Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

‘Clickable’ cyclopentadienyl iron carbonyl complexes for bioorthogonal conjugation of mid‐infrared labels to a model protein and PAMAM dendrimer

Owing to the intrinsic limitations of the conventional bioconjugation methods involving native nucleophilic functions of proteins, we sought to develop alternative approaches to introduce metallocarbonyl infrared labels onto proteins on the basis of the [3 + 2] dipolar azide‐alkyne cycloaddition (AAC). To this end, two cyclopentadienyl iron dicarbonyl (Fp) complexes carrying a terminal or a strained alkyne handle were synthesized. Their reactivity was examined towards a model protein and poly (amidoamine) (PAMAM) dendrimer, both carrying azido groups. While the copper (I)‐catalysed azide‐alkyne cycloaddition (CuAAC) proceeded smoothly with the terminal alkyne metallocarbonyl derivative, labelling by strain‐promoted azide‐alkyne cycloaddition (SPAAC) was less successful in terms of final coupling ratios. Infrared spectral characterization of the bioconjugates showed the presence of two bands in the 2000 cm^?1 region, owing to the stretching vibration modes of the carbonyl ligands of the Fp entities.     

Circular DNA by “Bis‐Click” Ligation: Template‐Independent Intramolecular Circularization of Oligonucleotides with Terminal Alkynyl Groups Utilizing Bifunctional Azides

A highly effective and convenient “bis‐click” strategy was developed for the template‐independent circularization of single‐stranded oligonucleotides by employing copper(I)‐assisted azide–alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7‐deaza‐2′‐deoxyadenosine and 2′‐deoxyuridine residues with different side chains were used in solid‐phase synthesis with phosphoramidite chemistry. The bis‐click ligation of linear 9‐ to 36‐mer oligonucleotides with 1,4‐bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9‐mers. Compared with linear duplexes, circular bis‐click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis‐click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.     

Artificial Formation and Tuning of Glycoprotein Networks on Live Cell Membranes: A Single‐Molecule Tracking Study

We present a method to artificially induce network formation of membrane glycoproteins and show the precise tuning of their interconnection on living cells. For this, membrane glycans are first metabolically labeled with azido sugars and then tagged with biotin by copper‐free click chemistry. Finally, these biotin‐tagged membrane proteins are interconnected with streptavidin (SA) to form an artificial protein network in analogy to a lectin‐induced lattice. The degree of network formation can be controlled by the concentration of SA, its valency, and the concentration of biotin on membrane proteins. This was verified by investigation of the spatiotemporal dynamics of the SA‐protein networks employing single‐molecule tracking. It was also proven that this network formation strongly influences the biologically relevant process of endocytosis as it is known from natural lattices on the cell surface.     

Effect of sialic acid content on glycoprotein pI analyzed by two‐dimensional electrophoresis

2‐DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2‐DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2‐DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2‐DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2‐DE pattern of three serum glycoproteins (haptoglobin, α1‐antitrypsin and ribonuclease 1), which N‐glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule.     

肝癌细胞蛋白差异凝胶电泳技术系统评估方法

利用二维差异凝胶电泳技术(two-d im ensional d ifferential in-gel electrophoresis,2D-D IGE)研究肝癌细胞与正常细胞的差异时应先对其蛋白样品进行标记系统评估,以确保正式实验的成功。在细胞水平上展开蛋白质组研究应尽可能提取到细胞的所有蛋白,为此本研究先比较了机械刮除法、胰酶消化法和PBSE消化法收获肝癌细胞后的细胞形态与蛋白得率,结果显示PBSE消化法收集肝癌细胞时细胞形态完整,提取的蛋白得率最高。然后将提取的细胞蛋白经D IGETM试剂盒的3重荧光(Cy2、Cy3、Cy5)标记后再进行聚丙烯酰胺凝胶电泳(SDS-PAGE)和双向电泳(2DE,two-d im ensional electrophoresis)。前者图像使用Im ageQuant软件进行分析,结果说明肝癌细胞蛋白提取液与该染料能相互兼容,样品得到了有效的标记。2DE的扫描图像使用DeCyder 5.0软件的胶内分析(d ifferential in-gel analysis,D IA)和胶间分析(b iological variation analysis,BVA)模式,结果表明,Cy3和Cy5标记的蛋白质定量结果基本一致,但该染料标记肝癌细胞蛋白后会造成极少量蛋白出现位置偏差,给蛋白定量带来一定的误差影响,而BVA中的反标策略能够有效纠正这种由于不同荧光标记带来的定量误差。总之,本研究首次报道了用同一肝癌样品进行不同荧光标记后进行2DE的评估方法,该方法支持了D IGE体系定量的可靠性,而且能给出定量的误差范围,完善了评估体系,为D IGE技术平台的质量控制提供了参考。     

Generation of Pseudocontact Shifts in Proteins with Lanthanides Using Small “Clickable” Nitrilotriacetic Acid and Iminodiacetic Acid Tags

Pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions provide unique long‐range structural information in nuclear magnetic resonance (NMR) spectra, but the site‐specific attachment of lanthanide tags to proteins remains a challenge. Here we incorporated p‐azido‐phenylalanine (AzF) site‐specifically into the proteins ubiquitin and GB1, and ligated the AzF residue with alkyne derivatives of small nitrilotriacetic acid and iminodiacetic acid tags using the Cu^I‐catalysed “click” reaction. These tags form lanthanide complexes with no or only a small net charge and produced sizeable PCSs with paramagnetic lanthanide ions in all mutants tested. The PCSs were readily fitted by single magnetic susceptibility anisotropy tensors. Protein precipitation during the click reaction was greatly alleviated by the presence of 150 mM NaCl.     

Sequential functionalization of janus‐type dendrimer‐like poly(ethylene oxide)s with camptothecin and folic acid

Janus‐type dendrimer‐like poly(ethylene oxide)s (PEOs) of 1st, 2nd, and 3rd generation carrying terminal hydroxyl functions on one side and cleavable ketal groups on the other were used as substrates to conjugate folic acid as a folate receptor and camptothecin (CPT) as a therapeutic drug in a sequential fashion. The conjugation of both FA and CPT was accomplished by “click chemistry” based on the 1,3 dipolar cycloaddition coupling reaction. First, the hydroxyl functions present at one face of Janus‐type dendrimer‐like PEOs were transformed into alkyne groups through a simple Williamson‐type etherification reaction. Next, the ketals carried by the other face of the dendrimer‐like PEOs were hydrolyzed, yielding twice as many hydroxyls which were subsequently subjected to an esterification reaction using 2‐bromopropionic bromide. Before substituting azides for the bromide of 2‐bromopropionate esters just generated in the presence of NaN₃, an azido‐containing amidified FA derivative was reacted through click chemistry with alkyne functions introduced on the other face of the dendrimer‐like PEOs. A purposely designed alkyne‐functionalized biomolecule derived from CPT was conjugated to the azido functions carried by the dendritic PEOs by a second “click reaction.” In this case, twice as many CPT as FA moieties were finally conjugated to the two faces of the Janus‐type dendrimer‐like PEOs, the numbers of folate and CPT introduced being 2 and 4, 4 and 8, and 8 and 16 for samples of 1st, 2nd, and 3rd generation, respectively (route A). An alternate route for functionalizing the dendrimer‐like PEO of 1st generation consisted, first, in conjugating the azido‐containing CPT onto the alkyne groups present on one face of the dendritic PEO scaffold. The alkyne‐functionalized FA was further introduced by click chemistry after the bromides of 2‐bromopropionate esters were chemically transformed into azido groups. The corresponding prodrug thus contains 2 CPT and 4 FA external moieties (route B). Every reaction step product was thoroughly characterized by ¹H NMR spectroscopy. A preliminary investigation into the water solution properties of these functionalized dendritic PEOs is also presented. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Quantitative proteomics by fluorescent labeling of cysteine residues using a set of two cyanine‐based or three rhodamine‐based dyes

Despite all remarkable progress in gel‐based proteomics in recent years, there is still need to further improve quantification by decreasing the detection limits and increasing the dynamic range. These criteria are achieved best by fluorescent dyes that specifically stain the proteins either by adsorption after gel electrophoresis (in‐gel staining) or covalent coupling prior to gel electrophoresis (in‐solution staining). Here we report a multiplex analysis of protein samples using maleimide‐activated cyanine‐based (Cy3 and Cy5) and rhodamine‐based dyes (Dy505, Dy535, and Dy635) to permanently label all thiol‐groups of cysteine‐containing proteins. The detection limits in SDS‐PAGE were about 10 ng per band and even 2 ng for BSA due to its high content of cysteine residues. Thus only 5 μg protein of a mouse brain homogenate were analyzed by 2‐DE. Both cyanine‐ and rhodamine‐based dyes also stained proteins that did not contain cysteines, probably by reaction with amino groups. This side reactivity did not limit the method and might even extend its general use to proteins missing cysteine residues, but at a lower sensitivity. The dynamic range was more than two orders of magnitude in SDS‐PAGE and the Dy‐fluorophores did not alter the mobility of the tested proteins. Thus, a mixture of Dy505‐, Dy555‐, and Dy635‐labeled Escherichia coli lysates were separated by 2‐DE in a single gel and the three spot patterns relatively quantified.     

Bioconjugatable Azo‐Based Dark‐Quencher Dyes: Synthesis and Application to Protease‐Activatable Far‐Red Fluorescent Probes

We describe the efficient synthesis and one‐step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher‐3 (BHQ‐3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α‐iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper‐catalyzed azide–alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water‐soluble (sulfonated derivative) and aldehyde‐modified BHQ‐3 dyes, the direct preparation of which failed by means of conventional azo‐coupling reactions. The aldehyde‐ and α‐iodoacetyl‐containing fluorescence quenchers were readily conjugated to aminooxy‐ and cysteine‐containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red‐ or far‐red‐emitting rhodamine or cyanine dyes through sequential and/or one‐pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis.     

Azide/alkyne‐“click”‐reactions on amino resin materials: An LC‐ESI‐TOF analysis

This article describes the reaction of amino resins with functional molecules using the azide/alkyne‐“click”‐reaction, opening a simple chemical modification of amino resins under aqueous conditions. Alkyne‐modified melamine‐formaldehyde resins are prepared via a direct cocondensation approach using propargylic alcohol (21.6–86.3 mmol) as additive. Subsequently, alkyne‐modified mono‐, bi‐, and trinuclear melamine‐species are identified via LC‐ESI‐TOF methods proving the covalent incorporation of alkyne‐moieties in amounts of up to 3.9 mol %. Subsequent modification of the alkyne‐modified resins was accomplished by reaction of functional azides (octyl azide (1), (azidomethyl)benzene (2), 1‐(6‐azidohexyl) thymine (3), and 4‐azido‐N‐(2,2,6,6‐tetramethylpiperidin‐4‐yl)benzamide (4)) with Cu(I)Br and DIPEA as a base. The formation of triazolyl‐modified MF‐resins was proven by LC‐ESI‐TOF methods, indicating the successful covalent modification of the amino resin with the azides 1 – 4 . © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Combining “click” chemistry and step‐growth polymerization for the generation of highly functionalized polyesters

Aliphatic polyesters bearing pendant alkyne groups were successfully prepared by step‐growth polymerization of different building blocks such as adipic acid and succinic acid in combination with an acetylene‐based diol, 2‐methyl‐2‐propargyl‐1,3‐propanediol, besides 1,4‐butanediol and ethylene glycol. It was demonstrated that the alkyne groups survive the high reaction temperatures (200 °C) in the presence of a radical inhibitor. The alkyne loading has been tuned by the ratio of the different monomers used, up to 25 mol % of alkyne groups. Subsequently, the alkyne groups have been reacted with azides by the copper‐catalyzed Huisgen 1,3‐dipolar cycloaddition reaction, a popular type of “click” chemistry. “Click” reactions have been performed quantitatively in the presence of benzyl azide and azide‐terminated poly(ethylene glycol), yielding brush copolymers in the latter case. Kinetic investigations about this click reaction have been performed by means of on‐line Fourier transform mid‐infrared spectroscopy, which was reported for the first time in the field of the click chemistry research. A whole range of functionalized polyesters, based on poly(ethylene succinate) and poly(butylene adipate), is available, the properties of which can be tailored by choosing the appropriate azide compound. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6552–6564, 2008     

Synthesis and photoresponsive behavior of azobenzene‐containing side‐chain liquid crystalline diblock polymers with polypeptide block

Well‐defined azobenzene‐containing side‐chain liquid crystalline diblock copolymers composed of poly[6‐(4‐methoxy‐azobenzene‐4′‐oxy) hexyl methacrylate] (PMMAZO) and poly(γ‐benzyl‐L ‐glutamate) (PBLG) were synthesized by click reaction from alkyne‐ and azide‐functionalized homopolymers. The alkyne‐terminated PMMAZO homopolymers were synthesized by copper‐mediated atom transfer radical polymerization with a bromine‐containing alkyne bifunctional initiator, and the azido‐terminated PBLG homopolymers were synthesized by ring‐opening polymerization of γ‐benzyl‐L ‐glutamate‐N‐carboxyanhydride in DMF at room temperature using an amine‐containing azide initiator. The thermotropic phase behavior of PMMAZO‐b‐PBLG diblock copolymers in bulk were investigated using differential scanning calorimetry and polarized light microscopy. The PMMAZO‐b‐PBLG diblock copolymers exhibited a smectic phase and a nematic phase when the weight fraction of PMMAZO block was more than 50%. Photoisomerization behavior of PMMAZO‐b‐PBLG diblock copolymers and the corresponding PMMAZO homopolymers in solid film and in solution were investigated using UV–vis. In solution, trans–cis isomerization of diblock copolymers was slower than that of the corresponding PMMAZO homopolymers. These results may provide guidelines for the design of effective photoresponsive anisotropic materials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

A Sequential Homologation of Alkynes and Aldehydes for Chain Elongation with Optional 13C‐Labeling

Terminal alkynes (RCCH) are homologated by a sequence of ruthenium‐catalyzed anti‐Markovnikov hydration of alkyne to aldehyde (RCH₂CHO), followed by Bestmann–Ohira alkynylation of aldehyde to chain‐elongated alkyne (RCH₂CCH). Inverting the sequence by starting from aldehyde brings about the reciprocal homologation of aldehydes instead. The use of ¹³C‐labeled Bestmann–Ohira reagent (dimethyl ((1‐¹³C)‐1‐diazo‐2‐oxopropyl)phosphonate) for alkynylation provides straightforward access to singly or, through additional homologation, multiply ¹³C‐labeled alkynes. The labeled alkynes serve as synthetic platform for accessing a multitude of specifically ¹³C‐labeled products. Terminal alkynes with one or two ¹³C‐labels in the alkyne unit have been submitted to alkyne–azide click reactions; the copper‐catalyzed version (CuAAC) was found to display a regioselectivity of >50 000:1 for the 1,4‐ over the 1,5‐triazine isomer, as shown analytically by ¹³C NMR spectroscopy.     

A novel approach to tag and identify geranylgeranylated proteins

A recently developed proteomic strategy, the “GG‐azide”‐labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido‐geranylgeranyl analog and chemoselective derivatization of azido‐geranylgeranyl‐modified proteins by the “click” chemistry, using a tetramethylrhodamine‐alkyne. The resulting conjugated proteins can be separated by 1‐D or 2‐D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC‐MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG‐azide”‐labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post‐translational modifications.     

Labeling Cell Surface GPIs and GPI‐Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives

Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI‐anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell‐surface GPIs and GPI‐anchored proteins by using inositol derivatives carrying an azido group. The azide‐labeled GPIs and GPI‐anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin‐conjugated dyes or other molecules. The strategy can be used to label GPI‐anchored proteins with various tags for biological studies.     

Facile Synthesis of Glycopolypeptides by Combination of Ring‐Opening Polymerization of an Alkyne‐Substituted N‐carboxyanhydride and Click “Glycosylation”

Synthetic glycopolypeptides have attracted much interest for application in biomedical field as they are structural mimics to the natural glycopeptides or glycoproteins. However, the synthesis methods toward glycopolypeptides are still few or less efficient. Herein, we present a facile route to preparation of glycopolypeptides with highly effective “glycosylation” by click postpolymerization modification. First, an alkyne‐substituted N‐carboxyanhydride (NCA) monomer was synthesized and subsequently polymerized to afford the polypeptide with “clickable” alkyne pendants. The alkyne‐functionalized polypeptide was then “glycosylated” by click reaction of different sugar azides to the alkyne pendants with high efficiency. All the obtained glycopolypeptides were soluble and preferred α‐helix conformation in water. Primary studies on the obtained glycopolypeptides binding with Con A lectin were assessed by turbidimetric assay. The more quantitive studies of the interactions between lectin proteins and the synthetic glycopolypeptides, and the application of these materials as the multivalent ligands are in progress.

     

V‐shaped graft copolymers via triple click reactions: Diels–alder,copper‐catalyzed azide–alkyne cycloaddition,and nitroxide radical coupling

We report here a simple and universal synthetic pathway covering triple click reactions, Diels–Alder, copper‐catalyzed azide–alkyne cycloaddition (CuAAC), and nitroxide radical coupling (NRC), to prepare well‐defined graft copolymers with V‐shaped side chains. The Diels–Alder click reaction between the furan protected‐maleimide‐terminated poly(ethylene glycol) (PEG) and a trifunctional core ( 1 ) carrying an anthracene, alkyne, and bromide was carried out to yield the corresponding α‐alkyne‐ and α‐bromide‐terminated PEG (PEG‐alkyne/Br) in toluene at 110 °C. Subsequently, the polystyrene or polyoxanorbornene with pendant azide functionality as a main backbone is reacted with the PEG‐alkyne/Br and 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO)‐terminated poly(ε‐caprolactone) using the CuAAC and NRC reactions in a one‐pot fashion in N,N′‐dimethylformamide at room temperature to result in the target V‐shaped graft copolymers. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013 , 51, 4667–4674     

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Methyltransferases have proven useful to install functional groups site‐specifically in different classes of biomolecules when analogues of their cosubstrate S‐adenosyl‐l ‐methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site‐specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell‐permeable, thus preventing implementation of this strategy in cells. We present a two‐step enzymatic cascade for site‐specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5′ cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.     

Targeted Ultrasound‐Assisted Cancer‐Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry

Metabolic sugar labeling followed by the use of reagent‐free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N‐azidoacetylmannosamine (Ac₄ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac₄ManAz‐loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor‐localized ultrasound pulses. The released Ac₄ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)‐Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac₄ManAz‐loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer‐selective labeling and targeted cancer therapies.