Benzylpyridinium reagent for mass spectrometry

Patent metadata

Field	Value
Publication number	US-12424426-B2
Application number	US-202217990714-A
Country	US
Kind code	B2
Filing date	Nov 20, 2022
Priority date	May 20, 2020
Publication date	Sep 23, 2025
Grant date	Sep 23, 2025

Abstract

The present invention relates to compounds which are suitable to be used in mass spectrometry as well as methods of mass spectrometric determination of analyte molecules using said compounds.

First claim

The invention claimed is:

1. A compound of formula I for mass spectrometric determination of an analyte:

wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q, which is capable of forming a covalent bond with the analyte,
wherein the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotope or derivative thereof,
wherein Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, amine or wherein Y1 and Y2 form a ring structure, which is selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic.

2. The compound of claim 1 , wherein the coupling group Q is bonded to X according to the following formula II:

wherein K is a reactive unit, which is capable of forming the covalent bond with the analyte,
wherein n is 0, 1, 2, 3, 4 or 5, and
wherein X is a binding carbon-atom of the pyridinium cation of formula I.

3. The compound of claim 2 , wherein K is capable of reacting with a carbonyl group, phenol group, amine, hydroxyl group or diene group of the analyte.

4. The compound of claim 2 , wherein K is selected from the group consisting of hydrazide, hydrazine, hydroxylamine, Br, F, 4-substituted 1,2,4-triazolin-3,5-dione (TAD) and carbamate.

5. The compound of claim 2 , wherein n =0 and K is Br or n=0 and K is F.

6. The compound of claim 1 , wherein Q is selected from the group consisting of methyl hydrazide, methyl hydrazine, methyl hydroxylamine, acetohydrazide, 4-substituted 1,2,4-triazoline-3,5-dione (TAD) and F.

7. The compound of claim 1 , wherein B1 is Q or B2 is Q or B3 is Q.

8. The compound of claim 1 , wherein the compound is permanent positively charged.

9. A composition comprising the compound of claim 1 .

10. A kit comprising the compound of claim 1 .

11. A complex for detecting an analyte using mass spectrometric determination comprising a binding analyte and a binding compound, which are covalently linked to each other, wherein the complex is formed by chemical reaction of the analyte and the compound of claim 1 , and wherein the analyte is selected from the group consisting of a nucleic acid, an amino acid, a peptide, a protein, a metabolite, a hormone, a fatty acid, a lipid, a carbohydrate, a steroid, a ketosteroid, a secosteroid, a molecule characteristic of a certain modification of another molecule, a substance that has been internalized by the organism, and a metabolite of such a substance, and combinations thereof.

12. The complex of claim 11 , wherein the binding compound comprises the formulae III and IV:

wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q*, which forms a covalent bond with the analyte,
wherein the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, isotope or derivative thereof,
wherein Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, amine or wherein Y1 and Y2 form a ring structure, which is selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, benzylic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic,
wherein n* is 0, 1, 2, 3, 4 or 5,
wherein the binding analyte is covalently bonded via K*, this is true for pyridinium-hydrazide and pyridinium-1,2,4-triazoline-3,5-dione, but in the case of fluoro-pyridinium K*=X* and n*=0, and
wherein X* is a binding carbon-atom of the pyridinium cation of formula III.

13. A method for mass spectrometric determination of an analyte comprising the steps of:
(a) reacting the analyte with the compound of formula I as defined in claim 1 , whereby a complex is formed,
(b) subjecting the complex from step (a) to a mass spectrometric analysis, wherein step (b) comprises:
(i) subjecting an ion of the complex to a first stage of mass spectrometric analysis, whereby the ion of the complex is characterized according to its mass/charge (m/z) ratio,
(ii) causing fragmentation of the complex ion, whereby a first entity is released and a daughter ion of the complex is generated, wherein the daughter ion of the complex differs in its m/z ratio from the complex ion, and
(iii) subjecting the daughter ion of the complex to a second stage of mass spectrometric analysis, whereby the daughter ion of the complex is characterized according to its m/z ratio, and/or
wherein (ii) may further comprise alternative fragmentation of the complex ion, whereby a second entity different from the first entity is released and a second daughter ion of the complex is generated, and
wherein (iii) may further comprise subjecting the first and second daughter ions of the complex to a second stage of mass spectrometric analysis, whereby the first and second daughter ions of the complex are characterized according to their m/z ratios.

14. A compound of formula V:

wherein one of the substituents B1, B2, B3, B4, B5 is a coupling group Q, which is capable of forming a covalent bond with the analyte,
wherein the other substituents A1, A2, A3, A4, A5, B1, B2, B3, B4, B5 are each independently selected from hydrogen, halogen, alkyl, N-acylamino, N,N-dialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, isotope or derivative thereof,
wherein Y1 and Y2 are each independently selected from hydrogen, methyl, ethyl, methoxy, substituted aromatic, unsubstituted aromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heteroaromatic, unsubstituted heteroaromatic, amine or wherein Y1 and Y2 form a ring structure, which is selected from substituted cycloalkyl, unsubstituted cycloalkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic.

Assignees

• Roche Diagnostics Operations Inc

Inventors

• Hahn Robert
• Heindl Dieter
• Hoffmann Thomas
• Josel Hans-Peter
• Kobold Uwe
• Kuchelmeister Hannes
• Loibl Simon Ferdinand
• Mazzier Daniela
• Milic Jelena
• Prencipe Giuseppe
• Rempt Martin
• Seidel Christoph

Classifications

• C07D401/04

  directly linked by a ring-member-to-ring-member bond · CPC title

• C07D213/86

  Hydrazides; Thio or imino analogues thereof · CPC title

• C07D213/61

  Halogen atoms or nitro radicals · CPC title

• C07D213/55

  Acids; Esters · CPC title

• H01J49/0031 Primary

  Step by step routines describing the use of the apparatus (H01J49/0081 takes precedence) · CPC title