WO2012053017A1 - Enzyme-catalyzed process for the preparation of sugar 6 -e sters - Google Patents

Abstract

The present invention deals with a process for the enzyme-catalysed selective esterif ication of sugar molecules. Said process may comprise one phase in which a lipase enzyme is encapsulated into a biopolymer scaffold to catalyse the selective esterif ication of a sugar substrate with an acylating agent, e.g., an acyl donor fatty acid ester such as a vinyl alkanoate, mostly at a fixed position of the sugar moiety, e.g. at the C-6 position thereof; and one phase in which said reaction is carried out preferably in an alkyl hydroxylic solvent, being both the enzyme and the sugar substrate immobilized or encapsulated within the biopolymer scaffold. This process is particularly useful for producing biodegradable surfactants, like fatty acid esters of suitable sugars, and for producing intermediates for the production of useful sugar derivatives, e.g. 6-O-acyl-sucrose, which is a key intermediate in a route to the production of the high-intensity sweetener Sucralose®.

Description

TITLE

ENZYME-CATALYZED PROCESS FOR THE PREPARATION

OF SUGAR 6-ESTERS

Technical Field of the invention

The present invention deals with a process for the enzyme-catalysed selective esterification of sugar molecules.

More specifically, the process of the present invention may comprise one phase in which an enzyme, e.g. a lipase, is encapsulated into a properly designed biopolymer scaffold to catalyse the selective esterification reaction of a sugar substrate, e.g. sucrose, with an acylating agent, e.g. an acyl donor fatty acid ester such as a vinyl alkanoate, at a fixed position of the sugar moiety, e.g. at the C-6 position thereof; and one phase in which said reaction is preferably carried out in an alkyl hydroxylic solvent, being both the enzyme and the sugar substrate immobilized or encapsulated within the biopolymer scaffold. This process is particularly useful for the production of biodegradable surfactants, for example, fatty acid esters of suitable sugars, as well as for the production of suitable intermediates for the production of useful sugar derivatives, e.g. 6-0-acyl- sucrose, which is a key intermediate in a route to the production of the high-intensity sweetener, 4,1', 6'- trichloro- , 1 ' , 6' -trideoxy galactosucrose (Sucralose®) .

Background Art of the invention

It is well known that surface active products are primarily produced from non-regenerable petrochemicals using a number of synthetic strategies; however with these products there are serious environmental issues such as eutrophication of lakes and rivers and concerns regarding human health. Hence, in recent years, impetus on research in regenerable resources, such as sucrose, starch and cellulose, has considerably increased. For example, sucrose is one of the leading world commodities: its annual production in all forms exceeds 150 million tonnes. The potential of this regenerable, almost ubiquitous, natural product as a chemical raw-material has been well recognized .

The long-chain fatty acid esters of sucrose are non- ionic, non-toxic and biodegradable, and compare well in overall performance with other surface active compounds in detergency, emulsification, and related properties. They have generally been produced by a chemical process of transesterification reaction, using, for example, methyl fatty acid esters as the acylating agents and a base such as potassium carbonate or sodium methoxide in an aprotic solvent such as N, N-dimethylformamide [J. C. Colbert, Sugar Esters: Preparation and Applications, Noyes Data Corporation, New Jersey, (1974); Riaz Khan and Paul A. Konowicz, Kirk-Othmer Encyclopaedia of Chemical Technology, Fourth Edition, Volume 23, ISBN 0-471-52692-4 (1997)]. The limitations in most of those processes are represented by the use of DMF, a high boiling aprotic organic solvent quite difficult to remove. A solvent-less process was also developed in which sucrose was heated with a triglyceride or a methyl fatty acid ester and potassium carbonate under stirring at 140° C to give a mixture containing unreacted sucrose, sucrose mono esters (-27%), higher esters (3%), and soaps (30%) [K. J. Parker, R. A. Khan, and K. S. Mufti, US Patent 3, 996,206 (December, 7, 1976)]. For example, purified sucrose mono fatty acid esters are produced and sold by Dai-Ichi Kogyo Seiyaku Company Ltd., in Kyoto and Mitsubishi Corporation in Tokyo, Japan; they have been approved by the FDA as food additives. Sucrose mono fatty acid esters are commonly used, for example, in food formulations; on fruits and vegetable as edible semi-permeable coatings to retard ripening and reduce wastage resulting from rotting; and, because of their excellent skin compatibility, find also application in shampoos and cosmetics.

Enzyme catalyzed acylation of monosaccharides in pyridine (for example, by porcine pancreatic lipase) has been shown to regio-selectively acylate the primary hydroxyl group thereof; however, the enzyme was virtually unreactive with di- and oligo-saccharides (M. Therisod and A. M. Klibanov, J. American Chemical Society, 108, 5638- 5640, 1986) .

By way of example, selective acylation of sucrose with an acylating agent, i.e., isopropenyl acylate, in the presence of excess of lipase P Amano in pyridine for 4 days at 60°C has been shown to afford a mixture of 6-0- acyl-sucrose in 33% yeld and 6, 4 ' -di-acylate-sucrose in 8% yield (J. S. Dordick, A. J. Hacking, and R. A. Khan, USP 5 128 248; July 7, 1992) . Unfortunately, a low yield of the mono-acylated product and the use of pyridine as a solvent is a substantial disadvantage of the process; not only the solvent is difficult to recover, but its residual presence render the product toxic or smelly.

Transesterification of sucrose with vinyl laurate in the presence of celite-immobilized Thermomyces lanuginosus (T. lanuginosus or T.l.) in a mixture of DMSO and t-amyl alcohol, at 40°C, at pH 7, provided 6-O-lauroyl-sucrose ( . Ferrer, M. A. Cruces, M. Bernabe, A. Ballesteros, F. J. Plou, "Lipase-catalyzed regioselective acylation of sucrose in two-solvent mixtures", Biotechnology and Bioengineering, 65, 10-16, 1999) . However, recyclability of the celite immobilized enzyme has not been fully demonstrated. The cost of the enzyme is the major item in this process so that discarding the enzyme after every reaction makes the enzymatic process less competitive than the other current chemical processes. In addition, a serious disadvantage is also given by the use of dimethylsulfoxide (DMSO) , a toxic, high boiling solvent, which must necessarily be completely removed from the end product .

Further, it was recently discovered that enzymes like, for example, CAL-B (Novozyme 435) and T. lanuginosus, can be encapsulated in a polysaccharide- polyethylene glycol (PEG) based scaffold. The scaffold encapsulated enzymes have been shown to be able to catalyse the reaction between sucrose and, for example, vinyl laurate in a mixture of DMSO (about 17% or more) and t-amyl alcohol under standard esterification reaction conditions to afford 6-0-lauroyl-sucrose in acceptable yield; and with the advantage of recyclability of the scaffold-encapsulated enzyme for the acylation reaction, thus reducing the cost of the enzyme and thus making the process, at least in principle, economically attractive.

However, the process still necessarily used a substantial amount of a toxic and high boiling, difficult to remove, aprotic solvent like DMSO to bring sucrose in solution; namely, if the acylation reaction was performed in only t-amyl alcohol, sucrose precipitation occurred and the reaction did not proceed to afford the desired 6-0- acyl-sucrose .

Technical need of the invention

As a matter of fact, no convenient, ecological and industrially applicable processes for carrying out the selective esterification reaction of suitable sugar molecules mostly at a desired, position thereof have been till now disclosed.

Hence, there still exists a strong request from the skilled technicians for a competitive and environmentally acceptable biotechnological process (green chemistry) for the production of selectively substituted sugar esters.

In particular, specifically, there is still a need for a process for the production of sucrose esters selectively esterified mostly or substantially completely at the C-6 position of the sucrose molecule.

Today, to the knowledge of the Inventors, said type of process is still lacking.

Therefore, it is a major aim of the present invention to give a proper response to the above described technical need.

Summary of the invention

Actually, the Inventors have now surprisingly found that by carrying out an enzyme-catalysed reaction of transesterification between a suitable sugar and a suitable acylating agent in the presence of only one solvent suitable for solubilizing the acylating agent, wherein both the sugar substrate, preferably in solid form, and the enzyme are immobilized or encapsulated into a properly designed porous biopolymer scaffold, it is possible to give the proper response to the above described technical problem.

Thus, it is an object of the present invention a process for selectively acylating, i.e. esterifying, a sugar mostly on the primary C-6 position, in which a donor acyl ester is reacted preferably in a tertiary hydroxylic solvent with said sugar, which is immobilized or encapsulated into a porous biopolymer scaffold along with an enzyme catalyzer, as disclosed in the appended independent claim. Further objects of the present invention are disclosed in the appended dependent claims.

Description of the invention

The present invention deals with a process for selectively acylating a sugar moiety substrate mostly, or even substantially completely, at the primary C-6 position thereof, said process comprising at least one step in which a reactive donor acyl ester is reacted, in a suitable solvent, with said sugar moiety, characterized in that the sugar moiety is immobilized or encapsulated into a porous biopolymer scaffold together with an enzyme catalyzer .

Preferably, the process of the present invention further comprises a preliminary step in which, before the esterification reaction, a biopolymer porous scaffold is produced with known methods, for example, preferably, with a commonly used photo cross-linking method, wherein said scaffold comprises, i.e. encapsulates, the desired sugar moiety substrate together with the desired enzyme catalyzer.

In a preferred embodiment, the invention relates to a process for the production of sucrose 6-O-acylates comprising the steps of:

(a) producing a stable biopolymer scaffold of appropriate pore size, by using a polysaccharide ( s ) or a combination of a polysaccharide and a polyethylene glycol (PEG);

(b) immobilizing or encapsulating both the enzyme (a lipase) and the substrate (sucrose), preferably in solid form, into the polysaccharide-PEG scaffold, preferably during the preparation step of said scaffold;

(c) reacting the sucrose, immobilized or encapsulated together with the enzyme into the scaffold, with an acylating reagent in t-amyl alcohol to selectively give mostly, or even totally, sucrose 6-O-esters.

For example, in a preferred embodiment according to the present invention, a process is provided for acylating sucrose or a mono- or a di- or a oligo-saccharide immobilized or encapsulated, preferably, in solid form, within a porous polysaccharide-PEG scaffold along with a lipase enzyme, e.g., T. lanuginosus or CAL-B (Novozyme 435), with a donor acyl ester, e.g., a vinyl alkanoate, in an alkyl hydroxylic solvent, e.g., t-amyl alcohol, to obtain the selective acylation at the primary C-6 position of the sugar moiety.

Preferably, the sugar moiety substrate is either sucrose or any other mono- or di- or oligo-saccharide; most preferably, sucrose is used as the starting substrate material for the esterification reaction.

The acylating reagent is a donor acyl ester, i.e. a reactive ester of an alkanoic acid, preferably a vinyl, isopropenyl or trihaloethyl ester. The alkanoic acid is preferably selected from C2-C24 alkanoic acids; most preferably, acetic, butyric, lauric, or palmitic acid. Preferably, the acylating reagent is a vinyl laurate, a vinyl palmitate, a vinyl butyrate, a vinyl acetate; more preferably, a vinyl palmitate or a vinyl acetate. In general, the reactive ester should be present in excess over the theoretical requirement for a mono substitution. Typically, a ratio of 1.5-15 molar equivalents (hereinafter, ME) of the reactive ester per mole of sucrose (or of the sugar moiety) is employed; preferably, of 2-13 ME of the reactive ester per mole of sucrose: most preferably, of 2.5-10 ME of the reactive ester per mole of sucrose .

The solvent of the esterification reaction is a solvent selected from the group comprising acetone, acetonitrile, toluene a tertiary alcohol; preferably, a tertiary alkyl hydroxylic solvent; more preferably, a tertiary alcohol; most preferably, t-amyl alcohol is the preferred solvent.

The enzyme is preferably an eukariotic bacterial or fungal lipase, said lipase being preferably selected from CAL-B from Candida antarctica and T. lanuginosus (T.I.); preferably, T. lanuginosus, because the catalytic conversion of sucrose to 6-O-acyl-sucrose is more efficient and selective than with CAL-B. The lipase should be present in an amount comprised from 0.25 units to 10 units per mg of sucrose; preferably, from 0.35 to 7.5 units; most preferably, from 0.5 to 5 units. The actual weight of the enzyme will depend on the purity of the enzyme; preferably, the enzyme is purified by dialysis and freeze-dried prior to its immobilisation or encapsulation into the scaffold.

Regarding the scaffold, biopolymer scaffolds have been generated in recent years to construct bioartificial tissues or organs for treatment of patients (J. B. Leach, K. A. Bivens, C. W. Patrick Jr., C. E. Schmidt, Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds, Biotechnologies and Bioengineering, 82, 578-589, 2003). Methodologies available to generate biopolymer scaffolds are known like, for example, fibber bonding, gas foaming, phase separation/emulsification, solvent casting/ particulate leaching, interpenetrating polymer net work, chemical cross-linking, and photo cross-linking.

The photo cross-linking methodology proved to be preferred for the production of the proper scaffolds to encapsulate the required enzymes. Polysaccharides such as chitosan, hyaluronic acid (HA) and curdlan (Cud) , cellulose or cellulose derivatives, alginate, carrageenan, agar, starch, and amylose, more preferably, chitosan, are preferably employed as the basic structural materials, either alone or, preferably, in combination with ■ polyethylene glycol (PEG), said polyethylene glycol having a molecular weight comprised in the range from 1 to 200 KDa, preferably, from 2 to 50 KDa, more preferably from 4 to 8 KDa, to produce the appropriate scaffold.

Accordingly, in a preferred embodiment, the thee- dimensional structure in the scaffold for the immobilization or encapsulation of enzymes and substrates is obtained by using the photo cross-linking methodology. Preferably, as a general procedure, the polysaccharide and the PEG are first converted into methacrylate derivatives and then irradiated with UV light (365nm), in water at pH 7, in the presence of N-vinyl-pyrrolidinone as a catalyst and IRGACURE as a photoinitiator (preferably, IRGACURE 2959, i.e. the bis ( 2 , 4 , 6-trimethylbenzoyl ) -phenylphosphine oxide - CAS Registry Number: 162881-26-7;), to afford a desired scaffold.

The biopolymer scaffolds of the present invention fulfil the following requirements: (i) the thee- dimensional structure is robust to withstand repeated use in organic solvents at the required temperature, (ii) they have appropriate porosity to retain the enzyme and the substrate within the structure, and (iii) they allow the influx of the acylating reagents and the efflux of the final products during the enzymatic reaction.

Chitosan-methylacrylate (methacrylate; C-MA) with different degree of methylacrylate substitution was prepared by treatment . with methacrylic anhydride (MMA) (from 1 to 5 equivalents, depending on the required degree of substitution) in 0.5% acetic acid solution (~pH 4) at room temperature with stirring for 17-24 h (depending on the preferred degree of substitution) . The products were precipitated from ethanol. After washing, the solid was dissolved in water and freeze-dried. Similarly, PEG- dimethacrylate (PEG-MA) was prepared in methylene dichloride in the presence of triethylamine as a catalyst. The degree of the methacrylation in chitosan was determined by ¹H-NMR and diffusion edited DOSY. As an example, five different Types (I-V) of chitosan-PEG based scaffolds were prepared by photo cross-linking (UV light, 365nm) an aqueous solution of chitosan methacrylate and PEG methacrylate in the presence of 0.5% IRGACURE as a photo initiator and 0.3% N-vinyl pyrrolidinone as a catalyst, maintaining pH 4 with HC1. gelation occurred in less than 15 min at room temperature (see, for example the following SCHEME 1) .

SCHEME 1 Reactions sequence leading to a biopolymer scaffold of the invention

OH OH

The scaffolds Types I to III differ in their degree of methacrylate substituents , resulting in different structures and pore sizes, assisted by PEG-methacrylate . The scaffolds Type IV and V represent self cross-linked scaffolds; they resulted less effective for encapsulating the enzyme, in particular the scaffold Type V from PEG- methacrylate alone (see the following TABLE 1).

TABLE 1 Different Types of hydrogels prepared by photo cross-linking

* The subscript number after MA indicates the % degree of methylacrylate group substitution in chitosan and the subscript number after PEG indicates the molecular weight.

The Authors identified the potential of the Biopolymer Scaffold Technology for the encapsulation (i.e., immobilisation) of the enzymes and the substrates of the present invention (lipases and sugar moieties) in order to use this technology for the acylation reaction of sugars moieties to selectively afford sugar 6-esters, which can be used, for example, to produce industrially important products such as sugar surfactants and/or a high-intensity low calorie sweetener, 4, 1' , 6' -trichloro- 4, 1' , 6' -trideoxy galactosucrose (Sucralose®) .

Preferably, the scaffold for the encapsulation of both the enzyme and the substrate is prepared starting from a biopolymer, in particular from a polysaccharide such as chitosan, hyaluronic acid, curdlan, cellulose or its derivatives, alginate, carrageenan, agar, starch, amylose of molecular weight (MW) within the range of 4 to 500 KDa. Conveniently, methacrylate derivatives of said polysaccharide are used in combination with PEG- methacrylate; then irradiation with UV light (365 nm) causes the cross-linking of the biopolymers to give the preferred scaffold. When this process is carried out in the presence of the preferred enzyme and substrate of the present invention, their encapsulation or immobilization in the scaffold occurs. In the desired scaffold of the invention the enzyme lipase is retained, is stable in an organic solvent, and is stable and active at temperatures comprised from 40°C to 60 °C for at least 3 week.

By way of a non-limiting example, advantageously, the enzyme-catalysed selective esterification reaction between sucrose and vinyl laurate can be achieved, mostly or almost completely at the 6 position of sucrose (up to 80-90% of the final compound) and also in excellent yield (up to 80% or even more) , in just one single solvent, preferably, a tertiary alcohol like t-amyl alcohol, only when both the lipase enzyme, preferably T. lanuginosus, and the sucrose substrate are encapsulated together into the porous polysaccharide-PEG scaffold prior to the esterification reaction. Unexpectedly, that represents a considerable process advantage as compared to the use of the above mentioned mixture of DMSO and t-amyl alcohol. Advantageously, the activity of the enzyme is retained for a longer period of time at temperatures higher than the native enzyme so that the preferred enzyme can be repeatedly used for several acylation reactions, in excess of more than four reactions; preferably, more than five reactions; most preferably more than six reactions; even most preferably, more than eight reactions, thus affording a number of process advantages, among which: (i) a reduced enzyme cost because of recyclability (at least from a reduced cost of four time to a reduced cost of eight times) ; (ii) the use of a single, less toxic, low boiling, organic solvent, i.e. t-amyl alcohol, which is easy to remove, recover and recycle; and (iii) the avoidance of the use of a highly toxic, highly boiling, and difficult to remove solvent like DMSO.

Preferably, the esterification reaction takes place at temperatures ranging from 25 °C to about 75 °C; preferably, from 28 °C to about 70 °C, more preferably, from 30°C to 50°C; even more preferably, from 30°C to 40^°C, for about 18-28 h, preferably, about 22-26 h, more preferably, about 24 h, to afford predominantly or even specifically the sucrose 6-ester. If the reaction is carried on further, higher esters can form. The product can be purified by a solvent-solvent extraction process, partitioning between water and cyclohexane-butanol (1:2) mixture and some brine and/or by chromatography on a silica gel column (eluent: chloroform:methanol 8:1, or acetone-water (9:1) vol/vol). The sucrose β-ester can further be^' chlorinated to provide Sucralose®, according to the methods reported in the literature (see R. A. Khan and K. S. Mufti, GB 2,079,749 B, 1982).

Preferred embodiments of the invention

The following experimental Examples have the only scope to further illustrate the invention in details, but do not absolutely limit the same:

EXAMPLE 1

Chitosan-PEG Scaffolds

(i) Preparation of the scaffolds;

(ii) Stability of the scaffolds;

(iii) Rheological properties of the scaffolds;

(iv) Encapsulation of the enzyme (CAL-B) ; and

(v) CAL-B activity in the scaffolds.

(i) Preparation of Chitosan-PEG scaffolds

Chitosan-methacrylate (C-MA, Substrate A) with different degree of methylacrylate substitution was prepared by treatment with methacrylic anhydride (MAA) (1.1-5.0 equivalent, depending on required degree of substitution) in 0.5% acetic acid solution (~pH 5) at 20°C under stirring for 17-24 h (depending on the required degree of substitution) . The products were precipitated from ethanol. After washing, the solid was dissolved in water and freeze-dried. The degree of substitution of methylacrylate group in chitosan, determined by ¹H-NMR and diffusion edited DOSY, was comprised from 10% to 60%.

PEG-methacrylate (P-MA, Substrate B) was synthesised by treatment of lOg of PEG with 819 μ]1 of methacrylic anhydride in 30 mL methylene dichloride (CH2CI2) in the presence of 765 pL of triethylamine at room temperature (20°C) for 48 h and then the product was precipitated from 4 volumes of diethyl ether. The precipitate was washed at least three times with ether and then dried under vacuum. The degree of methacrylation of the PEG, determined by ¹H- NMR was 100%.

Different types of hydrogels were prepared by photo cross-linking using the following general procedure: to a solution of 1% w/v Substrate A in water, maintaining pH 4 with HC1, were added 1 equivalent of Substrate B, 0.5% w/v IRGACURE as „a photo initiator, 0.3% v/v N-vinyl pyrrolidinone as a catalyst and the mixture was stirred for 30 min and irradiated with UV light (365nm) . gelation occurred in less than 15 min. In the case of Type IV and V only one substrate was used (see TABLE 1).

(ii) Stability of the Chitosan-PEG scaffolds in different organic solvents at different temperatures Standard freeze-dried samples of the Type I, Type II and Type III hydrogels were placed in different solvents and kept at different temperatures for 72 h and their stability was observed.

See the following TABLE 2) .

TABLE 2 Stability of the chitosan-PEG scaffolds in water and different organic solvents at room temperature

See also the following TABLE 3 TABLE 3 Stability of the Chitosan-PEG scaffolds in water and different organic solvents at room temperature and at a temperature of 70°C

The experiments with Types I, II and III scaffolds were also performed in n-heptane, acetone, n-ButOH, DMF, DMSO and pyridine at 25°C, 50°C and 100°C. At 25°C and 50°C all the scaffolds maintained their shape and structure. They maintained their stability in the organic solvents; in pyridine they tended to swell. The stability results at 100°C are shown in TABLE 4.

TABLE 4 Stability of the scaffolds at 100°C

Temp. 100 °C DMF DMSO PYRIDINE

Type I Structure does not change Swelling Melted

Type II Structure does not change Swelling Melted

Type III Swelling Swelling Swelling

Type IV Structure becomes rigid Melted Melted (iii) Rheological measurements of the Chitosan-PEG scaffolds

The scaffold mechanical properties were measured by Stress Sweep and Frequency Sweep tests using a rheometer HAAKE Rheostress 150. The tests were performed on a few millimetres thickness gel discs arranged between the rheometer plates. In the Stress Sweep test, the samples were stressed by an increasing tension pulse (τ) in order to determine the linear viscoelastic limits for the Frequency Sweep test. From the gel response, the elastic (G' ) and viscous (G' ' ) modules were calculated and plotted against deformation (γ) in logarithmic scale graph; G' represents the elastic component and G' ' the viscous component of the gel. After establishing the sample viscoelastic limits, it is possible to perform a Frequency Sweep test maintaining the applied stress inside these limits where G' and G' ' are independent from the applied stress. In the test, the samples were stressed by a frequency decreasing pulses (fixed stress) in order to determine the gels viscoelastic spectra. G' and G' ' were plotted in logarithmic scale against pulsation (ω) .

As there are small differences in the thickness between the samples, they do not permit to apply exactly the same compression to the gels. Hence, Short Stress Sweep pre-tests were performed in order to obtain a standard gel compression. The pre-tests consisted of a series of short Stress Sweep decreasing step by step the plates gap. The resulting elastic modules (G' ) were plotted against the gap obtaining a curve. The gap at the upper curve asymptote represents the maximum sample compression and was used in the Full Stress Sweep and Frequency Sweep tests, which allowed comparison between samples with different thickness under standard conditions .

From the obtained above mentioned graphs, it was possible to realise that the hydrogels hardness was in the following order: Type III > Type IV > Type II > Type I.

(iv) Encapsulation of the enzyme (CAL-B) in the Chitosan-PEG scaffolds

Chitosan methacrylate (C-MA_i5, 15%, Substrate A) was prepared by treatment of chitosan (3 g) with methacrylic anhydride (3 ml; 1.1 equiv. ) in water (300 ml) and 0.5% acetic acid (SIGMA) .

PEGKDa dimethacrylate (Substrate B) was prepared by treating PEG_4KDa (10) with 819 μΐ (2.2 equiv.) of methacrylic anhydride in dichloromethane and 765 μΐ (2.2 equiv.) of triethylamine .

The solution comprising Substrate A (100 mg) in water (10 ml, pH 5 with 1% HC1) , Substrate B (182 mg, 1 equiv.), IRGACURE (50 mg, 0.5% w/v) , and N-vinyl pyrrolidinone (30 μΐ) was stirred in dark for -20 min; CAL-B (150 mg) lipase was then added and then the solution was divided into 10 wells (1ml each) and exposed to the UV light source for 10-15 min, until complete gelation occurred. It was then first frosted at -20°C, then at - 80°C and then was freeze-dried for 16-20 hours to obtain 10 Chitosan-PEG-CAL-B scaffolds of about 1 ml size.

(v) Enzyme activity measurement of Chitosan-PEG- CAL-B scaffolds at different times and temperatures ( Protocol )

The enzyme (CAL-B, provided in glycerol solution) „was entrapped into the scaffold as provided or after purification by 24 h dialysis (by using a membrane with a cut off of 12 KDa) and freeze-drying . The enzyme activity of the scaffolds was measured according to the following protocol: , standard enzymatic esterification of the following mixture (identified as ENZYME MIX): 1.5 ml butyric acid, 3 ml n-butanol and 95.5 ml n-heptane. The standard esterification reactions were performed with a defined enzyme quantity on 3 ml of ENZYME MIX at 50 °C for 20 minutes (stopped by adding 2 ml of methanol); the butyric acid consumed during the reactions was titrated with NaOH (0, 025M). The activity unit (U) was defined as the μπιοΐ of butyric acid converted by the enzyme in 1 minute. Enzymatic activity (EA) was calculated according to the following equation:

_{EA =} (v» - v>)'o,02s* iooo, _[u/mgHu/mli

where :

VB = volume NaOH of blank titration;

Vs = volume NaOH of sample;

E = quantity of enzyme (mg or ml, if liquid) ;

t = reaction time (always 20 min) .

The esterification activity of the free CAL-B (i.e. non-immobilized) enzyme was measured in thee different forms: non-purified enzyme; enzyme purified by dialysis and freeze-drying; and non-purified enzyme treated with UV light for 20 min. The enzyme activity was measured according to the protocol above. The mean enzyme activity was 3.96 U/mg for the purified enzyme; and 57.78 U/mg for the unpurified enzyme and 55,95 U/mg for the unpurified enzyme treated with UV light.

Moreover, CAL-B lipase was immobilized in the scaffolds by dissolving into 1 ml of scaffold substrate solution (for Type II, see TABLE 1), 3 mg of purified CAL- B (or 0.1 ml of unpurified CAL-B), and exposing to UV light for^' 20 min. The enzyme activity was measured according to the protocol above. The resulting enzyme in the scaffold was freeze-dried and placed into 3 ml of ENZYME MIX for a standard esterification activity measurement; the blank was composed of 1 ml of freeze- dried scaffolds without enzyme. The enzyme activity was 1.15 U/mg for the purified enzyme; and 34.4 U/ml for the unpurified enzyme.

In order to identify the efficient recycling (or reuse) capability of the scaffold-encapsulated CAL-B, the enzyme activity of the scaffolds was determined at different temperatures and times. The purified CAL-B (3.3 mg) was entrapped into 1 ml of standard Type I and Type II hydrogels and then freeze-dried. The scaffold-encapsulated CAL-B was then used for the standard esterification activity reaction. After the reaction the scaffolds were left in pure heptane at 4°C, the enzyme activity measurements were performed every 24 h for 4 times, according to the protocol previously described.

It was unexpectedly found that, even with some variability, each scaffold-encapsulated CAL-B, purified or unpurified, had the activity similar to that of the free enzyme .

Further enzyme activity measurements were carried out with purified enzyme in Type I and Type II scaffolds. Five samples were used for each scaffold Types. It was found that, in both of the scaffold Types, the esterification activities were higher than the ones of the free enzyme (previously calculated as 3.96 U/mg). The activity was maintained constant even after 5 days of repeated measurements on the same scaffold; that demonstrated that the enzyme was efficiently retained inside the scaffold (see TABLES 5 and 6) . TABLE 5 Summary Enzyme Activity of CAL-B Scaffold Type I over 5 days period (storage conditions 4°C)

TABLE 6 Summary Enzyme Activity of CAL-B Scaffold Type II over 5 days period (storage conditions 4°C)

1st day 2nd day 3rd day 4th day 5th day

Exp. activity activity activity activity activity

(U/mg) (U/mg) (U/mg) (U/mg) (U/mg)

1 2,44 3,56 3,81 3, 77 3, 77

2 4, 12 4, 03 4,01 4,09 4,25

3 3,45 4, 03 4,31 4, 05 4, 05

4 3,86 4, 05 3, 92 4, 27 3, 79 5 4, 12 4,48 4,20 4, 22 4, 35

Average 3,60 4,03 4,05 4,08 4,04

The activity measurements of the scaffold entrapped enzyme stored at 4°C were performed on CAL-B scaffold Type I, once a week for six weeks. After six weeks only a small decrease in the activity was observed; thus confirming that retention of the enzyme in the scaffold was efficient. A comparative activity of the scaffold entrapped enzyme stored at 4 °C and at room temperature (RT) also confirmed the stability of the enzyme.

The CAL-B scaffold Type I (stored at 50°C) activity was measured twice a week and the average weekly data were recorded. These results revealed that, at 50°C, the enzyme remained active up to thee weeks, when, on its part, the native free enzyme lost its activity during the second week (see TABLE 7) . The free enzyme (stored at 50°C) activity was measured again to obtain activity data between week one and week two; the activity was completely lost after one week (see TABLE 8). TABLE 7 Summary of Enzyme Activity comparisons between CAL-B-Scaffold Type I and Free CAL-B stored at 50 °C

TABLE 8 Experimental Details of Free Enzyme Activity (storage conditions 50 °C) NaOH (ml) Activity (U/mg)

A 11, 60 2,60

1^st week activity

E 19, 30

B 18,40 0,00

2^nd week activity

E 16, 85

Wherein:

A, B = free enzyme samples;

E = reagents only EXAMPLE 2

Preparation of Chitosan-PEG^-Thermomyces lanuginosus (T. lanuginosus) scaffold Type I

To a solution of chitosan-methacrylate (100 mg, 15%; C-MA15) in 10 ml of water, pH 5 with HC1 (1% solution), were added 182 mg PEG_4KDa-dimethacrylate ( PEG4- A; 1 equiv.), 50 mg IRGACURE (0,5%) from CIBA, 30 μΐ N-vinyl- pyrrolidinone, and 500 mg of sucrose (final 50 mg/scaffold) . The solution was stirred in dark at ambient temperature for 10-20 min, then 150 mg of purified T. lanuginous lipase (SIGMA) were added, and then the resulting solution was divided into 10 wells (1ml each) and exposed to UV light for 10-15 min until gelation was complete. It was frosted at -20°C then at -80°C for final frosting, ^' and freeze-dried for 16-20 h to afford 10 scaffolds of about 1ml size each.

EXAMPLE 3

HA-PEG_4K-CAL-B scaffold

(i) .Preparation of HA-PEG_4K-CAL-B scaffold

To a solution of HA (Hyaluronic acid, 50 mg) in 5 ml of water, pH 5 with HC1 (1% solution), were added 137.5 mg of PEG_4KDa-dimethacrylate (PEG_4K-MA), 25 mg IRGACURE (0,5%) from CIBA, 15 μΐ N-vinyl-pyrrolidinone . The solution was stirred in dark at ambient temperature for 10-20 min, then 150 mg of purified CAL-B were added, and then the resulting solution was divided into 12 wells (1ml each) and exposed to UV light for 20 min until gelation was complete. It was frosted at -20°C then at -80°C for final frosting, and freeze-dried for 16-20 h to afford 12 scaffolds about 1 ml size each.

(ii) Swelling characteristics of the HA-PEG_4K-CAL-B scaffold

The 12 scaffolds were divided into the following four groups:

Group A (samples 1, 2 and 3) : the scaffolds were weighted (0.908, 0.939, 0.909 g, respectively), then were put in water for at least 5 days, and then weighted again (1.144, 1.254, 1.1634 g, respectively);

Group B (samples 4, 5 and 6) : the scaffolds were weighted (0.931, 0.94, 0.926 g, respectively), then were put in DMSO for at least 5 days, and then weighted again (0.801, 0.909, 0.844 g, respectively);

Group C (samples 7, 8 and 9) : the scaffolds were freeze-dried and weighted (0.043, 0.042, 0.043 g, respectively) , then were put in water for 5 days, and then weighted again (0.965, 0.879, 0.894 g, respectively);

Group D (samples 10, 11 and 12) : the scaffolds were freeze-dried and weighted (0.042, 0.043, 0.043 g, respectively) , then were put in DMSO for at least 5 days, and then weighted again (0.874, 0.79, 0.811 g, respectively) .

(iii) The enzyme activity of the HA-PEG_4K-CAL-B scaffold

The activity of CAL-B in the scaffold was measured, according to the protocol of EXAMPLE 1 (v) , and was found to be 2.56 U/mg. EXAMPLE 4

Curdlan-PEG_4K-CAL-B scaffold

(i) Preparation of Curdlan (Cud) -PEG_4K-CAL-B scaffold To a solution of Cud (120 mg) in 12 ml of water, pH

5 with HC1 (1% solution), were added 411.6 mg of PEG_4KDa - dimethacryiate (PEG_4K-MA, 0.72 equiv. in terms of reactive site), 60 mg IRGACURE (0,5%) from CIBA, 36 μΐ N-vinyl- pyrrolidinone . The solution was stirred in dark at ambient temperature for 10-20 min, then 150 mg of purified CAL-B were added, and then the resulting solution was divided into 12 wells (1 ml each) and exposed to UV light for 20 min until gelation was complete. It was frosted at -20°C then at -80°C for final frosting, and freeze-dried for 16- 20 h to afford 12 scaffolds about 1ml size each.

(ii) Swelling characteristics of the Cud-PEG₄K^_CAL-B scaffold

The 12 scaffolds were divided into the following four groups:

Group A (samples 1, 2 and 3) : the scaffolds were weighted (0.868, 0.917, 0.912 g, respectively), then were put in water for at least 5 days, and then weighted again (0.97, 1.029, 1.041 g, respectively);

Group B (samples 4, 5 and 6) : the scaffolds were weighted (0.759, 0.791 0.743 g, respectively), then were put in DMSO for at least 5 days, and then weighted again (0.919, 0.933, 0.927 g, respectively);

Group C (samples 7, 8 and 9) : the scaffolds were freeze-dried and weighted (0.049, 0.0506, 0.0512 g, respectively), then were put in water for 5 days, and then weighted again (0.866, 0.906, 0.957 g, respectively);

Group D (samples 10, 11 and 12) : the scaffolds were freeze-dried and weighted (0.050, 0.049, 0.046 g, respectively) , then were put in DMSO for at least 5 days, and then weighted again (0.734, 0.769, 0.762 g, respectively) .

(iii) The enzyme activity of the Cud-PEG_4K-CAL-B scaffold

The enzyme activity of the scaffold containing 3.1 mg of CAL-B was measured, according to the protocol of EXAMPLE 1 (v) , and was found to be 2.96 U/mg. EXAMPLE 5 (Comparative)

Preparation of 6-O-lauroyl-sucrose : Chitosan-PEG- CAL-B scaffold catalysed esterification of sucrose in organic solvent mixture .

As a general procedure, dry sucrose (50 mg) was treated with 3.8 ml (10 equiv. ) of vinyl laurate in DMSO (10.2 ml, 17%) and t-amyl alcohol (2-methyl-2-butanol, 47.9 ml) in the presence of the Chitosan-PEG-CAL-B (Novozyme 345) scaffold (1 ml), and molecular sieves (250 mg) with stirring at 40°C. After 16 h, an additional amount of vinyl laurate (5 equiv.) was added and the reaction continued for another 24 h. TLC on silica gel plate (chloroform: methanol 4:1 v/v) and then the plate, sprayed with 5% H₂S0₄ and heated to reveal a black (charred) spot with Rf value similar to the standard sucrose mono laurate (SML) , revealed a charred spot corresponding to -20% of the SML, along with some sucrose and fast moving products.

When the above reaction was performed at 60 °C, the yield of the SML was increased to about 40%, according to the TLC. The LC-ESI-MS revealed that actually the SML was a mixture of 6-O-lauroyl-sucrose (54%) and 6' -O-lauroyl- sucrose (41%); thus indicating low selectivity. EXAMPLE 6 (Comparative)

Preparation of 6-O-lauroyl-sucrose : Chitosan-PEG- T.l. scaffold catalyzed esterification of sucrose with vinyl laurate in 17% DMSO and t-amyl alcohol Dry sucrose (0.5 g) was treated with 3.8 ml (10 equiv. ) of vinyl laurate in DMSO (10.2 ml, 17%) and t-amyl alcohol ( 2-methyl-2-butanol , 47.9 ml) in the presence of the Chitosan-PEG-T.1. scaffold (10 ml) and molecular sieves (2 g) with stirring at 40°C. After 16 h, an additional, amount of vinyl laurate (5 equiv.) was added and the reaction continued for another 24 h. TLC on silica gel plate ( chloroform: methanol 4:1 v/v) revealed a spot corresponding to ~ 60% of the sucrose mono laurates mixture (SML) , along with some sucrose and minor fast moving products. The reaction was worked up by concentration under vacuum to afford an oil which was washed several times with hexane, the resulting semisolid material was partitioned between water and hexane-butanol (1:1) and some brine. The organic layer on concentration and drying afforded the sucrose mono laurates (SML; 450 mg) .

The purified SML was characterised by HPLC-UV, and LC-ESI-MS, and ¹H-NMR, ¹³C-NMR, 2DHSQC. The major isomer of the SML was 6-0-lauroyl-sucrose (-78%) , according to LC- ESI-MS (see TABLE 9), thus indicating higher selectivity for the C-6 position than with the CAL-B enzyme.

The high polarity of these compounds makes them amenable for detection by electrospray-ionisation mass spectrometry (ESI-MS) . As revealed by LC-ESI-MS, it was possible to identify each regio-isomer peak by its mass, of m/z 547, corresponding to the molecular ion peak. Moreover, performing the MS/MS experiments it was possible to follow the fragmentation pattern of the peaks that mainly produced fragments of m/ z 385 and m/z 367 as expected (see TABLE 9) .

TABLE 9 LC-ESI-MS results: sucrose mono laurate (SML) regio-isomers composition in samples

EXAMPLE 7 (Comparative)

Preparation of 6-0-lauroyl-sucrose : Chitosan-PEG_4K- T.l. scaffold catalyzed esterification of sucrose with vinyl laurate in 5% DMSO and t-amyl alcohol

A solution of sucrose (50 mg) in 300 μΐ of DMSO anhydrous (5%) and t-amyl alcohol (5.32 ml), 200-300 mg molecular sieves, and 1 ml scaffold (loaded with enzyme) was stirred using magnetic stirrer for 1 h at room temperature. Then 190 μΐ of vinyl laurate (5 equiv.) were added; the temperature was raised to 40°C and the reaction continued for 24 h. TLC on silica gel plate using chloroform/methanol 4:1 v/v revealed the presence of -70% sucrose 6-laurate in the resulting mixture of regioisomers ; the structure was confirmed by ¹H-NMR and ¹³C-NMR.

EXAMPLE 8

Preparation of 6-O-lauroyl-sucrose : Chitosan-PEG_4K- .1. scaffold catalyzed esterification of sucrose in t-amyl alcohol alone

(i) Preparation of the Chitosan-PEG_4K ^_T .1. -sucrose scaffold

Chitosan-methacrylate 15% (100 mg, C-MA15) was dissolved in 10 ml of water, pH 5 was maintained with HC1 (1% solution), and then were added, in the following sequence: PEG_4KDa-dimethacrylate (182 mg PEG_4K-MA, 1 equiv. , in terms of reactive site), 50 mg of IRGACURE (0,5%) from CIBA, 30 μΐ of N-vinyl-pyrrolidinone (SIGMA), 500mg (50 mg/scaffold) of sucrose. The solution was left under stirring in dark at ambient temperature for 10-20 min. and then 150 mg (final 15 mg/scaffold) of purified T. lanuginosus lipase (SIGMA) were added. The resulting solution was divided into 10 wells (1 ml each) and then exposed to UV light source for 10-15 min until complete gelation occurred. The Gel was frosted at -20°C then at - 80°C for final frosting and freeze-dried for 16-20 h to obtain 10 scaffolds around 1 ml size.

(ii) Preparation of 6-O-lauroyl-sucrose

The Chitosan-PEG_4K- .1. -sucrose scaffold (containing both enzyme and sucrose, 1 ml) was magnetically stirred (-200 rpm) in 5.62 ml t-amyl alcohol in the presence of 200-300 mg of molecular sieves for 1 h at room temperature and then added of 190 μΐ of vinyl laurate (5 equiv.). The temperature was then raised to 40°C and the reaction was carried out for 24 h. TLC using chloroform/methanol 4:1 v/v revealed the presence of -80% or more of sucrose 6- laurate and -15% sucrose di-laurate. The reaction was worked up 'by concentration under vacuum to afford an oil which was washed several times with hexane, the resulting semisolid material was partitioned between water and hexane-butanol (1:1) and some brine. The organic layer on concentration and drying afforded the sucrose 6-laurate (60.8 mg) , with good yield and excellent selectivity.

Sucrose 6-laurate was characterised by high resolution NMR spectroscopy

EXAMPLE 9

Recycling of Chitosan-PEG-T .1. -sucrose scaffold for the esterification reaction of sucrose with vinyl laurate in t-amyl alcohol

The general procedure for the re-use of the

Chitosan-PEG-T .1. -sucrose scaffold adopted was: after the esterification reaction shown above, the scaffold was taken out- of the reaction mixture and washed with heptane : butanol (1:1, 15 ml), dried in a vacuum oven (-1.5 h) , soaked overnight with a sucrose solution (100 mg/ml) to re-encapsulate sucrose into the scaffold, and then freeze-dried overnight. The scaffold was then used for the successive esterification reactions and worked up as described previously (see EXAMPLE 8) .

The Chitosan-PEG-T .1. -sucrose scaffold was recycled five times to catalyse the esterification reaction of sucrose with vinyl laurate in t-amyl-alcohol ; the yield of sucrose laurate was progressively reduced from the initial 80% to -60%.

EXAMPLE 10

Preparation of 6-O-palmitoyl-sucrose (on 5g scale) by using Chitosan-PEG_4K-T .1. -sucrose scaffold catalysed esterification of sucrose in t-amyl alcohol

(i) Preparation of the Chitosan-MAi₅

To a solution of chitosan in 200 ml of water containing 0.5% acetic acid (~pH 5) was added 1 equivalent of methacrylic anhydride (MAA, 1.8 ml) and the mixture was stirred at room temperature (20°C) for 17 h. The reaction product (C-MA15) was then precipitated from 3 volumes of 100% ethanol, filtered on Whatman paper filter grade 113 (30 μπι) and washed with 100% ethanol 3-4 times in order to remove the unreacted MAA. The C-MA15 was dried under vacuum for 1 h and then resuspended in a minimum amount of water and freeze-dried to obtain 1.8 g of C-MA15. The degree of methacrylation in C-MA15 was confirmed by ¹H-NMR and DOSY analysis.

(ii) Preparation of PEG_4K-dimethacrylate (PEG_4K-MA; 10 g scale)

To a solution of PEG_4K (10 g) in dichloromethane (30 ml) were added 2.2 equivalents of methacrylic anhydride (820 μΐ) and 2.2 equivalents of triethylamine (TEA, 766 μΐ); the mixture was then stirred at room temperature (~20°C) for 48h. The product PEG_4K-MA was then precipitated from 3 volumes of diethyl ether, and the precipitate was filtered on Whatman glass microfiber filter grade C (1,2 μπι) , washed with diethyl ether 2-3 times to remove the unreacted MAA, and dried over night under vacuum to afford PEG_4K-MA (9.4 g) . The structure of PEG_4K-dimethacrylate was confirmed by ¹H-NMR and DOSY analysis .

(iii) T. lanuginosus lipase purification by dialysis

In order to remove most of the glycerol and other preservatives from T. lanuginosus lipase (purchased from Sigma) the enzyme solution was dialyzed using a 12KDa cutoff dialysis membrane in milli-Q water at 4°C for 24 h and then freeze-dried for 16-20 h.

(iv) Sucrose and T. lanuginosus lipase immobilized in C-MAi₅-PEG_4K-MA scaffold (10 scaffolds, ~1 ml each) C-MA₁₅ (100 mg) was dissolved in 10 ml of water at ~pH 5; the pH was controlled during dissolution and then sucrose was added (1 g; final 100 mg/scaffold) . To this solution 1 equivalent of PEG₄K^_MA (182 mg) was added followed by addition of 50 mg I RGACURE ( C I BA ) and 30 μΐ N - vinyl-pyrrolidinone . The mixture was then stirred in dark conditions at room temperature for 10-20 min. and then added with 150 mg of freeze-dried T. lanuginosus lipase (final 15 mg/scaffold). After the enzyme was completely resuspended, the solution was divided into 10 wells (1 ml each, about 1 cm diameter) and then exposed to UV light source (Black light, 365 nm) for 10-15 min. , until complete gelation occurred. The gels were frosted at -20°C and then at -80°C for final frosting before freeze-drying for 16-20 h to obtain 10 scaffolds, about 1 ml size, embedded with the enzyme and sucrose.

(v) Preparation of sucrose 6-palmitate (5 g scale) To 50 scaffolds, prepared as described above and loaded with 750 mg enzyme and 5 g of sucrose in total, were added 450 ml of t-amyl alcohol (2-methyl-2-buthanol) and 16 g of activated molecular sieves. The mixture was stirred for 1 h at room temperature, then added with 5 equivalents of vinyl palmitate (10.3 g) and stirred at 30°C for 24 h. The reaction is followed by TLC (silica gel plate, using chloroform: methanol 4:1 v/v and developed by H₂S0₄) . TLC revealed that sucrose 6-palmitate was the major product along with a minor sucrose di-palmitate and a faster moving product. A comparison of the reaction of sucrose immobilized with T. lanuginosus in the chitosan scaffold (100 mg/scaffold) with vinyl palmitate in t-amyl alcohol with those in which sucrose was dispersed in the reaction mixture in t-amyl alcohol and 5-17% DMSO, revealed that the reaction without DMSO was indeed superior either for yield or selectivity.

At the end of the reaction, the scaffolds were recovered and the solution containing sucrose 6- monopalmitate was isolated and purified as described below.

(vi) Isolation and Purification of sucrose 6- monopalmitate

After the removal of the scaffolds, the reaction mixture was filtered on a Whatman glass microfiber filter grade F (0.7 μιτι) ; the solvent was concentrated on a rotary evaporator to obtain a semisolid white precipitate. It was then divided and placed into six 50 ml tubes; 30 ml of hexane were added to each tube and vigorously vortexed in order to wash out the unreacted vinyl-palmitate and fatty acids. The phases were separated by centrifugation (4000 rpm) and the upper hexane phase was removed. The hexane was repeatedly washed 6 times and then, 20 ml of diethyl ether were added to each tube and vigorously vortexed in order to remove the residual free fatty acids. Again, the phases were separated by centrifugation and the upper diethyl ether phase was removed. The diethyl ether was repeatedly washed 5 times; after the last washing, 20 ml of water were added to each tube and the semisolid phase was resuspended by vigorous vortexing. Then 25 ml of a cyclohexane : butanol (1:2) mixture were added to each tube and vigorously vortexed to extract the sucrose monopalmitate from the water layer. The phases were separated by centrifugation (4000 rpm) and the upper organic solvent phase was collected. The extraction was repeated several times (normally 5-7 times); the presence of sucrose monopalmitate was followed by TLC (chloroform: methanol 4:1 v/v) , which revealed the presence of about -80% of sucrose 6-palmitate and -15% of sucrose di- palmitate. The organic phases were collected and evaporated on a rotary evaporator to obtain the solid product, which was taken in an appropriate amount of water and then freeze-dried to afford, after silica gel column chromatography using chloroform-methanol (8:1, vol/vol) a yield of sucrose 6-palmitate of about 4 g.

The purity of sucrose 6-palmitate was evaluated by ¹H-NMR (considering the protons shift caused by the palmitic acid esterification on the sucrose molecule) , and also by HPLC on a C₁₈ column under water : acetonitrile gradient, and by TLC.

EXAMPLE 11

Preparation of 6-O-butyryl-sucrose : esterification of sucrose with vinyl butyrate using Chitosan-PEG-

T.l. -sucrose scaffold in t-amyl alcohol

The Chitosan-PEG-T .1. -sucrose scaffold (1 ml) was magnetically stirred (-200 rpm) in 5.8 ml of t-amyl alcohol in the presence of 200-300 mg of molecular sieves for 1 h at room temperature and then added with 93 μΐ of vinyl butyrate (5 equiv. ) . The temperature was then raised to 30°C and the reaction was carried out for 24 h. TLC using chloroform/methanol 4:1 v/v revealed a yield of -60% of sucrose 6-butyrate and -20% sucrose di-butyrate. The reaction was worked up by concentration under vacuum to afford an oil which was washed several times with hexane, then the resulting semisolid material was partitioned between water and hexane-butanol (1:1) and some brine. The organic layer upon concentration and drying afforded, after chromatography on silica gel column using aqueous acetone (9:1, v/v), 6-O-butyryl-sucrose (33 mg, 55%, in terms of sucrose conversion) . The ¹H-N R of 6-O-butyryl- sucrose was consistent with the literature report (J S Dordick, A J Hacking, and R A Khan, USP 5,128,248, July 7, 1992) .

EXAMPLE 12

Preparation of 6-O-acetyl-sucrose : esterification of sucrose with vinyl acetate using Chitosan-PEG-T .1. - sucrose scaffold in t-amyl alcohol

Chitosan-PEG-T .1. -sucrose scaffold (1 ml) was magnetically stirred (-200 rpm) in 5.9 ml t-amyl alcohol in the presence of 200-300 mg of molecular sieves for 1 h at room temperature and then added with 67.5 μΐ of vinyl acetate (5 equiv.) . The temperature was then raised to 30°C and the reaction was carried out for 24 h. TLC using chloroform: methanol 4:1 v/v revealed a yield of ~70% of sucrose 6-acetate and -25% sucrose di-acetate and some faster moving products. The reaction was worked up as described above to afford 6-O-acetyl-sucrose (33.6 mg, 60%, in terms of sucrose conversion, see TABLE 10) . The ^""^"H-NMR of 6-O-acetyl-sucrose was consistent with the literature report (R A Khan, Keith Smith, Andrew Pelter and Jin Zhao, USP 5,440,026, August 8, 1995; J S Dordick, A J Hacking, and R A Khan, USP 5,128,248, July 7, 1992).

TABLE 10 Esterification of sucrose with vinyl acetate using Chitosan-PEG-T .1. -sucrose scaffold in t-amyl alcohol

Final

t-Amyl Vinyl % Sucrose % yield

% Sucrose yield of

acetate 6-acetate of Sucrose alcohol di-acetate Sucrose 6- 6-acetate acetate

16.2 μΐ

5.9838 ml 40% 0% 22.4 mg 35%

(1. 2 eq)

33.75 μΐ

5.9676 ml 40% 5% 22.4 mg

(2.5 eq) 35%

67,5 μΐ

5.933 ml 50% 30%

(5eq) 25 mg 45%

101.3 μΐ

5.899 ml 70% 25%

(7.5eq) 33.6 mg 60%

Claims

A process for selectively acylating a sugar moiety substrate mostly at the primary C-6 position thereof, said process comprising at least one step in which a donor acyl ester is reacted in a solvent, with said sugar moiety, characterized in that the sugar moiety is immobilized or encapsulated, together with an enzyme catalyzer, into a porous biopolymer scaffold.

The process according to claim 1, in which said sugar moiety is selected from sucrose or a mono- or a di- or a oligo-saccharide; preferably, sucrose; more preferably, sucrose in solid form.

The process according to claims 1 and 2, in which said donor acyl ester is a reactive vinyl, isopropenyl or trihaloethyl ester of an alkanoic acid or a benzoic acid; preferably, of a C2-C2 alkanoic acid, an acetic, butyric, lauric, or palmitic acid; most preferably, of a palmitic or acetic acid.

The process according to claim 3, in which said donor acyl ester is a vinyl alkanoate; preferably a vinyl laurate, a vinyl acetate, a vinyl butyrate, a vinyl palmitate; most preferably, a vinyl palmitate or a vinyl acetate.

The process according to anyone of the preceding claims, in which said solvent is selected from the group comprising acetone, acetonitrile, toluene, a tertiary alkyl hydroxylic solvent; more preferably, a tertiary alcohol; most preferably, t-amyl alcohol.

6. The process according to anyone of the preceding claims, in which said enzyme catalyzer is an eukariotic bacterial or fungal lipase; preferably, selected from T. lanuginosus or CAL-B; most preferably, from T. lanuginosus.

7. The process according to anyone of the preceding claims, in which said biopolymer scaffold substantially is a porous polysaccharide-polyethylene based scaffold.

8. The process according to claim 7, in which said polysaccharide is selected from a chitosan, hyaluronic acid, curdlan, cellulose or a cellulose derivative, alginate, carrageenan, agar, starch, amylose; preferably, from a chitosan.

9. The process according to claim 7, in which said polyethylene glycol has a molecular weight comprised in the range from 1 to 10 KDa; preferably, from 4 to 8 KDa.

10. The process according to anyone of the preceding claims, in which the donor acyl ester is present in a ratio of 2.5 to 10 ME per mole of sugar moiety.

11. The process according to anyone of the preceding claims, in which the lipase is present in an amount comprised from 0.25 units to 10 units per mg of sucrose; preferably, from 0.35 to 7.5 units; more preferably, from 0.5 to 5 units; even more preferably, from 0.5 to 2.5 units.

12. The process according to anyone of the preceding claims, in which the esterification reaction takes place at temperatures ranging from 25°C to about 75°C; preferably, from 28 °C to about 70 °C, more preferably, from 30°C to 50°C; even more preferably, from 30°C to 40 °C, for about 18-28 h, preferably, about 22-26 h, more preferably, about 24 h.

13. The process according to anyone of the preceding claims, further comprising a preliminary step in which, before the esterification reaction, said biopolymer porous scaffold immobilising or encapsulating said sugar moiety substrate and said enzyme catalyzer is produced by submitting to photo cross-linking with UV light at 365 nm an aqueous mixture comprising: methacrylate derivatives of said polysaccharide and of said polyethylene glycol, and said sugar moiety substrate and said enzyme catalyzer.

14. The process according to anyone of the preceding claims, for producing biodegradable surfactants, as well as for the producing an intermediate, 6-O-acyl- sucrose, which is a key intermediate in a route to the production of the high-intensity sweetener, 4,1', 6'- trichloro-4, 1' , 6' -trideoxygalactosucrose (Sucralose®) .