Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Identification and characterization of emGalaseE, a ß-1,4 galactosidase from Elizabethkingia meningoseptica, and its application on living cell surface.

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37 °C) and pH (5-8) levels and can remove ß-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As this enzyme has a potential application for living cell glycan editing, we named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI). In summary, our findings lay the groundwork for further investigation by presenting a simple and effective approach for the removal of galactose moieties from cell surface.

Enzymatic biosynthesis of D-galactose derivatives: Advances and perspectives.

D-Galactose derivatives, including galactosyl-conjugates and galactose-upgrading compounds, provide various physiological benefits and find applications in industries such as food, cosmetics, feed, pharmaceuticals. Many research on galactose derivatives focuses on identification, characterization, development, and mechanistic aspects of their physiological function, providing opportunities and challenges for the development of practical approaches for synthesizing galactose derivatives. This study focuses on recent advancements in enzymatic biosynthesis of galactose derivatives. Various strategies including isomerization, epimerization, transgalactosylation, and phosphorylation-dephosphorylation were extensively discussed under the perspectives of thermodynamic feasibility, theoretical yield, cost-effectiveness, and by-product elimination. Specifically, the enzymatic phosphorylation-dephosphorylation cascade is a promising enzymatic synthesis route for galactose derivatives because it can overcome the thermodynamic equilibrium of isomerization and utilize cost-effective raw materials. The study also elucidates the existing challenges and future trends in enzymatic biosynthesis of galactose derivatives. Collectively, this review provides a real-time summary aimed at promoting the practical biosynthesis of galactose derivatives through enzymatic catalysis.

Selective Elimination of Senescent Cancer Cells by Galacto-Modified PROTACs.

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated ß-galactosidase (SA-ß-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-ß-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

A nanochitosan-D-galactose formulation increases the accumulation of primaquine in the liver.

Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.

Ultra-Galactocation to Sialic Acid on Tumor Cells with A Penta-Functional Dendritic Probe for Enhanced Immune-Killing.

Glycans on tumor cell surface have significant impacts in the immune-killing process. Here an ultra-galactocation to sialic acid (Sia) strategy is designed to hugely introduce galactose (Gal) to Sia and on tumor cells in vivo by using a penta-functional dendritic probe (Den@5F), which efficiently enhances the immune-killing of tumor cells. The Den@5F contains five different kinds of functional groups, including Gal, Cy5, amino, phenylboronic acid (PBA) and 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butanoate (mNB), which can be conveniently prepared through a two-step reaction. After injecting into the tumor-bearing mouse, Den@5F can efficiently block Sia through the specific recognition between PBA and Sia on tumor cells and hugely introduce Gal through the subsequent photo-crosslinking between mNB and amino groups to multiply conjugate excessive Den@5Fs. The comprehensively blocked Sia can prevent the immune escape, and the hugely introduced Gal can promote the immune stimulation of the immune cells, which lead to an efficient enhancement of the immune-killing. The proposed strategy provides a significant and promising tool to promote the clinical immunotherapy of tumor.

Structural characterization, chain conformation and immunomodulatory activity of a heteropolysaccharide from Inonotus hispidus.

A new polysaccharide (IHP-1aa) was isolated from the fruiting body of Inonotus hispidus by hot water extraction, ethanol precipitation and column chromatography. The molecular weight of IHP-1aa was 26.9 kDa. Structural analysis showed that IHP-1aa consisted of glucose (Glc), galactose (Gal), fucose (Fuc), mannose (Man) and contained a certain amount of 3-O-methylgalactose (3-O-Me-Gal). The structure was mainly composed of →6)-α/ß-D-Glcp-(1→, →6)-α-D-Galp-(1→, →6)-(3-O-Me)-α-D-Galp-(1→, →6)-α-D-Manp-(1 â†’ and →2, 6)-α-D-Galp-(1 â†’ as the main chain. Branched at O-2 with single ß-L-Fucp-(1 â†’ 6)-α-D-Galp-(1 â†’ 6)-α-D-Glcp-(1 â†’ as major the side chain. The results of SEM, XRD and AFM combined with Congo red indicated that IHP-1aa may be amorphous granular chain conformation. In addition, IHP-1aa stimulated macrophage function and improved phagocytic ability of RAW264.7, as well as promoted the secretion of NO, TNF-α and IL-6. IHP-1aa, a 3-O-methylgalactose-containing heteropolysaccharide, was isolated for the first time from the I. hispidus, which may be used as a potential immunomodulator in functional foods.

Exploring an l-arabinose isomerase from cryophile bacteria Arthrobacter psychrolactophilus B7 for d-tagatose production.

A novel l-arabinose isomerase (L-AI) from Arthrobacter psychrolactophilus (Ap L-AI) was successfully cloned and characterized. The enzyme catalyzes the isomerization of d-galactose into a rare sugar d-tagatose. The recombinant Ap L-AI had an approximate molecular weight of about 258 kDa, suggesting it was an aggregate of five 58 kDa monomers and became the first record as a homo-pentamer L-AI. The catalytic efficiency (kcat/Km) and Km for d-galactose were 0.32 mM-1 min-1 and 51.43 mM, respectively, while for l-arabinose, were 0.64 mM-1 min-1 and 23.41 mM, respectively. It had the highest activity at pH 7.0-7.5 and 60 °C in the presence of 0.250 mM Mn2+. Ap L-AI was discovered to be an outstanding thermostable enzyme that only lost its half-life value at 60 °C for >1000 min. These findings suggest that l-arabinose isomerase from Arthrobacter psychrolactophilus is a promising candidate for d-tagatose mass-production due to its industrially competitive temperature.

Invited review: Properties of ß-galactosidases derived from Lactobacillaceae species and their capacity for galacto-oligosaccharide production.

ß-galactosidase (enzymatic class 3.2.1.23) is one of the dairy industry's most important and widely used enzymes. The enzyme is part of a large family known to catalyze hydrolysis and transglycosylation reactions. Its hydrolytic activity is commonly used to decrease lactose content in dairy products, while its transglycosylase activity has recently been used to synthesize galacto-oligosaccharides (GOS). During the past couple of years, researchers have focused on studying ß-galactosidase isolated and purified from lactic acid bacteria. This review will focus on ß-galactosidase purified and characterized from what used to be the Lactobacillus genera. Furthermore, particular emphasis is given to its kinetics, biochemical characteristics, GOS production, market, and utilization by Lactobacilllaceae species.

Unveiling the Structural Characteristics and Bioactivities of the Polysaccharides Extracted from Endophytic Penicillium sp.

Polysaccharides are abundantly present in fungi and are gaining recognition for their exceptional bioactivities. Hence, the present study aimed to extract intracellular polysaccharides (IPS-1 and IPS-2) from the endophytic Penicillium radiatolobatum and compare their physicochemical and bioactive attributes. The monosaccharide composition analysis revealed the existence of galactose, glucose, and mannose in both the IPS, while a trace amount of xylose was found in IPS-1. Further, FT-IR, 1H NMR, and 13C NMR analysis suggested that the IPS-2 was mainly composed of the ß-(1→4)-D-Galactose and ß-(1→4)-D-Glucose as the main chain, with the ß-(1→6)-D-mannose as branched chains. Compared to IPS-1, the IPS-2 showed higher antioxidant activities with an IC50 value of 108 ± 2.5 µg/mL, 272 ± 4.0 µg/mL, and 760 ± 5.0 µg/mL for ABTS+ scavenging, DPPH radical scavenging, and ferric reducing power, respectively. In addition, the IPS-2 inhibited the viability of prostate cancer (PC-3) cells (IC50; 435 ± 3.0 µg/mL) via apoptosis associated with mitochondrial membrane potential collapse and altered morphological features, which was revealed by cellular staining and flow cytometric analysis. Moreover, no apparent cytotoxic effects were seen in IPS-2-treated (1000 µg/mL) non-cancerous cells (HEK-293 and NIH3T3). Overall, the findings of this study suggest that P. radiatolobatum could be a potent source of polysaccharides with promising antioxidant and anticancer activity.

Fluorescence probe technique for determining the hydrophobic interactions and critical aggregation concentrations of Gleditsia microphylla gum, circular Gleditsia sinensis gum, and tara gum.

Circular Gleditsia sinensis gum, Gleditsia microphylla gum, and tara gum are galactomannans (GMs) with similar mannose/galactose (M/G) molar ratios, which complicates the characterization of physicochemical properties using conventional methods. Herein, the hydrophobic interactions and critical aggregation concentrations (CACs) of the GMs were compared using a fluorescence probe technique, in which the I1/I3 ratio of pyrene indicated polarity changes. With increasing GM concentration, the I1/I3 ratio decreased slightly in dilute solutions below the CAC but decreased sharply in semidilute solutions above the CAC, indicating that the GMs formed hydrophobic domains. However, increases in temperature destroyed the hydrophobic microdomains and increased the CACs. Higher concentrations of salts (SO42-, Cl-, SCN-, and Al3+) promoted hydrophobic microdomain formation, and the CACs in Na2SO4 and NaSCN solutions were lower than those in pure water. Hydrophobic microdomain formation also occurred upon Cu2+ complexation. Although urea addition promoted hydrophobic microdomain formation in dilute solutions, the microdomains were destroyed in semidilute solutions and the CACs increased. The formation or destruction of hydrophobic microdomains depended on the molecular weight, M/G ratio and galactose distribution of GMs. Therefore, the fluorescent probe technique enables the characterization of hydrophobic interactions in GM solutions, which can provide valuable insight into molecular chain conformations.

Enzymatic synthesis of prebiotic galactooligosaccharides from galactose derived from gum arabic.

A novel enzymatic process was established for galactooligosaccharides (GOS) synthesis by using plant-derived galactose as substrate, without producing any byproducts. The galactose was prepared from the acid hydrolysate of gum arabic. The yeast Kluyveromyces lactis producing ß-galactosidase capable of catalyzing GOS synthesis from galactose was screened out. The synthesis conditions using the yeast cells as enzyme source were optimized by both single-factor experiment and response surface methodology, with the highest GOS yield reached 45%. The composition of reaction mixture contained only GOS and unreacted galactose, which could be easily separated by the cation exchange resin column. The structures of major GOS products were identified as Gal-ß-D-(1 â†’ 6)-Gal, Gal-ß-D-(1 â†’ 3)-Gal, and Gal-ß-D-(1 â†’ 6)-Gal-ß-D-(1 â†’ 6)-Gal by MS and NMR spectra. Moreover, the ß-galactosidase-containing cells can be recycled for at least 30 batches of GOS synthesis at 35 °C, with the enzyme activity remaining above 60%.

Bivalent non-human gal-α1-3-gal glycan epitopes in the Fc region of a monoclonal antibody model can be recognized by anti-Gal-α1-3-Gal IgE antibodies.

Monoclonal antibody (mAb) production using non-human cells can introduce non-human glycan epitopes including terminal galactosyl-α1-3-galactose (α1-3-Gal) moieties. Cetuximab is a commercial mAb associated with causing anaphylaxis in some patients due to the binding of endogenous anti-α1-3-Gal IgE to the Fab (containing bi-α1-3-galactosylated glycans) but not to the Fc region (containing mono-α1-3-galactosylated glycans). Despite being low in abundance in typical commercial mAbs, the inherent sensitivity of cell culture conditions on glycosylation profiles, and the development of novel glycoengineering strategies, novel antibody-based modalities, and biosimilars by various manufacturers with varying procedures, necessitates a better understanding of the structural requirements for anti-α1-3-Gal IgE binding to the Fc region. Herein, we synthesized mAb glycoforms with varying degrees and regioisomers of α1-3-galactosylation and tested their binding to two commercial anti-α1-3-Gal human IgE antibodies derived from a human patient with allergies to red meat (comprising α1-3-Gal epitopes), as well as to the FcγRIIIA receptor. Our results demonstrate that unexpectedly, anti-α1-3-Gal human IgE antibodies can bind to Fc glycans, with bi-α1-3-galactosylation being the most important factor, highlighting that their presence in the Fc region may be considered as a potential critical quality attribute, particularly when using novel platforms in mAb-based biotherapeutics.

Structural characteristics, functional properties, antioxidant and hypoglycemic activities of pectins from feijoa (Acca sellowiana) peel.

The structure characteristics, functional properties, antioxidant and hypoglycemic activities of pectins extracted from feijoa peel with water (FP-W), acid (FP-A) and alkali (FP-B) were investigated. Results showed that the feijoa peel pectins (FPs) were mainly composed of galacturonic acid, arabinose, galactose and rhamnose. FP-W and FP-A had higher proportion of homogalacturonan domain, degree of esterification and molecular weight (for main component) than FP-B; FP-B owned the highest yield, protein and polyphenol contents. FP-W had a compact and smooth surface morphology unlike FP-A and FP-B. FP-W and FP-A had better thermal stability than FP-B. The rheological analysis suggested that the FPs exhibited pseudoplastic fluid behavior, and the elastic characteristics were dominant. Results showed that FP-W and FP-B had superior antioxidant and hypoglycemic activities than FP-A. According to correlation analysis, monosaccharide composition, sugar ratios and degree of acetylation were chief factors affecting the functional properties, antioxidant and hypoglycemic activities of the FPs.

Tenuifolin in the prevention of Alzheimer's disease-like phenotypes: Investigation of the mechanisms from the perspectives of calpain system, ferroptosis, and apoptosis.

Polygala tenuifolia was documented to calm the mind and promote wisdom. However, its underlying mechanisms are still unclear. This study aimed to investigate the mechanisms underlying the effects of tenuifolin (Ten) on Alzheimer's disease (AD)-like phenotypes. We first applied bioinformatics methods to screen the mechanisms of P. tenuifolia in the treatment of AD. Thereafter, the d-galactose combined with Aß1-42 (GCA) was applied to model AD-like behaviors and investigate the action mechanisms of Ten, one active component of P. tenuifolia. The data showed that P. tenuifolia actioned through multi-targets and multi-pathways, including regulation of synaptic plasticity, apoptosis, and calcium signaling, and so forth. Furthermore, in vitro experiments demonstrated that Ten prevented intracellular calcium overload, abnormal calpain system, and down-regulation of BDNF/TrkB signaling induced by GCA. Moreover, Ten suppressed oxidative stress and ferroptosis in HT-22 cells induced by GCA. Calpeptin and ferroptosis inhibitor prevented the decrease of cell viability induced by GCA. Interestingly, calpeptin did not interrupt GCA-induced ferroptosis in HT-22 cells but blocked the apoptosis. Animal experiments further demonstrated that Ten prevented GCA-induced memory impairment in mice and increased synaptic protein expression while reducing m-calpain expression. Ten prevents AD-like phenotypes through multiple signaling by inhibiting oxidative stress and ferroptosis, maintaining the stability of calpain system, and suppressing neuronal apoptosis.

Glycan-Presenting Coacervates Derived from Charged Poly(active esters): Preparation, Phase Behavior, and Lectin Capture.

This study presents the preparation and phase behavior of glycan-functionalized polyelectrolytes for capturing carbohydrate-binding proteins and bacteria in liquid condensate droplets. The droplets are formed by complex coacervation of poly(active ester)-derived polyanions and polycations. This approach allows for a straightforward modular introduction of charged motifs and specifically interacting units; mannose and galactose oligomers are used here as first examples. The introduction of carbohydrates has a notable effect on the phase separation and the critical salt concentration, potentially by reducing the charge density. Two mannose binding species, concanavalin A (ConA) and Escherichia coli, are shown to not only specifically bind to mannose-functionalized coacervates but also to some degree to unfunctionalized, carbohydrate-free coacervates. This suggests non-carbohydrate-specific charge-charge interactions between the protein/bacteria and the droplets. However, when mannose interactions are inhibited or when non-binding galactose-functionalized polymers are used, interactions are significantly weakened. This confirms specific mannose-mediated binding functionalization and suggests that introducing carbohydrates reduces non-specific charge-charge interactions by a so far unidentified mechanism. Overall, the presented route toward glycan-presenting polyelectrolytes enables new functional liquid condensate droplets with specific biomolecular interactions.

Bifunctional and monofunctional α-neoagarooligosaccharide hydrolases from Streptomyces coelicolor A3(2).

Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: • SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. • SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. • SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.

Isolation, structural and bioactivities of polysaccharides from Anoectochilus roxburghii (Wall.) Lindl.: A review.

Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii), a valuable herbal medicine in China, has great medicinal and edible value. Polysaccharides, as one of the main active components of A. roxburghii, comprise glucose, arabinose, xylose, galactose, rhamnose, and mannose in different molar ratios and glycosidic bond types. By varying the sources and extraction methods of A. roxburghii polysaccharides (ARPS), different structural characteristics and pharmacological activities can be elucidated. ARPS has been reported to exhibit antidiabetic, hepatoprotective, anti-inflammatory, antioxidant, antitumor, and immune regulation activities. This review summarizes the available literature on the extraction and purification methods, structural features, biological activities, and applications of ARPS. The shortcomings of the current research and potential focus in future studies are also highlighted. This review provides systematic and current information on ARPS to promote their further exploitation and application.

Efficient production of d-tagatose via DNA scaffold mediated oxidoreductases assembly in vivo from whey powder.

Among the emerging sweeteners, d-tagatose occupies a significant niche due to its low calorific value, antidiabetic property and growth promoting effects on intestinal probiotics. Recently, the main approach for d-tagatose biosynthesis is l-arabinose isomerase-based isomerization reaction from galactose, which shows relatively low conversion rate because of unfavorable thermodynamic equilibria. Herein, oxidoreductases, d-xylose reductase and galactitol dehydrogenase, together with endogenous ß-galactosidase were employed to catalyze the biosynthesis of d-tagatose from lactose with a yield of 0.282 g/g in Escherichia coli. Then, a deactivated CRISPR-associated (Cas) proteins-based DNA scaffold system was developed, which were proved to be efficient for assembling the oxidoreductases in vivo and got a 1.44-folds increase in d-tagatose titer and yield. Further, by employing d-xylose reductase with higher galactose affinity and activity, as well as overexpressing pntAB genes, the d-tagatose yield from lactose (0.484 g/g) increased to 92.0 % of the theoretical value, 1.72-times as that of original strain. Finally, whey powder, a lactose-rich food by-product, was bifunctionally utilized as an inducer and substrate. In the 5 L bioreactor, d-tagatose titer reached 32.3 g/L with little galactose detected, and the yield from lactose approached 0.402 g/g, which was the highest from waste biomass in the literature. The strategies used here might provide new insights into the biosynthesis of d-tagatose in future.

Exploring the interaction sites in glucose and galactose using phenol as a probe.

Sugars, together with amino acids and nucleobases, are the fundamental building blocks of a cell. They are involved in many fundamental processes and they especially play relevant roles as part of the immune system. The latter is connected to their ability to establish a collection of intermolecular interactions, depending on the position of their hydroxyl groups. Here we explore how the position of the OH in C4, the anomeric conformation and the nature substituent affect the interaction with phenol, which serves as a probe of the preferred site for the interaction. Using mass-resolved excitation spectroscopy and density functional calculations, we unravel the structure of the dimers and compare their conformation with those found for similar systems. The main conclusion is that the hydroxymethyl group has a very strong influence, guiding the whole aggregation process and that the position of the substituent in C4 has a stronger influence on the final structure of the dimer than the anomeric conformation.