Orientation agent for NMR measurement

Abstract

The purpose of the present invention is to provide an orientation agent for NMR measurement that has excellent resistance to salts and the like. The present invention relates to an orientation agent for NMR measurement, the orientation agent being provided with a nanosheet and a coating agent that coats at least a portion of the nanosheet, wherein the coating agent contains a reaction product of polyoxyethylene monomethyl ether or a derivative thereof and a polyamine having a total of 3 to 30 amino groups and / or imino groups.

Description

Orienting agent for NMR measurement

[0001] This invention relates to an orientation agent for NMR measurement.

[0002] Established methods for analyzing protein structure include X-ray crystallography and nuclear magnetic resonance (NMR) analysis.

[0003] X-ray crystallography attempts to predict the three-dimensional structure of a protein by irradiating a crystallized sample with X-rays and measuring the resulting diffraction phenomenon. Since there are no restrictions on the molecular weight of the sample, very large compounds can be analyzed. However, this method cannot be applied to samples that cannot be crystallized. Furthermore, because X-ray crystallography is inherently designed to measure static structures with atomic-level precision, it is unsuitable for the dynamic analysis of biomolecules and other similar molecules.

[0004] On the other hand, structural analysis techniques using nuclear magnetic resonance (NMR) do not require the crystallization process and can non-destructively obtain information about the local structure and mobility around the nucleus. In particular, by measuring anisotropic terms such as residual dipole coupling (RDC), nuclear quadrupole interactions, and chemical shifts, it is possible to obtain information not only about the relative distance and angle of atoms in close proximity, but also about the angle and distance between atoms that are far apart, relative to an external magnetic field. This enables more precise three-dimensional analysis of proteins.

[0005] Normally, molecules being measured undergo random thermal motion in a solvent. Therefore, in order to measure anisotropic terms such as residual dipole coupling (RDC), it is necessary to orient the molecules being measured using a magnetic field. By using a media that has the property of aligning in a certain direction with respect to a static magnetic field (hereinafter also referred to as an "orienting agent"), the molecules being measured can be oriented, and the accuracy of NMR can be improved.

[0006] Generally available orientation agents include lipid aggregates called bicelles, which are disc-shaped micelles having a lipid bilayer structure, and orientation agents using fibrous phages called pf1. Furthermore, NMR measurement orientation agents in which nanosheets are coated with a predetermined compound have also been proposed (Patent Document 1).

[0007] Japanese Patent Publication No. 2018-200233

[0008] The NMR measuring orientation agent described in Patent Document 1 is said to be easy to prepare, excellent in handling, economy, and versatility, and capable of stable magnetic field orientation. Here, in dispersions containing the orientation agent prepared for NMR measurement, salts, buffers, surfactants, etc., may be added to adjust the pH, etc. However, even in the case of conventional NMR measuring orientation agents using nanosheets, when prepared as a dispersion in the presence of salts, buffers, surfactants, etc., the orientation performance tends to gradually decrease due to aggregation progressing over time. For this reason, there is a need for the development of an NMR measuring orientation agent that has excellent resistance to salts, etc., and can maintain a good dispersion state even when a dispersion containing salts, etc. is prepared.

[0009] The object of the present invention is to provide an NMR measurement orientation agent that has excellent resistance to salts and the like.

[0010] The invention made to solve the above problems is an NMR measuring orientation agent comprising a nanosheet and a coating agent covering at least a portion of the nanosheet, wherein the coating agent contains a reaction product of polyoxyethylene monomethyl ether or a derivative thereof and a polyamine having a total of 3 to 30 amino groups and / or imino groups.

[0011] Another invention made to solve the above problems is an NMR measuring orientation agent comprising a nanosheet and a coating agent covering at least a portion of the nanosheet, wherein the coating agent contains a compound having a polyoxyethylene chain and a polyamine chain containing three or more nitrogen elements.

[0012] The NMR measurement orientation agent of the present invention exhibits excellent resistance to salts and the like.

[0013] The following describes in detail an NMR orienting agent according to one embodiment of the present invention.

[0014] Unless otherwise specified, the upper and lower limits of numerical ranges described herein may be any combination of the disclosed values. Furthermore, when a numerical range is indicated using the symbol "~", it signifies that the range includes both the upper and lower limits. For example, "1~6" means "1 or greater and 6 or less".

[0015] <Orienting Agent for NMR Measurement> An NMR measuring agent according to one embodiment of the present invention (hereinafter also simply referred to as "orienting agent") comprises a nanosheet and a coating agent covering at least a portion of the nanosheet, wherein the coating agent contains a reaction product of polyoxyethylene monomethyl ether or a derivative thereof and a polyamine having a total of 3 to 30 amino groups and imino groups or both. An orienting agent according to another embodiment of the present invention comprises a nanosheet and a coating agent covering at least a portion of the nanosheet, wherein the coating agent contains a compound having a polyoxyethylene chain and a polyamine chain containing 3 or more nitrogen elements.

[0016] The orientation agent according to any embodiment of the present invention exhibits excellent resistance to salts, buffers, surfactants, deuterated organic solvents, etc. Therefore, because the nanosheet is coated with a predetermined coating agent, the dispersibility of the dispersion of the orientation agent does not easily decrease even in the presence of salts, buffers, surfactants, deuterated organic solvents, etc. As a result, by using this orientation agent, target molecules such as proteins can be magnetically oriented with high stability even in the presence of salts, buffers, surfactants, deuterated organic solvents, etc., and NMR measurements can be performed. Furthermore, because the nanosheet is coated with a predetermined coating agent, this orientation agent can also suppress non-specific adsorption during NMR measurements. The orientation agent will be described in detail below.

[0017] (Nanosheet) A "nanosheet" is a sheet with a thickness on the nanoscale. In principle, the thickness of a nanosheet can be reduced to the thickness of a single atom, but generally, it is difficult to fabricate nanosheets with a thickness of 0.5 nm or less. The thickness of the nanosheet, excluding the coating agent described later, is preferably 0.5 to 3 nm, and more preferably 1.0 to 2.5 nm. Furthermore, the thickness of the nanosheet coated with the coating agent (i.e., the orientation agent) is preferably 10 nm or less. The thickness of the nanosheet and the orientation agent refers to the value of the thickest part calculated from the cross-sectional profile observed by an atomic force microscope (AFM).

[0018] The D50 particle size of the nanosheet is preferably 100 nm or larger from the viewpoint of magnetic field orientation. While it is preferable for the D50 particle size of the nanosheet to be as large as possible in terms of dispersion in the solution, in practice, the upper limit is about 100 μm. That is, the D50 particle size of the nanosheet is preferably 100 nm to 100 μm. Note that the D50 particle size of the nanosheet and the orientation agent described later refers to the value calculated as the particle size that accounts for 50% of the cumulative frequency in the volume-based frequency distribution measured by dynamic light scattering.

[0019] Typical nanosheets have a D50 particle size of several micrometers and a thickness of several nanometers. The ratio of nanosheet thickness to D50 particle size (nanosheet thickness:D50 particle size) is, for example, 1:10. ２ from 1:10 ４ It may be within the range.

[0020] Orienting nanosheets typically exhibit anisotropic magnetic susceptibility to external magnetic fields. When an external magnetic field is applied to this type of nanosheet, the degree to which the nanosheet senses the magnetic field (magnetic susceptibility) differs depending on the direction of application of the magnetic field. As a result, the nanosheet orients in the energetically most stable direction, that is, the direction in which the interaction potential between the magnetic field and the nanosheet is minimized.

[0021] Such nanosheets can be manufactured using known methods. In particular, by exfoliating a layered precursor into individual layers, nanosheets with a large surface area can be obtained. Obtaining nanosheets with a large surface area enables greater magnetic field orientation. Such nanosheets can orient small molecules such as water and sugars like glucose in the state of uncoated nanosheets only. In addition, by coating the nanosheets with a predetermined coating agent, it becomes possible to magnetically orient proteins with a large molecular weight in solution.

[0022] The magnetic field orientation of nanosheets occurs when the sum of the magnetic anisotropy of the atoms constituting the nanosheets exceeds the free movement in the solution. Although there are differences in the magnitude of the anisotropy, since all nanosheets are composed of atoms with magnetic anisotropy, if the size of the nanosheets is sufficiently large, theoretically all nanosheets will orient regardless of the type and composition of the constituent atoms. Therefore, as nanosheets, any type and composition can be used as long as they have magnetic field orientation properties. For example, the following can be mentioned.

[0023] Ti ０．９１ O ２ 、Ti ０．８７ O ２ 、Ti ３ O ７ 、Ti ４ O ９ 、Ti ５ O １１ titanium oxide nanosheets such as etc.; TiNbO ５ titanium niobium oxide nanosheets such as etc.; Nb ３ O ８ 、Nb ６ O １７ niobium oxide nanosheets such as etc.; Ca ２ Nb ３ O １０ calcium niobium oxide nanosheets such as etc.; graphene oxide; graphene; boron nitride (h-BN); graphitic carbon nitride (g-C ３ N ４); nanosheets of polylactic acid, polythiophene, polystyrene, etc.; DNA (DNA origami); molecular organic frame (MOF) nanosheets; covalent organic frame (COF) nanosheets; black phosphorus nanosheets; peptoid nanosheets; hydroxyapatite (Ca ５ (PO ４ ) ３ (OH)), NbS ３ NbSe ３ NbTe ３ , Ti, Ti-Ni, Zn-Ti, Zn-Al, Pd, Co ９ See ８ TiS ３ TiSe ３ TiTe ３ , TaS ３ , TaSe ３ , TaTe ３ MnPS ３ , CdPS ３ NiPS ３ Mn ０．５ Fe ０．５ PS ３ MoCl ２ MoS ２ RuCl ２ , CrCl ２ , BiI ３ BiS ３ , PbCl ４ , V ２ O ５ MoO ３ , TaO ３ WO ３ HNbWO ６ , HTaWO ６ HNb ３ O ８ MnO ２ Na ｘ (Mn ４＋ , Mn ３＋ ) ２ O ４ , Sr ２ RuO ４ , KCa ２ Nb ３ O １０ H ２ W ２. ７ 、!b ２ . ７ 、a ０．９０ u ０．０５ b ２ . ７ 、.u ０．５６ ! ２ . ７ 、+ ２ __ ４ 、+ ３ fi ２ . ７ 、 ２ . ７ 、.i ２ (3) ２ . ９ 、+ ２ b ３ . １０ 、!!!! ３ . １０ 、|||| ２ () １０ 、a ２ () ２ : １０ 、! ５ ! ４ . １５ The following: ２ * ２ fi ２－δ 3 ２ ( * * * * , , # ) , 、 ２ * ２ fi ２ｍ－０．５ 3 ｍ＋１ (#))()、3). ４ * ３ . ７．５ 、u ２ (). ２ The ２ F、O ２ fi ２ 、Fe|o ４ pi? ４ 、.* ｎ＋ ｘ／ｎ ・yH ２ __ ４－ｘ * ｘ ()) ８)O ２０ (OH) ４ (M: cation), [(Mg ３ )(Si ２ O ５ ) ２ (OH) ２ , [Mg ６ Si ６ Al ２ O ２０ (OH) ４ [M ｎ＋ １／ｎ (Mg ６ )(M: cation), [(MgFe) ３ (Si ３ Al)O １０ (OH) ２ , [Mg ６ (Si ６ Al ２ )O ２０ (OH) ４ , [Mg １１／４ (Si ６ Al ２ )O ２０ F ４ [(M ２＋ ) ３／２ (M: cation)], [(Al ２ (Si ２ Al ２ )O １０ (OH) ２ , [(Al ２ (Si ３ Al)O １０ (OH) ２ , [Al ４ Si[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0024] Among the above, the nanosheet is preferably formed from at least one selected from the group consisting of titanium dioxide, titanium niobium dioxide, niobium oxide, and graphene oxide (at least one selected from the group consisting of titanium dioxide nanosheets, titanium niobium dioxide nanosheets, niobium oxide nanosheets, and graphene oxide nanosheets), more preferably from at least one selected from the group consisting of titanium dioxide, titanium niobium dioxide, and niobium oxide (at least one selected from the group consisting of titanium dioxide nanosheets, titanium niobium dioxide nanosheets, and niobium oxide nanosheets), and even more preferably from at least one selected from the group consisting of titanium dioxide and titanium niobium dioxide (at least one selected from the group consisting of titanium dioxide nanosheets and titanium niobium dioxide nanosheets). It is also preferable that the nanosheet is formed from an oxide containing the element titanium.

[0025] (Coating Agent) In the orientation agent according to one embodiment of the present invention, at least a portion of the surface of the nanosheet is coated with a predetermined coating agent, thereby enabling magnetic field orientation of proteins and the like for NMR measurement. Furthermore, the coating with the predetermined coating agent allows the dispersibility of the orientation agent to be maintained without being significantly affected by the presence of salts, buffers, surfactants, etc.

[0026] In one embodiment of the present invention, the coating agent comprises a reaction product of polyoxyethylene monomethyl ether or a derivative thereof and a polyamine having a total of 3 to 30 amino groups and / or imino groups. Hereinafter, polyoxyethylene monomethyl ether or a derivative thereof will also be referred to as "polyoxyethylene monomethyl ether (A)". That is, polyoxyethylene monomethyl ether (A) includes not only polyoxyethylene monomethyl ether but also derivatives of polyoxyethylene monomethyl ether. Furthermore, hereafter, a polyamine having a total of 3 to 30 amino groups and / or imino groups will also be referred to as "polyamine (B)". Hereinafter, the reaction product of polyoxyethylene monomethyl ether (A) and polyamine (B) will also be referred to as compound (X).

[0027] (Method for producing compound (X)) Compound (X) is obtained by reacting polyoxyethylene monomethyl ether (A) with polyamine (B).

[0028] Polyoxyethylene monomethyl ether (A) is polyoxyethylene monomethyl ether or a derivative thereof. Preferably, polyoxyethylene monomethyl ether (A) is a tosylated polyoxyethylene monomethyl ether. Tosylated polyoxyethylene monomethyl ethers are examples of derivatives of polyoxyethylene monomethyl ether, and other derivatives of polyoxyethylene monomethyl ether include, for example, polyoxyethylene monomethyl ether trifluoromethanesulfonic acid esters and polyoxyethylene monomethyl ether trifluoromethylbenzenesulfonic acid esters, which are sulfonic acid esters of polyoxyethylene monomethyl ether.

[0029] Polyoxyethylene monomethyl ether tosylated products are α-methyl-ω-tosylpolyoxyethylene obtained by tosyling polyoxyethylene monomethyl ether. "Tosyling" refers to the conversion of a hydroxyl group (-OH) to a p-toluenesulfonyloxy group (-OTs group).

[0030] Polyoxyethylene monomethyl ether can be any known type, and various molecular weights are industrially available, such as the Uniox M series from NOF Corporation, the MPG series from Nippon Emulsifier Co., Ltd., and the Reosolve PEM series from Lion Corporation.

[0031] The molecular weight of the polyoxyethylene monomethyl ether is preferably 200 to 100,000, more preferably 1,000 to 10,000. Having a molecular weight within this range enhances noise reduction and signal enhancement effects.

[0032] Known methods can be applied to tosylate polyoxyethylene monomethyl ether. For example, by reacting polyoxyethylene monomethyl ether with a p-toluenesulfonate, the hydrogen atoms of the terminal hydroxyl groups of the polyoxyethylene monomethyl ether are converted to tosyl groups. The p-toluenesulfonate is not particularly limited, but examples include p-toluenesulfonic acid chloride. Typically, this step is carried out by dissolving polyoxyethylene monomethyl ether in an organic solvent such as pyridine, dichloromethane, or acetonitrile, and then, if necessary, using an amine catalyst consisting of an amine compound, adding 1 to 5 mol of p-toluenesulfonic acid chloride per mol of polyoxyethylene monomethyl ether and reacting at room temperature for 10 minutes to 24 hours. As a result, α-methyl-ω-tosylpolyoxyethylene is obtained.

[0033] Here, it is preferable to use trimethylamine hydrochloride, trimethylamine hydrobromide, trimethylamine hydrofluoride, trimethylamine sulfate, trimethylamine nitrate, trimethylamine phosphate, triethylamine, tripropylamine, N,N,N',N'-tetramethylethylenediamine, etc., alone or in combination as the amine catalyst. Furthermore, it is particularly preferable to use trimethylamine hydrochloride in combination with other amine compounds in order to shorten the reaction time and improve the tosylation rate. The preferred amount of amine catalyst to use is 1.5 to 10 mol per mol of polyoxyethylene monomethyl ether when the amine catalyst is used alone, and 0.1 to 3 mol of trimethylamine hydrochloride and 1.5 to 10 mol of the other amine compound when trimethylamine hydrochloride is used in combination with other amine compounds. The α-methyl-ω-tosylpolyoxyethylene after the reaction may be purified by precipitation with diethyl ether, hexane, etc. Such precipitation purification can remove residual p-toluenesulfonate and suppress the following side reactions with polyamines.

[0034] Polyamine (B) has an amino group (-NH ２Polyamine (B) has a total of 3 to 30 amino groups and imino groups (-NH-). Polyamine (B) may have only amino groups, only imino groups, or both. The total number of amino groups and imino groups in polyamine (B) is 3 to 30. The upper limit of the total number of amino groups and imino groups in polyamine (B) is preferably 12, but may be 10 or 8. The lower limit of the total number of amino groups and imino groups in polyamine (B) is preferably 4, but may be 5 or 6.

[0035] When the total number of amino and imino groups in the polyamine (B) is three or more, the resulting compound (X) is more easily adsorbed onto the surface of the nanosheet, which increases the stability of the resulting orientation agent and reduces noise during measurement.

[0036] Polyamine (B) may have two amino groups. In this case, the lower limit of the number of imino groups that polyamine (B) has may be one, two, three, or four. The upper limit of the number of imino groups may be 29, 11, 10, 8, or 6.

[0037] Examples of polyamines (B) include polymethylene polyamines such as dimethylenetriamine, trimethylenetetramine, tetramethylenepentamine, pentamethylenehexamine, hexamethyleneheptamine, heptamethyleneoctamine, octamethylenenonamine, nonamethylenedecamine, decamethyleneundecamine, and undecamethylenedodecamine; polyethylene polyamines such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexamine, hexaethyleneheptamine, heptaethyleneoctamine, octaethylenenonamine, nonaethylenedecamine, decaethyleneundecamine, and undecaethylenedodecamine; and propylene-butylene amines such as spermine and spermidine. Polyamines (B) can be used individually or in combination of two or more.

[0038] As for the polyamine (B), polyethylene polyamine is preferred from the viewpoint of the solubility of the resulting compound (X) in water, etc., and the noise reduction effect of the resulting orientation agent, triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine is more preferred, and pentaethylenehexamine is even more preferred.

[0039] The reaction between polyoxyethylene monomethyl ether (A) and polyamine (B) can typically be carried out by dissolving 2 to 100 times the number of moles of polyamine (B) relative to the number of moles of polyoxyethylene monomethyl ether (A) in a solution, and then reacting the solution of polyoxyethylene monomethyl ether (A) dropwise over 1 to 24 hours at room temperature to 60°C. Suitable solvents for this step are water; aprotic polar solvents such as acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide; and a mixture of at least two solvents selected from the group consisting of water and polar solvents. Compound (X) is produced by the above steps.

[0040] The generated compound (X) is preferably purified by precipitation with an organic solvent such as diethyl ether or isopropyl alcohol. A mixed solvent of a non-polar solvent and a polar solvent, such as a mixed solvent of hexane and ethyl acetate, can also be suitably used as the organic solvent. As another purification method, the solution of the generated compound (X) may be dissolved in water, and low molecular weight compounds may be removed using a dialysis tube, dialyzer, accelerator, etc. In this case, the organic solvent may be removed with an evaporator before dissolving in water. Such purification can remove residual polyoxyethylene monomethyl ether (A), polyamine (B), catalyst, etc., and enhance the noise reduction effect.

[0041] (Structure of compound (X)) The typical structure of compound (X) is α-methyl-ω-polyamination polyoxyethylene.

[0042] In compound (X), the terminal end of the methoxypolyoxyethylene group derived from polyoxyethylene monomethyl ether (A) (hereinafter referred to as "the terminal end of polyoxyethylene monomethyl ether (A)") may be bonded to the nitrogen atom of the terminal primary amino group of polyamine (B) (hereinafter referred to as "the terminal primary amino group of polyamine (B)"), or the terminal end of polyoxyethylene monomethyl ether (A) may be bonded to the nitrogen atom of the non-terminal imino group of polyamine (B) (hereinafter referred to as "the non-terminal imino group of polyamine (B)").

[0043] In compound (X), it is sufficient that one or more molecules of polyoxyethylene monomethyl ether (A) are bonded to one molecule of polyamine (B). Furthermore, if two or more molecules of polyoxyethylene monomethyl ether (A) are bonded to one molecule of polyamine (B), both the terminal primary amino group and the non-terminal imino group of polyamine (B) may be bonded to the terminals of the polyoxyethylene monomethyl ether (A).

[0044] From the viewpoint of noise reduction and signal enhancement effects, it is preferable that compound (X) contains one molecule of polyoxyethylene monomethyl ether (A) and one molecule of polyamine (B). More preferably, compound (X) has a structure in which one molecule of polyamine (B) is bonded to the terminal end of one molecule of polyoxyethylene monomethyl ether (A). The number of polyoxyethylene monomethyl ether (A) molecules bonded to one molecule of polyamine (B) can be determined by molecular weight measurement using liquid chromatography or the like.

[0045] (Compound (Y)) In other embodiments, the coating agent comprises a compound having a polyoxyethylene chain and a polyamine chain containing three or more nitrogen elements. Hereinafter, the compound having a polyoxyethylene chain and a polyamine chain containing three or more nitrogen elements will also be referred to as compound (Y).

[0046] Polyoxyethylene chains, for example, have an alkoxy group at one end (-O-CH ２ -CH ２ -) ｍ１It has a linear structure represented by . m1 is, for example, an integer of 3 or more, preferably an integer between 4 and 2,000, and more preferably an integer between 20 and 200.

[0047] Polyamine chains are, for example, (-(CH ２ ) ｍ２ -NR-) ｍ３ It has a linear structure represented by . R is a hydrogen atom or a linker. m2 is an integer from 1 to 4, preferably 2. That is, the polyamine chain is preferably a polyethylene polyamine chain. m3 is an integer of 3 or more, preferably an integer from 3 to 30, more preferably an integer from 3 to 12, and even more preferably an integer from 4 to 8.

[0048] In compound (Y), the polyoxyethylene chain and the polyamine chain may be directly bonded. For example, the end of the polyoxyethylene chain may be bonded to the end of the polyamine chain, the end of the polyoxyethylene chain may be bonded to a part of the polyamine chain other than the end, or the ends of two or more polyoxyethylene chains may be bonded to the ends of the polyamine chain and to parts of the polyamine chain other than the end. Compound (Y) may have one polyoxyethylene chain and one polyamine chain. Compound (Y) may have a structure in which one polyoxyethylene chain and one polyamine chain are bonded, and the terminal groups may be an alkyl group (preferably a methyl group) and an amino group.

[0049] Compound (Y) can be produced in the same manner as compound (X) described above. However, in the production of compound (Y), polyoxyethylene alkyl ethers other than polyoxyethylene monomethyl ether (A) and their derivatives may be used, and polyamines other than polyamine (B) having a total of 31 or more amino groups and / or imino groups may be used.

[0050] A compound (X) in which one molecule of polyoxyethylene monomethyl ether (A) has one molecule of polyamine (B) bonded to its terminal, and a compound (Y) having one polyoxyethylene chain and one polyamine chain may be, for example, a compound represented by the following formula (1).

[0051]

[0052] In equation (1), n ​​is an integer between 4 and 2,000. １ and R ２ Each of these groups independently has 1 to 29 hydrogen atoms, or one or both of an amino group and / or an imino group. However, R １ and R ２ The total number of amino and imino groups contained in it is between 2 and 29.

[0053] In equation (1), n ​​is preferably an integer between 20 and 200.

[0054] In equation (1), R １ and R ２ Each of these is a group that independently has 1 to 11 hydrogen atoms, or one or both of an amino group and / or an imino group, R １ and R ２ The total number of amino groups and imino groups contained in is preferably 2 to 11. １ and R ２ Each of these is a group that independently has 1 to 7 hydrogen atoms, or one or both of an amino group and / or an imino group, R １ and R ２ It is more preferable that the total number of amino groups and imino groups contained in the compound is 3 to 7.

[0055] In equation (1), R １ and R ２ These may be groups represented by the following formulas (2a) and (2b), respectively.

[0056]

[0057]

[0058] In equations (2a) and (2b), x1 and x2 are integers from 1 to 4, respectively. y1 and y2 are integers from 0 to 29, except that y1 + y2 is an integer from 2 to 29.

[0059] In equations (2a) and (2b), x1 and x2 are preferably 2. Y1 and y2 are preferably integers from 0 to 11 (where y1 + y2 satisfies the range of 2 to 11), and more preferably integers from 0 to 7 (where y1 + y2 satisfies the range of 3 to 7).

[0060] The coating agent for covering the nanosheet may consist solely of compound (X) or compound (Y). The coating agent may also contain other components besides compound (X) and compound (Y). Examples of other components include, when the coating agent contains compound (X), polyoxyethylene monomethyl ether (A), polyamine (B), etc., which are residuals from the production of compound (X). The coating agent may further contain conventionally known compounds as coating agents, such as proteins, tannic acid, polydiallyldimethylamine chloride, chondroitin sulfate, etc. The content of compound (X) in the coating agent for covering the nanosheet is preferably 50% by mass or more, more preferably 70% by mass or more, and may be 80% by mass or more, 90% by mass or more, or 95% by mass or more. The content of compound (Y) in the coating agent for covering the nanosheet is preferably 50% by mass or more, more preferably 70% by mass or more, and may be 80% by mass or more, 90% by mass or more, or 95% by mass or more.

[0061] (Manufacturing of Orienting Agent) An orientation agent according to one embodiment of the present invention can be manufactured by coating a nanosheet with a coating agent. Coating of the nanosheet with the coating agent can be carried out by the following procedure.

[0062] The nanosheet and coating agent are mixed in a suitable solvent. This solvent may function as a dispersion medium. The solvent used is appropriately selected depending on the combination of the coating agent and the nanosheet. That is, it is appropriately selected from solvents that can dissolve or disperse both and do not react with each other. Examples of solvents include water, tetramethylammonium (TMA), and tetrabutylammonium (TBA). The mixing ratio of the nanosheet to the coating agent may be, for example, 1 to 10 times the amount of coating agent to nanosheet by mass, or 1.5 to 8 times.

[0063] The pH of the dispersion containing the nanosheet and coating agent may be adjusted by adding an acid, base, buffer solution, etc.

[0064] It is preferable to allow the dispersion of the nanosheet and coating agent to stand for a certain period of time. This allows for obtaining an orientation agent (dispersion containing the orientation agent) with sufficient coating agent adhering to the nanosheet surface. To remove any unadsorbed coating agent, the dispersion may be centrifuged. If necessary, the precipitate obtained after centrifugation may be redispersed in a suitable solvent, and the centrifugation and redispersion washing may be repeated.

[0065] (Size and Use of Orienting Agent) The D50 particle size of the orientation agent (nanosheet coated with a coating agent) according to one embodiment of the present invention is preferably 100 nm or larger, and more preferably 1 μm or larger, from the viewpoint of magnetic field orientation. The D50 particle size of the orientation agent is preferably as large as possible in terms of dispersion in the solution, but in practice, the upper limit is about 100 μm. That is, the D50 particle size of the orientation agent is preferably 100 nm to 100 μm, more preferably 1 μm to 100 μm, and may also be 1 μm to 60 μm.

[0066] An orientation agent according to one embodiment of the present invention is used as an orientation agent for NMR measurement. That is, it is used as an orientation agent for oriented a target molecule in a magnetic field when performing NMR measurement. The target molecule is not particularly limited, but biomacromolecules are preferred, and proteins are more preferred. The target molecule may be a cationic molecule, anionic molecule, amphoteric molecule having both cationic and anionic groups, or nonionic molecule. For example, if the target molecule is a cationic molecule, the orientation agent may further contain a cationic compound. Also, if the target molecule is anionic molecule, the orientation agent may further contain an anionic compound. Compounds (X) and (Y) contained in the orientation agent may have cationic or anionic groups introduced into them.

[0067] The orientation agent can be used in the same manner as conventionally known orientation agents for NMR measurement. For example, an NMR measurement sample can be prepared by mixing the target molecule into a solvent in which the orientation agent is dispersed in a suitable dispersion medium. After mixing, stirring may be performed as needed. NMR measurement can be performed using the prepared NMR measurement sample using conventional methods. Buffers, salts, surfactants, deuterated organic solvents, etc., may be added to the NMR measurement sample. Because the orientation agent has high stability, aggregation is less likely to occur even when buffers, salts, surfactants, deuterated organic solvents, etc., are added to the dispersion, allowing for good NMR measurement.

[0068] For example, a dispersion containing an orientation agent according to one embodiment of the present invention is also a preferred embodiment of the present invention. The dispersion may contain water as a dispersion medium. The dispersion may further contain at least one selected from the group consisting of buffers, salts, surfactants, and deuterated organic solvents. The dispersion remains highly stable even when it further contains at least one selected from the group consisting of buffers, salts, surfactants, and deuterated organic solvents together with the nanosheets. The dispersion can be suitably used as a dispersion for NMR measurement.

[0069] The following describes some examples. It should be noted that the following examples are representative examples of the present invention and should not be interpreted as narrowing the scope of the invention.

[0070] [Manufacturing Example 1] (Manufacturing of TiNS) In accordance with "Tanaka T, Ebina Y, Takada K, Kurashima K, Sasaki T. (2003) Chem Mater; 15:3564-3568", titanium oxide nanosheets (referred to as "TiNS"). Composition Ti ０．８７ O ２ ) was manufactured. Unless otherwise specified below, TiNS was dispersed in a diethylamine (DEA) solution (1 mM) and used for various measurements.

[0071] [Manufacturing Example 2] (Manufacturing of TiNbNS) Following "Takagaki A, Sugisawa M, Lu D, Kondo J, Hara M, Domen K, Hayashi S. (2003) J. Am. Chem. Soc; 125: 5479-5485", titanium oxide niobium nanosheets (referred to as "TiNbNS"). Composition: TiNbO ５ ) was manufactured. Unless otherwise specified below, TiNbNS was dispersed in a diethylamine (DEA) solution (1 mM) and used for various measurements.

[0072] [Evaluation of liquid properties of orientation agent (compound-coated nanosheet)] (1) Zeta potential measuring device: Zetasizer Nano Ultra (trade name), manufactured by Malvern Panalogical. Measurement conditions: Room temperature. Sample: An orientation agent dispersion with a nanosheet content adjusted to 0.04% by mass was used for measurement.

[0073] (2) Particle size D50 measurement device by laser diffraction: MT3300EXII (product name), manufactured by MicrotracBEL Co., Ltd. Measurement conditions: Room temperature Sample: An orientation agent dispersion with a nanosheet content adjusted to 0.4% by mass was used for measurement.

[0074] (3) pH measuring device: LAQUA F-2000PI (product name), manufactured by Horiba, Ltd. Measurement conditions: Room temperature Sample: An orientation agent dispersion with a nanosheet content adjusted to 0.8% by mass was used for measurement.

[0075] (4) NMR (Nuclear Magnetic Resonance) measurement device: Avance 400 (trade name), manufactured by Bruker. Measurement conditions: Room temperature, D using an orientation agent (compound coated nanosheet). ２ For the measurement of the quadrupole interaction of O, an orientation agent dispersion with a nanosheet content adjusted to 0.8% by mass was used.

[0076] [Synthesis Example 1] Synthesis of Compound X1 10 g of polyoxyethylene monomethyl ether with an average molecular weight of 4,000 (manufactured by NOF Corporation, trade name "Uniox M-4000") was dissolved in 100 g of pyridine and placed in a separable flask equipped with a stirrer. In a separate container, 2 g of p-toluenesulfonic acid chloride was dissolved in 20 g of pyridine to obtain a solution which was added dropwise to the above separable flask over 1 hour, and the reaction was continued at room temperature for 6 hours. The resulting reaction product was added dropwise to 1 L of diethyl ether and purified by precipitation to remove the remaining p-toluenesulfonate, obtaining 8 g of polyoxyethylene monomethyl ether tosylate. Next, 50 g of aqueous solution containing 2 g of pentaethylenehexamine was stirred and reacted dropwise over 2 hours at room temperature while adding 80 g of aqueous solution containing 8 g of tosylate dropwise. The resulting reaction product was added dropwise to 1 L of diethyl ether, and the residual pentaethylenehexamine was removed by precipitation and purification. Further vacuum drying yielded 6 g of compound X1 (reaction product of tosylated polyoxyethylene monomethyl ether and pentaethylenehexamine).

[0077] [Synthesis Example 2] Synthesis of Compound X2 100 g of polyoxyethylene monomethyl ether (Fluca) with an average molecular weight of 5,000, 5 g of trimethylamine hydrochloride, 8 g of tripropylamine, and 8 g of p-toluenesulfonic acid chloride were dissolved in 300 g of acetonitrile and placed in a separable flask equipped with a stirrer. The mixture was reacted at 30°C for 2 hours with stirring to obtain a solution of polyoxyethylene monomethyl ether tosylate. Next, 47 g of pentaethylenehexamine was dissolved in 230 g of acetonitrile and placed in another separable flask equipped with a stirrer. The mixture was kept at 40°C with stirring, and the above tosylate was added dropwise over 1 hour. The reaction was continued for a further 9 hours with stirring. After the reaction, the mixture was allowed to stand at room temperature for 16 hours, and the precipitated by-product was removed by decantation. The supernatant obtained by decantation was concentrated using an evaporator, dissolved in 500 g of water, filtered, and further purified using a dialyzer to obtain a 2% by mass aqueous solution of compound X2 (reaction product of tosylated polyoxyethylene monomethyl ether and pentaethylenehexamine).

[0078] [Example 1] Compound X1-coated titanium oxide nanosheet dispersion A mixture was prepared by adding 33 g of an aqueous solution containing 2.0% by mass of compound X1 and 20 g of an aqueous dispersion containing 2.0% by mass of TiNS from Production Example 1. The mixture was concentrated in an evaporator to obtain a dispersion with a TiNS content of 2.0% by mass. Tris-HCl buffer (1 M, pH = 7.0) and 0.05% by mass of dilute hydrochloric acid were added to adjust the pH to 7.0. ２ Add O, and H in mass ratio ２ O / D ２ The dilution ratio was set to 0 = 9 / 1, and Tris-HCl buffer (10 mM, pH = 7.0) was added to obtain a dispersion of titanium oxide nanosheets coated with compound X1. The TiNS content in the dispersion was adjusted to match the content required for various measurements.

[0079] (Measurement of liquid properties) The zeta potential, particle size D50, and pH of the obtained dispersion of titanium oxide nanosheets coated with compound X1 were measured using the method described above. The results are shown in Table 1.

[0080] (D ２ (Stability evaluation by time-dependent changes in the splitting peak of the quadrupole interaction) A mixture was prepared by combining 400 μL of a dispersion of compound X1-coated titanium oxide nanosheets containing 0.8% by mass of TiNS with 200 μL of a solution containing additives at three times the concentration of each measurement condition. The additives used were 50 mM pH buffer (tris hydrochloride or phosphate buffer (sodium dihydrogen phosphate / disodium hydrogen phosphate)), 300 mM inorganic salt (sodium chloride or potassium chloride), and 1% by mass surfactant (CHAPS, n-Dodecyl-β-D-maltoside, n-Octyl-β-D-glucoside, sodium cholate or MEGA-8). For each mixture, measurements were taken 1 hour after preparation and again 48 hours later. ２ D in H-NMR ２ The splitting peak of the O quadrupole interaction was measured. The value at 1 hour after preparation of the mixture was used as a baseline, and the degree of change in the value after 48 hours was examined and evaluated according to the following criteria: (Baseline) A: D after 48 hours ２ O: Change in quadrupole splitting width is less than 20% B: D after 48 hours ２O: Change in quadrupole splitting width is between 20% and 50% C: D after 48 hours ２ Change in the quadrupole splitting width of O is 50% or more.

[0081] (Stability evaluation by time-dependent changes in the splitting peak of quadrupole interaction in deuterated organic solvents) 400 μL of a dispersion of titanium oxide nanosheets coated with compound X1 containing 0.8% by mass of TiNS was completely dewatered using an evaporator, and a deuterated organic solvent (DMSO-d6 or MeOD-d4) was added until the TiNS concentration was 0.8% by mass. For each mixture, the following was observed: 1 hour after preparation of the mixture, and again 48 hours later. ２ The quadrupole splitting peaks of deuterated organic solvents were measured using 1H-NMR. The degree of change in the value after 48 hours was confirmed, using the value at 1 hour after preparation of the mixture as a baseline, and evaluated according to the following criteria: (Criteria) A: Change in quadrupole splitting width of deuterated organic solvent less than 20% after 48 hours B: Change in quadrupole splitting width of deuterated organic solvent between 20% and 50% after 48 hours C: Change in quadrupole splitting width of deuterated organic solvent more than 50% after 48 hours

[0082] [Example 2] Compound X1-coated titanium oxide nanosheet dispersion A mixture was prepared by combining 500 μL of an aqueous solution containing 2.0% by mass of compound X1 with 500 μL of an aqueous dispersion containing 0.4% by mass of TiNS from Production Example 1. After the mixture was allowed to stand for 60 minutes, it was subjected to sonication for 10 seconds. To remove unadsorbed compound X1, centrifugation (15,000 rpm) was performed at 4°C for 15 minutes using a centrifuge (product name CR22N, manufactured by Eppendorf Hi-Mac Technologies, Inc.). The resulting precipitate was dissolved in 1 mL of Tris-HCl buffer (10 mM, pH = 7.0, H ２ O / D ２ The mixture was redispersed in a solution of O=9 / 1 to obtain a dispersion of titanium oxide nanosheets coated with compound X1. The TiNS content in the dispersion was adjusted by controlling the amount of buffer solution used to disperse the final precipitate, so that it matched the content required for various measurements. Liquid properties and stability were measured and evaluated using the same method as in Example 1. The results are shown in Table 1.

[0083] [Example 3] Compound X2-coated titanium oxide nanosheet dispersion A compound X2-coated titanium oxide nanosheet dispersion was obtained in the same manner as in Example 1, except that 44 g of a 2.0% by mass aqueous solution of compound X2 was used instead of 33 g of a 2.0% by mass aqueous solution of compound X1. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0084] [Example 4] Compound X1-coated titanium niobium oxide nanosheet dispersion A dispersion of compound X1-coated titanium niobium oxide nanosheets was obtained in the same manner as in Example 1, except that TiNbNS from Production Example 2 was used instead of TiNS from Production Example 1. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0085] [Example 5] Compound X2-coated titanium niobium oxide nanosheet dispersion A dispersion of compound X2-coated titanium niobium oxide nanosheets was obtained in the same manner as in Example 3, except that TiNbNS from Production Example 2 was used instead of TiNS from Production Example 1. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0086] [Comparative Example 1] Casein-coated titanium oxide nanosheet dispersion A mixture was prepared by combining 500 μL of an aqueous solution containing 2.0% by mass of casein with 500 μL of an aqueous dispersion containing 0.4% by mass of TiNS from Production Example 1. After the mixture was allowed to stand for 60 minutes, it was subjected to sonication for 10 seconds. To remove unadsorbed casein, centrifugation (15,000 rpm) was performed at 4°C for 15 minutes using a centrifuge (product name CR22N, manufactured by Eppendorf Hi-Mac Technologies, Inc.). The resulting precipitate was redispersed in Tris-HCl buffer. This centrifugation and redispersion washing operation was repeated three times, and the final precipitate was dissolved in 500 μL of Tris-HCl buffer (1 mM, pH 7.7, H ２ O / D ２ The casein-coated titanium oxide nanosheet dispersion was obtained by redispersing the nanosheets in a solution of O=9 / 1. The TiNS content in the dispersion was adjusted by controlling the amount of buffer solution used to disperse the final precipitate, so that it matched the content required for various measurements. Liquid properties and stability were measured and evaluated using the same method as in Example 1. The results are shown in Table 1.

[0087] [Comparative Example 2] Lysozyme-coated titanium oxide nanosheet dispersion A lysozyme-coated titanium oxide nanosheet dispersion was obtained in the same manner as in Comparative Example 1, except that a 2.0% by mass aqueous solution of lysozyme was used instead of a 2.0% by mass aqueous solution of casein. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0088] [Comparative Example 3] Albumin-Coated Titanium Oxide Nanosheet Dispersion An albumin-coated titanium oxide nanosheet dispersion was obtained in the same manner as in Comparative Example 1, except that a 2.0% by mass aqueous solution of albumin was used instead of a 2.0% by mass aqueous solution of casein. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0089] [Comparative Example 4] A casein-coated titanium niobium nanosheet dispersion was obtained in the same manner as in Comparative Example 1, except that TiNbNS from Production Example 2 was used instead of TiNS from Production Example 1. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0090] [Comparative Example 5] A lysozyme-coated titanium niobium oxide nanosheet dispersion was obtained in the same manner as in Comparative Example 2, except that TiNbNS from Production Example 2 was used instead of TiNS from Production Example 1. The liquid properties were measured and the stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0091] [Comparative Example 6] An albumin-coated titanium niobium oxide nanosheet dispersion was obtained in the same manner as in Comparative Example 3, except that TiNbNS from Production Example 2 was used instead of TiNS from Production Example 1. Liquid properties were measured and stability was evaluated using the same method as in Example 1. The results are shown in Table 1.

[0092]

[0093] As shown in Table 1, the dispersion of nanosheets coated with compound X1 or compound X2 showed a stability rating of D after 48 hours, which was used as an indicator for stability evaluation. ２ ​The change in O quadrupole splitting width was small in all cases. Nanosheets coated with compound X1 or compound X2 showed excellent resistance to buffer solutions, salts, surfactants, and deuterated organic solvents, confirming their usefulness as oriented agents for NMR measurements.

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