ClickChemistry點擊化學(xué)的應(yīng)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(yīng)或以可預(yù)測的方式相互“點擊”以形成具有雜原子鏈（C-X-C）的生理穩(wěn)定產(chǎn)物的反應(yīng)。Click chemistry廣泛用于生物分子、表面、顆粒和有機(jī)化合物的改性，具有許多優(yōu)點1：

* 應(yīng)用范圍廣泛；

* 模塊化性質(zhì)；

* 在“小量”和“大量”反應(yīng)中均適用；

* 反應(yīng)條件溫和；

* 產(chǎn)品分離簡單（幾乎不需要純化）；

* 產(chǎn)率高，速度快；

* 無害副產(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(tǒng)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種不同類型的點擊反應(yīng)中，有幾種是在各種生命科學(xué)應(yīng)用中使用最頻繁，從“簡單”的生物分子標(biāo)記和檢測到先進(jìn)的CRISPER應(yīng)用。在此，我們重點介紹最重要的9種（最新）應(yīng)用：

* 生物分子標(biāo)記與檢測

*（固&液相）生物分子修飾/連接

* 構(gòu)建用于構(gòu)效關(guān)系分析的類似物庫

* 藥物先導(dǎo)化合物發(fā)現(xiàn)

* 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(biāo)記

* 新應(yīng)用，包括“點擊發(fā)布”

1.生物分子標(biāo)記與檢測

Click chemistry十分有用的功能之一是它能夠標(biāo)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(nèi)）10。在進(jìn)行標(biāo)記的兩步反應(yīng)中，首先用雙正交點擊手柄（如炔烴或疊氮化物）標(biāo)記目標(biāo)生物分子（酶、代謝11,12或合成（請見圖1）9,13）。然后當(dāng)一個分子上有熒光或親和基團(tuán)的互補(bǔ)點擊手柄與目標(biāo)分子發(fā)生點擊反應(yīng)時，就會發(fā)生檢測/可視化。

Click chemistry應(yīng)用生物分子標(biāo)記與檢測13

例如，在活體發(fā)育的斑馬魚中，表面聚糖以亞細(xì)胞分辨率被觀察到；依靠基因編碼的傳統(tǒng)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(jié)合，以揭示細(xì)胞表面表達(dá)、細(xì)胞內(nèi)運(yùn)輸和整個斑馬魚胚胎發(fā)生過程中聚糖組織分布的差異。類似的研究也在小鼠中進(jìn)行，以跟蹤移植細(xì)胞和測定細(xì)胞對肽的攝取情況，這有助于結(jié)構(gòu)-活性-通透性關(guān)系優(yōu)化研究15。兩個位點標(biāo)記生物分子（稱為雙位點標(biāo)記）有效的促進(jìn)了復(fù)雜生物系統(tǒng)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(xué)進(jìn)行修飾，幾乎無需使用保護(hù)基團(tuán)，也無需產(chǎn)品純化3,21,22?？傮w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(yōu)缺點23,24。

3. 類似物庫的建設(shè)

類似物庫可以通過點擊化學(xué)快速可靠地構(gòu)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(jìn)分子結(jié)構(gòu)-活性關(guān)系（SAR）分析，這是優(yōu)化和發(fā)現(xiàn)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）通過此方法被構(gòu)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(dǎo)化合物發(fā)現(xiàn)的原位點擊化學(xué)

原位點擊化學(xué)是一種（動力學(xué)）靶點導(dǎo)向合成方法，Sharpless及其同事于2002年第一次提出并應(yīng)用于發(fā)現(xiàn)一種有效的乙酰膽堿酯酶抑制劑26。這種方法使用目標(biāo)生物分子本身作為支架，如果使其足夠接近并以適當(dāng)?shù)姆较蚍磻?yīng)，則結(jié)合配體在其上進(jìn)行咔噠反應(yīng)。通過這種方式，可以從帶有互補(bǔ)反應(yīng)性官能團(tuán)的片段庫中篩選出能夠與目標(biāo)物形成穩(wěn)定絡(luò)合物的最佳配體27。無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(jì)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（通過磺基點擊化學(xué)）34、抗體樣蛋白質(zhì)捕獲劑35,36、轉(zhuǎn)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(shè)計的一個重要方面。點擊化學(xué)已用于構(gòu)建聚合物納米和微粒藥物遞送系統(tǒng)（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，從線性聚合物和接枝聚合物到更復(fù)雜結(jié)構(gòu)（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(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，點擊化學(xué)可以最大限度地減少氣泡、空穴和不規(guī)則的形成，就像其他縮聚反應(yīng)一樣，這些氣泡、空穴和不規(guī)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(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），該策略優(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(tuán)和可逆酮酰肼反應(yīng)的自交聯(lián)系統(tǒng)）。Click交聯(lián)聚合物薄膜的機(jī)械強(qiáng)度、硬度和耐水/溶劑性能顯著提高，為工業(yè)涂料應(yīng)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(yī)學(xué)47、藥物輸送48和基因治療領(lǐng)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(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(shè)計53,54、診斷55,56,57和基于病毒的傳遞系統(tǒng)58,59都使用了點擊化學(xué)。例如，通過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（QD），使用無銅點擊反應(yīng)來標(biāo)記包膜病毒（痘苗病毒（VACV）和A病毒（H9N2））。標(biāo)記效率達(dá)到80%以上，不干擾病毒的感染能力，熒光強(qiáng)度足以實現(xiàn)單個病毒粒子的跟蹤60。

8. CRISPER-sgRNA合成與靶基因標(biāo)記

Click chemistry現(xiàn)在可以在CRISPR工具箱中找到合成單個或多個單一導(dǎo)向RNA（sgRNA）的位置，繞過了與（更長）寡聚體長度相關(guān)的現(xiàn)有合成限制，并縮短sgRNA設(shè)計和應(yīng)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?。ǜ菀缀铣桑┑钠危阂粋€按需制備的高純度~20-mer（crRNA）靶向序列和一個通用的可大規(guī)模生產(chǎn)的79-mer CRISPR內(nèi)切酶蛋白（Cas9）序列（tracrRNA）。結(jié)果發(fā)現(xiàn)，帶有三唑鍵的~99-聚體能夠在體外和細(xì)胞內(nèi)有效地進(jìn)行Cas9介導(dǎo)的DNA切割，其靶向性與體外轉(zhuǎn)錄的sgRNA相當(dāng)61。

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

此外，點擊化學(xué)還用于設(shè)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(zhuǎn)染效率和較大的處理量63。

9. 包括“點擊發(fā)布”的新應(yīng)用

除了連接，點擊化學(xué)現(xiàn)在正在探索解封或“點擊釋放”應(yīng)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(yīng)在體外和腫瘤小鼠中激發(fā)阿霉素從抗體-藥物結(jié)合物（ADC）中的快速釋放67。

點擊化學(xué)還被用于開發(fā)新的的微芯片和毛細(xì)管系統(tǒng)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(xué)免疫傳感器以及在空間和時間上控制蛋白質(zhì)接合71,72,73。

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