Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals

Patent metadata

Field	Value
Publication number	US-10416166-B2
Application number	US-201214342131-A
Country	US
Kind code	B2
Filing date	Sep 28, 2012
Priority date	Sep 28, 2011
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

Abstract

Reagents comprising MS active, fluorescent molecules with an activate functionality for reaction with amines useful in rapid tagging of biomolecules such as N-glycans, proteins, peptides, and amino acids, and uses thereof are taught and described. These MS active, fluorescent molecules may have three functional components such as a tertiary amino group or other MS active atom, a highly fluorescent moiety, and a functional group that rapidly reacts with amines.

First claim

We claim:

1. A compound of Formula I

wherein
R 1 is O═C═N— or

R 2 is independently selected from —H, —C 1 -C 8 alkyl, —C 1 -C 8 cycloalkyl, halo, dialkylamino, CH 2 -dialkylamino, aminocarbonyl, alkoxycarbonyl, or alkoxy, but not Cl or O═C═N—; and
R 3 and R 4 are independently selected from —H, alkyl, alkyl amino, alkylsulfonic acid, alkyl phosphonic acid, wherein either R 3 or R 4 is alkylamino, alkyl phosphonic acid, or alkylsulfonic acid.

2. A compound of Formula II

wherein
m=0-9;
n=0-9;
R 1 is O═C═N—, S═C═N—, or

R 2 is independently selected from methylene, substituted nitrogen, oxygen, carbonyl, amide, ester, sulfur, sulfoxide, or sulfone;
R 3 and R 4 are independently selected from —H, alkyl, alkyl amino, alkylsulfonic acid, alkyl phosphonic acid, wherein either R 3 or R 4 is alkylamino, alkyl phosphonic acid, or alkylsulfonic acid; and
R 5 is independently selected from —H, —C 1 -C 8 alkyl, —C 1 -C 8 cycloalkyl, halo, dialkylamino, CH 2 -dialkylamino, aminocarbonyl, alkoxycarbonyl, or alkoxy, but not Cl when R 1 is O═C═N—, and when R 1 is S═C═N, R 5 is H.

3. A compound of the structural formula

4. A method for rapid tagging of a glycan comprising the steps of:
reacting a precursor glycan or precursor amino acid with the compound of Formula I

wherein
R 1 is O═C═N—, S═C═N—, or

R 2 is independently selected from —H, —C 1 -C 8 alkyl, —C 1 -C 8 cycloalkyl, halo, CH 2 -dialkylamino, dialkylamino, aminocarbonyl, alkoxycarbonyl, or alkoxy;
R 3 and R 4 are independently selected from —H, alkyl, alkyl amino, alkylsulfonic acid, alkyl phosphonic acid, wherein R 3 or R 4 is alkylamino, alkyl phosphonic acid, or alkylsulfonic acid, and wherein R 3 and R 4 together with the nitrogen to which they are attached may form an optionally substituted 5- to 8-membered saturated or partially unsaturated ring, and forming an MS active, fluorescent glycan derivative or an amino acid derivative.

5. A method for rapid tagging of a glycan comprising the steps of reacting a precursor glycan or precursor amino acid with the compound of Formula II

wherein
R 1 is O═C═N—, S═C═N, or

R 2 is independently selected from methylene, substituted nitrogen, oxygen, carbonyl, amide, ester, sulfur, sulfoxide, or sulfone;
R 3 and R 4 are independently selected from —H, alkyl, alkyl amino, alkylsulfonic acid, alkyl phosphonic acid, wherein R 3 or R 4 is alkylamino, alkyl phosphonic acid, or alkylsulfonic acid, and wherein R 3 and R 4 together with the nitrogen to which they are attached may form an optionally substituted 5- to 8-membered saturated or partially unsaturated ring; and
R 5 is independently selected from —H, —C 1 -C 8 alkyl, —C 1 -C 8 cycloalkyl, halo, CH 2 -dialkylamino, dialkylamino, aminocarbonyl, alkoxycarbonyl, or alkoxy, but not Cl or O═C═N—, and not when R 1 is S═C═N,
and forming an MS active, fluorescent glycan derivative or an amino acid derivative.

6. A method for analyzing a glycan in a sample containing at least one glycan by means of liquid chromatography and mass spectrometry comprising labeling the glycan in the sample by reacting the glycan with the compound according to claim 1 , 2 or 3 for a time and under conditions suitable to facilitate the labeling; providing a sample containing the glycan labeled with the compound; and subjecting the labeled compound to liquid chromatograph and mass spectrometry.

7. The compound of claim 1 , wherein R 3 and R 4 together with the nitrogen to which R 3 and R 4 are attached form a substituted or unsubstituted 5- to 8-membered saturated or partially unsaturated ring, and R 1 is not O═C═N—.

8. The compound of claim 2 , wherein R 3 and R 4 together with the nitrogen to which R 3 and R 4 are attached form a substituted or unsubstituted 5- to 8-membered saturated or partially unsaturated ring.

Assignees

• Waters Technologies Corp

Inventors

• Brousmiche Darryl W
• Yu Ying-Qing

Classifications

• C09B57/08

  Naphthalimide dyes; Phthalimide dyes · CPC title

• C09B57/02

  Coumarine dyes · CPC title

• C09B19/00

  Oxazine dyes · CPC title

• G01N2458/20

  Labels for detection by gas chromatography, e.g. haloaryl systems · CPC title

• Y10T436/143333

  Saccharide [e.g., DNA, etc.] · CPC title