JPH05339283A - 5-bromo-4-chloroindo-3-yl-2-sialic acid - Google Patents

Abstract

PURPOSE:To provide a new compound giving a chromogenic substrate useful for the retrieval of new sialidase-like enzyme. CONSTITUTION:The compound of formula I. The compound can be produced e.g. by reacting a compound of formula II with acetic anhydride in pyridine, chlorinating the 2-acetyl group of the resultant compound of formula III in a saturated HCl/acetyl chloride solution, reacting the product with a compound of formula IV in formamide in the presence of sodium hydride and treating the produced compound of formula V with 28% sodium methoxide and 1N sodium hydroxide in methanol.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel chromogenic substrate useful for searching and screening for enzymes. This novel chromogenic substrate is particularly useful for searching for a novel sialidase-like enzyme by recombinant DNA technology or screening of strains.

[0002]

2. Description of the Related Art Glycolipids and glycoproteins containing sialic acid are present on the surface of cells and are deeply involved in basic life phenomena such as cell differentiation, proliferation, canceration and immunity. ing. Further, it has been gradually revealed that those sugar chain portions function as receptors for viruses and toxins and are involved in cell recognition.
It is known that there are more than 200 kinds of glycolipid molecular species with different sugar chain structures, and if the glycolipid sugar chains can be selectively hydrolyzed, the physiological significance of each glycolipid will be clarified. can do. From the above viewpoints, various methods have been used to search for unknown sugar hydrolase (glycosidase) that specifically acts on the sugar chain sequence of glycolipids.

As a typical example, a glycosidase that selectively acts on the sugar chain sequence of glycolipid is searched for from a strain isolated from bovine brain which grows on a synthetic medium containing ganglioside as the sole carbon source. ing. Also recombination D
Expression of an enzyme having a new activity has been investigated by NA technology, but in each case, a great deal of labor is spent on purification of the enzyme and measurement of its activity, and development of a simple screening method is strongly desired. There is. In particular, if a method for efficiently screening on a medium an enzyme that specifically cleaves sialic acid located at the end of a specific sugar chain structure (hereinafter, this enzyme is referred to as sialidase-like enzyme) is developed,
It is considered that the technology can greatly contribute not only to the medical field but also to various fields such as research on physiological roles of glycolipids (gangliosides).

[0004]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies on a chromogenic substrate that can facilitate the search and screening of sialidase-like enzymes on the medium by strains or recombinant DNA technology, and as a result, It was found that the shown 5-bromo-4-chloroindo-3-yl-2-sialic acid can achieve its purpose, and the present invention has been completed.

[0005]

[Chemical 2]

The chromogenic substrate (1) according to the present invention is represented by the following reaction formula 1

[Chemical 3] As shown in Fig. 2, it has both a sialidase recognition site (A) and a chromogenic site (B), and in the presence of a sialidase-like enzyme, the glycosyl bond is hydrolyzed and released simultaneously with the indole type coloring site (B). Promptly undergoes air oxidation to form the indigo compound (2). The indigo compound (2) exhibits a blue color, and it becomes possible to search for a novel sialidase-like enzyme using this blue color as an index.

The chromogenic substrate (1) of the present invention has the following reaction formula 2

[Chemical 4] It can be synthesized according to the synthetic route shown in. That is,
The sialic acid (3) was reacted with acetic anhydride in pyridine to protect it as a hydroxyl group and an acetyl group, and then the compound (4) was obtained by converting the carboxyl group to an ester with diazomethane, and then the compound (4) was prepared in a saturated hydrogen chloride / acetyl chloride solution. 2) the acetyl group at the 2-position is a chloro group, which is separately treated with 3-acetoxy-
1-acetyl-5-bromo-4-chloro-indole
The condensate (7) is obtained by reacting 1-acetyl-5-bromo-4-chloro-indole (6) obtained by hydrolysis of (5) with formamide in the presence of sodium hydride. Thereafter, the condensate (7) is treated with 28% sodium methoxide and 1N sodium hydroxide in methanol to hydrolyze all the acetyl groups. The reaction residue can be purified by high performance liquid chromatography to obtain the chromogenic substrate (1).

The chromogenic substrate (1) thus obtained is
When reacted with natural sialidase (EC 3.2.1.18), the reaction proceeds promptly and the blue color peculiar to indigo compounds
(Absorption maximum 625 nm). From this experimental fact, the stereochemistry at the 2-position (COOH: β coordination, indole moiety: α coordination) of the compound of the present invention was determined.

[0009]

The action and effect of the present invention The reactivity and substrate specificity of the chromogenic substrate (1) of the present invention were investigated. The substrate (1) was rapidly decomposed by a natural sialidase (EC 3.2.1.18) and exhibited a blue color. This reaction is absorptiometry
(625 nm) followed to determine the kinetic amount of this substrate for sialidase (Km = 1.62 × 10 ⁻³ M). In addition, this substrate (1) is a natural β-galactosidase (β-Gala
It was found that it was not decomposed in the reaction with ctosidase; EC 3.2.1.23), and the substrate specificity of this substrate for sialidase was proved.

From the above, this substrate (1) can be used as an effective screening method for the search for a novel sialidase-like enzyme by recombinant DNA technology or the search for bacterial strains. Further, in the medical field, applicability as a clinical test reagent can be expected for measuring the in-vivo activity of natural sialidase. Hereinafter, the present invention will be described in more detail with reference to examples. The compound numbers in the examples are the same as the compound numbers shown in Reaction Scheme 2.

Example 1 Synthesis of acetyl derivative (4) Sialic acid (3) (2.05 g, 6.63 mmol) was added to pyridine (2).
The mixture was suspended in 5 ml), acetic anhydride (30 ml) was added under ice cooling, and after 1 hour, the mixture was returned to room temperature and stirred overnight. After concentration under reduced pressure, toluene was added to the residue and the mixture was concentrated under reduced pressure. The operation of adding toluene and concentrating under reduced pressure was repeated 3 times. The residue was dissolved in methanol (20 ml), an ether solution of diazomethane was added until the reaction solution became yellow, and the mixture was left for 5 minutes. Acetic acid was added to decompose excess diazomethane and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel: 50 g, elution solution: toluene-ethyl acetate (1: 1) -ethyl acetate).
The target fraction was concentrated under reduced pressure to obtain an oily substance. Yield: 3.2
8 g (92.7%).

Example 2 Synthesis of indole derivative (6) 3-acetoxy-1-acetyl-5-bromo-4-chloro-indole (1.00 g, 3.03 mmol) was replaced with argon, and then 80% under ice water. Sulfuric acid (5 ml) was added, followed by stirring at room temperature for 50 minutes. Ice was added to make the solution about 80 ml, and the precipitated crystals were collected by filtration. Yield: 844 mg (96.5)
%).

Example 3 Synthesis of Condensate (7) Acetyl derivative (4) (533 mg, 1.00 mmol) was converted to acetyl chloride.
It was dissolved in (10 ml), saturated hydrogen chloride-acetyl chloride solution (5 ml) was added under ice cooling, and the mixture was stirred at room temperature overnight. After concentration under reduced pressure, toluene was added to the residue and the mixture was concentrated. This operation was repeated 3 times to obtain crystals. The crystals thus obtained are D
The solution in MF (2.5 ml) was dissolved in indole derivative (6) (289 mg, 1.00 mmol) in DMF (2.5 ml), and sodium hydride (24 mg, 1.00 mmol) was added under ice cooling. The above solution was added, and the mixture was stirred under ice cooling for 1 hour and then at room temperature overnight. After concentration under reduced pressure, 10% aqueous citric acid solution and ethyl acetate were added, and the ethyl acetate layer was washed with water. The extract was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by a silica gel column to obtain a condensate (7). Yield: 547mg
(71.8%).

Example 4 Synthesis of chromogenic substrate (1) Compound (7) (260 mg, 341 μmol) was added to methanol (15 m).
28% sodium methoxide (45 μl) was added and the mixture was reacted at room temperature for 2 hours. Add Amberlist 15
It was adjusted to pH 7 and concentrated under reduced pressure. The residue was dissolved in water (3 ml), 1M sodium hydroxide (600 μmol) was added, and the mixture was stirred at room temperature for 30 min. After adding Amberlyst 15 to adjust to pH 2, the resin was immediately filtered, and 1 M ammonium acetate (pH 7.0) (10 ml) was added to the filtrate and freeze-dried. The residue is H
Purified by PLC (Cosmo Seal 5C ₁₈ , 10 × 2
50 mm, acetonitrile-50 mmol ammonium acetate buffer solution, gradient 10% -40% acetonitrile
(15 minutes), flow rate 3 ml / min, detection 254 nm, retention time 1
5.0 minutes). The peak with a retention time of 15.0 minutes was collected and freeze-dried. Ammonium acetate contained as a residue was removed by HPLC to obtain the desired chromogenic substrate (1).
(Cosmo Seal 5 C ₁₈ , 10 × 250 mm, acetonitrile-water, gradient 10% -80% acetonitrile
(10 min), flow rate 3 ml / min, detection 254 nm). Yield: 10
0 mg (54.5%). Melting point: 158-160 ° C. NMR
(Δ, ppm): 1.79 (dd, J = 12.0, 12.0H
z, 1H), 1.91 (s, 3H), 2.82 (dd, J = 1
2.0, 4.4 Hz, 1 H), 3.46 to 3.77 (m, 7
H), 7.13 (d, J = 8.8Hz, 1H), 7.28
(D, J = 8.8Hz, 1H). NMR was measured with D ₂ O (heavy water).

Example 5 Determination of kinetic amount Natural sialidase (0.04 unit) and bovine serum albumin
(0.03%) of 200 mM potassium acetate buffer (pH 4.6)
2) Keep 0.9 ml in a constant temperature layer at 25 ° C. To this, 0.1 ml of various concentrations (40 mM, 20 mM, 10 mM, 5 mM, 2.5 mM) of the substrate (1) was added, and after rapid shaking, the increase in absorption (625 nm) was monitored with an absorptiometer for 30 minutes. From the results obtained, the Km value of sialidase for this substrate (1) was 1.
It was determined to be 62 × 10 ⁻³ M.

Claims (1)

[Claims] 1. A compound represented by the following formula 1. [Chemical 1]