Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

BackgroundFollowing fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown.ResultsWe identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development.ConclusionsOur study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0493-0) contains supplementary material, which is available to authorized users.

The cleavage of aprotecting group from aprotein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity.Disclosed herein is that vinyl ethers serve as protecting groups for alcoholcontaining molecules and as reagents for bioorthogonal bondcleavage reactions.Avinyl ether moiety was installed in arange of molecules,including amino acids,amonosaccharide,afluorophore,a nd an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics.I mportantly,t he nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.Bioorthogonal chemistry for covalently conjugating synthetic molecules at ap redefined protein residue has been am ajor focus of research in the past two decades.[1] Very recently,focus has been placed on reactions which can instead cleave specific bonds under bioorthogonal conditions.[2] This strategy holds great potential for the precise spatiotemporal control of protein function in vivo. [1c,2] Fore xample,p hotodeprotection of agenetically encoded caged cysteinecould be used to reveal the active native protein in live cells.[3]Similarly,p alladium-mediated depropargylation, [4] phosphine-mediated Staudinger reduction, [5] and tetrazine-triggered inverse electron-demand Diels-Alder (IEDDA) elimination reactions [6] were successfully employed to restore the activity of proteins bearing acaged lysine residue in the active site.B ond-cleavage reactions are also attractive for drugdelivery applications.P alladium-catalyzed deprotection of a5 -fluoroacil prodrug was shown as am ethod for controlled drug release in vivo. [7] TheI EDDAr eaction between at etrazine and ac aged doxorubicin derivative efficiently releases the cytotoxic drug.[8] Strategies based on IEDDA elimination reactions with tetrazines are particularly attractive for decaging relevant molecules in cells and interrogating biology,b ecause of the favorable kinetics and the abiotic nature of tetrazines when compared to photo-and metalcatalyzed reactions.O ne limitation, however,h as been the breadth of protecting groups available for stable,y et conditionally reversible linkages.T ypically,I EDDAe limination reactions have been used with strained alkene protecting groups connected through ac arbamate,t hus resulting in ac ascade release of ap rimary amine (Figure 1a). [2,9] Furthermore,the reduced metabolic stability of strained alkenes constitutes am ajor caveat for its utility.F or instance, ciscyclooctene easily isomerizes to the non-reactive trans-cyclooctene,thus limiting the efficiencyofthe decaging process in cells. [10] Herein, we report the development of av inyl ether/ tetrazine system as IEDDAreaction partners for the traceless

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein’s structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.

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