Quantitative method for amino-group-containing compounds protected with Fmoc-based protecting groups

Abstract

In the present invention, it was discovered that an amino-group-containing compound protected by protecting groups having an Fmoc skeleton can be accurately quantified by removing the protecting groups having an Fmoc skeleton from said compound and measuring the resulting amount of dibenzofulvenes contained or the amount of dibenzofulvene derivatives contained.

Description

[Technical field]

[0001] The present invention relates to a method for quantifying an amino group-containing compound protected with a protecting group having an Fmoc skeleton. [Background technology]

[0002] In recent years, the development of drug discovery technology that enables drug discovery for tough targets such as protein-protein interaction inhibitors, agonists, and molecular chaperones using molecular peptide compounds (molecular weight 500-2000) has attracted attention. These compounds are generally synthesized on a solid phase by extending amino acids whose N-terminus is protected with an Fmoc group. In pharmaceutical manufacturing, it is very important to evaluate the content of raw materials from the viewpoint of quality control, and ICHQ11 requires that appropriate quality control items be established for raw materials set as starting materials under GMP. When Fmoc-amino acids are used as starting materials, their content is generally evaluated by titration, and quantitative NMR is also used. The titration method is most commonly used due to the simplicity of the test, but if the measured material contains acids such as carboxylic acids as impurities, the accuracy of the content measurement is lost. In addition, while quantitative NMR has the advantage of being able to directly and accurately measure the number of hydrogen atoms at the functional group level, it cannot separate multiple components, unlike chromatographic methods such as HPLC and GC.

[0003] The Fmoc group is a protecting group that is widely used as a protecting group for compounds containing an amino group. This Fmoc group has ultraviolet (UV) absorption, so it can be quantified by a chromatographic method. In addition, a method is known for quantifying the loading amount of Fmoc groups on polymer beads by utilizing dibenzofulvene (DBF) generated during removal of the Fmoc group (Non-Patent Documents 1, 2, 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] William SN et al. Biotechnol. Bioeng. 1998, 61, 55-60. [Non-Patent Document 2] Laszlo V. et al. J. Chromatogr. A 2000, 869, 171-179. [Non-Patent Document 3] Freeman CE et al. Talanta 2005, 65, 574-577. Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, the Fmoc group has ultraviolet (UV) absorption, so that it can be quantified by chromatography. However, the molar absorption coefficient at the same wavelength differs for each compound, and even if a compound has the same molar absorption coefficient under certain conditions, the molar absorption coefficient may differ if the solvent composition at the time of elution in chromatography changes. Therefore, it is necessary to prepare a standard sample of the compound whose content has been confirmed for each compound to be quantified. For example, when quantifying Fmoc-amino acids consisting of natural amino acids by chromatography, since the standard samples of the Fmoc-amino acids are already available from many manufacturers, it is possible to quantify the amount by comparing the standard samples with the Fmoc-amino acids by chromatography. On the other hand, in the case of non-natural amino acids and peptides protected by the Fmoc group, which have been used in recent years, it is difficult to obtain standard samples of the amino acids and peptides, so when measuring by chromatography, it is necessary to synthesize the standard samples one by one and confirm the content.

[0006] In addition, the above-mentioned prior art documents evaluate the loading amount of Fmoc groups on polymer beads, but this is a quantitative evaluation including other impurities having Fmoc groups. Furthermore, dibenzofulvene generated by removing Fmoc groups is an unstable molecule and decomposes over time, which may cause a decrease in the quantitative accuracy of amino group-containing compounds protected with a protecting group having an Fmoc skeleton.

[0007] The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a method for quantifying an amino group-containing compound protected with a protecting group having an Fmoc skeleton to the first decimal place, which is necessary for the production of pharmaceuticals, using only one sample of the amino group-containing compound protected with a protecting group having an Fmoc skeleton. [Means for solving the problem]

[0008] The present inventors have discovered the following means for solving the above problems, and have completed the present invention. 1) We have discovered a novel method for quantifying an amino-containing compound protected with a protecting group having an Fmoc skeleton by an indirect technique in which, instead of quantifying the compound itself, the protecting group having an Fmoc skeleton is removed from the compound and the content of dibenzofulvene or its derivative produced thereby is quantified. 2) It was found that the content of the amino-containing compound protected with a protecting group having an Fmoc skeleton can be calculated to one decimal place, as required for quality control of pharmaceutical raw materials, based on the purity of the amino-containing compound protected with a protecting group having an Fmoc skeleton in the crude product and the content of dibenzofulvene or its derivative produced after removal of the protecting group having an Fmoc skeleton. 3) By making the purity measurement conditions the same as those for dibenzofulvene or its derivatives, it became possible to calculate the content accurately with little error. 4) We found reaction conditions under which the deprotection reaction rate of the Fmoc protecting group was 100% and side reactions were almost 0%, enabling more accurate quantification. 5) It was found that by mixing dibenzofulvene or its derivative generated after removal of the protecting group having the Fmoc skeleton with an acid, the stability of dibenzofulvene or its derivative can be improved and a value closer to the true value can be calculated. That is, the present invention includes the following.

[0009] The present invention relates to a method for quantifying an amino group-containing compound protected with a protecting group having an Fmoc skeleton as follows: (A1) A method for quantifying the content of an amino group-containing compound protected with a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as a first Fmoc compound) contained in a crude product, comprising: The method comprises deprotecting a protecting group from a first Fmoc compound with a base in a solution (hereinafter sometimes referred to as a first solution), and quantifying the content of dibenzofulvene or a derivative thereof produced thereby. (A2) further comprises deprotecting a protecting group from an amino-group-containing compound having a protecting group with an Fmoc skeleton (hereinafter sometimes referred to as a second Fmoc compound) used as a standard in the solution (A2) (hereinafter sometimes referred to as a second solution) with a base, and quantifying the content of dibenzofulvene or a derivative thereof produced thereby, The method according to (A1), wherein the content (% by weight) of the first Fmoc compound relative to the second Fmoc compound is calculated by comparing the content of dibenzofulvene or a derivative thereof in the first solution with the content of dibenzofulvene or a derivative thereof in the second solution. (A3) The method according to (A2), wherein the deprotection reaction proceeds quantitatively in the first solution and the second solution. (A4) The method according to any one of (A1) to (A3), wherein the content of dibenzofulvene or a derivative thereof is quantified by measuring a peak area of ​​dibenzofulvene or a derivative thereof. (A5) further comprising correcting the content based on the Fmoc purity of the crude product; The method according to any one of (A2) to (A4), wherein the "Fmoc purity" refers to a ratio of the content of the first Fmoc compound to the total content of compounds protected with all types of protecting groups having an Fmoc skeleton contained in the crude product. (A6) The method according to (A5), wherein the Fmoc purity is calculated based on (i) the peak area of ​​the first Fmoc compound and (ii) each peak area of ​​a compound protected with a protecting group having an Fmoc skeleton other than the first Fmoc compound in the crude product. (A7) The method according to any one of (A2) to (A6), further comprising correcting the content using an internal standard substance. (A8) The method according to (A7), comprising comparing the content of an internal standard substance contained in the first solution (hereinafter sometimes referred to as a first internal standard substance) with the content of an internal standard substance contained in the second solution (hereinafter sometimes referred to as a second internal standard substance). (A9) The method according to (A8), wherein the contents of the first and second internal standards are quantified by measuring a peak area of ​​the first internal standard and a peak area of ​​the second internal standard, respectively. (A10) The method according to any one of (A8) to (A9), wherein the first and second internal standards are independently selected from the group consisting of anthracene, phenanthrene, naphthalene, benzene, toluene, triphenylene, and naphthacene. (A11) The method according to any one of (A8) to (A10), wherein the first and second internal standards are anthracene. (A12) The method according to any one of (A4), (A6), and (A9), wherein each peak area is measured by a chromatographic method. (A13) The method according to (A12), wherein the chromatography method is liquid chromatography or gas chromatography. (A14) The method according to (A12) or (A13), wherein each peak area is measured under the same measurement conditions. (A15) The method according to any one of (A1) to (A14), wherein the protecting group having an Fmoc skeleton is represented by the following formula (1): TIFF0007671251000001.tif39170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl. (A16) The method according to any one of (A1) to (A15), wherein the protecting group having an Fmoc skeleton is an Fmoc group, an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br) group, a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, an sm-Fmoc group, or an rm-Fmoc group. (A17) The method according to any one of (A1) to (A16), wherein the protecting group having an Fmoc skeleton is an Fmoc group. (A18) The method according to any one of (A1) to (A17), wherein the first Fmoc compound and / or the second Fmoc compound is not supported on a solid phase. (A19) The method according to any one of (A1) to (A18), wherein the first Fmoc compound and / or the second Fmoc compound is an amino acid, a peptide, or a small organic compound protected with a protecting group having an Fmoc skeleton. (A20) The method according to any one of (A1) to (A19), wherein the first Fmoc compound and / or the second Fmoc compound is an amino acid or a peptide protected with a protecting group having an Fmoc backbone. (A21) The method according to (A19) or (A20), wherein the amino acid is an α-amino acid or a β-amino acid. (A22) The method according to any one of (A19) to (A21), wherein the carboxy group in the main chain of the amino acid is a free carboxy group. (A23) The method according to (A19) or (A20), wherein the peptide comprises α-amino acids and / or β-amino acids or consists of α-amino acids and / or β-amino acids. (A24) The method according to any one of (A1) to (A23), wherein the base is an organic base. (A25) The method according to any one of (A1) to (A24), wherein the base is at least one selected from the group consisting of amines, bases having an amidine skeleton, and bases having a guanidine skeleton. (A26) The organic base is 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetramethyl The method according to (A24), wherein the aryl group is selected from the group consisting of guanidine, 1,8-bis(tetramethylguanidino)naphthalene, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, p-dimethylaminopyridine, piperidine, morpholine, dicyclohexylamine, p-dimethylaminopyridine, diisopropylethylamine, pyridine, piperazine, and tetrabutylammonium fluoride. (A27) The method according to any one of (A1) to (A26), wherein the base is an organic base containing one or more tertiary amines and containing no primary or secondary amines. (A28) The organic base containing one or more tertiary amines and not containing primary and secondary amines is 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H,-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2,2,2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-trimethyl-1H,5,7-triphenylamine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H,-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2,2,2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-triphenylamine, 1,5-diazabicyclo[4.3.0]-5-nonene, 1,5-diazabicyclo[4.3.0]-triphenylamine ... The method according to (A27), wherein the aryl group is selected from the group consisting of azabicyclo[4.4.0]dec-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,8-bis(tetramethylguanidino)naphthalene, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, and p-dimethylaminopyridine. (A29) The method according to (A27), wherein the organic base containing one or more tertiary amines and no primary or secondary amines is 1,8-diazabicyclo[5.4.0]-7-undecene. (A30) The method according to any one of (A1) to (A29), further comprising adding an acid to the first and / or second solution after removal of the protecting group having an Fmoc skeleton. (A31) The method according to (A30), wherein the acid is an organic acid and / or an inorganic acid. (A32) The method according to (A31), wherein the organic acid is selected from the group consisting of citric acid, oxalic acid, maleic acid, tetramethylammonium hydrogen sulfate, formic acid, acetic acid, trifluoroacetic acid, propionic acid, monofluoroacetic acid, difluoroacetic acid, trichloroacetic acid, monochloroacetic acid, dichloroacetic acid, and combinations thereof. (A33) The method according to (A31) or (A32), wherein the inorganic acid is selected from the group consisting of phosphoric acid, boric acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, phosphonic acid, and combinations thereof. (A34) The method according to any one of (A1) to (A33), wherein the solvent of the first and / or second solution is an aprotic polar solvent. (A35) The method according to (A34), wherein the aprotic polar solvent is DMF, DMA, NMP, DMSO, or acetonitrile. (A36) The method according to any one of (A1) to (A35), wherein the protecting group is deprotected at a reaction temperature of 0°C to 50°C. (A37) The content (wt%) is expressed by the following formula: TIFF0007671251000002.tif9170 (in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A Tis the peak area of ​​dibenzofulvene or its derivative in the first solution. Or the formula: TIFF0007671251000003.tif9170 (in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, IS S is the peak area of ​​the internal standard of the second solution, IS T is the peak area of ​​the internal standard of the first solution, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or its derivative in the first solution. or the following formula: TIFF0007671251000004.tif9170(in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or a derivative thereof in the first solution, P S is Fmoc purity.) Or the formula: TIFF0007671251000005.tif9170(in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, ISS is the peak area of ​​the internal standard of the second solution, IS T is the peak area of ​​the internal standard of the first solution, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or a derivative thereof in the first solution, P S is Fmoc purity.) The method according to any one of (A2) to (A36), wherein the calculation is based on the above.

[0010] The present invention also relates to the following method for stabilizing dibenzofulvene or a derivative thereof: (B1) A method for stabilizing dibenzofulvene or a derivative thereof contained in a crude product, the method comprising mixing dibenzofulvene or a derivative thereof with an acid. (B2) The method according to (B1), which comprises mixing dibenzofulvene or a derivative thereof produced by removing a protecting group having an Fmoc skeleton from an amino group-containing compound protected with the protecting group having an Fmoc skeleton (hereinafter, may be referred to as an Fmoc compound) with an acid. (B3) The method according to (B2), wherein the protecting group having an Fmoc skeleton is represented by the following formula (1): TIFF0007671251000006.tif39170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl. (B4) The method according to (B2) or (B3), wherein the protecting group having an Fmoc skeleton is an Fmoc group, an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br) group, a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, an sm-Fmoc group, or an rm-Fmoc group. (B5) The method according to any one of (B2) to (B4), wherein the protecting group having an Fmoc skeleton is an Fmoc group. (B6) The method according to any one of (B2) to (B5), wherein the Fmoc compound is a low molecular weight organic compound protected with a protecting group having an Fmoc skeleton that is not supported on a solid phase. (B7) The method according to any one of (B2) to (B6), wherein the Fmoc compound is an amino acid, a peptide, or a low molecular weight organic compound protected with a protecting group having an Fmoc skeleton. (B8) The method according to any one of (B2) to (B7), wherein the Fmoc compound is an amino acid or a peptide protected with a protecting group having an Fmoc skeleton. (B9) The method according to (B7) or (B8), wherein the amino acid is an α-amino acid or a β-amino acid. (B10) The method according to (B7) or (B8), wherein the peptide comprises α-amino acids and / or β-amino acids or consists of α-amino acids and / or β-amino acids. (B11) The method according to (B2), wherein the acid is an organic acid and / or an inorganic acid. (B12) The method according to (B11), wherein the organic acid is selected from the group consisting of citric acid, oxalic acid, maleic acid, tetramethylammonium hydrogen sulfate, formic acid, acetic acid, trifluoroacetic acid, propionic acid, monofluoroacetic acid, difluoroacetic acid, trichloroacetic acid, monochloroacetic acid, dichloroacetic acid, and combinations thereof. (B13) The method according to (B11), wherein the inorganic acid is selected from the group consisting of phosphoric acid, boric acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, phosphonic acid, and combinations thereof.

[0011] The present disclosure also relates to the following stabilizers: (C1) A stabilizer for stabilizing dibenzofulvene or a derivative thereof. (C2) The stabilizer according to (C1), which is an acid. (C3) The stabilizer according to (C2), wherein the acid is an organic acid and / or an inorganic acid. (C4) The stabilizer according to (C3), wherein the organic acid is selected from the group consisting of citric acid, oxalic acid, maleic acid, tetramethylammonium hydrogen sulfate, formic acid, acetic acid, trifluoroacetic acid, propionic acid, monofluoroacetic acid, difluoroacetic acid, trichloroacetic acid, monochloroacetic acid, dichloroacetic acid, and combinations thereof. (C5) The stabilizer according to (C3), wherein the inorganic acid is selected from the group consisting of phosphoric acid, boric acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, phosphonic acid, and combinations thereof.

[0012] Alternatively, the present disclosure also relates to the following quantitative method for an amino group-containing compound. (D1) A method for quantifying the content of an amino group-containing compound generated from an amino group-containing compound protected with a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as an Fmoc compound) contained in a crude product, comprising the steps of: The method includes quantifying the content of an amino group-containing compound produced by removing a protecting group having an Fmoc skeleton from an Fmoc compound. (D2) The method according to (D1), wherein the content of the amino group-containing compound is measured by a chromatographic method. (D3) The method according to (D2), wherein the chromatographic method is hydrophilic interaction chromatography (HILIC). (D4) The method according to any one of (D2) to (D3), wherein the chromatographic detector is a charged aerosol detector (CAD) and / or a mass spectrometer. (D5) The method according to any one of (D1) to (D4), wherein the protecting group having an Fmoc skeleton is represented by the following formula (1): TIFF0007671251000007.tif39170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl. (D6) The method according to any one of (D1) to (D5), wherein the protecting group having an Fmoc skeleton is an Fmoc group, an Fmoc(2,7tb) group, an Fmoc(1Me) group, an Fmoc(2F) group, an Fmoc(2,7Br) group, a mio-Fmoc group, a dio-Fmoc group, a tdf-Fmoc group, an Fmoc(2TMS) group, an Fmoc(2so3h) group, an sm-Fmoc group, or an rm-Fmoc group. (D7) The method according to any one of (D1) to (D6), wherein the protecting group having an Fmoc skeleton is an Fmoc group. (D8) The method according to any one of (D1) to (D7), wherein the Fmoc compound is not supported on a solid phase. (D9) The method according to any one of (D1) to (D8), wherein the Fmoc compound is an amino acid, a peptide, or a small organic compound protected with a protecting group having an Fmoc skeleton. (D10) The method according to any one of (D1) to (D9), wherein the Fmoc compound is an amino acid or a peptide protected with a protecting group having an Fmoc skeleton. (D11) The method according to (D9) or (D10), wherein the amino acid is an α-amino acid or a β-amino acid. (D12) The method according to (D9) or (D10), wherein the peptide comprises α-amino acids and / or β-amino acids or consists of α-amino acids and / or β-amino acids. (D13) The method according to any one of (D9) to (D12), wherein the carboxy group in the main chain of the amino acid is a free carboxy group. (D14) The method according to any one of (D1) to (D13), wherein the protecting group having an Fmoc skeleton is removed with a base. (D15) The method according to (D14), wherein the base is an organic base. (D16) The method according to (D14) or (D15), wherein the base is at least one kind of base selected from the group consisting of amines, bases having an amidine skeleton, and bases having a guanidine skeleton. (D17) The organic base is 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,1,3,3-tetramethylguanidine, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 15. The method according to claim 14, wherein the aryl group is selected from the group consisting of arylamine, 1,8-bis(tetramethylguanidino)naphthalene, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, p-dimethylaminopyridine, piperidine, morpholine, dicyclohexylamine, p-dimethylaminopyridine, diisopropylethylamine, pyridine, piperazine, and tetrabutylammonium fluoride. (D18) The method according to any one of (D14) to (D17), wherein the base is an organic base containing one or more tertiary amines and no primary or secondary amines. (D19) The organic base containing one or more tertiary amines and not containing primary and secondary amines is 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H,-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2,2,2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-triazabicyclo[4.4.0] The method according to (D18), wherein the aryl group is selected from the group consisting of dec-5-ene, 2-tert-butyl-1,1,3,3-tetramethylguanidine, 1,8-bis(tetramethylguanidino)naphthalene, 1,8-bis(tetramethylguanidino)naphthalene, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, and p-dimethylaminopyridine. (D20) The method according to (D18) or (D19), wherein the organic base containing one or more tertiary amines and no primary or secondary amines is 1,8-diazabicyclo[5.4.0]-7-undecene. Effect of the Invention

[0013] According to the method of the present invention, it is possible to quantitatively determine with high accuracy and in a simple manner the content of an amino-containing compound protected with a protecting group having an Fmoc skeleton contained in an analytical sample, and therefore the present invention can be a very useful method for quantifying the compound at a quality level required for the production of pharmaceuticals. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram showing a chromatogram of the standard solution (a solution obtained by treating Fmoc-Gly-OH with DBU) in Example 1. [Diagram 2] FIG. 2 is a chromatogram of a sample solution containing crude Fmoc-NMe-Val-OH in Example 1 after the sample solution was treated with DBU. [Diagram 3]FIG. 3 is a chromatogram of a sample solution containing crude Fmoc-Pro-OH in Example 1 after the sample solution was treated with DBU. [Figure 4] FIG. 4 is a chromatogram of the sample solution containing crude Fmoc-NMe-Ala-OH in Example 1 after the sample solution was treated with DBU. [Diagram 5] FIG. 5 is a chromatogram of the sample solution containing crude Fmoc-Ile-OH in Example 1 after the sample solution was treated with DBU. [Figure 6] FIG. 6 is a chromatogram of the sample solution containing crude Fmoc-NMe-Leu-OH in Example 1 after the sample solution was treated with DBU. [Figure 7] FIG. 7 is a chromatogram of the sample solution containing crude Fmoc-NMe-Gly-OH in Example 1 after the sample solution was treated with DBU. [Figure 8] FIG. 8 is a diagram showing a chromatogram of the standard solution (a solution obtained by treating Fmoc-Gly-OH with DBU) in Example 2. [Figure 9] FIG. 9 is a chromatogram of the sample solution containing crude Fmoc-Val-OH in Example 2 after the sample solution was treated with DBU. [Figure 10] FIG. 10 is a chromatogram of the sample solution containing crude Fmoc-Phe-OH in Example 2 after the sample solution was treated with DBU. [Figure 11] FIG. 11 is a chromatogram of the sample solution containing crude Fmoc-NMe-Val-OH in Example 2 after the sample solution was treated with DBU. [Figure 12-1] FIG. 12-1 is a comparison of chromatograms of the sample solutions containing crude Fmoc-Val-OH in Example 3 before and after treatment with DBU. [Figure 12-2] FIG. 12-2 is an enlarged view of a portion of FIG. 12-1. [Figure 13]FIG. 13 is a chromatogram of the sample solution containing crude Fmoc-Val-OH in Example 3 before the sample solution was treated with DBU. [Figure 14] FIG. 14 shows a chromatogram of the sample solution containing crude Fmoc-Val-OH in Example 3 after the sample solution was treated with DBU. [Figure 15-1] FIG. 15-1 is a diagram comparing chromatograms of the sample solutions containing crude Fmoc-Phe-OH in Example 3 before and after treatment with DBU. [Figure 15-2] FIG. 15-2 is an enlarged view of a portion of FIG. 15-1. [Figure 16] FIG. 16 is a chromatogram of the sample solution containing crude Fmoc-Phe-OH in Example 3 before the sample solution was treated with DBU. [Figure 17] FIG. 17 is a chromatogram of the sample solution containing crude Fmoc-Phe-OH in Example 3 after the sample solution was treated with DBU. [Figure 18-1] FIG. 18-1 is a diagram comparing chromatograms of the sample solutions containing crude Fmoc-NMe-Val-OH in Example 3 before and after treatment with DBU. [Figure 18-2] FIG. 18-2 is an enlarged view of a portion of FIG. 18-1. [Figure 19] FIG. 19 is a chromatogram of the sample solution containing crude Fmoc-NMe-Val-OH in Example 3 before treatment with DBU. [Figure 20] FIG. 20 is a chromatogram of a sample solution containing crude Fmoc-NMe-Val-OH in Example 3 after the sample solution was treated with DBU. [Figure 21] FIG. 21 is a diagram showing a comparison between the content of each Fmoc-amino acid calculated by the quantification method using DBF of the present invention in Example 5 and the content of each Fmoc-amino acid calculated by quantitative NMR. [Figure 22] FIG. 22 is a chromatogram of a standard solution of H-NMe-Gly-OH in Example 6. [Figure 23] FIG. 23 is a chromatogram of a sample solution containing crude Fmoc-NMe-Gly-OH in Example 6 after the sample solution was treated with DBU. [Figure 24] FIG. 24 is a comparison between the chromatogram of the standard solution of H-NMe-Gly-OH in Example 6 and the chromatogram of the sample solution containing crude Fmoc-NMe-Gly-OH after treatment with DBU. [Diagram 25] FIG. 25 is a chromatogram of the standard solution (a solution obtained by treating Fmoc-Gly-OH with DBU) in Example 7. [Figure 26] FIG. 26 is a chromatogram of a solution obtained by treating the sample solution containing crude Fmoc-Phe-Phe-OH in Example 7 with DBU. [Figure 27] FIG. 27 shows a chromatogram of a solution obtained by treating the sample solution containing crude Fmoc-Gly-Gly-OH in Example 7 with DBU. [Figure 28] FIG. 28 shows a chromatogram of a solution obtained by treating a sample solution containing crude Fmoc-Gly-Leu-OH in Example 7 with DBU. [Figure 29] FIG. 29 shows a chromatogram of a solution obtained by treating the sample solution containing crude Fmoc-Gly-bAla-OH in Example 7 with DBU. [Diagram 30] FIG. 30 is a diagram comparing chromatograms of the sample solutions containing crude Fmoc-Phe-Phe-OH in Example 7 before and after treatment with DBU. [Diagram 31] FIG. 31 is a comparison of chromatograms of the sample solutions containing crude Fmoc-Gly-Gly-OH in Example 7 before and after treatment with DBU. [Diagram 32]FIG. 32 is a diagram comparing chromatograms of the sample solutions containing crude Fmoc-Gly-Leu-OH in Example 7 before and after treatment with DBU. [Diagram 33] FIG. 33 is a diagram comparing chromatograms of the sample solutions containing crude Fmoc-Gly-bAla-OH in Example 7 before and after treatment with DBU. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] (Abbreviation) The symbols and abbreviations used in this specification have the following meanings. Fmoc: 9-fluorenylmethoxycarbonyl DBF: Dibenzofulvene DBU: 1,8-diazabicyclo[5.4.0]-7-undecene DMF: N,N-dimethylformamide DMA: N,N-Dimethylacetamide NMP: N-methylpyrrolidone DMSO: Dimethyl sulfoxide TFA: Trifluoroacetic acid TFE: 2,2,2-trifluoroethanol TMS: Trimethylsilyl CH3CN:Acetonitrile Na2SO4: Sodium sulfate Pip: piperidine HILIC: Hydrophilic Interaction Chromatography CAD: Charged Aerosol Detector HPLC: High-performance liquid chromatography GC: Gas chromatography PDA: Photodiode Array Gly: glycine Ala: Alanine Val: Valin Leu: Leucine Ile: Isoleucine Met: methionine Phe: Phenylalanine Tyr: Tyrosine Trp: Tryptophan His: Histidine Lys: Lysine Arg: arginine Ser: Serine Thr: Threonine Asp: Aspartic acid Glu: glutamic acid Asn: Asparagine Gln: glutamine Cys: Cysteine Pro: Proline Cha: Cyclohexylalanine cLeu: cycloleucine NMe: N-methyl bAla: β-alanine

[0016] In one embodiment, the present invention relates to a method for quantifying the content of an amino group-containing compound protected with a protecting group having an Fmoc skeleton (hereinafter, sometimes referred to as a first Fmoc compound) in a crude product or an analytical sample.

[0017] In one embodiment, the method includes deprotecting a protecting group from a first Fmoc compound with a base in a solution (hereinafter sometimes referred to as a first solution), and quantifying the content of dibenzofulvene or a derivative thereof produced thereby.

[0018] In one embodiment, the method further comprises deprotecting an amino group-containing compound having a protecting group with an Fmoc skeleton (hereinafter referred to as a second solution) used as a standard with a base, and quantifying the content of dibenzofulvene or its derivative thus produced in a solution (hereinafter referred to as a second solution) different from the first solution. In this case, the content (wt%) of the first Fmoc compound relative to the second Fmoc compound can be calculated by comparing the content of dibenzofulvene or its derivative in the first solution with the content of dibenzofulvene or its derivative in the second solution.

[0019] In the present invention, the term "analytical sample" refers to a sample containing an amino group-containing compound protected with a protecting group having an Fmoc skeleton. As the analytical sample, it is preferable to use a crude product from which impurities derived from the analytical sample, water, organic salts, inorganic salts, residual solvents, and other impurities have been removed by utilizing filtration, adsorption, recrystallization, distillation, extraction, chromatography, and the like.

[0020] The term "content" in the present invention means the amount of any substance contained in an analytical sample (crude product) or a solution, for example, the amount of an amino group-containing compound protected with a protecting group having an Fmoc skeleton, an amino group-containing compound (deprotected form), dibenzofulvene or a derivative thereof, an internal standard substance, etc.

[0021] In the present invention, the "amino group-containing compound" refers to any compound having a primary amino group and / or a secondary amino group. The number of amino groups contained in the amino group-containing compound is not particularly limited, but is preferably 1 to 2, more preferably 1. Examples of the amino group-containing compound include amino acids, peptides, and low molecular weight organic compounds. The amino group-containing compound may or may not be supported on a solid phase (e.g., a resin for solid phase synthesis).

[0022] In the present specification, the term "resin for solid-phase synthesis" is not particularly limited as long as it can be used in the synthesis of peptide compounds by the solid-phase method. Specific examples of such resins for solid-phase synthesis include CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), 4-methoxytrityl chloride resin (Mmt), and other resins that can be removed under acidic conditions. The resin can be appropriately selected according to the functional group of the amino acid used. For example, when a carboxylic acid (main chain carboxylic acid or side chain carboxylic acid represented by Asp or Glu) or a hydroxy group on an aromatic ring (phenol group represented by Tyr) is used as the functional group of the amino acid, it is preferable to use trityl chloride resin (Trt resin) or 2-chlorotrityl chloride resin (CTC resin). When an aliphatic hydroxy group (aliphatic alcohol group represented by Ser or Thr) is used as the functional group on the amino acid side, it is preferable to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin) or 4-methyltrityl chloride resin (Mtt resin) as the resin. In this specification, resin may also be referred to as resin.

[0023] The type of polymer constituting the resin is not particularly limited. In the case of a resin made of polystyrene, either 100-200 mesh or 200-400 mesh may be used. The cross-linking rate is also not particularly limited, but 1% DVB (divinylbenzene) cross-linking is preferred. Examples of the type of polymer constituting the resin include Tentagel and Chemmatrix.

[0024] As used herein, an “amino acid” may be either a “natural amino acid” or an “amino acid analog.” As used herein, an “amino acid,” a “natural amino acid,” and an “amino acid analog” may be referred to as an “amino acid residue,” a “natural amino acid residue,” and an “amino acid analog residue,” respectively.

[0025] "Natural amino acids" are α-aminocarboxylic acids (α-amino acids) and refer to the 20 types of amino acids contained in proteins, specifically, Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro.

[0026] The "amino acid analogue" includes, but is not limited to, β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, hydroxycarboxylic acids, and non-natural amino acids (amino acids whose side chains are different from natural ones; for example, non-natural α-amino acids, β-amino acids, and γ-amino acids). In the case of α-amino acids, they may be D-amino acids or α,α-dialkylamino acids. In the case of β-amino acids and γ-amino acids, any steric configuration is allowed, as in the case of α-amino acids. There are no particular limitations on the selection of the amino acid side chain, but in addition to hydrogen atoms, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups are freely selected. Each of them may have a substituent, and the substituent is also freely selected from any functional group including, for example, an N atom, an O atom, an S atom, a B atom, a Si atom, or a P atom (i.e., an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group, a cycloalkyl group, etc., which may be substituted).

[0027] The main chain amino group of an amino acid may be unsubstituted (NH2 group) or substituted (i.e., -NHR group: R is, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl, which may have a substituent, and the carbon chain bonded to the N atom and the carbon atom at the α position may form a ring, as in proline). Such amino acids in which the main chain amino group is substituted are referred to as "N-substituted amino acids" in this specification. As used herein, the "N-substituted amino acids" preferably include N-alkyl amino acids (e.g., N-C1-C6 alkyl amino acids, N-C1-C4 alkyl amino acids, N-methyl amino acids, etc.), N-cycloalkyl amino acids, N-cycloalkyl alkyl amino acids, N-aryl amino acids, N-aralkyl amino acids, etc., but are not limited thereto.

[0028] The main chain carboxy group of the amino acid may be unsubstituted (carboxy group: COOH group) or may form an ester group (i.e., -COOR group: R is, for example, an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, or cycloalkyl group that may be substituted). Such an amino acid in which the main chain carboxy group forms an ester group is referred to as an "amino acid ester" in this specification. As used herein, the "amino acid ester" refers to an amino acid C1- 10 Alkyl esters, amino acid benzyl esters, amino acids [2,4-di(C1-C 25 Alkoxy)phenyl]methyl esters, amino acids (3,4,5-trimethyl C1-C 25 Examples of the amino acid include, but are not limited to, (3,4,5-trioctadecoxyphenyl)methyl ester, amino acid methyl ester, amino acid ethyl ester, amino acid tert-butyl ester, amino acid trityl ester, amino acid (3,4,5-trioctadecoxyphenyl)methyl ester, amino acid [2-(12-docoxydodecoxy)-9-(3-fluorophenyl)fluoren-9-yl]ester, and [2,4-di(docoxy)phenyl]methyl ester.

[0029] The main chain carboxy group of the amino acid may form an amide group (i.e., -CONR1R2 group: R1 and R2 are, for example, independently selected from the group consisting of hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl). Such an amino acid in which the main chain carboxy group is substituted with an amide group is referred to herein as an "amino acid amide". As used herein, the "amino acid amide" includes amino acids C1-C 10 Examples include, but are not limited to, alkylamides, amino acid benzylamides, amino acid bis[4-(docoxy)phenyl]methylamides (AJIPHASE®), and the like.

[0030] As used herein, the term "side chain" is used in the context of the side chain of an amino acid or the side chain of the cyclic portion of a cyclic peptide, and means a portion that is not included in the respective main chain structures.

[0031] As used herein, the term "peptide" includes linear or cyclic peptides in which two or more amino acids are linked, and the amino acids may be either "natural amino acids" or "amino acid analogs."

[0032] The "natural amino acids" and "amino acid analogs" that constitute the peptides include all of their corresponding isotopes. Isotopes of "natural amino acids" and "amino acid analogs" are those in which at least one atom has been replaced with an atom that has the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes contained in the "natural amino acids" and "amino acid analogs" that constitute the peptides of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35 S, 18 F, 36 Cl etc. are included.

[0033] In the present invention, "organic compound" means a compound containing carbon.

[0034] In the present invention, the term "low molecular weight organic compound" refers to a carbon-containing compound having a small molecular weight, and the molecular weight is preferably 1500 daltons or less.

[0035] In the present invention, the term "protecting group having an Fmoc skeleton" refers to an Fmoc group or a group in which an arbitrary substituent has been introduced at an arbitrary position of the structural skeleton of the Fmoc group. Specific examples of such a protecting group having an Fmoc skeleton include a protecting group represented by the following formula (1): TIFF0007671251000008.tif39170 (in the formula, R1-R8 are independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfo, and trimethylsilyl; R9~R 10 are independently hydrogen or methyl.

[0036] More specifically, examples of the protecting group having an Fmoc skeleton include a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,7-di-tert-butyl-Fmoc (Fmoc(2,7tb)) group, a 1-methyl-Fmoc (Fmoc(1Me)) group, a 2-fluoro-Fmoc (Fmoc(2F)) group, a 2,7-dibromo-Fmoc (Fmoc(2,7Br)) group, a 2-monoisooctyl-Fmoc (mio-Fmoc) group, a 2,7-diisooctyl-Fmoc (dio-Fmoc) group, a 2,7-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc Examples of such protecting groups include (tdf-Fmoc), 2,7-bis(trimethylsilyl)-Fmoc (Fmoc(2TMS)), (2-sulfo-9H-fluoren-9-yl)methoxycarbonyl (Fmoc(2so3h)), [(1S)-1-(9H-fluoren-9-yl)ethoxy]carbonyl (sm-Fmoc), [(1R)-1-(9H-fluoren-9-yl)ethoxy]carbonyl (rm-Fmoc), etc. Reagents for introducing protecting groups having these Fmoc skeletons can be purchased or synthesized by known methods.

[0037] In the present invention, the term "amino group-containing compound protected with a protecting group having an Fmoc skeleton" means a compound in which at least one of the primary amino group and / or secondary amino group of the amino group-containing compound is protected with a protecting group having an Fmoc skeleton.

[0038] In the present invention, an "amino acid protected with a protecting group having an Fmoc skeleton" refers to a natural amino acid or an amino acid analog in which at least one amino group in the main chain and / or side chain is protected with a protecting group having an Fmoc skeleton.

[0039] In the present invention, "amino group-containing compound protected with an Fmoc group" means a compound in which at least one of the primary amino group and / or secondary amino group of the amino group-containing compound is protected with an Fmoc group.

[0040] In the present invention, an "amino acid protected with an Fmoc group" refers to a natural amino acid or amino acid analog in which at least one amino group in the main chain and / or side chain is protected with an Fmoc group. An amino acid protected with an Fmoc group, a natural amino acid protected with an Fmoc group, or an amino acid analog protected with an Fmoc group may be referred to as an "Fmoc-amino acid," "Fmoc-natural amino acid," or "Fmoc-amino acid analog," respectively.

[0041] Examples of Fmoc-protected amino acids that can be used in the present invention include, but are not limited to, the following. Many of these Fmoc-protected amino acids can be purchased with or without the side chains protected and the amine moiety protected or unprotected. Those that cannot be purchased can be synthesized by known methods. TIFF0007671251000009.tif100170

[0042] In the present invention, the term "peptide protected with a protecting group having an Fmoc skeleton" refers to a peptide containing the above-mentioned "amino acid protected with a protecting group having an Fmoc skeleton". Examples of such peptides include dipeptides and oligopeptides having two or more amino acids, including either or both of the above-mentioned natural amino acid protected with a protecting group having an Fmoc skeleton and an amino acid analog protected with a protecting group having an Fmoc skeleton.

[0043] In the present invention, the term "peptide protected with an Fmoc group" refers to a peptide containing the above-mentioned "amino acid protected with an Fmoc group". Examples of such peptides include dipeptides and oligopeptides having two or more amino acids, including either or both of the above-mentioned natural amino acid protected with an Fmoc group and an amino acid analog protected with an Fmoc group.

[0044] In this specification, "alkyl" refers to a monovalent group derived by removing any one hydrogen atom from an aliphatic hydrocarbon, does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton, and has a subset of hydrocarbyl or hydrocarbon group structures containing hydrogen and carbon atoms. The alkyl includes linear and branched ones. The alkyl includes those having 1 to 20 carbon atoms (C1-C 20 , hereinafter referred to as “C p -C q " means that the number of carbon atoms is p to q.) alkyl, preferably C1-C 10 Examples of the alkyl include alkyl, C1-C8 alkyl, C1-C6 alkyl, and C1-C4 alkyl. Specific examples of the alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, tert-butyl, sec-butyl, 1-methylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1,2-dimethylpropyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,1,2,2-tetramethylpropyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, isopentyl, and neopentyl.

[0045] In the present specification, "fluoroalkyl" refers to a group in which one or more hydrogen atoms of the "alkyl" are substituted with fluorine atoms, and preferably includes C1-C8 fluoroalkyl, C1-C6 fluoroalkyl, C1-C4 fluoroalkyl, C1-C2 fluoroalkyl, etc. Specific examples of fluoroalkyl include trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, 2,2,3,3-tetrafluoropropyl, heptafluoropropyl, trifluoromethoxy, 2,2,2-trifluoroethoxy, pentafluoroethoxy, 2,2,3,3-tetrafluoropropoxy, heptafluoropropoxy, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl, etc.

[0046] In the present specification, "cycloalkyl" refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including a monocyclic ring, a bicyclic ring, and a spiro ring. Preferred is C3-C8 cycloalkyl. Cycloalkyl may be partially unsaturated. Specific examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0047] In the present specification, "cycloalkylalkyl" refers to a group in which any hydrogen atom in the above-defined "alkyl" is replaced with the above-defined "cycloalkyl". Preferred examples of cycloalkylalkyl include C3-C8 cycloalkylC1-C6 alkyl and C3-C6 cycloalkylC1-C4 alkyl. Specific examples include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0048] As used herein, "aryl" refers to a monovalent aromatic hydrocarbon ring, preferably a C6-C 10 Specific examples of the aryl include phenyl and naphthyl (e.g., 1-naphthyl and 2-naphthyl).

[0049] In the present specification, the term "heteroaryl" refers to a monovalent group of an aromatic ring containing preferably 1 to 4 heteroatoms in the atoms constituting the ring (also referred to as "within the ring" in the present specification), and may be partially saturated. The ring may be a single ring or two condensed rings (for example, a bicyclic heteroaryl condensed with benzene or a single ring heteroaryl). The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered ring heteroaryl). Specific examples of heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, benzofuranyl, benzothienyl, quinolyl, isoquinolyl, quinazolinyl, and quinoxalinyl.

[0050] In the present specification, the term "aralkyl (arylalkyl)" refers to a group containing both aryl and alkyl, for example, a group in which at least one hydrogen atom of the alkyl is replaced by an aryl, and preferably refers to a group having a C6-C 10 Aryl C1-C6 alkyl". For example, benzyl, phenethyl, etc.

[0051] As used herein, "alkenyl" refers to a monovalent group having at least one double bond (two adjacent sp2 carbon atoms). Depending on the configuration of the double bonds and the substituents (if present), the geometry of the double bond can be in the Entgegen (E) or Zusammen (Z) configuration, cis or trans. Alkenyl includes linear or branched chains, including linear chains containing internal olefins. Preferably, C2-C 10 Alkenyl, more preferably C2-C6 alkenyl. Specific examples of such alkenyl include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans), 3-butenyl, pentenyl, and hexenyl.

[0052] As used herein, "alkynyl" refers to a monovalent group having at least one triple bond (two adjacent SP carbon atoms). It includes straight or branched chain alkynyl, including internal alkylene. It is preferably C2-C 10 Alkynyl is preferably C2-C6 alkynyl. Specific examples of alkynyl include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, and hexynyl.

[0053] The term "alkoxy" as used herein means an oxy group to which the above-defined "alkyl" is bonded, and examples thereof include alkoxy groups having 1 to 20 carbon atoms (C1-C 20 Specific examples of the alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, and (3,7,11,15-tetramethylhexadecyl)oxy.

[0054] Halogens include fluoro (-F), chloro (-Cl), bromo (-Br), iodo (-I), and the like.

[0055] As used herein, "sulfo" is a group represented by -SO3H.

[0056] As used herein, "trimethylsilyl" is a group represented by -Si(CH3)3 or TMS.

[0057] In one embodiment, the first solution contains a crude product containing an amino group-containing compound protected with a protecting group having an Fmoc skeleton (first Fmoc compound) whose content is to be calculated, a solvent, and a base. The first solution may contain an internal standard. In the first solution, a reaction for deprotecting the protecting group having an Fmoc skeleton occurs. When a peptide protected with a protecting group having an Fmoc skeleton is synthesized by a solid-phase method, the method of the present invention may include a step of cleaving a peptide compound supported on a resin for solid-phase synthesis from the resin to obtain the peptide prior to the deprotection step. In this specification, the first solution may be referred to as a "sample solution". In addition, in this specification, the sample solution before the deprotection reaction may be referred to as an "unreacted" sample solution, and when used together with this term, the term "sample solution" may refer to a solution after the deprotection reaction. It is preferable that the deprotection reaction in the first solution proceeds quantitatively.

[0058] In one embodiment, the second solution contains an amino-group-containing compound protected with a protecting group having an Fmoc skeleton (second Fmoc compound) used as a standard, a solvent, and a base. The second solution may contain an internal standard. The second Fmoc compound is not limited as long as its exact content can be known, and for example, one with a purity and / or content of 100% or close to 100% is used. Specific examples of the second Fmoc compound include, but are not limited to, glycine (Fmoc-Gly-OH) in which an amino group is protected with an Fmoc group, which is sold by many vendors. As in the first solution, a reaction to deprotect the protecting group having an Fmoc skeleton also occurs in the second solution. In this specification, the second solution may be referred to as a "standard solution". In addition, in this specification, the standard solution before the deprotection reaction may be referred to as an "unreacted" standard solution, and when used together with this term, the term "standard solution" may refer to the solution after the deprotection reaction. It is preferable that the deprotection reaction in the second solution proceeds quantitatively.

[0059] For the first solution and / or the second solution, any solvent that dissolves the second Fmoc compound used as the analytical sample and standard can be used, and for example, aprotic polar solvents such as acetonitrile, DMF, DMA, NMP, DMSO, etc. The solvents used for the first solution and / or the second solution may be the same or different, but it is preferable to use the same solvent.

[0060] As used herein, the terms "removal" and "deprotection" refer to converting a primary amino group and / or a secondary amino group protected with a protecting group having an Fmoc skeleton into a state where it is not protected by the protecting group.

[0061] In one embodiment, the protecting group having an Fmoc skeleton can be removed by either a solid-phase reaction or a liquid-phase reaction, with the liquid-phase reaction being preferred. Also, the amino-containing compound protected with a protecting group having an Fmoc skeleton may or may not be supported on a solid phase, with the one not supported on a solid phase being preferred.

[0062] In one embodiment, a protecting group having an Fmoc skeleton can be removed by stirring and / or leaving the reaction mixture for 1 minute to 5 days in the presence of a base at a temperature of 0° C. to about the boiling point of the solvent, preferably at a temperature of 0° C. to 80° C., and more preferably at a temperature of 0 to 50° C. An internal standard substance that is not involved in the removal reaction may or may not be added to this reaction.

[0063] In one embodiment, any base capable of removing a protecting group can be used to remove a protecting group having an Fmoc skeleton. Such bases include organic bases or inorganic bases, and organic bases are preferred.

[0064] In some embodiments, the organic base includes 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinolizine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]-5-nonene, 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, 1,1,3,3-tetramethylguanidine, 1,8-bis(tetramethylguanidino)naphthalene, 2-tert-butoxy-1,5-dihydro ... Examples of bases include bases containing tertiary amines such as 1,1,3,3-tetrmethylguanidine, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, and p-dimethylaminopyridine, and in addition, other bases include, but are not limited to, piperidine, morpholine, dicyclohexylamine, p-dimethylaminopyridine, diisopropylethylamine, pyridine, piperazine, and tetrabutylammonium fluoride. Among these, organic bases containing one or more tertiary amines and no primary or secondary amines are preferred, and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) is more preferred.

[0065] In one embodiment, the present invention can use any equivalent amount of base capable of completely removing a protecting group having an Fmoc skeleton. Specifically, the base can be used in an amount of 0.1 to 20 v / v %, preferably 1 to 10 v / v %, and more preferably 1 to 4 v / v % in the solution.

[0066] In the present specification, the term "dibenzofulvene or a derivative thereof" refers to a compound represented by the following formula (2): TIFF0007671251000010.tif38170 (in the formula, R1~R 10 R1 to R of a protecting group having an Fmoc skeleton represented by formula (1) 10 The compounds represented by R1 to R 10 The following formula (3) is a formula in which all are hydrogen: The compound represented by TIFF0007671251000011.tif25170 is "dibenzofulvene."

[0067] In an embodiment, in the present invention, the content (% by weight) of the first Fmoc compound relative to the second Fmoc compound can be calculated by comparing the content of dibenzofulvene or a derivative thereof in the first solution with the content of dibenzofulvene or a derivative thereof in the second solution.

[0068] In an embodiment, the dibenzofulvene or a derivative thereof in the first solution and / or the second solution can be separated from other components in the first solution and / or the second solution by a chromatographic method, and its content can be quantified by the same method.

[0069] The chromatography method may be any method capable of separating an amino group-containing compound protected with a protecting group having an Fmoc skeleton, an amino group-containing compound (deprotected), dibenzofulvene (DBF) or a derivative thereof, and an internal standard. Examples of such chromatography methods include liquid chromatography, supercritical fluid chromatography, gas chromatography, thin layer chromatography, hydrophilic interaction chromatography (HILIC), ion exchange chromatography (IEX), size exclusion chromatography (SEC), electrophoresis, ion mobility spectrometry (e.g., ion mobility), and the like, and liquid chromatography, gas chromatography, electrophoresis, or ion mobility spectrometry is preferred. In addition, the liquid chromatography method is preferably high performance liquid chromatography (HPLC) or ultra-high performance high resolution liquid chromatography (UPLC). These methods may be known.

[0070] Normal phase liquid chromatography can be carried out by known methods using, for example, a stationary phase such as silica or alumina, and a mobile phase such as hexane, ethyl acetate, methylene chloride, isopropyl alcohol, ethanol, methanol, tetrahydrofuran, and mixed solvents thereof.

[0071] Reverse phase liquid chromatography can be performed by a known method. For example, a packing material modified with a hydrophobic compound, specifically, for example, silica gel modified with a hydrophobic compound, can be used as the stationary phase of reverse phase liquid chromatography. For example, an organic solvent, an aqueous solution, and a mixed solvent thereof can be used as the mobile phase of reverse phase liquid chromatography. For example, acetonitrile, methanol, ethanol, isopropyl alcohol, etc. can be used as the organic solvent. An acid such as formic acid, trifluoroacetic acid, or acetic acid can be included in the organic solvent. For example, water, an aqueous solution of formic acid, an aqueous solution of ammonium formate, an aqueous solution of trifluoroacetic acid, an aqueous solution of acetic acid, an aqueous solution of ammonium acetate, an aqueous solution of ammonium bicarbonate, an aqueous ammonia solution, a buffer solution, etc. can be used as the aqueous solution. In addition, an ion pair reagent such as sodium alkylsulfonate and its salt, tetraalkylammonium or trialkylamine and its salt, etc. can be added to the solvent. This can improve the separation ability of the analytical column when peak separation is poor. In liquid chromatography, when the mobile phase is a mixed solvent, the mixing ratio may be changed stepwise or continuously. The temperature conditions and mobile phase flow rate of the liquid chromatography can be appropriately set depending on the properties of dibenzofulvene or its derivatives, the internal standard, and the like.

[0072] Any method can be used for identifying and quantifying the amino-containing compound protected with a protecting group having an Fmoc skeleton, dibenzofulvene (DBF) or its derivative, and the internal standard. Examples of such methods include methods using an ultraviolet-visible light absorption detector, a diode array detector (PDA), a fluorescence detector, a differential refractive index detector, an evaporative light scattering detector, a charged particle detector (CAD), a mass spectrometer, and the like. From the viewpoint of high sensitivity and high selectivity detection, it is preferable to use an ultraviolet-visible light absorption detector. Therefore, it is more preferable to perform the separation and quantification method including the method of the present invention using LC-UV, LC-PDA, and the like, which are a combination of liquid chromatography (LC) and an ultraviolet-visible light absorption detector.

[0073] The dibenzofulvene or its derivative in the first solution (sample solution) or the second solution (standard solution) can be quantified by measuring its peak area by the above-mentioned chromatographic method.

[0074] In one embodiment, the content of the first Fmoc compound relative to the second Fmoc compound can be corrected based on the internal standard. That is, the first and second Fmoc compounds can be more accurately quantified by including an internal standard in the first solution (sample solution) and the second solution (standard solution) as an internal standard for correcting the injection amount of these solutions into a chromatograph. Correction based on the internal standard is performed, for example, by adding an internal standard during preparation of the first solution (sample solution) and / or the second solution (standard solution) and allowing them to stand, and adding the content of the internal standard to the quantification result (actual measurement value) of dibenzofulvene or its derivative generated after removal of the protecting group. More specifically, for example, the content can be corrected by comparing the content of the internal standard (sometimes referred to as the first internal standard) contained in the first solution with the content of the internal standard (sometimes referred to as the second internal standard) contained in the second solution. The timing of adding the internal standard is not limited to the time of preparation of the first solution (sample solution) and / or the second solution (standard solution), and may be after removal of the protecting group.

[0075] Any substance can be used as the internal standard as long as it can be separated from dibenzofulvene (DBF) or its derivatives by chromatography. For example, commercially available substances can be used as such internal standard substances, and specific examples include anthracene, phenanthrene, naphthalene, benzene, toluene, triphenylene, naphthacene, etc., and among these, anthracene is preferred.

[0076] The content of the internal standard in the first solution (sample solution) or the second solution (standard solution) can be quantified by measuring its peak area by a chromatographic method. The peak area of ​​the internal standard is preferably measured under the same measurement conditions as those of dibenzofulvene (DBF) or its derivatives.

[0077] In one embodiment, the content (wt%) of the first Fmoc compound relative to the second Fmoc compound is 1) The peak area (A) of dibenzofulvene or its derivative in the first solution (sample solution) and the second solution (standard solution) T and A S )and, 2) The concentration of the crude product in the first solution (sample solution) when it is assumed that the crude product consists only of the first Fmoc compound (C T ) and the concentration of the second Fmoc compound in the second solution (standard solution) (C s ) can be calculated using the following formula: TIFF0007671251000012.tif9170(in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or its derivative in the first solution.

[0078] Specifically, C T can be calculated based on the weighed value of the crude product and the molecular weight of the first Fmoc compound.

[0079] In addition, when the first Fmoc compound is an Fmoc-peptide synthesized by a solid phase method, the Fmoc peptide compound supported on a resin for solid phase synthesis is regarded as the first Fmoc compound, and TSpecifically, for example, C can be calculated based on the weight of the Fmoc peptide compound supported on the resin for solid phase synthesis and the molecular weight of the Fmoc peptide. T can be calculated.

[0080] In one embodiment, the content (wt%) of the first Fmoc compound relative to the second Fmoc compound is calculated by subtracting the peak area (IS) of the internal standard from that of the first solution (sample solution) and the second solution (standard solution) when correction is performed using an internal standard. T and IS S ) can be further used to calculate the following formula: TIFF0007671251000013.tif9170 (in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, IS S is the peak area of ​​the internal standard of the second solution, IS T is the peak area of ​​the internal standard of the first solution, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or its derivative in the first solution.

[0081] In one embodiment, the content can be corrected by the Fmoc purity of the crude product. Here, "Fmoc purity" refers to the percentage (%) of the content of the first Fmoc compound relative to the total content of all types of compounds protected with protecting groups having an Fmoc skeleton contained in the crude product. By carrying out this correction, errors due to compounds (impurities) having a protecting group having an Fmoc skeleton other than the amino-group-containing compound protected with a protecting group having an Fmoc skeleton in the crude product (analysis sample) can be eliminated, and the content can be calculated more accurately.

[0082] Specifically, the Fmoc purity can be calculated based on (i) the peak area of ​​the first Fmoc compound and (ii) the peak area of ​​compounds protected with a protecting group having an Fmoc skeleton other than the first Fmoc compound in the crude product (other Fmoc compounds, or Fmoc-related impurities). When a plurality of Fmoc-related impurities are present in the crude product, it is preferable to use the peak areas of all detectable Fmoc-related impurities for calculating the Fmoc purity.

[0083] More specifically, for example, the Fmoc purity is (i) identifying peaks corresponding to the first Fmoc compound and Fmoc-related impurities by chromatographic methods; (ii) determining the peak areas of the first Fmoc compound and the Fmoc-related impurity and adding up the peak areas; and (iii) The peak area of ​​the first Fmoc compound can be determined by dividing the peak area by the combined value determined in (ii).

[0084] The peaks of Fmoc-related impurities can be identified by determining the presence or absence of a protecting group having an Fmoc skeleton from the UV chromatogram (254 nm) of chromatographic analysis.

[0085] When a first Fmoc compound (e.g., an Fmoc peptide) is produced by a solid phase method, the Fmoc purity may be determined by measuring the peak areas of the first Fmoc compound and Fmoc-related impurities using a solution (cleavage solution) used in cleaving the first Fmoc compound from a solid phase synthesis resin.

[0086] By determining the Fmoc purity through measuring the peak areas of the first Fmoc compound and Fmoc-related impurities under the same measurement conditions as those for the quantification of dibenzofulvene or its derivatives and internal standard, it becomes possible to calculate the content accurately with less error. In one embodiment, by using the same measurement equipment and measurement conditions for determining the purity as those for the quantification of dibenzofulvene or its derivatives, it becomes possible to calculate the content accurately with less error.

[0087] When correcting for Fmoc purity, the content can be calculated, for example, by the following formula. (When no correction using internal standards is used) TIFF0007671251000014.tif9170(in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, A S is the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or a derivative thereof in the first solution, P S is Fmoc purity.)

[0088] (When correction using internal standards is required) TIFF0007671251000015.tif9170(in the formula, C s is the molar concentration of the second Fmoc compound, C T is the molar concentration of the crude product assuming that the crude product consists solely of the first Fmoc compound, IS S is the peak area of ​​the internal standard of the second solution, IS T is the peak area of ​​the internal standard of the first solution, A Sis the peak area of ​​dibenzofulvene or a derivative thereof in the second solution, A T is the peak area of ​​dibenzofulvene or a derivative thereof in the first solution, P S is Fmoc purity.)

[0089] In some embodiments, an acid can be used to prevent decomposition of dibenzofulvene or its derivatives. The decomposition inhibition (stabilization) of dibenzofulvene or its derivatives is achieved by contacting the produced dibenzofulvene or its derivatives with an acid. The acid can preferably include organic acids and inorganic acids, and organic acids, inorganic acids, and combinations thereof are used.

[0090] When an acid is added, the acid can be added to the analytical sample (e.g., the first and / or second solution) after the removal of the protecting group having the Fmoc skeleton. In order to prevent decomposition of dibenzofulvene, it is preferable to add the acid immediately after the removal. The amount of acid can be any amount as long as the standard solution or the sample solution is more acidic than neutral. For example, this can be achieved by adding an equivalent amount of acid equal to or greater than the amount of base added to remove the protecting group. The acid may be added to the analytical sample as it is, or may be added to the analytical sample after dissolving it in an organic solvent or the like.

[0091] As the organic acid, citric acid, oxalic acid, maleic acid, tetramethylammonium hydrogen sulfate, formic acid, acetic acid, trifluoroacetic acid, propionic acid, monofluoroacetic acid, difluoroacetic acid, trichloroacetic acid, monochloroacetic acid, dichloroacetic acid, or a combination thereof may be used, with citric acid being preferred.

[0092] As the inorganic acid, phosphoric acid, boric acid, hydrochloric acid, nitric acid, sulfuric acid, hydrobromic acid, phosphonic acid, or a combination thereof can be used, with phosphoric acid being preferred.

[0093] In one embodiment, the present invention relates to a method for quantifying an amino group-containing compound protected with a protecting group having an Fmoc skeleton by quantifying the amino group-containing compound after deprotection.

[0094] In one embodiment, the present invention relates to a method for removing a protecting group having an Fmoc skeleton from an amino-group-containing compound protected with a protecting group having an Fmoc skeleton, and quantifying the amino-group-containing compound thus produced.

[0095] In one embodiment, the method can quantify the content of the amino group-containing compound by separating it from other components by chromatography and measuring the peak area.

[0096] In an embodiment, the separation of the amino group-containing compound by chromatography can be performed by any method capable of separating the amino group-containing compound protected with a protecting group having an Fmoc skeleton, dibenzofulvene (DBF) or its derivative, and the amino group-containing compound. Examples of such chromatography include liquid chromatography, supercritical fluid chromatography, gas chromatography, thin layer chromatography, hydrophilic interaction chromatography (HILIC), ion exchange chromatography (IEX), size exclusion chromatography (SEC), electrophoresis, and ion mobility spectrometry (e.g., ion mobility), and hydrophilic interaction chromatography (HILIC) is preferred. These methods may be known.

[0097] Any method can be used for the identification and quantification of amino group-containing compounds. Examples of such methods include a method using an ultraviolet-visible light absorption detector, a diode array detector, a fluorescence detector, a differential refractive index detector, an evaporative light scattering detector, a charged particle detector (CAD), a mass spectrometer, etc., and it is preferable to use a charged particle detector (CAD) and / or a mass spectrometer that can detect even if there is no absorption at ultraviolet-visible light wavelengths. Therefore, it is more preferable to use a separation and quantification method including the method of the present invention, such as HILIC-CAD, which is a combination of hydrophilic interaction chromatography (HILIC) and a charged particle detector (CAD).

[0098] The content of the amino group-containing compound in the first solution (sample solution) or the second solution (standard solution) can be quantified by measuring the peak area of ​​the amino group-containing compound obtained by the above-mentioned chromatography.

[0099] All prior art documents cited in this specification are hereby incorporated by reference. EXAMPLES

[0100] The present invention will be described in more detail below with reference to examples, but the contents of the present invention are not necessarily limited to the following examples.

[0101] The following Fmoc-amino acids were used in the quantification described in this example: Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-cLeu-OH, Fmoc-NMe-Gly-OH, Fmoc-NMe-Ala-OH, Fmoc-NMe-Leu-OH, Fmoc-NMe-Val-OH, Fmoc-NMe-Cha-OH, Fmoc-Aze-OH. These were purchased from raw material manufacturers such as Watanabe Chemical Industry. Unless otherwise stated, the same Fmoc-amino acids may be from different raw material manufacturers or lots.

[0102] Example 1 (Quantification of Fmoc-amino acids by DBF-1) Preparation of standard solutions Fmoc-Gly-OH (about 10 mg) was precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (20 mL) to prepare an Fmoc-Gly-OH solution (an acetonitrile solution containing Fmoc-Gly-OH at a concentration of 500 ug / mL). To this solution, an internal standard solution (an acetonitrile solution containing anthracene at a concentration of 250 ug / mL), a DBU solution (an acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to prepare acetonitrile solutions containing Fmoc-Gly-OH at a concentration of 100 ug / mL, anthracene at a concentration of 50 ug / mL, and DBU at a concentration of 4 v / v%. The solution was then stirred with a vortex mixer and allowed to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. In this example, the solution before the deprotection reaction is referred to as the "unreacted standard solution," and the solution after the deprotection reaction is simply referred to as the "standard solution." Note that Fmoc-Gly-OH (purity 99.8%, water content less than 0.10%) used in the preparation of the standard solution was purchased from Watanabe Chemical Industry.

[0103] Preparation of sample solutions Crude Fmoc-amino acids (about 10 mg) including the Fmoc-amino acids to be quantified were precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (20 mL) to prepare a crude Fmoc-amino acid solution (acetonitrile solution containing crude Fmoc-amino acids at a concentration of 500 ug / mL). An internal standard solution (acetonitrile solution containing anthracene at a concentration of 250 ug / mL), a DBU solution (acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to the crude Fmoc-amino acids at a concentration of 100 ug / mL, anthracene at a concentration of 50 ug / mL, and DBU at a concentration of 4 v / v%, respectively, to prepare acetonitrile solutions. The solution was then stirred with a vortex and then left to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. In this embodiment, the solution before the start of the deprotection reaction is sometimes referred to as the "unreacted sample solution," and the solution after the deprotection reaction is sometimes simply referred to as the "sample solution."

[0104] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) UV-Visible light absorption detector: ACQ-TUV Pump: ACQ-QSM Autosampler: ACQ-FTN

[0105] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (2.1 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.3 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 1

[0106] [Table 1]

[0107] The chromatogram of the standard solution is shown in FIG. 1, and the chromatograms of each sample solution are shown in FIGS. The content (w / w%) of each Fmoc-amino acid relative to Fmoc-Gly-OH can be calculated using the following formula: The results are shown in Table 2 (without correction of the injection amount by anthracene) and Table 3 (with correction of the injection amount by anthracene).

[0108] Calculation formula (When no anthracene injection correction is used) TIFF0007671251000017.tif9170C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L) C T : Concentration of each crude Fmoc-amino acid in each unreacted sample solution (umol / L) A S : DBF peak area of ​​standard solution A T : DBF peak area of ​​each sample solution

[0109] Calculation formula (When anthracene is used to correct the injection amount) TIFF0007671251000018.tif9170C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L) C T : Concentration of each crude Fmoc-amino acid in each unreacted sample solution (umol / L) IS S : Anthracene peak area of ​​standard solution IS T : Anthracene peak area of ​​each sample solution A S : DBF peak area of ​​standard solution A T : DBF peak area of ​​each sample solution

[0110] [Table 2]

[0111] [Table 3]

[0112] As a result of the above, it was found that while there are Fmoc-amino acids with a content rate of almost 100%, such as Fmoc-Ile-OH and Fmoc-NMe-Leu-OH, there are also Fmoc-amino acids with a content rate that does not reach 100%. Fmoc-amino acids with a content rate that does not reach 100% are thought to contain impurities that cannot be detected by UV, such as moisture, inorganic salts, and residual solvents. In addition, by correcting with the peak area of ​​the internal standard substance, quantitative calculation is possible even when there is a difference in the injection amount of the standard solution and each sample solution.

[0113] Example 2 (Quantification of Fmoc-amino acids by DBF-2) Preparation of standard solutions Fmoc-Gly-OH (about 10 mg) was precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (10 mL) to prepare an Fmoc-Gly-OH solution (an acetonitrile solution containing Fmoc-Gly-OH at a concentration of 1 mg / mL). To this solution, an internal standard solution (an acetonitrile solution containing anthracene at a concentration of 500 ug / mL), a DBU solution (an acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to prepare acetonitrile solutions containing Fmoc-Gly-OH at a concentration of 100 ug / mL, anthracene at a concentration of 50 ug / mL, and DBU at a concentration of 4 v / v%. The solution was then stirred with a vortex mixer and allowed to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. In this example, the solution before the deprotection reaction is sometimes referred to as the "unreacted standard solution," and the solution after the deprotection reaction is sometimes simply referred to as the "standard solution." Note that Fmoc-Gly-OH used to prepare the standard solution was purchased from Watanabe Chemical Industry Co., Ltd. (purity 99.8%, water content less than 0.10%).

[0114] Preparation of sample solutions Crude Fmoc-amino acids (about 10 mg) including the Fmoc-amino acids to be quantified were precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (10 mL) to prepare a crude Fmoc-amino acid solution (acetonitrile solution containing crude Fmoc-amino acids at a concentration of 1 mg / mL). An internal standard solution (acetonitrile solution containing anthracene at a concentration of 500 ug / mL), a DBU solution (acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to the crude Fmoc-amino acids at a concentration of 100 ug / mL, anthracene at a concentration of 50 ug / mL, and DBU at a concentration of 4 v / v%, respectively, to prepare acetonitrile solutions. The solution was then stirred with a vortex and then left to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. In this embodiment, the solution before the start of the deprotection reaction is sometimes referred to as the "unreacted sample solution," and the solution after the deprotection reaction is sometimes simply referred to as the "sample solution."

[0115] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) UV-Visible light absorption detector: ACQ-TUV Pump: ACQ-QSM Autosampler: ACQ-FTN

[0116] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (3.0 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.6 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 4

[0117] [Table 4]

[0118] The chromatogram of the standard solution is shown in FIG. 8, and the chromatograms of each sample solution are shown in FIGS.

[0119] In addition, the content (w / w%) of each Fmoc-amino acid relative to Fmoc-Gly-OH can be calculated using the formula described in Example 1. The results are shown in Table 5 (without correction of the injection amount by anthracene) and Table 6 (with correction of the injection amount by anthracene).

[0120] [Table 5]

[0121] [Table 6]

[0122] As a result, the content of the three Fmoc-amino acids was almost 100%. In addition, the content hardly changed depending on whether or not the injection amount was corrected by anthracene, so it is presumed that there was no difference between the injection amount of the standard solution and each sample solution in this example.

[0123] Example 3 (Content analysis corrected for purity of Fmoc-amino acids) Preparation of sample solutions A 1 mg / mL crude Fmoc-amino acid solution was diluted to prepare a sample solution (acetonitrile solution containing crude Fmoc-amino acid at a concentration of 100 ug / mL). The various crude Fmoc-amino acids used in the sample solutions were purchased from Watanabe Chemical Industry.

[0124] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) -UV-Visible light absorption detector: ACQ-TUV Pump: ACQ-QSM Autosampler: ACQ-FTN

[0125] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (3.0 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.6 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 7

[0126] [Table 7]

[0127] As shown in Figures 12 to 20, the peaks of Fmoc-amino acids that disappeared by DBU treatment can be confirmed by comparison with the chromatogram of Example 2. These peaks are considered to be peaks derived from compounds having an Fmoc group, and the purity of each Fmoc-amino acid calculated by integrating only these peaks is shown in Table 8. The content can be calculated by correcting for these purities and the injection amount of anthracene. The results are shown in Table 9.

[0128] Calculation formula TIFF0007671251000025.tif9170DBF assay sample : Content of each Fmoc-amino acid before correction for purity P S : Purity of each Fmoc-amino acid in a compound with an Fmoc group

[0129] [Table 8]

[0130] [Table 9]

[0131] Example 4 (Study of DBF stability) Preparation of sample solutions Fmoc-Gly-OH was dissolved in each solvent shown in Table 10 to prepare a sample solution (containing Fmoc-Gly-OH at a concentration of 100 ug / mL). The sample solution was stored at 10°C and analyzed.

[0132] [Table 10]

[0133] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) -UV-Visible light absorption detector: ACQ-PDA Pump: ACQ-QSM Autosampler: ACQ-FTN

[0134] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (2.1 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.3 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 11

[0135] [Table 11]

[0136] The ratios of the peak areas of DBF and its decomposition products in the sample solution on the first day and in the sample solution after storage for 1, 2, and 5 days are shown in Table 12. In addition, the results of comparing the peak areas of DBF in the sample solution on the first day and in the sample solution after storage for 5 days (storage for 5 days / first day) are shown in Table 13. This shows that the decomposition of DBF can be suppressed by treatment with citric acid.

[0137] [Table 12]

[0138] Note) DBF-Pip has the following structure. TIFF0007671251000031.tif27170Note) The structure of the DBF decomposition product is as follows. TIFF0007671251000032.tif20170

[0139] [Table 13]

[0140] Example 5 (Comparison with quantitative NMR) In order to evaluate the validity of the quantitative method (DBF assay) according to the present invention, various crude Fmoc-amino acids were treated with DBU to produce DBFs, which were quantified in the same manner as in Examples 1 and 2 (without correction by an internal standard substance and with correction by purity), and the quantified values ​​were compared with the quantified values ​​by quantitative NMR of the crude Fmoc-amino acids. Fmoc-Ile-OH (content by quantitative NMR: 99.18%) was used as the standard amino acid used to prepare the standard solution. As shown in FIG. 21 and Table 14, a good positive correlation was observed between the quantitative values ​​by the present invention and those by quantitative NMR, with a correlation coefficient of 0.95, supporting the validity of the present invention.

[0141] The analytical equipment and reagents are as follows: (Analyzer) NMR: JEOL 500MHz Royal HFX Probe Reagents: Fmoc-amino acids Solvent: DMSO-d6 Standard: 3,5-bis(trifluoromethyl)-benzoic acid (99.96% ±0.06%, quantitative NMR reagent)

[0142] [Table 14]

[0143] Correlation with qNMR demonstrated that this method is as effective as qNMR.

[0144] Example 6 (Amino acid quantification after DBU treatment of Fmoc-amino acids) When Fmoc-amino acid is deprotected with DBU, DBF and amino acid are generated. In Examples 1 to 4, DBF is quantified, but in this Example, amino acid is quantified. As a result of comparing the peak area of ​​H-NMe-Gly-OH generated by deprotecting Fmoc-NMe-Gly-OH with DBU and the peak area of ​​an amino acid standard solution using H-NMe-Gly-OH as an internal standard substance, H-NMeGly-OH generated by deprotection was obtained in a yield of 97.2% (Figures 22 to 24). This confirmed that the deprotection reaction by adding DBU proceeded quantitatively. In this example, measurements were performed using hydrophilic interaction chromatography (HILIC), with the peaks of highly hydrophobic compounds (e.g., DBF) appearing at early elution positions and the peaks of highly hydrophilic compounds (e.g., amino acids) appearing at late elution positions. In addition, since H-NMe-Gly-OH has almost no absorption in the ultraviolet and visible wavelengths, it was detected by CAD.

[0145] Preparation of amino acid standard solutions A solution containing H-NMeGly-OH (N-methylglycine) at a concentration of 100 ug / mL was prepared. H-NMe-Gly-OH (purity 99.7% (non-aqueous method)) was purchased from Tokyo Chemical Industry Co., Ltd.

[0146] Preparation of sample solutions A 2% DBU solution (acetonitrile solution containing DBU at a concentration of 2 v / v%) was added to Fmoc-NMeGly-OH, and then diluted with CH3CN / H2O (4:1) to prepare a sample solution (a solution containing Fmoc-amino acids at a concentration of 300 ug / mL). Fmoc-MeGly-OH was purchased from a raw material manufacturer.

[0147] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: 1200 series (Agilent) UV-Visible Light Detector: Agilemt 1200 MWD Pump: Agilent 1200 Binary pump-1, -2 Autosampler: Agilent 1200 Auto sampler

[0148] (Analysis conditions) Column: Imtakt Intrada Amino Acid (3 x 100 mm, 3 um) Mobile phase A: 0.1% formic acid / acetonitrile Mobile phase B: 100 mM ammonium formate / water Flow rate: 1.0 mL / min Column temperature: 35℃ Sample injection volume: 2 uL Detector: Charged Aerosol Detector (CAD) (Corona ULTRA) Gradient conditions: as shown in Table 15

[0149] [Table 15]

[0150] In addition to these conditions, an inverse gradient was set up so that the detector was always introduced with A:B = 50:50. Reverse gradient conditions: as shown in Table 16

[0151] [Table 16]

[0152] Example 7 (Quantification of Fmoc-peptides by DBF) Preparation of standard solutions Fmoc-Gly-OH (about 10 mg) was precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (20 mL) to prepare an Fmoc-Gly-OH solution (an acetonitrile solution containing Fmoc-Gly-OH at a concentration of 500 ug / mL). To this solution, an internal standard solution (an acetonitrile solution containing anthracene at a concentration of 250 ug / mL), a DBU solution (an acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to prepare acetonitrile solutions containing Fmoc-Gly-OH at a concentration of 50 ug / mL, anthracene at a concentration of 25 ug / mL, and DBU at a concentration of 2 v / v%. The solution was then stirred with a vortex mixer and allowed to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. In this example, the solution before the deprotection reaction is referred to as the "unreacted standard solution," and the solution after the deprotection reaction is simply referred to as the "standard solution." The Fmoc-Gly-OH (purity 98.6%) used in the preparation of the standard solution was purchased from Tokyo Chemical Industry Co., Ltd.

[0153] Preparation of sample solution-1 The crude Fmoc-Phe-Phe-OH (about 10 mg) to be quantified was precisely weighed according to the Japanese Pharmacopoeia and dissolved in acetonitrile (20 mL) to prepare a crude Fmoc-Phe-Phe-OH solution (acetonitrile solution containing Fmoc-Phe-Phe-OH at a concentration of 500 ug / mL). An internal standard solution (acetonitrile solution containing anthracene at a concentration of 250 ug / mL), a DBU solution (acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to the crude Fmoc-Phe-Phe-OH solution (acetonitrile solution containing Fmoc-Phe-Phe-OH at a concentration of 50 ug / mL), anthracene at a concentration of 25 ug / mL, and DBU at a concentration of 2 v / v% to prepare an acetonitrile solution. The solution was then stirred with a vortex and left at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction proceeded quantitatively by the UV chromatogram obtained by HPLC. The crude Fmoc-Phe-Phe-OH (purity 99.8%) used in preparing the solution was purchased from Watanabe Chemical Industry Co., Ltd. In this example, the solution before the start of the deprotection reaction may be referred to as the "unreacted sample solution," and the solution after the deprotection reaction may be simply referred to as the "sample solution."

[0154] Preparation of sample solution-2 (Crude Fmoc-peptide compound synthesized by solid-phase reaction and supported on resin) The crude Fmoc-peptide compound to be quantified, which was synthesized by solid-phase reaction and supported on a resin (hereinafter, sometimes simply referred to as "crude Fmoc-peptide compound supported on a resin") (approximately 30 mg) was precisely weighed according to the Japanese Pharmacopoeia, and dichloromethane (3 mL) was added and the mixture was left to stand for 15 minutes or more to cause swelling. In this Example and Example 8, CTC resin was used as the resin for solid-phase synthesis. After draining the dichloromethane, the resin was washed twice with dichloromethane (3 mL). 0.6 mL of TFA solution (dichloromethane solution containing TFA at a concentration of 1 v / v%) was added to the resin and shaken at room temperature to cleave the crude Fmoc-peptide from the resin. To this, an internal standard solution (acetonitrile solution containing anthracene at a concentration of 250 ug / mL), a DBU solution (acetonitrile solution containing DBU at a concentration of 10 v / v%), and acetonitrile were added to prepare an acetonitrile solution containing a crude Fmoc-peptide at a concentration of 1 mg / mL, anthracene at a concentration of 25 ug / mL, and DBU at a concentration of 2 v / v% as a crude Fmoc-peptide compound supported on a resin. The solution was then stirred with a vortex and allowed to stand at room temperature for 10 minutes or more to deprotect the Fmoc group. It was confirmed that the deprotection reaction had progressed quantitatively by a UV chromatogram obtained by HPLC. In this embodiment, the solution before the start of the deprotection reaction may be referred to as an "unreacted sample solution", and the solution after the deprotection reaction may be simply referred to as a "sample solution".

[0155] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) -UV-Visible light absorption detector: ACQ-TUV Pump: ACQ-QSM Autosampler: ACQ-FTN

[0156] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (2.1 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.3 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 17

[0157] [Table 17]

[0158] The chromatogram of the standard solution is shown in FIG. 25, and the chromatograms of each sample solution are shown in FIGS. The content (w / w%) of each Fmoc-peptide relative to Fmoc-Gly-OH can be calculated using the following formula. The results are shown in Table 18 (without correction of the injection amount by anthracene) and Table 19 (with correction of the injection amount by anthracene). The molecular weight of the Fmoc-peptide contained in the sample solution is calculated as a carboxylic acid having a carboxyl group at the end.

[0159] Calculation formula (When no anthracene injection correction is used) TIFF0007671251000038.tif9170C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L) C T : Concentration of each crude Fmoc-peptide in each unreacted sample solution (umol / L) A S : DBF peak area of ​​standard solution A T : DBF peak area of ​​each sample solution

[0160] Calculation formula (When anthracene is used to correct the injection amount) TIFF0007671251000039.tif9170C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L) C T : Concentration of each crude Fmoc-peptide in each unreacted sample solution (umol / L) IS S : Anthracene peak area of ​​standard solution IS T : Anthracene peak area of ​​each sample solution A S : DBF peak area of ​​standard solution A T : DBF peak area of ​​each sample solution

[0161] [Table 18]

[0162] [Table 19]

[0163] Example 8 (Content analysis corrected for purity of Fmoc-peptide) Preparation of sample solution-1 A 500 ug / mL crude Fmoc-Phe-Phe-OH solution was diluted to prepare a sample solution (an acetonitrile solution containing crude Fmoc-Phe-Phe-OH at a concentration of 50 ug / mL).

[0164] Preparation of sample solution-2 As in "Sample solution preparation-2" of Example 7, the cleavage solution was diluted with acetonitrile so as to contain the crude Fmoc-peptide as the resin-supported crude Fmoc-peptide compound at a concentration of 1 mg / mL, to prepare a sample solution.

[0165] The analytical equipment and conditions are as follows. (Analyzer) HPLC system: H-class (Waters) UV-Visible light absorption detector: ACQ-TUV Pump: ACQ-QSM Autosampler: ACQ-FTN

[0166] (Analysis conditions) Column: Osaka Soda CAPCELL CORE ADME (2.1 x 150 mm, 2.7 um) Mobile phase A: 0.05% TFA / water Mobile phase B: 0.05% TFA / acetonitrile Flow rate: 0.3 mL / min Column temperature: 40℃ Sample injection volume: 2 uL Measurement wavelength: 254 nm Gradient conditions: as shown in Table 20

[0167] [Table 20]

[0168] The purity of the crude Fmoc-peptide relative to the total Fmoc compounds for each sample solution was calculated. The results are shown in Table 21. The content of the Fmoc-peptide was calculated by correcting the calculated purity and anthracene. The results are shown in Table 22. For samples in which methyl esters were generated during storage of the measured samples, the purity was calculated assuming that the methyl esters were also the desired Fmoc-peptide.

[0169] Calculation formula TIFF0007671251000043.tif9170DBF assay sample : Content of each Fmoc-peptide before correction for purity P S : Purity of each Fmoc-peptide in compounds with Fmoc groups

[0170] [Table 21]

[0171] [Table 22]

[0172] The above results demonstrate that the quantitative method according to the present invention can be used for the analysis of Fmoc-peptide content, and also for the analysis of Fmoc-peptide content from Fmoc-peptide compounds synthesized by solid-phase reaction and supported on a resin. [Industrial Applicability]

[0173] The present invention provides a novel method for quantifying an amino-containing compound protected with a protecting group having an Fmoc skeleton. The present invention makes it possible to quantitatively determine the content of an amino-containing compound protected with a protecting group having an Fmoc skeleton contained in an analytical sample with high accuracy and in a simple manner.

Claims

1. A method for quantifying the content of an amino group-containing compound protected with a protecting group having an Fmoc skeleton (hereinafter referred to as a first Fmoc compound) contained in a crude product, comprising the steps of: removing a protecting group from a first Fmoc compound with a base in a solution (hereinafter referred to as a first solution), and quantifying the content of dibenzofulvene or a derivative thereof produced thereby; The above method, wherein the amino group-containing compound is selected from the group consisting of β-amino acids, γ-amino acids, D-amino acids, N-substituted amino acids, α,α-disubstituted amino acids, non-naturally occurring amino acids, and peptides.

2. The method according to claim 1, wherein the content of dibenzofulvene or a derivative thereof is quantified by measuring the peak area of ​​dibenzofulvene or a derivative thereof.

3. 3. The method of claim 2, wherein each peak area is measured by a chromatographic method.

4. The method according to claim 3, wherein each peak area is measured under the same measurement conditions.

5. the method further comprises deprotecting a protecting group from an amino-group-containing compound having a protecting group with an Fmoc skeleton (hereinafter referred to as a second Fmoc compound) used as a standard in the solution (hereinafter referred to as a second solution) with a base, and quantifying the content of dibenzofulvene or a derivative thereof produced thereby; The method according to any one of claims 1 to 4, wherein the content (% by weight) of the first Fmoc compound relative to the second Fmoc compound is calculated by comparing the content of dibenzofulvene or a derivative thereof in the first solution with the content of dibenzofulvene or a derivative thereof in the second solution.

6. The method according to claim 5 , wherein the first Fmoc compound and / or the second Fmoc compound is an amino acid, a peptide, or a small organic compound protected with a protecting group having an Fmoc skeleton.

7. The method according to claim 5 or 6, wherein the deprotection reaction proceeds quantitatively in the first solution and the second solution.

8. Further comprising correcting the content by the Fmoc purity of the crude product; The method according to any one of claims 5 to 7, wherein the "Fmoc purity" refers to a ratio of a content of the first Fmoc compound to a total content of compounds protected with all types of protecting groups having an Fmoc skeleton contained in the crude product.

9. The method according to claim 8, wherein the Fmoc purity is calculated based on (i) a peak area of ​​the first Fmoc compound and (ii) each peak area of ​​a compound protected with a protecting group having an Fmoc skeleton other than the first Fmoc compound in the crude product.

10. The method according to any one of claims 5 to 9, further comprising correcting the content rate using an internal standard substance.

11. The method according to claim 10, comprising comparing the content of an internal standard contained in the first solution (hereinafter referred to as the first internal standard) with the content of an internal standard contained in the second solution (hereinafter referred to as the second internal standard).

12. The method according to claim 11, wherein the contents of the first and second internal standards are quantified by measuring the peak area of ​​the first internal standard and the peak area of ​​the second internal standard, respectively.

13. The protecting group having an Fmoc skeleton is represented by the following formula (1): 【Chemistry 1】 (In the formula, R １ ~R ８ are independently hydrogen, C １ -C ８ Alkyl, C １ -C ８ selected from the group consisting of fluoroalkyl, halogen, sulfo, and trimethylsilyl; R ９ ~R １０ are independently hydrogen or methyl. The method according to any one of claims 1 to 12, wherein

14. The method according to any one of claims 1 to 13, wherein the base is an organic base.

15. The method according to any one of claims 1 to 14, further comprising adding an acid to the first and / or second solution after removal of the Fmoc skeleton-containing protecting group.

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