Purification of peptides containing reductively cleavable linkers

Abstract

Described are cleavable linkers comprising polypeptide affinity purification tags. Also described are methods for purifying polypeptides using the cleavable linkers described herein.

Description

Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325PURIFICATION OF PEPTIDES CONTAINING REDUCTIVELY CLEAVABLE LINKERSRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Application No. 63 / 722,284, filed November 19, 2024, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] Disclosed herein are cleavable linker groups comprising polypeptide affinity purification tags. Also disclosed are methods for purification of polypeptides for biotechnological and therapeutic applications.BACKGROUND

[0003] The purification of chemically synthesized polypeptides is typically performed through chromatographic separation of the crude reaction mixture over various types of polar or nonpolar stationary phases. High-pressure liquid chromatography (HPLC) using C4, C8 or C18-functionalized phases is the most widely used separation technology for polypeptides derived from solid-phase peptide synthesis (SPPS). HPLC, however, is a time- and resourceintensive process that suffers from inherent limitations in compound throughput and material scalability, typically representing one of the most cost- and process mass-intensive components of chemical peptide production at scale. Compound throughput is limited by the fact that peptides must be purified in sequence, typically over extended gradients, which severely limits the number of peptides that can be purified at one time using a single instrument.

[0004] In view of the limitations of chromatographic purifications of polypeptide materials, there is a need to develop selective, reversible reagents which enable purification of polypeptides through a “catch and release” method that takes advantage of the affinity interactions between a functionalized product and a capture resin.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 SUMMARY

[0005] In certain aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula I:I;wherein:PG1is a protecting group selected from tert-butyloxycarbonyl (Boc), tert-butyl, tertbutyldimethylsilyl, methoxymethyl, triisopropylsilyl, and tetrahydropyranyl;E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstitutedFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R1and R2is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR1and R2, taken together with the intervening atoms, form a cycloalkyl ring;R3is hydrogen, branched or unbranched alkyl, or an acid labile protecting group;R4is hydrogen or branched or unbranched alkyl;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S (0)2-, -C(O)-alkylene, -C(O)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

[0006] In certain preferred embodiments, PG1is tert-butyloxycarbonyl (Boc).

[0007] In certain aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula PI:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 Pi;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R1and R2is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR1and R2, taken together with the intervening atoms, form a cycloalkyl ring; R3is hydrogen, branched or unbranched alkyl, or an acid labile protecting group;Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 R4is hydrogen or branched or unbranched alkyl;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; and G and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, - C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene-C(O)-NH-, alkylene- NH-C(O)-, alkylene-OC(O)-NH-, alky lene-NH-C (0)0-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(0)-, alkylene-S(0)2-, alkylene- S(0)2-NH-, alkylene-NH-S(0)2-, -C(O)-alkylene, -C(O)-NH-alkylene, -NH-C(O)- alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, -OC(O)O-alkylene, -NH- alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(0)2-alkylene, -S(0)2-NH- alkylene, and -NH-S(0)2-alkylene.

[0008] In some aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula II:OII;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted -Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R5and R6is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR5and R6, taken together with the intervening atoms, form a cycloalkyl ring; Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S (0)2-, -C(O)-alkylene, -C(0)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, -Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(0)2- alkylene, -S(0)2-NH-alkylene, and -NH-S(0)2-alkylene.

[0009] In other aspects, the present disclosure provides a compound, or a salt thereof, having the structure:wherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoro alkyl; andG and G'are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alkylene- NHC(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(O)-, alkylene-S (0)2-, alkylene-S(0)2NH-, alky lene-NHS (0)2-, - C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -O(O)CNH-alkylene, - NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O- alkylene, -S(O)-alkylene, -S(0)2-alkylene, -S(0)2NH-alkylene, and -NHS(0)2- alkylene.

[0010] In certain aspects, the present disclosure provides a compound, or a salt thereof, having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325O; orOiiE G 0 HHOwherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)- alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl,Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoro alkyl;G and G'are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alkylene- NHC(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(O)-, alkylene-S (0)2-, alkylene-S(O)2NH-, alky lene-NHS (0)2-, - C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -OC(O)NH-alkylene, - NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O- alkylene, -S(O)-alkylene, -S(0)2-alkylene, -S(O)2NH-alkylene, and -NHS(O)2- alkylene; andPP is a polypeptide residue.

[0011] In certain aspects, the present disclosure provides a method of installing an enrichment tag on a target polypeptide, comprising combining a mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a compound having the structure of:O; orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 0 00wherein PP represents a residue of the target polypeptide.

[0012] In certain aspects, the present disclosure provides a method of purifying a target polypeptide, comprising:(a) combining a first mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a second mixture comprising an enrichment-tagged compound having the structure of:0 0" 11E^G^\ / ^0'' TP0wherein PP represents a residue of the target polypeptide;(b) contacting the second mixture with an affinity resin, thereby binding the enrichment-tagged compound to the affinity resin;wherein the affinity resin has an affinity for the enrichment tag;(c) washing the affinity resin; and(d) removing the enrichment tag from the enrichment-tagged compound under conditions sufficient to produce the purified target polypeptide.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts a schematic representation of a “catch and release” workflow using the reductively cleavable linker of the invention. The reductive decarboxylation is fully traceless affording the native polypeptide without any additional modifications.

[0014] FIG. 2 depicts a cleavable linker of the invention, comprising an internal 2-((2-aminoethyl)disulfaneyl)-2-methylpropyl carbamate. The carbamate (X = NH) may be substituted for a carbonate (X = O) to release alcohols instead of amines.

[0015] FIG. 3 describes the forward synthesis of the BTOXGLY linker 1.

[0016] FIG. 4 describes the forward synthesis of the BTSSME2 linker 2.

[0017] FIG. 5 describes the forward synthesis of the BT linker 3.

[0018] FIG. 6 describes the forward synthesis of the BTASE linker 4.

[0019] FIG. 7 describes the forward synthesis of the BTSSHME linker 5.

[0020] FIG. 8 describes the forward synthesis of the BTSSH2 linker 6.

[0021] FIG. 9 depicts a representative workflow using a biotin-capped reductively cleavable linker installed through base-promoted amination of the polypeptide N-terminus using a 4-nitrophenyl (pNP) carbonate following the completion of peptide elongation by solid phase peptide synthesis (SPPS).

[0022] FIG. 10 depicts a reaction scheme for cleaving the BTSSME2 linker 2 from the target polypeptide.

[0023] FIG. 11 depicts cleavage kinetics of a model BTSSME2-spgys-CONH2 pentapeptide 7 to liberate unmodified pentapeptide 8 at varied pH range of 1 - 9 in the presence of excess P-ME (3% v / v).

[0024] FIG. 12 depicts cleavage kinetics of a model BTSSME2-spgys-CONH2 pentapeptide 7 to liberate unmodified pentapeptide 8 at pH 9 in the presence of various equivalents of P-ME (0.09 - 11% v / v; 50 - 6300 equiv.) relative to linker.

[0025] FIG. 13 depicts cleavage kinetics of a model BTSSME2-spgys-CONH2 pentapeptide 7 to liberate unmodified pentapeptide 8 at pH 7 in the presence of various equivalents of P-ME (0.09 - 11% v / v; 50 - 6300 equiv.) relative to linker.

[0026] FIG. 14 depicts the purification of twenty representative peptides using the BTSSHME linker 5. Shown are the total ion chromatograms (TIC) corresponding to before (left, “Crude”) and after (right, “Purified”) affinity capture and release from streptavidinFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 agarose resin. Shaded bars indicate the presence of at least three peptide- associated ions coeluting in the TIC.

[0027] FIG. 15 depicts the purification of twenty representative peptides using the BTSSME2 linker 2. Shown are the total ion chromatograms (TIC) corresponding to before (left, “Crude”) and after (right, “Purified”) affinity capture and release from streptavidin agarose resin. Shaded bars indicate the presence of at least three peptide- associated ions coeluting in the TIC.

[0028] FIG. 16 depicts the purification of one representative peptide using the BTOXGLY linker 1. Shown are the total ion chromatograms (TIC) corresponding to before (left, “Crude”) and after (right, “Purified”) affinity capture and release from streptavidin agarose resin. Shaded bars indicate the presence of at least three peptide- associated ions coeluting in the TIC. The released peptide contains a residual N-terminal Gly residue.

[0029] FIG. 17 depicts the hypothesized cleavage mechanisms for each of the disclosed linker classes.DETAILED DESCRIPTION

[0030] The present invention is based on the discovery of a series of novel, cleavable linkers that can be appended to a variety of selective molecular tags for affinity purification and subsequent release. The release occurs rapidly and in a traceless fashion to afford the native or unmodified synthetic target. This new “catch and release” technology significantly improves on the selectivity, kinetics, and process mass intensity (PMI) of previously developed cleavable linkers. The purification methods described herein are significant in their ability to rapidly deliver assay-ready quantities of synthetic material in a highly parallelized fashion.

[0031] One such linker described herein, the reductively cleavable linker BTSSME2, is notable for its high stability during SPPS and in non-reducing aqueous buffers, and for its rapid and quantitative cleavage kinetics in the presence of reducing agent under weakly basic conditions.

[0032] The combination of these two features enables the linker BTSSME2 to be cleaved on command in a reductant and pH-dependent manner, while supporting assays that may require the use of the tagged compound (i.e., for compound immobilization) prior to tag removal. Furthermore, the linker chemistry has relevance to the manufacturing of polypeptides on a commercial scale, as the technology provides a method for rapid purification with significantFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 reductions in cost and usage of organic solvents, potentially supplanting or simplifying downstream HPLC purification.

[0033] Additionally, the linkers may be applied to polypeptide manufacturing, solid-phase macrocyclization and ligation, and reversible bioconjugation of drugs or polymers.Enrichment- Tagged Linker Reagents and Polypeptides

[0034] In certain aspects, the present disclosure provides a reagent that enables installation of an enrichment tag onto a polypeptide via a linker, which enrichment tag facilitates purification of the polypeptide. After purification, the linkers may be removed in a traceless fashion to afford the purified target polypeptide.

[0035] Accordingly, in certain aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula I:I;wherein:PG1is a protecting group selected from tert-butyloxycarbonyl (Boc), tert-butyl, tertbutyldimethylsilyl, methoxymethyl, triisopropylsilyl, and tetrahydropyranyl;E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R1and R2is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR1and R2, taken together with the intervening atoms, form a cycloalkyl ring;R3is hydrogen, branched or unbranched alkyl, or an acid labile protecting group;R4is hydrogen or branched or unbranched alkyl;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G’ are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S (0)2-, -C(O)-alkylene, -C(0)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(0)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0036] In certain preferred embodiments, PG1is tert-butyloxycarbonyl (Boc).

[0037] In certain aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula PI:Pi;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R1and R2is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR1and R2, taken together with the intervening atoms, form a cycloalkyl ring; R3is hydrogen, branched or unbranched alkyl, or an acid labile protecting group;R4is hydrogen or branched or unbranched alkyl;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S(O)2-, -C(O)-alkylene, -C(O)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

[0038] In some aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula II:OII;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R5and R6is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR5and R6, taken together with the intervening atoms, form a cycloalkyl ring; Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 G and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S(O)2-, -C(O)-alkylene, -C(O)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

[0039] In other aspects, the present disclosure provides a compound, or a salt thereof, having a structure represented by formula III:R7III;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;R7is hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G’ are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene-C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alky lene-NHC (0)0-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2NH-, alkylene -NHS(O)2-, -C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -OC(O)NH-alkylene, -NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(0)-alkylene, -S(O)2-alkylene, -S(O)2NH-alkylene, and -NHS(O)2-alkylene.

[0040] In certain especially preferred embodiments, X is O. In certain preferred embodiments, m is 0. In some embodiments, m is an integer from 1 to 5. In certain preferred embodiments, m is 4. In some embodiments, one of G and G' is absent. In certain embodiments, one of G and G' is alkylene. In certain embodiments, one of G and G' is -C(O)NH-alkylene. In some embodiments, one of G and G' is alkylene-C(O)NH-. In certainFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 preferred embodiments, G is absent; and wherein G' is alkylene. In some preferred embodiments, G is -C(O)NH-alkylene; and wherein G' is alkylene.

[0041] In certain embodiments, E is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl; and wherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl terminates in a substituent selected from alkyne, azide, aminooxy, and hydrazine. In some preferred embodiments, E is substituted or unsubstituted -NH-C(O)-alkyl. In certain especially preferred embodiments, E is

[0042] In some embodiments, E is substituted or unsubstituted heteroaryl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -NHC(O)-heteroaryl, substituted or unsubstituted -C(O)NH-heteroaryl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -NHS(O)2-heteroaryl. In certain preferred embodiments, E is substituted or unsubstituted 8-hydroxyquinoline. In some especially preferred embodiments,OZ’Eis i; and Z' is H or a hydroxyl protecting group. In certain such embodiments, Z'Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0043] In some preferred embodiments, Y is -O-Z; and Z is selected fromN(^2FF. In certain especially preferred embodiments, Z. In some embodiments, Z is In certain embodiments, Z is. In certain embodiments,Z is

[0044] In certain embodiments, R3is selected fromand1. In some preferred embodiments, R3is'. In certain embodiments, R4is H. In certain embodiments, R1and R2are each H. In some preferred embodiments, R5and R6are each methyl. In certain embodiments, R7is H.

[0045] In certain aspects, the present disclosure provides a compound, or a salt thereof, having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325wherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)- alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoro alkyl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alkylene- NHC(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(O)-, alkylene- S(O)2-, alkylene-S(O)2NH-, alkylene-NHS(O)2-, - C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -O(O)CNH-alkylene, - NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O- alkylene, -S(O)-alkylene, -S(O)2-alkylene, -S(O)2NH-alkylene, and -NHS(O)2- alkylene.

[0046] In certain embodiments, the present disclosure provides a compound having the Ostructure:N<-)2. In certain embodiments, the present disclosure provides a compound having the structure:N H INO2. In certain embodiments, the present disclosure provides a compound having the structure:In certain embodiments, the present disclosure provides a compound having the structure:N°2. In certain embodiments, the present disclosure provides a compound having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0047] In certain embodiments, the present disclosure provides a compound having the structure:. In certain especially preferredembodiments, E is. In certain embodiments, E is substituted or OZ'cunsubstituted 8-hydroxyquinoline. In some preferred embodiments, E is i; and Z' is H or a hydroxyl protecting group. In certain such embodiments, Z' is a hydroxyl protectinggroup selected from', \ >, 0 J, \and '. In some OHembodiments,E is '. In certain embodiments, E is O; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz). In some preferred embodiments, each Z" is Boc. In certain preferred embodiments, each Z" is H. In other preferred embodiments, one of Z" is H and the other is Boc. In certain embodiments, E is (Z")2N.N / ^N^H 11O; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz). In some preferred embodiments, each Z" is H. In certain preferred embodiments, each Z" is Boc. InFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 other preferred embodiments, one of Z" is H and the other is Boc. In certain embodiments, EH2N.In certain embodiments, E is

[0048] Also disclosed are compounds comprising a polypeptide bound to an enrichment tag via a cleavable linker. In some embodiments, the cleavable linker is bound to the N-terminus of a polypeptide. In other embodiments, the cleavable linker is bound to the C-terminus of a polypeptide. The linkers are covalently attached to an enrichment tag.

[0049] The cleavable linker is one that can be dissociated from the polypeptide via chemical means. The cleavable linkers include oxidatively cleavable linkers, reductively cleavable linkers, and base-cleavable linkers.

[0050] Accordingly, in certain aspects, the present disclosure provides a compound, or a salt thereof, having the structure:O; or0 011110wherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl,Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)- alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;G and G’ are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alkylene- NHC(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(O)-, alkylene-S (0)2-, alkylene-S(0)2NH-, alky lene-NHS (0)2-, - C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -OC(O)NH-alkylene, - NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O- alkylene, -S(O)-alkylene, -S(0)2-alkylene, -S(0)2NH-alkylene, and -NHS(0)2- alkylene; andPP is a polypeptide residue.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0051] In further aspects, the present disclosure provides a compound, or a salt thereof, having the structure:whereinPG1is a protecting group selected from tert-butyloxycarbonyl (Boc), tert-butyl, tertbutyldimethylsilyl, methoxymethyl, triisopropylsilyl, and tetrahydropyranyl; E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH- aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted - C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substitutedFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 or unsubstituted -NHC(O)-alkyl, substituted or unsubstituted -NH-C(O)- heterocycloalkyl, substituted or unsubstituted -NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH- C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)- heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted - S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH- heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted - S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2- aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, - NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;G and G are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)NH-, alkylene-NHC(O)-, alkylene-OC(O)NH-, alkylene- NHC(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene- S(O)-, alkylene-S (0)2-, alkylene-S(0)2NH-, alky lene-NHS (0)2-, - C(O)-alkylene, -C(O)NH-alkylene, -NHC(O)-alkylene, -OC(O)NH-alkylene, - NHC(O)O-alkylene, -OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O- alkylene, -S(O)-alkylene, -S(0)2-alkylene, -S(0)2NH-alkylene, and -NHS(0)2- alkylene; andPP is a polypeptide residue.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0052] In certain embodiments, the present disclosure provides a compound having theIn certain embodiments, the present disclosureprovides a compound having the structure:

[0053] In certain embodiments, the present disclosure provides a compound having theIn certain embodiments, the present disclosure Oprovides a compound having the structure:

[0054] In certain embodiments, the present disclosure provides a compound having theIn certain embodiments, the present disclosure provides a compound having the structure:

[0055] In certain especially preferred embodiments,E is certain embodiments, E is substituted or unsubstituted 8-hydroxyquinoline. In some preferred OZ'embodiments, Eis i; and Z' is H or a hydroxyl protecting group. In certain suchembodiments, Z' is a hydroxyl protecting group selected fromFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 OH>x< °9 / JVWP \,ANC[,. In some embodiments, E is '. In certain embodiments, E is(Z")2N O |j rO; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz). In some preferred embodiments, each Z" is Boc. In certain preferred embodiments, each Z" is H. In other preferred embodiments, one of Z" is H and the other is Boc. In certain embodiments, E (Z'')2N,N / ^N^H 11isO; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz). In some preferred embodiments, each Z" is H. In certain preferred embodiments, each Z" is Boc. In other preferred embodiments, one of Z" is H and the other is Boc. In some embodiments, E is H H O Y N v ZII H IIO. In certain embodiments, E is O

[0056] In certain embodiments, the polypeptide residue comprises from 5 to 100 amino acid residues. In certain embodiments, the polypeptide residue comprises from 5 to 55 amino acid residues. In certain embodiments, the polypeptide residue comprises from 25 to 55 amino acid residues. In some embodiments, the polypeptide residue comprises at least one D-amino acid residue. In certain embodiments, the target polypeptide comprises at least one alphadisubstituted amino acid residue (e.g., aminoisobutyric acid). In some embodiments, the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine). In certain such embodiments, the non-canonical amino acid further comprises a non-natural side chain. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in Table 1. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 80% to about 100% identity to any one of the amino acid sequences set forth in Table 1. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 90% to about 100% identity to any one of the amino acid sequences set forth in Table 1. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 95% to about 100% identity to anyFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 one of the amino acid sequences set forth in Table 1. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 98% to about 100% identity to any one of the amino acid sequences set forth in Table 1. In certain embodiments, the polypeptide residue comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, preferably about 95%, or more preferably about 98% identity to any one of the amino acid sequences set forth in Table 1.

[0057] In certain preferred embodiments, the polypeptide residue comprises one of the amino acid sequences with a SEQ. ID disclosed in Table 1.

[0058] In some embodiments, the reagents described herein may be useful for antibody purification. Accordingly, in some embodiments, the polypeptide residue is an antibody or an antigen-binding fragment thereof.Methods of Installing the Enrichment Tag via Cleavable Linker

[0059] Methods of making the enrichment-tagged polypeptide include methods of solidphase peptide synthesis (SPPS). Installation of the linker is typically achieved through an amide bond forming reaction with an activated carbonate following the completion of polypeptide elongation by SPPS.

[0060] An exemplary scheme of synthesizing a polypeptide attached to an enrichment tag via a cleavable linker is shown in FIG. 2. Examples of cleavable linkers are shown in FIG. 7.

[0061] In certain aspects, the present disclosure provides a method of installing an enrichment tag on a target polypeptide, comprising combining a mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a compound having the structure of:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325wherein PP represents a residue of the target polypeptide.

[0062] In certain aspects, the present disclosure provides a method of installing an enrichment tag on a target polypeptide, comprising combining a mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a compound having the structure of:E. G^S.SX^O¥PPO; or0 0E^G^\ / ^o'^PP0wherein PP represents a residue of the target polypeptide.

[0063] In certain embodiments, the enrichment tag and cleavable linker are covalently bound to the N-terminus of the polypeptide.

[0064] In certain embodiments, the mixture comprising the target polypeptide further comprises one or more non-target polypeptides. In some embodiments, the one or more nontarget polypeptides do not react with the compound of the present disclosure. In certain embodiments, the target polypeptide is covalently bound at its C-terminus to a solid phase support. In certain embodiments, the target polypeptide comprises from 5 to 55 amino acid residues. In certain embodiments, the target polypeptide comprises from 25 to 55 amino acidFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 residues. In certain embodiments, the target polypeptide comprises at least one D-amino acid residue. In certain embodiments, the target polypeptide comprises at least one alphadisubstituted amino acid residue (e.g., aminoisobutyric acid). In some embodiments, the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine). In certain such embodiments, the non-canonical amino acid further comprises a non-natural side chain.Methods of Purification

[0065] The disclosed enrichment-tagged linker reagents can be used in purification methods.

[0066] In some embodiments, the disclosed enrichment-tagged linker reagents are used in producing tagged polypeptides or tagged proteins. The disclosed enrichment-tagged linker reagents can be incorporated at the N-termini of recombinant or synthetic polypeptides and proteins to produce the tagged polypeptides or tagged proteins. The disclosed affinity tags can be incorporated at the C-termini of recombinant or synthetic polypeptides and proteins to produce the tagged polypeptides or tagged proteins. The tagged polypeptides or tagged proteins can be recombinantly or synthetically produced.

[0067] In some embodiments, the disclosed linker-tagged enrichment reagents are used in polypeptide or protein purification. The tagged recombinant polypeptides, tagged recombinant proteins, tagged synthetic polypeptides, and tagged synthetic proteins can be purified using the disclosed enrichment tags.

[0068] In some embodiments, the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins are in native form. In these aspects, the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins are maintained in native conditions with the use of one or more of neutral or pH-balanced buffers, optionally containing protease inhibitors, cofactor reagents, and reducing agents.

[0069] In some embodiments, the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins are in denatured form. In these aspects, the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins are maintained under denaturing conditions in buffers containing reducing agent, protease inhibitors, denaturants, and detergents.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0070] The tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins can then be purified with the use of affinity chromatography. In some embodiments, enrichment tag is a polyhistidine group. In certain such embodiments, the mode of affinity chromatography is immobilized metalaffinity chromatography (IMAC). The metal ion can be a transition metal ion, such as cobalt (Co2+), nickel (Ni2+), copper (Cu2+), or zinc (Zn2+). In some embodiments, the transition metal ion is immobilized on a substrate. Suitable substrates include resin column and resin beads, such as resin flow columns, resin spin column, or resin microbeads. In some embodiments, the metal ions are coupled to nitrilotriacetic acid (NTA). Metal ion-NTA complex (such as Ni-NTA, Co-NTA, Cu-NTA, or Zn-NTA) can then be coupled to agarose or magnetic beads for IMAC.

[0071] For example, the affinity-tagged polypeptides or proteins can be purified using the commercially available matrices nickel-nitrilotriacetic acid (Ni2+-NTA) and Co2+-carboxylmethylaspartate (Co2+-CMA), which are coupled to a solid support resin. These matrices securely coordinate metal ions through four coordination sites while leaving two of the transition metal coordination sites exposed to interact with histidine residues in the affinity tag. The stability of metal binding in these resins also allows the resins to be regenerated and reused several times. The Ni2+-NTA matrix (available from Qiagen®) has a binding capacity of 5-10 mg protein / ml of matrix resin and a high binding affinity for the six residue polyhistidine tag at pH 8.0. The Co2+-CMA matrix (Talon resin, available from Clontech®) exhibit less nonspecific protein binding than the Ni2+-NTA resin, resulting in higher elution product purity. The binding capacity of the Co2+-CMA resin is also about 5-10 mg of protein / ml of resin (Bornhorst and Falk, Methods Enzymol. 2000; 326: 245-254).

[0072] The purification of the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins via IMAC can include the following steps.1. equilibrating with equilibration buffer a resin having any one of metal ions Co2+, Ni2+, Cu2+, or Zn2+immobilized in the resin;2. loading the resin with solution containing the tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins;3. washing the resin with wash buffer(s);Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 4. releasing the affinity tag from the polypeptide through a selective chemical reaction promoted by the release buffer5. eluting bound tagged recombinant polypeptides, the tagged recombinant proteins, the tagged synthetic polypeptides, or the tagged synthetic proteins by passing elution buffer through the resin to obtain an eluate;6. concentrating the eluate to obtain concentrated eluate; and7. dialyzing the concentrated eluate against storage buffer.

[0073] In some embodiments, the resin is in a spin column format. In these aspects, each one of the steps of equilibrating, loading, washing, and eluting are followed by centrifuging the spin columns.

[0074] In some embodiments, the step of washing is repeated one or more times.

[0075] Equilibration buffer can be buffer suitable for storage and maintenance of tagged recombinant peptides, the tagged recombinant proteins, the tagged synthetic peptides, or the tagged synthetic proteins. In some embodiments, equilibration buffer comprises neutral or pH-balanced buffer containing salts.

[0076] In some embodiments, the equilibration buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the equilibration buffer can comprise 6 M urea. The equilibration buffer can comprise 8 M urea.

[0077] In some embodiments, the equilibration buffer can be a buffer comprising guanidine hydrochloride (Gdn. HCl), such as between 4 M and 6 M Gdn. HCl. For example, the equilibration buffer can comprise 6 M Gdn. HCl. The wash buffer can be any buffer comprising up to about 50 mM imidazole. For example, wash buffer can contain between about 1 mM and about 50 mM imidazole, such as between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 mM, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM imidazole. Wash buffer can comprise about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM imidazole.

[0078] In some embodiments, the wash buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the wash buffer can comprise 6 M urea. The wash buffer can comprise 8 M urea.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0079] In some embodiments, the wash buffer can be a buffer comprising Gdn. HCl, such as between 4 M and 6 M Gdn. HCl. For example, the wash buffer can comprise 6 M Gdn. HCl.

[0080] Elution buffer can be a buffer comprising up to about 500 mM imidazole, such as between about 200 mM and about 500 mM imidazole, such as about 250 mM imidazole.

[0081] In some embodiments, the elution buffer can be a buffer comprising urea, such as between 5 M and 10 M urea. For example, the elution buffer can comprise 6 M urea. The elution buffer can comprise 8 M urea.

[0082] In some embodiments, the elution buffer can be a buffer comprising Gdn. HCl, such as between 4 M and 6 M Gdn. HCl. For example, the elution buffer can comprise 6 M Gdn. HCl.

[0083] Storage buffer can be a buffer comprising neutral or pH-balanced buffer. Storage buffer can comprise salts and, optionally, protease inhibitors and cofactor reagents.n H oHNy^| [p

[0084] In further embodiments, the enrichment tagis H. In certain such embodiments, the affinity chromatography is conducted with immobilized streptavidin.

[0085] Accordingly, in certain aspects, the present disclosure provides a method of purifying a target polypeptide, comprising:(a) combining a first mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a second mixture comprising an enrichment-tagged compound having the structure of:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325wherein PP represents a residue of the target polypeptide;(b) contacting the second mixture with an affinity resin, thereby binding the enrichment-tagged compound to the affinity resin;wherein the affinity resin has an affinity for the enrichment tag;(c) washing the affinity resin; and(d) removing the enrichment tag from the enrichment-tagged compound under conditions sufficient to produce the purified target polypeptide.

[0086] In some aspects, the present disclosure provides methods of purifying a target polypeptide, comprising:(a) combining a first mixture comprising the target polypeptide with a compound of the present disclosure under conditions sufficient to form a second mixture comprising an enrichment-tagged compound having the structure of:o oA II PPO; oro oG- SE" 'G' I ^ O A PPO?wherein PP represents a residue of the target polypeptide;Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 (b) contacting the second mixture with an affinity resin, thereby binding the enrichment-tagged compound to the affinity resin;wherein the affinity resin has an affinity for the enrichment tag;(c) washing the affinity resin; and(d) removing the enrichment tag from the enrichment-tagged compound under conditions sufficient to produce the purified target polypeptide.

[0087] In certain embodiments, the first mixture further comprises one or more non-target polypeptides. In some embodiments, the one or more non-target polypeptides do not react with the compound of the present disclosure. In certain embodiments, the target polypeptide is covalently bound at its C-terminus to a solid phase support. In certain embodiments, the methods disclosed herein further comprise a step (a’) following step (a):(a’) removing the solid phase support from the target polypeptide.

[0088] In certain embodiments, the target polypeptide comprises from 5 to 55 amino acid residues. In certain embodiments, the target polypeptide comprises from 25 to 55 amino acid residues. In some embodiments, the target polypeptide comprises at least one D-amino acid residue. In certain embodiments, the target polypeptide comprises at least one alphadisubstituted amino acid residue (e.g., aminoisobutyric acid). In some embodiments, the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine). In certain such embodiments, the non-canonical amino acid further comprises a non-natural side chain.

[0089] In certain especially preferred embodiments, the enrichment tag isNH, and the affinity resin comprises streptavidin.

[0090] In certain embodiments, the enrichment tag is a peptide fragment, such as a polyhistidine moiety; and wherein the affinity resin comprises a metal ion.

[0091] In some embodiments, the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl; and wherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl terminates in an alkyne; and wherein the affinity resin comprises an azide moiety.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0092] In certain embodiments, the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted - C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH- S(0)2-alkyl; and wherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted - C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl, terminates in an azide; and wherein the affinity resin comprises an alkyne.

[0093] In certain embodiments, the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted - C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH- S(0)2-alkyl; and wherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted - C(O)-NH-alkyl, substituted -NHC(O)-alkyl, substituted -C(O)-alkyl, substituted -S(0)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl, terminates in an aminooxy or a hydrazine group; and wherein the affinity resin comprises an aldehyde or ketone moiety.4 r\tr> BOC J-lor O Bu O; and the conditions sufficient to produce the purified target polypeptide comprise an oxidizing agent. In some embodiments, the conditions sufficient to produce the purified target polypeptide comprise sodium periodate.

[0095] In certain embodiments, the enrichment-tagged compound is'•G"S" S4O; and the conditions sufficient to produce the purified target polypeptide comprise a reducing agent. In some embodiments, the conditions sufficient to produce the purified target polypeptide comprise a thiol (e.g., beta- mercaptoethanol). In certain embodiments, the conditions sufficient to produce the purified target further comprise a base.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0096] In certain embodiments, the enrichment-tagged compound isPPor; and the conditions sufficient to produce the purified target polypeptide comprise a base.Mechanism

[0097] Taking reductively cleavable linker BTSSME2 as an example, the mechanism of polypeptide purification and linker cleavage follows below. The resultant carbamate-linked polypeptide is stable to the routine acidolytic cleavage conditions used to liberate the synthetic target from its synthetic solid support. Following capture of the linker-bound crude polypeptide target on the desired capture resin, the linker, in the presence of reductant and under mildly basic conditions, undergoes a “self-immolative” reductive decarboxylation cascade reaction that releases the unmodified synthetic target in a traceless fashion (FIG. 2).The reductive decarboxylation also releases CO2 and dimethylthiirane as volatile byproducts, both of which can be removed through routine polypeptide lyophilization procedures.

[0098] The reductive cleavage mechanism of the linker is enabled by a novel internal 2-((2- aminoethyl)disulfaneyl)-2-methylpropyl carbamate (FIG. 9). Without being bound to theory, it is hypothesized that within the linker, gem-dimethylation a- to the disulfide affords a sterically hindered S-S bond which, by virtue of the local stereoelectronics, is consequently protected from cleavage during standard SPPS workup. Moreover, methylation adjacent to the disulfide bond provides a kinetic acceleration to the subsequent immolation reaction, which proceeds through a thiolate-mediated 3-exo-tet ring closure. Specifically, gem- dimethylation enforces Thorpe-Ingold bond angle compression, reducing the energetic barrier to cyclization and formation of the thiirane. At this point, the reaction is rendered irreversible through the decarboxylative release of CO2.

[0099] Traceless cleavage of the BTSSME2 linker 2 is performed through the addition of >1 equiv. of an appropriate reducing agent such as PPI13 or [3-ME at neutral to moderately basic pH (pH 7 - 9). In the case of an exogenous thiol reducing agent, preformation of the thiolate anion is required to cleave the sterically hindered gem-dimethyl disulfide and release the synthetic target. This affords significant process control over the release of the material, as demonstrated by a test cleavage of the model peptide (-HN-spgys-CONH2) pentapeptide 7Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 sequence linked to an N-terminal biotin- SSME2-pNP streptavidin capture linker 5 (FIG. 10).In the absence of reducing agent, the peptide linker complex 7 is completely stable over a wide pH range for >24 h. Moreover, the disulfide remains stable at pH < 1 even in a large excess of exogenous thiol (>1000 equiv). Under the appropriate conditions (pH 7 - 9, >100 equiv. of P-ME), release of the pentapeptide 8 is quantitative and complete within 10 min.

[0100] Cleavage of the BTOXGLY linker 1 can be performed through the addition of excess NaIO4, which selectively cleaves the 1,2-aminoalcohol moiety embedded in the linker. The oxidative cleavage of BTOXGLY liberates the linked peptide with an N-terminal glycine “scar”, but the sequence otherwise remains unmodified. Excess periodate is quenched through Na2SO3salt quench and MeONH2 nucleophilic trapping of Schiff-base intermediates arising from the periodate cleavage.

[0101] Similar to other affinity purification linkers known in the prior art, the invention finds application in the enrichment of a target polypeptide from crude SPPS reaction mixtures. The enrichment step positively selects for (i.e., “catches”) full-length product. Truncated peptides that arise due to incomplete couplings throughout the course of synthesis do not bear the linker, provided that a suitable N-terminal capping reagent (e.g. AC2O) is used between sequential coupling steps. As a result, these truncated peptides are not captured on the affinity resin and are therefore removed (FIG. 1). The generality of the 2-((2-aminoethyl)disulfaneyl)-2-methylpropyl core naturally lends itself to; 1) installation onto nucleophiles such as amines or alcohols through a variety of activated carbonates and, 2) a modular capture component compatible with multiple affinity enrichment strategies.

[0102] In a representative application of the technology, the capture component is biotin, which enables highly stringent enrichment over resin-immobilized streptavidin. Following rigorous washing of the resin, the affinity purified polypeptide is released into buffered media (pH 7 - 9) through the addition of a suitable disulfide reducing agent such as [3-mercaptoethanol (P-ME). Alternative affinity enrichment tags compatible with this linker include both alkyl and perfluoroalkyl (Rf) tags for selective enrichment over hydrocarbon or fluorocarbon media, propargyl- or azido-terminated moieties for covalent capture via [3+2] Huisgen-type triazole “click” chemistry (both Cu2+and strain-promoted), polyhistidine sequences for capture on widely used immobilized metal affinity chromatography (IMAC) resins, as well as aminooxy- or hydrazine-terminated moieties for capture over aldehyde / ketone resin via reversible hydrazone or oxime formation, respectively.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 Additionally, the capture moiety may consist of an N-terminal cysteine (Cys) residue for selective capture with ortho-formyl- arylboronic acids to form thiazolidines.

[0103] The yield of the cleavage reactions for the linkers of this disclosure may be determined through any suitable method known in the art, including LCMS analysis.Typically, the capture resin is loaded with an excess of cleavage mixture, such that the yield of peptide is set by the loading density of the capture resin. Sequences of representative affinity-tagged peptides having undergone successful affinity purification by catch and release are summarized in Table 1.Definitions

[0104] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0105] 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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0106] Further, the term “comprising” should be understood as having its open-ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B may be a composition that includes A, B, and other components, but may also be a composition made of A and B only.

[0107] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0108] As used herein, a “moiety” indicates a chemical group containing the indicated structural element. For example, a “biotin moiety” means a chemical group comprising a residue of biotin.

[0109] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which itFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0110] As used herein, an “enrichment tag” is a chemical moiety that facilitates purification of polypeptides, for example, through affinity interactions. In certain embodiments, the enrichment tag comprises a biotin residue. In certain embodiments, the enrichment tag comprises 8-hydroxyquinoline.

[0111] It is to be understood that the connectivity of groups, radicals, and linkages represented typographically, for example the divalent group alkylene-C(O)-NH-, shall map to their corresponding structural formulae in a left-to-right manner. For instance, the divalent Os^f^alk N^- group alkylene-C(O)-NH- shall be interpreted as having the structure H; wherein Ralkrepresents the alkylene group. Likewise, the divalent group -C(O)-NH-alkylene shall be O\ N e-interpreted as having the structure H

[0112] It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skilled person in the art to result chemically stable compounds which can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

[0113] As used herein, the term “alkyl” refers to saturated aliphatic groups, including but not limited to Ci-Cio straight-chain alkyl groups or Ci-Cio branched-chain alkyl groups.Preferably, the “alkyl” group refers to Ci-Ce straight-chain alkyl groups or Ci-Ce branched-chain alkyl groups. Most preferably, the “alkyl” group refers to C1-C4 straight-chain alkyl groups or C1-C4 branched-chain alkyl groups. Examples of “alkyl” include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1 -pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1 -hexyl, 2-hexyl, 3-hexyl, 1 -heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl and the like. The “alkyl” group may be optionally substituted. A “linear” alkyl group refers to a straight-chain alkyl group without a branching point. A “branched” alkyl group refers to an alkyl group having at least one branch point. Examples of branched alkyl groups include, e.g., isopropyl, sec-butyl, and tert-butyl.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0114] Moreover, the term “alkyl” as used throughout the specification, examples, and claims is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0115] The term “Cx.y” or “Cx-Cy”, when used in conjunction with a chemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups that contain from x to y carbons in the chain. A Cnealkyl group, for example, contains from one to six carbon atoms in the chain.

[0116] The term “alkylene” refers to a divalent alkyl group. The term (hydroxyalkyl)-O-alkylene-, for example, refers to a divalent alkyl group (i.e., an alkylene), appended to the parent molecule at one of its open valencies, and attached to an oxygen atom at the other of its open valencies, which oxygen atom is substituted by a hydroxyalkyl group.

[0117] The term "alkenyl" as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-l -heptenyl, and 3-decenyl. The unsaturated bond(s) of the alkenyl group can be located anywhere in the moiety and can have either the (Z) or the (E) configuration about the double bond(s). A “linear” alkenyl group refers to a straight-chain alkenyl group without a branching point. A “branched” alkenyl group refers to an alkenyl group having at least one branch point.

[0118] The term "alkynyl" as used herein means a straight or branched chain hydrocarbon radical containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl. A “linear” alkynyl group refers to a straight-chain alkynyl group without a branching point. A “branched” alkynyl group refers to an alkynyl group having at least one branch point.

[0119] The term “acyl” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0120] The term “acylamino” is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-, preferably alkylC(O)NH-.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0121] The term “acyloxy” is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0122] The term “alkoxy” refers to an alkyl group appended to the parent molecular moiety through an oxygen atom. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.

[0123] The term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula -alkyl-O-alkyl.

[0124] The term “alkylamino”, as used herein, refers to an amino group substituted with at least one alkyl group. A “dialkylamino” refers to an amino group substituted with two alkyl groups.

[0125] The term “alkylthio”, as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0126] The term “amide”, as used herein, refers to a groupR10wherein R9and R10each independently represent a hydrogen or hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. In some embodiments, “amido” refers to the group -C(O)NH2.

[0127] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented byR9R9f— N or |— N-Ri°R10R10'wherein R9, R10, and R10’ each independently represent a hydrogen or a hydrocarbyl group, or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure. In some embodiments, “amino” refers to the group -NH2.

[0128] The term “aminoalkyl”, as used herein, refers to an alkyl group substituted with an amino group.

[0129] The term “arylalkyl”, as used herein, refers to an alkyl group substituted with an aryl group.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0130] The term “aryl” as used herein include substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0131] The term “carbamate” is art-recognized and refers to a groupO Oc£ JI D10 jf 11 D10orN' XXR9R9wherein R9and R10independently represent hydrogen or a hydrocarbyl group.

[0132] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0133] The term “carbocycle” includes 5-7 membered monocyclic and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycle includes bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term “fused carbocycle” refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, is included in the definition of carbocyclic. Exemplary “carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-IH-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one or more positions capable of bearing a hydrogen atom.

[0134] The term “carbocyclylalkyl”, as used herein, refers to an alkyl group substituted with a carbocycle group.

[0135] The term “carbonate” is art-recognized and refers to a group -OCO2-.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0136] The term “carboxy” or “carboxyl”, as used herein, refers to a group represented by the formula -CO2H.

[0137] The term “cycloalkyl” includes substituted or unsubstituted non-aromatic single ring structures, preferably 4- to 8-membered rings, more preferably 4- to 6-membered rings. The term “cycloalkyl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is cycloalkyl and the substituent (e.g., R100) is attached to the cycloalkyl ring, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Cycloalkyl groups include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0138] The term “ester”, as used herein, refers to a group -C(O)OR9wherein R9represents a hydrocarbyl group.

[0139] The term “ether”, as used herein, refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical.Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include “alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.

[0140] The terms “halo” and “halogen” as used herein means halogen and includes chloro, fluoro, bromo, and iodo.

[0141] The terms “heteroaralkyl” and “heteroarylalkyl”, as used herein, refers to an alkyl group substituted with a heteroaryl group.

[0142] The term “heteroaryl” includes substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term “heteroaryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.

[0143] The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0144] The terms “heterocycloalkylalkyl” and “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.

[0145] The terms “heterocycloalkyl,” “heterocyclyl”, “heterocycle”, and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms “heterocycloalkyl,” “heterocyclyl”, “heterocycle”, and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls. Heterocycloalkyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0146] The term “hydrocarbyl”, as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbonhydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0147] The term “hydroxyalkyl”, as used herein, refers to an alkyl group substituted with a hydroxy group.

[0148] The term “lower” when used in conjunction with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant to include groups where there are ten or fewer atoms in the substituent, preferably six or fewer. A “lower alkyl”, for example, refers to an alkyl group that contains ten or fewer carbon atoms, preferably six or fewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents, such as in the recitations hydroxyalkyl and aralkyl (in which case, for example, the atoms within the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0149] The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls) inFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 which two or more atoms are common to two adjoining rings, e.g., the rings are “fused rings”. Each of the rings of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms in the ring, preferably from 5 to 7.

[0150] The term “sulfate” is art-recognized and refers to the group -OSO3H, or a pharmaceutically acceptable salt thereof.

[0151] The term “sulfonamide” is art-recognized and refers to the group represented by the general formulaewherein R9and R10independently represents hydrogen or hydrocarbyl.

[0152] The term “sulfoxide” is art-recognized and refers to the group-S(O)-.

[0153] The term “sulfonate” is art-recognized and refers to the group SO3H, or a pharmaceutically acceptable salt thereof.

[0154] The term “sulfone” is art-recognized and refers to the group -S(O)2-.

[0155] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include any substituents described herein, for example, a halogen, an alkyl, an alkenyl, an alkynyl, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano,Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamide, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0156] The term “thioalkyl”, as used herein, refers to an alkyl group substituted with a thiol group.

[0157] The term “thioester”, as used herein, refers to a group -C(O)SR9or -SC(O)R9

[0158] wherein R9represents a hydrocarbyl.

[0159] The term “thioether”, as used herein, is equivalent to an ether, wherein the oxygen is replaced with a sulfur.

[0160] The term “urea” is art-recognized and may be represented by the general formula OR9R9wherein R9and R10independently represent hydrogen or a hydrocarbyl.

[0161] The phrase “protecting group”, as used herein, means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 5th ed.; Wiley: New York, 2014). Protected forms of the inventive compounds are included within the scope of this invention. An “amino protecting group” means a protecting group that protects an -NH-, -NH2, or -N(R)H moiety, wherein R can be any hydrocarbyl or other substituent. Exemplary amino protecting groups include those that form carbamates such as 9-fluorenylmethyloxycarbonyl (Fmoc), tertbutyloxycarbonyl (Boc), methyloxycarbonyl, and benzyloxycarbonyl (Cbz); amides such as benzoyl (Bz); and N-sulfonyl derivatives such as N-toluenesulfonamide (“tosyl”; Ts) or methanesulfonamide (Ms). An “acid labile protecting group” means a protecting group that may be removed under acidic conditions.

[0162] A “peptide fragment” means a residue of polypeptide comprising at least two amino acids.

[0163] An “affinity resin” refers to a matrix comprising an affinity group that binds a molecule of interest.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0164] As used herein, “affinity” refers to the strength of the sum total of non-covalent interactions between a particular binding agent (e.g., a molecule of interest), and / or a binding site thereof, with a binding target (e.g., an affinity resin). Unless indicated otherwise, as used herein, “binding affinity” refers to a 1:1 interaction between a binding agent and a binding target thereof (e.g., a molecule of interest with an affinity resin). Those of skill in the art appreciate that a change in affinity can be described by comparison to a reference (e.g., increased or decreased relative to a reference), or can be described numerically. Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and / or equilibrium association constant (KA). The KD is the quotient of koff / kon, whereas KA is the quotient of kon / koff, where konrefers to the association rate constant of, e.g., a molecule of interest with an affinity resin, and koff refers to the dissociation of, e.g., a molecule of interest with an affinity resin. konand koff can be determined by techniques known to those of skill in the art, such as BIAcore® or KinExA.

[0165] The term “amino acid”, in its broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Canonical amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Non-canonical amino acid” refers to any amino acid, other than the canonical amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with a typical or canonical amino acid structure. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, sulfation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification can, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing anFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” can be used to refer to a free amino acid; in some embodiments it can be used to refer to an amino acid residue of a polypeptide.

[0166] The term “amino acid side chain”, as used herein, refers to a substituent that is typically connected to the a-carbon of an amino acid. Such a substituent may be, for example, a hydrogen atom, as in the case of glycine, or may be a naturally occurring or unnatural (i.e., synthetic) substituent, such as 4-fluorophenyl in the case of 4-fluorophenylalanine. In some embodiments, an amino acid side chain may be connected to the P-carbon, if present, as in the case of P-alanine. In certain embodiments, the amino acid side chain may be substituted or unsubstituted alkyl (such as hydroxyalkyl or aminoalkyl, as in the side chains of lysine, valine, threonine, and serine), substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aralkyl (e.g., a phenylalanine side chain), substituted or unsubstituted alkoxy, substituted or unsubstituted amido, substituted or unsubstituted heterocyclyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl (e.g., a histidine side chain).EXAMPLESExample 1. Exemplary synthesis of linker agents comprising an enrichment tag

[0167] The synthesis of the BTOXGLY linker agent (compound 1) containing a Boc- and ‘Bn-protected 1,2-aminoalcohol core linked to an p-NP activated glycine was accomplished over 10 consecutive steps (FIG. 3). Commencing with racemic methyl 3-amino-2-hydroxypropanoate 1A, the amine and secondary alcohol were protected with CbzCl and TBTA (tert-butyl 2,2,2-trichloroacetimidate) to afford the fully protected precursor 1C.Redox manipulation of the methyl ester was followed by reductive amination to form the amine-linked glycine methyl ester IF. Boc protection of the secondary amine with subsequent hydrogenation to remove the carboxybenzyl moiety returned 1H, which was competent in an amide coupling with Biotin-PEG4-COOH acid to afford the PEG-linked reagent II. Finally, LiOH saponification of the methyl ester and DCC-mediated esterification in the presence of / wz / -nitrophenol (p-NP) and catalytic DMAP provided the target compound 1 (1.30 g, 96% purity).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 CbzCI (1.10 eq)NaOH (1.00 eq)NaHCO3(2.50 eq) H2N Y O CbzHN Y OHCIOH H2O (10.0 V) OH0-25 °C, 13 hrsstep 1

[0168] To the solution of compound 1A (25.0 g, 130.1 mmol, 1.00 eq, HC1) in H2O (250 mL) was added NaOH (5.21 g, 130 mmol, 1.00 eq) and stirred for 30 min at 25 °C under N2 atmosphere, before NaHCO? (27.3 g, 325 mmol, 2.50 eq) was added at 25 °C. The solution was cooled to 0 °C, before CbzCI (24.4 g, 143 mmol, 1.10 eq) was added dropwise over 30 min. The resulting mixture was stirred at 25 °C for an additional 12 hrs. The reaction mixture was extracted with ethyl acetate (200 mL * 3), and the organic layer was concentrated under reduced pressure to give crude compound IB (31.0 g, 94.0% yield) as a colorless oil. The crude product was used into the next step without further purification.TBTA (3.00 eq)BF3Et2O (0.15 eq) CbzHN CbzHN Y ODCM (3.00 V)OfBun-Hexane (10.0 V)25 °C, 12 hrsstep 2

[0169] To a solution of compound IB (30.0 g, 118 mmol, 1.00 eq) in DCM (90.0 mL) and n-Hexane (300 mL) under N2 atmosphere at 25 °C was added BFs. Et2O (5.37 g, 17.7 mmol, 47.0% purity, 0.15 eq) in portions (inner temperature below 40 °C), followed by the solution of TBTA (tert-butyl 2,2,2-trichloroacetimidate, 77.6 g, 355 mmol, 3.00 eq in 70 mL DCM). The mixture was stirred at 25 °C for 12 hrs. TLC indicated compound IB was consumed completely and several new spots were observed. The reaction mixture was quenched by brine (100 mL) and extracted with ethyl acetate (200 mL * 3). The organic phase was concentrated at 45 °C to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 100 / 1 to 5 / 1) to afford compound 1C (48.0 g, crude) as a yellow solid. 'H NMR (400 MHz, DMSO-rfc): <57.39 - 7.30 (m, 5H), 5.02 (br d, J= 3.4 Hz, 2H), 4.10 (br t, J = 6.4 Hz, 1H), 3.60 (s, 3H), 3.27 - 3.14 (m, 1H), 3.03 (td, J = 6.6, 13.6 Hz, 1H), 1.07 (s, 8H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325LiBH4 / THF (1.50 eq, 2.00 M) CbzHN CbzHN OfBu THF (10.5 V)0-25 °C, 16 hrs1D 36.6%yieldstep 3

[0170] The solution of compound 1C (38.0 g, 122 mmol, 1.00 eq) in THF (400 mL) under N2 atmosphere at 25 °C was added LiBIHU / THF (2.00 M, 92.1 mL, 1.50 eq) dropwise at 0 °C, before the mixture was stirred at 25 °C for 12 hrs. LCMS showed compound 1C was consumed completely and one main peak with desired MS was detected. The reaction was quenched by H2O (500 mL) and extracted with DCM (500 mL * 3). The combined organic phase was washed with brine (500 mL) and concentrated at 45 °C to give a residue. The residue was purified by column chromatography (SiCL, Petroleum ether / Ethyl acetate = 100 / 1 to 2 / 1) to give compound ID (13.0 g, 36.6% yield) as a yellow oil.1H NMR (400 MHz, CHLOROFORM-7): S 7.36 (s, 5 H) 5.07 - 5.19 (m, 3H) 3.71 (q, 7=4.91 Hz, 1 H) 3.44 - 3.55 (m, 3 H) 3.17 (dt, 7=14.26, 5.13 Hz, 1 H) 2.77 (brt, 7=6.82 Hz, 1 H) 1.20 (s, 9 H).DMP (2.00 eq)CbzHNDCM (10.0 V) 0 °C, 16 hrs1E61.9%yieldstep 4

[0171] The solution of compound ID (13.0 g, 46.2 mmol, 1.00 eq) in DCM (130 mL) under N2 atmosphere was cooled to 0 °C, before DMP (39.2 g, 92.4 mmol, 2.00 eq) was added under N2 atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 16 hrs. TLC indicated compound ID was consumed completely and one new spot was formed. The solution was poured into aq. NaHCCh (400 mL) solution, the aqueous layer was separated and extracted with DCM (400 mL * 3). The combined organic layer was dried over anhydrous Na2SC>4, filtered, and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiCL, Petroleum ether / Ethyl acetate = 100 / 1 to 2 / 1) to give compound IE (8.00 g, 61.9% yield) as a yellow oil. 'H NMR (400 MHz, CHLOROFORM-7): S 9.46 - 9.97 (m, 1 H) 7.36 (brs, 5 H) 4.81 - 5.35 (m, 3 H) 4.01 (brs, 1H) 3.25 - 3.56 (m, 2 H) 1.23 (brs, 9 H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325H2N'xxY°MeHCI OCbzHN (2.00 eq) OMe - *• CbzHN N NaBH3CN (2.50 eq) OfBuHO AcOH (10.0 V)KOAc (2.00 eq)THF (12.5 V)25 °C, 18 hrsstep 5

[0172] The mixture of methyl glycinate hydrochloride (6.35 g, 57.2 mmol, 2.00 eq) and KOAc (5.68 g, 56.6 mmol, 2.00 eq) in THF (100 mL) was stirred for 2 hrs at 25 °C under N2 atmosphere. Compound IE (8.00 g, 28.5 mmol, 1.00 eq) was added at 25 °C, before AcOH (80 mL) and NaBHsCN (4.42 g, 73.9 mmol, 2.50 eq) were added under N2 atmosphere at 25 °C. The resulting mixture was stirred for 16 hrs at 25 °C. LCMS showed compound IE was consumed completely and one main peak with desired MS was detected. The reaction was quenched by H2O (500 mL) and extracted with ethyl acetate (200 mL * 3). The combined organic phase was concentrated under reduced pressure to give crude compound IF (9.26 g) as a colorless oil. The crude product was used into the next step without further purification.LCMS (ESI, IF): calc’d for C18H28N2O5 [M+H]+: 353.20, found: 353.0.BOC2O (2.00 eq)CbzHN CbzHN DIEA (2.00 eq) DMAP (0.20 eq)THF (10.7 V)25 °C, 16 hrs49.4%yieldstep 6

[0173] To the solution of compound IF (9.26 g, 27.8 mmol, 1.00 eq), DIEA (9.24g, 71.55 mmol, 2.00 eq) and DMAP (787 mg, 7.15 mmol, 0.20 eq) in THF (100 mL) under N2 atmosphere was added BOC2O (10.0 g, 71.5 mmol, 2.00 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 16 hrs. TLC indicated compound IF was consumed completely and one new spot was formed. The reaction mixture was concentrated under reduced pressure to afford a residue. The residue was diluted with H2O (100 mL), and extracted with DCM (200 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue wasFlagship Ref. No.: VL83003-W1 Attorney Docket No.: FBZ-00325 purified by prep-HPLC (Column: CD07-Daisogel SP-100-8-ODS-PK 150*25*10 um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 50%-80% B over 10 min) to afford compound1G (7.50 g, 49.4% yield) as a colorless oil. 'H NMR (400 MHz, CHLOROFORM- ): 37.46 - 7.28 (m, 5H), 5.97 - 5.01 (m, 3H), 4.25 - 3.92 (m, 2H), 3.82 - 3.61(m, 5H), 3.47 - 3.05 (m, 2H), 2.98 (br d, J = 10.8 Hz, 1H), 1.49 - 1.41 (m, 9H), 1.23 - 1.09 (m, 9H).H2(50 Psi)Pd / C (0.50 eq, 10%) OMe CbzHN H2N N Y THF (10.0 V) oteuBoco40 °C, 3 hrsStep 7

[0174] The mixture of compound 1G (3.00 g, 6.63 mmol, 1.00 eq) and Pd / C (3.00 g, 2.82 mmol, 10% purity, 0.50 eq) in THF (30 mL) was purged with H2 for 3 times and stirred at 40 °C for 3 hrs. TLC indicated compound 1G was consumed completely and one new spot was formed. The mixture was filtered over Celite, and the filtrate was concentrated under reduced pressure to give a crude compound 1H (2.11 g). The crude product was used into the next step without further purification.Biotin-PEG4-acid1H DMF (460 V)25 °C, 16 hrs 76 2%yield

[0175] To the solution of Biotin-PEG4-acid (3.26 g, 6.63 mmol, 1.00 eq), HBTU (2.51 g, 6.63 mmol, 1.00 eq) and DIEA (2.57 g, 19.8 mmol, 3.00 eq) in DMF (15 mL) under N2 atmosphere at 25 °C was added compound 1H (2.11 g, 6.63 mmol, 1.00 eq). The reaction mixture was stirred for 16 hrs at 25 °C. LCMS showed compound 1H was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (Column: YMC Triart C1870*250mm*7um; mobile phase: [water (FA)-ACN]; gradient: 25%-55% B over 25 min) to give compound II (4.00 g, 76.2% yield) as a yellow oil.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-003251J

[0176] The solution of compound II (4.00 g, 5.05 mmol, 1.00 eq) and L1OH. H2O (1.06 g, 25.2 mmol, 5.00 eq) in THF (40.0 mL) and H2O (20.0 mL) was stirred at 25 °C for 1 hr.LCMS showed compound II was consumed completely and one main peak with desired MS was detected. HC1 (1.00 N) was added to adjust pH = 4 and the mixture was extracted with ethyl acetate (50.0 mL * 3). The organic layer was concentrated at 45 °C to afford crude compound 1J (1.90 g) as a colorless oil. The crude product was used in the next step without further purification.(1.12 eq) DCC (1.10 eq) DMAP (0.10 eq) DCM (10.5 V) 25 °C, 16 hrs 69.2%yield step 1011.30 g, 96.4%

[0177] The solution of compound 1J (1.90 g, 2.44 mmol, 1.00 eq), DCC (554 mg, 2.69 mmol, 1.10 eq), DMAP (29.8 mg, 244 pmol, 0.10 eq) and 4-nitrophenol (380 mg, 2.73 mmol, 1.12 eq) in DCM (20 mL) under N2 atmosphere was stirred for 16 hrs at 25 °C. LCMS showed compound 1 J was consumed completely and one main peak with desired MS was detected. The reaction mixture was filtered and concentrated under reduced pressure to give aFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 residue. The residue was purified by prep-HPLC (Column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water (NH4HCO3) ACN]; gradient: 30%-60% B over 20 min) to afford compound 1 (1.52 g, 69.2% yield) as a white solid. LCMS (ESI): calc’d for C41H66N6O14S [M+H]+: 899.44, found: 899.3. 'H NMR (400 MHz, CHLOROFORM-7): 3 8.34 - 8.24 (m, 2H), 7.38 - 7.29 (m, 2H), 7.03 (br d, J = 1.6 Hz, 1H), 6.72 - 6.58 (m, 1H), 6.19 - 5.83 (m, 1H), 5.27 - 4.98 (m, 1H), 4.54 - 4.38 (m, 2H), 4.33 (br dd, 7= 4.4, 7.4 Hz, 1H), 4.27 - 4.16 (m, 1H), 3.86 - 3.75 (m, 4H), 3.66 - 3.62 (m, 12H), 3.57 (br t, 7= 4.9 Hz, 3H), 3.49 - 3.40 (m, 3H), 3.24 - 3.09 (m, 2H), 3.05 - 2.86 (m, 3H), 2.74 (br d, 7= 12.9 Hz, 1H), 2.57 - 2.44 (m, 2H), 2.31 - 2.16 (m, 3H), 1.79 - 1.59 (m, 6H), 1.46 (s, 8H), 1.24 (s, 9H).

[0178] The synthesis of the linker agent BTSSME2 (compound 2) containing a reductively cleavable 2-((2-aminoethyl)disulfaneyl)-2-methylpropyl core was accomplished through a 2-step synthesis (FIG. 4). The asymmetric pyridinyl disulfide 2A was first redox exchanged with 2-mercapto-methylpropan-l-ol in MeOH to afford the gem-dimethyl core 2B as its HC1 salt. This intermediate was then coupled to a desired affinity tag (Biotin-PEG4-COOH), followed by activation of the propanolic alcohol with 4-nitrophenyl-chloroformate to afford the activated carbonate BTSSME2 linker 2. The extent of methylation a to the disulfide can be varied by supplying differentially substituted variants of the 2-mercaptoethanol.Additionally, the intermediate HC1 salt 2B may be coupled to Fmoc-OSu or Fmoc-Cl prior to activation as the 4-nitrophenyl (p-NP) carbonate to permit tag diversification following on-resin installation.(0.67 eq) MeOH (25.2 V) HCI25 °C, 16 hrsStep 1 2B

[0179] The solution of compound 2A (8.90 g, 39.9 mmol, 1.00 eq) in MeOH (225 mL) was stirred for 15 min at 25 °C, before a solution of 2-mercapto-2-methylpropan-l-ol (2.84 g, 26.7 mmol, 0.67 eq in 45 mL MeOH) was added dropwise over 30 min at 25 °C. The reaction mixture was stirred for 16 hrs at 25 °C. TLC (DCM / MeOH = 5 / 1, Rf <2B) = 0.15) indicated compound 2A was remained and two new spots were detected. The mixture was concentrated under reduced pressure to give a residue. The residue was added to petroleum ether (50 mL)Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 and stirred for 10 min at 25 °C. The suspension was filtered, and the wet cake was washed with petroleum ether (10 mL). The filter cake was dried at 40 °C under reduced pressure to give compound 2B (9.00 g, crude, HC1) as a white solid. 'H NMR (400 MHz, METHANOL-6 / 4): 3 1.29 (s, 6 H) 2.93 (t, J = 6.69 Hz, 2 H) 3.27 (t, J = 6.69 Hz, 2 H) 3.50 (s, 2 H).1). Biotin-PEG4-OH (1.0 eq)HBTU (1.35 eq)DIEA (4.45 eq)DMF (3.0 V)25 °C, 16 hrsHCI 2). 4-nitrophenyl carbonochloridate (1.50 eq)25 °C, 12 hrs20.0% yieldStep 225 g, 95%

[0180] The solution of Biotin-PEG4-OH (14.9 g, 30.4 mmol, 1.00 eq), HBTU (15.6 g, 41.1 mmol, 1.35 eq) and DIEA (17.5 g, 135 mmol, 4.45 eq) in DMF (45 mL) was purged with N2 for 3 times, before compound 2B (8.89 g, 40.8 mmol, 1.34 eq, HCI) was added. The reaction mixture was stirred for 16 hrs at 25 °C under N2 atmosphere. LCMS showed 39.6% of desired MS was detected. 4-nitrophenyl carbonochloridate (9.21 g, 45.7 mmol, 1.50 eq) was added, purged with N2 for 3 times, and stirred for additional 12 hrs at 25 °C. LCMS showed 10.4% of compound 2 was detected. The mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (FA)-ACN]; gradient: 25%-55% B over 20 min). The resulting product was further purified by prep-NPLC (column: Welch Ultimate XB-CN 250*50*10 um; mobile phase: [Hexane-IPA]; gradient: 40%-70% B over 20 min) to afford compound 2 (5.00 g, 20.0% yield) as an orange gum. LCMS (ESI): calc’d for C34H53N5O12S3 [M+H]+: 820.29, found: 820.4. 'H NMR (400 MHz, CHLOROFORM- ): 3 1.40 (s, 6 H) 1.43 - 1.49 (m, 2 H) 1.64 - 1.79 (m, 4 H) 2.24 (br t, J = 7.32 Hz, 2 H) 2.49 (t, J = 5.82 Hz, 2 H) 2.75 (br d, J = 12.76 Hz, 1 H) 2.84 -2.95 (m, 3 H) 3.13 - 3.20 (m, 1 H) 3.40 - 3.47 (m, 2 H) 3.52 - 3.60 (m, 4 H) 3.61 - 3.72 (m, 12 H) 3.74 (t, J = 5.82 Hz, 2 H) 4.26 (s, 2 H) 4.34 (br dd, J = 7.50, 4.75 Hz, 1 H) 4.52 (dd, J =Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 7.19, 5.07 Hz, 1 H) 5.38 - 5.56 (m, 1 H) 6.29 (br s, 1 H) 6.71 (br s, 1 H) 7.03 (br s, 1 H) 7.41 (d, J = 9.01 Hz, 2 H) 8.30 (d, J = 9.13 Hz, 2 H).

[0181] The synthesis of a non-cleavable BT tag (compound 3) was accomplished in a single step through activation of a Biotin-PEG4-COOH acid and esterification with p-NP (FIG. 5).(2.00 eq) DCC (1 10 eq) DMAP (0.10 eq) DMF (100 V) 3 3A 25 °C, 16 hrs 100 g, 95%48.8%yield

[0182] To a solution of compound 3A (58.0 g, 118 mmol, 1.00 eq) in DMF (580 mL) was added DCC (26.8 g, 129 mmol, 1.10 eq) and DMAP (1.44 g, 11.8 mmol, 0.10 eq) under N2 atmosphere at 25 °C, before 4-nitrophenol (32.8 g, 236 mmol, 2.00 eq) was added. The resulting mixture was stirred at 25 °C for 16 hrs. LCMS indicated compound 3A was consumed completely. The mixture was filtered, and the filtrate was concentrated in vacuo to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 250 mm*100 mm*10 um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 17%-48% B over 25 min) to afford 3 (99.2 g, 48.8% yield) as an off-white solid. LCMS (ESI): calc’d for C27H40N4O10S [M+H]+: 613.25, found: 613.1. 'H NMR (400 MHz, CHLOROFORM- ): S 8.20 - 8.32 (m, 2 H) 7.28 - 7.33 (m, 2 H) 6.86 (br s, 1 H) 6.62 (br s, 1 H) 5.50 - 5.96 (m, 1 H) 4.49 (dd, J = 7.57, 4.82 Hz, 1 H) 4.29 (dd, J = 7.75, 4.63 Hz, 1 H) 3.87 (t, J = 6.13 Hz, 2 H) 3.58 - 3.70 (m, 12 H) 3.55 (t, J = 4.94 Hz, 2 H) 3.38 - 3.45 (m, 2 H) 3.12 (td, J = 7.16, 4.69 Hz, 1 H) 2.85 - 2.92 (m, 3 H) 2.73 (d, J = 12.76 Hz, 1 H) 2.22 (br t, J = 7.50 Hz, 2 H) 1.59 - 1.80 (m, 4 H) 1.37 - 1.50 (m, 2 H).

[0183] The synthesis of base-cleavable linker BTASE (compound 4) containing a 2-((2-aminoethyl)sulfonyl)ethane core was accomplished over 5 consecutive steps (FIG. 6).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325Br— OH_ (1.50 eq)H2N BocHN OH LiOH. H2O (2.50 eq)4A BOC2O (2.00 eq)4BEtOH (10.0 V)25-55 °C, 20 hrs46.1 %yieldStep 1

[0184] To the solution of compound 4A (20.0 g, 176 mmol, 1.00 eq) and 2-bromoethan-l-ol (33.0 g, 264 mmol, 1.50 eq) in EtOH (200 mL) was added LiOH. H2O (18.4 g, 440 mmol, 2.50 eq) at 25 °C under N2 atmosphere. The reaction mixture was stirred at 55 °C for 4 hrs, before BOC2O (76.8 g, 352 mmol, 2.00 eq) was added and stirred at 25 °C for additional 16 hrs. TLC indicated one new spot with lower polarity was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 3 / 1 to 1 / 1) to afford compound 4B (18.0 g, 46.1% yield) as a colorless oil. ‘H NMR (400 MHz, CHLOROFORM- ): d 3.75 (t, J = 5.9 Hz, 2H), 3.37 - 3.25 (m, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 6.6 Hz, 2H), 1.45 (s, 9H).Oxone (3.0 eq) BocHN^^^ Rnrl-IN'^''’ — —x / Xx(~>|_ i MeOH (11.1 V)BocHNo °H4B Q-25 °C, 3 hrs 36.4%yieldStep 2

[0185] The solution of compound 4B (18.0 g, 81.3 mmol, 1.00 eq) in MeOH (200 mL) under N2 atmosphere was cooled to 0 °C, before Oxone (41.0 g, 243 mmol, 3.00 eq) was added in portions under N2 atmosphere at 0 °C. The reaction mixture was stirred at 25 °C for 3 hrs. TLC indicated compound 4B was consumed completely and one new spot was formed. The reaction mixture was diluted with H2O (160 ml), and extracted with ethyl acetate (155 ml). The combined organic layer was washed with brine (100 ml), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate = 1 / 1) to give compound 4C (7.50 g, 36.4% yield) as a colorless oil. 'H NMR (400 MHz, CHLOROFORM-d): d 5.40 -4.94 (m, 1H), 4.18 - 4.08 (m, 2H), 3.71 - 3.60 (m, 2H), 3.35 (t, J = 6.1 Hz, 2H), 3.30 - 3.22 (m, 2H), 2.25 - 2.10 (m, 1H), 1.50 - 1.40 (m, 9H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325o II HCI (10.0 eq, 6.00 M) 9 2 HCI BocHN THF (26.6 V)4C 0-25 °C, 3 hrs 96.0%yieldStep 3

[0186] The solution of compound 4C (7.50 g, 29.6 mmol, 1.00 eq) in THF (200 mL) under N2 atmosphere was cooled to 0 °C, before HCI (6.00 M, 46.0 mL, 10.0 eq) was added at 0 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 3 hrs. TLC indicated compound 4C was consumed completely and one new spot was formed. The reaction mixture was concentrated under reduced pressure to give compound 4D (6.00 g, 96.0% yield, 2 HCI) as a colorless oil. 'H NMR (400 MHz, DMSO-rfc): <58.61 - 8.03 (m, 3H), 3.82 - 3.75 (m, 2H), 3.57 - 3.48 (m, 2H), 3.40 - 3.32 (m, 2H), 3.26 - 3.14 (m, 2H).Biotin-PEG4-OH Il 2 HCI (1.00 eq)2O HBTU (1.50 eq)DIEA (5.00 eq) 4D DMF (9.50 V) 4E 25 °C, 3 hrs 97.1%yieldStep 4

[0187] The solution of Biotin-PEG4-OH (10.5 g, 21.3 mmol, 1.00 eq), HBTU (12.1 g, 32.0 mmol, 1.50 eq) and DIEA (13.8 g, 106 mmol, 5.00 eq) in DMF (100 mL) under N2 atmosphere was added compound 4D (6.00 g, 26.5 mmol, 1.68 eq, 2 HCI) at 25 °C under N2 atmosphere. The reaction mixture was stirred at 25 °C for 3 hrs. LCMS showed compound 4D was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition, Column: YMC Triart C1870*250 mm*7 um; mobile phase:[water (FA)-ACN]; gradient: 0%-30% B over 25 min) to afford compound 4E (13.0 g, 97.1% yield) as a colorless oil. LCMS (ESI): calc’d for C25H46N4O10S2 [M+H]+: 627.27, found: 627.3.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325(2.00 eq) NMM (2.00 eq) DMF (10.0 V) 4E 25 °C, 16 hrs 43.1% yield Step 545.00 g, 95%

[0188] The solution of compound 4E (5.50 g, 8.77 mmol, 1.00 eq), NMM (1.78 g, 17.5 mmol, 2.00 eq) and bis(4-nitrophenyl) carbonate (5.34 g, 17.5 mmol, 2.00 eq) in DMF (55 mL) was stirred at 25 °C for 16 hrs under N2 atmosphere. LCMS showed compound 4E was consumed completely and one major peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition, Column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water (TFA)-ACN]; gradient: 15%-45% B over 30 min) to afford compound 4 (5.00 g, 43.1% yield) as a colorless oil. LCMS (ESI): calc’d for C32H49N5O14S2 [M+H]+: 792.27, found: 792.5. 'H NMR (400 MHz, CHLOROFORM- ): 38.30 (d, J = 9.1 Hz, 2H), 7.46 -7.40 (m, 2H), 6.88 - 6.44 (m, 2H), 6.16 - 5.84 (m, 2H), 4.75 (t, J = 5.6 Hz, 2H), 4.59 (dd, J = 4.7, 7.6 Hz, 1H), 4.40 (dd, J = 4.6, 7.8 Hz, 1H), 3.81 - 3.76 (m, 2H), 3.74 (t, J= 5.6 Hz, 2H), 3.66 (d, J = 6.9 Hz, 12H), 3.58 (t, J = 5.3 Hz, 4H), 3.49 - 3.39 (m, 4H), 3.26 - 3.15 (m, 1H), 2.99 - 2.91 (m, 1H), 2.77 (d, J = 13.1 Hz, 1H), 2.52 (t, J = 5.6 Hz, 2H), 2.34 - 2.23 (m, 2H), 1.79 - 1.64 (m, 4H), 1.57 - 1.41 (m, 2H).

[0189] The synthesis of reductively cleavable linker BTSSHME (compound 5) was initiated with symmetric pyridinyl disulfide 5A and completed over 7 consecutive steps (FIG. 7).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325(1.00 eq)MeOH (10.0 V)5A 25 °C, 12 hrs 5B62.3%yieldStep 1

[0190] The solution of compound 5A (47.8 g, 217 mmol, 2.00 eq) and 2-mercaptopropan-l-ol (10.0 g, 108 mmol, 1.00 eq) in MeOH (100 mL) was stirred at 25 °C for 12 hrs. LCMS showed 50 % of the desired compound was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was dissolved in ethyl acetate (50.0 mL * 3), washed with brine (50.0 mL * 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Daisogel C18 250*70 mm*10 um; mobile phase: [water (NHTfcO^ACN]; gradient: 20%-50% B over 5 min) to afford compound 5B (30.2 g, 62.3% yield) as a yellow oil. LCMS (ESI): calc’d for C8H11NOS2[M+H]+: 202.03, found: 202.0.B°C'N-^\ / SHH(1.93 eq)Py (1.00 V)5B MeOH (40.0 V) 5C25 °C, 16 hrs32.4%yieldStep 2

[0191] To a solution of compound 5B (30.6 g, 152 mmol, 1.00 eq), Pyridine (Py) (30.6 mL) in MeOH (1.22 L) was added a solution of tert-butyl (2-mercaptoethyl)carbamate (52.0 g, 293 mmol, 1.93 eq) dropwise at 25 °C. The mixture was stirred at 25 °C for 16 hrs. LCMS showed compound 5B was consumed completely and one major peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Ultimate XB-Diol 250*50*10 um; mobile phase: [Hexane-EtOH]; gradient: l%-28% B over 15 min) to afford compound 5C (13.2 g, 32.4% yield) as a yellow oil. LCMS (ESI): calc’d for C10H21NO3S2 [M+H]+: 268.10, found: 168.1 [M-100+H]+, 290.1 [M+Na]+. 'H NMR (400 MHz, CHLOROFORM- ): 3 1.30 (d, J = 7.00 Hz, 3 H) 1.44 (s, 9 H) 2.47 (br s, 1 H) 2.79 (t, J = 6.50 Hz, 2 H) 2.91 - 3.05 (m, 1 H) 3.45 (br d, J = 6.00 Hz, 2 H) 3.54 - 3.79 (m, 2 H) 4.94 (br s, 1 H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325TBSCI (1.35 eq)BOCOTBS'N imidazole (1.67 eq)DMF (10.0 V) SD 25 °C, 3 hrsStep 3

[0192] To the solution of compound 5C (9.70 g, 36.2 mmol, 1.00 eq) and imidazole (4.13 g, 60.6 mmol, 1.67 eq) in DCM (97.0 mL) was added TBSCI (7.39 g, 49.0 mmol, 1.35 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 3 hrs. TLC indicated one major new spot with lower polarity was detected. The reaction mixture was concentrated under reduced pressure to give crude compound 5D (13.9 g, crude) as a yellow oil. 'H NMR (400 MHz, DMSO-rfc): 30.05 (s, 6 H) 0.87 (s, 9 H) 1.21 (d, J = 6.88 Hz, 3 H) 1.32 - 1.42 (m, 9 H) 2.67 -2.78 (m, 2 H) 2.89 - 2.99 (m, 1 H) 3.18 (q, J = 6.63 Hz, 2 H) 3.57 (dd, 7= 10.44, 6.82 Hz, 1 H) 3.72 (dd, 7 = 10.38, 5.25 Hz, 1 H) 6.93 (br t, 7 = 5.19 Hz, 1 H).TMSI (1.50 eq) IBOC'N'^-'S'SZ%'OTBS- *• H, N ' ^0TB3H DCM (10.0 V)M2N b5D -78~20 °C, 16.5 hrs 5E89.3%yieldStep 4

[0193] The solution of compound 5D (17.2 g, 45.0 mmol, 1.00 eq) in DCM (172 mL) was cooled to -78 °C, before TMSI (13.5 g, 67.6 mmol, 1.50 eq) was added in portions. The reaction mixture was stirred at -70 °C for 30 min, warmed to 20 °C, and stirred for additional 16 hrs. TLC indicated one major new spot with lower polarity was detected. The reaction was quenched with the addition of a solution of MeOH / aq. NfDHCCh = 2.5 / 1 (35.0 mL), and extracted with DCM (100 mL * 3). The combined organic layer was washed with brine (50.0 mL * 3), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give compound 5E (11.3 g, 89.3% yield) as a yellow oil.1H NMR (400 MHz, DMSO-<7>): <5 0.03 - 0.08 (m, 5 H) 0.87 (s, 9 H) 1.19 - 1.26 (m, 3 H) 2.72 - 3.03 (m, 5 H) 3.58 (dd, J = 10.44, 6.69 Hz, 1 H) 3.72 (dd, 7= 10.38, 5.38 Hz, 1 H) 4.11 - 4.75 (m, 3 H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 Biotin-PEG4-acid (1 00 eq) HBTU (1.35 eq) 5E DIPEA (4.45 eq)DMF (100 V) 25 °C, 16 hrsStep 5

[0194] To the solution of Biotin-PEG4-acid (18.0 g, 36.6 mmol, 1.00 eq), HBTU (18.7 g, 49.4 mmol, 1.35 eq), DIPEA (21.0 g, 162 mmol, 4.45 eq) in DMF (180 mL) was added compound 5E (11.3 g, 40.2 mmol, 1.10 eq) over 15 min at 25 °C under N2 atmosphere. The reaction mixture was stirred for 16 hrs at 25 °C. TLC indicated one major new spot with lower polarity was detected. The reaction was quenched with the addition of H2O (200 mL), and extracted with DCM (100 mL * 3). The combined organic layer was washed with brine (50.0 mL * 3), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm,10 um); mobile phase: [water (HCl)-ACN]; gradient: 10%-40% B over 25 min) to afford compound 5F (32.5 g, crude) as a yellow oil. 'H NMR (400 MHz, DMSO-r / d):S 0.05 (s, 6 H) 0.87 (s, 9 H) 1.22 (d, J = 6.88 Hz, 3 H) 1.25 - 1.37 (m, 4 H) 1.44 - 1.55 (m, 3 H) 1.59 (br d, J = 9.51 Hz, 1 H) 2.06 (t, J = 7.44 Hz, 2 H) 2.26 - 2.34 (m, 2 H) 2.71 - 2.77 (m, 2 H) 2.82 (dd, J = 12.44, 5.07 Hz, 1 H) 2.91 - 3.00 (m, 1 H) 3.09 (br dd, 7= 4.13, 2.38 Hz, 1 H) 3.18 (br d, J = 5.75 Hz, 3 H) 3.36 - 3.42 (m, 4 H) 3.50 (s, 9 H) 3.54 - 3.64 (m, 4 H) 3.66 -3.76 (m, 1 H) 4.09 - 4.17 (m, 1 H) 4.24 - 4.36 (m, 1 H) 6.27 - 6.49 (m, 2 H) 7.73 - 7.88 (m, 1 H) 8.02 (brt, 7= 5.44 Hz, 1 H).Et3N-3HF (5.00 eq) DCM (3.07 V)0 °C, 3 hrs32.6%yield

[0195] To a solution of compound 5F (32.5 g, 43.0 mmol, 1.00 eq) in DCM (100 mL) was added EtsN-3HF (34.6 g, 215 mmol, 5.00 eq) in portions under N2 atmosphere at 0 °C. The resulting mixture was stirred at 0 °C for 3 hrs. LCMS showed compound 5F was consumedFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 completely and one major peak with desired MS was detected. The reaction was quenched with the addition of sat. aq. NaHCCh (50.0 mL), and extracted with DCM (100 mL * 3). The combined organic layer was washed with brine (50.0 mL * 3), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water (HCl)-ACN]; gradient: 10%-40% B over 25 min) to give compound 5G (9.00 g, 32.6% yield) was obtained as a yellow oil. LCMS (ESI): calc’d for C26H48N4O8S3 [M+H]+: 641.26, found: 641.3. 'H NMR (400 MHz, DMSO-rfc): 3 1.21 (d, J = 6.88 Hz, 3 H) 1.25 - 1.35 (m, 2 H) 1.39 - 1.54 (m, 3 H) 1.56 - 1.67 (m, 1 H) 2.06 (t, J = 7.38 Hz, 2 H) 2.30 (t, J = 6.50 Hz, 2 H) 2.57 (d, J = 12.38 Hz, 1 H) 2.69 - 2.77 (m, 2 H) 2.82 (dd, J = 12.51, 5.13 Hz, 1 H) 2.89 (td, J = 6.82, 5.63 Hz, 1 H) 3.09 (br dd, J = 4.44, 2.44 Hz, 1 H) 3.26 - 3.34 (m, 3 H) 3.36 - 3.42 (m, 4 H) 3.45 - 3.52 (m, 17 H) 4.12 (dd, J = 7.75, 4.50 Hz, 1 H) 4.30 (dd, J = 7.63, 4.50 Hz, 1 H) 6.41 (br s, 1 H) 7.83 (t, J = 5.50 Hz, 1 H) 8.04 (br t, J = 5.44 Hz, 1 H).(2.00 eq)NMM (2.00 eq)DMF (10.0 V)5G25 °C, 16 hrs55 g, 95%

[0196] To the solution of compound 5G (8.50 g, 13.2 mmol, 1.00 eq) in DMF (85.0 mL) was added NMM (2.68 g, 26.5 mmol, 2.00 eq) and bis(4-nitrophenyl) carbonate (8.07 g, 26.5 mmol, 2.00 eq) at 25 °C. The reaction mixture was stirred at 25 °C for 16 hrs. LCMS showed compound 5G was consumed completely and one major peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 um); mobile phase: [water (TFA)-ACN]; gradient: 25%-55% B over 25 min) to afford compound 5 (5.50 g, 51.4% yield) as a yellow oil. LCMS (ESI): calc’d for C33H51N5O12S3 [M+H]+: 806.27, found: 806.3. 'H NMR (400 MHz, DMSO-rfc): 3 1.13 - 1.38 (m, 5 H) 1.39 - 1.54 (m, 3 H)Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 1.55 - 1.68 (m, 1 H) 2.06 (t, J= 7.38 Hz, 2 H) 2.31 (t, J = 6.44 Hz, 2 H) 2.57 (d, J = 12.38 Hz, 1 H) 2.73 - 2.87 (m, 3 H) 3.05 - 3.12 (m, 1 H) 3.18 (q, J = 5.84 Hz, 2 H) 3.24 - 3.45 (m, 6 H) 3.46 - 3.55 (m, 11 H) 3.58 (t, J = 6.50 Hz, 2 H) 4.12 (dd, J = 7.63, 4.38 Hz, 1 H) 4.25 -4.35 (m, 2 H) 4.36 - 4.44 (m, 1 H) 6.15 - 6.63 (m, 2 H) 7.49 - 7.68 (m, 2 H) 7.82 (br t, J = 5.50 Hz, 1 H) 8.05 (br t, J = 5.50 Hz, 1 H) 8.23 - 8.44 (m, 2 H).

[0197] The synthesis of reductively cleavable linker BTSSH2 (compound 6) was initiated with asymmetric pyridinyl disulfide 6A and completed over 6 consecutive steps (FIG. 8).C. (1.00 eq) „ „.|l - ►Boc'N / '^s-s / '\^OH" 'S"S' / 'OH DCM (20.0 V)H6A 25 °C, 12 hrs6B58.7%yieldStep 1

[0198] To the solution of compound 6A (58.1 g, 310 mmol, 1.10 eq) in DCM (1.00 L) was added a solution of tert-butyl (2-mercaptoethyl)carbamate (50.0 g, 1.00 eq, in 1.00 L DCM) at 25 °C. The resulting mixture was stirred at 25 °C for 12 hrs. TLC (Petroleum ether / Ethyl acetate = 2 / 1, Rf (6A)= 0.76, Rf(6B) = 0.43) showed compound 6A was consumed completely and one new spot was formed. The reaction mixture was concentrated under reduced pressure to give a residue, and the residue was purified by column chromatography (SiCh, Petroleum ether / Ethyl acetate = 10 / 1) to give compound 6B (42.0 g, 58.7% yield) as a yellow oil. 'H NMR (400 MHz, CHLOROFORM- ): 34.95 (br s, 1H), 3.45 - 3.26 (m, 3H), 2.73 (t, J = 6.3 Hz, 3H), 1.38 (s, 13H).TBSCI (1.35 eq)Boc"N-g / ''^0TBSH IMIDAZOLE (1.67 eq)DMF (10.0 V) 6C 25 °C, 16 hrsStep 2

[0199] To the solution of compound 6B (10.0 g, 39.4 mmol, 1.00 eq) and imidazole (4.49 g, 66.0 mmol, 1.67 eq) in DCM (100 mL) was added TBSC1 (8.04 g, 53.3 mmol, 1.35 eq) at 25 °C. The resulting mixture was stirred at 25 °C for 16 hrs. TLC indicated one new spot with lower polarity was detected. The reaction mixture was concentrated under reduced pressureFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 to give compound 6C (14.0 g, 96.4% yield) as a yellow oil and used into the next step directly.'H NMR (400 MHz, CHLOROFORM- ): 30.07 (s, 5 H) 0.88 - 0.90 (m, 9 H) 1.43 (s, 9 H) 2.71 - 2.85 (m, 5 H) 3.45 (br d, J = 6.00 Hz, 2 H) 3.84 (t, J = 6.69 Hz, 2 H) 4.94 (br s, 1 H).Boc^, / \ / OTBS TMSI (1.10 eq)N S ► H2N / x^OTBSoNDCM (10.0 V)6C -78-20 °C, 16.5 hrs 6DStep 3

[0200] The solution of compound 6C (14.0 g, 38.0 mmol, 1.00 eq) in DCM (140 mL) was cooled to -78 °C, before TMSI (8.38 g, 41.8 mmol, 1.10 eq) was added in portions to the mixture. The resulting mixture was stirred at -70 °C for 30 min, warmed to 20 °C, and stirred for additional 16 hrs. TLC indicated one new spot with larger polarity was detected. The reaction was quenched with the addition of a solution of MeOH / aq. NH4HCO3 = 2.5 / 1 (35.0 mL). The resulting mixture was extracted with DCM (100 mL * 3), the combined organic layer was washed with brine (50.0 mL * 3), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give crude compound 6D (10.1 g, crude) as a yellow oil. The crude product was used into the next step directly. ’H NMR (400 MHz, DMSO-rfc): 3 0.02 - 0.09 (m, 6 H) 0.85 - 0.91 (m, 9 H) 2.69 - 2.76 (m, 2 H) 2.78 - 2.87 (m, 4 H) 3.12 (br s, 2 H) 3.81 (t, J = 6.38 Hz, 2 H).Biotin-PEG4-acid (1 00 eq) H2N—OTBSHBTU (1.35 eq)6D DIPEA (445 eq)DMF (100 V) 25 °C, 16 hrsStep 4

[0201] The solution of Biotin-PEG4-acid (17.0 g, 34.5 mmol, 1.00 eq) in DMF (170 mL) was stirred for 5 min at 25 °C, before purged with N2 for 3 times. HBTU (17.7 g, 46.6 mmol, 1.35 eq) and DIEA (19.8 g, 153 mmol, 4.45 eq) were added in portions over 5 min at 25 °C under N2 atmosphere, followed by compound 6D (10.1 g, 38.0 mmol, 1.10 eq). The resulting reaction mixture was stirred for 16 hrs at 25 °C. TLC indicated one new spot with lower polarity was detected. The reaction was quenched with the addition of water (200 mL), and extracted with DCM (100 mL * 3). The combined organic layer was washed with brine (50.0Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 mL * 3), dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure to give crude compound 6E (27.0 g, crude) as a yellow oil. The crude product was used into the next step directly. 'H NMR (400 MHz, DMSO-<7>): <50.05 (s, 6 H) 0.87 (s, 9 H) 1.22 - 1.33 (m, 4 H) 1.49 (br d, J = 8.00 Hz, 2 H) 1.55 - 1.67 (m, 1 H) 2.06 (t, J = 7.44 Hz, 2 H) 2.30 (t, J = 6.44 Hz, 2 H) 2.69 (s, 6 H) 2.76 (s, 2 H) 2.78 - 2.93 (m, 4 H) 3.05 - 3.13 (m, 1 H) 3.18 (br d, 7= 5.75 Hz, 2 H) 3.35 - 3.42 (m, 2 H) 3.48 - 3.54 (m, 8 H) 3.58 (t, 7= 6.44 Hz, 2 H) 3.81 (t, 7= 6.32 Hz, 2 H) 4.13 (td, 7= 4.85, 2.19 Hz, 1 H) 4.25 - 4.35 (m, 1 H) 6.26 - 6.51 (m, 2 H) 7.83 (br t, 7 = 5.57 Hz, 1 H) 8.02 (br t, 7 = 5.50 Hz, 1 H).Et3N-3HF (5.00 eq) DCM (3.38 V)0 °C, 3 hrs45.6%yield

[0202] The solution of compound 6E (20.7 g, 27.9 mmol, 1.00 eq) in DCM (70 mL) under N2 atmosphere was cooled to 0 °C, before EtsN-3HF (22.5 g, 139 mmol, 5.00 eq) was added dropwise under N2 atmosphere at 25 °C. The resulting reaction mixture was stirred at 0 °C for 3 hrs. LCMS showed compound 6E was consumed completely and one major peak with desired MS was detected. The reaction was quenched with the addition of aq. NaHCCh (50.0 mL), and extracted with DCM (100 mL * 3). The combined organic layer was washed with brine (50.0 mL * 3), dried over anhydrous Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC to afford compound 6F (8.00 g, 45.6% yield) as a yellow oil. LCMS (ESI): calc’d for C25H46N4O8S3 [M+H]+:627.25, found: 627.3. 'H NMR (400 MHz, DMSO-rfc): 3 1.28 - 1.35 (m, 2 H) 1.41 - 1.55 (m, 3 H) 1.56 - 1.66 (m, 1 H) 2.06 (s, 2 H) 2.31 (t, J = 6.44 Hz, 2 H) 2.71 - 2.82 (m, 6 H) 3.18 (br d, 7= 5.75 Hz, 3 H) 3.30 - 3.36 (m, 4 H) 3.36 - 3.40 (m, 3 H) 3.50 (s, 9 H) 3.58 - 3.63 (m, 4 H) 4.09 - 4.18 (m, 1 H) 4.27 - 4.35 (m, 1 H) 4.86 (br t, J = 5.13 Hz, 1 H) 6.30 - 6.46 (m, 2 H) 7.83 (br t, J = 5.32 Hz, 1 H) 8.03 (br t, J = 5.44 Hz, 1 H).Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325(2.00 eq)NMM (2.00eq)DMF (10.0 V)25 °C, 16 hrs65 g, 95%

[0203] The solution of compound 6F (10.0 g, 15.9 mmol, 1.00 eq), NMM (3.23 g, 31.9 mmol, 2.00 eq) and bis(4-nitrophenyl) carbonate (9.71 g, 31.9 mmol, 2.00 eq) in DMF (100 mL) was stirred for 16 hrs at 25 °C. LCMS showed compound 6F was consumed completely and one major peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 (250*70mm, 10 um); mobile phase: [water (TFA)-ACN]; gradient: 22%-52% B over 25 min) to afford compound 6 (5.10 g, 40.3% yield) as a yellow oil. LCMS (ESI): calc’d for C32H49N5O12S3 [M+H]+: 792.25, found: 792.3. 'H NMR (400 MHz, DMSO-d6) 3 1.22 - 1.34 (m, 2 H) 1.48 (br dd, J = 13.76, 5.88 Hz, 3 H) 1.60 (br dd, J= 8.63, 5.38 Hz, 1 H) 2.06 (t, 7= 7.38 Hz, 2 H) 2.31 (t, J = 6.44 Hz, 2 H) 2.57 (d, J = 12.51 Hz, 1 H) 2.75 - 2.86 (m, 3 H) 3.04 - 3.13 (m, 3 H) 3.18 (q, J = 5.88 Hz, 2 H) 3.28 - 3.45 (m, 5 H) 3.46 -3.50 (m, 11 H) 3.59 (t, J = 6.44 Hz, 2 H) 4.12 (br dd, J = 7.69, 4.44 Hz, 1 H) 4.30 (dd, J = 7.63, 4.75 Hz, 1 H) 4.48 (t, J = 6.19 Hz, 2 H) 6.41 (br s, 2 H) 7.49 - 7.65 (m, 2 H) 7.82 (br t, J = 5.57 Hz, 1 H) 8.05 (br t, J= 5.38 Hz, 1 H) 8.27 - 8.37 (m, 2 H).Example 2. Installation of enrichment tag on peptide during SPPS

[0204] Installation of the linker was achieved through an amide bond forming reaction with an activated carbonate linker (e.g. BTSSME2 linker compound 2) following the completion of polypeptide elongation by SPPS using DIC, HATU, HBTU, or PyBOP (5 - 10 equiv. relative to resin loading) as coupling reagents. Addition of Oxyma, HOAt, HOBt, or a similar compound was used as the racemization suppressant (5 - 10 equiv). Intermittent capping with AC2O was performed in between each monomer coupling step in order to minimize the growth of truncated chains throughout the synthesis. To 5 pmol of a peptide selected fromFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 Table 1 and elongated on TentaGel S RAM was added a mixture of BTSSME2 linker 2 (21 mg, 5 equiv.), Oxyma (4.3 mg, 6 equiv), and DIPEA (5.2 uL, 6 equiv.) in 0.5 - 1 mL of anhydrous amine-free DMF. The resin suspension was gently stirred for 18 h, drained, then washed sequentially with 5 x 1 mL DMF and 5 x 1 mL DCM.Example 3. SPPS cleavage and capture of the linker-tagged peptide

[0205] Resin-bound target peptide with linker installed was subject to cleavage from the TentaGel S RAM synthesis resin using a cocktail of TFA, TIPS, and H2O (95:2.5:2.5) for up to 3 hr. The suspension was filtered to remove the resin, and the TFA cleavage cocktail was removed over the course of 1 hr under a gentle stream of N2 gas. Cold ether was added to precipitate the linked peptide from the residual TFA, and the peptide pellet was isolated by centrifugation for 10 min at 4000 rpm and 4 °C. The centrifuged pellet was resuspended in DMSO at a concentration of 50 pL per 1 pmol of synthesis resin loading (e.g. 250 pL for a 5 pmol scale synthesis) with gentle shaking and heating at 37 °C for at least 1 hr. Once homogenized, the DMSO was diluted to a final concentration of 20% with the addition of 1 mL of PBS. Separately, Pierce High Capacity Streptavidin Agarose resin (Thermo Scientific, binding capacity ~ 274 nmol biotinylated peptide mL1) was loaded in a 2 mL deep well plate (250 - 500 pL resin per 5 pmol of synthesis resin) and washed 3X with 1 mL of PBS to equilibrate the capture resin. The DMSO peptide solution was then added directly to the Streptavidin resin, the plate sealed, and the resin incubated with the peptide solution with gentle shaking at 1500 rpm and 37 °C for 1 hour. The DMSO solution was then drained into a separate deep well collection plate, and the capture plate (containing captured biotinylated peptides bound to streptavidin) was washed 3 x 1 mL with PBS, 3 x 1 mL of 3 M GdnHCl pH 7.3, 3 x 1 mL of 2 M GdnHCl, 3 x 1 mL of 1 M GdnHCl, followed by 5 x 1 mL washes with 10 mM NH4CO3 pH 9.Example 4. Counterion exchange of the resin-bound peptide.

[0206] The resultant carbamate-linked polypeptide bound to streptavidin is stable to standard aqueous acidic and basic buffers, and can thus be subject to additional wash steps, or counterion exchange steps, prior to peptide release. Thus, the tagged polypeptide was washed with 10 x 1 mL washes of 50% MeCN in H2O supplemented with 0.1% NH4OH to remove residual TFA and other counterions, prior to release from capture resin. Subsequent releaseFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 with 1% P-ME then afforded the peptide with counterion associated with the final wash buffer (i.e., the ammonium carboxylate salt). If the peptide hydrochloride salt is desired, 0.1 M HC1 in 50% MeCN can be used as a final additional wash, following the wash with 0.1% NH4OH.Example 5. Traceless reductive release of peptide from the streptavidin capture resin using BTSSHME linker 5

[0207] Following capture of the crude polypeptide target bound to BTSSHME linker 5 on the desired capture resin, the linker was cleaved under reductive conditions. A self-immolative reductive decarboxylation cascade reaction was triggered by the addition of 1% (v / v) P-ME in 50% MeCN buffered by 10 mM NH4CO3 pH 9. The release buffer was added to a sealed 2 mL deep well containing the saturated streptavidin resin, and the resin incubated for 60 min at 37 °C with gentle shaking. After 60 min, the suspension was filtered to remove the capture resin and the filtrate was frozen and lyophilized to affinity purified peptides with some residual buffer salts as a hygroscopic, off-white powder. Following lyophilization, the peptides were dissolved in 5% MeCN in H2O supplemented with 0.1% formic acid, and analyzed by LCMS on a standard reverse-phase HPLC gradient. Total ion chromatograms before (crude) and after (purified) the catch and release revealed isolation of the target sequences with significant levels of enrichment (FIG. 14).Example 6. Traceless reductive release of peptide from the streptavidin capture resin using BTSSME2 linker 2

[0208] Following capture of the crude polypeptide target bound to BTSSME2 linker 2 on the desired capture resin, the linker was cleaved under reductive conditions. A self-immolative reductive decarboxylation cascade reaction was triggered by the addition of 1% (v / v) P-ME buffered by 10 mM NH4CO3 pH 9. The release buffer was added to a sealed 2 mL deep well containing the saturated streptavidin resin, and the resin incubated for 30 min at 37 °C with gentle shaking. After 30 min, the suspension was filtered to remove the capture resin and the filtrate was frozen and lyophilized to affinity purified peptides with some residual buffer salts as a hygroscopic, off-white powder. Following lyophilization, the peptides were dissolved in 5% MeCN in H2O supplemented with 0.1% formic acid, and analyzed by LCMS on a standard reverse-phase HPLC gradient. Total ion chromatograms before (crude) and afterFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 (purified) the catch and release revealed isolation of the target sequences with significant levels of enrichment (FIG. 15).Example 7. Oxidative release of peptide from streptavidin capture resin using BTOXGLY linker 1

[0209] Following capture of the crude polypeptide target bound to BTOXGLY linker 1 on streptavidin capture resin, the linker was cleaved under oxidative conditions. To 200 nmol of peptide bound to 1 mL of streptavidin agarose resin in a 2 mL deep well plate was added 500 pL of 20% MeCN in PBS. Freshly prepared NaIO4 (7.3 mg, 167 equiv.) in 500 pL of PBS was added to the well; the plate was covered and shaken at 800 rpm in the dark for 10 min. After 10 min, the oxidation reaction was quenched with the addition of Na2SO3(43.2 mg, 1670 equiv.) and MeONH2 pH 7.5 (28.6 mg, 1670 equiv.), filtered, and the filtrate lyophilized. LCMS analysis of the affinity purified product confirmed significant enrichment of the target peptide 20 containing an N-terminal Gly as the byproduct of the cleavage mechanism (FIG. 17).

[0210] Sequences of representative affinity-tagged peptides having undergone successful affinity purification by catch and release are summarized in Table 1.Table 1. Sequences tested for affinity purification by catch and release.PEPTIDE LINKER SEQID SEQUENCEastaqalarakeeaiadleaagitdpqvldwirklstveevrartaql Peptide 1 BTSSHME 1laehtsaylkelekakqeaieelkkhgitdpeiiqwikslpnvtgvreetq Peptide 2 BTSSHME 2kiiqehlsaeraelerakeealaalaahgikdpevlawirelptvadvrartrel Peptide 3 BTSSHME 3iaahlFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325h[D- Peptide 4 BTSSHME 4 Nle]haleltlaklrainelnehgitdpgilqwiltlptvadirnathn ilanylaayaaalatakreaiarlaaagltdpelldwirhletvdevkrftsitl Peptide 5 BTSSHME 5eihs[D- Peptide 6 BTSSHME 6 Nle]aaaeelkqakteavnrlkvkgitdpiilqwirnlptvedvrrr teeiieqteaqyqralteakaqaikrlqqhgitdpdvltwvqtlstvaevesatda Peptide 7 BTSSHME 7liqrhlslkesllkeakeravkelekagitdpavlewiknletveevvtstka Peptide 8 BTSSHME 8lldkhkgaaaaalaaareaaiaalrragitdpavlewirrlptveevvgttatll Peptide 9 BTSSHME 9aehagakaaalaaakaaaeaalaaagitdpevlnwvrslptvadvlaatn Peptide 10 BTSSHME 10allashan[D- Peptide 11 BTSSHME 11 Nle]ereallnaktkaearlrkhgitdpeilnwvrnlptveqvnhit eniisqheFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325qga[D- Peptide 12 BTSSHME 12 Nle]kdlkkakeeaieklkqhgitdpkiikwietlptveevkkrtk eiieehlgsysqqlsaakskaitelkkhgitdpeiiqwiqtletveqvyqqtk Peptide 13 BTSSHME 13qliekfs[D- Peptide 14 BTSSHME 14 Nle]keerqlqeakkraiarlrragitdpeilqwirdlptvdavrtrtr qllrrha[D- Peptide 15 BTSSHME 15 Nle]ekeeklkkakeraikklkekgitdpkiiewikslktveevik ktkeiiekyldkrkkalkeakrlaierlreigitdpevinwirrlstvaqvldfterlir Peptide 16 BTSSHME 16ehlsaaeqalaaartaaiarlraagitdpavlnwirvlptveevekrtrqll Peptide 17 BTSSHME 17aehlaayaaslaaakaeaiarlrkagitdpsildwvrrlptvavvnnttari Peptide 18 BTSSHME 18Inshlarraaalraakaralarlrragitdpdvlawiralptvaaveratrella Peptide 19 BTSSHME 19ahrFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325tidqwllknakedaiaelkkagitephvisfinhapyvshvnglk Peptide 20 BTSSHME 20nailkahaPeptide 21 BTSSME2 21 ttihvgdltlkydnpkkayeiakklakkynltvtikngkitvtPeptide 22 BTSSME2 22 gtvkvqgitikvpnpdkaykiakklakkynltvtrkgntikvePeptide 23 BTSSME2 23 gtlhlngvtvkvpslekaikaakkfakkynlevqvhgntvhvhttvhvngveyrfnnpeeaykfavrfD- Peptide 24 BTSSME2 24Nle] akeiggtvefhgeeihvePeptide 25 BTSSME2 25 htvhvngqkytfptlekaakfakklakklgltvevhgntiklePeptide 26 BTSSME2 26 ttqievddeeqakklakkwqkkgydahytqkngkwfvqfnPeptide 27 BTSSME2 27 ritfh [D -Nle] ngpeqakrlikwakkqnlrfqvrqengqiiididqftvtiegdea[D-Nle]kkfike[D- Peptide 28 BTSSME2 28Nle]kkrnlevhvrvengqwiahvsPeptide 29 BTSSME2 29 tttytyddkqaakeaaeewkkrgyevhltqkgnkwqvkvgPeptide 30 BTSSME2 30 dseqlkeeakkaykngneekakrilkkagldeseaeeflrrafFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325Peptide 31 BTSSME2 31 ddearrrakkllekgysedyireylkrqgvnpdeaedairwakeeevkkrirelakrgvspeqirewlrkqgyspdqakqlfeea[D- Peptide 32 BTSSME2 32Nle]Peptide 33 BTSSME2 33 seiekiaeklwrerpgskewvvevlkrlgvtedearkaaekaePeptide 34 BTSSME2 34 pdkkkkivkkllnkgldksevekearkngisddiveqaykewlPeptide 35 BTSSME2 35 sleealerakealkknqplevngyrfddprkayewareqleraPeptide 36 BTSSME2 36 tldearelverakkegtgvdvngqrfedwreaerwvreqeknkPeptide 37 BTSSME2 37 tekqeraieyirrlgyseeearrilkkangnpdlakeiarrigPeptide 38 BTSSME2 38 tideiikaleqavkdnkpiqvgnytvtsadeaeklakklkkeyPeptide 39 BTSSME2 39 ieevkkivseiqskqltritingntyqvnspeelveyvrkvfsPeptide 40 BTSSME2 40 dwreiaklaeealkkqqpvtvngqtfndpneikklletaqkaitidqwllknakedaiaelkkagitephvisfinhapyvshvnglk Peptide 20 BTOXGLY 20nailkahaExample 8. Semi-Preparative Purification with BTSSME2Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325

[0211] In one example of a semi-preparative purification, a 53mer peptide 20 (SEQ. ID 20) was synthesized by single-shot SPPS on the 10 pmol scale (single couplings and serial truncation product capping by AC2O), then labeled with cleavable linker 2 (see FIG. 9) before cleavage from the synthesis resin. The intended product of the synthesis, crude peptide-linker complex 20, was detected in low abundance in the LCMS chromatogram of the crude cleavage reaction. The crude reaction material was then dissolved in DMSO, captured over streptavidin agarose resin, the capture resin extensively washed, and the peptide subsequently released by the addition of 3% P-ME (v / v) in 20 mM NH4CO3 supplemented with 10% MeCN, shaking at 40 °C for 1 hr. LCMS analysis of the resultant affinity purified material (following elution and lyophilization) demonstrated significant levels of enrichment for the unmodified target peptide 20. Inspection of the integrated mass spectrum revealed no detectable amount of biotinylated material in the affinity purified sample, 1.6 mg of which was obtained from 0.75 mb of capture resin (0.27 pmol mL1capacity) directly following lyophilization from volatile NH4HCO3 cleavage buffer. This example and the other examples provided in Table 1 highlight the straightforward synthesis, chemical stability, and rapid release kinetics of this new reductively cleavable linker design.INCORPORATION BY REFERENCE

[0212] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.EQUIVALENTS

[0213] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 We claim:

1. A compound, or a salt thereof, having a structure represented by formula I:G',EI;wherein:PG1is a protecting group selected from tert-butyloxycarbonyl (Boc), tert-butyl, tertbutyldimethylsilyl, methoxymethyl, triisopropylsilyl, and tetrahydropyranyl;E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R1and R2is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR1and R2, taken together with the intervening atoms, form a cycloalkyl ring; R3is hydrogen, branched or unbranched alkyl, or an acid labile protecting group;R4is hydrogen or branched or unbranched alkyl;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S (0)2-, -C(O)-alkylene, -C(O)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

2. The compound of claim 1, wherein PG1is tert-butyloxycarbonyl (Boc).

3. A compound, or a salt thereof, having a structure represented by formula II:OII;Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -C(O)-alkyl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;each R5and R6is independently hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain; orR5and R6, taken together with the intervening atoms, form a cycloalkyl ring; Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)-NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S(O)2-, -C(O)-alkylene, -C(O)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

4. A compound, or a salt thereof, having a structure represented by formula III:R7Ill;wherein:E is an enrichment tag selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH-heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted -C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted -C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)-alkyl,Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted - C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted - C(O)-cycloalkyl, substituted or unsubstituted -S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2-alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted -S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH-cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, - C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2-NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)- CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;X is O or S;m is an integer from 0 to 10;R7is hydrogen, branched or unbranched alkyl, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, or an amino acid side chain;Y is selected from -Br, -Cl, -I, -F, -O-Z, -S-Z, -NH-Z, substituted or unsubstituted alkyl, and an amino acid side chain;Z is selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted heteroaryl; andG and G' are each independently absent or are selected from a alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, -OC(O)O-, - NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene- C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene- S(O)2-NH-, alkylene-NH- S (0)2-, -C(O)-alkylene, -C(0)- NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, - OC(O)O-alkylene, -NH-alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(0)2- alkylene, -S(O)2-NH-alkylene, and -NH-S(O)2-alkylene.

5. The compound of any one of claims 1-4, wherein X is O.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-003256. The compound of any one of claims 1-5, wherein m is 0.

7. The compound of any one of claims 1-5, wherein m is an integer from 1 to 5.

8. The compound of claim 7, wherein m is 4.

9. The compound of any one of claims 1-8, wherein one of G and G' is absent.

10. The compound of any one of claims 1-8, wherein one of G and G' is alkylene.

11. The compound of any one of claims 1-8, wherein one of G and G' is -C(O)NH- alkylene.

12. The compound of any one of claims 1-8, wherein one of G and G' is alkylene- C(O)NH-.

13. The compound of any one of claims 1-10, wherein G is absent; and wherein G' is alkylene.

14. The compound of any one of claims 1-8 and 10-11, wherein G is -C(O)NH-alkylene;and wherein G' is alkylene.

15. The compound of any one of claims 1-14, wherein E is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NH-C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl; andwherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH- alkyl, substituted -NH-C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2- alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl terminates in a substituent selected from alkyne, azide, aminooxy, and hydrazine.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 16. The compound of any one of claims 1-14, wherein E is substituted or unsubstituted -NH-C(O)-alkyl.

17. The compound of claim 15 or 16, whereinE is18. The compound of any one of claims 1-14, wherein E is substituted or unsubstituted heteroaryl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-heteroaryl, substituted or unsubstituted -C(O)-NH-heteroaryl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-heteroaryl.

19. The compound of claim 18, wherein E is substituted or unsubstituted 8- hydroxy quinoline.OZ'20. The compound of claim 18 or 19, wherein Eis and wherein Z' is H or a hydroxyl protecting group.

21. The compound of claim 20, wherein Z' is H.

22. The compound of claim 20, wherein Z' is a hydroxyl protecting group selected from23. The compound of any one of claims 1-22, wherein Y is -O-Z; and wherein Z isFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-0032524. The compound of claim 23, whereinZ is N02025. The compound of claim 23, wherein Z is26. The compound of claim 23, whereinZ is27. The compound of claim 23, whereinZ is F28. The compound of any one of claims 1-2 and 5-27, wherein R3is selected fromSi\29. The compound of claim 28, wherein R3is30. The compound of any one of claims 1-2 and 5-29, wherein R4is H.

31. The compound of any one of claims 1-2 and 5-30, wherein R1and R2are each H.

32. The compound of any one of claims 3 and 5-27, wherein R5and R6are each methyl.

33. The compound of any one of claims 4-27, wherein R7is H.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-0032534. A compound, or a salt thereof, having the structure:wherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)- alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl,Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted or unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoroalkyl; andG and G’ are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2-NH-, -NH-S(O)2-, alkylene-C(O)-, alkylene-C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene-S(O)2-NH-, alkylene-NH-S(O)2-, -C(O)-alkylene, -C(O)-NH-alkylene, -NH-C(O)-alkylene, -OC(O)-NH-alkylene, -NH-C(O)O-alkylene, -OC(O)O-alkylene, -NH- alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2-alkylene, -S(O)2- NH-alkylene, and -NH-S(O)2-alkylene.

35. The compound of claim 34, having the structure:

36. The compound of claim 34, having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 37. The compound of claim 34, having the structure:

38. The compound of claim 34, having the structure:O39. The compound of claim 34, having the structure:

40. The compound of claim 34, having the structure:

41. The compound of any one of claims 34-40, whereinE is42. The compound of any one of claims 34-40, wherein E is substituted or unsubstituted 8-hydroxyquinoline.OZ'43. The compound of claim 42, wherein Eis and wherein Z' is H or a hydroxyl protecting group.

44. The compound of claim 43, wherein Z' is H.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 45. The compound of claim 44, wherein Z' is a hydroxyl protecting group selected fromZK. 4 > X< °Q / \ \ £ 0?J \, and(Z")2N.O / ^N^46. The compound of any one of claims 34-40, wherein E is O; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz).

47. The compound of claim 46, wherein each Z" is independently H; and E isHO48. The compound of claim 46, wherein one of Z" is H and the other is tert- H Hbutyloxycarbonyl (Boc); and E is O(Z")2N,N / ^N^H 11 49. The compound of any one of claims 34-40, wherein E is0; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz).

50. The compound of claim 49, wherein each Z" is independently H; and E isHo51. The compound of claim 49, wherein one of Z" is H and the other is tert- H HBoc"N'N'^yNXbutyloxycarbonyl (Boc); and E is O52. A compound, or a salt thereof, having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325wherein E is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted -NH-alkyl, substituted or unsubstituted -NH- heterocycloalkyl, substituted or unsubstituted -NH-heteroaryl, substituted or unsubstituted -NH-aryl, substituted or unsubstituted -NH-cycloalkyl, substituted or unsubstituted -C(O)-NH-alkyl, substituted or unsubstituted - C(O)-NH-heterocycloalkyl, substituted or unsubstituted C(O)-NH-heteroaryl, substituted or unsubstituted -C(O)-NH-aryl, substituted or unsubstituted - C(O)-NH-cycloalkyl, substituted or unsubstituted -NH-C(O)-alkyl, substituted or unsubstituted -NH-C(O)-heterocycloalkyl, substituted or unsubstituted - NH-C(O)-heteroaryl, substituted or unsubstituted -NH-C(O)-aryl, substituted or unsubstituted -NH-C(O)-cycloalkyl, substituted or unsubstituted -C(O)- alkyl, substituted or unsubstituted -C(O)-heterocycloalkyl, substituted or unsubstituted -C(O)-heteroaryl, substituted or unsubstituted -C(O)-aryl, substituted or unsubstituted -C(O)-cycloalkyl, substituted or unsubstituted - S(O)2-heterocycloalkyl, substituted or unsubstituted -S(O)2-heteroaryl, substituted or unsubstituted -S(O)2-aryl, substituted or unsubstituted -S(O)2- alkyl, substituted or unsubstituted -S(O)2-cycloalkyl, substituted orFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 unsubstituted -S(O)2-NH-heterocycloalkyl, substituted or unsubstituted - S(O)2-NH-heteroaryl, substituted or unsubstituted -S(O)2-NH-aryl, substituted or unsubstituted -S(O)2-NH-alkyl, substituted or unsubstituted -S(O)2-NH- cycloalkyl, substituted or unsubstituted -NH-S(O)2-heterocycloalkyl, substituted or unsubstituted -NH-S(O)2-heteroaryl, substituted or unsubstituted -NH-S(O)2-aryl, substituted or unsubstituted -NH-S(O)2-alkyl, substituted or unsubstituted -NH-S(O)2-cycloalkyl, -C(O)-NH-CH2-O-NH2, -C(O)-NH-CH2- NH-NH2, -NHC(O)-CH2-O-NH2, -NH-C(O)-CH2-NH-NH2, a peptide fragment, and perfluoroalkyl;G and G’ are each independently absent or are selected from alkylene, alkenylene, alkynylene, -C(O)-, -C(O)NH-, -NH-C(O)-, -OC(O)-NH-, -NH-C(O)O-, - OC(O)O-, -NH-, -S-, -O-, -S(O)-, -S(O)2-, -S(O)2NH-, -NHS(O)2-, alkylene- C(O)-, alkylene-C(O)-NH-, alkylene-NH-C(O)-, alkylene-OC(O)-NH-, alkylene-NH-C(O)O-, alkylene-OC(O)O-, alkylene-NH-, alkylene-S-, alkylene-O-, alkylene-S(O)-, alkylene-S(O)2-, alkylene-S(O)2-NH-, alkylene- NH-S(O)2-, -C(O)-alkylene, -C(O)-NH-alkylene, -NH-C(O)-alkylene, - OC(O)-NH-alkylene, -NH-C(O)O-alkylene, -OC(O)O-alkylene, -NH- alkylene, -S-alkylene, -O-alkylene, -S(O)-alkylene, -S(O)2-alkylene, -S(O)2- NH-alkylene, and -NH-S(O)2-alkylene; andPP is a polypeptide residue.

53. The compound of claim 52, having the structure:

54. The compound of claim 52, having the structure:O O O55. The compound of claim 52, having the structure:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 56. The compound of claim 52, having the structure:O57. The compound of claim 52, having the structure:

58. The compound of claim 52, having the structure:

59. The compound of any one of claims 52-58, wherein E is60. The compound of any one of claims 52-58, wherein E is substituted or unsubstituted 8-hydroxyquinoline.OZ'61. The compound of claim 60, wherein Eis i; and wherein Z' is H or a hydroxyl protecting group.

62. The compound of claim 61, wherein Z' is a hydroxyl protecting group selected fromFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325(Z")2N.O / X^N^63. The compound of any one of claims 52-58, wherein E is O; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz).

64. The compound of claim 63, wherein each Z" is independently H; and E isHH2N,0o n65. The compound of claim 63, wherein one of Z" is H and the other is tert- H HBoc O Abutyloxycarbonyl (Boc); and E isO(Z")2N,N / ^N^H 11 66. The compound of any one of claims 52-58, wherein E is0; and each Z" is independently selected from H, tert-butyloxycarbonyl (Boc), benzyl, benzoyl, fluorenylmethoxy (Fmoc), and benzyloxycarbonyl (CBz).

67. The compound of claim 66, wherein each Z" is independently H; and E is68. The compound of claim 66, wherein one of Z" is H and the other is tert- H H BOC"N'N'^Y'N / butyloxycarbonyl (Boc); and E is O69. The compound of any one of claims 52-68, wherein the polypeptide residue comprises from 5 to 55 amino acid residues.

70. The compound of any one of claims 52-69, wherein the polypeptide residue comprises from 25 to 55 amino acid residues.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 71. The compound of any one of claims 52-70, wherein the polypeptide residue comprises at least one D-amino acid residue.

72. The compound of any one of claims 52-71, wherein the target polypeptide comprises at least one alpha-disubstituted amino acid residue (e.g., aminoisobutyric acid).

73. The compound of any one of claims 52-72, wherein the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine).

74. The compound of claim 73, wherein the non-canonical amino acid further comprises a non-natural side chain.

75. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 70% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.

76. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 80% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.

77. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 90% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.

78. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 95% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.

79. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 98% to about 100% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.

80. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 90%, preferably about 95%, or more preferably about 98% identity to any one of the amino acid sequences set forth in SEQ ID Nos: 1-40.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 81. The compound of any one of claims 52-71, wherein the polypeptide residue comprises an amino acid sequence selected from the following table:PEPTIDE SEQID SEQUENCEastaqalarakeeaiadleaagitdpqvldwirklstveevrartaql Peptide 1 1laehtsaylkelekakqeaieelkkhgitdpeiiqwikslpnvtgvreetq Peptide 2 2kiiqehlsaeraelerakeealaalaahgikdpevlawirelptvadvrartrel Peptide 3 3iaahlh[D- Peptide 4 4 Nle]haleltlaklrainelnehgitdpgilqwiltlptvadimathn ilanylaayaaalatakreaiarlaaagltdpelldwirhletvdevkrftsitl Peptide 5 5eihs[D- Peptide 6 6 Nle]aaaeelkqakteavnrlkvkgitdpiilqwirnlptvedvrrr teeiieqteaqyqralteakaqaikrlqqhgitdpdvltwvqtlstvaevesatda Peptide 7 7liqrhlFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325slkesllkeakeravkelekagitdpavlewiknletveevvtstka Peptide 8 8lldkhkgaaaaalaaareaaiaalrragitdpavlewirrlptveevvgttatll Peptide 9 9aehagakaaalaaakaaaeaalaaagitdpevlnwvrslptvadvlaatn Peptide 10 10allashan[D- Peptide 11 11 Nle]ereallnaktkaearlrkhgitdpeilnwvmlptveqvnhit eniisqheqga[D- Peptide 12 12 Nle]kdlkkakeeaieklkqhgitdpkiikwietlptveevkkrtk eiieehlgsysqqlsaakskaitelkkhgitdpeiiqwiqtletveqvyqqtk Peptide 13 13qliekfs[D- Peptide 14 14 Nle]keerqlqeakkraiarlrragitdpeilqwirdlptvdavrtrtr qllrrha[D- Peptide 15 15 Nle]ekeeklkkakeraikklkekgitdpkiiewikslktveevik ktkeiiekylFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325dkrkkalkeakrlaierlreigitdpevinwirrlstvaqvldfterlir Peptide 16 16ehlsaaeqalaaartaaiarlraagitdpavlnwirvlptveevekrtrqll Peptide 17 17aehlaayaaslaaakaeaiarlrkagitdpsildwvrrlptvavvnnttari Peptide 18 18Inshlarraaalraakaralarlrragitdpdvlawiralptvaaveratrella Peptide 19 19ahrtidqwllknakedaiaelkkagitephvisfinhapyvshvnglk Peptide 20 20nailkahaPeptide 21 21 ttihvgdltlkydnpkkayeiakklakkynltvtikngkitvtPeptide 22 22 gtvkvqgitikvpnpdkaykiakklakkynltvtrkgntikvePeptide 23 23 gtlhlngvtvkvpslekaikaakkfakkynlevqvhgntvhvhttvhvngveyrfnnpeeaykfavrfD- Peptide 24 24Nle] akeiggtvefhgeeihvePeptide 25 25 htvhvngqkytfptlekaakfakklakklgltvevhgntikleFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325Peptide 26 26 ttqievddeeqakklakkwqkkgydahytqkngkwfvqfnPeptide 27 27 ritfh[D-Nle]ngpeqakrlikwakkqnlrfqvrqengqiiididqftvtiegdea[D-Nle]kkfike[D- Peptide 28 28Nle]kkmlevhvrvengqwiahvsPeptide 29 29 tttytyddkqaakeaaeewkkrgyevhltqkgnkwqvkvgPeptide 30 30 dseqlkeeakkaykngneekakrilkkagldeseaeeflrrafPeptide 31 31 ddearrrakkllekgysedyireylkrqgvnpdeaedairwakeeevkkrirelakrgvspeqirewlrkqgyspdqakqlfeea[D- Peptide 32 32Nle]Peptide 33 33 seiekiaeklwrerpgskewvvevlkrlgvtedearkaaekaePeptide 34 34 pdkkkkivkkllnkgldksevekearkngisddiveqaykewlPeptide 35 35 sleealerakealkknqplevngyrfddprkayewareqleraPeptide 36 36 tldearelverakkegtgvdvngqrfedwreaerwvreqeknkPeptide 37 37 tekqeraieyirrlgyseeearrilkkangnpdlakeiarrigFlagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325Peptide 38 38 tideiikaleqavkdnkpiqvgnytvtsadeaeklakklkkeyPeptide 39 39 ieevkkivseiqskqltritingntyqvnspeelveyvrkvfsPeptide 40 40 dwreiaklaeealkkqqpvtvngqtfndpneikklletaqkai82. The compound of any one of claims 52-81, wherein the polypeptide residue is an antibody or an antigen-binding fragment thereof.

83. A method of installing an enrichment tag on a target polypeptide, comprising combining a mixture comprising the target polypeptide with a compound of any one of claims 34-51 under conditions sufficient to form a compound having the structure of:o; orO OG- S XE" 'G'H^O PPOwherein PP represents a residue of the target polypeptide.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 84. The method of claim 83, wherein the mixture comprising the target polypeptide further comprises one or more non-target polypeptides.

85. The method of claim 84, wherein the one or more non-target polypeptides do not react with the compound of any one of claims 34-51.

86. The method of any one of claims 83-85, wherein the target polypeptide is covalently bound at its C-terminus to a solid phase support.

87. The method of any one of claims 83-86, wherein the target polypeptide comprises from 5 to 55 amino acid residues.

88. The method of any one of claims 83-87, wherein the target polypeptide comprises from 25 to 55 amino acid residues.

89. The method of any one of claims 83-88, wherein the target polypeptide comprises at least one D-amino acid residue.

90. The method of any one of claims 83-89, wherein the target polypeptide comprises at least one alpha-disubstituted amino acid residue (e.g., aminoisobutyric acid).

91. The method of any one of claims 83-90, wherein the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine).

92. The method of claim 91, wherein the non-canonical amino acid further comprises a non-natural side chain.

93. A method of purifying a target polypeptide, comprising:(a) combining a first mixture comprising the target polypeptide with a compound of any one of claims 34-51 under conditions sufficient to form a second mixture comprising an enrichment-tagged compound having the structure of:Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325wherein PP represents a residue of the target polypeptide;(b) contacting the second mixture with an affinity resin, thereby binding the enrichment-tagged compound to the affinity resin;wherein the affinity resin has an affinity for the enrichment tag;(c) washing the affinity resin; and(d) removing the enrichment tag from the enrichment-tagged compound under conditions sufficient to produce the purified target polypeptide.

94. The method of claim 93, wherein the first mixture further comprises one or more non-target polypeptides.

95. The method of claim 94, wherein the one or more non-target polypeptides do not react with the compound of any one of claims 34-51.

96. The method of any one of claims 93-95, wherein the target polypeptide is covalently bound at its C-terminus to a solid phase support.

97. The method of claim 96, further comprising step (a’) following step (a):(a’) removing the solid phase support from the target polypeptide.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 98. The method of any one of claims 93-97, wherein the target polypeptide comprises from 5 to 55 amino acid residues.

99. The method of any one of claims 93-98, wherein the target polypeptide comprises from 25 to 55 amino acid residues.

100. The method of any one of claims 93-99, wherein the target polypeptide comprises at least one D-amino acid residue.

101. The method of any one of claims 93-100, wherein the target polypeptide comprises at least one alpha-disubstituted amino acid residue (e.g., aminoisobutyric acid).

102. The method of any one of claims 93-101, wherein the target polypeptide comprises at least one non-canonical amino acid (e.g., 4-fluorophenylalanine).

103. The method of claim 102, wherein the non-canonical amino acid further comprises a non-natural side chain.

104. The method of any one of claims 93-103, wherein the enrichment tag is, and the affinity resin comprises streptavidin.

105. The method of any one of claims 93-103, wherein the enrichment tag is a peptide fragment, such as a polyhistidine moiety; and wherein the affinity resin comprises a metal ion.

106. The method of any one of claims 93-103, wherein the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NH- C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH- alkyl, and substituted -NH-S(O)2-alkyl; andwherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH- alkyl, substituted -NH-C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2- alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl terminates in an alkyne; andwherein the affinity resin comprises an azide moiety.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325 107. The method of any one of claims 93-103, wherein the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NH- C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH- alkyl, and substituted -NH-S(O)2-alkyl; andwherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH- alkyl, substituted -NH-C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2- alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl, terminates in an azide; andwherein the affinity resin comprises an alkyne.

108. The method of any one of claims 93-103, wherein the enrichment tag is selected from substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH-alkyl, substituted -NH- C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2-alkyl, substituted -S(O)2-NH- alkyl, and substituted -NH-S(O)2-alkyl; andwherein the alkyl in substituted alkyl, substituted -NH-alkyl, substituted -C(O)-NH- alkyl, substituted -NH-C(O)-alkyl, substituted -C(O)-alkyl, substituted -S(O)2- alkyl, substituted -S(O)2-NH-alkyl, and substituted -NH-S(O)2-alkyl, terminates in an aminooxy or a hydrazine group; andwherein the affinity resin comprises an aldehyde or ketone moiety.

109. The method of any one of claims 93-108, wherein the enrichment-tagged compoundE'G-GY~'N-'YPPE'Gfo^GV''N'''YPPis OfBu B°co or4OfBu ^ocO; and the conditions sufficient to produce the purified target polypeptide comprise an oxidizing agent.

110. The method of claim 109, wherein the conditions sufficient to produce the purified target polypeptide comprise sodium periodate.

111. The method of any one of claims 93-108, wherein the enrichment-tagged compoundis O or O; and the conditions sufficient to produce the purified target polypeptide comprise a reducing agent.Flagship Ref. No.: VL83003-W1Attorney Docket No.: FBZ-00325112. The method of claim 111, wherein the conditions sufficient to produce the purified target polypeptide comprise a thiol (e.g., beta-mercaptoethanol).

113. The method of claim 111 or 112, wherein the conditions sufficient to produce the purified target further comprise a base.

114. The method of any one of claims 93-108, wherein the enrichment-tagged compound O PP; and the conditions sufficient to produce the purified target polypeptide comprise a base.

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