CN1160363C - A kind of simple preparation method of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6 - Google Patents

Abstract

The invention relates to the simple preparation of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6. Using mannose orthoester as raw material, condensing it can directly obtain α1-2-linked mannobiose, from which disaccharides can be prepared into α1-2-linked tetrasaccharides and trisaccharides, the former is linked with α1-3 mannose Mannan polysaccharide antigenic factor 4 can be obtained by double sugar coupling, and mannan polysaccharide antigenic factor 6 can be obtained by coupling the latter with 3,6 branched mannotriose.

Description

A kind of simple preparation method of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6

The invention belongs to the technical field of preparation of biologically active oligosaccharides, in particular to a simple preparation method of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6 which can be used for drug screening.

Oligosaccharides, polysaccharides and glycoconjugates (glycoproteins, glycolipids) are important information substances in organisms, participating in the contact process of all cells, communication, recognition and interaction of oligosaccharides on the cell surface between cells, embryogenesis , metastasis, plays an important role in signal transmission, cell motility and adhesion, and the interaction between pathogens and host cells. The latest research shows that oligosaccharides not only work with their conjugates, many oligosaccharides themselves have important physiological functions, some oligosaccharides can stimulate the immune system of plants, and some oligosaccharides can induce nitrogen fixation of rhizobia Function; some oligosaccharides can combine with glycoproteins on invading microorganisms to prevent these microorganisms from invading normal human cells, while some oligosaccharides have the function of heparin (haparin), and blood group-determining oligosaccharides are very useful Hopeful drug against cancer. Oligosaccharides have broad application prospects in agriculture and medicine. However, oligosaccharides and polysaccharides are directly used as medicines to eliminate diseases and improve health, which will lead to an update of the concept of disease prevention and treatment, and is also a major advance in life sciences. Emerging "glycoengineering" (research and development of drugs based on sugar) is at a new starting point of vigorous development. According to "Biotech News" (Biotech News) 1995, 14 (4), 7 reports, the market share of sugar drugs will increase from 13 percent in 1993 to 24 percent in 2000.

Both mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6 are components of polysaccharides present in the cell wall. They are the active sites of polysaccharides and are key pathogenic factors for certain skin diseases. Synthesizing them and further preparing vaccines can be made New drugs to treat skin diseases or other conditions caused by Candida.

The structure of mannan antigenic factor 4 is α-D-Man-1→3-(α-D-Man-1→6)-α-D-Man-1→2-α-D-Man-1→2- α-D-Man-1→2-D-Man and the structure of mannan antigenic factor 6 is α-D-Man-1→3-α-D-Man-1→2-α-D-Man-1→ 2-α-D-Man-1 → 2-α-D-Man-1 → 2-D-Man, so far there is no report on the synthesis of these two antigenic factors.

The purpose of the present invention is to adopt a brand-new idea to provide a method for preparing the two antigenic factors with simple steps, saving time and effort, and using orthoester of sugar as the starting material.

The object of the present invention is achieved like this: take the simplest bromoacyl mannose as raw material, first convert it into benzoylated orthoester, then condense under triflate catalysis to obtain 1- 2-linked disaccharides are converted into tetrasaccharide acceptors and trisaccharide acceptors through simple chemical conversion, the former is coupled with 1-3 linked mannobiose donors, and then deprotected to obtain antigenic factor 6, and then The latter is coupled with 3,6-branched mannotriose donors, and then deprotected to obtain antigenic factor 4.

The synthetic method of the present invention is: take bromoacetyl mannose (1) as raw material, prepare the orthoester (2) of mannose, take off the acetyl group in sodium methylate-methanol to obtain (3), use pyridine- Benzoyl chloride is benzoylated to give benzoylated orthoester (4), as shown in the following figure:

The orthoester (4) is condensed under the action of trimethylsilyl triflate (TMSOTf) or triethylsilyl triflate (TESOTf) to obtain the α1-2 linked disaccharide (5), As shown below:

The disaccharide (5) selectively removes the 1-position R group, and then activates to obtain the disaccharide donor (6), while the disaccharide (5) selectively removes the acetyl group to obtain the disaccharide acceptor (7), as shown in the figure below :

Disaccharide acceptor (7) couples with disaccharide donor (6) to give tetrasaccharide (8), while disaccharide acceptor (7) couples with monosaccharide donor (9) to give trisaccharide (10) ,As shown below:

Tetrasaccharide (8) is selectively deacetylated to obtain tetrasaccharide acceptor (11); and trisaccharide (10) is selectively deacetylated to obtain trisaccharide acceptor (12), as shown in the following figure:

In the above structural formula, R=alkyl or aryl, Bz=benzoyl;

A tetrasaccharide acceptor (11) is coupled with a disaccharide donor (13) to give a hexasaccharide (14); while a trisaccharide acceptor (12) is coupled to a trisaccharide donor (15) to give a hexasaccharide (16) ,As shown below:

In the above structural formula, R=alkyl or aryl, R'=acyl or alkyl, Bz=benzoyl, X=halogen, acyl or trichloroacetimide ester;

Remove the protective group of hexasaccharide (14) by conventional method to obtain mannan polysaccharide antigenic factor 6 (17); remove the protective group of hexasaccharide (16) by conventional method to obtain mannan polysaccharide antigenic factor 4 (18). As shown below:

In the above structural formula, R=H, alkyl or aryl

Described coupling reaction is carried out under Lewis acid catalysis, and used Lewis acid is silver salt, boron trifluoride, trimethylsilyl trifluoromethanesulfonate (TMSOTf), triethylsilyl trifluoromethanesulfonate (TESOTf).

The present invention will be described in detail below in conjunction with the examples.

Embodiment 1: Preparation of benzoylated orthoester 4 (3,4,6-tri-O-benzoyl-β-D-mannopyranose 1,2-allylorthoester)

Bromoacetyl mannose (tetra-O-acetyl-α-D-mannopyranosyl bromide) (1,8220 mg, 20 mmol) was dissolved in 40 ml of allyl alcohol (allyl alcohol), and 2,4 - Lutidine (2.3 ml, 20 mmol), the reaction was carried out at room temperature under stirring, and monitored by thin layer chromatography. After the reaction was completed, the reactant was concentrated to dryness to obtain the crude product 2, and 2 was suspended in 20 ml To anhydrous methanol, sodium methoxide (0.4 ml, 2 mol/L) was added, and the solution was stirred at room temperature overnight, and thin-layer chromatography analysis showed that the reaction was complete. The reaction solution was concentrated to dryness to obtain 4.7 g of 3, with a yield of 90%. 3 was dissolved in 30 ml of pyridine, and benzoyl chloride (8.2 ml, 70 mmol) was slowly added dropwise, and the reaction was carried out under stirring at room temperature. Thin-layer chromatography analysis and monitoring, completed after 3 hours, the reactant was processed by conventional methods, and the obtained residue was purified by silica gel column chromatography, and washed with ethyl acetate/petroleum ether (1/3) as eluent , and the corresponding components were collected to obtain 10 g of pure sugar ortho ester 4 with a yield of 97%. Melting point: 142°C Specific rotation [α] _D -12°

Example 2: α1-2 linked disaccharide 5 (All 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6- The preparation of tri-O-benzoyl-α-D-mannopyranoside)

4 was dried under vacuum for 2 hours, then dissolved in 80 milliliters of dry dichloromethane, at -40°C, 100 microliters of TMSOTf was added, and the reactant naturally returned to room temperature under stirring. After 1 hour, the reaction was complete. The reaction solution was neutralized with triethylamine, concentrated under reduced pressure, purified by silica gel column chromatography, rinsed with ethyl acetate/petroleum ether (1/3) as eluent, and collected corresponding components to obtain pure bis Sugar 5 6.72 g, 66% yield.

Example 3: Disaccharide donor 6 (2-O-Acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O- Preparation of benzoyl-α-D-mannopyranosyl trichloroacetimidate)

5 (2100 mg, 2 mmol) was added to 90% acetic acid (20 mL), then 590 mg (6 mmol) of sodium acetate and 0.18 mg ( ₁ mmol) of PdCl were added, and the reaction was completed after stirring at room temperature for 12 hours. The mixture was diluted with 60 ml of dichloromethane, washed with water and saturated aqueous sodium bicarbonate solution, the organic phase was evaporated to dryness under reduced pressure, and separated by silica gel column chromatography to obtain disaccharide 2-O-acetyl-3 with a free hydroxyl group in the 1 position , 4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-D-mannopyranose (1820 mg), this disaccharide was dissolved in 20 mL of dichloromethane, and then added CCl ₃ CN 0.4ml (8 mmol) and DBU (56 μ L, 0.72 mmol), the reaction was completed after stirring at room temperature for 2 hours, the reactant was concentrated, separated by column chromatography to obtain crystallized Disaccharide donor 6 1890 mg, 82% yield in two steps: mp 139-142°C; [α] _D -1.5° (c1.1, CHCl ₃ )

Example 4: Disaccharide receptor 7 (Allyl 3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D -mannopyranoside) preparation

Disaccharide 5 (2100 mg, 2 mmol) was added to 60 mL of anhydrous methanol, 1.8 mL of acetyl chloride was added to the solution at 0°C, stirred at room temperature for 10 hours, and 1.2 mL of acetyl chloride was added, detected by TLC react until the reaction is complete. The solution was neutralized to neutral with Et ₃ N, concentrated to dryness, and separated by column chromatography to obtain 7 (1870 mg, 93%): melting point 147-150°C; [α] _D -3.6° (c1.4, CHCl ₃ ) ;

Example 5: Tetrasaccharide 8 (Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl -α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α -D-mannopyranoside) preparation

Disaccharide donor 6 (1150 mg, 1 mmol) and disaccharide acceptor 7 (1010 mg, 1 mmol) were dried together under vacuum for 2 hours, then dissolved in 60 ml of anhydrous dichloromethane, and The solution was added TMSOTf (20 microliters, 0.10 mmol) at -42°C under nitrogen protection and stirring, and the reaction was carried out for 3 hours, and the temperature was naturally raised. TLC showed that the reaction was complete. The solution was neutralized to neutral with Et ₃ N, and concentrated to Dry, column chromatography to obtain 8 (1700 mg, 85%): melting point 140-145 ° C; [α] _D -2.2 ° (c1.3, CHCl ₃ ); ¹³ C NMR δ168.96,, 166.25, 166.18, 166.00，165.69，165.61，165.44，165.36，165.29，165.23，165.18，165.10，164.93，117.89，100.74，100.46，99.22，98.02，77.18，76.27，70.91，70.83，70.71，69.65，69.58，69.38，69.35，69.15， 68.88, 68.68, 67.76, 67.29, 67.13, 67.13, 63.75, 63.75, 63.54, 62.95;

Example 6: Trisaccharide 10 (Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl - Preparation of α-D-mannopyranosyl-(1-2)-3,4,6-tri-O-benzoyl-α-D-mannopyranoside)

According to the conditions of condensation of 6 and 7, monosaccharide donor 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl trichloroacetimidate (9,747 mg, 1.1 mmol) and disaccharide acceptor Condensation of 7 (980 mg, 1 mmol) afforded 101217 mg in 88% yield.

Example 7: Tetrasaccharide receptor 11 (Allyl 3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D -mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranoside ) preparation

According to the conditions of preparing 7 from 5, 890 mg of tetrasaccharide acceptor 11 was obtained from tetrasaccharide 8 (1000 mg, 0.5 mmol), with a yield of 91%: mp 137-141°C; [α] _D -0.4° (c1 .2, CHCl ₃ );

Example 8: Trisaccharide receptor 12 (Allyl 3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D Preparation of -mannopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranoside)

According to the conditions of preparing 7 from 5, trisaccharide acceptor 12 1112 mg, 86% was obtained from trisaccharide 10 (1220 mg, 0.5 mmol): melting point 132-134 ° C; [α] _D -1.7 ° (c1.0, CHCl ₃ );

Example 9: Disaccharide donor 13 (2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-, 2,4,6-tri-O-acetyl-α -D-mannopyranosyl trichloroacetimidate) preparation

From 2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl trichloroacetimidate (1290 mg, 2 mmol, its preparation see RRSchmidt, Adv.Carbohydr.Chem.Biochem., Vol 50, 1994, 21- 124) with 4,6-O-benzylidene-1,2-O-ethylidene-β-D-mannopyranose (560 mg, 2 mmol, for its preparation see Wang Weikong Fanzuo Carbohydr.Res., 151, 1999, 117-226 ) was condensed under standard conditions to obtain the disaccharide 2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl-(1→3)-4,6-O-benzylidene-1,2-O-ethylidene- β-D-mannopyranose 1570 mg, yield 90%: mp 128-132 ° C; [α] _D -1.5 ° (c1.0, CHCl ₃ ); %F ₃ CCOOH (5 mL) was treated at room temperature for 1 hour, the reaction solution was concentrated, and the residue was acetylated with pyridine (5 mL) and acetic anhydride (3 mL) at room temperature for 2 hours, and the reaction solution was processed according to a conventional method. The obtained residue and potassium carbonate (207 mg, 1.5 mmol) were dissolved in 10 ml of DMF, stirred at room temperature for 12 hours, the reaction solution was processed by conventional methods, the organic phase was concentrated, and the obtained 1 The disaccharide crude product at the free hydroxyl position was dissolved in dichloromethane (20 ml), and then CCl ₃ CN (0.3 ml, 3 mmol) and DBU (42 μl, 0.3 mmol) were added. The reaction mixture was heated at room temperature After one hour shift, the reaction was concentrated and separated by column chromatography to obtain 960 mg of disaccharide donor 13 in a total yield of 62% in four steps: mp 125-127°C; [α] _D -0.9° (c1.2, CHCl ₃ );

Example 10: Preparation of fully protected antigenic factor 6 hexasaccharide 14

According to the condensation conditions of 6 and 7, disaccharide donor 13 (103 mg, 0.1 mmol) was condensed with tetrasaccharide acceptor 11 (195 mg, 0.1 mmol) to obtain antigenic factor hexasaccharide 14, 212 mg, yield 75 %: mp142-146°C; [α] _D -2.9°(c1.1, CHCl ₃ ); ¹ H NMR δ8.05-7.26, 6.34, 6.29, 6.14, 6.00-5.70, 5.62, 5.56, 5.48, 5.30, 5.28, 5.23, 5.15, 5.13, 5.05, 4.92, 4.75-4.70, 4.68-4.50, 4.48, 4.45, 4.42-4.26, 3.88-3.84, 3.69-3.64, 3.59-3.55, 2.35, 2.24, 2.04.

Example 11: Preparation of fully protected antigenic factor 4 hexasaccharide 16

First use known method (Wang is Kong Fanzuo Angew.Chem.Int.Ed.Engl., 38,1999,1247-1249.) by the mannose Schmidt reagent of 1,2-ethylidene mannose and benzoylation Prepared 3,6 branched mannotriose (2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl(1-3)-[2,3,4,6-tetra-O- benzoyl-α-D-mannopyranosyl[1-6]]-1,2-O-ethylene-β-D-mannopyranose) its melting point is 141-144°C; [α] _D -1.4°(c1.1, CHCl ₃ ); 1 milligram of the trisaccharide thus obtained was treated with 90% trifluoroacetic acid at room temperature for 2 hours to remove the ethylidene group, then quantitatively acetylated it with pyridine-acetic anhydride, and then selectively removed the 1-position acetyl group, and then Activation with trichloroacetate-DBU gave the trisaccharide donor 15 (2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl(1-3)-[2,3,4,6 -tetra-O-benzoyl-α-D-mannopyranosyl[1-6]]-2,4-di-O-acetyl-α-D-mannopyranosyl trichloroacetimidate) 1048 mg, total yield 68% for the four-step reaction: mp125 -127°C; [α] _D -2.2°(c1.1, CHCl ₃ ); ¹ H NMR δ8.97, 8.07-7.26, 6.37, 6.22, 6.13, 5.89, 5.80, 5.69, 5.64, 5.54, 5.52, 5.42 , 5.11, 4.69-4.65, 4.64-4.60, 4.52-4.47, 4.26-4.22, 4.02-3.98, 3.77-3.74, 2.38, 2.30.

According to the method of condensation of 6 and 7, trisaccharide donor 15 (313 mg, 0.2 mmol) was condensed with trisaccharide acceptor 12 (291 mg, 0.2 mmol) to obtain antigenic factor hexasaccharide 16 437 mg with a yield of 77 %: mp126-129°C; [α] _D -3.8°(c1.1, CHCl ₃ ); ¹ H NMR δ8.08-7.24, 6.36, 6.12, 6.00-5.68, 5.61, 5.48, 5.41, 5.09, 5.08, 5.06, 4.98, 4.85-4.79, 4.61-4.40, 4.32-4.27, 4.25, 4.13, 4.02-3.98, 3.92, 3.85-3.81, 3.72-3.68, 3.62-3.58, 2.27, 2.04.

Example 12: Preparation of antigenic factor 6 hexaglycoside 17, antigenic factor 6 hexaglycoside 17a and antigenic factor 4 hexaglycoside 18:

14 (1410 mg, 0.5 mmol) or 16 (1432 mg, 0.5 mmol) was dissolved in 15 ml of methanol saturated with ammonia, reacted at room temperature for 6 days, the solvent was dried under reduced pressure, and washed with ethyl acetate As the residue, 410 mg of milky white powdered antigenic factor 6 hexaglycoside 17 was obtained from 14, with a yield of 79%: [α] _D +75°(c1.1, D ₂ O); ¹ HNMR δ5.90, 5.42, 5.34 , 5.10; Mass Spectrometry: m/z to C ₃₉ H ₆₆ O ₃₁ : theoretical value 1030; measured value: m/z 1053 (M+sodium); obtain milky white powdery antigenic factor 4 hexaglycoside 18 414 mg from 16, yield 80%: [α] _D +62°(c2.3, D ₂ O); ¹ H NMR δ5.20, 4.98, 3.41; mass spectrometry analysis: m/z vs. C ₃₉ H ₆₆ O ₃₁ : theoretical value 1030; measured Value: m/z 1053 (M+sodium).

14 (1410 mg, 0.5 mmol) was added to 90% acetic acid (10 ml), then 150 mg (1.4 mmol) of sodium acetate and 0.06 g (0.3 mmol) of PdCl ₂ were added, and the reaction was stirred at room temperature for 12 hours Complete, the allyl group at position 1 was removed, the reaction mixture was diluted with 20 ml of dichloromethane, washed with water and saturated aqueous sodium bicarbonate solution, the organic phase was evaporated to dryness under reduced pressure, and separated by silica gel column chromatography to obtain The hexasaccharide whose 1 position is a free hydroxyl group was dissolved in 15 ml of methanol saturated with ammonia, reacted at room temperature for 6 days, the solvent was dried under reduced pressure, and the residue was washed with ethyl acetate to obtain milky white powder Free hexasaccharide 17a 362 mg, yield 73%: mass spectrometry: m/z vs. C ₃₆ H ₆₂ O ₃₁ : theoretical 990; found: m/z 1013 (M+Na).

Claims (2)

1. a simple preparation method of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6, characterized in that: (1) Use bromoacetyl mannose (1) as raw material to prepare the orthoester (2) of mannose, remove the acetyl group in sodium methoxide-methanol to obtain (3), and use pyridine-benzoyl chloride benzene Formylation, to obtain benzoylated orthoester (4), as shown in the figure below:

(2) Orthoester (4) is condensed under the action of trimethylsilyl triflate or triethylsilyl triflate to obtain α1-2 linked disaccharide (5), as shown in the figure below Show:

(3) The disaccharide (5) selectively removes the 1-position R group, and then activates to obtain the disaccharide donor (6), while the disaccharide (5) selectively removes the acetyl group to obtain the disaccharide acceptor (7), as follows As shown in the figure:

(4) Disaccharide acceptor (7) is coupled with disaccharide donor (6) to obtain tetrasaccharide (8), while disaccharide acceptor (7) is coupled with monosaccharide donor (9) to obtain trisaccharide (10), as shown in the figure below:

(5) Tetrasaccharide (8) is selectively deacetylated to obtain tetrasaccharide acceptor (11); and trisaccharide (10) is selectively deacetylated to obtain trisaccharide acceptor (12), as shown in the figure below: In the above structural formula, R=alkyl or aryl, Bz=benzoyl; (6) Tetrasaccharide acceptor (11) is coupled with disaccharide donor (13) to obtain hexasaccharide (14); while trisaccharide acceptor (12) is coupled to trisaccharide donor (15) to obtain hexasaccharide (16), as shown in the figure below:

In the above structural formula, R=alkyl or aryl, R'=acyl or alkyl, Bz=benzoyl, X=halogen, acyl or trichloroacetimide ester; (7) take off the protecting group of hexasaccharide (14) by conventional method, obtain mannan polysaccharide antigenic factor 6 (17); take off the protecting group of hexasaccharide (16) by conventional method, obtain mannan polysaccharide antigenic factor 4 ( 18), as shown in the figure below:

In the above structural formula, R=H, alkyl or aryl. 2. the simple and easy preparation method of mannan polysaccharide antigenic factor 4 and mannan polysaccharide antigenic factor 6 as claimed in claim 1, it is characterized in that described coupling reaction is carried out under Lewis acid catalysis, and used Lewis acid is silver salt, three Boron Fluoride, Trimethylsilyl Triflate, Triethylsilyl Triflate.