PEPTIDE PURIFICATION METHOD USING SULFONATE COMPOUND

Information

  • Patent Application
  • 20220144888
  • Publication Number
    20220144888
  • Date Filed
    August 08, 2019
    5 years ago
  • Date Published
    May 12, 2022
    2 years ago
Abstract
A method for purifying a target peptide may include mixing a peptide product obtained through a peptide synthesis with a solvent in the presence of a sulfonic acid compound to obtain a solid; and performing a solid-liquid separation to collect the solid.
Description
TECHNICAL FIELD

The present application relates to a method for purifying a target peptide from peptide product obtained by peptide synthesis, and a medicament comprising the target peptide obtained by the purification method.


BACKGROUND ART

Peptides with relatively few amino acid residues (usually less than or equal to 30 residues) are commonly produced by organic chemical synthesis (for example, Patent Document 1). Examples of the organic chemical synthesis method include solid-phase and liquid-phase methods, in which peptide elongation reactions consisting of coupling reaction of N-protected amino acid with C-protected peptide (C-protected amino acid in a case of dipeptide formation) followed by N-terminal deprotection reaction are repeated. In the organic chemical synthesis methods, since various analogue peptides are also synthesized due to partial duplication, partial omission, etc. of the peptide elongation reaction, it is required to separate the generated target peptide from these analogue peptides. However, the physicochemical properties of analogue peptides are often very similar to those of the target peptide, making their separation very difficult. It is desired to provide a method for effectively separating a target peptide and analogue peptides in order to obtain a purified target peptide.


Peptides comprising more amino acid residues (usually over 30 residues) are commonly produced by fermentation (for example, Patent Document 2). In this case, analogue peptides, for example, having amino acid replacement(s) may contaminate the target one, and the separation of these peptides having very similar properties is a serious problem.


At present, even in the case where the scale is increased, the purification of peptide is generally performed by a preparative high performance liquid chromatography method, which requires a large cost. It is desired to provide a method for effectively separating/removing analogue peptide(s) from a mixture of a target peptide and analogue peptide(s) thereof, for replacing or supplementing the preparative high performance liquid chromatography.


PRIOR ART DOCUMENT
Patent Document



  • Patent Document 1: JP 2018-52933 A

  • Patent Document 2: JP 2016-190851 A



The disclosures of the prior art documents cited herein are incorporated herein by reference in their entirety.


SUMMARY
Technical Problem

An objective of the present application is to provide a method for purifying a target peptide comprising removing analogue peptide(s) from a mixture comprising the target peptide and the analogue peptide(s), obtained through peptide synthesis. Another objective of the present application is to provide a peptide purification method for replacing or supplementing conventional peptide purification method(s).


Solution to Problem

As a result of studies to solve the above problems, the present inventors have found that when a synthetic peptide product of a target peptide comprising an analogue peptide is mixed with a sulfonic acid compound having a certain structure in the presence of a solvent followed by a solid-liquid separation, the purity (absolute purity, except the sulfonic acid compounds) of the target peptide is improved and a solid product comprising the sulfonate salt of the target peptide having a reduced ratio of the analogue peptide is provided, thereby reaching the present invention.


The invention of the present application is explained in detail below.


[Aspect 1] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 1-1] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation one minute or more after the mixing to collect the solid.


[Aspect 1-2] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid, wherein the solvent comprises water; an alcohol compound; acetonitrile; an ether compound; a ketone compound; an ester compound; a hydrocarbon compound; DMSO; an amide compound; a halogenated hydrocarbon; or a mixture thereof;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 1-3] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation to collect the solid;


(3) washing the solid.


[Aspect 1-4] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound at 0 to 50° C. to provide a solid;


(2) performing a solid-liquid separation at 0 to 50° C. to collect the solid.


[Aspect 1-5] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid, wherein the weight ratio of the solvent to the target peptide (the weight of solvent/the weight of target peptide) is 1 to 100000;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 1-6] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid, wherein the molar ratio of the sulfonic acid compound to the target peptide is 0.1 to 50;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 1-7] A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a sulfonic acid compound in the presence of a solvent;


(2) separating the resulting solid from the liquid phase to collect the resulting solid.


[Aspect 2] The method according to any one of Aspects 1, and 1-1 to 1-7, wherein the sulfonic acid compound is represented by formula (I):




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wherein:


A is C6-14aryl (for example, C6-13aryl, C6-10aryl) which may be substituted, a bicyclic heterocyclic group which may be substituted, C2-3alkenyl, or C2-3alkynyl;


X is

    • (i) a single bond,
    • (ii) C1-6alkylene (for example, C1-5alkylene, C1-4alkylene) which may be substituted,
    • (iii) —CO—(CH2)n— wherein the CO binds to the A, or
    • (iv) C2-4 alkenylene; and


n is an integer selected from 1 to 3.


[Aspect 3] The method according to Aspect 2, wherein


A is C6-14aryl (for example, C6-13aryl, C6-10aryl) which may be substituted;


X is

    • (i) a single bond,
    • (ii) C1-5alkylene (for example, C1-5alkylene, C1-4alkylene, C1-3alkylene) which may be substituted,
    • (iii) —CO—(CH2)n— wherein the CO binds to the A, or
    • (iv) C2-3alkenylene; and


n is an integer selected from 1 to 3.


[Aspect 4] The method according to Aspect 2, wherein


A is a bicyclic heterocyclic group which may be substituted;


X is

    • (i) a single bond,
    • (ii) C1-6 alkylene (for example, C1-5alkylene, C1-4alkylene, C1-3alkylene),
    • (iii) —CO—(CH2)n— wherein the CO binds to the A, or
    • (iv) C2-3alkenylene; and


n is an integer selected from 1 to 3.


[Aspect 5] The method according to Aspect 2, wherein


A is C2-3 alkenyl; and


X is C1-4alkylene.


[Aspect 6] The method according to Aspect 2, wherein


A is C2-3alkynyl; and


X is C1-4alkylene.


[Aspect 7] The method according to any one of Aspects 1 to 6 and 1-1 to 1-7 wherein the target peptide has 5 to 31 amino acid residues.


[Aspect 8] The method according to any one of Aspects 1 to 7 and 1-1 to 1-7 wherein the peptide product comprises an analogue peptide.


[Aspect 9] The method according to any one of Aspects 1 to 8 and 1-1 to 1-7 wherein the molar ratio of the analogue peptide to the target peptide is 0.7 or less in the peptide product.


[Aspect 10] The method according to any one of Aspects 1 to 9 and 1-1 to 1-7 wherein the peptide synthesis is a solid phase peptide synthesis.


[Aspect 11] The method according to any one of Aspects 1 to 10 and 1-1 to 1-7 wherein the target peptide comprises at least one basic amino acid residue and may be free at the N-terminus.


[Aspect 12] The method according to any one of Aspects 2, 3, and 7 to 11, wherein


A is C6-14aryl (for example, C6-13aryl, C6-10aryl) which may be substituted with the same or different 1 to 5 substituents selected from the group consisting of C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and C6-10aryl.


[Aspect 13] The method according to any one of Aspects 2, 3, and 7 to 12, wherein


A is C6-13aryl selected from phenyl, naphthyl, fluorenyl, and indanyl wherein the C6-13aryl may be substituted with the same or different 1 to 5 substituents selected from the group consisting of C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and C6-10aryl.


[Aspect 13-1] The method according to any one of Aspects 2, 3, and 7 to 12, wherein


A is C6-10aryl selected from phenyl, naphthyl, and indanyl wherein the C6-10aryl may be substituted with the same or different 1 to 5 substituents selected from the group consisting of C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and C6-10aryl.


[Aspect 14] The method according to any one of Aspects 2, 4, and 7 to 11, wherein


A is a bicyclic heterocyclic group selected from the group consisting of 2,3,4,5-tetrahydro-1H-1-benzazepinyl, benzoxanyl, indolinyl, isoindolinyl, phthalazinyl, chromanyl, benzofuranyl, benzothiophenyl, pyrimidinyl, benzothiazolyl, quinolyl, isochromanyl, and benzotriazolyl wherein the bicyclic heterocyclic group may be substituted.


[Aspect 15] The method according to any one of Aspects 2, 4, 7 to 11, and 14, wherein


A is a bicyclic heterocyclic group which may be substituted with the same or different 1 to 5 substituents selected from the group consisting of C6-10aryl, C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and oxo.


[Aspect 16] The method according to any one of Aspects 2, 4, 7 to 11, 14 and 15, wherein


A is a bicyclic heterocyclic group selected from




embedded image


wherein the bicyclic heterocyclic group may be substituted with one phenyl group.


[Aspect 17] The method according to any one of Aspects 2 to 4, 7 to 16 and 13-1, wherein X is

    • (i) a single bond,
    • (ii) C1-4alkylene (for example, C1-3alkylene) which may be substituted with the same or different 1 to 3 substituents selected from the group consisting of methyl, benzyl, cyano, and phenyl, or
    • (iii) —CO—CH2— wherein the CO binds to the A.


      [Aspect 17-1] The method according to any one of Aspects 2 to 4, and 7 to 16 and 13-1, wherein X is
    • (i) a single bond,
    • (ii) C1-4alkylene (for example, C1-3alkylene) which may be substituted with one methyl group, one benzyl group, one cyano group, and/or one or two phenyl groups, or
    • (iii) —CO—CH2— wherein the CO binds to the A.


      [Aspect 18] The method according to Aspect 12, wherein X is
    • (i) a single bond,
    • (ii) C1-4alkylene (for example, C1-3alkylene) which may be substituted with the same or different 1 to 3 substituents selected from the group consisting of methyl, benzyl, cyano, and phenyl, or
    • (iii) —CO—(CH2)— wherein the CO binds to the A.


      [Aspect 18-1] The method according to Aspect 12, wherein X is
    • (i) a single bond,
    • (ii) C1-4alkylene (for example, C1-3alkylene) which may be substituted with one methyl group, one benzyl group, one cyano group, and/or one or two phenyl groups, or
    • (iii) —CO—(CH2)— wherein the CO binds to the A.


      [Aspect 19] The method according to any one of Aspects 2 to 4, 7 to 18, 13-1, 17-1, and 18-1, wherein X is a single bond.


      [Aspect 20] The method according to Aspect 16, wherein X is a single bond.


      [Aspect 21] The method according to any one of Aspects 1 to 20, 1-1 to 1-7, 13-1, 17-1, and 18-1, wherein the sulfonic acid compound is selected from:




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[Aspect 22] The method according to any one of Aspects 1 to 21, and 1-1 to 1-7, 13-1, 17-1, and 18-1, further comprising a step of removing the sulfonic acid compound.


[Aspect 23] The method according to any one of Aspects 1 to 22, 1-1 to 1-7, 13-1, 17-1, and 18-1, wherein the method is for increasing the molar ratio of the target peptide to an analogue peptide.


[Aspect 24] A method for producing a target peptide, comprising the method according to any one of Aspects 1 to 23, 1-1 to 1-7, 13-1, 17-1, and 18-1.


[Aspect 25] An agent comprising a target peptide purified by a method comprising the method according to any one of Aspects 1 to 23, 1-1 to 1-7, 13-1, 17-1, and 18-1.


[Aspect 26] An agent comprising a sulfonic acid compound for use in the method according to any one of Aspects 1 to 23, 1-1 to 1-7, 13-1, 17-1, and 18-1.


[Aspect 27] A compound selected from:




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[Aspect 28] An agent comprising a target peptide obtained through purification from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, and the purification comprises steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 28-1] An agent comprising a target peptide obtained through purification from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, and the purification comprises steps:


(1) mixing the peptide product with a sulfonic acid compound in the presence of a solvent;


(2) separating the resulting solid from the liquid phase to collect the resulting solid.


[Aspect 29] An agent comprising a sulfonic acid compound, for use in purification of a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, and the purification comprises steps:


(1) mixing the peptide product with a solvent in the presence of the sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation to collect the solid.


[Aspect 29-1] An agent comprising a sulfonic acid compound, for use in purification of a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, and the purification comprises steps:


(1) mixing the peptide product with the agent comprising the sulfonic acid compound in the presence of a solvent;


(2) separating the resulting solid from the liquid phase to collect the resulting solid.


Effect of the Invention

By the invention of the application, an increased purity of a target peptide is provided from a peptide product obtained through a peptide synthesis, which may comprise an analogue peptide.







DESCRIPTION OF EMBODIMENTS

In one aspect, the present application provides a method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;


(2) performing a solid-liquid separation to collect the solid.


In one aspect, the present application provides a method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, comprising steps:


(1) mixing the peptide product with a sulfonic acid compound in the presence of a solvent;


(2) separating the resulting solid from the liquid phase to collect the resulting solid.


The term “purify/purification” as used herein means that the purity of a substance is increased. The purity may mean both an absolute purity (the amount of a substance of interest relative to the total amount of the mixture comprising the substance of interest) and a relative purity (the amount of a substance of interest relative to the amount of another substance (for example, impurity), in a mixture).


For example, in the present invention, the term “purifying a target peptide” includes a case where the amount of the target peptide in the solid obtained after mixing with the sulfonic acid compound relative to the weight obtained by subtracting the portion corresponding to the sulfonic acid compound from the solid obtained after mixing with the sulfonic acid compound is increased as compared to the amount of the target peptide relative to the total amount of the peptide product before mixing with the sulfonic acid compound (for example, increase in % by weight). In the present invention, the term “purifying a target peptide” includes, for example, a case where the molar ratio of the target peptide to the analogue peptide in the resulting solid after the mixing with sulfonic acid compound is increased as compared with before the mixing.


In the present application, the method for determining the purity of the target peptide is not particularly limited, and the purity may be determined by a commonly used method (for example, HPLC method).


In the present application, the term “peptide synthesis” is not particularly limited as long as it is a method for synthesizing a peptide, and examples thereof include an organic chemical synthesis method (for example, solid phase and liquid phase methods) and fermentation methods, which may be performed by a commonly used method. In peptide synthesis, the synthesis conditions (for example, resin, protecting group, reaction temperature, reaction time, and solvent in the organic chemical synthesis methods; culture conditions, host, vector, nucleic acid sequence in the fermentation methods) are usually set so as to suppress the generation of impurities (for example, analogue peptide(s)). Also in the present application, the peptide synthesis is preferably conducted under synthesis conditions in which generation of analogue peptide(s) is suppressed, on the basis of the knowledge generally known to a person skilled in the art, and the amount of substance of the target peptide is preferably larger than that of the analogue peptide, in the peptide product obtained through the peptide synthesis.


For example, in the peptide product obtained through peptide synthesis, the molar ratio of the analogue peptide to the target peptide (analogue peptide/target peptide) is 0.7 or less (preferably 0.6 or less, more preferably 0.5 or less, still more preferably 0.45 or less, still more preferably 0.4 or less, and more preferably 0.2 or less). A lower molar ratio of the analogue peptide to the target peptide is preferable. Thus the lower limit of the range of the molar ratio is not particularly limited, but examples of the range of the molar ratio include 0.0001 to 0.7, 0.001 to 0.6, and 0.01 to 0.5. For example, an approximate value of the molar ratio (for example, area ratio) calculated based on a value obtained by HPLC may be used as the molar ratio.


In a peptide product obtained through a peptide synthesis, “the amount of substance of the target peptide is larger than that of the analogue peptide” means that the amount of substance of the target peptide is larger than the amount of substance of one kind of analogue peptide, and when a multiple kinds of analogue peptides are comprised, it means that the amount of substance of the target peptide is larger than that of each analogue peptide. In a peptide product obtained through a peptide synthesis, “the amount of substance of the target peptide is larger than that of the analogue peptide” may be indicated by the extremely high probability that in the peptide product the amount of substance of the analogue peptide is lower than that of the target peptide due to the fact that the amount of the target peptide relative to the total amount of the peptide product obtained through a peptide synthesis, the absolute purity, is large (for example, the concentration of the target peptide in the peptide product is 40 wt. % or more, 50 wt. % or more, 60 wt. % or more, 70 wt. % or more, 80 wt. % or more, or 90 wt. % or more).


In the present application, “target peptide” means a peptide which is produced by a peptide synthesis carried out for the purpose of producing the peptide. The structure of the target peptide is not particularly limited so long as: is free at the N-terminus; has at least 1 (for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1) basic amino acid residue; or the N-terminus is free and has at least 1 (for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, and 1) basic amino acid residue. The target peptide preferably has 5 to 31 amino acid residues, more preferably 5 to 25, still more preferably 5 to 20, and even more preferably 5 to 15 amino acid residues. The range of the number of amino acid residues of the target peptide may also be indicated by a combination of a lower limit selected from 5, 6, 7, and 8 and an upper limit selected from 31, 25, 20, 15, and 10.


The amino acid residues which may be comprised in the target peptide may be those derived from naturally occurring amino acids such as L-selenocysteine, 2-aminoisobutyric acid, D-isovaline, L-isovaline, L-norleucine, and L-ornithine in addition to the 20 amino acids listed in the table below (L-form except glycine, which does not have an asymmetric carbon) which usually constitute a protein; and those derived from unnatural amino acids which do not exist in nature or exist in small amounts, if any (therefore, it is usually produced by chemical synthesis, etc.).


Examples of unnatural amino acids include:

  • D forms of the 19 amino acids, excluding glycine, shown in the table below;
  • N-methylated forms of the 20 amino acids shown in the table below (for example, N-methylglycine, L-N-methylphenylalanine) and enantiomers of L-N-methylated forms of the 19 amino acids, excluding N-methylglycine, shown in the table below;
  • both enantiomers of α-methylated forms of the 18 amino acids, excluding glycine and alanine, shown in the table below (for example, α-methyllysine);
  • both enantiomers of α-ethylated forms of the 19 amino acids, excluding alanine, shown in the table below;
  • D-selenocysteine;
  • D-norleucine;
  • D-ornithine;
  • (S)- or (R)-2,3-diaminopropionic acid;
  • (S)- or (R)-2,4-diaminobutyric acid;
  • (S)- or (R)-pyroglutamic acid;
  • (S)- or (R)-α-methyl-orthofluorophenylalanine;
  • (S)- or (R)-α-(4-pentenyl)alanine;
  • (S)- or (R)-α-(7-octenyl)alanine;
  • (S)- or (R)-α-propargylalanine;
  • (S)- or (R)-α-allylalanine;
  • (S)- or (R)-indan-2-ylglycine;
  • (S)- or (R)-pyridin-3-ylmethylglycine, and derivatives thereof in which the pyridine ring may be substituted with phenyl group(s) which may be substituted with alkyl group(s);
  • (S)- or (R)-pyridin-2-ylmethylglycine;
  • (S)- or (R)-pipecolic acid; and
  • (S)- or (R)- and trans- or cis-4-hydroxyproline;
  • RC8 ((R)-α-(7-octenyl)alanine);
  • SC5 ((S)-α-(4-pentenyl)alanine).


















alanine
A



leucine
L



arginine
R



lysine
K



asparagine
N



methionine
M



aspartic acid
D



phenylalanine
F



cysteine
C



proline
P



glutamine
Q



serine
S



glutamic acid
E



threonine
T



glycine
G



tryptophan
W



histidine
H



tyrosine
Y



isoleucine
I



valine
V










In the present application, the term “basic amino acid residue” means an amino acid residue derived from an amino acid having basic moiety (moieties) other than the amino group (for example, D- or L-lysine, D- or L-arginine, D- or L-histidine, D- or L-ornithine, (S)- or (R)-2,3-diaminopropionic acid, and (S)- or (R)-2,4-diaminobutyric acid).


In the present application, the target peptide may be modified at the terminus (termini) with a protecting group and the like commonly used in peptide synthesis. For example, the N-terminus may be modified with Ac group (acetyl group), Fmoc group (9-fluorenylmethyloxycarbonyl group), or Boc group (tert-butoxycarbonyl group). For example, the C-terminus may be modified with an amide group or an ester group.


In the present application, the term “analogue peptide” refers to a peptide that may be produced by peptide synthesis for producing a target peptide and that is not the target peptide. Examples of the analogue peptide include peptide(s) in which:


(1) some of the amino acid residues (for example, continuous or discontinuous 1 to 3 amino acid residues, continuous or discontinuous 1 to 2 amino acid residues, or amino acid residue) in the target peptide are/is duplicated;


(2) some of the amino acid residues (for example, continuous or discontinuous 1 to 3 amino acid residues, continuous or discontinuous 1 to 2 amino acid residues, or 1 amino acid residue) in the target peptide are/is deleted.


(3) some of the amino acid residues (for example, continuous or discontinuous 1 to 3 amino acid residues, continuous or discontinuous 1 to 2 amino acid residues, or amino acid residue) in the target peptide are/is epimerized (for example, epimerization by isomerization at α-position of N-protected amino acid); and/or


(4) some of the amino acid residues (for example, continuous or discontinuous 1 to 3 amino acid residues, continuous or discontinuous 1 to 2 amino acid residues, and 1 amino acid residue) in the target peptide are/is replaced by the same number of amino acid residue(s).


The number of amino acid residues of the analogue peptide is, for example, the same as, 1 to 3 (preferably 1 to 2, more preferably 1) more than, or 1 to 3 (preferably 1 to 2, more preferably 1) less than the number of amino acid residues of the target peptide.


The “analogue peptide” may have basic moiety/moieties (for example, is free at the N-terminus/has at least 1 (for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) basic amino acid residue), or may not have such a basic moiety. As described above, in a peptide product obtained through a peptide synthesis, the amount (amount of substance) of a target peptide is usually larger than that of analogue peptide, and when the structures of the target peptide and the analogue peptide are similar, the solubility properties of the target peptide and the analogue peptide in a solvent may usually be similar. Therefore, in the solid obtained after mixing with the sulphonic acid compound, the ratio of the target peptide, which was originally contained more than the analogue peptide, may be increased although when the “analogue peptide” has a basic moiety, a solid may be formed by forming a salt with the sulphonic acid compound. However, the effect of the present invention is not limited to any specific mechanism.


In the present application, a solvent used in mixing with a sulfonic acid compound is not particularly limited, and may be appropriately selected in consideration of the purification efficiency of the target peptide. Examples of the solvent include:


water;


alcohol compounds (for example, C1-4 alcohol (for example, isopropyl alcohol, methanol, ethanol));


acetonitrile;


ether compounds (for example, C2-6 ether (for example, diethyl ether, MTBE, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, and CPME (cyclopentyl methyl ether)));


ketone compounds (for example, C3-6 ketone (for example, acetone), MEK (methyl ethyl ketone), methyl isopropyl ketone, MIBK (methyl isobutyl ketone));


ester compounds (for example, C3-6 ester (for example ethyl acetate, isopropyl acetate, t-butyl acetate));


hydrocarbon compounds (for example, 06-9 hydrocarbon (for example, toluene));


DMSO;


amide compounds (for example, DMF (N,N-dimethylformamide), DMA (N,N-dimethylacetamide), NMP (N-methylpyrrolidone));


halogenated hydrocarbons (for example, chloroform, dichloromethane, 1,2-dichloroethane); and


mixture thereof (wherein the mixture means any combination thereof). The solvent may further contain other substance(s).


Further examples of the solvent include water, isopropyl alcohol (IPA), methanol (MeOH), 1,4-dioxane, 1,2-dimethoxyethane (DME), tetrahydrofuran (THF), acetone, acetonitrile, ethanol, ethyl acetate, toluene, MTBE, DMSO, diethyl ether, and a mixture thereof (where the mixture means any combination thereof). The solvent may further contain other substance(s).


When a peptide product obtained through a peptide synthesis is mixed with a sulfonic acid compound in the presence of a solvent, the amounts of the solvent and the sulfonic acid compound(s) relative to the peptide product are not particularly limited and may be appropriately selected in consideration of the purification efficiency of the target peptide. The solvent may be used at the weight-by-weight ratio to the target peptide obtained by peptide synthesis in the range of 1 to 100000, preferably 1 to 10000, preferably 1 to 2000, and more preferably 10 to 1000, which may be based on the theoretical value of the weight of the target peptide.


For example, the molar ratio of the sulfonic acid compound to the target peptide may be in the range 0.1 to 50, preferably 0.2 to 40, more preferably 0.3 to 30, and even more preferably 0.5 to 20, which may be based on the theoretical value of the amount of substance of the target peptide.


Further, the sulphonic acid compound may be used at the molar ratio to the target peptide, which may be based on the theoretical value of the amount of substance of the target peptide, in the ranges:


when the sulfonic acid compound has one sulfo group, for example, in the range 0.5 to 20, preferably 0.8 to 10, more preferably 1 to 8, even more preferably 1.5 to 7;


when the sulfonic acid compound has 2 sulfo groups, for example, in the range 0.2 to 10, preferably 0.4 to 5, more preferably 0.5 to 4, even more preferably 0.75 to 3.5;


when the sulfonic acid compound has 3 sulfo groups, for example, in the range 0.15 to 6, preferably 0.3 to 3.3, more preferably 0.4 to 2.6, and even more preferably 0.5 to 2.3.


The theoretical value may mean a value calculated by assuming that the purity of the target peptide in the peptide synthesis product is 100%.


The method for separating the resulting solid from the liquid phase to collect the resulting solid (solid-liquid separation) after mixing with the sulfonic acid compound is not particularly limited, and may be appropriately selected depending on the production scale, etc., and for example, includes filtration, centrifugation, decant, etc.


Since the purity of the target peptide in the solid may be increased through solid-liquid equilibrium, the solid-liquid separation is preferably performed after a predetermined time (for example, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more) has elapsed after the mixing the peptide product with the sulfonic acid compound and the solvent.


In consideration of operation efficiency, it is preferable that the solid-liquid separation is performed, for example, within one day, within two days, within one week, or within two weeks, but the mixture may be stored without the solid-liquid separation, and then the solid-liquid separation may be performed accordingly.


The temperature at the time of mixing with the sulfonic acid compound and/or the solvent and at the time of separating the resulting solid from the liquid phase to collect the resulting solid (solid-liquid separation) is not particularly limited, and may be appropriately selected in consideration of the purification efficiency of the target peptide, for example. The examples include 0 to 50° C., 0 to 40° C., 0 to 30° C., and 10 to 25° C.


The solid obtained by the solid-liquid separation may be optionally washed. The liquid used for washing (the liquid for washing) is not particularly limited, and may be appropriately selected in consideration of the purification efficiency of the target peptide. Examples of the liquid for washing include:


water;


alcohol compounds (for example, C1-4alcohol (for example, isopropyl alcohol, methanol, ethanol));


acetonitrile;


ether compounds (for example, C2-6 ether (for example, diethyl ether, MTBE, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, CPME (cyclopentyl methyl ether)));


ketone compounds (for example, 03-6 ketone (for example, acetone), MEK (methyl ethyl ketone), methyl isopropyl ketone, MIBK (methyl isobutyl ketone));


ester compounds (for example, C3-6ester (for example, ethyl acetate, isopropyl acetate, t-butyl acetate));


hydrocarbon compounds (for example, 06-9hydrocarbon (for example, toluene));


DMSO;


amide compounds (for example, DMF (N,N-dimethylformamide), DMA (N,N-dimethylacetamide), NMP (N-methylpyrrolidone));


halogenated hydrocarbons (for example, chloroform, dichloromethane, 1,2-dichloroethane); and


mixture thereof (wherein the mixture means any combination thereof). The liquid for washing may further contain other substance(s).


Repeating the purification method of the present application on the solid obtained by the purification method of the present application is one embodiment of the purification method of the present application. The purity of the target peptide may be increased by repeating the purification method of the present application.


The purification method of the present application may be performed in combination with other purification method(s) (for example, preparative HPLC, recrystallization, dispersion and wash). For example, the solid obtained by the method of the present application may be subjected to another purification method, or a peptide product previously purified by another purification method may be subjected to the purification method of the present invention.


It is preferable that the sulfonic acid compound is removed from the solid obtained by the purification method of the present application in a further purification step. The method for removing the sulfonic acid compound is not particularly limited, and may be carried out by a usual method for removing the sulfonic acid compound (for example, an acid or alkali treatment, and a removal method using an ion exchange resin).


In the present application, the term “sulfonic acid compound” means an organic compound having sulfo group(s) (—SO3H). In the present application, the sulfonic acid compound is preferably those where a sulfo group is attached to a moiety having one or more unsaturated bonds through a single bond or a short linker (for example, C1-12, C1-6, C1-4, etc.).


Preferred examples of the sulfonic acid compound include a sulfonic acid compound of formula (I):




embedded image


wherein:


A is C6-14aryl (for example, C6-13aryl, C6-10aryl) which may be substituted, a bicyclic heterocyclic group which may be substituted, C2-3alkenyl, or C2-3alkynyl;


X is

    • (i) a single bond,
    • (ii) C1-6alkylene (for example, C1-5 alkylene, C1-4alkylene) which may be substituted,
    • (iii) —CO—(CH2)n-wherein the CO binds to the A, or (iv) C2-4alkenylene; and n is an integer selected from 1 to 3.


The term “C6-14aryl” as used herein alone or as part of another group refers to a 6 to 14 membered monocyclic, bicyclic or tricyclic aromatic hydrocarbon ring group and a ring group wherein one cycloalkane is fused to said aromatic hydrocarbon ring. Examples of C6-14aryl include phenyl, naphthyl, indanyl, tetrahydronaphthyl, and fluorenyl.


The term “C6-10 aryl” as used herein alone or as part of another group refers to a 6 to 10 membered monocyclic or bicyclic aromatic hydrocarbon ring group, and a ring group wherein one cycloalkane is fused to said aromatic hydrocarbon ring group. Examples of C6-10 aryl include phenyl, naphthyl, indanyl, and tetrahydronaphthyl.


Examples of “C6-14aryl (or C6-13aryl, C6-10aryl) which may be substituted” include C6-14 aryl (or C6-13 aryl, C6-10 aryl) which may be substituted with the same or different 1 to 5 (for example, 1 to 3, 1 to 2, and 1) substituents selected from the group consisting of C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and C6-10aryl.


The term “a bicyclic heterocyclic group” as used herein means a 8 to 11 membered bicyclic group wherein a monocyclic aromatic ring having 1 to 4 heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom (for example, pyridine, furan, thiophene, imidazole, pyrimidine, oxazole, thiazole, thiazine, triazole) or a monocyclic non-aromatic ring (tetrahydropyran, azepane, piperidine, etc.) is fused to a monocyclic aromatic ring (benzene, pyridine, etc.). The bicyclic heterocyclic group may be attached to another moiety at any heteroatom or carbon atom which results in a stable structure. The bicyclic heterocyclic group may comprise one or more (for example, 1 or 2) oxo groups. Examples of the bicyclic heterocyclic group include:




embedded image


2,3,4,5-tetrahydro-1H-1-benzazepinyl, benzoxanyl, indolinyl, isoindolinyl, phthalazinyl, chromanyl, benzofuranyl, benzothiophenyl, pyrimidinyl, benzothiazolyl, quinolyl, isochromanyl, and benzotriazolyl.


Examples of “a bicyclic heterocyclic group which may be substituted” as used herein include a bicyclic heterocyclic group which may be substituted with the same or different 1 to 5 (for example, 1 to 3, 1 to 2, and 1) substituents selected from the group consisting of C6-10aryl, C1-4alkyl, —CO—C6-10aryl, —OH, —O—C1-3alkyl, —NO2, —CO2H, —CO2—C1-4alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4alkyl, —CF3, and oxo.


As used herein, “02-3alkenyl” means a straight or branched unsaturated hydrocarbon group having 2 to 3 carbon atoms and comprising 1 or 2 double bonds, examples of which include vinyl, allyl, 1-propenyl, isopropenyl, allenyl.


As used herein, “02-3alkynyl” means a straight or branched unsaturated hydrocarbon group having 2 to 3 carbon atoms and comprising one triple bond, examples of which include ethynyl, propynyl (1-propynyl, 2-propynyl).


As used herein, “C1-4alkylene” means a divalent group derived from a straight chain C1-4alkyl. Examples of C1-4alkylene include methylene, ethylene, trimethylene, and tetramethylene.


As used herein, “C1-6alkylene” means a divalent group derived from a straight chain C1-6alkyl. Examples of C1-6alkylene include methylene, ethylene, trimethylene, tetramethylene, and pentamethylene.


As used herein, “C1-3alkylene” means a divalent group derived from a straight chain C1-3alkyl. Examples of C1-3alkylene include methylene, ethylene, and trimethylene.


As used herein, examples of “C1-6alkylene (or C1-5alkylene, C1-4alkylene, C1-3 alkylene) which may be substituted” include C1-6alkylene (or C1-5alkylene, C1-4alkylene, C1-3alkylene) which may be substituted with the same or different 1 to 3 substituents selected from the group consisting of methyl, benzyl, cyano, and phenyl (for example, C1-6alkylene (or C1-5alkylene, C1-4alkylene, C1-3alkylene) which may be substituted with one methyl group, one benzyl group, or one phenyl group)).


In the “—CO—(CH2)n—” as used herein, n is an integer of 1 to 3 (preferably 1 to 2), and preferred examples of —CO—(CH2)n— include —CO—CH2—.


As used herein, “C2-4alkenylene” means a divalent group derived from a straight chain C2-4alkenyl. As used herein, “C2-3alkenylene” means a divalent group derived from a straight chain C2-3alkenyl. Examples of C2-4alkenylene and C2-3alkenylene include vinylene(—CH═CH—), and propenylene(—CH2CH═CH—).


The term “C1-4alkyl” as used herein alone or as part of another group means a straight or branched saturated hydrocarbon group having 1 to 4 carbon atoms. Examples of C1-4alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.


The term “C1-3alkyl” as used herein alone or as part of another group means a straight or branched saturated hydrocarbon group having 1 to 3 carbon atoms. Examples of C1-3alkyl include methyl, ethyl, propyl, and isopropyl.


As used herein, examples of a “—CO—C6-10aryl” include —CO-phenyl.


As used herein, examples of the halogen include fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).


More preferred examples of the sulfonic acid compound include those listed in [Aspect 21] above.


One aspect of the present invention provides a method for producing a target peptide comprising the purification method of the present application.


One aspect of the present invention provides an agent comprising a target peptide purified by a method comprising the purification method of the present application. The agent may be prepared by a usual method.


One aspect of the present invention provides an agent comprising a sulfonic acid compound for use in the purification method of the present application. The agent may be prepared by a usual method.


EXAMPLE

The present invention is explained in further detail with reference to Test examples. However, the scope of the invention is not limited to these Examples.


Purification of Target Peptide
Outline of Examinations:

When a target peptide is synthesized by solid phase method, it is expected that analogue peptides are contained in the resulting crude peptide as impurities, but it is difficult to identify what kind of analogue peptides are actually contained. Therefore, the target peptide and the pseudo-analogue peptide having a known sequence as the analogue peptide are synthesized by solid phase method, and the resulting crude target peptide, the resulting crude pseudo-analogue peptide, and each acid compound are dissolved in a solvent, which provides a uniform sample to obtain a value before the treatment by HPLC, followed by lyophilization to remove the solvent. A solvent is added to the residue to provide a solid-liquid mixture, and then the supernatant is removed. The ratio of the amount of substance (mol) of the target peptide in the solid to the sum of the amount of substance (mol) of the target peptide and the amount of substance (mol) of the pseudo-analogue peptide in the solid is determined. Whether the target peptide is purified in the solid is evaluated by the change of the ratios.


1-1: Synthesis of Peptide

The target peptides and the analogue peptides (pseudo) shown in the tables below were respectively synthesized by the solid phase method described below.










TABLE 1-1





Target peptide
Analogue peptide







 (1) Ac—YFYPEL—NH2 (SEQ ID NO: 1)
 (1) Ac—YYPEL—NH2 (SEQ ID NO: 16)





(27) Ac—YHYPEL—NH2 (SEQ ID NO: 62)
 (2) H—YYPEL—NH2 (SEQ ID NO: 17)





(28) Ac—Y(MeK)YPEL—NH2 (SEQ ID NO: 63)
 (3) H—VIRALRALVALRALR—OH (SEQ ID NO: 18)





 (2) H—YFYPEL—NH2 (SEQ ID NO: 2)
 (4) H—ARLDVASEFRKKNKALSR—NH2



(SEQ ID NO: 19) 





 (3) H—VIRALRRALVALRALR—OH (SEQ ID NO: 3)
 (5) H—HAEGTFTSDVSSYLEGQAAKEFAWLVKGRG—OH



(SEQ ID NO: 20) 





 (4) H—ARLDVASEFRKKWNKALSR—NH2
 (6) H—AGCKNFFWKTFTSC—OH (SEQ ID NO: 21)


(SEQ ID NO: 4)






 (5) H—HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG—OH
(35) H—AGcKNFFWKTFTSC—OH


(SEQ ID NO: 5)






 (6) H—AGCKNFFWKTFTS—OH (SEQ ID NO: 6)
 (7) H—AQKLRAD—OH (SEQ ID NO: 22)





 (7) H—AQKLRASD—OH (SEQ ID NO: 7)
 (8) H—AKLRAD—OH (SEQ ID NO: 23)





 (8) H—YERAKSNM—OH (SEQ ID NO: 8)
(19) H—QKLRASD—OH (SEQ ID NO: 24)






(20) H—AQKLRSD—OH (SEQ ID NO: 25)






(21) H—AQKLRAS—OH (SEQ ID NO: 26)






(24) H—AQKRASD—OH (SEQ ID NO: 27)






(27) H—AQKLASD—OH (SEQ ID NO: 28)






 (9) H—YERAKNM—OH (SEQ ID NO: 29)






(13) H—YERKSNM—OH (SEQ ID NO: 30)






(14) H—YERASNM—OH (SEQ ID NO: 31)






(16) H—YERAKSM—OH (SEQ ID NO: 32)






(22) H—ERAKSNM—OH (SEQ ID NO: 33)






(23) H—YERAKSN—OH (SEQ ID NO: 34)






(28) H—YRAKSNM—OH (SEQ ID NO: 35)

















TABLE 1-2





Target peptide
Analogue peptide







 (9) H—FRVDEEFQSPFASQSRGYFLFRPRN—NH2
(10) H—FRVDEEFQSPFASQSRGYFLFRRN—NH2


(SEQ ID NO: 9)
(SEQ ID NO: 36)





(11) H—GGGRG—NH2 (SEQ ID NO: 10) 
(11) H—GGGG—NH2 (SEQ ID NO: 37)





(12) Ac—ALRAL—NH2 (SEQ ID NO: 11) 
(12) Ac—ALAL—NH2 (SEQ ID NO: 38)





(13) Ac—ALRAL—OH (SEQ ID NO: 12) 
(15) Ac—ALAL—OH (SEQ ID NO: 39)





(14) H—ALRALRALR—OH (SEQ ID NO: 13) 
(17) H—ALALRALR—OH (SEQ ID NO: 40)





(15) Ac—GGRGG—NH2 (SEQ ID NO: 14) 
(18) Ac—GGGG—NH2 (SEQ ID NO: 41)





(16) H—HSDGTFTSELSRLREGARLQRLLQGLV—NH2
(30) H—HSDGTFTSELSRLEGARLQRLLQGLV—NH2


(SEQ ID NO: 15)
(SEQ ID NO: 42)





(17) Pyr—HWSYlLRP—NH2
(26) Pyr—HWSYLLRP—NH2 (SEQ ID NO: 43)





(18) H—HVTTV—NH2 (SEQ ID NO: 44)
(31) Pyr—HWSYlLLRP—NH2





(19) H—YERAKSNM—NH2 (SEQ ID NO: 46)
(32) H—VTTV—NH2 (SEQ ID NO: 45)





(20) H—VKRESYSGVT—NH2 (SEQ ID NO: 48)
(33) H—hVTTV—NH2





(21) H—RAVLP—OH (SEQ ID NO: 49)
(34) H—YERAKSNL—NH2 (SEQ ID NO: 47)





(22) H—(RC8)RRR((Me)F)R(SC5)—NH2
(36) H—VKRESYSGV—NH2 (SEQ ID NO: 55)


(SEQ ID NO: 50)






(23) Ac—HYFYPEL—NH2 (SEQ ID NO: 51)
(37) H—RALP—OH (SEQ ID NO: 56) 





(24) H—WPVTLNAQTID—NH2 (SEQ ID NO: 52)
(38) H—(RC8)RRRR(SC5)—NH2



(SEQ ID NO: 57)





(25) H—T((Me)G)RK(Dap)H—OH 
(39) Ac—HYYPEL—NH2 (SEQ ID NO: 58) 


(SEQ ID NO: 53)






(26) Pyr—GPWLEEEEEAYGWMDF—NH2
(40) H—PVTLNAQTID—NH2 (SEQ ID NO: 59)


(SEQ ID NO: 54)







(41) H—((Me)G)RK(Dap)H—OH 



(SEQ ID NO: 60)






(42) Pyr—GPWLEEEEEYGWMDF—NH2



(SEQ ID NO: 61)









The apparatus used in Test examples are as follows.


Peptide synthesizer: “KMS-3” (KOKUSAN CHEMICAL Co., Ltd.).


Mass spectrometer: “6224 TOF LC/MS” and “1260 Infinity series” (Agilent). Ionization condition: ESI (capillary voltage: 3500 V, Fragmentor voltage: 100 V).


HPLC analyzer: “SPD-M20A”, “LC-20AD”, “SIL-20AC”, “DGU-20A”, “CTO-20AC”, “CBM-20A” (Shimadzu); or “SPD-M20A”, “LC-2010C” (Shimadzu); or “1200 series” (Agilent).


(1) Synthesis of Protected Peptide Resin

In syntheses of protected peptide resins, the α-amino group of each constituent amino acid was all protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group).


Regarding active side chains, the β-carboxyl group of aspartic acid and the γ-carboxyl group of glutamic acid was protected with a tert-butyl group (tBu group); the guanidino group of arginine was protected with a 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-ylsulfonyl group (Pbf group); the hydroxyl group of serine and threonine was protected with a tert-butyl group (tBu group); the thiol group of cysteine and the imidazole group of histidine was protected with a trityl group (Trt group); the phenolic hydroxyl group of tyrosine was protected with a tert-butyl group (tBu group); the carboxamide group of asparagine and glutamine was protected with a trityl group (Trt group); the ε-amino group of lysine, the β-amino group of Dap and the indole group of tryptophan were protected with a tert-butyloxycarbonyl group (Boc group).


Regarding resins, (2-chloro)trityl chloride resin (WATANABE CHEMICAL INDUSTRIES, LTD., approx. 1.6 mmol/g) was used for synthesizing a peptide sequence having a C-terminal carboxylic acid; and Fmoc-NH-SAL resin (WATANABE CHEMICAL INDUSTRIES, LTD., approx. 0.43 mmol/g) was used for synthesizing a peptide sequence having a C-terminal carboxamide. In the case of synthesizing the peptide sequence having the C-terminal carboxylic acid, in order to introduce the C-terminal amino acid, a solution of Fmoc amino acid (2.5 equivalents relative to the resin) in DMF, and N,N-diisopropylethylamine (2.5 equivalents relative to the resin) were added to the resin, followed by shaking and stirring for 2 hours or longer. In the case of synthesizing the peptide sequence having the C-terminal carboxamide, to remove the Fmoc group from the resin, a solution of 20% piperidine in DMF was added to the resin, followed by shaking and stirring for not less than 10 minutes and washing the resin 3 to 5 times with DMF, and then to introduce the C-terminal amino acid, a solution of Fmoc amino acid (2.5 equivalents relative to the resin) in DMF, a solution of 1-hydroxybenzotriazole (2.5 equivalents relative to the resin) in DMF, and DIC (2.5 equivalents relative to the resin) were added thereto, followed by shaking and stirring for not less than 40 minutes.


After the C-terminal amino acid was introduced to the resin, the excess amounts of reagents were removed by washing the resin 3 to 5 times with DMF, and then the Fmoc group, the protecting group of the terminal amino group, was removed by shaking and stirring with 20% piperidine at room temperature for 10 minutes or more. The piperidine was removed by washing the resin with DMF 3 to 5 times and then a condensation with the carboxyl group of the Fmoc-protected derivative of the next amino acid in the amino acid sequence of the peptide to be produced was performed. The condensation was performed by adding a solution of the Fmoc-amino acid (2.5 equivalents relative to the resin) in DMF, a solution of 1-hydroxybenztriazole (2.5 equivalents relative to the resin) in DMF, and DIC (2.5 equivalents relative to the resin). Removals of Fmoc group and condensation reactions with Fmoc amino acid were repeated in a similar way until the amino acid sequence of the peptide to be produced was formed to synthesize the protected peptide resin with amino acid sequence of the peptide to be produced.


In a case of synthesizing a peptide having a free amino group at the N-terminus, after the condensation of the Fmoc amino acid of the N-terminus, the Fmoc group was removed by shaking and stirring in a solution of 20% piperidine in DMF at room temperature for 10 minutes or more, and then the piperidine was removed by washing the resin 3 to 5 times with DMF to complete the synthesis of the protected peptide resin. In a case of synthesizing a peptide sequence whose N-terminus was protected by an Ac group, after the N-terminus was converted to a free amino group as described above, a condensation reaction was performed by adding acetic acid (5 equivalents), a solution of N-hydroxysuccinic acid (4.95 equivalents) in DMF, and DIC (5 equivalents) to introduce an Ac group to the N-terminus to synthesize the protected peptide resin having the amino acid sequence of the peptide to be produced. When the N-terminal amino acid residue was (S)- or (R)-pyroglutamic acid, a condensation reaction was carried out by shaking and stirring with (S)- or (R)-Fmoc-pyroglutamic acid chloride (5 equivalents) corresponding to the N-terminal Fmoc amino acid and pyridine (10 equivalents) for at least 40 minutes, and then the Fmoc group was removed by shaking and stirring with a solution of 20% piperidine in DMF for at least 10 minutes at room temperature, and then the piperidine was removed by washing the resin 3 to 5 times with DMF to complete the synthesis of the protected peptide resin.


(2) Synthesis of Crude Peptide Product

In a synthesis of the crude peptide product, the above protected peptide resin having the amino acid sequence of the peptide to be produced was subjected to deprotection and excision from the resin by treating with an acid. When the peptide to be produced had a peptide sequence comprising an amino acid(s) having a sulfur atom, such as cysteine or methionine, the deprotection and excision from the resin were performed by shaking and stirring with TFA/water/ethanedithiol/TIS (94/2.5/2.5/1) for 30 minutes or more at a temperature from cooling with ice to room temperature. When the peptide to be produced had a peptide sequence not comprising an amino acid having a sulfur atom, such as cysteine or methionine, the deprotection and excision from the resin were performed by shaking and stirring with TFA/water/TIS (95/2.5/2.5) for 30 minutes or more at a temperature from cooling with ice to room temperature. After the completion of the reaction, the resin was removed by filtration and washed 3 to 5 times with the solution for deprotection, and the filtrate combined with the washings was concentrated by an evaporator. MTBE was added to the residue, and the resulting precipitates were subjected to a solid-liquid separation by filtration or centrifugation, followed by multiple washes with MTBE to provide the crude peptide product of interest.


Method for Producing (8) H-YERAKSNM-OH (Method A)

(1) To a column made of PP, attached to the peptide synthesizer “KMS-3” was added 187.5 mg (0.30 mmol) of (2-chloro) trityl chloride resin (WATANABE CHEMICAL INDUSTRIES, LTD., approx. 1.6 mmol/g). Five milliliters of DMF was added thereto, and the mixture was shaken and stirred overnight. After removal of the DMF by suction filtration, to the residue were added 2 mL of a solution of Fmoc-Met-OH in DMF (375 mM) (0.75 mmol; 2.5 equivalents), 128 μL of N,N-diisopropylethylamine (0.75 mmol; 2.5 equivalents), and 1 mL of DMF. The resulting mixture was shaken and stirred for 6 hours. After removal of the liquid by suction filtration, the residue was washed with 3 mL of DMF (5 times). To the residue was added 3 mL of a solution of 20% piperidine in DMF, and the mixture was shaken and stirred for 35 minutes. The reaction mixture was filtered by suction, and the residue was washed 5 times with 3 mL of DMF.


(2) To the residue were added 2 mL of a solution of Fmoc-Asn(Trt)-OH in DMF (375 mM) (0.75 mmol; 2.5 equivalents), 1 mL of a solution of 1-hydroxybenzotriazole in DMF (750 mM) (0.75 mmol; 2.5 equivalents), and 116 μL of DIC (0.75 mmol; 2.5 equivalents), and then the mixture was shaken and stirred overnight. After removal of the liquid by suction filtration, the residue was washed 5 times with 3 mL of DMF. To the residue was added 3 mL of a solution of 20% piperidine in DMF, and the mixture was shaken and stirred for 35 minutes. The mixture was filtered by suction, and the residue was washed 5 times with 3 mL of DMF.


(3) With Reference to the step (2) above, the subsequent amino acids were sequentially combined. The order and reaction time of the subsequent amino acids were Fmoc-Ser(tBu)-OH 20 h, Fmoc-Lys(Boc)-OH 45 min, Fmoc-Ala-OH 3 h, Fmoc-Arg(Pbf)-OH 16 h, Fmoc-Glu(tBu)-OH 2.5 h, and Fmoc-Tyr(tBu)-OH 15 h.


(4) To the resulting protected peptide resin was added 3 mL of a solution for deprotection (TFA/water/ethanedithiol/TIS=94/2.5/2.5/1) under ice cooling, and the ice bath was removed and the mixture was shaken and stirred at ambient temperature for 3 hours. The resin was filtered off and washed three times with the solution for deprotection. The filtrate combined with the washings was concentrated by an evaporator. To the residue was added 5 mL of MTBE, and the mixture was stirred overnight under ice cooling. The entire amount of the mixture was transferred to a centrifuge tube, and centrifuged at 3000 rpm for 1 minute, and then the supernatant was removed. To the precipitate was added 2 mL of MTBE, and the mixture was shaken and stirred for 3 minutes, centrifuged at 3000 rpm for 1 minute, and then the supernatant was removed, which was repeated 3 times. The precipitate was dried in vacuo to give 179.5 mg of a crude peptide product of the desired sequence (H-YERAKSNM-OH). The resulting crude peptide product was subjected to confirmation by HPLC and mass spectrometry with an ESI-TOF/MS instrument.


ESI-MS: Calcd. for M: [M+H]+=998.47.

Found M: (m/z): [M+H]+=998.4739.


HPLC condition (Condition A)


Column: YMC-triart C18 3 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.7 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm for target peptide (8) and analogue peptide (22) in [Table 14-2], or 274 nm


Gradient: 2% B (0 min)→2% B (0.2 min)→6.4% B (0.5 min)→6.4% B (11 min)→95% B (15.5 min)→95% B (20.5 min)→2% B (20.6 min)→2% B (29 min)


Retention time: around 13.3 min


Method for producing (12) Ac-ALRAL-NH2 (Method B)


(1) To a column made of PP, attached to the peptide synthesizer “KMS-3” was added 348.8 mg (0.15 mmol) of Fmoc-NH-SAL resin (WATANABE CHEMICAL INDUSTRIES, LTD., approx. 0.43 mmol/g). Five milliliters of DMF was added thereto, and the mixture was shaken and stirred for 1 minute, and allowed to stand overnight. After removal of the DMF by suction filtration, to the residue was added 3 mL of a solution of 20% piperidine in DMF, and the mixture was shaken and stirred for 35 minutes. The liquid was filtered off by suction, and the residue was washed 5 times with 3 mL of DMF.


(2) To the residue were added 2 mL of a solution of Fmoc-Leu-OH in DMF (187.5 mM)(0.375 mmol; 2.5 equivalents), 1 mL of a solution of 1-hydroxybenzotriazole in DMF (375 mM) (0.375 mmol; 2.5 equivalents), and 58 μL of DIC (0.375 mmol; 2.5 equivalents), and then the mixture was shaken and stirred for 3 hours. After removal of the liquid by suction filtration, the residue was washed 5 times with 3 mL of DMF. To the residue was added 3 mL of a solution of 20% piperidine in DMF, and the mixture was shaken and stirred for 35 minutes. The liquid was filtered off by suction, and the residue was washed 5 times with 3 mL of DMF.


(3) With Reference to the step (2) above, the subsequent amino acids were sequentially coupled. The order and reaction time of the subsequent amino acids were Fmoc-Ala-OH 2.5 h, Fmoc-Arg(Pbf)-OH 15.5 h, Fmoc-Leu-OH 2.5 h, and Fmoc-Ala-OH 2.5 h. After synthesis of the protected peptide resin whose N-terminus is free amino group, 3 mL of a solution of N-hydroxysuccinic acid in DMF (0.744 mmol; 4.96 equivalents) (248 mM), 43 μL of acetic acid (0.75 mmol; 5 equivalents) and 116 μL of DIC (0.75 mmol; 5 equivalents) were added, and the mixture was shaken and stirred for 16 hours. The liquid was filtered off by suction, and the residue was washed 5 times with 3 mL of DMF and then washed 3 times with 3 mL of MTBE.


(4) To the resulting protected peptide resin was added 3 mL of a solution for deprotection (TFA/water/TIS=95/2.5/2.5) under ice cooling, and the ice bath was removed and the mixture was shaken and stirred at ambient temperature for 4 hours. The resin was filtered off and washed three times with the solution for deprotection. The filtrate combined with the washings was concentrated by an evaporator. To the residue was added 5 mL of MTBE, and the mixture was stirred overnight under ice cooling. The entire amount of the mixture was transferred to a centrifuge tube, and centrifuged at 3000 rpm for 1 minute, and then the supernatant was removed. To the precipitate was added 2 mL of MTBE, and the mixture was shaken and stirred for 3 minutes, centrifuged at 3000 rpm for 1 minute, and then the supernatant was removed, which was repeated 3 times. The precipitate was dried in vacuo to give 90.6 mg of a crude peptide product of the desired sequence (Ac-ALRAL-NH2). The resulting crude peptide product was subjected to confirmation by HPLC and mass spectrometry with an ESI-TOF/MS instrument.


ESI-MS: Calcd. for M: [M+H]+=584.39.

Found M: (m/z): [M+H]+=584.3879.


HPLC Condition (Condition B)
Column: YMC-PackProCl8 3 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 5% B (0 min)→5% B (5 min)→95% B (17 min)→5% B (17.1 min)→5% B (20.1 min)→5% B (24 min)


Retention time: around 10.6 min


Method for Producing (1) Ac-YFYPEL-NH2

According to the procedures of Method B, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, and Fmoc-Tyr(tBu)-OH were sequentially coupled to 233 mg of Fmoc-NH-SAL resin (0.1 mmol), followed by the Ac protection of the free amino group at the N-terminus to provide a protected peptide resin. Excision from the resin was performed using a solution for deprotection (TFA/Water/TIS=95/2.5/2.5), followed by a similar way to Method B to provide 90.6 mg of a crude peptide product of the desired sequence (Ac-YFYPEL-NH2). The resulting crude peptide product was subjected to confirmation by HPLC and mass spectrometry with an ESI-TOF/MS instrument.


ESI-MS: Calcd. for M: [M+H]+=872.42.

Found M: (m/z): [M+H]+=872.4203.


HPLC condition (Condition C)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 20% B (0 min)→20% B (12 min)→95% B (15 min)→95% B (19 min)→20% B (20 min)→20% B (28 min)


Retention time: around 10.2 min


Using the methods A or B described above, the target peptides in the tables below were synthesized by solid phase method, respectively. The HPLC analyses were performed with the following conditions in addition to the above HPLC conditions (Conditions A-C).


HPLC condition (Condition D)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 275 nm


Gradient: 15% B (0 min)→25% B (1 min)→25% B (12 min)→95% B (14.5 min)→95% B (17 min)→15% B (17.5 min)→15% B (28 min)


HPLC condition (Condition E)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.7 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 20% B (0 min)→35% B (17 min)→95% B (18 min)→95% B (20 min)→20% B (20.5 min)→20% B (28 min)


HPLC condition (Condition F)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 279 nm


Gradient: 36% B (0 min)→36% B (12.5 min)→95% B (13 min)→95% B (14.5 min)→36% B (15 min)→36% B (27 min)


HPLC condition (Condition G)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 281 nm


Gradient: 22% B (0 min)→25% B (2 min)→25% B (14 min)→95% B (17 min)→95% B (18.5 min)→22% B (19 min)→22% B (30 min)


HPLC condition (Condition H)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.65 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 2% B (0 min)→2% B (3 min)→5% B (12 min)→5% B (14 min)→8% B (16 min)→95% B (19 min)→95% B (19.9 min)→2% B (20 min)→2% B (28 min)


HPLC condition (Condition I)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 23% B (0 min)→25% B (2 min)→25% B (10 min)→35% B (15 min)→95% B (17 min)→95% B (19 min)→23% B (20 min)→23% B (28 min)


HPLC condition (Condition J)


Column: Waters Atlantis T3 3 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 1.0 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 20% B (0 min)→20% B (12 min)→95% B (17 min)→95% B (19 min)→20% B (19.1 min)→20% B (28 min)


HPLC condition (Condition K)


Column: Waters Xbridge Phenyl 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 1.0 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 5% B (0 min)→5% B (3 min)→95% B (20 min)→5% B (20.1 min)→5% B (26 min)


HPLC condition (Condition L)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 28% B (0 min)→38% B (12 min)→95% B (15 min)→95% B (19 min)→28% B (20 min)→28% B (28 min)


HPLC condition (Condition M)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.65 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 4% B (0 min)→4% B (3 min)→7% B (8 min)→95% B (15 min)→95% B (15.9 min)→4% B (16 min)→4% B (28 min)


HPLC condition (Condition N)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 1.0 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 0% B (0 min)→3% B (8 min)→95% B (25 min)→95% B (28 min)→0% B (28.5 min)→0% B (35 min)


HPLC condition (Condition 0)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 25% B (0 min)→25% B (14 min)→95% B (17 min)→95% B (21 min)→0% B (22 min)→0% B (32 min)


HPLC condition (Condition P)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 20% B (0 min)→25% B (12 min)→25% B (14 min)→95% B (17 min)→95% B (18.5 min)→20% B (19 min)→20% B (28 min)


HPLC condition (Condition Q)


Column: Waters Xbridge C18 3.5 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 3% B (0 min)→5% B (5 min)→5% B (16 min)→95% B (17 min)→95% B (19 min)→3% B (19.1 min)→3% B (28 min)


HPLC condition (Condition R)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.6 mL/min, Temperature: 30° C.


Detection wavelength: 275 nm


Gradient: 11% B (0 min)→11% B (10 min)→20% B (12 min)→95% B (15 min)→95% B (17.5 min)→11% B (18 min)→11% B (28 min)


HPLC condition (Condition S)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.6 mL/min, Temperature: 30° C.


Detection wavelength: 281 nm


Gradient: 29% B (0 min)→29% B (10 min)→40% B (12 min)→95% B (15 min)→95% B (17.5 min)→29% B (18 min)→29% B (28 min)


HPLC condition (Condition T)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.7 mL/min, Temperature: 30° C.


Detection wavelength: 276 nm


Gradient: 10.5% B (0 min)→10.5% B (10 min)→95% B (14 min)→95% B (17 min)→10.5% B (17.1 min)→10.5% B (28 min)


HPLC condition (Condition U)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.65 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 15% B (0 min)→15% B (10 min)→30% B (12 min)→95% B (15 min)→95% B (17.5 min)→15% B (17.6 min)→15% B (29 min)


HPLC condition (Condition V)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 1 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 17% B (0 min)→17% B (12 min)→95% B (17 min)→95% B (19 min)→17% B (19.1 min)→17% B (26 min)


HPLC condition (Condition W)


Column: YMC Triart C18 3 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 5% B (0 min)→95% B (17 min)→5% B (17.1 min)→5% B (23.5 min)


HPLC condition (Condition X)


Column: YMC-PackproC18RS 3 μm, 50×3 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.5 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 5% B (0 min)→95% B (12 min)→95% B (18 min)→5% B (18.5 min)→5% B (25 min)


HPLC condition (Condition Y)


Column: YMC Triart C18 3 μm, 50×3.0 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.65 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 2% B (0 min)→2% B (6 min)→5% B (8 min)→8% B (16 min)→95% B (19 min)→95% B (22 min)→2% B (22.1 min)→2% B (28 min)


HPLC condition (Condition Z)


Column: Waters Xbridge C18 3.5 μm, 150×4.6 mm

Mobile phase: A=0.1% aqueous solution of TFA; B=0.1% TFA in acetonitrile


Flow rate: 0.7 mL/min, Temperature: 30° C.


Detection wavelength: 205 nm


Gradient: 10% B (0 min)→10% B (0.2 min)→34% B (0.5 min)→34% B (14 min)→95% B (16 min)→95% B (19 min)→10% B (19.1 min)→10% B (29 min)













TABLE 2-1








Resin 


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Target peptide
of resin
method
Yield
Found M
for M
condition
RT





 (1) Ac—YFYPEL—NH2
Resin B
Method B
 78 mg
[M + H]+
[M + H]+
Condition
around



233 mg


872.4203
872.42
C
10.2 min





 (2) H—YFYPEL—NH2
Resin B
Method B
 83 mg
[M + H]+
[M + H]+
Condition
around



233 mg


830.4064
830.41
D
11.8 min





 (3) H—VIRALRRALVALR
Resin A
Method A
366 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


ALR—OH
187.5 mg


924.1176
924.11
E
14.5 min





 (4) H—ARLDVASEFRKKW
Resin B
Method B
267 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


NKALSR—NH2
233 mg


820.8034
820.80
C
9.2 min





 (5) H—HAEGTFTSDVSSY
Resin A
Method A
440 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


LEGQAAKEFIAWLVKGRG—
125 mg


1118.8891
1118.90
F
10.4 min


OH












 (6) H—AGCKNFFWKTFTS
Resin A
Method A
206 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


C—OH
125 mg


820.3680
820.37
G
16.6 min






[M + 2H]2+
[M + 2H]2+
Condition
around






820.3734
820.37
S
8.4 min





 (7) H—AQKLRASD—OH
Resin A
Method A
183 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


888.4879
888.49
H
17.2 min








Condition
around








M
9.2 min





 (8) H—YERAKSNM—OH
Resin A
Method A
179 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


888.4879
888.49
A
13.4 min





 (9) H—FRVDEEFQSPFAS
Resin B
Method B
313 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


QSRGYFLFRPRN—NH2
233 mg


1027.1906
1027.18
I
9.8 min





(12) Ac—ALRAL—NH2
Resin B
Method B
105 mg
[M + H]+
[M + H]+
Condition
around



348.8 mg


584.3876
584.39
B
10.4 min





(13) Ac—ALRAL—OH
Resin A
Method A
124 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


585.3720
585.37
B
10.6 min




















TABLE 2-2








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Target peptide
of resin
method
Yield
Found M
for M
condition
RT





(14) H—ALRALRALR—OH
Resin A
Method A
213 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


1039.6851
1039.68
J
9.7 min





(15) Ac—GGRGG—NH2
Resin B
Method B
115 mg
[M + H]+
[M + H]+
Condition
around



348.8 mg


444.2319
444.23
K
2.2 min





(16) H—HSDGTFTSELSRL
Resin B
Method B
313 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


REGARLQRLLQGLV—NH2
233 mg


1013.5576
1013.56
L
7.2 min





(11) H—GGGRG—NH2
Resin B
Method B
 72 mg
[M + H]+
[M + H]+
Condition
around



348.8 mg


402.2181
402.22
N
3.2 min





(17) Pyr—HWSYILRP—
Resin B
Method B
134 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


NH2
233 mg


591.3131
591.31
O
13.4 min








Condition
around








P
7.9 min





(18) H—HVTTV—NH2
Resin B
Method B
 67 mg
[M + H]+
[M + H]+
Condition
around



189 mg


555.3249
555.32
Q
8.7 min





(19) H—YERAKSNM—NH2
Resin B
Method B
134 mg
[M + H]+
[M + H]+
Condition
around



222 mg


997.4871
997.49
R
4.8 min




















TABLE 2-3








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Target peptide
of resin
method
Yield
Found M
for M
condition
RT





(20) H—VKRESYSGVT—
Resin B
Method
 101 mg
[M + H]+
[M + H]+
Condition
around


NH2
189 mg
B

1124.6058
1124.61
T
10.1 min





(21) H—RAVLP—OH
Resin A
Method
52.5 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg
A

555.3611
555.36
U
14.4 min





(22) H—(RC8)RRR((Me)
Resin B
Method
  99 mg
[M + H]+
[M + H]+
Condition
around


F)R(SC5)—NH2
222 mg
B

1123.7689
1123.77
W
7.6 min





(27) Ac—YHYPEL—NH2
Resin B
Method
  79 mg
[M + H]+
[M + H]+
Condition
around



222 mg
B

862.4088
862.41
V
12.8 min





(28) Ac—Y(MeK)YPEL—
Resin B
Method
  93 mg
[M + H]+
[M + H]+
Condition
around


NH2
222 mg
B

867.4607
867.46
V
11.7 min





(23) Ac—HYFYPEL—NH2
Resin B
Method
 106 mg
[M + H]+
[M + H]+
Condition
around



222 mg
B

1009.4756
1009.48
X
7.5 min





(24) H—WPVTLNAQTID—
Resin B
Method
 114 mg
[M + H]+
[M + H]+
Condition
around


NH2
222 mg
B

1256.6613
1256.66
X
7.3 min





(25) H—T((Me)G)RK
Resin A
Method
 163 mg
[M + H]+
[M + H]+
Condition
around


(Dap)H—OH
125 mg
A

698.4059
698.41
Y
9.7 min





(26) Pyr—GPWLEEEEEAY
Resin B
Method
 215 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


GWMDF—NH2
233 mg
B

1049.4321
1049.43
Z
16.0 min









Similarly, the analogue peptides shown in the tables below were synthesized by solid phase method using the above methods.













TABLE 3-1








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Analogue peptide
of resin
method
Yield
Found M
for M
condition
RT





 (1) Ac—YYPEL—NH2
Resin B
Method B
 65 mg
[M + H]+
[M + H]+
Condition
around



233 mg


725.3501
725.35
C
1.9 min








Condition
around








V
16.0 min





 (2) H—YYPEL—NH2
Resin B
Method B
 80 mg
[M + H]+
[M + H]+
Condition
around



233 mg


683.3391
683.34
D
8.5 min





 (3) H—VIRALRALVALRA
Resin A
Method A
354 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


LR—OH
187.5 mg


846.0698
846.07
E
13.6 min





 (4) H—ARLDVASEFRKKN
Resin B
Method B
258 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


KALSR—NH2
233 mg


758.7746
758.77
C
2.3 min





 (5) H—HAEGTFTSDVSSY
Resin A
Method A
464 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


LEGQAAKEFAWLVKGRG—OH
125 mg


1081.2033
1081.20
F
5.7 min





 (6) H—AGCKNFFWKTF
Resin A
Method B
382 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


TS—OH
125 mg


768.8683
768.87
G
14.2 min





 (7) H—AQKLRAD—OH
Resin A
Method A
127 mg
[M + H]+
[M + H]+
Condition
17.0 min



187.5 mg


801.4588
801.46
H






 (8) H—AKLRAD—OH
Resin A
Method A
115 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


673.4015
673.40
H
15.7 min





(19) H—QKLRASD—OH
Resin A
Method A
 45 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


817.4480
817.45
M
11.5 min





(20) H—AQKLRSD—OH
Resin A
Method A
151 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


817.4547
817.45
M
5.2 min





(21) H—AQKLRAS—OH
Resin A
Method A
201 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


773.4053
773.46
M
7.3 min




















TABLE 3-2








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Analogue peptide
of resin
method
Yield
Found M
for M
condition
RT





(24) H—AQKRASD—OH
Resin A
Method A
 42 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


775.4053
775.41
M
5.2 min





 (9) H—YERAKNM—OH
Resin A
Method A
152 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


911.4377
911.44
A
7.3 min





(13) H—YERKSNM—OH
Resin A
Method A
124 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


927.4348
927.44
A
11.4 min





(14) H—YERASNM—OH
Resin A
Method A
147 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


870.3745
870.38
A
12.4 min





(16) H—YERAKSM—OH
Resin A
Method A
150 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


884.4260
884.43
A
6.9 min





(22) H—ERAKSNM—OH
Resin A
Method A
 90 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


835.4087
835.41
A
4.3 min





(23) H—YERAKSN—OH
Resin A
Method A
175 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


867.4312
867.43
A
14.0 min





(10) H—FRVDEEFQSPFAS
Resin B
Method B
289 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


QSRGYFLFRRN—NH2
233 mg


994.8306
994.83
I
8.8 min





(12) Ac—ALAL—NH2
Resin B
Method B
 67 mg
[M + H]+
[M + H]+
Condition
around



348.8 mg


428.2874
428.29
B
10.6 min





(15) Ac—ALAL—OH
Resin A
Method A
 30 mg
[M + H]+
[M + H]+
Condition
around



1258 mg


429.2710
429.27
B
11.0 min





(17) H—ALALRALR—OH
Resin A
Method A
224 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg


883.5824
883.58
J
12.0 min





(18) Ac—GGGG—NH2
Resin B
Method B
 30 mg
[M + H]+
[M + H]+
Condition
around



233 mg


288.1300
288.13
K
2.3 min





(30) H—HSDGTFTSELSRL
Resin B
Method B
289 mg
[M + 3H]3+
[M + 3H]3+
Condition
around


EGARLQRLLQGLV—NH2
233 mg


961.5224
961.52
L
9.7 min





(11) H—GGGG—NH2
Resin B
Method B
 69 mg
[M + H]+
[M + H]+
Condition
around



348.8 mg


246.1180
246.12
N
1.9 min





(26) Pyr—HWSYLLRP—
Resin B
Method B
143 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


NH2
233 mg


591.3136
591.31
O
10.0 min




















TABLE 3-3








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Analogue peptide
of resin
method
Yield
Found M
for M
condition
RT





(31) Pyr—HWSYILLRP—
Resin B
Method B
129 mg
[M + H]+
[M + H]+
Condition
around


NH2
233 mg


1294.7055
1294.72
P
14.2 min





(32) H—VTTV—NH2
Resin B
Method B
 49 mg
[M + H]+
[M + H]+
Condition
around



233 mg


418.2660
418.28
Q
5.2 min





(33) H—hVTTV—NH2
Resin B
Method B
 61 mg
[M + H]+
[M + H]+
Condition
around



233 mg


555.3249
555.32
Q
10.9 min





(34) H—YERAKSNL—NH2
Resin B
Method B
119 mg
[M + H]+
[M + H]+
Condition
around



222 mg


979.5310
979.53
R
6.8 min





(35) H—AGcKNFFWKTPT
Resin A
Method A
129 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


SC—OH
125 mg


820.3865
820.37
S
7.4 min




















TABLE 3-4








Resin


Confirmation test













used


ESI-MS
HPLC















Amount
Synthesis


Calcd.
Analysis



Analogue peptide
of resin
method
Yield
Found M
for M
condition
RT





(36) H—VKRESYSGV—NH2
Resin B
Method
105 mg
[M + H]+
[M + H]+
Condition
around



233 mg
B

1023.5577
1023.56
T
8.8 min





(37) H—RALP—OH
Resin A
Method
 45 mg
[M + H]+
[M + H]+
Condition
around



187.5 mg
A

456.2926
456.29
U
5.8 min





(38) H—(RC8)RRRR
Resin B
Method
119 mg
[M + H]+
[M + H]+
Condition
around


(SC5)—NH2
222 mg
B

962.6858
962.68
W
6.6 min





(39) Ac—HYYPEL—NH2
Resin B
Method
 93 mg
[M + H]+
[M + H]+
Condition
around



222 mg
B

862.4114
862.41
X
6.9 min





(40) H—PVTLNAQTID—
Resin B
Method
119 mg
[M + H]+
[M + H]+
Condition
around


NH2
233 mg
B

1070.5823
1070.58
X
6.6 min





(41) H—((Me)G)RK
Resin A
Method
134 mg
[M + H]+
[M + H]+
Condition
around


(Dap)H—OH
125 mg
A

597.3584
597.36
Y
6.8 min





(42) Pyr—GPWLEEEEEY
Resin B
Method
222 mg
[M + 2H]2+
[M + 2H]2+
Condition
around


GWMDF—NH2
233 mg
B

1013.9123
1013.91
Z
14.2 min









1-2: Acid Compound

The following acid compounds were purchased to use.


Sulfuric acid: NACALAI TESQUE, INC., special grade;


Hydrochloric acid: NACALAI TESQUE, INC., 0.1 N special grade;




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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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NACALAI TESQUE, INC., special grade;




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Wako Pure Chemical, special grade;




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Tokyo Chemical Industry Co., Ltd.;



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NACALAI TESQUE, INC., first grade;




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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.;



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Tokyo Chemical Industry Co., Ltd.

Unless otherwise stated, the following sulfonic acids were synthesized by reacting the corresponding halides with sodium sulfite and subjecting the resulting sulfonic acid sodium salts to acidification with hydrochloric acid.


As a specific example, the synthesis of p-methylbenzyl sulfonic acid is described below:




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p-Methylbenzyl bromide 1.55 g (7 mmol) was dissolved in 4 mL of ethanol. To the mixture was added 0.97 g of sodium sulfite (7.7 mmol; 1.1 equivalents) and 8 mL of water. The mixture was heated at reflux overnight. After the reaction, the mixture was concentrated by an evaporator to remove ethanol, and the precipitated solid was filtered to give 1.09 g of p-methylbenzylsulfonic acid sodium salt. To 625 mg of the resulting solid were added 1 mL of concentrated hydrochloric acid and 1.5 mL of water, and the mixture was stirred overnight. The liquid was filtered off. The residue was washed 2 times with 0.5 mL of water to give 448 mg of p-methylbenzyl sulfonic acid. 1H NMR (400 MHz, D2O) 7.25 (d, J=8.0 Hz, 2H), 7.19 (d, J=7.9 Hz, 2H), 4.07 (s, 2H), 2.28 (s, 3H).




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was prepared from the corresponding alcohol compound as follows.


1. In a 50 mL 3-necked flask, 1.18 g (6 mmol) of 9-fluorenyl methanol was dissolved in 9 mL of ethyl acetate, and cooled to 0° C. in an ice bath. To the mixture was added 488 μL (6.3 mmol; 1.05 eq) of methanesulfonyl chloride. 920 μL (6.6 mmol; 1.05 eq) of triethylamine was added dropwise thereto at an internal temperature of 0 to 10° C. After stirring at 0° C. for 26 hours, 10 mL of water and 10 mL of MTBE were added to quench the reaction. The separated organic layer was washed sequentially with 15 mL of saturated aqueous sodium hydrogen carbonate, 15 mL of 0.5 M aqueous sodium hydrogen sulfate, and 15 mL of water. Then the resulting organic layer was concentrated to give 1.28 g of a crude 9-fluorenylmethoxymethanesulfonate. 1H NMR (400 MHz, CDCl3) δ: 7.78 (d, J=7.8 Hz, 2H), 7.62 (d, J=7.8 Hz, 2H), 7.43 (m, 4H), 4.50 (d, J=7.6 Hz, 2H), 4.31 (t, J=7.6 Hz, 1H), 2.91 (s, 3H).


2. To 1.28 g of the resulting crude 9-fluorenylmethoxymethanesulfonate was added 3 mL of ethanol, and then to the resulting solution were added 7 mL of water and 1.01 g (8 mmol) of sodium sulfite. The mixture was heated and stirred at an internal temperature of 80° C. for 90 h. After the reaction was completed, the reaction mixture was concentrated by an evaporator to remove ethanol, and the precipitated solid was filtered off. To the filtrate was added 1 mL of concentrated hydrochloric acid, and the mixture was evaporated to dryness. To the residue was added 2 mL of water, and the mixture was suspended for 1 hour, and filtered, and the resulting residue was washed with 0.5 mL of water 2 times to give 310 mg of 9-fluorenylmethylsulfonic acid (′H NMR (400 MHz) (CD3OD) 7.91 (d, J=7.6 Hz, 2H), 7.78 (d, J=7.6 Hz, 2H), 7.34 (m, 4H), 4.44 (t, J=6.0 Hz, 1H), 3.28 (m, 2H)).


According to the above synthesis of




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805 mg of 2-indanol (6 mmol) was used as a starting material to give 1.26 g of 2-indanyloxymethanesulfonate (1H NMR (400 MHz, CDCl3) δ: 7.22 (m, 4H), 5.53 (m, 1H), 3.37 (m, 1H), 3.21 (m, 2H), 3.02 (s, 3H)), and 1.26 g of the resulting 2-indanyloxymethanesulfonate was used to give 131 mg of 2-indanylsulfonic acid:




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(1H NMR (400 MHz) (CD3OD) 7.19 (m, 2H), 7.12 (m, 2H), 3.80 (m, 1H), 3.34 (m, 3H)).


According to the above procedure for the synthesis of p-methylbenzylsulfonic acid, the sulfonic acids shown in the tables below were synthesized using the corresponding halides.












TABLE 4-1





Sulfonic acid
Halide
Yield

1H NMR (400 MHz)










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0.15 g
(D2O) 7.71 (m, 4H), 7.66 (t, J = 7.6 Hz, 1H), 7.50 (m, 4H), 4.22 (s, 2H)







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0.95 g
(CD3OD) 7.23 (m, 4H), 7.15 (t, J = 7.0 Hz, 1H), 2.75 (m, 4H), 2.09 (m, 2H)







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0.21 g
(CD3OD) 8.02 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 4.48 (s, 2H), 2.43 (s, 3H)







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0.26 g
(CD3OD) 7.20 (t, J = 7.9 Hz, 1H), 7.00 (m, 2H), 6.81 (m, 1H), 4.02 (s, 2H), 3.79 (s, 3H)







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0.47 g
(CD3OD) 8.18 (d, J = 9.2 Hz, 2H), 7.65 (d, J = 9.2 Hz, 2H), 4.17 (s, 2H)



















TABLE 4-2









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0.32 g
(CD3OD) 7.96 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 4.12 (s, 2H), 3.89 (s, 3H)







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0.46 g
(CD3OD) 7.87 (m, 4H), 7.61 (m, 1H), 7.46 (m, 2H), 4.24 (s, 2H)







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0.16 g
(D2O) 8.17 (d, J = 7.6 Hz, 1H), 7.92 (t, J = 8.0 Hz, 2H), 7.55 (m, 4H), 4.66 (s, 2H)







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0.24 g
(D2O) 7.26 (m, 4H), 7.18 (m, 1H), 3.05 (m, 4H)







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0.13 g
(D2O) 7.33 (s, 4H), 4.09 (s, 4H)







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0.53 g
(CD3OD) 2.81 (t, J = 7.8 Hz, 2H), 2.21 (m, 3H), 1.90 (m, 2H), 1.64 (m, 2H)



















TABLE 4-3









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0.15 g
(DMSO-d6) 9.40 (s, 1H), 7.17 (m, 2H), 7.05 (t, J = 7.4 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 3.32 (dd, J = 8.6 Hz, 11.0 Hz, 1H), 2.67 (m, 2H), 2.39 (m, 2H)







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1.50 g
(CD3OD) 7.27 (m, 8H), 7.16 (m, 2H), 4.07 (t, J = 8.0 Hz, 1H), 2.72 (m, 2H), 2.53 (m, 2H)







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0.19 g
(DMSO-d6) 3.13 (s, 6H), 2.43 (s, 9H)







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67 mg
(CD3OD) 7.12 (d, J = 6.4 Hz, 2H), 6.82 (d, J = 6.8 Hz, 2H), 3.75 (s, 3H), 2.77 (m, 2H), 2.66 (t, J = 7.5 Hz, 2H), 2.05 (m, 2H)







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0.18 g
(CD3OD) 7.07 (d, J = 7.7 Hz, 1H), 6.99 (t, J = 7.8 Hz, 1H), 6.72 (m, 2H), 2.80 (m, 2H), 2.71 (t, J = 7.4 Hz, 2H), 2.08 (m, 2H)



















TABLE 4-4









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0.84 g
(CD3OD) 7.23 (m, 4H), 7.15 (t, J = 7.1 Hz, 1H), 6.72 (m, 2H), 2.83 (dd, J = 8.0 Hz, 7.4 Hz, 1H), 2.65 (t, J = 7.4 Hz, 2H), 1.86-1.73 (m, 4H)







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1.05 g
(CD3OD) 7.33 (m, 4H), 4.01 (s, 2H), 1.30 (s, 9H)







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67 mg
(CD3OD) 6.75 (m, 2H), 6.67 (m, 1H), 3.70 (s, 3H), 3.67 (s, 3H), 2.55 (m, 4H), 1.88 (m, 2H)







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 6 mg
(DMSO-d6) 6.98 (s, 2H), 5.06 (s, 1H), 2.85 (m, 2H), 2.68 (t, J = 7.3 Hz, 2H), 2.14 (m, 2H)




















TABLE 4-5









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7
mg
(CD3OD) 7.42 (m, 8H), 7.34 (m, 2H), 2.91 (m, 2H), 2.81 (m, 2H)







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126
mg
(DMSO-d6) 8.74 (dd, J = 8.1 Hz, 1.2 Hz, 1H), 7.48 (m, 4H), 7.30 (t, J = 6.4 Hz, 1H), 7.22 (t, J = 6.0 Hz, 1H), 7.12 (m, 7H)







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21
mg
(CD3OD) 8.03 (s, 1H), 7.99 (dd, J = 9.0 Hz, 2.7 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H), 4.30 (t, J = 5.2 Hz, 2H), 2.89 (t, J = 6.4 Hz, 2H), 2.05 (m, 2H)







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345
mg
(CD3OD) 7.43 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 2.74 (m, 4H), 2.09 (m, 2H)




















TABLE 4-6









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302
mg
(CD3OD) 8.15 (t, J = 1.8 Hz, 1H), 8.09 (m, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.55 (t, J = 7.9 Hz, 1H), 2.93 (t, J = 7.8 Hz, 2H), 2.85 (t, J = 7.6 Hz, 2H), 2.16 (m, 2H)







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338
mg
(CD3OD) 7.55 (s, 1H), 7.50 (m, 3H), 2.85 (m, 4H), 2.13 (m, 2H)







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85
mg
(CD3OD) 7.43 (s, 3H), 4.13 (s, 6H)







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833
mg
(CD3OD) 7.70 (dd, J = 6.5 Hz, 1.9 Hz, 2H), 7.50 (dd, J = 6.5 Hz, 1.9 Hz, 2H), 4.15 (s, 2H)




















TABLE 4-7









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80
mg
(CD3OD) 7.25 (m, 4H), 7.16 (t, J = 6.8 Hz, 1H), 2.83 (m, 3H), 2.35 (m, 1H), 1.76 (m, 1H), 1.37 (d, J = 6.8 Hz, 3H)







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40
mg
(CD3OD) 5.84 (m, 1H), 5.03 (m, 2H), 2.81 (t, J = 9.8 Hz, 2H), 2.20 (m, 2H), 1.90 (m, 2H)







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424
mg
(CD3OD) 7.44 (d, J = 7.2 Hz, 2H), 7.32 (m, 2H), 7.22 (m, 1H), 6.64 (d, J = 15.8 Hz, 1H), 6.40 (dd, J = 15.8 Hz, 7.8 Hz, 1H), 3.70 (d, J = 7.8 Hz, 2H)



















TABLE 4-8









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361 mg
(CD3OD) 7.32 (m, 4H), 7.24 (m, 6H), 4.74 (m, 1H), 2.93 (m, 4H)









Synthesis of Benzylsulfonic Acid



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To 1.71 g (10 mmol) of benzyl bromide were added 10 mL of ethanol and 0.76 g (10 mmol) of thiourea, and heated at reflux overnight. After the disappearance of the starting materials was confirmed by TLC, the mixture was concentrated and dried by an evaporator. To the residue was added 10 mL of acetonitrile to give a solution, and the solution was cooled in an ice bath. To the solution were added 2 mL of 2N hydrochloric acid (4 mmol) and 5.34 g of N-chlorosuccinimide (40 mmol) under ice cooling, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated by an evaporator, and to the residue was added 5 mL of ethyl acetate, and the separated organic layer was concentrated by an evaporator. To the residue were added 5 mL of MTBE and 5 mL of heptane, the precipitated solid was filtered off, and the filtrate was concentrated by an evaporator. The residue was purified by silica gel column chromatography (Silica gel: Fuji SILYSIA CHEMICAL LTD, PSQ-100B, 50 g; Mobile phase: hexane/ethyl acetate=20/1) to give 0.64 g of benzylsulfonyl chloride. To 0.59 g of the resulting benzylsulfonyl chloride was added 5.9 mL of water, and the mixture was heated at reflux for 5 days. The reaction mixture was concentrated to dryness and dried by a vacuum pump to give 0.39 g of benzylsulfonic acid. 1H NMR (400 MHz, CD3OD) 7.42 (m, 2H), 7.28 (m, 3H), 4.05 (s, 2H)


Synthesis of p-Carboxybenzylsulfonic Acid



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To 0.50 g of p-methoxycarbonylbenzylsulfonic acid synthesized above, 0.6 mL of concentrated hydrochloric acid and 0.6 mL of water were added and the mixture was heated to 90° C. The mixture was heated with stirring at 90° C. overnight, then cooled to room temperature, and then filtered under reduced pressure to collect the precipitated solid to give 0.37 g of p-carboxybenzylsulfonic acid. 1H NMR (400 MHz, CD3OD) 7.97 (d, J=8.3 Hz, 2H), 7.53 (d, J=8.2 Hz, 2H), 4.12 (s, 2H)


Abbreviations

TFA: trifluoroacetic acid


IPA: isopropyl alcohol


DMSO: dimethyl sulfoxide


DIC: diisopropylcarbodiimide


TIS: triisopropylsilane


MTBE: methyl tert-butyl ether


Test Example 1: Purification of Target Peptide-1 (for Removal of Analogue Peptide)

A crude target peptide and a crude analogue peptide prepared in the above 1-1 were dissolved in 1% TFA in water/acetonitrile=100 to 70/0 to 30, to give 20 mM crude target peptide solution and 20 mM crude analogue peptide solution, respectively (assuming 100% purity of each peptide). Poorly soluble peptides were diluted to about 5 mM (assuming 100% purity of each peptide) and dissolved.


Each acid compound was dissolved in water, or in 10% IPA aqueous solution or 10% DMSO aqueous solution in case where it was difficult to dissolve in water, to give 50 mM acid compound solution.


The crude target peptide solution (1 μmol, assuming 100% purity of the target peptide) and the crude analogue peptide solution (0.1 μmol, assuming 100% purity of the analogue peptide) were mixed. The mixture was mixed with an acid compound solution (1.5 μmol) (the resulting mixture was analyzed by HPLC to provide the value of “before treatment”). The mixture was dried by lyophilization.


To the residue was added 100 μL of IPA, and the mixture was shaken overnight at room temperature.


The resulting slurry was centrifuged (5000 to 15000 rpm, 1 to 5 min), and the resulting cake was washed with 150 μL of IPA 2 times. The filtrate and the washings were combined.


The resulting solid and the filtrate combined with the washings were analyzed by HPLC to provide the values of “solid obtained after treatment” and “filtrate obtained after treatment”.


HPLC instruments were the same as those described above.


Measurement conditions: the same as the HPLC conditions A to Z for peptide synthesis.


For peptides comprising aromatic amino acid residue(s), long-wavelength ultraviolet absorption can be attributed exclusively to the aromatic side chain(s). The HPLC detection wavelengths for analyzing such peptides were Condition A (for detection at 274 nm), Condition D, Conditions F to G, and Conditions R to T. Under condition A (for detection at 274 nm), condition R, and condition T, both of the target and analogue peptides had 1 aromatic amino acid residue each. Under condition F, both of the target and analogue peptides had 5 aromatic amino acid residues each. Under condition G and condition S, both of the target and analogue peptides had 4 aromatic amino acid residues each. Under condition D, the target peptide had 3 aromatic amino acid residues, and the analogue peptide had 2 aromatic amino acid residues. That is, the number of aromatic side chains contributing to the long-wavelength ultraviolet absorption at 274 to 281 nm was the same or 1 difference between the target peptide and the analogue peptide, and the ratio of the HPLC area values can be regarded as an approximate molar ratio.


On the other hand, a peptide not comprising an aromatic amino acid residue has no ultraviolet absorption on a long wavelength side, and a short wavelength ultraviolet absorption (205 nm) attributed to a carbonyl group can be used for detection. For example, the target peptide had 10 carbonyl groups and the analogue peptide had 9 carbonyl groups in condition A (for detection at 205 nm) and condition J. Similarly, the target peptide has 6 carbonyl groups and the analogue peptide has 5 carbonyl groups in condition B and condition K, the target peptide has 8 carbonyl groups and the analogue peptide has 7 carbonyl groups in condition C, the target peptide has 16 carbonyl groups and the analogue peptide has 15 carbonyl groups in condition E, the target peptide has 9 carbonyl groups and the analogue peptide has 6 or 8 carbonyl groups in condition H, the target peptide has 31 carbonyl groups and the analogue peptide has 30 carbonyl groups in condition I, the target peptide has 32 carbonyl groups and the analogue peptide has 31 carbonyl groups in condition L, the target peptide has 9 carbonyl groups and the analogue peptide has 8 carbonyl groups in condition M. The number of carbonyl groups that contribute to the short-wavelength ultraviolet absorption at 205 nm is only 1 to 3 different between the target peptide and the analogue peptide, accordingly the ratio of the HPLC area values of the target peptide and the analogue peptide can be regarded as the approximate molar ratio. Although the target peptides (9) and (10) have aromatic side chain(s), they are sufficiently long and contain a large number of carbonyl groups, so they were detected by ultraviolet absorption (205 nm) of carbonyl group in condition I, but can also be detected at the long wavelength side (Both of the target peptide and analogue peptide have six aromatic amino acid residues). Similarly, the target peptide (16) and the analogue peptide (25), each of which has 2 aromatic side chains, were detected at 205 nm.


In condition N, condition 0, condition P, condition Q, condition U, condition V, condition W, condition X, condition Y, and condition Z, the short-wavelength ultraviolet absorption (205 nm) of carbonyl group was similarly used for detection.


As shown in the following Equation 1), the ratio (%) of the area of the target peptide to the total area of the target peptide and the analogue peptide was calculated using the HPLC measurement values (Area). The above ratio was regarded as the approximate ratio (%) of the amount of substance (mol) of the target peptide to the total amounts of substance (mol) of the target peptide and the analogue peptide (hereinafter, referred to as “target peptide ratio”).










Target





peptide





ratio






(
%
)


=




Area





of





target





peptide






Area





of





target





peptide





+






Area





of





analouge





peptide





×
100




Tagret





peptide






(
mol
)







Tagret





peptide






(
mol
)


+






Analogue





peptide






(
mol
)












Equation





1

)







Examination 1: Investigation of Salt Formation

When a peptide is mixed with an acid compound, the N-terminal amino group or a side chain moiety in a basic amino acid (lysine K, arginine R, histidine H etc.) and the acid compound may form a salt.


Based on the change of target peptide ratio (%) in the solid using the following peptides, whether a salt with the peptide and the acid compound is formed was examined.


Target peptide H-YFYPEL-NH2, which does not comprise a basic amino acid, and its analogue peptide H-YYPEL-NH2 having one residue deletion; and


The corresponding peptides which are N-terminally protected with an acetyl group (Ac), target peptide Ac-YFYPEL-NH2, and analogue peptide Ac-YYPEL-NH2 having one residue deletion.


In addition, the change of target peptide ratio (%) in the solid was examined using the following peptides, which comprise neither a basic amino acid nor functional group capable of forming a salt:


Target peptide Pyr-GPWLEEEEEAYGWMDF-NH2 and its analogue peptide Pyr-GPWLEEEEEYGWMDF-NH2 having one residue deletion.


The change in the ratio of the target peptide (%) was calculated as “Increased point of target peptide ratio (%)” using Equation 2 below.





Increased point of target peptide ratio (%)=target peptide ratio (%) in the solid obtained after treatment−target peptide ratio (%) of before treatment.  Equation 2):


The results are shown in the tables below.















TABLE 5-1





Target peptide
Analogue peptide


No acid compound
Sulfuric acid


embedded image









(1) Ac-YF
(1) Ac-YYP
Before
Area of target peptide
3778581
3778581
3778581


YPEL—NH2
EL—NH2
treatment
Area of analogue peptide
 280840
 280840
 280840





Target peptide ratio (%)
93.1%
93.1%
93.1%




Solid
Area of target peptide
1214330
 938194
1191857




obtained
Area of analogue peptide
 70516
 48524
 59848




after
Target peptide ratio (%)
94.5%
95.1%
95.2%




treatment


















Increased point of
 1.4
2.0
2.1




target peptide ratio (%)
















(2) H-YFY
(2) H-YYPE
Before
Area of target peptide
 638016
 441924
 464222


PEL—NH2
L—NH2
treatment
Area of analogue peptide
 64491
 45406
 47540





Target peptide ratio (%)
90.8%
90.7%
90.7%




Solid
Area of target peptide
 41395
 102094
 58715




obtained
Area of analogue peptide
  4325
 10239
  3880




after
Target peptide ratio (%)
90.5%
90.9%
93.8%




treatment


















Increased point of
−0.3
0.2
3.1




target peptide ratio (%)






















TABLE 5-2





Target peptide
Analogue peptide


No acid compound
Hydro- chloric acid


embedded image









(26) Pyr-
(42) Pyr-
Before
Target
89.1%
87.5%
88.9%


GPWLEEEEEA
GPWLEEEEEYG
treatment
peptide





YGWMDF—NH2
WMDF—NH2

ratio








(%)







Solid
Target
89.0%
87.5%
88.9%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
−0.1
0
0




of target







peptide ratio







(%)





Pyr- =




embedded image








Regarding Table 5-1

Regarding the target peptide Ac-YFYPEL-NH2 and its analogue peptide Ac-YYPEL-NH2, wherein the N-terminuses are protected with Ac group, the increased points of target peptide ratio (%) are 1.4 points in the case of no acid compound, 2.0 points in the case of sulfuric acid, 2.1 points in the case of the sulfonic acid compound, indicating that there was no large difference in the increased points of target peptide rate (%) in the presence or absence of an acid compound.


On the other hand, regarding the target peptide: H-YFYPEL-NH2 and its analogue peptide: H-YYPEL-NH2, wherein the N-terminuses are free, the increased points of target peptide ratio (%) are −0.3 points in the case of no acid compound, 0.2 points in the case of sulfuric acid, and 3.1 points in the case of the sulfonic acid compound, indicating that the increased point for the sulfonic acid compound was greater than the increased points for no acid compound and sulfuric acid.


When structures of a target peptide and its analogue peptide are similar, the solubility properties of the target peptide and the analogue peptide in a solvent are usually similar. When a peptide forms a solid by forming a salt with a sulfonic acid compound, the solubility property of the solid in the solvent greatly changes as compared with that of the original peptide. Accordingly, in a case where the target peptide exists more than its analogue peptide, the possibility that the target peptide ratio in the solid separated by solid-liquid separation is increased is remarkably increased.


Thus, these results suggest that the sulfonic acid compound formed a salt with the N-terminal amino group of the peptides.


Regarding the case of the target peptide Ac-YFYPEL-NH2 and its analogue peptide Ac-YYPEL-NH2, wherein the N-terminuses are protected with Ac group, it is considered that the increase in target peptide ratio (%), regardless of the presence or absence of the acid compound, was caused by the difference in the dissolution behavior of these peptides in IPA.


Regarding Table 5-2

Regarding the case of the target peptide Pyr-GPWLEEEEEAYGWMDF-NH2 and its analogue peptide Pyr-GPWLEEEEEYGWMDF-NH2, both of which have no moiety capable of forming a salt, there is no difference of the increased points of target peptide ratio (%) between use of the acid compound and no use of the acid compound.


Examination 2: Effect of Sulfonic Acid Compound

The increased points of the target peptide ratio (%) in the obtained solid were calculated in the same way as described above, using the acid compounds, the target peptides, and the analogue peptides shown in the table below.


The results are shown in the table below.















TABLE 6





Target peptide
Analogue peptide


No acid compound


embedded image




embedded image









(3) H—VIRALRR
(3) H—VIRALRAL
Before
Target peptide
88.8%
89.0%
88.6%


ALVALRALR—OH
VALRALR—OH
treatment
ratio (%)







Solid obtained
Target peptide
88.6%
89.3%
89.0%




after treatment
ratio (%)

















Increased point of
−0.2 
0.3
0.4




target peptide ratio (%)
















(4) H—ARLDVAS
(4) H—ARLDVASSE
Before
Target peptide
88.5%
88.5%
88.5%


EFRKKWNKALSR—
FRKKNKALSR—
treatment
ratio (%)





NH2
NH2
Solid obtained
Target peptide
90.1%
91.9%
92.5%




after treatment
ratio (%)

















Increased point of
1.6
3.4
4.0




target peptide ratio (%)
















(5) H—HAEGTFT
(5) H—HAEGTFTS
Before
Target peptide
80.8%
81.2%
81.5%


SDVSSYLEGQAA
DVSSYLEGQAAKE
treatment
ratio (%)





KEFIAWLVGRG—
FAWLVKGRG—OH
Solid obtained
Target peptide
80.7%
82.4%
81.7%


OH

after treatment
ratio (%)

















Increased point of
−0.1 
1.2
0.2




target peptide ratio (%)
















(6) H—AGCKNFF
(6) H—AGCKNFFW
Before
Target peptide
89.9%
90.4%
90.6%


WKTFTSC—OH
KTFTS—OH
treatment
ratio (%)







Solid obtained
Target peptide
90.0%
92.5%
93.4%




after treatment
ratio (%)

















Increased point of
0.1
2.1
2.8




target peptide ratio (%)









In comparison with the case where no acid compound was used, use of the sulfonic acid compounds increased the increased points of the target peptide ratio (%). These results showed that the sulfonic acid compounds have excellent effects in separating the target peptide and the analogue peptide.


It is considered that this is because the dissolution behavior was changed by the salt formation with the sulfonic acid compounds, as suggested in the Examination 1.


Examination 3: Comparison with Inorganic Acids

The increased points of the ratio (%) of the target peptide in the obtained solid was calculated in the same way as described above, using the acid compounds, the target peptides, and the analogue peptides shown in the table below.


The results are shown in the table below.
















TABLE 7





Target peptide
Analogue peptide


Hydrochloric acid
Sulfuric acid


embedded image




embedded image









(7) H—AQK
(7) H—AQKL
Before
Target peptide
86.3%
86.4%
89.6%
87.1%


LRASD—OH
RAD—OH
treatment
ratio (%)








Solid obtained
Target peptide
85.4%
84.8%
89.9%
87.5%




after
ratio (%)








treatment




















Increased point of
−0.9
−1.6
0.3
0.4




target peptide ratio (%)



















(8) H—AKLR
Before
Target peptide
91.2%
91.2%
94.5%
90.9%



AD—OH
treatment
ratio (%)








Solid obtained
Target peptide
91.2%
91.7%
97.1%
94.4%




after
ratio (%)








treatment




















Increased point of
 0.0
 0.5
2.6
3.5




target peptide ratio (%)


















(8) H—YER
(9) H—YERA
Before
Target peptide
91.5%
91.2%
90.9%
91.0%


AKSNM—OH
KNM—OH
treatment
ratio (%)








Solid obtained
Target peptide
90.4%
89.0%
94.2%
93.0%




after
ratio (%)








treatment




















Increased point of
−1.1
−2.2
3.3
2.0




target peptide ratio (%)


















(9) H—FRV
(10) H—FRV
Before
Target peptide
93.6%
94.3%
85.2%
93.7%


DEEFQSPF
DEEFQSPFA
treatment
ratio (%)






ASQSRGYF
SQSRGYFLF
Solid obtained
Target peptide
92.9%
95.3%
96.8%
97.7%


LFRPRN—
RRN—NH2
after
ratio (%)






NH2

treatment




















Increased point of
−0.7
 1.0
11.6 
4.0




target peptide ratio (%)









The increase points of the target peptide ratio (%) were larger in the cases where the sulfonic acid compounds were used than in the cases where the hydrochloric acid and sulfuric acid were used, which indicates that the sulfonic acid compounds showed an excellent effect in the purification of the target peptide.


Examination 4: Comparison with Carboxylic Acid Compounds

Using the sulfonic acid compounds or carboxylic acid compounds and the target peptides and analogue peptides, shown in the tables below, the increased points of the target peptide ratio (%) in the obtained solid were calculated in the same way as described above.


The results are shown in the tables below.
















TABLE 8





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(9) H—FRVD
(10) H—FRVD
Before
Target
92.2%
95.0%
92.3%
95.6%


EEFQSPFAS
EEFQSPFASQ
treatment
peptide






QSRGYFLFR
SRGYFLFRRN—

ratio






PRN—NH2
NH2

(%)








Solid
Target
96.5%
93.6%
97.1%
93.3%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
4.3
−1.4
4.8
−2.3




target peptide ratio(%)





















TABLE 9





Target peptide
Analogue peptide




embedded image




embedded image









(9) H—FRVDEEF
(10) H—FRVDEEF
Before
Target peptide
91.8%
94.8%


QSPFASQSRGYF
QSPFASQSRGYFL
treatment
ratio (%)




LFRPRN—NH2
FRRN—NH2
Solid obtained
Target peptide
96.2%
93.4%




after treatment
ratio (%)















Increased point of
4.4
−1.4




target peptide ratio (%)









The increased points of the target peptide ratio (%) were larger in the cases where the sulfonic acid compounds were used than in the cases where the carboxylic acid compounds were used, which indicates that the sulfonic acid compounds showed an excellent effect in the purification of the target peptide.


Examination 5: Effects of Various Sulfonic Acid Compounds-(1)

When the sulfonic acid compounds, target peptides, and analogue peptides shown in the tables below were used, the target peptide ratio (%) in some of the solids could not be calculated due to analytical problems. Therefore, the change in the target peptide ratio (%) in the filtrate combined with the washings obtained after treatment was calculated as “decreased point of target peptide ratio (%)” using the following Equation (3). Since the concentrations of the target peptide and analogue peptide before and after the treatment are unknown, and the HPLC sensitivities of the target peptide and analogue peptide are not the same, it is not possible to convert the decreased point of target peptide ratio (%) in the filtrate to the increased point of the target peptide ratio (%) in the solid. However, the decrease of the target peptide ratio in the filtrate means the increase of the target peptide ratio in the solid.





Decreased point of target peptide ratio (%)=(Target peptide ratio (%) in filtrate obtained after treatment)−(Target peptide ratio (%) of before treatment)  Equation 3):


The results are shown in the table below.















TABLE 10





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(11) H—GG
(11) H—G
Before
Target peptide
77.0%
69.7%
72.9%


GRG—NH2
GGG—NH2
treatment
ratio (%)







Filtrate
Target peptide
70.2%
69.4%
67.0%




obtained
ratio (%)







after treatment


















Decreased point of
 6.8
 0.3
 5.9




target peptide ratio (%)
















(12) Ac-A
(12) Ac-
Before
Target peptide
94.5%
94.4%
92.4%


LRAL—NH2
ALAL—NH2
treatment
ratio (%)







Filtrate
Target peptide
86.4%
79.2%
82.4%




obtained
ratio (%)







after treatment


















Decreased point of
 8.1
15.2
10.0




target peptide ratio (%)
















(8) H—YER
(13) H—Y
Before
Target peptide
91.1%
91.8%
91.8%


AKSNM—OH
ERKSNM—
treatment
ratio (%)






OH
Filtrate
Target peptide
75.8%
76.5%
72.8%




obtained
ratio (%)







after treatment


















Decreased point of
15.3
15.3
19.0




target peptide ratio (%)

















(14) H—Y
Before
Target peptide
89.8%
90.7%
90.5%



ERASNM—
treatment
ratio (%)






OH
Filtrate
Target peptide
78.3%
82.5%
83.0%




obtained
ratio (%)







after treatment


















Decreased point of
11.5
 8.2
 7.5




target peptide ratio (%)






















TABLE 11





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(5) H—HAEG
(5) H—HAEG
Before
Target
80.9%
81.2%
80.8%


TFTSDVSSY
TFTSDVSSY
treatment
peptide





LEGQAAKEF
LEGQAAKEF

ratio (%)





IAWLVKGRG—
AWLVKGRG—
Filtrate
Target
80.2%
79.9%
79.6%


OH
OH
obtained
peptide







after treatment
ratio (%)

















Decreased point of
0.7
1.3
1.2




target peptide ratio (%)
















(6) H—AGCK
(6) H—AGCK
Before
Target
90.5%
90.0%
90.6%


NFFWKTFTS
NFFWKTFTS—
treatment
peptide





C—OH
OH

ratio (%)







Filtrate
Target
89.4%
88.0%
89.4%




obtained
peptide







after treatment
ratio (%)

















Decreased point of
1.1
2.0
1.2




target peptide ratio (%)























TABLE 12





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




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(12) Ac-
(12) Ac-
Before
Target peptide
84.0%
82.6%
82.6%
79.0%


ALRAL—
ALAL—
treatment
ratio (%)






NH2
NH2
Filtrate
Target peptide
73.5%
 8.0%
 4.5%
18.4%




obtained
ratio (%)








after treatment




















Decreased point of
10.5
74.6 
78.1 
60.6 




target peptide ratio (%)


















(13) Ac-
(15) Ac-
Before
Target peptide
83.9%
81.2%
82.3%
82.5%


ALRAL—
ALAL—
treatment
ratio (%)






OH
OH
Filtrate
Target peptide
78.1%
77.5%
76.7%
76.6%




obtained
ratio (%)








after treatment




















Decreased point of
 5.8
3.7
5.6
5.9




target peptide ratio (%)


















(8) H—Y
(8) H—Y
Before
Target peptide
90.9%
91.9%
91.9%
91.8%


ERAKSN
ERKSN
treatment
ratio (%)






M—OH
M—OH
Filtrate
Target peptide
78.3%
80.9%
77.3%
77.3%




obtained
ratio (%)








after treatment




















Decreased point of
12.6
11.0 
14.6 
14.5 




target peptide ratio (%)



















(14) H—
Before
Target peptide
92.3%
90.7%
91.0%
91.1%



YERASN
treatment
ratio (%)







M—OH
Filtrate
Target peptide
84.7%
84.0%
83.7%
81.6%




obtained
ratio (%)








after treatment




















Decreased point of
 7.6
6.7
7.3
9.5




target peptide ratio (%)



















(16) H—
Before
Target peptide
91.3%
91.3%
92.1%
92.0%



YERAKS
treatment
ratio (%)







M—OH
Filtrate
Target peptide
80.1%
89.5%
89.6%
87.0%




obtained
ratio (%)








after treatment




















Decreased point of
11.2
1.8
2.5
5.0




target peptide ratio (%)























TABLE 13





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(8) H—Y
(13) H—
Before
Target
91.9%
91.8%
91.6%
91.9%


ERAKSN
YERKSN
treatment
peptide






M—OH
M—OH

ratio (%)








Filtrate
Target
84.6%
71.3%
65.4%
75.5%




obtained after
peptide








treatment
ratio (%)



















Decreased point of
7.3
20.5
26.2
16.4 




target peptide ratio (%)



















(14) H—
Before
Target
90.4%
90.5%
92.5%
91.0%



YERASN
treatment
peptide







M—OH

ratio (%)








Filtrate
Target
88.4%
79.5%
74.1%
82.4%




obtained after
peptide








treatment
ratio (%)



















Decreased point of
2.0
11.0
18.4
8.6




target peptide ratio (%)


















(14) H—
(17) H—
Before
Target
89.5%
90.9%
90.0%
90.7%


ALRALR
ALALRA
treatment
peptide






ALR—OH
LR—OH

ratio (%)








Filtrate
Target
88.3%
85.7%
88.9%
85.9%




obtained after
peptide








treatment
ratio (%)



















Decreased point of
1.2
 5.2
 1.1
4.8




target peptide ratio (%)


















(6) H—A
(6) H—A
Before
Target
90.0%
90.5%
89.8%
90.5%


GCKNFF
GCKNFF
treatment
peptide






WKTFTS
WKTFTS—

ratio (%)






C—OH
OH
Filtrate
Target
85.9%
90.4%
88.9%
89.9%




obtained after
peptide








treatment
ratio (%)



















Decreased point of
4.1
 0.1
 0.9
0.6




target peptide ratio (%)






















TABLE 14-1





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(15) Ac-GGRGG—
(18) Ac-GGGG—
Before
Target peptide
93.7%
92.6%
91.2%


NH2
NH2
treatment
ratio (%)







Filtrate obtained
Target peptide
74.0%
69.3%
67.9%




after treatment
ratio (%)

















Decreased point of
19.7
22.3
23.3




target peptide ratio (%)
















(7) H—AQKLRAS
(19) H—QKLRAS
Before
Target peptide
92.8%
92.5%
91.8%


D—OH
D—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
36.5%
40.2%
20.6%




after treatment
ratio (%)

















Decreased point of
56.3
52.3
71.2




target peptide ratio (%)

















(20) H—AQKLRS
Before
Target peptide
92.8%
92.1%
92.5%



D—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
74.3%
73.3%
26.6%




after treatment
ratio (%)

















Decreased point of
18.5
18.8
65.9




target peptide ratio (%)

















(21) H—AQKLRA
Before
Target peptide
91.4%
91.0%
90.7%



S—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
66.8%
65.9%
46.6%




after treatment
ratio (%)

















Decreased point of
24.6
25.1
44.1




target peptide ratio (%)






















TABLE 14-2





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(8) H—YERAKSN
(22) H—ERAKSN
Before
Target peptide
94.3%
94.2%
94.0%


M—OH
M—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
91.7%
91.8%
93.7%




after treatment
ratio (%)

















Decreased point of
2.6
2.4
0.3




target peptide ratio (%)

















(13) H—YERKSN
Before
Target peptide
91.8%
91.9%
90.6%



M—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
71.6%
75.7%
79.4%




after treatment
ratio (%)

















Decreased point of
20.2 
16.2 
11.2 




target peptide ratio (%)

















(23) H—YERAKS
Before
Target peptide
87.8%
87.1%
84.8%



N—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
86.6%
85.5%
77.7%




after treatment
ratio (%)

















Decreased point of
1.2
1.6
7.1




target peptide ratio (%)
















(6) H—AGCKNFF
(6) H—AGCKNFF
Before
Target peptide
90.3%
90.4%
90.20% 


WKTFTSC—OH
WKTFTS—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
89.2%
87.2%
90.16% 




after treatment
ratio (%)

















Decreased point of
1.1
3.2
 0.04




target peptide ratio (%)






















TABLE 15-1





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(13) Ac-ALRAL—
(15) Ac-ALAL—
Before
Target peptide
82.3%
81.4%
82.5%


OH
OH
treatment
ratio (%)







Filtrate obtained
Target peptide
78.3%
76.8%
76.6%




after treatment
ratio (%)

















Decreased point of
4.0
 4.6
 5.9




target peptide ratio (%)
















(12) Ac-ALRAL—
(12) Ac-ALAL—
Before
Target peptide
81.5%
82.3%
79.0%


NH2
NH2
treatment
ratio (%)







Filtrate obtained
Target peptide
50.4%
63.4%
18.4%




after treatment
ratio (%)

















Decreased point of
31.1 
18.9
60.6




target peptide ratio (%)
















(7) H—AQKLRAS
(24) H—AQKRAS
Before
Target peptide
89.4%
89.9%
96.5%


D—OH
D—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
82.9%
78.1%
74.5%




after treatment
ratio (%)

















Decreased point of
6.5
11.8
22.0




target peptide ratio (%)
















(8) H—YERAKSN
(13) H—YERKSN
Before
Target peptide
91.5%
91.8%
91.8%


M—OH
M—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
76.1%
76.8%
77.3%




after treatment
ratio (%)

















Decreased point of
15.4 
15.0
14.5




target peptide ratio (%)

















(14) H—YERASN
Before
Target peptide
90.7%
90.6%
91.1%



M—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
83.8%
83.7%
81.6%




after treatment
ratio (%)

















Decreased point of
6.9
 6.9
 9.5




target peptide ratio (%)






















TABLE 15-2





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(3) H—VIRALRR
(3) H—VIRALRA
Before
Target peptide
89.2%
89.4%
 88.9%


ALVALRALR—OH
LVALRALR—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
87.8%
89.0%
 87.5%




after treatment
ratio (%)

















Decreased point of
1.4
0.4
1.4




target peptide ratio (%)
















(16) H—HSDGTF
(30) H—HSDGTF
Before
Target peptide
87.1%
87.3%
 82.9%


TSELSRLREGAR
TSELSRLEGARL
treatment
ratio (%)





LQRLLQGLV—NH2
QRLLQGLV—NH2
Filtrate obtained
Target peptide
36.5%
64.4%
 74.6%




after treatment
ratio (%)

















Decreased point of
50.6 
22.9 
8.3




target peptide ratio (%)
















(6) H—AGCKNFF
(6) H—AGCKNFF
Before
Target peptide
91.5%
90.2%
90.309%


WKTFTSC—OH
WKTFTS—OH
treatment
ratio (%)







Filtrate obtained
Target peptide
85.0%
87.6%
90.307%




after treatment
ratio (%)

















Decreased point of
6.5
2.6
 0.002




target peptide ratio (%)























TABLE 16-1





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(13) Ac-
(15) Ac-
Before
Target peptide
81.3%
78.6%

82.6%


ALRAL—
ALAL—
treatment
ratio (%)






OH
OH
Filtrate
Target peptide
78.5%
67.6%

76.6%




obtrained
ratio (%)








after treatment




















Decreased point of
 2.8
11.0

 6.0




target peptide ratio (%)


















(15) Ac-
(18) Ac-
Before
Target peptide
93.0%
93.5%
93.8%
92.7%


GGRGG—
GGGG—
treatment
ratio (%)






NH2
NH2
Filtrate
Target peptide
43.8%
77.9%
51.7%
56.8%




obtrained
ratio (%)








after treatment




















Decreased point of
49.2
15.6%
42.1
35.9




target peptide ratio (%)























TABLE 16-2





              Target peptide
              Analogue peptide


embedded image


embedded image


    embedded image


embedded image
























(7)H-
(24)H-
Before
Target

90.5%

88.5%


AQKLRASD-
AQKRASD-
treatment
peptide






OH
OH

ratio









(%)








Filtrate
Target

60.0%

82.0%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point

30.5%

6.5%




of target








peptide ratio








(%)



















(27)H-
Before
Target
94.2%
91.7%
90.2%
91.0%



AQKLASD-
treatment
peptide







OH

ratio









(%)








Filtrate
Target
83.3%
84.5%
77.2%
76.2%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
10.9
7.2
13.0
14.8




of target








peptide ratio








(%)



















(20)H-
Before
Target
92.0%

92.3%
92.1%



AQKLRSD-
treatment
peptide







OH

ratio









(%)








Filtrate
Target
85.5%

76.7%
85.8%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
6.5

15.6%
6.3




of target








peptide ratio








(%)



















(21)H-
Before
Target
90.7%
90.5%
90.5%
90.7%



AQKLRAS-
treatment
peptide







OH

ratio









(%)








Filtrate
Target
66.2%
48.9%
74.1%
59.3%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
24.5
41.6
16.4
31.4




of target








peptide ratio








(%)



















(8)H-
Before
Target
90.9%
92.2%
92.3%
92.5%



AKLRAD-
treatment
peptide







OH

ratio









(%)








Filtrate
Target
40.2%
≤88.5%
76.7%
50.4%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
50.7
≥3.7
15.6
42.1




of target








peptide ratio








(%)























TABLE 16-3





              Target peptide
              Analogue peptide


embedded image


embedded image


    embedded image


embedded image
























(8)H-
(28)H-
Before
Target
91.0%
91.4%
91.7%
91.3%


YERAKSNM-
YRAKSNM-
treatment
peptide






OH
OH

ratio (%)








Filtrate
Target
87.2%
≤66.4%
87.4%
82.1%




obtained
peptide








after
ratio (%)








treatment




















Decreased point of
3.8%
≥25.0%
4.3%
9.2%




target peptide ratio (%)



















(13)H-
Before
Target
91.8%
91.9%
91.7%
91.5%



YERKSNM-
treatment
peptide







OH

ratio (%)








Filtrate
Target
81.4%
81.6%
81.8%
79.9%




obtained
peptide








after
ratio (%)








treatment




















Decreased point of
10.4
10.3
9.9
11.6




target peptide








ratio (%)



















(16)H-
Before
Target
92.8%
90.7%
95.0%




YERAKSM-
treatment
peptide







OH

ratio (%)








Filtrate
Target
≤89.6%
≤63.4%
93.6%





obtained
peptide








after
ratio (%)








treatment




















Decreased point of
≥3.2
≥27.3
1.4





target peptide








ratio (%)



















(23)H-
Before
Target
88.3%
88.4%

88.6%



YERAKSN-
treatment
peptide







OH

ratio (%)








Filtrate
Target
76.1%
≤84.8%

86.8%




obtained
peptide








after
ratio (%)








treatment




















Decreased point of
12.2
≥3.6

1.8




target peptide








ratio (%)



























TABLE 16-4





              Target peptide
              Analogue peptide




embedded image


embedded image
      embedded image


embedded image
























(4)H-
(4)H-
Before
Target
88.5%





ARLDVASEFR
ARLDVASEFR
treatment
peptide






KKWNKALSR-
KKNKALSR-

ratio






NH2
NH2

(%)








Filtrate
Target
87 .8%







obtained
peptide








after
ratio








treatment
(%)



















Decreased point
0.7%







of target








peptide ratio (%)


















(16)H-
(30)H-
Before
Target
90.8%

83.6%



HSDGTFTSELSRLR
HSDGTFTSELSRL
treatment
peptide






EGARLQRLLQGLV-
EGARLQRLLQGLV-

ratio






NH2
NH2

(%)








Filtrate
Target
≤86.1%

83.4%





obtained
peptide








after
ratio








treatment
(%)



















Decreased point
≥4.7

0.2





of target








peptide ratio (%)


















(5)H-
(5)H-
Before
Target
81.9%
81.2%

80.5%


HAEGTFTSDVSSYLEG
HAEGTFTSDVSSYLEG
treatment
peptide






QAAKEFIAWLVKGRG-
QAAKEFAWLVKGRG-

ratio






OH
OH

(%)








Filtrate
Target
74.9%
80.7%

80.1%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
7.0
0.5

0.4




of target








peptide ratio (%)


















(6)H-
(6)H-
Before
Target
90.9%
90.7%
90.3%
90.3%


AGCKNFFWKTFTSC-
AGCKNFFWKTFTS-
treatment
peptide






OH
OH

ratio









(%)








Filtrate
Target
89.2%
88.6%
85.3%
88.6%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point
1.7
2.1
5.0%
1.7




of target








peptide ratio (%)


















(17)Pyr-
(26)Pyr-
Before
Target

89.9%
89.0%
89.0%


HWSYILRP-
HWSYLLRP-
treatment
peptide






NH2
NH2

ratio









(%)








Filtrate
Target

89.7%
87.5%
86.9%




obtained
peptide








after
ratio








treatment
(%)



















Decreased point

0.2
1.5
2.1




of target








peptide ratio (%)




















TABLE 17









embedded image


















De-




Target peptide
creased




ratio (%)
point














Filtrate
of





obtained
target




Before
after
peptide




treat-
treat-
ratio


Target peptide
Analogue peptide
ment
ment
(%)














(5)H-
(5)H-
80.6%
80.3%
0.3


HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG-
HAEGTFTSDVSSYLEGQAAKEFAWLVKGRG-





OH
OH





(6)H-AGCKNFFWKTFTSC-OH
(6)H-AGCKNFFWKTFTS-OH
90.3%
89.7%
0.6


(7)H-AQKLRASD-OH
(20)H-AQKLRSD-OH
92.4%
77.9%
14.5



(21)H-AQKLRAS-OH
90.1%
68.3%
21.8


(8)H-YERAKSNM-OH
(13)H-YERKSNM-OH
91.3%
73.8%
17.5



(14)H-YERASNM-OH
90.5%
82.3%
8.2



(16)H-YERAKSM-OH
92.0%
88.3%
3.7



(22)H-ERAKSNM-OH
94.3%
90.8%
3.5


(11)H-GGGRG-NH2
(11)H-GGGG-NH2
74.7%
72.3%
2.4









Examination 6: Effects of Various Sulfonic Acid Compounds-(2)

The increased points of the target peptide ratio (%) in the obtained solid were calculated in the same way as described above using the acid compounds, the target peptides, and the analogue peptides shown in the tables below.


The results are shown in the tables below.
















TABLE 18





              Target peptide
              Analogue peptide




embedded image


          embedded image
embedded image
        embedded image






















(9)H-
(10)H-
Before
Target
91.8%
93.0%
91.8%
94.6%


FRVDEEFQSPFAS
FRVDEEFQSPFAS
treatment
peptide






QSRGYFLFRPRN-
QSRGYFLFRRN-

ratio






NH2
NH2

(%)








Solid
Target
96.2%
94.9%
96.9%
94.8%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
4.4
1.9
5.1
0.2




of target








peptide ratio (%)


















(4)H-
(4)H-
Before
Target
88.5%
88.5%
88.5%
88.5%


ARLDVASEFRKKWNKALSR-
ARLDVASEFRKKNKALSR-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
92.6%
90.8%
92.5%
89.3%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
4.1
2.3
4.0
0.8




of target








peptide ratio (%)


















(6)H-
(6)H-
Before
Target
90.5%
89.9%
90.2%
90.3%


AGCKNFFWKTFTSC-
AGCKNFFWKTFTS-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
93.0%
90.4%
93.6%
90.3%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
2.5
0.5
3.4
0.0




of target








peptide ratio (%)





















TABLE 19





                    Target peptide
                    Analogue peptide




embedded image


    embedded image




















(6)H-
(6)H-
Before
Target
91 .8%
90.3%


AGCKNFFWKTFTSC-
AGCKNFFWKTFTS-
treatment
peptide




OH
OH

ratio (%)






Solid
Target
91.9%
93.7%




obtained
peptide






after
ratio






treatment
(%)















Increased point
0.1
3.4




of target






peptide ratio (%)






















TABLE 20







            Target peptide
            Analogue peptide


embedded image


embedded image




embedded image







(17)Pyr-
(26)Pyr-
Before
Target
90.0%
88.8%
89.1%


HWSYILRP-
HWSYLLRP-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
95.0%
93.6%
96.6%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
5.0
4.8
7.5




of target







peptide ratio (%)















          Target peptide
          Analogue peptide




embedded image


          embedded image





(17)Pyr-
(26)Pyr-
Before
Target
89.5%
89.3%


HWSYILRP-
HWSYLLRP-
treatment
peptide




NH2
NH2

ratio







(%)






Solid
Target
93.4%
90.6%




obtained
peptide






after
ratio






treatment
(%)















Increased point
3.9
1.3




of target






peptide ratio (%)





Pyr- =




embedded image

1 = D-Leu




















TABLE 21





                Target peptide
                Analogue peptide


embedded image


embedded image


              embedded image





















(17)Pyr-
(31)Pyr-
Before
Target
89.1%
88.7%
89.7%


HWSYILRP-
HWSYILLRP-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
94.8%
93.0%
92.9%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
5.7%
4.3%
3.2%




of target







peptide ratio (%)























TABLE 22





                      Target peptide
                      Analogue peptide


    embedded image
embedded image
        embedded image


embedded image
























(18)H-
(32)H-
Before
Target
94.7%
95.3%
94.8%
95.2%


HVTTV-
VTTV-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
98.5%
99.3%
97.6%
98.4%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
3.8
4.0
2.8
3.2




of target








peptide ratio (%)



















(33)H-
Before
Target
89.3%
88.9%
90.0%
93.4%



hVTTV-
treatment
peptide







NH2

ratio









(%)








Solid
Target
91.2%
92.6%
93.8%
98.0%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
1.9
3.7
3.8
4.6




of target








peptide ratio (%)





h = D-His




















TABLE 23





                Target peptide
                Analogue peptide


        embedded image
embedded image
      embedded image


embedded image
























(19)H-
(34)H-
Before
Target
91.2%
91.2%
90.7%
91.1%


YERAKSNM-
YERAKSNL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
92.2%
91.3%
93.5%
91.4%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
1.0
0.1
2.8
0.3




of target








peptide ratio (%)


















(6)H-
(35)H-
Before
Target
74.5%
76.6%
76.6%
74.0%


AGCKNFFWKTFTSC-
AGcKNFFWKTFTSC-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
76.6%
76.8%
77.1%
78.1%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
2.1
0.2
0.5
4.1




of target








peptide ratio (%)





c = D-Cys




















TABLE 24





              Target peptide
              Analogue peptide


          embedded image
embedded image
    embedded image


embedded image
























(19)H-
(34)H-
Before
Target
91.3
91.1
91.2
92.0


YERAKSNM-
YERAKSNL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
92.3
91.4
92.2
92.2




obtained
peptide








after
ratio








treatment
(%)



















Increased point
1.0
0.3
1.0
0.2




of target








peptide ratio (%)


















(6)H-
(35)H-
Before
Target
76.1
74.0
74.0
76.6


AGCKNFF
AGcKNFF
treatment
peptide






WKTFTSC-
WKTFTSC-

ratio






OH
OH

(%)








Solid
Target
76.8
78.1
77.7
77.2




obtained
peptide








after
ratio








treatment
(%)



















Increased point
0.7
4.1
3.7
0.6




of target








peptide ratio (%)






















TABLE 25





                    Target peptide
                    Analogue peptide




embedded image




embedded image




embedded image























(19)H-
(34)H-
Before
Target
91.2
91.1
91.3


YERAKSNM-
YERAKSNL-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
91.5
91.2
92.3




obtained
peptide







after
ratio







treatment
(%)

















Increased point
0.3
0.1
1.0




of target







peptide ratio (%)
















(6)H-
(35)H-
Before
Target
74.2
75.6
76.6


AGCKNFFWKTFTSC-
AGcKNFFWKTFTSC-
treatment
peptide





OH
OH

ratio








(%)







Solid
Target
77.2
79.2
77.8




obtained
peptide







after
ratio







treatment
(%)

















Increased point
3.0
3.6
1.2




of target







peptide ratio (%)





















TABLE 26





              Target peptide
              Analogue peptide




embedded image




embedded image






















(6)H-
(35)H-
Before
Target
76.9
74.9


AGCKNFFWKTFTSC-
AGcKNFFWKTFTSC-
treatment
peptide




OH
OH

ratio







(%)






Solid
Target
77.1
75.3




obtained
peptide






after
ratio






treatment
(%)















Increased point
0.2
0.4




of target






peptide ratio (%)
























TABLE 27





              Target peptide
              Analogue peptide


    embedded image


embedded image




embedded image























(20)H-
(36)H-
Before
Target
79.8%
80.2%
78.8%


VKRESYSGVT-
VKRESYSGV-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
88.1%
80.8%
84.5%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
8.3
0.6
5.7




of target







peptide ratio (%)
















(21)H-
(37)H-
Before
Target
92.9%
92.9%
93.0%


RAVLP-
RALP-
treatment
peptide





OH
OH

ratio








(%)







Solid
Target
93.7%
96.2%
95.1%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
0.8
3 .3
2.1




of target







peptide ratio (%)
















(27)Ac-
(1)Ac-
Before
Target
92.3%
91.7%
93.5%


YHYPEL-
YYPEL-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
99.1%
93.8%
99.0%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
6.8
2.1
5.5




of target







peptide ratio (%)
















(28)Ac-
(1)Ac-
Before
Target
90.7%
91.2%
90.9%


Y(MeK)YPEL-
YYPEL-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
93.0%
93.2%
98.1%




obtained
peptide







after
ratio







treatment
(%)

















Increased point
2.3
2.0
7.2




of target







peptide ratio (%)





MeK = α-Methyllysine




















TABLE 28





                    Target peptide
                    Analogue peptide




embedded image


      embedded image
embedded image
    embedded image






















(21)H-
(37)H-
Before
Target
92.9%
92.7%
94.5%
93.0%


RAVLP-
RALP-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
94.0%
96.8%
98.2%
94.3%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
1.1
4.1
3.7
1.3




of target








peptide ratio (%)


















(27)Ac-
(1)Ac-
Before
Target
92.3%
93.5%
93.5%
93.7%


YHYPEL-
YYPEL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
94.1%
95.4%
94.4%
95.9%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
1.8
1.9
0.9
2.2




of target








peptide ratio (%)


















(28)Ac-
(1)Ac-
Before
Target
91.0%
90.2%
90.7%
91.1%


Y(MeK)YPEL-
YYPEL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
91.5%
92.3%
92.0%
94.1%




obtained
peptide








after
ratio








treatment
(%)



















Increased point
0.5
2.1
1.3
3.0




of target








peptide ratio (%)





MeK = α-Methyllysine



















TABLE 29





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(22)H-
(38)H-
Before
Target
55.7%
66.6%
66.2%


(RC8)RRR
(RC8)RRRR
treatment
peptide ratio





((Me)F)R(SC5)-
(SC5)-NH2

(%)





NH2

Solid obtained
Target
67.7%
68.2%
77.3%




after
peptide ratio







treatment
(%)

















Increased point of target
12.0   
1.6  
11.1   




peptide ratio (%)
















(27)Ac-
(1)Ac-
Before
Target
92.3%
92.7%
92.8%


YHYPEL-NH2
YYPEL-
treatment
peptide ratio






NH2

(%)







Solid obtained
Target
93.7%
93.2%
94.2%




after
peptide ratio







treatment
(%)

















Increased point of target
1.4  
0.5  
1.4  




peptide ratio (%)
















(28)Ac-
(1)Ac-
Before
Target
91.3%
91.1%
91.0%


Y(MeK)YPEL-
YYPEL-
treatment
peptide ratio





NH2
NH2

(%)







Solid obtained
Target
91.8%
91.5%
94.5%




after
peptide ratio







treatment
(%)

















Increased point of target
0.5  
0.4  
3.5  




peptide ratio (%)







embedded image

(Me) F═N-Methylphenylalanine,





embedded image

MeK = α-Methyllysine





















TABLE 30





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(27)Ac-
(1)Ac-
Before
Target
92.5%
92.3%
92.8%
92.5%


YHYPEL-
YYPEL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
95.6%
98.7%
96.7%
95.7%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
3.1  
6.4  
3.9  
3.2  




target peptide








ratio (%)























TABLE 31





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(28)Ac-
(1)Ac-
Before
Target
91.9%
91.2%
91.1%
91.0%


Y(MeK)
YYPEL-
treatment
peptide






YPEL-
NH2

ratio






NH2


(%)








Solid
Target
99.2%
95.0%
94.4%
96.3%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
7.3  
3.8  
3.3  
5.3  




target peptide








ratio (%)





MeK = α-Methyllysine




















TABLE 32





Target peptide
Analogue peptide




embedded image




embedded image




embedded image




embedded image









(27)Ac-
(1)Ac-
Before
Target
93.6%
92.3%
92.5%
92.3%


YHYPEL-
YYPEL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
94.7%
94.1%
92.9%
93.8%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
1.1  
1.8  
0.4  
1.5  




target peptide








ratio (%)


















(28)Ac-
(1)Ac-
Before
Target
90.8%
92.4%
91.1%
91.9%


Y(MeK)
YYPEL-
treatment
peptide






YPEL-NH2
NH2

ratio









(%)








Solid
Target
91.3%
92.6%
91.3%
92.1%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
0.5  
0.2  
0.2  
0.2  




target peptide








ratio (%)


















(23)Ac-
(39)Ac-
Before
Target
93.1%
92.3%
92.4%
92.7%


HYFYPEL-
HYYPEL-
treatment
peptide






NH2
NH2

ratio









(%)








Solid
Target
95.2%
99.7%
95.0%
9.4%




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
2.1  
7.4  
2.6  
1.7  




target peptide








ratio (%)





MeK = α-Methyllysine



















TABLE 33





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(24)H-
(40)H-
Before
Target
93.7%
92.8%
92.4%


WPVTLNAQTID-
PVTLNAQTID-
treatment
peptide





NH2
NH2

ratio








(%)







Solid
Target
94.2%
93.2%
93.3%




obtained
peptide







after
ratio







treatment
(%)

















Increased point of
0.5  
0.4  
0.9  




target peptide







ratio (%)






















TABLE 34





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









(25)H-
(41)H-
Before
Target
95.5%
92.6%
92.9%


T((Me)G)
((Me)G)RK
treatment
peptide





RK(Dap)H-
(Dap)H-

ratio





OH
OH

(%)







Solid
Target
96.7%
94.3%
94.9%




obtained
peptide







after
ratio







treatment
(%)

















Increased point of
1.2  
1.7  
2.0  




target peptide







ratio (%)





(Me)G = N-Methylglycine




embedded image








As shown in Tables 5 to 34, the target peptide ratios in the obtained solid were increased (or the target peptide ratios in the obtained filtrate were decreased), and the sulfonic acid compounds showed excellent effects in the purification of various target peptides.


Test Example 2: Evaluation of Solvent-1

The crude target peptide ((8)H-YERAKSNM-OH) and the crude analogue peptide ((13)H-YERKSNM-OH) obtained in the above 1-1 were each dissolved in water/acetonitrile=1/2 to give a crude target peptide solution or a crude analogue peptide solution (10 mM, assuming 100% purity of each peptide).


The acid compound:




embedded image


was dissolved in water to give a 30 mM acid compound solution.


The crude target peptide solution (1 μmol, assuming 100% purity of the target peptide) and the crude analogue peptide solution (0.1 μmol, assuming 100% purity of the analogue peptide) were mixed.


The mixture was mixed with the acid compound solution (0.9 μmol) (the resulting mixture was subjected to HPLC analysis to give a measurement value of before treatment), and then the mixture was dried by lyophilization.


To the residue was added 100 μL of the solvent shown in the tables below and shaken overnight at room temperature. The resulting slurry was subjected to centrifugal filtration (14000 rpm, 1 min), and the cake was washed with 100 μL of the solvent 2 times. The filtrate was combined with the washings.


The resulting solid and the filtrate combined with the washings were analyzed by HPLC to give the values of “solid obtained after treatment” and “filtrate obtained after treatment”.


In the same manner as described above, the decreased points of target peptide ratio (%) in the obtained filtrate combined with the washings was calculated.


In addition, the HPLC area percentage of the target peptide was calculated using the HPLC analysis results of before treatment and the solid obtained after treatment, while the peak of sulfonic acid compound was excluded from the total area.










TABLE 35







Target peptide
(8) H-YERAKSNM-OH


Analogue peptide
(13) H-YERKSNM-OH














Before
Filtrate
Decreased



treatment
obtained
point of



Target
after treatment
target



peptide
Target peptide
peptide


Solvent
ratio (%)
ratio (%)
ratio (%)





IPA
91.6%
64.9%
26.7


MeOH
91.8%
91.5%
 0.3


1,4-Dioxane/Water (20/1)
91.2%
60.1%
31.1


DME/Water(20/1)
91.8%
74.9%
16.9


THF/Water (20/1)
91.8%
48.2%
43.6


IPA/Water (20/1)
91.7%
71.9%
19.8


Acetone/Water(20/1)
92.2%
70.3%
21.9


Acetonitrile/Water(20/1)
91.1%
64.4%
26.7


















TABLE 36






Before
Solid obtained



treatment
after treatment



Area of target
Area of target


Solvent
peptide (%)
peptide (%)







IPA
47.13
65.29


MeOH
47.47
54.74


1,4-Dioxane/Water(20/1)
49.07
70.66


DME/Water(20/1)
49.56
72.53


THF/Water (20/1)
49.61
68.26


IPA/Water(20/1)
49.47
66.80


Acetone/Water(20/1)
48.86
65.58


Acetonitrile/Water(20/1)
48.44
64.83





Area of target peptide (%) = Peak area of target peptide/(Total area − Peak area of sulfonic acid compound) × 100






As shown in Tables 35 and 36, the absolute purity and relative purity of the target peptide were increased. The results show that the sulfonic acid compound is effective in the purification of the target peptide using any of the solvents. That is, a solvent suitable for removing an analogue peptide can be selected by examining a constitution of solvents within the extent reasonably expected to do so.


Test Example 3: Scale-Up

The same target peptide, analogue peptide, and sulfonic acid compound as in Test Example 2 were used.


The crude target peptide, the crude analogue peptide, the sulfonic acid compound were each dissolved in water/acetonitrile=1/2 to give a crude target peptide solution (50 mM, assuming 100% purity of peptide), a crude analogue peptide solution (50 mM, assuming 100% purity of peptide), and a sulfonic acid compound solution (75 mM). The crude target peptide solution 1 mL (50 μmol), and the crude analogue peptide solution 0.1 mL (5 μmol) were mixed.


One milliliter of the sulfonic acid compound solution (75 μmol) was added thereto (the resulting mixture was subjected to HPLC analysis (Condition A) to give a measurement value of before treatment), and then the mixture was dried by lyophilization. To the residue was added 1 mL of IPA, the mixture was dispersed, and then lyophilized again to obtain a residue (67.35 mg).


To the residue was added 2 mL of IPA, the mixture was shaken at 700 rpm for 3 hours, and subjected to centrifugation (14000 rpm, 1 min). The solid and the supernatant were separated and each was lyophilized to give 60.74 mg of a residue derived from the solid portion, and 5.84 mg of a residue derived from the supernatant portion. Each was analyzed by HPLC (Condition A) to give the values of “after treatment”. The results are shown in the table below.










TABLE 37







Target peptide
(8) H-YERAKSNM-OH


Analogue peptide
(13) H-YERKSNM-OH










Solvent
Before treatment
Filtrate obtained
Decreased



Target peptide
after treatment
point of target



ratio
Target peptide
peptide



(%)
ratio (%)
ratio (%)


IPA
91.4%
63.5%
27.9









Even when the scale was increased, similar results as in Test example 2 were obtained.


Test Example 4: Removal of Sulfonic Acid Compound

The same target peptide, analogue peptide, and sulfonic acid compound as in Test example 3 were used.


The crude target peptide, the crude analogue peptide, the sulfonic acid compound were each dissolved in water/acetonitrile=1/2 to give a crude target peptide solution (50 mM, assuming 100% purity of peptide), a crude analogue peptide solution (50 mM, assuming 100% purity of peptide), and a sulfonic acid compound solution (75 mM). One milliliter of the crude target peptide solution (50 μmol) and 0.1 mL of the crude analogue peptide (5 μmol) were mixed.


One milliliter of the sulfonic acid compound solution (75 μmol) was added thereto (the resulting mixture was subjected to HPLC analysis (Condition A) to give a measurement value of before treatment), and then the mixture was dried by lyophilization. To the residue was added 1 mL of IPA, the mixture was dispersed, and then lyophilized again to give a residue (70.93 mg).


To the residue was added 5 mL of IPA, the mixture was shaken at 700 rpm for 3.5 hours, and subjected to centrifugation (14000 rpm, 1 min). The solid and the supernatant were separated. The solid was washed with 1 mL of IPA. The solid and the supernatant combined with the washing were respectively lyophilized to give 59.58 mg of a residue derived from the solid portion, and 5.40 mg of a residue derived from the supernatant portion.


Oasis WAX Cartridge (6 cc/150 mg) (Waters) was pretreated with 1 mL of 1N aqueous solution of sodium hydroxide, 7 mL of water (purified water), and 2 mL of water/MeOH (10/1) in this order.


Fifteen point seven four milligrams of the residue derived from the solid was dissolved in 1 mL of water/MeOH (10/1), (the resulting mixture was subjected to HPLC analysis (Condition A) to give a measurement value of after treatment, where the sulfonic acid was eluted at around 5.1 min). The mixture was loaded onto the pretreated Oasis WAX Cartridge (6 cc/150 mg), and 9 mL of water/MeOH (10/1) was passed through the cartridge. The solution passed through the cartridge was analyzed by HPLC (Condition A) to confirm removal of the sulfonic acid compound and give a measurement value after removal of sulfonic acid. The solution was lyophilized to give 9.47 mg of a residue.


The following table shows the HPLC area percentage of the target peptide in the HPLC analysis of the solution before the treatment with the sulfonic acid compound, of the solid obtained after the treatment, and of the sample after removal of the sulfonic acid compound.









TABLE 38







Area of Target peptide (%)











After removal of




sulfonic acid


Before treatment
After treatment
compound





42.85
61.59
76.44














Area





of





target





peptide






(
%
)


=



Peak





area





of





target





peptide





(


Total





area

-








Peak





area





of





sulfonic





acid





compound

)





×
100





Test Example 5: Evaluation of Solvent-2

The crude target peptide ((8)H-YERAKSNM-OH) and the crude analogue peptide ((23)H-YERAKSN-OH) obtained in the above 1-1 were each dissolved in water/acetonitrile=1/2 to give a crude target peptide solution and a crude analogue peptide solution (10 mM, assuming 100% purity of each peptide).


The sulfonic acid compound:




embedded image


was dissolved in water to give a 50 mM sulfonic acid compound solution.


The crude target peptide solution (1 μmol, assuming 100% purity of the target peptide) and the crude analogue peptide solution (0.1 μmol, assuming 100% purity of the analogue peptide) were mixed.


The mixture was mixed with the sulfonic acid compound solution (1.5 μmol) (the resulting mixture was subjected to HPLC analysis to give a measurement value of before treatment), and then the mixture was dried by lyophilization.


To the residue was added 100 μL of the solvent shown in the tables below and the mixture was shaken overnight at room temperature. The resulting slurry was subjected to centrifugal filtration (14000 rpm, 1 min), and the cake was washed with 100 μL of the solvent 2 times. The filtrate was combined with the washings.


The resulting solid and the filtrate combined with the washings were analyzed by HPLC to give the values of “solid obtained after treatment” and “filtrate obtained after treatment”.


In the same manner as described above, the decreased points of target peptide ratio (%) in the obtained filtrate combined with the washings were calculated.


In addition, the HPLC area percentage of the target peptide was calculated using the HPLC analysis results of before treatment and the solid obtained after treatment, while regarding the solid obtained after treatment the peak of sulfonic acid compound was excluded from the total area.


The results are shown in the tables below.


As shown in the tables below, the absolute purity and relative purity of the target peptide were increased. The results show that the sulfonic acid compound is effective in the purification of the target peptide using any of the solvents.










TABLE 39







Target peptide
(8) H-YERAKSNM-OH


Analogue peptide
(23) H-YERAKSN-OH










Solvent
Before
Filtrate
Decreased



treatment
obtained
point of



Target
after
target



peptide
treatment
peptide



ratio
Target
ratio



(%)
peptide
(%)




ratio (%)



IPA
  93.2%
61.3%
  31.9


MeOH
≥92.4%
89.8%
 ≥2.6


1,4-Dioxane/Water (20/1)
≥92.9%
77.3%
≥15.6


DME/Water (20/1)
≥92.5%
70.3%
≥22.2


IPA/Water (20/1)
≥92.7%
76.5%
≥16.2


Acetone/Water (20/1)
≥92.7%
47.1%
≥45.6


Acetonitrile/Water (20/1)
≥91.9%
32.4%
≥59.5


















TABLE 40






Before
Solid obtained



treatment
after treatment



Area of target
Area of target


Solvent
peptide (%)
peptide (%)







IPA
45.99
64.44


MeOH
43.91
51.47


1,4-Dioxane/Water (20/1)
46.03
65.65


DME/Water (20/1)
44.24
65. 46


THF/Water (20/1)
46.08
62.48


IPA/Water (20/1)
45.20
64.40


Acetone/Water(20/1)
46.07
60.27


Acetonitrile/Water (20/1)
43.31
62.53





Area of target peptide (%) = Peak area of target peptide/(Total area − Peak area of sulfonic acid compound) × 100






Test Example 6: Purification of Target Peptide-2

With respect to the target peptides, analogue peptides, and sulfonic acid compounds shown in the table below, the HPLC area percentage of the target peptide in the solution before treatment and in the solid obtained after treatment was calculated based on the results of the HPLC analysis performed in Test example 1, while the peak of sulfonic acid compound was excluded from the total area.













TABLE 41











Area of target peptide (%)













Analogue
Sulfonic acid
HPLC
Before
Solid obtained


Target peptide
peptide
compound
condition
treatment
after treatment





(14)H-ALRALRALR-OH
(17)H-ALALRALR-OH


embedded image


J
60.35
71.05





(8)H-YERAKSNM-OH
(23)H-YERAKSN-OH


embedded image


A
41.50
61.69





(8)H-YERAKSNM-OH
(23)H-YERAKSN-OH


embedded image


A
42.88
61.75





Area of target peptide (%) = Peak area of target peptide/(Total area-Peak area of sulfonic acid compound) × 100






Test Example 7: Purification of Target Peptide-3

(1) This test was performed in the same manner as Test Example 1 (paragraph [0141]), using H-AQKLRASD-OH (referred to as target peptide (7) in Table 1) as a crude analogue peptide, H-QKLRASD-OH (referred to as analogue peptide (19) in Table 1) as a crude target peptide, and the same sulfonic acid compounds as paragraph [0167](Table 14-1). The peptides were obtained in “1-1: Synthesis of Peptide (paragraphs [0082] to [0128])”.


The results are shown in the table below.















TABLE 42





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









H-
H-
Before
Target
83.3%
90.3%
91.3%


QKLRASD-
AQKLRASD-
treatment
peptide





OH
OH

ratio








(%)







Filtration
Target
83.0%
77.7%
32.5%




obtained
peptide







after
ratio







treatment
(%)

















Decreased point of
0.3  
12.6   
58.8   




target peptide ratio







(%)










(2) Similarly, this test was performed in the same manner as Test example 1 (paragraph [0141]), using H-AQKLRASD-OH (referred to as target peptide (7) in Table 1) as a crude analogue peptide, H-AQKLRSD-OH (referred to as analogue peptide (20) in Table 1) as a crude target peptide, and the same sulfonic acid compound as paragraph [0172] (Table 16-2). The results are shown in the table below.













TABLE 43





Target peptide
Analogue peptide




embedded image









H-
H-
Before
Target
76.6


AQKLRSD-
AQKLRASD-
treatment
peptide



OH
OH

ratio






(%)





Filtration
Target
74.5




obtained
peptide





after
ratio





treatment
(%)













Decreased point of
 2.1




target peptide ratio





(%)










(3) Similarly, this test was performed in the same manner as Test example 1 (paragraph [0141]), using H-YERAKSNM-OH (referred to as target peptide (8) in Table 1) as a crude analogue peptide, H-YERAKNM-OH (referred to as analogue peptide (9) in Table 1) as a crude target peptide, and the same sulfonic acid compounds as paragraph [0155] (Table 7). The results are shown in the table below.














TABLE 44





Target peptide
Analogue peptide




embedded image




embedded image









H-
H-
Before
Target
89.7
89.8


YERAKNM-
YERAKSNM-
treatment
peptide ratio




OH
OH

(%)






Solid
Target
91.1
91.3




obtained after
peptide ratio






treatment
(%)















Increased point of target
 1.4
 1.5




peptide ratio (%)










(4) Similarly, this test was performed in the same manner as Test example 1 (paragraph [0141]), using H-YERAKSNM-OH (referred to as target peptide (8) in Table 1) as a crude analogue peptide, H-YERAKSN-OH (referred to as analogue peptide (23) in Table 1) as a crude target peptide, and the same sulfonic acid compounds as paragraph [0168] (Table 14-2). The results are shown in the table below.















TABLE 45





Target peptide
Analogue peptide




embedded image




embedded image




embedded image









H-
H-
Before
Target
91.7
92.9
93.1


YERAKSN-
YERAKSNM-
treatment
peptide





OH
OH

ratio








(%)







Filtration
Target
81.5
91.3
92.5




obtained
peptide







after
ratio







treatment
(%)

















Decreased point of
10.2
 1.6
 0.6




target peptide ratio







(%)










(5) Similarly, this test was performed in the same manner as Test example 1 (paragraph [0141]), using H-AQKLRASD-OH (referred to as target peptide (7) in Table 1) as a crude analogue peptide, H-AQKRASD-OH (referred to as analogue peptide (24) in Table 1) as a crude target peptide, and the same sulfonic acid compound as paragraph [0172] (Table 16-2). The results are shown in the table below.













TABLE 46





Target peptide
Analogue peptide




embedded image









H-
H-
Before
Target
88.5


AQKLRSD-
AQKLRASD-
treatment
peptide



OH
OH

ratio






(%)





Filtration
Target
79.1




obtained
peptide





after
ratio





treatment
(%)













Decreased point of
 9.4




target peptide ratio





(%)









In Test Example 7, in order to further prove that the method of the present invention is a highly versatile peptide purification method, the molar ratio of the target peptide to the analogue peptide of Test Example 1 was reversed, that is, the analogue peptide of Test example 1 was used as a target peptide, and the target peptide of Test example 1 was used as an analogue peptide.


Specifically, Test example 1, paragraph [0167] [Table 14-1] shows the results of a model in which the target peptide (7)H-AQKLRASD-OH and analogue peptide (19)H-QKLRASD-OH were mixed in a molar ratio of about 10:1. On the other hand, Test example 7 was performed using the same sulfonic acid compounds, with a model of reversed molar ratio (theoretical value), i.e. (7)H-AQKLRASD-OH:(19)H-QKLRASD-OH=about 1:10.


In addition, regarding the following combinations of target peptide and analogue peptide in Test example 1, Test example 7 was performed using the reversed molar ratio (theoretical value), with the same sulfonic acid compounds as Test example 1:


target peptide (7)H-AQKLRASD-OH and analogue peptide (20)H-AQKLRSD-OH in paragraph [0172][Table 16-2];


target peptide (8)H-YERAKSNM-OH and analogue peptide (9)H-YERAKNM-OH in paragraph [0155][Table 7];


target peptide (8)H-YERAKSNM-OH and analogue peptide (23)H-YERAKSN-OH in paragraph [0168][Table 14-2];


target peptide (7)H-AQKLRASD-OH and analogue peptide (24)H-AQKLRSD-OH in paragraph [0172][Table 16-2].


The results of Test Example 7 clearly show that even when the mixing ratio was reversed, that is, even when the analogue peptide of Test Example 1 was used as a target peptide and the target peptide of Test Example 1 was used as an analogue peptide, the relative purity of the target peptide relative to the analogue peptide in the solid was increased (or reduced in the liquid).


Test Example 8: Effects of Various Sulfonic Acid Compounds (Removal of Multiple Analogue Peptides)

Using multiple crude analogue peptide solutions (0.1 μmol each, assuming 100% purity of peptide), the increased point of target peptide ratio (%) to each analogue peptide in the obtained solid was calculated in the same manner as in Test Example 1 (paragraph [0141]).


The results are shown in the tables below.
















TABLE 47





Target peptide
Analogue peptide




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H-
H-
Before
Target
95.4
95.8
95.8
96.0


AQKLRASD-
QKLRASD-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
95.7
96.8
96.6
96.4




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 0.3
 1.0
 0.8
 0.4




target peptide








ratio (%)



















H-
Before
Target
98.7
95.7
95.9
95.4



AQKLRSD-
treatment
peptide







OH

ratio









(%)








Solid
Target
99.4
97.8
97.6
97.0




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 0.7
 2.1
 1.7
 1.6




target peptide








ratio (%)























TABLE 48





Target peptide
Analogue peptide




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H-
H-
Before
Target
89.0
89.6
89.7
92.0


YERAKSNM-
YERASNM-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
91.5
91.4
91.3
93.8




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 2.5
 1.8
 1.6
 1.8




target peptide








ratio (%)



















H-
Before
Target
93.3
92.9
92.6
94.8



YERAKNM-
treatment
peptide







OH

ratio









(%)








Solid
Target
95.4
95.4
95.2
96.4




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 2.1
 2.5
 2.6
 1.6




target peptide








ratio (%)























TABLE 49





Target peptide
Analogue peptide




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H-
H-
Before
Target
88.1
88.8
90.1
88.6


YERAKSNM-
YERASNM-
treatment
peptide






OH
OH

ratio









(%)








Solid
Target
90.4
90.4
93.8
90.3




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 2.3
 1.6
 3.7
 1.7




target peptide








ratio (%)



















H-
Before
Target
92.5
92.5
93.6
93.2



YERAKNM-
treatment
peptide







OH

ratio









(%)








Solid
Target
96.3
95.0
96.5
95.0




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 3.8
 2.5
 2.9
 1.8




target peptide








ratio (%)



















H-
Before
Target
87.7
88.5
89.2
89.3



YERAKSN-
treatment
peptide







OH

ratio









(%)








Solid
Target
90.0
91.5
97.4
94.2




obtained
peptide








after
ratio








treatment
(%)



















Increased point of
 2.3
 3.0
 8.2
 4.9




target peptide








ratio (%)









As shown in Tables 47 to 49, the target peptide ratios in the obtained solid were increased, and the sulfonic acid compounds showed excellent effects in the purification of various target peptides.


INDUSTRIAL APPLICABILITY

The methods of the present application can be used to purify peptides.

Claims
  • 1. A method for purifying a target peptide from a peptide product obtained through a peptide synthesis, wherein the target peptide is free at the N-terminus and/or comprises at least one basic amino acid residue, the method comprising: (1) mixing the peptide product with a solvent in the presence of a sulfonic acid compound to provide a solid;(2) performing a solid-liquid separation to collect the solid.
  • 2-7. (canceled)
  • 8. The method of claim 1, wherein the sulfonic acid compound is of formula (I):
  • 9. The method of claim 8, wherein A is C6-10 aryl which may be substituted;X is(i) a single bond,(ii) an optionally substituted C1-4 alkylene,(iii) —CO—(CH2)n— wherein the CO binds to the A, or(iv) C2-3 alkenylene; andn is an integer in a range of from 1 to 3.
  • 10. The method of claim 8, wherein A is an optionally substituted bicyclic heterocyclic group;X is(i) a single bond,(ii) C1-4 alkylene,(iii) —CO—(CH2)n— wherein the CO binds to the A, or(iv) C2-3 alkenylene; andn is an integer in a range of from 1 to 3.
  • 11. The method of claim 8, wherein A is C2-3 alkenyl; andX is C1-4 alkylene.
  • 12. The method of claim 8, wherein A is C2-3 alkynyl; andX is C1-4 alkylene.
  • 13. The method of claim 1, wherein the target peptide has 5 to 31 amino acid residues.
  • 14. The method of claim 1, wherein the peptide product comprises a peptide analog.
  • 15. The method of claim 14, wherein the molar ratio of the peptide analog to the target peptide is 0.7 or less in the peptide product.
  • 16. The method of claim 1, wherein the peptide synthesis is a solid phase peptide synthesis.
  • 17. The method of claim 1, wherein the target peptide comprises a basic amino acid residue and is optionally free at the N-terminus.
  • 18. The method of claim 8, wherein A is a C6-14 aryl optionally comprising 1 to 5 substituents independently selected from the group consisting of C1-4 alkyl, —CO—C6-10 aryl, —OH, —O—C1-3 alkyl, —NO2, —CO2H, —CO2—C1-4 alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4 alkyl, —CF3, and C6-10 aryl.
  • 19. (canceled)
  • 20. The method of claim 8, wherein A is a C6-10 aryl selected from the group consisting of phenyl, naphthyl, and indanyl, wherein the C6-10 aryl is optionally substituted with 1 to 5 substituents independently selected from the group consisting of C1-4 alkyl, —CO—C6-10 aryl, —OH, —O—C1-3 alkyl, —NO2, —CO2H, —CO2—C1-4 alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4 alkyl, —CF3, and C6-10 aryl.
  • 21. The method of claim 8, wherein A is an optionally substituted bicyclic heterocyclic group selected from the group consisting of 2,3,4,5-tetrahydro-1H-1-benzazepinyl, benzoxanyl, indolinyl, isoindolinyl, phthalazinyl, chromanyl, benzofuranyl, benzothiophenyl, pyrimidinyl, benzothiazolyl, quinolyl, isochromanyl, and benzotriazolyl.
  • 22. The method of claim 8, wherein A is a bicyclic heterocyclic group optionally substituted with 1 to 5 substituents independently selected from the group consisting of C6-10 aryl, C1-4 alkyl, —CO—C6-10 aryl, —OH, —O—C1-3 alkyl, —NO2, —CO2H, —CO2—C1-4 alkyl, halogen, —NH2, —CH2—SO3H, —SO3H, —CN, —CO—C1-4 alkyl, —CF3, and oxo.
  • 23. The method of claim 8, wherein A is
  • 24. (canceled)
  • 25. The method of claim 8, wherein X is (i) a single bond,(ii) C1-4 alkylene optionally substituted with one methyl group, one benzyl group or one phenyl group, or(iii) —CO—CH2— wherein the CO binds to the A.
  • 26. (canceled)
  • 27. The method of claim 18, wherein X is (i) a single bond,(ii) C1-4 alkylene optionally substituted with one methyl group, one benzyl group, or one phenyl group, or(iii) —CO—(CH2)— wherein the CO binds to the A.
  • 28. The method of claim 8, wherein X is a single bond.
  • 29. The method of claim 23, wherein X is a single bond.
  • 30. The method of claim 1, wherein the sulfonic acid compound is:
  • 31. The method of claim 1, further comprising: removing the sulfonic acid compound.
  • 32. The method of claim 1, wherein the method increases the molar ratio of the target peptide to an peptide analog.
  • 33. A method for producing the target peptide, comprising the method of claim 1.
  • 34-36. (canceled)
Priority Claims (2)
Number Date Country Kind
2018-173042 Sep 2018 JP national
2019-008701 Jan 2019 JP national
PCT Information
Filing Document Filing Date Country Kind
PCT/JP2019/031405 8/8/2019 WO 00