A is a polycyclic fluorophore; and L 1 and L 2 is independently a linker selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof.

[0008] In some aspects, provided herein are labeled nucleotides, the labeled nucleotides include one or more nucleotides associated with the labeled biomolecule of the application. In some embodiments, the one or more nucleotides include a type of nucleotides selected from guanine, cytosine, adenine and thymine or uracil. In some embodiments, when under polymerization conditions, the one or more nucleotides are cut off from the labeled biomolecule by a polymerase. In some embodiments, the one or more nucleotides include polyphosphate nucleosides. In some embodiments, the one or more nucleotides include triphosphate nucleosides. In some embodiments, the one or more nucleotides include hexaphosphate nucleosides. In some embodiments, the one or more nucleotides (such as polyphosphate nucleosides) are connected to the labeled biomolecule by terminal phosphate. In some aspects, provided herein are compositions, the compositions include the labeled nucleotides of the application.

[0009] In some aspects, a nucleic acid sequencing reaction composition is provided herein, which includes two or more different types of labeled nucleotides in a reaction mixture. In some embodiments, at least one type of labeled nucleotide in the nucleic acid sequencing reaction composition is a labeled nucleotide according to the present application. In some embodiments, the nucleic acid sequencing reaction composition includes four different types of labeled nucleotides. In some embodiments, the nucleic acid sequencing reaction composition includes a first labeled nucleotide, a second labeled nucleotide, a third labeled nucleotide, and a fourth labeled nucleotide, wherein the first labeled nucleotide includes guanine, the second labeled nucleotide includes cytosine, the third labeled nucleotide includes adenine, and the fourth labeled nucleotide includes thymine or uracil.

[0010] In some aspects, the present invention provides a method for determining the sequence of a template nucleic acid. In some embodiments, the method includes exposing a complex of a target volume to a nucleic acid sequencing reaction composition of the present application, wherein the complex includes a template nucleic acid, a primer, and a polymerase. In some embodiments, the method also includes directing a series of pulses with one or more excitation energies to the vicinity of the target volume. In some embodiments, the method also includes detecting multiple photons emitted from luminescent labeled nucleotides during sequential incorporation into a nucleic acid comprising a primer. In some embodiments, the method also includes identifying the sequence of the incorporated nucleotides by determining the time and optional luminescence intensity of the emitted photons.

[0011] In some aspects, provided herein is a kit for sequencing a template nucleic acid. In some embodiments, the kit includes two or more different types of labeled nucleotides. In some embodiments, at least one of these two or more different types of labeled nucleotides includes a labeled nucleotide of the application.

[0012] In some aspects, the present application provides a compound of formula (II) or a salt thereof:

[0013]

[0014] Where: A is a multi-ring fluorophore; L 1 and L 2 P is independently a linker selected from the group consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof; P 1 is an oxygen protecting group; and R 1 is a reactive moiety. In some aspects, provided herein are compositions comprising a compound of formula (II).

[0015] In some aspects, provided herein are methods for preparing the labeled biomolecules of the present application. In some embodiments, the method comprises: (i) reacting a compound of formula Q 2 The monomeric or oligomeric biomolecule containing -OH or its salt is contacted with the compound of formula (II) or its salt to obtain a conjugate of the following formula:

[0016]

[0017] In some embodiments, the method further comprises: (ii) in a sufficient amount to cleave P 1 The conjugate formed in step (i) is deprotected under conditions in which the protecting group is removed and the conjugate of the formula:

[0018]

[0019] In some embodiments, the method further comprises: (iii) reacting the conjugate formed in step (ii) with a compound of formula Q under conditions sufficient to promote conjugation. 1 -OR 1 The monomeric or oligomeric biomolecule or a salt thereof is contacted to obtain a labeled biomolecule of formula (I).

[0020] As described below, details of certain embodiments of the invention are set forth in the detailed description of certain embodiments. Other features, objects and advantages of the invention will be apparent from the definitions, embodiments, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0022] Figures 1A-1G Various examples of labeled biomolecules of the present application are shown. Figure 1A A labeled biomolecule with an internal label is shown. Figure 1B A labeled biomolecule with two internal labels is shown. Figure 1C The circularized labeled biomolecule is shown. Figure 1D A labeled oligonucleotide is shown that includes an internally labeled strand that is hybridized to an unlabeled strand. Figure 1E A labeled oligonucleotide is shown that includes one internally labeled strand hybridized to another internally labeled strand. Figure 1F A labeled oligonucleotide is shown comprising an unlabeled strand hybridized to a labeled strand having two internal labels. Figure 1G A labeled oligonucleotide strand is shown which self-hybridizes to form a stem-loop motif.

[0023] Figures 2A-2C Examples of uses of labeled biomolecules of the present application are depicted. Figure 2A Internally labeled oligonucleotides that hybridize to a target nucleic acid sequence are described. Figure 2B An internally labeled biomolecule that binds to a target protein is described. Figure 2C Internally labeled antibodies that bind to target proteins are described.

[0024] Figure 3A-3B Examples of labeled biomolecules of the present application that are modified with functional moieties are described. Figure 3A Depicted are internally labeled oligonucleotides modified with a quenching moiety that prevents detection of the internal label unless cleaved from the biomolecule. Figure 3B Depicted are internally labeled biomolecules modified with a ligand that binds to a target protein, wherein binding of the ligand to the target protein allows detection of the internal label.

[0025] Figures 4A-4C Examples of labeled nucleotides bound to polymerases according to the present application are described. Figure 4A A polymerase is described that is bound to nucleotides that include an external label. Figure 4B A polymerase enzyme is described that is bound to nucleotides that include externally labeled biomolecules. Figure 4C A polymerase is described that is coupled to nucleotides that include internally labeled oligonucleotides.

[0026] Figure 5A-5B Shown is a comparison of sequencing analysis of externally labeled biomolecules and internally labeled biomolecules according to the present application. Figure 5A A set of externally labeled nucleotides and internally labeled nucleotides are generally depicted as they were prepared and further analyzed. Figure 5B Shown are the results of single-molecule sequencing reactions using externally labeled nucleotides and internally labeled nucleotides.

[0027] Figure 6 An alignment of the excitation and emission spectra of externally labeled nucleotides and internally labeled nucleotides is shown. DETAILED DESCRIPTION

[0028] In other aspects, the present disclosure provides labeled biomolecules, the labeled biomolecules comprising internally conjugated luminescent labels (e.g., internal labels). In some embodiments, the internal labels are configured with enhanced conformational constraints, thereby limiting rotation and blocking deactivation pathways that shorten the luminescence lifetime. In some embodiments, the labeled biomolecules are configured to provide a rigid molecular scaffold for the internal labels, thereby avoiding label-label interactions and other quenching effects that may reduce luminescence intensity or other emission characteristics.

[0029] Without wishing to be bound by any particular theory, the labeled biomolecules provided herein have many significant advantages, such as increased quantum yield and extended luminescence lifetime, increased luminescence intensity and / or brightness, and reduced exposure to a large number of solvent molecules, thereby limiting the formation of reactive species. Therefore, in some embodiments, labeled biomolecules and methods of use thereof are provided herein. In some embodiments, the present disclosure provides compositions and methods related to the preparation of labeled biomolecules. In some embodiments, the present disclosure provides compositions and methods related to the preparation of dual conjugated labels.

[0030] Internally labeled biomolecules

[0031] One aspect of the present invention relates to internally labeled biomolecules ("labeled biomolecules"). As described herein, labeled biomolecules (e.g., oligonucleotides, nucleic acids, polypeptides, proteins, polysaccharides) include internally conjugated luminescent compounds ("luminescent labels" or "labels", such as polycyclic fluorophores). "Internally labeled" or "internal conjugated" are used interchangeably herein to refer to a portion of the biomolecule conjugated to a first site of the luminescent compound and another portion of the biomolecule conjugated to a second site of the luminescent compound. Internal conjugation of luminescent compounds has multiple advantages. For example, conjugating a label to the interior of a biomolecule can change the lifetime and other photophysical properties of the label (e.g., by limiting the rotation of the label). As another example, internally conjugating a label can immobilize the label, separating it from other labels, thereby reducing self-quenching. In addition, internally conjugating a label may limit the label from contacting a large amount of solvent in the solution, thereby reducing the formation of free radicals, which may damage other components in the solution. Internal incorporation of labels has other advantages described herein. A set of non-limiting examples of labeled biomolecules is shown in Figures 1A-1G .

[0032] Figures 1A-1G Various configurations of labeled biomolecules of the present application are generally described. Each example is shown with an internal label and biomolecule (shown as a dot shape). Figure 1A is a labeled biomolecule that includes an internal label that conjugates one portion of the biomolecule to another portion of the biomolecule. In some embodiments, the labeled biomolecule includes two or more internal labels.

[0033] Figure 1B It is a labeled biomolecule comprising two internal labels. As shown, one label conjugates a first portion of the biomolecule to a second portion of the biomolecule, and another label conjugates the second portion of the biomolecule to a third portion of the biomolecule. In some embodiments, the luminescence intensity and / or brightness of a labeled biomolecule having two or more copies of the same internal label increases relative to a biomolecule having one copy of the internal label. In some embodiments, a labeled biomolecule having two or more different types of internal labels can provide two or more unique detectable signals. Examples of various configurations and uses of multi-labeled biomolecules are described elsewhere herein.

[0034] According to the present application, it has been demonstrated that increased rigidity of internally conjugated labels can enhance one or more luminescent properties of the labeled biomolecules, such as conformational constraints provided by the polycyclic fluorophores of the internal labels. Advantageously, the biomolecular scaffolds to which the internal labels are conjugated can be engineered to further promote rigidity and further enhance one or more luminescent properties in these systems.

[0035] Figure 1C Is a labeled biomolecule comprising an internal label conjugated to a cyclized biomolecule. As described in this example, in some embodiments, the internal label conjugates one end of the biomolecule to the other end of the biomolecule to cyclize the labeled biomolecule. Without wishing to be bound by any particular theory, it is believed that cyclization can promote the structural rigidity of the entire biomolecule backbone, which enhances the favorable luminescent properties of the internal labels provided herein. Examples of cyclic biomolecules include, but are not limited to, cyclic peptides, cyclic proteins, and cyclic nucleic acids (e.g., circular RNA, DNA plasmids).

[0036] The inventors have also recognized and appreciated that oligonucleotides (e.g., polynucleotides, nucleic acids) provide a rigid, highly tunable biomolecular scaffold for the internal markers of the present application. Various examples of internally labeled oligonucleotides are shown in Figure 1D-1G These exemplary constructs and other embodiments involving internally labeled oligonucleotides are described in detail elsewhere herein.

[0037] As in Figures 1A-1G As generally illustrated in the example structure shown, in some embodiments, as described herein, the internal marker 100 corresponds to A of formula (I). In some embodiments, as described herein, the biomolecule of the example structure (shown as a dot shape) corresponds to Q of formula (I) 1 and / or Q 2 In some embodiments, as described herein, internal marker 100 corresponds to A, and the biomolecule of the exemplary structure (shown as a dotted shape) corresponds to Q of formula (I) 1 and Q 2 .

[0038] In one aspect, provided herein are labeled biomolecules of formula (I):

[0039]

[0040] in:

[0041] Q 1 and Q 2 independently a monomeric or oligomeric biomolecule;

[0042] A is a polycyclic fluorophore; and

[0043] L 1 and L 2 is independently a linker selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof.

[0044] As stated in this article, (also described herein as "A") is a luminescent compound or dye. As shown in formula (I), A is composed of L 1 The linker represented by the part of the biomolecule (Q 1 Group) conjugated, and through the linker L 2 Another part of the biomolecule (Q 2 Q 1 and Q 2 together to form a biomolecule that is –OL 1 -AL 2 -O – The structure is broken and thus marked internally.

[0045] In some embodiments, A is a polycyclic fluorophore. In some embodiments, incorporation of a polycyclic fluorophore is advantageous because the polycyclic structure can provide greater rigidity to the system compared to linear or non-polycyclic fluorophores. 1 and L 2 Directly linked (e.g., by covalent bonds) to one or more rings (e.g., benzene or heteroaromatic rings) of a polycyclic structure. Such direct linkage can also impart greater rigidity to the system (e.g., by immobilization / restricted rotation of the label). In certain embodiments, L 1 and L 2 Direct attachment to different rings on A, this design feature can also provide greater rigidity to the system and / or aid in dye fixation.

[0046] In certain embodiments, A is a polycyclic cyanine, fluorone, acridine, phenoxazine, coumarin, or boron dipyrromethene (BODIPY) fluorophore. In certain embodiments, A is a porphyrin, phthalocyanine, or naphthalimide. These are non-limiting examples. In certain embodiments, A is a polycyclic cyanine fluorophore. In certain embodiments, A is a polycyclic fluorone fluorophore. In certain embodiments, A is a polycyclic acridine fluorophore. In certain embodiments, A is a polycyclic phenoxazine fluorophore. In certain embodiments, A is a polycyclic coumarin fluorophore. In certain embodiments, A is a polycyclic BODIPY fluorophore. In certain embodiments, A is a porphyrin. In certain embodiments, A is a phthalocyanine. In certain embodiments, A is a naphthalimide. Other embodiments of Ring A are described below and herein.

[0047] In certain embodiments, A is a polycyclic cyanine fluorophore. In certain embodiments, A is an optionally substituted Cy3B dye. In certain embodiments, A is Cy3B. In certain embodiments, A has the formula:

[0048]

[0049] Where X - is a counterion or is absent; and wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0050] In certain embodiments, A has the formula:

[0051]

[0052] Where X - is a counterion or is absent; and wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0053] Thus, in certain embodiments, the labeled biomolecule has the formula:

[0054]

[0055] Where X - is a counterion or is absent.

[0056] In certain embodiments, the labeled biomolecule has the formula:

[0057]

[0058] wherein n is independently an integer from 1 to 20, inclusive.

[0059] In certain embodiments, the labeled biomolecule has the formula:

[0060]

[0061] In certain embodiments, the labeled biomolecule has the formula:

[0062]

[0063] As described herein, in certain embodiments, ring A is a polycyclic fluorone fluorophore (e.g., fluorescein or rhodamine). In certain embodiments, A is a fluorone dye. In certain embodiments, A is a rhodamine dye. In certain embodiments, A has the formula:

[0064]

[0065] Wherein each R is independently hydrogen, halogen, –N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or –N(R N ) 2 In certain embodiments, the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0066] As defined herein, each R is independently hydrogen, halogen, -N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or –N(R N ) 2 In certain embodiments, R is hydrogen. In certain embodiments, R is halogen. In certain embodiments, R is -N 3 In certain embodiments, R is -CN. In certain embodiments, R is -NO 2 In certain embodiments, R is optionally substituted alkyl. In certain embodiments, R is optionally substituted alkenyl. In certain embodiments, R is optionally substituted alkynyl. In certain embodiments, R is optionally substituted carbocyclyl. In certain embodiments, R is optionally substituted aryl. In certain embodiments, R is optionally substituted heterocyclyl. In certain embodiments, R is optionally substituted heteroaryl. In certain embodiments, R is -ORO In certain embodiments, R is -SR S In certain embodiments, R is -N(R N ) 2 In certain embodiments, R is optionally substituted C 1-6 In certain embodiments, R is unsubstituted C 1-6 In certain embodiments, R is an optionally substituted C 1-3 In certain embodiments, R is unsubstituted C 1-3 In certain embodiments, R is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. In certain embodiments, R is methyl. In certain embodiments, each R is methyl.

[0067] In certain embodiments, A has the formula:

[0068]

[0069] Wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0070] In certain embodiments, A has the formula:

[0071]

[0072] Wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0073] In certain embodiments, A has the formula:

[0074]

[0075] Wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted.

[0076] Thus, in certain embodiments, the labeled biomolecule has the formula:

[0077]

[0078] In certain embodiments, the labeled biomolecule has the formula:

[0079]

[0080] in:

[0081] n is independently an integer from 1 to 20, inclusive; and

[0082] RN Each instance of is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group.

[0083] In certain embodiments, the labeled biomolecule has the formula:

[0084]

[0085] In certain embodiments, the labeled biomolecule has the formula:

[0086]

[0087] In certain embodiments, the labeled biomolecule has the formula:

[0088]

[0089] As described herein, in certain embodiments, A is a boron dipyrromethene (BODIPY) fluorophore. In certain embodiments, A has the following formula:

[0090]

[0091] wherein the structure is optionally substituted at any position.

[0092] In certain embodiments, A has the formula:

[0093]

[0094] wherein Ar is optionally substituted aryl or optionally substituted heteroaryl; and wherein the structure is optionally substituted at any position.

[0095] As defined herein, Ar is an optionally substituted aryl or an optionally substituted heteroaryl. In certain embodiments, Ar is an optionally substituted aryl. In certain embodiments, Ar is an optionally substituted heteroaryl. In certain embodiments, Ar is an optionally substituted phenyl. In certain embodiments, A is a polyfluorophenyl. In certain embodiments, Ar has the formula: In certain embodiments, Ar has the formula: In certain embodiments, Ar has the formula: In certain embodiments, Ar has the formula: wherein m is as defined herein.

[0096] As defined herein, R Ar is hydrogen, halogen, –N 3 、–CN、–NO2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or –N(R N ) 2 In certain embodiments, R Ar is hydrogen. In certain embodiments, R Ar is a halogen (—Cl, —I, —Br, —F). In certain embodiments, R Ar Yes – N 3 In certain embodiments, R Ar is -CN. In certain embodiments, R Ar Yes–No 2 In certain embodiments, R Ar is an optionally substituted alkyl. Ar is an optionally substituted alkenyl. In certain embodiments, R Ar is an optionally substituted alkynyl. Ar is an optionally substituted carbocyclyl. In certain embodiments, R Ar is an optionally substituted aryl. In certain embodiments, R Ar is an optionally substituted heterocyclyl. In certain embodiments, R Ar is an optionally substituted heteroaryl. In certain embodiments, R Ar Yes – OR O In certain embodiments, R Ar Yes – SR S In certain embodiments, R Ar Yes or –N(R N ) 2 In certain embodiments, R Ar is -F. In certain embodiments, R Ar Yes – S(CH 2 CH 2 O) m OCH 3 , wherein m is as defined herein.

[0097] As defined herein, m is an integer from 1 to 6, inclusive.

[0098] In certain embodiments, A has the formula:

[0099]

[0100] Where R Ar is hydrogen, halogen, –N 3、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or –N(R N ) 2 In certain embodiments, the structure is substituted at any position. In certain embodiments, the structure is unsubstituted.

[0101] Thus, in certain embodiments, the labeled biomolecule has the formula:

[0102]

[0103] Where R Ar is hydrogen, halogen, –N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or N(R N ) 2 .

[0104] In certain embodiments, the labeled biomolecule has the formula:

[0105]

[0106] In certain embodiments, the labeled biomolecule has one of the following formulae:

[0107]

[0108] In certain embodiments, the labeled biomolecule has the formula:

[0109]

[0110] In certain embodiments, the labeled biomolecule has one of the following formulae:

[0111]

[0112] As described herein, in certain embodiments, A is an acridine fluorophore. In certain embodiments, A is an acridine fluorophore of the formula:

[0113]

[0114] wherein the structure is optionally substituted.

[0115] As described herein, in certain embodiments, A is a phenoxazine fluorophore. In certain embodiments, A is a phenoxazine fluorophore of the formula:

[0116]

[0117] wherein the structure is optionally substituted.

[0118] As described herein, in certain embodiments, A is a coumarin fluorophore. In certain embodiments, A is a coumarin fluorophore of the formula:

[0119]

[0120] wherein the structure is optionally substituted. In certain embodiments, A is a coumarin fluorophore of the formula:

[0121]

[0122] wherein the structure is optionally substituted.

[0123] As defined herein, L 1 and L 2 is independently a linker selected from the group consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof. In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 In certain embodiments, L 1 is an optionally substituted C 1-20 In certain embodiments, L 1 is an optionally substituted C 1-10In certain embodiments, L 1 is an optionally substituted C 1-6 In certain embodiments, L 1 With the formula: wherein n is as defined herein. In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula: In certain embodiments, L 1 With the formula:

[0124] In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 In certain embodiments, L 2 is an optionally substituted C 1-20In certain embodiments, L 2 is an optionally substituted C 1-10 In certain embodiments, L 2 is an optionally substituted C 1-6 In certain embodiments, L 2 With the formula: wherein n is as defined herein. In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula: In certain embodiments, L 2 With the formula:

[0125] As defined herein, n is independently an integer from 1 to 20, inclusive. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, n is 7. In certain embodiments, n is 8. In certain embodiments, n is 9. In certain embodiments, n is 10. In certain embodiments, n is 11. In certain embodiments, n is 12. In certain embodiments, n is 13. In certain embodiments, n is 14. In certain embodiments, n is 15. In certain embodiments, n is 16. In certain embodiments, n is 17. In certain embodiments, n is 18. In certain embodiments, n is 19. In certain embodiments, n is 20.

[0126] As described in this article, Q 1 and Q2 are independently monomeric or oligomeric biomolecules. As described herein, Q 1 and Q 2 together to form oligomeric or polymeric biomolecules, which are 1 -AL 2 -O–, thereby being internally labeled (e.g., forming a labeled biomolecule).

[0127] In some embodiments, the labeled biomolecule is an oligomeric or polymeric biomolecule comprising at least 5 monomeric biomolecules (e.g., at least 5 nucleotides, at least 5 amino acids, at least 5 monosaccharides). In some embodiments, the oligomeric or polymeric biomolecule comprises at least 10 monomeric biomolecules. In some embodiments, the oligomeric or polymeric biomolecule comprises at least 10 and less than 200 monomeric biomolecules. For example, in some embodiments, the oligomeric or polymeric biomolecule comprises at least 10 and less than 150 monomeric biomolecules, at least 10 and less than 100 monomeric biomolecules, at least 10 and less than 50 monomeric biomolecules, at least 10 and less than 40 monomeric biomolecules, at least 10 and less than 30 monomeric biomolecules, or at least 10 and less than 20 monomeric biomolecules.

[0128] In certain embodiments, the labeled biomolecule is an oligonucleotide or a nucleic acid.

[0129] In certain embodiments, Q 1 and Q 2 is independently a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 is independently a nucleoside or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 is independently a nucleotide derivative or a fragment thereof. In certain embodiments, Q 1 and Q 2 is independently an oligonucleotide or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 are independently nucleic acids or derivatives or fragments thereof.

[0130] In certain embodiments, Q 1 and Q 2 are independently deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, locked nucleic acid, or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 is independently a deoxyribonucleic acid or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2is independently a ribonucleic acid or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 is independently a peptide nucleic acid or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 It is a locked nucleic acid or a derivative or fragment thereof.

[0131] As described in this article, Q 1 and Q 2 together to form oligomeric or polymeric biomolecules, which are 1 -AL 2 -O- is interrupted, thereby being labeled internally (e.g., forming a labeled biomolecule). In certain embodiments, the labeled biomolecule is a single-stranded nucleic acid. In this case, the single-stranded nucleic acid includes a first oligonucleotide strand (Q 1 and / or Q 2 In some embodiments, the labeled biomolecule includes a second oligonucleotide chain hybridized to the first oligonucleotide chain. For example, in some embodiments, the second oligonucleotide chain is hybridized to Q 1 and / or Q 2 Hybridization. A visual representation of this internal labeling system is as follows (where ---- represents a hybridization interaction (e.g., one or more Watson-Crick base interactions)):

[0132]

[0133] A visual representation of the system can also be found in Figure 1D .

[0134] Other examples of internally labeled oligonucleotides are shown in Figure 1E-1G . As shown in these and other examples described herein, various oligonucleotide chain hybridization strategies are provided to promote rigidity and / or shield internal markers from the influence of a large amount of solvents. Accordingly, oligonucleotide chain hybridization can be used as a general design strategy for preparing the labeled oligonucleotides of the application. In some embodiments, oligonucleotide chain hybridization relates to self-chain hybridization (e.g., self-hybridization in a single strand). In some embodiments, oligonucleotide chain hybridization relates to the hybridization of different oligonucleotide chains.

[0135] Figure 1DIt is a labeled oligonucleotide, which includes an internally labeled oligonucleotide chain, which is hybridized with an unlabeled oligonucleotide chain. In some embodiments, an unlabeled oligonucleotide chain is used to increase the rigidity of a specific region (e.g., a region comprising an internal marker) of a labeled oligonucleotide chain. In some embodiments, an unlabeled oligonucleotide chain is hybridized with an internally labeled oligonucleotide chain provided herein, and the internally labeled oligonucleotide chain comprises two or more internal markers.

[0136] Figure 1E It is a labeled oligonucleotide, which includes an internally labeled oligonucleotide chain, which is hybridized with another internally labeled oligonucleotide chain. In some embodiments, the internal label of an oligonucleotide chain includes a fluorophore identical to the internal label of another oligonucleotide chain. In some embodiments, the internal label of an oligonucleotide chain includes a fluorophore different from the internal label of another oligonucleotide chain. In some embodiments, one of the internally labeled oligonucleotide chains includes two or more internal labels of the present application. In some embodiments, both internally labeled oligonucleotide chains include two or more internal labels of the present application.

[0137] In some embodiments, oligonucleotide strand hybridization can promote the formation of one or more structural motifs, such as stem loops, knots, pseudoknots and double helices. According to the present application, structural motifs can be used to enhance the rigidity of labeled oligonucleotides and / or limit the extent of exposure of internal labels to bulk solvents. Figure 1F-1G Examples of labeled oligonucleotides having higher order structural motifs formed by strand hybridization are described.

[0138] Figure 1F It is a labeled oligonucleotide, which comprises an unlabeled oligonucleotide chain, which hybridizes with an oligonucleotide chain comprising two internal markers. As summarized in this example, the formation of a double helix can promote the separation of two internal markers of the same oligonucleotide chain. In some embodiments, the oligonucleotide chain of hybridization forms a double helix, and every turn has about 10 to 12 base pairs.

[0139] Therefore, in some embodiments, when two internal labels of the same oligonucleotide chain occupy about the same space as one nucleotide in the chain, the internal labels are separated by 5 to 6 nucleotides at least along the chain, so that the labels are on the approximately opposite sides of the double helix. In some embodiments, the internal labels are separated by 4 to 8 (e.g., 4, 5, 6, 7 or 8) nucleotides along the oligonucleotide chain. Without wishing to be bound by theory, since the intermediate helical structure absorbs all radiation and / or non-radiative decay, such a design strategy can be utilized to limit the degree of label-label interaction (e.g., quenching effect).

[0140] In some embodiments, internal marker of the present disclosure is integrated into the oligonucleotide backbone, so that the degree of marker-marker interaction is minimized.Therefore, in some embodiments, internal marker is separated by 1 to 3 (for example 1,2 or 3) or 9 to 13 (for example 9,10,11,12 or 13) nucleotide along the same oligonucleotide chain.As shown in this example, can design the oligonucleotide with label by considering the spiral structure of prediction or known, thus can manipulate an internal marker with respect to another internal marker through the relative position of space, to reach required application.Other examples of the structural motif that can be used when designing the oligonucleotide with label are known in the present technology, and are described herein.

[0141] Figure 1G It is an internally labeled oligonucleotide chain, which self-hybridizes to form a stem-loop motif. Stem-loop, or hairpin loop, is an unpaired nucleotide loop on an oligonucleotide chain, formed when the oligonucleotide chain folds and forms a base pair with another section of the same chain. In some embodiments, the unpaired loop of the stem-loop includes 3 to 10 nucleotides. Therefore, the stem-loop can be formed by two regions of an oligonucleotide chain with a reverse complementary sequence, wherein the reverse complementary sequence hybridizes to form a stem, wherein the two regions are separated by 3 to 10 nucleotides that form an unpaired loop. In some embodiments, the stem can be designed to have one or more G / C nucleotides, and compared with A / T / U nucleotides, G / C nucleotides can provide additional stability by forming additional hydrogen bonds. In some embodiments, the stem includes the G / C nucleotides next to the unpaired loop sequence. In some embodiments, the stem includes the G / C nucleotides in the first 2,3,4 or 5 nucleotides adjacent to the unpaired loop sequence.

[0142] As described herein, in some embodiments, an internal label conjugates one portion of an oligonucleotide chain to another portion of an oligonucleotide chain. Figure 1D-1GIn some embodiments, both portions of the oligonucleotide chain conjugated by the internal label are hybridized to the same oligonucleotide chain. Thus, the internal label can be adjacent to one or more (e.g., 1, 2, 3, 4, 5, or more) unpaired bases of the hybridized oligonucleotide chain.

[0143] In some embodiments, the internal marker can interact with the guanine nucleobase through radioactivity and / or non-radiative decay to affect the weakened luminescence lifetime. In some embodiments, one or more unpaired bases adjacent to the internal marker are designed to exclude guanine or minimize guanine. In some embodiments, the region around the internal conjugated marker is designed to exclude G / C content or minimize G / C. In some embodiments, the internal conjugated marker is at least separated from the G or C nucleotides on the oligonucleotide chain by 2 nucleotides (for example, separated from the G or C nucleotides by 2, 3, 4, 5, 6, 7, 8, 9 or more than 10 nucleotides). Therefore, in some embodiments, each internal marker has at least 2 continuous nucleotides selected from A or T / U on both sides.

[0144] The labels provided herein also have applications in systems other than oligonucleotides and nucleic acids. In certain embodiments, the labeled biomolecule is a polypeptide or protein. For example, in certain embodiments, Q 1 and Q 2 are independently amino acids, oligopeptides, polypeptides, proteins or fragments thereof. In certain embodiments, Q 1 and Q 2 are independently amino acids. In certain embodiments, Q 1 and Q 2 is independently an oligopeptide or a fragment thereof. 1 and Q 2 is independently a polypeptide or protein or a fragment thereof. In certain embodiments, Q 1 and Q 2 Connected together to form cyclic peptides or cyclic proteins.

[0145] Non-limiting examples of oligopeptides and polypeptides suitable for use as labeled biomolecules of the present application include, but are not limited to, oligopeptides, cyclic peptides, and small proteins (e.g., mini-proteins based on avian pancreatic peptides, such as described in Hodges, AM and Schepartz, A. (2007) J. Am. Chem. Soc. 129: 11024-11025). Methods for engineering structural constraints in polypeptides are well known in the art and are considered to be particularly useful, for example, to impart rigidity and enhance one or more luminescent properties described herein. For example, the proline content of a peptide amino acid sequence can be modified to control the shape of the peptide and impart rigidity (see, e.g., Kritzer, JA, et al. (2006) ChemBioChem 7: 29-31). Other non-limiting examples of peptide engineering techniques that can be used include peptide cyclization (see, e.g., Maltsev, OV, et al. (2016) Angewandte Chemie 55(4): 1535-1539), α-helical peptide constraints by stapling and / or H-bond surrogates (see, e.g., Douse, CH, et al. (2014) ACS Chem. Biol. 9: 2204-2209), peptide constraints by cyclic β-sheets and β-hairpin mimetics (see, e.g., Gibbs, AC, et al. (1998) Nat. Struc. Biol. 5: 284-288).

[0146] In certain embodiments, the labeled biomolecule is an oligosaccharide or a polysaccharide. For example, in certain embodiments, Q 1 and Q 2 are independently monosaccharides, oligosaccharides, polysaccharides or fragments thereof. 1 and Q 2 In certain embodiments, Q 1 and Q 2 is independently an oligosaccharide or a fragment thereof. 1 and Q 2 are independently polysaccharides or fragments thereof. Examples of oligosaccharides and polysaccharides suitable for use as labeled biomolecules in the present application are known in the art (eg, as described in Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries, Wiley 2001).

[0147] According to the present application, the labeled biomolecules provided herein include biomolecules that act as rigid scaffolds onto which the internal labels of the present application are incorporated. In some embodiments, the biomolecules contain one or more features that confer additional functionality that can be used in various detection, quantitative analysis, and imaging methods. For example, FIG. 2A to FIG. 2C Non-limiting examples of internally labeled biomolecules are shown, including biomolecules that interact with target molecules of interest.

[0148] In some embodiments, labeled biomolecules including oligonucleotides can be used as hybridization probes. Hybridization probes are labeled fragments of DNA or RNA (e.g., oligonucleotides) that can be added to a sample of known or unknown content to detect the presence of a target nucleic acid that is complementary to the hybridization probe. For example, Figure 2A Depicted is an internally labeled hybridization probe 200 hybridized to a target nucleic acid 210. As generally illustrated in this example, the internally labeled hybridization probe 200 forms base pairing interactions with the target nucleic acid 210, resulting in an increase in detectable luminescence from the internal label. However, in some embodiments, hybridization of the internally labeled hybridization probe 200 to the target nucleic acid 210 results in a decrease in detectable luminescence.

[0149] In some embodiments, the internally labeled hybridization probe of the present application comprises a sequence substantially complementary to the target nucleic acid sequence, so that the probe and the target form base pairing interactions under hybridization conditions. As described herein, the internal label conjugates a portion of a biomolecule to another portion of a biomolecule. Therefore, one or two portions of a biomolecule (e.g., an oligonucleotide) conjugated by an internal label can be designed to hybridize with a target nucleic acid sequence. In some embodiments, the target nucleic acid comprises RNA (e.g., mRNA). In some embodiments, the target nucleic acid comprises DNA (e.g., cDNA, genomic DNA, or a fragment thereof).

[0150] The internally labeled hybridization probe of the present application can be used in any method utilizing hybridization probe known in the art. Examples of such techniques include, but are not limited to, fluorescence in situ hybridization (FISH), Northern blotting, Southern blotting, and general technical analysis and clinical diagnostic analysis involving SNP detection, real-time nucleic acid detection, real-time PCR quantification, allele discrimination and identification, multiplex PCR.

[0151] The labeled biomolecules of the present application, in some embodiments, include biomolecules that act as protein ligands. For example, Figure 2BDepicted is a labeled biomolecule 201 bound by a target protein 211. As generally illustrated in this example, the target protein 211 associates (e.g., binds) with at least a portion of the biomolecules (shown as dashed lines) of the labeled biomolecule 201, resulting in an increase in detectable luminescence from the internal label. However, in some embodiments, the binding of the target protein 211 to the labeled biomolecule 201 results in a decrease in detectable luminescence.

[0152] In some embodiments, the labeled biomolecule 201 and the target protein 211 comprise a known binding pair. In some embodiments, the labeled biomolecule 201 can be added to a sample of known or unknown content to detect the presence of the target protein 211. In some embodiments, the target protein 211 is a receptor and the labeled biomolecule 201 comprises a receptor ligand. In some embodiments, the target protein 211 is an antibody specific to at least a portion of the labeled biomolecule 201. In some embodiments, the target protein 211 is an antibody and the labeled biomolecule 201 comprises an antigen. In some embodiments, the target protein 211 is a nucleic acid binding protein (e.g., a DNA binding protein) and the labeled biomolecule 201 comprises a nucleic acid. It is predicted that such labeled biomolecules are useful in methodologies (e.g., fluorescence polarization and other techniques known in the art or described herein) that employ labeled protein ligands to detect the presence of a target protein or to assess protein-ligand binding interactions.

[0153] In some embodiments, labeled biomolecules comprising polypeptides may be used as antibodies. Figure 2C An internally labeled antibody 202 is depicted that is bound to a target protein 212. As generally described in this example, the internally labeled antibody 202 includes a Fab region that is configured to specifically bind to the target protein 212, resulting in an increase in detectable luminescence from the internal label. However, in some embodiments, binding of the internally labeled antibody 202 to the target protein 212 results in a decrease in detectable luminescence.

[0154] The antibody of the internal labeling of the application can be used in the known method utilizing luminescent labeling antibody in the art.The example of such technology includes but is not limited to fluorescence in situ hybridization (FISH), Western blot, relates to the general method of immunolabeling such as immunocytochemistry and immunohistochemistry technique and other technology known in the technology or described herein.

[0155] As described above, the internal label of the present application can be conjugated with a biomolecule that interacts with a desired target molecule. In some embodiments, the internal label is conjugated with a protein that associates (e.g., binds) with a target ligand. In some embodiments, the protein is an antibody or an antigen-binding portion of an antibody. In some embodiments, the protein is an enzyme, such as a peptidase (e.g., an exopeptidase or an endopeptidase), a ribozyme, an aptamer (aptazyme), a ligase, a transferase, or a tRNA synthetase. In some embodiments, the internal label is conjugated with a nucleic acid that associates (e.g., binds) with a target ligand. In some embodiments, the nucleic acid is a nucleic acid aptamer (e.g., a DNA aptamer, an RNA aptamer, or a derivative or analog thereof).

[0156] In some embodiments, the labeled biomolecule comprises a biomolecule modified with one or more functional moieties. For example, Figure 3A-3B Non-limiting examples of labeled biomolecules comprising moieties that interact with target molecules and / or internal markers are shown.

[0157] According to the present application, the biomolecule scaffold provides a rigid marker scaffold, which is particularly beneficial for techniques that require a firmly defined position of internal markers. For example, Resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for studying conformational dynamics of biomolecules in vitro and in vivo. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore by either a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, such as used in photoinduced electron transfer (PET) FCS.

[0158] In some embodiments, the labeled biomolecule comprises one or more quenching moieties (e.g., fluorescent and / or non-fluorescent quenching moieties) that interact with the internal label of the labeled biomolecule. In some embodiments, such moieties can be used in real-time PCR, where the position of the internal label is well defined relative to the quencher cleaved by the exonuclease activity. An example of this process is shown in Figure 3A .

[0159] As shown in Figure 1, an internally labeled hybridization probe is hybridized to a target nucleic acid, and the internally labeled hybridization probe comprises a quenching portion 300. In some embodiments, the quenching portion 300 is a non-fluorescent quencher that absorbs the emission light from the internal label. In some embodiments, the quenching portion 300 is a fluorescent quencher that absorbs the emission light of one wavelength from the internal label and emits at another wavelength.

[0160] As shown in FIG. II, the quenching portion 300 has been cleaved from the internally labeled hybridization probe (e.g., by an exonuclease). This separation of the quenching portion 300 from the internal label can eliminate the distance-dependent quenching effect, thereby allowing the luminescence from the internal label to be detected. It should be understood that in some embodiments, the internally labeled hybridization probe includes an internal label that serves as a quencher for another label of the hybridization probe.

[0161] Hybridization probes modified by quenching moieties are known in the art and are contemplated to be used with the internal labels of the present application. Examples of such hybridization probes include, but are not limited to, molecular beacons, TaqMan probes, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHO) probes, and cyclic probe technology (CPT) probes.

[0162] Therefore, in some embodiments, multiple internal markers (e.g., two, three, four, five or more internal markers) can be incorporated into biomolecules according to the desired luminescent properties of the labeled biomolecules provided herein. For example, in some embodiments, the luminescent intensity and / or brightness of the labeled biomolecules with two or more internal markers is increased relative to the biomolecules with one internal marker. In some embodiments, the two or more internal markers are configured to provide independent reporter signals. In some embodiments, the two or more internal markers are configured to provide related reporter signals (e.g., donor markers and acceptor markers for FRET).

[0163] In some aspects, the application provides internal markers configured for conventional solid phase synthesis techniques, such as phosphoramidite analogs that can be used for oligonucleotide synthesis. Therefore, in some embodiments, the markers provided herein can be easily incorporated into biomolecules to produce labeled biomolecules with a certain number of internal markers, and the number of internal markers is only limited by the required biomolecule size. For example, in some embodiments, the labeled biomolecule comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) internal markers. In some embodiments, the labeled biomolecule includes 2 to 5, 2 to 10, 5 to 10, 5 to 15, 10 to 15, 15 to 20 or more internal markers.

[0164] In some embodiments, the labeled biomolecules provided herein further include one or more luminescent labels in addition to the polycyclic fluorophores of formula (I). For example, in some embodiments, the labeled biomolecules include at least one internal label according to formula (I), and one or more internal labels, the one or more internal labels including linear or non-polycyclic fluorophores. In some embodiments, the labeled biomolecules include at least one internal label according to formula (I), and one or more external labels.

[0165] In some embodiments, an external label refers to a label (e.g., a fluorophore) conjugated to a single site of a labeled biomolecule provided herein. In some embodiments, the external label is conjugated to the end of the labeled biomolecule. For example, in some embodiments, the external label is conjugated to the 5' or 3' end of a labeled oligonucleotide. In some embodiments, the external label is conjugated to the N- or C-terminus of a labeled polypeptide. In some embodiments, the external label is conjugated to the labeled biomolecule on the terminal monomer of the biomolecule (e.g., conjugated to the base of the terminal nucleotide in the oligonucleotide chain, conjugated to the side chain of the terminal amino acid in the polypeptide chain).

[0166] In some embodiments, the external label is conjugated to the labeled biomolecule at a non-terminal site of the biomolecule. In some embodiments, the external label is conjugated to the labeled biomolecule at a site between monomers of the biomolecule. In some embodiments, the external label is conjugated to an abasic site of a labeled oligonucleotide. In some embodiments, the external label is conjugated to the labeled biomolecule on a non-terminal monomer of the biomolecule (e.g., conjugated to a base of a non-terminal nucleotide in an oligonucleotide chain, conjugated to a side chain of a non-terminal amino acid in a polypeptide chain).

[0167] In some embodiments, the labeled biomolecules of the present application include biomolecules modified with one or more moieties, wherein the one or more moieties act as protein ligands. For example, Figure 3B A method is described in which a labeled biomolecule comprising a ligand portion is detectably bound by a target protein. As shown in FIG. 1 , a target protein is exposed to a labeled biomolecule comprising a ligand portion 301 configured to bind to the target protein. In the absence of binding between the target protein and the ligand portion 301, the internal label of the labeled biomolecule does not emit a detectable signal.

[0168] As shown in FIG. II, the target protein associates (e.g., binds) with the ligand portion 301, resulting in an increase in detectable luminescence from the internal marker. However, in some embodiments, the binding of the target protein to the ligand portion 301 can result in a decrease in detectable luminescence. In some embodiments, the change in detectable luminescence upon binding is a result of the internal marker biomolecule being constrained to the observation area (e.g., fixed on the surface for a period of time sufficient to allow detection). In some embodiments, the change in luminescence upon binding is a result of a FRET interaction (e.g., an acceptor / donor conjugated to the target protein or surface).

[0169] In some embodiments, labeled biomolecules comprising one or more ligand moieties can be used, for example, for the purpose of immobilizing the labeled biomolecules to a surface or material, for detecting a target protein in a known or unknown sample, for detecting (e.g., quantifying) a binding interaction between a target protein and a ligand moiety that is known or unknown to bind to the target protein.

[0170] In certain embodiments, the labeled biomolecule is associated with a reagent that is configured to serve as a reaction substrate. For example, in certain embodiments, Q of Formula (I) 1 and Q 2 Independently optionally associated with a reactant configured to be used as a reaction substrate. For example, in the case of an oligonucleotide or nucleic acid system, the first and second oligonucleotide chains are independently optionally associated with a reactant configured to be used as a reaction substrate. In some embodiments, the reactant is configured to be used as a substrate for polymerization. In some embodiments, when under polymerization conditions, the reactant is cut off from the labeled biomolecule by a polymerase. For example, in some embodiments, the reactant is a nucleotide (e.g., for use in a nucleic acid sequencing method).

[0171] Labeled nucleotides

[0172] Also provided herein are labeled nucleotides comprising one or more nucleotides associated with a labeled biomolecule described herein. In some embodiments, the one or more nucleotides comprise a type of nucleotide selected from guanine, cytosine, adenine, and thymine or uracil. In some embodiments, the one or more nucleotides are cleaved from the labeled biomolecule by a polymerase when subjected to polymerization reaction conditions.

[0173] Without wishing to be bound by any particular theory, in addition to the advantages described elsewhere herein, the labeled nucleotides provided herein have many distinct advantages over those currently used in sequencing reactions, such as increased read length and increased accuracy. Figures 4A-4C Several features of the labeled nucleotides of the present disclosure are highlighted.

[0174] Figures 4A-4C Each of the nucleotides 400 depicts a nucleotide 400 bound to a polymerase 410. Figure 4A The nucleotide 400 is conjugated to a luminescent marker on the outside, and Figure 4B and 4C The nucleotides are associated with various labeled biomolecules described herein. Figure 4A As shown, the external conjugated label is relatively close to the polymerase, and the degree of proximity to a large number of solvent molecules is relatively high. In some embodiments, such characteristics are not conducive to polymerization. For example, in some embodiments, the shorter distance between the label and the polymerase can cause damage to the polymerase caused by the label through radioactive and / or non-radiative decay (as shown in path (i)). In certain embodiments, the higher the degree to which the label contacts a large number of solvent molecules, the higher the incidence of reactive oxygen species (ROS) formation can be caused. Once formed, ROS can destroy the polymerase, thereby adversely affecting the enzyme activity (shown as path (ii)).

[0175] Figure 4B Depicted are nucleotides associated with labeled biomolecules, such as labeled oligonucleotide chains. As shown relative to externally conjugated labels, the biomolecule increases the separation between the label and the polymerase. In addition, the biomolecule portion between the nucleotide and the internal label provides a protective barrier between the label and the polymerase. Therefore, due to the separation of the label-polymerase and / or due to the biomolecule absorbing all decays emitted from the label (shown as path (iii)), the occurrence of label-induced damage to the polymerase can be reduced.

[0176] In addition, relative to Figure 4A As shown, internal dyes are integrated into Figure 4B The exposure of the label to the bulk solvent molecules is reduced in the biomolecules shown in . Thus, the incidence of ROS formation is reduced due to the reduced access of the label to the bulk solvent, and / or due to the absorption of all ROS-induced damage by the biomolecule, and / or due to the decay of free radicals over the label-polymerase separation distance (as shown in path (iv)).

[0177] Figure 4C Depicts a nucleotide associated with a labeled oligonucleotide. As shown, the labeled oligonucleotide is hybridized to an unlabeled oligonucleotide strand. According to the present application, such a construct confers a high degree of rigidity, enhancing the ability to target Figure 4B The labeled nucleotides have each of the advantages described above.

[0178] For example, the hybridized strands provide increased rigidity, meaning less overall flexibility, which further facilitates label-polymerase separation. The hybridized strands also provide another barrier between the label and the polymerase that can absorb any label-induced decay (shown as path (v)). In addition, because the hybridized strands further limit the label's exposure to bulk solvent, and / or because the hybridized strands absorb any damage caused by ROS, and / or because free radicals decay as the label-polymerase separation distance increases, damage caused by ROS is further reduced.

[0179] Therefore, in each of the above examples, the advantages provided by the labeled biomolecules of the present application increase the read length in the sequencing reaction by limiting the extent of light-induced polymerase damage. In addition, for example, due to the enhancement of one or more emission characteristics, the labeled nucleotides of the present application provide improved accuracy.

[0180] It should be understood that in the context of a labeled biomolecule, a "nucleotide" or "polynucleoside" attached thereto refers to one or more nucleotides (e.g., nucleoside polyphosphates) configured to be incorporated into a growing nucleic acid chain (e.g., during a sequencing reaction). In some embodiments, the one or more nucleotides include one or more nucleoside monophosphates or nucleoside polyphosphates. In some embodiments, examples of polyphosphate nucleosides include: di- or tri-phosphate nucleosides, or nucleosides with more than three 5' phosphates, such as hexaphosphate nucleosides. In some embodiments of any composition or method described in the present application, the phosphate portion (e.g., polyphosphate portion) of the nucleotide includes one or more phosphates (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate groups) or variants thereof. For example, in some embodiments, the phosphate portion (e.g., polyphosphate portion) of the nucleotide may include phosphate, thioester, aminophosphoester, alkylphosphonate bond, other suitable bonds or multiple such modifications, or a combination of two or more thereof.

[0181] The labeled nucleotides may be terminal phosphate-labeled nucleotides such that the labeled biomolecules of the present application are linked to the terminal phosphate of the nucleotides. For example, in some embodiments, one or more nucleotides may be linked to a biomolecule (e.g., Q of Formula (I)) that forms part of a labeled biomolecule as described herein via a terminal phosphate. 1 and / or Q 2 ). Therefore, in some embodiments, the "labeled nucleotide" of the present application refers to a nucleotide connected to a labeled biomolecule of formula (I). In some embodiments, one or more nucleotides can be connected to an oligonucleotide (e.g., an unlabeled oligonucleotide chain) that forms a part of a labeled biomolecule as described in the present application through a terminal phosphate.

[0182] The biomolecule with label can be connected to the terminal phosphate of nucleotide by a linker. The linker may include, for example, at least one or more hydroxyl groups, sulfhydryl groups, amino groups or haloalkyl groups, which may be suitable for forming, for example, phosphate, thioester, phosphoramidate or alkylphosphonate bonds on the terminal phosphate of natural or modified nucleotides. The linker may be cleavable so that, for example, the marker is separated from the terminal phosphate by means of a polymerase. Examples of nucleotides and linkers are provided in U.S. Patent No. 7,041,812, which is incorporated herein by reference in its entirety. In some embodiments, the linker includes optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene and combinations thereof. Other examples of linkers that can be used to attach labels to nucleotides can be found in co-pending US Patent Application No. 15 / 600,979, the relevant portions of which are incorporated herein by reference in their entirety.

[0183] Nucleotide (for example, polyphosphate nucleoside) can include any one of adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or its variant. Nucleotide (for example, polyphosphate nucleoside) can include methylated nucleobase. For example, methylated nucleotide can be a nucleotide comprising one or more methyl groups connected to nucleobase (for example, directly connected to the ring of nucleobase, connected to the substituent of the ring of nucleobase). Exemplary methylated nucleobase includes 1-methylthymine, 1-methyluracil, 3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N6-methyladenine, N6, N6-dimethyladenine, 1-methylguanine, 7-methylguanine, N2-methylguanine and N2, N2-dimethylguanine.

[0184] As used herein, the term "nucleic acid" generally refers to a molecule comprising one or more nucleic acid subunits. Nucleic acid may include one or more subunits selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. In some examples, nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or derivatives thereof. In some embodiments, nucleic acid is a modified nucleic acid, including but not limited to locked nucleic acid (LNA), peptide nucleic acid (PNA), triazole-linked nucleic acid, 2'-F modified nucleic acid, and derivatives and analogs thereof. Nucleic acid can be single-stranded or double-stranded. In some embodiments, nucleic acid generally refers to any polymer of nucleotides.

[0185] Emission characteristics

[0186] As described herein, conjugating a label to the interior of a biomolecule can change the photophysical properties of the label (e.g., by restricting rotation and fixation of the label within the biomolecule). Thus, in certain embodiments, one or more emission characteristics of the labeled biomolecule are altered (e.g., increased) relative to an unconjugated molecule comprising the label. As described herein, a "non-conjugated molecule" comprising a label does not include Q 1 and Q 2 One or both of the emission characteristics changed by internal conjugation may include, but are not limited to, luminescence lifetime, luminescence intensity, brightness, maximum emission wavelength, luminescence quantum yield, and photostability.

[0187] In certain embodiments, the luminescence lifetime of the labeled biomolecule is increased relative to the unconjugated molecule. In certain embodiments, the luminescence lifetime of the labeled biomolecule is increased by at least 10% relative to the unconjugated molecule. In certain embodiments, the luminescence lifetime of the labeled biomolecule is increased by about 10% to 50% (e.g., about 10% to 25%, about 10% to 15%, about 25% to 50%, about 40% to 50%) relative to the unconjugated molecule. Examples of increased luminescence lifetime are shown in Figure 5A-5B .

[0188] according to Figure 5A A set of labeled biomolecules comprising nucleotides were prepared using constructs generally described in . Labeled nucleotides (1) and (2) represent conjugates in which the Cy3B dye is externally conjugated to a DNA linker at either the end or branch point of the oligonucleotide, respectively. In contrast, labeled nucleotide (3) exchanges one of the bases in the oligonucleotide chain for the Cy3B dye itself, thereby becoming internally integrated into the DNA backbone. These labeled nucleotides were used in sequencing experiments, and lifetime measurements were obtained for each conjugate. The externally conjugated Cy3B constructs (1) and (2) produced lifetimes of approximately 2.2 nanoseconds, while the internally conjugated Cy3B construct (3) had a measured lifetime of 2.6 nanoseconds—an increase of approximately 15-20% in the lifetime of the internally conjugated dye.

[0189] In certain embodiments, the luminescence intensity of the labeled biomolecule is increased relative to the unconjugated molecule. In certain embodiments, the luminescence intensity of the labeled biomolecule is increased by about 5% and 25% (e.g., about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 25%, about 15% to 25%, about 20% to 25%) relative to the unconjugated molecule. In some embodiments, the luminescence intensity of the labeled biomolecule is increased by about 5%, about 10%, about 15%, about 20%, about 25% or more relative to the unconjugated molecule.

[0190] In certain embodiments, the brightness of the labeled biomolecule is increased relative to the unconjugated molecule. In certain embodiments, the brightness of the labeled biomolecule is increased by about 5% to 10% relative to the unconjugated molecule. In certain embodiments, the brightness of the labeled biomolecule is increased by about 5% and 25% (e.g., about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 25%, about 15% to 25%, about 20% to 25%) relative to the unconjugated molecule. In some embodiments, the brightness of the labeled biomolecule is increased by about 5%, about 10%, about 15%, about 20%, about 25% or more relative to the unconjugated molecule.

[0191] In certain embodiments, the maximum emission wavelength of the labeled biomolecule is increased by at least 1% relative to the unconjugated molecule. In certain embodiments, the maximum emission wavelength of the labeled biomolecule is increased by about 1% and 10% (e.g., about 1% to 5%, about 5% to 10%) relative to the unconjugated molecule. An example of an increase in the maximum emission wavelength is shown in Figure 6 In some embodiments, the maximum emission wavelength of the labeled biomolecule is increased by about 1%, about 2%, about 5%, about 10% or more relative to the unconjugated molecule.

[0192] A large amount of fluorescence data obtained for internally conjugated Cy3B and externally conjugated Cy3B are shown in Figure 2. Figure 6 As shown, the excitation spectrum of the internal dye is red-shifted by 8 nm relative to the external dye, which brings the vibrational shoulder (hump on the left side of the trace) closer to 532 nm. In addition, as shown, the emission spectrum of the internal dye is red-shifted, which is found to advantageously increase the detectable signal because more light is allowed to pass through the filter.

[0193] In certain embodiments, the luminescence quantum yield of the labeled biomolecule is increased relative to the unconjugated molecule. In certain embodiments, the luminescence quantum yield of the labeled biomolecule is increased by about 5% and 25% (e.g., about 5% to 20%, about 5% to 15%, about 5% to 10%, about 10% to 25%, about 15% to 25%, about 20% to 25%) relative to the unconjugated molecule. In some embodiments, the luminescence quantum yield of the labeled biomolecule is increased by about 5%, about 10%, about 15%, about 20%, about 25% or more relative to the unconjugated molecule.

[0194] In certain embodiments, the photostability of the labeled biomolecule is increased relative to the unconjugated molecule. As used herein, in some embodiments, photostability refers to the ability of a luminescent molecule to continue to fluoresce over time. In some embodiments, photostability can be assessed by measuring the photobleaching rate. For example, in some embodiments, the photobleaching rate of a labeled biomolecule (e.g., a biomolecule with an internally conjugated label) can be measured and compared to the photobleaching rate measured for the unconjugated molecule. A decrease in the measured photobleaching rate will indicate an increase in photostability. Methods for measuring photobleaching rates are known in the art, for example, as described in Wüstner, D., et al. (2014) Molecules, 9: 11096-11130; Brakenhoff, GJ, et al. (1994) Journal of Microscopy, 175 (2): 154-161; and Song, L., et al. (1995) Biophys J. 68 (6): 2588-2600. In some embodiments, the photobleaching of the labeled biomolecule is reduced relative to the unconjugated molecule, as measured by fluorescence recovery after photobleaching assay (FRAP), for example, as described in Meyvis, T., et al. (1999) Pharmaceutical Research, 16 (8): 1153-1162. In some embodiments, photobleaching of the labeled biomolecule is reduced relative to the unconjugated molecule, as measured by fluorescence loss in photobleaching (FLIP), e.g., as described in Wüstner, D., et al. (2012) BMC Bioinformatics, 13:296.

[0195] In some embodiments, the disclosure provides novel compositions for identifying these molecules based on one or more luminescent properties of a single molecule. In some embodiments, molecules (e.g., nucleotides of luminescent labels) are identified based on the brightness, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, luminescence intensity, or a combination of two or more thereof of a molecule. Identification may refer to the exact molecular identity of a specified molecule, or may refer to distinguishing or distinguishing a specific molecule from a group of possible molecules. In some embodiments, multiple single molecules can be distinguished from each other based on different brightness, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, luminescence intensity, or a combination of two or more thereof. In some embodiments, by exposing a single molecule to a series of independent light pulses, and assessing the time or other properties of each photon emitted from the molecule, the molecule is identified (e.g., distinguished from other molecules). In some embodiments, the information of multiple photons emitted from a single molecule sequence is gathered and evaluated, thereby identifying the molecule. In some embodiments, the luminescence lifetime of a molecule is determined by multiple photons emitted from a molecule sequence, and the luminescence lifetime is used to identify the molecule. In some embodiments, the luminescence intensity of a molecule is determined by multiple photons emitted from a molecule sequence, and the luminescence intensity is used to identify the molecule. In some embodiments, the luminescence lifetime and luminescence intensity of a molecule are determined from multiple photons emitted sequentially from the molecule, and the luminescence lifetime and luminescence intensity are used to identify the molecule.

[0196] Therefore, in some aspects of the present application, the reaction sample is exposed to multiple independent light pulses, and a series of emitted photons are detected and analyzed. In some embodiments, this series of emitted photons can provide information about a single molecule present in the reaction sample that does not change during the experimental time. However, in some embodiments, this series of emitted photons provides information about a series of different molecules present in the reaction sample at different times (e.g., as the reaction or process proceeds).

[0197] The determination of the luminescence lifetime of a molecule can be performed using any suitable method (e.g., by measuring the lifetime using a suitable technique, or by determining the time-dependent characteristics of the emission). In some embodiments, determining the luminescence lifetime of a molecule includes determining the lifetime relative to one or more molecules (e.g., different luminescent-labeled nucleotides in a sequencing reaction). In some embodiments, determining the luminescence lifetime of a molecule includes determining the lifetime relative to a reference. In some embodiments, determining the luminescence lifetime of a molecule includes measuring the lifetime (e.g., fluorescence lifetime). In some embodiments, determining the luminescence lifetime of a molecule includes determining one or more time characteristics indicating the lifetime. In some embodiments, the luminescence lifetime of a molecule can be determined based on the distribution of multiple emission events (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more emission events) occurring within one or more time windows relative to an excitation pulse. For example, the luminescence lifetime of a single molecule can be distinguished from multiple molecules with different luminescence lifetimes based on the distribution of photon arrival times measured relative to the excitation pulse.

[0198] It should be understood that the luminescence lifetime of a single molecule indicates the time of the photons emitted after the single molecule reaches an excited state, and a single molecule can be distinguished by information indicating the photon time. Some embodiments may include distinguishing a molecule from a plurality of molecules based on the luminescence lifetime of the molecule by measuring the time associated with the photons emitted by the molecule. The distribution of time can provide an indication of the luminescence lifetime, and the luminescence lifetime can be determined from the distribution. In some embodiments, a single molecule can be distinguished from a plurality of molecules based on the distribution of time, for example, by comparing the distribution of time with a reference distribution corresponding to a known molecule. In some embodiments, the value of the luminescence lifetime is determined by the distribution of time.

[0199] As used herein for single molecules, luminescence intensity refers to the number of emission photons emitted per unit time by a molecule that is excited by delivering pulsed excitation energy. In some embodiments, luminescence intensity refers to the detected number of emission photons emitted per unit time by a molecule that is excited by delivering pulsed excitation energy and detected by one or a group of specific sensors.

[0200] In some aspects, the present disclosure provides methods and compositions related to labeled biomolecules with enhanced emission brightness. As used herein, in some embodiments, "brightness" (and its variations, e.g., "brightness," "brightness," etc.) refers to a parameter that reports the average emission intensity of each labeled reactant molecule. Therefore, in some embodiments, "emission intensity" can be used to refer to the brightness of a composition comprising a bright labeled reactant. In some embodiments, the brightness of a labeled reactant is equal to the product of its quantum yield and the extinction coefficient. In some embodiments, the labeled biomolecules of the present disclosure are designed to maximize the quantum yield to promote increased brightness.

[0201] Luminescence quantum yield refers to the fraction of excitation events that result in emission events at a given wavelength or within a given spectral range, and is generally less than 1. In some embodiments, the luminescence quantum yield of a molecule described herein is from 0 to about 0.001, from about 0.001 to about 0.01, from about 0.01 to about 0.1, from about 0.1 to about 0.5, from about 0.5 to 0.9, or from about 0.9 to 1. In some embodiments, a molecule is identified by determining or estimating the luminescence quantum yield.

[0202] In some embodiments, the internal labels described herein allow for the addition of sequential luminescent labels to a labeled biomolecule to increase brightness and / or luminescence intensity. In some embodiments, an internally labeled biomolecule comprising two or more luminescent labels is labeled according to the formula L n (x) exhibits brightness and / or luminous intensity, where L n =Where is equal to the total number of luminescent labels on the labeled reagent, and x is equal to the measured brightness or fluorescence intensity of the corresponding single labeled reagent. Thus, in some embodiments, the brightness and / or luminescence intensity of the dual dye labeled reaction component is twice that of the single dye labeled analog. In some embodiments, the brightness and / or luminescence intensity of the triple or quadruple dye labeled reaction component is three or four times that of the single dye labeled analog, respectively. In some embodiments, the brightness and / or luminescence intensity exhibited by the bright labeled reagents described herein is L n (x) at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the predicted value.

[0203] Nucleic acid sequencing reaction composition

[0204] Also provided herein are nucleic acid sequencing reaction compositions comprising two or more different types of labeled nucleotides in the reaction mixture, wherein at least one type of labeled nucleotide is a labeled nucleotide comprising a labeled biomolecule described herein.

[0205] In some embodiments, the nucleic acid sequencing reaction composition includes two or more (e.g., two, three, four, five or more) different types of labeled nucleotides. In some embodiments, the nucleic acid sequencing reaction composition includes four different types of labeled nucleotides. In some embodiments, the four different types of labeled nucleotides include a first labeled nucleotide, a second labeled nucleotide, a third labeled nucleotide, and a fourth labeled nucleotide, wherein the first labeled nucleotide includes guanine, the second labeled nucleotide includes cytosine, the third labeled nucleotide includes adenine, and the fourth labeled nucleotide includes thymine or uracil.

[0206] In some embodiments, each type of labeled nucleotide in the nucleic acid sequencing reaction composition is present at a concentration of about 100 to 1000 nM (eg, about 100 to 800 nM, about 150 to 700 nM, about 200 to 600 nM, or about 250 to 500 nM).

[0207] In some embodiments, the nucleic acid sequencing reaction composition comprises a sequencing template, and the sequencing template comprises a polymerase complexed with the target nucleic acid. In some embodiments, the complex also comprises a primer oligonucleotide, and the primer oligonucleotide has a sequence complementary to a portion of the target nucleic acid. In some embodiments, the concentration of the complex is about 10pM to 10nM (e.g., about 25pM to 5nM, about 50pM to 2nM, about 50pM to 1nM, about 50pM to 500pM, about 50pM to 100pM, about 250pM to 5nM, about 250pM to 2nM, about 250pM to 1nM or about 250pM to 500pM.

[0208] In some embodiments, the nucleic acid sequencing reaction composition comprises one or more buffers (e.g., MES, MOPS, MOPSO, HEPES, Tris, TAPS, and other such suitable buffers known in the art). In some embodiments, the one or more buffers include MOPS. In some embodiments, the buffer is present at a concentration of about 25 to 100 mM (e.g., about 25 to 75 mM, about 50 to 75 mM, or about 65 mM).

[0209] In some embodiments, the nucleic acid sequencing reaction composition includes divalent cations (e.g., magnesium ions, calcium ions). In some embodiments, the divalent cations include magnesium or calcium salts (e.g., salts including magnesium or calcium and acetate, chloride, phosphate, sulfate). In some embodiments, the salt is magnesium acetate. In some embodiments, the divalent cations are present at a concentration of about 5 to 50 mM (e.g., about 10 to 40 mM, about 15 to 35 mM, about 20 to 30 mM, or about 25 mM).

[0210] In some embodiments, the nucleic acid sequencing reaction composition comprises one or more monovalent salts (e.g., sodium salts or potassium salts, such as sodium chloride, sodium acetate, potassium chloride, or potassium acetate). In some embodiments, the monovalent salt is present at a concentration of about 10 to 200 mM (e.g., about 25 to 150 mM, about 25 to 40 mM, about 50 to 150 mM, about 100 to 150 mM). In some embodiments, the nucleic acid sequencing reaction composition includes about 40 mM monovalent salt, such as 40 mM sodium chloride. In some embodiments, the nucleic acid sequencing reaction composition includes about 120 mM monovalent salt, such as 120 mM potassium acetate.

[0211] In some embodiments, the nucleic acid sequencing reaction composition comprises one or more light stabilizers (e.g., one or more light protection additives, such as antioxidants, oxygen scavengers, three-state quenchers, and similar energy-absorbing additives known in the art). In some embodiments, the light stabilizer comprises protocatechuic acid (PCA). In some embodiments, the light stabilizer comprises 4-nitrobenzyl alcohol (NBA). In some embodiments, the light stabilizer comprises trolox or a derivative thereof. In some embodiments, the light stabilizer is present at a concentration of about 0.1 mM to about 20 mM. In some embodiments, the concentration of Trolox is about 5 mM. In some embodiments, the concentration of PCA is about 3 mM. In some embodiments, the concentration of PCA is about 8 mM. In some embodiments, the concentration of NBA is about 3 mM. The mixture containing the light stabilizer (e.g., PCA) may also include an enzyme (e.g., protocatechuate dioxygenase (PCD)) for regenerating the light stabilizer. In some embodiments, the concentration of PCD is about 0.3 mM. In some embodiments, the concentration of PCD is about 0.5 mg / mL.

[0212] In some embodiments, the nucleic acid sequencing reaction composition comprises one or more reducing agents. For example, in some embodiments, the nucleic acid sequencing reaction composition comprises about 10 to 100 mM DTT (eg, about 40 mM DTT).

[0213] Sequencing

[0214] Some aspects of the present application can be used for sequencing biopolymers, such as nucleic acids and proteins. In some aspects, the compositions and techniques described in the present application can be used to identify a series of nucleotides or amino acid monomers incorporated into nucleic acids or proteins (e.g., by detecting the time course of incorporation of a series of labeled nucleotides or amino acid monomers). In some embodiments, the compositions and techniques described in the present application can be used to identify a series of nucleotides incorporated into a template-dependent nucleic acid sequencing reaction product synthesized by a polymerase.

[0215] Therefore, the present invention also provides a method for determining the sequence of a template nucleic acid using the nucleic acid sequencing reaction composition of the present application. In some embodiments, the sequencing method includes the following steps: (i) exposing a target volume of a complex to a nucleic acid sequencing reaction composition of the present invention (e.g., at least one labeled nucleotide containing a labeled biomolecule described herein), wherein the complex includes a template nucleic acid, a primer, and a polymerase, (ii) directing a series of pulses with one or more excitation energies toward the vicinity of the target volume; (iii) detecting multiple photons emitted from a luminescent labeled nucleotide during sequential incorporation into a nucleic acid containing a primer; and (iv) identifying the sequence of the incorporated nucleotide by determining the time and optional luminescence intensity of the emitted photons.

[0216] In some embodiments, as used herein, excitation energy is a light pulse from a light source. In some embodiments, the excitation energy is in the visible spectrum. In some embodiments, the excitation energy is in the ultraviolet spectrum. In some embodiments, the excitation energy is in the infrared spectrum. In some embodiments, the excitation energy is in or near the absorption maximum of the molecule (to detect multiple emission photons from this molecule) of the luminescent label. In some embodiments, the excitation energy is about 500nm to about 700nm (for example, about 500nm to about 600nm, about 600nm to about 700nm, about 500nm to about 550nm, about 550nm to about 600nm, about 600nm to about 650nm or about 650nm to about 700nm). In some embodiments, the excitation energy can be monochromatic or limited to a spectral range. In some embodiments, the spectral range is about 0.1nm to about 1nm, about 1nm to about 2nm or about 2nm to about 5nm. In some embodiments, the spectral range is about 5nm to about 10nm, about 10nm to about 50nm or about 50nm to about 100nm.

[0217] When base pairing occurs between the nucleobase of the target nucleic acid and the complementary polyphosphate nucleoside (e.g., dNTP), the polymerase incorporates the dNTP into the newly synthesized nucleic acid chain by forming a phosphodiester bond between the 3' hydroxyl end of the newly synthesized nucleic acid chain and the α-phosphate of the dNTP. In the example where the luminescent molecule conjugated to the dNTP (e.g., a labeled biomolecule as described herein) comprises a fluorophore, its presence is signaled by excitation, and an emission pulse is detected during and / or after the incorporation step. For luminescent molecules conjugated to the terminal (γ) phosphate of the dNTP (e.g., a labeled biomolecule), incorporation of the dNTP into the newly synthesized chain results in the release of β and γ phosphates and luminescent molecules, which diffuse freely in the sample well, resulting in a reduction in emission detected from the fluorophore.

[0218] In certain embodiments, the template-dependent nucleic acid sequencing product is carried out by a naturally occurring nucleic acid polymerase. In some embodiments, the polymerase is a mutant or modified variant of a naturally occurring polymerase. In some embodiments, the template-dependent nucleic acid sequencing product will include one or more nucleotide segments complementary to the template nucleic acid chain. In one aspect, the application provides a method for determining the sequence of a template (or target) nucleic acid chain by determining the sequence of its complementary nucleic acid chain.

[0219] As used herein, the term "polymerase" generally refers to any enzyme (or polymerase) that can catalyze polymerization. Examples of polymerases include, but are not limited to, nucleic acid polymerases, transcriptases, or ligases. A polymerase can be a polymerase. Embodiments for single-molecule nucleic acid extension (e.g., for nucleic acid sequencing) can use any polymerase that can synthesize a nucleic acid complementary to a target nucleic acid molecule. In some embodiments, the polymerase can be a DNA polymerase, an RNA polymerase, a reverse transcriptase, and / or one or more thereof, a mutant or altered form.

[0220] Examples of polymerases include, but are not limited to, DNA polymerases, RNA polymerases, thermostable polymerases, wild-type polymerases, modified polymerases, Escherichia coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase (psi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerase with 3' to 5' exonuclease activity and variants, modified products and derivatives thereof. In some embodiments, the polymerase is a single subunit polymerase. Non-limiting examples of DNA polymerases and their properties are described in detail in DNA Replication 2nd edition, Kornberg and Baker, WH Freeman, New York, NY (1991), among others.

[0221] On the other hand, the application provides a method for sequencing a target nucleic acid by sequencing a plurality of nucleic acid fragments, wherein the target nucleic acid comprises these fragments. In certain embodiments, the method comprises merging a plurality of fragment sequences to provide a sequence or partial sequence of the parental target nucleic acid. In some embodiments, the merging step is performed by computer hardware and software. Method described herein can allow a group of related target nucleic acids, such as whole chromosome or genome to be sequenced.

[0222] During sequencing, polymerase can be coupled (e.g., connected) to the primer position of the target nucleic acid molecule. The primer position can be a primer complementary to a part of the target nucleic acid molecule. As another option, the primer position is the space or gap provided in the double-stranded segment of the target nucleic acid molecule. The length of the space or gap can be 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or 40 nucleotides. The gap can provide a break in a chain of the double-stranded sequence, and can provide a primer position for a polymerase such as a strand displacement polymerase.

[0223] In some cases, sequencing primers can be annealed to target nucleic acid molecules, which may or may not be fixed on a solid support.Solid support can include, for example, sample wells (e.g., nanopores, reaction chambers) on a chip for nucleic acid sequencing.In some embodiments, sequencing primers can be fixed to a solid support, and the hybridization of target nucleic acid molecules also fixes target nucleic acid molecules to a solid support.In some embodiments, polymerase is fixed to a solid support, and soluble primers and target nucleic acid are contacted with polymerase.However, in some embodiments, a complex comprising polymerase, target nucleic acid and primer is formed in a solution, and the complex is fixed to a solid support (e.g., by fixation of polymerase, primer and / or target nucleic acid).In some embodiments, any component in a sample well (e.g., nanopore, reaction chamber) is not fixed to a solid support.For example, in some embodiments, a complex comprising polymerase, target nucleic acid and primer is formed in a solution, and the complex is not fixed to a solid support.

[0224] Under appropriate conditions, a polymerase in contact with the annealed primer / target nucleic acid can add or incorporate one or more nucleotides to the primer, and the nucleotides can be added to the primer in a 5' to 3', template-dependent manner. This process of incorporating nucleotides into the primer (e.g., by the action of a polymerase) can generally be referred to as a primer extension reaction. Each nucleotide can be associated with a detectable marker, which can be detected and identified (e.g., based on its luminescence lifetime and / or other characteristics) during the nucleic acid extension reaction to determine each nucleotide incorporated into the extension primer, thereby determining the sequence of the newly synthesized nucleic acid molecule. The sequence of the target nucleic acid molecule can also be determined by the sequence complementarity of the newly synthesized nucleic acid molecules. In some cases, annealing of the sequencing primer to the target nucleic acid molecule and binding of the nucleotide to the sequencing primer can occur under similar reaction conditions (e.g., the same or similar reaction temperature) or under different reaction conditions (e.g., different reaction temperatures). In some embodiments, sequencing by synthesis can include: there is a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and / or amplification of the target nucleic acid to obtain a population of target nucleic acids. However, in some embodiments, the sequence of the single molecule in each reaction being evaluated is determined by synthetic sequencing (and nucleic acid amplification is not required to prepare the target template for sequencing). In some embodiments, according to aspects of the present application, multiple single molecule sequencing reactions are performed in parallel (e.g., on a single chip). For example, in some embodiments, multiple single molecule sequencing reactions are performed in separate reaction chambers (e.g., nanopores, sample wells) on a single chip.

[0225] Embodiments are capable of sequencing single nucleic acid molecules with high accuracy and long read lengths, e.g., an accuracy of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, and / or a read length of greater than or equal to about 10

[0226] BY20IM3317FGPC-CN.Telephone OA base pairs (bp), 50bp, 100bp, 200bp, 300bp, 400bp, 500bp, 1000bp, 10,000bp, 20,000bp, 30,000bp, 40,000bp, 50,000bp or 100,000bp. In some embodiments, the target nucleic acid molecule used in single molecule sequencing is a single-stranded target nucleic acid (e.g., deoxyribonucleic acid (DNA), DNA derivative, ribonucleic acid (RNA), RNA derivative) template, which is added or fixed to a sample well (e.g., nanopore) containing at least one additional sequencing reaction component (e.g., polymerase such as DNA polymerase, sequencing primer), and the additional component is fixed or connected to a solid support such as the bottom or side wall of the sample well. The target nucleic acid molecule or polymerase can be directly or via a linker connected to the sample wall, for example, at the bottom or side wall of the sample well. The sample well (e.g., nanopore) can also contain any other reagents required for synthesizing nucleic acids by a primer extension reaction, for example, suitable buffers, cofactors, enzymes (e.g., polymerases), and deoxynucleoside polyphosphates such as deoxynucleoside triphosphates, including deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, including luminescent markers, such as the luminescent markers of the labeled biomolecules provided herein.

[0227] In some embodiments, each type of dNTPs (e.g., adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs (e.g., dUTPs), and thymosin-containing dNTPs (e.g., dTTP)) is conjugated to a luminescent molecule that comprises different luminescent properties, such that detection of light emitted from the luminescent molecule can indicate the identity of the dNTP incorporated into the newly synthesized nucleic acid. Emitted light from the luminescent molecule (e.g., emitted light from a labeled biomolecule comprising at least one luminescent label) can be detected by any suitable device and / or method and attributed to its appropriate luminescent molecule (and therefore, to the associated dNTP). The luminescent molecule can be conjugated to the dNTP at any position such that the presence of the luminescent molecule (e.g., the labeled biomolecule of the present application) does not inhibit the incorporation of the dNTP into the newly synthesized nucleic acid chain or does not inhibit the activity of the polymerase. In some embodiments, the luminescent molecule is conjugated to the terminal phosphate (eg, gamma phosphate) of a dNTP.

[0228] In some embodiments, the single-stranded target nucleic acid template can be contacted with a sequencing primer, dNTPs, a polymerase, and other reagents required for nucleic acid synthesis. In some embodiments, all suitable dNTPs can be contacted with the single-stranded target nucleic acid template at the same time (e.g., all dNTPs are present at the same time), so that the incorporation of dNTPs can be performed continuously. In other embodiments, dNTPs can be contacted with the single-stranded target nucleic acid template in sequence, wherein the single-stranded target nucleic acid template is contacted with each suitable dNTP respectively, and there is a washing step between the single-stranded target nucleic acid template and the different dNTPs. For each continuous base position of the single-stranded target nucleic acid template to be identified, such a cycle can be repeated, that is, the single-stranded target nucleic acid template is contacted with each dNTP respectively, and then washed.

[0229] In some embodiments, the sequencing primer is annealed to the single-stranded target nucleic acid template, and the polymerase continuously incorporates dNTPs (or other polyphosphate nucleosides) into the primer according to the single-stranded target nucleic acid template. The unique luminescent molecule associated with each incorporated dNTP, such as the labeled biomolecule described herein, can be excited with appropriate excitation light during or after the dNTP is incorporated into the primer, and its emission can be subsequently detected using any suitable device and / or method. Specific light emission (e.g., with a specific emission lifetime, intensity, spectrum and / or combination thereof) is detected and can be attributed to the specific dNTP incorporated. Then, a sequence is obtained from the set of detected luminescent molecules, and the sequence of the single-stranded target nucleic acid template can be determined by sequence complementarity using the sequence.

[0230] In some embodiments, the present disclosure provides methods that may be used in co-pending U.S. Patent Application Nos. 14 / 543,865, 14 / 543,867, 14 / 543,888, 14 / 821,656, 14 / 821,686, 14 / 821,688, 15 / 161,067, 15 / 161,088, 15 / 161,125, 15 / 255,245, 15 / 255,303, 15 / 255,624, 15 / 261,688, , 697, 15 / 261,724, 15 / 600,979, 15 / 846,967, 15 / 847,001, 62 / 289,019, 62 / 296,546, 62 / 310,398, 62 / 339,790, 62 / 343,997, 62 / 344,123, 62 / 426,144 and 62 / 505,525 (each of which is incorporated herein by reference).

[0231] Reagent test kit

[0232] Also provided herein is a kit for sequencing a template nucleic acid, the kit comprising two or more different types of labeled nucleotides, wherein at least one type of labeled nucleotide is a labeled nucleotide comprising a labeled biomolecule described herein. In some embodiments, the kit comprises two or more (e.g., two, three, four, five or more) different types of labeled nucleotides. In some embodiments, the kit comprises four different types of labeled nucleotides. In some embodiments, the kit comprises a polymerase. In some embodiments, the kit comprises a primer complementary to the template nucleic acid.

[0233] In some embodiments, the kit includes multiple types of labeled nucleotides, including labeled biomolecules described herein. In some embodiments, at least one type (e.g., two, three, four, five or more types) of labeled nucleotides includes a labeled biomolecule of the present application, the labeled biomolecule having two or more internally conjugated labels. In some embodiments, the plurality of nucleotides is selected from Figures 1A-1G , 4A-4C, 5A and 5C. In some embodiments, the kit further comprises a polymerase (e.g., a DNA polymerase as described elsewhere herein). In some embodiments, the kit further comprises a primer complementary to the template nucleic acid being sequenced.

[0234] Dual conjugated labels

[0235] In another aspect, the present invention provides compounds. The compounds provided herein can be used as markers, that is, used in conjugation reactions to form labeled biomolecules as described herein. For example, compounds of formula (II) are provided herein:

[0236]

[0237] and salts thereof, wherein:

[0238] A is a multicyclic fluorophore;

[0239] L 1 and L 2 is independently a linker selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof;

[0240] P 1 is an oxygen protecting group; and

[0241] R 1It is the reactive part.

[0242] As described herein, compounds of formula (II) and salts thereof are bifunctional (eg, "asymmetric"). 1 is a reactive moiety that can be used as a reactive handle in a conjugation reaction. 1 is an oxygen protecting group. 1 After the conjugation reaction, R can be cleaved or removed to reveal a free -OH group, which can then be used as a reactive moiety in a subsequent conjugation reaction. 1 is a reactive moiety (e.g., phosphoramidite) that is reactive in nucleoside coupling reactions. 1 Various oxygen protecting groups are used at the positions of , examples of which are provided herein.

[0243] In this paper, L 1 and L 2 , and exemplary embodiments are provided. In addition, exemplary embodiments of A are provided herein. All definitions and embodiments provided herein, including but not limited to the definitions and embodiments provided in the internally labeled biomolecule section, apply to the compounds provided herein.

[0244] As defined herein, P 1 is an oxygen protecting group. Several examples of oxygen protecting groups are provided herein. In certain embodiments, P 1 is an optionally substituted triphenyl protecting group (e.g., trityl). 1 is a trityl group having the formula: In certain embodiments, P 1 is 4-monomethoxytrityl (MMT) having the formula: In certain embodiments, P 1 is 4,4-dimethoxytrityl (DMT) having the formula:

[0245] In certain embodiments, R 1 is the reactive part. For example, R 1 It is a reactive handle that can be used for polynucleotide synthesis, polypeptide synthesis, polysaccharide synthesis, etc. The reactive portion can be a reactive portion that can react with a second reactive portion (e.g., –OH or –NH 2 A person skilled in the art will know which reactive moieties can be used to form bonds in polynucleotide synthesis, polypeptide synthesis, polysaccharide synthesis, etc. In certain embodiments, R 1 is a moiety that can be used for polynucleotide synthesis (e.g., phosphoramidite). 1is a phosphoramidite. In certain embodiments, R 1 Is of the formula: The phosphoramidite, where R N1 and R 2 As defined herein. In certain embodiments, R 1 Is of the formula: In certain embodiments, R 1 Is of the formula: of phosphoramidites.

[0246] As defined herein, R 2 is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or an oxygen protecting group. In certain embodiments, R 2 is an optionally substituted alkyl. 2 is an optionally substituted C 1-6 In certain embodiments, R 2 is an optionally substituted C 1-3 In certain embodiments, R 2 With the formula:

[0247] As defined herein, R N1 Each instance of is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group; optionally, wherein two R bonded to the same nitrogen N1 and the middle atom are connected together to form an optionally substituted heterocyclic group or an optionally substituted heteroaryl group. N1 is an optionally substituted alkyl. N1 is an optionally substituted C 1-6 In certain embodiments, R N1 is unsubstituted C 1-6 In certain embodiments, R N1 is an optionally substituted C 1-3 In certain embodiments, R N1 is unsubstituted C 1-3 In certain embodiments, R N1 is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. N1- In certain embodiments, R N1In both cases it is isopropyl.

[0248] In certain embodiments, R 1 is a reactive moiety in nucleoside coupling reactions, such as a phosphoramidite. In certain embodiments, the compound of formula (II) has the following formula:

[0249]

[0250] or a salt thereof, wherein:

[0251] R 2 is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or an oxygen protecting group;

[0252] R N1 Each instance of is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group; optionally, wherein two R bonded to the same nitrogen N1 The intermediate atoms are linked together to form an optionally substituted heterocyclic group or an optionally substituted heteroaryl group.

[0253] In certain embodiments, the compound of formula (II) has the following formula:

[0254]

[0255] or a salt thereof.

[0256] In certain embodiments, the compound of formula (II) has the following formula:

[0257]

[0258] or a salt thereof.

[0259] In certain embodiments, the compound of formula (II) has the following formula:

[0260]

[0261] or a salt thereof, wherein:

[0262] Each instance of n is an integer from 1 to 20, inclusive.

[0263] In certain embodiments, the compound of formula (II) has the following formula:

[0264]

[0265] or a salt thereof, wherein:

[0266] Each instance of n is an integer from 1 to 20, inclusive.

[0267] In certain embodiments, the compound of formula (II) has the following formula:

[0268]

[0269] or a salt thereof.

[0270] In certain embodiments, the compound of formula (II) has the following formula:

[0271]

[0272] or a salt thereof.

[0273] As described herein, Ring A can be a polycyclic cyanine, such as Cy3B. In certain embodiments, the compound of formula (II) has the following formula:

[0274]

[0275] or a salt thereof, wherein X - is a counterion or is absent.

[0276] In certain embodiments, the compound of formula (II) has the following formula:

[0277]

[0278] or a salt thereof.

[0279] In certain embodiments, the compound of formula (II) has the following formula:

[0280]

[0281] or a salt thereof.

[0282] In certain embodiments, the compound of formula (II) has the following formula:

[0283]

[0284] or a salt thereof.

[0285] In certain embodiments, the compound of formula (II) has the following formula:

[0286]

[0287] or a salt thereof.

[0288] In certain embodiments, the compound of formula (II) has the following formula:

[0289]

[0290] or a salt thereof.

[0291] In certain embodiments, the compound of formula (II) has the following formula:

[0292]

[0293] or a salt thereof.

[0294] In certain embodiments, the compound of formula (II) has the following formula:

[0295]

[0296] or a salt thereof.

[0297] In certain embodiments, the compound of formula (II) has the following formula:

[0298]

[0299] or a salt thereof.

[0300] In certain embodiments, the compound of formula (II) has the following formula:

[0301]

[0302] or a salt thereof.

[0303] In certain embodiments, A can be a fluorone fluorophore, such as fluorescein or rhodamine. In certain embodiments, the compound of formula (II) has the following formula:

[0304]

[0305] or a salt thereof, wherein:

[0306] Each R is independently hydrogen, halogen, –N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or N(R N ) 2 .

[0307] In certain embodiments, the compound of formula (II) has the following formula:

[0308]

[0309] or a salt thereof, wherein:

[0310] n is independently an integer from 1 to 20, inclusive.

[0311] In certain embodiments, the compound of formula (II) has the following formula:

[0312]

[0313] or a salt thereof, wherein:

[0314] n is independently an integer from 1 to 20, inclusive; and

[0315] R N Each instance of is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group.

[0316] In certain embodiments, the compound of formula (II) has the following formula:

[0317]

[0318] or a salt thereof.

[0319] In certain embodiments, the compound of formula (II) has the following formula:

[0320]

[0321] or a salt thereof.

[0322] In certain embodiments, the compound of formula (II) has the following formula:

[0323]

[0324] or a salt thereof.

[0325] In certain embodiments, the compound of formula (II) has the following formula:

[0326]

[0327] or a salt thereof.

[0328] In certain embodiments, the compound of formula (II) has the following formula:

[0329]

[0330] or a salt thereof.

[0331] As described herein, in certain embodiments, A is a BODIPY fluorophore. Thus, in certain embodiments, the compound of formula (II) has the following formula:

[0332]

[0333] or a salt thereof, wherein:

[0334] Each R is independently hydrogen, halogen, –N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or N(R N ) 2 ;and

[0335] Ar is optionally substituted aryl or optionally substituted heteroaryl.

[0336] In certain embodiments, the compound of formula (II) has the following formula:

[0337]

[0338] or a salt thereof.

[0339] In certain embodiments, the compound of formula (II) has the following formula:

[0340]

[0341] or a salt thereof, wherein:

[0342] R Ar is hydrogen, halogen, –N 3 、–CN、–NO 2 , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -OR O ,–SR S or N(R N ) 2 .

[0343] As generally defined in this article, R O is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or an oxygen protecting group. In certain embodiments, R O is hydrogen. In certain embodiments, R Ois an optionally substituted alkyl. O is an optionally substituted C 1-6 In certain embodiments, R O is unsubstituted C 1-6 In certain embodiments, R O is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. O is an optionally substituted alkenyl. In certain embodiments, R O is an optionally substituted alkynyl. O is an optionally substituted carbocyclyl. In certain embodiments, R O is an optionally substituted heterocyclyl. In certain embodiments, R O is an optionally substituted aryl. In certain embodiments, R O is an optionally substituted heteroaryl. In certain embodiments, R O is an optionally substituted acyl group. O is an oxygen protecting group. O It is –C(=O)Ph.

[0344] As generally defined in this article, R N each instance of is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group; or optionally, both R N and the middle atom are connected together to form an optionally substituted heterocyclic group or an optionally substituted heteroaryl group. N is hydrogen. In certain embodiments, R N is an optionally substituted alkyl. N is an optionally substituted C 1-6 In certain embodiments, R N is unsubstituted C 1-6 In certain embodiments, R N is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. N is an optionally substituted alkenyl. In certain embodiments, R N is an optionally substituted alkynyl. N is an optionally substituted carbocyclyl. In certain embodiments, R N is an optionally substituted heterocyclyl. In certain embodiments, R Nis an optionally substituted aryl. In certain embodiments, R N is an optionally substituted heteroaryl. In certain embodiments, R N is an optionally substituted acyl group. N is a nitrogen protecting group. N The intermediate atoms are linked together to form an optionally substituted heterocyclic group or an optionally substituted heteroaryl group.

[0345] As generally defined in this article, R S is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a sulfur protecting group. In certain embodiments, R S is hydrogen. In certain embodiments, R S is an optionally substituted alkyl. S is an optionally substituted C 1-6 In certain embodiments, R S is unsubstituted C 1-6 In certain embodiments, R S is selected from the group consisting of: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. S is an optionally substituted alkenyl. In certain embodiments, R S is an optionally substituted alkynyl. S is an optionally substituted carbocyclyl. In certain embodiments, R S is an optionally substituted heterocyclyl. In certain embodiments, R S is an optionally substituted aryl. In certain embodiments, R S is an optionally substituted heteroaryl. In certain embodiments, R S is an optionally substituted acyl group. S is a sulfur protecting group. S is PEG (polyethylene glycol). In certain embodiments, R S Yes – (CH 2 CH 2 O) m OCH 3 In certain embodiments, –SR S Yes – S(CH 2 CH 2 O) m OCH 3 .

[0346] In certain embodiments, the compound of formula (II) has the following formula:

[0347]

[0348] or a salt thereof.

[0349] In certain embodiments, the compound of formula (II) has the following formula:

[0350]

[0351] or a salt thereof.

[0352] In certain embodiments, the compound of formula (II) has the following formula:

[0353]

[0354] or a salt thereof.

[0355] In certain embodiments, the compound of formula (II) has the following formula:

[0356]

[0357] or a salt thereof, wherein n is independently an integer from 1 to 20, inclusive.

[0358] In certain embodiments, the compound of formula (II) has the following formula:

[0359]

[0360] or a salt thereof.

[0361] In certain embodiments, the compound of formula (II) has the following formula:

[0362]

[0363] or a salt thereof.

[0364] In certain embodiments, the compound of formula (II) has the following formula:

[0365]

[0366] or a salt thereof.

[0367] In certain embodiments, the compound of formula (II) has the following formula:

[0368]

[0369] or a salt thereof.

[0370] In certain embodiments, the compound of formula (II) has the following formula:

[0371]

[0372] or a salt thereof.

[0373] Methods for preparing internally labeled biomolecules

[0374] Also provided herein are methods for preparing the internally labeled biomolecules described herein (eg, biomolecules of Formula (I)). Typically, the method comprises two subsequent conjugation steps involving the bifunctional compounds provided herein (eg, compounds of Formula (II)).

[0375] Thus, provided herein is a method for preparing a labeled biomolecule, the method comprising:

[0376] (i) subjecting a compound of formula Q to 2 -OH monomer or oligomeric biomolecule or its salt is contacted with the compound of formula (II) or its salt to obtain the conjugate or its salt of the following formula:

[0377]

[0378] (ii) sufficient to cleave P 1 The conjugate formed in step (i) is deprotected under conditions where the protecting group is removed and the conjugate or a salt thereof is produced:

[0379]

[0380] (iii) reacting the conjugate formed in step (ii) with a compound of formula Q under conditions sufficient to promote conjugation 1 -OR 1 A monomeric or oligomeric biomolecule or a salt thereof is contacted to obtain a labeled biomolecule of formula (I):

[0381]

[0382] For example, in certain embodiments, when preparing internally labeled oligonucleotides, the compound of formula (II) can be reacted with Q using standard phosphoramidite chemistry. 2 -OH coupling. In addition, in certain embodiments, when preparing internally labeled oligonucleotides, the unprotected conjugate with the hydroxyl moiety formed in step (ii) is coupled with an alkyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, such as used in standard oligonucleotide synthesis. The reaction can be carried out in the presence of an activator such as 1H-tetrazole, ethylthiotetrazole, benzylthiotetrazole, dicyanoimidazole or other suitable weak acid. Then, in certain embodiments, step (iii) can be carried out using standard phosphoramidite chemistry.

[0383] Method for preparing double conjugated labels

[0384] In another aspect, the present invention provides synthetic methods for preparing the dual conjugated labels described herein (eg, compounds of formula (II)).

[0385] General reaction parameters

[0386] The following embodiments apply to all synthetic methods described herein.

[0387] The reaction provided and described herein may involve one or more reagents. In certain embodiments, reagent can be present in catalytic amount. In certain embodiments, catalytic amount is 0-1mol%, 0-5mol%, 0-10mol%, 1-5mol%, 1-10mol%, 5-10mol%, 10-20mol%, 20-30mol%, 30-40mol%, 40-50mol%, 50-60mol%, 60-70mol%, 70-80mol%, 80-90mol% or 90-99mol%. In certain embodiments, reagent can be present in stoichiometric amount (e.g., about 1 equivalent). In certain embodiments, reagent can be present in excess (e.g., greater than 1 equivalent). In certain embodiments, the excess is about 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, or 20 equivalents. In certain embodiments, the excess is about 1.1-2, 2-3, 3-4, 4-5, 1.1-5, 5-10, 10-15, 15-20, or 10-20 equivalents. In certain embodiments, the excess is greater than 20 equivalents.

[0388] The reactions described herein can be carried out at any temperature. In certain embodiments, the reaction is carried out at room temperature (rt) (21 ° C or 70 ° F) or its approximate value. In certain embodiments, the reaction is carried out below room temperature (e.g., -100 ° C to 21 ° C). In certain embodiments, the reaction is carried out at -78 ° C or approximately -78 ° C. In certain embodiments, the reaction is carried out at -10 ° C or approximately -10 ° C. In certain embodiments, the reaction is carried out at 0 ° C or approximately 0 ° C. In certain embodiments, the reaction is carried out at a temperature higher than room temperature. In certain embodiments, the reaction is carried out at 30, 40, 50, 60, 70, 80, 110, 120, 130, 140 or 150 ° C. In certain embodiments, the reaction is carried out at a temperature higher than 150 ° C.

[0389] The reaction described herein can be carried out in a solvent or a solvent mixture (e.g., a cosolvent). The solvent can be polar or non-polar, proton or aproton. Any solvent can be used in the reaction described herein, and the reaction is not limited to a specific solvent or a combination of solvents. Common organic solvents that can be used for the methods described herein include, but are not limited to, acetone, acetonitrile, benzene, benzonitrile, 1-butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide, carbon tetrachloride, chlorobenzene, 1-chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-dichlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N,N-dimethylacetamide N,N-dimethylformamide (DMF), 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidone (DMPU), 1,4-dioxane, 1,3-dioxane, ether, 2-ethoxyethyl ether, ethyl acetate, ethanol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexane alkane, hexamethylphosphoramide (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methanol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidone, dimethyl sulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahydrofuran (THF), 2-methyltetrahydrofuran, toluene, trichlorobenzene, 1,1,2-trichlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N,N-diisopropylethylamine, diisopropylamine, water, o-xylene, p-xylene.

[0390] The reactions described herein can be carried out in any amount of time. In certain embodiments, the reactions can be carried out for a few seconds, minutes, hours or days.

[0391] Methods described herein can be used to prepare compounds with any chemical yield. In certain embodiments, compounds are produced with a yield of 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100%. In certain embodiments, the yield is the percentage yield after a synthesis step. In certain embodiments, the yield is the percentage yield after more than one synthesis step (e.g., 2, 3, 4 or 5 synthesis steps).

[0392] The methods described herein may further include one or more purification steps. For example, in certain embodiments, the compounds produced by the methods described herein may be purified by chromatography, extraction, filtration, precipitation, crystallization, or any other method known in the art. In certain embodiments, the compound or mixture may be used directly in the next synthesis step (e.g., crude product) without purification.

[0393] Preparation method of Cy3B-based marker

[0394] The present invention provides a method for preparing a compound of formula (III) or a salt thereof:

[0395]

[0396] The method comprises, in the presence of palladium, Compound or its salt, with formula Compounds or salts thereof are coupled to obtain compounds of formula (III) or salts thereof, wherein:

[0397] X 1 is a halogen or a leaving group;

[0398] B(R B ) 2 is borane, boric acid or boric ester;

[0399] L 1 is a linker selected from the group consisting of optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof; and

[0400] R O It is an oxygen protecting group.

[0401] In certain embodiments, the reaction to produce a compound of formula (III) is carried out in the presence of a metal other than palladium. For example, the reaction can be catalyzed by palladium, or by a different metal. In certain embodiments, the metal is a transition metal.

[0402] As defined herein, the group -B(R B ) 2 is borane, boronic acid or a boronic ester. In certain embodiments, -B(R B ) 2 is borane. In certain embodiments, –B(R B ) 2 is boronic acid. In certain embodiments, –B(R B ) 2 is a borate ester. In certain embodiments, –B(R B ) 2 Is the formula: As defined herein, R B Each instance of is independently optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -OH, or -OR O Optionally, two R BThe intermediate atoms are connected together to form an optionally substituted carbocyclic group or an optionally substituted heterocyclic group. B ) 2 Borane is any suitable borane, boronic acid or boronate ester useful in metal-promoted or metal-catalyzed cross-coupling reactions.

[0403] Linking base L 1 As defined herein.

[0404] As defined herein, X 1 is a halogen or a leaving group. 1 In certain embodiments, X 1 is a leaving group. In certain embodiments, X 1 is -Cl, -Br or -I. In certain embodiments, X 1 Yes-I.

[0405] In certain embodiments of the coupling reaction, the compound of formula (III) has the following formula or a salt thereof:

[0406]

[0407] The chemical formula of the starting material is therefore:

[0408]

[0409] or a salt thereof.

[0410] In certain embodiments, the compound of formula (III) has the following formula or a salt thereof:

[0411]

[0412] wherein n is an integer from 1 to 20, including 1 and 20; thus, the starting material has the formula:

[0413]

[0414] or a salt thereof.

[0415] As described above, the coupling reaction is carried out in the presence of palladium. In certain embodiments, the palladium is a palladium complex. In certain embodiments, the palladium complex is a palladium (II) complex. In certain embodiments, the palladium complex is PdCl 2 (dppf). In certain embodiments, the palladium is present in a catalytic amount. In other embodiments, the palladium is present in a stoichiometric amount or in excess.

[0416] In certain embodiments, the coupling reaction is carried out in the presence of a base. In certain embodiments, the base is a carbonate base. In certain embodiments, the base is Cs 2 CO3 .

[0417] In certain embodiments, the coupling reaction is carried out in a solvent. In certain embodiments, the solvent is THF, DMF or a mixture thereof.

[0418] The coupling reaction can be carried out at any temperature. In certain embodiments, the reaction is carried out at room temperature. In certain embodiments, the reaction is carried out at a temperature higher than room temperature (i.e., high temperature). In certain embodiments, the reaction is carried out between room temperature and 100 ° C. In certain embodiments, the reaction is carried out between 50 ° C and 100 ° C. In certain embodiments, the reaction is carried out at about 70 ° C.

[0419] In certain embodiments, the method further comprises the steps of: Compound or its salt, alkylating the compound of formula (III) to obtain Compound, or its tautomer: or a salt thereof, wherein:

[0420] X 2 is a halogen or a leaving group; and

[0421] Each R 3 are independently optionally substituted alkyl, optionally substituted acyl or an oxygen protecting group; or optionally, two R 3 The atoms in the middle are linked together to form an optionally substituted heterocyclic group.

[0422] In certain embodiments, the product is of the formula: A compound, or a tautomer thereof: or a salt thereof.

[0423] In certain embodiments, the alkylation step is carried out in the presence of a base. In certain embodiments, the base is a halide salt. In certain embodiments, the base is an iodide salt. In certain embodiments, the base is KI. In certain embodiments, the alkylation step is carried out in a solvent. In certain embodiments, the solvent is acetonitrile (MeCN). In certain embodiments, the reaction is carried out at room temperature. In certain embodiments, the reaction is carried out at high temperature. In certain embodiments, the reaction is carried out at about 100 ° C.

[0424] As defined herein, X 2 is a halogen or a leaving group. 2 In certain embodiments, X 2 is -Cl, -Br or -I. In certain embodiments, X 2 is -Br. In certain embodiments, X 2 It is a leaving group.

[0425] As defined herein, each R 3 are independently optionally substituted alkyl, optionally substituted acyl or an oxygen protecting group; or optionally, two R 3 and the middle atom are connected together to form an optionally substituted heterocyclic group. 3 is an optionally substituted alkyl. 3 is an optionally substituted acyl group. 3 is an oxygen protecting group. 3 With the middle atom connected together, form the optionally substituted heterocyclic group. In certain embodiments, two R 3 Joined together with the middle atom to form: In certain embodiments, both R 3 Joined together with the middle atom to form:

[0426] In certain embodiments, the method further comprises: in the presence of formamidine, A compound or a tautomer thereof or its salt to obtain the formula In certain embodiments, the reaction requires (i) reacting a compound of formula (IV) or a salt thereof in the presence of formamidine to form an intermediate; and (ii) reacting the intermediate formed in step (i) with another compound of formula (IV) or a salt thereof to obtain the above product.

[0427] In certain embodiments, the formamidine in step (i) is diphenylformamidine. In certain embodiments, the reaction in step (i) is carried out in the presence of a base. In certain embodiments, the base is a pyridine base. In certain embodiments, the base is DMAP. In certain embodiments, the reaction in step (i) is carried out in the presence of an anhydride. In certain embodiments, the anhydride is acetic anhydride (Ac 2 In some embodiments, the reaction in step (i) is carried out at an elevated temperature (e.g., about 125° C.). In some embodiments, the reaction is carried out in a solvent. In some embodiments, the reaction in step (ii) is carried out in the presence of a base. In some embodiments, the base is an amine base (e.g., a trialkylamine base). In some embodiments, the base is Et 3 N. wherein the reaction is carried out in the presence of a base. In certain embodiments, the reaction is carried out in a solvent. In certain embodiments, the solvent is EtOH. In certain embodiments, the reaction is carried out at an elevated temperature (eg, about 80°C).

[0428] In certain embodiments, the product has the formula:

[0429]

[0430] or a salt thereof.

[0431] In certain embodiments, the method further comprises: in the presence of an acid, The compound or its salt is cyclized to obtain Compound or a salt thereof. In certain embodiments, the acid is sulfonic acid. In certain embodiments, the acid is sulfuric acid. In certain embodiments, the reaction is carried out in a solvent. In certain embodiments, the solvent is CH 2 Cl 2 In certain embodiments, the reaction is carried out at an elevated temperature (eg, about 60°C).

[0432] In certain embodiments, the product has the formula:

[0433]

[0434] or a salt thereof.

[0435] In certain embodiments, the method further comprises the following steps: deprotecting the compound or its salt,

[0436] The following compound is obtained:

[0437]

[0438] or a salt thereof.

[0439] In certain embodiments, the compound has the formula:

[0440]

[0441] or a salt thereof.

[0442] In certain embodiments, the method further comprises the following steps:

[0443] (i) Protective A compound or a salt thereof of the formula

[0444] or a salt thereof; and

[0445] (ii) sufficient to form The compound prepared in step (i) is reacted under the conditions of: a compound or a salt thereof.

[0446] definition

[0447] Chemical Definition

[0448] Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th ed., inside pages), and specific functional groups are generally defined as described herein. Additionally, Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd General principles of organic chemistry as well as specific functional moieties and reactivities are described in Organic Chemistry: Organic Chemistry: Organic Chemistry Edition, Cambridge University Press, Cambridge, 1987.

[0449] The compounds described herein may contain one or more asymmetric centers and may therefore exist in various stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention further includes compounds in the form of individual isomers substantially free of other isomers, or in the form of various isomeric mixtures.

[0450] Unless otherwise stated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. 18 F instead 19 F or use 13 C or 14 C instead 12 C, compounds having the present structure are within the scope of the present invention. Such compounds can be used, for example, as analytical tools or probes in biological assays.

[0451] When a range of values ​​is listed, it is intended to include every value and sub-range within that range. For example, "C 1-6 The term "alkyl" is intended to encompass C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1-6 , C 1-5 , C1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0452] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term "heteroaliphatic" refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

[0453] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 10 carbon atoms ("C 1-10 In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C 1-9 In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C 1-8 In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C 1-7 In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C 1-6 In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C 1-5 In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C 1-4 In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C 1-3 In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C 1-2 In some embodiments, an alkyl group has 1 carbon atom (“C 1 In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2-6 C 1-6 Examples of alkyl groups include methyl (C 1 ), ethyl (C 2 ), propyl (C 3 ) (e.g., n-propyl, isopropyl), butyl (C 4 ) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C 5 ) (e.g., n-pentyl, 3-pentyl, pentyl, neopentyl, 3-methyl-2-butyl, tert-pentyl) and hexyl (C 6) (e.g., n-hexyl). Other examples of alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ) etc. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (“unsubstituted alkyl”) or substituted (“substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, an alkyl group is an unsubstituted C 1-10 Alkyl (e.g., unsubstituted C 1-6 Alkyl groups, such as -CH 3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, such as unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, such as unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is substituted C 1-10 Alkyl (e.g. substituted C 1-6 Alkyl, such as -CF 3 , Bn).

[0454] The term "haloalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a halogen, such as fluorine, bromine, chlorine, or iodine. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C 1-6 In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C 1-4 In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C 1-3 In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C 1-2 Examples of haloalkyl groups include -CHF 2 、-CH 2 F, -CF 3 、-CH 2 CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 , -CCl 3 , -CFCl 2 , -CF 2 Cl et al.

[0455] The term "heteroalkyl" refers to an alkyl group that also includes at least one (e.g., 1, 2, 3, or 4 heteroatoms) heteroatom selected from oxygen, nitrogen, or sulfur, wherein the heteroatom is within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or is placed at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group ("heteroalkyl"). 1-10 In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-9 In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-8 In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-7 In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-6 In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroalkyl”). 1-5 In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroalkyl”). 1-4 In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroalkyl”). 1-3 In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroalkyl”). 1-2 In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC 1 In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroalkyl”). 2-6 Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, a heteroalkyl group is an unsubstituted heteroalkyl group. 1-10 In certain embodiments, the heteroalkyl group is a substituted heteroC 1-10 alkyl.

[0456] The term "alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C 2-9 In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C 2-8 In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C 2-7 In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C 2-6 In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C 2-5 In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C 2-4 In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C 2-3 In some embodiments, an alkenyl group has 2 carbon atoms (“C 2 The one or more carbon-carbon double bonds may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). 2-4 Examples of alkenyl groups include vinyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadiene (C 4 ) etc. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkenyl groups and pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ) and the like. Other examples of alkenyl groups include heptenyl (C 7 ), octenyl (C 8 ), octatrienyl (C 8 ) etc. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, an alkenyl group is an unsubstituted C 2-10 In certain embodiments, an alkenyl group is a substituted C 2-10 In an alkenyl group, a C=C double bond whose stereochemistry is not specified (e.g., -CH=CHCH 3 or ) can be an (E)- or (Z)-double bond.

[0457] The term "heteroalkenyl" refers to an alkenyl group that also includes at least one (e.g., 1, 2, 3, or 4 heteroatoms) heteroatom selected from oxygen, nitrogen, or sulfur, which is within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or placed at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkenyl group is a group having 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms in the parent chain ("heteroalkenyl"). 2-10 In some embodiments, a heteroalkenyl group is a group having 2 to 9 carbon atoms, at least one double bond, and 1 or more heteroatoms in the parent chain (“heteroC 2-9 In some embodiments, a heteroalkenyl group is a group having 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms in the parent chain (“heteroC 2-8 In some embodiments, a heteroalkenyl group is a group having 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms in the parent chain (“heteroC 2-7 In some embodiments, a heteroalkenyl group is a group having 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms in the parent chain (“heteroC 2-6 In some embodiments, a heteroalkenyl group is a group having 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain (“heteroC 2-5 In some embodiments, a heteroalkenyl group is a group having 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain (“heteroC 2-4 In some embodiments, a heteroalkenyl group is a group having 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom in the parent chain (“heteroC 2-3 In some embodiments, a heteroalkenyl group is a group having 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms in the parent chain (“heteroC 2-6 Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, a heteroalkenyl group is an unsubstituted heteroC 2-10 In certain embodiments, a heteroalkenyl group is a substituted heteroC 2-10 Alkenyl.

[0458] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical ("C 2-10 In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C 2-9In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C 2-8 In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C 2-7 In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C 2-6 In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C 2-5 In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C 2-4 In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C 2-3 In some embodiments, an alkynyl group has 2 carbon atoms (“C 2 The one or more carbon-carbon triple bonds may be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). 2-4 Examples of alkynyl groups include, but are not limited to, ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ) etc. 2-6 Examples of the alkynyl group include the above-mentioned C 2-4 Alkynyl and pentynyl (C 5 ), hexynyl (C 6 ) and the like. Other examples of alkynyl groups include heptynyl (C 7 ), octynyl (C 8 ) and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, an alkynyl group is an unsubstituted C 2-10 In certain embodiments, the alkynyl group is a substituted C 2-10 Alkynyl.

[0459] The term "heteroalkynyl" refers to an alkynyl group that also includes at least one (e.g., 1, 2, 3, or 4 heteroatoms) heteroatom selected from oxygen, nitrogen, or sulfur, which is within the parent chain (e.g., inserted between adjacent carbon atoms of the parent chain) and / or placed at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms in the parent chain ("heteroalkynyl"). 2-10 In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“hetero C 2-9In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“hetero C 2-8 In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“hetero C 2-7 In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“hetero C 2-6 In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“hetero C 2-5 In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“hetero C 2-4 In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom (“hetero C 2-3 In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“hetero C 2-6 Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, a heteroalkynyl group is an unsubstituted heteroalkynyl. 2-10 In certain embodiments, a heteroalkynyl group is a substituted heteroC 2-10 Alkynyl.

[0460] The term "carbocyclyl" or "carbocyclic" refers to a non-aromatic ring system having 3 to 14 ring carbon atoms (C 3-14 In some embodiments, the carbocyclyl group has 3 to 10 ring carbon atoms ("C 3-10 In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C 3-8 In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C 3-7 In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C 3-6 In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C 4-6 In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C 5-6 In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10 "Carbocyclyl"). Exemplary C 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ) etc. Exemplary C 3-8 Carbocyclyl groups include but are not limited to the above C 3-6 Carbocyclyl groups and cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptyl(C 7 ), bicyclo[2.2.2]octyl(C 8 ) etc. Exemplary C 3-10 Carbocyclyl groups include but are not limited to the above C 3-8 Carbocyclyl groups and cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As shown in the above examples, in certain embodiments, the carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused ring, a bridged ring or a spirocyclic system, such as a bicyclic system ("bicyclic carbocyclyl") or a tricyclic system ("tricyclic carbocyclyl")), may be saturated, and may contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which the carbocyclyl ring as defined herein is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in this case, the number of carbons continues to represent the number of carbons in the carbocyclic ring system. Unless otherwise indicated, each instance of the carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted ("substituted carbocyclyl") with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C 3-14 In certain embodiments, a carbocyclyl group is a substituted C3-14 Carbocyclic group.

[0461] In some embodiments, "carbocyclyl" is a monocyclic saturated carbocyclyl ("C 3-14 In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C 3-10 In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C 3-8 In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C 3-6 In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C 4-6 In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C 5-6 In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C 5-10 Cycloalkyl”) C 5-6 Examples of cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ). 3-6 Examples of the cycloalkyl group include the above-mentioned C 5-6 Cycloalkyl groups and cyclopropyl (C 3 ) and cyclobutyl (C 4 ). C 3-8 Examples of the cycloalkyl group include the above-mentioned C 3-6 Cycloalkyl groups and cycloheptyl (C 7 ) and cyclooctyl (C 8 Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, a cycloalkyl group is an unsubstituted C 3-14 In certain embodiments, a cycloalkyl group is a substituted C 3-14 Cycloalkyl.

[0462] The term "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("3-14 membered heterocyclyl"). In heterocyclyl radicals that include one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, where valence permits. Heterocyclyl radicals may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged or spirocyclic systems, such as bicyclic systems ("bicyclic heterocyclyl") or tricyclic systems ("tricyclic heterocyclyl")), may be saturated, and may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic systems may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the heterocyclyl ring as defined herein is fused to one or more carbocyclyl groups, where the point of attachment is on the carbocyclyl or heterocyclyl ring, or includes ring systems in which the heterocyclyl ring as defined herein is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring, and in this case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") with one or more substituents. In certain embodiments, heterocyclyl groups are unsubstituted 3-14 membered heterocyclyls. In certain embodiments, heterocyclyl groups are substituted 3-14 membered heterocyclyls.

[0463] In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heterocyclyl”). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-8 membered heterocyclyl”). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0464] Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolyl, phthalimidyl, naphthylimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydro pyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

[0465] The term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared electrons in the cyclic array) having 6-14 ring carbon atoms and zero heteroatoms in the aromatic ring system ("C 6-14 In some embodiments, an aryl group has 6 ring carbon atoms (“C 6 In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl" also includes ring systems in which an aryl ring, as defined herein, is fused to one or more carbocyclyl or heterocyclyl groups, wherein the radical or point of attachment is on the aryl ring, and in such a case, the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, an aryl group is an unsubstituted C 6-14 In certain embodiments, an aryl group is a substituted C 6-14 Aryl.

[0466] The term "heteroaryl" refers to a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared electrons in the cyclic array) having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system. Where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). Where valence permits, in heteroaryl groups that include one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined herein is fused to one or more carbocyclic or heterocyclic groups, wherein the point of attachment is on the heteroaryl ring, and in this case, the number of ring members continues to represent the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in this case, the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. For polycyclic heteroaryl groups in which one ring does not contain heteroatoms (e.g., indolyl, quinolyl, carbazolyl, etc.), the point of attachment can be on either ring, for example, the ring with the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

[0467] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-10 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted ("substituted heteroaryl") with one or more substituents. In certain embodiments, a heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, a heteroaryl group is a substituted 5-14 membered heteroaryl.

[0468] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups including 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthrenylpyridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

[0469] The term "unsaturated bond" refers to a double bond or a triple bond.

[0470] The term "unsaturated" or "partially unsaturated" refers to a moiety that includes at least one double or triple bond.

[0471] The term "saturated" refers to a moiety that includes no double or triple bonds, ie, the moiety includes only single bonds.

[0472] The prefix "-ene" appended to a group indicates that the group is a divalent moiety, for example, alkylene is a divalent moiety of alkyl, alkenylene is a divalent moiety of alkenyl, alkynylene is a divalent moiety of alkynyl, heteroalkylene is a divalent moiety of heteroalkyl, heteroalkenylene is a divalent moiety of heteroalkenyl, heteroalkynylene is a divalent moiety of heteroalkynyl, carbocyclylene is a divalent moiety of carbocyclyl, heterocyclylene is a divalent moiety of heterocyclyl, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0473] Unless otherwise expressly stated, a group is optionally substituted. The term "optionally substituted" refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclic radical, heterocyclic radical, aryl and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a group that can be substituted or unsubstituted (for example, "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclic radical, "substituted" or "unsubstituted" heterocyclic radical, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl groups). Typically, the term "substituted" refers to at least one hydrogen present on a group being replaced by a permitted substituent, for example, a substituent that produces a stable compound when substituted, such as a compound that will not spontaneously undergo transformation by, for example, rearrangement, cyclization, elimination, or other reactions. Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent may be the same or different at each position. It is contemplated that the term "substituted" includes substitution by all permitted substituents of an organic compound, and includes any substituent described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to obtain a stable compound. For purposes of the present invention, heteroatoms such as nitrogen may have a hydrogen substituent and / or any suitable substituent described herein that satisfies the heteroatom valence and results in the formation of a stable portion. The present invention is not intended to be limited in any way to the exemplary substituents described herein.

[0474] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO 2 、-N 3 、-SO 2 H, -SO 3 H, -OH, -OR aa 、-ON(R bb ) 2 、-N(R bb ) 2 、-N(R bb ) 3 + X - 、-N(OR cc )R bb , -SH, -SR aa 、-SSR cc 、-C(=O)R aa , -CO 2 H, -CHO, -C(ORcc ) 3 、-CO 2 R aa 、-OC(=O)R aa 、-OCO 2 R aa 、-C(=O)N(R bb ) 2 、-OC(=O)N(R bb ) 2 、-NR bb C(=O)R aa 、-NR bb CO 2 R aa 、-NR bb C(=O)N(R bb ) 2 、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb ) 2 、-OC(=NR bb )N(R bb ) 2 、-NR bb C(=NR bb )N(R bb ) 2 、-C(=O)NR bb SO 2 R aa 、-NR bb SO 2 R aa 、-SO 2 N(R bb ) 2 、-SO 2 R aa 、-SO 2 OR aa 、-OSO 2 R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa ) 3 、-OSi(R aa ) 3 -C(=S)N(R bb) 2 、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)(R aa ) 2 、-P(=O)(OR cc ) 2 、-OP(=O)(R aa ) 2 、-OP(=O)(OR cc ) 2 、-P(=O)(N(R bb ) 2 ) 2 、-OP(=O)(N(R bb ) 2 ) 2 、-NR bb P(=O)(R aa ) 2 、-NR bb P(=O)(OR cc ) 2 、-NR bb P(=O)(N(R bb ) 2 ) 2 、-P(R cc ) 2 、-P(OR cc ) 2 、-P(R cc ) 3 + X - 、-P(OR cc ) 3 + X - 、-P(R cc ) 4 、-P(OR cc ) 4 、-OP(R cc ) 2 、-OP(R cc ) 3 + X - 、-OP(OR cc ) 2 、-OP(OR cc )3 + X - 、-OP(R cc ) 4 ,

[0475] BY20IM3317FGPC-CN.Telephone OA-OP(OR cc ) 4 ,-B(R aa ) 2 、-B(OR cc ) 2 , -BR aa (OR cc ), C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution; wherein X - is the counter ion;

[0476] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb ) 2 , =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O) 2 R aa , =NR bb or = NOR cc replace;

[0477] R aa Each case of is independently selected from C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14Aryl and 5-14 membered heteroaryl, or two R aa The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0478] R bb Each instance of is independently selected from hydrogen, -OH, -OR aa 、-N(R cc ) 2 、-CN、-C(=O)R aa 、-C(=O)N(R cc ) 2 , -CO 2 R aa 、-SO 2 R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc ) 2 、-SO 2 N(R cc ) 2 、-SO 2 R cc 、-SO 2 OR cc 、-SOR aa 、-C(=S)N(R cc ) 2 、-C(=O)SR cc 、-C(=S)SR cc ,-P(=O)(R aa ) 2 、-P(=O)(OR cc ) 2 、-P(=O)(N(R cc ) 2 ) 2 , C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R bbThe groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution; wherein X - is the counter ion;

[0479] R cc Each instance of is independently selected from hydrogen, C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R cc The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R dd Group substitution;

[0480] R dd Each instance of is independently selected from halogen, -CN, -NO 2 、-N 3 、-SO 2 H, -SO 3 H, -OH, -OR ee 、-ON(R ff ) 2 、-N(R ff ) 2 、-N(R ff ) 3 + X - 、-N(OR ee )R ff , -SH, -SR ee 、-SSR ee 、-C(=O)R ee , -CO 2 H, -CO 2 R ee 、-OC(=O)R ee 、-OCO 2 R ee 、-C(=O)N(R ff ) 2 、-OC(=O)N(R ff )2 , -NR ff C(=O)R ee , -NR ff CO 2 R ee , -NR ff C(=O)N(R ff ) 2 , -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(R ff ) 2 , -OC(=NR ff )N(R ff ) 2 , -NR ff C(=NR ff )N(R ff ) 2 , -NR ff SO 2 R ee , -SO 2 N(R ff ) 2 , -SO 2 R ee , -SO 2 OR ee , -OSO 2 R ee , -S(=O)R ee , -Si(R ee ) 3 , -OSi(R ee ) 3 , -C(=S)N(R ff ) 2 , -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)(OR ee ) 2 , -P(=O)(R ee ) 2 , -OP(=O)(R ee ) 2 , -OP(=O)(OR ee ) 2 , C 1-6 alkyl, C 1-6 perhaloalkyl, C 2-6Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, 3-10 membered heterocyclic group, C 6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may be linked to form =O or =S; where X - is the counter ion;

[0481] R ee Each case of is independently selected from C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, C 6-10 aryl, 3-10 membered heterocyclyl and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution;

[0482] R ff Each instance of is independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, 3-10 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, or two R ff The groups are connected to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R gg group substitution; and

[0483] R gg Each instance of is independently selected from halogen, -CN, -NO 2 、-N 3、-SO 2 H, -SO 3 H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 alkyl) 2 、-N(C 1-6 alkyl) 2 、-N(C 1-6 alkyl) 3 + X - 、-NH(C 1-6 alkyl) 2 + X - 、-NH 2 (C 1-6 alkyl) + X - 、-NH 3 + X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 Alkyl), -CO 2 H, -CO 2 (C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO 2 (C 1-6 alkyl), -C(=O)NH 2 、-C(=O)N(C 1-6 alkyl) 2 、-OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO 2 (C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl) 2 、-NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH 2 、-C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C1-6 alkyl) 2 、-C(=NH)NH(C 1-6 alkyl), -C(=NH)NH 2 、-OC(=NH)N(C 1-6 alkyl) 2 、-OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH 2 、-NHC(=NH)N(C 1-6 alkyl) 2 、-NHC(=NH)NH 2 、-NHSO 2 (C 1-6 Alkyl), -SO 2 N(C 1-6 alkyl) 2 、-SO 2 NH(C 1-6 Alkyl), -SO 2 NH 2 、-SO 2 (C 1-6 Alkyl), -SO 2 O(C 1-6 Alkyl), -OSO 2 (C 1-6 Alkyl), -SO(C 1-6 Alkyl), -Si(C 1-6 alkyl) 3 、-OSi(C 1-6 alkyl) 3 -C(=S)N(C 1-6 alkyl) 2 、C(=S)NH(C 1-6 alkyl), C(=S)NH 2 、-C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)(OC 1-6 alkyl) 2 ,-P(=O)(C 1-6 alkyl) 2 ,-OP(=O)(C 1-6 alkyl) 2 ,-OP(=O)(OC 1-6 alkyl) 2 , C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, C 6-10 Aryl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl; or two geminal R gg Substituents may be linked to form =O or =S; where X - It is a counter ion.

[0484] In certain embodiments, carbon atom substituents include: halogen, -CN, -NO 2 、-N 3 、-SO 2 H, -SO 3 H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 alkyl) 2 、-N(C 1-6 alkyl) 2 、-N(C 1-6 alkyl) 3 + X - 、-NH(C 1-6 alkyl) 2 + X - 、-NH 2 (C 1-6 alkyl) + X - 、-NH 3 + X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 Alkyl), -CO 2 H, -CO 2 (C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO 2 (C 1-6 alkyl), -C(=O)NH 2 、-C(=O)N(C 1-6 alkyl) 2 、-OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C1-6 Alkyl), -NHCO 2 (C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl) 2 、-NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH 2 、-C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 alkyl) 2 、-C(=NH)NH(C 1-6 alkyl), -C(=NH)NH 2 、-OC(=NH)N(C 1-6 alkyl) 2 、-OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH 2 、-NHC(=NH)N(C 1-6 alkyl) 2 、-NHC(=NH)NH 2 、-NHSO 2 (C 1-6 Alkyl), -SO 2 N(C 1-6 alkyl) 2 、-SO 2 NH(C 1-6 Alkyl), -SO 2 NH 2 、-SO 2 (C 1-6 Alkyl), -SO 2 O(C 1-6 Alkyl), -OSO 2 (C 1-6 Alkyl), -SO(C 1-6 Alkyl), -Si(C 1-6 alkyl) 3 、-OSi(C 1-6 alkyl) 3 -C(=S)N(C 1-6 alkyl) 2 、C(=S)NH(C 1-6 alkyl), C(=S)NH 2 、-C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6Alkyl, -P(=O)(OC 1-6 alkyl) 2 ,-P(=O)(C 1-6 alkyl) 2 ,-OP(=O)(C 1-6 alkyl) 2 ,-OP(=O)(OC 1-6 alkyl) 2 , C 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, C 6-10 Aryl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl; or two geminal R gg Substituents may be linked to form =O or =S; where X - It is a counter ion.

[0485] The term "halo" or "halogen" refers to fluorine (F, -F), chlorine (Cl, -Cl), bromine (Br, -Br), or iodine (I, -I).

[0486] The term "hydroxy" refers to the group -OH. By extension, the term "substituted hydroxy" refers to a hydroxy group in which the oxygen atom directly attached to the parent molecule is replaced by a group other than hydrogen, and includes groups selected from: -OR aa 、-ON(R bb ) 2 、-OC(=O)SR aa 、-OC(=O)R aa 、-OCO 2 R aa 、-OC(=O)N(R bb ) 2 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-OC(=NR bb )N(R bb ) 2 、-OS(=O)R aa 、-OSO 2 R aa 、-OSi(R aa ) 3 、-OP(R cc ) 2 、-OP(R cc )3 + X - 、-OP(OR cc ) 2 、-OP(OR cc ) 3 + X - ,-OP(=O)(R aa ) 2 ,-OP(=O)(OR cc ) 2 and-OP(=O)(N(R bb ) 2 ) 2 , where X - , R aa , R bb and Rcc are as defined herein.

[0487] The term "amino" refers to the group -NH 2 By extension, the term "substituted amino" refers to mono-substituted amino, di-substituted amino or tri-substituted amino. In certain embodiments, "substituted amino" is mono-substituted amino or di-substituted amino.

[0488] The term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by one hydrogen and one group other than hydrogen, and includes groups selected from the group consisting of: -NH(R bb )、-NHC(=O)R aa 、-NHCO 2 R aa 、-NHC(=O)N(R bb ) 2 、-NHC(=NR bb )N(R bb ) 2 、-NHSO 2 R aa 、-NHP(=O)(OR cc ) 2 and -NHP(=O)(N(R bb ) 2 ) 2 , where R aa , R bb and R cc As defined herein, and wherein the group -NH(R bb ) bb Not hydrogen.

[0489] The term "disubstituted amino" refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with two groups other than hydrogen, and includes groups selected from: -N(R bb ) 2 、-NR bb C(=O)R aa 、-NR bb CO 2 R aa 、-NR bb C(=O)N(R bb ) 2 、-NR bb C(=NR bb )N(R bb ) 2 、-NR bb SO 2 R aa 、-NR bb P(=O)(OR cc ) 2 and-NR bb P(=O)(N(R bb ) 2 ) 2 , where R aa , R bb and R cc As defined herein, provided that the nitrogen atom directly attached to the parent molecule is not replaced by hydrogen.

[0490] The term "trisubstituted amino" refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted by three groups and includes groups selected from: -N(R bb ) 3 and -N(R bb ) 3 + X - , where R bb and X - As defined herein.

[0491] The term "sulfonyl" refers to a group selected from: -SO 2 N(R bb ) 2 ,–SO 2 R aa and–SO 2 OR aa , where R aa and R bb As defined herein.

[0492] The term "sulfinyl" refers to the group -S(=O)R aa , where Raa As defined herein.

[0493] The term "acyl" refers to an acyl group having the general formula -C(=O)R X1 、-C(=O)OR X1 、-C(=O)-OC(=O)R X1 、-C(=O)SR X1 、-C(=O)N(R X1 ) 2 、-C(=S)R X1 、-C(=S)N(R X1 ) 2 、-C(=S)O(R X1 )、-C(=S)S(R X1 ),-C(=NR X1 )R X1 、-C(=NR X1 )OR X1 、-C(=NR X1 )SR X1 and -C(=NR X1 )N(R X1 ) 2 , where R X1 is hydrogen; halogen; substituted or unsubstituted hydroxy; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain alkyl; cyclic or acyclic, substituted or unsubstituted, branched or straight-chain alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphaticthio, heteroaliphaticthio, alkylthio, heteroalkylthio, arylthio, heteroarylthio, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two R X1 The groups together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO 2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates and ureas. Acyl substituents include, but are not limited to, any substituent described herein that results in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo (thiooxo), cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halogen, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkoxy, aryloxy, heteroaryloxy, aliphatic thiooxy, heteroaliphatic thio, alkylthio, heteroalkylthio, arylthio, heteroarylthio, acyloxy, etc., each of which may or may not be further substituted).

[0494] The term "carbonyl" refers to a group in which the carbon directly attached to the parent molecule is replaced by sp 2 hybridized and substituted with an oxygen, nitrogen or sulfur atom, for example, a group selected from the group consisting of: ketone (e.g. -C(=O)R aa ), carboxylic acids (e.g. –CO 2 H), aldehydes (–CHO), esters (such as CO 2 R aa 、–C(=O)SR aa , –C(=S)SR aa ), amides (e.g. –C(=O)N(R bb ) 2 、–C(=O)NR bb SO 2 R aa 、-C(=S)N(R bb ) 2 ) and imines (e.g. –C(=NR bb )R aa , –C(=NR bb )OR aa ),–C(=NR bb )N(R bb ) 2 ), where R aa and R bb As defined herein.

[0495] The term "silyl" refers to the group -Si(R aa ) 3 , where R aa As defined herein.

[0496] The term "oxo" refers to the group =0 and the term "thioxo" refers to the group =S.

[0497] The nitrogen atoms may be substituted or unsubstituted where valence permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc ) 2 、-CN、-C(=O)R aa 、-C(=O)N(R cc ) 2 , -CO 2 R aa 、-SO 2 R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc ) 2 、-SO 2 N(R cc ) 2 、-SO 2 R cc 、-SO 2 OR cc 、-SOR aa 、-C(=S)N(R cc ) 2 、-C(=O)SR cc 、-C(=S)SR cc 、-P(=O)(OR cc ) 2 ,-P(=O)(R aa ) 2 、-P(=O)(N(R cc ) 2 ) 2 , C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl, or two R groups attached to the N atom cc The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R dd group substituted, and wherein Raa , R bb , R cc and R dd As defined above.

[0498] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an "amino protecting group"). Nitrogen protecting groups include, but are not limited to, -OH, -OR aa 、-N(R cc ) 2 、-C(=O)R aa 、-C(=O)N(R cc ) 2 , -CO 2 R aa 、-SO 2 R aa 、-C(=NR cc )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc ) 2 、-SO 2 N(R cc ) 2 、-SO 2 R cc 、-SO 2 OR cc 、-SOR aa 、-C(=S)N(R cc ) 2 、-C(=O)SR cc 、-C(=S)SR cc , C 1-10 Alkyl (e.g., aralkyl, heteroaralkyl), C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R dd group substituted, and wherein R aa , R bb , R cc and R ddAs defined herein. Nitrogen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999 (which is incorporated herein by reference).

[0499] For example, a nitrogen protecting group such as an amide group (e.g., -C(=O)R aa ) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrobenzamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyamido)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0500] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR aa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthenyl)]methylcarbamate (DBD-Tmoc), 4-methoxybenzoylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (h Z), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-bicyclo 1-Hexylcarboxamido)ethylcarbamate, tert-butyl carbamate (BOC or Boc), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), allylcarbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamylcarbamate (Coc), 4-nitrocinnamylcarbamate (Noc), 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p-nitrobenzylcarbamate, p-bromobenzylcarbamate , p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-Dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate carbamate, Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarbamoylamino)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarbamoylamino)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isoborynylcarbamate, isobutylcarbamate, isonicotinoylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenyl carbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate and 2,4,6-trimethylbenzylcarbamate. ,

[0501] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O) 2 R aa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylsulfonamide.

[0502] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N′-toluenesulfonylaminoacyl derivatives, N′-phenylaminosulfonyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilaziridine adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-ones, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-ones, 1-substituted 3,5-dinitro- 4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide ( N'-oxide), N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)m-tolyl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylidineamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenyl Boric acid derivatives, N-[phenyl(pentacylchromium or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, N-amine oxides, diphenylphosphinamide (Dpp), dimethylphosphinamide (Mpt), diphenylphosphinamide (Ppt), dialkylphosphinamides, dibenzylphosphinamides, diphenylphosphinamides, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).In certain embodiments, the nitrogen protecting group is benzyl (Bn), tert-butoxycarbonyl (BOC), carbonylbenzyloxy (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethoxycarbonyl (Troc), trityl (Tr), tosyl (Ts), bromobenzenesulfonyl (Bs), nitrobenzenesulfonyl (nosyl, Ns), methanesulfonyl (Ms), trifluoromethanesulfonyl (triflyl, Tf) or dansyl (dansyl, Ds).

[0503] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to herein as a "hydroxy protecting group"). Oxygen protecting groups include, but are not limited to, -R aa 、-N(R bb ) 2 、-C(=O)SR aa 、-C(=O)R aa , -CO 2 R aa 、-C(=O)N(R bb ) 2 、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )N(R bb ) 2 、-S(=O)R aa 、-SO 2 R aa 、-Si(R aa ) 3 、-P(R cc ) 2 、-P(R cc ) 3 + X - 、-P(OR cc ) 2 、-P(OR cc ) 3 + X - ,-P(=O)(R aa ) 2 、-P(=O)(OR cc ) 2 and -P(=O)(N(R bb ) 2 ) 2 , where X- , R aa , R bb and R cc As defined herein. Oxygen protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999 (incorporated herein by reference).

[0504] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), o-methoxyphenolmethyl (guaiacolmethyl, GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (S EMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methylbenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl- 1-Benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylseleno)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridylmethyl, 4-pyridylmethyl, 3-methyl-2-pyridylmethyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzocycloheptyl (5- dibenzosuberyl), triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromobenzoyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"'-tris(levulinyloxyphenyl)methyl, 4,4',4"'-tris(levulinoyloxyphenyl)methyl, 4,4',4"'-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-Bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S, S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenyl Silyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoic acid (levulinic acid ester), 4,4-(ethylenedithio)pentanoic acid (levulinic acid dithioacetaldehyde), pivalate, adamantanoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2 , 4,6-trimethylbenzoate, methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphino)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, tert-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate carbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamino ester, alkyl N-phenyl carbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, mesylate, benzyl sulfonate and tosylate (Ts). In certain embodiments, the oxygen protecting group is a silyl group. In certain embodiments, the oxygen protecting group is tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxy ethyl (EE), 2-methoxy-2-propyl (MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP), trityl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl, p-methoxybenzyl (PMB), tert-butyl, benzyl (Bn), allyl or pivaloyl (Piv). ,

[0505] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a "thiol protecting group"). Sulfur protecting groups include, but are not limited to: -R aa 、-N(R bb ) 2 、-C(=O)SR aa 、-C(=O)R aa , -CO 2 R aa 、-C(=O)N(R bb ) 2 、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )N(R bb ) 2 、-S(=O)R aa 、-SO 2 R aa 、-Si(R aa ) 3 、-P(Rcc ) 2 、-P(R cc ) 3 + X - 、-P(OR cc ) 2 、-P(OR cc ) 3 + X - ,-P(=O)(R aa ) 2 、-P(=O)(OR cc ) 2 and -P(=O)(N(R bb ) 2 ) 2 , where R aa , R bb and R cc As defined herein. Sulfur protecting groups are well known in the art and include those described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3 rd edition, John Wiley & Sons, 1999 (incorporated herein by reference). In certain embodiments, the sulfur protecting group is acetamidomethyl, tert-butyl, 3-nitro-2-pyridinesulfinyl, 2-pyridine-sulfinyl or triphenylmethyl.

[0506] A "counterion" or "anionic counterion" is a negatively charged group that associates with a positively charged group to maintain electronic neutrality. Anionic counterions can be monovalent (i.e., include one formal negative charge). Anionic counterions can also be multivalent (i.e., include more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halides (e.g., F – , Cl – Br – ,I – ), NO 3 – , ClO 4 – OH – , H 2 PO 4 – , HCO 3 - , HSO 4 –, sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.), carboxylate ions (e.g., acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, etc.), BF 4 - PF 4 – PF 6 – , AsF 6 – , SbF 6 – , B[3,5-(CF 3 ) 2 C 6 H 3 ] 4 ] – , B(C 6 F 5 ) 4 - BPh 4 – 、Al(OC(CF 3 ) 3 ) 4 – and carborane anions (e.g., CB 11 H 12 – or (HCB 11 Me 5 Br 6 ) – ). Exemplary counterions that may be multivalent include CO 3 2- , HPO 4 2- ,PO 4 3- , B 4 O 7 2- 、SO 4 2- , S 2 O 3 2- , carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelaate, sebacate, salicylate, phthalate, aspartic acid, glutamic acid, etc.), and carborane.

[0507] The term "leaving group" has its usual meaning in the field of synthetic organic chemistry and refers to an atom or group that can be substituted by a nucleophilic reagent. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Examples of suitable leaving groups include, but are not limited to, halogen (e.g., F, Cl, Br or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arylsulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N, O-dimethylhydroxylamino, pixyl and haloformate. In some cases, the leaving group is a sulfonate such as tosylate (-OTs), mesylate (-OMs), p-bromobenzenesulfonyloxy (bromoformate, -OBs), -OS (=O) 2 (CF 2 ) 3 CF 3 In some cases, the leaving group is a bromoformate, such as bromobenzenesulfonyloxy. In some cases, the leaving group is a nonanoate, such as 2-nitrobenzenesulfonyloxy. The leaving group can also be a phosphine oxide (e.g., formed during the Mitsunobu reaction) or an internal leaving group, such as an epoxide or a cyclic sulfate. Other non-limiting examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties. Other exemplary leaving groups include, but are not limited to, halogens (e.g., chlorine, bromine, iodine) and activated substituted hydroxyl groups (e.g., –OC(=O)SR aa 、–OC(=O)R aa , –OCO 2 R aa 、–OC(=O)N(R bb ) 2 、–OC(=NR bb )R aa 、–OC(=NR bb )OR aa 、–OC(=NR bb )N(R bb ) 2 、–OS(=O)R aa , –OSO 2 R aa ,–OP(R cc ) 2 ,–OP(R cc ) 3 , –OP(=O) 2 R aa ,–OP(=O)(R aa )2 ,–OP(=O)(OR cc ) 2 , –OP(=O) 2 N(R bb ) 2 and –OP(=O)(NR bb ) 2 , where R aa , R bb and R cc As defined herein.

[0508] As used herein, use of the phrase "at least one instance" refers to 1, 2, 3, 4 or more instances, but also encompasses ranges such as 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, or 3 to 4 instances, inclusive.

[0509] "Non-hydrogen group" refers to any non-hydrogen group defined for a particular variable.

[0510] The following definitions are of more general terms used throughout this application.

[0511] As used herein, the term "salt" refers to any and all salts, including pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to salts suitable for contact with human and lower animal tissues without excessive toxicity, irritation, allergic reactions, etc. within the scope of reasonable medical judgment, and they are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19 describes pharmaceutically acceptable salts in detail, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino groups with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or salts formed using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 Salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, where appropriate.

[0512] It is also understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

[0513] Stereoisomers that are not mirror images of one another are termed "diastereomers," while stereoisomers that are non-superimposable mirror images of one another are termed "enantiomers." When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers are characterized by the absolute configuration of their asymmetric center and are described by the R- and S-ordering rules of Cahn and Prelog, or by the way the molecules rotate the plane of polarized light and are termed right- or left-handed (i.e., (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is termed a "racemic mixture."

[0514] The term "catalysis" refers to the increase in the rate of a chemical reaction due to the participation of a substance called a "catalyst". In certain embodiments, the amount and properties of the catalyst remain essentially unchanged during the reaction. In certain embodiments, the catalyst is regenerated after the reaction, or the properties of the catalyst are substantially restored. A catalyst can participate in a variety of chemical transformations. The effect of a catalyst may vary due to the presence of other substances called inhibitors or poisons (reducing catalytic activity) or promoters (increasing activity). A catalytic reaction has a lower activation energy (rate-limiting free energy of activation) than a corresponding uncatalyzed reaction, thereby having a higher reaction rate at the same temperature. The catalyst may favorably affect the reaction environment, combine with a reagent to polarize a bond, form a specific intermediate that is not usually produced by an uncatalyzed reaction, or cause the reagent to dissociate into a reactive form.

[0515] The term "solvent" refers to a substance that can dissolve one or more solutes, thereby producing a solution. A solvent can serve as a medium for any reaction or transformation described herein. A solvent can dissolve one or more reactants or reagents in a reaction mixture. A solvent can promote the mixing of one or more reagents or reactants in a reaction mixture. A solvent can also serve to increase or decrease the reaction rate relative to a reaction in a different solvent. A solvent can be polar or non-polar, affinic or asexual. Common organic solvents useful in the methods are described herein.

[0516] Example

[0517] Synthesis of dual conjugated labels

[0518] Indolenine benzoate 2

[0519]

[0520] A 0.5M THF solution of 9-borane bicyclo [3.3.1] nonane (34.1 mL, 17.1 mmol) was added to a flask containing 3-butan-1-ol benzoate (3.0 g, 17.1 mmol). The clear colorless solution was stirred at room temperature for 3 hours. In another flask equipped with a condenser, iodoindoline (1, 3.4 g, 11.9 mmol), [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II) (0.7 g, 0.8 mmol) and cesium chloride (6.0 g, 18.5 mmol) were loaded. DMF (20 mL) was added to the flask, and the dark suspension was bubbled with argon for 10 minutes. After adding the benzoate borane / THF solution, the reaction was heated to 70 ° C for 12 hours. After this time, HPLC showed that the starting indolenine 1 had been completely converted. The reaction was cooled to room temperature, diluted with EtOAc (50 mL) and hexanes (50 mL), and filtered through celite. The organic layer was washed with water (3x) and dried over magnesium sulfate. After filtration and evaporation, the crude residue was purified by normal phase chromatography (0→50% EtOAc / hexanes, SiO 2 ) to give indolenine 2 (3.0 g, 75% yield) as a viscous yellow oil. 22 H 26 NO 2 (M+H) + The HRMS (ESI) calculated value was 336.1964 and the found value was 336.1962.

[0521] Alkylated indolenine 3

[0522]

[0523] Potassium iodide (2.0 g, 12.0 mmol) was added to a schlenk flask and flushed with argon. Indolenine (2, 2.0 g, 6.0 mmol) was added as a solution in acetonitrile (8.0 mL). 2-(2-bromoethyl)-1,3-dioxolane (1.4 mL, 12.0 mmol) was added and the sealed container was heated to 100 °C for 14 hours. The reaction was diluted with dichloromethane (20 mL) and the suspension was filtered through a sintered glass funnel. The reaction mixture was purified by normal phase chromatography (0 → 7% MeOH / DCM, SiO 2 ) The filtrate was directly purified to give indolenine 3 (1.9 g, 57% yield) as a beige solid. 27 H 34 NO 4 (M) + The HRMS (ESI) calculated value was 436.2488 and the found value was 436.2487.

[0524] Trimethylamine cyanine 4

[0525]

[0526] Acetic anhydride (5mL) was added to a mixture of indolenine (3, 0.96g, 1.7mmol), diphenylformamidine (0.40g, 2.1mmol) and DMAP (21mg, 0.17mmol). The brown mixture was heated to 120°C for 1 hour. After cooling to room temperature, the volatiles were concentrated in vacuo. Another portion of indolenine (3, 1.6g, 2.8mmol) was added to the crude intermediate, followed by ethanol (5mL) and trimethylamine (1.2mL, 8.6mmol). The reaction was heated to reflux for 1 hour under argon. The reactant was diluted with sodium chloride aqueous solution and extracted with DCM. The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo. The product was purified by normal phase chromatography (100% EtOAc, then 0 → 10% MeOH / DCM, SiO 2 ) was used to purify the crude residue to afford Cy34 (0.9 g, 57% yield) as a dark purple solid. 55 H 65 N 2 O 8 (M) + The HRMS (ESI) calculated value was 881.4735 and the found value was 881.4717.

[0527] Cy3B analog 5

[0528]

[0529] Chloroform (6 mL) and sulfuric acid (4 mL, 50% v / v water) were added to a flask containing Cy3 4 (400 mg, 0.437 mmol). The two-phase mixture was vigorously stirred at 60 ° C for 30 minutes, changing from deep red to purple. After cooling to room temperature, the reaction was diluted with water (20 mL) and extracted with EtOAc (50 mL). The organic layer was washed with saturated sodium chloride aqueous solution, dried over magnesium sulfate, filtered, and concentrated in vacuo. The product was purified by normal phase chromatography (100% EtOAc, then 0→15% MeOH / DCM, SiO 2 ) was used to purify the crude residue to afford Cy3B5 (177 mg, yield 50%) as a dark purple solid. 51 H 55 N 2 O 5 (M) + The HRMS (ESI) calculated value was 775.4105 and the found value was 775.4091.

[0530] Cy3B diol 6

[0531]

[0532] To a solution of Cy3B dibenzoate 5 (150 mg, 0.185 mmol) in methanol (4 mL) was added sodium methoxide (0.37 mL, 0.74 mmol, 2.0 M in MeOH). The reaction was heated to 70 °C for 1 hour. The reaction was quenched by adding solid ammonium chloride (66 mg) and stirred at room temperature for 30 minutes. The volatiles were concentrated in vacuo and the crude product was redissolved in DCM (10 mL). The suspension was filtered and concentrated in vacuo. The residue was purified by normal phase chromatography (100% EtOAc, then 0 → 25% MeOH / DCM, SiO 2 ) was used to purify the crude residue to afford Cy3B6 (99 mg, yield 86%) as a dark purple solid. 37 H 47 N 2 O 3 (M) + The HRMS (ESI) measured value was 567.3581 and the observed value was 567.3568

[0533] Cy3B Mono-MMT 7

[0534]

[0535] To a solution of Cy3B diol 6 (100 mg, 0.166 mmol) and monomethoxytrityl chloride (62 mg, 0.20 mmol) in dichloromethane was added pyridine (0.26 mL, 3.3 mmol). The dark purple reaction was stirred at room temperature for 30 minutes. The reaction was diluted with DCM and washed sequentially with water and saturated aqueous sodium chloride solution. The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The residue was purified by normal phase chromatography (100% EtOAc, then 0→25% 1% EtOAc). 3 N in MeOH / DCM, SiO 2 ) to afford Cy3B7 (43 mg, 30% yield) as a dark purple solid. 57 H 63 N 2 O 4 (M) + The HRMS (ESI) calculated value was 839.4782 and the found value was 839.4751.

[0536] Phosphoramidite 8

[0537]

[0538] 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (9.4 mg, 0.040 mmol) was added to a solution of Cy3B7 (35 mg, 0.036 mmol) and N,N-diisopropylethylamine (14 μL, 0.079 mmol) in anhydrous dichloromethane at room temperature. The reaction was diluted with deoxygenated DCM, washed with aqueous potassium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by normal phase chromatography (0→2% containing 1% Et 3 N in MeOH / DCM, basic alumina). The product was redissolved in DCM (1 mL) and precipitated into hexane (25 mL). Drying under high vacuum gave Cy3B8 (20 mg, 51% yield) as a dark purple solid. 66 H 80 N 4 O 5 P(M) + The HRMS (ESI) calculated value was 1039.5861 and the found value was 1039.5835.

[0539] Equivalents and scope

[0540] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless stated to the contrary or obvious from the context. If one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, a statement or description including "or" between one or more of the group members is deemed to be satisfied unless stated to the contrary or obvious from the context. The invention includes embodiments in which exactly one of the group members is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which multiple or all of the group members are present in, used in, or otherwise relevant to a given product or process.

[0541] In addition, the present invention encompasses all variations, combinations and permutations, wherein one or more limitations, elements, clauses and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same basic claim. In the case where an element is presented as a list, for example, in the form of a Markush group, each subgroup of these elements is also disclosed, and any element can be removed from the group. It should be understood that, generally, in the case where the present invention or aspects of the present invention are referred to as including specific elements and / or features, certain embodiments of the present invention or aspects of the present invention include, or are substantially composed of, these elements and / or features. For the purpose of simplification, these embodiments are not specifically set forth herein.

[0542] The phrase "and / or" as used in this specification and claims should be understood to refer to "one or both" of the elements so connected, i.e., elements that are present jointly in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so connected. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to only A (optionally including elements other than B) in one embodiment; to only B (optionally including elements other than A) in another embodiment; to both A and B (optionally including other elements) in another embodiment; and so on.

[0543] As used herein in the specification and claims, "or" shall be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one of a number of elements or a list of elements, but also including a plurality, and, optionally, additional unlisted items. Inclusion of exactly one of a number of elements or a list of elements is indicated only when explicitly indicated to the contrary, such as "only one" or "exactly one", or when "consisting of..." is used in a claim. In general, the term "or" as used herein shall be interpreted as indicating an exclusive selection (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either of the two", "one of them", "only one of them", or "exactly one of them". "Essentially consisting of...", as used in a claim, shall have its ordinary meaning as used in the field of patent law.

[0544] As used herein in the specification and claims, the phrase "at least one" when referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows that in addition to the elements specifically identified in the list of elements referred to by the phrase "at least one", other elements may optionally be present, whether or not they are related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B", or, equivalently, "at least one of A and / or B") may refer to at least one, optionally including more than one A, without B present (and optionally including elements other than B), in one embodiment; to at least one, optionally including more than one B, without A present (and optionally including elements other than A), in another embodiment; to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements), in another embodiment; and so on.

[0545] It should also be understood that, unless explicitly indicated to the contrary, in any method of the claims, including more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0546] In the claims and the above description, all transitional phrases such as "comprise", "include", "with", "have", "contain", "involve", "hold", "compose of", etc. should be understood to be open-ended, that is, including but not limited to them. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases as specified in Section 2111.03 of the Manual of Patent Examining Procedures of the United States Patent Office, respectively. It should be understood that the embodiments described herein using open transitional phrases (e.g., "comprising") are also considered to be "consisting of" and "consisting essentially of" the features described by the open transitional phrases in other embodiments. For example, if the disclosure describes a "composition comprising A and B", the disclosure also contemplates other embodiments "composition consisting of A and B" and "composition consisting essentially of A and B".

[0547] Where ranges are given, the endpoints are included. In addition, unless otherwise indicated or apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges may assume any specific value or sub-range within the range in different embodiments of the present invention, unless the context clearly dictates otherwise, the tenth of the unit of the lower limit of the range.

[0548] This application relates to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, the present specification shall prevail. In addition, any specific embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more claims. Because these embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if they are not explicitly stated to be excluded. Any specific embodiment of the present invention may be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[0549] Those skilled in the art will recognize or be able to use no more than routine experiments to determine many equivalents of the specific embodiments described herein. The scope of the embodiments of the present invention described herein is not intended to be limited to the above description, but is set forth in the appended claims. It will be understood by those of ordinary skill in the art that various changes and modifications may be made to this description without departing from the spirit or scope of the present invention as defined in the appended claims.

[0550] The listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

Claims

1. A labeled biomolecule of the following formula: in: Q 1 and Q 2 independently a monomeric or oligomeric biomolecule; X - is a counterion or is absent; and L 1 and L 2 is independently a linker selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof, Where L 1 and L 2 Directly attached to one or more rings of the polycyclic structure.

2. The labeled biomolecule according to claim 1, wherein L 1 is an optionally substituted alkylene group.

3. The labeled biomolecule according to claim 2, wherein L 1 is unsubstituted C 1-20 Alkylene.

4. The labeled biomolecule according to claim 1 or 2, wherein L 2 is an optionally substituted alkylene group.

5. The labeled biomolecule according to claim 4, wherein L 2 is unsubstituted C 1-20 Alkylene.

6. The labeled biomolecule of claim 1, wherein the labeled biomolecule has the following formula: wherein n is independently an integer from 1 to 20, inclusive.

7. The labeled biomolecule according to claim 1 or 2, wherein the labeled biomolecule has the following formula:

8. The labeled biomolecule according to claim 1 or 2, wherein Q 1 and Q 2 are independently a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid or a derivative or fragment thereof.

9. The labeled biomolecule according to claim 8, wherein Q 1 and Q 2 Independently, it is deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, locked nucleic acid or a derivative or fragment thereof.

10. The labeled biomolecule according to claim 8, wherein the labeled biomolecule is a single-stranded nucleic acid comprising a first oligonucleotide strand.

11. The labeled biomolecule according to claim 10, further comprising a second oligonucleotide chain hybridized to the first oligonucleotide chain.

12. The labeled biomolecule according to claim 11, wherein the second oligonucleotide strand is 1 and Q 2 Hybridization.

13. The labeled biomolecule according to claim 11, wherein the second oligonucleotide strand is 1 or Q 2 Hybridization.

14. The labeled biomolecule according to claim 1 or 2, wherein Q 1 and Q 2 are independently amino acids, oligopeptides, polypeptides, proteins or fragments thereof.

15. The labeled biomolecule of claim 14, wherein Q 1 and Q 2 Connected together to form cyclic peptides or cyclic proteins.

16. The labeled biomolecule according to claim 1 or 2, wherein Q 1 and Q 2 are independently monosaccharides, oligosaccharides, polysaccharides or fragments thereof.

17. The labeled biomolecule of claim 1 or 2, wherein one or more emission properties of the labeled biomolecule are increased relative to an unconjugated molecule comprising a polycyclic fluorophore.

18. The labeled biomolecule of claim 17, wherein the one or more emission characteristics are selected from the group consisting of: luminescence lifetime, luminescence intensity, luminescence quantum yield, brightness, and maximum emission wavelength.

19. The labeled biomolecule of claim 18, wherein the luminescence lifetime of the labeled biomolecule is increased by at least 10% relative to the unconjugated molecule.

20. The labeled biomolecule of claim 19, wherein the luminescence lifetime of the labeled biomolecule is increased by 10% to 25% relative to the unconjugated molecule.

21. The labeled biomolecule of claim 19, wherein the luminescence lifetime of the labeled biomolecule is increased by 25% to 50% relative to an unconjugated molecule.

22. The labeled biomolecule of claim 18, wherein the maximum emission wavelength of the labeled biomolecule is increased by at least 1% relative to the unconjugated molecule.

23. The labeled biomolecule of claim 22, wherein the maximum emission wavelength of the labeled biomolecule is increased by 1% to 10% relative to the unconjugated molecule.

24. The labeled biomolecule of claim 17, wherein the unconjugated molecule does not include Q 1 and Q 2 One or either.

25. The labeled biomolecule according to claim 1 or 2, wherein Q 1 and Q 2 Independently optionally associated with one or more fluorophores other than the polycyclic fluorophore.

26. The labeled biomolecule of claim 25, wherein each fluorophore is at least 5 angstroms away from any other fluorophore.

27. The labeled biomolecule according to claim 1 or 2, wherein Q 1 and Q 2 Independently, optionally associated with a reactant configured to serve as a reaction substrate.

28. The labeled biomolecule of claim 11, wherein the first oligonucleotide strand and the second oligonucleotide strand are independently optionally associated with a reactant configured to serve as a reaction substrate.

29. The labeled biomolecule of claim 27, wherein the reaction is a polymerization reaction.

30. The labeled biomolecule of claim 27, wherein the reagent is cleaved from the labeled biomolecule by a polymerase when subjected to polymerization conditions.

31. A labeled nucleotide comprising one or more nucleotides associated with the labeled biomolecule of any one of claims 1-30.

32. The labeled nucleotides of claim 31, wherein the one or more nucleotides comprise a type of nucleotide selected from guanine, cytosine, adenine, and thymine or uracil.

33. The labeled nucleotide of claim 31, wherein the one or more nucleotides are cleaved from the labeled biomolecule by a polymerase when subjected to polymerization conditions.

34. The labeled nucleotide of claim 31, wherein the one or more nucleotides comprise nucleoside triphosphates.

35. The labeled nucleotide of claim 31, wherein the one or more nucleotides comprise a nucleoside hexaphosphate.

36. The labeled nucleotide of claim 34, wherein the one or more nucleotides are linked to the labeled biomolecule via a terminal phosphate.

37. A composition comprising the labeled nucleotide of any one of claims 31-36.

38. A nucleic acid sequencing reaction composition comprising two or more different types of labeled nucleotides in a reaction mixture, wherein at least one type of labeled nucleotide is a labeled nucleotide according to any one of claims 31-36.

39. The nucleic acid sequencing reaction composition of claim 38, comprising four different types of labeled nucleotides.

40. The nucleic acid sequencing reaction composition of claim 39, wherein the four different types of labeled nucleotides include a first labeled nucleotide, a second labeled nucleotide, a third labeled nucleotide, and a fourth labeled nucleotide, wherein the first labeled nucleotide includes guanine, the second labeled nucleotide includes cytosine, the third labeled nucleotide includes adenine, and the fourth labeled nucleotide includes thymine or uracil.

41. A method for determining a template nucleic acid sequence, the method comprising: include: (i) exposing the target volume of the complex to a nucleic acid sequencing reaction composition according to any one of claims 38 to 40, wherein the complex comprises a template nucleic acid, a primer and a polymerase; (ii) directing a series of pulses having one or more excitation energies toward the vicinity of the target volume; (iii) detecting a plurality of photons emitted from the luminescently labeled nucleotide during sequential incorporation into a nucleic acid comprising a primer; as well as (iv) identifying the sequence of the incorporated nucleotide by determining the timing and, optionally, the intensity of the emitted photons.

42. A kit for sequencing a template nucleic acid, the kit comprising: include: Two or more different types of labeled nucleotides, wherein at least one type of labeled nucleotides comprises a labeled nucleotide according to any one of claims 31-36.

43. The kit of claim 42, comprising four different types of labeled nucleotides.

44. The kit of claim 42, further comprising a polymerase.

45. The kit of claim 42, further comprising a primer complementary to the template nucleic acid.

46. ​​A compound of the formula: in: X - is a counterion or is absent; L 1 and L 2 is independently a linker selected from optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted heteroalkenylene, optionally substituted heteroalkynylene, optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, and combinations thereof; P 1 is an oxygen protecting group; and R 1 is the reactive part, Where L 1 and L 2 Directly attached to one or more rings of the polycyclic structure.

47. The compound according to claim 46, wherein R 1 is a phosphoramidite group.

48. The compound according to claim 46 or 47, wherein L 1 is an optionally substituted alkylene group.

49. The compound according to claim 48, wherein L 1 is unsubstituted C 1-20 Alkylene.

50. The compound according to claim 46 or 47, wherein L 2 is an optionally substituted alkylene group.

51. The compound according to claim 50, wherein L 2 is unsubstituted C 1-20 Alkylene.

52. The compound according to claim 46 or 47, wherein P 1 is an optionally substituted trityl protecting group.

53. The compound according to claim 52, wherein P 1 It is 4-methoxytrityl (MMT).

54. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

55. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

56. The compound according to claim 55, wherein R 2 is an optionally substituted C 1 -C 6 alkyl.

57. The compound according to claim 56, wherein R 2 yes:

58. The compound according to claim 55, wherein each R N All are optionally substituted C 1-6 alkyl.

59. The compound according to claim 58, wherein each R N They are all isopropyl.

60. The compound of claim 55, wherein each n is 4.

61. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

62. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

63. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

64. The compound of claim 46 or 47, wherein the compound has the formula: or a salt thereof.

65. A composition comprising the compound of claim 46 or 47.

66. A method for preparing a labeled biomolecule according to claim 1, wherein the method include: (i) subjecting a compound of formula Q to 2 -OH monomer or oligomeric biomolecule or its salt is contacted with the compound or its salt according to claim 46 or 47 to obtain a conjugate of the following formula: (ii) sufficient to cleave P 1 The conjugate formed in step (i) is deprotected under conditions that remove the protecting group and produce a conjugate of the formula: (iii) reacting the conjugate formed in step (ii) with a compound of formula Q under conditions sufficient to promote conjugation 1 -OR 1 The labeled biomolecule according to claim 1 is contacted with a monomeric or oligomeric biomolecule or a salt thereof to obtain the labeled biomolecule according to claim 1.

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