JPS62207299A - Novel compound izupeptin a and b and production thereof - Google Patents

Abstract

NEW MATERIAL:The compound (or its salt) having the following physical and chemical properties. Elemental analysis, composed of carbon, hydrogen, nitrogen and chlorine; molecular weight, 1475 by secondary ionization mass-spectrometry; decomposition point, 260-280 deg.C; specific rotation, [alpha]D<25>=-26.0 deg. (C=0.1, H2O); UV spectrum, having a peak at 280nm (E1cm<1> 38.0) in acidic or neutral state and at 304nm (E1cm<1> 40.3) in basic state; IR spectrum peaks; 3450, 2980, 1640, 1380 and 1220cm<-1>; solubility, soluble in water and dimethyl sulfoxide and insol uble in methanol, ethanol and acetone; color reaction, positive to phosphomolybdic acid, 80% sulfuric acid and anisaldehyde sulfuric acid reaction; ampholytic substance; color, white. USE:Antibacterial agent. PREPARATION:Izupeptin A and B can be produced by culturing actinomycetes capable of producing izupeptin A and B in a medium and separating the produced and accumulated substance from the culture product.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to novel compounds Izpeptin A and B and their pharmaceutically acceptable salts, and to processes for the preparation of said compounds. Compounds and salts according to the invention are useful as antimicrobial agents.

Background Art Vancomycin, ristocetin, etc. are known as glycopeptide antibiotics, and these glycopeptide antibiotics are effective against infections caused by Durham-positive bacteria, such as pseudomembranous colitis and methicillin-resistant staphylococci. It is known.

The present invention is based on the knowledge that actinomycetes isolated from soil by the present inventors have the ability to produce new glycopeptide-based compounds, and that this compound has strong antibacterial activity against Durham-positive bacteria. I'm there.

PROBLEM TO BE SOLVED BY THE INVENTION The objects of the present invention are: 1. Izpeptins A and B, named by the inventors. The object of the present invention is to provide novel compounds and physiologically acceptable salts thereof useful as antibacterial agents, as well as methods for producing the compounds.

Means to Solve the Problems The present invention provides novel compounds ispeptin and B and pharmaceutically acceptable salts thereof.

The novel compounds Izpeptin A and B provided by the present invention can be obtained by culturing actinomycetes capable of producing Izpeptin A and B in a medium, and injecting Izpeptin A and B into the culture.
It is produced by producing, accumulating, and collecting it.

Next, the physicochemical properties and biological properties of the present compounds Izpeptin A and B will be described.

(I) Physical and chemical properties Izpeptin A ■Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine.

An example of elemental analysis values is as follows. becomes: C5'1.68%.

H-5, 23%, N8.22%.

(J5.23% ■Molecular weight: Secondary ionization mass conflict spectrum
According to on maSS spectrometry, (M+H) is 1474, so the molecular weight of 2 is approximately 1475.
It is.

■Decomposition point: 260-280℃ ■Specific optical rotation: [α15=-26,0° (C=0.1.H2O>) ■Ultraviolet absorption spectrum: Maximum value 280 nm (E1%1
cm38. O) In basicity, maximum value 304 nm (E1om40.3) ■Infrared absorption spectrum (KBr tablet method) = 3450.
2980, 1640, 1490°1380.1220c
It has an absorption band at m-1.

■Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone ■Color reaction reaction of niline molybdenum! , 80% sulfuric acid.

Positive for anisaldehyde sulfuric acid reaction■ Distinction between basic, acidic, and neutral: Amphoteric substance [phase] An example of the pale white elemental analysis value of the substance is as follows: C51.68%.

H5, 23%, N8.22%.

C value 5.23% ■Silica gel thin layer chromatogram: Rf=0.12 (Carrier Merck silica gel, 60F
254. Thickness 0.2s, Art 5554), developing solvent: isopropyl alcohol: hexane: concentrated ammonia water (4:1:2) ■Constituent sugars and constituent amino acidsContaining asparagine, N- It was shown to contain methyl-leucine and one amino acid whose structure has not been determined other than leucine. The relative retention time in HPLC of these loose aromatic amino acids was consistent with that in the hydrolyzate of vancomycin under the same conditions. It also contains one amino sugar in addition to glucose as a constituent sugar, which is different from vancosamine or ristosamine.

Izpeptin B ■Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine.

An example of elemental analysis values is as follows. Becomes: C46, 85%.

H5.26%, N7.03%.

C26, 70% ■Molecular weight: Secondary ionization mass spectrum (secondary 1oniza-tiO
According to n mass SpectrOmeter, the molecular weight is about 1460.

■Decomposition point: 280-300℃ ■Specific optical rotation: [α]D=-16.0° (C=0.1.H2O) ■Ultraviolet absorption spectrum: Maximum value 280 nm (E
36.2> 1c@ Basic maximum value 304 nm (Elom 39.5> ■Infrared absorption spectrum (KBr tablet method): 3450,
2980, 1660, 1480゜1410.1240c
It has an absorption band at m-1.

■Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone. ■Color reaction Niline molybdic acid, 80% sulfuric acid.

Positive for anisaldehyde sulfuric acid reaction ■ Distinction between basic, acidic, and neutral: Amphoteric substance [Co., Ltd.] Color of substance: White C Silica gel thin layer chromatogram: Rf = 0.56 (Carrier: silica gel manufactured by Merck & Co., Ltd.).

60F254. Thickness 0.2mm, Art 5554)
, Developing solvent: Isopropyl alcohol: Hexane: Concentrated ammonia water (4:1:2>) From the results of amino acid analysis of the C-constituent sugar and constituent amino acid acid hydrolyzate, it contains asparagine, and the structure other than N-methyl-leucine and leucine is undetermined. In addition, it contained other aromatic amino acids, but the relative retention time of these in HPLC was consistent with that in the hydrolyzate of vancomycin under the same conditions.Also, as a constituent sugar, glucose and one other amine sugar, which is different from vancosamine or ristosamine.

(II) Biological Properties 1) Antibacterial Spectrum Minimum inhibitory concentration (MIC) values of the activities of the antibiotics ispeptin A and B against various bacteria are measured by the agar dilution method.

Bacteria MIC (μg/ml) B Staphylococcus aureus 3KB
34 (FDA 209P) 3.12
1.58KB 142 (FS-1277, PC-
G') 6.25 3.12KB 199 (E
,TC! ) 3.12 1.56 to 82
83 (No, 1)IR3A) 3.12
1.56KB 284 (No, 1-I MISA
) 3.12 1.58KB 285 (N
o, 108) IR3A) 3.12 1.
56KB 286 (No, 108-I MISA)
>100 >100 Micrococcus luteus (Hicrococcus 1uteus) KB 2
12 (ATCC9341)
1.56 0.78 Bacillus subtilis (Bacillus 5ubti l 1s) KB
211 (ATCC6633) 0.78
B 143 to 0.78 Bacillus cereus
(IFO3001) 3.12 3.
12 Bacillus megaterium KB
144 (IFo 12108) 0.7
8 0.4 Mycobacterium smegmatis
KB 42 (ATCC607) >100
>100 Escherichia coli (Escherichia coi 1) KB 213
(NIHJ) >100 >1
00 Salmonella typhimurium K
B 20 ＞100 ＞
100 Proteus vulgaris (proteus vulgaris) KB 127
(IFO3167) >100 >1
00 Pseudomonas aeruginosa K
B 115 (IFO3080) >100
>100 TalebSiel Ia pneumoniae K
B 214 (IFO10031) >100
>100 Clostridium perfringens
) to B 129 (ATCCa624)
12.5 6.25 Clostridium cifficile K
B 258 (ATCC9689) 6.25
6.25 Bacteroides fragilis K
B 169 (ATCC237/15) 10
0 100 Fusobacterium varium KB
234 (ATCC8501) >100
>100 Peptococcus variabi I 1s
)KB 235 (ATCC14955) 3
．． 12 3.122) Toxicity Izpeptin A and B 200 ma/kg were each administered intraperitoneally to mice and observed up to 2 weeks later, but no abnormalities were observed.

The strains used to produce the novel compound ispeptin A and B substances of the present invention include - As an example, 1 actinomycete AM-5289 strain was newly isolated by the present inventors from the soil of the Kasaki coast in Shizuoka prefecture. can be mentioned.

The mycological properties of this strain are as follows.

(I) Morphological properties The vegetative hyphae develop well on various agar media, and single divisions are observed. Aerial mycelia are moderately epiphytic on yeast/malt agar or starch inorganic salt agar, and exhibit a linear shape. However, when observed under a microscope, no chains of spores are observed. or,
Sclerotia, sporangia and zoospores are not produced.

(II) Properties of E.B. shearing (E, B, 5hirli) on various media
n (J) and Gottlieb (D, Gottlie)
Method b) (International Journal of
Systematic Bacteriology, Volume 16, 31
3 pages.

The following table shows the culture properties of this producing bacterium, which were investigated by (1966). The color tone was determined as a standard color using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and the code was written in parentheses along with the name of the two-color chart. The following are the results of observations in each culture medium at 27°C for 2 weeks unless otherwise specified.

Culture properties (III) Physiological properties (1) Production of melanin pigment (a) Tyrosine agar negative (b) Peptone/yeast iron agar negative (c) Glucose/peptone/gelatin medium negative (21-23°C) (d) ) Tryptone yeast solution positive (2) Tyrosinase reaction negative (3) lliiit
Hydrogen production Negative (4) Reduction of nitrate Negative (5) Liquefaction of gelatin (21-23°C) Positive (glucose/peptone/gelatin medium) (6) Oral degradation of starch False positive (7)
) Coagulation of skim milk (37°C) Negative (8
) Peptonization of skim milk (37°C) Positive (9)
Growth temperature range 15-40'C ('10)
Utilization of carbon sources (Priedum-Gotlieb agar medium) Utilize: D-glucose, D-mannitol, xylose, 1-inositol, raffinose, fructose.

Some melichiose is used; arabinose, sucrose is not used; L-rhamnose, chemical composition of cellulose (IV) Diaminopimelic acid in the cell wall is of the meso type, and the sugar composition of the whole cell contains galactose and arabinose.

The mycological properties of the above two bacteria are summarized as follows. Diaminopimelic acid in the cell wall is mes.

type, it grows on aerial hyphae and has a linear shape, but no chains of spores are observed. As the sugar composition of the whole bacterial cell,
Contains galactose and arabinose. Regarding the properties of the culture, vegetative hyphae exhibit a light brownish or purple tone, and aerial hyphae exhibit a pinkish or white tone. Produces a light purple soluble pigment.

These results indicate that the two strains contain meso-diaminopimelic acid, galactose, and arabinose, and that they are morphologically immature aerial hyphae with fragmented vegetative hyphae, indicating that they are actinomycetes closely related to the genus Nocaldea. be.

The two strains have been entrusted to the Institute of Microbiology, Agency of Industrial Science and Technology as strain AM-5289.
No. 56), mutant strains of the above strains can also be used in the method of the present invention as long as they have the ability to produce ispeptin A and B.

As the medium, a synthetic medium or a natural medium containing carbon sources, nitrogen sources, inorganic substances, and, if necessary, other nutrients suitable for the cultivation of ordinary actinomycetes, can be used.

The carbon source and nitrogen source used in the culture medium may be any type that is available to the strain used.

That is, various carbohydrates such as glucose, glycerol, fruc-1-2, maltose, mannitol-2-xylose, galactose, lipose, starch or its hydrolyzate can be used as the carbon source. The concentration is usually preferably 0.1 to 5% based on the medium. Also gluconic acid and pyruvic acid.

Various organic acids such as lactic acid and acetic acid, glycine, glutamic acid,
Various amino acids such as alanine, alcohols such as methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and various vegetable or animal fats and oils can also be used.

Examples of nitrogen sources include ammonia, ammonium chloride, ammonium phosphate, and ammonium sulfate.

Ammonium salts of various inorganic or organic acids such as ammonium nitrate, urea, peptone, NZ-amine,
Nitrogen-containing organic substances such as meat extract, yeast extract, dried yeast, corn steep liquor, casein hydrolyzate, fitschmeal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, hydrolysed product, etc. Furthermore, various amino acids such as glycine, glutamic acid, and alanine can be used.

Examples of inorganic substances include various phosphates, magnesium sulfate,
Very small amounts of heavy metal salts, such as table salt, are used.

Also, when using a mutant strain that exhibits auxotrophy.

Naturally, substances that satisfy the nutritional requirements must be added to the medium, but such nutrients may not need to be added when using a medium containing natural products.

Cultivation is usually carried out under aerobic conditions such as photography or aerated agitation culture. Practically speaking, deep aeration agitation culture is preferred. The pH of the medium is, for example, 5.0 to 8.0, preferably around neutral. The culture temperature is, for example, 20 to 40°C, but usually 26 to 32°C (preferably 27°C).
(near C).

The culture time is usually 1 to 8 days in the case of liquid culture, and the culture is terminated when the concentrations of ispeptin A and BS in the culture reach the maximum. These culture conditions, such as medium composition, liquid properties of the medium, culture temperature, stirring rate, and aeration mother, are adjusted and selected as appropriate depending on the type of bacterial strain used, external conditions, and the like. If foaming occurs in liquid culture, use 1, for example, silicone oil.

Antifoaming agents such as vegetable oil and surfactants are used as appropriate.

The present izpeptins A and B accumulated in the culture thus obtained are usually produced in the culture filtrate.

However, about 10% of the production amount may be produced in the bacterial body.

To collect ispeptin A and B from culture filtrate 1
The means normally used to collect metabolites from microbial cultures may be used alone, in combination in any order, or repeatedly. Examples include filtration, centrifugation, dialysis, drying, freeze-drying, adsorption, desorption, and methods that utilize differences in solubility in various solvents (e.g., precipitation,
Crystallization.

Methods such as recrystallization, dissolution, countercurrent distribution, etc.) and chromatography are used.

Since ispeptin A and B are mainly produced and accumulated in the culture filtrate, in order to separate and collect one compound, it is preferable to collect it from the culture filtrate after removing the bacterial cells from the culture solution. For example, for chromatography, cation exchangers and anion exchangers (eg ion exchange resins, Sephadex ion exchangers) are used.

(ion exchange cellulose, etc.), gel filtration media, activated carbon.

High porous polymers (e.g. Amberlite XAD-
2 (manufactured by Rohm and Haas) and Diaion HP
Chromatography using -20 (manufactured by Mitsubishi Chemical Industries, Ltd.), aluminum 9 florisil, silica gel, cellulose, etc. is advantageously used.

Representative suitable salts of ispeptin A and B include:
Organic and inorganic acids (e.g. sulfuric acid, phosphoric acid.

Hydrochloric acid, acetic acid, succinic acid, citric acid, lactic acid, maleic acid,
Acid addition salts produced by reacting with fumaric acid, etc.) and strong bases (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, ammonium hydroxide, jetanolamine, etc.) in a conventional manner. This includes salts produced by Since the manufacturing method of these salts is well known, the explanation thereof will be omitted.

Effect of the invention The novel compounds according to the invention are useful as antibacterial agents.

EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited thereby.

Example In a 500ml Yosaka Lough flask, glucose 0.1%, potato starch 2.4%, tryptone 0.5%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.4
Dispense 100 ml of liquid medium (pH 7,0) containing %
Steam sterilized at 121°C for 15 minutes. This was inoculated with one platinum loop from the preserved slant.

Two seedlings were obtained by culturing at 27°C for 3 days. 2.0% soluble starch per 50Q jar fermenter
A liquid medium (pH 7,0) containing 0% soybean flour, 0.3% sodium chloride, and 0.3% calcium carbonate was prepared and steam sterilized at 121°C for 30 minutes. 900ml of the above seed mother was transplanted to this, stirring speed 25Or, l)
, m,, Aeration stirring culture was carried out for 68 hours at 27°C under conditions of maximum aeration of 15Q and 7 minutes.

Centrifuge the culture solution using a Sharpless centrifuge (10,00
Or, f), m,) to separate the bacterial cells and culture supernatant. Approximately 25% of the obtained culture supernatant was filtered using Celite as a filtration aid, and then adsorbed through an anion exchange resin Diaion PA-416 [OH] (manufactured by Mitsubishi Kasei, 4.5Q) through a column. After washing with (11), it was eluted with a 0.5N acetic acid aqueous solution (13λ).The obtained eluate was
After concentrating to 0.00 ml, the mixture was freeze-dried to obtain 6.7 g of assembled material. This composite material was suspended in methanol, the insoluble portion was filtered off, and the suspension was dried under reduced pressure to obtain 5.7C1. 40m
Polyamide C-200 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was dissolved in dimethyl sulfoxide.

A 400 m column was used and developed with water. The eluted active portion was analyzed by silica gel A-layer chromatography (Improper tool: hexane: concentrated aqueous ammonia = 4:1:2), and A (Rf = 0.12) and B (Rf = 0.56) were analyzed.
It was fractionated into two components. After concentrating each of them under reduced pressure, they were freeze-dried to obtain 480 mq of component A and 540 mq of component B. 1Nl each of the two components! 2 dissolved in an aqueous solution, and a preparative column for high performance liquid chromatography (Yamamura Chemical Laboratory W: A-32
4 (ODS); '10x300mm), O, I
M-1! Elution was performed with ammonium buffer (10% to 30% acetonitrile aqueous concentration gradient system) at a flow rate of 3 ml/min.

The active portion was collected and lyophilized to yield 310 mg of component A.

330 mq of component B was obtained as a white powder.

Claims (3)

[Claims] (1) A novel compound ispeptin A and its pharmaceutically acceptable salts having the following physicochemical properties. [1] Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine. [2] Molecular weight: Secondary ionization mass spectrum
According to ion mass spectrometry, (M+H)^+ is 1474, so the molecular weight is approximately 147.
It is 5. [3] Decomposition point: 260-280°C [4] Specific rotation: [α]^2^5_D=-26.0°(
C=0.1, H_2O) [5] Ultraviolet absorption spectrum: In acidic or neutral, maximum value 280 nm (E^1^%_1_c_m38.0
) In basicity, the maximum value is 304 nm (E^1^%_1_
c_m40.3) [6] Infrared absorption spectrum (KBr tablet method): 345
0, 2980, 1640, 1490, 1380, 122
It has an absorption band at 0cm^-^1. [7] Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone [8] Color reaction: Positive for phosphomolybdic acid, 80% sulfuric acid, anisaldehyde sulfuric acid reaction [9] Basic, Distinction between acidic and neutral: Amphoteric substance [10] Color of substance: white [11] Silica gel thin layer chromatogram: Rf = 0.1
2 (Carrier Merck silica gel, 60F_2_5_4,
Thickness 0.2 mm, Art5554), developing solvent: isopropyl alcohol: hexane: concentrated ammonia water (4:1
:2) [12] The results of amino acid analysis of the constituent sugar and constituent amino acid acid hydrolysates showed that they contained asparagine, N-methyl-leucine, and one amino acid of undetermined structure other than leucine. Furthermore, the relative retention times of these other aromatic amino acids in HPLC matched those in the hydrolyzate of vancomycin under the same conditions. It also contains glucose and one other amino sugar as constituent sugars, which is different from vancosamine or ristosamine. (2) A novel compound ispeptin B and its pharmaceutically acceptable salts having the following physicochemical properties. [1] Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine. [2] Molecular weight: Secondary ionization mass spectrum
According to ion mass spectrometry, the molecular weight is about 1460. [3] Decomposition point: 280-300°C [4] Specific rotation: [α]^2^7_D=-16.0°(
C=0.1, H_2O) [5] Ultraviolet absorption spectrum: In acidic or neutral, maximum value 280 nm (E^1^%_1_c_m36.2
) In basicity, the maximum value is 304 nm (E^1^%_1_
c_m39.5) [6] Infrared absorption spectrum (KBr tablet method): 345
0, 2980, 1660, 1480, 1410, 124
It has an absorption band at 0cm^-^1. [7] Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone [8] Color reaction: Positive for phosphomolybdic acid, 80% sulfuric acid, anisaldehyde sulfuric acid reaction [9] Basic, Distinction between acidic and neutral: amphoteric substance [10] Color of substance: white [11] Silica gel thin layer chromatogram: Rf = 0.5
6 (Carrier Merck silica gel, 60F_2_5_4,
Thickness: 0.2 mm, Art5554), Developing solvent: Isopropyl alcohol: Hexane: Concentrated ammonia water (4:1:2) [12] From the results of amino acid analysis of the constituent sugar and constituent amino acid acid hydrolysates, it contains asparagine, N -Methyl-leucine and one amino acid whose structure has not been determined other than leucine were shown to be included. Furthermore, the relative retention times of these other aromatic amino acids in HPLC matched those in the hydrolyzate of vancomycin under the same conditions. It also contains glucose and one other amino sugar as constituent sugars, which is different from vancosamine or ristosamine. (3) It is characterized by culturing actinomycetes having the ability to produce new compounds izpeptin A and B having the following physical and chemical properties in a medium, producing and accumulating the compounds izpeptin A and B, and collecting the same. Method for producing novel compounds izpeptin A and B. Izpeptin A [1] Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine. [2] Molecular weight: Secondary ionization mass spectrum
According to ion mass spectrometry, (M+H)^+ is 1474, so the molecular weight is approximately 147.
It is 5. [3] Decomposition point: 260-280°C [4] Specific rotation: [α]^2^5_D=-26.0°(
C=0.1, H_2O) [5] Ultraviolet absorption spectrum: In acidic or neutral, maximum value 280 nm (E^1^%_1_c_m38.0
) In basicity, the maximum value is 304 nm (E^1^%_1_
c_m40.3) [6] Infrared absorption spectrum (KBr tablet method): 345
0, 2980, 1640, 1490, 1380, 122
It has an absorption band at 0cm^-^1. [7] Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone [8] Color reaction: Positive for phosphomolybdic acid, 80% sulfuric acid, anisaldehyde sulfuric acid reaction [9] Basic, Distinction between acidic and neutral: Amphoteric substance [10] Color of substance: white An example of elemental analysis values is as follows: C 51.68%, H 5.23%, N 8.22%, Cl 5.23% [11] Silica gel thin layer chromatogram: Rf=0.1
2 (Carrier Merck silica gel, 60F_2_5_4,
Thickness: 0.2 mm, Art5554), Developing solvent: Isopropyl alcohol: Hexane: Concentrated ammonia water (4:1:2) [12] From the results of amino acid analysis of the constituent sugars and constituent amino acids acid hydrolyzates, it contains asparagine, N -Methyl-leucine and one amino acid whose structure has not been determined other than leucine were shown to be included. Furthermore, the relative retention times of these other aromatic amino acids in HPLC matched those in the hydrolyzate of vancomycin under the same conditions. It also contains glucose and one other amino sugar as constituent sugars, which is different from vancosamine or ristosamine. Izpeptin B [1] Elemental analysis: Consists of carbon, hydrogen, nitrogen, and chlorine. [2] Molecular weight: Secondary ionization mass spectrum
According to ion mass spectrometry, the molecular weight is about 1460. [3] Decomposition point: 280-300°C [4] Specific rotation: [α]^2^7_D=-16.0°(
C=0.1, H_2O) [5] Ultraviolet absorption spectrum: In acidic or neutral, maximum value 280 nm (E^1^%_1_c_m36.2
) In basicity, the maximum value is 304 nm (E^1^%_1_
c_m39.5) [6] Infrared absorption spectrum (KBr tablet method): 345
0, 2980, 1660, 1480, 1410, 124
It has an absorption band at 0cm^-^1. [7] Solubility in solvents: Soluble in water and dimethyl sulfoxide, insoluble in methanol, ethanol, acetone [8] Color reaction: Positive for phosphomolybdic acid, 80% sulfuric acid, anisaldehyde sulfuric acid reaction [9] Basic, Distinction between acidic and neutral: amphoteric substance [10] Color of substance: white [11] Silica gel thin layer chromatogram: Rf = 0.5
6 (Carrier Merck silica gel, 60F_2_5_4,
Thickness 0.2 mm, Art5554), developing solvent: isopropyl alcohol: hexane: concentrated ammonia water (4:1
:2) [12] The results of amino acid analysis of the constituent sugar and constituent amino acid acid hydrolysates showed that they contained asparagine, N-methyl-leucine, and one amino acid whose structure had not been determined other than leucine. Furthermore, the relative retention times of these other aromatic amino acids in HPLC matched those in the hydrolyzate of vancomycin under the same conditions. It also contains glucose and one other amino sugar as constituent sugars, which is different from vancosamine or ristosamine.