Developing a Library of Mannose-Based Mono- and Disaccharides: A General Chemoenzymatic Approach to Monohydroxylated Building Blocks

Regioselective deprotection of acetylated mannose-based mono- and disaccharides differently functionalized in anomeric position was achieved by enzymatic hydrolysis. Candida rugosa lipase (CRL) and Bacillus pumilus acetyl xylan esterase (AXE) were immobilized on octyl-Sepharose and glyoxyl-agarose, respectively. The regioselectivity of the biocatalysts was affected by the sugar structure and functionalization in anomeric position. Generally, CRL was able to catalyze regioselective deprotection of acetylated monosaccharides in C6 position. When acetylated disaccharides were used as substrates, AXE exhibited a marked preference for the C2, or C6 position when C2 was involved in the glycosidic bond. By selecting the best enzyme for each substrate in terms of activity and regioselectivity, we prepared a small library of differently monohydroxylated building blocks that could be used as intermediates for the synthesis of mannosylated glycoconjugate vaccines targeting mannose receptors of antigen presenting cells.

of MR role in antigen up-take stimulated the research towards the development of mannosylated antigens with the aim to design vaccines with improved immunogenicity by targeting MR [5].
Several studies regarding natural high-mannosylated glycoprotein120 (GP120) fragments as potential human deficiency virus (HIV) vaccine were previously reported [6,7]. In HIV, high-mannose glycans with α-(1→6) and α-(1→2) motifs are included in the important envelope glycoprotein GP120, involved in the mediation of the infection process by interacting with MR exposed on APC cells surface. Unfortunately, this approach resulted poorly efficient for the development of an effective HIV vaccine candidate, but it suggested the idea that mannosylation with natural polymannan or its analogues, being well-recognized by MR of APC, represents a possible strategy to improve the antigenic activity of peptides or proteins. Similarly, different synthetic α-(1→6) polymannan, mimicking Mycobacterium tuberculosis polysaccharides, were synthesized and characterized showing good binding affinity for lectins [8].
However, the structural diversity and complexity of natural oligosaccharides, including polymannan, make their production from natural sources and characterization extremely complicated. Thus, chemical and/or enzymatic synthesis can represent a valid alternative for the production of structure-defined oligosaccharides analogues in high purity, by exploiting efficient and scalable protocols [9]. Automatized methods either chemical or enzymatic have been developed over the years for the synthesis of oligosaccharides [10,11], including an α-(1→6)-30mer-mannoside [12]. However, the preparation of sugar acceptor building blocks bearing only one free hydroxyl group in the desired position represents the main bottleneck in the synthesis of oligosaccharides. Usually, the chemical synthesis of monodeprotected monosaccharides and disaccharides is performed through orthogonal multistep processes, which frequently result in low yields [13,14].
The use of enzyme-catalyzed reactions provides, instead, a more straightforward route: hydrolases have been successfully employed as catalysts in the regioselective deprotection of peracetylated mono- [15][16][17][18] and disaccharides [19][20][21] under mild reaction conditions. Hence, lipases (EC 3.1.1.3) and esterases (EC 3.1.1.x) represent valuable tools for a simple and efficient chemoenzymatic approach in the preparation of sugar building blocks involving the use of acetyl moiety as the only protecting group [21,22]. In particular, the availability of synthetic tools for the production of acetylated mannose building blocks with only one free hydroxyl group and different reactive groups in anomeric position would facilitate the preparation of polymannan analogues with α-(1→6) and α-(1→2) motifs and the subsequent biological evaluation of their corresponding glycoconjugates derivatives.
In this context, the use of immobilized hydrolases is highly sought since peracetylated sugars, especially disaccharides, are scarcely soluble in aqueous medium; thus, the use of organic co-solvents is required. Stabilization of enzymes via immobilization techniques is a valuable strategy to enhance the stability of biocatalysts in the presence of organic co-solvents and their easy recovery from reaction mixture and re-use [23]. The immobilization of lipases on hydrophobic supports, by means of interfacial adsorption (e.g., on octyl-Sepharose or octadecyl-Sepabeads), is a well-established methodology for obtaining highly active biocatalysts [24] with good stability in the presence of organic co-solvents [25].
Conversely, this simple methodology is not suited for esterases due to their different 3D-architecture and kinetics [26,27]. However, the esterase fraction from the crude extract of Aspergillus niger lipase (ANL) and acetyl xylan esterase from Bacillus pumilus (AXE) were successfully immobilized by covalent interaction on acrylic carriers and employed for the deprotection of acetylated mono-and disaccharides [15,19,21].
In the present work we report on a comparison between immobilized Candida rugosa lipase (CRL) and acetyl xylan esterase from Bacillus pumilus (AXE) in the synthesis of monohydroxylated sugar building blocks. A new immobilization protocol based on agarose carrier was developed for AXE to obtain a robust biocatalyst under a wide range of experimental conditions. An extensive screening of acetylated mannose-based mono-and disaccharides differently functionalized in C1 position provided a library of monodeprotected intermediates useful for the synthesis of mannosylated glycoproducts. Thus, a new immobilization protocol was set-up for AXE considering its multimeric three-dimensional structure. In fact, AXE has a complex hexameric quaternary structure formed by a dimer of trimers showing a "doughnut-shaped" assembly with the six active centers disposed towards a central pore [28,29]. When working with multimeric enzymes, the immobilization technique must be able to bind all the subunits in order to keep the quaternary structure of the enzyme unmodified (or poorly modified) under the selected experimental conditions. The potential dissociation into the single constitutive monomers can cause the loss of activity. This dissociative process is usually enhanced by extreme pH values, high temperature, and the presence of organic co-solvents, conditions often necessary to ensure the solubility of poorly water soluble substrates [30].
We decided to use glyoxyl-agarose (GLX-AG), a hydrophilic carrier activated with aldehyde functional groups, for AXE immobilization. GLX-AG has a wide hydrophilic superficial area and pores of such dimensions to harbor proteins in a wide range of molecular weights. In addition, the presence of several hydroxyl groups on its surface, that can be easily activated, allows for the formation of a high number of bonds between the enzyme and the carrier resulting in a three-dimensional network of covalent multipoint interactions with consequent stabilization effects Figure 1. Stability of soluble acetyl xylan esterase from Bacillus pumilus (AXE) (A) and immobilized AXE on acrylic epoxy resin (B) in different organic co-solvents at different percentages v/v: 15% acetonitrile (purple circles), 30% acetonitrile (green squares), 50% acetonitrile (black triangles), 90% ethanol (red triangles), 90% tert-butanol (blue rhombus). The measurements were performed in duplicate.
Immobilized AXE on acrylic epoxy resin was already successfully employed for the selective deprotection of peracetylated lactose and N-acetyl glucosamine derivatives [15,21]; however, a low stability profile of the biocatalyst during the reaction course was observed. As reported in Figure 1B, the residual activity of immobilized AXE on acrylic epoxy resin after 48 h incubation is lower than 60% in all conditions tested, except 90% tert-butanol, in which the enzyme maintained between 70%-80% residual activity.
Thus, a new immobilization protocol was set-up for AXE considering its multimeric three-dimensional structure. In fact, AXE has a complex hexameric quaternary structure formed by a dimer of trimers showing a "doughnut-shaped" assembly with the six active centers disposed towards a central pore [28,29]. When working with multimeric enzymes, the immobilization technique must be able to bind all the subunits in order to keep the quaternary structure of the enzyme unmodified (or poorly modified) under the selected experimental conditions. The potential dissociation into the single constitutive monomers can cause the loss of activity. This dissociative process is usually enhanced by extreme pH values, high temperature, and the presence of organic co-solvents, conditions often necessary to ensure the solubility of poorly water soluble substrates [30].
We decided to use glyoxyl-agarose (GLX-AG), a hydrophilic carrier activated with aldehyde functional groups, for AXE immobilization. GLX-AG has a wide hydrophilic superficial area and pores of such dimensions to harbor proteins in a wide range of molecular weights. In addition, the presence of several hydroxyl groups on its surface, that can be easily activated, allows for the formation of a high number of bonds between the enzyme and the carrier resulting in a three-dimensional network of covalent multipoint interactions with consequent stabilization effects [31]. Successful immobilization procedures for multimeric proteins stabilization based on this carrier have been widely described [32,33].
Different parameters were screened, such as temperature, time, and protein loading in order to optimize AXE immobilization on GLX-AG. As shown in Table 1, when using a loading of 150 mg/g and 25 • C, AXE immobilized on acrylic epoxy resin showed higher immobilization yields in terms of quantity of immobilized protein (39%) and activity (38%) when compared to GLX-AG derivative (28% of protein and 33% of activity immobilized); however, the activity of this derivative was almost four-fold lower (107 U/g vs. 383 U/g). Thus, it seems that the hydrophobic microenvironment of acrylic surface around the enzyme negatively influences its activity, while the hydrophilic nature of agarose seems to be preferred. Moreover, the multipoint interaction of the enzyme with the three-dimensional network of agarose fibers may prevent subunits dissociation maintaining the correct quaternary structure of the protein and, thus, its activity. In addition, the short time incubation needed for GLX-AG immobilization protocol compared to acrylic resin immobilization (3 h instead of 24 h) can explain the better performances of GLX-AG biocatalyst. We further tried to optimize GLX-AG immobilization procedure by lowering the temperature to 4 • C in order to prevent subunits dissociation during the immobilization process. However, as shown in Table 1, we were able to slightly increase the immobilization yield in terms of activity (48%), but the final activity expressed by the derivative was lower compared to the immobilization procedure performed at 25 • C (283 U/g vs. 383 U/g). The longer incubation time (18 h instead of 3 h) at pH 10 may negatively influence the stability of the enzyme. Finally, as in all the conditions tested we have observed low protein binding to the supports (26%-39% of the total protein used), we decided to decrease the immobilization loading from 150 mg/g to 50 mg/g. In these conditions, we obtained a two-fold increase in the % of immobilized protein (61% vs. 28%) and an improvement of immobilized activity (43% vs. 33%). Thus, the best immobilization conditions on GLX-AG were 25 • C, 3 h using 50 mg/g of protein. This biocatalyst was further used in all the experiments reported in this paper. The stability of AXE immobilized on GLX-AG was evaluated in 50% acetonitrile and phosphate buffer (100 mM; pH 7.0), and it was compared to that of the derivative immobilized on acrylic resin. As shown in Figure 2, AXE immobilized on GLX-AG showed higher stability compared to the acrylic resin derivative: GLX-AG derivative maintained 80% of its activity after 48 h of incubation, while the acrylic resin derivative lost almost 50% of its activity, in the same conditions.
Compound 8 was selectively hydrolyzed by using AXE immobilized on GLX-AG, and the results were in agreement with those previously reported using the enzyme immobilized on acrylic epoxy carrier [15,35].
These monodeprotected products are useful building blocks in the synthesis of oligosaccharides and glycoconjugates. For example, compounds obtained from 2a and 8a can be used to conjugate antigenic proteins through the thiocyanomethyl group in anomeric position. Compound 3a can be used both as acceptor or donor in further glycosylation reactions because, once used as acceptor, the product obtained can become a donor due to the good properties of S-Tol group as leaving group. Furthermore, compounds obtained by hydrolysis of 5 and 7 can be used as intermediates to prepare glycoproducts by click chemistry, thanks to their propargyl and azido groups in the anomeric position.

Enzymatic Hydrolysis of Peracetylated Disaccharides (10-18)
The disaccharides prepared as previously reported (see Section 2.3) were subsequently tested as substrates for enzymatic hydrolysis with the aim to obtain selectively deprotected disaccharides that can be further used as advanced building blocks for the synthesis of complex linear and branched oligosaccharides. Both CRL and AXE were tested as catalysts (Table 4), but only AXE showed a relevant activity towards the different disaccharides 10-18 (CRL was almost completely inactive towards all these substrates). The hydrolysis catalyzed by AXE-GLX-AG derivative provided different results depending from the substrate as shown in Table 4. The fully acetylated man(1→6)man 10 (Scheme 3) was deprotected at the anomeric position yielding product 10a with 50% of maximum yield after 48 h of reaction (63% of substrate consumption), in agreement with the results previously reported for lactose bearing an acetoxy group in anomeric position [21]. By introducing an alkyl or aryl glycoside or thioglycoside group (substrates 11-15 in Scheme 3) in anomeric position, the selectivity of AXE moved towards the C2 position allowing the production of compounds 11a-15a with yields ranging from 23% to 52%. When man(1→6)GluNHAc 16 was submitted to hydrolysis, AXE was not able to hydrolyze the acetamido group of the anomeric sugar (GluNAc); thus, the selectivity of the enzyme moved further towards C2 position of the mannose unit at the non-reducing end (Scheme 3). However, the reaction proceeded very slowly (168 h for achieving 40% of substrate consumption) and low yields (about 20% of product 16a).
referenced to the solvent signals (δ H 7.28, δ C 77.00). Signal multiplicities are abbreviated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; b, broad. Structures assignment was performed by means of 2D-COSY and HSQC and, in some cases, 2D-NOESY. Spectra analyses were carried out using Mestrenova reader software. For compounds 5a, 5b, 14, and 18 high resolution mass spectra (HRMS) were recorded with a Bruker Micro-TOF spectrometer in electrospray ionization (ESI) mode, using Tuning-Mix as reference. For all other compounds, mass spectra were recorded on an LCQ-DECA Thermo Finnigan Spectrometer by the ESI (Electron Spray Ionization) ionization method with an ionic source and with use of Xcalibur 2.2 software (Thermo-Finnigan, San Jose, CA, USA). Analyses were run under positive modality, and the experimental conditions were: voltage of the source 5.0 kV, voltage of the capillary 14 V, flow of the gas 35 (arbitrary units), and temperature 200 • C.

Determination of Enzymatic Activity
The activity of the enzymes was determined following a standard protocol by using an automatic titrator pH-Stat. The hydrolytic activity was calculated based on NaOH consumption (mL of NaOH/min).

Standard Activity Assay with Acetyl Xylan Esterase from Bacillus pumilus (AXE)
The activity of AXE was determined using 1-naphtyl acetate as standard substrate [21]. The standard reaction mixture was composed of 2 mL of acetonitrile, 2 mL of 1-naphtyl acetate (50 mM in acetonitrile), and 16 mL of phosphate buffer (25 mM, pH 7.0). The reaction was started through the addition of 100 µL soluble enzyme (49 mg/mL) or 10-15 mg of immobilized enzyme. The mixture was mechanically stirred and pH was maintained at 7.0 using 100 mM NaOH as titrant. Experiments were done at least in duplicate.

Standard Activity Assay with Candida rugosa Lipase (CRL)
The activity of CRL was determined using tripropionin as standard substrate [36]. The standard reaction mixture was composed of 0.6 mL of acetonitrile, 1 mL of tripropionin, and 18.4 mL of Tris-HCl (25 mM, pH 7.0). The reaction was started through the addition of 100 µL soluble enzyme (10 mg/mL) or 10-15 mg of immobilized enzyme. The mixture was mechanically stirred and pH was maintained at 7.0 using 100 mM NaOH as titrant. Experiments were done at least in duplicate.

AXE Immobilization on Glyoxyl-Agarose (GLX-AG)
GLX-AG was prepared as reported in literature [37]. Briefly, Sepharose TM CL-6B (agarose, 5 g) was suspended in deionized H 2 O (1.4 mL) and NaOH (1.7 M, 2.4 mL) containing NaBH 4 (28.4 mg/mL). Subsequently, glycidol (1.7 mL) was added dropwise, keeping the vessel at 4 • C in an ice bath. The reaction was kept under gently stirring overnight at 25 • C. After the incubation period, the suspension was filtered, and the carrier was washed abundantly with deionized H 2 O. Oxidation was initiated by adding NaIO 4 (100 mM, 34 mL). The reaction was carried out for 2 h at room temperature, and then the carrier was filtered under reduced pressure and washed abundantly with deionized H 2 O and stored at 4 • C.
Immobilization of AXE on GLX-AG was performed following a standard protocol [38,39]. Briefly, glyoxyl-agarose was washed abundantly with NaHCO 3 buffer (50 mM, pH 10) and then filtered under reduced pressure until dryness. Soluble enzyme (50 mg or 150 mg loading of protein per gram of carrier) was solubilized into NaHCO 3 buffer (50 mM, pH 10). Then, the carrier was added, and the suspension was allowed to stir at 25 • C or 4 • C. Finally, NaBH 4 (1 mg for each 100 mg of carrier) was added to the mixture and incubated for 30 min to allow imino bonds reduction. The immobilized enzyme was then filtered, rinsed thoroughly with distilled water, and stored at 4 • C till use.

AXE Immobilization on Sepabeads EC-EP/M
Immobilization of AXE on Sepabeads EC-EP/M was performed following a standard protocol [38,39]. Briefly, Sepabeads EC-EP/M was allowed to hydrate for 1 h in water on a rolling shaker at 25 • C, and then it was filtered under reduced pressure until dryness. Soluble enzyme (150 mg loading of protein per gram of carrier) was solubilized into KH 2 PO 4 buffer (1 M, pH 8). Then, the carrier was added and the suspension was allowed to stir for 24 h at 25 • C. Subsequently, the epoxy groups were quenched with 3 M glycine in KH 2 PO 4 buffer (1 M, pH 8) for 18 h at 25 • C. The immobilized enzyme was then filtered, rinsed thoroughly with distilled water, and stored at 4 • C till use.

CRL Immobilization on Octyl-Sepharose
The crude extract of CRL (1.5 g; loading 2500 UI per gram of carrier) was suspended in KH 2 PO 4 buffer (25 mM, pH 7.0). The mixture was allowed to stir on the rolling shaker for 30 min. Then, octyl-Sepharose ® (3 g), previously conditioned with the same buffer, was added and the suspension was stirred at room temperature overnight. The enzyme derivative was filtered under reduced pressure on a Büchner funnel, rinsed thoroughly with distilled water, and stored at 4 • C till use. (2) was synthesized as previously reported [40].

Enzymatic Deprotection of Monosaccharides 1-8
The deacetylated monosaccharides were produced following a general procedure of hydrolysis. The substrates (10 mM final concentration) were dissolved in acetonitrile (20%-30% v/v depending on substrate solubility) under magnetic stirring, and then phosphate buffer (50 mM, pH 4.0-5.0) was added slowly. The reaction was started through the addition of immobilized CRL and/or AXE, previously conditioned with reaction buffer. The reactions were performed at 25 • C under mechanical stirring; the pH of the solution was maintained constant by automatic titration. Reaction course was monitored by TLC.
After complete consumption of the starting substrate or before an excessive formation of undesired products, the reactions were stopped by enzymatic derivative filtration on Büchner funnel. Acetonitrile was evaporated under reduced pressure, and the solution was brined and extracted with ethyl acetate. The organic layers were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The mixture obtained was purified by flash chromatography.

Enzymatic Deprotection of Disaccharides 10-18
The deacetylated disaccharides were produced following a general procedure of hydrolysis. The substrates (10 mM final concentration; 5 mM for 12) were dissolved in acetonitrile (20%-30% v/v depending on substrate solubility) under magnetic stirring, and then phosphate buffer (50 mM, pH 4.0-5.8) was added slowly. The reaction was started through the addition of immobilized CRL and/or AXE, previously conditioned with reaction buffer. The reactions were performed at 25 • C under mechanical stirring; the pH of the solution was maintained constant by automatic titration. Reaction course was monitored by TLC.
After complete consumption of the starting substrate or before an excessive formation of undesired products, the reactions were stopped by enzymatic derivative filtration on Büchner funnel. Acetonitrile was evaporated under reduced pressure, and the solution was brined and extracted with ethyl acetate. The organic layers were dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. The mixture obtained was purified by flash chromatography.