ClickChemistry點(diǎn)擊化學(xué)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(diǎn)擊”以形成具有雜原子鏈（C-X-C）的生理穩定產(chǎn)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(yōu)點(diǎn)1：

* 應用范圍廣泛；

* 模塊化性質(zhì)；

* 在“小量”和“大量”反應中均適用；

* 反應條件溫和；

* 產(chǎn)品分離簡(jiǎn)單（幾乎不需要純化）；

* 產(chǎn)率高，速度快；

* 無(wú)害副產(chǎn)品生成（遵循綠色化學(xué)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(xué)選擇性修飾，幾乎不受干擾）2。

在大約10種不同類(lèi)型的點(diǎn)擊反應中，有幾種是在各種生命科學(xué)應用中使用最頻繁，從“簡(jiǎn)單”的生物分子標記和檢測到先進(jìn)的CRISPER應用。在此，我們重點(diǎn)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(guān)系分析的類(lèi)似物庫

* 藥物先導化合物發(fā)現

* 藥物輸送

* 材料優(yōu)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(diǎn)擊發(fā)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(zhì)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(jìn)行標記的兩步反應中，首先用雙正交點(diǎn)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(jiàn)圖1）9,13）。然后當一個(gè)分子上有熒光或親和基團的互補點(diǎn)擊手柄與目標分子發(fā)生點(diǎn)擊反應時(shí)，就會(huì )發(fā)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(fā)育的斑馬魚(yú)中，表面聚糖以亞細胞分辨率被觀(guān)察到；依靠基因編碼的傳統分子成像方法通常無(wú)法看見(jiàn)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(gè)斑馬魚(yú)胚胎發(fā)生過(guò)程中聚糖組織分布的差異。類(lèi)似的研究也在小鼠中進(jìn)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(guān)系優(yōu)化研究15。兩個(gè)位點(diǎn)標記生物分子（稱(chēng)為雙位點(diǎn)標記）有效的促進(jìn)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(guò)固相或液相點(diǎn)擊化學(xué)進(jìn)行修飾，幾乎無(wú)需使用保護基團，也無(wú)需產(chǎn)品純化3,21,22?？傮w來(lái)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(yōu)缺點(diǎn)23,24。

3. 類(lèi)似物庫的建設

類(lèi)似物庫可以通過(guò)點(diǎn)擊化學(xué)快速可靠地構建，無(wú)需太多的合成工作，然后通過(guò)原位高通量篩選（HTS）來(lái)促進(jìn)分子結構-活性關(guān)系（SAR）分析，這是優(yōu)化和發(fā)現生物活性分子所必需的。已經(jīng)有許多基于click（三唑）骨架的片段庫（聚焦組合）通過(guò)此方法被構建出來(lái)25，例如Janus激酶抑制劑ruxolitinib衍生的三唑文庫，它被用來(lái)評估JAK3抑制劑24 。

4. 用于先導化合物發(fā)現的原位點(diǎn)擊化學(xué)

原位點(diǎn)擊化學(xué)是一種（動(dòng)力學(xué)）靶點(diǎn)導向合成方法，Sharpless及其同事于2002年第一次提出并應用于發(fā)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(jìn)行咔噠反應。通過(guò)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(luò )合物的最佳配體27。無(wú)需事先對文庫成員進(jìn)行合成、純化和生化評估，即可快速且經(jīng)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(zhì)-蛋白質(zhì)相互作用（通過(guò)磺基點(diǎn)擊化學(xué)）34、抗體樣蛋白質(zhì)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(jìn)入人體的控制給藥是有效藥物設計的一個(gè)重要方面。點(diǎn)擊化學(xué)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(zhì)體、膠囊、碳納米管等6,39。

6. 材料優(yōu)化（聚合物改性）

在材料制造領(lǐng)域，從線(xiàn)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(shù)狀聚合物）的合成，再到表面和界面的功能化40，點(diǎn)擊化學(xué)都產(chǎn)生了巨大的影響。例如，由于不產(chǎn)生小分子副產(chǎn)物，點(diǎn)擊化學(xué)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(huì )破壞新合成熱固性材料的外觀(guān)和性能41。

CuAAC click chemistry還被用作一種高效、環(huán)保的交聯(lián)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?xún)?yōu)于其他可用的交聯(lián)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(lián)系統）。Click交聯(lián)聚合物薄膜的機械強度、硬度和耐水/溶劑性能顯著(zhù)提高，為工業(yè)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(xué)47、藥物輸送48和基因治療領(lǐng)域49也越來(lái)越受到重視。

7. 病毒研究探針

在過(guò)去幾十年中50，與病毒相關(guān)的研究，包括病毒（蛋白質(zhì)、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(diǎn)擊化學(xué)。例如，通過(guò)將疊氮化物修飾的病毒粒子連接到由二苯并環(huán)辛烯（DBCO）衍生的量子點(diǎn)（QD），使用無(wú)銅點(diǎn)擊反應來(lái)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(shí)現單個(gè)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(gè)或多個(gè)單一導向RNA（sgRNA）的位置，繞過(guò)了與（更長(cháng)）寡聚體長(cháng)度相關(guān)的現有合成限制，并縮短sgRNA設計和應用之間的時(shí)間。

Click chemistry（被稱(chēng)為“分裂和點(diǎn)擊”）不是一次性制造整個(gè)sgRNA，而是簡(jiǎn)單地連接兩個(gè)更?。ǜ菀缀铣桑┑钠危阂粋€(gè)按需制備的高純度~20-mer（crRNA）靶向序列和一個(gè)通用的可大規模生產(chǎn)的79-mer CRISPR內切酶蛋白（Cas9）序列（tracrRNA）。結果發(fā)現，帶有三唑鍵的~99-聚體能夠在體外和細胞內有效地進(jìn)行Cas9介導的DNA切割，其靶向性與體外轉錄的sgRNA相當61。

點(diǎn)擊化學(xué)也被用于標記靶基因（稱(chēng)為sgRNA點(diǎn)擊（sgR CLK））62。該技術(shù)包括在體外轉錄的CRISPR-sgRNA的3′端安裝點(diǎn)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(guò)與炔烴對應物的點(diǎn)擊反應實(shí)現該三元絡(luò )合物的功能化。

此外，點(diǎn)擊化學(xué)還用于設計一種柔性樹(shù)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(diǎn)擊發(fā)布”的新應用

除了連接，點(diǎn)擊化學(xué)現在正在探索解封或“點(diǎn)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(fā)阿霉素從抗體-藥物結合物（ADC）中的快速釋放67。

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

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