JP2005069998A - Method for analyzing sugar utilizing vcd - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technology for conveniently measuring α, β arrangements of a glycoside bond of sugar. <P>SOLUTION: In an infrared region partially including a region of approximately 1,170 cm<SP>-1</SP>-1,130 cm<SP>-1</SP>as specified absorption, due to the glycoside bond of the α arrangement of the sugar, a sugar containing material having the particular glycoside bond in the sample can be detected by measuring a circular dichroic spectrum of a target sample. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to sugar analysis.

  The α and β configurations of sugars are one large parameter that determines the three-dimensional structure of sugars, and differences in the configuration can affect biological activity. Therefore, it is important to determine the configuration of sugar linkages.

  With analytical methods such as nuclear magnetic resonance (NMR) and mass spectrometry (MS) that are commonly used for sugar analysis, it is difficult to determine the three-dimensional structure of the sugar, and if possible, the structure is simple. It is limited to.

  Various methods have been reported for the purpose of determining the arrangement of sugar linkages such as the α-configuration, but they all use complicated procedures and are often inaccurate.

  Optical measurement of optical rotation may be used to determine the three-dimensional structure of a substance, as in transient optical spectroscopy, but optical rotation measurement is a technique that compares the reference data with various methods, but about chirality. Can hardly get any information. Therefore, useful information cannot be obtained regarding the α and β configuration of the sugar.

  When linearly polarized light passes through the optically active substance, the transmitted light that has emerged is elliptically polarized at the same time as its plane of polarization is rotated by an optical rotation angle α. This is because a difference occurs in the absorption intensity for left and right-handed polarized light. This phenomenon is called circular dichroism (CD).

  As an example of CD, there is a method of measuring circular dichroism (ultraviolet dichroism = ECD, Electronic Circular Dichroism). Although ECD can be used for analysis of chirality, sugars themselves do not absorb in the ultraviolet region that ECD normally measures. Therefore, in order to determine the steric structure of the sugar, it is necessary to perform measurement using other foreign substances such as chromophore, which requires complicated work.

As another example of CD, absorption based on transition of molecular vibrational state observed by irradiating optically active material with circularly polarized infrared light is absorbed in infrared region circular dichroism (vibrational circular dichroism = Vibrational Circular). Dichroism (VCD). By measuring VCD, inverted absorption is observed between the enantiomers, so that information on the absolute configuration of the molecule and the conformation in the solution can be obtained. However, regarding saccharides, the results of measuring VCD are only reported in Non-Patent Documents 1 and 2, etc., and there is no description regarding specific absorption and arrangement of glycosidic bonds.
Carbohydr. Res., 133 (1984) 163-167, Carbohydr. Res., 319 (1999) 172-183 PKBose, et al., J. Am. Chem. Soc., 1999, 121, 6094-6095

  An object of the present invention is to provide a technique for simply measuring the α and β configurations of glycosidic bonds of sugars.

The above problems have been solved in the present invention by the inventors unexpectedly finding specific absorption due to glycoside bonds in the α configuration of sugars in a specific infrared region. Accordingly, the present invention provides the following.
(1) a specific glycosidic bond in the sample, comprising a step of measuring a circular dichroism spectrum of a sample of interest in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ^−1. A method for detecting a sugar-containing substance having
(2) The method according to item 1, wherein the sample is substantially removed except for the sugar-containing substance.
(3) The area of about ^{1170cm -1 ~1130cm} -1 comprises at least a portion of the area of at least about ^{1154cm -1 ~1137cm} -1, The method of claim 1.
(4) region of the about ^{1170cm -1 ~1130cm} -1 when resolution of ^{2 cm -1,} about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, about 1142cm ^-1 and about 2. The method of item 1, comprising at least one region selected from the group consisting of 1138 cm ⁻¹ .
(5) The method according to item 1, further comprising a step of allowing the sample to stand after the dissolution in a solvent and before the measurement.
(6) The method according to item 5, wherein the standing is at least 6 hours.
(7) The method according to Item 1, wherein the sugar-containing substance includes at least one sugar-containing substance selected from the group consisting of simple sugars and complex sugars.
(8) The sugar-containing substance is at least one sugar-containing substance selected from the group consisting of simple sugars, glycoproteins, glycolipids, sugar nucleotides, proteoglycans, glycopeptides, glycoside antibiotics, and glycoside organic compounds. The method according to item 1, comprising:
(9) The above measurement is carried out from the group consisting of DMSO, water, carbon disulfide, carbon tetrachloride, dichloromethane, acetone, chloroform, aliphatic alcohol, acetonitrile, heavy atom substitution products thereof, and mixed solvents thereof. Item 2. The method according to Item 1, wherein the selected solvent is used.
(10) The method according to item 1, wherein the sample is prepared so that the concentration of the sugar-containing substance is 0.001M to 5M.
(11) The method according to item 1, wherein the measurement uses 1700 cm ⁻¹ to 800 cm ⁻¹ as the wave number of the modulation frequency.
(12) absorbing in the infrared region including at least part of the area of the approximately ^{1170cm -1 ~1130cm} -1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample, in the item 1 The method described.
(13) The method according to Item 1, wherein the specific glycosidic bond includes an α-configuration glycosidic bond.
(14) The method according to Item 1, wherein the specific glycosidic bond includes an α-configuration and a β-configuration glycosidic bond.
(15) The method according to item 1, further comprising a step of measuring IR of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ .
(16) containing a sugar having a specific glycosidic bond in the sample, including a step of measuring IR of a sample of interest in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ Substance detection method.
(17) The following steps:
(A) a step of measuring an infrared region circular dichroism spectrum of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ;
(B) Absorption in the infrared region including at least part of the region of about 1170-1130 cm ⁻¹ of the target sample and at least one of the region of about 1170-1130 cm ⁻¹ of the sugar-containing substance to be measured. Comparing the absorption in the infrared region including the part,
A method for quantifying a specific glycosidic bond, comprising
(18) Item 17 in which the absorption in the infrared region including at least part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured is measured during the measurement step (A) The method described.
(19) The method according to item 17, wherein the absorption in the infrared region including at least a part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured is a measurement value obtained in advance.
(20) The method according to Item 17, wherein the specific glycosidic bond includes an α-configuration glycosidic bond.
(21) The method according to Item 17, wherein the quantification includes quantification of a ratio of glycoside bond in α configuration to glycoside bond in β configuration.
(22) The method according to item 17, wherein the sample is substantially removed except for the sugar-containing substance.
(23) The area of about ^{1170cm -1 ~1130cm} -1 comprises at least a portion of the area of at least about ^{1154cm -1 ~1137cm} -1, method of claim 18.
(24) region of the about ^{1170cm -1 ~1130cm} -1 when resolution of ^{2 cm -1,} about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, about 1142cm ^-1 and about 18. A method according to item 17, comprising at least one region selected from the group consisting of 1138 cm- ¹ .
(25) The method according to item 17, further comprising a step of allowing the sample to stand before the measurement after being dissolved in a solvent.
(26) The method according to item 25, wherein the standing is at least 6 hours.
(27) The method according to item 17, wherein the sugar-containing substance includes at least one sugar-containing substance selected from the group consisting of simple sugars and complex sugars.
(28) The sugar-containing substance is at least one sugar-containing substance selected from the group consisting of simple sugars, glycoproteins, glycolipids, sugar nucleotides, proteoglycans, glycopeptides, glycoside antibiotics, and glycoside organic compounds. 18. A method according to item 17, comprising:
(29) The above measurement is carried out from the group consisting of DMSO, water, carbon disulfide, carbon tetrachloride, dichloromethane, acetone, chloroform, aliphatic alcohol, acetonitrile, heavy atom substitution products thereof, and mixed solvents thereof. Item 2. The method according to Item 1, wherein the selected solvent is used.
(30) The method according to item 17, wherein the sample is prepared so that the concentration of the sugar-containing substance is 0.001M to 5M.
(31) The method according to item 17, wherein the measurement uses 1700 cm ⁻¹ to 800 cm ⁻¹ as the wave number of the modulation frequency.
(32) absorbing in the infrared region including at least part of the area of the approximately ^{1170cm -1 ~1130cm} -1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample, in the item 1 The method described.
(33) The method according to item 17, further comprising a step of measuring IR of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ .
(34) The following steps:
(A) a step of measuring IR of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ;
(B) IR absorption in the infrared region including at least a part of the region of about 1170-1130 cm ⁻¹ of the target sample, and at least of the region of about 1170-1130 cm ⁻¹ of the sugar-containing substance to be measured. Comparing the IR absorption in the infrared region including a part,
A method for quantifying a specific glycosidic bond, comprising
(35) The following:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) sample cell; and E) detecting means for measuring a circular dichroism spectrum of infrared region including at least part of the area of about ^{1170cm -1 ~1130cm} -1,
Wherein the absorption in the infrared region comprising at least a portion of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ indicates the presence of a sugar-containing substance having a glycosidic bond in the α configuration in the sample.
A device that detects a sugar-containing substance of a specific glycoside bond.
(36) The apparatus according to item 35, further comprising means for substantially removing the sample other than the sugar-containing substance.
(37) region of the about ^{1170cm -1 ~1130cm} -1 comprises at least a portion of the area of at least about ^{1154cm -1 ~1137cm} -1, device of claim 35.
(38) region of the about ^{1170cm -1 ~1130cm} -1 when resolution of ^{2 cm -1,} about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, about 1142cm ^-1 and about 36. The apparatus of item 35, comprising at least one region selected from the group consisting of 1138 cm ⁻¹ .
(39) The above measurement is carried out from the group consisting of DMSO, water, carbon disulfide, carbon tetrachloride, dichloromethane, acetone, chloroform, aliphatic alcohol, acetonitrile, heavy atom substitution products thereof, and mixed solvents thereof. 36. An apparatus according to item 35, wherein the selected solvent is used.
(40) The apparatus according to item 35, wherein the measurement uses 1700 cm ⁻¹ to 800 cm ⁻¹ as the wave number of the modulation frequency.
(41) Absorption in an infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ is determined and displayed when a sugar-containing substance having a glycosidic bond of α configuration is present in the sample. Means, an apparatus according to item 35.
(42) E-2) The apparatus according to item 35, further comprising means for measuring IR in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ .
(43) The apparatus according to item 35, further comprising at least one further means selected from the group consisting of a recording medium for storing the detection results and a means for displaying the detection results.
(44) The following:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) sample cell; and E) detecting means for measuring the IR of infrared region including at least part of the area of about ^{1170cm -1 ~1130cm} -1,
Provided, where, IR absorption in the infrared region including at least part of the area of the approximately ^{1170cm -1 ~1130cm} -1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample ,
A device that detects a sugar-containing substance of a specific glycoside bond.
(45) Below:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) Sample cell;
E) detection means for measuring a circular dichroism spectrum in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ; and F) absorption detected by the E means and the object of measurement. Means for comparing the absorption in the infrared region including at least part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance, and calculating the abundance of the sugar-containing substance to be measured;
An apparatus for quantitatively determining a sugar-containing substance having a specific glycosidic bond.
(46) The apparatus according to item 45, further comprising means for substantially removing the sample other than the sugar-containing substance.
(47) region of the about ^{1170cm -1 ~1130cm} -1 comprises at least a portion of the area of at least about ^{1154cm -1 ~1137cm} -1, device of claim 45.
(48) region of the about ^{1170cm -1 ~1130cm} -1 when resolution of ^{2 cm -1,} about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, about 1142cm ^-1 and about 46. Apparatus according to item 45, comprising at least one region selected from the group consisting of 1138 cm ⁻¹ .
(49) The above measurement is carried out from the group consisting of DMSO, water, carbon disulfide, carbon tetrachloride, dichloromethane, acetone, chloroform, aliphatic alcohol, acetonitrile, heavy atom substitution products thereof, and mixed solvents thereof. 46. The apparatus according to item 45, wherein the selected solvent is used.
(50) The apparatus according to item 45, wherein the measurement uses 1700 cm ⁻¹ to 800 cm ⁻¹ as the wave number of the modulation frequency.
(51) Absorption in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ is determined and displayed when a sugar-containing substance having a glycosidic bond of α configuration is present in the sample. Means, an apparatus according to item 45.
(52) E-2) The apparatus according to item 45, further comprising means for measuring IR in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ .
(53) The apparatus according to item 45, further comprising recording means for storing information relating to the sugar-containing substance to be measured.
(54) The apparatus according to item 45, further comprising at least one further means selected from the group consisting of a recording medium for storing the detection results and a means for displaying the detection results.
(55) The following:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) Sample cell;
E) detection means for measuring an IR spectrum in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ; and F) IR absorption detected in the E means, and sugar content to be measured Means for comparing the IR absorption in the infrared region including at least a part of the region of about 1170-1130 cm ⁻¹ of the substance, and calculating the abundance of the sugar-containing substance to be measured;
An apparatus for quantitatively determining a sugar-containing substance having a specific glycosidic bond.

  Preferred embodiments of the present invention will be described below. However, those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and the accompanying drawings, as well as well-known and common techniques in the field, and the effects of the present invention. It should be appreciated that and understand the effect easily.

  According to the present invention, the α and β configurations of glycoside bonds of sugars can be easily measured, and the quantification of stereoisomers related to the α and β configurations of sugar-containing substances can be easily performed. . If this technology is applied, it can also be applied to purity tests of sugar-containing substances such as pharmaceuticals (for example, simple sugars, glycolipids, glycoproteins, etc.) without using other substances.

  The present invention will be described below. Throughout this specification it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

(the term)
Listed below are definitions of terms particularly used in the present specification.

  In the present specification, “sugar” and “sugar chain” are used interchangeably and refer to a compound in which one or more unit sugars (monosaccharides and / or derivatives thereof) are connected. When two or more unit sugars are connected, the unit sugars are usually bonded by dehydration condensation using a glycosidic bond. Examples of such sugar chains include monosaccharides (glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), xylose (Xyl), N-acetylglucosamine (GlcNAc) contained in the living body. ), N-acetylgalactosamine (GalNAc), sialic acid (SA) and complexes and derivatives thereof), and polysaccharides, oligosaccharides and the like in which a plurality of these monosaccharides are bonded, but are not limited thereto. In this specification, in the narrow sense, the term “sugar” refers to a substance (simple sugar) composed only of unit sugars. Therefore, in the present specification, sugar can be used interchangeably with “oligosaccharide”, “polysaccharide”, “sugar”, and “carbohydrate”. However, in general, the term “sugar” includes, but is not limited to, a wide range of glycoproteins, proteoglycans, glycosaminoglycans, glycolipids and other complex sugars, and sugar chains decomposed or derived therefrom. Therefore, unless otherwise specified, in the present specification, “sugar” and “sugar chain” may be used interchangeably with a sugar-containing substance.

As used herein, “monosaccharide” refers to polyhydroxy aldehyde or polyhydroxy ketone and derivatives thereof containing at least one hydroxyl group and at least one aldehyde group or ketone group. Usually monosaccharides, the general formula C _n H _2n O is represented by _n is not limited to, fucose (deoxyhexose), also include such N- acetylglucosamine. Where n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively That's it. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. When specifically mentioned herein, a monosaccharide derivative refers to a substance that results from the substitution of one or more hydroxyl groups on an unsubstituted monosaccharide with another substituent. Examples of such monosaccharide derivatives include sugars having a carboxyl group (for example, aldonic acid in which C-1 position is oxidized to carboxylic acid (for example, D-gluconic acid in which D-glucose is oxidized), terminal Uronic acid in which C atom of carboxylic acid becomes carboxylic acid (D-glucuronic acid in which D-glucose is oxidized), sugar having amino group or amino group derivative (for example, acetylated amino group) (for example, N- Acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc.), sugars having both amino group and carboxyl group (for example, N-acetylneuraminic acid (NANA, a kind of sialic acid), N-acetylmuramic acid Etc.), deoxylated sugars (eg, 2-deoxy-D-ribose), sulfated sugars containing sulfate groups, and phosphorylated sugars containing phosphate groups. In the present specification, the term “monosaccharide” as used herein includes the above-described derivatives, or, in a saccharide that has formed a hemiacetal structure, a glycoside having an acetal structure by reacting with an alcohol is also within the range of the monosaccharide. Oligosaccharides or polysaccharides are a combination of a plurality of these monosaccharides, usually dehydration condensation by glycosidic bonds, but α and β depending on the direction of the binding of sugars on the non-reducing end side, It is known that the physical properties are greatly affected by the position of the carbon bond in the position of the sugar chain. There is no clear distinction between oligosaccharides and polysaccharides, but the boundary is about 10-50 sugars unless otherwise stated in this specification, oligosaccharides and polysaccharides are interchangeable Used for

  As used herein, “sugar-containing substance” refers to a substance containing sugar. Many such sugar-containing substances are found in the living body. For example, in addition to simple sugars such as monosaccharides, oligosaccharides and polysaccharides contained in the living body, glycoproteins, proteoglycans, glycosaminoglycans, sugars Examples include, but are not limited to, complex sugars such as lipids and sugar chains that are decomposed or derived from these complex biomolecules.

  As used herein, “simple sugar” refers to a substance that is substantially composed only of sugar. Examples of such simple sugars include, but are not limited to, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Simple sugars may also be linked to commonly substituted substituents such as N acetyl groups and methyl groups.

  As used herein, “complex sugar” refers to a substance composed of a narrowly defined sugar and a portion other than the narrowly defined sugar. Any part other than such sugar may be used. In the present specification, examples of the complex sugar include, but are not limited to, glycoprotein, glycolipid, sugar nucleotide, mucin, mucopolysaccharide, proteoglycan, GPI anchor, and the like.

  As used herein, “glycoprotein” refers to a protein comprising at least one sugar chain, and preferably having such a sugar chain bound thereto. Thus, unless otherwise stated, glycoproteins include glycopeptides. However, when a glycopeptide is distinguished from a glycoprotein, the glycopeptide may refer to a polypeptide smaller than the glycoprotein. Usually, glycoproteins have sugar chains necessary for exerting functions in higher organisms. Examples of such glycoproteins include, but are not limited to, N-linked sugar chains, O-linked sugar chains, C-linked sugar chains, or glycoproteins having a plurality of types thereof. Such glycoproteins are enzymes (eg, oxidoreductase (reductase), transferase (transferase), hydrolase (hydrolase), eliminase (lyase), isomerase (isomerase), synthase (ligase). Hormones (eg, peptide hormones (insulin, erythropoietin, glucagon, pituitary hormones, parathyroid hormone, etc.)); cytokines (eg, hematopoietic factors such as interleukins, chemokines, colony stimulating factors, Tumor necrosis factor, interferons, etc.); growth factors (eg, platelet derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth Factor (VEGF) etc.); binding factor; antibody; vaccine; receptor; Click proteins; encompasses the like serum proteins, but not limited to.

  In this specification, the “structure” of a sugar includes a configuration. The configuration to be measured by the present invention includes, but is not limited to, the α configuration and β configuration of glycoside bonds of sugars. Other sugar structures include, for example, asparagine-linked glycoprotein sugar chains (N-linked sugar chains), mucins and mucin-linked glycoprotein sugar chains (O-linked sugar chains), glycolipids, sugar nucleotides, Examples include the sugar chains contained in proteoglycans, GPI anchors and the like, as well as the structures of free sugar chains and their precursors, but these other structures can also be analyzed additionally in the measurement of the present invention. Such other structural analysis methods can be carried out using methods known in the art.

  In this specification, “α configuration” and “β configuration” are used in the same meaning as used in the art, and isomers having this configuration are also referred to as α anomer and β anomer, respectively. Anomers refer to a pair of isomers that differ only in the configuration of C1. When a monosaccharide is drawn by Fischer projection, an anomeric center and an anomeric reference atom with the same orientation (right or left) are called α-anomers, and those with the opposite orientation are called β-anomers. For example, the following is exemplified.

┌ ---- ┐
│ │
H-C-OH │ ← Rightward │ │
H-C-OH │
│ │
HO-C-H │
│ │
H-C-OH │
｜ ｜
HC-O-┘ ← Rightward ｜
CH ₂ OH

α-D-Glucopyranose [IUPAC-IUBMB / CAS]
α-D-Glucopyranose

┌ ------ ┐
│ │
HO-C-CH ₂ OH │ ← Leftward │ │
HO-C-H │
│ │
H-C-OH │
｜ ｜
HC-O ---- ┘ ← Rightward ｜
CH ₂ OH

β-D-Fructofuranose [IUPAC-IUBMB / CAS]
β-D-fructofuranose.

  It is known that glucose in the α configuration and glucose in the β configuration can be transferred to each other through the following hemiacetal bond.

In the present specification, the sample may be any sample as long as the purpose is to detect a sugar-containing substance having a specific glycoside bond, and examples thereof include biological substances and pharmaceutical products. The sample to be measured can be used without pretreatment, but preferably a substance that interferes with the wave number region to be measured (typically, a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ) is substantially present. It is advantageous to have been removed. Here, being substantially removed means that it has been removed to such an extent that measurement is not affected. Examples of such a substance capable of interfering include any substance other than a sugar-containing substance having absorption in the specific region. Such interfering substances can be determined by prior measurement of potential substances alone or substantially purified preparations with VCD and / or IR to determine whether there is absorption in a particular wavenumber region. This can also be identified.

  If there are substances to be removed, the sample can be removed by pretreatment. For such pretreatment, any purification and separation method known in the art can be used. Examples of such a method include chromatography and electrophoresis. Examples of such chromatography include HPLC, column chromatography such as medium speed chromatography, paper chromatography, affinity chromatography using a structure specific to sugar (eg, lectin, antibody, etc.), thin layer, etc. Examples include, but are not limited to chromatography.

(Measurement of VCD)
In this specification, “circular dichroism” (CD) is also referred to as circularly polarized dichroism, which is a phenomenon observed in chiral substances and having different absorption for circularly polarized light between right polarized light and left polarized light. The circular dichroism can be typically represented by a differential absorbance (ΔA) that is a difference in absorption between left circularly polarized light and right circularly polarized light. ΔA _is therefore can be represented by A L -A _R, where _{A L} is the left-handed circularly polarized light, _{A R} is a right circularly polarized light.

  In this specification, “vibrating circular dichroism” (VCD) is a technique reported in RK Dukor et al., Encyclopedia of Analytical Chemictry, John Wiley & Sons, New York, 2000, pp. 662-675, etc. It is a detection method based on measurement using molecular vibration instead of conventional circular dichroism measurement using electronic excitation. In VCD, electromagnetic waves in the infrared region are used to cause molecular vibration transitions. In VCD, light having a wavelength of 800 nm or more can usually be observed. VCD is based on the transition of molecular vibrational state observed by irradiating an optically active substance with circularly polarized infrared light. By measuring VCD, inverted absorption is observed between the enantiomers, so that information on the absolute configuration of the molecule and the conformation in the solution can be obtained.

  A measuring apparatus for measuring circular dichroism in the present invention typically includes a light beam emitting means, a polarizing means, a circularly polarized light generating means, a sample cell, and a detecting means. In this measuring apparatus, the sample cell can be arranged at an arbitrary position through which the measuring light passes.

  As the luminous flux emitting means, one that can emit a desired monochromatic light can be typically used, but is not limited thereto. Any light flux emitting means can be used.

  As the polarization unit, one that can convert the monochromatic light from the light beam emitting unit into linearly polarized light in a predetermined vibration direction can be used, but is not limited thereto. Any such polarizing means can be used.

  As the circularly polarized light generating means, an arbitrary photoelastic modulation element can be used which alternately generates right and left circularly polarized light from linearly polarized light of the polarizing means, but is not limited thereto. As such circularly polarized light generating means, any means can be used. Any photoelastic modulation element can be used.

  A sample cell may be used in which a sample containing or presumed to contain a sugar-containing substance is placed and left and right circularly polarized light from the photoelastic modulation element is arranged to pass through the sample. In this measuring apparatus, the detection means detects the amount of transmitted light from the sample. In order to stabilize the passage of light, a holding means for holding the sample cell may be provided. As the sample cell, any material may be used, but it is preferable to use a cell that is suitable for the sample. Examples of such a material include, but are not limited to, barium fluoride, calcium fluoride, and quartz. The sample cell preferably has a desired optical path length. Such an optical path length is typically 1 μm to 100 μm (for example, 72 μm), but is not limited thereto.

In the apparatus of the present invention, any detection means can be used as long as it can measure a desired specific wave number (typically, an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ). A detector may be used.

  Preferably, the apparatus of the present invention may include means (for example, a display, a printer, etc.) for displaying the detected measurement value.

  In another embodiment, the apparatus of the present invention preferably includes a recording medium that stores information necessary for measurement (for example, information on a sugar-containing substance to be measured) and / or measured information. Examples of such a recording medium include, but are not limited to, a hard disk, a flexible disk, an MO, a CD-ROM, a CD-R, a DVD-ROM, a DVD-R, or a transmission medium on a network.

  In the present invention, it is preferable that the detection means, the means for displaying the measurement value, the recording medium or the drive for reading and writing it, the CPU for calculating, and the like are connected by a bus.

  Here, the measurement of circular dichroism is performed using such a measuring apparatus or another circular dichroism spectrometer (CD spectrometer), and a differential absorbance (ΔA) or the like can be obtained as a measurement value.

  The difference absorbance (ΔA) is an absorbance difference obtained by subtracting the absorbance (AR) for right circularly polarized light from the absorbance (AL) for left circularly polarized light, and is defined by the following equation (1).

ΔA = A _L −A _R = Δε × c × L (1)
Here, Δε is the difference in molar extinction coefficient, c is the concentration (mol / 1), and L is the optical path length (cm). ΔA can be obtained at each wavelength, and Δε varies depending on the wavelength. Thus, the difference in molar extinction coefficient can also be utilized in the detection of the present invention.

  As described above, in the present invention, absorption in a specific region can be observed by using either Δε or ΔA. However, when Δε or ΔA is used, evaluation can be performed using an absolute value. Evaluation can also be made using a control (that is, a value when a sample containing no sugar-containing substance is used) as a reference and a relative value thereof.

In this specification, the absorption of a specific wavelength can represent a measured value by the wavelength itself, but a wave number that is the reciprocal thereof (typically, a unit of cm ⁻¹ is used) may be used. Here, when the circular dichroism is measured, the wave number is obtained by multiplying the reciprocal of the wavelength by 10,000.

The measurement method of the present invention can also be applied to quantitative analysis of sugar-containing substances having a specific configuration. When quantification is performed, either the photometric value ellipticity (θ) or the difference absorbance (ΔA) of CD can be used, and the quantification can be performed using the equation (1) typically at a certain wavelength. In the present invention, in the infrared region including a specific wave number, typically at least part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ , preferably at least one of the region of at least about 1154 cm ^{−1 to} 1137 cm ^−1. By measuring the circular dichroism spectrum in the part and detecting the presence of absorption, a sugar-containing substance having a glycosidic bond in the α configuration can be detected. In the measurement method of the present invention, when the spectra overlap each other and the contribution of other components cannot be ignored, or when each component interferes with each other, the spectrum data is subjected to multiple regression analysis, partial minimum two Multivariate analysis such as multiplication (PLS = Partial Least Squares) can be used.

The sample to be measured is preferably dissolved in a solvent suitable for the measurement. In the present invention, any solvent can be used as long as it can be used in the measurement. Examples of such a solvent include DMSO, water, carbon disulfide, carbon tetrachloride, and dichloromethane. , Acetone, chloroform, aliphatic alcohol, acetonitrile, a heavy atom substitution product thereof, and a solvent selected from the group consisting of a mixed solvent thereof, but are not limited thereto. Preferred solvents include _{DMSO-d 6.} This is because there is almost no interference in the measurement region. Here, the target sample is preferably measured for VCD or IR after being dissolved in a solvent suitable for measurement and allowed to stand for a certain period of time. This is because the vibration state of the molecule is stabilized by allowing it to stand for a certain period of time, and more accurate measurement can be performed. Examples of such standing conditions include, but are not limited to, at least 1 hour, preferably at least 3 hours, more preferably at least 6 hours. Such a minimum numerical value for a certain period of time and a preferable numerical value may vary depending on a target substance, a solvent, and the like.

  The sample to be measured preferably contains at least a sugar-containing substance to be measured at a concentration of 0.001 M, but is not limited thereto. This is because it can usually be detected at such a concentration. Therefore, when such a minimum concentration is not reached, the sample may be concentrated before detection.

The sample to be measured can be any wave number as long as it is suitable for the object of the present invention as the wave number of the modulation frequency, and is usually 1700 cm ^{−1 to} 800 cm ⁻¹ , typically, for example, Approximately 1200 cm ⁻¹ can be used, but is not limited thereto, and wave numbers outside this range can also be used arbitrarily.

(IR measurement)
In another aspect, the present invention also measures infrared spectroscopy (IR) itself in a specific region (typically an infrared region that includes at least a portion of the region from about 1170 cm ^{−1 to} 1130 cm ⁻¹ ), A method and apparatus for detecting a sugar-containing substance having a glycosidic bond in the α configuration in a sample due to the presence of absorption is provided.

  Infrared spectroscopic measurement is a well-known method in this field, and in this specification, measurement can be performed using any method and apparatus (a commercially available apparatus from Shimadzu Corporation, JASCO, etc.).

(Quantitative measurement)
The present invention provides a method and apparatus for quantifying a sugar-containing substance having a specific glycosidic bond. Here, “quantitative” refers to determining, in this specification, how much sugar-containing substance having a specific glycoside bond is contained in a certain sample.

  In this specification, quantification is based on the measurement value obtained for the sugar-containing substance to be measured by obtaining a measurement value (also referred to as a standard value or a reference value) regarding absorption of VCD and / or IR by the method of the present invention. Thus, a calibration curve can be created and extrapolated from the calibration curve and the measured value of VCD and / or IR of the sample to be measured. Here, the “sugar-containing substance to be measured” refers to a sugar-containing substance having a known amount of a specific glycoside bond (for example, α configuration).

  As used herein, quantification. It involves quantifying the proportion of a specific amount of glycosidic bonds. Such quantification typically includes, but is not limited to, the ratio of glycoside bonds in the α configuration to glycoside bonds in the β configuration.

(Description of a preferred embodiment for carrying out the present invention)
The best mode of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

In one specific aspect, the present invention provides a method for detecting a sugar-containing substance having a specific glycosidic bond (for example, a simple sugar, a complex sugar such as a glycoprotein) in a sample. The method of the present invention has found that a sugar-containing substance having an α configuration has absorption and a sugar-containing substance having a β configuration has a tendency to inhibit the absorption at a wavelength or wave number in a specific infrared region. Achieved by. Therefore, the method of the present invention is characterized by including the step of measuring the circular dichroism spectrum of the target sample in this specific region. In the present invention, the specific wave number is typically an infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ , but the infrared absorption in the vicinity thereof also has an α configuration. It can be appreciated that it may be used to measure sugar-containing materials. Here, “including at least a part” includes that a circular dichroism spectrum is measured at a wave number of at least one point. Since this region shows a specific absorption of a sugar-containing substance having an α configuration, the absorption in this region is interpreted as indicating the presence of a sugar-containing material having an α configuration in the present invention.

Preferably, the infrared absorption wavenumber region in the method of the present invention comprises at least a portion of a region of at least about 1154 cm ^{−1 to} 1137 cm ⁻¹ . In this region, since the characteristic absorption peak of simple sugars (for example, monosaccharides, disaccharides, etc.) having an α configuration is observed in particular, by measuring the circular dichroism spectrum in this region, Highly accurate measurement can be performed.

In a more preferred embodiment, when the resolution is ^{2 cm -1,} the region of the about ^{1170cm -1 ~1130cm} -1 of the present invention, about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, It may include at least one point selected from the group consisting of about 1142 cm ⁻¹ and about 1138 cm ⁻¹ . This specific wave number is a numerical value when the resolution is 2 cm ⁻¹ , and it can be understood that a peak can be found at a point in between as the resolution further increases. In this particular region, there is a characteristic absorption peak of simple sugars (eg monosaccharides, disaccharides, etc.), especially with an α configuration, so measuring the circular dichroism spectrum at these particular points Allows a more efficient measurement of a sugar-containing substance (typically having an α configuration) of a particular glycosidic bond.

  In the present invention, simple sugars to which a substituent that can be generally substituted such as N acetylation or methylation is bonded can also be measured.

  The method of the present invention can measure complex sugars in addition to simple sugars. Since the absorption due to a specific glycoside bond is usually similar to that of the corresponding simple sugar in complex sugars, the above specific region should also apply to the measurement of specific glycoside bonds in complex sugars. Can do. In the present invention, examples of such complex sugars include, but are not limited to, arbitrary biological substances and pharmaceuticals, and examples thereof include glycoproteins, glycolipids, and sugar nucleotides.

In the method of the present invention, the absorption in the infrared region including at least part of the area of about 1170cm ^-1 ~1130cm ^-1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample. Without being bound by theory, sugar-containing materials with β-configuration glycosidic bonds appear to have little absorption in this region. Therefore, in the present invention, the specific glycoside bond to be measured usually includes an α-configuration glycoside bond, but is not limited thereto, and in another embodiment, the specific glycoside bond has an α-configuration and a β-configuration. It can contain both glycosidic bonds. In such a case, for example, by calculating the total amount of sugar-containing substances in both the α configuration and the β configuration, the amounts of the α configuration and the β configuration can be determined.

In a preferred embodiment, the method of the present invention, in addition to the measurement of the VCD, in the infrared region including at least part of the area of about ^{1170cm -1 ~1130cm} -1, further the step of measuring the IR of samples of interest It may be included. IR can also be used to ensure detection of the method of the invention, since it also shows absorption from specific glycosidic bonds in similar regions.

Alternatively, in the present invention, a specific glycosidic bond in a sample is determined by using only a process of measuring IR of a target sample in an infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ^−1. It is also possible to detect the sugar-containing substance that it has.

In another aspect, the present invention provides (A) a step of measuring an infrared region circular dichroism spectrum of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ^−1. ; (B) and the absorption in the infrared region including at least part of the area that purpose become approximately 1170～1130Cm ^-1 sample, at least in the region of 1170～1130Cm ^-1 of sugar-containing substances to be measured A method for quantifying a specific glycosidic bond comprising comparing the absorption with a portion of the infrared region is included. Here, the step (A) can be performed in the same manner as the measurement method described above. In the step (B), the absorption in the infrared region including at least a part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured can be measured simultaneously with the measurement of (A). Alternatively, a standard curve may be created in advance. When measuring in large quantities, the absorption in the infrared region including at least a part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured is measured in advance as a standard, and a new one is prepared for each step A. Comparison can be made without measuring a standard calibration curve. When a more accurate measurement is desired, or when the measurement conditions have changed or may have been changed, it is preferable to newly measure the standard calibration curve.

  In the above-described quantification method of the present invention, a sugar-containing substance containing a glycoside bond having an α configuration can be quantified. In another embodiment, the quantification of the present invention can perform a quantification of the ratio of glycoside bonds in the α configuration to glycoside bonds in the β configuration. Without being bound by theory, a sugar-containing substance having an α configuration has a specific absorption peak in the specific infrared region, while a sugar-containing substance having a β configuration has a specific absorption peak in the same infrared region. This is because there seems to be no influence at least on a specific absorption peak.

In another aspect, the present invention provides an apparatus for implementing the above method of the present invention. Accordingly, the present invention provides A) a light beam emitting means; B) a polarizing means; C) a circularly polarized light generating means; D) a sample cell; and E) an infrared including at least a portion of the region from about 1170 cm ^{−1 to} 1130 cm ^−1. There is provided an apparatus for detecting a sugar-containing substance of a specific glycoside bond, comprising a detection means for measuring a circular dichroism spectrum of a region. Here, the absorption in the infrared region including at least part of the area of the approximately ^{1170cm -1 ~1130cm} -1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample. In the present invention, any light flux emitting means, polarizing means, circularly polarized light generating means, sample cell, and detecting means can be used as long as the method of the present invention can be realized. The arrangement of these means is usually arranged in the order of luminous flux emitting means, polarizing means, circularly polarized light generating means, sample cell, and detecting means.

In another aspect, the present invention includes at least a portion of a region of A) luminous flux emitting means; B) polarizing means; C) circularly polarized light generating means; D) sample cell; E) about 1170 cm ^{−1 to} 1130 cm ^−1. Detection means for measuring a circular dichroism spectrum in the infrared region; and F) absorption detected by the E means and at least a part of a region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured. There is provided a quantification apparatus for a sugar-containing substance having a specific glycoside bond, comprising means for comparing absorption in the infrared region and calculating the abundance of the sugar-containing substance to be measured.

  Preferably, the device of the present invention may include means for measuring IR and / or means for detecting the presence of a specific sugar-containing substance based on IR, in addition to means for measuring VCD.

  In another aspect, the present invention provides an apparatus for quantifying a sugar-containing substance having a specific glycoside bond using a means for measuring IR and / or a means for detecting the presence of a sugar-containing substance of a specific target based on IR. To do.

  The above-mentioned apparatus specifically used in the present invention may optionally use means for realizing a more preferable embodiment for realizing the above-described method of the present invention.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited only to the embodiments, but only by the claims.

  Examples of the present invention will be described below. In the examples, first, a commercially available sugar was used.

(Example 1: VCD and IR measurement of monosaccharide)
In this example, by way of example, monosaccharides were measured as simple sugars.
VCD and IR were measured for the following monosaccharides (1) to (10) under the following conditions. The results are shown in FIG. 1 (FIGS. 1A to 1B). Monosaccharides (1) to (10) can be purchased as reagents, and in this example, those purchased from Wako Pure Chemicals, Sigma or Aldrich were used.

[Monosaccharide used]
(1) D-glucose
(2) D-galactose (3) D-GlcNAc
(4) D-mannose (5) D-xylose (6) D-GalNAc
(7) L-Fucose (8) Neu5Ac
(9) D-glucuronic acid (10) D-lyxose.

[Measurement condition]
Equipment used: Chiralir (Bomem / BioTools)
Concentration: 0.16M
Cell used: Barium fluoride window plate cell (optical path length 72 micrometers)
Measurement time: 3 hours (accumulation count 13500 times)
Resolution: 8cm ^-1
modulationfrequency (optimum wavelength): 1200cm ^-1 center.
Solvent used: DMSO-d ₆ (measured after dissolving the sample for 6 hours or more).

The results measured under the above conditions are shown in FIG. The compound used in this example becomes a mixture of α and β anomers by equilibrium, so that there is no sugar having a glycosidic bond of α configuration, and a part of the compound has α configuration by equilibrium. Exist as a sugar mixture. As is clear from FIG. 1, it was found that the mixture containing sugars having a glycosidic bond in the α configuration has characteristic absorption of VCD and IR in the wave number in the region of about 1170-1130 cm ⁻¹ . Furthermore, the measurement results are shown in FIG. 2 in comparison with the α anomer and the β anomer. As can be seen from FIG. 2, a sugar having a glycosidic bond in the 1-position at the 1-position is shown as 1150, 1146, 1144, 1142, 1138 cm ^-1 when the resolution is 2 cm ^-1 as in this example. A characteristic absorption peak of VCD and IR was shown in the wave number, and it was found that there was a characteristic absorption of VCD and IR in the wave number in the region of about 1170-1130 cm ⁻¹ .

(Example 2: Measurement of monosaccharides in water)
When the same measurement was performed using H ₂ O instead of DMSO-d ₆ as the solvent used under the same conditions as in Example 1, a similar peak was observed, and the wave number in the region of about 1170-1130 cm ^−1. It was found that there is a characteristic absorption.
(Example 3: VCD and IR measurement of disaccharide)
VCD and IR of the following disaccharides (11) to (21) were measured under the following conditions. The structure of the disaccharide used is shown in FIG. The result is shown in FIG. As disaccharides (11) to (21), those purchased from Aldrich, Sigma, Wako Pure Chemical Industries, Ltd. and the like were used as reagents.

[Disaccharide used]
(11) α, α-trehalose (12) α, β-trehalose (neotrehalose)
(13) β, β-trehalose (isotrehalose)
(14) Kojibiose
(15) Sophorose
(16) Nigerose
(17) Laminaribiose
(18) Maltose
(19) Cellobiose
(20) Isomaltose
(21) Gentiobiose
[Measurement condition]
Equipment used: Chiralir (Bomem / BioTools)
Concentration: 0.08M
Cell used: Barium fluoride window plate cell (optical path length 72 micrometers)
Measurement time: 3 hours (accumulation count 13500 times)
Resolution: 8cm-1
modulationfrequency (optimum wavelength): 1200cm ^-1 center.
Solvent used: DMSO-d ₆ (Measured after dissolving the sample for 6 hours or more.)
As can be seen from FIG. 4, a sugar having a glycosidic bond in the α-position at the 1-position. 1154, 1150, 1146, 1142, 1138 cm ^-1 wavenumbers with characteristic absorption peaks of VCD and IR, and VCD and IR characteristic absorption peaks in the region of about 1170-1130 cm- ¹ I found out that

(Example 4: Measurement of disaccharide using water)
When the same measurement was performed using H ₂ O instead of the solvent used in Example 3, a characteristic absorption was observed at the same peak as in Example 3. Therefore, it can be seen that the present invention can measure not only monosaccharides but also polysaccharides and any solvent.

  Therefore, from the results of Examples 1 to 4,

It can be seen that the sugar represented by the formula shows a characteristic absorption around around 1145 cm ⁻¹ , in particular, 1170 to 1130 cm ⁻¹ , particularly 1154 to 1137 cm ⁻¹ .

(Example 5: Measurement of polysaccharides)
In the measurement in Examples 1 to 4, an experiment is performed using a larger molecule (polysaccharide) as a target. As a result, similar peaks can be observed in the regions in Examples 1 to 4.

(Example 6: Observation of complex sugar)
In the measurements in Examples 1 to 4, experiments are performed using complex sugars (here, glycoproteins and glycolipids) as targets. As a result, similar peaks can be observed in the regions in Examples 1 to 4.

Example 7: Determination of sugar diastereoselectivity
Solutions prepared in the following maltose 0.25M and cellobiose 0.25M were prepared at 0: 100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80: Mixed solutions were prepared in ratios of 20, 90:10, and 100: 0. About each solution, VCD was measured on condition of the following. The result is shown in FIG. In this example, sugars purchased from Aldrich, Sigma, Wako Pure Chemical Industries, etc. were used.

[Measurement condition]
Equipment used: Chiralir (Bomem / BioTools)
Cell used: Barium fluoride window plate cell (optical path length 72 micrometers)
Measurement time: 1 hour (4500 times of integration)
Resolution: 8cm ^-1
modulationfrequency (optimum wavelength): 1200cm ^-1 center.
Solvent used: DMSO-d ₆ (measured after dissolving the sample for 6 hours or more).

  The ratio of maltose based on the intensity of Glycosideband in VCD of each composition, based on the intensity of the bands (also referred to herein as Glycoside bands) when the ratio of maltose: cellobiose is 0: 100 and 100: 0 The expected value of was calculated. The relationship between the proportion of maltose actually contained and the expected value is shown in Table 1 and FIG.

  As can be seen from Table 1 and FIG. 6, the proportion of maltose expected from the VCD measurement results is almost the same as the proportion of maltose actually contained. Therefore, by using the measurement result of VCD, it is possible to predict the content of sugar having an α bond contained in the solution.

(Example 8: Quantification of unknown sample)
Next, the unknown sample was quantified.

  Using solutions prepared to maltose 0.25M shown below, 0.25M, 0.225M, 0.2M, 0.175M, 0.15M, 0.125M, 0.1M, 0.075M, 0.05M, 0.025M and 0M solutions were prepared. Randomly about 0.11M and 0.29M maltose solutions were prepared as unknown samples. About each solution, VCD was measured on condition of the following. In this example, sugars purchased from Aldrich, Sigma, Wako Pure Chemical Industries, etc. were used.

[Measurement condition]
Equipment used: Chiralir (Bomem / BioTools)
Cell used: Barium fluoride window plate cell (optical path length 72 micrometers)
Measurement time: 1 hour (4500 times of integration)
Resolution: 8cm ^-1
modulationfrequency (optimum wavelength): 1200cm ^-1 center.
Solvent used: DMSO-d ₆ (Measured after dissolving the sample for 6 hours or more.)
A calibration curve was prepared from the maltose concentration and the intensity of the glycoside band. When the predicted values of the maltose ratio were calculated from the measured values of the unknown sample, they were 0.114M and 0.295M, respectively. Therefore, it was demonstrated that the absolute amount of the sugar-containing substance having a glycoside bond in the α configuration can be actually quantified in the present invention.

  As mentioned above, although this invention was illustrated using the preferable embodiment and Example of this invention, it is understood that the scope of this invention should be interpreted only by a claim. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, the α and β configurations of glycoside bonds of sugars can be easily measured, and the quantification of stereoisomers related to the α and β configurations of sugar-containing substances can be easily performed. . If this technology is applied, it can also be applied to purity tests of sugar-containing substances such as pharmaceuticals (for example, simple sugars, glycolipids, glycoproteins, etc.) without using other substances.

Examples of VCD and IR spectrum measurement results of monosaccharides are shown. FIG. 1A shows the measurement results of D-glucose, D-galactose, D-mannose, D-xylose, L-fucose and D-lyxose. Examples of VCD and IR spectrum measurement results of monosaccharides are shown. FIG. 1B shows the measurement results of Neu5Ac, D-GlcNAc, D-glucuronic acid and D-GalNAc. The example of the spectrum comparison of VCD / IR between (alpha) anomer and (beta) anomer is shown. In FIG. 2A, D-glucose, 1-methyl-D-glucose and 1-methyl-D-galactose are shown from the left. The upper row shows the α anomer, and the lower row shows the β anomer. The example of the spectrum comparison of VCD / IR between (alpha) anomer and (beta) anomer is shown. In FIG. 2B, D-mannose and 1-methyl-D-GlcNAc are shown from the left. The upper row shows the α anomer, and the lower row shows the β anomer. As an example of a disaccharide that is one embodiment of the present invention, the structure of glucobiose measured in the examples and the glycoside binding mode thereof are shown. The measurement example of the VCD / IR spectrum of the glucobiose measured in the Example as an illustration of the disaccharide which is one embodiment of the present invention is shown. The measurement result for quantification which is another embodiment of this invention is shown. The VCD measurement result of a maltose / Serbiose mixture is shown. 6 shows a standard calibration curve based on the results of FIG.

Claims (18)

A sugar having a specific glycosidic bond in the sample, comprising measuring a circular dichroism spectrum of the sample of interest in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ^−1. Method for detecting contained substances. The region of about ^{1170cm -1 ~1130cm} -1 comprises at least a portion of the area of at least about ^{1154cm -1 ~1137cm} -1, The method of claim 1. The region of about ^{1170cm -1 ~1130cm} -1 when resolution of ^{2 cm -1,} about 1154cm ^-1, about 1150 cm ^-1, about 1146cm ^-1, about 1144cm ^-1, about 1142cm ^-1 and about 1138cm ^-1 The method of claim 1, comprising at least one region selected from the group consisting of: The method according to claim 1, wherein the sugar-containing substance includes at least one sugar-containing substance selected from the group consisting of simple sugars and complex sugars. The sugar-containing substance includes at least one sugar-containing substance selected from the group consisting of simple sugars, glycoproteins, glycolipids, sugar nucleotides, proteoglycans, glycopeptides, glycoside antibiotics and glycoside organic compounds, The method of claim 1. Absorption in the infrared region including at least part of the area of the approximately 1170cm ^-1 ~1130cm ^-1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample, according to claim 1 Method. The method of claim 1, wherein the specific glycosidic bond comprises an α-configuration glycosidic bond. The method of claim 1, wherein the specific glycosidic bond comprises an α-configuration and a β-configuration glycosidic bond. The method of claim 1, further comprising measuring the IR of the sample of interest in an infrared region including at least a portion of the region from about 1170 cm ^{−1 to} 1130 cm ⁻¹ . The following steps:
(A) a step of measuring an infrared region circular dichroism spectrum of a target sample in an infrared region including at least a part of a region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ;
(B) Absorption in the infrared region including at least a part of the region of about 1170-1130 cm ⁻¹ of the target sample and at least one of the region of about 1170-1130 cm ⁻¹ of the sugar-containing substance to be measured Comparing the absorption in the infrared region including the part,
A method for quantifying a specific glycosidic bond, comprising The method according to claim 10, wherein the absorption in the infrared region including at least a part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance to be measured is a measurement value obtained in advance. 11. The method of claim 10, wherein the specific glycosidic bond comprises an alpha configuration glycosidic bond. 11. The method of claim 10, wherein the quantification comprises quantifying the ratio of glycoside bonds in the α configuration to glycoside bonds in the β configuration. Less than:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) sample cell; and E) detecting means for measuring a circular dichroism spectrum of infrared region including at least part of the area of about ^{1170cm -1 ~1130cm} -1,
Provided, where the absorption in the infrared region including at least part of the area of該約^{1170cm -1 ~1130cm} -1 indicates that the sugar-containing substance having a glycosidic bond of α arranged is present in the sample,
A device that detects a sugar-containing substance of a specific glycoside bond. E-2) The apparatus of claim 14, further comprising means for measuring IR in the infrared region including at least a portion of the region from about 1170 cm- ^{1 to} 1130 cm- ¹ . Less than:
A) luminous flux emitting means;
B) Polarization means;
C) Circularly polarized light generating means;
D) Sample cell;
E) detection means for measuring a circular dichroism spectrum in the infrared region including at least a part of the region of about 1170 cm ^{−1 to} 1130 cm ⁻¹ ; and F) absorption detected by the E means and the object of measurement. Means for comparing the absorption in the infrared region including at least a part of the region of about 1170 to 1130 cm ⁻¹ of the sugar-containing substance, and calculating the abundance of the sugar-containing substance to be measured;
An apparatus for quantitatively determining a sugar-containing substance having a specific glycosidic bond. E-2) The apparatus of claim 16, further comprising means for measuring IR in the infrared region including at least a portion of the region from about 1170 cm- ^{1 to} 1130 cm- ¹ . The apparatus according to claim 16, further comprising recording means for storing information on the sugar-containing substance to be measured.