Quantitative method for compounds containing an amino group protected by a protecting group having an Fmoc skeleton

Abstract

It has been found herein that a compound containing an amino group protected with a protecting group having an Fmoc skeleton can be accurately quantified by removing the protecting group having an Fmoc skeleton from the compound and measuring the content of dibenzofulvene or a derivative thereof thus produced.

Description

Technical Field

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

[0002] In recent years, the development of innovative drug technology has attracted attention. Using molecular peptide compounds (molecular weight 500-2000), innovative drugs can inhibit protein-protein interactions, target agonists, and difficult targets represented by molecular chaperones. These compounds are usually synthesized on a solid phase by extending amino acids protected with Fmoc groups at the N-terminus. In drug preparation, it is very important to evaluate the content of raw materials from the perspective of quality management; ICHQ11 requires the formulation of appropriate quality management items for raw materials set as starting materials under GMP. When Fmoc-amino acids are used as starting materials, their content is usually evaluated by titration; in addition to this, quantitative NMR is also used. Titration is the most commonly used method due to the simplicity of the test; however, when its quantification object contains acids such as carboxylic acids as impurities, there is a disadvantage of losing the accuracy of content determination. In addition, although quantitative NMR has the advantage of being able to directly and accurately measure the number of hydrogen nuclei at the functional group level, it cannot separate multiple components unlike chromatography methods such as HPLC or GC.

[0003] The Fmoc group is often used as a protecting group for compounds containing amino groups. Since the Fmoc group has ultraviolet (UV) absorption, it can be quantified by chromatography. In addition, it is known that a method for quantifying the amount of Fmoc group loaded on polymer beads is used to remove dibenzofulvene (DBF) produced when the Fmoc group is removed (Non-Patent Documents 1, 2, 3).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1. William SN et al., Biotechnol. Bioeng. 1998, 61, 55-60.

[0007] Non-patent literature 2. Laszlo V. et al., J. Chromatogr. A 2000, 869, 171-179.

[0008] Non-patent literature 3. Freeman CE et al., Talanta 2005, 65, 574-577. SUMMARY OF THE INVENTION

[0010] Problems to be solved by the invention

[0011] As described above, the Fmoc group can be quantified by chromatography because it has ultraviolet (UV) absorption. However, the molar absorption coefficient at the same wavelength varies from compound to compound. In addition, even for compounds with the same molar absorption coefficient under certain conditions, when the solvent composition during elution in chromatography changes, there are also cases where the molar absorption coefficient is different. Therefore, for each compound to be quantified, it is necessary to prepare an authentic sample (authentic sample) of the compound whose content has been confirmed. For example, in the case of quantifying Fmoc-amino acids composed of natural amino acids by chromatography, since authentic samples of its Fmoc-amino acids have been sold by many manufacturers, it is possible to quantify by comparing the authentic samples with Fmoc-amino acids by chromatography. On the other hand, in the case of non-natural amino acids and peptides protected with Fmoc groups that are being used in recent years, it is difficult to obtain authentic samples of their amino acids or peptides. Therefore, when measuring by chromatography, it is necessary to synthesize these authentic samples one by one and confirm the content.

[0012] In addition, the above-mentioned prior art document evaluates the loading amount of the Fmoc group on the polymer beads, but it is a quantitative evaluation of other impurities containing the Fmoc group. Furthermore, dibenzofulvene generated by removing the Fmoc group is an unstable molecule and degrades over time, which becomes a cause of reduced quantitative accuracy of compounds containing an amino group protected by a protecting group having an Fmoc skeleton.

[0013] The present invention has been completed in view of such circumstances, and one of the objects of the present invention is to provide a method for quantifying a compound containing an amino group protected by a protecting group having an Fmoc skeleton to the first decimal place required for the preparation of a drug, as long as there is an authentic sample of the compound containing an amino group protected by a protecting group having an Fmoc skeleton.

[0014] Means for solving problems

[0015] As means for solving the above-mentioned problems, the present inventors have found the following and have completed the present invention.

[0016] 1) A new method for quantifying a compound containing an amino group protected by a protecting group having an Fmoc skeleton has been discovered, which is not a method for quantifying the compound containing an amino group protected by a protecting group having an Fmoc skeleton itself, but an indirect method for quantifying the content of dibenzofulvene or a derivative thereof produced by removing the protecting group having an Fmoc skeleton from the compound.

[0017] 2) It was found that based on the purity of the compound containing an amino group protected by a protecting group having an Fmoc skeleton in the crude product and the content of dibenzofulvene or its derivative produced after removing the protecting group having an Fmoc skeleton, the content of the compound containing an amino group protected by a protecting group having an Fmoc skeleton can be calculated to the first decimal place required for the quality management of pharmaceutical raw materials.

[0018] 3) By making the purity measurement conditions the same as those for dibenzofulvene or its derivative, the accurate content can be calculated with a small error.

[0019] 4) Reaction conditions were found under which the reaction rate of the deprotection reaction of the protecting group having an Fmoc skeleton was 100% and the side reaction was almost 0%, enabling more accurate quantification.

[0020] 5) It was found that by mixing dibenzofulvene or its derivative produced by removing the protecting group having an Fmoc skeleton with an acid, the stability of dibenzofulvene or its derivative is improved, thereby enabling calculation of a value closer to the true value.

[0021] That is, the present invention includes the following contents.

[0022] The present invention relates to a method for quantitatively determining the following compound containing an amino group protected with a protecting group having an Fmoc skeleton.

[0023] (A1) A method for quantifying the content of a compound containing an amino group protected by a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as a first Fmoc compound) contained in a crude product, the method comprising:

[0024] The protecting group is deprotected from the first Fmoc compound in a solution (hereinafter sometimes referred to as the first solution) with a base, and the content of dibenzofulvene or a derivative thereof produced thereby is quantified.

[0025] (A2) the method according to (A1), further comprising deprotecting the protecting group from a compound having an amino group with a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as a second Fmoc compound) used as an authentic sample in a solution (hereinafter sometimes referred to as a second solution) with a base, and quantifying the content of dibenzofulvene or a derivative thereof produced thereby,

[0026] Here, the content rate (weight %) of the first Fmoc compound relative to the second Fmoc compound is 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.

[0027] (A3) The method according to (A2), wherein the deprotection reaction is carried out quantitatively in the first solution and the second solution.

[0028] (A4) The method according to any one of (A1) to (A3), wherein the content of dibenzofulvene or its derivative is quantified by measuring the peak area of ​​dibenzofulvene or its derivative.

[0029] (A5) The method described in any one of (A2) to (A4), further comprising correcting the content rate using the Fmoc purity of the crude product, wherein "Fmoc purity" is the ratio of the content of the first Fmoc compound to the sum of the contents of all types of compounds protected with a protecting group having an Fmoc skeleton contained in the crude product.

[0030] (A6) The method described in (A5), wherein the Fmoc purity is calculated based on (i) the peak area of ​​the first Fmoc compound and (ii) each peak area of ​​compounds protected with a protecting group having an Fmoc skeleton other than the first Fmoc compound in the crude product.

[0031] (A7) The method according to any one of (A2) to (A6), further comprising correcting the content ratio using an internal standard substance.

[0032] (A8) The method described in (A7), comprising comparing the content of an internal standard substance contained in a first solution (hereinafter sometimes referred to as a first internal standard substance) and the content of an internal standard substance contained in a second solution (hereinafter sometimes referred to as a second internal standard substance).

[0033] (A9) The method described in (A8), wherein the contents of the first and second internal standard substances are quantified respectively by measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance.

[0034] (A10) The method of any one of (A8) to (A9), wherein the first and second internal standard substances are independently selected from anthracene, phenanthrene, naphthalene, benzene, toluene, triphenylene and tetracene.

[0035] (A11) The method according to any one of (A8) to (A10), wherein the first and second internal standard substances are anthracene.

[0036] (A12) The method according to any one of (A4), (A6) and (A9), wherein each peak area is measured by chromatography.

[0037] (A13) The method according to (A12), wherein the chromatography is liquid chromatography or gas chromatography.

[0038] (A14) The method according to (A12) or (A13), wherein each peak area is measured under the same measurement conditions.

[0039] (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):

[0040]

[0041] (Where,

[0042] R 1 ~R 8 are independently selected from hydrogen, C 1 -C 8 Alkyl, C 1 -C 8 Fluoroalkyl, halogen, sulfonyl and trimethylsilyl,

[0043] R 9 ~R 10 are independently hydrogen or methyl).

[0044] (A16) The method described in any one of (A1) to (A15), wherein the above-mentioned 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, a sm-Fmoc group, or a rm-Fmoc group.

[0045] (A17) The method according to any one of (A1) to (A16), wherein the protecting group having an Fmoc skeleton is an Fmoc group.

[0046] (A18) The method according to any one of (A1) to (A17), wherein the first Fmoc compound and / or the second Fmoc compound are not supported on a solid phase.

[0047] (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 low molecular weight organic compound protected by a protecting group having an Fmoc skeleton.

[0048] (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 skeleton.

[0049] (A21) The method according to (A19) or (A20), wherein the amino acid is an α-amino acid or a β-amino acid.

[0050] (A22) The method according to any one of (A19) to (A21), wherein the amino acid main chain carboxyl group is a free carboxyl group.

[0051] (A23) The method according to (A19) or (A20), wherein the peptide comprises or consists of α-amino acids and / or β-amino acids.

[0052] (A24) The method according to any one of (A1) to (A23), wherein the base is an organic base.

[0053] (A25) The method according to any one of (A1) to (A24), wherein the base is at least one base selected from the group consisting of amines, bases having an amidine skeleton, and bases having a guanidine skeleton.

[0054] (A26) The method of (A24), wherein the organic base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinoline, 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, 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.

[0055] (A27) The method according to any one of (A1) to (A26), wherein the base is an organic base comprising one or more tertiary amines but not primary amines and secondary amines.

[0056] (A28) The method of (A27), wherein the organic base containing one or more tertiary amines but not primary and secondary amines is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinoline, 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, 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.

[0057] (A29) The method as described in (A27), wherein the organic base comprising one or more tertiary amines but excluding primary amines and secondary amines is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0058] (A30) The method according to any one of (A1) to (A29), further comprising adding an acid to the first and / or second solution after removing the protecting group having an Fmoc skeleton.

[0059] (A31) The method according to (A30), wherein the acid is an organic acid and / or an inorganic acid.

[0060] (A32) The method described in (A31), wherein the above-mentioned 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 or a combination thereof.

[0061] (A33) The method described in (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 or a combination thereof.

[0062] (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.

[0063] (A35) The method described in (A34), wherein the aprotic polar solvent is DMF, DMA, NMP, DMSO or acetonitrile.

[0064] (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.

[0065] (A37) The method according to any one of (A2) to (A36), wherein the content (weight %) is calculated by the following formula:

[0066]

[0067] (Where,

[0068] C s is the molar concentration of the second Fmoc compound;

[0069] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0070] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0071] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution);

[0072] Or calculated by the following formula:

[0073]

[0074] (Where,

[0075] C s is the molar concentration of the second Fmoc compound;

[0076] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0077] IS S is the peak area of ​​the internal standard substance in the second solution;

[0078] IS T is the peak area of ​​the internal standard substance of the first solution;

[0079] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0080] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution);

[0081] Or calculated by the following formula:

[0082]

[0083] (Where,

[0084] C s is the molar concentration of the second Fmoc compound;

[0085] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0086] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0087] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution;

[0088] P S is Fmoc purity);

[0089] Or calculated by the following formula:

[0090]

[0091] (Where,

[0092] C s is the molar concentration of the second Fmoc compound;

[0093] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0094] IS S is the peak area of ​​the internal standard substance in the second solution;

[0095] IS T is the peak area of ​​the internal standard substance of the first solution;

[0096] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0097] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution;

[0098] P S is Fmoc purity).

[0099] In addition, the present invention also relates to the following method for stabilizing dibenzofulvene or its derivatives.

[0100] (B1) A method for stabilizing dibenzofulvene or a derivative thereof contained in a crude product, the method comprising mixing dibenzofulvene or a derivative thereof and an acid.

[0101] (B2) The method described in (B1), comprising mixing dibenzofulvene or a derivative thereof produced by removing a protecting group having an Fmoc skeleton from a compound containing an amino group protected by a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as an Fmoc compound) and an acid.

[0102] (B3) The method described in (B2), wherein the protecting group having an Fmoc skeleton is represented by the following formula (1):

[0103] (Where,

[0104] R 1 ~R 8 are independently selected from hydrogen, C 1 -C 8 Alkyl, C 1 -C 8 Fluoroalkyl, halogen, sulfonyl and trimethylsilyl,

[0105] R 9 ~R 10 are independently hydrogen or methyl).

[0106] (B4) The method described in (B2) or (B3), wherein the above-mentioned 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, a sm-Fmoc group, or a rm-Fmoc group.

[0107] (B5) The method according to any one of (B2) to (B4), wherein the protecting group having an Fmoc skeleton is an Fmoc group.

[0108] (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 and not supported on a solid phase.

[0109] (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 by a protecting group having an Fmoc skeleton.

[0110] (B8) The method according to any one of (B2) to (B7), wherein the Fmoc compound is an amino acid or a peptide protected by a protecting group having an Fmoc skeleton.

[0111] (B9) The method according to (B7) or (B8), wherein the amino acid is an α-amino acid or a β-amino acid.

[0112] (B10) The method according to (B7) or (B8), wherein the peptide comprises or consists of α-amino acids and / or β-amino acids.

[0113] (B11) The method according to (B2), wherein the acid is an organic acid and / or an inorganic acid.

[0114] (B12) The method described in (B11), wherein the above-mentioned 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 or a combination thereof.

[0115] (B13) The method described in (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 or a combination thereof.

[0116] In addition, the present disclosure relates to the following stabilizers.

[0117] (C1) A stabilizer for stabilizing dibenzofulvene or a derivative thereof.

[0118] (C2) The stabilizer described in (C1), which is an acid.

[0119] (C3) The stabilizer described in (C2), wherein the acid is an organic acid and / or an inorganic acid.

[0120] (C4) The stabilizer described in (C3), wherein the above-mentioned 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 or a combination thereof.

[0121] (C5) The stabilizer described in (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, or a combination thereof.

[0122] Alternatively, the present disclosure relates to the following quantitative determination method of a compound containing an amino group.

[0123] (D1) A method for quantifying the content of an amino group-containing compound produced from a compound containing an amino group protected by a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as an Fmoc compound) contained in a crude product, the method comprising: quantifying the content of the amino group-containing compound produced by removing the protecting group having an Fmoc skeleton from the Fmoc compound.

[0124] (D2) The method according to (D1), wherein the content of the amino group-containing compound is measured by chromatography.

[0125] (D3) The method according to (D2), wherein the chromatography is hydrophilic interaction chromatography (HILIC).

[0126] (D4) The method according to any one of (D2) to (D3), wherein the detector of the chromatography is a charged particle detector (CAD) and / or a mass spectrometer.

[0127] (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):

[0128]

[0129] (Where,

[0130] R 1 ~R 8 are independently selected from hydrogen, C 1 -C 8Alkyl, C 1 -C 8 Fluoroalkyl, halogen, sulfonyl and trimethylsilyl,

[0131] R 9 ~R 10 are independently hydrogen or methyl).

[0132] (D6) The method described in any one of (D1) to (D5), wherein the above-mentioned 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, a sm-Fmoc group, or a rm-Fmoc group.

[0133] (D7) The method according to any one of (D1) to (D6), wherein the protecting group having an Fmoc skeleton is an Fmoc group.

[0134] (D8) The method according to any one of (D1) to (D7), wherein the Fmoc compound is not supported on a solid phase.

[0135] (D9) The method according to any one of (D1) to (D8), 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.

[0136] (D10) The method according to any one of (D1) to (D9), wherein the Fmoc compound is an amino acid or a peptide protected by a protecting group having an Fmoc skeleton.

[0137] (D11) The method according to (D9) or (D10), wherein the amino acid is an α-amino acid or a β-amino acid.

[0138] (D12) The method according to (D9) or (D10), wherein the peptide comprises or consists of α-amino acids and / or β-amino acids.

[0139] (D13) The method according to any one of (D9) to (D12), wherein the amino acid main chain carboxyl group is a free carboxyl group.

[0140] (D14) The method according to any one of (D1) to (D13), wherein the protecting group having an Fmoc skeleton is removed using a base.

[0141] (D15) The method according to (D14), wherein the base is an organic base.

[0142] (D16) The method according to (D14) or (D15), wherein the base is at least one base selected from the group consisting of amines, bases having an amidine skeleton, and bases having a guanidine skeleton.

[0143] (D17) The method described in (D15), wherein the organic base is selected from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinoline, 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, 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.

[0144] (D18) The method according to any one of (D14) to (D17), wherein the base is an organic base comprising one or more tertiary amines but not primary amines and secondary amines.

[0145] (D19) The method described in (D18), wherein the organic base containing one or more tertiary amines but not primary amines and secondary amines is selected from the group consisting of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinoline, 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, 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.

[0146] (D20) The method described in (D18) or (D19), wherein the organic base comprising one or more tertiary amines but not primary amines and secondary amines is 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0147] Effects of the Invention

[0148] According to the method of the present invention, the content of the compound containing an amino group protected by a protecting group having an Fmoc skeleton contained in a sample can be easily and quantitatively analyzed with high accuracy. Therefore, the present invention can be a very useful technology for quantifying the above-mentioned compound at a quality level required for manufacturing a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] [ Figure 1 ] Figure 1 This is a chromatogram showing the standard solution in Example 1 (a solution in which Fmoc-Gly-OH was treated with DBU).

[0151] [ Figure 2 ] Figure 2 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Val-OH in Example 1 after being treated with DBU.

[0152] [ Figure 3 ] Figure 3 : is a chromatogram showing the sample solution containing crude Fmoc-Pro-OH in Example 1 after being treated with DBU.

[0153] [ Figure 4 ] Figure 4 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Ala-OH in Example 1 after being treated with DBU.

[0154] [ Figure 5 ] Figure 5 : is a chromatogram showing the sample solution containing crude Fmoc-Ile-OH in Example 1 after being treated with DBU.

[0155] [ Figure 6 ] Figure 6 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Leu-OH in Example 1 after being treated with DBU.

[0156] [ Figure 7 ] Figure 7 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Gly-OH in Example 1 after being treated with DBU.

[0157] [ Figure 8 ] Figure 8 This is a chromatogram showing the standard solution in Example 2 (a solution in which Fmoc-Gly-OH was treated with DBU).

[0158] [ Fig. 9 ] Fig. 9: is a chromatogram showing the sample solution containing crude Fmoc-Val-OH in Example 2 after being treated with DBU.

[0159] [ Fig.10 ] Fig.10 : is a chromatogram showing the sample solution containing crude Fmoc-Phe-OH in Example 2 after being treated with DBU.

[0160] [ Fig.11 ] Fig.11 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Val-OH in Example 2 after being treated with DBU.

[0161] [ Figure 12-1 ] Figure 12-1 This is a chromatogram comparing the sample solution containing crude Fmoc-Val-OH in Example 3 before and after the DBU treatment.

[0162] [ Figure 12-2 ] Figure 12-2 yes Figure 12-1 A magnified view of a portion of the interval.

[0163] [ Fig.13 ] Fig.13 3 is a chromatogram showing the sample solution containing crude Fmoc-Val-OH in Example 3 before the DBU treatment.

[0164] [ Fig.14 ] Fig.14 : is a chromatogram showing the sample solution containing crude Fmoc-Val-OH in Example 3 after being treated with DBU.

[0165] [ Figure 15-1 ] Figure 15-1 This is a chromatogram comparing the sample solution containing crude Fmoc-Phe-OH in Example 3 before and after the DBU treatment.

[0166] [ Figure 15-2 ] Figure 15-2 yes Figure 15-1 A magnified view of a portion of the interval.

[0167] [ Fig.16 ] Fig.16 3 is a chromatogram showing the sample solution containing crude Fmoc-Phe-OH in Example 3 before the DBU treatment.

[0168] [ Fig.17 ] Fig.173 is a chromatogram showing the sample solution containing crude Fmoc-Phe-OH in Example 3 after being treated with DBU.

[0169] [ Figure 18-1 ] Figure 18-1 This is a chromatogram comparing the sample solution containing crude Fmoc-NMe-Val-OH in Example 3 before and after the DBU treatment.

[0170] [ Figure 18-2 ] Figure 18-2 yes Figure 18-1 A magnified view of a portion of the interval.

[0171] [ Fig.19 ] Fig.19 3 is a chromatogram showing the sample solution containing crude Fmoc-NMe-Val-OH in Example 3 before DBU treatment.

[0172] [ Fig. 20 ] Fig. 20 3 is a chromatogram showing the sample solution containing crude Fmoc-NMe-Val-OH in Example 3 after being treated with DBU.

[0173] [ Fig.21 ] Fig.21 This is a graph showing the content of each Fmoc-amino acid calculated using the quantitative method of DBF of the present invention in Comparative Example 5 and the content of each Fmoc-amino acid calculated by quantitative NMR.

[0174] [ Fig. 22 ] Fig. 22 : is a chromatogram showing a standard solution of H-NMe-Gly-OH in Example 6.

[0175] [ Fig.23 ] Fig.23 : is a chromatogram showing the sample solution containing crude Fmoc-NMe-Gly-OH in Example 6 after being treated with DBU.

[0176] [ Fig.24 ] Fig.24 This is a diagram comparing 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 DBU treatment.

[0177] [ Fig.25 ] Fig.25 It is a chromatogram showing the standard solution in Example 7 (a solution in which Fmoc-Gly-OH was treated with DBU).

[0178] [ Fig.26 ] Fig.26 : is a chromatogram showing a solution of the sample solution containing crude Fmoc-Phe-Phe-OH in Example 7 treated with DBU.

[0179] [ Fig. 27 ] Fig. 27 : is a chromatogram showing a solution of the sample solution containing crude Fmoc-Gly-Gly-OH in Example 7 treated with DBU.

[0180] [ Fig.28 ] Fig.28 : is a chromatogram showing a solution of the sample solution containing crude Fmoc-Gly-Leu-OH in Example 7 treated with DBU.

[0181] [ Fig.29 ] Fig.29 : is a chromatogram showing a solution of the sample solution containing crude Fmoc-Gly-bAla-OH in Example 7 treated with DBU.

[0182] [ Fig.30 ] Fig.30 This is a chromatogram comparing the sample solution containing crude Fmoc-Phe-Phe-OH in Example 7 before and after the DBU treatment.

[0183] [ Fig.31 ] Fig.31 This is a chromatogram comparing the sample solution containing crude Fmoc-Gly-Gly-OH in Example 7 before and after the DBU treatment.

[0184] [ Fig.32 ] Fig.32 This is a chromatogram comparing the sample solution containing crude Fmoc-Gly-Leu-OH in Example 7 before and after the DBU treatment.

[0185] [ Fig.33 ] Fig.33 This is a chromatogram comparing the sample solution containing crude Fmoc-Gly-bAla-OH in Example 7 before and after the DBU treatment. DETAILED DESCRIPTION

[0186] (abbreviation)

[0187] The meanings of the symbols and abbreviations used in this specification are as follows.

[0188] Fmoc: 9-fluorenylmethoxycarbonyl

[0189] DBF: dibenzofulvene

[0190] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0191] DMF: N,N-dimethylformamide

[0192] DMA: N,N-dimethylacetamide

[0193] NMP: N-methylpyrrolidone

[0194] DMSO: dimethyl sulfoxide

[0195] TFA: trifluoroacetic acid

[0196] TFE: 2,2,2-trifluoroethanol

[0197] TMS: trimethylsilyl

[0198] CH 3 CN: Acetonitrile

[0199] Na 2 SO 4 :Sodium sulfate

[0200] Pip: piperidine

[0201] HILIC: Hydrophilic Interaction Chromatography

[0202] CAD: Charged Aerosol Detector

[0203] HPLC: High Performance Liquid Chromatography

[0204] GC: Gas chromatography

[0205] PDA: Photodiode Array

[0206] Gly: Glycine

[0207] Ala: Alanine

[0208] Val: valine

[0209] Leu: Leucine

[0210] Ile: Isoleucine

[0211] Met: methionine

[0212] Phe: Phenylalanine

[0213] Tyr: tyrosine

[0214] Trp: Tryptophan

[0215] His:Histidine

[0216] Lys: lysine

[0217] Arg: Arginine

[0218] Ser:serine

[0219] Thr:Threonine

[0220] Asp: Aspartic acid

[0221] Glu: Glutamic acid

[0222] Asn: Asparagine

[0223] Gln: Glutamine

[0224] Cys: Cysteine

[0225] Pro: Proline

[0226] Cha: Cyclohexylalanine

[0227] cLeu: cycloleucine

[0228] NMe: N-methyl

[0229] bAla: beta-alanine

[0230] In a certain embodiment, the present invention relates to a method for quantifying the content of a compound having an amino group protected with a protecting group having an Fmoc skeleton (hereinafter sometimes referred to as a first Fmoc compound) in a crude product or an analysis sample.

[0231] In a certain embodiment, the above method comprises: deprotecting a protecting group from a first Fmoc compound in a solution (hereinafter sometimes referred to as a first solution) using a base, and quantifying the content of dibenzofulvene or a derivative thereof produced thereby.

[0232] In a certain embodiment, the above method further comprises: deprotecting the protecting group from a compound having an amino group with a protecting group having an Fmoc skeleton (hereinafter referred to as a second Fmoc compound) used as an authentic sample in a solution different from the first solution (hereinafter sometimes referred to as a second solution) with a base, and quantifying the content of dibenzofulvene or a derivative thereof produced thereby. In this case, the content rate (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.

[0233] The "analysis sample" in the present invention refers to a sample containing a compound containing an amino group protected by a protecting group having an Fmoc skeleton. As the analysis sample, it is preferred to use a crude product from which impurities such as impurities and water, organic salts, inorganic salts, and residual solvents derived from the analysis sample are removed by filtration or adsorption, recrystallization, distillation, extraction, chromatography, etc.

[0234] The "content" in the present invention refers to the amount of any substance contained in the analysis sample (crude product) or in the solution, for example, the amount of a compound containing an amino group protected by a protecting group having an Fmoc skeleton, a compound containing an amino group (deprotected form), dibenzofulvene or its derivatives, an internal standard substance, etc.

[0235] The "amino group-containing compound" in the present invention refers to any compound having a primary amino group and / or a secondary amino group. There is no particular limitation on the number of amino groups contained in the amino group-containing compound, but it is preferably 1 to 2, and more preferably 1 amino group. 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).

[0236] In the present specification, there is no particular limitation on the "resin for solid phase synthesis" as long as it can be used to synthesize a peptide compound by a solid phase method. As such a resin for solid phase synthesis, specifically, resins that can be removed under acidic conditions such as CTC resin, Wang resin, SASRIN resin, trityl chloride resin (Trt resin), 4-methyltrityl chloride resin (Mtt resin), 4-methoxytrityl chloride resin (Mmt) can be exemplified. The resin can be appropriately selected according to the functional group on the amino acid side used. For example, when a carboxylic acid (main chain carboxylic acid or a side chain carboxylic acid represented by Asp or Glu) or a hydroxyl group on an aromatic ring (a phenolic group represented by Tyr) is used as the functional group on the amino acid side, it is preferred to use a trityl chloride resin (Trt resin) or a 2-chlorotrityl chloride resin (CTC resin) as the resin. When an aliphatic hydroxyl group (an aliphatic alcohol group represented by Ser or Thr) is used as the functional group on the amino acid side, it is preferred to use trityl chloride resin (Trt resin), 2-chlorotrityl chloride resin (CTC resin) or 4-methyltrityl chloride resin (Mtt resin) as the resin. In addition, in this specification, there is a case where the resin is described as "resin".

[0237] There is no particular restriction on the type of polymer constituting the resin. In the case of a resin composed of polystyrene, either 100-200 mesh or 200-400 mesh can be used. In addition, there is no particular restriction on the crosslinking rate, but those crosslinked with 1% DVB (divinylbenzene) are preferred. In addition, the type of polymer constituting the resin can be exemplified by Tentagel or Chemmatrix.

[0238] In the present specification, "amino acid" may be "natural amino acid" or "amino acid analog". In the present specification, "amino acid", "natural amino acid" and "amino acid analog" are sometimes referred to as "amino acid residue", "natural amino acid residue" and "amino acid analog residue", respectively.

[0239] "Natural amino acids" refer to the 20 amino acids contained in proteins, which are α-aminocarboxylic acids (α-amino acids). Specifically, they refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro.

[0240] There are no particular restrictions on "amino acid analogs", and they include β-amino acids, γ-amino acids, D-type amino acids, N-substituted amino acids, α,α-disubstituted amino acids, hydroxycarboxylic acids, and non-natural amino acids (amino acids with side chains different from those of natural ones: for example, non-natural α-amino acids, β-amino acids, γ-amino acids). In the case of α-amino acids, they may be D-type amino acids or α,α-dialkyl amino acids. In the case of β-amino acids or γ-amino acids, as with α-amino acids, any stereo configuration is also allowed. There are no particular restrictions on the selection of amino acid side chains, and in addition to hydrogen atoms, they are freely selected from, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and cycloalkyl. They may also have substituents, and these substituents may also be freely selected from any functional group, including, for example, N atoms, O atoms, S atoms, B atoms, Si atoms, and P atoms (i.e., optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, cycloalkyl, etc.).

[0241] The amino group of the amino acid backbone can be unsubstituted (NH 2 The amino acid in which the main chain amino group is substituted is referred to as "N-substituted amino acid" in the present specification. As the "N-substituted amino acid" in the present specification, N-alkyl amino acids (e.g., NC 1 -C6 Alkyl amino acid, NC 1 -C 4 The amino acids include, but are not limited to, N-alkyl amino acids, N-methyl amino acids, etc.), N-cycloalkyl amino acids, N-cycloalkylalkyl amino acids, N-aryl amino acids, N-arylalkyl amino acids, etc.

[0242] The main chain carboxyl group of an amino acid may be unsubstituted (carboxyl group: COOH group), or may form an ester group (i.e., -COOR group: R is, for example, an optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl or cycloalkyl group). Such an amino acid in which the main chain carboxyl group forms an ester group is referred to as "amino acid ester" in this specification. Examples of "amino acid ester" in this specification include amino acid C 1-10 Alkyl esters, amino acid benzyl esters, amino acid [2,4-di(C 1 -C 25 Alkoxy)phenyl]methyl ester, amino acid (3,4,5-tri-C 1 -C 25 The present invention also includes, but is not limited to, amino acid (3,4,5-tris-octadecyl) methyl ester, amino acid (2-(12-docosanyloxydodecyloxy)-9-(3-fluorophenyl)fluoren-9-yl) ester, [2,4-di(docosanyloxy)phenyl] methyl ester, and the like.

[0243] The main chain carboxyl groups of amino acids can also form amide groups (i.e., -CONR 1 R 2 Base: R 1 and R 2 (independently selected from, for example, hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl and cycloalkyl). Such an amino acid in which the main chain carboxyl group is substituted with an amide group is referred to as "amino acid amide" in this specification. As "amino acid amide" in this specification, amino acid C 1 -C 10 Alkylamide, amino acid benzylamide, amino acid bis[4-(docosyloxy)phenyl]methylamide (AJIPHASE (registered trademark)) and the like are examples, but are not limited thereto.

[0244] In the present specification, "side chain" is used in the context of a side chain of an amino acid or a side chain of a cyclic portion of a cyclic peptide, and refers to a portion not included in each main chain structure.

[0245] In the present specification, the term "peptide" includes a linear or cyclic peptide in which two or more amino acids are linked, and the amino acids may be "natural amino acids" or "amino acid analogs".

[0246] The "natural amino acids" and "amino acid analogs" constituting the peptides include all isotopes corresponding to them. The isotopes of "natural amino acids" and "amino acid analogs" are those in which at least one atom is replaced by an atom having the same atomic number (number of protons) but a different mass number (the sum of the number of protons and the number of neutrons). Examples of isotopes contained in the "natural amino acids" and "amino acid analogs" constituting the peptides of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., respectively. 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 32 P. 35 S. 18 F. 36 Cl et al.

[0247] In the present invention, "organic compound" refers to a compound containing carbon.

[0248] In the present invention, "low molecular weight organic compound" refers to a compound containing carbon having a small molecular weight, and the molecular weight is preferably 1500 Daltons or less.

[0249] In the present invention, "a protecting group having an Fmoc skeleton" refers to an Fmoc group or a group in which an arbitrary substituent is introduced at an arbitrary position constituting the skeleton of the Fmoc group. As such a protecting group having an Fmoc skeleton, specifically, a protecting group represented by the following formula (1) can be exemplified:

[0250]

[0251] (Where,

[0252] R 1 ~R 8 are independently selected from hydrogen, C 1 -C 8 Alkyl, C 1 -C 8 Fluoroalkyl, halogen, sulfonyl, and trimethylsilyl;

[0253] R 9 ~R 10 are independently hydrogen or methyl).

[0254] More specifically, the protecting group having an Fmoc skeleton includes, for example, a 9-fluorenylmethoxycarbonyl (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-mono-isooctyl-Fmoc (mio-Fmoc) group, a 2,7-di-isooctyl-Fmoc (dio-Fmoc) group, and a 2,7-di-isooctyl-Fmoc (dio-Fmoc) group. 7-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-Fmoc (tdf-Fmoc) group, 2,7-bis(trimethylsilyl)-Fmoc (Fmoc (2TMS)) group, (2-sulfo-9H-fluorene-9-yl)methoxycarbonyl (Fmoc (2so3h)), [(1S) -1- (9H-fluorene-9-yl)ethoxy] carbonyl (sm-Fmoc), [(1R) -1- (9H-fluorene-9-yl)ethoxy] carbonyl (rm-Fmoc), etc. Reagents for introducing these protective groups having an Fmoc skeleton can be purchased or synthesized by a known method.

[0255] In the present invention, the "compound containing an amino group protected with a protecting group having an Fmoc skeleton" refers to a compound in which at least one of the primary and / or secondary amino groups contained in the amino group-containing compound is protected with a protecting group having an Fmoc skeleton.

[0256] In the present invention, "amino acid protected with a protecting group having an Fmoc skeleton" refers to a natural amino acid or amino acid analog in which at least one of the amino groups in the main chain and / or side chain is protected with a protecting group having an Fmoc skeleton.

[0257] In the present invention, the "compound containing an amino group protected with an Fmoc group" refers to a compound in which at least one of the primary and / or secondary amino groups of the amino group-containing compound is protected with an Fmoc group.

[0258] In the present invention, "amino acid protected with an Fmoc group" refers to a natural amino acid or an amino acid analog in which at least one of the amino groups in the main chain and / or the side chain is protected with an Fmoc group. There are cases where an amino acid protected with an Fmoc group, a natural amino acid protected with an Fmoc group, and an amino acid analog protected with an Fmoc group are respectively referred to as "Fmoc-amino acid", "Fmoc-natural amino acid", and "Fmoc-amino acid analog".

[0259] The following are examples of amino acids protected with Fmoc groups that can be used in the present invention, but are not limited thereto. Many of these amino acids protected with Fmoc groups can be purchased with or without protected side chains and with or without protected amine sites. Amino acids protected with Fmoc groups that are not available for purchase can be synthesized by known methods.

[0260]

[0261] In the present invention, "a 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". Such a peptide may be, for example, a dipeptide or oligopeptide having two or more amino acids, containing either or both of the above-mentioned natural amino acids protected with a protecting group having an Fmoc skeleton and amino acid analogs protected with a protecting group having an Fmoc skeleton.

[0262] In the present invention, "peptide protected with an Fmoc group" refers to a peptide containing the above-mentioned "amino acid protected with an Fmoc group". Examples of such a peptide include dipeptides or oligopeptides having two or more amino acids, containing either or both of the above-mentioned natural amino acids protected with an Fmoc group and amino acid analogs protected with an Fmoc group.

[0263] In this specification, "alkyl" refers to a monovalent group derived from an aliphatic hydrocarbon by removing any hydrogen atom, and includes a hydrocarbon group or a subset of hydrocarbon groups containing hydrogen and carbon atoms, which does not contain heteroatoms (atoms other than carbon and hydrogen atoms) or unsaturated carbon-carbon bonds in the skeleton. The alkyl group includes straight-chain or branched alkyl groups. Alkyl groups are groups with 1 to 20 carbon atoms (C 1 -C 20 , hereinafter, “C p -C q " refers to an alkyl group having p to q carbon atoms, preferably C 1 -C 10 Alkyl, C 1 -C 8 Alkyl, C 1 -C 6 Alkyl, C 1 -C 4Alkyl, etc. Specifically, the alkyl group can be exemplified by 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, neopentyl, etc.

[0264] The "fluoroalkyl group" in this specification refers to a group in which one or more hydrogen atoms of the above-mentioned "alkyl group" are substituted with fluorine atoms, and preferably C 1 -C 8 Fluoroalkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 4 Fluoroalkyl, C 1 -C 2 Fluoroalkyl, etc. Specifically, the fluoroalkyl group includes 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.

[0265] The term "cycloalkyl" as used herein refers to a saturated or partially saturated cyclic monovalent aliphatic hydrocarbon group, including a monocyclic, bicyclic, or spirocyclic ring. 3 -C 8 Cycloalkyl. The cycloalkyl group may be partially unsaturated. Specifically, the cycloalkyl group may be exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0266] The "cycloalkylalkyl" in this specification refers to a group in which any hydrogen atom in the "alkyl" defined above is replaced by the "cycloalkyl" defined above. Preferably, the cycloalkylalkyl can be exemplified by C 3 -C 8 Cycloalkyl C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl C 1 -C 4 Specific examples thereof include cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl.

[0267] The "aryl group" in this specification refers to a monovalent aromatic hydrocarbon ring, preferably C 6 -C 10 Aryl. Specifically, the aryl group includes, for example, phenyl, naphthyl (eg, 1-naphthyl, 2-naphthyl) and the like.

[0268] The "heteroaryl" in this specification refers to a monovalent group of an aromatic ring preferably containing 1 to 4 heteroatoms among the atoms constituting the ring (also referred to as "in the ring" in this specification), which may be partially saturated. The ring may be a monocyclic ring or a condensed ring of two monocyclic rings (for example, a bicyclic heteroaryl condensed with benzene or a monocyclic heteroaryl). The number of atoms constituting the ring is preferably 5 to 10 (5- to 10-membered heteroaryl). Specifically, the heteroaryl group can be exemplified by furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, benzofuranyl, benzothienyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, and the like.

[0269] The term "aralkyl" in the present specification refers to a group containing both an aryl group and an alkyl group, for example, a group in which at least one hydrogen atom of the alkyl group is replaced by an aryl group. Preferably, "C 6 -C 10 Aryl C 1 -C 6 "Alkyl" includes, for example, benzyl, phenethyl, etc.

[0270] In this specification, "alkenyl" is a monovalent group having at least one double bond (two adjacent sp2 carbon atoms). Depending on the arrangement of the double bond and the substituent (if present), the double bond geometry may be entgegen (E) or zusammen (Z), cis or trans arrangement. Examples of alkenyl include straight chain or branched alkenyl, including straight chain containing internal olefins, etc. Preferably, C 2 -C 10 Alkenyl, more preferably C 2 -C 6 Alkenyl. Specifically, such alkenyl groups include vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl (including cis and trans forms), 3-butenyl, pentenyl, hexenyl and the like.

[0271] In the present specification, "alkynyl" is a monovalent group having at least one triple bond (two adjacent SP carbon atoms). Examples include straight-chain or branched alkynyl groups, including internal alkylene groups. Preferably, examples include C 2 -C 10 Alkynyl, more preferably C 2 -C 6Alkynyl. Specifically, examples of the alkynyl group include ethynyl, 1-propynyl, propargyl, 3-butynyl, pentynyl, and hexynyl.

[0272] The "alkoxy group" in the present specification refers to an oxy group bonded to the "alkyl group" defined above, and examples thereof include 1 to 20 carbon atoms (C 1 -C 20 ) . Specifically, the alkoxy group includes, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, (3,7,11,15-tetramethylhexadecyl)oxy, and the like.

[0273] Examples of the halogen include fluorine (—F), chlorine (—Cl), bromine (—Br), and iodine (—I).

[0274] The term "sulfo" in this specification is represented by -SO 3 The group represented by H.

[0275] The "trimethylsilyl group" in this specification is -Si(CH 3 ) 3 Or a group represented by TMS.

[0276] In a certain embodiment, the first solution comprises a crude product, a solvent, and a base, and the crude product comprises a compound (the first Fmoc compound) containing an amino group protected by a protecting group having an Fmoc skeleton, the content of which is to be calculated. The first solution may contain an internal standard substance. A deprotection reaction of a protecting group having an Fmoc skeleton occurs in the first solution. In the case of synthesizing a peptide protected by a protecting group having an Fmoc skeleton by a solid phase method, the method of the present invention may include a step of cutting out a peptide compound loaded on a resin for solid phase synthesis from a resin before the deprotection step to obtain the above-mentioned peptide. In this specification, the first solution is sometimes referred to as a "sample solution". In addition, in this specification, the sample solution before the deprotection reaction is sometimes referred to as an "unreacted" sample solution, and when used together with this term, the term "sample solution" sometimes refers to a solution after the deprotection reaction. The deprotection reaction in the first solution is preferably carried out quantitatively.

[0277] In a certain embodiment, the second solution comprises a compound (second Fmoc compound) containing an amino group protected with a protecting group having an Fmoc skeleton, a solvent and a base, which is used as a credible sample. The second solution may contain an internal standard substance. The second Fmoc compound is not limited as long as its accurate content can be determined, for example, a compound with a purity and / or content of 100% or close to 100% is used. Specifically, the second Fmoc compound can be exemplified by those sold by many suppliers. Glycine (Fmoc-Gly-OH) with an amino group protected with an Fmoc group, but is not limited thereto. As with the first solution, the second solution also undergoes a deprotection reaction of a protecting group having an Fmoc skeleton. In this specification, the second solution is sometimes referred to as a "standard solution". In addition, in this specification, the standard solution before the deprotection reaction is sometimes referred to as an "unreacted" standard solution, and when used together with this term, the term "standard solution" sometimes refers to a solution after the deprotection reaction. The deprotection reaction in the second solution is preferably carried out quantitatively.

[0278] Any solvent that can dissolve the analysis sample and the second Fmoc compound used as an authentic sample can be used in the first solution and / or the second solution, and preferably, aprotic polar solvents such as acetonitrile, DMF, DMA, NMP, DMSO, etc. can be used. The solvents used in the first solution and / or the second solution can be the same or different, but preferably, the same solvent is used.

[0279] In the present specification, "removal" and "deprotection" refer to a state in which a primary amino group and / or a secondary amino group protected with a protecting group having an Fmoc skeleton is no longer protected by the protecting group.

[0280] In one embodiment, the protecting group having an Fmoc skeleton can be removed by a solid phase reaction or a liquid phase reaction. In addition, the compound containing an amino group protected by a protecting group having an Fmoc skeleton may be supported on a solid phase or may not be supported on a solid phase, but preferably is not supported on a solid phase.

[0281] In a certain embodiment, the protective group having an Fmoc skeleton can be removed by stirring and / or standing the reaction mixture for 1 minute to 5 days in the presence of a base at a temperature of 0° C. to near the boiling point of the solvent, preferably at a temperature of 0° C. to 80° C., more preferably at a temperature of 0 to 50° C. In this reaction, an internal standard substance that does not participate in the removal reaction may or may not be added.

[0282] In a certain embodiment, the removal of the protecting group having the Fmoc skeleton can be carried out using any base capable of removing the protecting group. Such a base can be exemplified by an organic base or an inorganic base, and an organic base is preferred.

[0283] In one embodiment, the organic base can be exemplified by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,3,6,7-tetrahydro-1H,5H-9-azabenzo[ij]quinoline, 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-butyl-1,1,3,3-tetramethylguanidine, triethylamine, trimethylamine, 1-methylpiperidine, N,N'-dimethylpiperazine, N-methylmorpholine, N-ethylmorpholine, p-dimethylaminopyridine and other bases containing tertiary amines. In addition to these, organic bases can also include piperidine, morpholine, dicyclohexylamine, p-dimethylaminopyridine, diisopropylethylamine, pyridine, piperazine, tetrabutylammonium fluoride and the like, but are not limited thereto. Among these organic bases, organic bases containing one or more tertiary amines but not primary amines and secondary amines are preferred, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) is more preferred.

[0284] In a certain embodiment, the present invention can use any equivalent amount of base that can completely remove the protecting group having the Fmoc skeleton. Specifically, 0.1-20 v / v%, preferably 1-10 v / v%, more preferably 1-4 v / v% of the base can be used in the solution.

[0285] In the present specification, "dibenzofulvene or its derivative" refers to a compound represented by the following formula (2) produced when a protecting group having an Fmoc skeleton is removed:

[0286]

[0287] (Where R 1 ~R 10 Each R of the protecting group having an Fmoc skeleton represented by formula (1) 1 ~R 10 Synonymous). 1 ~R 10 The compound represented by the following formula (3) in which all the atoms are hydrogen is "dibenzofulvene":

[0288]

[0289] In one embodiment, in the present invention, the content rate (weight %) 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.

[0290] In a certain embodiment, dibenzofulvene or its derivative 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 chromatography, and its content can be quantified by the same method.

[0291] Chromatography can use any method that can separate the compound containing the amino protected by the protecting group with Fmoc skeleton, the compound containing amino (deprotection body), dibenzofulvene (DBF) or its derivatives and internal standard substance. Such chromatography can exemplify the chromatography such as 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 (for example, ion mobility), etc.; preferably liquid chromatography, gas chromatography, electrophoresis or ion mobility spectrometry. In addition, liquid chromatography is preferably high performance liquid chromatography (HPLC), ultra high performance separation liquid chromatography (UPLC). These methods can be known.

[0292] Normal phase liquid chromatography can be performed by a known method using, for example, a stationary phase such as silica or alumina and a mobile phase such as hexane, ethyl acetate, dichloromethane, isopropanol, ethanol, methanol, tetrahydrofuran, or a mixed solvent thereof.

[0293] Reversed phase liquid chromatography can be performed by a known method. The stationary phase of reversed phase liquid chromatography can use, for example, a filler modified with a hydrophobic compound, specifically, for example, a silica gel modified with a hydrophobic compound. The mobile phase of reversed phase liquid chromatography can use, for example, an organic solvent, an aqueous solution, and a mixed solvent thereof. Examples of organic solvents include acetonitrile, methanol, ethanol, isopropanol, etc. Acids such as formic acid, trifluoroacetic acid, and acetic acid can also be included in the organic solvent. Examples of aqueous solutions include water, formic acid aqueous solution, ammonium formate aqueous solution, trifluoroacetic acid aqueous solution, acetic acid aqueous solution, ammonium acetate aqueous solution, ammonium bicarbonate aqueous solution, ammonia, and buffer solutions. In addition, ion pair reagents such as sodium alkyl sulfonate and its salt, tetraalkyl ammonium or trialkyl amine and its salt can also be added to the solvent. Thus, in the case of poor peak separation, the separation ability of the analytical column can be improved. In liquid chromatography, when the mobile phase is a mixed solvent, the mixing ratio can be changed in stages or continuously. The temperature conditions of liquid chromatography and the flow rate of the mobile phase can be appropriately set according to the properties of dibenzofulvene or its derivative, the internal standard substance, and the like.

[0294] Any method can be used to identify and quantify compounds containing amino groups protected by protecting groups having an Fmoc skeleton, dibenzofulvene (DBF) or its derivatives, and internal standard substances. Such methods can be exemplified by methods such as using ultraviolet visible absorption detectors, diode array detectors (PDA), fluorescence detectors, differential refractive index detectors, evaporative light scattering detectors, charged particle detectors (CAD), mass spectrometers, etc. From the perspective of high sensitivity and high selectivity detection, ultraviolet visible absorption detectors are preferably used. Therefore, the separation and quantification method included in the method of the present invention is more preferably carried out using LC-UV, LC-PDA, etc., which are combined with liquid chromatography (LC) and ultraviolet visible absorption detectors.

[0295] The dibenzofulvene or its derivative in the first solution (sample solution) or the second solution (standard solution) can be quantified by measuring the peak area thereof by the above-mentioned chromatography.

[0296] In a certain embodiment, based on the internal standard (internal standard), the content rate of the first Fmoc compound relative to the second Fmoc compound is corrected. That is, as an internal standard for correcting the injection amount of the first solution (sample solution) and the second solution (standard solution) to the chromatogram, the first and second Fmoc compounds can be more accurately quantified by including an internal standard substance in these solutions. The correction based on the internal standard can be implemented by, for example, adding an internal standard substance and standing when preparing the first solution (sample solution) and / or the second solution (standard solution), and considering the content of the internal standard substance in the quantitative result (measured value) of dibenzofulvene or its derivatives produced after the protective group is removed. More specifically, for example, the content rate can be corrected by comparing the content of the internal standard substance (sometimes referred to as the first internal standard substance) contained in the first solution with the content of the internal standard substance (sometimes referred to as the second internal standard substance) contained in the second solution. The timing of adding the internal standard substance is not limited to the preparation of the first solution (sample solution) and / or the second solution (standard solution), and the internal standard substance can also be added after removing the protective group.

[0297] As long as it can be separated from dibenzofulvene (DBF) or its derivatives by chromatography, any substance can be used as the internal standard substance. Such internal standard substances, for example, commercially available substances can be used, specifically, anthracene, phenanthrene, naphthalene, benzene, toluene, triphenylene, tetracene, etc. can be cited. Among them, anthracene is preferred.

[0298] The content of the internal standard substance in the first solution (sample solution) or the second solution (standard solution) can be quantified by measuring its peak area by chromatography. The peak area of ​​the internal standard substance is preferably measured under the same measurement conditions as the peak area of ​​dibenzofulvene (DBF) or its derivatives.

[0299] In one embodiment, the content (wt%) of the first Fmoc compound relative to the second Fmoc compound is

[0300] 1) The peak area (A) of dibenzofulvene or its derivatives of the first solution (sample solution) and the second solution (standard solution) T and A S ),and

[0301] 2) The crude product concentration (C T ) and the concentration of the second Fmoc compound in the second solution (standard solution) (C s ),

[0302] It can be calculated by the following formula:

[0303]

[0304] (Where,

[0305] C s is the molar concentration of the second Fmoc compound;

[0306] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0307] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0308] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution).

[0309] Specifically, C can be calculated based on the weight of the crude product and the molecular weight of the first Fmoc compound. T .

[0310] When the first Fmoc compound is an Fmoc-peptide synthesized by a solid phase method, the Fmoc peptide compound supported by the resin for solid phase synthesis is regarded as the first Fmoc compound, and C can be calculated. T Specifically, C can be calculated based on, for example, the weight of the Fmoc peptide compound supported by the resin for solid phase synthesis and the molecular weight of the Fmoc peptide. T .

[0311] In one embodiment, the content rate (wt%) of the first Fmoc compound relative to the second Fmoc compound can be further calculated using the peak area (IS %) of the internal standard substance of the first solution (sample solution) and the second solution (standard solution) after calibration with the internal standard substance. T and ISS ), calculated by the following formula:

[0312]

[0313] (Where,

[0314] C s is the molar concentration of the second Fmoc compound;

[0315] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0316] IS S is the peak area of ​​the internal standard substance in the second solution;

[0317] IS T is the peak area of ​​the internal standard substance of the first solution;

[0318] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0319] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution).

[0320] In one embodiment, the above content can be corrected by the Fmoc purity of the crude product. Here, "Fmoc purity" refers to the ratio (%) of the content of the first Fmoc compound relative to the sum of the contents of all types of compounds protected with a protective group having an Fmoc skeleton contained in the crude product. By implementing this correction, the error caused by compounds (impurities) having a protective group having an Fmoc skeleton other than compounds containing an amino group protected with a protective group having an Fmoc skeleton in the crude product (analysis sample) can be eliminated, thereby more accurately calculating the content.

[0321] Specifically, the Fmoc purity can be calculated based on (i) the peak area of ​​the first Fmoc compound and (ii) the peak areas of compounds protected with a protecting group having an Fmoc skeleton other than the first Fmoc compound in the crude product (sometimes referred to as other Fmoc compounds or Fmoc-related impurities). When a plurality of Fmoc-related impurities are present in the crude product, the Fmoc purity is preferably calculated using the peak areas of all detectable Fmoc-related impurities.

[0322] More specifically, for example, the purity of Fmoc can be determined by

[0323] (i) Identifying the peaks corresponding to the first Fmoc compound and the Fmoc-related impurities by chromatography

[0324] (ii) finding the peak areas of the first Fmoc compound and the Fmoc-related impurities and summing these values; and

[0325] (iii) dividing the peak area of ​​the first Fmoc compound by the sum obtained in (ii),

[0326] And obtain.

[0327] The presence or absence of a protecting group having an Fmoc skeleton can be determined by the UV chromatogram (254 nm) in the chromatographic analysis, and whether or not it is an Fmoc-related impurity peak can be identified.

[0328] When the first Fmoc compound (e.g., Fmoc peptide) is prepared by a solid phase method, the Fmoc purity can be determined by determining the peak areas of the first Fmoc compound and Fmoc-related impurities using the solution (cut solution) used to cut out the first Fmoc compound from the solid phase synthesis resin.

[0329] By measuring the peak areas of the first Fmoc compound and the Fmoc-related impurities to obtain the Fmoc purity under the same measurement conditions as those for the quantitative determination of dibenzofulvene or its derivatives or the internal standard substance, the accurate content rate can be calculated with a small error. In a certain embodiment, by making the measurement instrument and measurement conditions used for the purity determination the same as those used for the quantitative determination of dibenzofulvene or its derivatives, the accurate content rate can be calculated with a smaller error.

[0330] When correction is performed using the Fmoc purity, the content can be determined, for example, by the following calculation formula.

[0331] (When not using internal standard material for calibration)

[0332]

[0333] (Where,

[0334] C s is the molar concentration of the second Fmoc compound;

[0335] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0336] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0337] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution;

[0338] P S is Fmoc purity).

[0339] (When using internal standard material for calibration)

[0340]

[0341] (Where,

[0342] C s is the molar concentration of the second Fmoc compound;

[0343] C T is the molar concentration of the crude product assuming that the crude product consists only of the first Fmoc compound;

[0344] IS S is the peak area of ​​the internal standard substance in the second solution;

[0345] IS T is the peak area of ​​the internal standard substance of the first solution;

[0346] A S is the peak area of ​​dibenzofulvene or its derivative of the second solution;

[0347] A T is the peak area of ​​dibenzofulvene or its derivative of the first solution;

[0348] P S is Fmoc purity).

[0349] In a certain embodiment, an acid can be used to prevent the degradation of dibenzofulvene or its derivatives. The degradation inhibition (stabilization) of dibenzofulvene or its derivatives can be achieved by contacting the produced dibenzofulvene or its derivatives with an acid. The acid can preferably include organic acids, inorganic acids, and organic acids, inorganic acids, and combinations thereof can be used.

[0350] In the case of adding acid, it is possible to remove the protecting group with the Fmoc skeleton and then add acid to the analysis sample (e.g., the first and / or second solution). Preferably, acid is added immediately after removal to prevent dibenzofulvene from degrading. The amount of acid can be used in any amount, as long as the standard solution or sample solution is relatively neutral and slightly acidic, for example, adding the same or more equivalent acid as the base added to remove the protecting group can achieve this. Acid can be added directly to the analysis sample or can be added to the analysis sample after the acid is dissolved in an organic solvent, etc.

[0351] 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 can be used, and citric acid is preferably used.

[0352] 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, and phosphoric acid is preferably used.

[0353] In a certain embodiment, the present invention relates to a method for quantifying a compound containing an amino group protected with a protecting group having an Fmoc skeleton by quantifying the compound containing the amino group after deprotection.

[0354] In a certain embodiment, the present invention relates to a method for removing a protecting group having an Fmoc skeleton from a compound having an amino group protected with a protecting group having an Fmoc skeleton, and quantifying the amino group-containing compound thus produced.

[0355] In a certain embodiment, the above method can separate the amino group-containing compound from other components by chromatography and measure the peak area to quantify the content thereof.

[0356] In a certain embodiment, for separating compounds containing amino groups by chromatography, any method capable of separating compounds containing amino groups protected by protecting groups having an Fmoc skeleton, dibenzofulvene (DBF) or its derivatives and compounds containing amino groups can be used. Such chromatography can exemplify chromatography such as 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), etc.; preferably hydrophilic interaction chromatography (HILIC). These methods may be well known.

[0357] For the identification and quantitative method of the compound containing amino group, any method can be used. Such method can exemplify the method using ultraviolet visible absorption detector, diode array detector, fluorescence detector, differential refractive index detector, evaporative light scattering detector, charged particle detector (CAD), mass spectrometer, etc. Preferably, a charged particle detector (CAD) and / or mass spectrometer that can detect the compound even when the compound does not absorb ultraviolet visible light wavelength is used. Therefore, the separation and quantitative method included in the method of the present invention is more preferably carried out using HILIC-CAD combined with hydrophilic interaction chromatography (HILIC) and charged particle detector (CAD).

[0358] 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.

[0359] In addition, all prior art documents cited in this specification are incorporated into this specification by reference.

[0360] Example

[0361] The present invention is described in more detail below by way of examples, but the present invention is not limited to the following examples.

[0362] The following Fmoc-amino acids were used in the quantification described in this example.

[0363] 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. They were purchased from raw material manufacturers such as Watanabe Chemical Industries. Unless otherwise stated, the same Fmoc-amino acid included different raw material manufacturers and batches.

[0364] Example 1 (Quantification of Fmoc-amino acid by DBF-1)

[0365] Preparation of standard solutions

[0366] Fmoc-Gly-OH (about 10 mg) was accurately weighed according to the Japanese Pharmacopoeia, and 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 a Fmoc-Gly-OH solution (an acetonitrile solution containing Fmoc-Gly-OH at a concentration of 500 ug / mL) dissolved in acetonitrile (20 mL) 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%. Next, the solution was vortex-stirred and allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was carried out quantitatively. In this embodiment, the above-mentioned solution before the start of the deprotection reaction is referred to as the "unreacted standard solution", and the above-mentioned solution after the deprotection reaction is only referred to as the "standard solution". In addition, 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.

[0367] Preparation of sample solution

[0368] The crude Fmoc-amino acid (about 10 mg) containing the quantitative object Fmoc-amino acid was accurately weighed according to the Japanese Pharmacopoeia, and an internal standard solution (an acetonitrile solution containing anthracene at a concentration of 250ug / mL), a DBU solution (an acetonitrile solution containing DBU at a concentration of 10v / v%) and acetonitrile were added to the crude Fmoc-amino acid solution (an acetonitrile solution containing a crude Fmoc-amino acid at a concentration of 500ug / mL) prepared by dissolving in acetonitrile (20mL) to prepare acetonitrile solutions containing crude Fmoc-amino acid at a concentration of 100ug / mL, anthracene at a concentration of 50ug / mL, and DBU at a concentration of 4v / v%. Next, after vortex stirring the solution, it was allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was quantitatively carried out. In this embodiment, the above solution before the start of the deprotection reaction is referred to as the "unreacted sample solution", and the above solution after the deprotection reaction is only referred to as the "sample solution".

[0369] The analysis apparatus and analysis conditions are as follows.

[0370] (Analysis device)

[0371] HPLC system: H-class (Waters)

[0372] UV-visible absorption detector: ACQ-TUV

[0373] Pump: ACQ-QSM

[0374] Automatic sample injector: ACQ-FTN

[0375] (Analysis conditions)

[0376] Column: OSAKA SODA CAPCELL CORE ADME (2.1x150mm, 2.7um)

[0377] Mobile phase A: 0.05% TFA / water

[0378] Mobile phase B: 0.05% TFA / acetonitrile

[0379] Flow rate: 0.3mL / min

[0380] Column temperature: 40°C

[0381] Sample injection volume: 2uL

[0382] Measurement wavelength: 254nm

[0383] Gradient conditions: see Table 1

[0384] [Table 1]

[0385] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0386] Figure 1 shows the chromatogram of the standard solution, Figure 2 to Figure 7 The chromatogram of each sample solution is shown.

[0387] In addition, the content ratio (w / w%) of each Fmoc-amino acid relative to Fmoc-Gly-OH can be calculated using the following mathematical formula: Table 2 (without correction of the injection amount by anthracene) and Table 3 (with correction of the injection amount by anthracene) show the results.

[0388] Calculation formula (When the injection amount is not corrected with anthracene)

[0389]

[0390] C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L)

[0391] C T : Concentration of each crude Fmoc-amino acid in each unreacted sample solution (umol / L)

[0392] A s : DBF peak area of ​​standard solution

[0393] A T : DBF peak area of ​​each sample solution

[0394] Calculation formula (When the injection amount is corrected using anthracene)

[0395]

[0396] C s :Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L)

[0397] C T :Concentration of each crude Fmoc-amino acid in each unreacted sample solution (umol / L)

[0398] IS S : Anthracene peak area of ​​standard solution

[0399] IS T : Anthracene peak area of ​​each sample solution

[0400] A S : DBF peak area of ​​standard solution

[0401] A T : DBF peak area of ​​each sample solution

[0402] [Table 2]

[0403] (Injection volume not corrected with anthracene)

[0404]

[0405] [Table 3]

[0406] (Use anthracene to calibrate the injection volume)

[0407]

[0408] From the above results, it can be seen that there are Fmoc-amino acids with a content rate of almost 100%, such as Fmoc-Ile-OH and Fmoc-NMe-Leu-OH, and there are also Fmoc-amino acids with a content rate of less than 100%. It is believed that Fmoc-amino acids with a content rate of less than 100% contain impurities such as water, inorganic salts, and residual solvents that cannot be detected by UV. In addition, even when the injection amount of the standard solution and each sample solution is different, quantitative calculation can be performed by correcting the peak area using the internal standard substance.

[0409] Example 2 (Quantification of Fmoc-amino acid by DBF-2)

[0410] Preparation of standard solutions

[0411] Fmoc-Gly-OH (about 10 mg) was accurately weighed according to the Japanese Pharmacopoeia, and an internal standard solution (an acetonitrile solution containing 500ug / mL anthracene), a DBU solution (an acetonitrile solution containing 10v / v% DBU) and acetonitrile were added to a Fmoc-Gly-OH solution (an acetonitrile solution containing 1mg / mL Fmoc-Gly-OH) dissolved in acetonitrile (10mL) to prepare an acetonitrile solution containing 100ug / mL Fmoc-Gly-OH, 50ug / mL anthracene, and 4v / v% DBU, respectively. Next, the solution was vortex-stirred and allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was carried out quantitatively. In this embodiment, the above-mentioned solution before the start of the deprotection reaction is referred to as the "unreacted standard solution", and the above-mentioned solution after the deprotection reaction is only referred to as the "standard solution". In addition, Fmoc-Gly-OH used in the preparation of the standard solution was purchased from Watanabe Chemical Industry (purity 99.8%, water content less than 0.10%).

[0412] Preparation of sample solution

[0413] The crude Fmoc-amino acid (about 10 mg) containing the quantitative object Fmoc-amino acid is accurately weighed according to the Japanese Pharmacopoeia, and the internal standard solution (acetonitrile solution containing 500ug / mL anthracene), DBU solution (acetonitrile solution containing 10v / v% DBU) and acetonitrile are added to the crude Fmoc-amino acid solution (acetonitrile solution containing 1mg / mL crude Fmoc-amino acid) dissolved in acetonitrile (10mL) to prepare acetonitrile solutions containing 100ug / mL crude Fmoc-amino acid, 50ug / mL anthracene, and 4v / v% DBU, respectively. Next, after vortex stirring the solution, let it stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was quantitatively carried out. In this embodiment, the above solution before the start of the deprotection reaction is referred to as the "unreacted sample solution", and the above solution after the deprotection reaction is only referred to as the "sample solution".

[0414] The analysis apparatus and analysis conditions are as follows.

[0415] (Analysis device)

[0416] HPLC system: H-class (Waters)

[0417] UV-visible absorption detector: ACQ-TUV

[0418] Pump: ACQ-QSM

[0419] Automatic sample injector: ACQ-FTN

[0420] (Analysis conditions)

[0421] Column: OSAKA SODA CAPCELL CORE ADME (3.0x 150mm, 2.7um)

[0422] Mobile phase A: 0.05% TFA / water

[0423] Mobile phase B: 0.05% TFA / acetonitrile

[0424] Flow rate: 0.6mL / min

[0425] Column temperature: 40°C

[0426] Sample injection volume: 2uL

[0427] Measurement wavelength: 254nm

[0428] Gradient conditions: see Table 4

[0429] [Table 4]

[0430] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0431] Figure 8 shows the chromatogram of the standard solution, Figures 9 to 11 The chromatogram of each sample solution is shown.

[0432] In addition, the content (w / w%) of each Fmoc-amino acid relative to Fmoc-Gly-OH can be calculated using the mathematical formula shown 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).

[0433] [Table 5]

[0434] (Injection volume not corrected with anthracene)

[0435]

[0436] [Table 6]

[0437] (Use anthracene to calibrate injection volume)

[0438]

[0439] As can be seen from the above results, the content of the three Fmoc-amino acids is almost 100%. In addition, the content is almost unchanged by whether or not the injection amount is corrected with anthracene, so it is estimated that there is no difference between the injection amount of the standard solution in this example and the injection amount of each sample solution.

[0440] Example 3 (Content analysis using Fmoc-amino acid purity correction)

[0441] Preparation of sample solution

[0442] The sample solution (acetonitrile solution containing 100 ug / mL crude Fmoc-amino acid) was prepared by diluting a 1 mg / mL crude Fmoc-amino acid solution. In addition, various crude Fmoc-amino acids used in the sample solution were purchased from Watanabe Chemical Industry.

[0443] The analysis apparatus and analysis conditions are as follows.

[0444] (Analysis device)

[0445] HPLC system: H-class (Waters)

[0446] UV-visible absorption detector: ACQ-TUV

[0447] Pump: ACQ-QSM

[0448] Automatic sample injector: ACQ-FTN

[0449] (Analysis conditions)

[0450] Column: OSAKA SODA CAPCELL CORE ADME (3.0x150mm, 2.7um)

[0451] Mobile phase A: 0.05% TFA / water

[0452] Mobile phase B: 0.05% TFA / acetonitrile

[0453] Flow rate: 0.6mL / min

[0454] Column temperature: 40°C

[0455] Sample injection volume: 2uL

[0456] Measurement wavelength: 254nm

[0457] Gradient conditions: see Table 7

[0458] [Table 7]

[0459] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0460] As shown in Figure 12~ Fig. 20 As shown in the figure, from the comparison with the chromatogram of Example 2, the peak of Fmoc-amino acid that disappeared due to DBU treatment can be confirmed. This peak is considered to be a peak from a compound having an Fmoc group, and Table 8 shows the purity of each Fmoc-amino acid calculated by integrating only these peaks. Using these purity corrections and anthracene correction injection amount, the content rate can be calculated. Table 9 shows the results.

[0461] Calculation formula

[0462]

[0463] DBF assay 样品 : The content of each Fmoc-amino acid before purity correction

[0464] P S : Purity of each Fmoc-amino acid in compounds with Fmoc groups

[0465] [Table 8]

[0466] Purity of each Fmoc amino acid

[0467]

[0468] [Table 9]

[0469] Content of each Fmoc amino acid (with purity correction)

[0470] content Fmoc-Val-OH 101.2％ Fmoc-Phe-OH 100.6％ Fmoc-NMe-Val-OH 100.8％

[0471] Example 4 (Study on DBF Stability)

[0472] Preparation of sample solution

[0473] 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.

[0474] [Table 10]

[0475]

[0476] The analysis apparatus and analysis conditions are as follows.

[0477] (Analysis device)

[0478] HPLC system: H-class (Waters)

[0479] UV-Vis Absorption Detector: ACQ-PDA

[0480] Pump: ACQ-QSM

[0481] Automatic sample injector: ACQ-FTN

[0482] (Analysis conditions)

[0483] Column: OSAKA SODA CAPCELL CORE ADME (2.1x150mm, 2.7um)

[0484] Mobile phase A: 0.05% TFA / water

[0485] Mobile phase B: 0.05% TFA / acetonitrile

[0486] Flow rate: 0.3mL / min

[0487] Column temperature: 40°C

[0488] Sample injection volume: 2uL

[0489] Measurement wavelength: 254nm

[0490] Gradient conditions: see Table 11

[0491] [Table 11]

[0492] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0493] Table 12 shows the ratio of the peak areas of DBF and its degradation products in the sample solution on the first day and the sample solution after storage for 1 day, 2 days, and 5 days. In addition, Table 13 shows the results of the comparison of the DBF peak areas in the sample solution on the first day and the sample solution after storage for 5 days (storage for 5 days / first day). It can be seen that DBF degradation can be inhibited by citric acid treatment.

[0494] [Table 12]

[0495] Peak ratio of DBF and its degradation products in stability test

[0496]

[0497] Note) DBF-Pip has the following structure.

[0498]

[0499] Note) The DBF degradation product has the following structure.

[0500]

[0501] [Table 13]

[0502] Comparison of the DBF peak area in the sample solution on the first day and after 5 days of storage

[0503]

[0504] Example 5 (Comparison with quantitative NMR)

[0505] In order to evaluate the validity of the quantitative method (DBF determination method) of the present invention, DBF generated by treatment with DBU was quantified for various crude Fmoc-amino acids in the same manner as in Example 1 or 2 (not calibrated with an internal standard substance, but calibrated with purity), and compared with the quantitative values ​​of the crude Fmoc-amino acids measured by quantitative NMR. In addition, Fmoc-Ile-OH (content ratio by quantitative NMR was 99.18%) was used as an authentic sample amino acid for preparing a standard solution. Fig.21 As shown in Table 14, a good positive correlation was observed between the quantitative value of the present invention and the quantitative value of quantitative NMR, and the correlation coefficient was 0.95, which proved the validity of the present invention.

[0506] The analytical apparatus and reagents are as follows.

[0507] (Analysis device)

[0508] NMR:JEOL 500MHz Royal HFX Probe

[0509] Reagents: Fmoc-amino acids

[0510] Solvent:DMSO-d6

[0511] Standard: 3,5-bis(trifluoromethyl)-benzoic acid (99.96%±0.06%, quantitative NMR reagent)

[0512] [Table 14]

[0513]

[0514] The correlation with qNMR showed that the present quantitative method is an effective quantitative method like qNMR.

[0515] Example 6 (Amino Acid Quantification after DBU Treatment of Fmoc-Amino Acid)

[0516] When Fmoc-amino acid is deprotected with DBU, DBF and amino acid are generated. DBF was quantified in Examples 1 to 4, but amino acid was quantified in this Example. The peak area of ​​H-NMe-Gly-OH generated by deprotecting Fmoc-NMe-Gly-OH with DBU was compared with 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 at a yield of 97.2% ( Figures 22-24 ). Thus, it was confirmed that the deprotection reaction by adding DBU proceeded quantitatively.

[0517] In addition, in this embodiment, the measurement was carried out by hydrophilic interaction chromatography (HILIC), and the peak of the highly hydrophobic compound (such as DBF) appeared at an earlier elution position, while the peak of the highly hydrophilic compound (such as amino acid) appeared at a later elution position. In addition, H-NMe-Gly-OH hardly absorbs ultraviolet visible light wavelengths, so it was detected by CAD.

[0518] Preparation of amino acid standard solution

[0519] 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.

[0520] Preparation of sample solution

[0521] After adding 2% DBU solution (acetonitrile solution containing 2 v / v% DBU concentration) to Fmoc-NMeGly-OH, CH 3 CN / H 2The sample solution (containing a solution with a concentration of 300 ug / mL Fmoc-amino acid) was prepared by diluting the mixture with 1% Fmoc-MeGly-OH (4:1). Fmoc-MeGly-OH was purchased from the raw material manufacturer.

[0522] The analysis apparatus and analysis conditions are as follows.

[0523] (Analysis device)

[0524] HPLC system: 1200 series (Agilent)

[0525] UV-visible absorption detector: Agilemt 1200MWD

[0526] Pump: Agilent 1200 Binary Pump-1, -2

[0527] Autosampler: Agilent 1200 Autosampler

[0528] (Analysis conditions)

[0529] Column: Imtakt Intrada Amino Acid (3x 100mm, 3um)

[0530] Mobile phase A: 0.1% formic acid / acetonitrile

[0531] Mobile phase B: 100 mM ammonium formate / water

[0532] Flow rate: 1.0mL / min

[0533] Column temperature: 35°C

[0534] Sample injection volume: 2uL

[0535] Detector: Charged Aerosol Detector (CAD) (Corona ULTRA)

[0536] Gradient conditions: see Table 15

[0537] [Table 15]

[0538] Time (minutes) A(％) B(％) 0 86 14 3 86 14 10 0 100 10.01 86 14 14 86 14

[0539] A reverse gradient setting different from this condition is implemented, and A:B=50:50 is usually introduced into the detector.

[0540] Reverse gradient conditions: see Table 16

[0541] [Table 16]

[0542] Time (minutes) A(％) B(％) 0 14 86 3 14 86 10 100 0 10.01 14 86 14 14 86

[0543] Example 7 (Quantification of Fmoc-peptide by DBF)

[0544] Preparation of standard solutions

[0545] According to the Japanese Pharmacopoeia, Fmoc-Gly-OH (about 10 mg) was accurately weighed, and 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 the Fmoc-Gly-OH solution (an acetonitrile solution containing Fmoc-Gly-OH at a concentration of 500 ug / mL) prepared by dissolving in acetonitrile (20 mL) 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%. Next, the solution was vortex-stirred and allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was carried out quantitatively. In this embodiment, the above-mentioned solution before the start of the deprotection reaction is referred to as the "unreacted standard solution", and the above-mentioned solution after the deprotection reaction is only referred to as the "standard solution". In addition, Fmoc-Gly-OH (purity: 98.6%) used in the preparation of the standard solution was purchased from Tokyo Chemical Industry.

[0546] Preparation of sample solution-1

[0547] According to the Japanese Pharmacopoeia, crude Fmoc-Phe-Phe-OH (about 10 mg) of the quantitative object was accurately weighed, and 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 500 ug / mL) prepared by dissolving it in acetonitrile (20 mL) to prepare acetonitrile solutions containing crude 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%. Next, after vortex stirring the solution, it was allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction proceeded quantitatively. The crude Fmoc-Phe-Phe-OH (purity 99.8%) used in the preparation of the solution was purchased from Watanabe Chemical Industry Co., Ltd. In this example, the solution before the deprotection reaction is referred to as "unreacted sample solution", and the solution after the deprotection reaction is referred to as simply "sample solution".

[0548] Preparation of sample solution-2 (Crude Fmoc-peptide compound synthesized by solid phase reaction and supported on resin)

[0549] The quantitative object was precisely weighed according to the Japanese Pharmacopoeia, which was a crude Fmoc-peptide compound synthesized by solid phase reaction and loaded on the resin (hereinafter sometimes referred to as "crude Fmoc-peptide compound loaded on the resin") (about 30 mg), and dichloromethane (3 mL) was added and allowed to stand for more than 15 minutes to swell. In addition, in this example and Example 8, CTC resin was used as a resin for solid phase synthesis. After the dichloromethane was discharged, the resin was washed twice with dichloromethane (3 mL). 0.6 mL of TFA solution (a dichloromethane solution containing TFA at a concentration of 1 v / v%) was added to the resin and shaken at room temperature to cut out the crude Fmoc-peptide from the resin. An internal standard solution (an acetonitrile solution containing an anthracene concentration of 250 μg / mL), a DBU solution (an acetonitrile solution containing a DBU concentration of 10 v / v%) and acetonitrile were added thereto to prepare an acetonitrile solution containing a crude Fmoc-peptide, which contained a crude Fmoc-peptide at a concentration of 1 mg / mL as a crude Fmoc-peptide compound loaded on the resin, anthracene at a concentration of 25 ug / mL, and DBU at a concentration of 2 v / v%. Next, the solution was vortex-stirred and allowed to stand at room temperature for more than 10 minutes to deprotect the Fmoc group. The UV chromatogram obtained by HPLC confirmed that the deprotection reaction was carried out quantitatively. In this embodiment, the above solution before the start of the deprotection reaction is referred to as the "unreacted sample solution", and the above solution after the deprotection reaction is only referred to as the "sample solution".

[0550] The analysis apparatus and analysis conditions are as follows.

[0551] (Analysis device)

[0552] HPLC system: H-class (Waters)

[0553] UV-visible absorption detector: ACQ-TUV

[0554] Pump: ACQ-QSM

[0555] Automatic sample injector: ACQ-FTN

[0556] (Analysis conditions)

[0557] Column: OSAKA SODA CAPCELL CORE ADME (2.1x150mm, 2.7um)

[0558] Mobile phase A: 0.05% TFA / water

[0559] Mobile phase B: 0.05% TFA / acetonitrile

[0560] Flow rate: 0.3mL / min

[0561] Column temperature: 40°C

[0562] Sample injection volume: 2uL

[0563] Measurement wavelength: 254nm

[0564] Gradient conditions: see Table 17

[0565] [Table 17]

[0566] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0567] Fig.25 shows the chromatogram of the standard solution, Figure 26 to Figure 29 The chromatogram of each sample solution is shown.

[0568] In addition, the content rate (w / w%) of each Fmoc-peptide relative to Fmoc-Gly-OH can be calculated using the following mathematical formula. Table 18 shows the results (without correction of the injection amount by anthracene), and Table 19 shows the results (with correction of the injection amount by anthracene). In addition, the molecular weight of the Fmoc-peptide contained in the sample solution was calculated as a carboxylic acid body having a carboxyl group at the end.

[0569] Calculation formula (When the injection amount is not corrected with anthracene)

[0570]

[0571] C s : Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L)

[0572] C T : Concentration of each crude Fmoc-peptide in each unreacted sample solution (umol / L)

[0573] A S : DBF peak area of ​​standard solution

[0574] A T : DBF peak area of ​​each sample solution

[0575] Calculation formula (When the injection amount is corrected using anthracene)

[0576]

[0577] C s :Concentration of Fmoc-Gly-OH in the unreacted standard solution (umol / L)

[0578] C T :Concentration of each crude Fmoc-peptide in each unreacted sample solution (umol / L)

[0579] IS S : Anthracene peak area of ​​standard solution

[0580] IS T : Anthracene peak area of ​​each sample solution

[0581] A S : DBF peak area of ​​standard solution

[0582] A T : DBF peak area of ​​each sample solution

[0583] [Table 18]

[0584] (Injection volume not corrected with anthracene)

[0585]

[0586] [Table 19]

[0587] (Use anthracene to calibrate the injection volume)

[0588]

[0589] Example 8 (Content Analysis Corrected by Purity of Fmoc-Peptide)

[0590] Preparation of sample solution-1

[0591] The 500 ug / mL crude Fmoc-Phe-Phe-OH solution was diluted to prepare a sample solution (an acetonitrile solution containing 50 ug / mL crude Fmoc-Phe-Phe-OH).

[0592] Preparation of sample solution-2

[0593] The cut solution was diluted with acetonitrile in the same manner as in "Preparation of sample solution-2" of Example 7 so that the crude Fmoc-peptide was contained at a concentration of 1 mg / mL as a crude Fmoc-peptide compound supported on the resin, and this was used as a sample solution.

[0594] The analysis apparatus and analysis conditions are as follows.

[0595] (Analysis device)

[0596] HPLC system: H-class (Waters)

[0597] UV-visible absorption detector: ACQ-TUV

[0598] Pump: ACQ-QSM

[0599] Automatic sample injector: ACQ-FTN

[0600] (Analysis conditions)

[0601] Column: OSAKA SODA CAPCELL CORE ADME (2.1x150mm, 2.7um)

[0602] Mobile phase A: 0.05% TFA / water

[0603] Mobile phase B: 0.05% TFA / acetonitrile

[0604] Flow rate: 0.3mL / min

[0605] Column temperature: 40°C

[0606] Sample injection volume: 2uL

[0607] Measurement wavelength: 254nm

[0608] Gradient conditions: see Table 20

[0609] [Table 20]

[0610] Time (minutes) B(％) 0 30 20 70 22 100 24 100 24.1 30 28 30

[0611] The purity of the crude Fmoc-peptide relative to the total Fmoc compound of each sample solution was calculated. The results are shown in Table 21. The calculated purity and correction with anthracene were used to calculate the content rate of the Fmoc-peptide. The results are shown in Table 22. For samples in the form of methyl esters generated during the storage of the measured samples, the purity was calculated using the methyl ester form as the target Fmoc-peptide.

[0612] Calculation formula

[0613]

[0614] DBF assay 样品 : The content of each Fmoc-peptide before purity correction

[0615] P S : Purity of each Fmoc-peptide in compounds with Fmoc group

[0616] [Table 21]

[0617]

[0618] [Table 22]

[0619] Content rate Fmoc-Phe-Phe-OH 96.6％ Fmoc-Gly-Gly-O-resin 4.3％ Fmoc-Gly-Leu-O-resin 13.6％ Fmoc-Gly-bAla-O-resin 15.4％

[0620] The above results show that the quantitative method of the present invention can be used for the content analysis of Fmoc-peptide and the content analysis of Fmoc-peptide in Fmoc-peptide compounds supported on resins synthesized by solid phase reaction.

[0621] Industrial Availability

[0622] The present invention provides a novel quantitative method for compounds containing an amino group protected by a protective group having an Fmoc skeleton. According to the present invention, the content of a compound containing an amino group protected by a protective group having an Fmoc skeleton contained in a sample can be quantitatively analyzed with high accuracy and in a simple manner.

Claims

1. A method for quantifying the content of a first Fmoc compound contained in a crude product, wherein the first Fmoc compound contains an amino group protected by a protecting group having an Fmoc skeleton, the method comprising: deprotecting the protecting group from the first Fmoc compound in the first solution with a base, and quantifying the content of dibenzofulvene or its derivative produced thereby, The method further comprises adding an acid to the first solution after removing the protecting group having the Fmoc skeleton.

2. The method according to claim 1, further comprising deprotecting a protecting group from a second Fmoc compound with a base, the second Fmoc compound used as an authentic sample in the second solution containing an amino group having a protecting group of an Fmoc skeleton, and quantifying the content of dibenzofulvene or its derivatives produced thereby, in, 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, the content rate of the first Fmoc compound relative to the second Fmoc compound is calculated, and the unit of the content rate is weight % relative to the second Fmoc compound.

3. The method according to claim 2, wherein: The deprotection reaction is carried out quantitatively in the first solution and the second solution.

4. The method according to claim 1, wherein: The content of dibenzofulvene or its derivatives is quantified by measuring the peak area of ​​dibenzofulvene or its derivatives.

5. The method according to claim 2, further comprising correcting the content rate using the Fmoc purity of the crude product, wherein: “Fmoc purity” is the ratio of the content of the first Fmoc compound to the sum of the contents of all kinds of compounds protected with a protecting group having an Fmoc skeleton contained in the crude product. 6 . The method according to claim 5 , 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. The method according to claim 2 , further comprising correcting the content ratio using an internal standard substance. 8 . The method according to claim 7 , comprising comparing the content of a first internal standard substance contained in the first solution and the content of a second internal standard substance contained in the second solution.

9. The method according to claim 8, wherein: By measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance, the contents of the first and second internal standard substances are quantified respectively.

10. The method according to claim 4, wherein: The area of ​​each peak was determined by chromatography.

11. The method according to claim 10, wherein: The area of ​​each peak was measured under the same measurement conditions.

12. The method according to claim 1, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

13. The method according to claim 1, 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 skeleton.

14. The method according to claim 1, wherein: The base is an organic base.

15. The method according to claim 1, wherein: The acid is an organic acid and / or an inorganic acid.

16. The method according to claim 15, 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 or a combination thereof.

17. The method according to claim 15, 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 or a combination thereof.

18. The method according to claim 2, wherein: The content of dibenzofulvene or its derivatives is quantified by measuring the peak area of ​​dibenzofulvene or its derivatives.

19. The method according to claim 3, wherein: The content of dibenzofulvene or its derivatives is quantified by measuring the peak area of ​​dibenzofulvene or its derivatives.

20. The method according to claim 3, further comprising correcting the content rate using the Fmoc purity of the crude product, wherein: “Fmoc purity” is the ratio of the content of the first Fmoc compound to the sum of the contents of all kinds of compounds protected with a protecting group having an Fmoc skeleton contained in the crude product.

21. The method according to claim 18, further comprising correcting the content ratio using the Fmoc purity of the crude product, wherein: “Fmoc purity” is the ratio of the content of the first Fmoc compound to the sum of the contents of all kinds of compounds protected with a protecting group having an Fmoc skeleton contained in the crude product.

22. The method according to claim 20, 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.

23. The method according to claim 21, 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. The method according to claim 3 , further comprising correcting the content ratio using an internal standard substance. The method according to claim 4 , further comprising correcting the content ratio using an internal standard substance. The method according to claim 5 , further comprising correcting the content ratio using an internal standard substance. The method according to claim 6 , further comprising correcting the content ratio using an internal standard substance.

28. The method according to claim 24, comprising comparing the content of a first internal standard substance contained in the first solution and the content of a second internal standard substance contained in the second solution.

29. The method according to claim 25, comprising comparing the content of a first internal standard substance contained in the first solution and the content of a second internal standard substance contained in the second solution.

30. The method according to claim 26, comprising comparing the content of a first internal standard substance contained in the first solution and the content of a second internal standard substance contained in the second solution.

31. The method according to claim 27, comprising comparing the content of a first internal standard substance contained in the first solution and the content of a second internal standard substance contained in the second solution.

32. The method of claim 28, wherein: By measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance, the contents of the first and second internal standard substances are quantified respectively.

33. The method of claim 29, wherein: By measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance, the contents of the first and second internal standard substances are quantified respectively.

34. The method of claim 30, wherein: By measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance, the contents of the first and second internal standard substances are quantified respectively.

35. The method of claim 31, wherein: By measuring the peak area of ​​the first internal standard substance and the peak area of ​​the second internal standard substance, the contents of the first and second internal standard substances are quantified respectively.

36. The method according to claim 6, wherein: The area of ​​each peak was determined by chromatography.

37. The method of claim 9, wherein: The area of ​​each peak was determined by chromatography.

38. The method of claim 36, wherein: The area of ​​each peak was measured under the same measurement conditions.

39. The method of claim 37, wherein: The area of ​​each peak was measured under the same measurement conditions.

40. The method of claim 2, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

41. The method of claim 3, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

42. The method of claim 4, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

43. The method of claim 5, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

44. The method of claim 6, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

45. The method of claim 7, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

46. ​​The method of claim 8, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

47. The method of claim 9, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

48. The method of claim 10, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

49. The method of claim 11, wherein: The protecting group having an Fmoc skeleton is represented by the following formula (1): In the formula, R1 to R8 are independently selected from hydrogen, C1-C8 alkyl, C1-C8 fluoroalkyl, halogen, sulfonyl and trimethylsilyl, R9~R 10 is independently hydrogen or methyl.

50. The method of claim 2, 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 skeleton.

51. The method of claim 3, 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 skeleton.

52. The method of claim 4, 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 skeleton.

53. The method of claim 5, 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 skeleton.

54. The method of claim 6, 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 skeleton.

55. The method of claim 7, 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 skeleton.

56. The method of claim 8, 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 skeleton.

57. The method of claim 9, 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 skeleton.

58. The method of claim 10, 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 skeleton.

59. The method of claim 11, 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 skeleton.

60. The method of claim 12, 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 skeleton.

61. The method of claim 2, wherein: The base is an organic base.

62. The method of claim 3, wherein: The base is an organic base.

63. The method of claim 4, wherein: The base is an organic base.

64. The method of claim 5, wherein: The base is an organic base.

65. The method of claim 6, wherein: The base is an organic base.

66. The method of claim 7, wherein: The base is an organic base.

67. The method of claim 8, wherein: The base is an organic base.

68. The method of claim 9, wherein: The base is an organic base.

69. The method of claim 10, wherein: The base is an organic base.

70. The method of claim 11, wherein: The base is an organic base.

71. The method of claim 12, wherein: The base is an organic base.

72. The method of claim 13, wherein: The base is an organic base.

73. The method of claim 16, 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 or a combination thereof.

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