Biconjugable labels and methods of use

Abstract

Provided herein is a biomolecule comprising a fluorescent marker. The label is Cy3B having the formula (I): 1 and Q 2 are independently monomeric or oligomeric biomolecules.

Description

[Technical field]

[0001] The present invention relates to biconjugable labels and methods of use. [Background technology]

[0002] Labeled probes are widely used in methods to detect biological analytes and analyze biological processes. Some of these techniques involve monitoring biological reactions in real time using luminescently labeled reaction components. The label is illuminated with a light source and emits light, which is detected by a photodetector. These events can be recorded and analyzed to identify individual reaction components based on their corresponding emission characteristics. In identifying a specific type of labeled molecule among multiple types, it is important that each type exhibits unique and easily distinguishable emission characteristics. However, the inherent sensitivity of complex biological processes must be carefully considered when designing labeled probes for use in these systems. Summary of the Invention [Means for solving the problem]

[0003] Aspects of the present disclosure relate to labeled biomolecules that include an internally-conjugated luminescent label. In some embodiments, the present disclosure provides labeled biomolecules that include a substrate configured for use in a reaction. In some embodiments, the present disclosure provides labeled biomolecules that include a nucleotide configured for use in a polymerization reaction. In some embodiments, the present disclosure provides a method of sequencing using the labeled nucleotides described herein. In some embodiments, the present disclosure provides biconjugatable luminescent labels and methods of making the same described herein.

[0004] In some embodiments, the present application provides a labeled biomolecule of formula (I):

[0005] [ka]

[0006] In the formula, Q 1 and Q 2 are independently monomeric or oligomeric biomolecules; A is a polycyclic fluorophore; and L 1 and L 2 is a linker independently 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.

[0007] In some embodiments, provided herein is a labeled nucleotide comprising one or more nucleotides associated with a labeled biomolecule according to the present application. In some embodiments, the one or more nucleotides comprise one 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. In some embodiments, the one or more nucleotides comprise a nucleoside polyphosphate. In some embodiments, the one or more nucleotides comprise a nucleoside triphosphate. In some embodiments, the one or more nucleotides comprise a nucleoside hexaphosphate. In some embodiments, the one or more nucleotides (e.g., nucleoside polyphosphate) are attached to the labeled biomolecule via a terminal phosphate. In some embodiments, provided herein is a composition comprising a labeled nucleotide according to the present application.

[0008] In some embodiments, provided herein is a nucleic acid sequencing reaction composition comprising two or more different types of labeled nucleotides in a reaction mixture.In some embodiments, at least one type of labeled nucleotide of the nucleic acid sequencing reaction composition is a labeled nucleotide according to the present application.In some embodiments, the nucleic acid sequencing reaction composition comprises four different types of labeled nucleotides.In some embodiments, the nucleic acid sequencing reaction composition comprises a first labeled nucleotide comprising guanine, a second labeled nucleotide comprising cytosine, a third labeled nucleotide comprising adenine, and a fourth labeled nucleotide comprising thymine or uracil.

[0009] In some aspects, provided herein is a method for determining the sequence of a template nucleic acid. In some embodiments, the method comprises exposing a complex in a target volume, the complex comprising a template nucleic acid, a primer and a polymerase, to a nucleic acid sequencing reaction composition according to the present application. In some embodiments, the method further comprises directing a series of one or more pulses of excitation energy near the target volume. In some embodiments, the method further comprises detecting a plurality of emitted photons from the luminescently labeled nucleotides during their successive incorporation into the nucleic acid comprising the primer. In some embodiments, the method further comprises identifying the sequence of the incorporated nucleotides by determining the timing and, optionally, the emission intensity of the emitted photons.

[0010] In some embodiments, the present specification provides a kit for determining the sequence of template nucleic acid.In some embodiments, the kit comprises two or more different types of labeled nucleotides.In some embodiments, at least one of the two or more different types of labeled nucleotides of the nucleic acid sequencing reaction composition comprises the labeled nucleotide according to the present application.

[0011] In some embodiments, the present application provides a compound of formula (II) or a salt thereof:

[0012] [ka]

[0013] wherein A is a polycyclic fluorophore; and L 1 and L 2 is a linker independently 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 embodiments, provided herein are compositions comprising a compound of formula (II).

[0014] In some aspects, provided herein is a method for preparing a labeled biomolecule of the present application. In some embodiments, the method comprises: (i) reacting a compound of formula Q 2 The method includes contacting a -OH monomeric or oligomeric biomolecule, or a salt thereof, with a compound of formula (II), or a salt thereof, under conditions sufficient to promote conjugation to produce a conjugate of the formula:

[0015] [ka]

[0016] In some embodiments, the method further comprises: (ii) P 1 The method further comprises deprotecting the conjugate formed in step (i) under conditions sufficient to cleave the protecting group and produce a conjugate of the formula:

[0017] [ka]

[0018] 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 to produce a labeled biomolecule of formula (I). 1 -OR 1 or a salt thereof.

[0019] The details of certain embodiments of the invention are set forth in the detailed description of specific embodiments set forth below. Other features, objects, and advantages of the invention will become apparent from the definition, examples, drawings, and claims. [Brief description of the drawings]

[0020] [Figure 1A] Figures 1A-1G show various examples of labeled biomolecules according to the present application: Figure 1A shows a labeled biomolecule with one internal label. [Figure 1B] Figures 1A-1G show various examples of labeled biomolecules in accordance with the present application: Figure 1B shows a labeled biomolecule with two internal labels. [Figure 1C] Figures 1A-1G show various examples of labeled biomolecules in accordance with the present application: Figure 1C shows a circularly labeled biomolecule. [Figure 1D] Figures 1A-1G show various examples of labeled biomolecules in accordance with the present application: Figure 1D shows a labeled oligonucleotide comprising an internally labeled strand hybridized to an unlabeled strand. [Figure 1E] Figures 1A-1G show various examples of labeled biomolecules in accordance with the present application: Figure 1E shows a labeled oligonucleotide comprising one internally labeled strand hybridized to another internally labeled strand. [Figure 1F] Figures 1A-1G show various examples of labeled biomolecules in accordance with the present application. Figure 1F shows a labeled oligonucleotide comprising an unlabeled strand hybridized to a labeled strand having two internal labels. [Figure 1G] Figures 1A-1G show various examples of labeled biomolecules according to the present application: Figure 1G shows a labeled oligonucleotide strand that self-hybridizes to form a stem-loop motif. [Figure 2A] Figures 2A-2C show examples of the use of labeled biomolecules in accordance with the present application: Figure 2A shows an internally labeled oligonucleotide hybridized to a target nucleic acid sequence. [Figure 2B] Figures 2A-2C show examples of the use of labeled biomolecules in accordance with the present application: Figure 2B shows an internally labeled biomolecule bound by a target protein. [Figure 2C] Figures 2A-2C show examples of the use of labeled biomolecules in accordance with the present application: Figure 2C shows an internally labeled antibody bound to a target protein. [Figure 3A] Figures 3A-3B show examples of labeled biomolecules modified with functional moieties in accordance with the present application: Figure 3A shows an internally labeled oligonucleotide modified with a quenching moiety that prevents detection of the internal label unless cleaved from the biomolecule. [Figure 3B] Figures 3A-3B show examples of labeled biomolecules modified with functional moieties in accordance with the present application. Figure 3B shows an internally labeled biomolecule modified with a ligand that is bound by a target protein, such that binding of the ligand to the target protein allows for detection of the internal label. [Figure 4A] Figures 4A-4C show examples of labeled nucleotides attached by polymerases in accordance with the present application: Figure 4A shows a polymerase attached to a nucleotide that includes an external label. [Figure 4B] Figures 4A-4C show examples of labeled nucleotides attached by polymerases in accordance with the present application, and Figure 4B shows a polymerase attached to a nucleotide containing internally labeled biomolecule. [Figure 4C] Figures 4A-4C show examples of labeled nucleotides attached by polymerases in accordance with the present application. Figure 4C shows a polymerase attached to a nucleotide that comprises an internally labeled oligonucleotide. [Figure 5A] 5A-5B show comparative sequencing analyses of externally and internally labeled biomolecules in accordance with the present application. Figure 5A generally shows sets of externally and internally labeled nucleotides that were prepared and subjected to further analysis. [Figure 5B] 5A-5B show comparative sequencing analyses of externally and internally labeled biomolecules in accordance with the present application. Figure 5B shows the results of single molecule sequencing reactions performed using externally and internally labeled nucleotides. [Figure 6] FIG. 6 shows the alignment of excitation and emission spectra of externally and internally labeled nucleotides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The accompanying drawings, which form a part of this specification, illustrate several embodiments of the invention and together with the description serve to explain the principles of the invention. Among other aspects, the present disclosure provides labeled biomolecules that include internally-conjugated luminescent labels (e.g., internal labels). In some embodiments, the internal labels are configured with enhanced conformational restraints that restrict rotation and block inactivation pathways that shorten luminescence lifetimes. In some embodiments, the labeled biomolecules are configured to provide a rigid molecular scaffold for the internal labels that avoids label-label interactions and other quenching effects that can reduce luminescence intensity or other luminescence properties.

[0022] Without wishing to be bound by any particular theory, the labeled biomolecules provided herein provide a number of distinct advantages, such as improved quantum yield and extended emission lifetime, increased emission intensity and / or brightness, and reduced exposure to bulk solvent molecules that limit the formation of reactive species.Thus, in some embodiments, provided herein are labeled biomolecules and methods of use.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 biconjugatable labeled biomolecules.

[0023] Internally labeled biomolecules 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) contain internally conjugated luminescent compounds ("luminescent labels" or "labels", e.g., polycyclic fluorophores). "Internally-labeled" or "internally conjugated" are used interchangeably herein and refer to when a portion of a biomolecule is conjugated to a first site of a luminescent compound and another portion of the biomolecule is conjugated to a second site of the luminescent compound. Internal conjugation of luminescent compounds has several advantages. For example, internal conjugation of a label to a biomolecule may alter the lifetime and other photophysical properties of the label (e.g., by limited rotation of the label). As another example, internal conjugation of a label may immobilize the label and separate it from other labels to mitigate self-quenching. Furthermore, internal conjugation of the label may limit the access of the label to the bulk solvent in the solution to mitigate radical formation that may damage other components in the solution. Internal incorporation of the label has other advantages that are described herein. Figures 1A-1G show a non-limiting set of examples of labeled biomolecules.

[0024] 1A-1G generally depict various configurations of labeled biomolecules in accordance with the present application. Each example is shown as having an internal label and a biomolecule (displayed as a stippled shape). FIG. 1A is a labeled biomolecule that includes one 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.

[0025] FIG. 1B shows a labeled biomolecule with 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, labeled biomolecules with two or more copies of the same internal label exhibit increased emission intensity and / or brightness compared to labeled biomolecules with one copy of the internal label. In some embodiments, labeled biomolecules with two or more different types of internal labels provide two or more unique detectable signals. Examples of various configurations and uses of multi-labeled biomolecules are described elsewhere herein.

[0026] In accordance with the present application, increasing the rigidity of an internally conjugated label has been shown to enhance one or more luminescence properties of the labeled biomolecule, for example, via the conformational constraint provided by the polycyclic fluorophore of the internal label. Advantageously, the biomolecular scaffold to which the internal label is conjugated can be engineered to further promote rigidity and further enhance one or more luminescence properties in these systems.

[0027] FIG. 1C is a labeled biomolecule that includes an internal label that is conjugated to a circularized biomolecule. As shown by this example, in some embodiments, the internal label is conjugated at one end of the biomolecule to another end of the biomolecule, so that the labeled biomolecule is circularized. Without wishing to be bound by a particular theory, it is believed that circularization promotes structural rigidity throughout the backbone of the biomolecule, which enhances the favorable luminescence properties of the internal label provided herein. Examples of circular biomolecules include, but are not limited to, circular peptides, circular proteins, and circular nucleic acids (e.g., circular RNA, DNA plasmids).

[0028] The inventors have further recognized and appreciated that oligonucleotides (e.g., polynucleotides, nucleic acids) provide a robust and highly tunable biomolecular scaffold for the internal labeling of the present application. Various examples of internally labeled oligonucleotides are shown in Figures 1D-1G. These exemplary constructs and additional embodiments relating to internally labeled oligonucleotides are described in detail elsewhere herein.

[0029] As generally shown in the exemplary structures depicted in Figures 1A-1G, in some embodiments, the internal label 100 corresponds to A of formula (I) as depicted herein. In some embodiments, the biomolecule of the exemplary structure (shown as a stippled shape) corresponds to Q of formula (I) as depicted herein. 1 and / or Q 2 In some embodiments, as shown herein, the internal label 100 corresponds to A and the biomolecule of the exemplary structure (shown as a stippled shape) corresponds to Q of formula (I). 1 and Q 2 Corresponds to.

[0030] In one embodiment, provided herein is a labeled biomolecule of formula (I):

[0031] [ka]

[0032] During the ceremony: Q 1 and Q 2 are independently monomeric or oligomeric biomolecules; A is a polycyclic fluorophore; and L 1 and L 2is a linker independently 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.

[0033] As described herein,

[0034] [ka]

[0035] (also referred to herein as "A") is a light-emitting compound or dye. As represented by formula (I), A is L 1 A portion of a biomolecule (Q 1 group) and is conjugated to a linker L 2 via another part of the biomolecule (Q 2 Q 1 and Q 2 together form a biomolecule, which has the structure -OL 1 -AL 2 It is interrupted by -O-, which causes it to be labeled internally.

[0036] In certain embodiments, A is a polycyclic fluorophore. In certain embodiments, the incorporation of a polycyclic fluorophore is advantageous insofar as the polycyclic structure can impart greater rigidity to the system compared to linear or non-polycyclic fluorophores. In certain embodiments, L 1 and L 2is directly (e.g., via a covalent bond) linked to one or more rings (e.g., benzenoid or heteroaromatic rings) of the polycyclic structure. This direct linkage can also provide greater rigidity to the system (e.g., via immobilization / restricted rotation of the label). In certain embodiments, L 1 and L 2 are directly linked to different rings on A, a design feature that may also impart greater rigidity to the system and / or help immobilize the dye.

[0037] 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.

[0038] 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:

[0039] [ka]

[0040] In the formula, X - is a counterion or is absent, and the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted. In certain embodiments, A has the formula:

[0041] [ka]

[0042] In the formula, X - is a counterion or is absent, and the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted. Thus, in certain embodiments, the labeled biomolecule is of the formula:

[0043] [ka]

[0044] In the formula, X - is a counter ion or is absent. In certain embodiments, the labeled biomolecule has the formula:

[0045] [ka]

[0046] In the formula, n is independently an integer of 1 to 20. In certain embodiments, the labeled biomolecule is of the formula:

[0047] [ka]

[0048] In certain embodiments, the labeled biomolecule is of the formula:

[0049] [ka]

[0050] As described herein, in certain embodiments, ring A is a polycyclic 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 is of the formula:

[0051] [ka]

[0052] wherein each R is independently hydrogen, halogen, -N, -CN, -NO, 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.

[0053] As defined herein, each R is independently hydrogen, halogen, -N, -CN, -NO, 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 -N3. In certain embodiments, R is -CN. In certain embodiments, R is -NO2. 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 -OR O In certain embodiments, R is -SR S In certain embodiments, R is -N(R N In certain embodiments, R is optionally substituted C 1-6 In certain embodiments, R is an unsubstituted C 1-6 In certain embodiments, R is optionally substituted C 1-3 In certain embodiments, R is an 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.

[0054] In certain embodiments, A has the formula:

[0055] [ka]

[0056] wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted. In certain embodiments, A has the formula:

[0057] [ka]

[0058] wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted. In certain embodiments, A has the formula:

[0059] [ka]

[0060] wherein the structure is optionally substituted at any position. In certain embodiments, the structure is unsubstituted. Thus, in certain embodiments, the labeled biomolecule is of the formula:

[0061] [ka]

[0062] In certain embodiments, the labeled biomolecule has the formula:

[0063] [ka]

[0064] During the ceremony: n is independently an integer from 1 to 20, and 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.

[0065] In certain embodiments, the labeled biomolecule is of the formula:

[0066] [ka]

[0067] In certain embodiments, the labeled biomolecule is of the formula:

[0068] [ka]

[0069] In certain embodiments, the labeled biomolecule is of the formula:

[0070] [ka]

[0071] As described herein, in certain embodiments, A is a boron-dipyrromethene (BODIPY) fluorophore. In certain embodiments, A is of the formula:

[0072] [ka]

[0073] wherein the structure is optionally substituted at any position. In certain embodiments, A has the formula:

[0074] [ka]

[0075] wherein Ar is an optionally substituted aryl or an optionally substituted heteroaryl, and the structure is optionally substituted at any position. 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 is of the formula:

[0076] [ka]

[0077] In certain embodiments, Ar is of the formula:

[0078] [ka]

[0079] In certain embodiments, Ar is of the formula:

[0080] [ka]

[0081] In certain embodiments, Ar has the formula:

[0082] [ka]

[0083] where m is as defined herein. As defined herein, R Aris hydrogen, halogen, -N3, -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 In certain embodiments, R Ar is hydrogen. In certain embodiments, R Ar is halogen (-Cl, -I, -Br, -F). In certain embodiments, R Ar is -N3. In certain embodiments, R Ar is -CN. In certain embodiments, R Ar is -NO2. In certain embodiments, R Ar is optionally substituted alkyl. In certain embodiments, R Ar is optionally substituted alkenyl. In certain embodiments, R Ar is optionally substituted alkynyl. In certain embodiments, R Ar is optionally substituted carbocyclyl. In certain embodiments, R Ar is optionally substituted aryl. In certain embodiments, R Ar is optionally substituted heterocyclyl. In certain embodiments, R Ar is optionally substituted heteroaryl. In certain embodiments, R Ar -OR O In certain embodiments, R Ar Ha-SR S In certain embodiments, R Ar -N(R N In certain embodiments, R Ar is -F. In certain embodiments, R Ar -S(CH2CH2O) m OCH3, and m is as defined herein.

[0084] As defined herein, m is an integer from 1 to 6. In certain embodiments, A has the formula:

[0085] [ka]

[0086] In the formula, R Ar is hydrogen, halogen, -N3, -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, the structure is substituted at any position. In certain embodiments, the structure is unsubstituted.

[0087] Thus, in certain embodiments, the labeled biomolecule is of the formula:

[0088] [ka]

[0089] In the formula, R Ar is hydrogen, halogen, -N3, -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.

[0090] In certain embodiments, the labeled biomolecule is of the formula:

[0091] [ka]

[0092] In certain embodiments, the labeled biomolecule is of the formula:

[0093] [ka]

[0094] In certain embodiments, the labeled biomolecule is of the formula:

[0095] [ka]

[0096] In certain embodiments, the labeled biomolecule is of the formula:

[0097] [ka]

[0098] As described herein, in certain embodiments, A is an acridine fluorophore. In certain embodiments, A is an acridine fluorophore of the following formula:

[0099] [ka]

[0100] wherein the structure is optionally substituted. As described herein, in certain embodiments, A is a phenoxazine fluorophore. In certain embodiments, A is a phenoxazine fluorophore of the following formula:

[0101] [ka]

[0102] wherein the structure is optionally substituted. As described herein, in certain embodiments, A is a coumarin fluorophore. In certain embodiments, A is a coumarin fluorophore of the formula:

[0103] [ka]

[0104] wherein the structure is optionally substituted. In certain embodiments, A is a coumarin fluorophore of the formula:

[0105] [ka]

[0106] wherein the structure is optionally substituted. As defined herein, L 1 and L 2 is a linker independently 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 includes an optionally substituted alkylene. 1 In certain embodiments, L includes optionally substituted alkenylene. 1 In certain embodiments, L includes optionally substituted alkynylene. 1 In certain embodiments, L includes optionally substituted heteroalkylene. 1 In certain embodiments, L includes optionally substituted heteroalkenylene. 1In certain embodiments, L includes optionally substituted heteroalkynylene. 1 In certain embodiments, L includes an optionally substituted carbocyclylene. 1 In certain embodiments, L includes optionally substituted heterocyclylene. 1 In certain embodiments, L includes an optionally substituted arylene. 1 In certain embodiments, L includes optionally substituted heteroarylene. 1 is an optionally substituted C 1-20 In certain embodiments, L 1 is an optionally substituted C 1-10 In certain embodiments, L 1 is an optionally substituted C 1-6 In certain embodiments, L 1 consists of the following formula:

[0107] [ka]

[0108] wherein n is as defined herein. In certain embodiments, L 1 consists of the following formula:

[0109] [ka]

[0110] In certain embodiments, L 1 consists of the following formula:

[0111] [ka]

[0112] In certain embodiments, L 1 consists of the following formula:

[0113] [ka]

[0114] In certain embodiments, L 1 consists of the following formula:

[0115] [ka]

[0116] In certain embodiments, L 1 consists of the following formula:

[0117] [ka]

[0118] In certain embodiments, L 1 consists of the following formula:

[0119] [ka]

[0120] In certain embodiments, L 1 consists of the following formula:

[0121] [ka]

[0122] In certain embodiments, L 1 consists of the following formula:

[0123] [ka]

[0124] In certain embodiments, L 1 consists of the following formula:

[0125] [ka]

[0126] In certain embodiments, L 1 consists of the following formula:

[0127] [ka]

[0128] In certain embodiments, L 1 consists of the following formula:

[0129] [ka]

[0130] In certain embodiments, L 1 consists of the following formula:

[0131] [ka]

[0132] In certain embodiments, L 2 In certain embodiments, L includes an optionally substituted alkylene. 2 In certain embodiments, L includes optionally substituted alkenylene. 2 In certain embodiments, L includes optionally substituted alkynylene. 2 In certain embodiments, L includes optionally substituted heteroalkylene. 2 In certain embodiments, L includes optionally substituted heteroalkenylene. 2 In certain embodiments, L includes optionally substituted heteroalkynylene. 2 In certain embodiments, L includes an optionally substituted carbocyclylene. 2 In certain embodiments, L includes optionally substituted heterocyclylene. 2In certain embodiments, L includes an optionally substituted arylene. 2 In certain embodiments, L includes optionally substituted heteroarylene. 2 is an optionally substituted C 1-20 In 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 consists of the following formula:

[0133] [ka]

[0134] wherein n is as defined herein. In certain embodiments, L 2 consists of the following formula:

[0135] [ka]

[0136] In certain embodiments, L 2 consists of the following formula:

[0137] [ka]

[0138] In certain embodiments, L 2 consists of the following formula:

[0139] [ka]

[0140] In certain embodiments, L 2 consists of the following formula:

[0141] [ka]

[0142] In certain embodiments, L 2 consists of the following formula:

[0143] [ka]

[0144] In certain embodiments, L 2 consists of the following formula:

[0145] [ka]

[0146] In certain embodiments, L 2 consists of the following formula:

[0147] [ka]

[0148] In certain embodiments, L 2 consists of the following formula:

[0149] [ka]

[0150] In certain embodiments, L 2 consists of the following formula:

[0151] [ka]

[0152] In certain embodiments, L 2 consists of the following formula:

[0153] [ka]

[0154] In certain embodiments, L 2 consists of the following formula:

[0155] [ka]

[0156] In certain embodiments, L 2 consists of the following formula:

[0157] [ka]

[0158] As defined herein, n is independently an integer from 1 to 20. 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.

[0159] As described herein, Q 1 and Q 2are independently monomeric or oligomeric biomolecules. As described herein, Q 1 and Q 2 together form an oligomeric or polymeric biomolecule, which is -OL 1 -AL 2 -O, thereby being internally labeled (e.g., to form a labeled biomolecule).

[0160] In some embodiments, the labeled biomolecule is an oligomeric or polymeric biomolecule that comprises 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.

[0161] In certain embodiments, the labeled biomolecule is an oligonucleotide or a nucleic acid. In certain embodiments, Q 1 and Q 2 is independently a nucleoside, nucleotide, oligonucleotide, nucleic acid, or derivative or fragment thereof. 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 or a derivative or fragment thereof.1 and Q 2 is independently an oligonucleotide or a derivative or fragment thereof. In certain embodiments, Q 1 and Q 2 are independently a nucleic acid or a derivative or fragment thereof.

[0162] In certain embodiments, Q 1 and Q 2 is independently a deoxyribonucleic acid, a ribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a derivative or fragment thereof. 1 and Q 2 are independently deoxyribonucleic acid, or derivatives or fragments thereof. In certain embodiments, Q 1 and Q 2 is 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. 1 and Q 2 is a locked nucleic acid, or a derivative or fragment thereof.

[0163] As described herein, Q 1 and Q 2 together form an oligomeric or polymeric biomolecule, which is -OL 1 -AL 2 -O, thereby internally labeling (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 is a first oligonucleotide strand (Q 1 and / or Q 2 In certain embodiments, the labeled biomolecule comprises a second oligonucleotide strand hybridized to the first oligonucleotide strand. For example, in certain embodiments, the second oligonucleotide strand comprises Q 1and / or Q 2 A visual representation of this internally labeled system is shown below (where ---- represents hybridization interactions (e.g., one or more Watson-Crick base interactions)):

[0164] [ka]

[0165] A visual representation of this system can also be found in FIG. 1D. Further examples of internally labeled oligonucleotides are shown in Figures 1E-1G. As illustrated by these and other examples described herein, various oligonucleotide strand hybridization strategies are provided to promote rigidity and / or protect the internal label from bulk solvent. Thus, oligonucleotide strand hybridization can be used as a general design strategy in preparing labeled oligonucleotides of the present application. In some embodiments, oligonucleotide strand hybridization includes self-strand hybridization (e.g., self-hybridization within a single strand). In some embodiments, oligonucleotide strand hybridization includes hybridization of different oligonucleotide strands.

[0166] FIG. 1D is a labeled oligonucleotide comprising an internally labeled oligonucleotide strand hybridized to an unlabeled oligonucleotide strand.In some embodiments, an unlabeled oligonucleotide strand is used to increase the rigidity of a specific region of a labeled oligonucleotide strand (e.g., the region that comprises an internal label).In some embodiments, an unlabeled oligonucleotide strand is hybridized to an internally labeled oligonucleotide strand that comprises two or more internal labels as provided herein.

[0167] FIG. 1E is a labeled oligonucleotide comprising one internally labeled oligonucleotide strand hybridized to another internally labeled oligonucleotide strand. In some embodiments, the internal label of one oligonucleotide strand comprises the same fluorophore as the internal label of the other oligonucleotide strand. In some embodiments, the internal label of one oligonucleotide strand comprises a different fluorophore than the internal label of the other oligonucleotide strand. In some embodiments, one of the internally labeled oligonucleotide strands comprises two or more internal labels of the present application. In some embodiments, both of the internally labeled oligonucleotide strands comprise two or more internal labels of the present application.

[0168] In some embodiments, oligonucleotide strand hybridization promotes the formation of one or more structural motifs, such as stem loops, junctions, pseudoknots, and double helices. Structural motifs according to the present application are useful for enhancing the rigidity of the labeled oligonucleotide and / or limiting the extent to which the internal label is exposed to bulk solvent. Figures 1F-1G show examples of labeled oligonucleotides with higher order structural motifs formed by strand hybridization.

[0169] FIG. 1F is a labeled oligonucleotide comprising an unlabeled oligonucleotide strand hybridized to an oligonucleotide strand containing two internal labels. As generally shown in this example, the formation of a double helix may facilitate the separation of the two internal labels of the same oligonucleotide strand. In some embodiments, the hybridized oligonucleotide strands form a double helix with about 10-12 base pairs per turn.

[0170] Thus, in some embodiments, when two internal labels of the same oligonucleotide strand occupy approximately the same amount of space as one nucleotide in the strand, the internal labels are separated by a minimum of 5-6 nucleotides along the strand such that the same labels are located on approximately opposite sides of the double helix. In some embodiments, the internal labels are separated by 4-8 (e.g., 4, 5, 6, 7, or 8) nucleotides along the oligonucleotide strand. Without wishing to be bound by theory, such design strategies may be utilized to limit the extent of label-label interactions (e.g., quenching effects) resulting from intervening helical structures that absorb radioactive and / or non-radioactive decay.

[0171] In some embodiments, the internal labels of the present disclosure are incorporated into the oligonucleotide backbone to minimize the extent of label-label interactions. Thus, in some embodiments, the internal labels are separated by 1-3 (e.g., 1, 2, or 3) or 9-13 (e.g., 9, 10, 11, 12, or 13) nucleotides along the same oligonucleotide strand. As illustrated by this example, labeled oligonucleotides can be designed with predicted or known helical structures in mind, such that the relative position of one internal label to another through space can be manipulated for a desired application. Further examples of structural motifs useful in the design of labeled oligonucleotides are known in the art and described herein.

[0172] FIG. 1G is an internally labeled oligonucleotide strand that self-hybridizes to form a stem-loop motif. A stem-loop, or hairpin loop, is an unpaired loop of nucleotides on an oligonucleotide strand that is formed when the oligonucleotide strand folds back and base pairs with another portion of the same strand. In some embodiments, the unpaired loop of the stem-loop comprises 3-10 nucleotides. Thus, a stem-loop can be formed by two regions of an oligonucleotide strand with reverse complementary sequences that hybridize to form a stem, the two regions being separated by 3-10 nucleotides that form an unpaired loop. In some embodiments, the stem can be designed to have one or more G / C nucleotides, which may provide additional stability with additional hydrogen bond interactions formed compared to A / T / U nucleotides. In some embodiments, the stem comprises a G / C nucleotide immediately adjacent to the unpaired loop sequence. In some embodiments, the stem comprises a G / C nucleotide within the first 2, 3, 4, or 5 nucleotides adjacent to the unpaired loop sequence.

[0173] As described herein, in some embodiments, an internal label conjugates a portion of an oligonucleotide strand to another portion of the oligonucleotide strand. As generally shown in the exemplary structures of Figures 1D-1G, in some embodiments, both portions of the oligonucleotide strand conjugated by an internal label are hybridized to the same oligonucleotide strand. Thus, the internal label may be adjacent to one or more (e.g., 1, 2, 3, 4, 5, or more) unpaired bases of the hybridized oligonucleotide strand.

[0174] In some embodiments, the internal label can interact with guanine nucleobases through radioactive and / or non-radioactive decay, resulting in a decrease in luminescence lifetime. In some embodiments, one or more unpaired bases adjacent to the internal label are designed to eliminate or minimize guanine. In some embodiments, the region surrounding the internally conjugated label is designed to eliminate or minimize G / C content. In some embodiments, the internally conjugated label is separated from G or C nucleotides on the oligonucleotide chain by at least two nucleotides (e.g., separated from G or C nucleotides by 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides). Thus, in some embodiments, each internal label is flanked on either side by at least two consecutive nucleotides selected from A or T / U.

[0175] The labels provided herein 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 is independently an amino acid, an oligopeptide, a polypeptide, a protein, or a fragment thereof. 1 and Q 2 is independently an amino acid. In certain embodiments, Q 1 and Q 2 is independently an oligopeptide, or a fragment thereof. 1 and Q 2 is independently a polypeptide, protein, or fragment thereof. In certain embodiments, Q 1 and Q 2 are linked together to form a cyclic peptide or protein.

[0176] Oligopeptides and Polypeptides Suitable for Use in Labeled Biomolecules of the Present Application Non-limiting examples of peptides include, but are not limited to, oligopeptides, cyclic peptides, and small proteins (e.g., avian pancreatic peptide-based miniature proteins as described in Hodges, AM and Schepartz, A., (2007), J. Am. Chem. Soc., 129:1104-21025). Methods for engineering structural constraints into polypeptides are well known in the art and are envisioned to be particularly useful, for example, to impart rigidity and enhance one or more of the luminescence properties discussed 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). Further non-limiting examples of useful peptide engineering techniques include peptide cyclization (see, e.g., Maltsev, OV et al., (2016), Angewandte Chemie, 55(4):1535-1539), α-helical peptide constraining via stapling and / or H-bond surrogates (see, e.g., Douse, CH et al., (2014), ACS Chem. Biol., 9:2204-2209), peptide constraining via cyclic β-sheet and β-hairpin mimetics (see, e.g., Gibbs, AC et al., (1998), Nat. Struc. Biol., 5:284-288).

[0177] In certain embodiments, the labeled biomolecule is an oligosaccharide or polysaccharide. For example, in certain embodiments, Q 1 and Q 2 is independently a monosaccharide, an oligosaccharide, a polysaccharide, or a fragment thereof. 1 and Q 2 is independently a monosaccharide. In certain embodiments, Q 1 and Q 2is independently an oligosaccharide or a fragment thereof. 1 and Q 2 are independently a polysaccharide or a fragment thereof. Examples of oligosaccharides and polysaccharides suitable for use in the labeled biomolecules of the present application are known in the art (e.g., as described in Solid Support Oligosugar Synthesis and Combinatorial Carbohydrate Libraries (Wiley, 2001)).

[0178] According to the present application, the labeled biomolecules provided herein include biomolecules that function as a rigid scaffold into which the internal labels of the present application are incorporated. In some embodiments, the biomolecules include one or more features that provide additional functionality useful for various methods of detection, quantitative analysis, and imaging. For example, Figures 2A-2C show non-limiting examples of internally labeled biomolecules that include biomolecules that interact with a desired target molecule.

[0179] In some embodiments, labeled biomolecules, including oligonucleotides, can function as hybridization probes. Hybridization probes are labeled fragments of DNA or RNA (e.g., oligonucleotides) that can be added to samples of known or unknown content to detect the presence of a desired target nucleic acid that is complementary to the hybridization probe. For example, FIG. 2A shows an internally labeled hybridization probe 200 hybridized to a target nucleic acid sequence 210. As generally illustrated by this example, the internally labeled hybridization probe 200 forms a base-pairing interaction with the target nucleic acid 210, which results in a detectable increase in light emission from the internal label. However, in some embodiments, hybridization of the internally labeled hybridization probe 200 with the target nucleic acid 210 results in a detectable decrease in light emission.

[0180] In some embodiments, the internally labeled hybridization probe of the present application comprises a sequence that is substantially complementary to the target nucleic acid sequence, such that the probe and the target form base-pairing interactions under hybridization conditions. As described herein, the internal label conjugates one portion of a biomolecule to another portion of a biomolecule. Thus, one or both 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 fragments thereof).

[0181] The internally labeled hybridization probes of the present application can be utilized in any methodology known in the art that utilizes hybridization probes. Examples of such techniques include, but are not limited to, fluorescent in situ hybridization (FISH), Northern blots, Southern blots, and common techniques including SNP detection, real-time nucleic acid detection, real-time PCR quantification, allele discrimination and identification, multiplex PCR assays, and diagnostic clinical assays.

[0182] The labeled biomolecules of the present application, in some embodiments, include biomolecules that function as protein ligands. For example, FIG. 2B shows an internally labeled biomolecule 201 bound by a target protein 211. As generally illustrated by this example, the target protein 211 associates with (e.g., binds to) at least a portion of the biomolecule (shown as a dashed line) of the labeled biomolecule 201, resulting in a detectable increase in luminescence from the internal label. However, in some embodiments, binding of the target protein 211 to the labeled biomolecule 201 results in a detectable decrease in luminescence.

[0183] 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. Such labeled biomolecules are believed to be useful in methodologies that use labeled protein ligands to detect the presence of a target protein or to assess protein-ligand binding interactions (e.g., fluorescence polarization and other techniques known in the art or described herein).

[0184] In some embodiments, a labeled biomolecule, including a polypeptide, can function as an antibody. Figure 2C shows an internally labeled antibody 202 bound to a target protein 212. As generally illustrated by this example, the internally labeled antibody 202 includes a Fab region configured to specifically bind to the target protein 212, which results in a detectable increase in light emission from the internal label. However, in some embodiments, binding of the internally labeled antibody 202 to the target protein 212 results in a detectable decrease in light emission.

[0185] The antibodies labeled within the present application can be utilized in any manner known in the art that utilizes luminescently labeled antibodies. Examples of such techniques include, but are not limited to, common methodologies involving immunolabeling, such as fluorescent in situ hybridization (FISH), Western blotting, immunocytochemistry and immunohistochemistry techniques, as well as other techniques known in the art or described herein.

[0186] As described above, the internal label of the present application can be conjugated to a biomolecule that interacts with a desired target molecule. In some embodiments, the internal label is conjugated to a protein that associates with (e.g., binds to) 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 aptazyme, a ligase, a transferase, or a tRNA synthetase. In some embodiments, the internal label is conjugated to a nucleic acid that associates with (e.g., binds to) 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).

[0187] In some embodiments, the labeled biomolecule comprises a biomolecule modified with one or more functional moieties. For example, Figures 3A-3B show non-limiting examples of labeled biomolecules that include moieties that interact with a target molecule and / or an internal label.

[0188] In accordance with the present application, the biomolecular scaffold provides a robust labeling scaffold that can be particularly beneficial in techniques where a strongly defined location of the internal label is desired. For example, Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) have become important tools for the in vitro and in vivo study of conformational dynamics in biomolecules. These methods rely on distance-dependent quenching of the fluorescence signal of a donor fluorophore by either a fluorescent acceptor fluorophore (FRET) or a non-fluorescent quencher, as used in FCS by photoinduced electron transfer (PET).

[0189] In some embodiments, the labeled biomolecule contains one or more quenching moieties (e.g., fluorescent and / or non-fluorescent quenching moieties) that interact with an internal label of the labeled biomolecule. In some embodiments, such moieties can be useful in real-time PCR, where the position of the internal label is clearly defined with respect to the quencher that is cleaved by exonuclease activity. An example of this process is shown in Figure 3A.

[0190] As shown in panel I, an internally labeled hybridization probe containing a quenching moiety 300 is hybridized to a target nucleic acid. In some embodiments, the quenching moiety 300 is a non-fluorescent quencher that absorbs emission from the internal label. In some embodiments, the quenching moiety 300 is a fluorescent quencher that absorbs emission from the internal label at one wavelength and emits at another wavelength.

[0191] As shown in panel II, the quenching moiety 300 has been cleaved (e.g., by an exonuclease) from the internally labeled hybridization probe. This separation of the quenching moiety 300 from the internal label eliminates distance-dependent quenching effects and allows for detection of emission from the internal label. It should be understood that in some embodiments, an internally labeled hybridization probe includes an internal label that functions as a quencher for another label on the hybridization probe.

[0192] Quencher-modified hybridization probes are known in the art and are believed to be useful for internal labeling in the present application. Examples of such hybridization probes include, but are not limited to, molecular beacons, TaqMan probes, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, and cycling probe technology (CPT) probes.

[0193] Thus, in some embodiments, multiple internal labels (e.g., 2, 3, 4, 5, or more internal labels) can be incorporated into a biomolecule according to the desired luminescence properties of the labeled biomolecule provided herein. For example, in some embodiments, a labeled biomolecule with two or more internal labels exhibits increased luminescence intensity and / or brightness compared to a biomolecule with one internal label. In some embodiments, the two or more internal labels are configured to provide independent reporter signals. In some embodiments, the two or more internal labels are configured to provide dependent reporter signals (e.g., donor and acceptor labels of a FRET pair).

[0194] In some aspects, the present application provides internal labels configured for use with conventional solid phase synthesis techniques, e.g., phosphoramidite analogs useful in oligonucleotide synthesis. Thus, in some embodiments, the labels provided herein can be readily incorporated into biomolecules to generate labeled biomolecules with several internal labels that can be limited only by the desired size of the biomolecule. For example, in some embodiments, the labeled biomolecule contains two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) internal labels. In some embodiments, the labeled biomolecule contains between 2-5, 2-10, 5-10, 5-15, 10-15, 15-20, or more internal labels.

[0195] In some embodiments, the labeled biomolecules provided herein further comprise one or more luminescent labels other than the polycyclic fluorophore of formula (I). For example, in some embodiments, the labeled biomolecules comprise at least one internal label according to formula (I) and one or more internal labels comprising a linear or non-polycyclic fluorophore. In some embodiments, the labeled biomolecules comprise at least one internal label according to formula (I) and one or more external labels.

[0196] In some embodiments, external label refers to a label (e.g., a fluorophore) that is conjugated to a single site of the labeled biomolecule provided herein. In some embodiments, the external label is conjugated to the labeled biomolecule at its end. For example, in some embodiments, the external label is conjugated to the 5' or 3' end of the labeled oligonucleotide. In some embodiments, the external label is conjugated to the N- or C-terminus of the labeled polypeptide. In some embodiments, the external label is conjugated to the labeled biomolecule at the terminal monomer of the biomolecule (e.g., conjugated to the base of the terminal nucleotide of the oligonucleotide chain, conjugated to the side chain of the terminal amino acid of the polypeptide chain).

[0197] 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 the monomers of the biomolecule. In some embodiments, the external label is conjugated to the abasic site of the labeled oligonucleotide. In some embodiments, the external label is conjugated to the labeled biomolecule at a non-terminal monomer of the biomolecule (e.g., conjugated to the base of a non-terminal nucleotide of an oligonucleotide chain, conjugated to the side chain of a non-terminal amino acid of a polypeptide chain).

[0198] The labeled biomolecules of the present application, in some embodiments, include biomolecules modified with one or more moieties that function as protein ligands. For example, FIG. 3B illustrates a process in which a labeled biomolecule containing a ligand moiety is detectably bound by a target protein. As shown in panel I, the target protein is exposed to a labeled biomolecule containing a ligand moiety 301 configured to bind to the target protein. In the absence of binding between the target protein and the ligand moiety 301, the internal label of the labeled biomolecule does not emit a detectable signal.

[0199] As shown in panel II, the target protein associates with (e.g., binds to) the ligand moiety 301, resulting in a detectable increase in emission from the internal label. However, in some embodiments, binding of the target protein to the ligand moiety 301 results in a detectable decrease in emission. In some embodiments, the change in detectable emission upon binding occurs as a result of confinement of the internally labeled biomolecule to the observation region (e.g., immobilization to a surface for a period of time sufficient to allow detection). In some embodiments, the change in emission upon binding occurs as a result of a FRET interaction (e.g., interaction with the target protein or an acceptor / donor conjugated at the surface).

[0200] In some embodiments, labeled biomolecules comprising one or more ligand moieties may be used, for example, for the purpose of immobilizing the labeled biomolecule to a surface or material, for detection of a target protein in a known or unknown sample, for detection (e.g., quantification) of a binding interaction between a target protein and a ligand moiety that is known or unknown to bind to the target protein.

[0201] In certain embodiments, the labeled biomolecule is associated with a reactant configured for use as a substrate in a reaction. For example, in certain embodiments, Q 1 and Q 2 is independently, optionally associated with a reactant configured for use as a substrate in a reaction. In the case of an oligonucleotide or nucleic acid system, for example, the first and second oligonucleotide strands are independently, optionally associated with a reactant configured for use as a substrate in a reaction. In some embodiments, the reactant is configured for use as a substrate in a polymerization reaction. In some embodiments, the reactant is cleaved from the labeled biomolecule by a polymerase when subjected to polymerization reaction conditions. For example, in some embodiments, the reactant is a nucleotide (e.g., for use in a method of sequencing a nucleic acid).

[0202] Labeled nucleotides Also provided herein is a labeled nucleotide, which comprises one or more nucleotides related to the labeled biomolecule described herein.In some embodiments, the one or more nucleotides comprise one 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 polymerase when subjected to polymerization reaction conditions.

[0203] Without wishing to be bound by any particular theory, the labeled nucleotides provided herein offer a number of distinct advantages over those currently used in sequencing reactions, such as increased read length and increased accuracy, in addition to the advantages described elsewhere herein. Figures 4A-4C highlight several features of the labeled nucleotides of the present disclosure.

[0204] Each of Figures 4A-4C shows a nucleotide 400 bound by a polymerase 410. The nucleotide 400 in Figure 4A is externally conjugated to a luminescent label, while the nucleotides in Figures 4B and 4C are associated with differently labeled biomolecules as described herein. As shown in Figure 4A, the externally conjugated label is in relatively close proximity to the polymerase and has a relatively high degree of access to bulk solvent molecules. In some embodiments, such characteristics are detrimental to the polymerization reaction. For example, in some embodiments, a short distance between the label and the polymerase may result in label-induced damage to the polymerase via radioactive and / or non-radioactive decay (shown as pathway (i)). In some embodiments, a high degree of access of the label to bulk solvent molecules may result in a high incidence of reactive oxygen species (ROS) formation. Once ROS are formed, they may damage the polymerase and adversely affect enzymatic activity (shown as pathway (ii)).

[0205] FIG. 4B shows a nucleotide associated with a labeled biomolecule, such as a labeled oligonucleotide strand. As shown in comparison to an externally conjugated label, the biomolecule increases the separation between the label and the polymerase. Furthermore, the portion of the biomolecule between the nucleotide and the internal label provides a protective barrier between the label and the polymerase. Thus, the occurrence of label-induced damage to the polymerase due to label-polymerase separation and / or the biomolecule absorbing decay released from the label can be reduced (shown as pathway (iii)).

[0206] Also, as shown in relation to Figure 4A, integration of an internal dye into the biomolecule shown in Figure 4B reduces the extent to which the label is exposed to bulk solvent molecules. Thus, ROS-induced damage can be reduced by a lower incidence of ROS formation as a result of reduced access of the label to bulk solvent, and / or by the biomolecule absorbing ROS-induced damage, and / or by free radical decay across the label-polymerase separation distance (shown as pathway (iv)).

[0207] Figure 4C shows a nucleotide associated with a labeled oligonucleotide. As shown, the labeled oligonucleotide is hybridized with an unlabeled oligonucleotide strand. In accordance with the present application, such a construct provides a high degree of rigidity that enhances each of the above advantages described for the labeled nucleotide of Figure 4B.

[0208] For example, the hybridized strand provides increased rigidity, meaning less overall flexibility, which further facilitates label-polymerase separation. The hybridized strand also provides another barrier between the label and the polymerase, and can absorb label-induced decay (shown as path (v)). Thus, ROS-induced damage is further reduced due to the hybridized strand further restricting the access of the label to the bulk solvent, and / or the hybridized strand absorbing ROS-induced damage and / or free radical decay, which increased the label-polymerase separation distance.

[0209] Thus, in each of the above examples, the advantages provided by the labeled biomolecules of the present application provide increased read length in sequencing reactions by limiting the extent of light-induced damage to the polymerase. Additionally, the labeled nucleotides of the present application provide improved accuracy, for example, as a result of enhanced one or more light-emitting properties.

[0210] In the context of a labeled biomolecule, a "nucleotide" or "nucleoside polyphosphate" attached thereto should be understood to refer to one or more nucleotides (e.g., nucleoside polyphosphates) that are configured to be incorporated into a growing nucleic acid chain (e.g., during a sequencing reaction). In some embodiments, the one or more nucleotides comprise one or more nucleoside monophosphates or nucleoside polyphosphates. Examples of nucleoside polyphosphates include, in some embodiments, nucleosides having more than three 5'-phosphates, such as nucleoside diphosphates or triphosphates, or nucleoside hexaphosphates. In some embodiments of any of the compositions or methods described in this application, the phosphate portion (e.g., polyphosphate portion) of the nucleotide comprises 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 of a nucleotide (e.g., a polyphosphate portion) can include a phosphate ester, a thioester, a phosphoramidate, an alkylphosphonate linkage, other suitable linkage, or two or more such modifications, or a combination of two or more thereof.

[0211] The labeled nucleotide may be a terminal phosphate labeled nucleotide, in which a labeled biomolecule of the present application is attached to the terminal phosphate of the nucleotide. For example, in some embodiments, one or more nucleotides are attached to a biomolecule (e.g., Q of formula (I)) that forms part of the labeled biomolecule, as described herein. 1 and / or Q 2 ) via a terminal phosphate. Thus, in some embodiments, a "labeled nucleotide" in this application refers to a nucleotide attached to a labeled biomolecule of formula (I). In some embodiments, one or more nucleotides can be attached via a terminal phosphate to an oligonucleotide (e.g., an unlabeled oligonucleotide strand) that forms part of a labeled biomolecule as described in this application.

[0212] The labeled biomolecule can be linked to the terminal phosphate of the nucleotide via a linker. The linker can include, for example, at least one hydroxyl group, sulfhydryl group, amino group, or haloalkyl group, which can be suitable for forming, for example, a phosphate ester, a thioester, a phosphoramidate, or an alkylphosphonate bond at the terminal phosphate of a natural or modified nucleotide. The linker can be cleavable to separate the label from the terminal phosphate, such as with the aid of a polymerase. Examples of nucleotides and linkers are provided in U.S. Pat. No. 7,041,812, which is incorporated herein by reference in its entirety. In some embodiments, the linker includes an optionally substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted heteroalkylene, an optionally substituted heteroalkenylene, an optionally substituted heteroalkynylene, an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, and combinations thereof. Further examples of linkers useful for attaching labels to nucleotides can be found in co-pending US patent application Ser. No. 15 / 600,979, the relevant portions of which are incorporated herein by reference in their entirety.

[0213] Nucleotides (e.g., nucleoside polyphosphates) may include adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or any of their variants. Nucleotides (e.g., nucleoside polyphosphates) may include methylated nucleobases. For example, a methylated nucleotide may be a nucleotide that includes one or more methyl groups attached to the nucleobase (e.g., directly attached to the ring of the nucleobase, attached to a substituent of the ring of the nucleobase). Exemplary methylated nucleobases include 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.

[0214] The term "nucleic acid" as used herein generally refers to a molecule comprising one or more nucleic acid subunits. A nucleic acid may comprise one or more nucleic acid subunits selected from adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. In some examples, a nucleic acid is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a derivative thereof. In some embodiments, a 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. A nucleic acid may be single-stranded or double-stranded. In some embodiments, a nucleic acid generally refers to any polymer of nucleotides.

[0215] Luminous properties As described herein, internal conjugation of a label to a biomolecule can alter the photophysical properties of the label (e.g., via restricted rotation or immobilization of the label within the biomolecule). Thus, in certain embodiments, one or more luminescence properties of a labeled biomolecule are altered (e.g., increased) compared to an unconjugated molecule that includes a label. As described herein, an "unconjugated molecule" that includes a label can be expressed as a Q 1 and Q 2 The one or more luminescence properties that are altered by internal conjugation can include, but are not limited to, luminescence lifetime, luminescence intensity, brightness, emission maximum, luminescence quantum yield, and photostability.

[0216] In certain embodiments, the emission lifetime of the labeled biomolecule is increased compared to the unconjugated molecule. In certain embodiments, the emission lifetime of the labeled biomolecule is increased by at least 10% compared to the unconjugated molecule. In certain embodiments, the emission lifetime of the labeled biomolecule is increased by between about 10% and 50% (e.g., between about 10% and 25%, between about 10% and 15%, between about 25% and 50%, between about 40% and 50%) compared to the unconjugated molecule. Examples of increased emission lifetimes are shown in Figures 5A-5B.

[0217] A set of labeled biomolecules containing nucleotides were prepared according to the constructs generally shown in Figure 5A. Labeled nucleotides (1) and (2) represent conjugates in which the Cy3B dye is externally conjugated to a DNA linker at the terminus or branch point of the oligonucleotide, respectively. In contrast, labeled nucleotide (3) exchanges bases within the oligonucleotide strand, allowing the Cy3B dye itself to be internally incorporated into the DNA backbone. These labeled nucleotides were used in sequencing experiments to obtain lifetime measurements for each conjugate. The externally conjugated Cy3B constructs (1) and (2) yielded a lifetime of approximately 2.2 nanoseconds, while the lifetime measured for the internally conjugated Cy3B construct (3) was 2.6 nanoseconds, which is an approximately 15-20% increase in lifetime for the internally conjugated dye.

[0218] In certain embodiments, the emission intensity of the labeled biomolecule is increased compared to the unconjugated molecule. In certain embodiments, the emission intensity of the labeled biomolecule is increased by about 5%-25% (e.g., about 5%-20%, about 5%-15%, about 5%-10%, about 10%-25%, about 15%-25%, about 20%-25%) compared to the unconjugated molecule. In some embodiments, the emission intensity of the labeled biomolecule is increased by about 5%, about 10%, about 15%, about 20%, about 25%, or more compared to the unconjugated molecule.

[0219] In certain embodiments, the brightness of the labeled biomolecule is increased compared to the unconjugated molecule. In certain embodiments, the brightness of the labeled biomolecule is increased by about 5%-10% compared to the unconjugated molecule. In certain embodiments, the brightness of the labeled biomolecule is increased by about 5%-25% (e.g., about 5%-20%, about 5%-15%, about 5%-10%, about 10%-25%, about 15%-25%, about 20%-25%) compared 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 compared to the unconjugated molecule.

[0220] In certain embodiments, the maximum emission of the labeled biomolecule is increased by at least 1% compared to the unconjugated molecule. In certain embodiments, the maximum emission of the labeled biomolecule is increased by between about 1% and 10% (e.g., between about 1% and 5%, between about 5% and 10%) compared to the unconjugated molecule. An example of increased maximum emission is shown in FIG. 6. In some embodiments, the maximum emission of the labeled biomolecule is increased by about 1%, about 2%, about 5%, about 10%, or more compared to the unconjugated molecule.

[0221] Bulk fluorescence data obtained for internally and externally conjugated Cy3B is shown in Figure 6. As shown, the excitation spectrum of the internal dye is red-shifted by 8 nm relative to the external dye, resulting in a vibronic shoulder (the hump on the left side of the trace) located closer to 532 nm. Also shown, the emission spectrum of the internal dye is red-shifted, which was found to advantageously increase the detectable signal as more light was passed through the filter.

[0222] In certain embodiments, the luminescence quantum yield of the labeled biomolecule is increased compared to the unconjugated molecule. In certain embodiments, the luminescence quantum yield of the labeled biomolecule is increased by about 5% to 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%) compared 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 compared to the unconjugated molecule.

[0223] In certain embodiments, the photostability of the labeled biomolecule is increased compared 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 rate of photobleaching. For example, in some embodiments, the rate of photobleaching may be measured for a labeled biomolecule (e.g., a biomolecule with an internally conjugated label) and compared to the rate of photobleaching measured for an unconjugated molecule. A decrease in the rate of photobleaching measured would indicate an increase in photostability. Methods for measuring photobleaching rates are known in the art and are described, for example, in Wuestner, 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 labeled biomolecule exhibits reduced photobleaching compared to the unconjugated molecule, as measured by fluorescence recovery after photobleaching (FRAP), for example, as described in Meyvis, T. et al., (1999), Pharmaceutical Research, 16(8):1153-1162. In some embodiments, the labeled biomolecule exhibits reduced photobleaching compared to the unconjugated molecule, as measured by fluorescence loss in photobleaching (FLIP), e.g., as described in Wuestner, D. et al., (2012), BMC Bioinformatics, 13:296.

[0224] In some embodiments, the present disclosure provides novel compositions for identifying single molecules based on one or more luminescence properties of those molecules. In some embodiments, molecules (e.g., luminescently labeled nucleotides) are identified based on their brightness, luminescence lifetime, absorption spectrum, emission spectrum, luminescence quantum yield, luminescence intensity, or a combination of two or more thereof. Identifying may mean assigning the exact molecular identity of a molecule, or may mean distinguishing or identifying a particular molecule from a set 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, single molecules are identified (e.g., distinguished from other molecules) by exposing the molecules to a series of separate light pulses and evaluating the timing or other properties of each photon emitted from the molecule. In some embodiments, information of multiple photons emitted sequentially from a single molecule is compiled and evaluated to identify the molecule. In some embodiments, the luminescence lifetime of a molecule can be determined from multiple photons emitted sequentially from the molecule, and the luminescence lifetime can be used to identify the molecule. In some embodiments, the luminescence intensity of a molecule can be determined from a plurality of photons sequentially emitted from the molecule, and the molecule can be identified using the luminescence intensity. In some embodiments, the luminescence lifetime and luminescence intensity of a molecule can be determined from a plurality of photons sequentially emitted from the molecule, and the molecule can be identified using the luminescence lifetime and luminescence intensity.

[0225] Thus, in some aspects of the present application, the reaction sample is exposed to multiple separate light pulses and the sequence of emitted photons is detected and analyzed. In some embodiments, the sequence of emitted photons provides information about a single molecule that is present in the reaction sample during the experiment and does not change. However, in some embodiments, the sequence of emitted photons provides information about a sequence of different molecules that are present at different times in the reaction sample (e.g., as a reaction or process progresses).

[0226] Determining the emission lifetime of a molecule can be performed using any suitable method (e.g., by measuring the lifetime using a suitable technique or by determining a time-dependent characteristic of the emission). In some embodiments, determining the emission lifetime of a molecule includes determining the lifetime for one or more molecules (e.g., differently luminescently labeled nucleotides in a sequencing reaction). In some embodiments, determining the emission lifetime of a molecule includes determining the lifetime relative to a reference. In some embodiments, determining the emission lifetime of a molecule includes measuring a lifetime (e.g., a fluorescence lifetime). In some embodiments, determining the emission lifetime of a molecule includes determining one or more time characteristics indicative of the lifetime. In some embodiments, the emission lifetime of a molecule can be determined based on a 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 over one or more time-gated windows relative to an excitation pulse. For example, based on the distribution of photon arrival times measured for an excitation pulse, the emission lifetime of a single molecule can be distinguished from multiple molecules with different emission lifetimes.

[0227] It should be appreciated that the luminescence lifetime of a single molecule is indicative of the timing of photons emitted after the single molecule reaches an excited state, and single molecules can be distinguished by information indicative of the timing of the photons. 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 times may provide an indication of the luminescence lifetime, which may be determined from the distribution. In some embodiments, a single molecule can be distinguished from a plurality of molecules based on the distribution of times, such as by comparing the distribution of times to a reference distribution corresponding to known molecules. In some embodiments, a luminescence lifetime value is determined from the distribution of times.

[0228] As used herein for a single molecule, luminescence intensity refers to the number of emission photons per unit time emitted by a molecule excited by delivery of pulsed excitation energy. In some embodiments, luminescence intensity refers to the detected number of emission photons per unit time emitted by a molecule excited by delivery of pulsed excitation energy and detected by a particular sensor or set of sensors.

[0229] In some aspects, the present disclosure provides methods and compositions relating to labeled biomolecules with enhanced luminescence brightness. As used herein, in some embodiments, "brightness" (and variations thereof, e.g., "bright", "brightly", etc.) refers to a parameter that reports the average luminescence intensity per labeled reactant molecule. Thus, in some embodiments, "luminescence intensity" may be used generally to refer to the brightness of a composition that includes a brightly labeled reactant. In some embodiments, the brightness of a labeled reactant is equal to the product of its quantum yield and extinction coefficient. In some embodiments, the labeled biomolecules of the present disclosure are engineered to maximize quantum yield to facilitate increased brightness.

[0230] Luminescence quantum yield refers to the proportion of excitation events at a given wavelength or within a given spectral range that result in a luminescence event, and is typically less than 1. In some embodiments, the luminescence quantum yield of a molecule described herein is between 0 and about 0.001, between about 0.001 and about 0.01, between about 0.01 and about 0.1, between about 0.1 and about 0.5, between about 0.5 and 0.9, or between about 0.9 and 1. In some embodiments, a molecule is identified by determining or estimating the luminescence quantum yield.

[0231] In some embodiments, the internal labels described herein allow for the addition of successive luminescent labels to a labeled biomolecule to increase brightness and / or luminescence intensity. In some embodiments, an internally labeled biomolecule containing two or more luminescent labels has the formula L n(x) indicates the luminance and / or luminous intensity according to n where L is the total number of luminescent labels on the labeled reactant, and x is the measured brightness or fluorescence intensity of the corresponding single-labeled reactant. Thus, in some embodiments, a two-dye labeled reaction component has twice the brightness and / or luminescence intensity compared to its one-dye labeled analog. In some embodiments, a three- or four-dye labeled reaction component has three or four times the brightness and / or luminescence intensity, respectively, compared to its one-dye labeled analog. In some embodiments, the brightly labeled reactants described herein have a L n exhibits a luminance and / or luminescence intensity that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the value predicted by (x).

[0232] Nucleic Acid Sequencing Reaction Compositions Also provided herein are nucleic acid sequencing reaction compositions comprising two or more different types of labeled nucleotides in a reaction mixture, where at least one type of labeled nucleotide is a labeled nucleotide comprising a labeled biomolecule described herein.

[0233] In some embodiments, the nucleic acid sequencing reaction composition comprises 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 comprises four different types of labeled nucleotides. In some embodiments, the four different types of labeled nucleotides comprise a first labeled nucleotide that comprises a guanine, a second labeled nucleotide that comprises a cytosine, a third labeled nucleotide that comprises an adenine, and a fourth labeled nucleotide that comprises a thymine or uracil.

[0234] In some embodiments, each type of labeled nucleotide in a nucleic acid sequencing reaction composition is present at a concentration of about 100 nM to 1000 nM (e.g., about 100 nM to 800 nM, about 150 nM to 700 nM, about 200 nM to 600 nM, or about 250 nM to 500 nM).

[0235] In some embodiments, the nucleic acid sequencing reaction composition includes a sequencing template that includes a polymerase in a complex with a target nucleic acid. In some embodiments, the complex further includes a primer oligonucleotide having a sequence complementary to a portion of the target nucleic acid. In some embodiments, the complex is present at a concentration of about 10 pM to 10 nM (e.g., about 25 pM to 5 nM, about 50 pM to 2 nM, about 50 pM to 1 nM, about 50 pM to 500 pM, about 50 pM to 100 pM, about 250 pM to 5 nM, about 250 pM to 2 nM, about 250 pM to 1 nM, or about 250 pM to 500 pM).

[0236] In some embodiments, the nucleic acid sequencing reaction composition comprises one or more buffering agents (e.g., MES, MOPS, MOPSO, HEPES, Tris, TAPS, and other such suitable buffering agents known in the art). In some embodiments, the one or more buffering agents comprise MOPS. In some embodiments, the buffering agent is present at a concentration of between about 25-100 mM (e.g., between about 25-75 mM, between about 50-75 mM, or about 65 mM).

[0237] In some embodiments, the nucleic acid sequencing reaction composition includes a divalent cation (e.g., magnesium ions, calcium ions). In some embodiments, the divalent cation includes a magnesium salt or a calcium salt (e.g., a salt including magnesium or calcium and acetate, chloride, phosphate, sulfate). In some embodiments, the salt is magnesium acetate. In some embodiments, the divalent cation is present at a concentration of between about 5-50 mM (e.g., between about 10-40 mM, between about 15-35 mM, between about 20-30 mM, or about 25 mM).

[0238] In some embodiments, the nucleic acid sequencing reaction composition includes one or more monovalent salts (e.g., a sodium or potassium salt, such as sodium chloride, sodium acetate, potassium chloride, or potassium acetate). In some embodiments, the monovalent salt is present at a concentration of between about 10-200 mM (e.g., between about 25-150 mM, between about 25-40 mM, between about 50-150 mM, between about 100-150 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.

[0239] In some embodiments, the nucleic acid sequencing reaction composition includes one or more photo stabilizers (e.g., one or more photoprotective additives, such as antioxidants, oxygen scavengers, triplet state quenchers, and similar energy absorbing additives known in the art). In some embodiments, the photo stabilizer includes protocatechuic acid (PCA). In some embodiments, the photo stabilizer includes 4-nitrobenzyl alcohol (NBA). In some embodiments, the photo stabilizer includes Trolox or a derivative thereof. In some embodiments, the photo stabilizer is present at a concentration between about 0.1 mM and 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 with the photo stabilizer (e.g., PCA) may also include an enzyme for regenerating the photo stabilizer (e.g., protocatechuate dioxygenase (PCD)). 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.

[0240] In some embodiments, the nucleic acid sequencing reaction composition includes one or more reducing agents, for example, in some embodiments, the nucleic acid sequencing reaction composition includes about 10-100 mM DTT (e.g., about 40 mM DTT).

[0241] Sequencing Some aspects of the present application are useful for sequencing biological polymers such as nucleic acids and proteins. In some aspects, the compositions and techniques described herein can be used to identify a series of nucleotides or amino acid monomers that are incorporated into a nucleic acid or protein (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 herein can be used to identify a series of nucleotides that are incorporated into a template-dependent nucleic acid sequencing reaction product synthesized by a polymerase enzyme.

[0242] Therefore, the present application also provides a method for determining the sequence of template nucleic acid using the nucleic acid sequencing reaction composition of the present application.In some embodiments, the method for sequencing includes the following steps: (i) exposing a complex in a target volume, the complex comprising template nucleic acid, primer and polymerase, to the nucleic acid sequencing reaction composition of the present disclosure (e.g., at least one labeled nucleotide comprising the labeled biomolecule described herein); (ii) directing a series of one or more pulses of excitation energy to the vicinity of the target volume; (iii) detecting a plurality of photons emitted from the luminescent labeled nucleotide during its successive incorporation into nucleic acid comprising the primer; (iv) determining the timing and optionally the luminescence intensity of the emitted photons to identify the sequence of the incorporated nucleotide.

[0243] In some embodiments, as used herein, excitation energy is a pulse of light 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 at or near the absorption maximum of a luminescently labeled molecule from which multiple emission photons are to be detected. In certain embodiments, the excitation energy is between about 500 nm and about 700 nm (e.g., between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 500 nm and about 550 nm, between about 550 nm and about 600 nm, between about 600 nm and about 650 nm, or between about 650 nm and about 700 nm). In certain embodiments, the excitation energy may be monochromatic or may be limited to a spectral range. In some embodiments, the spectral range has a range between about 0.1 nm and about 1 nm, between about 1 nm and about 2 nm, or between about 2 nm and about 5 nm. In some embodiments, the spectral range has a range between about 5 nm and about 10 nm, between about 10 nm and about 50 nm, or between about 50 nm and about 100 nm.

[0244] Upon base pairing between the nucleobase of the target nucleic acid and the complementary nucleoside polyphosphate (e.g., dNTP), the polymerase incorporates the dNTP into the newly synthesized nucleic acid strand by forming a phosphodiester bond between the 3' hydroxyl end of the newly synthesized strand and the alpha phosphate of the dNTP. In examples where the luminescent molecule (e.g., the labeled biomolecule described herein) conjugated to the dNTP contains a fluorophore, its presence is signaled by excitation, and a pulse of light emission is detected during and / or after the incorporation step. In the case of a luminescent molecule (e.g., a labeled biomolecule) conjugated to the terminal (gamma) phosphate of the dNTP, incorporation of the dNTP into the newly synthesized strand results in the release of the beta and gamma phosphates and the luminescent molecule, which freely diffuses into the sample well, reducing the light emission detected from the fluorophore.

[0245] In certain embodiments, the template-dependent nucleic acid sequencing product is performed 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 that are complementary to the template nucleic acid strand. In one aspect, the present application provides a method for determining the sequence of a template (or target) nucleic acid strand by determining the sequence of its complementary nucleic acid strand.

[0246] The term "polymerase" as used herein generally refers to any enzyme (or polymerizing enzyme) capable of catalyzing a polymerization reaction. Examples of polymerases include, but are not limited to, nucleic acid polymerases, transcriptases, or ligases. A polymerase can be a polymerizing enzyme. An embodiment directed to single molecule nucleic acid extension (e.g., for nucleic acid sequencing) can use any polymerase capable of synthesizing 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 mutant or modified forms thereof.

[0247] Examples of polymerases include, but are not limited to, DNA polymerase, RNA polymerase, thermostable polymerase, wild-type polymerase, modified polymerase, E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase ψ29 (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 Included are Taq polymerase, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutbo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, polymerases with 3' to 5' exonuclease activity, and their mutants, modified products and derivatives. In some embodiments, the polymerase is a single subunit polymerase. Non-limiting examples of DNA polymerases and their properties are described in detail in, among others, DNA Replication, 2nd Edition, Kornberg and Baker, WH, Freeman, New York, NY (1991).

[0248] In another aspect, the present application provides a method for sequencing a target nucleic acid by sequencing a plurality of nucleic acid fragments, the target nucleic acid comprising fragments. In certain embodiments, the method includes combining a plurality of fragment sequences to provide a sequence or subsequence of a parent target nucleic acid. In some embodiments, the combining step is performed by computer hardware and software. The methods described herein may allow for sequencing a set of related target nucleic acids, such as an entire chromosome or genome.

[0249] During sequencing, a polymerizing enzyme can bind (e.g., attach) to a priming position of a target nucleic acid molecule. The priming position can be a primer that is complementary to a portion of the target nucleic acid molecule. Alternatively, the priming position is a gap or nick provided in a double-stranded segment of the target nucleic acid molecule. The gap or nick can be 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. The nick can provide a break in one strand of a double-stranded sequence, which can provide a priming position for a polymerizing enzyme, such as, for example, a strand-displacing polymerase enzyme.

[0250] In some cases, the sequencing primer can anneal to the target nucleic acid molecule, which may or may not be immobilized on a solid support. The solid support can include, for example, a sample well (e.g., nanoaperture, reaction chamber) on a chip used for nucleic acid sequencing. In some embodiments, the sequencing primer can be immobilized on a solid support, and hybridization of the target nucleic acid molecule also immobilizes the target nucleic acid molecule on the solid support. In some embodiments, the polymerase is immobilized on a solid support, and the soluble primer and the target nucleic acid are contacted with the polymerase. However, in some embodiments, a complex including the polymerase, the target nucleic acid, and the primer is formed in solution, and the complex is immobilized on the solid support (e.g., via immobilization of the polymerase, the primer, and / or the target nucleic acid). In some embodiments, none of the components in the sample well (e.g., nanoaperture, reaction chamber) are immobilized on a solid support. For example, in some embodiments, a complex including the polymerase, the target nucleic acid, and the primer is formed in solution, and the complex is not immobilized on a solid support.

[0251] Under appropriate conditions, a polymerase enzyme contacted with the annealed primer / target nucleic acid can add or incorporate one or more nucleotides onto the primer, and the nucleotides can be added 5'→3' to the primer in a template-dependent manner. Such incorporation of nucleotides into the primer (e.g., via the action of a polymerase) can generally be referred to as a primer extension reaction. Each nucleotide can be detected and identified during the nucleic acid extension reaction (e.g., based on its luminescence lifetime and / or other properties) and associated with a detectable label that can be used to determine each nucleotide incorporated into the extension primer, and thus the sequence of the newly synthesized nucleic acid molecule. Depending on the sequence complementarity of the newly synthesized nucleic acid molecule, the sequence of the target nucleic acid molecule can also be determined. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule and the incorporation of the nucleotide into the sequencing primer can occur under similar reaction conditions (e.g., the same or similar reaction temperature) or different reaction conditions (e.g., different reaction temperatures). In some embodiments, the sequencing by synthesis method may include a step of determining the presence of a population of target nucleic acid molecules (e.g., copies of the target nucleic acid) and / or amplifying the target nucleic acid to achieve a population of target nucleic acids. However, in some embodiments, sequencing by synthesis is used to determine the sequence of a single molecule in each reaction being evaluated (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 each performed in a separate reaction chamber (e.g., nanoaperture, sample well) on a single chip.

[0252] Embodiments are capable of sequencing a single nucleic acid molecule with high accuracy and long read lengths, such as at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% accuracy and / or read lengths of about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1,000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or 100,000 bp or more. 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 derivatives, ribonucleic acid (RNA), RNA derivatives) template that is added or immobilized to a sample well (e.g., nanoaperture) that contains at least one additional component of the sequencing reaction (e.g., a polymerase such as a DNA polymerase, a sequencing primer) that is immobilized or attached to a solid support, such as the bottom or side wall of the sample well. The target nucleic acid molecule or polymerase can be attached directly or via a linker to a sample wall, such as the bottom or side wall of the sample well. The sample well (e.g., nanoaperture) can also contain any other reagents necessary for nucleic acid synthesis via a primer extension reaction, such as appropriate buffers, cofactors, enzymes (e.g., polymerases), and deoxyribonucleoside polyphosphates, including deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxythymidine triphosphate (dTTP) dNTPs, which contain a luminescent label, such as, for example, the luminescent label of a labeled biomolecule provided herein.

[0253] In some embodiments, each class of dNTP (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., dUTP), and thymine-containing dNTPs (e.g., dTTP)) is conjugated to a luminescent molecule that contains a distinct luminescent property, such that detection of light emitted from the luminescent molecule indicates the identity of the dNTP incorporated into the newly synthesized nucleic acid. The light emitted from the luminescent molecule (e.g., from a labeled biomolecule that contains at least one luminescent label) can be detected via any suitable device and / or method and attributed to the appropriate luminescent molecule (and thus 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., a labeled biomolecule of the present application) does not inhibit the incorporation of the dNTP into the newly synthesized nucleic acid strand or the activity of the polymerase. In some embodiments, the luminescent molecule is conjugated to the terminal phosphate (e.g., the gamma phosphate) of the dNTP.

[0254] In some embodiments, the single-stranded target nucleic acid template can be contacted with sequencing primers, dNTPs, polymerase, and other reagents required for nucleic acid synthesis. In some embodiments, all appropriate dNTPs can be contacted with the single-stranded target nucleic acid template simultaneously (e.g., all dNTPs are present at the same time) so that the incorporation of dNTPs can occur sequentially. In other embodiments, dNTPs can be contacted with the single-stranded target nucleic acid template sequentially, and the single-stranded target nucleic acid template is contacted with each appropriate dNTP separately, with a washing step between contacting the single-stranded target nucleic acid template with different dNTPs. Such cycles of contacting the single-stranded target nucleic acid template with each dNTP separately and then washing can be repeated for each successive base position of the single-stranded target nucleic acid template to be identified.

[0255] In some embodiments, the sequencing primer anneals to the single-stranded target nucleic acid template, and the polymerase sequentially incorporates dNTPs (or other nucleoside polyphosphates) into the primer based on the single-stranded target nucleic acid template. A unique luminescent molecule associated with each incorporated dNTP, such as the labeled biomolecules described herein, can be excited with appropriate excitation light during or after the incorporation of the dNTP into the primer, and the emission can then be detected using any suitable device and / or method. The detection of a specific emission of light (e.g., having a specific emission lifetime, intensity, spectrum, and / or combination thereof) can be attributed to the specific dNTP incorporated. The sequence obtained from the collection of detected luminescent molecules can then be used to determine the sequence of the single-stranded target nucleic acid template via sequence complementarity.

[0256] In some embodiments, the present disclosure may be used in conjunction with co-pending U.S. patent application Ser. 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 / 261,697, 15 / 261,7 The present invention provides methods and compositions that can be advantageously utilized in the techniques described in US Pat. Nos. 24, 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, the contents of each of which are incorporated herein by reference.

[0257] kit Also provided herein is a kit for sequencing a template nucleic acid, comprising two or more different types of labeled nucleotides, where 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 polymerizing enzyme. In some embodiments, the kit comprises a primer complementary to the template nucleic acid.

[0258] In some embodiments, the kit comprises a plurality of types of labeled nucleotides, including a labeled biomolecule described herein. In some embodiments, at least one type (e.g., two, three, four, five, or more types) of labeled nucleotides comprises a labeled biomolecule having two or more internally conjugated labels in accordance with the present application. In some embodiments, the plurality of nucleotides is selected from the labeled nucleotides shown in Figures 1A-1G, 4A-4C, 5A, and 5C. In some embodiments, the kit further comprises a polymerizing enzyme (e.g., a DNA polymerase as described elsewhere herein). In some embodiments, the kit further comprises a primer complementary to a template nucleic acid to be sequenced.

[0259] Biconjugatable Labels In another aspect, the present invention provides compounds. The compounds provided herein can be used as labels - i.e., can be used in conjugation reactions to form labeled biomolecules as described herein. For example, provided herein are compounds of formula (II) and salts thereof:

[0260] [ka]

[0261] During the ceremony: A is a polycyclic fluorophore; L 1 and L 2 is a linker independently 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 the reactive moiety.

[0262] As described herein, the compounds of formula (II) and their salts are bifunctional (e.g., "asymmetric"). 1 is a reactive moiety that can be used as a reactive handle in conjugation reactions. 1 is an oxygen protecting group, and R 1 is cleaved or removed following a conjugation reaction involving R, thereby revealing a free -OH group, which can be used as a reactive moiety in a subsequent conjugation reaction. 1 is a reactive moiety (e.g., a phosphoramidite) that is reactive in a nucleoside coupling reaction. 1 A variety of oxygen protecting groups can be used at positions corresponding to:

[0263] L 1 and L 2is defined herein and exemplary embodiments are provided. Additionally, exemplary embodiments of A are provided herein. All definitions and embodiments provided herein, including but not limited to those provided in the "Internally Labeled Biomolecules" section, are applicable to the compounds provided herein.

[0264] As defined herein, P 1 is an oxygen protecting group. Some 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 of the formula:

[0265] [ka]

[0266] In certain embodiments, P 1 is 4-monomethoxytrityl (MMT) of the formula:

[0267] [ka]

[0268] In certain embodiments, P 1 is 4,4-dimethoxytrityl (DMT), which has the formula:

[0269] [ka]

[0270] In certain embodiments, R 1 is a reactive moiety. For example, R 1is a reactive handle useful in polynucleotide synthesis, polypeptide synthesis, polysaccharide synthesis, and the like. The reactive moiety can be any group that can react with a second reactive moiety (e.g., an -OH or -NH group) to form a covalent bond. One of skill in the art will know which reactive moieties can be used to form bonds in polynucleotide synthesis, polypeptide synthesis, polysaccharide synthesis, and the like. In certain embodiments, R 1 is a moiety useful in polynucleotide synthesis (e.g., a phosphoramidite). In certain embodiments, R 1 is a phosphoramidite. In certain embodiments, R 1 is a phosphoramidite of the formula:

[0271] [ka]

[0272] In the formula, R N1 and R 2 is as defined herein. In certain embodiments, R 1 is a phosphoramidite of the formula:

[0273] [ka]

[0274] In certain embodiments, R 1 is a phosphoramidite of the formula:

[0275] [ka]

[0276] As defined herein, R 2is 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. 2 is optionally substituted alkyl. In certain embodiments, R 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 consists of the following formula:

[0277] [ka]

[0278] 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 including two R N1 are joined together with the intervening atoms to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl. In certain embodiments, R N1 is optionally substituted alkyl. In certain embodiments, R 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 1-3In 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 is isopropyl. In certain embodiments, R N1 An example of both is isopropyl.

[0279] In certain embodiments, R 1 is a moiety that is reactive in nucleoside coupling reactions, such as phosphoramidites. In certain embodiments, the compound of formula (II) has the following formula:

[0280] [ka]

[0281] or a salt thereof, wherein: 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; 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 including two R N1 are joined together with the intervening atoms to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl.

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

[0283] [ka]

[0284] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0285] [ka]

[0286] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0287] [ka]

[0288] or a salt thereof, wherein: Each instance of n is an integer from 1 to 20. In certain embodiments, the compound of formula (II) has the following formula:

[0289] [ka]

[0290] or a salt thereof, wherein: Each instance of n is an integer from 1 to 20. In certain embodiments, the compound of formula (II) has the following formula:

[0291] [ka]

[0292] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0293] [ka]

[0294] or a salt thereof. 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:

[0295] [ka]

[0296] or a salt thereof, wherein X - is a counter ion or is absent. In certain embodiments, the compound of formula (II) has the following formula:

[0297] [ka]

[0298] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0299] [ka]

[0300] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0301] [ka]

[0302] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0303] [ka]

[0304] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0305] [ka]

[0306] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0307] [ka]

[0308] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0309] [ka]

[0310] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0311] [ka]

[0312] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0313] [ka]

[0314] or a salt thereof. In certain embodiments, A can be a fluorophore, such as, for example, fluorescein or rhodamine. In certain embodiments, the compound of formula (II) has the following formula:

[0315] [ka]

[0316] or a salt thereof, wherein: Each R is independently hydrogen, halogen, -N, -CN, -NO, 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.

[0317] In certain embodiments, the compound of formula (II) has the following formula:

[0318] [ka]

[0319] or a salt thereof, wherein: n is independently an integer of 1 to 20. In certain embodiments, the compound of formula (II) has the following formula:

[0320] [ka]

[0321] or a salt thereof, wherein: n is independently an integer from 1 to 20, and R NEach 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.

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

[0323] [ka]

[0324] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0325] [ka]

[0326] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0327] [ka]

[0328] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0329] [ka]

[0330] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0331] [ka]

[0332] or a salt thereof. As described herein, in certain embodiments, A is a BODIPY fluorophore. Thus, in certain embodiments, the compound of formula (II) has the following formula:

[0333] [ka]

[0334] or a salt thereof, wherein: Each R is independently hydrogen, halogen, -N, -CN, -NO, 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 Ar is optionally substituted aryl or optionally substituted heteroaryl.

[0335] In certain embodiments, the compound of formula (II) has the following formula:

[0336] [ka]

[0337] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0338] [ka]

[0339] or a salt thereof, wherein: RAr is hydrogen, halogen, -N3, -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.

[0340] As generally defined herein, 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. O is hydrogen. In certain embodiments, R O is optionally substituted alkyl. In certain embodiments, R O is an arbitrarily 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 optionally substituted alkenyl. In certain embodiments, R O is optionally substituted alkynyl. In certain embodiments, R O is optionally substituted carbocyclyl. In certain embodiments, R O is optionally substituted heterocyclyl. In certain embodiments, R O is optionally substituted aryl. In certain embodiments, R O is optionally substituted heteroaryl. In certain embodiments, R Ois optionally substituted acyl. In certain embodiments, R O is an oxygen protecting group. In certain embodiments, R O is -C(=O)Ph.

[0341] As generally defined herein, 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, optionally including two R N are joined together with the intervening atoms to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl. In certain embodiments, R N is hydrogen. In certain embodiments, R N is optionally substituted alkyl. In certain embodiments, R N is an arbitrarily 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 optionally substituted alkenyl. In certain embodiments, R N is optionally substituted alkynyl. In certain embodiments, R N is optionally substituted carbocyclyl. In certain embodiments, R N is optionally substituted heterocyclyl. In certain embodiments, R N is optionally substituted aryl. In certain embodiments, R N is optionally substituted heteroaryl. In certain embodiments, R N is optionally substituted acyl. In certain embodiments, RN is a nitrogen protecting group. In certain embodiments, two R N is joined together with the intervening atoms to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl.

[0342] As generally defined herein, 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. S is hydrogen. In certain embodiments, R S is optionally substituted alkyl. In certain embodiments, R S is an arbitrarily 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 optionally substituted alkenyl. In certain embodiments, R S is optionally substituted alkynyl. In certain embodiments, R S is optionally substituted carbocyclyl. In certain embodiments, R S is optionally substituted heterocyclyl. In certain embodiments, R S is optionally substituted aryl. In certain embodiments, R S is optionally substituted heteroaryl. In certain embodiments, R S is optionally substituted acyl. In certain embodiments, R S is a sulfur protecting group. In certain embodiments, R S is PEG (polyethylene glycol). In certain embodiments, RS H - (CH2CH2O) m In certain embodiments, -SR S -S(CH2CH2O) m It is OCH3.

[0343] In certain embodiments, the compound of formula (II) has the following formula:

[0344] [ka]

[0345] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0346] [ka]

[0347] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0348] [ka]

[0349] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0350] [ka]

[0351] or a salt thereof, wherein n is independently an integer of 1 to 20. In certain embodiments, the compound of formula (II) has the following formula:

[0352] [ka]

[0353] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0354] [ka]

[0355] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0356] [ka]

[0357] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0358] [ka]

[0359] or a salt thereof. In certain embodiments, the compound of formula (II) has the following formula:

[0360] [ka]

[0361] or a salt thereof. Methods for preparing internally labeled biomolecules Also provided herein is a method for preparing an internally labeled biomolecule (e.g., a biomolecule of formula (I)) as described herein. In general, the method includes two subsequent conjugation steps involving a bifunctional compound (e.g., a compound of formula (II)) as provided herein.

[0362] Thus, provided herein is a method for preparing a labeled biomolecule, the method comprising: (i) Formula Q 2 -OH monomeric or oligomeric biomolecules or salts thereof may be catalyzed by conjugation to the following formula:

[0363] [ka]

[0364] or a salt thereof, with a compound of formula (II) under conditions sufficient to produce a conjugate thereof; (ii) P 1 The protecting group is cleaved to give the compound of the formula:

[0365] [ka]

[0366] or a salt thereof; and deprotecting the conjugate formed in step (i) under conditions sufficient to produce a conjugate of: (iii) treating the conjugate formed in step (ii) with a compound of formula Q under conditions sufficient to promote conjugation to produce a labeled biomolecule of formula (I). 1 -OR 1 or a salt thereof.

[0367] [ka]

[0368] For example, in certain embodiments, when internally labeled oligonucleotides are prepared, compounds of formula (II) can be synthesized using standard phosphoramidite chemistry, such as Q 2-OH. Furthermore, in certain embodiments, when an internally labeled oligonucleotide is prepared, the unprotected conjugate with hydroxyl moiety formed in step (ii) is coupled to an alkyl-(2-cyanoethyl)-N,N-diisopropyl)-phosphoramidite, as used in standard oligonucleotide synthesis. The reaction can be carried out in the presence of an activating agent, such as 1H-tetrazole, ethylthiotetrazole, benzylthiotetrazole, dicyanoimidazole, or other suitable weak acid. Step (iii) can then be carried out in certain embodiments using standard phosphoramidite chemistry.

[0369] Methods for preparing biconjugable labels In yet another aspect, the present invention provides synthetic methods for preparing the biconjugable labels described herein (eg, compounds of formula (II)).

[0370] General reaction parameters The following embodiments apply to all synthetic methods described herein. The reactions provided and described herein may include one or more reagents. In certain embodiments, the reagent may be present in a catalytic amount. In certain embodiments, the catalytic amount is 0-1 mol%, 0-5 mol%, 0-10 mol%, 1-5 mol%, 1-10 mol%, 5-10 mol%, 10-20 mol%, 20-30 mol%, 30-40 mol%, 40-50 mol%, 50-60 mol%, 60-70 mol%, 70-80 mol%, 80-90 mol%, or 90-99 mol%. In certain embodiments, the reagent may be present in a stoichiometric amount (e.g., about 1 equivalent). In certain embodiments, the reagent may be present in an excess amount (e.g., more 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.

[0371] The reactions described herein can be carried out at any temperature. In certain embodiments, the reactions are carried out at or near room temperature (rt) (21° C. or 70° F.). In certain embodiments, the reactions are carried out below room temperature (e.g., between −100° C. and 21° C.). In certain embodiments, the reactions are carried out at or near −78° C. In certain embodiments, the reactions are carried out at or near −10° C. In certain embodiments, the reactions are carried out at or near 0° C. In certain embodiments, the reactions are carried out at temperatures greater than room temperature. In certain embodiments, the reactions are carried out at 30, 40, 50, 60, 70, 80, 110, 120, 130, 140, or 150° C. In certain embodiments, the reactions are carried out at temperatures greater than 150° C.

[0372] The reactions described herein can be carried out in a solvent or in a mixture of solvents (e.g., a co-solvent). The solvent can be polar or non-polar, protic or aprotic. Any solvent can be used in the reactions described herein, and the reactions are not limited to a particular solvent or combination of solvents. Common organic solvents useful in 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-pyrimidinone (DMPU), 1,4-dioxane, 1,3-dioxane, diethyl ether, 2-ethoxyethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, and n-hexane. , hexanes, hexamethylphosphoramide (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidinone, dimethylsulfoxide (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, and p-xylene.

[0373] The reactions described herein can be carried out for any amount of time, in certain embodiments, the reactions can be carried out for seconds, minutes, hours, or days. The methods described herein can be used to prepare compounds in any chemical yield. In certain embodiments, compounds are produced in 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 percent yield after one synthetic step. In certain embodiments, the yield is the percent yield after two or more synthetic steps (e.g., 2, 3, 4, or 5 synthetic steps).

[0374] 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 can be purified by chromatography, extraction, filtration, precipitation, crystallization, or any other method known in the art. In certain embodiments, the compounds or mixtures are carried forward to the next synthetic step without purification (e.g., crude).

[0375] Methods for preparing Cy3B-based labels Provided herein is a compound of formula (III):

[0376] [ka]

[0377] or a salt thereof, the method comprising:

[0378] [ka]

[0379] or a salt thereof,

[0380] [ka]

[0381] or a salt thereof, in the presence of palladium to produce a compound of formula (III) or a salt thereof, X 1 is a halogen or a leaving group; B(R B 2 is a borane, a boronic acid, or a boronic ester; 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 R O is an oxygen protecting group.

[0382] 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 may be catalyzed by palladium or by a different metal. In certain embodiments, the metal is a transition metal.

[0383] As defined herein, the group -B(R B )2 is a borane, a boronic acid, or a boronic ester. In certain embodiments, -B(R B )2 is borane. In certain embodiments, -B(R B )2 is a boronic acid. In certain embodiments, -B(R B )2 is a boronic ester. In certain embodiments, -B(R B )2 is a borane with the formula:

[0384] [ka]

[0385] 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 B is joined together with the intervening atoms to form an optionally substituted carbocyclyl or an optionally substituted heterocyclyl. B ) 2 is any suitable borane, boronic acid, or boronic ester useful in metal-promoted or metal-catalyzed cross-coupling reactions.

[0386] Linker L 1 is as defined herein. As defined herein, X 1 is a halogen or a leaving group. In certain embodiments, X 1 is halogen. In certain embodiments, X 1 is a leaving group. In certain embodiments, X 1 is -Cl, -Br, or -I. In certain embodiments, X 1 is -I.

[0387] In a particular embodiment of the coupling reaction, the compound of formula (III) has the following formula:

[0388] [ka]

[0389] or a salt thereof, and thus the starting material consists of a compound of the formula:

[0390] [ka]

[0391] or a salt thereof. In certain embodiments, the compound of formula (III) has the following formula:

[0392] [ka]

[0393] or a salt thereof, wherein n is an integer from 1 to 20, and therefore the starting material comprises a compound of the formula:

[0394] [ka]

[0395] or a salt thereof. 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 PdCl2(dppf). In certain embodiments, the palladium can be present in a catalytic amount. In other embodiments, the palladium is present in a stoichiometric or excess amount.

[0396] 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 Cs2CO3.

[0397] In certain embodiments, the coupling reaction is carried out in a solvent. In certain embodiments, the solvent is THF, DMF, or a mixture thereof. 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., an elevated temperature). In certain embodiments, the reaction is carried out at room temperature to 100°C. In certain embodiments, the reaction is carried out at 50 to 100°C. In certain embodiments, the reaction is carried out at about 70°C.

[0398] In certain embodiments, the method comprises reacting a compound of formula (III) with a compound of the following formula:

[0399] [ka]

[0400] or a salt thereof to form a compound of the formula:

[0401] [ka]

[0402] or its tautomer;

[0403] [ka]

[0404] or a salt thereof, wherein: X 1 is a halogen or a leaving group; and Each R 3 are independently an optionally substituted alkyl, an optionally substituted acyl, or an oxygen protecting group; optionally, two R 3 is attached to an intervening atom to form an optionally substituted heterocyclyl.

[0405] In certain embodiments, the product is a compound of the formula:

[0406] [ka]

[0407] or its tautomer;

[0408] [ka]

[0409] or a salt thereof. 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. According to 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 an elevated temperature. In certain embodiments, the reaction is carried out at about 100° C.

[0410] As defined herein, X 2 is a halogen or a leaving group. In certain embodiments, X 2 is halogen. In certain embodiments, X 2 is -Cl, -Br, or -I. In certain embodiments, X 2 In certain embodiments, X is -Br. 2 is a leaving group.

[0411] As defined herein, each R 3 are independently an optionally substituted alkyl, an optionally substituted acyl, or an oxygen protecting group; optionally, two R 3 is attached to an intervening atom to form an optionally substituted heterocyclyl. 3 is optionally substituted alkyl. In certain embodiments, R 3 is optionally substituted acyl. In certain embodiments, R O is an oxygen protecting group. In certain embodiments, two R 3 is joined together with the intervening atoms to form an optionally substituted heterocyclyl. In certain embodiments, two R 3 is bonded with the intervening atom

[0412] [ka]

[0413] In certain embodiments, two R 3 is bonded with the intervening atom

[0414] [ka]

[0415] Form. In certain embodiments, the method comprises administering to a subject a compound of the formula:

[0416] [ka]

[0417] or its tautomer;

[0418] [ka]

[0419] or a salt thereof, in the presence of formamidine to produce a compound of the formula:

[0420] [ka]

[0421] In certain embodiments, the reaction involves (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 product.

[0422] 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 (Ac2O). In certain embodiments, the reaction in step (i) is carried out at elevated temperature (e.g., about 125°C). In certain embodiments, the reaction is carried out in a solvent. In certain embodiments, the reaction in step (ii) is carried out in the presence of a base. In certain embodiments, the base is an amine base (e.g., a trialkylamine base). In certain embodiments, the base is Et3N and 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 elevated temperature (e.g., about 80°C).

[0423] In certain embodiments, the product has the following formula:

[0424] [ka]

[0425] or a salt thereof. In certain embodiments, the method comprises reacting, in the presence of an acid, a compound of the formula:

[0426] [ka]

[0427] or a salt thereof, by cyclization to form a compound of the formula:

[0428] [ka]

[0429] or a salt thereof. In certain embodiments, the acid is a 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 CH2Cl2. In certain embodiments, the reaction is carried out at elevated temperature (e.g., about 60°C).

[0430] In certain embodiments, the product has the following formula:

[0431] [ka]

[0432] or a salt thereof. In certain embodiments, the method comprises administering to a subject a compound of the formula:

[0433] [ka]

[0434] or a salt thereof to provide a compound of the formula:

[0435] [ka]

[0436] or a salt thereof. In certain embodiments, the compound has the formula:

[0437] [ka]

[0438] or a salt thereof. In certain embodiments, the method comprises: (i) a compound of the formula:

[0439] [ka]

[0440] or a salt thereof to form a compound of the formula:

[0441] [ka]

[0442] or a salt thereof; (ii) treating the compound produced in step (i) with a compound of the formula:

[0443] [ka]

[0444] or a salt thereof. definition chemistry definition 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, inside cover of the 75th edition of the "Handbook of Chemistry and physics", and specific functional groups are generally defined as described herein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th 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, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0445] The compounds described herein may contain one or more asymmetric centers and therefore may exist in various stereoisomers, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures 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 can 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 (E.L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972). The invention further encompasses the compounds as individual isomers substantially free of other isomers or as mixtures of various isomers.

[0446] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of hydrogen with deuterium or tritium. 19 F 18 Substitution for F, or 12 C 13 C or 14 Compounds having this structure, except for the substitution at C, are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

[0447] When a range of values ​​is listed, it is intended to include each value and sub-range within the range. For example, "C 1-6 Alkyl" is C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , 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 is intended to be included.

[0448] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Similarly, the term "heteroaliphatic" refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

[0449] The term "alkyl" refers to the radical of a linear or branched 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 1-4In 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 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C 2-6 "Alkyl"). C 1-6 Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C6) (e.g., n-hexyl). Further examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (e.g., halogens such as F) ("substituted alkyl"). In certain embodiments, an alkyl group is an unsubstituted C 1-10 Alkyl (e.g., unsubstituted C 1-6 Alkyl, for example, -CH3 (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, for example, unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr), unsubstituted butyl (Bu, for example, 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 a substituted C 1-10 Alkyl (e.g., substituted C 1-6 alkyl, for example, -CF3, Bn).

[0450] The term "haloalkyl" refers to a substituted alkyl group in which one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. 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 -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, -CCl3, -CFCl2, -CF2Cl, and the like.

[0451] The term "heteroalkyl" refers to an alkyl group that contains at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, either internally (e.g., inserted between adjacent carbon atoms of a parent chain) and / or at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-10 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 9 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-9 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 8 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-8 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 7 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-7In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 6 carbon atoms and one or more heteroatoms in the parent chain ("heteroC 1-6 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1-5 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 1-4 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 3 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1-3 In some embodiments, a heteroalkyl group refers to a saturated group having 1 to 2 carbon atoms and 1 heteroatom in the parent chain ("heteroC 1-2 In some embodiments, heteroalkyl refers to a saturated group having one carbon atom and one heteroatom in the parent chain ("heteroC1 alkyl"). In some embodiments, heteroalkyl refers to a saturated group having 2-6 carbon atoms and 1 or 2 heteroatoms in the parent chain ("heteroC 2-6 Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted ("unsubstituted heteroalkyl") or substituted ("substituted heteroalkyl") with one or more substituents. In certain embodiments, a heteroalkyl group is an unsubstituted heteroC 1-10 In certain embodiments, the heteroalkyl group is a substituted heteroalkyl group. 1-10 It is an alkyl.

[0452] The term "alkenyl" refers to the radical of a straight-chain or branched 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, an alkenyl group has 2 to 9 carbon atoms ("C 2-9In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C 2-3 In some embodiments, an alkenyl group has two carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as 2-butenyl) or terminal (such as 1-butenyl). 2-4 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Further examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, an alkenyl group is an unsubstituted C 2-10 In certain embodiments, the alkenyl group is a substituted C 2-10 In an alkenyl group, a C=C double bond with unspecified stereochemistry (e.g., -CH=CHCH3 or

[0453] [ka]

[0454] ) can be an (E)- or a (Z)-double bond. The term "heteroalkenyl" refers to an alkenyl group that contains at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of a parent chain) and / or further at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having 2 to 10 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-10 In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-9 In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-8 In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-7 In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and one or more heteroatoms in the parent chain ("heteroC 2-6 In some embodiments, heteroalkenyl groups have 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, heteroalkenyl groups have 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 has 2 to 3 carbon atoms, at least one double bond, and one heteroatom in the parent chain ("heteroC2-3 In some embodiments, heteroalkenyl groups have 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 ("unsubstituted heteroalkenyl") or substituted ("substituted heteroalkenyl") with one or more substituents. In certain embodiments, a heteroalkenyl group is an unsubstituted heteroC 2-10 In certain embodiments, the heteroalkenyl group is a substituted heteroC 2-10 It is alkenyl.

[0455] The term "alkynyl" refers to the radical of a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (see "C 2-10 In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C 2-9 In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, the alkynyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C 2-3 In some embodiments, an alkynyl group has two carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as 2-butynyl) or terminal (such as 1-butynyl). 2-4Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkynyl groups include pentynyl (C5), hexynyl (C6), and the like. Further examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, an alkynyl group is an unsubstituted C 2-10 In certain embodiments, the alkynyl group is a substituted C 2-10 It is alkynyl.

[0456] The term "heteroalkynyl" refers to an alkynyl group that contains at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of a parent chain) and / or further 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 one or more heteroatoms in the parent chain ("heteroC 2-10 In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-9 In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-8 In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-7In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and one or more heteroatoms in the parent chain ("heteroC 2-6 In some embodiments, heteroalkynyl groups have 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-5 In some embodiments, heteroalkynyl groups have 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-4 In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and one heteroatom in the parent chain ("heteroC 2-3 In some embodiments, heteroalkynyl groups have 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms in the parent chain ("heteroC 2-6 Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted ("unsubstituted heteroalkynyl") or substituted ("substituted heteroalkynyl") with one or more substituents. In certain embodiments, a heteroalkynyl group is an unsubstituted heteroC 2-10 In certain embodiments, the heteroalkynyl group is a substituted heteroC 2-10 It is alkynyl.

[0457] The term "carbocyclyl" or "carbocyclic" refers to a ring structure having 3 to 14 ring carbon atoms ("C 3-14 In some embodiments, a 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 3-8In 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 ("C 5-10 Carbocyclyl). Exemplary C 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C 3-8 Carbocyclyl groups include, but are not limited to, those described above for C 3-6 Included are carbocyclyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Exemplary C 3-10 Carbocyclyl groups include, but are not limited to, those described above for C 3-8 Carbocyclyl groups, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10As the preceding examples illustrate, in certain embodiments, a carbocyclyl group is either monocyclic ("monocyclic carbocycle") or polycyclic (including, for example, fused, bridged, or spiro ring systems such as bicyclic ("bicyclic carbocyclyl") or tricyclic ("tricyclic carbocyclyl") rings) and may be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups whose points of attachment are on the carbocyclyl ring, in such cases the number of carbons continues to indicate the number of carbons in the carbocyclyl ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, a carbocyclyl group is an unsubstituted C 3-14 In certain embodiments, the carbocyclyl group is a substituted C 3-14 It is a carbocyclyl.

[0458] In some embodiments, "carbocyclyl" refers to a monocyclic saturated carbocyclyl group having 3 to 14 ring carbon atoms ("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 5-6Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). 3-6 Examples of cycloalkyl groups include the aforementioned C 5-6 Cycloalkyl groups include cyclopropyl (C3) and cyclobutyl (C4). 3-8 Examples of cycloalkyl groups include the aforementioned C 3-6 Cycloalkyl groups include cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted ("substituted cycloalkyl") with one or more substituents. In certain embodiments, a cycloalkyl group is an unsubstituted C 3-14 In certain embodiments, the cycloalkyl group is a substituted C 3-14 It is cycloalkyl.

[0459] The term "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and one to four ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems such as bicyclic ("bicyclic heterocyclyl") or tricyclic ("tricyclic heterocyclyl") systems), may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, the point of attachment being either on the carbocyclyl ring or on the heterocyclyl ring, or a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, the point of attachment being on the heterocyclyl ring, in which case the number of ring members continues to indicate the number of ring members in the heterocyclyl ring system. Unless otherwise indicated, each instance of heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, a heterocyclyl group is an unsubstituted C 3-14 In certain embodiments, the heterocyclyl group is a substituted C 3-14 It is a 3-membered heterocyclyl.

[0460] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 5- to 6-membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0461] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazomethane, 1H-benzo[e][1,4]dihydropyrrole ... zepinyl, 1,4,5,7-tetrahydropyrano[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.

[0462] The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 pi electrons in a cyclic array) having 6 to 14 ring carbon atoms and zero heteroatoms in the aromatic ring system (see "C 6-14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl"; e.g., 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl"; e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the radical or point of attachment is on the aryl ring, and in such cases the number of carbon atoms continues to indicate the number of carbons in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, an aryl group is an unsubstituted C 6-14 In certain embodiments, the aryl group is a substituted C 6-14 It is aryl.

[0463] The term "heteroaryl" refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 pi electrons in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups in which the point of attachment is on the heteroaryl ring, in such cases the number of ring members continues to indicate 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 where the point of attachment is on either the aryl or heteroaryl ring, and in such cases the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, e.g., a ring with a heteroatom (e.g., 2-indolyl) or a ring that does not contain a heteroatom (e.g., 5-indolyl).

[0464] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being 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, each heteroatom being 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, each heteroatom being 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 with one or more substituents ("substituted cycloaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

[0465] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one 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, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0466] The term "unsaturated bond" refers to a double bond or a triple bond. The terms "unsaturated" or "partially unsaturated" refer to a moiety that contains at least one double or triple bond.

[0467] The term "saturated" refers to a moiety that does not contain any double or triple bonds, ie, the moiety contains only single bonds. The addition of the suffix "-ene" indicates that the group is a divalent moiety, for example, alkylene is a divalent moiety of an alkyl, alkenylene is a divalent moiety of an alkenyl, alkynylene is a divalent moiety of an alkynyl, heteroalkylene is a divalent moiety of a heteroalkyl, heteroalkenylene is a divalent moiety of a heteroalkenyl, heteroalkynylene is a divalent moiety of a heteroalkynyl, carbocyclylene is a divalent moiety of a carbocyclyl, heterocyclylene is a divalent moiety of a heterocyclyl, arylene is a divalent moiety of an aryl, and heteroarylene is a divalent moiety of a heteroaryl.

[0468] Unless otherwise specified, a group is optionally substituted. The term "optionally substituted" refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. "Optionally substituted" refers to a group that may be substituted or unsubstituted (e.g., "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" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl group). In general, the term "substituted" means that at least one hydrogen present on a group is replaced with an acceptable substituent, e.g., a substituent that upon substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction. Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when multiple positions of any given structure are substituted, the substituents are either the same or different at each position. The term "substituted" is considered to include substitution with all acceptable substituents of organic compounds, including any of the substituents described herein that result in the formation of a stable compound. The present invention contemplates all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety. It is not intended that the present invention be limited in any manner by the exemplary substituents described herein.

[0469] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 , -COH, -CHO, -C(OR cc )3, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R 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 SO2R aa , -NR bb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(Raa )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、-OP(OR cc )4、-B(R aa )2、-B(OR cc )2、-BR aa (OR cc )、C 1-10 アルキル、C 1-10 ペルハロアルキル、C 2-10 アルケニル、C2-10 Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C 3-10 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5-14 membered heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd group; where X - is a counterion; Or the two geminal hydrogens on a carbon atom are =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb , or =NOR cc substituted with a group; R aa Each instance of 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 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R aa groups join to form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R dd is substituted with a group; Rbb Examples of each are hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)(OR cc )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 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R bb groups join to form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R dd group; where X - is the counterion; R cc Each instance of is independently hydrogen, C 1-10 Alkyl, C 1-10 Perhaloalkyl, C 2-10 Alkenyl, C 2-10Alkynyl, Hetero C 1-10 Alkyl, Hetero C 2-10 Alkenyl, Hetero C 2-10 Alkynyl, C 3-10 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14 aryl, and 5- to 14-membered heteroaryl, or two R cc groups join to form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R dd is substituted with a group; R dd Each example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R 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 SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R 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 2-6 Alkenyl, C 2-6 Alkynyl, Hetero C 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, 3-10 membered heterocyclyl, C 6-10 aryl, and 5- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg or two geminal R dd The substituents may combine to form =O or =S; where X - is the counterion; R ee Each instance of 1-6 Alkyl, C 1-6 Perhaloalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, Hetero C1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, C 6-10 aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg is substituted with a group; R ff Each instance of is independently 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 Carbocyclyl, 3-10 membered heterocyclyl, C 6-10 aryl, and 5- to 10-membered heteroaryl, or two R ff groups join to form a 3- to 10-membered heterocyclyl or 5- to 10-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R gg is substituted with a group; and R gg Each example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 - , -NH2(C 1-6 Alkyl) + X- , -NH3 + 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), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -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), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -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)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH2, -NHC(=NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2(C 1-6 alkyl), -SO2O(C 1-6 alkyl), -OSO2(C 1-6 alkyl), -SO(C 1-6Alkyl), -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)NH2, -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 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, C 6-10 aryl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl; or two geminal R gg The groups can be linked to form =O or =S, where X - is the counter ion.

[0470] In certain embodiments, carbon atom substituents include halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -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 - , -NH2(C 1-6 Alkyl) + X - , -NH3 + X - , -N(OC 1-6 Alkyl)(C1-6 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), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -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), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -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)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH2, -NHC(=NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2(C 1-6 alkyl), -SO2O(C 1-6 alkyl), -OSO2(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 1-6alkyl)2, C(=S)NH(C 1-6 alkyl), C(=S)NH2, -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 1-6 Alkyl, Hetero C 2-6 Alkenyl, Hetero C 2-6 Alkynyl, C 3-10 Carbocyclyl, C 6-10 aryl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl; or two geminal R gg groups can be linked to form =O or =S, where X - is the counter ion.

[0471] The term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I). The term "hydroxyl" or "hydroxy" refers to an -OH group. The term "substituted hydroxyl" or "substituted hydroxyl" refers, by extension, to a hydroxyl group in which the oxygen atom directly attached to the parent molecule has been replaced with a group other than hydrogen, such as -OR aa , -ON(R bb )2, -OC(=O)SR aa , -OC(=O)R aa , -OCO2R aa , -OC(=O)N(R bb )2, -OC(=NR bb )R aa , -OC(=NR bb ) OR aa , -OC(=NRbb )N(R bb )2, -OS(=O)R aa , -OSO2R 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 R cc is as defined herein.

[0472] The term "amino" refers to the group -NH. The term "substituted amino" extends to mono-, di-, or tri-substituted amino. In certain embodiments, a "substituted amino" is a mono- or di-substituted amino group.

[0473] The term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced with one hydrogen and one non-hydrogen group, -NH(R bb ), -NHC(=O)R aa , -NHCO2R aa , -NHC(=O)N(R bb )2, -NHC(=NR bb )N(R bb )2, -NHSO2R aa , -NHP(=O)(OR cc )2, and -NHP(=O)(N(R bb 2)2), where R aa , R bb and R cc is as defined herein, and -NH(R bb ) group Rbb is not hydrogen.

[0474] The term "disubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced with two groups other than hydrogen, -N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -NR bb SO2R aa , -NR bb P(=O)(OR cc )2, and -NR bb P(=O)(N(Rbb)2)2, where R aa , R bb and R cc is as defined herein, provided that the nitrogen atom directly attached to the parent molecule is not replaced with hydrogen.

[0475] The term "trisubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is substituted with three groups, -N(R bb )3 and -N(R bb )3 + X - where R bb and X - is as defined herein.

[0476] The term "sulfonyl" means -SO2N(R bb )2, -SO2R aa , and -SO2OR aa where R aa and R bb is as defined herein.

[0477] The term "sulfinyl" means -S(=O)Raa refers to the group, where R aa is as defined herein. The term "acyl" refers to a 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 hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, 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 X1The groups taken together form a 5- to 6-membered heterocycle. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroaryl amino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, etc., each of which may be further substituted or unsubstituted).

[0478] The term "carbonyl" refers to the carbon that is directly attached to the parent molecule and has a sp 2 Hybridized groups substituted with oxygen, nitrogen or sulfur atoms, such as ketones (e.g., -C(=O)R aa ), carboxylic acids (e.g., -COH), aldehydes (-CHO), esters (e.g., -COR aa , -C(=O)SR aa , -C(=S)SR aa ), amides (e.g., -C(=O)N(R bb )2, -C(=O)NR bb SO2R 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 Raa and R bb is as defined herein.

[0479] The term "silyl" refers to the group -Si(R aa )3, where R aa is as defined herein. The term "oxo" refers to the group =O and the term "thioxo" refers to the group =S.

[0480] Nitrogen atoms may be substituted or unsubstituted where valence permits, including 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, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR 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 Carbocyclyl, 3-14 membered heterocyclyl, C6-14 Aryl, 5- to 14-membered heteroaryl, or two R attached to a N atom cc groups linked to form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R dd group, where R aa , R bb , R cc and R dd The groups are as defined above.

[0481] In certain embodiments, the substituent present on a 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)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR 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 Carbocyclyl, 3-14 membered heterocyclyl, C 6-14aryl, and 5- to 14-membered heteroaryl groups, where each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R dd group, where R aa , R bb , R cc and R dd The groups are as defined above. Nitrogen protecting groups are well known in the art and include those described in detail in "Protecting Groups in Organic Synthesis", TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0482] For example, an amide group (e.g., -C(=O)R aa Nitrogen protecting groups such as acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0483] Carbamate group (e.g. -C(=O)OR aaNitrogen protecting groups such as, but not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate, (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-Methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (A Allyl carbamate (Ipaoc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, 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 methylcarbamate (Peoc), 2-triphenylphosphonioiisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4-di Methoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N -dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborinyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropyl methyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0484] Sulfonamide groups (e.g., -S(=O)R aa Nitrogen protecting groups such as, but 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 (Mc), 2,4,6-trimethylbenzenesulfonamide (Md), 2,4,6-trimethylbenzenesulfonamide (Ms), 2,4,6-trimethylbenzenesulfonamide (Mtb ... sulfonamides (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 phenacylsulfonamide.

[0485] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethylphenylphenylamine, N-phenylpropanediol ... Methyldisilylazacyclopentane adducts (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-pyridones, N-methylamines, N-allylamines, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline- 3-yl)amine, quaternary ammonium salt, 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-1,1-dimethylamine thiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaylchromium or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidates, diphenyl phosphoramidates, benzenesulfenamides, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide and 3-nitropyridine sulfenamide (Npys). In certain embodiments, the nitrogen protecting group includes benzyl (Bn), tert-butyloxycarbonyl (BOC), carbobenzyloxy (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc), triphenylmethyl (Tr), tosyl (Ts), brosyl (Bs), nosyl (Ns), mesyl (Ms), triflyl (Tf), or dansyl (Ds).

[0486] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to herein as a "hydroxyl 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 , -CO2R aa , -C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb ) OR aa , -C(=NRbb )N(R bb )2, -S(=O)R aa , -SO2R 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)(R bb )2)2, where X - , R aa , R bb and R cc is as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in "Protecting Groups in Organic Synthesis", TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, incorporated herein by reference.

[0487] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), 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-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethane ethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-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-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenyloxymethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)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, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethysilyl diacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), 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-(triphenylphosphonio)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-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, methyldithiocarbonate, 2-iodobenzoic acid, 4-azidobutyrate, 4-nitro-4-methylpentanoic acid, o-(dibromomethyl)benzoic acid, 2-formylbenzyl carbonate, Examples of suitable salts include benzenesulfonic acid, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoic acid, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid, chlorodiphenylacetic acid, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). In certain embodiments, the oxygen protecting group is silyl. In certain embodiments, the oxygen protecting group is t-butyldiphenylsilyl (TBDPS), t-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-ethoxyethyl (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), triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl, p-methoxybenzyl (PMB), t-butyl, benzyl (Bn), allyl, or pivaloyl (Piv).

[0488] In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to herein as a "thio 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 , -CO2R 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 , -SO2R 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 R aa , R bb and R ccis as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in "Protecting Groups in Organic Synthesis", TW Greene and PGM Wuts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference. In certain embodiments, the sulfur protecting group is acetamidomethyl, t-butyl, 3-nitro-2-pyridinesulfenyl, 2-pyridinesulfenyl, or triphenylmethyl.

[0489] A "counterion" or "anionic counterion" is a negatively charged group associated with a positively charged group to maintain electronic neutrality. Anionic counterions can be monovalent (i.e., containing one formal negative charge). Anionic counterions can also be multivalent (i.e., containing multiple formal negative charges), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F - , Cl - , Br - , I - ), NO3 - , ClO4 - , O.H. - , H2PO4 - , HCO3 - , HSO4 - , 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, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, etc.), BF4 - , PF4 - , PF6 - , AsF6 - , SbF6 - , B[3,5-(CF3)2C6H3]4] - , B(C6F5)4 -, BPh4 - , Al(OC(CF3)3)4 - and carborane anions (e.g., CB 11 H 12 - or (HCB 11 Me5Br6) - Exemplary counterions, which may be multivalent, include CO3 2- , HPO4 2- , PO4 3- , B4O7 2- , SO4 2- , S2O3 2- , carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalate, aspartate, glutarate, etc.) and carboranes.

[0490] The term "leaving group" is given its ordinary meaning in the field of organic synthetic chemistry and refers to an atom or group that can be displaced by a nucleophile. See, for example, Smith, March Advanced Organic Chemistry, 6th Edition, pp. 501-502. Examples of suitable leaving groups include, but are not limited to, halogens (such as F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, and haloformates. In some cases, the leaving group is a sulfonate ester, such as toluenesulfonate (tosylate, -OT), methanesulfonate (mesylate, -OM), p-bromobenzenesulfonyloxy (brosylate, -OB), -OS(=O)2(CF2)3CF3 (nonaflate, -ONf), or trifluoromethanesulfonate (triflate, -OTf). In some cases, the leaving group is a brosylate, such as p-bromobenzenesulfonyloxy. In some cases, the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. The leaving group may be an internal leaving group, such as a phosphine oxide (formed, for example, during the Mitsunobu reaction) or 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. Further exemplary leaving groups include, but are not limited to, halo (e.g., chloro, bromo, iodo) and activated substituted hydroxyl groups (e.g., -OC(=O)SR aa , -OC(=O)R aa , -OCO2R 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)Raa , -OSO2R aa , -OP(R cc )2, -OP(R cc )3, -OP(=O)2R aa , -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -OP(=O)2N(R bb )2, and -OP(=O)(NR bb )2), where R aa , R bb , and R cc is as defined herein.

[0491] As used herein, use of the phrase "at least one instance" refers to 1, 2, 3, 4, or more instances, but also encompasses ranges of, for example, 1-4, 1-3, 1-2, 2-4, 2-3, or 3-4 instances.

[0492] "Non-hydrogen group" refers to any group defined for a particular variable that is not hydrogen. The following definitions are of more general terms used throughout this application.

[0493] As used herein, the term "salt" refers to any and all salts, including pharma- ceutically acceptable salts. The term "pharma-ceutically acceptable salts" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharma-ceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (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 pharma- ceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or using other methods known in the art, such as ion exchange. Other pharma- ceutically 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. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N-type salts such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 108, 109, 109, 109, 1 + (C 1-4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed, where appropriate, with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates and arylsulfonates.

[0494] It should also be 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 referred to as "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers."

[0495] Stereoisomers that are not mirror images of one another are called "diastereomers" and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers". For example, if a compound has an asymmetric carbon atom that is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and described by the R- and S-sequence rules of Cahn and Prelog, or by the way the molecule rotates the plane of polarized light and are referred to as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0496] The terms "catalysis," "catalyze," or "catalytic" refer to an increase in the rate of a chemical reaction through the participation of a substance called a "catalyst." In certain embodiments, the amount and nature of the catalyst remain essentially unchanged during the reaction. In certain embodiments, the catalyst is regenerated or the catalytic properties are essentially restored after the reaction. Catalysts can participate in multiple chemical transformations. The effectiveness of a catalyst can be altered by the presence of other substances known as inhibitors or poisons (which decrease catalytic activity) or promoters (which increase activity). Catalytic reactions have lower activation energies (rate-limiting free energies) than the corresponding uncatalyzed reactions, resulting in higher reaction rates at the same temperature. Catalysts can favorably influence the reaction environment, bind to reagents and polarize the bonds, form specific intermediates not typically produced by uncatalyzed reactions, or cause dissociation of reagents into reactive forms.

[0497] The term "solvent" refers to a substance that dissolves one or more solutes to provide 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 facilitate mixing of one or more reagents or reactants in a reaction mixture. A solvent can also serve to increase or decrease the rate of a reaction compared to a reaction in a different solvent. A solvent can be polar or non-polar, protic or aprotic. Common organic solvents useful in this method are described herein. EXAMPLES

[0498] Synthesis of bifunctional labels Indolenine benzoate 2

[0499] [ka]

[0500] To a flask containing 3-buten-1-ol benzoate (3.0 g, 17.1 mmol) was added a 0.5 M solution of 9-borabicyclo[3.3.1]nonane (34.1 mL, 17.1 mmol) in THF. The clear, colorless solution was stirred at room temperature for 3 h. In a separate flask fitted with a condenser, iodoindolenine (1, 3.4 g, 11.9 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride (0.7 g, 0.8 mmol), and cesium chloride (6.0 g, 18.5 mmol) were added. DMF (20 mL) was charged to the flask, and the dark suspension was sparged with argon for 10 min. After the addition of the borane benzoate / THF solution, the reaction was heated to 70 °C for 12 h. After this time, HPLC showed complete conversion of the starting indolenine 1. 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), transferred and dried over magnesium sulfate. After filtration and evaporation, the crude residue was purified by normal phase chromatography (0→50% EtOAc / hexanes, SiO2) to give indolenine 2 (3.0 g, 75% yield) as a viscous yellow oil. HRMS (ESI) showed C 22 H 26 NO2(M+H) + Calculated for 336.1964 and observed value was 336.1962.

[0501] Alkylated indolenine 3

[0502] [ka]

[0503] A Schlenk flask was charged with potassium iodide (2.0 g, 12.0 mmol) 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 vessel was heated to 100 °C for 14 h. The reaction was diluted with dichloromethane (20 mL) and the suspension was filtered through a fritted glass funnel. The filtrate was directly purified by normal phase chromatography (0 → 7% MeOH / DCM, SiO2) to give indolenine 3 (1.9 g, 57% yield) as a beige solid. HRMS (ESI) showed C 27 H 34 NO4(M) + Calculated for 436.2488 and observed value was 436.2487.

[0504] Trimethine Cyanine 4

[0505] [ka]

[0506] To a mixture of indolenine (3, 0.96 g, 1.7 mmol), diphenylformamidine (0.40 g, 2.1 mmol), and DMAP (21 mg, 0.17 mmol) was added acetic anhydride (5 mL). The brown mixture was heated to 120 °C for 1 h. After cooling to room temperature, the volatiles were concentrated in vacuo. To the crude intermediate was added an additional portion of indolenine (3, 1.6 g, 2.8 mmol), followed by ethanol (5 mL) and trimethylamine (1.2 mL, 8.6 mmol). The reaction was heated to reflux under argon for 1 h. The reaction was diluted with aqueous sodium chloride and extracted with DCM. The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by normal phase chromatography (100% EtOAc, then 0 → 10% MeOH / DCM, SiO2) to give Cy3 4 (0.9 g, 57% yield) as a dark purple solid. HRMS(ESI) is C 55 H 65 N2O8(M) +Calculated against 881.4735, observed value was 881.4717.

[0507] Cy3B Analog 5

[0508] [ka]

[0509] To a flask containing Cy3B4 (400 mg, 0.437 mmol) were added chloroform (6 mL) and sulfuric acid (4 mL, 50% v / v water). The biphasic mixture was stirred vigorously at 60 °C for 30 min, changing from deep red to purple in color. 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 aqueous sodium chloride, dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by normal phase chromatography (100% EtOAc, then 0 → 15% MeOH / DCM, SiO2) to give Cy3B5 (177 mg, 50% yield) as a dark purple solid. HRMS (ESI) showed C 51 H 55 N2O5(M) + Calculated for 775.4105 and observed value was 775.4091.

[0510] Cy3B Diol 6

[0511] [ka]

[0512] To a solution of Cy3B bibenzoate 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 h. The reaction was quenched by the addition of solid ammonium chloride (66 mg) and stirred at room temperature for 30 min. 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 crude residue was purified by normal phase chromatography (100% EtOAc, then 0 → 25% MeOH / DCM, SiO2) to give Cy3B 6 (99 mg, 86% yield) as a dark purple solid. HRMS (ESI) showed C 37 H 47 N2O3(M) + Calculated for 567.3581, observed value was 567.3568.

[0513] Cy3B mono-MMT7

[0514] [ka]

[0515] 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 min. The reaction was diluted with DCM and washed successively with water and saturated aqueous sodium chloride. The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude residue was purified by normal phase chromatography (100% EtOAc, then 0→25% MeOH / DCM (with 1% Et3N, SiO2)) to give Cy3B 7 (43 mg, 30% yield) as a dark purple solid. HRMS (ESI) showed C 57 H 63 N2O4(M) + Calculated for 839.4782, observed value was 839.4751.

[0516] Phosphoramidite 8

[0517] [ka]

[0518] To a solution of Cy3B7 (35 mg, 0.036 mmol) and N,N-diisopropylethylamine (14 μL, 0.079 mmol) in anhydrous dichloromethane was added 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (9.4 mg, 0.040 mmol) at room temperature. The reaction was diluted with deoxygenated DCM, washed with aqueous potassium chloride, dried over magnesium sulfate, filtered, and concentrated under vacuum. The crude residue was purified by normal phase chromatography (0→2% MeOH / DCM, 1% Et3N, with basic alumina). The product was redissolved in DCM (1 mL) and precipitated into hexanes (25 mL). Drying under high vacuum gave Cy3B8 (20 mg, 51% yield) as a dark purple solid. HRMS (ESI) showed C 66 H 80 N4O5P(M) + Calculated for 1039.5861, observed value was 1039.5835.

[0519] Equivalents and Scope In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or clear from the context. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, utilized in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The invention includes embodiments in which exactly one member of a group is present in, utilized in, or otherwise relevant to a given product or process. The invention includes embodiments in which two or more, or all of the members of a group are present in, utilized in, or otherwise relevant to a given product or process.

[0520] Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in the other claims that are dependent on the same base claim. When elements are presented as a list, for example in Markush group format, each subgroup of the same elements is also disclosed, and any element can be removed from the group. In general, when the invention or aspects of the invention are said to include certain elements and / or features, it is to be understood that the particular embodiment of the invention or aspects of the invention include or consist essentially of such elements and / or features. For simplicity, these embodiments are not specifically described in detail herein.

[0521] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so connected, i.e., elements that are conjunctively present in some cases and non-conjunctively present in other cases. Elements listed with "and / or" should be interpreted in the same manner, i.e., as "one or more" of the elements so connected. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a description "A and / or B," when used in conjunction with open-ended language, such as "comprising," may refer in one embodiment to only A (optionally including elements other than B); in another embodiment to only B (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements), etc.

[0522] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as being inclusive, i.e., including at least one, but not less than one, and optionally including additional unlisted items, of a number or list of elements. "Only one of," "exactly one of," or, when used in the claims, "consisting of," refers to the inclusion of exactly one element of a number or list of elements, unless the term clearly indicates to the contrary. In general, the term "or" as used herein shall be construed to indicate exclusive alternative terms (i.e., "one or the other but not both") only when preceded by terms of exclusivity such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0523] As used herein and in the 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 not necessarily including at least one of each and every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to such 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") refers to, in one embodiment, that A is at least one, optionally including two or more, and B is absent (optionally including elements other than B); in another embodiment, that B is at least one, optionally including two or more, and A is absent (optionally including elements other than A); in yet another embodiment, that A is at least one, optionally including two or more, and B is at least one, optionally including two or more (optionally including other elements), etc.

[0524] It is also to be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the steps or acts are recited.

[0525] In the claims and the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are understood to be open-ended, i.e., meaning including, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as defined in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be recognized that embodiments described herein that use open-ended transitional phrases (e.g., "comprising") also contemplate in alternative embodiments the feature "consisting of" and "consisting essentially of" the feature described by the open-ended transitional phrase. For example, if the disclosure describes a "composition comprising A and B," the disclosure also contemplates alternative embodiments such as a "composition consisting of A and B" and a "composition consisting essentially of A and B."

[0526] When ranges are specified, the endpoints are included. Additionally, unless otherwise specified or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges are to be understood as contemplated to the tenth of the unit of the lower limit of that range, unless the context clearly dictates otherwise, for any particular value or subrange within the stated range in different embodiments of the invention.

[0527] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, the specification shall control. Furthermore, any particular embodiment of the present invention that falls within the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are deemed known to those of skill in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0528] Those skilled in the art will recognize, or be able to ascertain or recognize using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the invention, as defined in the following claims.

[0529] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The description of an embodiment of a variable herein includes that embodiment in any single embodiment or in combination with any other embodiment or portion thereof. The description of an embodiment herein includes that embodiment in any single embodiment or in combination with any other embodiment or portion thereof. The technical ideas that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] A labeled biomolecule of formula (I), [ka] During the ceremony: Q 1 and Q 2 is independently a monomeric or oligomeric biomolecule; A is a polycyclic fluorophore, and L 1 and L 2 is a linker independently 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. [Appendix 2] 2. The labeled biomolecule of claim 1, wherein A is a cyanine, fluorone, acridine, phenoxazine, coumarin, BODIPY, porphyrin, phthalocyanine, naphthalimide, or squaraine fluorophore. [Appendix 3] L 1 is optionally substituted alkylene. [Appendix 4] L 1 is unsubstituted C 1-20 4. The labeled biomolecule of claim 3, wherein the alkylene is [Appendix 5] L 2 5. The labeled biomolecule according to any one of claims 1 to 4, wherein is optionally substituted alkylene. [Appendix 6] L 2 is unsubstituted C 1-20 6. The labeled biomolecule of claim 5, wherein the alkylene is [Appendix 7] 7. The labeled biomolecule according to any one of claims 1 to 6, wherein A is a polycyclic cyanine. [Appendix 8] 8. The labeled biomolecule according to any one of claims 1 to 7, wherein A is Cy3B. [Appendix 9] The labeled biomolecule has the formula:

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Claims

1. A labeled biomolecule, comprising: formula: 【Chemistry 1】 A first oligonucleotide strand having the formula: During the ceremony: Q １ and Q. ２ are independently an oligonucleotide, a nucleic acid, or a fragment thereof; X － is a counter ion or is absent, and L １ and L ２ is independently an optionally substituted alkylene; and a second oligonucleotide strand hybridized to the first oligonucleotide strand; Including, Labeled biomolecules.

2. L １ is unsubstituted C １－２０ 2. The labeled biomolecule of claim 1, which is an alkylene.

3. L ２ is unsubstituted C １－２０ 3. The labeled biomolecule of claim 1 or claim 2, which is an alkylene.

4. The first oligonucleotide strand has the formula: 【Chemistry 2】 It consists of: During the ceremony, The labeled biomolecule according to any one of claims 1 to 3, wherein n is independently an integer of 1 to 20.

5. The first oligonucleotide strand has the formula: 【Chemistry 3】 It consists of: During the ceremony, The labeled biomolecule of any one of claims 1 to 4, wherein n is independently an integer from 1 to 20.

6. Q １ and Q. ２ The labeled biomolecule of any one of claims 1 to 5, wherein: is independently a deoxyribonucleic acid, a ribonucleic acid, a peptide nucleic acid, a locked nucleic acid, or a fragment thereof.

7. The second oligonucleotide strand is Q １ and Q. ２ The labeled biomolecule of any one of claims 1 to 6, which hybridizes to

8. The second oligonucleotide strand is Q １ Or Q ２ The labeled biomolecule of any one of claims 1 to 6, which hybridizes to

9. 9. The labeled biomolecule of any one of claims 1 to 8, wherein the first and second oligonucleotide strands are independently optionally bound to a reactant configured for use as a substrate in a reaction, optionally the reaction is a polymerization reaction, and further optionally the reactant is cleaved from the labeled biomolecule by a polymerase when subjected to polymerization reaction conditions.

10. 10. The labeled biomolecule of claim 1, wherein one or more luminescence properties of the labeled biomolecule are increased compared to an unconjugated molecule comprising a Cy3B fluorophore.

11. 11. The labeled biomolecule of claim 10, wherein the one or more luminescence properties are selected from the group consisting of luminescence lifetime, luminescence intensity, luminescence quantum yield, brightness, and maximum luminescence.

12. 12. The labeled biomolecule of claim 11, wherein the emission lifetime of the labeled biomolecule is increased by at least 10% compared to the unconjugated molecule, optionally the emission lifetime of the labeled biomolecule is increased by about 10%-25% compared to the unconjugated molecule, or the emission lifetime of the labeled biomolecule is increased by about 25%-50% compared to the unconjugated molecule.

13. 13. The labeled biomolecule of claim 11 or claim 12, wherein the maximum emission of the labeled biomolecule is increased by at least 1% compared to the unconjugated molecule, and optionally the maximum emission of the labeled biomolecule is increased by about 1% to 10% compared to the unconjugated molecule.

14. The unconjugated molecule is Q １ and Q. ２ The labeled biomolecule according to any one of claims 10 to 13, which does not contain one or any of the following:

15. Q １ and Q. ２ is independently optionally linked to one or more other fluorophores, optionally each fluorophore being separated from any other fluorophore by at least 5 angstroms.

16. Q １ and Q. ２ is independently optionally associated with a reactant configured for use as a substrate in a reaction, optionally said reaction being a polymerization reaction, and further optionally said reactant being cleaved from said labeled biomolecule by a polymerase when subjected to polymerization reaction conditions.

17. A labeled nucleotide comprising one or more nucleotides attached to a labeled biomolecule according to any one of claims 1 to 16, optionally comprising: (i) the one or more nucleotides comprise one type of nucleotide selected from guanine, cytosine, adenine, and thymine or uracil; (ii) the one or more nucleotides are cleaved from the labeled biomolecule by a polymerase when subjected to polymerization reaction conditions; (iii) the one or more nucleotides comprise a nucleoside triphosphate, and optionally the one or more nucleotides are attached to the labeled biomolecule via a terminal phosphate; and / or (iv) a labeled nucleotide, wherein the one or more nucleotides comprise a nucleoside hexaphosphate, and optionally, the one or more nucleotides are attached to the labeled biomolecule via a terminal phosphate.

18. A composition comprising the labeled nucleotide of claim 17.

19. 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 claim 17.

20. 20. The nucleic acid sequencing reaction composition of claim 19, comprising four different types of labeled nucleotides.

21. 21. The nucleic acid sequencing reaction composition of claim 20, wherein the four different types of labeled nucleotides comprise a first labeled nucleotide that comprises a guanine, a second labeled nucleotide that comprises a cytosine, a third labeled nucleotide that comprises an adenine, and a fourth labeled nucleotide that comprises a thymine or uracil.

22. 1. A method for determining a sequence of a template nucleic acid, the method comprising: (i) exposing a complex in a target volume, the complex comprising a template nucleic acid, a primer and a polymerizing enzyme, to a nucleic acid sequencing reaction composition according to any one of claims 19 to 21; (ii) directing a series of one or more pulses of excitation energy proximate the target volume; (iii) detecting a plurality of photons emitted from the light-emitting labeled nucleotides during their successive incorporation into the nucleic acid containing the primer; and (iv) identifying the sequence of the incorporated nucleotide by determining the timing and optionally the luminescence intensity of the emitted photons; The method includes:

23. A kit for determining a sequence of a template nucleic acid, the kit comprising:

20. The method of claim 18, further comprising: providing a nucleic acid sequence comprising: a) a nucleic acid sequence including at least one of said nucleic acid sequences; and b) a nucleic acid sequence including at least one of said nucleic acid sequences ... (i) contains four different types of labeled nucleotides; (ii) further comprising a polymerase; and / or (iii) a primer complementary to the template nucleic acid.

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