Fluorescent probes and uses thereof

Abstract

Disclosed herein are methods for identifying the terminal and the penultimate amino acids of a polypeptide in a single read, sequencing polypeptides with error correction, identifying modified (such as post translational modification) and unnatural / non-natural amino acids, and for barcoding target analyte molecules for detection and identification.

Description

Atty. Dkt. No. 701586-000165 WO PTFLUORESCENT PROBES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application 63 / 759,657 filed on February 18, 2025, the content of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The technology described herein relates generally to methods for identifying the terminal and the penultimate amino acids of a polypeptide in a single read, sequencing polypeptides with error correction, identifying modified (such as post translational modification) and unnatural / non-natural amino acids, and for barcoding target analyte molecules for detection and identification.BACKGROUND

[0003] There is need in the art for single molecule fluoro-sequencing of peptides and proteins and identification of post translational modification. The present disclosure addresses these needs.SUMMARY

[0004] In one aspect, provided herein is a method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a polypeptide, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide, wherein the probe exhibits different fluorescent spectral properties when conjugated to different pairs of terminal amino acid and penultimate amino acid; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid; and (c) identifying the terminal amino acid and the penultimate amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different terminal and penultimate amino acid pair. In some embodiments, the method further comprises a step (d) of cleaving the terminal amino acid, and repeating steps (a)-(c) one or more times.

[0005] Thus, in another aspect provided herein is a method for sequencing a polypeptide, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide, wherein the probe exhibits different fluorescent4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTspectral properties when conjugated to a first pair of terminal amino acid and penultimate amino acid, and when conjugated to a second pair of terminal amino acid and penultimate amino acid, wherein at least one amino acid in the first pair is different from at least one amino acid in the second pair; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid to determine the identity of the terminal amino acid and the penultimate amino acid; (c) cleaving the terminal amino acid of the polypeptide; and (d) sequentially repeating steps (a) to (c) one or more times to determine the sequence of at least a portion of the polypeptide.

[0006] In still another aspect, provided herein is a method for identifying a modified or nonnatural amino acid, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a modified or non-natural amino acid, wherein the probe exhibits different fluorescent spectral properties when conjugated to different modified or nonnatural amino acids; (b) detecting one or more fluorescent spectral properties of the probe conjugated to the modified or non-natural amino acid; and (c) identifying the modified or nonnatural amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different modified or non-natural amino acid.

[0007] In yet still another aspect, provided herein is a method for barcoding a plurality of target analytes, the method comprising: ligating a barcode peptide to target analytes in a plurality of target analytes via a terminal amido acid in the barcode peptide, wherein the terminal amino acid is a modified or non-natural amino acid, and wherein a barcode peptide conjugated to a first analyte in the plurality is distinguishable from a barcode peptide conjugated to a second analyte in the plurality, and wherein the first and second analyte are different.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 : Post-translationally modified AADCs. Fluorescence lifetime (ns) versus peak wavelength (nm) for BDP088, BDP103, BDP045, and BDP104 Amino Acids and Post-Translational Modifications (PTMs) defined in legend.

[0009] FIG. 2 : Post-translationally modified AADCs. Highlighted effect of phosphorylation. Left: Average lifetime (ns) versus emission peak (nm) for: Monomer S, PTM none; Monomer S, PTM p; Monomer T, PTM none; Monomer T, PTM p; Monomer Y, PTM none; Monomer Y, PTM p. Right: intensity versus wavelength (nm) and time (ns) plots for each4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTmonomer and PTM. AADC measurements were taken in 1 M Tris buffer at 1 pM, excitation wavelength 440 nm.

[0010] FIG. 3: Peptide-dye conjugate combinatorial library. Left: Fluorescence lifetime (ns) versus emission peak (nm) for the terminal amino acid (X1). Right: Fluorescence lifetime (ns) versus emission peak (nm) for penultimate amino acid (X2). Dye-peptide conjugate measurements were taken in 1 M Tris buffer at 1 pM.

[0011] FIG. 4: Peptide-dye conjugate combinatorial library: UMAP Dimensional Reduction. Dye-peptide conjugate measurements were taken in 1 M Tris buffer at 1 pM.

[0012] FIG. 5: Schematic of one embodiment of an overall workflow of the technology described herein.

[0013] FIG. 6: Description of utilization of unnatural amino acids for UAA barcode and sortase tag.

[0014] FIG. 7 : (Left) Structure of dye (4.1) and (Right) two-dimensional plot of fluorescent emission versus lifetime for amino acid monomers reacted with probe (4.2) with corresponding histograms. All measurements were taken at 1 pM in 1 M Tris buffer.

[0015] FIG. 8: Two-dimensional plot of fluorescent emission versus lifetime for a) ultimate amino acids and b) penultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4.1) with corresponding histograms. All measurements were taken at 1 pM in 1 M Tris buffer.

[0016] FIGs. 9A-9B: Two-dimensional plot of fluorescent emission versus photon counts for FIG.9A: ultimate amino acids and FIG.9B: penultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4.1) with corresponding histograms. All measurements were taken at 1 pM in 1 M Tris buffer.

[0017] FIGs. 10A-10B: Two-dimensional plot of fluorescent lifetime versus photon counts for FIG. 10A: ultimate amino acids and FIG. 10B: penultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4.1) with corresponding histograms. All measurements were taken at 1 pM in 1 M Tris buffer.

[0018] FIGs. 11A-11B: Swarm charts of fluorescent emission of each peptide of FIG.11 A: ultimate amino acids and FIG. 11B: penultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4-1).

[0019] FIGs. 12A-12B: Swarm charts of fluorescent lifetime of each peptide of FIG. 12A: ultimate amino acids and FIG. 12B: penultimate amino acids of 400 dipeptide AA4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTcombinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4-1).

[0020] FIG. 13: Supervised UMAP dimensionality reduction for ultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4.1) with corresponding histograms. All measurements were taken at 1 mM in 1 M Tris buffer.

[0021] FIG. 14: Supervised UMAP dimensionality reduction for penultimate amino acids of 400 dipeptide AA combinations of peptide H2N-X1X2GRAHEARG-OH (SEQ ID NO: 1) reacted with probe (4.1) with corresponding histograms. All measurements were taken at 1 mM in 1 M Tris buffer.

[0022] FIG. 15: Emission spectra of example peptide-dye conjugates with probe (4.1) compared to library peptide-dye conjugate with probe (4.1) with the same ultimate and penultimate amino acids. All measurements were taken at 1 pM in 1 M Tris.

[0023] FIG. 16: Emission spectra of example peptide-dye conjugates with probe (4.1) compared to library peptide-dye conjugate with probe (4.1) with the same ultimate and penultimate amino acids. All measurements were taken at 1 pM in 8 M Tris.

[0024] FIG. 17: Change in Pearson correlation between peptide fragment-dye conjugate and its corresponding reference peptide conjugated to probe (4.1) at increasing concentration of urea.

[0025] FIG. 18: Change in Pearson correlation between peptide fragment-dye conjugate and its corresponding reference peptide conjugated to probe (4.1) at increasing concentration of guanidinium chloride.

[0026] FIG. 19: Cartoon representation of peptides with a) truncated iteratively removed N-terminal amino acids, and b) circularly permutated N-terminal amino acids.

[0027] FIG. 20: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for circularly permutated KRAS peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0028] FIG. 21: Photon counts for dye-peptide conjugates of circularly permutated KRAS fragments reacted with probe (4.1). All measurements were taken at 1 pM in either 1 M Tris or 8 M Urea buffer.

[0029] FIG. 22 : Photon counts for dye-peptide conjugates of peptide fragments reacted with probe (4.1). All measurements were taken at 1 pM.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0030] FIG. 23: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for circularly permutated Shc-SH2 peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0031] FIG. 24: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for circularly permutated Src-SH2 peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0032] FIG. 25: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for circularly permutated Src-SH2 peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0033] FIG. 26: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for circularly permutated Angiotensin 1 peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0034] FIG. 27: Two-dimensional plot of fluorescent emission max (nm) versus lifetime (ns) for iteratively removed KRAS peptide fragments reacted with probe (4.1) with connecting line between step-wise shifts in sequence. All measurements were taken at 1 pM in 8 M Urea buffer.

[0035] FIG. 28: Normalized emission spectra of KRAS peptide fragments of both the circularly permutated and truncated series plotted according to matching last two N-terminal residues. All dye conjugates were excited at 440 nm at 1 pM in 8 M Urea buffer.

[0036] FIG. 29: Pearson coefficient between KRAS peptide fragments of both the circularly permutated and truncated series plotted according to matching last two N-terminal residues.

[0037] FIG. 30: Swarm chart of photon counts for all dye-peptide conjugates in 1 M Tris buffer (left) and 8 M Urea buffer (right). All measurements were taken at 1 pM.

[0038] FIG. 31: Scheme of reversible reaction of N-terminus with probe (4.1).

[0039] FIG. 32: Proposed mechanism of photoinduced scission of N-terminal amine from bound BODIPY dye probe.

[0040] FIG. 33 A: Absorbance curves for dye (4.1) reacted with methylamine at 100 pM in lx PBS irradiated with 365 nm LEDs over two hours.

[0041] FIG. 33B: Absorbance curves for dye (4.1) reacted with methylamine at 100 pM in degassed lx PBS with 1 mM Trolox irradiated with 365 nm LEDs over two hours.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0042] FIG. 33C: Decay curves for change in absorbance for dye (4.1) reacted with methylamine at 100 pM.

[0043] FIG. 34: Graphic representation of experimental design in which DBCO fimctionalized passivated glass surface is reacted with azido-peptide followed by dye conjugation.

[0044] FIG. 35: Two-dimensional plot of interpolated fluorescent emission max (nm) versus lifetime (ns) for circularly permutated KRAS peptide fragments reacted with probe (4.1) bound to glass surface with connecting line between step-wise shifts in sequence and 95% confidence intervals circling each point. All measurements were taken in lx PBS with 1 mM Trolox.

[0045] FIG. 36A: Structures of degrader probe (4.12) and AlexaFluor488-NHS (4.13).

[0046] FIG. 36B: Graphic depiction of on-binding kinetics assay where degrader attached to N-termini accumulates over time leading to a loss in fluorescence when then reacted with (4.13).

[0047] FIG. 36C: Graphic depiction of off-binding kinetics creating new N-termini following photoactivated N-degradation leading to greater abundance of new N-termini for (4.13) to react with causing greater fluorescent signal.

[0048] FIG. 37A: Change in fluorescent signal from surface bound objects following reaction of surface bound peptide SAKTRSYGIPFIETK(N3) (SEQ ID NO: 29) with probe (4.12) at 10 pM in pH 9.2 bicarbonate buffer, then reacted with (4.13) in lx PBS at 10 pM for 1 h. All measurements were taken in lx PBS with 1 mM Trolox.

[0049] FIG. 37B: Change in fluorescent signal from surface bound objects following reaction of surface bound peptide SAKTRSYGIPFIETK(N3) (SEQ ID NO: 29) pre-reacted with probe (4.12) then irradiated with 365 nm LEDs for 5 m in pH 10 buffer. Following this time, peptides were then reacted with (4.13) in lx PBS at 10 pM for 1 h. All measurements were taken in lx PBS with 1 mM Trolox.

[0050] FIG. 38A: Interpolated fluorescent emission maximum (nm) of objects following a single N-degradation (right, +) and no degradation (left, -) once reacted with probe (4.1).Series is paired so the next circular permutation is to the right of the previous permutation. All measurements were taken in lx PBS with 1 mM Trolox.

[0051] FIG. 38B: Average fluorescent lifetime (ns) of objects following a single N-degradation (right, +) and no degradation (left, -) once reacted with probe (4.1). Series is paired so the next circular permutation is to the right of the previous permutation. All measurements were taken in lx PBS with 1 mM Trolox.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0052] FIG. 38C: Paired interpolated fluorescent emission maxima (nm) of non-degraded and degraded peptide (same color) matched with their subsequent circular permutation. All measurements were taken in lx PBS with 1 mM Trolox.

[0053] FIG. 38D: Paired average fluorescent lifetime (ns) of non-degraded and degraded peptide (same color) matched with their subsequent circular permutation. All measurements were taken in lx PBS with 1 mM Trolox.

[0054] FIG. 39: (Top) Graphic representation of reading frame from each optical read following an N-degradation cycle and dye conjugation. (Bottom)Two-dimensional plot of fluorescent emission max (nm) versus fluorescent lifetime (ns) for circularly permutated KRAS peptide fragments reacted with (4.1) with an excitation at 440 nm. All measurements were taken in 1 M Tris buffer at 1 pM.

[0055] FIG. 40: Site selective photocatalyzed C-terminal decarboxylation and Michael addition followed by surface immobilization via copper catalyzed click reaction developed by MacMillan and Anslyn.

[0056] FIG. 41: Graphic representation of cellular digestion with clostripain followed by conjugation of C-terminal arginines with 4-azidophenyl glyoxal (4.18).

[0057] FIG. 42: Proposed synthetic route to make dipodal siloxy esters for glass passivation and functionalization.

[0058] FIG. 43: Graphical representation of intracellular fluoro-sequencing using expansion microscopy.DETAILED DESCRIPTION

[0059] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Simultaneously identifying the terminal and penultimate amino acids in a polypeptide4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0060] In one aspect, provided herein is a method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a polypeptide. The method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid; and (c) identifying the terminal amino acid and the penultimate amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties. Without wishing to be bound by a theory, the probe comprising the dipyrromethane-BFi derivative exhibits different fluorescent spectral properties when conjugated to different pairs of terminal amino acid and penultimate amino acid. Stated in another way, the probe exhibits at least one fluorescent spectral property when conjugated to a first pair of terminal and penultimate amino acids that is different from the same fluorescent spectral property when conjugated to a second pair of terminal and penultimate amino acids, when at least one amino acid is the first and second pair is different. It is noted that the reference fluorescent spectral property is representative of the probe conjugated to a different terminal and penultimate amino acid pair.

[0061] It is noted that the terminal and the penultimate amino acids can be that N-terminal or the C-terminal of the polypeptide. In some embodiments, the terminal and the penultimate amino acids are the N-terminal of the polypeptide. In some other embodiments, the terminal and the penultimate amino acids are the C-terminal of the polypeptide.

[0062] In some embodiments of any of the aspects, the method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a polypeptide can be used to detect / identify the terminal and penultimate amino acids of more than one polypeptide simultaneously, e.g., in a multiplex format. For example, the method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid of a plurality of polypeptides comprises conjugating a probe comprising the dipyrromethane-BFi derivative to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other, detecting one or more spectral properties of the probes conjugated to the terminal amino acids; and identifying the terminal and the penultimate amino acids by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties.

[0063] Thus, in another aspect, provided herein is a method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a plurality of polypeptides. The method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a plurality of polypeptides; (b) detecting one or more spectral4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTproperties of the probes conjugated to the terminal amino acid; and (c) identifying the terminal and the penultimate amino acids of the plurality of polypeptides by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties.

[0064] It is noted that probes conjugated to the terminal amino acid of the polypeptides in the plurality can be the same, i.e., the probes conjugated to the terminal amino acids of different polypeptide are identical. Stated in another way, a single probe is used to identify / detect the terminal and penultimate amino acids of two or more different polypeptides simultaneously, i.e., in a multiplex format.

[0065] In some embodiments, probes conjugated to the terminal amino acid of the polypeptides in the plurality are different, i.e., a probe conjugated with a first polypeptide is different from a probe conjugated with a second polypeptide, and wherein the first and second polypeptides are not identical. Stated in another way, a plurality of probes are used to identify / detect the terminal and penultimate amino acids of two or more different polypeptides simultaneously, i.e., in a multiplex format, and wherein at least two probes in the plurality are different.Sequencing of polypeptides

[0066] The method described herein for detecting / identifying a terminal or a penultimate amino acid of polypeptide can be used for sequencing, e.g., at least a part of a polypeptide. Generally, the method comprises, after the step of identifying the terminal amino acid and the penultimate amino acid, cleaving the terminal amino acid and repeating the steps of conjugating the probe (to the new terminal amino acid, which was previously the penultimate amino acid), detecting one or more fluorescent spectral properties of the probe conjugated to the terminal amino acid, and identifying the terminal amino acid and the penultimate amino acid. The steps of cleaving the terminal amino acid, conjugating the probe to the new terminal amino acid, detecting one or more fluorescent spectral properties of the probe conjugated to the terminal amino acid, and identifying the terminal amino acid and the penultimate amino acid can be repeated as needed to sequence, at least a part of, the polypeptide.

[0067] Accordingly, in another aspect provided herein is a method for sequencing, e.g., at least a part of a polypeptide. Generally, the method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid; (c) identifying the terminal amino acid and the penultimate amino acid by comparing the4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTfluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties; (d) cleaving the terminal amino acid; and (e) repeating steps (a)-(d) one or more times to determine the sequence of at least a portion of the polypeptide. As described herein, the probe comprising the dipyrromethane-BFi derivative exhibits different fluorescent spectral properties when conjugated to different pairs of terminal amino acid and penultimate amino acid, i.e., the probe exhibits at least one fluorescent spectral property when conjugated to a first pair of terminal and penultimate amino acids that is different from the same fluorescent spectral property when conjugated to a second pair of terminal and penultimate amino acids, when at least one amino acid is the first and second pair is different. Further, the reference fluorescent spectral property is representative of the probe conjugated to a different terminal and penultimate amino acid pair.

[0068] It is noted that the penultimate amino acid is identified twice, as the penultimate amino acid in a first read, and as the terminal amino acid in the second read. Without wishing to be bound by a theory, this provides error correction in identifying said amino acid and, thus, in sequencing the polypeptide.

[0069] Methods of cleaving a terminal amino acid of a polypeptide are well known in the art and available to one of skill in the art. Such method can include enzymatic cleavage or chemical cleavage. Accordingly, in some embodiments of any one of the aspects described herein, the step of cleaving the terminal amino acid comprises enzymatic cleavage. In some other embodiments of any one of the aspects described herein, the step of cleaving the terminal amino acid comprises chemical cleavage. For example, the N-terminal amino acid of a polypeptide can be cleaved using Edman, or related, chemical degradation. Alternatively, the N-terminal amino acid of a polypeptide can be cleaved enzymatically with a protease, such as an aminopeptidase. Similarly, the C-terminal amino acid of a polypeptide can be cleaved enzymatically with a protease such as a carboxypeptidase.

[0070] The method described herein for sequencing a polypeptide can be used for sequencing a plurality of polypeptides simultaneously, e.g., in a multiplex format. For example, the method for sequencing a plurality of polypeptides comprises conjugating a probe comprising the dipyrromethene-BFi derivative to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other, detecting one or more spectral properties of the probes conjugated to the terminal amino acids; and identifying the terminal and the penultimate amino acids of the plurality of polypeptides by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0071] Thus, in another aspect, provided herein is a method for sequencing a plurality of polypeptides simultaneously, e.g., in a multiplex format. The method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a plurality of polypeptides; (b) detecting one or more spectral properties of the probes conjugated to the terminal amino acids; (c) identifying the terminal and the penultimate amino acids of the plurality of polypeptides by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties; (d) cleaving the terminal amino acids; and (e) repeating steps (a)-(d) one or more times to determine the sequence of at least a portion of the polypeptides.

[0072] The probes conjugated to the terminal amino acid of the polypeptides in the plurality can be different, i.e., a probe conjugated with a first polypeptide is different from a probe conjugated with a second polypeptide, and wherein the first and second polypeptides are not identical. Stated in another way, a plurality of probes are used to identify / detect the terminal and penultimate amino acids of two or more different polypeptides simultaneously, i.e., in a multiplex format. Alternatively, the probes conjugated to the terminal amino acid of the polypeptides in the plurality can be the same, i.e., the probes conjugated to the terminal amino acids of different polypeptide are identical. Stated in another way, a single probe is used to identify / detect the terminal and penultimate amino acids of two or more different polypeptides simultaneously, i.e., in a multiplex format.

[0073] The methods described herein can be used to sequence a polypeptide in situ. For example, the method described herein can be used to sequence a polypeptide present in or on a biological sample, such as a tissue, cell, lipid membrane or intracellular organelle, or sample thereof.

[0074] In some embodiments, the terminal or the penultimate amino acid is a modified or non-natural amino acid.Detection / identification of modified or non-natural amino acids

[0075] In another aspect, provided herein is a method for identifying a modified or non-natural amino acid. The method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a modified or non-natural amino acid, wherein the probe exhibits different fluorescent spectral properties when conjugated to different modified or non-natural amino acids; (b) detecting one or more fluorescent spectral properties of the probe conjugated to the modified or non-natural amino acid; and (c) identifying the modified or non-natural amino acid by comparing the fluorescent spectral properties of the conjugated probe to4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTa plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different modified or nonnatural amino acid.

[0076] The modified or non-natural amino acid can be comprised in a polypeptide. For example, the modified or non-natural amino acid is at an N-terminal of a polypeptide. In another non-limiting example, the modified or non-natural amino acid is at a C-terminal of a polypeptide. In yet another non-limiting example, the modified or non-natural amino acid is at an internal position of a polypeptide. In some embodiments of any of the aspects, the polypeptide is immobilized on a surface.

[0077] The method described herein for identifying a modified or non-natural amino acid can be used detecting or identifying a plurality of amino acids simultaneously, e.g., in a multiplex format, where at least two amino acids in the plurality are different and at least one of the amino acids in the plurality is a modified or non-natural amino acid. For example, the method for detecting or identifying a plurality of amino acids comprises: conjugating a probe comprising the dipyrromethane-BFi derivative to a plurality of amino acids, wherein at least two amino acids in the plurality are different and at least one of the amino acids in the plurality is a modified or non-natural amino acid, detecting one or more spectral properties of the probes conjugated to the amino acids; and identifying the amino acids by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties.

[0078] Thus, in another aspect, provided herein is a method for detecting or identifying a plurality of amino acids simultaneously, e.g., in a multiplex format. The method comprises: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a plurality of amino acids, wherein at least two amino acids in the plurality are different and at least one of the amino acids in the plurality is a modified or non-natural amino acid; (b) detecting one or more spectral properties of the probes conjugated to the amino acids; and (c) identifying the plurality of amino acids by comparing the fluorescent spectral properties of the conjugated probes to a plurality of reference fluorescent spectral properties. It is noted that the probes conjugated to the amino acids in the plurality can be different, i.e., a probe conjugated with a first amino acid is different from a probe conjugated with a second amino acid, and wherein the first and second amino acids are not identical. Stated in another way, a plurality of probes are used to identify / detect two or more different amino acids simultaneously, i.e., in a multiplex format. Alternatively, the probes conjugated to the amino acids in the plurality can be the same, i.e., the probes conjugated to the different amino acids in the plurality are identical. Stated in4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTanother way, a single probe is used to identify / detect the two or more different amino acids simultaneously, i.e., in a multiplex format.

[0079] In some embodiments of any of the aspects, at least one amino acid in the plurality is a modified or non-natural amino acid and at least one amino acid in the plurality is a natural amino acid.Barcoding

[0080] In yet another aspect, provided herein is a method for barcoding a plurality of target analytes. The method for barcoding comprises: ligating a barcode peptide to target analytes in a plurality of target analytes via a terminal amido acid in the barcode peptide, wherein a barcode peptide conjugated to a first analyte in the plurality is distinguishable from a barcode peptide conjugated to a second analyte in the plurality, and wherein the first and second analyte are different. Generally, the terminal amino acid through which the barcode peptide is conjugated with the analyte is a modified or non-natural amino acid, and wherein a barcode peptide conjugated to a first analyte in the plurality is distinguishable from a barcode peptide conjugated to a second analyte in the plurality, and wherein the first and second analyte are different.

[0081] As used herein, a “barcode peptide”, refers to a polypeptide comprising a defined amino acid sequence that functions as an identifiable molecular tag. The barcode peptide is characterized by a sequence that is distinguishable from other barcode peptides in a population based on the amino acid sequence. In certain embodiments, the barcode peptide comprises a sequence of from about 4 to about 30 amino acids that is substantially unique within a defined set of barcode peptides and is configured to permit multiplexed detection and differentiation, for example by sequencing, such as a sequencing method described herein. The barcode peptide may optionally comprise one or more modifications, linker regions, cleavage sites, affinity tags, or reactive groups to facilitate conjugation to a target molecule, surface, particle, or complex. In some embodiments, the barcode peptide does not confer biological activity independent of its identification fimction and serves primarily as a molecular identifier.

[0082] In some embodiments of any of the aspects, the barcode peptide is conjugated to the analyte via a linker. In some embodiments of any of the aspects, the linker is a peptide linker. In some embodiments of any of the aspects, the peptide linker comprises a recognition amino acid sequence for a peptide ligase. As used herein, a “recognition amino acid sequence for a peptide ligase” refers to a contiguous sequence of two or more amino acid residues within a polypeptide that is specifically recognized by a peptide ligase enzyme and that directs,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTenables, or enhances ligation of the polypeptide to a second polypeptide or molecule. The recognition amino acid sequence comprises the minimal motif, consensus sequence, or structural determinant required for binding by the peptide ligase and for catalysis of peptide bond formation at, within, or adjacent to the sequence. In certain embodiments, the recognition amino acid sequence is positioned at or near a terminus of the linker connecting the barcode peptide with the analyte, such as within about 1-20 residues of the N-terminus or C-terminus, and may include residues that define a cleavage site, acyl-enzyme intermediate formation site, nucleophilic attack site, or other catalytic feature required for ligation. The recognition amino acid sequence can comprise a naturally occurring motif, a consensus sequence derived therefrom, or a variant thereof that retains the ability to be recognized and processed by the peptide ligase. In some embodiments, recognition is sequence-specific, structure-dependent, or both, and may tolerate conservative substitutions that do not substantially impair ligase binding or catalytic efficiency.

[0083] Exemplary ligases include, but are not limited to, sortases, asparaginyl endopeptidase-derived ligases, subtilisin-derived ligases, inteins, ATP-dependent peptide ligases, and cyanobactin macrocyclases. In some embodiments of any one of the aspects, the ligase is a sortase. For example, the ligase is sortase A.

[0084] In certain embodiments, the recognition amino acid sequence for a peptide ligase comprises a short peptide motif that is specifically recognized and processed by the ligase to mediate peptide bond formation. Non-limiting examples include motifs recognized by sortase enzymes, such as the LPXTG motif (where Xaa is any amino acid) that is cleaved and ligated between the threonine and glycine residues; asparaginyl endopeptidase-derived ligases, including motifs comprising Asn-His-Val (NHV), Asn-Gly-Leu (NGL), Asn-Gly-Val (NGV), or a consensus Asn-Xaa-Leu / Val / Ile sequence; and transglutaminase-recognized motifs comprising a glutamine residue, such as Gln-Xaa-Pro, wherein the glutamine serves as an acyl donor in ligation to a lysine-containing acceptor substrate. In farther embodiments, the recognition amino acid sequence comprises residues positioned at or near a terminus of a polypeptide and may include activated C-terminal groups (e.g., esters) or sequence contexts favoring ligation by engineered ligases. Functional variants of the foregoing motifs that retain the ability to be recognized and catalytically processed by the corresponding peptide ligase are also encompassed.

[0085] In some embodiments of any of the aspects, the recognition amino acid sequence for a peptide ligase comprises the amino acid sequence LPXTG, Asn-Xaa-Xaa, YRH, or Arg-Leu.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0086] In some embodiments of any of the aspects, the barcode peptide is conjugated by its C-terminal amino acid. In some embodiments of any of the aspects, the barcode peptide is conjugated by its N-terminal amino acid.

[0087] In some embodiments of any of the aspects, the analytes are polypeptides, amino acids, nucleic acids, or cells.

[0088] In some embodiments, the method for barcoding farther comprises sequencing the barcode peptides conjugated with the analytes. For example, the method for barcoding farther comprises a step of sequencing the barcode peptides conjugated with the analytes using a sequencing method described herein.

[0089] In some embodiments of any of the aspects, the analytes are immobilized on a surface. For example, the analytes are immobilized on a surface after conjugating with the barcode peptide. In another non-limiting example, the analytes are immobilized on a surface prior to conjugating with the barcode peptide.Spectral properties

[0001] As used herein, the term “spectral properties” refers to a detectable change in a spectral property (e.g., fluorescence emission wavelength, excitation wavelength, emission intensity, fluorescence lifetime, polarization (polarity / anisotropy), absorbance, Raman shift, or Forster resonance energy transfer (FRET) efficiency) at a single wavelength or at a plurality of wavelengths of a probe conjugated to a first pair of terminal and penultimate amnio acids relative to one or more different pairs of terminal and penultimate amnio acids. In the context of modified or non-natural amino acids, the term “spectral properties” refers to a detectable change in a spectral property (e.g., fluorescence emission wavelength, excitation wavelength, emission intensity, fluorescence lifetime, polarization (polarity / anisotropy), absorbance, Raman shift, or Forster resonance energy transfer (FRET) efficiency) at a single wavelength or at a plurality of wavelengths of a probe conjugated a modified or non-natural amino acid relative to one or more different amino acids, which can be natural, modified or non-natural amino acids. Exemplary spectral properties 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 property is a fluorescence spectral property.

[0002] As the data disclosed herein show (FIGS. 1-4 and 7-15 and Tables 1 and 2) the fluorescent spectra of exemplary BODIPY probes conjugated to different pairs of terminal and penultimate amino acids or different modified or non-natural amino acids exhibited distinctive spectral properties. Comparing the spectra of the conjugated probe can therefore be used to4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTidentify / detect the terminal and penultimate amino acid of the polypeptide to which the probe is conjugated or the amino acid to which the probe is conjugated.

[0003] In some embodiments of any one of the aspects described herein, the step of measuring or detecting a spectral property comprises measuring or detecting one or more fluorescent spectral properties of the probe. For example, the step of measuring or detecting a spectral property comprises detecting or measuring fluorescence emission wavelength, excitation wavelength, emission intensity, fluorescence lifetime, polarization (polarity / anisotropy), absorbance, Raman shift, or Forster resonance energy transfer (FRET) efficiency. In some embodiments, the step of measuring or detecting a spectral property comprises measuring or detecting emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime. For example, the step of measuring or detecting a spectral property comprises detecting or measuring the emission intensity and fluorescence lifetime.Reference spectral properties

[0004] Embodiments of the various aspects described herein include comparing the spectral properties of a conjugated probe to a plurality of reference spectral properties. In some embodiments of any one of the aspects described herein, each reference spectral property is representative of the probe conjugated to a different pair of terminal and penultimate amino acids. In some embodiments, embodiments of any one of the aspects described herein, each reference spectral property is representative of the probe conjugated to a different amino acid. In some embodiments of any one of the aspects described herein, comparing the spectral properties of the conjugated probe to the plurality of reference spectral properties comprises comparing the spectra of the conjugated probe to a plurality of reference spectra. In some embodiments of any one of the aspects described herein, the reference spectra are spectra of the probe bound to known amino acids or terminal and penultimate amino acid pairs. In some embodiments of any one of the aspects described herein, the method comprises identifying the closest match between the spectra of the conjugated probe and the reference spectra, thereby identifying the terminal and the penultimate amino acid pair or the modified / non-natural amino acid. Various statistical methods known in the art can be used to compare the spectra of the conjugated probe and reference spectra in order to identify the closest match and the amino acid conjugated with the probe.

[0005] In some embodiments of any one of the aspects described herein, the reference data are emission intensity and / or fluorescence lifetime of the probe bound to known amino acids or terminal and penultimate amino acid pairs, such as the data shown in FIGS. 1-4 and 7-15 and4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTTables 1 and 2. In some embodiments of any one of the aspects described herein, the method comprises identifying the closest match between the emission intensity and / or fluorescence lifetime of the conjugated probe and the reference emission intensity and / or fluorescence lifetime data, such as the data shown in FIGS. 1-4 and 7-15 and Tables 1 and 2, thereby identifying the terminal and the penultimate amino acid pair or the modified / non-natural amino acid. Various statistical methods known in the art can be used to compare the emission intensity and / or fluorescence lifetime of the conjugated probe and reference data in order to identify the closest match and the amino acid(s) conjugated with the probe.

[0006] In some embodiments of any one of the aspects described herein, suitable methods generate a quantitative measure of similarity or difference between the spectra and the reference spectra. In some embodiments of any one of the aspects described herein, the methods used herein for comparing the spectral properties of conjugated probe and a reference / control conjugate use one or more probabilistic algorithms. For example, a probabilistic algorithm can be trained to identify different pairs of terminal and penultimate amino acids or modified / non-natural amino acids conjugated to probes described herein using the spectral data provided herein associating specific spectra with specific pairs of terminal and penultimate amino acids or specific modified or non-natural amino acids. In some embodiments of any one of the aspects described herein, machine learning, genetic algorithms, or principle component analysis (PCA) can be used for comparing spectra and reference spectra.Detection of spectral properties

[0090] Embodiments of the various aspects described herein include detecting or measuring spectral properties of a probe conjugated to a polypeptide or an amino acid. Methods and systems for measuring / detecting spectral properties of molecule 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 brightness changes induced by quenching / dequenching of fluorescence, or fluorescence energy transfer.

[0091] In some embodiments of any one of the aspects described herein, the detecting the spectral properties of a conjugated probe comprises optical detection. Exemplary optical detection systems include, but are not limited to, a charge- coupled device (CCD), electron multiplying CCD (EMCCD), near-field scanning microscopy, far-field confocal microscopy,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTwide-field epi-illumination, light scattering, dark field microscopy, photoconversion, single and / or multiphoton excitation, spectral wavelength discrimination, fluorophore identification, evanescent wave illumination, total internal reflection fluorescence (TIRF) microscopy, superresolution fluorescence microscopy, single-molecule localization microscopy, and singlemolecule spectroscopy.

[0092] In some embodiments of any one of the aspects described herein, the method comprises detection of laser-activated fluorescence using a microscope equipped with a camera, sometimes referred to as high-efficiency photon detection system. Suitable photon detection systems include, but are not limited to, photodiodes and intensified CCD cameras.

[0093] It is noted that different techniques known in the art can be used to detect spectral properties of different conjugated probes at spatially resolved locations. For example, super resolution microscopy can be used to detect one or more spectral properties of a conjugated probe conjugated at a particular location within a sample. In some embodiments of any one of the aspects described herein, the methods described herein use stochastic optical reconstruction microscopy (STORM).

[0094] In some embodiments of any one of the aspects described herein, the detecting the spectral properties of a conjugated probe includes ultrasensitive detection systems that are able to repeatedly detect signals from precisely the same co-ordinates in a sample, thereby assigning the detected spectral information to a unique molecule, e.g., a polypeptide.

[0095] Embodiments of the various aspects described herein include detecting or measuring one or more spectral properties of a probe conjugated with an amino acid. In some embodiments of any one of the aspects described herein, detecting or measuring one or more spectral properties of a probe conjugated with an amino acid comprises measuring or detecting fluorescence of the conjugated probe.

[0096] In some embodiments, the method comprises detecting / identifying one or more spectral properties for each probe conjugated to the terminal amino acid of each of the plurality of polypeptides at spatially resolved locations in a sample comprising the plurality of polypeptides. In some embodiments, the method comprises detecting / identifying one or more spectral properties for each probe conjugated to the penultimate amino acid of each of the plurality of polypeptides at spatially resolved locations in a sample comprising the plurality of polypeptides.

[0097] In an embodiment, detection of one or more spectral properties comprises detecting fluorescence of the conjugated probe. In an embodiment, detecting one or more spectral properties of the conjugated probe comprises detecting fluorescence emission4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTintensity, polarity / anisotropy or lifetime. In another embodiment, detecting the emission intensity, polarity / anisotropy or lifetime is at a single wavelength. In a farther embodiment, detecting the emission intensity, polarity / anisotropy or lifetime is at a plurality of wavelengths.

[0098] In some embodiments of any one of the aspects described herein, detection of one or more spectral properties of the conjugated probe comprises super resolution microscopy. In some farther embodiments, the super resolution microscopy comprises stochastic optical reconstruction microscopy (STORM).

[0007] In some embodiments, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 500 nm to about 850 nm. For example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 500 nm to about 750 nm. In another non-limiting example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 500 nm to about 650 nm. In still another non-limiting example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 650 nm to about 750 nm. In yet still another non-limiting example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 650 nm to about 800 nm. In still another non-limiting example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 500 nm to about 600 nm. In yet another non-limiting example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 600 nm to about 700 nm. In some embodiments, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and / or lifetime at one or more wavelengths from about 500 nm to about 570 nm.

[0008] Typical excitation (absorption) wavelengths for BODIPY (boron-dipyrromethene) dyes are in the range from about 495 nm to about 750 nm. For example, for an unsubstituted BODIPY dye typical excitation maximum is from about 495 to about 505 nm, for alkyl substituted typical excitation maximum is from about 500 to about 540 nm, for ^-Extended (e.g., styryl-substituted) typical excitation maximum is from about 550 nm to about 650 nm,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTand for Aza-BODIPY typical excitation maximum is from about 600 nm to about 750 nm. The inventors h discovered inter alia that exciting the conjugated probe with electromagnetic radiation at a wavelength lower than 496 nm provide a detectable change in a spectral property at a single wavelength or at a plurality of wavelengths of a probe conjugated to a first pair of terminal and penultimate amnio acids relative to one or more different pairs of terminal and penultimate amnio acids, and / or a probe conjugated to a modified or non-natural amino acid relative to one or more different amino acids, which can be natural, modified or non-natural amino acids. Thus, in some embodiments of any one of the methods described herein, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises exciting the conjugated probe with electromagnetic radiation at a wavelength from about 430 nm to about 450 nm. For example, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises exciting the conjugated probe with electromagnetic radiation at a wavelength from about 435 nm to about 445 nm. In some embodiments, the step of measuring or detecting one or more spectral properties of the conjugated probe comprises exciting the conjugated probe with electromagnetic radiation at a wavelength of about 440 nm.

[0009] Without wishing to be bound by a theory, inter- and intramolecular interactions can adversely affect optical properties of the probe conjugated with a polypeptide. The inventors have discovered inter alia that conformationally driven interactions between a probe and the polypeptide it is conjugated to can be unexpectedly, surprisingly significantly reduced or even nullified by denaturation of the polypeptide, and the reduction of interaction between the probe and the polypeptide it is conjugated to as achieved through denaturation results in higher emission and brightness of the dyes. Accordingly, in some embodiments of any of the aspects, the method farther comprises a step of denaturing the polypeptide to which the probe is conjugated prior to the step of detecting one or more spectral properties of the conjugated probe. It is noted that denaturing of the polypeptide can be achieved by changing the temperature, pH, or solvent. In some aspect of any of the embodiments, said detecting one or more spectral properties of the conjugated probe is in the presence of a denaturing agent. Exemplary denaturing agents include, but are not limited to, chaotropic agents, detergents, organic solvents, acids, bases, reducing agents, oxidizing agents, and heavy metal ions. In some embodiments, the the denaturing agent is selected from the group consisting of urea, guanidine hydrochloride, guanidine thiocyanate, lithium perchlorate, sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100, NP-40, Tween-20, CHAPS, CHAPSO, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide (DMSO), trifluoroethanol (TFE),4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PThydrochloric acid, trifluoroacetic acid (TFA), acetic acid, sodium hydroxide, dithiothreitol (DTT), P-mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), hydrogen peroxide, performic acid, Hg2+, Ag+and Pb2+. In some embodiments of any of the aspects, the denaturing agent is urea or guanidine hydrochloride.

[0099] When used, the denaturing agent can be present at a concentration sufficient to denature the polypeptide. For example, the denaturing agent can be present at a concentration from about 0.1 M to about 10 M, such as the concentration of the denaturing agent can be about 0.5 M, about 1 M, about 1.5 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, about 5 M, about 5.5 M, about 6 M, about 6.5 M, about 7 M, about 7.5 M, about 8 M, about 8.5 M, about 9 M, about 9.5 M, or about 10 M. In some embodiments of any of the aspects, the concentration of the denaturing agent is 6 M. In some embodiments of any of the aspects, the concentration of the denaturing agent is about 8 M.

[0100] In some embodiments of any of the aspects, the polypeptide or analyte is immobilized on a surface. Methods for immobilizing molecules on a surface are well known in the art and available to one of skill in the art. Polypeptide and analytes can be immobilized on a surface by covalent or non-covalent attachment techniques. In some embodiments, the surface comprises glass, silica, a polymer, a hydrogel, a membrane, or a functionalized solid support bearing reactive groups such as amine, carboxyl, hydroxyl, epoxy, aldehyde, maleimide, N-hydroxy succinimide (NHS) ester, or thiol moieties. Immobilization can be achieved through covalent coupling between complementary reactive groups on the surface and the polypeptide or analyte, for example via amide bond formation, thiol-maleimide conjugation, click chemistry, or carbodiimide-mediated coupling. Alternatively, non-covalent strategies can be employed, including adsorption, affinity interactions (e.g., biotinstreptavidin, His-tag-metal chelate), electrostatic interactions, or hydrophobic interactions. The immobilization can be site-specific or random and can optionally employ linker molecules or spacer arms to enhance accessibility and reduce steric hindrance.

[0101] It is noted that the polypeptide can be immobilized prior to conjugating the probe. Alternatively, the polypeptide can be immobilized after conjugating the probe. Similarly, the analyte can be immobilized prior to conjugating with the barcode peptide or after conjugating with the barcode peptide.Modified / non-natural amino acids

[0102] As used herein, a “modified or non-natural amino acid” refers to an amino acid residue that differs structurally from any of the 20 canonical proteinogenic L-amino acids4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTencoded by the standard genetic code. A modified or non-natural amino acid may include (i) an amino acid having a side chain that is substituted, derivatized, or otherwise chemically altered relative to a naturally occurring amino acid; (ii) an amino acid comprising a non-native fimctional group, including but not limited to alkyl, alkenyl, alkynyl, aryl, halo, azido, nitro, cyano, keto, aldehyde, ester, amide, thiol, thioether, sulfonyl, boronate, or bioorthogonal reactive moieties; (iii) an amino acid having altered backbone structure, including 0-, y-, 5-, or other extended backbone amino acids; (iv) a D-amino acid or other stereoisomer not commonly found in naturally occurring proteins; (v) an amino acid analog comprising non-carbon atoms or heteroatoms within the side chain; or (vi) an amino acid bearing a post-translational modification or synthetic modification, such as phosphorylation, glycosylation, methylation, acetylation, lipidation, pegylation, or isotopic labeling. The term encompasses amino acids incorporated during chemical synthesis, ribosomal incorporation via expanded genetic code technology, or post-synthetic modification, provided that the residue is not one of the standard unmodified L-a-amino acids.

[0103] Exemplary modified or non-natural amino acids, amenable to methods described herein include, but are not limited to, 2-Aminoadipic acid, 3 -Aminoadipic acid, beta-Alanine, beta-Aminoproprionic acid, 2-Aminobutyric acid, 4-Aminobutyric acid, Piperidinic acid, 6- Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, t-butylalanine, Citrulline, Cyclohexylalanine, 2,4-Diaminobutyric acid, Desmosine, 2,2'-Diaminopimelic acid, 2,3-Diaminoproprionic acid, N-Ethylglycine, N-Ethylasparagine, Homoarginine, Homocysteine, Homoserine, Hydroxy lysine, Allo-Hydroxy lysine, 3 -Hydroxyproline, 4-Hydroxyproline, Isodesmosine, allo-Isoleucine, Methionine sulfoxide, N-Methylglycine, sarcosine, N-Methylisoleucine, 6-N-Methyllysine, N-Methylvaline, 2-Naphthylalanine, Norvaline, Norleucine, Ornithine, 4-Chlorophenylalanine, 2-Fluorophenylalanine, 3 -Fluorophenylalanine, 4-Fluorophenylalanine, Phenylglycine, and Beta-2 -thienylalanine. Additional modified or non-natural amino acids amenable to the methods described herein are described in the art in, for example, Sharma et al., Journal of Medicinal Chemistry, 67(22): 19932-19965 (2024), the contents of which are incorporated herein in its entirety.Conjugation of the probe

[0104] It is noted that the probe can be conjugated to an amino, a carboxylic, a hydroxyl or a thiol group of the amino acid. Accordingly, in some embodiments of any of the aspects, the probe is conjugated to an amino group of the amino acid. In some other embodiments of4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTany of the aspects, the probe is conjugated to a carboxylic group of the amino acid. In yet some other embodiments of any of the aspects, the probe is conjugated to a side chain of the amino acid.Sample

[0105] The polypeptide, the modified or non-natural amino acid or the analyte can be present in a sample. As used herein the term “sample” includes any material that contains the polypeptide, modified or non-natural amino acid or analyte to be detected / identified. The sample can be a biological sample, such as animal or plant tissues, biopsies, organs, cells, membrane vesicles, plasma membranes, organelles, cell extracts, secretions, urine or mucous, tissue extracts or other biological specimens both natural or synthetic in origin. The term sample also includes single cells, organelles or intracellular materials isolated from a biological specimen, or viruses, bacteria, frmgus or isolates 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 polypeptide, modified or non-natural amino acid or analyte be detected / identified.

[0106] In some embodiments of any of the aspects, the sample is a biological sample. In some embodiments of any of the aspects, the biological sample is a biological fluid, a tissue, an organ, or a cell.Probes

[0107] Embodiments of the various aspect described herein include a probe, e.g., a probe comprising a dipyrromethane-BFi derivative, e.g., the probe is a modified or unmodified BODIPY dye. In some embodiments of any of the aspects, the probe comprises a modified 8-thioether-BODIPY dye. Exemplary probes comprising dipyrromethane-BFi derivative are described, for example, in US Patent No. 12,181,479, the contents of which are incorporated herein in its entirety. In some embodiments of the various aspects described herein, the probe is a compound of Formula (I):(Formula I).4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0108] In compounds of Formula (I), R1, R2, R3, R4, R5, R6, R7and R8are 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, cynao, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, optionally at least one of R1, R2, R3, R4, R5, R6, R7and R8, is a labile or leaving group. It is noted that any 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) independently selected substituents from the group consisting of halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.

[0109] In some embodiments of any one of the aspects described herein, only one of R1, R2, R3, R4, R5, R6, R7and R8is a labile or leaving group. As used herein, a “labile group” or a “leaving group” refers to a group that can be substituted 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, a halide (fluoride, chloride, bromide, and iodide), azide, a sulfonate (e.g., an optionally substituted Ci-Ce alkanesulfonate, such as methanesulfonate and trifluoromethanesulfonate, or an optionally substituted C7-C12 alkylbenzenesulfonate, such as p-toluenesulfonate), succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, a carboxylate, an aminocarboxylate (carbamate) and an alkoxy carboxy late (carbonate). For substitutions at saturated carbon, halides and sulfonates are preferred leaving groups. For substitutions at a carbonyl carbon a halide, succinimide-N-oxide, p-nitrophenoxide, pentafluorophenoxide, tetrafluorophenoxide, a carboxylate, or an alkoxycarboxylate (carbonate) may for example be used as a leaving group.

[0110] In some embodiments of any one of the aspects described herein, the labile or leaving group is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydxoryl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, or diazonium. For example, the labile or leaving group is halogen or an alkylthio.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0111] In some embodiments of any one of the aspects described herein, the labile or leaving group is -SRL, wherein RLis optionally substituted alkyl, perhaloalkyl, optionally substituted alkenyl, optionally cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl. For example, RLis substituted Cl-C6alkyl, Cl-C6perhaloalkyl optionally substituted Ci-Cealkenyl, optionally substituted aryl or optionally substituted heteroaryl. In some embodiments of any one of the aspects described herein, RLis methyl, allyl, phenyl, 4-methoxyphenyl, 4-nitrophenyl, benzyl, or 4-methoxybenzyl. In some preferred embodiments, RLis methyl, i.e., the labile or leaving groups is -SMe.

[0112] In some embodiments of any one of the aspects described herein, R1, R2, R3, R4, R5, R6and R7are 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, cynao, nitro, thiol, optionally substituted alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido; and R8is a labile or leaving group.

[0113] In some embodiments of any one of the aspects described herein, a compoundof Formula (I) is of Formula (I-A):optionally substituted alkyl, optionally substituted alkenyl, optionally cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl, and R1, R2, R3, R4, R5, R6are R7are as defined for compounds of Formula (I).

[0114] In some embodiments of any one of the aspects described herein, R8Sis an optionally substituted aryl. For example, is R8San optionally substituted phenyl. Accordingly, in some embodiments of any one of the aspects described herein, a compound of Formula (I),4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTe.g., of Formula (I-A) is of Formula (I-B),R6are R7are as defined for compounds of Formula (I); and each of R81, R82, R83, R84and R85is 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, cynao, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido.

[0115] In some embodiments of any one of the aspects described herein, a compoundFormula (I); and each of R31, R32, R33, R34and R35can be 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, cynao, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PToptionally, a vicinal pair of R31, R32, R33, R34and R35, together with the carbon atoms they are attached to form an optionally substituted aryl; and R8Sis optionally substituted alkyl, optionally substituted alkenyl, optionally cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl.

[0116] In some embodiments of any one of the aspects described herein, a compoundof Formula (I) is of Formula (I-E),are as defined for compounds of Formula (I-C) or (I-D); and R81, R82, R83, R84and R85are as defined for compounds of Formula (I-B).4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0117] In some embodiments of any one of the aspects described herein, a compoundof Formula (I) is of Formula (I-G),defined for compounds of Formula (I); R31, R32, R33, R34and R35are as defined for compounds of Formula (I-C) or (I-D); and each of R41, R42, R34, R44and R45can be 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, cynao, nitro, thiol, alkylthio, sulfinyl, sulfonyl, sulfonate, carbamoyl, isocyanato, thiocyanato, isothiocyanato, and ureido, optionally, a vicinal pair of R41, R42, R34, R44and R45, together with the carbon atoms they are attached to form an optionally substituted aryl.

[0118] In some embodiments of any one of the aspects described herein, a compoundof Formula (I) is of Formula (I-I),4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT5are as defined for compounds of Formula (I-C) or (I-D); R41, R42, R34, R44and R45are as defined for compounds of Formula (I-G) or (I-H); and R81, R82, R83, R84and R85are as defined for compounds of Formula (I-B).

[0119] In some embodiments, the probe is a compound described in FIGS. 2-6 of US Patent No. 12,181,479, the contents of which are incorporated herein in its entirety."

[0120] In some embodiments, the probe is of structure:

[0121] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or fimctions are presented in a given order, alternative embodiments may perform fimctions in a different order, or fimctions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, fimctions and concepts of the above references and application to provide yet fiirther embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0122] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTembodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0123] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. 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 belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The definitions and terminology used herein are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

[0124] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.Some selected definitions

[0125] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0126] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to 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 invention, the methods, devices, and materials in this regard are described herein.

[0127] 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.), W. W. 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, N.Y., 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 047150338X, 9780471503385), 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, ADAM Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0128] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used to described the present invention, in connection with percentages means ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, or ±5%.

[0129] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0130] As used herein the terms “comprising” or “comprises” means “including” or “includes” and are used in reference to compositions, methods, systems, and respective component(s) thereof, that are useful to the invention, yet open to the inclusion of unspecified elements, whether useful or not.

[0131] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0132] The term “consisting of’ refers to compositions, methods, systems, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0133] The singular terms “a,” “an,” and “the” include plural referents unless 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 can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0134] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0135] The terms “significantly different than,”, “statistically significant,” and similar phrases refer to comparisons between data or other measurements, wherein the differences between two compared data or other measurements are evidently or reasonably different to the trained observer, or statistically significant (if the phrase includes the term “statistically” or if there is some indication of statistical test, such as a p-value, or if the data, when analyzed, produce a statistical difference by standard statistical tests known in the art).

[0136] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0137] As used herein, “polypeptide” refers to two or more amino acids linked together by a peptide bond. The term “polypeptide” includes proteins, or protein digests, that have a C-terminal end and an N-terminal end as generally known in the art and may be synthetic in origin or naturally occurring. As used herein “at least a portion of the polypeptide” refers to 2 or more amino acids of the polypeptide. Optionally, a portion of the polypeptide includes at least: 5, 10,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT20, 30, or 50 amino acids, either consecutive or with gaps, of the complete amino acid sequence of the polypeptide, or the full amino acid sequence of the polypeptide.

[0138] The phrase “N-terminal amino acid” refers to an amino acid that has a free amine group and is only linked to one other amino acid by a peptide amide bond in the polypeptide. Optionally, the “N-terminal amino acid” may be an “N-terminal amino acid derivative”. As used herein, an “N-terminal amino acid derivative” refers to a N-terminal amino acid residue that has been chemically modified, for example by an Edman reagent or other chemical in vitro or inside a cell via a natural post-translational modification (e.g. phosphorylation) mechanism.

[0139] The phrase “C-terminal amino acid” refers to an amino acid that has a free carboxylic group and is only linked to one other amino acid by a peptide amide bond in the polypeptide. Optionally, the “C-terminal amino acid” may be an “C-terminal amino acid derivative”. As used herein, an “C-terminal amino acid derivative” refers to a C-terminal amino acid residue that has been chemically modified in vitro or inside a cell via a natural post-translational modification (e.g. phosphorylation) mechanism.

[0140] 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 for a polypeptide.

[0141] As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “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 can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes 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 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.

[0142] A “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2 group to4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTan NH group or an O group). The term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfiir, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom(s) is placed 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-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

[0143] As used herein, the term “alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl andheptadec-8,ll-dien-l-yl.

[0144] As used herein, the term “alkynyl” refers to an alkyl group containing a carboncarbon triple bond. The alkynyl group can 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 the lower alkynyl groups.

[0145] As used herein, the term “cycloalkyl” refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and / or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

[0146] “Heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0147] “Aryl” refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms. The aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes 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, rylthio, 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.

[0148] “Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12 membered fiised bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.

[0149] Exemplary aryl and heteroaryls 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, benzofiiranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydroiuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofiiranyl, 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, oxindolyl, 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, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTthienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent.

[0150] As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

[0151] 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 described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.

[0152] The term “haloalkyl” as used herein refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (Ci-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).

[0153] The term “perhalo alkyl” means, unless otherwise stated, alkyl substituted with (2n+l) halogen atoms, where n is the total number of carbon atoms in the alkyl group. For example, the term “perhalo(Ci- -C4)alkyl” or “Ci-C4perhaloalkyl” includes trifluoromethyl, pentachloroethyl, 1,1,1- trifhioro-2-bromo-2-chloroethyl, and the like.

[0154] The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (Ci-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2, 2, 2-trifluoro- 1,1 -dichloroethyl, and the like).

[0155] As used herein, the term “amino” means -NH2. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl). The term “dialkylamino” means a nitrogen moiety having at 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” means a nitrogen moiety having at least one aryl radical attached to the nitrogen.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTFor example, -NHaryl, and — N(aryl)2. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl)2. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but is not limited to, NH(Ci-Cioalkyl), such as — NHCH3, — NHCH2CH3, — NHCH2CH2CH3, and — NHCH(CH3)2. Exemplary dialkylamino includes, but is not limited to, — N(Ci-Cioalkyl)2, such as N(CH3)2, — N(CH2CH3)2, — N(CH2CH2CH3)2, and — N(CH(CH3)2)2.

[0156] The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl. For example, an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

[0157] The terms “hydroxy” and “hydroxyl” mean the radical — OH.

[0158] The terms “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, wherein aryl and heteroaryl are as defined herein. The 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, O-n-propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec- butyl, O-tert-butyl, O-pentyl, O- hexyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.

[0159] As used herein, the term “carbonyl” means the radical — C(O) — . 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.

[0160] As used herein, the term “oxo” means double bonded oxygen, i.e., =O.

[0161] The term “carboxy” means the radical — C(O)O — . It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0162] The term “ester” refers to a chemical moiety with formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.

[0163] The term “cyano” means the radical — CN.

[0164] The term “nitro” means the radical — NO2.

[0165] The term, “heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include — N=, — NRN— , — N+(O )=, — O — , — S — or — S(O)2— , — OS(O)2 — , and — SS — , wherein RNis H or a further substituent.

[0166] The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with 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 groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups.

[0167] The term “sulfinyl” means the radical — SO — . 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.

[0168] The term “sulfonyl” means the radical — SO2 — . 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.

[0169] The term “thiocarbonyl” means the radical — C(S) — . It is noted that the thiocarbonyl radical can be fiirther substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

[0170] “Acyl” refers to an alkyl-CO — group, wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.

[0171] “Aroyl” means an aryl-CO — group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

[0172] “Arylthio” refers to an aryl-S — group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

[0173] “Aralkyl” refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0174] “Aralkyloxy” refers to an aralkyl-O — group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.

[0175] “Aralkylthio” refers to an aralkyl-S — group, wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.

[0176] “Alkoxycarbonyl” refers to an alkyl-O — CO — group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.

[0177] “Aryloxycarbonyl” refers to an aryl-O — CO — group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0178] “Aralkoxycarbonyl” refers to an aralkyl-O — CO — group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0179] “Carbamoyl” refers to an H2N — CO — group.

[0180] “Alkylcarbamoyl” refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.

[0181] “Dialkylcarbamoyl” refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.

[0182] “Acyloxy” refers to an acyl-O — group, wherein acyl is as previously described. “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.

[0183] The term “optionally substituted” means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. The term “substituents” refers to a group “substituted” on a substituted group at any atom of the substituted group. Suitable substituents include, without limitation, halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, 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 to can form a ring.

[0184] For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2, 3, 4 or 5 groups selected from OH, CN, -SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (Ci-C8)alkyl, O(Ci-4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTCs)alkyl, O(Ci-C8)haloalkyl, (C2-Cs)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— NH2or CH2-aryl-alkoxy; “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0185] In some embodiments, an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.

[0186] An “isocyanato” group refers to a NCO group.

[0187] A “thiocyanato” group refers to a CNS group.

[0188] An “isothiocyanato” group refers to a NCS group.

[0189] “Alkoyloxy” refers to a RC(=O)O- group.

[0190] “Alkoyl” refers to a RC(=O)- group.

[0191] All structures of any of the compounds are provided herein for illustrative purpose and disclose a particular isomer. However, one of ordinary skill in the art will recognize all possible isomers of the structures of any of the compounds described herein. Therefore, other isomers such as enantiomers of any of Formula (I) and (II) are considered to fall within the scope of the invention. As used herein, the term “isomer” refers to a compound having the same molecular formula but differing in structure. Isomers which differ only in configuration and / or conformation are referred to as “stereoisomers.” The term “isomer” is also used to refer to an enantiomer.

[0192] The term “enantiomer” is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable. The designations “R” and “S” are used to denote the absolute configuration of the molecule about its chiral center. The designations may appear as a prefix or as a suffix; they may or may not be separated from the isomer by a hyphen; they may or may not be hyphenated; and they may or may not be surrounded by parentheses. The designations “(+)” and are employed to designate the4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTsign of rotation of plane-polarized light by the compound, with (-) meaning that the compound is levorotatory (rotates to the left). A compound prefixed with (+) is dextrorotatory (rotates to the right). Other terms used to designate or refer to enantiomers include “stereoisomers” (because of the different arrangement or stereochemistry around the chiral center; although all enantiomers are stereoisomers, not all stereoisomers are enantiomers) or “optical isomers” (because of the optical activity of pure enantiomers, which is the ability of different pure enantiomers to rotate planepolarized light in different directions). Enantiomers generally have identical physical properties, such as melting points and boiling points, and also have identical spectroscopic properties. Enantiomers can differ from each other with respect to their interaction with plane-polarized light and with respect to biological activity.

[0193] In various embodiments, compounds of Formula (I) or (II) include enantiomers, derivatives, prodrugs, and pharmaceutically acceptable salts thereof.

[0194] The term “derivative” as used herein refers to a chemical substance related structurally to another, i.e., an “original” substance, which can be referred to as a “parent” compound. A “derivative” can be made from the structurally-related parent compound in one or more steps. The general physical and chemical properties of a derivative are also similar to the parent compound.

[0195] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0196] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to 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 can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fiilfilling the written description of all Markush groups used in the appended claims.

[0197] Aspects of the present technology can be defined in any of the following numbered embodiments:

[0198] Embodiment 1 : A method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a polypeptide, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTwherein the probe exhibits different fluorescent spectral properties when conjugated to different pairs of terminal amino acid and penultimate amino acid; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid; and (c) identifying the terminal amino acid and the penultimate amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different terminal and penultimate amino acid pair.

[0199] Embodiment 2: The method of Embodiment 1, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other.

[0200] Embodiment 3: The method of any one of Embodiments 1-2, wherein the method further comprises a step (c) of cleaving the terminal amino acid, and, and repeating steps (a) to (b), thereby providing error correction.

[0201] Embodiment 4: A method for sequencing a polypeptide, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide, wherein the probe exhibits different fluorescent spectral properties when conjugated to a first pair of terminal amino acid and penultimate amino acid, and when conjugated to a second pair of terminal amino acid and penultimate amino acid, wherein at least one amino acid in the first pair is different from at least one amino acid in the second pair; (b) detecting one or more spectral properties of the probe conjugated to the terminal amino acid to determine the identity of the terminal amino acid and the penultimate amino acid; (c)cleaving the terminal amino acid of the polypeptide; and (d) sequentially repeating steps (a) to (c) one or more times to determine the sequence of at least a portion of the polypeptide.

[0202] Embodiment 5: The method of Embodiment 4, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi derivative to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other, and wherein the probes conjugated to the terminal amino acid of the polypeptides in the plurality are identical.

[0203] Embodiment 6: The method of any one of Embodiments 1-5, wherein the terminal amino acid is at an N-terminal of the polypeptide.

[0204] Embodiment 7: The method of Embodiment 1-5, wherein the terminal amino acid is at C-terminal of the polypeptide.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0205] Embodiment 8: The method of any one of Embodiments 3-5, wherein the step of cleaving the terminal amino acid comprises enzymatic cleavage.

[0206] Embodiment 9: The method of any one of Embodiments 3-5, wherein the step of cleaving the terminal amino acid comprises chemical cleavage.

[0207] Embodiment 10: The method of any one of Embodiments 1-6, wherein polypeptide is in a sample.

[0208] Embodiment 11: The method any one of Embodiments 1-10, wherein at least one (e.g., 1 or both) of the terminal and penultimate amino acids are non-natural or modified amino acids.

[0209] Embodiment 12: The method of any one of Embodiments 1-11, wherein the polypeptide is immobilized on a surface.

[0210] Embodiment 13 : A method for identifying a modified or non-natural amino acid, the method comprising: (a) conjugating a probe comprising a dipyrromethane-BFi derivative to a modified or non-natural amino acid, wherein the probe exhibits different fluorescent spectral properties when conjugated to different modified or non-natural amino acids; (b) detecting one or more fluorescent spectral properties of the probe conjugated to the modified or non-natural amino acid; and (c) identifying the modified or non-natural amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different modified or non-natural amino acid.

[0211] Embodiment 14: The method of Embodiment 13, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi derivative to a plurality of amino acids, wherein at least two amino acids in the plurality are different from each other and at least one amino acid is a modified or non-natural amino acid, and wherein the probes conjugated to the amino acids in the plurality are identical.

[0212] Embodiment 15: The method of Embodiment 14, wherein at least one amino acid in the plurality is a modified or non-natural amino acid and at least one amino acid in the plurality is a natural amino acid.

[0213] Embodiment 16, The method of any one of Embodiments 13-15, wherein the modified or non-natural amino acid is in a sample.

[0214] Embodiment 17: The method of any one of Embodiments 13-16, wherein the amino acid is in a polypeptide.

[0215] Embodiment 18: The method of Embodiment 17, wherein the modified or non-natural amino acid is at an N-terminal of the polypeptide.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0216] Embodiment 19: The method of Embodiments 17, wherein the modified or nonnatural amino acid is at a C-terminal of the polypeptide.

[0217] Embodiment 20: The method of Embodiments 17, wherein the modified or nonnatural amino acid is at an internal position of the polypeptide.

[0218] Embodiment 21: The method of any one of Embodiments 13-20, wherein the polypeptide is immobilized on a surface.

[0219] Embodiment 22: The method of any one of Embodiments 1-21, wherein the probe is conjugated to an amino, a carboxylic, a hydroxyl or a thiol group of the amino acid.

[0220] Embodiment 23: The method of any one of Embodiments 1-22, wherein the probe is conjugated to an amino group of the amino acid.

[0221] Embodiment 24: The method of any one of Embodiments 1-22, wherein the probe is conjugated to a carboxylic group of the amino acid.

[0222] Embodiment 25: The method of any one of Embodiments 1-22, wherein the probe is covalently conjugated with the amino acid.

[0223] Embodiment 25: The method of any one of Embodiments 1-25, wherein the method comprises a step of denaturing the polypeptide to which the probe is conjugated prior to the step of detecting one or more spectral properties of the conjugated probe.

[0224] Embodiment 27: The method of any one of Embodiments 1-26, wherein said detecting one or more spectral properties of the conjugated probe is in the presence of a denaturing agent.

[0225] Embodiment 28: The method of Embodiment 27, wherein the denaturing agent is selected from the group consisting of a chaotropic agent, a detergent, an organic solvent, an acid, a base, a reducing agent, an oxidizing agent, a heavy metal ion.

[0226] Embodiment 29: The method of Embodiment 27 or 28, wherein the denaturing agent is selected from the group consisting of urea, guanidine hydrochloride, guanidine thiocyanate, lithium perchlorate, sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100, NP-40, Tween-20, CHAPS, CHAPSO, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide (DMSO), trifluoroethanol (TFE), hydrochloric acid, trifluoroacetic acid (TFA), acetic acid, sodium hydroxide, dithiothreitol (DTT), P-mercaptoethanol, tris(2-carboxyethyljphosphine (TCEP), hydrogen peroxide, performic acid, Hg2+, Ag+and Pb2+.

[0227] Embodiment 30: The method of any one of Embodiments 1-29, wherein said detecting one or more spectral properties of the conjugated probe comprises super resolution microscopy.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0228] Embodiment 31: The method of Embodiment 30, wherein the super resolution microscopy comprises stochastic optical reconstruction microscopy (STORM).

[0229] Embodiment 32: The method of any one of Embodiments 1-31, wherein detecting one or more spectral properties comprises detecting fluorescence of the conjugated probe.

[0230] Embodiment 33: The method of any one of Embodiments 1-32, wherein detecting one or more spectral properties of the conjugated probe comprises detecting fluorescence emission wavelength, excitation wavelength, emission intensity, fluorescence lifetime, polarization (polarity / anisotropy), absorbance, Raman shift, or Forster resonance energy transfer (FRET) efficiency.

[0231] Embodiment 34: The method of any one of Embodiments 1-33, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime.

[0232] Embodiment 35: The method of any one of Embodiments 1-35, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime.

[0233] Embodiment 36: The method of Embodiment 35, wherein said detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime is at a single wavelength.

[0234] Embodiment 37: The method of Embodiment 35, comprising said detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime is at a plurality of wavelengths.

[0235] Embodiment 38: The method of any one of Embodiments 1-37, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and fluorescence lifetime.

[0236] Embodiment 39: The method of any one of Embodiments 1-38, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and lifetime at one or more wavelengths from about 500 nm to about 570 nm.

[0237] Embodiment 40: The method of any one of Embodiments 1-39, wherein detecting one or more spectral properties of the conjugated probe comprises exciting the conjugated probe with electromagnetic radiation at a wavelength from about 430 nm to about 450 nm, preferably from about 435 nm to about 445 nm, more preferably a wavelength of about 440 nm.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0238] Embodiment 41: The method of any one of Embodiments 1-40, wherein detecting one or more spectral properties of the conjugated probe comprises: (i) exciting the conjugated probe with electromagnetic radiation at a wavelength from about 430 nm to about 450 nm, preferably from about 435 nm to about 445 nm, more preferably a wavelength of about 440 nm; and (ii) detecting the emission intensity and / or lifetime at one or more wavelengths in the range of from about 500 nm to about 570 nm.

[0239] Embodiment 42: The method of any one of Embodiments 1-41, wherein the dipyrromethane-BFi derivative comprises a labile or reactive group.

[0240] Embodiment 43: The method of any one of Embodiments 1-42, wherein the dipyrromethane-BFi derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

[0241] Embodiment 44: The method of Embodiment 1-43, wherein the probe is conjugated via position 8 (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

[0242] Embodiment 45: The method of Embodiment 44, wherein the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative comprises a labile or reactive group at position 8 (meso position).

[0243] Embodiment 46: The method of any one of Embodiments 1-45, wherein the>probe is of Formula, wherein: R1, R2, R3, R4, R5, R6, R7and R8are 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, cynao, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, optionally at least one of R1, R2, R3, R4, R5, R6, R7and R8, is a labile or leaving group, and any 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) independently selected substituents from the group consisting of halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTacylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.

[0244] Embodiment 47: The method of any one of Embodiments 1-46, wherein the probe comprises a modified 8-thioether-BODIPY dye.

[0245] Embodiment 48: The method of any one of Embodiments 1-47, wherein theprobe is of structure:.

[0246] Embodiment 49: A method for barcoding a plurality of target analytes, the method comprising: ligating a barcode peptide to target analytes in a plurality of target analytes via a terminal amido acid in the barcode peptide, wherein the terminal amino acid is a modified or non-natural amino acid, and wherein a barcode peptide conjugated to a first analyte in the plurality is distinguishable from a barcode peptide conjugated to a second analyte in the plurality, and wherein the first and second analyte are different.

[0247] Embodiment 50: The method of Embodiment 49, wherein the barcode peptide is conjugated to the analyte via a linker.

[0248] Embodiment 51 : The method of Embodiment 50, wherein the linker is a peptide linker.

[0249] Embodiment 52: The method of Embodiment 52, wherein the peptide linker comprises a recognition amino acid sequence for a peptide ligase.

[0250] Embodiment 53: The method of Embodiment 53, where the peptide ligase is selected from the group consisting of a sortase, an asparaginyl endopeptidase-derived ligase, a subtilisin-derived ligase, an intein, an ATP-dependent peptide ligase, and a cyanobactin macrocyclase, preferably the ligase is a sortase, more preferably the ligase is sortase A.

[0251] Embodiment 54: The method of any one of Embodiments 49-53, wherein the barcode peptide is conjugated by its C-terminal amino acid.

[0252] Embodiment 55: The method of any one of Embodiments 49-53, wherein the barcode peptide is conjugated by its N-terminal amino acid.

[0253] Embodiment 56: The method of any one of Embodiments 49-55, wherein the analytes are polypeptides, amino acids, nucleic acids, cells, barcodes, or any combinations thereof.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0254] Embodiment 57: The method of any one of Embodiments 49-56, wherein the modified or non-natural amino acid is in a sample.

[0255] Embodiment 58: The method of any one of Embodiments 49-57, wherein the analytes are immobilized on a surface.

[0256] Embodiment 59: The method of any one of Embodiments 49-58, wherein the analytes are immobilized on a surface after conjugating with the probe.

[0257] Embodiment 59: The method of any one of Embodiments 49-58, wherein the analytes are immobilized on a surface prior to conjugating with the probe.

[0258] Embodiment 60: The method of any one Embodiments 49-60, further comprising sequencing the barcode peptides conjugated with the analytes using the method of any one of Embodiments 4-12 or 22-48.

[0259] Embodiment 61 : The method of any one of Embodiments 10, 16 or 57, wherein the sample is a biological sample.

[0260] Embodiment 62: The method of Embodiment 61, wherein the biological sample is a biological fluid, a tissue, an organ, or a cell.

[0261] Embodiment 64: The method of Embodiment 12 or 21, wherein the method comprises a step of immobilizing the polypeptide on the surface prior to conjugating with the probe.

[0262] Embodiment 65: The method of Embodiment 12 or 21, wherein the method comprises a step of immobilizing the polypeptide on the surface after conjugating with the probe.

[0263] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLESExample 1

[0264] Post-translational modifications: Post-translational modifications (PTMs) are covalent changes to one or more amino acid side chains on a polypeptide after protein biosynthesis. PTMs impact protein structure, function, regulation, interactions, and stability. Most proteins expressed by human cells contain at least one PTM, significantly expanding proteoform diversity. The inventors have discovered inter alia modified 8-thioether-BODIPY dyes unexpectedly and surprisingly are capable of differentiating naturally occurring amino acids using fluorescence. The inventors have unexpectedly and surprisingly discovered that 8-4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTthioether-BODIPY dyes are capable of binding to the N-termini of PTM amino acid monomers, and display unique fluorescent signatures different from their unmodified amino acid counterparts. These results expand the utility of the 8-thioether-BODIPY dyes for unbiased N-terminal recognition probes for fluoro-sequencing.

[0265] Peptide reference library: The inventors have tested 8-thioether-BODIPY dyes reacted to a library of 400 different peptide fragments of the sequence: H2N-X1-X2-GRAHEARG-OH (SEQ ID NO: 1), where X1and X2are permutated with all twenty naturally occurring amino acids. Through this library study, the inventors ascertained the influence of each amino acid at the N-terminal ultimate and penultimate positions with respect to fluorescent emission, fluorescent lifetime, and brightness. As both positions effect the fluorescent response, this data set was then used as a training set for a machine learning model to predict both positions’ identities. Without wishing to be bound by a theory, degrading the N-terminal amino acid and reading the next amino acid in the sequence builds in an error correction as there are two independent attempts at assigning every amino acid within the sequence.

[0266] Multiplexing: Multiplexing protein extract different cell type samples allows for both the higher throughput of analysis on a single sample plate, but also spatial biology within a tissue sample. The use of small unnatural amino acid (UAA)-based peptide fragments that can be ligated onto the N-termini of proteins extracted from any particular cell sample can give a unique fluorescent signal separate from the natural amino acids of the proteome while allowing the same use of dye and degrader probe. By varying the length of the UAA barcode sequence, one can multiplex hundreds to thousands of cell types simultaneously while maintaining a single dye probe. Barcoded samples can then be attached covalently to a flow cell for analysis.Example 2

[0267] In this assay, the inventors measured the fluorescence emission, lifetime, and photon counts for each peptide combination. When observing fluorescent emission versus lifetime (FIGs. 8A-8B), most peptide combinations cluster together, except for those with glycine, proline, and tryptophan as the ultimate amino acid. Peptides with these amino acids at the N-terminus display a significant blue-shift in emission overall. Without wishing to be bound by theory, since glycine and proline are commonly found in peptide turn sequences, this emission change may be attributed to the dye interacting with the peptide residues due to its orientation being directed more into the peptide rather than away from it. Without wishing to be bound by4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTtheory, tryptophan, though not known for inducing specific turns in peptide structure, may cause the blue-shift due to it-stacking interactions between the dye and the indole ring of tryptophan, as suggested by the relatively low photon counts observed in the dye-peptide conjugates (FIGs. 9A-9B, 10A-10B). Other aromatic amino acids, such as tyrosine and phenylalanine, also exhibit lower photon counts, indicating that ^-stacking interactions between the ultimate amino acid and the bound dye significantly influence the optical properties of dye-peptide conjugates.

[0268] When evaluating the effects from the penultimate position (FIGs. 8B, 9B, 10B), a few significant trends were observed in the two-dimensional cluster plots. However, tyrosine and tryptophan at the penultimate position consistently blue-shifted the fluorescent emission and reduced the fluorescent lifetime of the peptide-dye conjugates compared to the average. These trends become more apparent when the data are parsed into individual ultimate and penultimate amino acid types for fluorescent emission (FIG.11) and fluorescent lifetime (FIG.12).

[0269] In these one- and two-dimensional plots, the inventors observe a significant amount of clustering among peptide-dye conjugate combinations. However, when applying dimensionality reduction analysis such as uniform manifold approximation and projection (UMAP), a commonly used tool in biology, the inventors detect more distinct clustering of peptides with the same ultimate N-terminal amino acid (FIG. 13).

[0270] Conjugates that could not be easily separated using two-dimensional plots now show clear clustering, such as those with methionine (M), aspartate (D), and histidine (H) at the ultimate N-terminus. Similarly, amino acids that exhibited separation in two- dimensional plots, such as glycine (G), proline (P), tryptophan (W), and tyrosine (Y), remain discernable in this analysis, farther supporting the clustering by ultimate N-terminal residues. In contrast, when analyzing the penultimate position using supervised UMAP (FIG.14), no clear clustering is observed. Without wishing to be bound by theory, this suggests that the N-terminal residue primarily dictates the optical properties of the dye-peptide conjugates. While the inventors did detect some influence from the penultimate position, the comprehensive data show that the optical identification of the N-terminal amino acid is possible. Additionally, these results indicate that a machine learning algorithm could be employed to better predict amino acid identity based on the fluorescent properties of the dye-peptide conjugates.Protein Fragment and Peptide Analysis

[0271] Since the penultimate amino acid displayed significant effects on the overall fluorescent emission and lifetime, the inventors wanted to assess the influence of the rest of the4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTpeptide on the optical readout of the dye conjugate. Protein secondary structures are highly variable and sensitive to their sequence and local environment. The inventors had already observed that proline and glycine, when at the N-terminus, significantly affect emission and lifetime due to their turn-inducing nature, which forces greater interaction between the dye and the peptide through proximity. While this effect was reduced when these residues were in the penultimate position, it was necessary to investigate how the conformational shape of a peptide affects the fluorescent profile of the dye-peptide conjugate.

[0272] The inventors initiated this study by comparing signals obtained from protein fragments or peptides to our initial peptide-dye conjugate library, using commercially available peptide fragments with the same ultimate and penultimate sequence: GRGDS, RGDS, bradykinin, bradykinin 2-9, a KRAS fragment, and angiotensin 1. When measuring the fluorescent emission of these fragments conjugated with probe (4.1) in Tris buffer, the inventors observed significant differences in the emission between the peptide-dye conjugates and their corresponding reference peptides with the same two final amino acids, except for the KRAS fragment (FIG. 15).

[0273] Without wishing to be bound by theory, it is expected that these spectral variations between dye-peptide conjugates are due to conformational changes in the peptide caused by other amino acids in the sequence. Computational analysis using DFT geometry optimization of some dye-peptide conjugates revealed key reasons for the differences in emission spectra (data not shown).

[0274] In the geometry optimization of dye-peptide conjugates of probe (4.1) with GRGDS and the reference peptide GRGRAHEARG (SEQ ID NO: 2) (data not shown), both peptides hydrogen bond with one of the central fluorines of the dye core. However, GRGDS achieves this with the alcohol group of serine, while GRGRAHEARG (SEQ ID NO: 2) uses the penultimate arginine for hydrogen bonding. This is particularly striking, as both peptides share the first three amino acids, unlike most other peptides in this study, which share only the first two. Despite these similarities, the method of conformational binding to achieve hydrogen bonding differs, with GRGDS relying on the C-terminal amino acid.

[0275] To overcome these conformation-based interactions, the inventors investigated whether denaturing the peptide could reduce their influence on the conjugated dye by forcing the peptide into its most linearized state. The inventors tested increasing concentrations of denaturing agents: urea and guanidinium chloride. As the concentration of these denaturants increased, the emission spectra of the dye-peptide fragments and their corresponding reference peptides converged (FIG. 16).4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0276] At the highest concentrations, 8 M urea and 6 M guanidinium chloride, the peaks completely overlapped, and the Pearson correlation between the peaks was nearly or fully correlated (FIGs. 17 and 18).

[0277] These results are promising; without wishing to be bound by theory, they suggest that conformationally driven interactions between the peptide and the bound dye can be significantly reduced or even nullified by adding a denaturant. This allows for the inference that the reference dataset of dye-peptide conjugates from the library of 400 different peptide combinations is representative of most peptides that end with those specific ultimate and penultimate residues. Therefore, this dye-peptide library can be utilized as a reference dataset in a machine learning platform for peptide identification via fluoro-sequencing of the N-terminus over several rounds of optical reads and degradation cycles.

[0278] Following the denaturation study, the inventors performed fluoro-sequencing and pseudo-fluoro-sequencing by conjugating probe (4.1) to engineered peptide fragments that were either truncated by iteratively removing N-terminal amino acids (FIG. 19A) or circularly permutated, where the N-terminal amino acid was iteratively shifted to the C-terminus (FIG.19B).Dye-peptide conjugates -with circularly permutated KRAS fragments

[0279] The inventors examined dye-peptide conjugates with circularly permutated peptide fragments of KRAS reacted with probe (4.1) in both 8 M urea and 1 M Tris for comparison. Both buffers displayed good separation between conjugates, even in two-dimensional analysis of fluorescent emission versus fluorescent lifetime (FIGs. 20 and 40). However, since the inventors previously observed similar emission profiles between peptides of interest and the dipeptide library, they decided to continue our optical analysis with Urea buffer.

[0280] When observing the KRAS peptide fragments in 8 M urea, the inventors identified key trends and similarities with both the circularly permutated dye-peptide fragments and the dipeptide library. The first two dye-peptide fragments, SYGIPFIETSAKTR (SEQ ID NO: 3) and YGIPFIETSAKTRS (SEQ ID NO: 4), both exhibited relatively low lifetimes due to the presence of tyrosine (Y) at the penultimate and ultimate positions, respectively. Once the sequence moves the tyrosine to the C-terminus for fragment GIPFIETSAKTRSY (SEQ ID NO: 5), the lifetime extends, but the emission shifts blue by more than 10 nm. A similar effect was observed in the dipeptide library when glycine (G) was the ultimate residue. The next dye-peptide conjugate, IPFIETSAKTRSYG (SEQ ID NO: 6), red-shifts the emission by more than 15 nm with no significant change in lifetime. The subsequent conjugate, PFIETSAKTRSYGI4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT(SEQ ID NO: 7), drastically blue-shifts the fluorescent emission and decreases the lifetime. This trend mirrors that of the dipeptide library. From then on, in the sequence iteration, the dye-peptide conjugates cluster around 548-557 nm for fluorescent emission and 4-4.8 ns for fluorescent lifetime. These observed trends align well with those seen in the dipeptide library.

[0281] Another trend observed between the dye-peptide conjugates in different buffers was that the conjugates tended to be significantly brighter in 8 M urea than in 1 M Tris (FIG. 21).The on-plate controls, (4.1) reacted with alanine and (4.1) reacted with water, show nearly identical photon counts between the two buffers, suggesting that this difference is not due to dye-dye aggregation. Without wishing to be bound by theory, it is possible that this increase in intensity is due to the urea breaking up intra- and inter-peptide dye interactions. When revisiting the otherpeptide conjugates tested with denaturing buffer conditions, the inventors continue to observe that the 8 M urea buffer tends to increase the brightness of the dye-peptide conjugates (FIG.22). Interestingly, 6 M guanidinium chloride also increased the brightness of the conjugates, but not to the same extent. Additionally, lowering the buffer concentration caused a precipitous decline in photon counts for most conjugates tested. Therefore, moving forward, the inventors continued using 8 M urea as the buffer for denaturing conditions.Dye-peptide conjugates -with circularly permutated Shc-SH2 fragment

[0282] The inventors then proceeded to test other circularly permutated peptide fragments to investigate whether the observed similarities between these dye-fragment conjugates and the dipeptide library persisted. The next peptide conjugate series we tested was Shc-SH2, reacted again with probe (4.1). Similar to the dipeptide library and the KRAS circularly permutated series, N-terminal glycine conjugates GEPWFHGKLAEQLR (SEQ ID NO: 8) and GKLAEQLRGEPWFH (SEQ ID NO: 9) displayed significant blue shifts from the usual cluster around 550-555 nm (FIG. 23). Additionally, the N-terminal proline conjugate, PWFHGKLAEQLRG (SEQ ID NO: 10), was also blue-shifted and had a reduced lifetime. Interestingly, the lifetime was even lower than that of the KRAS fragment PFIETSAKTRSYGI (SEQ ID NO: 7). Without wishing to be bound by theory, this is most likely due to the penultimate tryptophan (W), which exhibited a similar lifetime-lowering effect in the dipeptide library. The additive effects of proline and tryptophan significantly reduced the lifetime, making it the lowest among the dye-peptide conjugates in this series. Furthermore, tryptophan at the ultimate position in the peptide WFHGKLAEQLRGEP (SEQ ID NO: 11) also blue-shifted the emission and drastically lowered the lifetime.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTDye-peptide conjugates -with circularly permutated Src-SH2 fragments

[0283] Dye-peptide conjugates of the circularly permutated fragments of Src-SH2 (FIG.24) displayed interesting optical properties due to the sequence containing a tryptophan and tyrosine adjacent to one another. These residues have been shown in multiple cases to lower the fluorescent lifetime of the species (tyrosine) and blue-shift the emission spectrum (tryptophan). The inventors observe their influence even before their occurrence at the ultimate or penultimate positions. For instance, EEWYFGKITRREQA (SEQ ID NO: 12) is significantly blue-shifted and is similar to the conjugates of EWYFGKITRREQAE (SEQ ID NO: 13) and WYFGKITRREQAEE (SEQ ID NO: 14), despite the tryptophan and tyrosine being in the third and fourth positions from the N-terminus, respectively. When the tryptophan is moved to the C-terminus in the conjugate YFGKITRREQAEEW (SEQ ID NO: 15), the emission red-shifts to roughly 555 nm, which aligns with what we have observed for other N-terminal tyrosine conjugates in both KRAS and the dipeptide library. From the N-terminal tyrosine, the lifetime is extended, and the emission is blue-shifted as phenylalanine (F) and then glycine (G) are cycled through as the N-terminal amino acid, followed by a subsequent red-shift in emission after the glycine shift to the C-terminus in KITRREQAEEWYFG (SEQ ID NO: 16).Dye-peptide conjugates -with circularly permutated Src-SH3 fragments

[0284] Dye-peptide conjugates of circularly permutated Src-SH3 fragments display trends similar to those observed in other circularly permutated peptide series and the dipeptide library (FIG. 25). The initial conjugates, TFVALYDYESRTET-ALYDYESRTET (SEQ ID NO: 17), exhibit 5 nm fluctuations in emission and small changes to the fluorescent lifetime. However, when tyrosine occupies the penultimate position, we observe a dramatic decrease in fluorescent lifetime, which is maintained while a tyrosine is in the penultimate or ultimate positions (LYDYESRTETTFVA-YESRTETTFVALYD) (SEQ ID NO: 18). There are also spectral shifts between conjugates during this portion of the series, allowing for discernment between the conjugated species. Once the tyrosines have been moved from the N-terminus, the fluorescent lifetime extends again, approximately remaining stable until conjugate ETTFVALYDYESRT (SEQ ID NO: 19). This aberrant optical signal for this conjugate is significantly blue-shifted in emission and has a low lifetime.Dye-peptide conjugates -with circularly permutated Angiotension 1 fragments4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0285] Dye-peptide conjugates reacted with circularly permutated fragments of angiotensin 1 displayed one of the best optical separations between each stepwise shift (FIG. 26). The inventors observed good spectral and lifetime separation between initial conjugates as tyrosine quickly cycles through the penultimate and ultimate positions. Following the movement of the tyrosines to the C-terminus, the inventors see the typical increase in lifetime for IHPFLDRVY. For conjugate HPFLDRVY, there is a large spectral red-shift of 10 nm, followed by an immediate 18 nm blue-shift for PFLDRVYH. Additionally, the presence of proline at the ultimate position decreased the lifetime by more than a nanosecond, which corresponds well with other ultimate proline conjugates. The final three conjugates, FHLDRVYIHP-LDRVYIHPFH (SEQ ID NO: 20), exhibited good spectral separation of 5-10 nm per step.Dye-peptide conjugates -with continuously truncated KRAS fragments

[0286] The final series of peptide fragments tested in solution with probe (4.1) were continuously truncated fragments of KRAS (FIG. 27), in which the N-terminal amino acid is iteratively removed from the sequence rather than shifted to the C-terminus, as was done with the circularly permutated conjugates previously studied. This model provides a more accurate portrayal of what iterative fluoro-sequencing of a peptide’s N-terminus would look like. The conjugates display trends similar to those observed in the circularly permutated KRAS dye-peptide conjugates. Conjugates SYGIPFIETSAKTRQRVEDAFYTLV (SEQ ID NO: 21) and YGIPFIETSAKTRQRVEDAFYTLV (SEQ ID NO: 22) exhibited lower fluorescent lifetimes, followed by a rise in lifetime and a blue-shift in emission for the next conjugate, GIPFIETSAKTRQRVEDAFYTLV (SEQ ID NO: 23), which bears glycine at the N-terminus. Conjugate PFIETSAKTRQRVEDAFYTLV (SEQ ID NO: 24) showed both a significant blueshift in emission and a reduction in lifetime, consistent with other N-terminal proline conjugates. Interestingly, we observed similar emission for the final conjugate, RQRVEDAFYTLV (SEQ ID NO: 25), compared to its counterpart in the circularly permutated KRAS fragment, RSYGIPFIETSAKT (SEQ ID NO: 26), but with an increased lifetime. Without wishing to be bound by theory, this change is likely attributed to the shift in penultimate residue, which affected the lifetime of the species and aligns with observations from the RQ peptide library conjugate.

[0287] When comparing the emission spectra of the two KRAS fragment conjugates with similar ultimate and penultimate residues, excluding RSYGIPFIETSAKT (SEQ ID NO: 26), and RQRVEDAFYTLV RQRVEDAFYTLV (SEQ ID NO: 25), we observe good agreement among most conjugates (FIG.28). While some differences exist between the conjugate series,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTparticularly TSAKTRQRVEDAFYTLV (SEQ ID NO: 27) and TSAKTRSYGIPFIE (SEQ ID NO: 28), the overall similarity is substantial. However, the Euclidean distance between the dye conjugates indicates that the correlation among almost every conjugate pair with the same N-terminus, except for TS, is very low, with most being below 0.5 (FIG. 29). Additionally, the inventors observed consistent trends between the two with tyrosine, proline, and glycine N-terminal species.Discussion of dye-peptide conjugates in solution

[0288] Following the analysis of the dye-peptide conjugate series in solution, several trends were observed based on dye-peptide interactions. One key trend was the difference in brightness between conjugates in 1 M Tris buffer versus 8 M Urea buffer. Earlier observations indicated that the use of a denaturing buffer like 8 M Urea helped maintain the consistency of optical signals between peptides with similar ultimate and penultimate amino acids at then- respective N-termini. Furthermore, the inventors noted a consistent trend where dye-peptide conjugates are generally brighter in 8 M Urea than in 1 M Tris (FIG. 30).Without wishing to be bound by theory, this increase in brightness may result from a reduction in inter- and intramolecular through-space interactions between the dye and the peptide, achieved through the denaturation of the peptide. Such interactions can adversely affect the optical properties of the dye, causing changes not only in emission but also in brightness.

[0289] Without wishing to be bound by theory, since there is good agreement between emission values from different peptides with the same N-terminal ultimate and penultimate amino acids in 8 M Urea, it appears that the optical signal obtained arises from the direct inductive and through-space effects of those residues on the dye. The large separation between different peptide conjugates provides additional evidence that the dye probe (4.1) is sensitive to the intramolecular interactions between the dye and the N-terminus of the conjugated peptide, rather than to intermolecular interactions.

[0290] Each pseudo-sequencing and sequencing peptide fragment series displayed significant changes in fluorescent emission and lifetime with each step change within the sequence. While some changes were more dramatic than others, prominent changes occurred at almost every step within a series. The most notable changes came from a select group of amino acids at the ultimate or penultimate positions. As observed in the dipeptide conjugate library, proline and glycine at the ultimate positions drastically blue-shifted the emission due to their conformational flexibility in proteins. Proline conjugates also reduced the lifetime of the conjugate because the geometry of proline pushes the dye closer to the rest of the peptide.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTAromatic residues, such as tyrosine and tryptophan, significantly reduced the fluorescent lifetimes of the conjugate at both the ultimate and penultimate positions. Without wishing to be bound by theory, this lifetime reduction suggests that these residues affect the dye photophysically and likely quench the excited dye species due to proximity. Tryptophan also blue-shifts the emission, indicating possible it-stacking interactions between the tryptophan indole and the dye. When examining the dye-peptide conjugates in a series, it is clear when these specific residues occupy the penultimate or ultimate positions, as well as when they are removed from the N-terminus. The fluorescent vertex maps resulting from the changes in emission and lifetime for each peptide series provide a fingerprint for the peptide sequence, demonstrating that fluoro-sequencing of peptides with a single fluorescent probe is possible.Photoinduced Degradation ofN-terminal Bound Probe

[0291] A key aspect of the proposed platform for fluoro-sequencing of polypeptides is the iterative optical readout of the N-terminal amino acid by our BODIPY-based probes, followed by degradation using our photocaged amidine probes. However, both of these probes require a free N-terminal amine to conjugate properly.

[0292] While the degradation probes can generate a new N-terminal amine via N-degradation of the N-terminal amino acid, the BODIPY-based probes require a method for removal from the N-terminus to allow the degradation probe to bind. Fortunately, the inventors discovered through mass spectrometry that the dye probes can be removed from the N-terminus and the amine recovered upon photoinduced scission in reaction with molecular oxygen (FIG.31).

[0293] BODIPY-based dyes have been shown to photodegrade in the presence of molecular oxygen and can release their substitution at the meso position via a beta scission reaction. Without wishing to be bound by theory, in the proposed mechanism (FIG.32), the dye-peptide conjugate (4.3) performs energy transfer from its triplet state to its ground state onto molecular oxygen, converting it into singlet oxygen. The singlet oxygen then undergoes a [2+2] cycloaddition at the 7- and 8-positions of the dye-peptide conjugate, forming the 1,2-dioxetane (4.4). This dioxetane subsequently undergoes a retro- [2+2], oxidatively cleaving the two halves of the dye-conjugate to form amide (4.5) and lactam (4.6).

[0294] Amide (4.5) then reacts with an additional singlet oxygen molecule in a [4+2] cycloaddition onto the pyrrole to form the bridged intermediate (4.7). This intermediate then undergoes dual -scission of both the amide connected to the peptide and the remaining aryl group. This scission reaction forms the maleimide (4.8) and the carbamoyl radical (4.9). The4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTinventors believe that the carbamoyl radical (4.9) homolytically cleaves to release the amino radical (4.10) and carbon monoxide. Carbamoyl radicals have been shown to undergo this pathway for carbon monoxide release, forming the more stable alkyl amino radical instead of abstracting hydrogen from the surrounding area to create a formamide. Additionally, the inventors do not observe formamide formation at the N-terminus via LC-MS, indicating that carbon monoxide release is most likely occurring. Finally, the alkyl amino radical (4.10) abstracts a hydrogen from the surroundings to regenerate the N-terminal amine (4.11).

[0295] To substantiate these results, the inventors performed a photobleaching experiment with probe (4.1) reacted with methylamine, both in the presence and absence of oxygen. We irradiated the dye-methylamine conjugate with 365 nm LEDs over two hours and measured the absorbance of the species at various time points. The inventors observed that when the dye conjugate was irradiated in PBS alone, there was a clear decrease in absorbance at 425 nm, indicating photobleaching occurred (FIG. 33A). However, when we degassed the PBS with nitrogen and added the triplet quencher and antioxidant Trolox, we observed little to no change in the absorbance at 425 nm of the dye conjugate (FIG.33B).

[0296] Without wishing to be bound by theory, this finding further validates the proposed mechanism for oxidative photodegradation of BODIPY dye-peptide conjugates, providing a method not only for removing the dye from the N-terminal amine using oxygen but also for extending the longevity of the dye-peptide conjugate through oxygen scavengers or low-oxygen systems. In doing so, the inventors can increase the amount of photophysical data gathered from dye conjugates, enhancing the confidence in the accuracy of each optical read.Surface testing of optimized dye and degrader probes conjugated to peptides

[0297] Solution data for circularly permutated and truncated dye-peptide conjugates showed promising proof-of-concept for the dye's ability to identify N-termini by their ultimate and penultimate residues. However, the ultimate goal for this technology is to fluoro-sequence extracted, unknown peptides, and measuring the resulting mixture of dye-peptide conjugates in solution would obfuscate the obtained signal. To remedy this issue, the inventors sought to immobilize the peptides onto a solid support. This way, the peptide of interest will remain in a known location throughout the sequencing process. Additionally, the inventors could obtain multiple signals from different peptides bound elsewhere on the solid surface using a hyperspectral camera. Since functionalized glass as a solid support is commonly used for hyperspectral and multicolor fluorescence measurements, as well as single-molecule testing,4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTwe decided to use fiinctionalized cover glass to begin our studies of peptide fluoro-sequencing using our probes.Optical separation of circularly permutated peptides on glass surface

[0298] We initiated our study of the fluoro-sequencing platform on glass surfaces using a modified version of circularly permutated KRAS, where the C-terminus contains an azido lysine. As the sequence permutates, the N-terminal amino acid shifts to the C-terminal penultimate position rather than the ultimate position, as studied in solution. The C-terminal azido lysine was chosen because it can easily be used to orthogonally conjugate to a fiinctionalized surface via a strain-promoted azide-alkyne click reaction (SPAAC), while leaving the remaining peptide sequence free.

[0299] In a proof-of-concept experiment, the inventors aimed to verify that optical separation is observable between the different dye-peptide conjugates on the glass surface, and to compare these results to the solution-phase data of the circularly permutated KRAS conjugates. To passivate and fimctionalize the surface, the inventors used a 1:1,000,000 ratio of DBCO-PEG4-triethylsilane to mPEG5 -triethylsilane on a precleaned glass surface, employing modem single-molecule surface passivation chemistry. The inventors then conjugated the peptide to the DBCO molecules bound to the surface and subsequently reacted with probe (4.1), followed by washing to remove excess probe (FIG.34).

[0300] From the experiment, the inventors observed clear and distinct optical shifts for each dye-peptide conjugate. Similar to the solution-phase data, we found that glycine and proline at the N-terminus drastically blue-shift the emission spectra. Additionally, tyrosine at the ultimate and penultimate positions lowers the fluorescent lifetime. One clear difference between the solution phase and surface-bound conjugates is that the surface-bound versions are significantly blue-shifted, and their lifetime values have been reduced relative to their solutionphase counterparts. This optical shift is most likely due to the electrostatic interactions and the proximity of the dye-peptide conjugate to the glass surface, which could be affecting the photophysics of the bound dye. These interactions have been shown to reduce fluorescent lifetime for other fluorescent dyes bound to glass surfaces via a linker. While these interactions could potentially be minimized by optimizing passivation chemistry or by extending the PEG spacer between the dye-peptide conjugate and the glass surface, the initial data gathered displayed promising results, indicating that optical fingerprinting of a peptide bound to the surface could be achieved.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTN-degradation on surface bound peptides

[0301] Following optical separation experiment of circularly permutated KRAS peptides bound to a glass surface, the inventors turned to performing N-degradation using our degradation probe (4.12) on immobilized proteins. The inventors first conducted a model experiment to determine the on-binding kinetics of probe (4.12) to a peptide’s N-terminus, as well as the off-binding kinetics of the N-degradation on glass-bound peptides. The inventors tested these parameters using an amine-reactive dye, AlexaFluor488-NHS (4.13), measuring the loss of fluorescence in the on-binding experiment and the gain in fluorescence in the off-binding experiment (FIGs. 36A-36C). As N-termini become progressively occupied by the conjugated degrader during the on-binding experiment, the inventors expect a drop in the bound fluorescent dye. Conversely, in the off-binding experiment, the inventors generate new N-termini as the experiment progresses, thereby increasing the available locations for a fluorescent dye to bind.

[0302] From the on-binding experiment with probe (4.12) and surface-bound peptide SAKTRSYGIPFIETK(N3) (SEQ ID NO: 29), the inventors observed that a minimum was reached at approximately 20-30 minutes when the concentration was 10 pM in pH 9.2 bicarbonate buffer (FIG.37 A). These conditions and concentrations were chosen as they match those used in the dye probe binding experiments. Even after 5 minutes, there was a rapid drop in fluorescent signal compared to the initial starting point. While the fluorescence did not drop entirely to zero, without wishing to be bound by theory, the inventors believe that the plateau observed after 20 minutes indicates that the reaction had reached its limit under the given conditions, and the remaining signal could be attributed to non-specific adsorption of (4.13) to the surface. This fast on-binding time is very promising, as it shows that we can quickly conjugate our degrader probe to a surface-bound peptide.

[0303] Following the on-binding experiment, the inventors performed an off-binding experiment where the peptide, SAKTRSYGIPFIETK(N3) (SEQ ID NO: 29), was pre-reacted with probe (4.12) prior to surface conjugation to ensure consistent starting material across experiments. The conjugated surface-bound probe-peptide was then irradiated with 365 nm LEDs for 5 minutes in pH 10 buffer and allowed to react for incrementally increasing amounts of time (FIG. 37B). At the 5-minute time point, the inventors observed that the fluorescent signal increased due to the N-degradation reaction, which forms new N-termini following cyclization. Additionally, there was little to no signal at the zero-time point. Without wishing to be bound by theory, this suggests that (4.13) is not reactive to the guanidinylated N-termini. The fluorescent signal continued to increase until it dropped precipitously at 60 minutes, after4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTwhich it remained stable for the remaining time points. Without wishing to be bound by theory, this drop may be due to fluorescent quenchingcaused by the high abundance of (4.13) bound to the surface proteins. This quenching suggests that many liberated N-termini from degradation were available for reaction with the fluorescent probe, although it also means that the inventors cannot accurately determine the kinetic rate of degradation from this assay.

[0304] Following these preliminary degradation assays, the inventors proceeded with a single degradation event using degradation probe (4.12), followed by N-terminal labeling and optical identification with dye probe (4.1). For this assay, the inventors again used peptides pre-reacted with probe (4.12) prior to surface conjugation. The surface-bound probe-peptide conjugates were then irradiated with 365 nm LEDs for 5 minutes and incubated in pH 10 buffer for 5 hours. Afterward, the degraded peptides were reacted with dye probe (4.1). As a control, peptides not reacted with degrader probe (4.12) were subjected to the same conditions and reacted with dye (4.1). Since the inventors used the circularly permutated KRAS peptides, the control served a dual purpose: showing the change in optical signal following degradation, and providing a comparison for what the degraded peptide should look like. For instance, the degraded peptide YGIPFIETSAKTRSK(N3) (SEQ ID NO: 30) should appear different from its non-degraded counterpart, but similar to the non-degraded peptide GIPFIETSAKTRSYK(N3) (SEQ ID NO: 31).

[0305] From this study, the inventors clearly observe significant shifts in fluorescent emission (FIG. 37 A) and lifetime (FIG. 37B) following degradation, suggesting that N-degradation of the surface-bound proteins did occur. Additionally, the optical signals after degradation show good agreement with the circular permutation, indicating that the signal obtained from the peptide is likely a result of a single degradation event, which corresponds well with the sequence (FTGs.38A-38D).

[0306] There are, however, a few exceptions that display different emission and lifetime values, the most notable being the degraded GIPFIETSAKTRSYK(N3) (SEQ ID NO: 31) and the non-degraded IPFIETSAKTRSYGK(N3) (SEQ ID NO: 32). This could be due to the measurements being taken in PBS, which does not minimize dye-peptide interactions as effectively as denaturing buffers like urea or guanidinium chloride. Alternatively, it could be the result of an incomplete reaction of degrader probe (4.12) with the peptide GIPFIETSAKTRSYK(N3) (SEQ ID NO: 31). Since the peaks were averaged across all objects, this may represent a combination of the more blue-shifted GIPFIETSAKTRSYK(N3) (SEQ ID NO: 31) and the more red-shifted IPFIETSAKTRSYK(N3) (SEQ ID NO: 32), which is why the result falls roughly between the two.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0307] This degradation and optical fingerprinting proof-of-concept assay is highly promising, as it demonstrates that the platform for fluoro-sequencing can be applied to surfacebound proteins.

[0308] From the solution-phase dye-peptide data, the inventors have demonstrated that the optimized probe (4.1) can optically distinguish N-terminal amino acids of a protein. These effects were particularly noticeable with certain N-terminal amino acids. Glycine caused a blue shift in the emission of the dye-conjugate, while proline not only blue-shifted the emission but also reduced the fluorescent lifetime. Without wishing to be bound by theory, these shifts are most likely due to the altered geometric conformation of the dye in relation to the rest of the peptide. Since both glycine and proline are involved in secondary structure peptide turns, it is reasonable that the geometric conformation of the dye bound to these peptides differs from its interaction with other amino acid subtypes. Proline also likely decreases the lifetime due to the increased conformational restriction caused by its secondary amine. This restriction forces the bound dye to interact more closely with the peptide, leading to an increase in through-space interactions, which could explain the reduced fluorescent lifetime.

[0309] Interestingly, proline in its monomeric form did not exhibit these photophysical effects, further supporting the hypothesis that the reduction in lifetime results from forced interaction with the peptide. Additionally, tyrosine and tryptophan both decreased the lifetime of the bound dye when present at the N-terminal or penultimate positions. Without wishing to be bound by theory, this reduction in lifetime is likely due to the photo-quenching effects of these residues when in close proximity. Tryptophan also showed a more significant blue shift in emission compared to tyrosine, suggesting stronger through-space interaction between the indole ring and the bound dye.

[0310] While tyrosine and tryptophan caused the most significant differences from the penultimate positions, the inventors observed clear influences from all combinations of ultimate and penultimate amino acids on the bound dye probe. These effects can be exploited in the case of the dye-dipeptide library, where the emission and lifetime of the conjugates can serve as a reference dataset for a machine learning algorithm to identify both the N-terminal and penultimate amino acids. Since the inventors observed that high denaturing conditions reduce inter- and intramolecular interactions between the bound dye and the ultimate and penultimate amino acids, this method can be used as a dual identification factor following degradation. For example, if the N-terminal sequence of a peptide, SYGIPFIETSAKTR (SEQ ID NO: 3), most likely corresponds to SY, then the subsequent sequence should also correspond4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTto YG (FIG.39). This dual identification of tyrosine provides greater confidence in identifying any single amino acid within a polypeptide sequence.

[0311] The circularly permutated and iteratively truncated series of dye-peptide conjugates revealed significant optical differences between iterations of a peptide sequence, even in this proof-of-concept model. These differences in fluorescent emission and lifetime between each dye-peptide conjugate generated a vertex map as we permutated through the sequence for each respective peptide series. These maps also serve as identifiers for the peptide sequence. In combination with the dual identification reading frame of the peptide's identity, these maps help build a more comprehensive picture of the overall peptide’s primary sequence. Furthermore, they serve as an error-correction tool by helping to identify discrepancies or point differences observed from the dipeptide library, such as the TS peptide signals between KRAS fragments.

[0312] The inventors continued to observe these maps forming from optical separation on surface-bound peptides, further demonstrating the optical separation of our dye probe bound to peptides and the ability to optically fluoro-sequence peptides. Since the inventors have also demonstrated single degradation of peptides bound to the surface followed by dye labeling and identification, the inventors believe that, with optimization, this platform using two-probe technology for iterative N-terminal degradation and fluorescent fingerprinting can be used for fluoro-sequencing of unknown peptides from cellular extracts.Discussion

[0313] From the solution-phase dye-peptide data, the inventors have demonstrated that our optimized probe (4.1) can optically distinguish N-terminal amino acids of a protein. These effects were particularly noticeable with certain N-terminal amino acids. Glycine caused a blue shift in the emission of the dye-conjugate, while proline not only blue- shifted the emission but also reduced the fluorescent lifetime. Without wishing to be bound by theory, these shifts are most likely due to the altered geometric conformation of the dye in relation to the rest of the peptide. Since both glycine and proline are involved in secondary structure peptide turns, it is reasonable that the geometric conformation of the dye bound to these peptides differs from its interaction with other amino acid subtypes. Proline also likely decreases the lifetime due to the increased conformational restriction caused by its secondary amine. This restriction forces the bound dye to interact more closely with the peptide, leading to an increase in through-space interactions, which could explain the reduced fluorescent lifetime.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0314] Interestingly, proline in its monomeric form did not exhibit these photophysical effects, farther supporting the hypothesis that the reduction in lifetime results from forced interaction with the peptide. Additionally, tyrosine and tryptophan both decreased the lifetime of the bound dye when present at the N-terminal or penultimate positions. This reduction in lifetime is due to the photo-quenching effects of these residues when in close proximity. Tryptophan also showed a more significant blue shift in emission compared to tyrosine, suggesting stronger through-space interaction between the indole ring and the bound dye.

[0315] While tyrosine and tryptophan caused the most significant differences from the penultimate positions, the inventors observed clear influences from all combinations of ultimate and penultimate amino acids on the bound dye probe. These effects can be exploited in the case of the dye-dipeptide library, where the emission and lifetime of the conjugates can serve as a reference dataset for a machine learning algorithm to identify both the N-terminal and penultimate amino acids. Since the inventors observed that high denaturing conditions reduce inter- and intramolecular interactions between the bound dye and the ultimate and penultimate amino acids, this method can be used as a dual identification factor following degradation. For example, without wishing to be bound by theory, if the N-terminal sequence of a peptide, SYGIPFIETSAKTR (SEQ ID NO: 3), most likely corresponds to SY, then the subsequent sequence should also correspond to YG (FIG. 39). This dual identification of tyrosine provides greater confidence in identifying any single amino acid within a polypeptide sequence.

[0316] The circularly permutated and iteratively truncated series of dye-peptide conjugates revealed significant optical differences between iterations of a peptide sequence, even in this proof-of-concept model. These differences in fluorescent emission and lifetime between each dye-peptide conjugate generated a vertex map as we permutated through the sequence for each respective peptide series. These maps also serve as identifiers for the peptide sequence. In combination with the dual identification reading frame of the peptide's identity, these maps help build a more comprehensive picture of the overall peptide’s primary sequence. Furthermore, they serve as an error-correction tool by helping to identify discrepancies or point differences observed from the dipeptide library, such as the TS peptide signals between KRAS fragments.

[0317] The inventors continued to observe these maps forming from optical separation on surface-bound peptides, further demonstrating the optical separation of our dye probe bound to peptides and the ability to optically fluoro-sequence peptides. Since the inventors have also demonstrated single degradation of peptides bound to the surface followed by dye labeling and4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTidentification, the inventors believe that, with optimization, this platform using two-probe technology for iterative N-terminal degradation and fluorescent fingerprinting can be used for fluoro-sequencing of unknown peptides from cellular extracts.Further applications

[0318] One key observation noted as the inventors transitioned from solution-phase fluorescent measurements to surface-bound dye-peptide conjugates was the difference in signal obtained. While the trends observed in the fluorescent signal from surface-bound dye-peptide conjugates were similar to those in solution phase, the emission was significantly blue-shifted, and the lifetimes were shorter. This change in signal could be due to electrostatic interactions between the bound dye and the surface frmctionalization, but it may also result from the inherent difference between bound and free-floating dye-peptide conjugates.

[0319] Thus, a key next step in determining peptide identities via fluoro-sequencing will be to remeasure the dipeptide library on C-terminally bound peptides. Ideally, the inventors would continue our study with peptides containing C-terminal azido-lysine for easy conjugation to DBCO fr ctionalities on the glass surface. From this study, we can reestablish trends that arise due to specific N-terminal ultimate and penultimate amino acid residues.Chemically modifying native peptides for surface immobilization

[0320] Azido-lysines do not naturally occur in the human proteome. Therefore, a method for selectively conjugating peptides to a functionalized glass surface must be established. One option is to use the method MacMillan and Anslyn, where peptides are selectively modified at their C-terminus using a photocatalyzed decarboxylation and Michael addition. The Michael acceptor contains a reactive handle, such as an alkyne, which can then undergo copper-assisted click chemistry with an azide-frmctionalized glass surface.

[0321] The C-terminal decarboxylation chemistry has been shown to work well and be site-selective based on the redox potential of the C-terminus compared to aspartate and glutamate.

[0322] An alternative option for C-terminal residue-specific peptide immobilization is to exploit the reactivity of specific amino acids. Arginine is one such residue, as it can be selectively conjugated via a condensation reaction with glyoxals. Additionally, clostripain is an endopeptidase that cleaves at the C-terminus of arginine within a peptide. By coupling these two techniques, the inventors can digest cellular extract with clostripain to ensure that all C-terminal residues are arginines (FIG. 42). Then, by reacting the arginines with 4-azidophenyl4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTglyoxal (4.18), the inventors can create an azide handle (4.19) to proceed with the SPAAC chemistry previously established on a fiinctionalized glass surface.

[0323] A protein digestion step will be necessary to liberate new N-termini from proteins, as most N-terminal amines are acylated. This approach also provides an initial identification read for our peptides, as the inventors can infer that the amino acid preceding the new N-terminus was arginine.Surface passivation for prolonged silanization

[0324] Currently, surface passivation and fimctionalization chemistry rely on a single attachment point to the glass surface via a siloxy ester. Unfortunately, the dye and degrader probe conjugation, as well as degradation cyclization, occur in basic aqueous media, which hydrolyzes siloxy esters. This loss of attachment results in the gradual loss of immobilized peptides, especially with prolonged exposure to basic aqueous media.

[0325] To address this issue, multiple attachment points to the glass surface using siloxy dendrimers can be used. Polypodal siloxy esters have been shown to substantially increase surface stability. The inventors can synthesize early dipodal versions to test stability (FIG. 43) by coupling the DBCO-PEG or mPEG as an NHS ester (4.20) to a secondary amine with two siloxy pendant chains attached (4.21). The resulting amide will have two siloxy groups for surface attachment, allowing for direct measurement the loss of surface-bound fluorescent material over time.Fluoro-sequencing -within expanded cells

[0326] The ultimate goal of this technology is to perform fluoro-sequencing of proteins and peptide fragments within a cell. This method could provide spatial information on the proteome architecture at the single-cell level, offering a level of detail not achievable with immunofluorescence assays alone. Each protein, along with its location and quantity, can be accurately mapped, leading to a deeper understanding of cellular biology, disease states within cells, and transient protein post-translational modifications.

[0327] To achieve intracellular fluoro-sequencing, similar to glass-bound fluoro-sequencing, proteins must remain immobilized throughout the iterative reads and degradation steps to maintain confidence in protein identification. Additionally, spatial separatation of proteins from each other is necessary, as cells typically contain 103-104 proteins per square micron. To meet both of these requirements, expansion microscopy, which linearly expands cells while preserving relative spatial integrity can be used.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0328] Expansion microscopy has been used not only for intracellular genomic sequencing but more recently for spatial, single-cell protein identification via immunostaining and protein immobilization. This technique maintains the spatial integrity of the proteome by anchoring proteins to a hydrogel formed through polymerizable monomers on surface-exposed lysine side chains. Once the hydrogel forms, it expands in the presence of water, linearly separating the bound proteins. Depending on the method used, expansion microscopy can expand the cell to 20-50 times its original linear volume, reducing the number of proteins per square micron to 100-101.

[0329] Using this method, one can expand a cell, lyse the anchored, immobilized proteins to create new N-termini, and then iteratively fluoro-sequence each protein within the cell (FIG.44).

[0330] Without wishing to be bound by a theory, the N-terminal reactive dye probe and the N-terminal degrader probe, is the only fluoro-sequencing technique that is selective and mild enough to fully sequence, identify, and map the entire intracellular proteome using expansion microscopy. The combination of these techniques could truly revolutionize our understanding of the proteome within a cell.ExperimentalGeneral information

[0331] Unless otherwise noted, all reagents were purchased from commercial suppliers without further purification. Dipeptide library peptide fragments of the sequence “H2N-X1X2GRAHEARG-OH” (SEQ ID NO: 1) were purchased from Vivitide as trifluoroacetic acid salts. Circularly permutated and iteratively truncated peptide series including C-terminal azide series were purchased from Biomatik as trifluoroacetic acid salts. All other peptides tested were purchased from Sigma Aldrich as formic acid salts.

[0332] Analytical UPLC-MS experiments were performed using a Waters Acquity (ultraperformance liquid chromatography) with a binary solvent manager, an SQ mass spectrometer, a Waters 2996 photodiode array (PDA) detector, and an evaporative lightscattering detector (ELSD). The column used was an ACQUITY UPLC BEH Cl 8 Column, 130A, 1.7 pm, 2.1 mmX 50 mm.Synthetic ProceduresDye-peptide conjugation4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0333] In a 1536 polypropylene well plate, 1 pL of 10 mM stock solution of dye (4.1) was added to 6 pL 100 mM pH 9.2 sodium bicarbonate buffer followed by 3 pL 50 mM peptide solution in deionized water. This dilution resulted in the final concentration of 1 mM of dye in each well. After addition of all reagents to their respective wells, the multiwell plate was sealed with PCR film and stirred on a BioShake iQ plate shaker at 2000 rpm at ambient temperature for 16 hours. After 16 hours, most dyes were fully consumed and conjugated to their respective peptides. There is a stark optical shift of the solution from pink to yellow-green upon full reaction of the dye with the peptide. 1 pL of each of the reacted dye-peptide conjugate were pipetted into 99 pL of 1 M pH 7.6 Tris buffer or 8 M pH 7.6 Urea buffer in a 384-glass bottom well plate. This dilution changes the concentration of the AADC to 10 pM. 95 pL of each AADC solution was discarded, and 45 pL of 1 M pH 7.6 Tris or 8 M pH 7.6 Urea buffer was added back to each well making the final concentration of dye-peptide conjugate at 1 pM.Spectral and lifetime imaging in solution

[0334] Samples containing the dye conjugated with peptides were pipetted into 384 well glass bottom plates with a #1.5H cover glass (Cellvis, P384-1.5H-N). The imaging of these plates was performed with a Leica stellaris SP8 confocal microscope with a white light laser (WLL) source, 20x 0.75 NA objective (Model: HC PL APO CS2), and the FALCON time correlated single photon counting (TCSPC) fluorescence lifetime add-on. For all the dye-peptide conjugates, spectral acquisition was performed with 440 nm laser excitation, at 85.4 pW and repetition rate of 80 MHz, with a lOnm bandwidth notch filter at 445 nm was used to eliminate detection of excitation light. Fluorescence emission spectrums were acquired over a 1502 pm2region of interest (128 px by 128 px) with 30.78 ps dwell time, at 10 nm optical bandwidth and 5 nm step sizes over the 450 nm to 650 nm range. Buffer-only measurements were also made for background subtraction.

[0335] For fluorescence lifetime measurements, the arrival times of the photons were counted between 450-650 nm using 440 nm excitation and 20 MHz repetition rate (9.41 pW). The lower power eliminates photon pileup errors and the lower repetition rate better captures the full-time binned fluorescence decay curve.Extraction and analysis of solution data

[0336] Following the 128x128 xyX image acquisition, 5-by-5 super-pixels were generated for each image and then averaged, before background subtraction of buffer-only solution spectra. The resulting spectra was smoothed using a Savitzky-Golay filter and emission peaks were calculated using interpolation. Peaks and spectra shown here represent the average of 54910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTseparate ROIs per well. Observed brightness is the sum of the photon counts for each spectral bin. Spectral data analysis was completed in Python. The time-binned fluorescence decay data was fitted to a tri-exponential decay curve, and the mean intensity-weighted lifetime was extracted for each sample. Fluorescence lifetime fitting was computed using the Leica FALCON software and exported to an excel spreadsheet. Fluorescence lifetime was aligned with spectral results using Python.

[0337] Surface passivation and peptide conjugation: 384 well glass bottom plates with a #1.5H cover glass (Cellvis, P384-1.5H-N) were first cleaned with 100 pL per well of 5 M NaOH solution. The solution was allowed to clean the glass surface for 1 h prior to washing 3x with 100 pL of ddFLO. Then 100 pL of Optima grade methanol was added and allowed to sit for an additional 30 min. Following this time, the methanol was removed and the surface was dried under nitrogen stream.

[0338] Passivation and functionalization of the cleaned glass surface was performed by adding 100 pL of silanization mixture (Per 1 mL: 939 pL of Optima grade methanol, 50 pL of HPLC grade acetic acid, 10 pL of 1 M mPEG5 -triethoxy silane (Broadpharm Cat. No. BP-24080) in DMSO, and 1 pL of 1 pM DBCO-PEG4-triethoxysilane (Broadpharm Cat. No. BP-24137)) to wells required for experiment. The multiwell plate was sealed using a 384 well silicon cap mat, and stirred on a BioShake iQ plate shaker at 750 rpm at ambient temperature for 2 hours. Following this time, the silanization mixture was removed from the wells, and the wells were washed with 100 pL of Optima grade methanol 3x then dried under nitrogen stream.

[0339] Peptide conjugation and immobilization were performed by adding 100 pL of 1 pM peptide-azide solution in lx PBS to the frmctionalized well. The solution was allowed to shake under darkness on a BioShake iQ plate shaker at 750 rpm at ambient temperature for 1 hour. The conjugated wells were then washed with 100 pL of lx PBS 5x.Dye conjugation on surface immobilized peptides

[0340] To the pre-conjugated peptide surface containing wells of a frmctionalized glass backed 384 multiwell plate was added 100 pL of a 1 pM solution of dye probe (4.1) in pH 9.2 bicarbonate buffer. The solution was allowed to shake under darkness on a BioShake iQ plate shaker at 750 rpm at ambient temperature for 1 hour. The dye-peptide conjugated wells were then washed with 100 pL of Optima grade methanol 3x then 100 pL of lx PBS 3x. Then 50 pL of the final imaging buffer (ImM Trolox in lx PBS) was then added to the wells.Degrader conjugation and N-degradation on surface immobilized peptides4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT

[0341] To the pre-conjugated peptide surface containing wells of a ftmctionalized glass backed 384 multiwell plate was added 100 pL of a 1 pM solution of degrader probe (4.12) in pH 9.2 bicarbonate buffer. The solution was allowed to shake under darkness on a BioShake iQ plate shaker at 750 rpm at ambient temperature for 1 hour. Following this time, the wells were washed with 100 pL of lx PBS 5x.

[0342] The degrader-peptide conjugate wells were then taken up in pH 10 bicarbonate buffer, and irradiated with 10 W 365 nm LEDs from 3 cm away for 5 minutes. Following this time, the solution was allowed to shake on a BioShake iQ plate shaker at 750 rpm at ambient temperature for 5 hours. Following this time, the wells were washed with 100 pL of lx PBS 5x, and were ready for the next dye conjugation step.Protocol for surface measurementsAcquisition

[0343] Prepared Cell Vis 384- well, 1.5H glass bottom plates were images on a Leica Stellaris 8 Confocal Microscope with a 40x air immersion objective (0.95 NA). Regions of Interest (ROIs) within each well were chosen randomly using the Leica Navigator software feature, collecting 0.5 MP (512x512 pixels) with 25nm step size to yield an approximate FOV size of 12.7 microns by 12.7 microns. Initial focus on the glass surface was determined first by XZ confocal scanning in reflection mode, then optimized in traditional XY mode to maximize contrast for observable spots in the field of view (FOV). Autofocusing ensured consistent imaging of the glass surface at each ROI position. Simultaneous spectral-lifetime imaging proceeded with excitation at 440nm with 5% laser power and a 20MHz pulse repetition rate. Spectral acquisition ranged from 450nm-640nm in 5nm steps with lOnm bin width for a total of 39 steps. Confocal pinhole diameter was set to 2 AU using 550nm estimation. Line scan rate was 200 Hz for a pixel dwell time of approximately 7.7 microseconds per pixel.Data Processing

[0344] Image processing was completed using a custom hyperspectral package in Python, and save in HFD5 files. Hyperspectral datasets were first decoded from the Leica image format (.lif files) into 5 dimensional ND- Arrays [lambda , X, Y, p, t]. Lambda represents the spectral bin center (+ / - 5nm as described above). X and Y represent spatial cartesian positions. Lifetime characteristics are divided into to dimensions where p is the photon type, and t is the estimated arrival time. Since photon types other than type 1 may lead to inaccurate estimations of arrival4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTtimes, only type 1 photons were used for lifetime analysis. All photon types were used in spectral analysis.

[0345] Following image decoding, each 5D hyperspectral image array was split into 3 separate arrays representing [lambda, X, Y], [X, Y, t], and [X, Y] for spectral emission, emission lifetime and image projection analyses, respectively. Single molecules and microdomains were identified through the image projection, where object identification parameters were first tuned using an interactive module, then applied evenly across all images. Object maps were evaluated qualitatively for precision in locating molecules and microdomains as objects for downstream analyses.

[0346] Obj ect level hyperspectral were extracted iteratively using the obj ect maps generated in the previous step. Whole FOV spectra and lifetime traces were also extracted at the same time. The characterization of all object-level and FOV-level traces was identical for all data. For spectral emission data, precise emission peaks were interpolated from data following a savgol-filter to reduce noise. Average emission lifetimes for decay traces were estimated using 3 component exponential decay tail-fitting over a specified window (1.4ns - 44ns) to minimize artifacts introduced from the IRF. Best fit parameters were optimized via the Chi-square and the Scipy.minimize method. For some lifetime decays of single molecules / microdomains / objects, arrival time bins that contained zero photon counts were masked to ensure convergence. Average lifetimes were estimated by intensity-weighting each fitted lifetime component.

[0347] To ensure object quality, additional classification and characterization was used to identify erroneously processed objects that were sometimes identified. For each identified object, 2D gaussian PSFs were fitted to the image projections data, as well as object intensity, peak amplitude, area, and other morphological parameters. These general statistics were utilized to remove objects before downstream processing, and were applied evenly across the entire dataset. Data visualization was completed using a combination of matplotlib and seaborn packages in Python.

[0348] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTvirtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.4910-7494-6957.5Table 1:&4910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.5Table 2:&4910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.54910-7494-6957.5

Claims

Atty. Dkt. No. 701586-000165 WO PTCLAIMSWhat is claimed is:

1. A method for identifying, in a single read, both a terminal amino acid and a penultimate amino acid in a polypeptide, the method comprising:a. conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide, wherein the probe exhibits different fluorescent spectral properties when conjugated to different pairs of terminal amino acid and penultimate amino acid; andb. detecting one or more spectral properties of the probe conjugated to the terminal amino acid; andc. identifying the terminal amino acid and the penultimate amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different terminal and penultimate amino acid pair.

2. The method of claim 1, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other, and wherein the probes conjugated to the terminal amino acid of the polypeptides in the plurality are identical same.

3. The method of claim 1 , wherein the method further comprises a step (c) of cleaving the terminal amino acid, and, and repeating steps (a) to (b), thereby providing error correction.

4. A method for sequencing a polypeptide, the method comprising:a. conjugating a probe comprising a dipyrromethane-BFi derivative to a terminal amino acid of a polypeptide, wherein the probe exhibits different fluorescent spectral properties when conjugated to a first pair of terminal amino acid and penultimate amino acid, and when conjugated to a second pair of terminal amino acid and penultimate amino acid, wherein at least one amino acid in the first pair is different from at least one amino acid in the second pair;4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTb. detecting one or more spectral properties of the probe conjugated to the terminal amino acid to determine the identity of the terminal amino acid and the penultimate amino acid;c. cleaving the terminal amino acid of the polypeptide; andd. sequentially repeating steps (a) to (c) one or more times to determine the sequence of at least a portion of the polypeptide.

5. The method of claim 4, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi derivative to a terminal amino acid of a plurality of polypeptides, wherein at least two polypeptides in the plurality are different from each other, and wherein the probes conjugated to the terminal amino acid of the polypeptides in the plurality are identical.

6. The method of claim 1 or 4, wherein the terminal amino acid is at an N-terminal of the polypeptide.

7. The method of claim 1 or 4, wherein the terminal amino acid is at C-terminal of the polypeptide.

8. The method of claim 3 or 4. wherein the step of cleaving the terminal amino acid comprises enzymatic cleavage.

9. The method of claim 3 or 4, wherein the step of cleaving the terminal amino acid comprises chemical cleavage.

10. The method of claim 1 or 4, wherein polypeptide is in a sample.

11. The method of claim 1 or 4, wherein at least one of the terminal and penultimate amino acid is a non-natural or modified amino acids.

12. The method of claim 1 or 4, wherein the polypeptide is immobilized on a surface.

13. A method for identifying a modified or non-natural amino acid, the method comprising:a. conjugating a probe comprising a dipyrromethane-BFi derivative to a modified or non-natural amino acid, wherein the probe exhibits different fluorescent spectral properties when conjugated to different modified or non-natural amino acids;b. detecting one or more fluorescent spectral properties of the probe conjugated to the modified or non-natural amino acid; andc. identifying the modified or non-natural amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PTproperty is representative of the probe conjugated to a different modified or nonnatural amino acid.

14. The method of claim 13, wherein the method comprises conjugating the probe comprising the dipyrromethane-BFi derivative to a plurality of amino acids, wherein at least two amino acids in the plurality are different from each other, and wherein the probes conjugated to the amino acids in the plurality are identical.

15. The method of claim 14, wherein at least one amino acid in the plurality is a modified or non-natural amino acid and at least one amino acid in the plurality is a natural amino acid.

16. The method of claim 13 , wherein the modified or non-natural amino acid is in a sample.

17. The method of claim 13, wherein the modified or non-natural amino acid is in a polypeptide.

18. The method of claim 17, wherein the modified or non-natural amino acid is at an N- terminal of the polypeptide.

19. The method of claims 17, wherein the modified or non-natural amino acid is at a C- terminal of the polypeptide.

20. The method of claims 17, wherein the modified or non-natural amino acid is at an internal position of the polypeptide.

21. The method of claim 17, wherein the polypeptide is immobilized on a surface.

22. The method of claim 1, 4 or 13, wherein the probe is conjugated to an amino, a carboxylic, a hydroxyl or a thiol group of the amino acid.

23. The method of claim 1, 4 or 13, wherein the probe is conjugated to an amino group of the amino acid.

24. The method of claim 1, 4 or 13, wherein the probe is conjugated to a carboxylic group of the amino acid.

25. The method of claim 1, 4 or 13, wherein the probe is covalently conjugated with the amino acid.

26. The method of claim 1 , 4 or 13, wherein the method comprises a step of denaturing the polypeptide to which the probe is conjugated prior to the step of detecting one or more spectral properties of the conjugated probe.

27. The method of claim 1, 4 or 13, wherein said detecting one or more spectral properties of the conjugated probe is in the presence of a denaturing agent.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT28. The method of claim 27, wherein the denaturing agent is selected from the group consisting of a chaotropic agent, a detergent, heavy metal ions, an organic solvent, an acid, a base, a reducing agent, and an oxidizing agent.

29. The method of claim 28, wherein the denaturing agent is selected from the group consisting of urea, guanidine hydrochloride, guanidine thiocyanate, lithium perchlorate, sodium dodecyl sulfate (SDS), sodium deoxycholate, Triton X-100, NP-40, Tween-20, CHAPS, CHAPSO, methanol, ethanol, isopropanol, acetonitrile, dimethyl sulfoxide (DMSO), trifluoroethanol (TFE), hydrochloric acid, trifluoroacetic acid (TFA), acetic acid, sodium hydroxide, dithiothreitol (DTT), P-mercaptoethanol, tris(2- carboxyethyl)phosphine (TCEP), hydrogen peroxide, performic acid, Hg2+, Ag+and Pb2+.

30. The method of claim 1, 4 or 13, wherein said detecting one or more spectral properties of the conjugated probe comprises super resolution microscopy.

31. The method of claim 30, wherein the super resolution microscopy comprises stochastic optical reconstruction microscopy (STORM).

32. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties comprises detecting fluorescence of the conjugated probe.

33. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises detecting fluorescence emission wavelength, excitation wavelength, emission intensity, fluorescence lifetime, polarization (polarity / anisotropy), absorbance, Raman shift, or Forster resonance energy transfer (FRET) efficiency.

34. The method of any one of claims **, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime.

35. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime.

36. The method of claim 35, wherein said detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime is at a single wavelength.

37. The method of claim 35, comprising said detecting the emission intensity, polarization (polarity / anisotropy) and / or fluorescence lifetime is at a plurality of wavelengths.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT38. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and fluorescence lifetime.

39. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises detecting the emission intensity and lifetime at one or more wavelengths from about 500 nm to about 570 nm.

40. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises exciting the conjugated probe with electromagnetic radiation at a wavelength from about 430 nm to about 450 nm, preferably from about 435 nm to about 445 nm, more preferably a wavelength of about 440 nm.

41. The method of claim 1, 4 or 13, wherein detecting one or more spectral properties of the conjugated probe comprises: (i) exciting the conjugated probe with electromagnetic radiation at a wavelength from about 430 nm to about 450 nm, preferably from about 435 nm to about 445 nm, more preferably a wavelength of about 440 nm; and (ii) detecting the emission intensity and / or lifetime at one or more wavelengths in the range of from about 500 nm to about 570 nm.

42. The method of claim 1, 4 or 13, wherein the dipyrromethane-BFi derivative comprises a labile or reactive group.

43. The method of claim 1, 4 or 13, wherein the dipyrromethane-BFi derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

44. The method of claim 41 , wherein the probe is conjugated via position 8 (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

45. The method of claim 42, wherein the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative comprises a labile or reactive group at position 8 (meso position).

46. The method of claim 1, 4 or 13, wherein the probe is of Formula (I):wherein:R1, R2, R3, R4, R5, R6, R7and R8are each independently selected from the group consisting of hydrogen, halogen, alkyl, perhaloalkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino,4910-' -6957.5Atty. Dkt. No. 701586-000165 WO PTdialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cynao, nitro, thiol, alkylthio, sulfonate, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, optionally at least one of R1, R2, R3, R4, R5, R6, R7and R8, is a labile or leaving group, and any 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) independently selected substituents from the group consisting of halogen, hydroxy, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.

47. The method of claim 1, 4 or 13, wherein the probe comprises a modified 8-thioether-BODIPY dye.

48. The method of claim 1, 4 or 13, wherein the probe has the structure:.

49. A method for barcoding a plurality of target analytes, the method comprising: ligating a barcode peptide to target analytes in a plurality of target analytes via a terminal amido acid in the barcode peptide, wherein the terminal amino acid is a modified or nonnatural amino acid, and wherein a barcode peptide conjugated to a first analyte in the plurality is distinguishable from a barcode peptide conjugated to a second analyte in the plurality, and wherein the first and second analyte are different.

50. The method of claim 49, wherein the barcode peptide is conjugated to the analyte via a linker.

51. The method of claim 50, wherein the linker is a peptide linker.

52. The method of claim 51 , wherein the peptide linker comprises a recognition amino acid sequence for a peptide ligase.

53. The method of claim 52, where the peptide ligase is selected from the group consisting of a sortase, an asparaginyl endopeptidase-derived ligase, a subtilisin-derived ligase, an intein, an ATP-dependent peptide ligase, and a cyanobactin macrocyclase, preferably the ligase is a sortase, more preferably the ligase is sortase A.4910-7494-6957.5Atty. Dkt. No. 701586-000165 WO PT54. The method of claim 49, wherein the barcode peptide is conjugated by its C-terminal amino acid.

55. The method of claim 49, wherein the barcode peptide is conjugated by its N-terminal amino acid.

56. The method of claim 49, wherein the analytes are polypeptides, amino acids, nucleic acids, cells, barcodes, or any combinations thereof.

57. The method of claim 49, wherein the modified or non-natural amino acid is in a sample.

58. The method of claim 49, wherein the analytes are immobilized on a surface.

59. The method of claim 58, wherein the analytes are immobilized on the surface after conjugating with the barcode peptide.

60. The method of claim 58, wherein the analytes are immobilized on the surface prior to conjugating with the barcode peptide61. The method of claim 49, fiirther comprising sequencing the barcode peptides conjugated with the analytes using the method of any one of claims 4-12 and 22-48.

62. The method of claim 10, 16 or 57, wherein the sample is a biological sample.

63. The method of claim 62, wherein the biological sample is a biological fluid, a tissue, an organ, or a cell.

64. The method of claim 12 or 21, wherein the method comprises a step of immobilizing the polypeptide on the surface prior to conjugating with the probe.

65. The method of claim 12 or 21, wherein the method comprises a step of immobilizing the polypeptide on the surface after conjugating with the probe.4910-7494-6957.5

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