JP5232769B2 - C−反応性タンパク質についての結合剤 - Google Patents
C−反応性タンパク質についての結合剤 Download PDFInfo
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- JP5232769B2 JP5232769B2 JP2009504167A JP2009504167A JP5232769B2 JP 5232769 B2 JP5232769 B2 JP 5232769B2 JP 2009504167 A JP2009504167 A JP 2009504167A JP 2009504167 A JP2009504167 A JP 2009504167A JP 5232769 B2 JP5232769 B2 JP 5232769B2
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 1
- 239000002594 sorbent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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Description
− 配列番号1に記載の配列を有するポリペプチドを合成する工程;
− リジンへの、ホスホコリン誘導体の付着に適切な条件下で、ホスホコリン誘導体を、当該ポリペプチドの未ブロックのリジンと接触させる工程;
− 必要に応じて、リジンへの、レポーター基の付着に適切な条件下で、レポーター基を、当該ポリペプチドの未ブロックのリジン基と接触させる工程、を包含する。
OP(OH)2O(CH2)2N+(CH3)2(CH2)nCOOR1 (I)
ここで1≦n≦12であり、R1は、約6〜8のpKaを伴う脱離基である。活性なエステルの合成(ここで、nは4、6、または11であり、およびR1はp−ニトロ−フェニルである)は、以下の実施例の節において記載される。
本発明の1つの局面は、本発明の結合剤を使用する結合アッセイに、ならびにより詳細には、生物学的サンプル中のリウマチ様因子およびヒト抗マウス抗体(HAMA)から生じる干渉を排除するための方法に関する。
− サンプルを、請求項1〜6のいずれかに記載の第1のポリペプチド二量体と接触させる工程であって、該第1のポリペプチドは固体支持体に結合される工程;
− サンプルを、請求項3〜6のいずれかに記載の、これに付着されるレポーター基を有する、第2のポリペプチド二量体と接触させる工程;および
− 該第2のポリペプチド二量体上のレポーター基の存在または不在を検出する工程、を包含する。
ポリペプチド足場に付着されるべきホスホコリン誘導体は、上述で説明されるように、1〜12個の炭素原子のスペーサーを有し得る。これらはホスホコリンについて、PCと表わされ、スペーサーの長さを与える番号が続き、すなわちPC6は、6個の炭素原子のスペーサーを有する。
PC6−pニトロフェニルエステル合成
リン酸2−ブロモ−エチルエステルジエチルエステル。乾燥ジクロロメタン(25mL)中の2−ブロモエタノール(1.42mL、20mmol)および乾燥ピリジン(3.23mL、40mmol)の溶液を、0℃に冷却した。ジエチルクロロホスファート(3.36mL、23mmol)を滴下して添加し、そして反応混合物を室温にて24時間攪拌した。ジエチルエーテル(40mL)および1N HCl(40mL)を添加し、そして有機層を分離し、続いて1N HClおよび飽和化NaHCO3で洗浄し、そしてMgSO4上で乾燥した。溶媒の蒸発後、残渣をカラムクロマトグラフィー(シリカゲル、酢酸エチル/ペンタン 1:1)により精製して、生成物(4.85g、18.6mmol、93%)を、淡黄色がかった油として得た。
Fmoc−ウンデカン酸(635mg、1.5mmol)を乾燥DMF(2ml)中に溶解し、そして乾燥ジクロロメタン(10ml)中のジイソプロピルカルボジイミド(0.12ml、0.75mmol)の溶液を添加した。得られる黄色の混合物を、0℃にて20分間攪拌した。揮発性ジクロロメタンの除去後、対称無水物の溶液をWang樹脂(250mg、1.2mmol/g、0.3mmol)に添加した。DMAP(18mg、0.15mmol)を添加し、そして反応混合物を一晩攪拌した(W.C.Chan,P.D.White Fmoc固相ペプチド合成、実践的なアプローチにおいて(W.C.Chan,P.D.White編)Oxford University Press、Oxford 2000、55〜56頁)。樹脂をDMF(5×2分間)、ジクロロメタン(5×2分間)で洗浄し、そしてジエチルエーテルで収縮した。手順を1回反復し、そして0.8mmol/g(66%)の最終置換レベルを、Fmoc−ピペリジン付加物のUV分光光度法に基づいて決定した(同書、62〜63頁)。
乾燥アセトニトリル(5ml)中のカルボン酸Xの溶液に、トリエチルアミン(0.03ml、0.2mmol)、p−ニトロフェニルクロロホルマート(80mg、0.39mmol)、およびDMAP(7mg)を続けて添加した。4時間後、水中の0.1%TFAの添加により反応を停止し、そして続いてRP HPLC(Supelco Discovery C18、21.2mm×15cm、5μm、A:IPA/H2O 5:95中の0.1%TFA、B:IPA/H2O 90:10中の0.1%TFA、15〜45% B 20分間にわたる、tR=16.5分)により精製して、p−ニトロフェニルエステルXを無色の油として得た(4mg、0.007mmol、%)。LC−MS(Phenomenex Gemini、C18、5μm、150×3.0mm、A:水中の0.1%ギ酸、B:アセトニトリル中の0.1%ギ酸、10〜90% B 10分間にわたる)、tR=5.2分(UVシグナル)、M=531.5(M+)。
保護化p−ニトロフェニルエステル(4mg、0.007mmol)を、乾燥アセトニトリル(3ml)中に溶解し、そしてトリメチルシリルブロミド(0.11ml、0.07mmol)を、0、12、および24時間後に、それぞれ添加した。反応混合物を室温にて、合計36時間攪拌した。水中(1ml)の0.1% TFAの添加後、溶液をrp HPLC(Hichrom C8、21.2mm×25cm、10μm、A:ACN/H2O 10:90中の0.1%TFA、B:ACN/H2O 90:10中の0.1%TFA、40〜60% B 15分間にわたる、tR=14.2分)により精製して、ホスホコリン誘導体(1.1mg、0.002mmol、27%)をTFA塩として得た。
PC6についてと同じ手順
メチルエステルX(516mg、3.5mmol)を、乾燥アセトニトリル(8ml)中に溶解し、そしてK2CO3(484mg、3.5mmol)を添加した。この懸濁液を窒素ガス下で24時間、還流した。室温への冷却後、水を添加し、そしてpHをTFAの添加により2に調節した。溶液をrp HPLC(Supelco Discovery C18、21.2mm×15cm、5μm、A:H2O中の0.1% TFA、5% IPA、B:IPA中の0.1%TFA、10% H2O、0〜10% B 10分間にわたる、tR=8.8分)により精製し、そして凍結乾燥して、第4級アミンのTFA塩を、淡黄色の油として得た。
PC6についてと同じ手順。
乾燥アセトニトリル(5ml)中のカルボン酸の溶液に、トリエチルアミン、p−ニトロフェニルクロロホルマート、およびDMAPを連続的に添加した。反応を、水中の0.1%TFAの添加により4時間後に停止し、続いてRP HPLC(Supelco Discovery C18、21.2mm×15cm、5μm、A:H2O中の0.1% TFA、5% IPA、B:IPA中の、0.1%TFA、10% H2O、10〜30% B 13分間にわたる、tR=11.8分)により精製して、p−ニトロフェニルエステルを、無色の油として得た。
優先年の間に、ホスホコリンリガンドPC6を調製するための代替のストラテジーが開発された。これは以下に概説される。PC6(化合物9)の調製のためのこのストラテジーは、スキーム3において概説される。リン酸化は、この基が、保護化形態にあっても、他の必要な転換工程を通じて保有するには問題があると考慮されたので、最終工程として選択された。
水性ホルムアルデヒド(37%、40mL)中の6−アミノヘキサン酸(2、13.2g)の溶液を、Pd/C(1.0g)と混合し、混合物を、磁性攪拌を備えるParrステンレススチール装置において、一晩、室温および50バールにて水素化した。TLC(酢酸エチル−メタノール−酢酸−水、6:3:3:2、ニンヒドリン検出)による確認は、開始材料(rf 0.7)からよりゆっくりと移動する生成物(rf 0.5、茶味を帯びた色)へのほとんど完全な変換を明らかにした。反応混合物を、Celiteの層を介して濾過し、フィルター層を水(20mL)で洗浄し、希釈し(60mL)、そして溶液をDowex−50 W×2−100メッシュのカラム(H+ホルム、0.7meq/mL、150mL、ミリ−Q水で注意深く予洗した)を介して、ゆっくりと通過させた。次いで、カラムをミリ−Q水で洗浄し(200+100mL)、次いで生成物を2%アンモニア水溶液(400mL)で溶出した。溶出物を、TLC(酢酸エチル−メタノール−酢酸−水、6:3:3:2、ニンヒドリン検出)によりモニターした。適切な画分を部分的に蒸発し、次いで凍結乾燥して、半固体の残渣の状態にした(13.7g、86%)。直接導入ES−MSは、m/z 160.2(M+H)での強力なピークを明らかにし、N,N−ジメチル化生成物に対応した(3)。
16個の42残基ペプチドのライブラリー(図1)を、固相ペプチド合成により調製した。ライブラリーの成分は、荷電される残基の数およびリガンドの取り込みの部分に関して変化した。合成手順は、各場合において同じであり、そして3−C15L8Cys24(TA4Cys(Acm))の手順により本明細書中で例示される。
ジスルフィド結合形成(H.Tamamura、A.Otaka、J.Nakamura、K.Okubo、T.Koide、K.Ikeda、T.Ibuka、N.Fuji Int.J.Peptide Protein Res.1995、45、312−319):
ペプチド(2.4mg(0.51μmol)の3−C15L8Cys(Acm)24)を、TFA(200μl)中に溶解し、そして銀トリフラート(5mg、20μmol)およびアニソール(1滴)を添加した。反応混合物を、冷蔵庫中、4℃にて24時間、インキュベートした。ジエチルエーテルの添加後、ペプチドは沈殿し、そして冷却ジエチルエーテルで3回洗浄した。沈殿物にDMSO(0.25ml)および1N HCl(1ml)を添加した。反応混合物を室温にて22時間攪拌した。懸濁液を遠心分離し、そして沈殿物を廃棄した。液体を希釈NaOHで中和化し、そしてRP−HPLC(分析等級:Varian C18、150×4.6mm、5μm、A:H2O中の0.1%TFA、10%ACN、B:ACN中の0.1%TFA、10% H2O、30〜60% B 30分間にわたる、tR=23.4分)によって解析した。
これは、ホスホコリン誘導体をペプチドに取り込むためのプロトコルの具体例であり、この場合において、TA4+は、Cys−残基上に保護基Acmを備える。100μLの0.1Mリン酸緩衝液 pH8中のTA4 +Cys(Acm)ペプチド(500μg、0.1μモル)の溶液に、DMSO中の2.8μLの0.1M PC6−pニトロフェニルエステル(0.3μモル、3当量)を添加した。反応混合物を、4℃にてインキュベートした。LC−MS(Phenomenex Gemini、C18、5μm、150×3.0mm、A:水中の0.1%ギ酸、B:アセトニトリル中の0.1%ギ酸、10〜90% B、10分間にわたる)、tR=4.1分(UVシグナル)、m/z=[M+8H]8+について670.5、[M+7H]7+について766.1、[M+6H]6+について893.3、[M+5H]5+について1071.9。
18μLの0.1Mリン酸緩衝液pH8中のTA4+PC6Cys(Acm)ペプチド(90μg、18nmol)の溶液に、水中の50%MeOH中の1.8μLの5mg/mLヨウ素(8.6μg、34nmol、チオール基に比較して2倍過剰)を添加した。反応混合物を、25℃にて20分間インキュベートし、PepClean C18カラム(Pierce)で洗浄し、そしてリン酸緩衝液中の10mgのF108−PDSコート化多孔性粒子を含有する50μL懸濁液に直接的に添加した。
18μLの0.1Mリン酸緩衝液pH8中のTA4+PC6Cys(Acm)ペプチド(90μg、18nmol)の溶液に、水中の50%MeOH中の1.8μLの5mg/mLヨウ素(8.6μg、34nmol、チオール基に比較して2倍過剰)を添加した。反応混合物を、25℃にて20分間インキュベートし、PepClean C18カラム(Pierce)で洗浄し、そしてリン酸緩衝液中の1.5mLの1.3mg/mlのキナーゼ−PDSに直接的に添加した。
TA4Cys(Acm)とPC6−pニトロフェニルエステルとの反応。1mLの0.1Mリン酸緩衝液pH8中のTA4Cys(Acm)ペプチド(1mg、0.2μmol)の溶液に、10μLのDMSO中の0.1MPC6−pニトロフェニルエステル(0.8μmol、5当量)を添加した。反応混合物を4℃にて2日間インキュベートし、NAP−10カラム(GE Healthcare)を使用するゲル濾過により精製して、水中の1.5mLのTA4PC6Cys(Acm)を得、そして最後に凍結乾燥した。LC−MS(Phenomenex Gemini、C18、5μm、150×3.0mm、A:水中の0.1%ギ酸、B:アセトニトリル中の0.1%ギ酸、10〜90% B、10分間にわたる)、tR=4.1分(UVシグナル)m/z=[M+6H]6+について856.0、[M+5H]5+について1026.8、[M+4H]4+について1282.6。
TA4+Cys(Acm)とPC6−pニトロフェニルエステルとの反応。1mLの0.1Mリン酸緩衝液pH8中のTA4+Cys(Acm)ペプチド(1mg、0.2μmol)の溶液に、DMSO中の10μLの0.1M PC6−pニトロフェニルエステル(0.8μmol、5当量)を添加した。反応混合物を4℃にて一晩インキュベートし、NAP−10カラム(GE Healthcare)を使用するゲル濾過により精製して、水中の1.5mLのTA4+PC6Cys(Acm)を有し、そして最後に凍結乾燥した。LC−MS(Phenomenex Gemini、C18、5μm、150×3.0mm、A:水中の0.1%ギ酸、B:アセトニトリル中の0.1%ギ酸、10〜90% B、10分間にわたる)、tR=4.0分(UVシグナル)、m/z=[M+8H]8+について670.3、[M+7H]7+について766.1、[M+6H]6+について893.4。
ホスホコリン誘導体の取り込みのためのより一般的なプロトコルが以下に与えられる。当業者は、このプロトコルを、適用され得る特定の状態についてこれを最適化するために、適合し得る。
マイクロタイタープレートのウェルの別個のセットにおいて、C−反応性タンパク質(CRP)を、pH7.4にて、10mM HEPES、150mM NaCl、および5mM CaCl2中に500nMの濃度に溶解した。リガンドおよび蛍光因子で官能化されたポリペプチドのそれぞれを、希釈し、そして標的タンパク質を含有する各ウェルに添加して、500nMのペプチドの最終濃度を得た。
固相に結合される本発明の結合剤を有するCRP検出アッセイの1つの実施態様は、図3において模式的に示される。
239nmの直径を備える単分散ポリスチレンラテックス粒子を、高分子界面活性剤Pluronic F108(チオールを含有するリガンドの連結を許容するためにピリジルジスルフィド(PDS)結合化末端基を含有する)を含有する超純粋(MilliQ)水中に懸濁した。界面活性剤分子は、それらの疎水性中心ブロックを介して、粒子を吸着する14。各粒子により、一晩の吸着を介して、取り込まれるPluronic分子の数は、沈降場流動分画、SdFFFにより、16500であると決定した15。次いで、PDS基を、それぞれCys含有CRP結合剤により置き換えた。結合剤(Ta4+)を固定化するためのプロトコルは上述に与えられる。SdFFFによる解析は、各粒子が2560の結合剤を取り込んだことを示した。
13PA Fowler、A.H.Haines、R.J.K.Taylor、E.J.T.Chrystal、M.B.Gravestock:ノジリマイシンの非環状類似体の合成および生物学的活性、JCS Perkin 1(1994)2229−35。
Claims (14)
- 配列番号1に記載の配列を有するポリペプチドであって、ここで少なくとも1つのホスホコリン誘導体が該ポリペプチドに結合されたポリペプチド。
- 請求項1に記載の2つの二量体化ポリペプチドからなり、ここで2つのプロトマーは、同じであるかまたは異なるアミノ酸配列を有するポリペプチド。
- ホスホコリン誘導体が、プロトマーのうちの1つの8位、17位、22位、または34位においてリジンに結合された請求項1または2に記載のポリペプチド。
- 前記ポリペプチドに結合されたレポーター基をさらに有する請求項1〜3のいずれかに記載のポリペプチド。
- レポーター基が、プロトマーのうちの1つの10位、15位、25位、または37位においてリジンに結合された請求項4に記載のポリペプチド。
- ホスホコリン誘導体が、1〜12個の炭素原子の長さを備える炭素鎖を含むスペーサーを介してポリペプチドに結合された請求項1〜5のいずれかに記載のポリペプチド。
- C−反応性結合タンパク質について、100ナノモル未満の結合親和性を有する請求項1〜6のいずれかに記載のポリペプチド。
- 前記プロトマーが同じ配列または異なる配列を有し、そして該配列が配列番号3〜18から選択される、請求項1〜7のいずれかに記載のポリペプチド。
- サンプル中のC−反応性タンパク質の濃度をアッセイするための方法であって、該方法は、サンプルを、請求項1〜8のいずれかに記載のポリペプチドと接触させる工程を包含する方法。
- サンプルが哺乳動物患者に由来する請求項9に記載の方法。
- 前記ポリペプチド二量体が、固体支持体に結合される請求項9〜10のいずれかに記載の方法。
- 請求項11に記載の方法であって、以下の工程
− サンプルを、請求項1〜8のいずれかに記載の第1のポリペプチドと接触させる工程であって、該第1のポリペプチドは固体支持体に結合される工程;
− サンプルを、請求項4〜8のいずれかに記載の、これに結合されたレポーター基を有する、第2のポリペプチドと接触させる工程;および
− 該第2のポリペプチド上のレポーター基の存在または不在を検出する工程、
を包含する方法。 - C−反応性タンパク質を含む組成物を、請求項1〜8のいずれかに記載のポリペプチドと接触させる工程を包含するC−反応性タンパク質の精製のための方法。
- 請求項1〜8のいずれかに記載のポリペプチドを含む診断用組成物。
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