Method of generating linkage-specific di-and polyprotein probes
AssigneeUniverstiy of Delaware
InventorsZhihao ZHUANG, Guorui LI
Life of this patent
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Abstract
Deubiquitinating enzyme (DUB) probes are provided that resemble native diubiquitin (diUB) with a similar linkage size and that may contain a Michael acceptor for trapping the DUB active-site cysteine. For example, both K63- and K48-linked diubiquitin probes are generated using a facile chemical ligation method, utilizing the linker compound 3-(2-(bromomethyl)-1,3-dioxolan-2-yl)prop-2-en-1-amine. The diUb probes are capable of labelling DUBs from different families and may be employed to reveal intrinsic linkage specificities of DUBs.
Claims35
A compound having a structure in accordance with Formula (I):
- H2NCH2—R—C(Diox)CH2X (I)
- wherein R is —CH2CH2— or —CH═CH—, Diox is —O(CH2)nO— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH2)nO— are both bonded, and X is Br, Cl or I.
A di- or polyprotein comprising a first polypeptide moiety covalently bonded to a second polypeptide moiety through a linkage having a structure corresponding to —[C(═O)NHCH2C(═O)NHCH2—R—C(═O)CH2SCH2]—, wherein R is —CH2CH2— or —CH═CH—.
A method of making a di- or polyprotein, comprising linking a first polypeptide molecule and a second polypeptide molecule using a compound having a structure in accordance with Formula (I):
- H2NCH2—R—C(Diox)CH2X (I)
- wherein R is —CH2CH2— or —CH═CH—, Diox is —O(CH2)nO— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH2)nO— are both bonded, and X is Br, Cl or I.
A method of making a di- or polyprotein, comprising the steps of:
- a) reacting PP1-C(═O)—S—R1, wherein PP1 is a first polypeptide and —C(═O)—S—R1 is a thioester group, with a compound having a structure in accordance with Formula (I):
- H2NCH2—R—C(Diox)CH2X (I)
- wherein R is —CH2CH2— or —CH═CH—, Diox is —O(CH2)nO— with n=2 or 3 and forms a 1,3-dioxolane ring or 1,3-dioxane ring which includes the carbon atom to which the oxygen atoms of —O(CH2)nO— are both bonded, and X is Br, Cl or I to provide PP1-C(═O)NHCH2—R—C(Diox)CH2X;
- b) converting the 1,3-dioxolane ring or 1,3-dioxane ring to a ketone to provide PP1-C(═O)NH—CH2—R—C(═O)CH2X;
- and
- c) reacting the PP1-C(═O)NH—CH2—R—C(═O)CH2X with a second polypeptide bearing a cysteine residue.
Dependencies are inferred from the claim text.