ClickChemistry點擊化學的應用

Click Chemistry由K.Barry Sharpless、Hartmuth C.Kolb和M.G.Finn于2001年提出，用于描述快速選擇性反應或以可預測的方式相互“點擊”以形成具有雜原子鏈（C-X-C）的生理穩定產物的反應。Click chemistry廣泛用于生物分子、表面、顆粒和有機化合物的改性，具有許多優點1：

* 應用范圍廣泛；

* 模塊化性質；

* 在“小量”和“大量”反應中均適用；

* 反應條件溫和；

* 產品分離簡單（幾乎不需要純化）；

* 產率高，速度快；

* 無害副產品生成（遵循綠色化學的12項原則）；

* 兼容性良好，尤其在生命系統中（允許生物分子的化學選擇性修飾，幾乎不受干擾）2。

在大約10種不同類型的點擊反應中，有幾種是在各種生命科學應用中使用最頻繁，從“簡單”的生物分子標記和檢測到先進的CRISPER應用。在此，我們重點介紹最重要的9種（最新）應用：

* 生物分子標記與檢測

*（固&液相）生物分子修飾/連接

* 構建用于構效關系分析的類似物庫

* 藥物先導化合物發現

* 藥物輸送

* 材料優化（聚合物改性）

* 病毒研究探針

* CRISPER sgRNA合成和靶基因標記

* 新應用，包括“點擊發布”

1.生物分子標記與檢測

Click chemistry十分有用的功能之一是它能夠標記和可視化生物分子，如脂質3、肽4、聚糖5、糖蛋白6、核酸和合成分子7,8（如紫杉醇9），并且具有最小的生理干擾性（體外和體內）10。在進行標記的兩步反應中，首先用雙正交點擊手柄（如炔烴或疊氮化物）標記目標生物分子（酶、代謝11,12或合成（請見圖1）9,13）。然后當一個分子上有熒光或親和基團的互補點擊手柄與目標分子發生點擊反應時，就會發生檢測/可視化。

Click chemistry應用生物分子標記與檢測13

例如，在活體發育的斑馬魚中，表面聚糖以亞細胞分辨率被觀察到；依靠基因編碼的傳統分子成像方法通常無法看見14。在這項研究中，Bertozzi等人將代謝糖工程與多色檢測策略相結合，以揭示細胞表面表達、細胞內運輸和整個斑馬魚胚胎發生過程中聚糖組織分布的差異。類似的研究也在小鼠中進行，以跟蹤移植細胞和測定細胞對肽的攝取情況，這有助于結構-活性-通透性關系優化研究15。兩個位點標記生物分子（稱為雙位點標記）有效的促進了復雜生物系統的研究16,17,18,19,20。

2.(固相和液相）生物分子修飾/連接

肽、核苷酸、小分子、超分子等都可以通過固相或液相點擊化學進行修飾，幾乎無需使用保護基團，也無需產品純化3,21,22。總體來講，固相合成更快，且需要更少的后處理，但是每種方法都各有優缺點23,24。

3. 類似物庫的建設

類似物庫可以通過點擊化學快速可靠地構建，無需太多的合成工作，然后通過原位高通量篩選（HTS）來促進分子結構-活性關系（SAR）分析，這是優化和發現生物活性分子所必需的。已經有許多基于click（三唑）骨架的片段庫（聚焦組合）通過此方法被構建出來25，例如Janus激酶抑制劑ruxolitinib衍生的三唑文庫，它被用來評估JAK3抑制劑24 。

4. 用于先導化合物發現的原位點擊化學

原位點擊化學是一種（動力學）靶點導向合成方法，Sharpless及其同事于2002年第一次提出并應用于發現一種有效的乙酰膽堿酯酶抑制劑26。這種方法使用目標生物分子本身作為支架，如果使其足夠接近并以適當的方向反應，則結合配體在其上進行咔噠反應。通過這種方式，可以從帶有互補反應性官能團的片段庫中篩選出能夠與目標物形成穩定絡合物的最佳配體27。無需事先對文庫成員進行合成、純化和生化評估，即可快速且經濟高效地篩選大量化合物28,29。

碳酸酐酶30、HIV蛋白酶31、幾丁酶32、核苷酸配體33、蛋白質-蛋白質相互作用（通過磺基點擊化學）34、抗體樣蛋白質捕獲劑35,36、轉錄因子37、通道38等的抑制劑也已被表征。

5. 藥物輸送

藥物進入人體的控制給藥是有效藥物設計的一個重要方面。點擊化學已用于構建聚合物納米和微粒藥物遞送系統（DDS），如聚合物膠束、脂質體、膠囊、碳納米管等6,39。

6. 材料優化（聚合物改性）

在材料制造領域，從線性聚合物和接枝聚合物到更復雜結構（如星形聚合物、嵌段共聚物和樹狀聚合物）的合成，再到表面和界面的功能化40，點擊化學都產生了巨大的影響。例如，由于不產生小分子副產物，點擊化學可以最大限度地減少氣泡、空穴和不規則的形成，就像其他縮聚反應一樣，這些氣泡、空穴和不規則會破壞新合成熱固性材料的外觀和性能41。

CuAAC click chemistry還被用作一種高效、環保的交聯策略，以改善適用于涂料和粘合劑的水性聚合物的性能42（下圖2）。廣泛適用于聚氨酯（WPU）、聚酯分散體（PED）和聚丙烯酸酯乳液（PAE），該策略優于其他可用的交聯策略（包括基于N-羥甲基丙烯酰胺（NMA）、懸垂乙酰乙酸基團和可逆酮酰肼反應的自交聯系統）。Click交聯聚合物薄膜的機械強度、硬度和耐水/溶劑性能顯著提高，為工業涂料應用中使用硬化劑提供了一種有可能降低成本的替代品。

Formation of click cross-linked waterborne polymers42

此外，各種（1D、2D、3D）生物材料（如水凝膠）的合成在組織工程43,44,45,46再生醫學47、藥物輸送48和基因治療領域49也越來越受到重視。

7. 病毒研究探針

在過去幾十年中50，與病毒相關的研究，包括病毒（蛋白質、核酸或病毒粒子）追蹤51,52、抗病毒設計53,54、診斷55,56,57和基于病毒的傳遞系統58,59都使用了點擊化學。例如，通過將疊氮化物修飾的病毒粒子連接到由二苯并環辛烯（DBCO）衍生的量子點（QD），使用無銅點擊反應來標記包膜病毒（痘苗病毒（VACV）和A病毒（H9N2））。標記效率達到80%以上，不干擾病毒的感染能力，熒光強度足以實現單個病毒粒子的跟蹤60。

8. CRISPER-sgRNA合成與靶基因標記

Click chemistry現在可以在CRISPR工具箱中找到合成單個或多個單一導向RNA（sgRNA）的位置，繞過了與（更長）寡聚體長度相關的現有合成限制，并縮短sgRNA設計和應用之間的時間。

Click chemistry（被稱為“分裂和點擊”）不是一次性制造整個sgRNA，而是簡單地連接兩個更小（更容易合成）的片段：一個按需制備的高純度~20-mer（crRNA）靶向序列和一個通用的可大規模生產的79-mer CRISPR內切酶蛋白（Cas9）序列（tracrRNA）。結果發現，帶有三唑鍵的~99-聚體能夠在體外和細胞內有效地進行Cas9介導的DNA切割，其靶向性與體外轉錄的sgRNA相當61。

點擊化學也被用于標記靶基因（稱為sgRNA點擊（sgR CLK））62。該技術包括在體外轉錄的CRISPR-sgRNA的3′端安裝點擊手柄，以形成疊氮化物標記的三元復合物（由dCas9、sgRNA和靶基因組成）。然后通過與炔烴對應物的點擊反應實現該三元絡合物的功能化。

此外，點擊化學還用于設計一種柔性樹枝狀聚合物，用于傳遞鋅指、TALEs和CRISPR/dCas9平臺。使用該方法具有高轉染效率和較大的處理量63。

9. 包括“點擊發布”的新應用

除了連接，點擊化學現在正在探索解封或“點擊釋放”應用，這使得探針激活和治療傳遞的新策略成為可能64,65,66。例如，利用逆電子需求Diels-Alder-噠嗪消除反應在體外和腫瘤小鼠中激發阿霉素從抗體-藥物結合物（ADC）中的快速釋放67。

點擊化學還被用于開發新的的微芯片和毛細管系統68，如微流控“點擊芯片”69和基于石墨烯的“點擊芯片”70。此外，“電點擊”接合方法已被用于固定酶（用于生物傳感器）、制備電化學免疫傳感器以及在空間和時間上控制蛋白質接合71,72,73。

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