JP4468174B2 - ペプチドをアシル化するための方法 - Google Patents
ペプチドをアシル化するための方法 Download PDFInfo
- Publication number
- JP4468174B2 JP4468174B2 JP2004538774A JP2004538774A JP4468174B2 JP 4468174 B2 JP4468174 B2 JP 4468174B2 JP 2004538774 A JP2004538774 A JP 2004538774A JP 2004538774 A JP2004538774 A JP 2004538774A JP 4468174 B2 JP4468174 B2 JP 4468174B2
- Authority
- JP
- Japan
- Prior art keywords
- glp
- amide
- arg
- lys
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 99
- 238000000034 method Methods 0.000 title claims abstract description 83
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 21
- 239000002798 polar solvent Substances 0.000 claims abstract description 32
- 150000002148 esters Chemical class 0.000 claims abstract description 28
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 23
- 125000003277 amino group Chemical group 0.000 claims abstract description 14
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 14
- 239000001257 hydrogen Substances 0.000 claims abstract description 14
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims abstract description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 7
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 87
- 102100040918 Pro-glucagon Human genes 0.000 claims description 31
- 239000011541 reaction mixture Substances 0.000 claims description 29
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical group CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 23
- TWSALRJGPBVBQU-PKQQPRCHSA-N glucagon-like peptide 2 Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O)[C@@H](C)CC)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=CC=C1 TWSALRJGPBVBQU-PKQQPRCHSA-N 0.000 claims description 19
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 18
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 10
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 7
- 102000004877 Insulin Human genes 0.000 claims description 7
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 7
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
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- JUFFVKRROAPVBI-PVOYSMBESA-N chembl1210015 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N[C@H]1[C@@H]([C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@]3(O[C@@H](C[C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C3)C(O)=O)O2)O)[C@@H](CO)O1)NC(C)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 JUFFVKRROAPVBI-PVOYSMBESA-N 0.000 claims description 5
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- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 4
- 125000002843 carboxylic acid group Chemical group 0.000 claims description 4
- 125000004185 ester group Chemical group 0.000 claims description 4
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 4
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- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 125000004149 thio group Chemical group *S* 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Description
a)以下の一般式Iのアシル化剤と少なくとも1つの遊離のアミノ基を有するペプチドを、水性混合溶媒において塩基性条件下で、反応させることと;
nは、0〜8であり;
R1は、COOR4であり;
R2は、親油性部分であり;
R3は、R3が付着されるカルボキシル基と共に、反応性のエステルまたは反応性のN-ヒドロキシイミドエステルを示し;および、
R4は、水素、C1-12-アルキル、およびベンジルから選択され;
b)R4が水素でない場合は、塩基性条件下でアシル化されたペプチドのエステル基(COOR4)をけん化することと;
c)N-アシル化されたペプチドを単離することと、
の工程を含み、前記a)の水性混合溶媒は、10未満%w/w非プロトン性極性溶媒を含むことによって特徴づけられる方法を提供する。
ペプチドおよびタンパク質
本発明は、インビボでのクリアランス速度を減少させるために、いずれかのペプチド(または、タンパク質)に、親油性のアシル基を導入するために有用である。このようなペプチドおよびタンパク質の例は、ACTH、副腎皮質刺激ホルモン放出因子、アンジオテンシン、カルシトニン、exendinおよびこれらの類似体、インスリンおよびこれらの類似体、グルカゴンおよびこれらの類似体、グルカゴン様ペプチド-1およびこれらの類似体、グルカゴン様ペプチド-2およびこれらの類似体、インシュリン様成長因子-1、インシュリン様成長因子-2、胃抑制性ペプチド、成長ホルモン放出因子、下垂体アデニル酸シクラーゼ活性化ペプチド、セクレチン、エンテロガストリン、ソマトスタチン、ソマトトロピン、ソマトメジン、副甲状腺ホルモン、トロンボポエチン、エリスロポエチン、視床下部放出因子、プロラクチン、甲状腺刺激ホルモン、エンドルフィン、エンケファリン、バソプレッシン、オキシトシン、オピオイドおよびこれらの類似体、スーパーオキシドジスムターゼ、インターフェロン、アスパラギナーゼ、アルギナーゼ、アルギニンデアミナーゼ、アデノシンデアミナーゼ、およびリボヌクレアーゼである。
本発明に従った方法において、少なくとも1つの遊離のアミノ基を有するペプチド(または、タンパク質)を一般式Iのアシル化剤と作用させる。
式Iのアシル化剤とペプチドまたはタンパク質との間の反応は、10%w/w未満の非プロトン性極性溶媒を含む水溶液中において塩基性条件下で行われる。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
表1に、実施例1〜5の実験のArg34-GLP-17-37をアシル化するために使用する実験条件を要約してあり、生じる不純物Arg34Lys26-[N-ε-(α-Glu(N-ヘキサデカノイル))]-GLP-17-37(α-gluと省略)の含量を一覧表に記載してある。安定剤としてH2SO4を伴わないと、低濃度の非プロトン性極性溶媒は、α-glu不純物の量を減少させる際に有効なことが明らかである。安定剤を添加すると、有意な量のα-glu不純物が、有意に高い濃度、すなわち28%w/w以上の濃度の非プロトン性極性溶媒で始まる。
ナトリウム結晶化したdes(B30)-ヒトインスリン(1.74gの等電沈殿によって単離した凍結ペプチド材料、約0.088mmol)を室温で0.1mol/kgのトリエチルアミン(10.85ml)に溶解した。溶液のpHは、10.9であった。次いで、N-メチル-2-ピロリドン(1.25ml)に溶解したメチル(2S)-5-[(2,5-ジオキソ-1-ピロリジニル)オキシ]-2-{[(3a,5b)-3-ヒドロキシ-9-メチル-24-オキソコラン-24-イル]アミノ}-5-オキソペンタノアート(55.1mg、0.089mmol)を添加した。室温で30分後に、水(5℃、85ml)を添加した。
Arg34GLP-1(7-37)を従来の組換えDNA技術によって(たとえば、国際公開公報第98/08871に記載したように)、酵母(S.cerevisiae)に発現した。次いで、培養液中のArg34GLP-1(7-37)を従来の逆相クロマトグラフィーによって精製し、その後ペプチドの等電pHで、すなわちpH5.4で沈殿させた。沈殿を遠心分離によって単離し、凍結した。
Claims (31)
- N-アシル化されたペプチドを生成するための方法であって、該方法は:
a)以下の一般式Iのアシル化剤と少なくとも1つの遊離のアミノ基を有するペプチドを、水性混合溶媒において塩基性条件下で、反応させることと;
nは、0〜8であり;
R1は、COOR4であり;
R2は、親油性部分であり;
R3は、R3が付着されるカルボキシル基と共に、反応性のエステルまたは反応性のN-ヒドロキシイミドエステルであって、アミンをアシル化するために適したカルボン酸基のエステルを官能化させた形態のエステルを示し;および、
R4は、水素、C1-12-アルキル、およびベンジルから選択され;
b)R4が水素でない場合は、塩基性条件下でアシル化されたペプチドのエステル基(COOR4)をけん化することと;
c)N-アシル化されたペプチドを単離することと、
の工程を含み、前記a)の水性混合溶媒は、7未満%w/w非プロトン性極性溶媒を含むことによって特徴づけられる方法。 - アシル化剤が固体として反応混合物に添加される、請求項1に記載の方法。
- 請求項1または2に記載の方法であって、前記工程a)の反応は、非プロトン性極性溶媒の存在下において生じ、かつ前記非プロトン性極性溶媒は、N-メチル-2-ピロリドン、テトラヒドロフラン、およびジメチルスルホキシドからなる群より選択される方法。
- 非プロトン有機溶媒の全てが、アシル化剤のための溶媒として反応混合物に添加される、請求項3に記載の方法。
- アシル化剤が、酸を添加することによって安定化される溶液として反応混合物に添加される、請求項3または4に記載の方法。
- 前記酸が、0.01%w/w〜1%w/wの濃度で非プロトン性極性溶媒に添加される、請求項5に記載の方法。
- 前記酸が、0.05%w/w〜0.5%w/wの濃度で非プロトン性極性溶媒に添加される、請求項5に記載の方法。
- 前記酸が、硫酸、メタンスルホン酸、およびトリフルオロ酢酸からなる群より選択される、請求項5〜7のいずれか1項に記載の方法。
- 前記工程a)の反応が、非プロトン性極性溶媒の非存在下で生じる、請求項1または2に記載の方法。
- R4が水素である、請求項1〜9のいずれか1項に記載の方法。
- R4がC1-8-アルキルおよびベンジルから選択される、請求項1〜9のいずれか1項に記載の方法。
- R3は、R3が付着されるカルボキシル基と共に、反応性のN-ヒドロキシイミドエステルを示す、請求項1〜11のいずれか1項に記載の方法。
- アシル化されたペプチドエステルが、10〜14の範囲のpH値でけん化される、請求項1〜9のいずれか1項に記載の方法。
- アシル化されたペプチドエステルが、9〜13のpH範囲でけん化される、請求項1〜9のいずれか1項に記載の方法。
- 前記工程a)の反応混合物のpHが、pH9〜pH13である、請求項1〜14のいずれか1項に記載の方法。
- 前記工程a)の反応混合物のpHが、pH10〜pH12である、請求項1〜14のいずれか1項に記載の方法。
- 前記工程a)の反応混合物のpHが、pH11.0〜pH11.5である、請求項1〜14のいずれか1項に記載の方法。
- 前記工程a)の反応混合物の温度が、0〜50℃の範囲である、請求項1〜17のいずれか1項に記載の方法。
- 前記工程a)の反応混合物の温度が、5〜40℃の範囲である、請求項1〜17のいずれか1項に記載の方法。
- 前記工程a)の反応混合物の温度が、10〜30℃の範囲である、請求項1〜17のいずれか1項に記載の方法。
- R2が、C3-39-アルキル、C3-39-アルケニル、C3-39-アルカジエニル、およびステロイド残基から選択される、請求項1〜20のいずれか1項に記載の方法。
- R2-C(=O)-が、リトコロイルおよびヘキサデカノイルからなる群より選択される、請求項21に記載の方法。
- 工程a)の出発原料として使用する前記ペプチドが、RP-HPLCによって決定すると少なくとも80%のペプチド純度を有する、請求項1〜22のいずれか1項に記載の方法。
- 工程a)の出発原料として使用する前記ペプチドが、RP-HPLCによって決定すると少なくとも90%のペプチド純度を有する、請求項1〜22のいずれか1項に記載の方法。
- 工程a)の出発原料として使用する前記ペプチドが、RP-HPLCによって決定すると少なくとも93%のペプチド純度を有する、請求項1〜22のいずれか1項に記載の方法。
- 工程a)の出発原料として使用する前記ペプチドが、RP-HPLCによって決定すると少なくとも95%のペプチド純度を有する、請求項1〜22のいずれか1項に記載の方法。
- 工程a)の出発原料として使用する前記ペプチドが、RP-HPLCによって決定すると97%のペプチド純度を有する、請求項1〜22のいずれか1項に記載の方法。
- 前記ペプチドが、GLP-1、exendin-4、GLP-2、グルカゴン、インスリン、GLP-1および以下のものなどの切断された類似体:Arg26-GLP-1(7-37); Arg34-GLP-1(7-37); Lys36-GLP-1(7-37); Arg26,34Lys36-GLP-1(7-37); Arg26,34Lys38GLP-1(7-38); Arg26,34Lys39-GLP-1(7-39); Arg26,34Lys40-GLP-1(7-40); Arg34Lys36-GLP-1(7-37); Arg26Lys39-GLP-1(7-39); Arg34Lys40-GLP-1(7-40); Arg26,34Lys36,39-GLP-1(7-39); Arg26,34Lys36,40-GLP-1(7-40); Gly8Arg26-GLP-1(7-37); Gly8Arg34-GLP-1(7-37); Gly8Lys36-GLP-1(7-37); Gly8Arg26,34Lys36-GLP-1(7-37); Gly8Arg26,34Lys39-GLP-1(7-39); Gly8Arg26,34Lys40-GLP-1(7-40); Gly8Arg26Lys36-GLP-1(7-37); Gly8Arg34Lys36-GLP-1(7-37); Lys36-GLP-1(7-37); Arg26,34Lys36-GLP-1(7-37); Arg26,34-GLP-1(7-37); Arg26,34Lys40-GLP-1(7-37); Arg26Lys36-GLP-1(7-37); Arg34Lys36-GLP-1(7-37); Val8Arg22-GLP-1(7-37); Met8Arg22-GLP-1(7-37);Gly8His22-GLP-1(7-37); Val8His22-GLP-1(7-37); Met8His22-GLP-1(7-37);His37-GLP-1(7-37); Gly8-GLP-1(7-37); Val8-GLP-1(7-37); Met8-GLP-1(7-37);Gly8Asp22-GLP-1(7-37); Val8Asp22-GLP-1(7-37); Met8Asp22-GLP-1(7-37);Gly8Glu22-GLP-1(7-37); Val8Glu22-GLP-1(7-37); Met8Glu22-GLP-1(7-37); Gly8Lys22-GLP-1(7-37); Val8Lys22-GLP-1(7-37); Met8Lys22-GLP-1(7-37); Gly8Arg22-GLP-1(7-37); Val8Lys22His37-GLP-1(7-37); Gly8Glu22His37-GLP-1(7-37); Val8Glu22His37-GLP-1(7-37); Met8Glu22His37-GLP-1(7-37);Gly8Lys22 His37-GLP-1(7-37); Met8Lys22His37-GLP-1(7-37);Gly8Arg22His37-GLP-1(7-37); Val8Arg22His37-GLP-1(7-37); Met8Arg22His37-GLP-1(7-37); Gly8His22His37-GLP-1(7-37); Val8His22His37-GLP-1(7-37); Met8His22His37-GLP-1(7-37); Gly8His37-GLP-1(7-37); Val8His37-GLP-1(7-37); Met8His37-GLP-1(7-37);Gly8Asp22 His37-GLP-1(7-37); Val8Asp22His37-GLP-1(7-37); Met8Asp22His37-GLP-1(7-37); Arg26-GLP-1(7-36)-アミド; Arg34-GLP-1(7-36)-アミド; Lys36-GLP-1(7-36)-アミド; Arg26,34Lys36-GLP-1(7-36)-アミド; Arg26,34-GLP-1(7-36)-アミド; Arg26,34Lys40-GLP-1(7-36)-アミド; Arg26Lys36-GLP-1(7-36)-アミド; Arg34Lys36-GLP-1(7-36)-アミド; Gly8-GLP-1(7-36)-アミド; Val8-GLP-1(7-36)-アミド; Met8-GLP-1(7-36)-アミド;Gly8Asp22-GLP-1(7-36)-アミド; Gly8Glu22His37-GLP-1(7-36)-アミド; Val8Asp22-GLP-1(7-36)-アミド; Met8Asp22-GLP-1(7-36)-アミド;Gly8Glu22-GLP-1(7-36)-アミド; Val8Glu22-GLP-1(7-36)-アミド; Met8Glu22-GLP-1(7-36)-アミド; Gly8Lys22-GLP-1(7-36)-アミド; Val8Lys22-GLP-1(7-36)-アミド; Met8Lys22-GLP-1(7-36)-アミド; Gly8His22His37-GLP-1(7-36)-アミド; Gly8Arg22-GLP-1(7-36)-アミド; Val8Arg22-GLP-1(7-36)-アミド; Met8Arg22-GLP-1(7-36)-アミド;Gly8His22-GLP-1(7-36)-アミド; Val8His22-GLP-1(7-36)-アミド; Met8His22-GLP-1(7-36)-アミド;His37-GLP-1(7-36)-アミド; Val8Arg22His37-GLP-1(7-36)-アミド; Met8Arg22His37-GLP-1(7-36)-アミド; Gly8His37-GLP-1(7-36)-アミド; Val8His37-GLP-1(7-36)-アミド; Met8His37-GLP-1(7-36)-アミド;Gly8Asp22 His37-GLP-1(7-36)-アミド; Val8Asp22His37-GLP-1(7-36)-アミド; Met8Asp22His37-GLP-1(7-36)-アミド; Val8Glu22His37-GLP-1(7-36)-アミド; Met8Glu22His37-GLP-1(7-36)-アミド;Gly8Lys22 His37-GLP-1(7-36)-アミド; Val8Lys22His37-GLP-1(7-36)-アミド; Met8Lys22His37-GLP-1(7-36)-アミド;Gly8Arg22His37-GLP-1(7-36)-アミド; Val8His22His37-GLP-1(7-36)-アミド; Met8His22His37-GLP-1(7-36)-アミド、並びにHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2(ZP-10)からなる群より選択される、請求項1〜27のいずれか1項に記載の方法。
- 前記ペプチドは、exendin-3、exendin-4、Arg34-GLP-1(7-37)、Gly8-GLP-1(7-36)-アミド、Gly8-GLP-1(7-37)、Val8-GLP-1(7-36)-アミド、Val8-GLP-1(7-37)、Val8Asp22-GLP-1(7-36)-アミド、Val8Asp22-GLP-1(7-37)、Val8Glu22-GLP-1(7-36)-アミド、Val8Glu22-GLP-1(7-37)、Val8Lys22-GLP-1(7-36)-アミド、Val8Lys22-GLP-1(7-37)、Val8Arg22-GLP-1(7-36)-アミド、Val8Arg22-GLP-1(7-37)、Val8His22-GLP-1(7-36)-アミド、Val8His22-GLP-1(7-37)、des(B30)ヒトインスリンからなる群より選択される、請求項1〜28のいずれか1項に記載の方法。
- 前記工程a)の反応混合物が、反応の間に一定のpHを維持するために適している緩衝液を含む、請求項1〜17のいずれか1項に記載の方法。
- 前記ペプチドが、インスリン・ペプチドでない、請求項1〜30のいずれか1項に記載の方法。
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