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高分子期刊

高分子期刊. 題目名稱： Spatially Addressed Synthesis of Amino- and Amino-Oxy-Substituted 1,3,5-Triazine Arrays on Polymeric Membranes 原作者及期刊： Dirk Scharn,† Holger Wenschuh,*,‡ Ulrich Reineke,‡ Jens Schneider-Mergener,†,‡ and Lothar Germeroth*,ACS, J. Comb. Chem. 2000, 2, 361-369 姓名：黃郁庭 學號： 49740057.

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高分子期刊

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  1. 高分子期刊 • 題目名稱：Spatially Addressed Synthesis of Amino- and Amino-Oxy-Substituted 1,3,5-Triazine Arrays on Polymeric Membranes • 原作者及期刊：Dirk Scharn,† Holger Wenschuh,*,‡ Ulrich Reineke,‡ Jens Schneider-Mergener,†,‡ and • Lothar Germeroth*,ACS, J. Comb. Chem. 2000, 2, 361-369 • 姓名：黃郁庭 • 學號：49740057

  2. 摘要 • 利用SPOT合成技術在纖維素與聚丙烯膜上，有效運用平行空間組合來達成trisamino-和amino-oxy-1,3,5-triazines的合成。除了運用胺和酚鹽離子形成適合的鍵結之外，還能在一氯三嗪型的膜鍵結上，發展高效能的微波輔助親核取代反應系統。1,3,5-triazines利用TFA蒸發在固態中能被平行斷鍵，能給予膜表面吸附物質用以分析和篩選。運用於組成8000纖維素與1,3,5-triazines的鍵結反應條件為要研究並辨識出表位模擬anti-transforming growth factor-α與表2中monoclonal antibody的水平鍵結。

  3. 簡介 • 最主要的化學組成是集中注意於雜環系統中，結構小的有機化合物系統。1,3,5-Triazines特別在於易於合成且有電療治療法的價值，像是治療癌症及憂鬱症等。這些從cyanuric chloride合成的化合物，是氯原子利用不同的親核試劑與通過溫度轉換的優點達成位移。之前有報導在合成cyanuric chloride的衍生化合物中，利用在固相中“one bead one compound” 得到混合物或在溶液相中使用高精密儀器設備做相對更嚴苛的實驗條件及更長的反應時間。 • 不同以往，本次研究是開發SPOT合成技術是允許單體在室溫條件下快速生產進行多變化的鍵結。雖然SPOT合成技術已能直接在纖維素膜上合成及篩選縮氯酸，但用於集合小的有機分子並未發表過。要能高效率的合成小的有機分子，必須建立可行的SPOT合成技術。像是合成1,3,5-Triazines的先決條件是(i)在合適的基材上做運用(ii)在乾燥條件下，脂化合物是能被斷結的(iii)有開發更好的合成方法(圖1)。

  4. 實驗方法 　 洗滌液A為在水中含量為0.05%的TFA，洗滌液B為在乙腈溶液中含有0.05%的TFA，λ＝220 nm；流速0.3 mL/min。 • 含氨官能基的PP膜 • 含Rink Linker的PP膜 • 乙醛薄膜 • 對位硝基酚薄膜 • 含氨官能基的薄膜 • 二氯均三嗪的薄膜 • 一氯均三嗪的薄膜 • 1,3,5-三嗪的薄膜 • 8000-Compound Library • Tab2抗體的鍵結測試 • 檢測抗體的方法 • 材料：纖維素紙、PP膜、 溶劑採用德國Sigma-Aldrich GmbH 、 氨基酸衍生物由德國Calbiochem-Novabiochem GmbH提供。 • 清洗：所有清洗膜的步驟都要在不鏽鋼中進行，每一步驟所使用的培養皿必須用DMF 、 MeOH和DCM各清洗三次。 • 分裂方式：乾燥器放置一個玻璃盤，盤內放5毫升TFA且以每小時蒸發700毫巴的速率進行。把含有化合物的膜用Rink或氨基甲酸酯連接器固定後，放置在充滿TFA蒸氣的乾燥氣中30分鐘。最後，使膜在真空下120分鐘吸收TFA。 • 分析：用質譜法與LCQ-MS儀器在配有ESI源下作標準分析。質譜法實驗必須在VYDAC C-18柱下使用線性梯度檢測。

  5. 結果與討論 • 首先，為了檢測薄膜的化學合成上的作用，用2種薄膜做測試。除了纖維素在早期就證實了適用於合成縮胺酸和異構物的鍵結測試，開發了9種改質的PP膜，表現出高的化學性和機械性，而且減少過量官能基團的纖維素可能會出現的各種合成操作干擾。為了使其穩定含氨官能基的薄膜最為適合，可避免在聚合的過程因為強親核試劑而發生裂解。根據Scheme1羧酸或甲基酯官能團轉換成相對應的胺基。 • 以羧基基團做為活化薄膜鍵結，進行了測試(表1)。活化能在通過PCl5其中的氯酸，可能到最好的薄膜載荷(表1) 。 • 在液相篩選中要生成裂解化合物和質量控制，首先要有條件建立適當的鍵結（圖1中的A） • 1,3,5 - 三嗪能在固態基材上並通過三氟乙酸（TFA）達到有效蒸發，由於化合物仍然吸附住，含有化合物的薄膜篩選化合物特性與控制純度，在DCM溶液中做含80%的TFA 蒸氣的單薄膜裂解持續30分鐘，得到了類似的產物，驗證TFA蒸汽裂解的完整性，可由圖4中產物在HPLC的表現量。

  6. 抑制常數(表4)是根據實驗結果計算。兩種化合物都有些微弱，但在上部micromolar範圍有顯著的抑製作用。抑制常數(表4)是根據實驗結果計算。兩種化合物都有些微弱，但在上部micromolar範圍有顯著的抑製作用。

  7. 圖4、資料顯示由TFA 蒸氣使得1,3,5-三嗪有不同的取代裂解。 　　　　 (a-c為纖維素薄膜；d-f為pp薄膜)

  8. 結論 • 開發一個有效的合成系統，為了能夠用SPOT合成技術使大量的1,3,5-triazines在一平面上做延伸，聚合成雜環衍生化合物。採用2種不同的不穩定的酸做鍵結，在室溫條件下利用微波輔助進行親核取代反應，得到固態基材上未曾被研究過的amino-oxy-1,3,5-triazines。一般情況下，氯單體會選擇性的取代二氯-1,3,5 - 三嗪在膜上的鍵結，能使胺達到超過50℃的沸點和在NMP中得到溶解度達1M或更高。弱親核試劑像是4 - 硝基苯胺或有難鍵結的胺及醇會導致製程出現低產率和低飽和度及溫度易上升。加入三甲基氯矽烷（胺）或使用銫鹽（醇和酚）會增加產量。在室溫條件下，生成物可利用TFA蒸氣達到裂解，使生成物吸附在聚合基材上進行鍵結，研究此合成條件為適用於高度聚合的8000 cellulosebound 1,3,5-triazines，可能會出現表面不均勻的篩選。所以此方法適合於篩選高活性的高分子薄膜。

  9. 參考文獻 (6) Gustafson, G. R.; Baldino, C. M.; O’Donnell, M. E.; Sheldon, A.; Tarsa, R. J.; Verni, C. J.; Coffen, D. Incorporation of carbohydrates and peptides into large triazine-based screening libraries using automated parallel synthesis. Tetrahedron 1998, 54, 4051-4065. (7) Falorni, M.; Giacomelli, G.; Mameli, L.; Porcheddu, A. New 1,3,5- triazine derivatives as templates for the homogeneous phase synthesis of chemical libraries. Tetrahedron Lett. 1998, 39, 7607-7610. (8) Frank, R. SPOT-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 1992, 48, 9217-9232. (9) Kramer, A.; Schuster, A.; Reineke, U.; Malin, R.; Volkmer-Engert, R.; Landgraf, C.; Schneider-Mergener, J. Combinatorial cellulosebound peptide libraries: screening tools for the identification of peptides that bind ligands with predefined specifity. METHODS (San Diego) 1994, 6, 388-410. (10) Ast, T.; Heine, N.; Schneider-Mergener, J.; Germeroth, L.; Wenschuh, H. Efficient assembly of peptomers on continuous surfaces. Tetrahedron Lett. 1999, 40, 4317-4318. (1) Nefzi, A.; Ostresh, J. M.; Houghten, R. A. The current status of heterocyclic combinatorial libraries. Chem. ReV. 1997, 97, 449-472. (2) Dhainaut, A.; Regnier, G.; Tizot, A.; Pierre, A.; Leonce, S.; Guilbaud, N.; Kraus-Berthier, L.; Atassi, G. New purines and purine analogues as modulators of multidrug resistance. J. Med. Chem. 1996, 39, 4099-4108. (3) Whitten, J. P.; Xie, Y. F.; Erickson, P. E.; Webb, T. R.; DeSouza, E.B.;Grigoriasdis, D. E.; McCarty J. R. Rapid microscale synthesis, a new method for lead optimization using robotics and solution phase chemistry: application to the synthesis and optimization of corticotropin- releasing factor1 receptor antagonists. J. Med. Chem. 1996, 39, 4354-4357. (4) Schaefer, F. C.; Thurston, J. T.; Dudley, J. R. Cyanuric chloride derivatives. J. Am. Chem. Soc. 1951, 73, 2900-2992 and references therein. (5) Stankova, M.; Lebl, M. Library generation through successive substitution of trichlorotriazine. Mol. DiVersity 1996, 2, 75 80.

  10. (11) Wenschuh, H.; Schmidt, M.; Germeroth, L.; Reineke, U.; Scharn, D.; Heine, N.; Hummel, G.; Jobron, L.; Matuschewski, H.; Ulbricht, M.; Schedler, U.; Schneider-Mergener, J.; Schulz, M. Spatially addressed SPOT-synthesis on novel polymeric membranes. In InnoVation and PerspectiVes in Solid-Phase Synthesis & Combinatorial Libraries; Epton, R., Ed.; Mayflower Worldwide Ltd.: Birmingham, 1999; in press. (12) Rink, H. Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methyl ester resin. Tetrahedron Lett. 1987, 28,3787-3790. (13) For a complete list of applied building blocks, refer to the Supporting Information. (14) Conversion was studied as follows: (i) Pipetting of 2-5 M solutions (depending on the solubility) of amines onto dichlorotriazines immobilized with the Rink linker on pp-membrane and cellulose was followed by washing of the membranes after 30 min. (ii) To use UV quantification of purities and conversions, all remaining chlorines (nonreacted chlorines of dichlorotriazines and chlorines of monochlorotriazines) were quenched with either benzylamine (when aromatic amines were tested in step i) or piperidine (when aliphatic amines were tested in step i) to give compounds with similar extinction coefficients. Membranes were immersed in the corresponding amine solution and exposed to microwave irradiation for 2 3 min to ensure complete substitution. (iii) After washing and drying of the membranes, compounds were cleaved from the membranes with TFA vapor, and conversion and purity were analyzed by LC MS. (15) Several aniline substituted triazines were synthesized on Rink linker modified cellulose and pp-membranes under comparable conditions. Analysis of the cleaved products revealed average purities of 80% from the pp-membrane case compared to 50% purity from cellulose. (16) Salmoria, G. V.; D’allOglio, E.; Zucco, C. Aromatic nucleophilic substitution under microwave irradiation. Tetrahedron Lett. 1998, 39, 2471-2474. (17) Hoeprich, P. D., Jr.; Langton, B. C.; Zhang, J. W.; Tam, J. P. Identification of immunodominant regions of transforming growth factor R. J. Biol. Chem. 1989, 264, 19086-19091.

  11. (18) Reineke, U.; Sabat, R.; Kramer, A.; Stigler, R. D.; Seifert, M.; Michel, T.; Volk, H. D.; Schneider-Mergener, J. Mapping protein-protein contact sites using cellulose-bound peptide scans. Mol. DiVersity 1996, 1, 141-148. (19) Kramer, A.; Reineke, U.; Dong, L.; Hoffmann, B.; Hoffmu¨ller, U.; Winkler, D.; Volkmer-Engert, R.; Schneider-Mergener, J. SPOTsynthesis: observations and optimizations. J. Peptide Res. 1999, 54, 319-327. (20) Friguet, B.; Chaffotte, A. F.; Djavadi-Ohaniance, L.; Goldberg, M. E. Measurement of the true affinity constant in solutions of antigenantibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Meth. 1985, 77, 305-319. (21) Wilson, M. B.; Nakane, P. K. Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In Immunofluorescence related staining techniques; Knapp, W., Holubar, K., Wick, G., Eds.; Elsevier: Amsterdam, 1978; pp 215-224.

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