WO2012171966A1

WO2012171966A1 - Method for the regioselective hydrolysis of monosaccharides

Info

Publication number: WO2012171966A1
Application number: PCT/EP2012/061222
Authority: WO
Inventors: Marco Terreni; Teodora Bavaro; Alassal Qais ABU
Original assignee: Universita' Degli Studi Di Pavia Dipartimento Di Scienze Del Farmaco
Priority date: 2011-06-13
Filing date: 2012-06-13
Publication date: 2012-12-20
Also published as: ITMI20111064A1

Abstract

The present invention relates to a method for the preparation of disaccharides and polysaccharides by means of the regioselective hydrolysis of monosaccharide units in the primary position.

Description

DESCRIPTION

"Method for the regioselective hydrolysis of

monosaccharides"

[0001] The present invention relates to a method for the regioselective hydrolysis of monosaccharides having a thiocyanomethyl (-SCH₂CN) group in the anomeric position.

[0002] The possibility of obtaining activated disaccharides and/or polysaccharides in the anomeric position is the key step for performing the semi- synthesis of glycoprotein by glycosylation of reactive groups, such as the free lysine amino groups (neo-glycoproteins ) .

The synthesis of the sole monosaccharide, cyanomethyl-l-thio- -D-mannopyranoside acetylate has been described by Y. C. Lee (Lee Biochemistry 15, 1976 3956-3962). In particular, the first step consists of the direct acetylation and bromuration in the anomeric position (C-l) of -D-mannopyranoside . The substitution of the halogen with thiourea and the subsequent reaction with chloroacetonitrile allows the corresponding cyanomethyl 2 , 3 , 4 , 6-tetra-O-acetyl- 1-thio- -D-mannopyranoside to be obtained.

The same synthesis method is reported in US 2007/0253942 and US 2008/0069802. These documents disclose the preparation and claim the application of various monosaccharides activated in C-l with a specific linker, 2-imino-2-methoxyethyl , (IME- thioglycosides ) for the glycosylation of biomolecules . In US 2007/0253942, these compounds have been conjugated to the wild type deglycosylated naringinase to obtain glycocon ugates potentially useful as the recognition sites of specific membrane proteins such as lectins. In US 2008/0069802, activated thioglycosides in C-l are conjugated to amine groups of superficial lysine residues of the adenovirus capsid to alter the cellular tropism of the virus .

IME-thiomannopyranoside synthesis is also reported in WO 2010/087612 according to the above method. The monosaccharide thus activated is conjugated to the human serum albumin in an aqueous media at room temperature for 1.5 hours. The same author describes the application of the glycoconjugate obtained in the radiopharmaceutical field .

The preparation of oligomannans activated with the IME group or with its precursor thiocyanomethyl has not been reported in literature. Only a few efficient chemical approaches have been reported in literature for the preparation of disaccharides bearing in the anomeric position the IME group needed for the glycosylation of protein. The few methods for preparing oligosaccharides activated in the anomeric position with the IME group or with its precursor thiocyanomethyl is due to the fact that the protection and deprotection methods using classical chemical approaches are poorly compatible with the stability of the thiocyanomethyl group or IME in anomeric position.

With the purpose of obtaining simple and efficient methods for obtaining protected sugars bearing a single free hydroxyl, lipase and other hydrolases have previously been used in the hydrolysis of monosaccharides completely protected with acetylic groups or bearing an alkyl group in anomeric position. The regioselectivity of these enzymes is highly influenced by the type of substituent present in the anomeric position.

[0003] However, an efficient process for the preparation of di- and polysaccharides starting from monosaccharides bearing a thiocyanomethyl group in anomeric position has not yet been described.

SUMMARY OF THE INVENTION

[0004] It has surprisingly been found that some hydrolases, in particular the lipase from Candida rugosa, are able to selectively hydrolyse the protective acyl group in only one position of monosaccharides bearing a thiocyanomethyl group in anomeric position.

OBJECT OF THE INVENTION

[0005] A first object of the present invention is therefore represented by a process for the selective hydrolysis of one acetyl group in a fully acethylated monosaccharide bearing a thiocyanomethyl group in anomeric position. In particular said monosaccharide is mannose.

[0006] According to a further object, a process is described for the preparation of di- and polysaccharides starting from the monosaccharide residue bearing the only one free hydroxyl group and obtained according to the process of the invention.

[0007] Further, the present invention describes the products cyanomethyl-6-O- ( 2 , 3 , 4 , 6-tetra-O-acetyl- -D-mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside, cyanomethyl ( -D-2 ' , 3 ' , 5 ' -tri-O- acetyl-arabinofuranosyl ) - ( 1→6 ) -2 , 3 , 4-tri-O-acetyl- - D- thiomannopyranoside and cyanomethyl ( -D- 2' ',3' ',5' '-tri-0-acetyl-arabinofuranosyl)-( 1→6 ) - ( - D-2 ' , 3 ' , 4 ' -tri-O-acetyl-mannopyranosyl ) - ( 1→6 ) -2 , 3 , 4- tri-O-acetyl-oi-D- thiomannopyranoside .

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 shows the preparation of cyanomethyl 6-0- (2,3,4, 6-tetra-O-acetyl- -D- mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside according to the present invention;

Figures 2, 3 and 4 show particular embodiments of the invention .

DETAILED DESCRIPTION OF THE INVENTION

[0009] As said above, a first object of the present invention is represented by a process for the regioselective hydrolysis of one protecting group in a monosaccharide fully protected with acyl groups and bearing a thiocyanomethyl group (-SCH₂CN) in anomeric position .

[0010] In particular said residue is mannose.

[0011] The reaction of hydrolysis may therefore be represented as follows:

wherein R is a protective group, thus showing that the acyl protecting group in position C6 has been hydrolysed.

According to a preferred aspect, the mannose is protected at the hydroxyl groups 2, 3, 4 and 6 with an R group =acyl (that is 2 , 3 , 4 , 6-tetra-O-acyl- mannose) .

In particular the acyl group is preferably represented by the acetyl group.

[0012] According to the process disclosed, the hydrolysis is performed using suitable enzymes, in particular hydrolase enzymes such as, for example, lipase and esterase.

According to a preferred aspect of the invention, the lipase is obtained from Candida rugosa (CRL) .

In particular for the purposes of the present invention a CRL ( Sigma-Aldrich srl, Milan, Italy) was used having a specific activity equal to 2.3 U/mg (116 U/g of support for the immobilised enzyme) .

[0013] In one embodiment of the invention, the enzymes are previously immobilised onto a suitable solid carrier.

[0014] For example, the lipases can be immobilised onto octyl-agarose or on decaoctyl Sepabeads .

[0015] As for the immobilisation phase, this is performed according to standard methods known to those skilled in the art. [0016] In the particular case of lipase, supports such as octyl-agarose gel (Octyl Sepharose^® CL-4B) or polymethacrylic-based and butyl (Sepabeads FP-BU) or decaoctyl (Sepabeads EC-OD/S) type resins, completely derivatized with hydrophobic groups, (respectively butyl and decaoctyl chains) may be used.

Alternatively, the immobilisation may take place on a macroporous matrix of silica or silicates, on a matrix formed of acrylic type adsorbent resins, even cross-linked (Amberlite^® XAD-8 or Lewatit^® E 2001/85), by an amphiphilic substrate containing lipophilic chains, on a matrix of styrene and divinylbenzene optionally containing epoxy groups (Lewatit^® R 259 K or R 260 K or Diaion^® HP-40), on a polyacrylic resin containing epoxy groups (FP 4000) or, optionally, on a polymethacrylic resin containing epoxy groups (Sepabeads^® FP-EP or Eupergit^® C) suitably derivatized with hydrophobic groups.

[0017] As for the conditions of the hydrolysis reaction, this is performed in an aqueous environment, optionally buffered to a pH of between 3 to 6 and preferably 4 to 5.

[0018] Buffers which can be usefully used include the TRIS buffer and the phosphate buffer (solution of KH₂PO₄ 10-100 mM, preferably 50 mM) or other commonly used buffers.

[0019] Moreover, the use of an organic co-solvent is possible, preferably chosen from among acetonitrile or acetone.

Preferably, up to 50% of the co-solvent may be added to the reaction mixture and, preferably, it is between about 10-30% (v/v the reaction mixture) .

[0020] During the reaction, the temperature is preferably kept constant in the range of about 0- 25°C.

[0021] While developing the present invention several tests were performed varying some of the parameters of the hydrolysis reaction, such as the concentration of the substrate, the pH and the substrate type used for the immobilisation.

[0022] The results of the tests are shown in the table below.

[0023] In particular, for performing such tests acetonitrile was used at 20% as the co-solvent, except in tests 3 and 6, in which the percentage was increased to 30%.

3 20 ECOD 5 7 0,14 90 20

4 5 ECOD 4 8 0,75 95 70

5 10 ECOD 4 7 0,81 95 50

6 20 ECOD 4 7 0,81 95 50

7 5 § 5 7 0,12 89 26

OAg=octyl-agarose (Octyl Sepharose CL-4B)

ECOD=decaoctyl sepabeads (Sepabeads EC-OD/S)

¹ Conversion % = Percentage of substrate transf

² product: cyanomethyl-2 , 3 , 4-tri-O-acetyl-a-D- thiomannopyranoside ;

yield % (6-OH) = Percentage of product (2)

hydrolised in position C-6 obtained in HPLC

§ the enzyme used is AXE

[0024] According to another embodiment of the invention, it has been unexpectedly found that under the same reaction conditions, apart from the C6 deprotected monosaccharide, there is also recovered the monosaccharide deprotected at the position C2

as a further product of the hydrolysis process of the invention .

[0025] According to another embodiment of the invention, a process for the preparation of di- and polysaccharides starting from the mannose selectively deprotected in one position and bearing a thiocyanomethyl group (-SCH₂CN) in anomeric position is described.

[0026] In particular, the mannose obtained from the regioselective hydrolysis reaction described above is conjugated with a suitably protected saccharide residue which is activated in the anomeric position, so as to obtain di- or polysaccharide units .

[0027] For instance, the conjugation may be performed with a unit of mannopyranose bearing a protective group in position 2,3,4 and 6; in this way oligomannans may be obtained.

[0028] For said purposes, the hydroxyl groups can be suitably protected with acyl groups, such as acetyl groups .

[0029] On the other side, the activator group is preferably chosen from the outgoing groups commonly used in the glycosylation reactions, such as groups active in anomeric position of the sugar donor, such as halogen groups or preferably the trichloroacetamide group.

[0030] The conjugation reaction is performed according to methods known to persons skilled in the art and is followed by appropriate isolation and purification steps of the di- or polysaccharide obtained .

[0031] According to a particularly preferred embodiment, the present invention allows to obtain the products: cyanomethyl 6-0- ( 2 , 3 , 4 , 6-tetra-O- acetyl- -D-mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside

3,

the product

and the product

s

For instance, the first product is prepared starting from cyanomethyl-2 , 3 , 4-tri-O-acetyl- -D- thiomannopyranoside, obtained by the selective hydrolysis of cyanomethyl 2 , 3 , 4-tri-O-acetyl- -D- thiomannopyranoside, subsequently conjugated with 2,3,4, 6-Tetra-O-acetyl- -D-mannopyranose

trichloroacetimidate .

[0032] In order to perform the conjugation with suitable groups of the protein, such as for example the amine groups of lysine, the di- or polysaccharide obtained according to the present invention, protected and functionalised in the anomeric position with the thiocyanomethyl group, has to be deprotected by the protective groups and activated in position CI. Commonly, said operation is performed in a single step with NaOMe/NaOH so as to obtain the IME- thioglycoside ready for conjugation to the protein. The coupling with the protein is then performed in phosphate buffer 20 mM at pH 9 and a ratio of protein: thioglycoside of 1:1. The reaction is kept under magnetic stirring at room temperature. The glycoprotein is subsequently purified by molecular exclusion chromatography and can be analysed using the MALDI technique.

EXAMPLE 1

Preparation of cyanomethyl 2,3,4-tri-O- acetyl-a-D-thiomannopyranoside (2 )

1.5 g of lipase from Candida Rugosa immobilised on decaoctyl sepabeads (Sepabeads ED/OD) was added to a solution 20mM of cyanomethyl 2 , 3 , 4 , 6-tetra-O- acetyl- -D-thiomannopyranoside (prepared as reported in literature by Y. C. Lee, Biochemistry 15, 1976 3956-3962) in phosphate buffer 50 mM containing 30% of acetonitrile and at constant pH 4. The solution was left under mechanical stirring at room temperature checking progress by means of HPLC and the pH was kept constant at a value of 4 by automatic titration. After 7 hours of incubation a conversion of 50% of the substrate was observed. The enzyme was removed by means of filtration and the product cyanomethyl-2 , 3, 4-tri-O-acetyl- -D- thiomannopyranoside thus obtained was isolated using conventional methods eliminating any residual organic solvent. The product was extracted from the aqueous solution with ethyl acetate. After evaporation of the combined organic extracts at reduced pressure, the residue was purified by means of a silica gel chromatographic column using a mixture (hexane- ethyl acetate 40:60) as the eluent thus obtaining the cyanomethyl-2 , 3, 4-tri-O-acetyl- -D- thiomannopyranoside with an overall yield of 30%.

The purified product was identified by ¹H-NMR and COSY 2D NMR analysis recorded in CDCI₃ (5=ppm) using as instrumentation a Bruker AMX 400.

¹R NMR (400 MHz, CDC1₃, 25 °C): 6 =2.03, 2.12, 2.19 (3s, 9H, CH3), 3.35 (d, 1H, J = 17.23 Hz, -SCH₂-) , 3.49 (d, 1H, J = 17.22 Hz, -SCH₂-), 3.69 (dd, 1H, H-6a ), 3.82 (dd, 1H, H-6b) , (dd, 1H, H-3), 4.1-4.18 (m, 1H, H-5), 5.27 (dd, 1H, H-3), 5.35 (t, 1H, H-4), 5.40 (dd, 1H, H-2), 5.50 (s, 1H, H-l) .

EXAMPLE 2

Preparation of cyanomethyl 6-0- (2, 3, 4, 6-tetra-O- acetyl-a-D-mannopyranosil) -2,3, 4-tri-O-acetyl-a-D- thiomannopyranoside (3)

2,3,4, 6-Tetra-O-acetyl- -D-mannopyranose

trichloroacetimidate (0.136 g; 0.276 mmol) and cyanomethyl-2, 3, 4-tri-O-acetyl- -D- thiomannopyranoside (0.05; 0.138 mmol) obtained from Example 1 were dissolved in anhydrous CH₂CI₂ (5 ml) and the solution brought to 0°C. Subsequently, boron trifluoride diethyl etherate (0.5 g) was added in the presence of activated molecular sieves (4 A), after which the mixture was left under magnetic stirring under nitrogen. After 6 hours of stirring the reaction mixture was neutralised with the addition of triethylamine . The solvent was removed in a vacuum and the residue was purified by means of a silica gel chromatographic column using a mixture (hexane- ethyl acetate 50:50) as eluent, thus supplying the cyanomethyl 6-0- (2,3,4, 6-tetra-O-acetyl- -D- mannopyranosil ) -2,3, 4-tri-O-acetyl- -D- thiomannopyranoside with a yield of 70%.

¹R NMR (400 MHz, CDC1₃, 25 ° C ) : δ = 1.95-2.22 (7s, 21H, COCH₃), 3.36 (d, 1H, J = 17.29 Hz, -SCH₂-) , 3.54 (d, 1H, J = 17.03 Hz, -SCH₂-), 3.58 (dd, 1H, H-6^'a ), 3.85 (dd, 1H, H-6'b), 4.00-4.09 (m, 1H, H-5'), 4.13 (dd, 1H, H-6a), 4.25-4.35 (m, 2H, H-5, H-6b) , 4.86 (s, 1H, H-l'), 5.20-5.35 (m, 5H, H-4 ' , H-3 ' , H-2 ' , H-4, H-3), 5.39 (dd, 1H, H-2), 5.48 (s, 1H, H-l) .

The pH during hydrolysis was controlled using a Metrohm automatic pH-Stat 718 Stat Tritino (Herisau, Switzerland) . The HPLC analysis was conducted using a HPLC Merck Hitachi L-7100 (E.Merck, Darmstadt, Germany) provided with detector UV L-7400 and an injection valve with 20 pL loop. A Gemini RP Cig (250 x 4.6 mm, 5 pm; Phenomenex, Castel Maggiore (BO), Italy) column was used. The analyses were performed at room temperature (λ=210 nm) . A mobile phase 30% of acetonitrile in KH₂PO₄ 10 mM buffer at pH 4, previously filtered and degassed, was used to monitor the hydrolysis reactions. The flow speed applied was 1 ml/min.

Monitoring was performed by mean of TLC on silica gel 60 (0.25 mm, E.Merck, Darmstadt, Germany) .

Example 3

Synthesis of cyanomethyl (a-D-2 ' , 3 ' , 5 ' -tri-O-acetyl- arabinofuranosyl) - (1→6) -2,3, 4-tri-O-acetyl-a-D- thiomannopyranoside (5)

As represented in Figure 2, 2 , 3 , 5-Tri-O-acetyl- -D- arabinofuranosyl trichloroacet imidate (0.373 g, 0.886 mmol) 4, synthesized in according to the literature (European Journal of Medicinal Chemistry 45 (2010) 2713-2718), and cyanomethyl 2 , 3 , 4-tri-O-acetyl- -D- thiomannopyranoside (0.16 g, 0.443 mmol) 2 prepared according to Example 1, were dissolved in anhydrous CH₂CI₂ (20 mL) and the mixture was stirred to room temperature. Boron trifluoride diethyl etherate (1 eq) was added in the presence of activated molecular sieves 4 A, and the solution was left to stir under nitrogen. After stirring for 1 hour the reaction mixture was neutralized by the addition of triethylamine . The solvent was removed in vacuo, and the residue was purified by flash column chromatography (hexane/EtOAc, 1:1) afforded the pure product with a yield of 62%.

¹H NMR (400 MHz, CDC1₃): δ = 2.00-2.18 (6s, 18H, COCH₃), 3.32 (d, 1H, CH₂CN), 3.58 (d, 1H, CH₂CN) , 3.98 (dd, 1H, H-5^'a ), 3.66 (dd, 1H, H-5'b), 3.62 (dd, 1H, H-6b) , 4.06 (dd, 1H, H-6a) , 4.24-4.26 (m, 1H, H-4'), 4.53 (d, 1H, H-2), 5.06 (dd, 1H, H- 3), 5.18 (dd, 1H, H-4), 5.22 (dd, 1H, H-3 ' ) , 5.27-5.36 (m, 3H, H-l', H-2', H-5), 5.46 (s, 1H, H-l).

¹³C NMR (400 MHz, CDC1₃): δ = 15.74 (CH₂CN), 21.40 (COCH₃), 63.75 (C-6), 66.99 (C-3'), 67.40 (C-5'), 68.11 (C-l'), 69.67 (C-5), 70.05 (C-4), 70.25 (C-2'), 70.57 (C-3), 71.46 (C-4'), 82.02 (C- 1), 101.31 (C-2), 170.45 (CO).

MS: ml z = 642.33 [M + Na⁺] (calcd. 642.59).

Example 4 a) Synthesis of acetyl a-D-2 ' , 3 ' , 5 ' -tri-O-acetyl- arabinofuranosyl- (1→6) 2,3, 4-tri-O-acetyl-a-D- mannopyranoside (6)

As represented in Figure 3, 2 , 3 , 5-Tri-O-acetyl- -D- arabinofuranosyl trichloroacetimidate 4 prepared according to the literature (European Journal of Medicinal Chemistry 45 (2010) 2713-2718) (2.41 g, 5.78 mmol) and 1 , 2 , 3 , 4-tetra-O-acetyl- -D- mannopyranoside (1 g, 2.89 mmol) were dissolved in anhydrous CH₂CI₂ (80 mL) and the mixture was stirred to room temperature. Boron trifluoride diethyl etherate (1 eq) was added in the presence of activated molecular sieves 4 A, and the solution was left to stir under nitrogen. After stirring for 1 hour the reaction mixture was neutralized by the addition of triethylamine . The solvent was removed in vacuo, and the residue was purified by flash column chromatography (hexane/EtOAc, 1:1) afforded the pure product with a yield of 70%.

¹R NMR (400 MHz, CDC1₃): δ = 2.00-2.10 (7s, 21H, COCH₃), 3.64 (dd, 1H, H-6b ), 3.61-3.86 (m, 2H, H-5'ab), 3.98 (dd, 1H, H-6a) , 4.02 (m, 1H, H-5), 4.62 (d, 1H, H-l ' ) , 5.07 (dd, 1H, H-3 ' ) , 5.18 (dd, 1H, H-2 ' ) , 5.23-5.34 (m, 2H, Η-4', H-2 ) , 5.37 (dd, 1H, H-4), 5.45 (dd, 1H, H-3 ) , 6.09 (s, 1H, H-l) .

¹³C NMR (400 MHz, CDC1₃): δ = 21.40 (COCH₃), 63.13 (C-6), 66.37 (C-3), 66.91 (C-5'), 68.01 (C-2), 69.09 (C-4'), 69.56 (C-2'), 69.62 (C-4), 70.47 (C-3'), 72.71 (C-5), 91.03 (C-l), 100.80 (C-l'), 116.56 (CH₂CN), 170.53 (CO) .

MS: ml z = 645.17 [M + K⁺] (calcd. 645.53) . b) Synthesis of cyanomethyl (a-D-2^{1 1} , 3^{1 1} , 5^{1 1} -tri-O- acetyl-arabinofuranosyl) - (1→6) - (a-D-2 ' , 3 ' , 4 ' -tri-O- acetyl-mannopyranosyl) - (1→6) -2,3, 4-tri-O-acetyl-a-D- thiomannopyranoside (8)

As represented in Figure 4, a-D-2 ' , 3 ' , 5 ' -Tri-O- acetyl-arabinofuranosyl ) - ( 1→6 ) -1, 2, 3, 4-tetra-0- acetyl-a-D-mannopyranoside 6 was activated in anomeric position to a-D-2 ' , 3 ' , 5 ' -tri-O-acetyl- arabinofuranosyl)-(l→6)-2,3, 4-tri-O-acetyl-a-D- mannopyranosyl trichloroacetimidate 7 by hydrazinolysis followed by reaction with trichloroacetonitrile . Mass confirmed by ES-MS. a-D-2 ',3',5'-Tri-0-acetyl-arabinofuranosyl)-( 1→6 ) -

2, 3, 4-tri-O-acetyl-a-D- mannopyranosyl trichloroacetimidate 7 (0.117 g, 0.186 mmol) and 2 , 3 , 4-tri-O-acetyl- -D-mannopyranoside 2 prepared according to Example 1 (0.03 g, 0.083 mmol) were dissolved in anhydrous CH₂CI₂ (20 mL) and the mixture was stirred to room temperature. Boron trifluoride diethyl etherate (1.5 eq) was added in the presence of activated molecular sieves 4 A, and the solution was left to stir under nitrogen. After stirring for 4 hours the reaction mixture was neutralized by the addition of triethylamine . The solvent was removed in vacuo, and the residue was purified by flash column chromatography (hexane/EtOAc, 4:6) afforded the pure product with a yield of 70%.

¹R NMR (400 MHz, CDC1₃): δ = 2.00-2.18 (9s, 27H, COCH₃), 3.40 (d, 1H, CH₂CN) , 3.55 (d, 1H, CH₂CN) , 3.53-3.86 (m, 4H, H-5^"ab, H-6'ab), 3.63- 4.03 (m, 2H, H-6ab) , 3.96 (m, 1H, H-5^'), 4.29 (m, 1H, H-4 " ) , 4.66 (d, 1H, H-2'), 4.86 (s, 1H, H-l " ) , 5.08 (d, 1H, H-2 " ) , 5.19 (dd, 1H, H-3"), 5.20 (d, 1H, H-l'), 5.25 (dd, 1H, H-5), 5.28 (m, 2H, H-4, H- 3'), 5.31 (dd, 1H, H-3), 5.38 (m, 1H, H-4'), 5.40 (dd, 1H, H-2), 5.50 (s, 1H, H-l) .

¹³C NMR (400 MHz, CDC1₃): δ = 15.19 (CH₂CN), 20.52-21.54 (COCH₃), 60.34- 77.33 (14C, ring carbons), 81.23 (C-l), 97.15 (C-l"), 100.16 (C-2'), 115.65 (CH₂CN) , 169.28-170.18 (CO) .

MS: ml z = 930.58 [M + Na⁺] (calcd. 930.83) . Example 5

Preparation of cyanomethyl 2, 3, 4-tri-O-acetyl-a- D-thiomannopyranoside and cyanomethyl 3,4,6-tri-O- acetyl-a-D-thiomannopyranoside

1.5 g of lipase from Candida Rugosa immobilised on decaoctyl sepabeads (Sepabeads ED/OD) was added to a solution 20 mM of cyanomethyl 2 , 3 , 4 , 6-tetra-0- acetyl- -D-thiomannopyranoside (prepared as reported in literature by Y. C. Lee, Biochemistry 15, 1976 3956-3962) in phosphate buffer 50 mM containing 30% of acetonitrile and at constant pH 4. The solution was left under mechanical stirring at room temperature checking progress of the reaction by means of HPLC and the pH was kept constant at a value of 4 by automatic titration. After 7 hours of incubation a conversion of 50% of the substrate was observed. The enzyme was removed by means of filtration and the products cyanomethyl-2 , 3 , 4-tri-O- acetyl- -D-thiomannopyranoside and cyanomethyl 3,4,6- tri-O-acetyl- -D-thiomannopyranoside thus obtained were isolated using conventional methods eliminating any residual organic solvent. The mixture of products was extracted from the aqueous solution with ethyl acetate. After evaporation of the combined organic extracts at reduced pressure, the residue was purified by means of a silica gel chromatographic column using a mixture of hexane-ethyl acetate 40:60 as the eluent thus obtaining the cyanomethyl-2 , 3 , 4- tri-O-acetyl- -D-thiomannopyranoside and cyanomethyl 3 , 4 , 6-tri-O-acetyl- -D-thiomannopyranoside with an overall yield of 30%.

The assays of above paragraph [0022] have been repeated obtaining the following results:

OAg=octylagarose (Octyl Sepharose CL-4B)

ECOD=decaottyl sepabeads (Sepabeads EC-OD/S)

¹ Conversion % = Percentage of transformed substrate

² Yield % (6-OH) = Percentage of product hydrolyzed at position C-6 obtained by HPLC ³ Yield % (2-OH) = Percentage of product hydrolyzed at position C-2 obtained by HPLC

Compound : cyanomethyl 2,3, 4-tri-O-acetyl- -D- thiomanno-pyranoside

¹R NMR (400 MHz, CDC1₃, 25 °C): 6=2.03, 2.12, 2.19 (3s, 9H, CH3), 3.35 (d, 1H, J = 17.23 Hz, -SCH₂-) , 3.49 (d, 1H, J = 17.22 Hz, -SCH₂- ), 3.69 (dd, 1H, H-6a ), 3.82 (dd, 1H, H-6b) , (dd, 1H, H-3), 4.1- 4.18 (m, 1H, H-5), 5.27 (dd, 1H, H-3), 5.35 (t, 1H, H-4), 5.40 (dd, 1H, H-2) , 5.50 (s, 1H, H-l) .

Compound : cyanomethyl 3, 4, 6-tri-O-acetyl- -D- thiomannopyranoside

¹R NMR (400 MHz, CDC1₃, 25 °C): 6=2.06, 2.12, 2.16 (3s, 9H, CH3), 3.35 (d, 1H, J = 17.23 Hz, -SCH₂-) , 3.48 (d, 1H, J = 17.22 Hz, -SCH₂- ), 4.15-4.22 (m, 2H, H-2, H-6b) , 4.30-4.40 (m, 2H, H-5, H-6a) , 5.18 (dd, 1H, H-3), 5.42 (dd, 1H, H-4), 5.55 (s, 1H, H-l) .

[0033] From the description given above of the regioselective hydrolysis process of monosaccharide units bearing a thiocyanomethyl group in anomeric position, a person skilled in the art may make several modifications and additions, replacing elements with others functionally equivalent so as to satisfy specific contingent requirements while remaining within the scope of the appended claims. Each of the characteristics described as belonging to a possible embodiment may be realised independently of the other embodiments described.

Claims

1. A process for the hydrolysis of one acyl group in a fully acylated monosaccharide bearing a thiocyanomethyl group in anomeric position, comprising the use of lipase obtained from Candida rugosa .

2. A process according to claim 1, wherein said hydrolysed acyl group is the acyl group in position C-6.

3. The process according to claim 1, wherein said monosaccharide residue is mannose.

4. The process according to any one of the previous claims, wherein said monosaccharide is a fully acetylated monosaccharide.

5. The process according to any one of the previous claims, wherein said lipase is immobilised on a solid support.

6. The process according to claim 5, wherein said support is chosen in the group comprising octyl- agarose and decaoctyl sepabeads .

7. The process according to any one of the previous claims, wherein said hydrolysis reaction is conducted at a pH of 3 to 6.

8. The process according to claim 7, wherein the pH value is preferably 4 to 5.

9. The process according to any one of the previous claims, wherein the concentration of the monosaccharide bearing a thiocyanomethyl group in anomeric position in the reaction mixture is 5 to 50 mM.

10. The process according to claim 9, wherein the concentration of the monosaccharide bearing a thiocyanomethyl group in anomeric position in the reaction mixture is preferably 5 to 20 mM.

11. The process according to any one of the previous claims, wherein the reaction is performed in an aqueous environment, optionally in the presence of a buffer.

12. The process according to the previous claim, wherein said buffer is chosen from the group which comprises the TRIS buffer and phosphate buffer.

13. The process according to claim 11 or 12, wherein said buffer is present in the reaction mixture in a concentration of 10-100 mM.

14. The process according to any one of the previous claims, wherein the reaction mixture further comprises an organic co-solvent chosen from among acetonitrile and acetone.

15. The process according to claim 14, wherein said co-solvent is present in a percentage of up to 50%.

16. The process according to claim 14 or 15, wherein said co-solvent is present in the concentration of 10-30% (v/v reaction mixture).

17. The process according to any one of the previous claims, wherein the hydrolysis reaction is performed on cyanomethyl-2 , 3 , 4 , 6-tetra-O-acetyl- -D- thiomannopyranoside .

18. A process for the preparation of di- and polysaccharides which comprises the steps of:

a) selectively hydrolysing one protected hydroxyl group in a fully acylated mannose bearing a thiocyanomethyl group in anomeric position;

b) conjugating the deprotected mannose obtained from phase a) with a mono or polysaccharide residue bearing a suitable activator group in anomeric position;

wherein step a) is conducted with the use of lipase obtained from Candida rugosa.

19. The process according to claim 18, wherein the monosaccharide of step a) is cyanomethyl-2 , 3 , 4 , 6- tetra-O-acyl- -D-thiomannopyranoside .

20. The process according to any one of claims 18 or 19, wherein the monosaccharide of step a) is the cyanomethyl-2, 3, 4, 6-tetra-O-acetyl- -D- thiomannopyranoside .

21. The process according to claim 18, wherein the residue of step a) is obtained according to the process of any one of claims from 1 to 17.

22. The process according to any one of claims 18 to 21, wherein in the step b) the activator group in anomeric position of the mono- or polysaccharide residue is a trichloroacetamidate .

23. The process according to claim 18, wherein said one hydrolysed acyl group in the fully acetylated mannose is the one in position C2 or C6.

24. The process according to any one of the claims 18 to 23, wherein in step b) the residue bearing an activator group in anomeric position is 2,3,4, 6-tetra-O-acetyl- -D-thiomannopyranoside or 2, 3, 5-tri-O-acetyl- -D-arabinofuranosyl

trichloroacetimidate or -D-2 ' , 3 ' , 5 ' -tri-O-acetyl- arabinofuranosyl)-(l→6)-2,3, 4-tri-O-acetyl- -D- mannopyranosyl trichloroacetimidate .

25. The compound having the formula (I)

wherein R is an acyl or H group.

26. The compound of formula (I) according to the claim 25, wherein R is an acetyl group.

27. A compound of formula:

8 wherein Ac is an acyl group.

28. The compounds of the preceding claim, wherein Ac is the acetyl group.

29. The compound according to claim 25 to 28 obtained according to the process of any one of the claims 18 to 24.

30. Use of lipase from Candida rugosa for the regioselective deprotection of a hydroxyl group protected with an acyl group, preferably acetyl, in position 6 of the mannose bearing a thiocyanomethyl (-SCH₂CN) group in anomeric position.

31. A process according to claim 1, wherein said hydrolysed acyl group is the acyl group in position C-2.