IP Library › Granted Patent US 12674804
Granted Patent B2
US 12674804 · App. 17/628,808 · Granted Jul 7, 2026

Modular chemical probe for detection of amino acid citrulline in physiological samples

Inventors: Dmitry V. Kadnikov (Woodbury, MN); Jennifer E. Grant (Menomonie, WI)
Assignee: WiSys Technology Foundation, Inc.
G01N33/6812C07C47/277C07C49/255C07C49/258C07C49/755C07C49/757G01N33/533G01N33/54326G01N33/6848
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Quick Facts
Patent No.
US 12674804
App. No.
17/628,808
Granted
Jul 7, 2026
Kind
B2
Abstract

An improved chemical probe for the detection of the amino acid citrulline combines: 1) a reactive head formed of 1,3-dicarbonyl moiety that reacts with a citrulline side chain in an improved manner compared to currently used 1,2-dicarbonyl moieties; and 2) a modular action of the probe where citrulline side chains are labeled first using reactive heads described above, and attachment of a read-out subunit or tag, be it a fluorophore, a nanoparticle, or an antigen is performed separately. The modular nature of the chemical probe increases the sensitivity of the probes due to their smaller size. Additionally, the chemical probes of the present disclosure allow the same sample to be analyzed using a variety of read-out methods.

Claims (48)

1 . A chemical probe for the detection of the amino acid citrulline comprising a reactive head,

wherein the reactive head is formed of 1,3-dicarbonyl moiety, and

wherein reactive head is selected from the group consisting of:

wherein R is selected from the group consisting of: an alkyne group, an alkene group, an alkyl group, or an oxygen-containing function group, including, but not limited to a C═O-alkyl group or (C═O)—O-alkyl group, and a PEG-based linker —(OCH 2 CH 2 ) n , where n=2 to 12.

2 . A chemical probe for the detection of the amino acid citrulline comprising a reactive head,

wherein the reactive head is formed of a 1,3-dicarbonyl moiety at one end of the reactive head, and

wherein the reactive head includes a triple bond located at the other end of the reactive head opposite the 1, 3-dicarbonyl moeity.

3 . The probe of claim 2 wherein the 1,3-dicarbonyl moiety includes a ketone-ketone, a ketone-aldehyde, or an aldehyde-aldehyde structure.

4 . The probe of claim 3 wherein the reactive head has a structure selected from the group consisting of:

5 . The probe of claim 3 wherein the reactive head has the structure:

wherein R is selected from the group consisting of: any alkyl group, or an oxygen-containing function group, including, but not limited to a C═O-alkyl group or (C═O)—O-alkyl group, and a PEG-based linker —(OCH 2 CH 2 ) n , where n=2 to 12.

6 . The probe of claim 3 wherein the reactive head has the structure:

7 . The probe of claim 3 wherein the reactive head has the structure selected from the group consisting of:

wherein n=1 to 10.

8 . The probe of claim 3 wherein the reactive head has a structure selected from the group consisting of:

9 . The probe of claim 3 wherein the reactive head has a structure selected from the group consisting of:

10 . The probe of claim 1 or 2 , further comprising a detection tag bonded to reactive head.

11 . The probe of claim 10 wherein the detection tag comprises:

a. a probe linker attached to the reactive head; and

b. a read-out tag attached to the probe linker.

12 . The probe of claim 11 wherein the read-out tag is a fluorescent read-out tag.

13 . The probe of claim 11 wherein the read-out tag is a biotinylated read-out tag.

14 . The probe of claim 11 wherein the read-out tag is a superparamagnetic nanoparticle read-out tag.

15 . The probe of claim 2 , further comprising a detection tag bonded to reactive head, wherein the detection tag comprises:

a. a probe linker attached to the reactive head; and

b. a read-out tag attached to the probe linker, and

wherein the probe linker is an azide-modified probe linker.

16 . The probe of claim 2 , further comprising a detection tag bonded to the reactive head, wherein the detection tag comprises:

a. a probe linker attached to the reactive head; and

b. a read-out tag attached to the probe linker, and

wherein the read-out tag is a fluorescent read-out tag having the following formula:

17 . The probe of claim 2 , further comprising a detection tag bonded to the reactive head, wherein the detection tag comprises:

a. a probe linker attached to the reactive head; and

b. a read-out tag attached to the probe linker, and

wherein the read-out tag is a biotinylated read-out tag having the following formula:

18 . The probe of claim 2 , further comprising a detection tag bonded to the reactive head, wherein the detection tag comprises:

a. a probe linker attached to the reactive head; and

b. a read-out tag attached to the probe linker, and

wherein the read-out tag is a superparamagnetic nanoparticle read-out tag having the following formula:

19 . A method for detecting the presence of the amino acid citrulline in proteins from biological samples, the method comprising the steps of:

a) providing the detection probe of claim 1 or 2 ;

b) reacting the reactive head of the probe with citrulline to bond the probe to the citrulline;

c) bonding a detection tag to the reactive head; and

d) detecting the detection tag of the probe bonded to the citrulline.

20 . The method of claim 19 , wherein the detection tag comprises:

a) a probe linker attached to the reactive head; and

b) a read-out tag attached to the probe linker, and

wherein the read-out tag is selected from the group consisting of: a fluorescent read-out tag, a biotinylated read-out tag, and a superparamagnetic nanoparticle read-out tag.