图1 基于醛或酮类的生物正交反应［13］醛和酮可以与胺类化合物［氨基氧化合物（上）、酰肼化合物（下）］发生缩合反应形成稳定的肟键或腙键

Fig. 1 Bioorthogonal reactions based on aldehydes or ketones[13]［Aldehydes or ketones can condense with aminooxy compounds （top） or hydrazide compounds （bottom） to form stable oxime or hydrazine linkages， respectively］

图2 生物大分子与配体通过无痕施陶丁格反应偶联［13］［通过膦（1）与叠氮基团自发的反应生成亚胺磷烷（2），进一步发生结构重排（3），之后水解得到酰胺键偶联产物（4）和氧化膦副产物］

Fig. 2 Biomacromolecules coupling with ligands by the traceless Staudinger ligation reaction[13].[The phosphine-modified biomacromolecule (1) could react with the azide-linked ligand by the Staudinger ligation reaction to yield iminophosphorane (2), which rearranges to produce the intermediate (3) for hydrolysis to generate the coupling product (4) and a phosphine oxide by-product.]

图3 叠氮基团与炔基发生环加成反应，生成三氮唑产物［13］［Cu（Ⅰ）催化的叠氮-炔基环加成反应（上）和张力推进的叠氮-炔基环加成反应（下）］

Fig. 3 Bioorthogonal cycloadditions of azides and alkynes to form triazoles[13][Terminal alkynes are activated by Cu (Ⅰ) to undergo cycloaddition with azides (top). Cyclooctynes react with azides through a strain-promoted cycloaddition (bottom).]

图4 流感病毒样颗粒包膜蛋白的体内位点特异性标记［71］将叠氮基团修饰的非天然氨基酸——AHA添加到培养基中，在细胞中后续血凝素蛋白扩增的过程中，将AHA引入新生的血凝素蛋白（Hemagglutinin，HA）中，带有叠氮基团的血凝素蛋白被融合到细胞分泌的囊泡表面，进而可利用环炔修饰的Alexa 488通过张力促进的叠氮-炔基环加成反应，实现囊泡表面血凝素蛋白的位点特异性标记，构建成可发荧光的流感病毒样颗粒

Fig. 4 Site-specific in vivo labeling of enveloped influenza VLPs[71]During intracellular protein synthesis， the AHA is incorporated into the nascent HA proteins. The modified HA is further incorporated into the vesicles’ envelope that can secret from the cells， carrying the chemical modification. Addition of the Alexa 488-cyclooctyne reagent allows the site-specific modification of HA by strain-promoted alkyne-azide cycloaddition（SPAAC）

图5 利用叠氮化的糖通过宿主细胞膜蛋白的糖基化过程实现对包膜麻疹病毒糖蛋白的修饰［85］

Fig. 5 Modification of an enveloped measles virus can be achieved by the metabolic incorporation of azido sugars into the host cells through the protein glycosylation process[85]

图6 宿主细胞内的病毒包膜与核酸的双荧光标记［91］［叠氮化物胆碱（Azide-Cho）和［Ru（phen）2（dppz）］2+存在下，利用VACV感染宿主细胞。首先Azide-Cho利用宿主细胞中磷脂的生物合成途径掺入细胞膜中（①）；然后在VACV感染细胞2 h后，加入［Ru（phen）2（dppz）］2+，使其能通过病毒导致细胞病理效应进入细胞，并嵌入病毒核酸中（②）；在病毒感染24 h后，将DBCO-Fluor 525荧光探针加入培养基中，用于VACV包膜的标记（③）；继续感染24 h后，双标记的病毒粒子被组装并释放］

Fig. 6 Dual-fluorescent labeling of the viral envelope and nucleic acid in host cells[91] [The vaccinia virus (VACV) propagates in the presence of both azide-Cho and [Ru(phen)2(dppz)]2+ in the host cell. The biosynthesis and incorporation of azide-Cho-containing phospholipids in the host cells are carried out at first (①);then the cells are infected with VACV, and the [Ru(phen)2(dppz)]2+ is added into the medium at 2 h after the infection, which could enter the cells through the permeable cytomembrane due to the cytopathogenic effect of the virus to label the nucleic acid of the virions (②); at 24 h after the infection, DBCO-Fluor 525 is added into the medium to label the envelope of the VACV (③); after another 24 h with the infection, the dual-labeled virions are finally assembled and released.]

图7 宿主细胞内的病毒蛋白与核酸的双荧光标记［99］（叠氮基衍生的病毒蛋白可通过SPAAC反应与环炔修饰荧光探针偶联；乙烯基衍生的病毒核酸可通过IEDDA反应与四嗪修饰荧光探针偶联）

Fig. 7 Dual-fluorescent labeling of viral protein and nucleic acid in host cells[99]（AHA-containing protein is labeled by SPAAC reaction，and the VdU-containing nucleic acid is labeled by IEDDA reaction）

图8 叶酸配体对腺病毒纤维蛋白的功能化示意图［84］［O-GlcNAz掺入腺病毒的纤维蛋白（掺入位点用红圈表示），进而通过Click（1）或Staudinger（2）反应将叶酸分子与腺病毒偶联］

Fig. 8 A cartoon illustration for incorporating the O-GlcNAz residue with the adenoviral fiber protein and subsequent chemical modification with the ligand[84]［Potential sites for the O-GlcNAz incorporation are indicated with red circles. Either “Click”（1）or Staudinger ligation（2）chemistry is used to decorate metabolically labeled adenovirus.］