2D NMR Spectroscopy Research Articles

“Novel chemo-enzymatic synthesis, structural elucidation and first antiprotozoal activity profiling of the atropoisomeric dimers of trans-8-Hydroxycalamenene”

Leishmaniasis and malaria are two debilitating protozoan diseases affecting millions globally, particularly in tropical and subtropical regions. Current therapeutic options face significant challenges due to emerging drug-resistant strains, necessitating the discovery of novel antiprotozoal agents. This study explores, for the first time, the antiprotozoal potential of calamenenes and their dimers, naturally occurring sesquiterpenes found in essential oils, through a novel chemo-enzymatic synthesis approach. Using the laccase from Trametes versicolor, atropoisomeric dimers of (−)- and (+)-8-trans-hydroxycalamenene were synthesized from commercially available (−)- and (+)-menthol. Structural elucidation was achieved via 2D-NMR spectroscopy, electronic circular dichroism, and DFT calculations. In vitro profiling against Leishmania spp and drug-resistant Plasmodium falciparum revealed that calamenene dimers exhibited significantly higher antiprotozoal activity compared to their monomeric counterparts, highlighting the potential of dimeric terpenoids as promising antiprotozoal agents. This work lays the foundation for developing novel antimalarial drugs based on calamenene scaffolds, encouraging further interactome studies to optimize their pharmacological properties.

Comparative metabolic profiling and quantitative analysis of metabolites in different tissues of Ajuga turkestanica by ESI-UHPLC-QqTOF-MS and NMR

Ajuga turkestanica preparations are used as anti-aging cosmeceuticals and for medicinal purposes. Herein we describe the characterization and quantification of its metabolites in different organs using UHPLC-MS and NMR spectroscopy. A total of 51 compounds belonging to various phytochemical classes (11 flavonoids, 10 ecdysteroids, 9 diterpenes, 6 fatty acids, 5 iridoids, 3 phenylpropanoids, 3 sugars, 2 phenolics, 1 coumarin, 1 triterpene) were annotated and tentatively identified by UHPLC-ESI-QqTOF-MS/MS of methanolic extracts obtained separately from the organs. 1D and 2D NMR spectroscopy independently confirmed the identity of six major compounds. The abundances of these main constituents in flowers, fruits, leaves, roots, seeds, and stems were compared and quantified using 1H NMR. The results showed that 8-O-acetylharpagide, 20-hydroxyecdysone (ecdysterone) and ajugachin B were the most abundant constituents in the species. The two major compounds, 8-O-acetylharpagide and 20-hydroxyecdysone, were chosen as the markers for the quality assessment of A. turkestanica material. The methanolic extract of the aerial parts of A. turkestanica showed no noteworthy anthelmintic (antihelmintic), antifungal, or cytotoxic effect in in vitro assays.

Synthesis of Betulonic and Betulinic Acids Derivatives with Sulfonamide Fragment Linked to the Triterpene Core by Amidoalkane Spaser

A series of potentially biologically active sulfonamides of betulonic and betulinic acids with a sulfonamide fragment containing residues of dibutylamine or N-heterocycles, which is linked to a triterpene core by an amidoethane or amidopropane spacer, was synthesized. Betulonic acid sulfonamides were obtained by chlorohydride conjugation of acid with 2-aminoethane and 3-aminopropanesulfonamides and used as precursor compounds for the transition to similar betulinic acid derivatives by selective reduction of the 3-keto group with the action of NaBH4 in EtOH. The structure of all synthesized triterpene sulfonamides was confirmed by 1D and 2D NMR spectroscopy and mass spectrometry.

First oxidative coupling of cyclazine heterocycle via regioselective dimerization of 1,2-dicarbomethoxy-3-phenylcycl[3.2.2]azine: Synthesis, theoretical aspects and physicochemical studies

The first covalently linked dimer has been prepared for cyclazine systems by regioselective oxidative homocoupling of 1,2-dicarbomethoxy-3-phenylcycl[3.2.2]azine. The regioselectivity of this reaction at 4-position, having been confirmed by X-ray diffraction analysis and NMR spectroscopy, has been also reliably predicted within the model of average local ionization energy on the molecular surface of the starting monomer at the BP86/def2-TZVP level of theory. Due to the planar π-π interaction of the cycl[3.2.2]azine subunits, the dimer is a green fluorophore (λem = 527 nm, toluene), characterized by a bathochromic shift of the main bands in the UV–vis and fluorescence spectra relative to the monomer by 41 and 71 nm, respectively. Furthermore, the dimer demonstrates an increased fluorescence quantum yield relative to the monomer (55 % vs. 35 % in toluene), and according to the data of X-ray diffraction analysis, DFT calculations and variable temperature 1D and 2D 1H NMR spectroscopy, is characterized by hindered rotation of the S1-state-involved cycl[3.2.2]azine cores along the C4–C4′ bond axis. Such prerequisites determine good application potential of the 4-4′ coupled cycl[3.2.2]azine derivatives as turn-on fluorescent, i.e. fluorogenic probes for advanced bioimaging in living systems. Finally, unlike the monomer, the dimer shows reversibility of both one- and two-electron reduction and oxidation processes, and therefore can become the basis of both n- and p-type semiconductors.

Synthesis of Sulfonamide-Based Schiff Bases as Potent Anticancer Agents: Spectral Analyses, Biological Activity, Molecular Docking, ADME, DFT, and Pharmacophore Modelling Studies.

The current study focuses on the synthesis and characterization of six benzenesulfonamide-based Schiff base derivatives (7-12) with various electron-withdrawing and electron-donating substituents (-F, -CI, -Br, -CH3, and -OCH3) and the assessment of their antiproliferative activities against human lung (A549) and liver (HepG2) cancer cell lines using in vitro and in silico approaches. The structures of the synthesized compounds (7-12) were elucidated by elemental analysis and FT-IR, 1D (1H, 13C, APT, and DEPT-135), and 2D (HMQC and HMBC) NMR spectroscopies. The cytotoxic activities of the targeted compounds were determined at various concentrations against these cancer cell lines for 72 h, using the MTT method. The targeted molecules (7-12) demonstrated remarkable antiproliferative activities, with IC50 values ranging from 6.032-9.533 μM against the A549 cell line and 5.244-9.629 μM against the HepG2 cell line. These compounds showed activities at lower or very similar concentrations to cisplatin against the A549 cell line and at much lower concentrations than cisplatin against the HepG2 cell line. Among them, compounds 10 and 12 were found to be more effective against A549 and HepG2 cells, respectively, than cisplatin. These compounds were analyzed by interacting with the 1BNA, 4HJO, and 4ASD crystal structures in molecular docking studies. The docking score of 4ASD-compound 12 interaction was calculated as -4.045 kcal/mol, 4HJO-compound 10 interaction was calculated as -5.179 kcal/mol and 1BNA-compound 10 interaction was calculated as -8.571 kcal/mol and it was determined that these compounds were theoretically better than Cisplatin. In the present study, ADME data were estimated using the web tool SwissADME. With ADME, it was calculated that the logP value of compounds 7-12 was less than 5, the HBD number was 1, the HBA number was 7 or 8, and the molecular weight was less than 500. Properties such as the electrophilic index and chemical hardness of the designed compounds were examined by density functional theory (DFT) using B3LYP/6-311G**. In conclusion, these compounds have emerged as promising new anti-cancer drug candidates.

Structure Characterization of Four New Sesquiterpene Pyridine Alkaloids from Tripterygium wilfordii Hook. f. and Anti-Inflammatory Activity Evaluations.

Sesquiterpene pyridine alkaloids (SPAs), as a main class of components in Tripterygium wilfordii Hook. f., possess a variety of bioactivities, such as immunosuppressive, insecticidal, and anti-tumor activities. SPAs can be structurally classed into four subtypes: wilfordate-, evoninate-, iso-wilfordate-, and iso-evoninate types. Our previous study unveiled ten new wilfordate-type SPAs, named wilfordatine A-J, isolated from the roots of Tripterygium wilfordii Hook. f., several of which exhibited significant immunosuppressive activities. As an extension and augmentation of the previous findings, we have now isolated one new iso-wilfordate-type SPA, wilfordatine K (1), alongside three new iso-evoninate-type SPAs, wilfordatines L-N (3-5), and six known analogs. Their structures were characterized by the extensive use of 1D and 2D NMR spectroscopic analysis, as well as HRMS data. Interestingly, compounds 4 and 6 were found to exhibit potent inhibitory effects on the nuclear factor-kappa B (NF-κB) pathway in lipopolysaccharide (LPS)-induced HEK293/NF-κB-Luc cells, with IC50 values of 1.64 μM and 9.05 μM, respectively. Notably, these two compounds had no influence on the cell viability at a concentration of 100 μM. Consequently, they hold significant promise as potential anti-inflammatory candidates for further exploration and development.

A new dammarane-type triterpenoid saponin from the leaves of sour jujube

A new dammarane-type triterpenoid saponin, suanzaoside A (1) along with one known analogue 3-O-[α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-]-30-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-3β,25,30-trihydroxy-16-one-20R,24R-epoxydammarane (2), were isolated from the leaves of sour jujube. The structure of these isolated compounds was elucidated by HRESIMS, 1D and 2D NMR spectroscopy, and literature comparison. In addition, the anti-inflammatory activities were further tested in LPS-induced RAW264.7 macrophage.

P-Terphenyl and Orsellinic Acid Derivatives from the European Polyporales Terana coerulea and Sparassis brevipes.

Investigation of the secondary metabolites of the solid-state rice cultures from the European basidiomycetes Terana coerulea and Sparassis brevipes afforded three previously undescribed secondary metabolites identified as two p-terphenyl derivatives (1 and 2) and one orsellinic acid congener (3) in addition to another known, isoeverninic acid (4). Chemical structures of the isolated compounds were elucidated through comprehensive 1D and 2D NMR spectroscopic analyses, coupled with HR-MS and other spectral methods. The isolated compounds were assessed for their cytotoxic and antimicrobial activities. Compound 1 revealed weak antimicrobial properties, whereas the inseparable mixture of 3 and 4 featured moderate cytotoxic and antimicrobial effects.

Pyrrole-hybridized spiro[chromeno[4,3-b]cycloalka[4,5]thieno[3,2-e]pyridine]cycloalkanes as modified tacrine system: Synthesis, structure, and photophysical evaluation

This paper reports the synthesis, structural elucidation, and investigation of the photophysical properties of a novel heterocyclic system, such as pyrrole-hybridized polyhydro-spiro[chromeno[4,3-b]cycloalka[4,5]thieno[3,2-e]pyridine]cycloalkanes (3), where the spiro-cycloalkanes are cyclopentane, cyclohexane, and cycloheptane; and the cycloalka[4,5]thieno are cyclopenta-, cyclohexa-, and cyclohepta-. These hybrid scaffolds 3 resulted from N-derivatization reactions of a series of twelve examples of modified 7-amino-substituted precursors 1, which, via the Clauson-Kaas reaction using 2,5-dimethoxy-tetrahydrofuran (2,5-DMOTHF) as a CCCC block, were applied to enable the construction and isolation of twelve mentioned pyrrole derivatives 3 in yields of 66 – 95%. Compounds 3 were structurally fully characterized by melting point, 1H-, 13C-NMR, and 1H-13C 2D-NMR (HSQC and HMBC) spectroscopy, X-ray diffraction, and HRMS analysis. The novel pyrroles 3 had their photophysical properties investigated (absorption and emission) in solution in several solvents. As a result, it was found that these pyrroles, where the transition bands were observed in the UV region, exhibit moderate luminescent and shorter lifetime decay values, which are related to and dependent on the spiro moiety and/or cycloalkanes directly fused to the thiophene ring of this new tacrine-modified system.

Study the effect of Convolvulus pilosellifolius Desr. as antialzheimer and identification of its active compounds

The phenolic pattern of the plant using chromatographic and spectroscopic methods. Three flavonoid and two phenolic compounds were isolated for first time from the alcoholic plant extract which identified as; Quercetin-3’-galactoside, Quercetin-7-glucoside, Quercetin −3-glucoside and two phenolic compounds identified as Methyl-trans-dihydroxy cinnamic acid and Methyl-cis-dihydroxy cinnamic acid, they were isolated and purified using classical and modern chromatographic methods; (PC, TLC, CC and HPLC); identified by Rf, UV,1H NMR,13C NMR and 2D NMR (HMQC) spectroscopy. The activity of total alcoholic extract of Convolvulus pilosellifolius as antialzheimer was investigated concerning its activity as acetylcholine esterase inhibitor. The recorded IC50 was 0.391 and 0.131 µg/mL for the extract and donepezil respectively. The activity of isolated compounds as acetylcholine esterase inhibitor were evaluated by Autodock Vina software to perform the docking protocol. This revealed that the most active compound was Quercetin-3Ì· galactoside.

Synthesis and Docking Studies of Novel Spiro[5,8-methanoquinazoline-2,3'-indoline]-2',4-dione Derivatives.

In this study, a set of spiro[5,8-methanoquinazoline-2,3'-indoline]-2',4-dione derivatives 3a-p were synthesized starting from unsubstituted and N-methyl-substituted diendo- and diexo-2-aminonorbornene carboxamides, as well as various substituted isatins. The typical method involves a condensation reaction of alicyclic aminocarboxamide and isatin in the presence of a catalyst, using a solvent and an acceptable temperature. We developed a cost-effective and ecologically benign high-speed ball milling (HSBM), microwave irradiation (MW), and continuous flow (CF) technique to synthesize spiroquinazolinone molecule 3a. The structures of the synthesized compounds 3a-p were determined using 1D and 2D NMR spectroscopies. Furthermore, docking studies and absorption, distribution, metabolism, and toxicity (ADMET) predictions were used in this work. In agreement with the corresponding features found in the case of both the SARS-CoV-2 main protease (RCSB Protein Data Bank: 6LU7) and human mast cell tryptase (RCSB Protein Data Bank: 2ZA5) based on the estimated total energy and binding affinity, H bonds, and hydrophobicity in silico, compound 3d among our 3a-g, 3i-k, and 3m derivatives was found to be our top-rated compound.

Bioactive alkaloids from Nepalese Corydalis chaerophylla D.C. acting on the regulation of PCSK9 and LDL-R in vitro.

Four new alkaloids Chaeronepaline-A (1), Chaeronepaline-B (2), Chaeronepaline-C (3), and Chaeronepaline-D (4) were isolated from Corydalis chaerophylla D.C. collected from Nepal and their structures were elucidated by spectroscopic data, 1D, 2D NMR and mass spectrometry. The structures were established as 3,12- Dimethoxy-5,6-dihydroisoquinolino [2,1-b] isoquinolin- 7- ium- 2, 9- diol (1), 7-methyl-5, 6, 7, 8-tetrahydroisoquinoline- 2, 3- methylenedioxy- (8-> 9)- 10, 12- methylenedioxy- benzoic-16-acid (2), 7- methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline (3) and 7- methyl-5, 6, 7, 8- tetrahydro- 8H-spiro-9,14-dihydroxy-11,12-methylenedioxy-indane-isoquinoline-N-oxide (4). The new alkaloids were tested in human hepatoma cell line to assess their ability to modulate the expression of low-density lipoprotein receptor (LDL-R), of proprotein convertase subtilisin/kexin 9 (PCSK9) and to affect cellular cholesterol biosynthesis with the aim to evaluate their potential hypocholesterolemic effect. Results indicated that compounds 2 and 3 upregulate the LDLR, and inhibited the cholesterol biosynthesis with compound 2, which also reduced the secretion of PCSK9 by Huh7 cells. These in vitro data indicated a potential hypocholesterolemic effect of compound 2 that requires further in vivo validation.

Revisiting the Cyclocephagenols via Astragalus condensatus: Structural Insights and Configurational Revision.

The phytochemical investigation of the MeOH extract of Astragalus condensatus roots led to the discovery of a new tetrahydropyran cycloartane-type triterpenoid, astracondensatol A (1), alongside six known cyclocephagenol derivatives (2, 3, 20, 32, 35, and 36). Elucidation of their structures involved 1D and 2D-NMR spectroscopy and mass data analysis. Upon comparing NMR spectroscopic data with prior literature, several carbon shift anomalies, particularly at C-24, prompted a reevaluation using quantum chemical calculations, resulting in the revision of the 24S to 24R absolute configuration for compound 2 and 38 other reported cyclocephagenol-type triterpenoids. X-ray crystallography data further supported the analysis in establishing the absolute configuration of compound 2. Ambiguous NOE correlations and publication bias could have played a significant role in miss-assigning the C-24 absolute configuration in tetrahydropyran cycloartane-type triterpenoids.

Mapping Natural Sugars Metabolism in Acute Myeloid Leukaemia Using 2D Nuclear Magnetic Resonance Spectroscopy.

Metabolism plays a central role in cancer progression. Rewiring glucose metabolism is essential for fulfilling the high energy and biosynthetic demands as well as for the development of drug resistance. Nevertheless, the role of other diet-abundant natural sugars is not fully understood. In this study, we performed a comprehensive 2D NMR spectroscopy tracer-based assay with a panel of 13C-labelled sugars (glucose, fructose, galactose, mannose and xylose). We assigned over 100 NMR signals from metabolites derived from each sugar and mapped them to metabolic pathways, uncovering two novel findings. First, we demonstrated that mannose has a semi-identical metabolic profile to that of glucose with similar label incorporation patterns. Second, next to the known role of fructose in driving one-carbon metabolism, we explained the equally important contribution of galactose to this pathway. Interestingly, we demonstrated that cells growing with either fructose or galactose became less sensitive to certain one-carbon metabolism inhibitors such as 5-Flurouracil and SHIN1. In summary, this study presents the differential metabolism of natural sugars, demonstrating that mannose has a comparable profile to that of glucose. Conversely, galactose and fructose contribute to a greater extent to one-carbon metabolism, which makes them important modulators for inhibitors targeting this pathway. To our knowledge, this is the first NMR study to comprehensively investigate the metabolism of key natural sugars in AML and cancer.

Water-Soluble Curcumin Derivatives Including Aza-Crown Ether Macrocycles as Enhancers of their Cytotoxic Activity.

The synthesis of three novel curcumin derivative compounds, featuring aza-crown ether macrocycles of various sizes (aza-12-crown-4, aza-15-crown-5, and aza-18-crown-6), is described. The incorporation of these aza-crown macrocycles significantly enhances their water solubility, positioning them as groundbreaking instances of curcumin derivatives that are fully soluble in aqueous environments. These curcumin ligands (L1, L2, and L3) were then reacted with zinc acetate to afford the coordination metal complexes (L1-Zn, L2-Zn, and L3-Zn). Comprehensive characterization of all compounds was achieved using various analytical techniques, including 1D and 2D NMR spectroscopy, ATR-FTIR spectroscopy, mass spectrometry (ESI+), elemental analysis and UV-Vis spectroscopy. The in vitro cytotoxic activity of both, ligands and complexes were evaluated on three human cancer cell lines (U-251, MCF-7, and SK-LU-1). Compared to conventional curcumin, these compounds demonstrated improved antiproliferative potential. Additionally, a wound healing assay was conducted to assess their antimigration properties. The obtained results suggest that these modifications to the curcumin structure represent a promising approach for developing therapeutic agents with enhanced cytotoxic properties.

Melt Polycondensation Strategy to Access Unexplored l-Amino Acid and Sugar Copolymers.

Biodegradable polymers from bioresources are highly in demand for the development of sustainable polymer platforms for commodity plastics and in the biomedical field. Here, an elegant one-pot synthetic strategy is developed, for the first time, to access unexplored hybrid polymers from two naturally abundant resources: carbohydrates (sugars) and l-amino acids. A bottleneck in the synthetic strategy is overcome by tailor-making d-mannitol-based six- and five-membered bicyclic acetalized diols, and their structures are confirmed by single-crystal X-ray diffraction and 2D NMR spectroscopy. l-Amino acids are converted into ester-urethane functional monomers, and they are polymerized with sugar-diols under solvent-free melt polycondensation to yield biodegradable poly(ester-urethane)s. Acid-catalyzed deprotection yielded amphiphilic polymers having exclusively alternating residues of sugar and l-amino acid in the polymer backbone. The polymer is self-assembled into 200 ± 10 nm sized nanoparticles that can encapsulate fluorescent dyes, are nontoxic to cells up to 250 μg/mL, and are readily endocytosed for lysosomal enzymatic biodegradation at the cellular level.

Chemical structures of polyketides and alkaloids isolated from a culture of fungus Curvularia moringae

Seven new polyketides [moringols I–VII (1–7)], a new alkaloid [moringamine I (8)], and seven known compounds (9–15) were isolated from the fungus Curvularia moringae JKYM-KR4. The planar chemical structures and relative configurations of the new compounds were elucidated by high-resolution mass spectrometry, 1D and 2D NMR spectroscopy, and DP4+ analysis using the calculated 13C NMR chemical shifts. For moringols I and II (1 and 2), the planar chemical structures and relative configurations were confirmed using X-ray crystallography. The absolute configurations of 1–6 and 8 were determined by ECD calculations. Among the isolated compounds, terpestacin (14) moderately inhibited the proliferation of HT-29 cells.

Modular Synthesis of Monoanionic PN Ligands Leads to Unexpected Structural Diversity in Lanthanum Chemistry.

We report a new synthetic entry to a series of N-substituted anilidophosphine ligands (short HPNR, R = pTol (HPNTol), 3,5-dimethylphenyl (HPN3,5Me), 3,5-bis(trifluoromethyl)phenyl (HPN3,5CF3), 2-methoxyphenyl (HPNOMe), diisopropylphenyl (HPNDipp), and adamantyl (HPNAd)), allowing a detailed tuning of their steric (and electronic) properties. HPNR could be converted into their lithium salts LiPNR, which are effective precursors for salt metathesis reactions. The new ligands are used for the synthesis of an array of lanthanide complexes using LaCl3(THF)1.2 as a precursor. Depending on the steric bulk of the anilidophosphine ligand, either chloride-bridged dimers of the general formula [(PNR)2La(μ-Cl2)La(PNR)2] (R = pTol (3f), 3,5-dimethylphenyl (3d) and adamantyl (3a)) or mononuclear complexes of the general formula (PNR)2LaCl (R = diisopropylphenyl (3e)) are observed, if the complexation reaction is carried out in toluene. Contrary, if salt metathesis reactions are carried out in dimethoxyethane (DME) as a coordinating solvent, -ate complexes of the general formula [(PNR)2La(μ-Cl2)Li(DME)] (R = Adamantyl (4a) and R = 2-methoxyphenyl (4d)) or [Li(DME)3][(PNR)2LaCl2] (R = 3,5-bis(trifluoromethyl)phenyl (4b), R = pTol (4f) and R = 3,5-dimethylphenyl (4d)) are observed. All ligands and complexes have been thoroughly characterized by 1D and 2D NMR spectroscopy, IR, and X-ray crystallography. Finally, the steric demand of the new anilidophosphine ligands is evaluated using SambVca simulations.

Intrinsically Flame‐Retardant Polyamide 6 by Anionic Ring‐Opening Copolymerization with a Phosphorous‐Containing Comonomer

AbstractA three‐step synthetic route is established for the synthesis of a phosphorus‐containing ε‐caprolactam derivative, starting from cyclohex‐2‐enone and diphenylphosphinous chloride, followed by a Beckmann rearrangement. 2D NMR spectroscopy reveals that the phosphorus moiety is selectively localized at the 4‐position of the caprolactam ring. Different amounts of the phosphorus‐containing comonomer are systematically incorporated into polyamide 6 by anionic ring‐opening copolymerization with ε‐caprolactam at elevated temperatures. Polymerization reactions are initiated by a latent, CO2 protected N‐heterocyclic carbene in the presence of N‐acetyl caprolactam and MgCl2. The phosphorus content is determined by inductively‐coupled plasma‐optical emission spectroscopy (ICP‐OES) analysis. Limiting oxygen indices up to 23.5 are found for polymers containing ≈1 wt.‐% of phosphorus. The thermal properties of the copolymers are determined and compared to those of polyamide 6.

Structural characterization of arabinogalactan-II and pectin from Urtica cannabina

Comparative analysis of extracellular and cell wall glycans from Urtica cannabina leaves was performed using chemical methods, GC, GC–MS, 1D, and 2D NMR spectroscopy. The structures of extracellular AG-II and cell wall AG-II are similar. The units are typical for AG-IIs: β-GlcpA-4-OMe-(1→, Rhap-(1 → 4)-β-GlcpA-(1→, attached to β-Galp at O-6, as well as arabinan chains attached to β-Galp at O-3. A single Araf and a trisaccharide formed by 2,5-Araf and two terminal Araf form short arabinan side chains in AG-II. 1,5-arabinan with a backbone substituted by a single Araf at O-3 was identified only in the side chains of cell wall AG-II. The side chains can be attached to O-3 and O-6 of the same β-Galp to form a bifurcated AG side chain. The backbone of AG-II is formed by 1,6- rather than 1,3-linked Galp, although it does include some 1,3-Galp. The high content of 3,6-Galp shows the highly branched nature of the AG carbohydrate chains. From the cell wall, AGP was extracted together with pectin, the simultaneous elution of which from both DEAE-cellulose and Sepharose may indicate a link between them.