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. 2026 Jul 2;6(4):423–431. doi: 10.1021/acsbiomedchemau.6c00086

Mechanistic Insights into β‑1,6-Branch Recognition and Orientation-Selective Hydrolysis by Endo-β-1,3-Glucanase Using Synthetic β‑Glucan Probes

Atsushi Miyagawa 1,*, Takahiro Yamaguchi 1, Masayuki Oda 2, Hatsuo Yamamura 1
PMCID: PMC13495021  PMID: 42630976

Abstract

Understanding how glycoside hydrolases structurally heterogeneous polysaccharides remains a central challenge due to the limited availability of well-defined substrates. Here, we establish a chemically defined, probe-based platform to systematically dissect the hydrolytic specificity of an endo-β-1,3-glucanase using a systematically designed library of branched β-1,3-glucan oligosaccharides with precisely controlled branch position and length. Synthetic access to these glycans enabled their conversion into fluorescent probes for sensitive, site-specific analysis of enzymatic cleavage. Our results revealed that β-1,6-linked branching acts as a key structural determinant governing both cleavage efficiency and positional selectivity in a branched length-dependent manner. While monosaccharide branches are broadly accommodated, disaccharide branches introduce steric constraints that significantly suppress hydrolysis. Notably, analysis using an extended octasaccharide substrate uncovered a dominant cleavage at the central β-1,3-glycosidic bond, allowing refinement and expansion of the enzyme subsite model beyond that accessible with shorter substrates. Collectively, these findings define branch architecture as a critical regulator of substrate orientation and cleavage-site selection, and establish chemically defined glycan libraries as a generalizable platform for mechanistic interrogation of glycoside hydrolases.

Keywords: beta-glucan, beta-1, 3-glucanase, oligosaccharide, hydrolysis, fluorescence, branch

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1. Introduction

Branched polysaccharides such as β-1,3-glucans are structurally and functionally important polysaccharides in fungi and other microorganisms, with broad relevance in immunology, biomaterials, and industrial biotechnology. − Their β-1,3-linked backbones are frequently decorated with β-1,6-linked branches, generating structurally heterogeneous architectures that influence solubility, higher-order assembly, and biological activity. − In fungal cell walls, β-1,3-glucans form polymorphic networks through interactions with chitin and mannan, and branching patterns are believed to modulate both mechanical properties and enzymatic accessibility. , Despite their biological and industrial importance, the structural complexity of branched β-1,3-glucans has limited precise investigation of how branching architecture governs enzymatic recognition, substrate orientation, and cleavage-site selection.

β-1,3-Glucanases hydrolyze β-1,3-glycosidic bonds in the glucan backbone while typically leaving β-1,6-linked branches intact. These enzymes participate in diverse physiological processes, including fungal cell-wall remodeling, plant defense responses, and microbial metabolism. − Although structural and biochemical studies have elucidated catalytic mechanisms and subsite organization in several β-1,3-glucanases, their hydrolytic specificity has largely been evaluated using natural polysaccharides or linear oligosaccharides. − Consequently, how defined β-1,6-linked branching influences cleavage efficiency, positional selectivity, and subsite occupancy remains insufficiently understood. Direct investigation of branching effects is particularly challenging because natural substrates exhibit heterogeneous branch distributions that obscure precise mapping of cleavage sites. Systematic interrogation of branching effects therefore requires structurally defined glycans with precise control over branch position and lengthmaterials that are scarcely accessible from natural sources. Chemical synthesis provides a powerful platform to construct such well-defined branched oligosaccharides, enabling controlled evaluation of how branch architecture modulates enzymatic recognition. However, a chemically defined strategy that allows systematic and site-specific interrogation of branch-dependent hydrolysis remains underdeveloped. − The endo-β-1,3-glucanase used in this study contains a glycoside hydrolase family 16 (GH16) catalytic domain and a carbohydrate-binding module family 13 (CBM13). Previous studies demonstrated efficient hydrolysis of linear β-1,3-glucans such as laminarin and laminarioligosaccharides, whereas the influence of β-1,6-linked branching on substrate recognition and cleavage-site selection remains largely unexplored. This enzyme was therefore selected as a model system to evaluate branch-dependent hydrolysis using structurally defined substrates.

Here, we establish a chemically defined, probe-based platform enabled by a systematically designed library of branched β-1,3-glucan oligosaccharides. To systematically investigate the effects of branch position and length on enzymatic cleavage, a library of fluorescent β-1,3-glucan probes was designed, including probes bearing mono- or disaccharide β-1,6-branches at the nonreducing end, internal position, or reducing end, together with corresponding linear controls (Figure S1). Using these synthetic glycans as fluorescent probes, we enable precise mapping of enzymatic cleavage and demonstrate that β-1,6-linked branching governs cleavage efficiency and orientation selectivity in an endo-β-1,3-glucanase from Cellulosimicrobium cellulans. Beyond this specific system, our approach provides a generalizable framework for dissecting how branch architecture regulates substrate recognition and cleavage-site selection in glycoside hydrolases. Notably, analysis using extended oligosaccharides reveals a dominant cleavage corresponding to the – 1/+1 scissile bond within a – 4 to +3 subsite architecture, providing insights that cannot be obtained from shorter substrates. This work highlights the utility of chemically defined glycan libraries for dissecting branch-dependent recognition and advancing mechanistic studies of glycoside hydrolases.

2. Results and Discussion

2.1. Synthesis of Branched β-1,3-Glucan Oligosaccharides

The β-1,3-glucan oligosaccharides were synthesized via stepwise glycosylation using glycosyl donors for both branched and unbranched units. After constructing trisaccharides, branches were introduced at the deprotected 6-position to generate a library of branched β-1,3-glucan oligosaccharides. As shown in Scheme , β-thiomethyl glucoside was converted to monosaccharide donors 1 and 2, as described in a previous study. The thiomethyl group of monosaccharide 3 was smoothly deprotected and converted into a trichloroacetimidate moiety to afford glycosyl donor 4. Donor 4 was then reacted with p-methoxyphenol to generate the reducing-end unit 5 containing a stable glycoside linkage. Selective cleavage of the benzylidene group of 5 produced intermediate 6 in high yield. Subsequent protection of the 6-hydroxy group was used to differentiate nonbranching from branching positions. Specifically, an acetyl group was introduced at the nonbranching site to give 7, while a selectively removable monochloroacetyl group was introduced at the branching site to yield 8. Deprotection of the silyl groups using BF3·Et2O produced glycosyl acceptors 9 and 10, which were subsequently coupled with glycosyl donors 1 and 2 to construct the desired disaccharides, as detailed below.

1. Synthesis of Monosaccharide Derivatives.

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As depicted in Scheme , glycosyl donor 1 was coupled with glycosyl acceptor 9 to produce disaccharide 11, lacking a branching position, in 76% yield. Similarly, disaccharides 12 and 13, containing a branching position, were synthesized in a β-selective manner via glycosylation using either donor 2 with acceptor 9 or donor 1 with acceptor 10. Additionally, acetylated glycosyl donor 17 was glycosylated with acceptor 6 to generate β-1,6-disaccharide 18 as a branched unit. Disaccharides 11–13 were converted to glycosyl acceptors 14–16 by desilylation with BF3·Et2O, giving the corresponding products in yields of 76–94%. Furthermore, β-1,6-disaccharide 18 was modified by substitution of its acetyl group with a TBDMS group, followed by removal of the p-methoxyphenyl group and subsequent conversion to disaccharide donor 19 via the introduction of a trichloroacetimidate moiety, achieving a yield of 95% over four steps.

2. Synthesis of Disaccharide Donors and Acceptors.

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As shown in Scheme , glycosyl acceptor 16, which lacks a chloroacetyl group, was coupled with donor 2 bearing a branching point to afford the trisaccharide 22 with a branch at the nonreducing end in 85% yield. Disaccharide acceptors 14 and 15, bearing chloroacetyl groups as branching points, were elongated via glycosylation with glycosyl donor 1 to afford trisaccharides 20 and 21 bearing branches at the reducing end and internal positions, respectively. Subsequently, trisaccharides 20–22 underwent selective deprotection of the monochloroacetyl group at the 6-position by heating with 1,8-diazabicyclo[5.4.0]­undec-7-ene (DBU) in ethanol, yielding glycosyl acceptors 23–25 for the subsequent introduction of β-1,6-branches.

3. Synthesis of Trisaccharides.

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Finally, trisaccharide acceptors 23–25 were glycosylated with acetylated glycosyl imidate 17 to provide the desired tetrasaccharides 26–28, each bearing a branched monosaccharide residue at the 6-position (49–75% yields). Tetrasaccharide 27, bearing a branched monosaccharide at the internal position, was obtained in the highest yield, contrary to expectations based on steric hindrance. Moreover, disaccharide donor 19 was coupled with trisaccharide acceptors 23–25 to construct pentasaccharides 29–31 bearing a branched disaccharide residue in 76–90% yields. Surprisingly, the disaccharide donor provided higher yields than the monosaccharide donor. Pentasaccharide 30, bearing a branch at the internal position, was obtained in the highest yield, similar to the trend observed during tetrasaccharide synthesis. These reactions are detailed in Scheme .

4. Construction of Tetrasaccharides and Pentasaccharides.

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2.2. Synthesis of the β-1,3-Glucan Oligosaccharide Probes

As shown in Scheme , branched β-1,3-glucan oligosaccharides 26–31 were first deprotected by removal of the TBDMS groups followed by acetylation, except for oligosaccharides 26, 27, and 29. Subsequently, their benzyl groups were removed by hydrogenation using Pd­(OH)2 under a H2 atmosphere, and the resulting free hydroxy groups were acetylated to afford the corresponding acetates bearing p-methoxyphenyl groups at their anomeric positions (76–100% overall yields). The p-methoxyphenyl groups of acetates 32–37 were then removed, and the resulting compounds were converted into glycosyl imidates 38–43 at the 1-position. A commercially available nonbranched linear trisaccharide was also acetylated and converted into acetyl imidate 44 following a reported procedure. Glycosyl imidates 38–44 were coupled with 5-(Fmoc amino)-1-pentanol to introduce a protected amino group at the ω-position, affording glycosides 45–51 bearing protected amino alkyl aglycones in a β-selective manner (38–84% yields). Glycosides 45–51 were then subjected to simultaneous deprotection of their acetyl and Fmoc groups in ammonium solution, and the resulting free amino groups were reacted with dansyl chloride to afford fluorescent β-1,3-glucan oligosaccharide probes 52–58 in 59–97% yields following purification by reversed-phase chromatography.

5. Synthesis of the Trisaccharide, Tetrasaccharide, and Pentasaccharide Probes.

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2.3. Evaluation of the Hydrolytic Specificity of Endo-β-1,3-Glucanase Using the Synthesized Probes

The synthesized β-1,3-glucan oligosaccharide probes were used to evaluate the hydrolytic specificity of endo-β-1,3-glucanase. To this end, the probes were incubated with endo-β-1,3-glucanase, and the reaction mixtures were analyzed by HPLC (HILIC column) and mass spectrometry (MS) at defined time points. ,− As detailed in Figure , tetrasaccharide probe 52, which bears a branch at its reducing end, was cleaved to give a β-1,6-disaccahride and a β-1,3-disaccahride; the fluorescent β-1,6-disaccharide was observed at m/z 683.2314. Additionally, tetrasaccharide probe 53, which bears a branch on its internal position, was cleaved at the anomeric position to yield a β-1,3-trisaccharide and the corresponding aglycone, the latter appearing as an earlier retention-time peak corresponding to the aglycone. Furthermore, tetrasaccharide probe 54bearing a branch at the nonreducing endand linear trisaccharide probe 58 were cleaved to give a monosaccharide with a fluorescent aglycone and the remaining oligosaccharide fragment. The peak corresponding to the degradation product from probe 54 was collected and characterized by MS (m/z 521.1873) and 1H NMR analysis (Figure S2), confirming the structure as a fluorescently labeled monosaccharide. The corresponding degradation product from probe 58 exhibited the expected MS signal (m/z 521.1949), consistent with assignment as the same monosaccharide species. Of the branched pentasaccharide probes 55–57, only probe 55 underwent cleavage, yielding a β-1,6-trisaccharide and a β-1,3-disaccharide. The peak corresponding to the fluorescent β-1,6-trisaccharide was collected and its mass was observed at m/z 845.3114, consistent with the trisaccharide structure. The hydrolysis rates followed the order: 54 > 58 > 52 ≫ 55 ≈ 53 (Figure h). Notably, the presence of a disaccharide branch markedly hindered enzymatic activity, and hydrolysis occurred only when the branch was positioned adjacent to the cleavage site. In contrast, monosaccharide branches were tolerated at all positions examined, although the enzyme exhibited enhanced activity when the branch was located near the scissile bond. These observations indicate that β-1,6-linked branching modulates both cleavage efficiency and positional selectivity in a branch length–dependent manner. While short branches can be accommodated within the catalytic cleft, elongation of the branch imposes steric constraints that restrict productive subsite occupancy. This behavior cannot be explained solely by steric hindrance, but instead reflects defined positioning of the substrate within the catalytic cleft. These results suggest that branch architecture governs substrate positioning within the active site, thereby controlling access to the scissile bond, and may represent a general mechanism by which branching modulates enzymatic accessibility in structurally heterogeneous polysaccharides. Based on these findings, a refined model of the enzymatic subsites of endo-β-1,3-glucanase is proposed (Figure ).

1.

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Cleavage of β-1,3-glucan oligosaccharide probes by endo-β-1,3-glucanase. HPLC profiles for the cleavage of (a) 52, (b) 53, (c) 54, (d) 58, (e) 55, (f) 56, and (g) 57. (h) Time-dependent cleavage of β-1,3-glucan oligosaccharide probes. The apparent peak splitting observed in (c) and (d) is attributed to HILIC chromatographic behavior.

2.

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Proposed enzymatic subsite of endo-β-1,3-glucanase.

2.4. Synthesis of a β-1,3-Glucan Octasaccharide Probe

To further validate the proposed enzymatic subsite model, a longer oligosaccharide was synthesized and subjected to hydrolysis. As shown in Scheme , tetrasaccharide 27 was converted into glycosyl donor 59 and acceptor 60 by introducing a trichloroacetimidate group at the anomeric position and removal of the TBDMS group at the 4-position of the glucose residue at the nonreducing end. Donor 59 and acceptor 60 were subsequently coupled to afford octasaccharide 61 in 31% yield after two-step purification by normal- and reversed-phase silica gel column chromatography. Octasaccharide 61 was then deprotected, acetylated, and converted into octasaccharide donor 63 via trichloroacetimidate formation at the anomeric position, giving a 78% yield over two steps. Donor 63 was subsequently coupled with 5-(Fmoc amino)-1-pentanol to afford 64 in 66% yield. Deprotection of the acetyl and Fmoc groups, followed by dansylation of the ω-amino group, yielded octasaccharide probe 65 bearing two monosaccharide branches within its repeating structure. Moreover, octasaccharide 61 was also converted into glycosyl donor and acceptor derivatives to afford a hexadecasaccharide via glycosylation. The resulting hexadecasaccharide was deprotected and functionalized with an aglycone to afford a hexadecasaccharide probe. However, this probe was insoluble in buffer and exhibited resistance to enzymatic cleavage. ,− Therefore, only octasaccharide probe 65 was treated with endo-β-1,3-glucanase to evaluate its hydrolytic specificity.

6. Synthesis of the Octasaccharide Probe.

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2.5. Evaluation of the Hydrolytic Specificity of endo-β-1,3-Glucanase Using Octasaccharide Probe 65

Finally, octasaccharide probe 65 was subjected to enzymatic hydrolysis under the same conditions using endo-β-1,3-glucanase. Short oligosaccharide substrates are insufficient to fully resolve subsite architecture, necessitating the use of extended glycans. This limitation can lead to incomplete or misleading interpretation of cleavage specificity when only short substrates are used. As shown in Figure , probe 65 was almost completely digested within 1 h, and two new peaks appeared after the reaction. The first peak (16.2 min) corresponded to a trisaccharide bearing a branch at the nonreducing end, and its structure was confirmed by MS analysis (m/z 845.2969). The second peak (18.6 min) was identified as having the same structure as probe 53, as confirmed by MS analysis (m/z 1007.3583). A minor peak corresponding to the fluorescent aglycone was also detected. This product is likely generated through secondary hydrolysis of probe 53, which is formed during degradation of probe 65. These results indicate that probe 65 is cleaved at two positions, with preferential hydrolysis occurring at the central glycosidic bond of the octasaccharide. Importantly, this dominant cleavage corresponding to the β-1,3-glycosidic bond located one glucose residue away from the branch-bearing glucose unit, consistent with productive binding across the – 4 to +3 subsites, as illustrated in the revised subsite model (Figure ). In contrast, shorter oligosaccharide substrates are insufficient to resolve this positional preference. This limitation can lead to incomplete or misleading interpretation of cleavage specificity when only short substrates are used. A secondary cleavage event was observed at the β-1,3 linkage immediately adjacent to the branch-bearing residue, consistent with the behavior observed for shorter probes. These findings demonstrate that substrate length is critical for capturing productive binding orientation and reveal a branch-dependent cleavage mechanism that becomes apparent only in extended glycans.

3.

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Cleavage of β-1,3-glucan octasaccharide probe 65 by endo-β-1,3-glucanase. (a) HPLC profiles recorded during the reaction. (b) Time course of cleavage.

4.

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Revised enzymatic subsite model of endo-β-1,3-glucanase.

The systematic analysis of structurally defined branched β-1,3-glucan probes reveals that branch architecture critically influences the hydrolytic specificity of endo-β-1,3-glucanase. Monosaccharide β-1,6 branches were accommodated within the catalytic cleft, whereas disaccharide branches imposed steric constraints that restricted productive binding. Importantly, extension of the substrate to an octasaccharide was required to unambiguously identify the dominant cleavage site. In the longer probe, preferential hydrolysis occurred at the β-1,3-glycosidic bond located one glucose residue away from the branch-bearing glucose unit; this scissile bond corresponds to the – 1 and +1 position within a – 4 to +3 subsite architecture. A secondary cleavage event was observed at the β-1,3 linkage immediately adjacent to the branch-bearing residue. These results enable refinement of the subsite model of endo-β-1,3-glucanase and clarify how β-1,6 branching governs cleavage-site selection in extended substrates and provide design principles for interpreting enzyme specificity in branched glycans. Previous crystallographic and computational studies of this enzyme established the overall architecture of the substrate-binding cleft and proposed a binding mode for linear β-1,3-glucan substrates. However, structurally defined branched β-1,3-glucan substrates suitable for systematically evaluating the effects of β-1,6-linked branching were not readily available in those studies. The present study therefore extends these earlier investigations by providing direct experimental evidence for branch-dependent substrate recognition, productive substrate orientation, and cleavage-site selection using a systematic library of synthetic branched β-1,3-glucan probes. More broadly, these findings establish branch architecture as a key structural determinant governing substrate orientation and cleavage-site selection in glycoside hydrolases. Beyond the specific system examined here, this chemically defined approach provides a generalizable framework for systematically dissecting how structural heterogeneity in polysaccharide regulates enzymatic recognition and catalysis.

3. Conclusions

This study establishes a chemically defined probe-based strategy to decipher the hydrolytic specificity of endo-β-1,3-glucanase using precisely branched β-1,3-glucan oligosaccharides. By overcoming the intrinsic structural heterogeneity of natural polysaccharides, this approach enables direct correlation between substrate structure and enzymatic cleavage behavior. Systematic variation of branch position and length reveals that endo-β-1,3-glucanase recognition is highly sensitive to β-1,6-linked branching, with disaccharide branches imposing pronounced steric constraints, whereas monosaccharide branches are preferentially accommodated near the catalytic center. Notably, extension to an octasaccharide substrate reveals dominant cleavage at the centrally positioned β-1,3-glycosidic bond, enabling expansion and validation of the – 4 to +3 subsite model that cannot be resolved using shorter substrates. Collectively, these findings establish branch architecture as a key structural determinant governing substrate orientation, enzymatic accessibility, and cleavage-site selection. More broadly, this work provides design principles for interpreting enzyme specificity in branched glycans and highlights the utility of structurally defined glycan probes for mechanistic investigation of carbohydrate-active enzymes. Beyond the specific system examined here, this chemically defined approach is expected to be broadly applicable to glycoside hydrolases acting on structurally heterogeneous polysaccharides, offering a generalizable framework for dissecting enzyme–substrate recognition in complex glycan systems.

4. Experimental Section

4.1. General Glycosylation Procedure

Glycosylation reactions were performed using protected glycosyl donors and acceptors in dry dichloromethane in the presence of activated molecular sieves 4Å. After cooling to the desired temperature, TMSOTf was added as the promoter, and the reaction mixture was stirred under the specified conditions. The reaction was quenched by dilution with dichloromethane, filtered through Celite, washed successively with saturated aqueous sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate, and concentrated. The crude products were purified by silica gel column chromatography. Detailed reaction conditions and characterization data for each compound are provided in the Supporting Information.

4.2. General Procedure for Probe Preparation

Acetylated β-1,3-glucan oligosaccharide donors were coupled with 5-(Fmoc-amino)-1-pentanol by TMSOTf-promoted glycosylation in dry dichloromethane in the presence of activated molecular sieves 4Å. Following purification by silica gel chromatography, acetylated glycosides with Fmoc-protected aglycons were deprotected with aqueous ammonia in methanol. The resulting amino-functionalized glycosides were labeled with dansyl chloride in methanol in the presence of sodium bicarbonate and purified by reversed-phase silica gel chromatography to afford the fluorescent β-1,3-glucan probes. Detailed synthetic procedures and characterization data are provided in the Supporting Information.

4.3. General Procedure for Enzymatic Hydrolysis

Hydrolysis assays were performed using recombinant endo-β-1,3-glucanase (25 nM), prepared as previously reported, and fluorescent β-1,3-glucan probes (50 μM). Reactions were carried out in 40 mM potassium phosphate buffer (pH 7.0) at 40 °C. Aliquots were withdrawn at appropriate time intervals, and reaction mixtures were analyzed by HPLC using a TSK-GEL Amide-80 column with fluorescence detection (λex = 325 nm, λem = 525 nm). Hydrolysis products were identified by HRMS analysis of the collected fractions. Detailed enzymatic assay procedures are provided in the Supporting Information.

Supplementary Material

bg6c00086_si_001.pdf (39.1MB, pdf)

Acknowledgments

This work was supported by JSPS KAKENHI (20K05602) and the Iketani Science and Technology Foundation awarded to A.M.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsbiomedchemau.6c00086.

  • Experimental procedures and characterization data for all compounds; schematic overview of the fluorescent β-1,3-glucan probe library; fully expanded structures corresponding to Schemes –; 1H and 13C NMR spectra; HRMS spectra; characterization of the fluorescently labeled monosaccharide isolated from probe 54 (PDF)

A.M.: Conceptualization, Supervision, Data curation, Project administration, Writing - Original Draft. T.Y.: Investigation, Experiments, Data curation, Validation. M.O.: Supervision, Writing - Review & Editing. H.Y.: Supervision, Writing - Review & Editing.

The authors declare no competing financial interest.

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