Compounds and methods for selective c-terminal labeling

Patent metadata

Field	Value
Publication number	US-2021221839-A1
Application number	US-202117153490-A
Country	US
Kind code	A1
Filing date	Jan 20, 2021
Priority date	Jan 21, 2020
Publication date	Jul 22, 2021
Grant date	—

Abstract

The present disclosure relates to compounds and methods for selective C-terminal functionalization of peptides. In certain embodiments, the compounds have improved water-solubility, and are suitable for use in connection with peptide sequencing methodologies.

First claim

1 . A method of C-terminal functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (I):
P—R(CO 2 H) n    (I)

or a salt thereof, wherein
each P independently is a peptide;
each R(CO 2 H) n independently is an amino acid residue having n carboxylate moieties; and
n is 1 or 2;
with a compound of Formula (II):
HX-L 1 -R 1    (II)

wherein HX is nucleophilic moiety that is capable of being acylated;
H is a proton;
X is a heteroatom;
L 1 is a linker; and
R 1 is a click chemistry handle;
to obtain a plurality of compounds of Formula (III):

b. reacting the plurality of compounds of Formula (III) with a compound of Formula (IV):
R 2 -L 2 -Z   (IV)

wherein R 2 is a click chemistry handle that is complementary to R 1 ;
L 2 is a linker or is absent; and
Z is a water-soluble moiety;

to afford a plurality of compounds of Formula (V):

wherein Y is a moiety resulting from the click reaction of R 1 and R 2 .

2 . The method of claim 1 , wherein the plurality of peptides of Formula (I) is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (I).

3 . The method of claim 2 , wherein the enzymatic digestion comprises cleaving the C-terminal bonds of aspartic acid and/or glutamic acid residues of the protein.

4 - 5 . (canceled)

6 . The method of claim 2 , wherein the enzymatic digestion comprises cleaving the C-terminal bonds of lysine and/or arginine residues of the protein.

7 - 30 . (canceled)

31 . The method of claim 1 , wherein Formula (II) is of Formula (IIa):

wherein m is 1-25.

32 . (canceled)

33 . The method of claim 1 , wherein Formula (III) is of Formula (IIIa):

34 - 42 . (canceled)

43 . The method of claim 1 , wherein Z comprises single-stranded DNA.

44 - 47 . (canceled)

48 . A method of C-terminal functionalization of a peptide, comprising:
a. reacting a plurality of peptides of Formula (VI):

or a salt thereof, wherein
each P independently is a peptide;
with a compound of Formula (VII):

wherein
L 3 is a linker;
R 3 is a click chemistry handle; and
R 4 is substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;

to obtain a plurality of compounds of Formula (VIII):

and
b. reacting the plurality of compounds of Formula (VIII) with a compound of Formula (IX):
R 5 -L 4 -Z 1    (IX)

wherein R 5 is a click chemistry handle that is complementary to R 3 ;
L 4 is a linker or is absent; and
Z 1 is a water-soluble moiety;

to afford a plurality of compounds of Formula (X):

wherein Y is a moiety resulting from the click reaction of R 3 and R 5 .

49 . The method of claim 48 , wherein the plurality of peptides of Formula (VI) is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (VI).

50 - 66 . (canceled)

67 . The method of claim 48 , wherein the compound of Formula (VII) is selected from:

68 . The method of claim 48 , wherein Formula (VIII) is of Formula (VIIIa) or Formula (VIIIb):

69 - 75 . (canceled)

76 . The method of claim 48 , wherein Z comprises single-stranded DNA.

77 - 81 . (canceled)

82 . A method of selective N-functionalization of a peptide, comprising reacting a plurality of peptides of Formula (XI):

or a salt thereof, wherein each P independently is a peptide having an N-terminal amine, with a compound of Formula (XII):

under conditions (a), comprising Cu 2+ , or a precursor thereof, and a buffer having a pH of about 7-8.5; to obtain a plurality of N-terminal azido compounds of Formula (XIIIa):

or
under conditions (b), comprising Cu 2+ , or a precursor thereof, and a buffer having a pH of about 10-11; to obtain a plurality of ε-azido compounds of the Formula (XIIIb):

83 - 86 . (canceled)

87 . The method of claim 82 , wherein the N-terminal:ε selectivity under conditions (b) is at least about 90%.

88 . The method of claim 82 , wherein the plurality of peptides of Formula (XI) is obtained by subjecting a protein to enzymatic digestion to obtain a digestive mixture comprising the plurality of peptides of Formula (XI).

89 - 93 . (canceled)

94 . The method of claim 82 , further comprising reacting the plurality of compounds of Formula (XIIIb) with a compound of Formula (XIV):
R 6 -L 5 -Z 2    (XIV)
wherein R 6 is a moiety comprising an alkyne or a strained alkene;
L 5 is a linker or is absent; and
Z 2 is a water-soluble moiety;
to obtain a plurality of compounds of Formula (XV):

wherein Y is a moiety resulting from a click reaction with the azide moiety of Formula (XIIIb) and R 6 .

95 - 98 . (canceled)

99 . The method of claim 94 , wherein Z comprises single-stranded DNA.

100 - 101 . (canceled)

102 . A compound of Formula (II):
HX-L 1 -R 1    (II)
or a salt thereof, wherein
HX is nucleophilic moiety that is capable of being acylated;
H is a proton;
X is a heteroatom;
L 1 is a linker; and
R 1 is a click chemistry handle; or
a compound of Formula (III):

or a salt thereof, wherein
n is 1 or 2;
P is a peptide;
R is an aspartic acid or glutamic acid residue;
X is a heteroatom;
L 1 is a linker; and
R 1 is a click chemistry handle; or

a compound of Formula (IV):
R 2 -L 2 -Z   (IV)

or a salt thereof, wherein
R 2 is a click chemistry handle;
L 2 is a linker or is absent; and
Z is a water-soluble moiety; or

a compound of Formula (V):

Assignees

• Quantum Si Inc

Inventors

• Huang Haidong
• Nani Roger
• Ad Omer
• Reed Brian
• Dyer Matthew
• Boer Robert E

Classifications

• C07K1/34

  by filtration, ultrafiltration or reverse osmosis · CPC title

• C07K1/12

  by hydrolysis {, i.e. solvolysis in general} · CPC title

• C07K1/1077 Primary

  by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids · CPC title

• C07K1/128 Primary

  sequencing · CPC title

• C07K14/195

  from bacteria · CPC title