US 2022/0227889 A1 · METHODS AND REAGENTS FOR CLEAVAGE OF THE N-TERMINAL AMINO ACID FROM A POLYPEPTIDE

Abstract

The present invention relates to methods of cleaving the N-terminal amino acid from a polypeptide, which may be in free form or conjugated to a carrier or surface, such as a bead. It provides methods to activate the N-terminal amine of a polypeptide to promote formation of a cyclic adduct of the N-terminal amino acid, resulting in cleavage of the N-terminal amino acid from the polypeptide. The method can be used to sequence and/or analyze a polypeptide. For example, the methods can be combined with methods described herein for sequencing and/or analysis that employ barcoding and nucleic acid encoding of molecular recognition events, and/or detectable labels. The invention also provides compounds and kits useful for practicing these methods.

Claims39

Dependent claims

A method to cleave an N-terminal amino acid residue from a peptidic compound of Formula (I)

wherein the method comprises:
(1) converting the peptidic compound to a guanidinyl derivative of Formula (II):
or a tautomer thereof;
and
(2) contacting the guanidinyl derivative with a suitable medium to produce a compound of Formula (III)
wherein:
R1 is R6, NHR3, —NHC(O)—R3, or —NH—SO2—R3
R2 is H or R4;
R3 is H or R6, wherein R6 is an optionally substituted group selected from phenyl, 5-membered heteroaryl, 6-membered heteroaryl, C1-3 haloalkyl, and C1-6 alkyl,
wherein optional substituents of the optionally substituted group are one to three members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, CON(R′)2, phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl, wherein the phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl are each optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, and CON(R′)2;
where each R′ is independently H or C1-3 alkyl;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
and wherein two R′ or two R″ on the same nitrogen can optionally be taken together to form a 4-7 membered heterocycle optionally containing an additional heteroatom selected from N, O and S as a ring member, wherein the 4-7 membered heterocycle is optionally substituted with one or two groups selected from halo, OH, OCH3, CH3, oxo, NH2, NHCH3 and N(CH3)2;
RAA1 and RAA2 are each independently selected amino acid side chains;
and the dashed semi-circle connecting RAA1 and/or RAA2 to the nearest N atom indicates that RAA1 and/or RAA2 can optionally cyclize onto the designated N atom;
and
Z is —COOH, CONH2, or an amino acid or a polypeptide that is optionally attached to a carrier or solid support.

15 more elements

A compound of the Formula:

wherein:
R2 is H or R4;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
ring A and ring B are each independently a 5-membered heteroaryl ring containing up to three N atoms as ring members and is optionally fused to an additional phenyl or a 5-6 membered heteroaryl ring, and wherein the 5-membered heteroaryl ring and optional fused phenyl or 5-6 membered heteroaryl ring are each optionally substituted with one or two groups selected from C1-4 alkyl, C1-4 alkoxy, —OH, halo, C1-4 haloalkyl, NO2, COOR, CONR2, —SO2R*, —NR2, phenyl, and 5-6 membered heteroaryl;
wherein each R is independently selected from H and C1-3 alkyl optionally substituted with OH, OR*, —NH2, —NHR*, or —NR*2;
and
each R* is C1-3 alkyl, optionally substituted with OH, oxo, C1-2 alkoxy, or CN;
wherein two R, or two R″, or two R* on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
with the proviso that Ring A and Ring B are not both unsubstituted imidazole and that Ring A and Ring B are not both unsubstituted benzotriazole;
or a salt thereof.

8 more elements

A compound of Formula (II):

or a tautomer thereof,
wherein:
R1 is R6, NHR3, —NHC(O)—R3, or —NH—SO2—R3;
R2 is H or R4;
R3 is H or R6, wherein R6 is an optionally substituted group selected from phenyl, 5-membered heteroaryl, 6-membered heteroaryl, C1-3 haloalkyl, and C1-6 alkyl,
wherein optional substituents of the optionally substituted group are one to three members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, CON(R′)2, phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl, wherein the phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl are each optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, and CON(R′)2;
where each R′ is independently H or C1-3 alkyl;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
wherein two R′ or two R″ on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
RAA1 and RAA2 are each independently selected from H and C1-6 alkyl optionally substituted with one or two groups independently selected from —OR5, —N(R5)2, —SR5, —SeR5, —COOR5, CON(R5)2, —NR5—C(═NR5)—N(R5)2, phenyl, imidazolyl, and indolyl, where phenyl, imidazolyl and indolyl are each optionally substituted with halo, C1-3 alkyl, C1-3 haloalkyl, —OH, C1-3 alkoxy, CN, COOR5, or CON(R5)2;
each R5 is independently selected from H and C1-2 alkyl;
and Z is —COOH, CONH2, or an amino acid or polypeptide that is optionally attached to a carrier or surface;
or a salt thereof.

11 more elements

A compound of Formula (IV):

wherein:
R2 is H or R4;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
wherein two R″ on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
ring A is a 5-membered heteroaryl ring containing up to three N atoms as ring members and is optionally fused to an additional phenyl or a 5-6 membered heteroaryl ring, and wherein the 5-membered heteroaryl ring and optional fused phenyl or 5-6 membered heteroaryl ring are each optionally substituted with one or two groups selected from C1-4 alkyl, C1-4 alkoxy, —OH, halo, C1-4 haloalkyl, NO2, COOR, CONR2, —SO2R*, —NR2, phenyl, and 5-6 membered heteroaryl;
wherein each R is independently selected from H and C1-3 alkyl optionally substituted with OH, OR*, —NH2, —NHR*, or —NR*2;
and
each R* is C1-3 alkyl, optionally substituted with OH, oxo, C1-2 alkoxy, or CN;
wherein two R, or two R″, or two R* on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
RAA1 and RAA2 are each independently selected amino acid side chains;
and the dashed semi-circle connecting RAA1 and/or RAA2 to the nearest N atom indicates that RAA1 and/or RAA2 can optionally cyclize onto the designated N atom;
and
Z is —COOH, CONH2, or an amino acid or a polypeptide that is optionally attached to a carrier or solid support;
or a salt thereof.

12 more elements

A method to identify the N-terminal amino acid residue of a peptidic compound of the Formula (I):

wherein the method comprises:
(1) converting the compound of Formula (I) to a guanidinyl derivative of Formula (II) or a tautomer thereof:
wherein:
R1 is R6, NHR3, —NHC(O)—R3, or —NH—SO2—R3
R2 is H or R4;
R3 is H or R6, wherein R6 is an optionally substituted group selected from phenyl, 5-membered heteroaryl, 6-membered heteroaryl, C1-3 haloalkyl, and C1-6 alkyl,
wherein optional substituents of the optionally substituted group are one to three members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, CON(R′)2, phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl, wherein the phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl are each optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, and CON(R′)2;
where each R′ is independently H or C1-3 alkyl;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
wherein two R′ or two R″ on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
RAA1 and RAA2 are each independently selected amino acid side chains;
and the dashed semi-circle connecting RAA1 and/or RAA2 to the nearest N atom indicates that RAA1 and/or RAA2 can optionally cyclize onto the designated N atom;
and
and Z is —COOH, CONH2, or an amino acid or polypeptide that is optionally attached to a carrier or surface;
(2) contacting the guanidinyl derivative with a suitable medium to induce elimination of the modified N-terminal amino acid and produce at least one cleavage product selected from:
(wherein R1 is NHR3, —NHC(O) R3, or —NH—SO2—R3, respectively) or a tautomer thereof;
and
determining the structure or identity of the at least one cleavage product to identify the N-terminal amino acid of the compound of Formula (I).

16 more elements

A method for analyzing a polypeptide, comprising the steps of:

(a) providing the polypeptide optionally associated directly or indirectly with a recording tag;
(b) functionalizing the N-terminal amino acid (NTAA) of the polypeptide with a chemical reagent, wherein the chemical reagent is either:
(b1) a compound of Formula (AA):
wherein:
R2 is H or R4;
R4 is C1-6 alkyl, which is optionally substituted with one or two members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR″, and CON(R″)2,
where each R″ is independently H or C1-3 alkyl;
each ring A is a 5-membered heteroaryl ring containing up to three N atoms as ring members and is optionally fused to an additional phenyl or a 5-6 membered heteroaryl ring, and wherein the 5-membered heteroaryl ring and optional fused phenyl or 5-6 membered heteroaryl ring are each optionally substituted with one or two groups selected from C1-4 alkyl, C1-4 alkoxy, —OH, halo, C1-4 haloalkyl, NO2, COOR, CONR2, —SO2R*, —NR2, phenyl, and 5-6 membered heteroaryl;
wherein each R is independently selected from H and C1-3 alkyl optionally substituted with OH, OR*, —NH2, —NHR*, or —NR*2;
and
each R* is C1-3 alkyl, optionally substituted with OH, oxo, C1-2 alkoxy, or CN;
wherein two R, or two R″, or two R* on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
or
(b2) a compound of the formula R3—NCS;
wherein R3 is H or an optionally substituted group selected from phenyl, 5-membered heteroaryl, 6-membered heteroaryl, C1-3 haloalkyl, and C1-6 alkyl,
wherein the optional substituents are one to three members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, CON(R′)2, phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl, wherein the phenyl, 5-membered heteroaryl, 6-membered heteroaryl, and C1-6 alkyl are each optionally substituted with one or two members selected from halo, —OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR′, —N(R′)2, and CON(R′)2;
where each R′ is independently H or C1-3 alkyl;
wherein two R′ on the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, or CN;
to provide an initial NTAA functionalized polypeptide;
optionally treating the initial NTAA functionalized polypeptide with an amine of Formula R2—NH2 or with a diheteronucleophile to form a secondary NTAA functionalized polypeptide;
and optionally treating the initial NTAA functionalized polypeptide or the secondary NTAA functionalized polypeptide with a suitable medium to eliminate the NTAA and form an N-terminally truncated polypeptide;
(c) contacting the polypeptide with a first binding agent comprising a first binding portion capable of binding to the polypeptide, or to the initial NTAA functionalized polypeptide, or to the secondary NTAA functionalized polypeptide, or to the N-terminally truncated polypeptide;
and either
(c1) a first coding tag with identifying information regarding the first binding agent, or
(c2) a first detectable label;
(d) (d1) transferring the information of the first coding tag to the recording tag to generate an extended recording tag and analyzing the extended recording tag, or
(d2) detecting the first detectable label.

24 more elements

Dependencies are inferred from the claim text.

Methods and reagents for cleavage of the n-terminal amino acid from a polypeptide

Life of this patent

Methods and reagents for cleavage of the n-terminal amino acid from a polypeptide

Life of this patent

Abstract

Claims39