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

Patent metadata

Field	Value
Publication number	US-10227287-B2
Application number	US-201514985720-A
Country	US
Kind code	B2
Filing date	Dec 31, 2015
Priority date	Dec 31, 2014
Publication date	Mar 12, 2019
Grant date	Mar 12, 2019

Abstract

Described is a method of detecting and/or determining levels of incorporation of monolignol ferulate conjugate esters into lignin by derivatizing the lignin, reductively cleaving the lignin, labeling the cleavage products, and then detecting cleavage products derived from monolignol ester conjugates via differential attachment of the label.

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 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), wherein the detecting the labeled lignin fragments comprises:
isolating the labeled lignin fragments, wherein each lignin fragment comprises a monolignol ester conjugate, wherein each monolignol ester conjugate comprises a monolignol moiety and a carboxylate moiety, wherein the isolated labeled lignin fragments comprise two 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; and

detecting a relative amount of 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 with respect to 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.

2. The method of claim 1 , wherein the labeled acetic anhydride is deuterium-labeled acetic anhydride.

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

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

5. The method of claim 1 , wherein the carboxylate moiety is selected from the group consisting of p-hydroxybenzoate, p-coumarate, ferulate, and sinapate.

6. The method of claim 1 , wherein the detecting the relative amount of 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 with respect to 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 comprises:
fragmenting each of the isolated labeled lignin fragments into sub-fragments, wherein each sub-fragment comprises a first sub-fragment and a second sub-fragment, wherein each first sub-fragment comprises at least a portion of the monolignol moiety and is a labeled first sub-fragment or an un-labeled first sub-fragment, wherein each second sub-fragment comprises at least a portion of the carboxylate moiety and is a labeled second sub-fragment or an un-labeled second sub-fragment; and
detecting a relative amount of the labeled first sub-fragments with respect to the un-labeled first sub-fragments, a relative amount of the labeled second sub-fragments with respect to the un-labeled second sub-fragments, or a relative amount of the labeled first sub-fragments with respect to the un-labeled first sub-fragments and a relative amount of the labeled second sub-fragments with respect to the un-labeled second sub-fragments.

Assignees

• Wisconsin Alumni Res Found

Inventors

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

Classifications

• C07C69/007

  Esters of unsaturated alcohols having the esterified hydroxy group bound to an acyclic carbon atom · CPC title

• C07C69/92

  with etherified hydroxyl groups · CPC title

• C07C69/732

  of unsaturated hydroxy carboxylic acids · CPC title

• C07C69/73 Primary

  of unsaturated acids · CPC title

• C07C69/16

  of dihydroxylic compounds · CPC title