BODIPY-Based Dyes for Digital Spatial Proteomics

Abstract

The present disclosure provides compositions, kits and methods for detecting / identifying amino acids and determining the sequence of polypeptide molecules. The reagents, kits and methods described herein are useful for detecting / identifying amino acids. An amino acid can be a part of a polypeptide. Thus, the reagents, kits and methods described herein are also useful for determining the sequence of a single polypeptide molecule, a plurality of molecules that are a single polypeptide, or a plurality of different single polypeptide molecules.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 292,683, filed December 22, 2021, and U.S. Provisional Application No. 63 / 339,137, filed May 6, 2022, the contents of both of which are incorporated by reference in their entireties herein.

[0002] Technical Field The field of the invention relates to compositions, kits and methods for detecting / identifying amino acids and for determining the sequence of polypeptide molecules. [Background technology]

[0003] background Proteins underlie cellular functions and their expression, and their location and abundance are important for identifying healthy and diseased cells. Rapid intracellular protein identification will enable a new paradigm in biomedical research, allowing scientists to determine how disease and aging affect protein networks in cells.

[0004] The invention described herein comprises novel dyes and methods for detecting and determining the concentrations of proteins and amino acids in solution and in biological environments (e.g., blood, plasma, serum, cells, tissues, etc.).

[0005] There remains a need for reagents, methods and assays for simultaneously detecting / identifying amino acids and determining the sequence of polypeptide molecules, and for identifying, quantitating and imaging large numbers of different proteins in complex biological samples. Summary of the Invention [Means for solving the problem]

[0006] summary In one aspect, a method for identifying amino acids is provided herein.Generally, the method comprises conjugating a probe that shows different spectral characteristics, for example, different fluorescence spectral characteristics, when conjugated to different amino acids.Once the probe is conjugated to amino acids, one or more spectral characteristics, for example, fluorescence spectral characteristics, of the probe conjugated to the amino acid are measured or detected.The amino acid is identified by comparing the fluorescence spectral characteristics, for example, the fluorescence spectral characteristics of the probe, with a plurality of reference spectral characteristics, for example, fluorescence spectral characteristics, each of which is representative of the probe conjugated to different amino acids.

[0007] The probe can be conjugated at any possible position of the amino acid.For example, the probe can be conjugated, for example, covalently linked, to the amino group, carboxyl group, hydroxyl group or thiol group of the amino acid.In some embodiments of any one of the aspects described herein, the probe is conjugated, for example, covalently linked, to the amino group of the amino acid.In some other embodiments of any one of the aspects described herein, the probe is conjugated, for example, covalently linked, to the carboxyl group of the amino acid.

[0008] The probes, when conjugated to amino acids, utilize different spectral properties, e.g., different fluorescent spectral properties, in the method for identifying or detecting, so that the methods described herein can be used to simultaneously detect / identify more than one amino acid, e.g., in a multiplex format. Thus, in another aspect, a multiplex method for detecting or identifying multiple amino acids is provided herein. In general, the method includes conjugating multiple probes to multiple amino acids, where the probes exhibit different spectral properties, e.g., different fluorescent spectral properties, when conjugated to different amino acids. Once the probes are conjugated to the amino acids, one or more spectral properties, e.g., fluorescent spectral properties, of the probes conjugated to the amino acids are detected or measured. The amino acids are identified or detected by comparing the multiple fluorescent spectral properties of the probes to multiple reference fluorescent spectral properties, each of which is representative of a probe conjugated to a different amino acid. In some embodiments, the multiple probes are identical.

[0009] The amino acid to be identified / detected can be part of a polypeptide.When an amino acid is part of a polypeptide, it can be anywhere in the polypeptide.For example, it can be at the N-terminus of the polypeptide.In some other non-limiting examples, it can be at the C-terminus of the polypeptide.In some other non-limiting examples, it can be at the interior of the polypeptide, i.e., at a non-terminal position.

[0010] When the amino acid to be identified / detected is present at a terminal position of a polypeptide, the method described herein can be used to identify / detect the terminal amino acid of the polypeptide. Thus, in another aspect, a method for identifying / detecting the terminal amino acid of a polypeptide is provided herein. In general, the method includes a step of conjugating a probe to the terminal amino acid of the polypeptide, where the probe exhibits different spectral properties, e.g., different fluorescent spectral properties, when conjugated to different amino acids. Once the probe is conjugated to the terminal amino acid, one or more spectral properties, e.g., fluorescent spectral properties, of the probe conjugated to the amino acid are measured or detected. The amino acid is identified by comparing the fluorescent spectral properties, e.g., the fluorescent spectral properties of the probe, with a plurality of reference spectral properties, e.g., fluorescent spectral properties, each of which is representative of a probe conjugated to a different amino acid.

[0011] In some embodiments of any one of the aspects described herein, the polypeptide is immobilized on a substrate prior to conjugating the probe to an amino acid in the polypeptide.

[0012] It is noted that the terminal amino acid to be detected / identified can be present at one end of the polypeptide.Thus, in some embodiments of any one of the aspects described herein, the terminal amino acid is present at the N-terminus of the polypeptide.In some embodiments of any one of the aspects described herein, the terminal amino acid is present at the C-terminus of the polypeptide.

[0013] The methods described herein for detecting / identifying terminal amino acids can be used to detect / identify terminal amino acids of more than one polypeptide simultaneously, e.g., in a multiplexed format. Thus, in another aspect, a multiplexed method for detecting / identifying terminal amino acids of multiple polypeptides is provided herein. In general, the method includes conjugating multiple probes to the terminal amino acids of each of the multiple polypeptides, where each probe in the multiple probes exhibits different spectral properties, e.g., different fluorescent spectral properties, when conjugated to a different amino acid. Once the probes are conjugated to the terminal amino acids, one or more spectral properties, e.g., fluorescent spectral properties, of each probe conjugated to the amino acid are measured or detected. The terminal amino acids are identified by comparing the fluorescent spectral properties, e.g., the fluorescent spectral properties of the probes, with multiple reference spectral properties, e.g., fluorescent spectral properties, each of which is representative of a probe conjugated to a different amino acid.

[0014] In the methods described herein, the probe in the multiple probes can be the same or different.Thus, in some embodiments of any one of the multiplex methods described herein, the probe in the multiple probes is the same.In some other embodiments of any one of the multiplex methods described herein, at least two probes in the multiple probes are different.

[0015] The method of identifying / detecting a terminal amino acid of a polypeptide can be used to determine the sequence of the polypeptide. For example, after a terminal amino acid is detected / identified, the terminal amino acid can be cleaved from the polypeptide and a new terminal amino acid can be detected. Such steps of cleaving the terminal amino acid after detection / identification and detecting / identifying the next terminal amino acid can be repeated one or more times, for example, to determine the sequence of at least a portion of the polypeptide. This can be done in a multiplexed format to simultaneously determine the sequence of at least a portion of multiple polypeptides.

[0016] Methods for cleaving terminal amino acids of polypeptides are well known in the art and are available to those skilled in the art. Such methods can include enzymatic cleavage or chemical cleavage. Thus, in some embodiments of any one of the aspects described herein, cleaving terminal amino acids comprises enzymatic cleavage. In some other embodiments of any one of the aspects described herein, cleaving terminal amino acids comprises chemical cleavage.

[0017] The amino acid or polypeptide may be present in a sample. The sample may be a biological sample. For example, the sample may be a biological fluid, tissue, organ and / or cell.

[0018] It is noted that any probe that can exhibit different spectral characteristics, e.g., fluorescent spectral characteristics, can be utilized. In some embodiments of any one of the aspects described herein, the probe is a dipyrromethane-BF2 derivative. For example, the probe is a dipyrromethane-BF2 derivative that includes a labile group or a reactive group. In some embodiments of any one of the aspects described herein, the probe is a dipyrromethane-BF2 derivative that includes a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative. Any position of the dipyrromethane-BF2 derivative can be used for conjugation with an amino acid. For example, the probe can be conjugated via the 8-position (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative. Thus, in some embodiments of any one of the aspects described herein, the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative includes a labile or reactive group at the 8-position (meso position).

[0019] In some embodiments of any one of the aspects described herein, the probe comprises a compound of Formula (I): [ka] It is.

[0020] In the compound of formula (I), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 At least one of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be substituted with halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarboxyl, aryloxy ... It is noted that the aryl group may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0021] In some embodiments of any one of the aspects described herein, R 8 is a labile or leaving group.

[0022] Also disclosed herein are probes conjugated to amino acids. Thus, in another aspect, a compound of formula (II): [ka] is provided herein.

[0023] In the compound of formula (II), R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and amino acid; and optionally R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28at least one of is an amino acid. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0024] In some embodiments of any one of the aspects described herein, R 28 is an amino acid.

[0025] It is noted that the amino acid may be linked to the remainder of formula (II) through its alpha amino group, carboxyl group or a functional group in the side chain. In some preferred embodiments, the amino acid is linked to the remainder of formula (II) through its alpha amino group.

[0026] In yet another aspect, provided herein are kits that include one or more compounds described herein. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic showing the reactivity of an exemplary 8-thioether BODIPY with the N-terminus of a protein via the SNAr reaction.

[0028] [Diagram 2] 2-6 illustrate some exemplary compounds (probes) that were synthesized using the exemplary synthetic methods described herein. [Diagram 3] Same as above. [Figure 4] Same as above. [Diagram 5] Same as above. [Figure 6] Same as above.

[0029] [Figure 7] 7-22 show the fluorescence of exemplary BODIPY dyes, namely BDP-001 (FIG. 7), BDP-002 (FIG. 8), BDP-003 (FIG. 9), Bodipy-004 (FIG. 10), Bodipy-005 (FIG. 11), Bodipy-006 (FIG. 12), Bodipy-007 (FIG. 13), BDP-008 (FIG. 14), BDP-009 (FIG. 15), BDP-010 (FIG. 16), BDP-011 (FIG. 17), BDP-012 (FIG. 18), Bodipy-013 (FIG. 19), Bodipy-014 (FIG. 20), BDP-015 (FIG. 21) and Bodipy-016 (FIG. 22), conjugated to different amino acids. [Figure 8] Same as above. [Figure 9] Same as above. [Figure 10] Same as above. [Figure 11] Same as above. [Figure 12] Same as above. [Figure 13] Same as above. [Figure 14] Same as above. [Figure 15] Same as above. [Figure 16] Same as above. [Figure 17] Same as above. [Figure 18] Same as above. [Figure 19] Same as above. [Figure 20] Same as above. [Figure 21] Same as above. [Figure 22] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. Moreover, the section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] The present disclosure provides reagents, kits and methods for detecting / identifying amino acids and polypeptides. The reagents, kits and methods described herein are useful for detecting / identifying amino acids. An amino acid can be a part of a polypeptide. Thus, the reagents, kits and methods described herein are also useful for determining the sequence of a single polypeptide molecule, a plurality of molecules that are a single polypeptide, or a plurality of different single polypeptide molecules.

[0032] Therefore, in one aspect, a method for detecting or identifying an amino acid is provided herein, comprising the steps of: (a) conjugating a probe to an amino acid, the probe comprising a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to the amino acid; (b) detecting one or more fluorescence spectral properties of the probe conjugated to the amino acid; and (c) identifying the amino acid by comparing the fluorescence spectral properties of the probe with a plurality of reference fluorescence spectral properties, each of which is representative of a probe conjugated to a different amino acid.

[0033] The method described herein for detecting / identifying amino acids can be used to detect / identify more than one amino acid simultaneously, for example, in a multiplex format.Therefore, in another aspect, a multiplex method for detecting or identifying multiple amino acids is provided herein.The method includes the steps of: (a) conjugating multiple probes to the amino, carboxyl, hydroxyl or thiol groups of multiple amino acids, wherein each probe in the multiple probes comprises a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral characteristics when conjugated to different amino acids; (b) detecting one or more fluorescence spectral characteristics for each probe conjugated to the multiple amino acids; and (c) identifying the amino acid by comparing the multiple fluorescence spectral characteristics of the probes with multiple reference fluorescence spectral characteristics, wherein each reference fluorescence spectral characteristic is representative of the probe conjugated to different amino acids.

[0034] probe

[0023] In some embodiments of the various aspects described herein, the probe comprises a compound of formula (I): [ka] It is.

[0035] In the compound of formula (I), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 At least one of is a labile group or a leaving group. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0036] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R3 , R 4 , R 5 , R 6 , R 7 and R 8 Only one of the is a labile or leaving group.

[0037] As used herein, a "labile group" or "leaving group" refers to a group that may be replaced by another group in a reaction, e.g., a substitution reaction, such as a nucleophilic substitution reaction. Exemplary labile or leaving groups include, but are not limited to, halides (fluoride, chloride, bromide, and iodide), azide, sulfonates (e.g., optionally substituted C1-C6 alkanesulfonates, such as methanesulfonate and trifluoromethanesulfonate, or optionally substituted C7-C8 alkanesulfonates, such as arylsulfonates, ... 12 Examples of leaving groups include alkylbenzenesulfonates (such as p-toluenesulfonate), succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, carboxylates, aminocarboxylates (carbamates) and alkoxycarboxylates (carbonates). In the case of substitution at a saturated carbon, halides and sulfonates are preferred leaving groups. In the case of substitution at a carbonyl carbon, halides, succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, carboxylates or alkoxycarboxylates (carbonates) may be used, for example, as leaving groups.

[0038] In some embodiments of any one of the aspects described herein, the labile group or leaving group is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, or diazonium. For example, the labile group or leaving group is halogen or alkylthio.

[0039] In some embodiments of any one of the aspects described herein, the labile group or leaving group is -SR L and R L is optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. For example, R L is substituted C1-C6 alkyl, C1-C6 perhaloalkyl, optionally substituted C2-C6 alkenyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments of any one of the aspects described herein, R L is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl. In some preferred embodiments, R L is methyl, i.e. the labile or leaving group is -SMe.

[0040] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7are each independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; R 8 is a labile or leaving group.

[0041] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has the formula (IA): [ka] It is.

[0042] In the compound of formula (IA), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7each can be independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; R 8S is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0043] In some embodiments of any one of the aspects described herein, R 8S is optionally substituted aryl. For example, R 8Sis optionally substituted phenyl. Thus, in some embodiments of any one of the aspects described herein, the compound of Formula (I), e.g., Formula (IA), has the formula (IB): [ka] It is.

[0044] In the compound of formula (IB), R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7 each can be independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; R 81 , R 82 , R 83 , R 84 and R 85are each independently hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0045] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has Formula (IC) or Formula (ID): [ka] It is.

[0046] In the compounds of formula (IC) and (ID), R 31 , R 32 , R 33 , R34 and R 35 each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 8Sis optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0047] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has Formula (IE) or Formula (IF): [ka] It is.

[0048] In the compounds of formula (IE) and (IF), R 31 , R 32 , R 33 , R 34 and R 35each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 81 , R 82 , R 83 , R 84 and R 85are each independently hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0049] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has Formula (IG) or Formula (IH): [ka] It is.

[0050] In the compounds of formula (IG) and (IH), R 31 , R 32 , R 33 , R34 and R 35 each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 41 , R 42 , R 34 , R 44 and R 45 each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 41 , R 42 , R 34 , R 44 and R 45 together with the carbon atom to which they are attached form an optionally substituted aryl; 8Sis optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0051] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has Formula (II) or Formula (IJ): [ka] [ka] It is.

[0052] In the compounds of formula (II) and (IJ), R 31 , R 32 , R 33 , R 34 and R 35each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 41 , R 42 , R 34 , R 44 and R 45 each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 41 , R 42 , R 34 , R 44 and R 45 together with the carbon atom to which they are attached form an optionally substituted aryl; 81 , R 82 , R 83 , R 84 and R 85are each independently hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0053] R 8 In some embodiments of any one of the aspects described herein, R 8 is a labile or leaving group. For example, R 8is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, or diazonium. In some embodiments of any one of the aspects described herein, R 8 is alkylthio or halogen.

[0054] In some embodiments of any one of the aspects described herein, R 8 -SR 8S and R 8S is optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. For example, R 8S is optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl, optionally substituted C2-C6 alkenyl, optionally substituted aryl or optionally substituted heteroaryl.

[0055] In some embodiments of any one of the aspects described herein, R 8S is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl.

[0056] In some embodiments of any one of the aspects described herein, R 8S is methyl. For example, R 8 is -SMe.

[0057] In some embodiments of any one of the aspects described herein, R 8 is a halogen. For example, R 8 is Cl, Br, I or F.

[0058] R 1 In the compound of formula (I), R 1 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 1 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0059] In some embodiments of any one of the aspects described herein, R 1is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 1 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0060] In some embodiments of any one of the aspects described herein, R 1 is H, halogen or optionally substituted alkyl. For example, R 1 is H. In some other non-limiting examples, R 1 is an optionally substituted C1-C6 alkyl. 1 Exemplary alkyl include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0061] In some embodiments of any one of the aspects described herein, R 1 is H or methyl.

[0062] In some embodiments of any one of the aspects described herein, R 1 is a labile or leaving group.

[0063] R 2 In the compound of formula (I), R 2 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 2 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0064] In some embodiments of any one of the aspects described herein, R 2is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 2 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0065] In some embodiments of any one of the aspects described herein, R 2 is H, halogen, optionally substituted alkyl, perhaloalkyl, carbonyl, carboxyl, optionally substituted acyl or sulfonyl. For example, R 2 can be H, halogen, optionally substituted C1-C6 alkyl, -C(O)H or -SO3H.

[0066] In some embodiments of any one of the aspects described herein, R 2 is H, Br, Cl, -C(O)H or -SOH.

[0067] In some embodiments of any one of the aspects described herein, R 2 is a labile or leaving group.

[0068] R 3 In the compound of formula (I), R3 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 3 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0069] In some embodiments of any one of the aspects described herein, R 3is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 3 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0070] In some embodiments of any one of the aspects described herein, R 3 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, or optionally substituted aryl. For example, R 3 is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl or optionally substituted aryl.

[0071] In some embodiments of any one of the aspects described herein, R 3 is H.

[0072] In some embodiments of any one of the aspects described herein, R 3 is an optionally substituted C1-C6 alkyl. 3Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl. 3 is methyl.

[0073] In some embodiments of any one of the aspects described herein, R 3 is an optionally substituted C2-C6 alkenyl. For example, R 3 is -CH=CH-R 9A and R 9A is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl or optionally substituted heterocyclyl.

[0074] In some embodiments of any one of the aspects described herein, R 3 is -CH=CH-R 9A and R 9A is optionally substituted aryl or optionally substituted heteroaryl. For example, R 3 is -CH=CH-R 9A and R 9A R is optionally substituted aryl. 9AExemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, anthracyl, indenyl, azulenyl, and fluorenyl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0075] In some embodiments of any one of the aspects described herein, R 3 is -CH=CH-R 9A and R 9A is optionally substituted phenyl. For example, R 3 is -CH=CH-R 9A and R 9A is phenyl or phenyl substituted with a substituent (e.g., 1, 2, or 3 substituents) selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, and C1-C6 haloalkyl. 3 is -CH=CH-R 9A and R 9A is 4-fluoropheyl, 4-trifluoromethylphenyl or 4-cyanophenyl.

[0076] In some embodiments of any one of the aspects described herein, R 3 is a halogen. For example, R 3is Br, Cl or F. In some embodiments, R 3 is Br or Cl.

[0077] In some embodiments of any one of the aspects described herein, R 3 is optionally substituted aryl or optionally substituted heteroaryl. For example, R 3 R is optionally substituted aryl. 3 Exemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, anthracyl, indenyl, azulenyl, and fluorenyl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0078] In some embodiments, R 3 is phenyl or naphthyl, each of which may be optionally substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, or aryl. For example, R 3is phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylpehnyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0079] In some embodiments of any one of the aspects described herein, R 3 is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0080] In some embodiments of any one of the aspects described herein, R 3 is a labile or leaving group.

[0081] R 5 In the compound of formula (I), R 5can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 5 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0082] In some embodiments of any one of the aspects described herein, R 5is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 5 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0083] In some embodiments of any one of the aspects described herein, R 5 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, or optionally substituted aryl. For example, R 5 is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl or optionally substituted aryl.

[0084] In some embodiments of any one of the aspects described herein, R 5 is H.

[0085] In some embodiments of any one of the aspects described herein, R 5 is an optionally substituted C1-C6 alkyl. 5Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl. 5 is methyl.

[0086] In some embodiments of any one of the aspects described herein, R 5 is an optionally substituted C2-C6 alkenyl. For example, R 5 is -CH=CH-R 9B and R 9B is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl or optionally substituted heterocyclyl.

[0087] In some embodiments of any one of the aspects described herein, R 5 is -CH=CH-R 9B and R 9B is optionally substituted aryl or optionally substituted heteroaryl. For example, R 5 is -CH=CH-R 9B and R 9B R is optionally substituted aryl. 9BExemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, anthracyl, indenyl, azulenyl, and fluorenyl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0088] In some embodiments of any one of the aspects described herein, R 5 is -CH=CH-R 9B and R 9B is optionally substituted phenyl. For example, R 5 is -CH=CH-R 9B and R 9B is phenyl or phenyl substituted with a substituent (e.g., 1, 2, or 3 substituents) selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, and C1-C6 haloalkyl. 5 is -CH=CH-R 9B and R 9B is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl.

[0089] In some embodiments of any one of the aspects described herein, R 5 is a halogen. For example, R 5 is Br, Cl or F. In some embodiments, R5 is Br or Cl.

[0090] In some embodiments of any one of the aspects described herein, R 5 is optionally substituted aryl or optionally substituted heteroaryl. For example, R 5 R is optionally substituted aryl. 5 Exemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, anthracyl, indenyl, azulenyl, and fluorenyl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0091] In some embodiments, R 5 is phenyl or naphthyl, each of which may be optionally substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, or aryl. For example, R 5 is phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0092] In some embodiments of any one of the aspects described herein, R 5 is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0093] In some embodiments of any one of the aspects described herein, R 3 is a labile or leaving group.

[0094] R 3 and R 5 It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 3 and R 5 In some other embodiments of any one of the aspects described herein, R 3 and R 5 is different.

[0095] R 6 In the compound of formula (I), R 6can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 6 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0096] In some embodiments of any one of the aspects described herein, R 6is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 6 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0097] In some embodiments of any one of the aspects described herein, R 6 is H, halogen, optionally substituted alkyl, perhaloalkyl, carbonyl, carboxyl, optionally substituted acyl, sulfonate or sulfonyl. For example, R 6 can be H, halogen, optionally substituted C1-C6 alkyl, -C(O)H or -SO3H.

[0098] In some embodiments of any one of the aspects described herein, R 6 is H, Br, Cl, -C(O)H or -SOH.

[0099] In some embodiments of any one of the aspects described herein, R 6 is a labile or leaving group.

[0100] R 2 and R 6It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 2 and R 6 In some other embodiments of any one of the aspects described herein, R 2 and R 6 is different.

[0101] R 7 In the compound of formula (I), R 7 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or a labile or leaving group. For example, R 7can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0102] In some embodiments of any one of the aspects described herein, R 7 is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 7 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0103] In some embodiments of any one of the aspects described herein, R 7is H, halogen or optionally substituted alkyl. For example, R 7 is H. In some other non-limiting examples, R 7 is an optionally substituted C1-C6 alkyl. 7 Exemplary alkyl include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0104] In some embodiments of any one of the aspects described herein, R 7 is H or methyl.

[0105] In some embodiments of any one of the aspects described herein, R 7 is a labile or leaving group.

[0106] R 1 and R 7 It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 1 and R 7 In some other embodiments of any one of the aspects described herein, R 1 and R 7 is different.

[0107] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R 3 , R 5 , R 6 and R 7 At least one of R is not H. In some non-limiting examples, R 1 is not H. In some other non-limiting examples, R 2 is not H. In some other non-limiting examples, R 3 is not H. In some other non-limiting examples, R 5 is not H. In some other non-limiting examples, R 6is not H. In some other non-limiting examples, R 7 is not H.

[0108] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R 3 , R 5 , R 6 and R 7 At least one of R is an electron withdrawing group (EWG). The term "electron withdrawing group" is art-recognized and refers to an atom or group that acts to withdraw electrons from adjacent atoms, i.e., the substituent is electronegative with respect to adjacent atoms. Exemplary electron withdrawing groups include, but are not limited to, halo (e.g., bromo, fluoro, chloro, and iodo), nitro, carboxy, ester, formyl, keto, azo, amidocarbonyl, amidosulfonyl, carboxamido, sulfonoxy, sulfonamido, ureido, and aryl. In some non-limiting examples, R 1 In some other non-limiting examples, R 2 In some other non-limiting examples, R 3 In some other non-limiting examples, R 5 In some other non-limiting examples, R 6 In some other non-limiting examples, R 7 is EWG.

[0109] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R 3 , R 5 , R 6 and R 7At least one of the is an electron donating group (EDG). The term "electron donating group" is recognized in the art and refers to an atom or group that acts to donate electrons to adjacent atoms by electronegativity differences (inductive effect) and / or by donating pi electrons through conjugation (mesometric effect). Exemplary electron donating groups include, but are not limited to, hydroxyl, lower alkoxy (e.g., methoxy, ethoxy, etc.), lower alkyl (methyl, ethyl, etc.), amino, lower alkylamino, di-lower alkylamino, aryloxy (phenoxy, etc.), arylalkoxy (phenoxy, etc.), aminoaryl (p-dimethylaminophenyl, etc.), mercapto, and alkylthio. In some non-limiting examples, R 1 In some other non-limiting examples, R 2 In yet some other non-limiting examples, R 3 In yet some other non-limiting examples, R 5 In yet some other non-limiting examples, R 6 In yet some other non-limiting examples, R 7 is EDG.

[0110] In some embodiments of any one of the aspects described herein, R 1 , R 2 and R 3 At least two of the are the same. For example, R 1 and R 2 is the same. In some other cases, R 1 and R 3 are the same. In some other examples, R 2 and R 3 are the same. In some embodiments, R 1 , R 2 and R 3 is the same.

[0111] In yet some other embodiments, R 1 , R 2 and R 3 is different.

[0112] In some embodiments of any one of the aspects described herein, R 5 , R 6 and R 7 At least two of the are the same. For example, R 5 and R 6 is the same. In some other cases, R 5 and R 7 are the same. In some other examples, R 6 and R 7 are the same. In some embodiments, R 5 , R 6 and R 7 is the same.

[0113] In yet some other embodiments, R 5 , R 6 and R 7 is different.

[0114] In some embodiments of any one of the aspects described herein, R 1 and R 7 are different or R 2 and R 6 are different or R 3 and R 5 is different.

[0115] In some embodiments of any one of the aspects described herein, R 1 and R 7 is the same as R 2 and R 6 is the same as R 3 and R 5 is the same.

[0116] In some embodiments of any one of the aspects described herein, R 1 , R 2 , R 3 , R 5 , R 6 and R 7 are the same. For example, R1 , R 2 , R 3 , R 5 , R 6 and R 7 is H.

[0117] In some embodiments of the various aspects described herein, R 8S is methyl, (a) R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H, (b) R 1 , R 2 , R 6 and R 7 is R, not H. 3 and R 5 is not methyl, ethyl, or p-nitrophenyl; (c) R 1 , R 3 , R 5 and R 7 is not methyl, but R 2 and R 6 is not H, (d) R 1 and R 7 is R, not H. 2 , R 3 , R 5 and R 6 is neither Cl nor Br, (e)R 1 , R 3 , R 5 , R 6 and R 7 is R, not H. 2 is not -C(O)H, (f)R 1 is not p-nitrophenyl, but R 2 , R 6 and R 7 is R, not H. 3 and R 5 is not methyl, and / or (g) R 1 , R 2 , R 5 , R 6 and R 7 is R, not H. 3is not phenyl, p-nitrophenyl, p-iodophenyl, p-carboxylicphenyl, or p-methoxyphenyl.

[0118] In some embodiments of the various aspects described herein, R 8S is allyl, ethyl, propyl, butyl, t-butyl, n-dodecyl, phenyl, 2,6-dimethylphenyl, p-methylphenyl, p-methoxyphenyl, p-nitrophenyl, or benzyl (-CHPH), then R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H.

[0119] In some embodiments of the various aspects described herein, R 8 If is Cl, then (a) R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H, (b) R 1 , R 3 , R 5 , R 6 and R 7 is R, not H. 2 is not Cl, (c) R 1 , R 3 , R 5 and R 7 is R, not H. 2 and R 5 is not Cl, (d) R 1 , R 5 and R 7 is R, not H. 2 , R 3 and R 5 is not Cl and / or (e)R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not Cl.

[0120] In some embodiments of any one of the aspects described herein, the compound of formula (I) exhibits different fluorescence spectral properties when covalently or non-covalently conjugated to an amino acid. Without being limited by theory, it is believed that the atomic interactions induced by the side chains of different amino acids affect the electronic (ground or activated) state of the probe conjugated to a particular amino acid, thereby resulting in different fluorescence spectral properties.

[0121] R 31 In some embodiments of any one of the aspects described herein, R 31 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 31 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 31 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 31 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 31 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 31is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 31 is hydrogen, methyl or ethyl.

[0122] R 32 In some embodiments of any one of the aspects described herein, R 32 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 32 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 32 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 32 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 32 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 32 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 32 is hydrogen or methyl.

[0123] R 33 In some embodiments of any one of the aspects described herein, R 33is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 33 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 33 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 33 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 33 is hydrogen, F, Br, Cl, I, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 33 is hydrogen, F, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 33 is hydrogen, F, methyl, trifluoromethyl or phenyl.

[0124] In some embodiments, R 32 and R 33 together with the carbon atom to which they are attached form an optionally substituted aryl.

[0125] R 34 In some embodiments of any one of the aspects described herein, R 34is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 34 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 34 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 34 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 34 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 34 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 34 is hydrogen or methyl.

[0126] In some embodiments, R 33 and R 34 together with the carbon atom to which they are attached form an optionally substituted aryl.

[0127] R 35 In some embodiments of any one of the aspects described herein, R 35is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 35 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 35 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 35 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 35 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 35 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 35 is hydrogen, methyl or ethyl.

[0128] R 41 In some embodiments of any one of the aspects described herein, R 41 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 41is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 41 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 41 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 41 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 41 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 41 is hydrogen, methyl or ethyl.

[0129] R 42 In some embodiments of any one of the aspects described herein, R 42 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 42 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 42is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 42 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 42 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 42 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 42 is hydrogen or methyl.

[0130] R 43 In some embodiments of any one of the aspects described herein, R 43 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 43 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 43 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 43 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 43is hydrogen, F, Br, Cl, I, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 43 is hydrogen, F, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 43 is hydrogen, F, methyl, trifluoromethyl or phenyl.

[0131] In some embodiments, R 42 and R 43 together with the carbon atom to which they are attached form an optionally substituted aryl.

[0132] R 44 In some embodiments of any one of the aspects described herein, R 44 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 44 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 44 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 44 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 44is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 44 is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 44 is hydrogen or methyl.

[0133] In some embodiments, R 43 and R 44 together with the carbon atom to which they are attached form an optionally substituted aryl.

[0134] R 45 In some embodiments of any one of the aspects described herein, R 45 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 45 is hydrogen, halogen, alkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 45 is hydrogen, halogen, alkoxyl, aryl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 45 is hydrogen, halogen, alkoxyl, aryl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 45 is hydrogen, methyl, ethyl, trifluoromethyl, cyano, hydroxyl, methoxy or phenyl. For example, R 45is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, or cyano. 45 is hydrogen, methyl or ethyl.

[0135] R 81 In some embodiments of any one of the aspects described herein, R 81 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 81 is hydrogen, halogen, alkyl, perhaloalkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 81 is hydrogen, halogen, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 81 is hydrogen, halogen, alkoxyl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 81 is hydrogen.

[0136] R 82 In some embodiments of any one of the aspects described herein, R 82is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 82 is hydrogen, halogen, alkyl, perhaloalkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 82 is hydrogen, halogen, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 82 is hydrogen, halogen, alkoxyl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 82 is hydrogen.

[0137] R 83 In some embodiments of any one of the aspects described herein, R 83 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 83 is hydrogen, halogen, alkyl, perhaloalkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate.83 is hydrogen, halogen, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 83 is hydrogen, halogen, alkoxyl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 83 is halogen or alkoxyl. For example, R 83 is F, Cl, Br, I, methoxy or ethoxy. 83 is fluoro or methoxy.

[0138] R 84 In some embodiments of any one of the aspects described herein, R 84 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 84 is hydrogen, halogen, alkyl, perhaloalkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 84 is hydrogen, halogen, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 84 is hydrogen, halogen, alkoxyl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 84 is hydrogen.

[0139] R 85 In some embodiments of any one of the aspects described herein, R 85 is hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, or ureido. For example, R 85 is hydrogen, halogen, alkyl, perhaloalkyl, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carbonyl, carboxyl, ester, cyano, nitro, thiol, alkylthio, or sulfonate. 85 is hydrogen, halogen, alkoxyl, amino, alkylamino, dialkylamino, hydroxyl, carboxyl, cyano, nitro, thiol, alkylthio, or sulfonate. For example, R 85 is hydrogen, halogen, alkoxyl, amino, hydroxyl, carboxyl, cyano, nitro, or thiol. 85 is hydrogen.

[0140] BOPIDY Amino Acid (Peptide, Polypeptide) Conjugates In another embodiment, the compound of formula (II): [ka] is provided herein.

[0141] In the compound of formula (II), R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and amino acid; and optionally R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 at least one of is an amino acid. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0142] As used herein, the term "amino acid" refers to any organic molecule that contains at least one amino group and at least one carboxyl group. Usually, at least one amino group is in the alpha position relative to the carboxyl group. As used herein, the term "amino acid" includes natural amino acids, L-amino acids, D-amino acids, synthetic amino acids, modified amino acids (such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine), as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, such as R groups attached to the alpha carbon, carboxyl group, amino group, and hydrogen (such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function similarly to natural amino acids.

[0143] It is noted that the amino acid can be a single amino acid, or part of an oligopeptide (e.g., containing 2-20 amino acids, such as di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, or deca-peptide), a polypeptide (e.g., containing 21 or more amino acid peptides). Those skilled in the art are well aware that polypeptides containing more than approximately 50 amino acids are known as proteins. Thus, the term "polypeptide" as used herein includes proteins. Furthermore, the amino acid may be linked to the remainder of formula (II) by its alpha amino group, carboxyl group, or a functional group in the side chain. In some preferred embodiments, the amino acid is linked to the remainder of formula (II) by its alpha amino group.

[0144] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 24 , R25 , R 26 , R 27 and R 28 Only one of them is an amino acid.

[0145] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 24 , R 25 , R 26 and R 27 are each independently selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; R 28 is an amino acid.

[0146] R 28 In some embodiments of any one of the aspects described herein, R 28 is an amino acid. For example, R 28 is -NHCH(R AS )C(O)-OR AC or -OC(O)CH(R AS )NHR AN and R ACare H, an amino acid, a peptide, a polypeptide, or a hydroxyl protecting group, and R AS is the side chain of an amino acid, and R AN is H, an amino acid, a peptide, a polypeptide, or an amino protecting group.

[0147] In some embodiments, R 28 is -NHCH(R AS )C(O)-OR AC and R AC is H, an amino acid, a peptide, a polypeptide, or a hydroxyl protecting group; R AS is the side chain of an amino acid. For example, R AC is H or a hydroxyl protecting group. Preferably, R AC is H.

[0148] In some embodiments, R 28 or -OC(O)CH(R AS )NHR AN and R AS is the side chain of an amino acid, and R AN is H, an amino acid, a peptide, a polypeptide, or an amino protecting group. For example, R AN is H or an amino protecting group. AN is H.

[0149] In some embodiments of any one of the aspects described herein, the compound of Formula (II) has the formula (II-a) or (II-b): [ka] It is.

[0150] In the compounds of formula (II-a) and (II-b), R AC are H, an amino acid, a peptide, a polypeptide, or a hydroxyl protecting group, and R AS is the side chain of an amino acid, and R AN is H, an amino acid, a peptide, a polypeptide, or an amino protecting group.

[0151] R 21 In the compound of formula (II), R 21 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 21 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0152] In some embodiments of any one of the aspects described herein, R 21is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 21 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0153] In some embodiments of any one of the aspects described herein, R 21 is H, halogen or optionally substituted alkyl. For example, R 21 is H. In some other non-limiting examples, R 21 is an optionally substituted C1-C6 alkyl. 21 Exemplary alkyl include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0154] In some embodiments of any one of the aspects described herein, R 21 is H or methyl.

[0155] In some embodiments of any one of the aspects described herein, R 21 is an amino acid.

[0156] R 22 In the compound of formula (II), R 22 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 22 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0157] In some embodiments of any one of the aspects described herein, R 22is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 22 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0158] In some embodiments of any one of the aspects described herein, R 22 is H, halogen, optionally substituted alkyl, perhaloalkyl, carbonyl, carboxyl, optionally substituted acyl, sulfonate or sulfonyl. For example, R 22 can be H, halogen, optionally substituted C1-C6 alkyl, -C(O)H or -SO3H.

[0159] In some embodiments of any one of the aspects described herein, R 22 is H, Br, Cl, -C(O)H or -SOH.

[0160] In some embodiments of any one of the aspects described herein, R 22 is an amino acid.

[0161] R 23 In the compound of formula (II), R 23 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 23 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0162] In some embodiments of any one of the aspects described herein, R 23is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 23 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0163] In some embodiments of any one of the aspects described herein, R 23 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, or optionally substituted aryl. For example, R 23 is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl or optionally substituted aryl.

[0164] In some embodiments of any one of the aspects described herein, R 23 is H.

[0165] In some embodiments of any one of the aspects described herein, R 23 is an optionally substituted C1-C6 alkyl. 23Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl. 23 is methyl.

[0166] In some embodiments of any one of the aspects described herein, R 23 is an optionally substituted C2-C6 alkenyl. For example, R 23 is -CH=CH-R 29 and R 29 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl or optionally substituted heterocyclyl.

[0167] In some embodiments of any one of the aspects described herein, R 23 is -CH=CH-R 29A and R 29A is optionally substituted aryl or optionally substituted heteroaryl. For example, R 23 is -CH=CH-R 29A and R 29A R is optionally substituted aryl. 29AExemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl and phenanthryl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, perhaloalkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0168] In some embodiments of any one of the aspects described herein, R 23 is -CH=CH-R 29A and R 29A is optionally substituted phenyl. For example, R 23 is -CH=CH-R 29A and R 29A is phenyl or phenyl substituted with a substituent (e.g., 1, 2, or 3 substituents) selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, and C1-C6 haloalkyl. 23 is -CH=CH-R 29A and R 29A is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl.

[0169] In some embodiments of any one of the aspects described herein, R 23 is a halogen. For example, R 23 is Br, Cl or F. In some embodiments, R 23 is Br or Cl.

[0170] In some embodiments of any one of the aspects described herein, R 23 is optionally substituted aryl or optionally substituted heteroaryl. For example, R 23 R is optionally substituted aryl. 23 Exemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl and phenanthryl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0171] In some embodiments, R 23 is phenyl or naphthyl, each of which may be optionally substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, or aryl. For example, R 23 is phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0172] In some embodiments of any one of the aspects described herein, R23 is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0173] In some embodiments of any one of the aspects described herein, R 23 is an amino acid.

[0174] R 25 In the compound of formula (II), R 25 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 25can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0175] In some embodiments of any one of the aspects described herein, R 25 is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 25 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0176] In some embodiments of any one of the aspects described herein, R 25is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, or optionally substituted aryl. For example, R 25 is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl or optionally substituted aryl.

[0177] In some embodiments of any one of the aspects described herein, R 25 is H.

[0178] In some embodiments of any one of the aspects described herein, R 25 is an optionally substituted C1-C6 alkyl. 25 Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl. 25 is methyl.

[0179] In some embodiments of any one of the aspects described herein, R 25 is an optionally substituted C2-C6 alkenyl. For example, R 25 is -CH=CH-R 29B and R 29B is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl or optionally substituted heterocyclyl.

[0180] In some embodiments of any one of the aspects described herein, R 25 is -CH=CH-R 29B and R 29B is optionally substituted aryl or optionally substituted heteroaryl. For example, R 25 is -CH=CH-R 29B and R 29BR is optionally substituted aryl. 29B Exemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl and phenanthryl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, perhaloalkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0181] In some embodiments of any one of the aspects described herein, R 25 is -CH=CH-R 29B and R 29B is optionally substituted phenyl. For example, R 25 is -CH=CH-R 29B and R 29B is phenyl or phenyl substituted with a substituent (e.g., 1, 2, or 3 substituents) selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, and C1-C6 haloalkyl. 25 is -CH=CH-R 29B and R 29B is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl.

[0182] In some embodiments of any one of the aspects described herein, R 25 is a halogen. For example, R 25is Br, Cl or F. In some embodiments, R 25 is Br or Cl.

[0183] In some embodiments of any one of the aspects described herein, R 25 is optionally substituted aryl or optionally substituted heteroaryl. For example, R 25 R is optionally substituted aryl. 25 Exemplary aryl for the group include, but are not limited to, phenyl, naphthyl, anthryl and phenanthryl, each of which may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0184] In some embodiments, R 25 is phenyl or naphthyl, each of which may be optionally substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, or aryl. For example, R 25 is phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0185] In some embodiments of any one of the aspects described herein, R 25 is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthyl.

[0186] R 23 and R 25 It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 23 and R 25 In some other embodiments of any one of the aspects described herein, R 23 and R 25 is different.

[0187] In some embodiments of any one of the aspects described herein, R 25 is an amino acid.

[0188] R 26 In the compound of formula (II), R 26can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 26 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0189] In some embodiments of any one of the aspects described herein, R 26is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 26 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0190] In some embodiments of any one of the aspects described herein, R 26 is H, halogen, optionally substituted alkyl, perhaloalkyl, carbonyl, carboxyl, optionally substituted acyl, sulfonate or sulfonyl. For example, R 26 can be H, halogen, optionally substituted C1-C6 alkyl, -C(O)H or -SO3H.

[0191] In some embodiments of any one of the aspects described herein, R 26 is H, Br, Cl, -C(O)H or -SOH.

[0192] R 22 and R 26 It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 22 and R 26In some other embodiments of any one of the aspects described herein, R 22 and R 26 is different.

[0193] In some embodiments of any one of the aspects described herein, R 26 is an amino acid.

[0194] R 27 In the compound of formula (II), R 27 can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted arylamino, optionally substituted heteroarylamino, hydroxyl, optionally substituted acyl, optionally substituted acyloxy, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, or amino acid. For example, R 27can be hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, amino, optionally substituted alkylamino, optionally substituted dialkylamino, hydroxyl, optionally substituted acyl, carbonyl, carboxyl, optionally substituted ester, optionally substituted alkoxyl, cyano, thiol, optionally substituted alkylthio, sulfonate, sulfinyl or sulfonyl.

[0195] In some embodiments of any one of the aspects described herein, R 27 is selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, cyano, amino, alkylamino, dialkylamino, nitro, carbonyl, carboxyl, hydroxyl, isocyanato, thiocyanato, isothiocyanato, sulfonate, and sulfonyl. For example, R 27 is H, halogen, optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxyl, carbonyl, optionally substituted acyl, carboxyl, hydroxyl, cyano, amino, nitro, sulfonate or sulfonyl.

[0196] In some embodiments of any one of the aspects described herein, R 27is H, halogen or optionally substituted alkyl. For example, R 27 is H. In some other non-limiting examples, R 27 is an optionally substituted C1-C6 alkyl. 27 Exemplary alkyl include, but are not limited to, methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0197] In some embodiments of any one of the aspects described herein, R 27 is H or methyl.

[0198] In some embodiments of any one of the aspects described herein, R 27 is an amino acid.

[0199] R 21 and R 27 It is noted that R may be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 21 and R 27 In some other embodiments of any one of the aspects described herein, R 21 and R 27 is different.

[0200] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 25 , R 26 and R 27 At least one of R is not H. In some non-limiting examples, R 21 is not H. In some other non-limiting examples, R 22 is not H. In some other non-limiting examples, R 23 is not H. In some other non-limiting examples, R 25 is not H. In some other non-limiting examples, R 26is not H. In some other non-limiting examples, R 27 is not H.

[0201] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 25 , R 26 and R 27 At least one of R is an electron withdrawing group (EWG). 21 In some other non-limiting examples, R 22 In some other non-limiting examples, R 23 In some other non-limiting examples, R 25 In some other non-limiting examples, R 26 In some other non-limiting examples, R 27 is EWG.

[0202] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 25 , R 26 and R 27 At least one of R is an electron donating group (EDG). 21 In some other non-limiting examples, R 22 In yet some other non-limiting examples, R 23 In yet some other non-limiting examples, R 25 In yet some other non-limiting examples, R 26 In yet some other non-limiting examples, R 27 is EDG.

[0203] In some embodiments of any one of the aspects described herein, R 21 , R 22 and R 23At least two of the are the same. For example, R 21 and R 22 are the same. In some other examples, R 21 and R 23 are the same. In some other examples, R 22 and R 23 are the same. In some embodiments, R 21 , R 22 and R 23 is the same.

[0204] In yet some other embodiments, R 21 , R 22 and R 23 is different.

[0205] In some embodiments of any one of the aspects described herein, R 25 , R 26 and R 27 At least two of the are the same. For example, R 25 and R 26 are the same. In some other examples, R 25 and R 27 are the same. In some other examples, R 26 and R 27 are the same. In some embodiments, R 25 , R 26 and R 27 is the same.

[0206] In yet some other embodiments, R 25 , R 26 and R 27 is different.

[0207] In some embodiments of any one of the aspects described herein, R 21 and R 27 are different or R 22 and R 26 are different or R 23 and R 25 is different.

[0208] In some embodiments of any one of the aspects described herein, R 21 and R 27 is the same as R 22 and R 26 is the same as R 23 and R 25 is the same.

[0209] In some embodiments of any one of the aspects described herein, R 21 , R 22 , R 23 , R 25 , R 26 and R 27 are the same. For example, R 21 , R 22 , R 23 , R 25 , R 26 and R 27 is H.

[0210] In some embodiments of any one of the aspects described herein, the compound of Formula (II) has Formula (II-C) or Formula (II-D): [ka] It is.

[0211] In the compounds of formula (II-C) and (II-D), R 28 is an amino acid, and R 31 , R 32 , R 33 , R 34 and R 35each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 8Sis optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0212] In some embodiments of any one of the aspects described herein, the compound of Formula (II) has Formula (II-E) or Formula (II-F): [ka] It is.

[0213] In the compounds of formula (IG) and (IH), R 28 is an amino acid, and R 31 , R 32 , R 33 , R 34 and R 35each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 31 , R 32 , R 33 , R 34 and R 35 together with the carbon atom to which they are attached form an optionally substituted aryl; 41 , R 42 , R 34 , R 44 and R 45 each can independently be hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; 41 , R 42 , R 34 , R 44 and R 45 together with the carbon atom to which they are attached form an optionally substituted aryl; 8Sis optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. It is noted that any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio may be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0214] Spectral characteristics As used herein, the term "spectral characteristic" refers to a detectable change in emission intensity, polarity / anisotropy or lifetime of a probe conjugated to an amino acid for one or more different amino acids at a single wavelength or multiple wavelengths. Exemplary spectral characteristics include, but are not limited to, spectral shape or peak intensity, and / or polarity. In some embodiments of any one of the aspects described herein, the spectral characteristic is a fluorescence spectral characteristic. As shown in Figures 7-22, the fluorescence spectra of exemplary BODIPY probes conjugated to different amino acids exhibited distinctive spectral characteristics. Thus, comparison of the spectra of amino acids can be used to identify / detect the amino acid to which the probe is conjugated.

[0215] In some embodiments of any one of the aspects described herein, measuring or detecting the spectral property comprises measuring or detecting one or more fluorescence spectral properties of the probe. For example, measuring or detecting the spectral property comprises measuring or detecting fluorescence intensity, polarity / anisotropy or lifetime.

[0216]

[0023] Embodiments of the various aspects described herein include comparing the spectral characteristics of the conjugated probe to a plurality of reference spectral characteristics. In some embodiments of any one of the aspects described herein, each of the reference spectral characteristics is representative of a probe conjugated to a different amino acid. In some embodiments of any one of the aspects described herein, comparing the spectral characteristics of the conjugated probe to a plurality of reference spectral characteristics includes comparing the spectrum of the conjugated probe to a plurality of reference spectra. In some embodiments of any one of the aspects described herein, the reference spectrum is a spectrum of a probe bound to a known amino acid, such as the spectra shown in Figures 7-22. In some embodiments of any one of the aspects described herein, the method includes identifying a closest match between the spectrum of the conjugated probe and the reference spectrum, thereby identifying the amino acid. Various statistical methods known in the art can be used to compare the spectrum of the conjugated probe to the reference spectrum to identify the closest match and the amino acid conjugated to the probe.

[0217] In some embodiments of any one of the aspects described herein, a suitable method produces a quantitative measure of similarity or difference between the spectrum and a reference spectrum. In some embodiments of any one of the aspects described herein, the method described herein uses one or more probabilistic algorithms to compare the spectral characteristics of the minimal amino acid-probe conjugates with the reference / control conjugates. For example, the probabilistic algorithm can be trained to identify different amino acids conjugated to the probes described herein using the spectral data presented in Figures 7-22, and associate a particular spectrum with a particular amino acid. In some embodiments of any one of the aspects described herein, machine learning, genetic algorithms, or principal component analysis (PCA) can be used to compare the spectrum with the reference spectrum.

[0218] Detecting Spectral Features Various aspects of the embodiments described herein include detecting or measuring the spectral properties of the probe conjugated to amino acid.Methods and systems for measuring / detecting the spectral properties of molecules are well known in the art.Exemplary such methods include, but are not limited to, confocal laser (scanning) microscopy, wide-field microscopy, near-field microscopy, fluorescence lifetime imaging microscopy, fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, measuring the brightness change induced by fluorescence quenching / dequenching or fluorescence energy transfer.

[0219] In some embodiments of any one of the aspects described herein, the detection of the spectral characteristics of the conjugated probe comprises optical detection.Exemplary optical detection systems include, but are not limited to, charge-coupled device (CCD), electron multiplying CCD (EMCCD), scanning near-field microscopy, far-field confocal microscopy, wide-field epi-illumination, light scattering, dark-field microscopy, photoconversion, single-photon and / or multi-photon excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, total internal reflection fluorescence (TIRF) microscopy, super-resolution microscopy, single-molecule localization microscopy and single-molecule spectroscopy.

[0220] In some embodiments of any one of the aspects described herein, the method includes detecting laser-activated fluorescence using a microscope equipped with a camera, sometimes referred to as a high-efficiency photon detection system. Suitable photon detection systems include, but are not limited to, photodiodes and intensifier CCD cameras.

[0221] It is noted that various techniques known in the art can be used to detect the spectral characteristics of various conjugated probes at spatially resolved positions.For example, super-resolution microscopy can be used to detect one or more spectral characteristics of the conjugated probes that are conjugated to specific positions in a sample.In some embodiments of any one of the aspects described herein, the method described herein uses stochastic reconstruction optical microscopy (STORM).

[0222] In some embodiments of any one of the aspects described herein, detecting the spectral properties of the conjugated probe comprises an ultrasensitive detection system capable of repeatedly detecting signals from the exact same coordinates in the sample, thereby assigning the detected spectral information to a unique molecule, e.g., a polypeptide.

[0223] Various embodiments of the aspects described herein include detecting or measuring one or more spectral properties of the probe conjugated to the amino acid. In some embodiments of any one of the aspects described herein, detecting or measuring one or more spectral properties of the probe conjugated to the amino acid includes measuring or detecting the fluorescence of the conjugated probe.

[0224] In some embodiments, the method comprises detecting / identifying one or more spectral properties of each probe conjugated to a terminal amino acid of each of a plurality of polypeptides at spatially resolved positions in a sample comprising a plurality of polypeptides.

[0225] In an embodiment, the detection of one or more spectral properties comprises detecting the fluorescence of the conjugated probe. In an embodiment, the detection of one or more spectral properties of the conjugated probe comprises detecting the fluorescence emission intensity, polarity / anisotropy or lifetime. In another embodiment, the detection of the emission intensity, polarity / anisotropy or lifetime is at a single wavelength. In a further embodiment, the detection of the emission intensity, polarity / anisotropy or lifetime is at multiple wavelengths.

[0226] In some embodiments of any one of the aspects described herein, detecting one or more spectral properties of the conjugated probe comprises super-resolution microscopy. In some further embodiments, the super-resolution microscopy comprises stochastic reconstruction optical microscopy (STORM).

[0227] Polypeptides The amino acid to be identified / detected can be part of a polypeptide. When an amino acid is part of a polypeptide, it can be anywhere in the polypeptide. For example, the amino acid can be at the N-terminus of the polypeptide. In some other non-limiting examples, the amino acid can be at the C-terminus of the polypeptide. In some other non-limiting examples, the amino acid can be at the interior of the polypeptide, i.e., at a non-terminal position. It is noted that the terminal amino acid can be modified or derivatized before being conjugated with the probe.

[0228] In some embodiments, the method further comprises immobilizing the polypeptide on a substrate prior to conjugation with the probe. For example, the C-terminus of the polypeptide can be conjugated to the substrate, optionally via a linker.

[0229] Sequencing The method for detecting / identifying the terminal amino acid of a polypeptide can be used to determine at least a portion of the sequence of the polypeptide. In general, the method includes the following steps: (a) conjugating a probe to a terminal amino acid, such as the N-terminal amino acid, of the polypeptide, the probe comprising a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to different terminal amino acids; (b) detecting one or more fluorescence spectral properties of the probe conjugated to the terminal amino acid by detecting the fluorescence of the probe bound to the terminal amino acid of the polypeptide; (c) identifying the corresponding terminal amino acid of the polypeptide by comparing the fluorescence spectral properties of the probe with a plurality of reference fluorescence spectral properties, each of which is representative of a probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of the polypeptide; and (e) sequentially repeating steps (a)-(d) one or more times to determine at least a portion of the sequence of the polypeptide.

[0230] Methods for cleaving terminal amino acids while leaving the remainder of the polypeptide are known in the art.For example, the N-terminal amino acid of a polypeptide can be cleaved using Edman degradation or related degradation, chemical degradation.Alternatively, the N-terminal amino acid of a polypeptide can be enzymatically cleaved using a protease such as an aminopeptidase.Similarly, the C-terminal amino acid of a polypeptide can be enzymatically cleaved using a protease such as a carboxypeptidase.

[0231] In some embodiments, the methods described herein for determining the sequence of a polypeptide include comparing the sequence obtained for the polypeptide to a reference protein sequence database. Without wishing to be bound by theory, small fragments containing 10-20 or fewer aligned amino acid residues consecutively or with breaks can be useful for detecting the identity of a polypeptide in a sample.

[0232] The methods and probes described herein are useful for labeling and sequencing multiple polypeptides in parallel.Therefore, in another aspect, a multiplex method of sequencing multiple polypeptides is provided herein.The method includes the following steps: (a) conjugating multiple probes to the terminal amino acid of each of the multiple polypeptides, where each probe in the multiple probes exhibits different fluorescence spectral characteristics when conjugated to a different terminal amino acid; (b) detecting one or more fluorescence spectral characteristics of each probe conjugated to the terminal amino acid of each of the multiple polypeptides; (c) identifying the corresponding terminal amino acid of each of the multiple polypeptides by comparing the fluorescence spectral characteristics of the probe with multiple reference fluorescence spectral characteristics, where each reference fluorescence spectral characteristic is representative of the probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of each of the multiple polypeptides; and (e) sequentially repeating steps (a) to (d) one or more times to determine at least a portion of the sequence of each of the multiple polypeptides.

[0233] The methods described herein can be used to sequence polypeptides in situ. For example, the methods described herein can be used to determine the sequence of a polypeptide present in or on a biological sample, such as a tissue, a cell, a lipid membrane, or a subcellular organelle, or a sample thereof.

[0234] sample The amino acid or polypeptide may be present in a sample. As used herein, the term "sample" includes any material that contains the amino acid to be detected / identified or a polypeptide that contains said amino acid. The sample can be a biological sample, such as animal or plant tissue, biopsy, organ, cell, membrane vesicle, plasma membrane, organelle, cell extract, secretion, urine or mucus, tissue extract, or other biological specimen of both natural or synthetic origin. The term sample also includes single cells, organelles or intracellular material isolated from biological specimens, or viruses, bacteria, fungi or isolates derived therefrom. The sample can also be an environmental sample, such as a water sample or soil sample, or a sample of any artificial or natural material that contains the amino acid to be detected / identified or a polypeptide that contains said amino acid.

[0235] kit In another aspect, the present disclosure provides a kit comprising one or more compounds described herein.Thus, in some embodiments, the kit comprises a compound of formula (I).In some embodiments, the kit comprises a compound of formula (II).In some embodiments, the kit comprises a compound of formula (I) and a compound of formula (II).

[0236] In certain embodiments, such kits are intended for therapeutic and / or diagnostic use. In certain embodiments, such kits are intended for research use. The kits can be useful in the methods described herein.

[0237] The kits described herein may further comprise one or more reagents / components for conjugating amino acids, peptides or polypeptides to the compounds of formula (I).

[0238] In some embodiments of any of the aspects, the kit further comprises instructions for use.

[0239] In some embodiments of any one of the aspects described herein, the kit further includes one or more reagents for conjugating an amino acid to the compound of Formula (I).

[0240] In some embodiments of any of the aspects, the kit further comprises reagents for isolating, or at least partially isolating, amino acids, peptides, or polypeptides from a sample, e.g., a biological sample.

[0241] The components described herein may be provided as a kit, either alone or in any combination. Such a kit includes the components described herein and their packaging materials. In addition, the kit may optionally include informational material. The informational material may be explanatory, instructional, promotional or other material related to the methods described herein and / or the use of the entities related to the methods described herein. For example, the informational material may describe how to use the kits provided herein to perform assays to detect amino acids, peptides or polypeptides. The kit may also include an empty container and / or a dosing device that may be used, for example, to dosing or preparing a test sample in the test container.

[0242] The informational material of the kit is not limited in its form. In many cases, the informational material, e.g., instructions for use, is provided in printed material, e.g., printed text, drawings and / or photographs, e.g., labels or printed sheets. However, the informational material can also be provided in other formats, such as Braille, computer-readable material, video recording or audio recording. In another embodiment, the informational material of the kit is link information or contact information, e.g., physical address, email address, hyperlink, website or phone number, so that the user of the kit can obtain practical information regarding the formulation and / or its use in the method described herein. Of course, the informational material can also be provided in any combination of formats.

[0243] In some embodiments, the kit can include separate containers, dividers, or compartments for each component and informational material. For example, each of the various components can be contained within a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the individual elements of the kit are contained within a single, undivided container. For example, a collection of magnetic particles is contained within a bottle, vial, or syringe that has informational material in the form of a label attached thereto.

[0244] In some embodiments, the kit includes a carrier to organize and protect the components in the kit during shipping or storage. The carrier can be in any form, including a bag, box, or case, including handles, straps, and wheels, that is convenient for moving or storage.

[0245] definition For convenience, certain terms used herein in the specification, examples, and appended claims are summarized here. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings presented below. Unless otherwise stated or evident from the context, the following terms and phrases do not exclude the meanings that these terms or phrases have acquired in the art to which they pertain. Definitions are presented to help explain certain embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Moreover, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0246] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the present invention, the methods, devices, and materials in this regard are described herein.

[0247] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition (published by Merck Sharp & Dohme Corp., 2018) (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine (published by Blackwell Science Ltd., 1999-2012) (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (published by VCH Publishers, Inc., 1995) (ISBN 1-56081-569-8); Immunology by Werner Luttmann (published by Elsevier, 2006); Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WW Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI (published by Jones & Bartlett Publishers, 2014) (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entireties.

[0248] Except in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When the term "about" is used to describe the invention in connection with percentages, it means ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5% or ±5%.

[0249] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0250] As used herein, the terms "comprising" or "comprises" mean "including" or "includes" and are used in reference to compositions, methods, systems and their individual components that are useful for the present invention, but which may include unspecified elements that may or may not be useful.

[0251] As used herein, the term "consisting essentially of" refers to those elements necessary for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of such embodiment of the invention.

[0252] The term "consisting of" refers to compositions, methods, systems, and individual components thereof, as described herein, which exclude any element not recited in such description of an embodiment.

[0253] The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further noted that the claims may be drafted to exclude any necessary elements. Thus, this statement is intended to serve as a predicate to the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0254] The abbreviation "eg" is derived from the Latin exempli gratia, and is used herein to denote a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0255] The terms "significantly different from," "statistically significant," and similar phrases refer to a comparison of data or other measurements where the difference between the two compared data or other measurements is apparent or different to a reasonable degree to a trained observer, or is statistically significant (when the phrase includes the term "statistically," or when there is some indication of a statistical test, such as a p-value, or when the data under analysis yields a statistical difference by standard statistical tests known in the art).

[0256] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2 SD) or greater.

[0257] As used herein, "polypeptide" refers to two or more amino acids linked together by peptide bonds. The term "polypeptide" includes proteins or protein digests with C-terminus and N-terminus as generally known in the art, and may be of synthetic or natural origin. As used herein, "at least a portion of a polypeptide" refers to two or more amino acids of a polypeptide. Optionally, a portion of a polypeptide includes at least 5, 10, 20, 30 or 50 amino acids of the entire amino acid sequence of a polypeptide, either consecutively or with a break, or includes the entire amino acid sequence of a polypeptide.

[0258] The phrase "N-terminal amino acid" refers to an amino acid that has a free amine group and is linked to only one other amino acid in a polypeptide by a peptide amide bond. Optionally, the "N-terminal amino acid" may also be an "N-terminal amino acid derivative". As used herein, the "N-terminal amino acid derivative" refers to an N-terminal amino acid residue that has been chemically modified, for example, in vitro by Edman's reagent or other chemicals, or inside a cell by natural post-translational modification (e.g., phosphorylation) mechanisms.

[0259] The phrase "C-terminal amino acid" refers to an amino acid that has a free carboxyl group and is linked to only one other amino acid in a polypeptide by a peptide amide bond. Optionally, the "C-terminal amino acid" may also be a "C-terminal amino acid derivative." As used herein, a "C-terminal amino acid derivative" refers to a C-terminal amino acid residue that has been chemically modified in vitro or inside a cell by natural post-translational modification (e.g., phosphorylation) mechanisms.

[0260] As used herein, "sequencing a polypeptide" refers to determining the amino acid sequence of a polypeptide. The term also refers to determining the sequence of a segment of a polypeptide, or determining partial sequence information about a polypeptide.

[0261] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group having 1 to about 60 carbon atoms in the chain, preferably about 6 to about 50 carbons in the chain, which may be straight-chained or branched. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms. The alkyl group may be optionally substituted with one or more alkyl group substituents, which may be the same or different, where "alkyl group substituents" include halo, amino, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo, and cycloalkyl. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl, or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, and hexadecyl. Useful alkyl groups include branched or straight chain alkyl groups of from 6 to 50 carbons, including lower alkyl groups of from 1 to about 4 carbons, and higher alkyl groups of from about 12 to about 16 carbons.

[0262] A "heteroalkyl" group is an alkyl group in which any one of the carbons is replaced by a heteroatom to which the appropriate number of hydrogen atoms are attached (e.g., a CH group is replaced by an NH group or an O group). The term "heteroalkyl" includes optionally substituted alkyl, alkenyl, and alkynyl radicals, which have one or more skeletal atoms selected from atoms other than carbon, such as oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom is located at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3.

[0263] As used herein, the term "alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group may be optionally substituted with one or more "alkyl group substituents". Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-1-yl and heptadeca-8,11-dien-1-yl.

[0264] As used herein, the term "alkynyl" refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group may be optionally substituted with one or more "alkyl group substituents". Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl. Useful alkynyl groups include lower alkynyl groups.

[0265] As used herein, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic ring system of about 3 to about 12 carbon atoms. Cycloalkyl groups can be optionally partially unsaturated. Cycloalkyl groups can also be optionally substituted with aryl group substituents, oxo and / or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful polycyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane and noradamantyl.

[0266] "Heterocyclyl" refers to a non-aromatic 3-8 membered monocyclic ring system, 8-12 membered bicyclic ring system, or 11-14 membered tricyclic ring system having 1-3 heteroatoms in the monocyclic ring, 1-6 heteroatoms in the bicyclic ring, or 1-9 heteroatoms in the tricyclic ring, the heteroatoms being selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic ring, respectively). x Heterocyclyl and C x ~C y Heterocyclyl is commonly used, with X and Y indicating the number of carbon atoms in the ring system. In some embodiments, one, two or three hydrogen atoms in each ring may be replaced by a substituent. Exemplary heterocyclyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, and the like.

[0267] "Aryl" refers to an aromatic carbocyclic radical containing from about 3 to about 13 carbon atoms. The aryl group may be optionally substituted with one or more aryl group substituents, which may be the same or different, where the "aryl group substituents" include alkyl, perhaloalkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NRR', where R and R' are each independently hydrogen, alkyl, perhaloalkyl, aryl, and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl, and substituted or unsubstituted naphthyl.

[0268] "Heteroaryl" refers to an aromatic 3-8 membered monocyclic ring system, 8-12 membered fused bicyclic ring system, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms in the monocyclic, 1-6 heteroatoms in the bicyclic, or 1-9 heteroatoms in the tricyclic, wherein the heteroatoms are selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S in the monocyclic, bicyclic, or tricyclic, respectively).

[0269] Exemplary aryl and heteroaryl include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzoisoxazolyl, benzoisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b] tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-Oxadiazolyl, oxazolidinyl, oxazolyl, oxindryl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidinyl, lolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. In some embodiments, one, two, three, or four hydrogen atoms of each ring may be replaced by a substituent.

[0270] As used herein, the term "halogen" or "halo" refers to an atom selected from fluorine, chlorine, bromine and iodine. The term "halogen radioisotope" or "haloisotope" refers to a radioactive nuclide of an atom selected from fluorine, chlorine, bromine and iodine.

[0271] A "halogen-substituted moiety" or "halo-substituted moiety" as an isolated group or part of a larger group means an aliphatic, alicyclic, or aromatic moiety, as such terms are defined in this application, that is substituted with one or more "halo" atoms.

[0272] The term "haloalkyl" as used herein refers to alkyl and alkoxy structures having at least one substituent of fluorine, chlorine, bromine or iodine, or a combination thereof. In embodiments, when more than one halogen is included in the group, the halogens are the same or different. The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, where the halo is fluorine. Exemplary halo-substituted alkyls include haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl, and the like (e.g., halo-substituted (C1-C3) alkyls include chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

[0273] The term "perhaloalkyl," unless otherwise specified, means an alkyl substituted with (2n+1) halogen atoms, where n is the total number of carbon atoms in the alkyl group. For example, the term "perhalo(C1-C4)alkyl" or "C1-C4 perhaloalkyl" includes trifluoromethyl, pentachloroethyl, 1,1,1-trifluoro-2-bromo-2-chloroethyl, and the like.

[0274] The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, where halo is fluorine. Exemplary halo-substituted alkyls include haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl, and the like (e.g., halo-substituted (C1-C3) alkyls include chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

[0275] As used herein, the term "amino" refers to -NH2. The term "alkylamino" refers to a nitrogen moiety with one straight or branched unsaturated aliphatic, cyclyl or heterocyclyl radical attached to the nitrogen, e.g., -NH(alkyl). The term "dialkylamino" refers to a nitrogen moiety with two straight or branched unsaturated aliphatic, cyclyl or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl). The term "alkylamino" includes "alkenylamino", "alkynylamino", "cyclylamino" and "heterocyclylamino". The term "arylamino" refers to a nitrogen moiety with at least one aryl radical attached to the nitrogen, e.g., -NHaryl and -N(aryl)2. The term "heteroarylamino" refers to a nitrogen moiety with at least one heteroaryl radical attached to the nitrogen, e.g., -NHheteroaryl and -N(heteroaryl)2. If necessary, the two substituents taken together with the nitrogen can also form a ring. Unless otherwise indicated, compounds described herein that contain an amino moiety can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylaminos include NH(C1-C2) groups, such as, but not limited to, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, and -NHCH(CH3)2. 10 Exemplary dialkylaminos include -N(C1-C alkyl), such as, but not limited to, N(CH3)2, -N(CH2CH3)2, -N(CH2CH2CH3)2, and -N(CH(CH3)2). 10 alkyl)2.

[0276] The term "aminoalkyl" refers to alkyl, alkenyl, and alkynyl as defined above, except that one or more substituted or unsubstituted nitrogen atoms (-N-) are positioned between the carbon atoms of the alkyl, alkenyl, or alkynyl. For example, a (C2-C6)aminoalkyl refers to a chain containing between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

[0277] The terms "hydroxy" and "hydroxyl" refer to the --OH radical.

[0278] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group as defined above having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or -O-heteroaryl, where aryl and heteroaryl are as defined herein. Alkoxy and aroxy groups can be substituted as described above for alkyl. Exemplary alkoxy groups include, but are not limited to, O-methyl, O-ethyl, On-propyl, O-isopropyl, On-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O-hexyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl, and the like.

[0279] As used herein, the term "carbonyl" refers to a -C(O)- radical. It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.

[0280] As used herein, the term "oxo" refers to a double bonded oxygen, ie, =O.

[0281] The term "carboxy" refers to the -C(O)O- radical. It is noted that compounds described herein containing a carboxy moiety can include protected derivatives thereof, i.e., where the oxygen is replaced with a protecting group. Suitable protecting groups for a carboxy moiety include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., a carboxyl group.

[0282] The term "ester" refers to a chemical moiety having the formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, and heterocycloalkyl.

[0283] The term "cyano" refers to the -CN radical.

[0284] The term "nitro" refers to the --NO2 radical.

[0285] The term "heteroatom" refers to an atom that is not a carbon atom. Specific examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and the halogens. A "heteroatom moiety" includes a moiety that is bonded by an atom other than carbon. Examples of heteroatom moieties include -N=, -NR N -, -N + (O - )=, -O-, -S- or -S(O)2-, -OS(O)2- and -SS-, R N is H or a further substituent.

[0286] The terms "alkylthio" and "thioalkoxy" refer to an alkoxy group, as defined above, in which an oxygen atom is replaced by a sulfur. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term "alkylthio" also encompasses cycloalkyl, alkene, and cycloalkene groups, and alkyne groups. "Arylthio" refers to an aryl or heteroaryl group.

[0287] The term "sulfinyl" means a -SO- radical. It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.

[0288] The term "sulfonyl" means a -SO2- radical. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.

[0289] The term "thiocarbonyl" means a -C(S)- radical. It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

[0290] "Acyl" refers to an alkyl-CO- group, where alkyl is as previously described. Exemplary acyl groups include alkyl groups of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl, and palmitoyl.

[0291] "Aroyl" means an aryl-CO- group, where aryl is as previously described. Exemplary aroyl groups include benzoyl, and 1- and 2-naphthoyl.

[0292] "Arylthio" refers to an aryl-S- group, in which the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

[0293] "Aralkyl" refers to an aryl-alkyl- group in which the aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0294] "Aralkyloxy" refers to an aralkyl-O- group in which the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.

[0295] "Aralkylthio" refers to an aralkyl-S- group in which the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.

[0296] "Alkoxycarbonyl" refers to an alkyl-O-CO- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.

[0297] "Aryloxycarbonyl" refers to an aryl-O-CO- group. Exemplary aryloxycarbonyl groups include phenoxy-carbonyl and naphthoxy-carbonyl.

[0298] "Aralkoxycarbonyl" refers to the group aralkyl-O-CO-. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0299] "Carbamoyl" refers to the group H2N-CO-.

[0300] "Alkylcarbamoyl" refers to the group R'RN-CO-, where one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.

[0301] "Dialkylcarbamoyl" refers to the group R'RN-CO- where R and R' are each independently alkyl as previously described.

[0302] "Acyloxy" refers to the group acyl-O-, where acyl is as previously described. "Acylamino" refers to the group acyl-NH-, where acyl is as previously described. "Aroylamino" refers to the group aroyl-NH-, where aroyl is as previously described.

[0303] The term "optionally substituted" means that the specified group or moiety is unsubstituted or substituted with one or more (usually 1, 2, 3, 4, 5, or 6) substituents independently selected from the group of substituents listed below in the definition for "substituent" or otherwise specified. The term "substituent" refers to a group that is "substituted" on a substituted group at any atom of the substituted group. Suitable substituents include, but are not limited to, halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido. In some cases, two substituents, together with the carbons to which they are attached, can form a ring.

[0304] For example, any of alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl, or aryl can be OH, CN, -SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1 ~C8) alkyl, O(C1~C8) alkyl, O(C1~C8) haloalkyl, (C2~C8) alkenyl, (C2~C8) alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p-OH, CH2-[CH(OH)] m -(CH2) p and "m" and "p" are independently 1, 2, 3, 4, 5, or 6.

[0305] In some embodiments, the optionally substituted group is substituted with one substituent. In some other embodiments, the optionally substituted group is substituted with two independently selected substituents that can be the same or different. In some other embodiments, the optionally substituted group is substituted with three independently selected substituents that can be the same, different, or any combination of the same and different. In yet some other embodiments, the optionally substituted group is substituted with four independently selected substituents that can be the same, different, or any combination of the same and different. In yet some other embodiments, the optionally substituted group is substituted with five independently selected substituents that can be the same, different, or any combination of the same and different.

[0306] An "isocyanato" group refers to an NCO group.

[0307] A "thiocyanato" group refers to a CNS group.

[0308] An "isothiocyanato" group refers to an NCS group.

[0309] "Alkoyloxy" refers to the group RC(=O)O-.

[0310] "Alkoyl" refers to the group RC(=O)--.

[0311] All of the structures of any of the compounds are presented herein for illustrative purposes and disclose specific isomers. However, one skilled in the art will recognize all possible isomers of any of the structures of the compounds described herein. Therefore, other isomers, such as enantiomers of any of formulas (I) and (II), are considered to fall within the scope of the present invention. As used herein, the term "isomer" refers to compounds that have the same molecular formula but different structures. Isomers that differ only in configuration and / or conformation are referred to as "stereoisomers". The term "isomer" is also used to refer to enantiomers.

[0312] The term "enantiomer" is used to describe one of a pair of molecular isomers that are mirror images of each other and are not superimposable. The designations "R" and "S" are used to denote the absolute configuration of a molecule about its chiral center. The designations may appear as prefixes or as suffixes. The designations may or may not be separated from the isomer by a hyphen. The designations may or may not be joined by a hyphen. The designations may or may not be enclosed in parentheses. The designations "(+)" and "(-)" are used to denote the sign of rotation of plane polarized light by a compound, with (-) meaning that the compound is levorotatory (rotates to the left). A compound prefixed by (+) is dextrorotatory (rotates to the right). Other terms used to denote or refer to enantiomers include "stereoisomers" (because of the different configurations or stereochemistry around the chiral center; all enantiomers are stereoisomers, but not all stereoisomers are enantiomers) or "optical isomers" (because pure enantiomers are optically active, which is the ability of different pure enantiomers to rotate plane-polarized light in different directions). Enantiomers generally have the same physical properties, such as melting points and boiling points, and also the same spectroscopic properties. Enantiomers can differ from each other with respect to interactions involving plane-polarized light, and with respect to biological activity.

[0313] In various embodiments, the compounds of formula (I) or (II) include enantiomers, derivatives, prodrugs, and pharma- ceutically acceptable salts thereof.

[0314] The term "derivative" as used herein refers to a chemical that is structurally related to another or "original" substance, which may be referred to as a "parent" compound. A "derivative" can be made from a structurally related parent compound in one or more steps. The general physical and chemical properties of a derivative are still similar to the parent compound.

[0315] Specified elements of any of the above-described embodiments may be combined with or substituted for elements in other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of the present disclosure.

[0316] Grouping of alternative elements or embodiments of the invention disclosed herein should not be interpreted as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or excluded from a group for reasons of convenience and / or patentability. When any such inclusion or exclusion is made, the specification is deemed to include the modified group herein, thereby satisfying the written description of all Markush groups used in the appended claims.

[0317] Aspects of the present technology may be defined in any of the following numbered embodiments:

[0318] Embodiment 1: A method of identifying an amino acid comprising the steps of: (a) conjugating a probe to an amino acid, the probe comprising a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to the amino acid; (b) detecting one or more fluorescence spectral properties of the probe conjugated to the amino acid; and (c) identifying the amino acid by comparing the fluorescence spectral property of the probe to a plurality of reference fluorescence spectral properties, each of the reference fluorescence spectral properties being representative of a probe conjugated to a different amino acid.

[0319] Embodiment 2: A multiplex method for identifying a plurality of amino acids, comprising the steps of: (a) conjugating a plurality of probes to amino, carboxyl, hydroxyl or thiol groups of a plurality of amino acids, wherein each probe in the plurality of probes comprises a dipyrrolomethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to a different amino acid; (b) detecting one or more fluorescence spectral properties of each probe conjugated to the plurality of amino acids; and (c) identifying the amino acids by comparing the plurality of fluorescence spectral properties of the probes to a plurality of reference fluorescence spectral properties, wherein each of the reference fluorescence spectral properties is representative of a probe conjugated to a different amino acid.

[0320] Embodiment 3: The method of embodiment 2 or embodiment 3, wherein the probe is covalently conjugated to an amino acid.

[0321] Embodiment 4: The method of any one of embodiments 1 to 3, wherein the probe is conjugated to an amino group, a carboxyl group, a hydroxyl group, or a thiol group of an amino acid.

[0322] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the probe is conjugated to an amino group of an amino acid.

[0323] Embodiment 6: The method of any one of embodiments 1 to 4, wherein the probe is conjugated to a carboxyl group of an amino acid.

[0324] Embodiment 7: The method of any one of embodiments 1 to 6, wherein the amino acid is present in a sample.

[0325] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the amino acid is present in a polypeptide.

[0326] Embodiment 9: The method of embodiment 8, wherein the amino acid is present at the N-terminus of the polypeptide.

[0327] Embodiment 10: The method of embodiment 8, wherein the amino acid is present at the C-terminus of the polypeptide.

[0328] Embodiment 11: A method for determining the sequence of a polypeptide, comprising: (a) conjugating a probe to a terminal amino acid of the polypeptide, wherein the probe comprises a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to different terminal amino acids; (b) detecting one or more fluorescence spectral properties of the probe conjugated to the terminal amino acid by detecting fluorescence of the probe bound to the terminal amino acid of the polypeptide; (c) identifying a corresponding terminal amino acid of the polypeptide by comparing the fluorescence spectral property of the probe to a plurality of reference fluorescence spectral properties, each of which is representative of a probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of the polypeptide; and (e) sequentially repeating steps (a)-(d) one or more times to determine the sequence of at least a portion of the polypeptide.

[0329] Embodiment 12: A multiplex method for determining a sequence of a plurality of polypeptides, comprising: (a) conjugating a plurality of probes to a terminal amino acid of each of the plurality of polypeptides, wherein each probe in the plurality of probes comprises a dipyrromethane-BF2 derivative that exhibits a different fluorescence spectral characteristic when conjugated to a different terminal amino acid; (b) detecting one or more fluorescence spectral characteristics of each probe conjugated to a terminal amino acid of each of the plurality of polypeptides; (c) identifying a corresponding terminal amino acid of each of the plurality of polypeptides by comparing the fluorescence spectral characteristic of the probe to a plurality of reference fluorescence spectral characteristics, each of which is representative of a probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of each of the plurality of polypeptides; and (e) sequentially repeating steps (a)-(d) one or more times to determine the sequence of at least a portion of each of the plurality of polypeptides.

[0330] Embodiment 13: The method of embodiment 2 or 12, wherein the probes in the plurality of probes are the same.

[0331] Embodiment 14: The method of embodiment 2 or 12, wherein at least two probes in the plurality of probes are different.

[0332] Embodiment 15: The method of any one of embodiments 11 to 14, wherein the terminal amino acid is at the N-terminus of the polypeptide.

[0333] Embodiment 16: The method of any one of embodiments 11 to 14, wherein the terminal amino acid is at the C-terminus of the polypeptide.

[0334] Embodiment 17: The method of any one of embodiments 11 to 16, wherein the step of cleaving the terminal amino acid comprises enzymatic cleavage.

[0335] Embodiment 18: The method of any one of embodiments 11 to 16, wherein the step of cleaving the terminal amino acid comprises chemical cleavage.

[0336] Embodiment 19: The method of any one of embodiments 11 to 15, wherein the polypeptide is present in a sample.

[0337] Embodiment 20: The method of embodiment 19, comprising detecting one or more spectral properties of each probe conjugated to a terminal amino acid of each of the plurality of polypeptides at spatially resolved positions in a sample comprising the plurality of polypeptides.

[0338] Embodiment 21: The method of any one of embodiments 7, 19 or 20, wherein the sample is a biological sample.

[0339] Embodiment 22: The method of embodiment 21, wherein the biological sample is a biological fluid, tissue, organ, or cell.

[0340] Embodiment 23: The method of any one of embodiments 1 to 22, wherein said detecting one or more spectral properties of the conjugated probe comprises super-resolution microscopy.

[0341] Embodiment 24: The method of embodiment 23, wherein the super-resolution microscopy comprises stochastic reconstruction optical microscopy (STORM).

[0342] Embodiment 25: The method of any one of embodiments 1 to 25, wherein the step of detecting one or more spectral properties comprises detecting fluorescence of the conjugated probe.

[0343] Embodiment 26: The method of any one of embodiments 1 to 26, wherein the step of detecting one or more spectral properties of the conjugated probe comprises detecting fluorescence emission intensity, polarity / anisotropy or lifetime.

[0344] Embodiment 27: The method of embodiment 26, wherein said detecting luminescence intensity, polarity / anisotropy or lifetime is at a single wavelength.

[0345] Embodiment 28: The method of embodiment 26, wherein said detecting luminescence intensity, polarity / anisotropy or lifetime is at multiple wavelengths.

[0346] Embodiment 29: The method of any one of embodiments 1 to 28, wherein the dipyrromethane-BF2 derivative comprises a labile or reactive group.

[0347] Embodiment 30: The method of any one of embodiments 1 to 29, wherein the dipyrromethane-BF2 derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

[0348] Embodiment 31: The method of embodiment 30, wherein the probe is conjugated via the 8-position (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

[0349] Embodiment 32: The method of embodiment 31, wherein the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative comprises a labile or reactive group at the 8-position (meso position).

[0350] Embodiment 33: The probe is represented by formula (I), where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; and optionally R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 at least one of the following is a labile group or a leaving group; and any of alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio is independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, and the like. , amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0351] Embodiment 34: R 8 34. The method of embodiment 33, wherein is a labile group or a leaving group.

[0352] Embodiment 35: R 8 The method of embodiment 33 or 34, wherein is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, and diazonium.

[0353] Embodiment 36:R 8 But, -SR 8S or halogen, R 8S is optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl.

[0354] Embodiment 37: R 8S is optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C2-C6 alkenyl, optionally substituted aryl or optionally substituted heteroaryl.

[0355] Embodiment 38: R 8S is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl, and optionally R 8S The method of embodiment 37, wherein is methyl.

[0356] Embodiment 39: R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The method of any one of embodiments 33-38, wherein at least one of is not H.

[0357] Embodiment 40:R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The method of any one of embodiments 33-39, wherein at least one of is an electron withdrawing group (EWG).

[0358] Embodiment 41:R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The method of any one of embodiments 33-39, wherein at least one of is an electron donating group (EDG).

[0359] Embodiment 42: R 1 , R 2 and R 3 42. The method of any one of embodiments 33-41, wherein at least two of are the same.

[0360] Embodiment 43: R 1 , R 2 and R 3 42. The method of any one of embodiments 33-41, wherein at least two of are different.

[0361] Embodiment 44: R 1 and R 7 are different or R 2 and R 6 are different or R 3 and R 5 The method of any one of embodiments 33 to 43, wherein:

[0362] Embodiment 45:R 1 and R 7 is the same, and R 2 and R 6 is the same, and R 3 and R 5 The method of any one of embodiments 33 to 43, wherein

[0363] Embodiment 46:R1 The method of any one of embodiments 33-45, wherein is H or optionally substituted C1-C6 alkyl.

[0364] Embodiment 47:R 1 The method of embodiment 46, wherein is H or methyl.

[0365] Embodiment 48:R 2 The method of any one of embodiments 33-47, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0366] Embodiment 49:R 2 The method of embodiment 48, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0367] Embodiment 50:R 3 The method of any one of embodiments 33-49, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted napthelene.

[0368] Embodiment 51: R 3 H, Br, Cl, methyl, -CH=CH-R 9A , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl), and R 9A The method of any one of embodiments 33-50, wherein is optionally substituted aryl or heteroaryl.

[0369] Embodiment 52: R 3The method of any one of embodiments 33-51, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0370] Embodiment 53: R 3 is H, methyl or -CH=CH-R 9A and R 9A The method of any one of embodiments 33-51, wherein is optionally substituted phenyl.

[0371] Embodiment 54: R 9A is phenyl or phenyl substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl.

[0372] Embodiment 55: R 9A 55. The method of embodiment 54, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0373] Embodiment 56: R 5 The method of any one of embodiments 33-55, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted naphthalene.

[0374] Embodiment 57: R 5 H, Br, Cl, methyl, -CH=CH-R 9B, phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl), and R 9B The method of any one of embodiments 33-56, wherein is optionally substituted aryl or heteroaryl.

[0375] Embodiment 58: R 5 The method of any one of embodiments 33-57, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0376] Embodiment 59: R 5 is H, methyl or -CH=CH-R 9B and R 9B The method of any one of embodiments 33-57, wherein is optionally substituted phenyl.

[0377] Embodiment 60: R 9B is phenyl or phenyl substituted with a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl.

[0378] Embodiment 61: R 9B The method of embodiment 60, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0379] Embodiment 62: R 6The method of any one of embodiments 33-61, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0380] Embodiment 63: R 6 The method of embodiment 62, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0381] Embodiment 64: R 7 The method of any one of embodiments 33-63, wherein is H or optionally substituted C1-C6 alkyl.

[0382] Embodiment 65: R 7 The method of embodiment 64, wherein is H or methyl.

[0383] Embodiment 66:R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The method of any one of embodiments 33-65, wherein is H.

[0384] Embodiment 67: R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; and optionally R 1 , R 2, R 3 , R 4 , R 5 , R 6 , R 7 and R 8 at least one of the following is a labile group or leaving group; and any of the following may be selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio: halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryl, heterocyclyl ... and optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of: aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido, with the proviso that, as appropriate, (i) R 8S is methyl, (a) R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H, (b) R 1 , R 2 , R 6 and R 7 is R, not H. 3 and R 5 is not methyl, ethyl, or p-nitrophenyl; (c) R 1 , R 3 , R 5 and R 7 is not methyl, but R 2 and R 6 is not H, (d) R 1 and R 7is R, not H. 2 , R 3 , R 5 and R 6 is neither Cl nor Br, (e)R 1 , R 3 , R 5 , R 6 and R 7 is R, not H. 2 is not -C(O)H, (f)R 1 is not p-nitrophenyl, but R 2 , R 6 and R 7 is R, not H. 3 and R 5 is not methyl, and (g) R 1 , R 2 , R 5 , R 6 and R 7 is R, not H. 3 is not phenyl, p-nitrophenyl, p-iodophenyl, p-carboxyphenyl, or p-methoxyphenyl, and / or (ii) R 8S is allyl, ethyl, propyl, butyl, t-butyl, n-dodecyl, phenyl, 2,6-dimethylphenyl, p-methylphenyl, p-methoxyphenyl, p-nitrophenyl, or benzyl (-CHPH), then R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H, and / or (iii) R 8 is Cl, (a) R 1 , R 2 , R 3 , R 5 , R 6 and R 7 is not H, (b) R 1 , R 3 , R 5 , R 6 and R 7 is R, not H. 2 is not Cl, (c) R 1 , R 3, R 5 and R 7 is R, not H. 2 and R 5 is not Cl, (d) R 1 , R 5 and R 7 is R, not H. 2 , R 3 and R 5 is not Cl, and (e) R 1 , R 2 , R 3 , R 5 , R 6 and R 7 A compound of formula (I), provided that is not Cl.

[0385] Embodiment 68: R 8 68. The compound of embodiment 67, wherein is a labile group or a leaving group.

[0386] Embodiment 69: R 8 The compound of embodiment 67 or 68, wherein is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, and diazonium.

[0387] Embodiment 70:R 8 But, -SR 8S or halogen, R 8S is optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0388] Embodiment 71:R 8Sis optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C2-C6 alkenyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0389] Embodiment 72:R 8S is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl, and optionally R 8S The compound of embodiment 72, wherein is methyl.

[0390] Embodiment 73: R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The compound of any one of embodiments 67-72, wherein at least one of is not H.

[0391] Embodiment 74: R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The compound of any one of embodiments 67-73, wherein at least one of is an electron withdrawing group (EWG).

[0392] Embodiment 75:R 1 , R 2 , R 3 , R 5 , R 6 and R 7 The compound of any one of embodiments 67-73, wherein at least one of is an electron donating group (EDG).

[0393] Embodiment 76:R 1 , R 2 and R 3 The compound of any one of embodiments 67-75, wherein at least two of are the same.

[0394] Embodiment 77: R1 , R 2 and R 3 The compound of any one of embodiments 67-75, wherein at least two of are different.

[0395] Embodiment 78:R 1 and R 7 are different or R 2 and R 6 are different or R 3 and R 5 The compound of any one of embodiments 66-77, wherein:

[0396] Embodiment 79: R 1 and R 7 is the same, and R 2 and R 6 is the same, and R 3 and R 5 The compound of any one of embodiments 67-78, wherein

[0397] Embodiment 80:R 1 The compound of any one of embodiments 67-79, wherein is H or optionally substituted C1-C6 alkyl.

[0398] Embodiment 81: R 1 is H or methyl.

[0399] Embodiment 82:R 2 The compound of any one of embodiments 67-81, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0400] Embodiment 83: R 2 The compound of embodiment 82, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0401] Embodiment 84:R 3The compound of any one of embodiments 67-83, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted naphthalene.

[0402] Embodiment 85:R 3 H, Br, Cl, methyl, -CH=CH-R 9 , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl), and R 9 The compound of any one of embodiments 67-84, wherein is optionally substituted aryl or heteroaryl.

[0403] Embodiment 86:R 3 The compound of any one of embodiments 67-85, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0404] Embodiment 87:R 3 is H, methyl or -CH=CH-R 9A and R 9 The compound of any one of embodiments 67-86, wherein is optionally substituted phenyl.

[0405] Embodiment 88:R 9AThe compound of embodiment 87, wherein is phenyl or phenyl substituted with substituents selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl.

[0406] Embodiment 89:R 9A The compound of embodiment 88, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0407] Embodiment 90:R 5 The compound of any one of embodiments 67-89, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted naphthalene.

[0408] Embodiment 91:R 5 H, Br, Cl, methyl, -CH=CH-R 9B , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl), and R 9B The compound of any one of embodiments 67-90, wherein is optionally substituted aryl or heteroaryl.

[0409] Embodiment 92:R 5The compound of any one of embodiments 67-91, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0410] Embodiment 93:R 5 is H, methyl or -CH=CH-R 9B and R 9B The compound of any one of embodiments 67-91, wherein is optionally substituted phenyl.

[0411] Embodiment 94:R 9B The compound of embodiment 93, wherein is phenyl or phenyl substituted with substituents selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl.

[0412] Embodiment 95:R 9B 95. The compound of embodiment 94, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0413] Embodiment 96:R 6 The compound of any one of embodiments 67-95, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0414] Embodiment 97:R 6 The compound of embodiment 96, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0415] Embodiment 98:R 7The compound of any one of embodiments 67-97, wherein is H or optionally substituted C1-C6 alkyl.

[0416] Embodiment 99:R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28 are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and amino acids; and optionally R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 and R 28is an amino acid, and any of the alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio are optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

[0417] Embodiment 100:R 28 is an amino acid.

[0418] Embodiment 101:R 21 , R 22 , R 23 , R 25 , R 26 and R 27 The compound of any one of embodiments 99-100, wherein at least one of is not H.

[0419] Embodiment 102:R 21 , R 22 , R 23 , R 25 , R 26 and R 27 The compound of any one of embodiments 99-101, wherein at least one of is an electron withdrawing group (EWG).

[0420] Embodiment 103:R 21 , R22 , R 23 , R 25 , R 26 and R 27 The compound of any one of embodiments 99-102, wherein at least one of is an electron donating group (EDG).

[0421] Embodiment 104:R 21 , R 22 and R 23 The compound of any one of embodiments 99-103, wherein at least two of are the same.

[0422] Embodiment 105:R 21 , R 22 and R 23 The compound of any one of embodiments 99-103, wherein at least two of are different.

[0423] Embodiment 106:R 21 and R 27 are different or R 22 and R 26 are different or R 23 and R 25 The compound of any one of embodiments 99-105, wherein:

[0424] Embodiment 107:R 21 and R 27 is the same, and R 22 and R 26 is the same, and R 23 and R 25 The compound of any one of embodiments 99-105, wherein

[0425] Embodiment 108:R 21 The compound of any one of embodiments 99-107, wherein is H or optionally substituted C1-C6 alkyl.

[0426] Embodiment 109:R 21 is H or methyl.

[0427] Embodiment 110:R 22 The compound of any one of embodiments 99-109, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0428] Embodiment 111:R 22 The compound of embodiment 110, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0429] Embodiment 112:R 23 The compound of any one of embodiments 99-111, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted naphthalene.

[0430] Embodiment 113:R 23 H, Br, Cl, methyl, -CH=CH-R 29A , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl and C1-C6 haloalkyl), and R 29A The compound of any one of embodiments 99-113, wherein is optionally substituted aryl or heteroaryl.

[0431] Embodiment 114:R 23 The compound of any one of embodiments 99-113, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0432] Embodiment 115:R 23is H, methyl or -CH=CH-R 29A and R 29A The compound of any one of embodiments 99-113, wherein is optionally substituted phenyl.

[0433] Embodiment 116:R 29A is phenyl or phenyl substituted with substituents selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl C1-C6 haloa nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl C1-C6 haloalkyl.

[0434] Embodiment 117:R 29A The compound of embodiment 116, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0435] Embodiment 118:R 25 The compound of any one of embodiments 99-117, wherein is H, halogen, optionally substituted C1-C6 alkyl, C1-C6 perhaloalkyl optionally substituted C1-C6 alkenyl, optionally substituted phenyl, or optionally substituted naphthalene.

[0436] Embodiment 119:R 25 H, Br, Cl, methyl, -CH=CH-R 29B , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (substituted by a substituent selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl, nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl), and R29B The compound of any one of embodiments 99-118, wherein is optionally substituted aryl or heteroaryl.

[0437] Embodiment 120:R 25 The compound of any one of embodiments 99-119, wherein is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl, or naphthalene.

[0438] Embodiment 121:R 25 is H, methyl or -CH=CH-R 29B and R 29B The compound of any one of embodiments 99-119, wherein is optionally substituted phenyl.

[0439] Embodiment 122:R 29B The compound of embodiment 121, wherein is phenyl or phenyl substituted with substituents selected from the group consisting of nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C1-C6 alkyl, C1-C6 perhaloalkyl, C1-C6 haloalkyl.

[0440] Embodiment 123:R 29B The compound of embodiment 122, wherein is 4-fluorophenyl, 4-trifluoromethylphenyl, or 4-cyanophenyl.

[0441] Embodiment 124:R 26 The compound of any one of embodiments 99-123, wherein is H, halogen, optionally substituted C1-C6 alkyl, -C(O)H, or -SO3H.

[0442] Embodiment 125:R 26 The compound of embodiment 124, wherein is H, Br, Cl, -C(O)H or -SO3H.

[0443] Embodiment 126:R 27 The compound of any one of embodiments 99-125, wherein is H or optionally substituted C1-C6 alkyl.

[0444] Embodiment 127:R 7 is H or methyl.

[0445] It should be understood that the present disclosure is not limited to the specific methodology, protocols, and reagents, etc., presented herein, and therefore may vary. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present disclosure, which is defined only by the claims. The present invention is further illustrated by the following examples, which should not be construed as further limiting. EXAMPLES

[0446] The following examples illustrate embodiments of the present invention and are not intended to limit the scope of the invention.

[0447] Example 1 Synthesis of Exemplary Compound BOPIDY In one embodiment, the inventors have developed the use of boron-dipyrromethane dyes (BODIPY) (Figure 1), which can be directly conjugated to the N-terminal amino acid. This conjugation affects the photophysical properties of the dye, which in turn results in different spectral signals that are amino acid dependent. As outlined in (Figure 6), the BODIPY dyes can distinguish, in this example, almost all amino acids by fluorescence emission.

[0448] The novel BODIPY derivatives are synthesized using known synthetic methods from readily available starting materials (Scheme 1 and Scheme 2). The dyes can be subjected to amino acids or polypeptides in solution in microarrays or in fixed cells under normal PBS or similar buffer conditions, resulting in conjugation to the nitrogen of the amino acid (Figure 1). Spectral properties are measured and the information is used to determine the identity of the directly conjugated amino acid (Figure 7).

[0449] Method 1: Synthesis of 8-thioether BODIPY core Exemplary 8-thioether BODIPY dyes were synthesized using the method shown in Scheme 1. [ka] Scheme 1: General synthesis of 8-thioether BODIPY dye cores

[0450] The synthesis began with the formation of a thioketone. In anhydrous THF under an inert atmosphere, the pyrrole derivative (2 equiv.) was added and cooled to 0° C. using an ice-water bath. Thiophosgene (1 equiv.) was then added and the reaction was stirred at 0° C. for 15 min. The reaction was quenched with methanol, concentrated, and purified by flash chromatography (5-20% ethyl acetate / hexanes).

[0451] The thioketone was then dissolved in anhydrous DCM under an inert atmosphere and an alkyl halide (10 equiv.) was added. The reaction was monitored by TLC. Upon completion, the crude mixture was concentrated to dryness under reduced pressure to remove excess alkyl halide and resuspended in anhydrous DCM. To this solution was added trimethylamine (5 equiv.). The solution was stirred at room temperature under an inert atmosphere for 30 min. After 30 min, BF3·OEt2 was added and the solution was stirred until the starting material was consumed by TLC. The reaction was concentrated and purified by flash chromatography (5-20% ethyl acetate / hexanes) to yield the 8-thioether BODIPY. An exemplary BODIPY dye made by this method is shown in Figure 2.

[0452] Method 2: 8-Chloro and 8-aryl thioether BODIPY synthesis Exemplary 8-chloro and 8-aryl thioethers BODIPY were synthesized as shown in Scheme 2. [ka] Scheme 2: General synthesis of 8-chloro and 8-aryl thioether BODIPYs

[0453] Pyrrole (1 eq) and diisopropylethylamine (0.5 eq) were added in anhydrous DCE under an inert atmosphere and the solution was cooled to 0° C. using an ice-water bath. Triphosgene (0.33 eq) was added to the solution and stirred at 0° C. until complete conversion by TLC. Once consumed, an additional equivalent of pyrrole was added and the solution was then refluxed until complete conversion to the ketone. The solution was then cooled, diluted with diethyl ether and washed with water. The organic layer was dried, concentrated and purified by flash chromatography (20% ethyl acetate / DCM).

[0454] The 8-chloro BODIPY was then displaced to form the 8-aryl thioether BODIPY. This was accomplished by two different methods:

[0455] 1. 8-ChloroBODIPY (1 eq.), arylthiol (1 eq.) and trimethylamine (1 eq.) were added to anhydrous DCM. The reaction was stirred under ambient conditions until complete by TLC. The reaction was concentrated and purified by flash chromatography.

[0456] 2. 8-ChloroBODIPY (1 eq.), aryl disulfide (1 eq.) and triphenylphosphine (1 eq.) were added to anhydrous DCM. The reaction was stirred under ambient conditions until complete by TLC. The reaction was concentrated and purified by flash chromatography.

[0457] Method 3: Tetrahalogenation of 8-thioether BODIPY Tetrahalogenated 8-thioether BODIPYs were synthesized using the method shown in Scheme 3. [ka] Scheme 3: Synthesis of tetrahalogenated 8-thioether BODIPYs

[0458] Unsubstituted BODIPY (BDP-001) was added to THF and cooled to -40 C. To this solution was added N-halosuccinimide (5 equiv.) slowly and monitored by TLC. Upon completion, the reaction was warmed to room temperature, concentrated, and purified by flash chromatography (10% ethyl acetate / hexanes).

[0459] Method 4: Vilsmeier-Haack addition of aldehydes to 8-thioether BODIPY β-Formyl-8-thioether BODIPY was synthesized as shown in Scheme 4. [ka] Scheme 4: Synthesis of beta-formyl substituted 8-thioether BODIPY

[0460] DMF was added to an equal portion of POCI2 and stirred in an ice bath under inert atmosphere for 5 min. After 5 min, the solution was warmed to room temperature and stirred for an additional 30 min. To the reaction mixture was added unsubstituted BODIPY (BDP-001) in DCE, the temperature was raised to 50° C., and the mixture was stirred for an additional 2 h. After 2 h, the solution was poured into ice water and extracted with DCM. The organic layer was dried, filtered, concentrated, and purified by flash chromatography to yield the β-formyl substituted 8-thioether BODIPY.

[0461] Method 5: Elongation of conjugated dyes by Knoevenagel condensation Elongation of the conjugates with mono- and bis-styryl BODIPY was achieved via Knoevenagel condensation, as shown in Scheme 5. [ka] Scheme 5: General synthesis of fused 8-thioether BODIPy dyes

[0462] α,α'-Dimethyl 8-thioester BODIPY (BDP-002) (1 equiv.) was mixed with aryl aldehyde (10 equiv.), anhydrous toluene, and 4 Å molecular sieves. Acetic acid (0.1 equiv.) and piperidine (0.1 equiv.) were added and the solution was refluxed. Once the starting material was consumed, the reaction was filtered, concentrated, and purified by flash chromatography to give the mono-styryl and bis-styryl 8-thioether BODIPY dyes.

[0463] Exemplary BODIPY dyes made by this method are shown in FIG.

[0464] Method 6: Sulfonation of 8-thioether BODIPY dyes by electrophilic aromatic substitution As shown in Scheme 6, electrophilic aromatic substitution of the 8-thioether BODIPY was carried out to introduce the β-sulfonate. [ka] Scheme 6: General synthesis of fused 8-thioether BODIPy dyes

[0465] Under inert conditions, α,α'-dimethyl 8-thioester BODIPY (BDP-002) (1 eq.) was added to anhydrous DCM and cooled to -40 C. Chlorosulfonic acid (3.7 eq.) in anhydrous DCM was added dropwise to the reaction and stirred at -40 C for 20 min. The reaction was then warmed to -15 C for 30 min and then warmed to room temperature. The solution was stirred at room temperature for an additional hour. The precipitate that formed was triturated several times with DCM. The solid was dried under reduced pressure to give β-β'-sulfonated 8-thioether BODIPY.

[0466] Exemplary BODIPY dyes made by this method are shown in FIG.

[0467] Method 7: Pyrrole biaryl synthesis via Suzuki coupling Suzuki coupling (Scheme 7) can be used to generate the bis-biaryl 8-thioether BODIPY, followed by the formation of a biarylpyrrole that can then be used as a starting material in Method 1 above. [ka] Scheme 7: General synthesis of pyrrole biaryls

[0468] To a mixture of aryl halide (1.1 eq), sodium carbonate (2 eq) and palladium tetrakistriphenylphosphine (0.2 eq) in water / THF was added Bo-pyrrole-2-boronic acid (1 eq). The solution was degassed with nitrogen and heated to 70 C until complete conversion was observed by TLC. The reaction was diluted with water and extracted with DCM. The organic layer was dried, filtered, concentrated and purified by flash chromatography.

[0469] The Boc-biaryl (1 eq.) was then dissolved in methanol, sodium methoxide (10 eq.) was added and heated to 50 C. The reaction was monitored by TLC. Upon complete formation of the product, the reaction was quenched with aqueous saturated ammonium chloride and washed with DCM. The organic layer was dried, filtered, concentrated and purified by flash chromatography to yield the biaryl which could be used as the starting material in Method 1.

[0470] The biaryls produced by this method are used as starting materials in Method 1, and exemplary BODIPY dyes are shown in FIG.

[0471] Method 8: Synthesis of unsymmetrical biaryl dyes by photoactivation [ka] Scheme 8: General synthesis of unsymmetrical pyrrole biaryls To generate the unsymmetrical bis-biaryl 8-thioether BODIPY, unsubstituted BODIPY (BDP-001) (1 equiv.) and an aryl diazonium tetrafluoroborate salt (1–2 equiv.) were mixed in anhydrous acetone and sparged with nitrogen gas for 15 min (Scheme 8). The solution was sealed and exposed to a 370 nm LED until complete conversion to the mono-biaryl. The mono-biaryl was purified by flash chromatography (30–50% toluene / hexane). The mono-biaryl was then mixed with a different aryl diazonium tetrafluoroborate salt (1–2 equiv.) in anhydrous acetone. The solution was sealed and sparged with nitrogen gas for 15 min and exposed to a 370 nm LED until complete conversion to the unsymmetrical biaryl 8-thioether BODIPY. The bis-biaryl was purified by flash chromatography (20–50% DCM / hexane).

[0472] Method 9: Synthesis of unsymmetrical biaryl-styryl dyes by photoactivation [ka] Scheme 9: General synthesis of unsymmetrical biaryl-styryl BODIPY dyes To generate the unsymmetrical biaryl-styryl 8-thioether BODIPY, for example, the unsymmetrical α-methyl 8-thioester BODIPY is first synthesized by Method 1 using equal amounts of pyrrole and 2-methylpyrrole for thioketone formation. The mono-biaryl is then synthesized by Method 8. Following this, the unsymmetrical biaryl-styryl is formed using the Knoevenagel condensation of Method 5.

[0473] Example 2 Conjugation of BODIPY dyes to amino acids Material / Equipment: - Make 10 mM stock solutions of the 20 natural amino acids in DMSO and store in a -20°C freezer. - Make a stock solution of the dye in the range of 10-100 mM in anhydrous DMSO and store in a freezer at -20° C. Dilute this solution to 111 μM for use in the testing process. - Cellvis brand black 384-well glass bottom black plate (part number: P384-1.5HN) with #1.5 high performance cover glass (0.17±0.005mm) (other 384-well plates will work as well, the above plate is what we currently have on hand). - Molecular Devices SpectraMax Gemini EM Microplate Reader - Bioshaker requires part number or model.

[0474] Plate Pipetting Protocol: 1. A 384-well plate was opened, lid removed, and oriented A1 to the top left. 2. Lights in this section of the laboratory were dimmed to the minimum level necessary to safely complete the following steps (this is to minimize photobleaching from any overhead lights). 3. Remove the dye and amino acid solutions and allow to thaw at room temperature. 4. Next, transfer the required amount of amino acid solution (6 μl x number of wells to be filled, i.e. 96 μl is needed for 16 wells) into a multichannel pipette reservoir and deposit 5 μl of this solution into the appropriate row of 16 wells. This process is then repeated for the remaining 19 amino acids in a new multichannel pipette reservoir. 5. In two of the remaining rows of 16 wells, pipette 5 μl of DMSO as a control into one row and 5 μl of water as a control into the other row. The remaining two rows can be used for any other controls of interest, but are left empty for these tests. 6. Next, gently tap the plate to ensure that all of the solution is at the bottom of each well and check the wells to ensure that none are empty. 7. After this, make an appropriate amount of a 111 μM solution of the dye to be tested in a falcon tube from the dye stock solution and anhydrous DMSO (the amount of solution made is approximately 50 μl x the number of wells to be filled). 8. This 111 μM solution is then placed into a multichannel pipette reservoir and 45 μl is added to the appropriate wells for a total volume of 50 μl. There is still a 10-fold excess of amino acid molecules compared to dye molecules. 9. The plate is then taken, wrapped in aluminum foil and placed in a bioshaker at 20° C. and 1500 RPM. 10. The plate was stirred under these conditions for 2 hours and then harvested and measured (kinetic studies showed that under these conditions the reactions were >95% complete at the 2 hour mark except for proline, which reacts at a slower rate due to steric factors).

[0475] The results are shown in Figures 7-22.

[0476] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided merely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or indication of the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

Claims

1. 1. A method for identifying an amino acid, said method comprising: (a) conjugating a probe to an amino acid, said probe comprising a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to said amino acid; (b) detecting one or more fluorescent spectral properties of the probe conjugated to the amino acid; and (c) identifying the amino acid by comparing the fluorescence spectral signature of the probe with a plurality of reference fluorescence spectral signatures, each reference fluorescence spectral signature being representative of the probe conjugated to a different amino acid. A method comprising:

2. 1. A multiplex method for identifying a plurality of amino acids, said method comprising: (a) conjugating a plurality of probes to amino, carboxyl, hydroxyl or thiol groups of a plurality of amino acids, wherein each probe in the plurality of probes comprises a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to a different amino acid; (b) detecting one or more fluorescent spectral properties of each probe conjugated to said plurality of said amino acids; and (c) identifying the amino acid by comparing the plurality of fluorescent spectral characteristics of the probe with a plurality of reference fluorescent spectral characteristics, each reference fluorescent spectral characteristic being representative of the probe conjugated to a different amino acid. Multiplex methods including: (i) the probe is covalently conjugated to the amino acid, and optionally The probe is conjugated to an amino group, a carboxyl group, a hydroxyl group or a thiol group of the amino acid, preferably the probe is conjugated to the amino group of the amino acid; or the probe is conjugated to a carboxyl group of the amino acid; and / or (ii) the amino acid is present in a sample, optionally the sample is a biological sample, optionally the biological sample is a biological fluid, tissue, organ or cell; and / or (iii) the amino acid is present in a polypeptide, and optionally:

3. The method of claim 1 or 2, wherein the amino acid is at the N-terminus of the polypeptide or at the C-terminus of the polypeptide.

4. 1. A method for determining the sequence of a polypeptide, comprising: (a) conjugating probes to terminal amino acids of the polypeptide, the probes comprising dipyrromethane-BF2 derivatives that exhibit different fluorescence spectral properties when conjugated to different terminal amino acids; (b) detecting one or more fluorescence spectral properties of the probe conjugated to the terminal amino acid of the polypeptide by detecting the fluorescence of the probe bound to the terminal amino acid; (c) identifying the corresponding terminal amino acid of the polypeptide by comparing the fluorescence spectral signature of the probe with a plurality of reference fluorescence spectral signatures, each reference fluorescence spectral signature being representative of the probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of the polypeptide; and (e) repeating steps (a) through (d) one or more times sequentially to determine the sequence of at least a portion of said polypeptide. A method comprising:

5. 1. A multiplex method for determining the sequence of a plurality of polypeptides, comprising: (a) conjugating a plurality of probes to the terminal amino acids of each of said plurality of polypeptides, each probe in said plurality of probes comprises a dipyrromethane-BF2 derivative that exhibits different fluorescence spectral properties when conjugated to a different terminal amino acid; (b) detecting one or more fluorescent spectral properties of each probe conjugated to the terminal amino acid of each of the plurality of polypeptides; (c) identifying the corresponding terminal amino acid of each of the plurality of said polypeptides by comparing the fluorescence spectral signature of the probe with a plurality of reference fluorescence spectral signatures, each reference fluorescence spectral signature being representative of the probe conjugated to a different terminal amino acid; (d) cleaving the terminal amino acid of each of the plurality of polypeptides; and (e) sequentially repeating steps (a) through (d) one or more times to determine the sequence of at least a portion of each of said plurality of polypeptides. Multiplex methods including: (i) the probes in the plurality of probes are the same; or The method of claim 2 or 5, wherein at least two probes in the plurality of probes are different. (i) the terminal amino acid is present at the N-terminus of the polypeptide; or the terminal amino acid is at the C-terminus of the polypeptide; and / or (ii) the step of cleaving the terminal amino acid comprises enzymatic or chemical cleavage; and / or (iii) the polypeptide is present in a sample, and optionally 6. The method of claim 5, wherein the method comprises detecting one or more spectral characteristics of each probe conjugated to the terminal amino acid of each of the plurality of polypeptides at spatially resolved positions in the sample comprising the plurality of polypeptides, optionally wherein the sample is a biological sample, and optionally wherein the biological sample is a biological fluid, tissue, organ, or cell. (i) detecting one or more spectral properties of the conjugated probe, (a) super-resolution microscopy, optionally including: The super-resolution microscopy method comprises stochastic reconstruction optical microscopy (STORM); (b) detecting the fluorescence of the conjugated probe; (c) detecting fluorescence emission intensity, polarity / anisotropy or lifetime, optionally including: the detecting of the luminescence intensity, polarity / anisotropy or lifetime is at a single wavelength; or the detecting of the luminescence intensity, polarity / anisotropy or lifetime is at multiple wavelengths; and / or (ii) the dipyrromethane-BF2 derivative contains a labile or reactive group; and / or (iii) the dipyrromethane-BF2 derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative, and optionally The probe is conjugated via the 8-position (meso-position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative, preferably The method according to any one of claims 1 to 2 and 4 to 5, wherein the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative contains an unstable group or a reactive group at the 8-position (meso-position).

9. The probe has the formula (I): 【Chemistry 24】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ is a labile group or a leaving group; Any of alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio can be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido. The method according to any one of claims 1 to 2 and 4 to 5,

10. (i) R ８ is a labile group or a leaving group, and / or (ii) R ８ is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester and diazonium, and optionally R ８ But, -SR ８Ｓ or halogen, R ８Ｓ is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl, and optionally R ８Ｓ is optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ alkenyl, optionally substituted aryl or optionally substituted heteroaryl, preferably R ８Ｓ is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl, and optionally R ８Ｓ is methyl, and / or (iii) R １ , R ２ , R ３ , R ５ , R ６ and R ７ is not H, and / or (iv) R １ , R ２ , R ３ , R ５ , R ６ and R ７ is an electron withdrawing group (EWG); and / or (v) R １ , R ２ , R ３ , R ５ , R ６ and R ７ at least one of which is an electron donating group (EDG); and / or (vi) R １ , R ２ and R ３ at least two of are the same, or R １ , R ２ and R ３ at least two of are different, and / or (vii) (a) R １ and R ７ are different, or R ２ and R ６ are different, or R ３ and R ５ are different, or (b) R １ and R ７ are the same, and R ２ and R ６ are the same, and R ３ and R ５ are the same, and / or (viii) R １ is H or optionally substituted C １ ~C ６ alkyl, and optionally R １ is H or methyl, and / or (ix) R ２ is H, halogen, and optionally, R ２ is H, Br, Cl, —C(O)H or —SO ３ H and / or (x)R ３ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ３ is H, Br, Cl, methyl, -CH=CH-R ９Ａ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl), and R ９Ａ is optionally substituted aryl or heteroaryl, preferably R ３ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, more preferably R ３ is H, methyl or —CH═CH—R ９Ａ and R ９Ａ is optionally substituted phenyl, and optionally R ９Ａ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ９Ａ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xi) R ５ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ５ is H, Br, Cl, methyl, -CH=CH-R ９Ｂ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl), and R ９Ｂ is optionally substituted aryl or heteroaryl, preferably R ５ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, more preferably R ５ is H, methyl or —CH═CH—R ９Ｂ and R ９Ｂ is optionally substituted phenyl, and optionally R ９Ｂ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ９Ｂ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xii) R ６ is H, halogen, optionally substituted C １ ~C ６ Alkyl, —C(O)H or —SO ３ H, and if necessary, R ６ is H, Br, Cl, —C(O)H or —SO ３ H and / or (xiii) R ７ is H or optionally substituted C １ ~C ６ alkyl, and optionally R ７ is H or methyl, and / or (xiv) R １ , R ２ , R ３ , R ５ , R ６ and R ７ The method of claim 9 , wherein is H.

11. Compounds of formula (I): 【Chemistry 25】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group; １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ and R ８ is a labile group or a leaving group; Any of alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio can be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; however, (i) R ８Ｓ If is methyl, a.R １ , R ２ , R ３ , R ５ , R ６ and R ７ is not H, b.R １ , R ２ , R ６ and R ７ is not H, but R ３ and R ５ is not methyl, ethyl or p-nitrophenyl, c.R １ , R ３ , R ５ and R ７ is not methyl, but R ２ and R ６ is not H, d.R １ and R ７ is not H, but R ２ , R ３ , R ５ and R ６ is neither Cl nor Br, e.R. １ , R ３ , R ５ , R ６ and R ７ is not H, but R ２ is not —C(O)H, f.R １ is not p-nitrophenyl, R ２ , R ６ and R ７ is not H, but R ３ and R ５ is not methyl, and g.R １ , R ２ , R ５ , R ６ and R ７ is not H, but R ３ is not phenyl, p-nitrophenyl, p-iodophenyl, p-carboxylic phenyl, or p-methoxyphenyl, (ii) R ８Ｓ is allyl, ethyl, propyl, butyl, t-butyl, n-dodecyl, phenyl, 2,6-dimethylphenyl, p-methylphenyl, p-methoxyphenyl, p-nitrophenyl, or benzyl (—CH ２ PH), then R １ , R ２ , R ３ , R ５ , R ６ and R ７ is not H, and (iii) R ８ When is Cl, a.R １ , R ２ , R ３ , R ５ , R ６ and R ７ is not H, b.R １ , R ３ , R ５ , R ６ and R ７ is not H, but R ２ is not Cl, c.R １ , R ３ , R ５ and R ７ is not H, but R ２ and R ５ is not Cl, d.R １ , R ５ and R ７ is not H, but R ２ , R ３ and R ５ is not Cl, and e.R. １ , R ２ , R ３ , R ５ , R ６ and R ７ is not Cl).

12. (i) R ８ is a labile group or a leaving group, and / or (ii) R ８ is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester and diazonium, and optionally R ８ But, -SR ８Ｓ or halogen, R ８Ｓ is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl, preferably R ８Ｓ is optionally substituted C １ ~C ６ Alkyl, optionally substituted C ２ ~C ６ alkenyl, optionally substituted aryl or optionally substituted heteroaryl, more optionally R ８Ｓ is methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl or 4-methoxybenzyl, and most preferably R ８Ｓ is methyl, and / or (iii) R １ , R ２ , R ３ , R ５ , R ６ and R ７ is not H, and / or (iv) R １ , R ２ , R ３ , R ５ , R ６ and R ７ is an electron withdrawing group (EWG); and / or (v) R １ , R ２ , R ３ , R ５ , R ６ and R ７ at least one of which is an electron donating group (EDG); and / or (vi) R １ , R ２ and R ３ at least two of are the same, and / or (vii) R １ , R ２ and R ３ at least two of are different, and / or (viii) R １ and R ７ are different, or R ２ and R ６ are different, or R ３ and R ５ are different and / or (ix) R １ and R ７ are the same, and R ２ and R ６ are the same, and R ３ and R ５ are the same, and / or (x)R １ is H or optionally substituted C １ ~C ６ alkyl, and optionally R １ is H or methyl, and / or (xi) R ２ is H, halogen, optionally substituted C １ ~C ６ Alkyl, —C(O)H or —SO ３ H, and if necessary, R ２ is H, Br, Cl, —C(O)H or —SO ３ H and / or (xii) R ３ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ３ is H, Br, Cl, methyl, -CH=CH-R ９ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl), and R ９ is optionally substituted aryl or heteroaryl, more optionally R ３ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, and even more optionally R ３ is H, methyl or —CH═CH—R ９Ａ and R ９ is optionally substituted phenyl, and optionally R ９Ａ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ９Ａ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xiii) R ５ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ５ is H, Br, Cl, methyl, -CH=CH-R ９Ｂ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl), and R ９Ｂ is optionally substituted aryl or heteroaryl, preferably R ５ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, more preferably R ５ is H, methyl or —CH═CH—R ９Ｂ and R ９Ｂ is optionally substituted phenyl, and optionally R ９Ｂ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ９Ｂ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xiv) R ６ is H, halogen, optionally substituted C １ ~C ６ Alkyl, —C(O)H or —SO ３ H, and if necessary, R ６ is H, Br, Cl, —C(O)H or —SO ３ H and / or (xv) R ７ is H or optionally substituted C １ ~C ６ The compound of claim 11 , wherein the compound is alkyl.

13. Compound of formula (II): 【Chemistry 26】 (In the formula, R ２１ , R ２２ , R ２３ , R ２４ , R ２５ , R ２６ , R ２７ and R ２８ are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and amino acids; ２１ , R ２２ , R ２３ , R ２４ , R ２５ , R ２６ , R ２７ and R ２８ is an amino acid; Any of alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio can be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents independently selected from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido.

14. (i) R ２８ is an amino acid, and / or (ii) R ２１ , R ２２ , R ２３ , R ２５ , R ２６ and R ２７ is not H, and / or (iii) R ２１ , R ２２ , R ２３ , R ２５ , R ２６ and R ２７ is an electron withdrawing group (EWG); and / or (iv) R ２１ , R ２２ , R ２３ , R ２５ , R ２６ and R ２７ at least one of which is an electron donating group (EDG); and / or (v) R ２１ , R ２２ and R ２３ at least two of are the same, and / or (vi) R ２１ , R ２２ and R ２３ at least two of are different, and / or (vii) R ２１ and R ２７ are different, or R ２２ and R ２６ are different, or R ２３ and R ２５ are different and / or (viii) R ２１ and R ２７ are the same, and R ２２ and R ２６ are the same, and R ２３ and R ２５ are the same, and / or (ix) R ２１ is H or optionally substituted C １ ~C ６ alkyl, and optionally R ２１ is H or methyl, and / or (x)R ２２ is H, halogen, optionally substituted C １ ~C ６ Alkyl, —C(O)H or —SO ３ H, and if necessary, R ２２ is H, Br, Cl, —C(O)H or —SO ３ H and / or (xi) R ２３ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ２３ is H, Br, Cl, methyl, -CH=CH-R ２９Ａ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, C １ ~C ６ haloalkyl), and R ２９Ａ is optionally substituted aryl or heteroaryl, preferably R ２３ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, more preferably R ２３ is H, methyl or —CH═CH—R ２９Ａ and R ２９Ａ is optionally substituted phenyl, and optionally R ２９Ａ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ２９Ａ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xii) R ２５ is H, halogen, optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkenyl, optionally substituted phenyl or optionally substituted naphthalene, and optionally R ２５ is H, Br, Cl, methyl, -CH=CH-R ２９Ｂ , phenyl, bisphenyl (4-phenylphenyl), naphthalene or phenyl (nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl), and R ２９Ｂ is optionally substituted aryl or heteroaryl, preferably R ２５ is H, methyl, phenyl, 4-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 2,4,6-trimethylphenyl, bisphenyl or naphthalene, more preferably R ２５ is H, methyl or —CH═CH—R ２９Ｂ and R ２９Ｂ is optionally substituted phenyl, and optionally R ２９Ｂ is phenyl, or nitro, cyano, halogen, amino, alkylamino, dialkylamino, carboxyl, C １ ~C ６ Alkyl, and C １ ~C ６ haloalkyl, preferably R ２９Ｂ is 4-fluorophenyl, 4-trifluoromethylphenyl or 4-cyanophenyl, and / or (xiii) R ２６ is H, halogen, optionally substituted C １ ~C ６ Alkyl, —C(O)H or —SO ３ H, and if necessary, R ２６ is H, Br, Cl, —C(O)H or —SO ３ H and / or (xiv) R ２７ is H or optionally substituted C １ ~C ６ alkyl, and optionally R ７ is H or methyl.

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