Methods for detecting and determining levels of monolignol ester conjugates incorporated into lignin and compounds relating thereto

Patent metadata

Field	Value
Publication number	US-11414372-B2
Application number	US-201916252915-A
Country	US
Kind code	B2
Filing date	Jan 21, 2019
Priority date	Dec 31, 2014
Publication date	Aug 16, 2022
Grant date	Aug 16, 2022

Abstract

Methods of detecting and, optionally, determining a level of incorporation of monolignol ester conjugates into lignin. The methods include derivatizing lignin to acylate at least a portion of free phenolic and aliphatic hydroxyls and to halogenate at least a portion of benzylic alcohols present in the lignin to yield derivatized lignin, treating the derivatized lignin with a reducing agent to cleave at least a portion of the derivatized lignin to yield lignin cleavage products, acetylating at least a portion of free hydroxyl groups in the lignin cleavage products with a labeled acetylation agent to yield labeled lignin fragments, and detecting the labeled lignin fragments.

First claim

What is claimed is:

1. A method of detecting and, optionally, determining a level of incorporation of monolignol ester conjugates into lignin, the method comprising:
(a) derivatizing lignin to acylate at least a portion of free phenolic and aliphatic hydroxyls and to halogenate at least a portion of benzylic alcohols present in the lignin, to yield derivatized lignin;
(b) treating the derivatized lignin of step (a) with a reducing agent to cleave at least a portion of the derivatized lignin, to yield lignin cleavage products;
(c) acetylating at least a portion of free hydroxyl groups in the lignin cleavage products of step (b) with a labeled acetylation agent, to yield labeled lignin fragments; and
(d) detecting the labeled lignin fragments produced in step (c).

2. The method of claim 1 wherein step (d) comprises determining the level of incorporation of monolignol ester conjugates in the lignin by measuring an amount or a concentration of labeled lignin fragments produced in step (c).

3. The method of claim 1 , wherein step (d) comprises determining the level of incorporation of monolignol ester conjugates in the lignin by measuring an amount or a concentration of labeled lignin fragments produced in step (c) using liquid or gas chromatography and mass spectrometry.

4. The method of claim 1 , wherein the labeled acetylation agent is labeled acetic anhydride.

5. A method of detecting and, optionally, determining a level of incorporation of monolignol ester conjugates into lignin, the method comprising:
(a) derivatizing lignin to acylate at least a portion of free phenolic and aliphatic hydroxyls and to halogenate at least a portion of benzylic alcohols present in the lignin, to yield derivatized lignin;
(b) treating the derivatized lignin of step (a) with a reducing agent to cleave at least a portion of the derivatized lignin, to yield lignin cleavage products;
(c) acetylating at least a portion of free hydroxyl groups in the lignin cleavage products of step (b) with a labeled acetylation agent, to yield labeled lignin fragments, wherein the labeled acetylation agent is labeled acetic anhydride, wherein the labeled acetic anhydride is labeled with an isotope; and
(d) detecting the labeled lignin fragments produced in step (c).

6. The method of claim 4 , wherein the labeled acetic anhydride is deuterium-labeled acetic anhydride.

7. The method of claim 1 , wherein step (a) comprises derivatizing the lignin with an acetyl halide.

8. The method of claim 1 , wherein step (a) comprises derivatizing extracted cell wall material or isolated lignin from plants, plant parts, or plant cells.

9. The method of claim 1 , wherein the labeled lignin fragments of step (c) comprise monolignol ester conjugates comprising a monolignol moiety and a carboxylate moiety, and wherein both the monolignol moiety and the carboxylate moiety are independently acetylated with the labeled acetylation agent.

10. The method of claim 9 , wherein the carboxylate moiety is selected from the group consisting of p-hydroxybenzoate, p-coumarate, ferulate, and sinapate.

11. The method of claim 1 , wherein the detecting the labeled lignin fragments comprises isolating the labeled lignin fragments.

12. The method of claim 11 , wherein the isolated lignin fragments comprise monolignol ester conjugates.

13. The method of claim 12 , wherein the monolignol ester conjugates comprise a monolignol moiety and a carboxylate moiety and the isolated labeled lignin fragments comprise one or more of:
a first type in which the monolignol moiety is a labeled monolignol moiety and the carboxylate moiety is an un-labeled carboxylate moiety;
a second type in which the monolignol moiety is an un-labeled monolignol moiety and the carboxylate moiety is a labeled carboxylate moiety; and
a third type in which the monolignol moiety is a labeled monolignol moiety and the carboxylate moiety is a labeled carboxylate moiety.

14. The method of claim 13 , wherein the isolating the labeled lignin fragments comprises isolating at least one of the first type of isolated labeled lignin fragment, the second type of isolated labeled lignin fragment, and the third type of isolated labeled lignin fragment from another of the first type of isolated labeled lignin fragment, the second type of isolated labeled lignin fragment, and the third type of isolated labeled lignin fragment.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Ralph John
• Karlen Steven D
• Lu Fachuang
• Padmakshan Dharshana

Classifications

• G01N2560/00

  Chemical aspects of mass spectrometric analysis of biological material · CPC title

• G01N2458/15

  Non-radioactive isotope labels, e.g. for detection by mass spectrometry · CPC title

• C07C69/86

  with esterified hydroxyl groups · CPC title

• C07C69/73 Primary

  of unsaturated acids · CPC title

• C07B2200/05

  Isotopically modified compounds, e.g. labelled · CPC title