ペプチド合成
Peptide Synthesis

　ペプチドとはアミノ酸のカルボキシル基とアミノ基がアミド結合でつながった化合物です。10アミノ酸単位程度までを低分子ペプチド，100アミノ酸単位程度までを（ポリ）ペプチドと呼びます。ペプチド単位はタンパク質だけでなく，例えば甘味料であるアスパルテームにも見出すことができます。ペプチドは生体内で様々な機能を発揮しており、特にペプチドホルモンは生体内の情報伝達において重要な役割を持っています。また，各種の合成ペプチドは生化学研究用試薬，医薬品として用いられています1)。
　生体から得られる生理活性ペプチドは比較的微量のものが多いこと，またペプチドに対応する遺伝子の配列が未知であることから，より詳細な研究のためにしばしばペプチド合成を行って機能解析に供します。残基数が数十までのペプチドであれば固相合成で得られるようになりました。また，残基数が少ないものでは，液相で大量に調製することも可能です。本項ではペプチド合成に用いられる試薬をご紹介します。

●固相合成
　Boc(t-Butoxycarbonyl)法，Fmoc(9-Fluorenylmethoxycarbonyl)法が知られていますが，脱保護条件の温和さ，樹脂からの切り出しの簡便さからFmoc法が広く使われています2)。ご使用になるペプチド合成機に付属のプロトコールに従い，簡便に合成を行うことができます。

　合成後はクリベージカクテルで樹脂から切り出し・脱保護を行い，メタノールで沈殿させます。その後はHPLCで精製し，目的ペプチドを得るのが一般的な方法です。

【必要な試薬】
・樹脂（Fmoc-アミノ酸のC末端を結合させたプレロード型）
・Fmoc-アミノ酸（側鎖保護型をおすすめします）
・縮合剤：1H-ベンゾトリアゾール-1-イルオキシトリピロリジノホスホニウムヘキサフルオロホスファート
・活性化剤：1-ヒドロキシベンゾトリアゾール
・溶媒： N,N-ジメチルホルムアミド（低水分用が望ましい）
・Fmoc基の脱保護：ピペリジン
・後処理剤（樹脂からの切り出し・側鎖の脱保護）：トリフルオロ酢酸，各種還元剤（残基により選択）

Fmoc-アミノ酸（側鎖保護型） / Fmoc-Amino Acids (Side Chain Protected Type)

縮合剤・活性化剤・脱保護剤 / Condensing Agents, Activating Agents & Deprotecting Agents

後処理剤 / After-treatment Agents

Fmoc-アミノ酸（側鎖保護型） / Fmoc-Amino Acids (Side Chain Protected Type)

B3150 B3150 B3167 B3167 F0669 F0669
F0305 F0305 F0294 F0294 F0295 F0295
F0296 F0296 F0297 F0297 F0298 F0298
F0299 F0299 F0652 F0652 F0293 F0293
F0507 F0507 F0508 F0508 F0505 F0505
F0506 F0506 F0653 F0653 B3072 B3072
B3168 B3168
B3150 4-tert-Butyl N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-aspartate
B3167 5-tert-Butyl N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-glutamate Hydrate
F0669 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-2-phenylglycine
F0305 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-alanine Monohydrate
F0294 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-isoleucine
F0295 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-leucine
F0296 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-methionine
F0297 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-phenylalanine
F0298 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-proline
F0299 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-L-valine
F0652 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-S-(triphenylmethyl)-L-cysteine
F0293 N-[(9H-Fluoren-9-ylmethoxy)carbonyl]glycine
F0507 Nalpha-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N1-tert-butoxycarbonyl-L-tryptophan
F0508 Nalpha-[(9H-Fluoren-9-ylmethoxy)carbonyl]-Ngamma-trityl-L-asparagine
F0505 Nalpha-[(9H-Fluoren-9-ylmethoxy)carbonyl]-O-tert-butyl-L-threonine
F0506 Nalpha-[(9H-Fluoren-9-ylmethoxy)carbonyl]-O-tert-butyl-L-tyrosine
F0653 Nalpha-[(9H-Fluoren-9-ylmethoxy)carbonyl]-tele-(triphenylmethyl)-L-histidine
B3072 Nepsilon-(tert-Butoxycarbonyl)-Nalpha-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine
B3168 O-tert-Butyl-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serine
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縮合剤・活性化剤・脱保護剤 / Condensing Agents, Activating Agents & Deprotecting Agents

H0468 H0468 B1774 B1774 P0453 P0453
H0468 1-Hydroxybenzotriazole Monohydrate
B1774 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate [Coupling Reagent for Peptide]
P0453 Piperidine
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後処理剤 / After-treatment Agents

E0032 E0032 D0944 D0944 B0041 B0041
D3479 D3479 E0143 E0143 M0097 M0097
B0991 B0991 T0191 T0191 T0431 T0431
E0032 1,2-Ethanedithiol
D0944 5,5'-Dithiobis(2-nitrobenzoic Acid) [for Determination of SH groups]
B0041 Benzenethiol
D3479 Diethyl Ether Anhydrous (stabilized with BHT)
E0143 Ethyl Methyl Sulfide
M0097 Methanol
B0991 tert-Butyl Methyl Ether
T0191 Thioanisole
T0431 Trifluoroacetic Acid
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その他のアミノ酸につきましては「アミノ酸」をご覧ください。

アミノ酸 / Amino Acids

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●液相合成
　通常の有機合成反応のように，バッチ式で保護アミノ酸どうしを縮合剤でカップリングする方法です3)。縮合後は後処理，応需精製を行い，次のアミノ酸との縮合反応を行います。縮合剤，活性化剤，保護基を合成目的に応じて柔軟に選択することが可能です。また，市販されていない保護アミノ酸を合成する際の保護基導入剤も本項にてご紹介します4)。

縮合剤 / Condensing Agents

保護剤 / Protecting Agents

縮合剤 / Condensing Agents

C0119 C0119 O0200 O0200 B2771 B2771
C1379 C1379 C1375 C1375 C0793 C0793
E0363 E0363 D1601 D1601 H0468 H0468
H1208 H1208 B1774 B1774 B1651 B1651
D1114 D1114 N0634 N0634 C1408 C1408
C1639 C1639 C1651 C1651 C0903 C0903
C1676 C1676 D2039 D2039 D3262 D3262
T1673 T1673 D2201 D2201 D2919 D2919
N0220 N0220 B1213 B1213 D1662 D1662
C1242 C1242 D2159 D2159 C1415 C1415
D1672 D1672 D0436 D0436 D0437 D0437
D0254 D0254 T2224 T2224 E0219 E0219
H0528 H0528 H0395 H0395 B0249 B0249
C1988 C1988 A1861 A1861 B1657 B1657
B1658 B1658 P0919 P0919 P1319 P1319
P1320 P1320
C0119 1,1'-Carbonyldiimidazole [Coupling Agent for Peptides Synthesis]
O0200 1,1'-Oxalyldiimidazole
B2771 1,3-Bis(2,2-dimethyl-1,3-dioxolan-4-ylmethyl)carbodiimide (This product is only available for selling domestically in Japan)
C1379 1-(Chloro-1-pyrrolidinylmethylene)pyrrolidinium Hexafluorophosphate
C1375 1-(Chloro-1-pyrrolidinylmethylene)pyrrolidinium Tetrafluoroborate
C0793 1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide Metho-p-toluenesulfonate [for Peptide Synthesis]
E0363 1-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
D1601 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride [Coupling Agent for Peptides Synthesis]
H0468 1-Hydroxybenzotriazole Monohydrate
H1208 1-Hydroxybenzotriazole-6-carboxamidomethyl Polystyrene Resin cross-linked with 1% DVB (50-100mesh) (1.3-1.5mmol/g)
B1774 1H-Benzotriazol-1-yloxytripyrrolidinophosphonium Hexafluorophosphate [Coupling Reagent for Peptide]
B1651 1H-Benzotriazol-1-yloxytris(dimethylamino)phosphonium Hexafluorophosphate [Coupling Reagent for Peptide]
D1114 2,2'-Dipyridyl Disulfide [for Peptide Synthesis]
N0634 2-(5-Norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium Tetrafluoroborate [Coupling Reagent for Peptide]
C1408 2-Chloro-1,3-dimethylimidazolinium Chloride
C1639 2-Chloro-1,3-dimethylimidazolinium Chloride (ca. 25% in Dichloromethane)
C1651 2-Chloro-1,3-dimethylimidazolinium Hexafluorophosphate
C0903 2-Chloro-1-methylpyridinium Iodide
C1676 2-Chloro-4,6-dimethoxy-1,3,5-triazine
D2039 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
D3262 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
T1673 3H-1,2,3-Triazolo[4,5-b]pyridin-3-ol [Peptide Coupling Additive]
D2201 4,6-Diphenylthieno[3,4-d]-1,3-dioxol-2-one 5,5-Dioxide
D2919 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium Chloride
N0220 4-Nitrophenol
B1213 Bis(2-oxo-3-oxazolidinyl)phosphinic Chloride
D1662 Di(N-succinimidyl) Carbonate
C1242 Diethyl Cyanophosphonate
D2159 Dimethylthiophosphinoyl Chloride
C1415 Diphenylphosphinic Chloride
D1672 Diphenylphosphoryl Azide
F0726 Fluoro-N,N,N',N'-tetramethylformamidinium Hexafluorophosphate
D0436 N,N'-Dicyclohexylcarbodiimide [for Peptide Synthesis]
D0437 N,N'-Dicyclohexylcarbodiimide (25% in Pyridine, ca. 1.2mol/L)
D0254 N,N'-Diisopropylcarbodiimide
T2224 N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium Tetrafluoroborate
E0219 N-Ethyl-5-phenylisoxazolium-3'-sulfonate
H0528 N-Hydroxy-5-norbornene-2,3-dicarboximide [for Peptide Synthesis]
H0395 N-Hydroxyphthalimide
B0249 N-Hydroxysuccinimide [Coupling Reagent for Peptide]
C1988 O-(6-Chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium Hexafluorophosphate
A1861 O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium Tetrafluoroborate
B1657 O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium Hexafluorophosphate [Coupling Reagent for Peptide]
B1658 O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium Tetrafluoroborate [Coupling Reagent for Peptide]
P0919 Pentafluorophenol
P1319 Propylphosphonic Acid Anhydride (Cyclic Trimer) (50% in Ethyl Acetate, ca. 1.7mol/L)
P1320 Propylphosphonic Acid Anhydride (Cyclic Trimer) (48% in N,N-Dimethylformamide, ca. 1.6mol/L)
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保護剤 / Protecting Agents

B1969 B1969 D1463 D1463 B0988 B0988
B1089 B1089 N0363 N0363 F0197 F0197
F0239 F0239 B3021 B3021 C0176 C0176
C1591 C1591 C1574 C1574 D1547 D1547
D3878 D3878 D3879 D3879 D3880 D3880
P1277 P1277 P1281 P1281 C1131 C1131
C1124 C1124 C0683 C0683 B0916 B0916
C0933 C0933
B1969 1-tert-Butoxycarbonyl-1,2,4-triazole
D1463 2,4-Dinitrophenylsulfenyl Chloride
B0988 2-(tert-Butoxycarbonyloxyimino)-2-phenylacetonitrile
B1089 2-Boc-thio-4,6-dimethylpyrimidine [Boc Agent for Peptides Synthesis]
N0363 2-Nitrophenylsulfenyl Chloride [N-Protecting Agent for Peptides Research]
F0197 9-Fluorenylmethyl Chloroformate [N-Protecting Agent for Peptides Research]
F0239 9-Fluorenylmethyl N-Succinimidyl Carbonate
B3021 Benzyl Chloroformate
C0176 Benzyl Chloroformate (30-35% in Toluene)
C1591 Carbonic Acid Benzyl 4-Nitrophenyl Ester
C1574 Carbonic Acid tert-Butyl 2,4,5-Trichlorophenyl Ester
D1547 Di-tert-butyl Dicarbonate [Boc-reagent for Amino Acid]
D3878 Di-tert-butyl Dicarbonate (ca. 30% in Dioxane)
D3879 Di-tert-butyl Dicarbonate (ca. 30% in Tetrahydrofuran)
D3880 Di-tert-butyl Dicarbonate (ca. 30% in Toluene)
P1277 Diallyl Pyrocarbonate
P1281 Dibenzyl Pyrocarbonate
C1131 N-(2-Chlorobenzyloxycarbonyloxy)succinimide
C1124 N-Carbobenzoxyoxysuccinimide
C0683 N-Ethoxycarbonylphthalimide [for Peptide Synthesis]
B0916 N-tert-Butoxycarbonylimidazole
C0933 tert-Butyl Carbazate
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文献

1) V. Marx, Chem. Eng. News 2005, 83, 17; Handbook of Biologically Active Peptides, ed. by A. J. Kastin, Academic Press, Boston, 2006.
2) Fmoc solid phase peptide synthesis: a practical approach, ed. by W. C. Chan, P. D. White Eds., Oxford University Press, New York, 2000.
3) 泉屋 信夫, ペプチド合成の基礎と実験, 丸善, 1985.
4) A. Isidro-Llobet, M. Álvarez, F. Albericio, Chem. Rev. 2009, 109, 2455 [DOI].


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