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

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.