JP2002080488A - Method for producing oligosaccharide chain/ phenylenediamine complex, oligosaccharide chain linkered compound, sulfated oligosaccharide chain/ phenylenediamine complex compound linkered by linker compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a sulfated oligosaccharide/phenylenediamine complex by using a terminal phenylene diamine group containing polyfunctional amine linkable simply plural oligosaccharide chains controlling the number of the chains. SOLUTION: This method for producing an oligosaccharide/phenylenediamine complex collected by reacting a reduction end of an oligosaccharide chain of a saccharide having a reduction level at a low pH condition by using a phenylenediamine group-containing polyfunctional amine compound of formula (1) or (2) (n is an integer of 2-7; m is an integer of 1-3; X is OH or H) as a linker compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for assembling oligosaccharide chains simply and without damaging functional sites of the oligosaccharide chains. More specifically, the present invention relates to a method for producing an oligosaccharide-phenylenediamine complex compound capable of being assembled without impairing the functional site of the oligosaccharide, an oligosaccharide-chain-assembled linker compound, and an assembly by a linker compound. To a sulfated oligosaccharide chain / phenylenediamine complex compound.

[0002]

2. Description of the Related Art There is an example in which an oligosaccharide chain is bound to a protein or a polymer matrix by utilizing a reducing amino acid conversion reaction to enhance the biological activity of the oligosaccharide chain. However,
Oligosaccharide-linked proteins and polymer matrices are heterogeneous, far from the concept of a single molecule, the number of linked oligosaccharide chains is not uniform, and the product is the average It is only sought.

[0003]

SUMMARY OF THE INVENTION In order to elucidate the mechanism of action of oligosaccharide chains at the molecular level and to develop functional molecules such as new drugs on the basis thereof, it is necessary to adjust the number of oligosaccharide chains and to use a simple method. Therefore, a method for obtaining a single molecule was required. However, there has been almost no known example in which a plurality of sugar chains are completely bound to a polyvalent matrix to obtain a single compound having a clear structure.

The present inventors have considered that it is optimal to use a polyvalent amine compound as a linker compound, and molecularly design and produce a polyvalent linker compound, and use these linker compounds to provide a functional oligosaccharide. We worked diligently to assemble the chains. Previously, the present inventors have clarified a partial structure involved in platelet binding in sulfated polysaccharide heparin and established a method for synthesizing a partial structure including the partial structure. Based on this finding, intensive studies were conducted to assemble predetermined oligosaccharide chains to improve platelet binding activity.

[0005]

SUMMARY OF THE INVENTION A first object of the present invention is to provide:
It is an object of the present invention to provide a method for producing an aggregated oligosaccharide-phenylenediamine composite compound by using a polyvalent amine having a phenylenediamine group as a linker compound and reacting it with a reducing end under low pH conditions.
A second object of the present invention is to provide a polyvalent amine compound having a phenylenediamine group at a terminal, which makes it possible to easily assemble a plurality of oligosaccharide chains while adjusting the number thereof. A third object of the present invention is to provide a sulfated oligosaccharide / phenylenediamine complex compound aggregated by the polyamine having the phenylenediamine group as a linker compound.

The method for producing an oligosaccharide-phenylenediamine complex compound according to the present invention comprises the following formula (19) or (2) having a phenylenediamine group as a linker compound.
It is characterized in that the polysaccharide is aggregated by reacting the reducing ends of oligosaccharide chains of sugars having a reducing position under a low pH condition by a reductive amination reaction using the polyvalent amine compound of 0).

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Embedded image In the formulas (19) and (20), n represents an integer of 2 to 7, m represents an integer of 1 to 3, and X represents OH or H. In addition,
The degree of polymerization of the oligosaccharide chain is up to 5.

The oligosaccharide chain having a reducing position is represented by the following formula (2)
1) or (22), wherein the oligosaccharide / phenylenediamine complex compound is represented by the following formulas (23), (24),
It is preferable to have (25) or (26).

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The linker compound according to the present invention is a polyvalent amine compound having a phenylenediamine moiety at the terminal and represented by the following formula (27) or (28).

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Embedded image In the formulas (27) and (28), n represents an integer of 2 to 7, m represents an integer of 1 to 3, and X represents OH or H.

The oligosaccharide-phenylenediamine complex compound according to the present invention is a compound having the following formula (2) having a phenylenediamine group at the reducing end and the terminal of the oligosaccharide having a reducing position.
It is obtained by reacting a polyvalent amine compound represented by 9) or (30) by a reductive amination reaction.

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Embedded image However, in Formulas (29) and (30), n represents an integer of 2 to 7, m represents an integer of 1 to 3, and X represents OH or H.

In the sulfated oligosaccharide chain-phenylenediamine complex compound according to the present invention, the oligosaccharide chain having a reducing position is represented by the following formula (31) or (32), and the following formulas (33) and (34) , (35) or (36).

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In the present invention, oligosaccharide chains having biological activity can be easily assembled while adjusting the number thereof.
Further, according to the present invention, it becomes possible to form an aggregate of sulfated oligosaccharide chains that form a partial structure involved in platelet binding in sulfated polysaccharide heparin. With one molecule of oligosaccharide chain having a heparin partial structure, it is possible to obtain a compound having extremely low biological activity. That is, it is considered that the function of glycosaminoglycan which is a sulfated polysaccharide in vivo is expressed by a specific structure of a partial oligosaccharide in the molecule and its assembly. In an interaction involving a saccharide in a living body, the binding activity of each sugar chain is generally low, so that aggregation is a particularly important factor. According to the present invention, it becomes possible to efficiently assemble sulfated oligosaccharide chains and synthesize an aggregate having a structure having excellent platelet activity.

[0012]

【Example】

Example 1 (1) Production of Linker Compound The linker compound according to the present invention was produced by the following method. (1-1) Amide condensation reaction between m-phenylenediamine and dicarboxylic acid such as succinic acid or pimelic acid (see formula (A) in FIG. 1) (1-2) Hydroxypolyamide such as citric acid Amide condensation reaction with carboxylic acid (Formula (B) in FIG. 1)

In the reaction (A), 10 equivalents of m-phenylenediamine and 1 equivalent of succinic acid or pimelic acid are used.
The equivalent was reacted with 3 equivalents of WSCI (Water Soluble Carbodi Imide: water-soluble carbodiimide) · HCl and 3 equivalents of butyl alcohol in an inert solvent DMF at room temperature to obtain a linker compound (A1). % Indicates the respective yield when n = 2.5. The obtained linker compound was identified by NMR and mass spectrometry.

In the above reaction (B), 15 equivalents of m-phenylenediamine and 1 equivalent of citric acid are combined with WSCl / HCl.
The mixture was reacted with 4.5 equivalents and 4.5 equivalents of butyl alcohol in an inert solvent DMF at room temperature to obtain a linker compound (B1). % Indicates the yield. The obtained linker compound was identified by NMR and mass spectrometry.

[0015]

Example 2 (2) Production of Sulfated Oligosaccharide Chain Having Reducing Position (2-1) Production Method of Sulfated Oligosaccharide Chain Having Reducing Position in FIGS. 2 and 3 Only the 6th position is protected. No glucose derivative (70) and L-
The acyl-based protecting group was removed from the disaccharide obtained by condensing the idose derivative and sulfated (89), and the benzyl protecting group was catalytically reduced to remove (90). The yields are shown respectively. More specifically, a glucose derivative (70), which was synthesized from D-glucose in 7 steps, except for the 6-position, which was protected with a benzyl group, and L-form, which was activated as an imidate at the 1-position
-Condensation with the -idose derivative in 1,2-dichloromethane using trifluorotin as the activator to give the disaccharide (79), followed by removal of the acetyl protecting group with sodium methoxide (8
0). Then, using N, N-dimethylformamide as a solvent, a sulfur trioxide-pyridine complex is allowed to act to sulfonate the hydroxyl group to give (89), and then tetrahydrofuran / acetic acid / water (1: 1) using 10% palladium-carbon as a catalyst. 7 kg / cm ² of hydrogen gas was allowed to act in the mixed solvent of 1) to remove all benzyl protecting groups to obtain (90).

(2-2) Production of a sulfated oligosaccharide chain having a reducing position shown in FIGS. 4 and 5 By protecting the disaccharide 80 shown in FIG. 3 at the 4′-6 ′ position, 81 is obtained. After protecting the 2'-position with an acetyl group, the protecting group at the 4'-6'-position was removed to give 83. The free 6'-position primary hydroxyl group was selectively oxidized to a carboxylic acid and then methyl-esterified to obtain 71. 71 and imidate 24 of an azido sugar synthesized in 9 steps from 1,6-anhydroglucose
After condensing in toluene using rt-butyldimethylsilyl triflate as an activator to obtain trisaccharide 72, the acetyl protecting group was removed with sodium methoxide to obtain 84. Then, using N, N-dimethylformamide as a solvent, a sulfur trioxide-pyridine complex is allowed to act to sulphate the hydroxyl group, and further saponify the methyl ester to 86, and then use 10% palladium-carbon as a catalyst to prepare tetrahydrofuran / Water (2:
Azide groups were reduced to amino groups by the action of hydrogen gas at a pressure of 1 kg / cm2 in the mixed solvent of 1) to give 87. Then, while keeping the pH of the system at 9.5, a sulfur trioxide pyridine complex was allowed to act to sulfonate the amino group to obtain 88, and then 10%
Using palladium-carbon as a catalyst, hydrogen gas was allowed to act in a mixed solvent of acetic acid / water (5: 1) at a pressure of 7 kg / cm 2 to remove all benzyl protecting groups to obtain D.

[0017]

Example 3 (3) Preparation of Sulfated Oligosaccharide Chain / Phenylenediamine Complex Compound (3-1) Glucose is Linked to the Reducing Terminal of the Sulfated Linker Compound of Formulas A1 and B1 in FIG. The sulfated oligosaccharide chain-phenylenediamine complex compound E1, E2, E
3 was obtained (see FIGS. 6 and 7). The yields were 59% and 49%, respectively, based on the linker compound. In the figure,
For example, to explain the mass spectrometry of E2 (n = 5), m / z =
735.20 [(M-6Na + 4H) 2-], which means that all 6 Nas were removed from the structural formula, and instead 4 Hs were added. it can. 2- is this. Measurement in negative mode shows the mass of negatively charged molecules. The exchange of Na <> H is a common behavior of sulfated sugars. M / z of E3 = 719.14
[(M-9Na + 6H) 3-] removes 9 Na from the molecule and replaces 6
It means that the mass of the molecule charged with trivalent as a whole in which the number H has been entered (six have been exchanged) has been detected. Data for compound E1: δ 7.17 (2H, t, J = 8.0 Hz), 6.83 (2H,
s), 6.78 (2H, d, J = 7.6 Hz), 6.61 (2H, d, J = 7.6H
z), 4.96 (2H, s), 4.43 (2H, dd, J = 2.3Hz, J = 8.8Hz),
4.25-4.16 (8H, m), 4.12 (2H, dd, J = 8.9Hz, J = 11.8Hz),
3.90 (2H, m), 3.84 (2H, m), 3.74 (2H, m), 3.70 (2H, d
d, J = 2.4Hz, J = 7.6Hz), 3.65-3.58 (2H, m), 3.57
(2H, dd, J = 6.1Hz, J = 10.7Hz), 3.31 (2H, b), 3.08 (2
H, b), 2.68 (4H, s) Data for compound E3: δ7.15 (3H, t, J = 8.1Hz), 6.
76-6.71 (6H, m), 6.62-6.57 (3H, m), 4.99 (1H, s)
, 4.98 (2H, s), 4.47-4.43 (3H, m), 4.25-4.17 (1
2H, m), 4.16-4.ll (3H, m), 3.97-3.90 (3H, m), 3.
90-3.80 (6H, m), 3.74 (3H, m), 3.71-3.65 (3H, m), 3.
57 (3H, dd, J = 6.OHz, J = 11.4Hz), 3.26 (2H, ddd, J = 3.
4Hz, J = 4.4Hz, J = 13.4Hz), 3.19 (1H, dd, J = 3.4Hz, J =
13.4Hz), 3.07-2.94 (overlap with tablet peak at 7H, m, 3.04 ppm), 3.04 (d, 14.1Hz), 2.84 (4H, d,
J = 14.5Hz)

(3-2) The sulfated oligosaccharide of the present invention is reacted with a sulfated sugar derivative D in which glucose is bound to the reducing end of a sugar obtained by sulfating a linker compound represented by the formulas A1 and B1 in FIG. Sugar chain / phenylenediamine complex compound E
4, E5 and E6 were obtained (see FIGS. 8 and 9). The yields were 60% (E3, E4) and 43% (E5), respectively, based on the linker compound. In the case of E4 (ES
I-MS (Neg.), M / z = 903.23 [(M-
8Na + 6H) ^2- ], E5 (ESI-MS (N
eg. ), M / z = 924.24 [(M-8Na + 6
H) ^2- ], and m / z = 719.14 [(M
-9Na + 6H) ^3- . 10 and FIG.
ESI-MS spectrum for E4 and 600M
3 shows a Hz NMR spectrum. The numerical values of the NMR spectrum are shown below.

Data for Compound E4: ¹ H NMR (600 MHz, D
₂ O), d 7.15 (2H, t, J = 8.2 Hz), 6.78-6.75 (4H,
m), 6.58 (2H, d, J = 6.9 Hz), 5.26 (2H, d, J = 3.6
Hz), 5.03 (2H, s), 4.42 (2H, d, J = 2.2 Hz), 4.20
(2H, d, J = 2.5 Hz), 4.19 (2H, dd, J = 2.6 Hz, J
= 5.2 Hz), 4.10 (2H, d, J = 2.8 Hz), 4.09 (2H, d,
J = 2.8 Hz), 3.97 (2H, t, J = 3.0 Hz), 3.89 (4H,
m), 3.87 (2H, m), 3.77 (2H, m), 3.72 (2H, dd, J =
5.5 Hz, J = 2.2 Hz), 3.69 (2H, dd, J = 2.2 Hz, J =
8.5 Hz), 3.61 (2H, t, J = 9.8 Hz), 3.58 (2H, dd, J
= 5.5 Hz, J = 11.0 Hz), 3.47 (6H, s), 3.29 (2H, d
d, J = 13.5 Hz, J = 3.8 Hz), 3.25 (2H, t, J = 9.8
Hz), 3.15 (2H, dd, J = 3.5 Hz, J = 10.6 Hz), 3.01
(2H, dd, J = 8.7 Hz, J = 13.6 Hz), 2.68 (4H, s).

Data for Compound E5: ¹ H NMR (600 MHz, D ₂
O), d 7.12 (2H, t, J = 8.0 Hz), 6.74-6.70 (4H, m),
6.57 (2H, d, J = 6.6 Hz), 5.26 (2H, d, J = 3.3 H
z), 5.03 (2H, d, J = 2.8 Hz), 4.42 (2H, d, J = 2.2
Hz), 4.21 (2H, s), 4.19 (2H, dd, J = 4.4 Hz), 4.1
1 (2H, s), 4.09 (2H, m), 3.97 (2H, t, J = 3.0 Hz),
3.89 (4H, m), 3.87 (2H, m), 3.77 (2H, m), 3.72 (2
H, dd, J = 5.8 Hz, J = 2.2 Hz), 3.69 (2H, dd, J =
2.1 Hz, J = 8.4 Hz), 3.61 (2H, t, J = 9.8 Hz), 3.5
8 (2H, dd, J = 5.5 Hz, J = 11.0 Hz), 3.47 (6H, s),
3.28 (2H, dd, J = 12.6 Hz, J = 3.9 Hz), 3.25 (2H,
t, J = 9.6 Hz), 3.15 (2H, dd, J = 3.6 Hz, J = 10.4
Hz), 3.00 (2H, dd, J = 8.7 Hz, J = 13.4 Hz), 2.32
(4H, t, J = 7.3 Hz), 1.62 (4H, dd, J = 7.6 Hz), 1.
34 (2H, t, J = 7.5 Hz).

Data for Compound E6: ¹ H NMR (600 MHz, D ₂
O), δ 7.21 (3H, m), 6.95-6.89 (6H, m), 6.73 (3H,
m), 5.25 (3H, d, J = 3.6 Hz), 5.05 (3H, s), 4.48
(3H, s), 4.20 (3H, s), 4.18 (3H, s), 4.13 (3H, m),
4.09 (3H, d, J = 11.0 Hz), 3.98 (3H, s), 3.87 (6H,
m), 3.84 (3H, m), 3.76 (3H, m), 3.71 (3H, m), 3.66
(3H, d, J = 10.4 Hz), 3.62 (3H, t, J = 9.6 Hz),
3.56 (3H, m), 3.47 (9H, s), 3.36-3.21 (6H, b, 3.2
3.17-3.02 (10H, b, 3.
Overlap with peaks at 15 and 3.05 ppm), 2.85 (4H,
d, J = 14.3 Hk)

[0022]

Example 4 Sulfated Oligosaccharide / Phenylenediamine Complex Compounds E4, E5, E
Platelet binding activity of 6, E7 and sulfated disaccharide one unit D was examined by a competitive inhibition test. Hereinafter, the test method will be described in more detail. Platelet-rich plasma is collected from the peripheral blood of healthy individuals and platelets are isolated therefrom. HBSS (Hank's Balanced Sa) containing 0.2% ovalbumin
lt Solution) buffer, and adjust to a concentration of 2,000,000 cells / microliter. Prepare a sample solution (prepared by diluting with the above buffer) to measure the activity prepared at each concentration.
Add 50 microliters and 100 microliters of the platelet suspension described above and gently stir at room temperature for 30 minutes.
Next, 50 microliters of tritium-labeled heparin solution (prepared by diluting with the above buffer) was added (final concentration was about
100 nM) Stir slowly at room temperature for another 1 hour. Take 75 microliters from this reaction mixture and place on silicone oil in a slender plastic tube (make two). The tube is centrifuged at 3000 rpm for 10 minutes to allow the platelets to settle. Cut off the sedimented platelets together with the plastic tube and transfer to a glass vial. To this, 500 microliters of SOLUENE-350 (manufactured by Packard) is added and heated at 60 ° C. for 30 minutes. After cooling the solution, 150 microliters of hydrogen peroxide was added, and the mixture was further heated and stirred at 60 ° C. for 30 minutes to dissolve the platelets. After cooling the solution, 5 milliliters of a scintillation solution (Hionic-Fluor, manufactured by Packard) was added, and the mixture was stirred well, and the tritium in the solution was measured with a liquid scintillation counter. The result is shown in FIG.

In the case of compound D having only one unit of sulfated disaccharide as a heparin partial structure, even when added to a concentration as high as 1 mM, binding of tritium-labeled heparin to platelet cells is not hindered at all, and compound D It can be seen that the platelet binding activity of is very low. On the other hand, compound E4 having two units
E5 exhibited a binding inhibitory activity of about 50% at a concentration of 1 mM, and an inhibitory activity of E6 having 3 units was observed from 20 μM. At 1 mM, the binding of tritium-labeled heparin was completely inhibited. This result indicates that the platelet binding activity was dramatically increased by increasing the number of sulfated disaccharides in one molecule. That is, it was revealed that a strong biological activity was expressed by assembling sugar chains.

[0024]

According to the present invention, by using a linker compound with a polyvalent amine having a phenylenediamine group at a terminal capable of assembling so as not to impair the functional site of the oligosaccharide chain, It is possible to obtain a sulfated oligosaccharide-phenylenediamine complex compound in which a plurality of oligosaccharide chains assembled by a compound are easily assembled while adjusting the number thereof. Further, it becomes possible to form a sulfated oligosaccharide chain aggregate having a partial structure involved in platelet binding in the sulfated polysaccharide heparin.

[Brief description of the drawings]

FIG. 1 (A) shows an amide condensation reaction between m-phenylenediamine and a dicarboxylic acid such as succinic acid or pimelic acid, and FIG. 1 (B) shows a reaction between m-phenylenediamine and a hydroxypolycarboxylic acid such as citric acid. 1 shows an amide condensation reaction.

FIG. 2 shows the first half of a method for producing a sulfated oligosaccharide chain having a reducing position in the case of two sugar chains.

FIG. 3 shows the latter half of the method for producing a sulfated oligosaccharide having a reducing position in the case of a disaccharide.

FIG. 4 shows the first half of a method for producing a sulfated oligosaccharide chain having a reducing position in the case of three sugar chains.

FIG. 5 shows the latter half of the method for producing a sulfated oligosaccharide having a reducing position in the case of a trisaccharide.

FIG. 6 shows the reaction of a sulfated oligosaccharide chain / phenylenediamine complex compound of the present invention by reacting with a sulfated sugar derivative C in which glucose is bound to the reducing end of a sugar obtained by sulfating a linker compound represented by the formula A1 in FIG. A method for obtaining E1 and E2 will be described.

FIG. 7: A sulfated oligosaccharide chain / phenylenediamine complex compound of the present invention by reacting with a sulfated sugar derivative C in which glucose is bound to the reducing end of a sugar obtained by sulfating the linker compound represented by formula B1 in FIG. A method for obtaining E3 will be described.

FIG. 8: A sulfated oligosaccharide chain / phenylenediamine complex compound of the present invention by reacting with a sulfated sugar derivative D in which glucose is bound to the reducing terminal side of a sugar obtained by sulfating the linker compound represented by formula A1 in FIG. A method for obtaining E4 and E5 will be described.

FIG. 9: A sulfated oligosaccharide chain / phenylenediamine complex compound of the present invention by reacting with a sulfated sugar derivative D in which glucose is bound to the reducing end of a sugar obtained by sulfating the linker compound represented by the formula B1 in FIG. A method for obtaining E6 will be described.

FIG. 10 shows an ESI-MS spectrum for E4.

FIG. 11 shows a 600 MHz NMR spectrum for E4.

FIG. 12 shows the results of a competition inhibition test of platelet binding activity performed on sulfated oligosaccharide chains / phenylenediamine complex compounds E4, 5, 6 and sulfated disaccharide one unit D, which are heparin partial structure aggregates.

Continuation of the front page (72) Inventor Michael Sobel United States of America 13066 Fayteville Limekirn Road 9640 F term (reference) 4C057 AA17 BB03 BB04 DD01 HH06 JJ23 4H006 AA01 AB84 BJ50 BN10 BU46 BV25

Claims (5)

[Claims] 1. Using a polyamine compound of the following formula (1) or (2) having a phenylenediamine group as a linker compound, reducing the reducing end of an oligosaccharide chain of a saccharide having a reducing position under low pH conditions. A method for producing an oligosaccharide / phenylenediamine complex compound assembled by reacting by an amination reaction. Embedded image Embedded image However, in the formulas (1) and (2), n represents an integer of 2 to 7,
m represents an integer of 1 to 3, and X represents OH or H. 2. The oligosaccharide chain having a reducing position is represented by the following formula (3) or (4), and the oligo-phenylenediamine sugar chain complex compound is represented by the following formula (5), (6) or (7).
Or the method for producing an oligosaccharide / phenylenediamine complex compound according to claim 1, having (8). Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image 3. A polyamine compound having a phenylenediamine moiety at a terminal and represented by the following formula (9) or (10). Embedded image Embedded image However, in Formulas (9) and (10), n represents an integer of 2 to 7, m represents an integer of 1 to 3, and X represents OH or H. 4. A reaction in which a reducing end of an oligosaccharide chain having a reducing position and a polyvalent amine compound having a phenylenediamine group at the terminal represented by the following formula (11) or (12) are reacted by a reductive amination reaction. Sulfated oligosaccharide chain / phenylenediamine complex compound obtained by the above method. Embedded image Embedded image However, in Formulas (11) and (12), n represents an integer of 2 to 7, m represents an integer of 1 to 3, and X represents OH or H. 5. The oligosaccharide chain having a reducing position is represented by the following formula (1)
3) or (14), and the following equations (15) and (1)
The oligosaccharide chain / phenylenediamine complex compound according to claim 4, having 6), (17) or (18). Embedded image Embedded image Embedded image Embedded image Embedded image Embedded image