Reductive-cleavage Method Research Articles

Lignin, extractives and structural carbohydrate characteristics of Thinopyrum intermedium biomass reveal additional valorization opportunities for dual-crop utilization.

Thinopyrum intermedium (Host) Barkworth & D.R. Dewey, or intermediate wheat grass (IWG), is being developed as the first widely-available perennial grain candidate. However, because the crop is still in development, grain yields are lower than those of traditional cereals. Utilization of its non-grain biomass (e.g. for biofuel production and as a source of fine chemicals) would increase the economic value of its cultivation. The present study provides a structural characterization of the lignin and cell wall carbohydrates in IWG biomass and qualitative profiling of biomass extractives and compares them to those of annual wheat (Triticum aestivum) biomass grown in the same location and growing season. The monosaccharide composition and ester-linked phenolic acid contents of vegetative biomass material from annual wheat and IWG were similar. IWG vegetative biomass is rich in feruloylated arabinoxylans (AX) with a very low substitution rate, whereas the AX from IWG bran have a slightly higher substitution rate. The structure of IWG lignin was investigated using both the quantitative derivatization followed by reductive cleavage method and 2D-NMR analysis, revealing an H:G:S lignin that incorporates tricin and is acylated with coumaric acid and smaller amounts of ferulates. IWG and wheat extractives contained fatty acids, various free phenolic compounds (tricin, monolignols and phenolic acids), phenolic conjugates and phytosterols. The present study provides firm support for the further exploration of T. intermedium biomass as a carbohydrate feedstock (e.g, abundant in lightly substituted AX and cellulose polymers) for biofuel production and source of high-value fine chemicals, such as tricin. © 2024 Society of Chemical Industry.

Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.

Reductive Cleavage Method for Quantitation of Monolignols and Low‐Abundance Monolignol Conjugates

AbstractInvited for this month's cover is the group of Prof. John Ralph at the University of Wisconsin–Madison. The image shows the fragmentation of various lignins into monomeric components using the derivatization followed by reductive cleavage (DFRC) method, with the included mechanism providing the details for the derivatization of these lignins with acetyl bromide and their subsequent reductive cleavage using elemental zinc. The Communication itself is available at 10.1002/cssc.201800617.

Reductive Cleavage Method for Quantitation of Monolignols and Low-Abundance Monolignol Conjugates.

As interest in biomass utilization has grown, the manipulation of lignin biosynthesis has received significant attention, such that recent work has demanded more robust lignin analytical methods. As the derivatization followed by reductive cleavage (DFRC) method is particularly effective for structurally characterizing natively acylated lignins, we used an array of synthetic β‐ether γ‐acylated model compounds to determine theoretical yields for all monolignol conjugates currently known to exist in lignin, and we synthesized a new set of deuterated analogs as internal standards for quantification using GC–MS/MS. Yields of the saturated ester conjugates ranged from 40 to 90 %, and NMR analysis revealed the presence of residual unsaturated conjugates in yields of 20 to 35 %. In contrast to traditional selected‐ion‐monitoring, we demonstrated the superior sensitivity and accuracy of multiple‐reaction‐monitoring detection methods, and further highlighted the inadequacy of traditional standards relative to isotopically labeled analogs.

Maturation-related changes of carrot lignins.

Lignified cell walls are important factors for textural and physiological properties of plant-based foods. However, carrot lignins and their modifications during maturation are poorly described. The objective of this study was to describe carrot lignins in detail and to study lignin structural alterations at later stages of maturity. Klason and acetyl bromide soluble lignin contents of insoluble fibers of carrots harvested at different times (26, 29 and 35 weeks after seeding) ranged between 46.38 and 62.68 g kg-1 and between 19.79 and 28.08 g kg-1 , respectively. As determined by both 2D-nuclear magnetic resonance and the derivatization followed by reductive cleavage method, coniferyl alcohol heavily dominated the traditional monolignol composition in carrot lignins, independently of harvest times. By using 2D-nuclear magnetic resonance experiments on isolated lignins, p-hydroxybenzoate was identified as a less common lignin constituent, attached to lignin γ-hydroxyl groups and being increasingly incorporated with maturation. β-Aryl ethers, phenylcoumaran, resinol and dibenzodioxocin structures were identified as lignin interunit linkages, largely independent of harvest times and with β-aryl ethers being expectedly dominant. Carrots contain guaiacyl-rich lignins that incorporate increasing amounts of p-hydroxybenzoate with maturation. All other lignin characteristics appear to be widely independent of harvest times. © 2017 Society of Chemical Industry.

Putative Cationic Cell-Wall-Bound Peroxidase Homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, Are Involved in Lignification

The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls.

Elucidation of structural condensation in lignin of eastern cottonwood

The chemical structure and condensation of eastern cottonwood (Populus deltoides) enzymatic lignin were investigated by wet-chemical and spectroscopic techniques. A cocktail of carbohydrate digesting enzymes were used to hydrolyze the polysaccharides of P. deltoides wood. The chemical structure of biochemical purified lignin was elucidated by 2D 1H–13C HSQC correlation NMR and quantitative 13C NMR analysis. The monomeric compositions of enzymatic lignin were analyzed by derivatization followed by a reductive cleavage method to allow a quantitative determination of different monomeric products in lignin. The results indicated that enzymatic lignin had more uncondensed structures compared to milled wood lignin which could be a consequence of lower oxidation during the extraction process. The contribution of H:G:S units in P. deltoides were in the order of 5:60:35, respectively. Also, the amount of β-O-4/α-OH structures as the prominent moieties of lignin in enzymatic lignin was determined as 0.43/C9.

ChemInform Abstract: Characterization of Complex Carbohydrates by the Reductive-Cleavage Method

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins

Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its beta-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure.

Evaluation of complex forming ability of hydroxypropyl-β-cyclodextrins

The complex forming ability of hydroxypropyl-β-cyclodextrins (HP-β-CDs) is highly influenced by the distribution of substituents and the average degree of substitution (DS), both the size of the cavity and the reactivity of CDs are altered when the hydroxyl groups are substituted. On the other hand, the guests themselves influence these interactions by their sizes and configurations. In the present study, 9 HP-β-CDs with different substitution patterns and DS, which have been investigated by the reductive-cleavage method and methylation analysis, were chosen. The interactions among HP-β-CDs and phenolphthalein (as a model for ‘larger spheriform’ guests) or p-methyl red (as a model for ‘smaller linear’ guests) were studied for determining the complex forming ability of HP-β-CDs. The results indicated that, compared with parent β-CD, HP-β-CDs have a lower ability to form inclusion complexes with the ‘larger spheriform’ guest molecules. With regard to the ‘smaller linear’ guest molecules, HP-β-CDs have a higher complex forming ability, especially the low DS value (<6.5) HP-β-CDs which have a ratio of DS (2 + 3) to DS (6) close to 1.

Aerobic biodegradability of hydroxypropyl-β-cyclodextrins in soil

Hydroxylpropyl-β-cyclodextrins (HP-β-CDs), hydroxyalkyl derivatives of β-CD, used in a broad range of applications in food, pharmaceutical, agriculture and bioremediation of soil because of their specific chemical properties. The possibility of varying the biodegradation rate of HP-β-CDs by changing the DS and substitution pattern makes HP-β-CDs suitable for various applications. Therefore, their biodegradation fate has been of great concern. In this study, the biodegradation of various HP-β -CDs, which have different degrees and patterns of substitution in different soil ecosystems, was investigated. The degree and pattern of substitution of HP-β-CDs were determined by the reductive-cleavage method and methylation analysis. Two common soils and a contaminated soil were used in the biodegradation test. All CDs were found to be more or less biodegradable. Increasing the degree of substitution (DS) had negative effect on the biodegradation rate of HP-β-CDs. The substitution pattern affected the biodegradation, too. The biodegradation rates of CDs in the contaminated soil were higher than that obtained in the uncontaminated soils. The contamination removing ability of CDs was highly affected by their own biodegradation fate in soil.

Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan

A fructan named CoPS3 was isolated from Cyathula officinalis Kuan. The structure of CoPS3 was determined by methylation, by the reductive–cleavage method combined with GC–MS analysis, and both 1D and 2D 1H and 13C NMR spectroscopy. These results show that CoPS3 is a graminans-type fructan that is comprised of a β- d-fructofuranosyl backbone having residues linked (2→1)- and (2→6) with branches and an α- d-glucopyranose residue on the nonreducing end of the fructan chain. Each branch is terminated by a β- d-Fru f residue. Bioassay showed that it could inhibit growth of Lewis pulmonary carcinoma implanted in mice.

Location of O-acetyl groups in S-657 using the reductive-cleavage method

A two-step procedure is described in this paper to identify the position of O-acetyl groups in S-657 polysaccharide. Reductive-cleavage experiments performed on the fully methylated (base-catalyzed) polysaccharide, followed by acetylation of anhydroalditols, identified individual sugar residues and their position of linkage. In a second experiment, the polysaccharide was methylated under neutral conditions leaving native acetate groups intact. Reductive cleavage of the neutral methylated polysaccharide using CF 3SO 3SiMe 3 as a catalyst, followed by acetylation in situ, identified sugar residues containing native acetate groups and established their position of substitution. Using this two-step procedure of analysis, S-657 polysaccharide is shown to contain O-acetyl groups on the 2-position and the 2,6-positions of 3-linked glucopyranosyl residues.

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-β- d-glucopyranoside

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-β- d-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-β- d-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri- O-methyl derivative. Also reported for the acetates and the tri- O-methyl derivative are their linear temperature-programmed gas–liquid chromatography (GLC) retention indices on three different capillary columns.

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydroribitol

The eight positional isomers of methylated and benzoylated 1,5-anhydroribitol were generated simultaneously from 1,5-anhydroribitol by sequential partial methylation and benzoylation, and the four meso isomers and two enantiomeric pairs of isomers so-formed were isolated in chemically pure form by high-performance liquid chromatography. The corresponding acetates were obtained by debenzoylation of the pure isomers and acetylation. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri- O-methyl derivative. Also reported for the acetates and the tri- O-methyl derivative are their linear temperature-programmed gas–liquid chromatography retention indices on three different capillary columns.

Authentic standards for the reductive-cleavage method: the positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro- l-arabinitol

Described herein is the synthesis of the eight positional isomers of methylated and acetylated or benzoylated 1,5-anhydro- l-arabinitol. The compounds were generated simultaneously from 1,5-anhydro- l-arabinitol by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography. The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri- O-methyl derivative. Also reported for the acetates and the tri- O-methyl derivative are their linear temperature-programmed gas–liquid chromatography retention indices on three different capillary columns.

Analysis of the positions of substitution of acetate and butyrate groups in cellulose acetate–butyrate by the reductive-cleavage method

The degree of substitution (ds) and the distribution pattern of the two ester substituents in commercial samples of cellulose acetate–butyrate (CAB) were determined by sequential neutral methylation, direct reductive cleavage and in situ acetylation. When the reductive-cleavage reaction was conducted with 35 equiv (per anhydroglucose unit) of Et 3SiH, 70 equiv of MeSO 3SiMe 3, and 14 equiv of BF 3·OEt 2 at room temperature for seven days, the O-acetyl groups were converted to O-ethyl groups and the O-butyryl groups were converted to O-butyl groups concurrent with reductive cleavage of the glycosidic linkages. Acetylation of the products gave 27 partially methylated, ethylated, and butylated 4- O-acetyl-1,5-anhydro- d-glucitol derivatives that were identified by GLC–CIMS (NH 3) and GLC–EIMS. Integration of the GLC profile and correction for molar response gave the mole percent of each product. From these data, the fractional degree of substitution for each ester at each position of the anhydroglucose unit was determined. The combined fractional degree of substitution of both esters at each position and the overall ds were also determined by sequential neutral methylation, acyl–ethyl exchange, and reductive cleavage, and the values so obtained were in good agreement with those derived by sequential neutral methylation and direct reductive cleavage.

Analysis of the positions of substitution of acetate and propionate groups in cellulose acetate–propionate by the reductive-cleavage method

The degree of substitution (ds) and the distribution pattern of the two ester substituents in commercial samples of cellulose acetate–propionate (CAP) were determined by sequential neutral methylation, direct reductive cleavage, and in situ acetylation. When the reductive-cleavage reaction was conducted with 35 equiv (per anhydroglucose unit) of Et 3SiH, 70 equiv of MeSO 3SiMe 3, and 14 equiv of BF 3·OEt 2 at room temperature for seven days, the O-acetyl groups were converted to O-ethyl groups, and the O-propionyl groups were converted to O-propyl groups concurrent with reductive cleavage of the glycosidic linkages. Acetylation of the products gave 27 partially methylated, ethylated, and propylated 4- O-acetyl-1,5-anhydro- d-glucitol derivatives that were identified by GLC–CIMS (NH 3) and GLC–EIMS. Integration of the GLC profile and correction for molar response gave the mole percent of each product. From these data, the fractional degree of substitution for each ester at each position of the anhydroglucose unit was determined. The combined fractional degree of substitution of both esters at each position and the overall ds were also determined by sequential neutral methylation, acyl–ethyl exchange, and reductive cleavage, and the values so obtained were in good agreement with those derived by sequential neutral methylation and direct reductive cleavage.

Analysis of the linkage positions in polydextrose by the reductive cleavage method

The structure of the synthetic carbohydrate polymer Polydextrose ® was investigated by a reliable and rapid method of methylation, combined with reductive cleavage and acetylation. Monomeric derivatives were identified by gas-liquid chromatography (GLC) retention data and electron impact (EI)- and NH 3 chemical ionization (CI)-mass spectrometry (MS) data. Ratios of permethylated, once or twice acetylated derivatives confirmed that Polydextrose ® is a highly branched material.

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro- l-rhamnitol

Described herein is an efficient method for the synthesis of the eight positional isomers of methylated and acetylated or benzoylated 1,5-anhydro- l-rhamnitol. The compounds are generated simultaneously from 1,5-anhydro- l-rhamnitol by sequential partial methylation and benzoylation. The individual isomers are obtained in pure form by high-performance liquid chromatography. Debenzoylation and acetylation yielded the desired acetates. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri- O-methyl derivative. Also reported for the acetates and the tri- O-methyl derivative are their linear temperature programmed gas-liquid chromatography retention indices on three different capillary columns. The title compounds were obtained in pure form from 1,5-anhydro- l-rhamnitol by sequential methylation, benzoylation, and fractionation by HPLC. Debenzoylation and acetylation yielded the corresponding acetates.