“Tag and Modify” Protein Conjugation with Dynamic Covalent Chemistry

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This article references 53 other publications.

1. 1

   Dozier, J. K. and Distefano, M. D. (2015) Site-Specific PEGylation of Therapeutic Proteins. Int. J. Mol. Sci. 16, 25831– 25864,  DOI: 10.3390/ijms161025831

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   Site-specific pegylation of therapeutic proteins

   Dozier, Jonathan K.; Distefano, Mark D.

   International Journal of Molecular Sciences (2015), 16 (10), 25831-25864CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)

   The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the no. of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degrdn. and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunol. response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG) group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chem. methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymic labeling.
2. 2

   Krall, N., da Cruz, F. P., Boutureira, O., and Bernardes, G. J. (2016) Site-selective protein-modification chemistry for basic biology and drug development. Nat. Chem. 8, 103– 113,  DOI: 10.1038/nchem.2393

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   Site-selective protein-modification chemistry for basic biology and drug development

   Krall, Nikolaus; da Cruz, Filipa P.; Boutureira, Omar; Bernardes, Goncalo J. L.

   Nature Chemistry (2016), 8 (2), 103-113CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)

   A review. Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-detd. sites. These reactions are now used to selectively install particular modifications on proteins for many biol. and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to det. their exact biol. roles. Labeling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiol. parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chem. site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.
3. 3

   Kuan, S. L., Wang, T., and Weil, T. (2016) Site-Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome. Chem. - Eur. J. 22, 17112– 17129,  DOI: 10.1002/chem.201602298

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   Site-Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

   Kuan, Seah Ling; Wang, Tao; Weil, Tanja

   Chemistry - A European Journal (2016), 22 (48), 17112-17129CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)

   A review. The synthetic transformation of polypeptides with mol. accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site-directed chem. to install addnl. features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site-selective modifications since most proteins contain disulfide bonds. In this Review, we present the chem. design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications.
4. 4

   Cho, H., Daniel, T., Buechler, Y. J., Litzinger, D. C., Maio, Z., Putnam, A.-M. H., Kraynov, V. S., Sim, B.-C., Bussell, S., Javahishvili, T., Kaphle, S., Viramontes, G., Ong, M., Chu, S., Becky, G. C., Lieu, R., Knudsen, N., Castiglioni, P., Norman, T. C., Axelrod, D. W., Hoffman, A. R., Schultz, P. G., DiMarchi, R. D., and Kimmel, B. E. (2011) Optimized clinical performance of growth hormone with an expanded genetic code. Proc. Natl. Acad. Sci. U. S. A. 108, 9060– 5,  DOI: 10.1073/pnas.1100387108

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   Optimized clinical performance of growth hormone with an expanded genetic code

   Cho, Ho; Daniel, Tom; Buechler, Ying Ji; Litzinger, David C.; Maio, Zhenwei; Putnam, Anna-Maria Hays; Kraynov, Vadim S.; Sim, Bee-Cheng; Bussell, Stuart; Javahishvili, Tsotne; Kaphle, Sami; Viramontes, Guillermo; Ong, Mike; Chu, Stephanie; Becky, G. C.; Lieu, Ricky; Knudsen, Nick; Castiglioni, Paola; Norman, Thea C.; Axelrod, Douglas W.; Hoffman, Andrew R.; Schultz, Peter G.; DiMarchi, Richard D.; Kimmel, Bruce E.

   Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (22), 9060-9065, S9060/1-S9060/3CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

   The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacol. properties. We report here the first clin. study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clin. studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chem. to optimize conventional natural products, low mol. wt. drugs, and peptides.
5. 5

   Elliott, T. S., Townsley, F. M., Bianco, A., Ernst, R. J., Sachdeva, A., Elsässer, S. J., Davis, L., Lang, K., Pisa, R., Greiss, S., Lilley, K. S., and Chin, J. W. (2014) Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat. Biotechnol. 32, 465– 472,  DOI: 10.1038/nbt.2860

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   Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal

   Elliott, Thomas S.; Townsley, Fiona M.; Bianco, Ambra; Ernst, Russell J.; Sachdeva, Amit; Elsaesser, Simon J.; Davis, Lloyd; Lang, Kathrin; Pisa, Rudolf; Greiss, Sebastian; Lilley, Kathryn S.; Chin, Jason W.

   Nature Biotechnology (2014), 32 (5), 465-472CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)

   Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chem. modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed std. food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddn. reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.
6. 6

   Boutureira, O. and Bernardes, G. J. L. (2015) Advances in chemical protein modification. Chem. Rev. 115, 2174– 2195,  DOI: 10.1021/cr500399p

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   Advances in Chemical Protein Modification

   Boutureira, Omar; Bernardes, Goncalo J. L.

   Chemical Reviews (Washington, DC, United States) (2015), 115 (5), 2174-2195CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)

   A review. Transition metal-free and -mediated approaches are covered.
7. 7

   Junutula, J. R., Raab, H., Clark, S., Bhakta, S., Leipold, D. D., Weir, S., Chen, Y., Simpson, M., Tsai, S. P., Dennis, M. S., Lu, Y., Meng, Y. G., Ng, C., Yang, J., Lee, C. C., Duenas, E., Gorrell, J., Katta, V., Kim, A., McDorman, K., Flagella, K., Venook, R., Ross, S., Spencer, S. D., Lee Wong, W., Lowman, H. B., Vandlen, R., Sliwkowski, M. X., Scheller, R. H., Polakis, P., and Mallet, W. (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925– 932,  DOI: 10.1038/nbt.1480

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   Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

   Junutula, Jagath R.; Raab, Helga; Clark, Suzanna; Bhakta, Sunil; Leipold, Douglas D.; Weir, Sylvia; Chen, Yvonne; Simpson, Michelle; Tsai, Siao Ping; Dennis, Mark S.; Lu, Yanmei; Meng, Y. Gloria; Ng, Carl; Yang, Jihong; Lee, Chien C.; Duenas, Eileen; Gorrell, Jeffrey; Katta, Viswanatham; Kim, Amy; McDorman, Kevin; Flagella, Kelly; Venook, Rayna; Ross, Sarajane; Spencer, Susan D.; Wong, Wai Lee; Lowman, Henry B.; Vandlen, Richard; Sliwkowski, Mark X.; Scheller, Richard H.; Polakis, Paul; Mallet, William

   Nature Biotechnology (2008), 26 (8), 925-932CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)

   Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogeneous conjugates with relatively narrow therapeutic index (max. tolerated dose/curative dose). Using leads from the authors' previously described phage display-based method to predict suitable conjugation sites, the authors engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb Ig folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepd. by conventional approaches. The favorable in vivo properties of the near-homogeneous compn. of this conjugate suggest that this strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
8. 8

   Stephan, J.-P., Chan, P., Lee, C., Nelson, C., Elliott, J. M., Bechtel, C., Raab, H., Xie, D., Akutagawa, J., Baudys, J., Saad, O., Prabhu, S., Wong, W. L. T., Vandlen, R., Jacobson, F., and Ebens, A. (2008) Anti-CD22-MCC-DM1 and MC-MMAF Conjugates: Impact of Assay Format on Pharmacokinetic Parameters Determination. Bioconjugate Chem. 19, 1673– 1683,  DOI: 10.1021/bc800059t

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   Anti-CD22-MCC-DM1 and MC-MMAF Conjugates: Impact of Assay Format on Pharmacokinetic Parameters Determination

   Stephan, Jean-Philippe; Chan, Pamela; Lee, Chien; Nelson, Christopher; Elliott, James Michael; Bechtel, Charity; Raab, Helga; Xie, David; Akutagawa, Jon; Baudys, Jakub; Saad, Ola; Prabhu, Saileta; Wong, Wai Lee T.; Vandlen, Richard; Jacobson, Fred; Ebens, Allen

   Bioconjugate Chemistry (2008), 19 (8), 1673-1683CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)

   CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To det. the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters detn., we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately det. the ADC PK parameters.
9. 9

   Chalker, J. M., Bernardes, G. J. L., and Davis, B. G. (2011) A “Tag-and-Modify” Approach to Site-Selective Protein Modification. Acc. Chem. Res. 44, 730– 741,  DOI: 10.1021/ar200056q

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   A "Tag-and-Modify" Approach to Site-Selective Protein Modification

   Chalker, Justin M.; Bernardes, Goncalo J. L.; Davis, Benjamin G.

   Accounts of Chemical Research (2011), 44 (9), 730-741CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)

   A review. Covalent modification can expand a protein's functional capacity. Fluorescent or radioactive labeling, for instance, allows imaging of a protein in real time. Labeling with an affinity probe enables isolation of target proteins and other interacting mols. At the other end of this functional spectrum, protein structures can be naturally altered by enzymic action. Protein-protein interactions, genetic regulation, and a range of cellular processes are under the purview of these post-translational modifications. The ability of protein chemists to install these covalent addns. selectively has been crit. for elucidating their roles in biol. Frequently the transformations must be applied in a site-specific manner, which demands the most selective chem. In this Account, we discuss the development and application of such chem. in our lab. A centerpiece of our strategy is a "tag-and-modify" approach, which entails sequential installation of a uniquely reactive chem. group into the protein (the "tag") and the selective or specific modification of this group. The chem. tag can be a natural or unnatural amino acid residue. Of the natural residues, cysteine is the most widely used as a tag. Early work in our program focused on selective disulfide formation in the synthesis of glycoproteins. For certain applications, the susceptibility of disulfides to redn. was a limitation and prompted the development of several methods for the synthesis of more stable thioether modifications. The desulfurization of disulfides and conjugate addn. to dehydroalanine are two routes to these modifications. The dehydroalanine tag has since proven useful as a general precursor to many modifications after conjugate addn. of various nucleophiles; phosphorylated, glycosylated, peptidylated, prenylated, and even mimics of methylated and acetylated lysine-contg. proteins are all accessible from dehydroalanine. While cysteine is a useful tag for selective modification, unnatural residues present the opportunity for bio-orthogonal chem. Azide-, arylhalide-, alkyne-, and alkene-contg. amino acids can be incorporated into proteins genetically and can be specifically modified through various transformations. These transformations often rely on metal catalysis. The Cu-catalyzed azide-alkyne addn., Ru-catalyzed olefin metathesis, and Pd-catalyzed cross-coupling are examples of such transformations. In the course of adapting these reactions to protein modification, we learned much about the behavior of these reactions in water, and in some cases entirely new catalysts were developed. Through a combination of these bio-orthogonal transformations from the panel of tag-and-modify reactions, multiple and distinct modifications can be installed on protein surfaces. Multiple modifications are common in natural systems, and synthetic access to these proteins has enabled study of their biol. role. Throughout these investigations, much has been learned in chem. and biol. The demands of selective protein modification have revealed many aspects of reaction mechanisms, which in turn have guided the design of reagents and catalysts that allow their successful deployment in water and in biol. milieu. With this ability to modify proteins, it is now possible to interrogate biol. systems with precision that was not previously possible.
10. 10

    Zhang, X., Lu, W., Kwan, K., Bhattacharyya, D., and Wei, Y. (2017) Dual-Functional-Tag-Facilitated Protein Labeling and Immobilization. ACS Omega 2, 522– 528,  DOI: 10.1021/acsomega.6b00512

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    Rybak, J.-N., Scheurer, S. B., Neri, D., and Elia, G. (2004) Purification of biotinylated proteins on streptavidin resin: A protocol for quantitative elution. Proteomics 4, 2296– 2299,  DOI: 10.1002/pmic.200300780

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    Purification of biotinylated proteins on streptavidin resin: A protocol for quantitative elution

    Rybak, Jascha-N.; Scheurer, Simone B.; Neri, Dario; Elia, Giuliano

    Proteomics (2004), 4 (8), 2296-2299CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)

    The interaction between streptavidin and biotin is one of the most widely used tools in chem. and biol. However, the release of biotinylated proteins from streptavidin resins remains a major problem, due to the extraordinary stability of this complex. We present a new protocol for the quant. elution of biotinylated proteins from streptavidin Sepharose, featuring harsh elution conditions and competition with free biotin. The usefulness of the method was demonstrated by the quant. recovery of biotinylated proteins from organ homogenates, obtained from mice perfused with a reactive ester deriv. of biotin.
12. 12

    Campos, S. K., Parrott, M. B., and Barry, M. A. (2004) Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector. Mol. Ther. 9, 942– 54,  DOI: 10.1016/j.ymthe.2004.03.006

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    Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector

    Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

    Molecular Therapy (2004), 9 (6), 942-954CODEN: MTOHCK; ISSN:1525-0016. (Elsevier)

    While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, the authors have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10-15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10-7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purifn. on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a mol. screening tool to assess the utility of different viral capsid proteins for ligand display and the biol. and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purifn. of adenoviral vectors.
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    Bornhorst, J. A. and Falke, J. J. (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol. 326, 245– 254,  DOI: 10.1016/S0076-6879(00)26058-8

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    Purification of proteins using polyhistidine affinity tags

    Bornhorst, Joshua A.; Falke, Joseph J.

    Methods in Enzymology (2000), 326 (Applications of Chimeric Genes and Hybrid Proteins, Pt. A), 245-254CODEN: MENZAU; ISSN:0076-6879. (Academic Press)

    A review with 30 refs. The expression and subsequent purifn. of recombinant proteins are widely employed in biochem. studies. A powerful purifn. method involves the use of peptide affinity tags, which are fused to the protein of interest and used to expedite protein purifn. via affinity chromatog. A widely employed method utilizes immobilized metal-affinity chromatog. (IMAC) to purify recombinant proteins contg. a short affinity tag consisting of polyhistidine residues. IMAC is a versatile method that can be utilized to rapidly purify polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purifn. step. Affinity-tagged protein purities can be achieved at up to 95% purity by IMAC in high yield. Protocols for the method are described in detail. (c) 2000 Academic Press.
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    Kimple, M. E., Brill, A. L., and Pasker, R. L. (2013) Overview of Affinity Tags for Protein Purification. Curr. Protoc Protein Sci. 73, 608– 616,  DOI: 10.1002/0471140864.ps0909s73

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    Fexby, S. and Bü Low, L. (2004) Hydrophobic peptide tags as tools in bioseparation. Trends Biotechnol. 22, 511– 516,  DOI: 10.1016/j.tibtech.2004.08.005

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    Jin, Y., Yu, C., Denman, R. J., and Zhang, W. (2013) Recent advances in dynamic covalent chemistry. Chem. Soc. Rev. 42, 6634– 6654,  DOI: 10.1039/c3cs60044k

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    Recent advances in dynamic covalent chemistry

    Jin, Yinghua; Yu, Chao; Denman, Ryan J.; Zhang, Wei

    Chemical Society Reviews (2013), 42 (16), 6634-6654CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)

    A review. Dynamic covalent chem. (DCvC) has been strongly integrated into diverse research fields, and has enabled easy access to a variety of combinatorial libraries, 2-D macrocycles, and 3-D mol. cages that target many important applications, such as drug discovery, biotechnol., mol. sepn., light harvesting, etc. DCvC relies on the reversible formation and breaking of rather strong covalent bonding within mols. Therefore it combines the error-correction capability of supramol. chem. and the robustness of covalent bonding. Compared to those supramol. interactions, dynamic covalent reactions usually have slower kinetics and require the assistance of catalysts to achieve rapid equil. Although the scope of dynamic covalent reactions is rapidly expanding, the reversible reactions suitable for DCvC are still very limited. The identification and development of new dynamic reactions and catalysts would be crit. for the further advancement of DCvC. This review covers the recent development of dynamic covalent reactions as well as their applications.
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    Bartolami, E., Bessin, Y., Gervais, V., Dumy, P., and Ulrich, S. (2015) Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters. Angew. Chem., Int. Ed. 54, 10183– 10187,  DOI: 10.1002/anie.201504047

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    Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters

    Bartolami, Eline; Bessin, Yannick; Gervais, Virginie; Dumy, Pascal; Ulrich, Sebastien

    Angewandte Chemie, International Edition (2015), 54 (35), 10183-10187CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    We report herein the implementation of a dynamic covalent chem. approach to the generation of multivalent clusters for DNA recognition. We show that biomol. clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titrn. calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chem. effector can be used to trigger DNA release through component exchange reactions.
18. 18

    Reuther, J. F., Dees, J. L., Kolesnichenko, I. V., Hernandez, E. T., Ukraintsev, D. V., Guduru, R., Whiteley, M., and Anslyn, E. V. (2017) Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner. Nat. Chem. 10, 45– 50,  DOI: 10.1038/nchem.2847

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    Diehl, K. L., Kolesnichenko, I. V., Robotham, S. A., Bachman, J. L., Zhong, Y., Brodbelt, J. S., and Anslyn, E. V. (2016) Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor. Nat. Chem. 8, 968– 973,  DOI: 10.1038/nchem.2601

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    Click and chemically triggered declick reactions through reversible amine and thiol coupling via a conjugate acceptor

    Diehl, Katharine L.; Kolesnichenko, Igor V.; Robotham, Scott A.; Bachman, J. Logan; Zhong, Ye; Brodbelt, Jennifer S.; Anslyn, Eric V.

    Nature Chemistry (2016), 8 (10), 968-973CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)

    The coupling and decoupling of mol. units is a fundamental undertaking of org. chem. Herein we report the use of a very simple conjugate acceptor, derived from Meldrum's acid, for the sequential 'clicking' together of an amine and a thiol in aq. conditions at neutral pH. Subsequently, this linkage can be 'declicked' by a chem. trigger to release the original amine and thiol undisturbed. The reactivity differs from that of other crosslinking agents because the selectivity for sequential functionalization derives from an altering of the electrophilicity of the conjugate acceptor on the addn. of the amine. We describe the use of the procedure to modify proteins, create multicomponent libraries and synthesize oligomers, all of which can be declicked to their starting components in a controlled fashion when desired. Owing to the mild reaction conditions and ease of use in a variety of applications, the method is predicted to have wide utility.
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    Wang, T., Ng, D. Y. W., Wu, Y., Thomas, J., TamTran, T., and Weil, T. (2014) Bis-sulfide bioconjugates for glutathione triggered tumor responsive drug release. Chem. Commun. 50, 1116– 1118,  DOI: 10.1039/C3CC47003B

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    Bis-sulfide bioconjugates for glutathione triggered tumor responsive drug release

    Wang, Tao; Ng, David Y. W.; Wu, Yuzhou; Thomas, Jessica; TamTran, Thuy; Weil, Tanja

    Chemical Communications (Cambridge, United Kingdom) (2014), 50 (9), 1116-1118CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)

    The reaction of bis-sulfone conjugation reagents with disulfide bonds allows the site-specific modification of various peptides and proteins. Herein, we present the intracellular disintegration of bis-sulfide contg. somatostatin bioconjugates under controlled, tumor-relevant glutathione levels. GSH responsive release is demonstrated, which offers high potential for designing tumor responsive therapeutics.
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    Brustad, E., Bushey, M. L., Lee, J. W., Groff, D., Liu, W., and Schultz, P. G. (2008) A Genetically Encoded Boronate-Containing Amino Acid. Angew. Chem., Int. Ed. 47, 8220– 8223,  DOI: 10.1002/anie.200803240

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    A genetically encoded boronate-containing amino acid

    Brustad, Eric; Bushey, Mark L.; Lee, Jae Wook; Groff, Dan; Liu, Wenshe; Schultz, Peter G.

    Angewandte Chemie, International Edition (2008), 47 (43), 8220-8223CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    A biol. boronate: An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved for the genetic incorporation of a boronic acid into proteins. This amino acid has been used to purify proteins in a one-step scarless purifn. procedure as well as for the site-specific labeling of proteins using various boronic acid chemistries.
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    Arzt, M., Seidler, C., Ng, D. Y. W., and Weil, T. (2014) Reversible Click Reactions with Boronic Acids to Build Supramolecular Architectures in Water. Chem. - Asian J. 9, 1994,  DOI: 10.1002/asia.201402061

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    Reversible Click Reactions with Boronic Acids to Build Supramolecular Architectures in Water

    Arzt, Matthias; Seidler, Christiane; Ng, David Y. W.; Weil, Tanja

    Chemistry - An Asian Journal (2014), 9 (8), 1994-2003CODEN: CAAJBI; ISSN:1861-4728. (Wiley-VCH Verlag GmbH & Co. KGaA)

    A review. The interaction of boronic acids with various bifunctional reagents offers great potential for the prepn. of responsive supramol. architectures. Boronic acids react with 1,2-diols yielding cyclic boronate esters that are stable at pH>7.4 but can be hydrolyzed at pH<5.0. The phenylboronic acid (PBA)-salicylhydroxamic acid (SHA) system offers ultra-fast reaction kinetics and high binding affinities. This Focus review summarizes the current advances in exploiting the bioorthogonal interaction of boronic acids to build pH-responsive supramol. architectures in water.
23. 23

    Jackson, T. R., Springall, J. S., Rogalle, D., Masumoto, N., Ching Li, H., D’Hooge, F., Perera, S. P., Jenkins, T. A., James, T. D., Fossey, J. S., and van den Elsen, J. M. H. (2008) Boronate affinity saccharide electrophoresis: A novel carbohydrate analysis tool. Electrophoresis 29, 4185– 4191,  DOI: 10.1002/elps.200800178

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24. 24

    Ng, D. Y. W., Arzt, M., Wu, Y., Kuan, S. L., Lamla, M., and Weil, T. (2014) Constructing Hybrid Protein Zymogens through Protective Dendritic Assembly. Angew. Chem., Int. Ed. 53, 324– 328,  DOI: 10.1002/anie.201308533

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    Constructing Hybrid Protein Zymogens through Protective Dendritic Assembly

    Ng, David Y. W.; Arzt, Matthias; Wu, Yuzhou; Kuan, Seah Ling; Lamla, Markus; Weil, Tanja

    Angewandte Chemie, International Edition (2014), 53 (1), 324-328CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    The modulation of protein uptake and activity in response to physiol. changes forms an integral part of smart protein therapeutics. We describe herein the self-assembly of a pH-responsive dendrimer shell onto the surface of active enzymes (trypsin, papain, DNase I) as a supramol. protecting group to form a hybrid dendrimer-enzyme complex. The attachment is based on the interaction between boronic acid and salicyl hydroxamate, thus allowing the macromol. assembly to respond to changes in pH between 5.0 and 7.4 in a highly reversible fashion. Catalytic activity is efficiently blocked in the presence of the dendrimer shell but is quant. restored upon shell degrdn. under acidic conditions. Unlike the native proteases, the hybrid constructs are shown to be efficiently taken up by A549 cells and colocalized in the acidic compartments. The programmed intracellular release of the proteases induced cytotoxicity, thereby uncovering a new avenue for precision biotherapeutics.
25. 25

    Seidler, C., Ng, D. Y. W., Wu, Y., and Weil, T. (2016) pH responsive supramolecular core-shell protein hybrids. Supramol. Chem. 28, 742– 746,  DOI: 10.1080/10610278.2016.1140764

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    pH responsive supramolecular core-shell protein hybrids

    Seidler, Christiane; Ng, David Y. W.; Wu, Yuzhou; Weil, Tanja

    Supramolecular Chemistry (2016), 28 (9-10), 742-746CODEN: SCHEER; ISSN:1029-0478. (Taylor & Francis Ltd.)

    PEGylation of proteins remains an integral part of macromol. therapeutics due to its well-known benign effects and pharmacokinetic enhancement properties. We report herein that PEGylation can be taken to the next level of complexity and dynamic behavior by introducing highly stable but responsive supramol. handles. By attaching small boronic acid groups onto proteins and salicylhydroxamate moiety to end-functionalise PEG chains, we demonstrate a comprehensive study on the facile assembly/disassembly of a core-shell protein-polymer architecture using fluorescence and microscale thermophoresis on a macromol. level. In addn., we demonstrate that both the activity and cellular transfer of functional proteins remained conserved throughout the assembly process thus establishing a rapid and orthogonal strategy towards protein PEGylation.
26. 26

    Swaminathan, R., Ravi, V. K., Kumar, S., Kumar, M. V. S., and Chandra, N. (2011) Lysozyme: A model protein for amyloid research. Adv. Protein Chem. Struct. Biol. 84, 63– 111,  DOI: 10.1016/B978-0-12-386483-3.00003-3

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    Lysozyme: A model protein for amyloid research

    Swaminathan, Rajaram; Ravi, Vijay Kumar; Kumar, Satish; Kumar, Mattaparthi Venkata Satish; Chandra, Nividh

    Advances in Protein Chemistry and Structural Biology (2011), 84 (), 63-111CODEN: APCSG7; ISSN:1876-1623. (Elsevier Ltd.)

    A review. Ever since lysozyme was discovered by Fleming in 1922, this protein has emerged as a model for investigations on protein structure and function. Over the years, several high-resoln. structures have yielded a wealth of structural data on this protein. Extensive studies on folding of lysozyme have shown how different regions of this protein dynamically interact with one another. Data is also available from numerous biotechnol. studies wherein lysozyme has been employed as a model protein for recovering active recombinant protein from inclusion bodies using small mols. like L-arginine. A variety of conditions have been developed in vitro to induce fibrillation in hen lysozyme. They include (a) acidic pH at elevated temp., (b) concd. solns. of ethanol, (c) moderate concns. of guanidinium hydrochloride at moderate temp., and (d) alk. pH at room temp. This review aims to bring together similarities and differences in aggregation mechanisms, morphol. of aggregates, and related issues that arise using the different conditions mentioned above to improve our understanding. The alk. pH condition (pH 12.2), discovered and studied extensively in our lab, shall receive special attention. More than a decade ago, it was revealed that mutations in human lysozyme can cause accumulation of large quantities of amyloid in liver, kidney, and other regions of gastrointestinal tract. Understanding the mechanism of lysozyme aggregation will probably have therapeutic implications for the treatment of systemic nonneuropathic amyloidosis. Numerous studies have begun to focus attention on inhibition of lysozyme aggregation using antibody or small mols. The enzymic activity of lysozyme presents a convenient handle to quantify the native population of lysozyme in a sample where aggregation has been inhibited. The rich information available on lysozyme coupled with the multiple conditions that have been successful in inducing/inhibiting its aggregation in vitro makes lysozyme an ideal model protein to investigate amyloidogenesis.
27. 27

    Rambaran, R. N. and Serpell, L. C. (2008) Amyloid fibrils: abnormal protein assembly. Prion 2, 112– 7,  DOI: 10.4161/pri.2.3.7488

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    Amyloid fibrils: abnormal protein assembly

    Rambaran Roma N; Serpell Louise C

    Prion (2008), 2 (3), 112-7 ISSN:.

    Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is insoluble and is structurally dominated by beta-sheet structure. Unlike other fibrous proteins it does not commonly have a structural, supportive or motility role but is associated with the pathology seen in a range of diseases known as the amyloidoses. These diseases include Alzheimer's, the spongiform encephalopathies and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality. Not surprisingly, research into the physicochemical properties of amyloid and its formation is currently intensely pursued. In this chapter we will highlight the key scientific findings and discuss how the stability of amyloid fibrils impacts on bionanotechnology.
28. 28

    Mankar, S., Anoop, A., Sen, S., and Maji, S. K. (2011) Nanomaterials: amyloids reflect their brighter side. Nano Rev. 2, 6032,  DOI: 10.3402/nano.v2i0.6032

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    Meier, C. and Welland, M. E. (2011) Wet-Spinning of Amyloid Protein Nanofibers into Multifunctional High-Performance Biofibers. Biomacromolecules 12, 3453– 3459,  DOI: 10.1021/bm2005752

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    29

    Wet-Spinning of Amyloid Protein Nanofibers into Multifunctional High-Performance Biofibers

    Meier, Christoph; Welland, Mark E.

    Biomacromolecules (2011), 12 (10), 3453-3459CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)

    Amyloid nanofibers derived from hen egg white lysozyme were processed into macroscopic fibers in a wet-spinning process based on interfacial polyion complexation using a polyanionic polysaccharide as crosslinker. As a result of their amyloid nanostructure, the hierarchically self-assembled protein fibers have a stiffness of up to 14 GPa and a tensile strength of up to 326 MPa. Fine-tuning of the polyelectrolytic interactions via pH allows to trigger the release of small mols., as demonstrated with riboflavin-5'-phosphate. The amyloid fibrils, highly oriented within the gellan gum matrix, were mineralized with calcium phosphate, mimicking the fibrolamellar structure of bone. The formed mineral crystals are highly oriented along the nanofibers, thus resulting in a 9-fold increase in fiber stiffness.
30. 30

    Wang, T., Riegger, A., Lamla, M., Wiese, S., Oeckl, P., Otto, M., Wu, Y., Fischer, S., Barth, H., Kuan, S. L., and Weil, T. (2016) Water-soluble allyl sulfones for dual site-specific labelling of proteins and cyclic peptides. Chem. Sci. 7, 3234– 3239,  DOI: 10.1039/C6SC00005C

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    30

    Water-soluble allyl sulfones for dual site-specific labelling of proteins and cyclic peptides

    Wang, Tao; Riegger, Andreas; Lamla, Markus; Wiese, Sebastian; Oeckl, Patrick; Otto, Markus; Wu, Yuzhou; Fischer, Stephan; Barth, Holger; Kuan, Seah Ling; Weil, Tanja

    Chemical Science (2016), 7 (5), 3234-3239CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)

    Water-sol. allyl sulfones provide convenient site-specific disulfide rebridging of native proteins and cyclic peptides. The site-selective functionalization of (a) the peptide hormone somatostatin, (b) the interchain disulfide of bovine insulin and (c) functionalization of the proteins GFP and lysozyme with allyl sulfones proceeds in aq. soln. Allyl sulfones offer three functionalizable sites that react with thiol contg. mols. in a step-wise fashion. Dual labeling of proteins and cyclic peptides is achieved attachment of chromophore and affinity tag in a single reaction step, which is of great significance for the construction of precise multifunctional peptide and protein conjugates.
31. 31

    Brocchini, S., Balan, S., Godwin, A., Choi, J.-W., Zloh, M., and Shaunak, S. (2006) PEGylation of native disulfide bonds in proteins. Nat. Chem. Biol. 1, 2241– 2252,  DOI: 10.1038/nprot.2006.346

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    Robinson, E., Nunes, J. P. M., Vassileva, V., Maruani, A., Nogueira, J. C. F., Smith, M. E. B., Pedley, R. B., Caddick, S., Baker, J. R., and Chudasama, V. (2017) Pyridazinediones deliver potent, stable, targeted and efficacious antibody–drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody. RSC Adv. 7, 9073– 9077,  DOI: 10.1039/C7RA00788D

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    Pyridazinediones deliver potent, stable, targeted and efficacious antibody-drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody

    Robinson, Eifion; Nunes, Joao P. M.; Vassileva, Vessela; Maruani, Antoine; Nogueira, Joao C. F.; Smith, Mark E. B.; Pedley, R. Barbara; Caddick, Stephen; Baker, James R.; Chudasama, Vijay

    RSC Advances (2017), 7 (15), 9073-9077CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)

    Herein we report the use of pyridazinediones to functionalize the native solvent accessible interstrand disulfide bonds in trastuzumab with monomethyl auristatin E (MMAE). This method of conjugation delivers serum stable antibody-drug conjugates (ADCs) with a controlled drug loading of 4. Moreover, we demonstrate that the MMAE-bearing ADCs are potent, selective and efficacious against cancer cell lines in both in vitro and in vivo models.
33. 33

    Greene, M. K., Richards, D. A., Nogueira, J. C. F., Campbell, K., Smyth, P., Fernández, M., Scott, C. J., and Chudasama, V. (2018) Forming next-generation antibody–nanoparticle conjugates through the oriented installation of non-engineered antibody fragments. Chem. Sci. 9, 79– 87,  DOI: 10.1039/C7SC02747H

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    Forming next-generation antibody-nanoparticle conjugates through the oriented installation of non-engineered antibody fragments

    Greene, Michelle K.; Richards, Daniel A.; Nogueira, Joao C. F.; Campbell, Katrina; Smyth, Peter; Fernandez, Marcos; Scott, Christopher J.; Chudasama, Vijay

    Chemical Science (2018), 9 (1), 79-87CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)

    The successful development of targeted nanotherapeutics is contingent upon the conjugation of therapeutic nanoparticles to target-specific ligands, with particular emphasis being placed on antibody-based ligands. Thus, new methods that enable the covalent and precise installation of targeting antibodies to nanoparticle surfaces are greatly desired, esp. those which do not rely on costly and time-consuming antibody engineering techniques. Herein we present a novel method for the highly controlled and oriented covalent conjugation of non-engineered antibody F(ab) fragments to PLGA-PEG nanoparticles using disulfide-selective pyridazinedione linkers and strain-promoted alkyne-azide click chem. Exemplification of this method with trastuzumab and cetuximab showed significant improvements in both conjugation efficiency and antigen binding capability, when compared to commonly employed strategies for antibody-nanoparticle construction. This new approach paves the way for the development of antibody-targeted nanomedicines with improved paratope availability, reproducibility and uniformity to enhance both biol. activity and ease of manuf.
34. 34

    Trivedi, M. V., Laurence, J. S., and Siahaan, T. J. (2009) The role of thiols and disulfides on protein stability. Curr. Protein Pept. Sci. 10, 614– 25,  DOI: 10.2174/138920309789630534

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    The role of thiols and disulfides on protein stability

    Trivedi, Maulik V.; Laurence, Jennifer S.; Siahaan, Teruna J.

    Current Protein and Peptide Science (2009), 10 (6), 614-625CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)

    A review. There has been a tremendous increase in the no. of approved drugs derived from recombinant proteins; however, their development as potential drugs has been hampered by their instability that causes difficulty to formulate them as therapeutic agents. It has been shown that the reactivity of thiol and disulfide functional groups could catalyze chem. (i.e., oxidn. and β-elimination reactions) and phys. (i.e., aggregation and pptn.) degrdn. of proteins. Because most proteins contain a free Cys residue and/or a disulfide bond, this review is focused on their roles in the phys. and chem. stability of proteins. The effect of introducing a disulfide bond to improve phys. stability of proteins and the mechanisms of degrdn. of disulfide bond were discussed. The qual./quant. methods to det. the presence of the thiol in the Cys residue and various methods to derivatize the thiol group for improving protein stability were also illustrated.
35. 35

    Rosen, C. B. and Francis, M. B. (2017) Targeting the N terminus for site-selective protein modification. Nat. Chem. Biol. 13, 697– 705,  DOI: 10.1038/nchembio.2416

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    Targeting the N terminus for site-selective protein modification

    Rosen, Christian B.; Francis, Matthew B.

    Nature Chemical Biology (2017), 13 (7), 697-705CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)

    A review. The formation of well-defined protein bioconjugates is crit. for many studies and technologies in chem. biol. Tried-and-true methods for accomplishing this typically involve the targeting of cysteine residues, but the rapid growth of contemporary bioconjugate applications has required an expanded repertoire of modification techniques. One very powerful set of strategies involves the modification of proteins at their N termini, as these positions are typically solvent exposed and provide chem. distinct sites for many protein targets. Several chem. techniques can be used to modify N-terminal amino acids directly or convert them into unique functional groups for further ligations. A growing no. of N-terminus-specific enzymic ligation strategies provided addnl. possibilities. This Perspective provides an overview of N-terminal modification techniques and the chem. rationale governing each. Examples of specific N-terminal protein conjugates are provided, along with their uses in a no. of diverse biol. applications.
36. 36

    Tanaka, K., Fukase, K., and Katsumura, S. (2011) Exploring a Unique Reactivity of 6p-Azaelectrocyclization to Enzyme Inhibition, Natural Products Synthesis, and Molecular Imaging: An Approach to Chemical Biology by Synthetic Chemists. Synlett 2011, 2115– 2139,  DOI: 10.1055/s-0030-1261192

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    Ban, H., Nagano, M., Gavrilyuk, J., Hakamata, W., Inokuma, T., and Barbas, C. F. (2013) Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 24, 520– 532,  DOI: 10.1021/bc300665t

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    37

    Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

    Ban, Hitoshi; Nagano, Masanobu; Gavrilyuk, Julia; Hakamata, Wataru; Inokuma, Tsubasa; Barbas, Carlos F., III

    Bioconjugate Chemistry (2013), 24 (4), 520-532CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)

    The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivs. in peptide and protein chem., we synthesized PTAD derivs. possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivs. and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decompn. resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addn. of a small amt. of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD deriv. linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small mols. The tyrosine click linkage demonstrated stability to extremes of pH, temp., and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small mols., peptides, and proteins under mild aq. conditions over a broad pH range using a wide variety of biol. acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compns.
38. 38

    Santos, F. M. F., Rosa, J. N., Candeias, N. R., Carvalho, C. P., Matos, A. I., Ventura, A. E., Florindo, H. F., Silva, L. C., Pischel, U., and Gois, P. M. P. (2016) A Three-Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes). Chem. - Eur. J. 22, 1631– 1637,  DOI: 10.1002/chem.201503943

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    A Three-Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes)

    Santos, Fabio M. F.; Rosa, Joao N.; Candeias, Nuno R.; Carvalho, Catia Parente; Matos, Ana I.; Ventura, Ana E.; Florindo, Helena F.; Silva, Liana C.; Pischel, Uwe; Gois, Pedro M. P.

    Chemistry - A European Journal (2016), 22 (5), 1631-1637CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)

    The modular assembly of boronic acids with Schiff-base ligands enabled the construction of innovative fluorescent dyes [boronic acid salicylidenehydrazone (BASHY)] with suitable structural and photophys. properties for live cell bioimaging applications. This reaction enabled the straightforward synthesis (yields up to 99 %) of structurally diverse and photostable dyes that exhibit a polarity-sensitive green-to-yellow emission with high quantum yields of up to 0.6 in nonpolar environments. These dyes displayed a high brightness (up to 54 000 M-1 cm-1). The promising structural and fluorescence properties of BASHY dyes fostered the prepn. of non-cytotoxic, stable, and highly fluorescent poly(lactide-co-glycolide) nanoparticles that were effectively internalized by dendritic cells. The dyes were also shown to selectively stain lipid droplets in HeLa cells, without inducing any appreciable cytotoxicity or competing plasma membrane labeling; this confirmed their potential as fluorescent stains.
39. 39

    Cal, P. M. S. D., Sieglitz, F., Santos, F. M. F., Parente Carvalho, C., Guerreiro, A., Bertoldo, J. B., Pischel, U., Gois, P. M. P., and Bernardes, G. J. L. (2017) Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells. Chem. Commun. 53, 368– 371,  DOI: 10.1039/C6CC08671C

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    39

    Site-selective installation of BASHY fluorescent dyes to Annexin V for targeted detection of apoptotic cells

    Cal, Pedro M. S. D.; Sieglitz, Florian; Santos, Fabio M. F.; Parente Carvalho, Catia; Guerreiro, Ana; Bertoldo, Jean B.; Pischel, Uwe; Gois, Pedro M. P.; Bernardes, Goncalo J. L.

    Chemical Communications (Cambridge, United Kingdom) (2017), 53 (2), 368-371CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)

    Fluorophores are indispensable for imaging biol. processes. The authors report the design and synthesis of azide-tagged boronic acid salicylidenehydrazone (BASHY) dyes and their use for site-selective labeling of Annexin V. The Annexin V-BASHY conjugate maintained function and fluorescence as demonstrated by the targeted detection of apoptotic cells.
40. 40

    Ng, D. Y. W., Vill, R., Wu, Y., Koynov, K., Tokura, Y., Liu, W., Sihler, S., Kreyes, A., Ritz, S., Barth, H., Ziener, U., and Weil, T. (2017) Directing intracellular supramolecular assembly with N-heteroaromatic quaterthiophene analogues. Nat. Commun. 8, 1850,  DOI: 10.1038/s41467-017-02020-2

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    Directing intracellular supramolecular assembly with N-heteroaromatic quaterthiophene analogues

    Ng David Y W; Wu Yuzhou; Koynov Kaloian; Tokura Yu; Liu Weina; Weil Tanja; Vill Roman; Tokura Yu; Liu Weina; Sihler Susanne; Kreyes Andreas; Ziener Ulrich; Weil Tanja; Ritz Sandra; Barth Holger

    Nature communications (2017), 8 (1), 1850 ISSN:.

    Self-assembly in situ, where synthetic molecules are programmed to organize in a specific and complex environment i.e., within living cells, can be a unique strategy to influence cellular functions. Here we present a small series of rationally designed oligothiophene analogues that specifically target, locate and dynamically self-report their supramolecular behavior within the confinement of a cell. Through the recognition of the terminal alkyl substituent and the amphiphilic pyridine motif, we show that the cell provides different complementary pathways for self-assembly that can be traced easily with fluorescence microscopy as their molecular organization emits in distinct fluorescent bands. Importantly, the control and induction of both forms are achieved by time, temperature and the use of the intracellular transport inhibitor, bafilomycin A1. We showcase the importance of both intrinsic (cell) and extrinsic (stimulus) factors for self-organization and the potential of such a platform toward developing synthetic functional components within living cells.
41. 41

    Najera, C., Baldo, B., and Yus, M. (1988) Regio- and Stereo-selective Synthesis of β-Sulphonyl-a,β- Unsaturated Carbonyl Compounds. J. Chem. Soc., Perkin Trans. 1 1029,  DOI: 10.1039/P19880001029

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    Guez, V., Roux, P., Navon, A., and Goldberg, M. E. (2002) Role of individual disulfide bonds in hen lysozyme early folding steps. Protein Sci. 11, 1136– 51,  DOI: 10.1110/ps.3960102

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    Role of individual disulfide bonds in hen lysozyme early folding steps

    Guez, Valerie; Roux, Pascale; Navon, Amiel; Goldberg, Michel E.

    Protein Science (2002), 11 (5), 1136-1151CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)

    To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, variants with 2 mutations, C30A/C115A, C64A/C80A, and C76A/C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-HCl-unfolded proteins with their 3 disulfide bonds intact showed that, for the 3 variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and Trp residue fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on Cys-6 and Cys-127, detailed anal. of the kinetics showed that: (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the 2 intra-α-domain disulfides (Cys-6-Cys-127 and Cys-30-Cys-115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population obsd. in WT lysozyme; and (3) the intra-β-domain (Cys-64-Cys-80) and the inter-αβ-domains (Cys-76-Cys-94) disulfide bonds did not affect the kinetics of formation of the trapped intermediate but were involved in its stability.
43. 43

    Springsteen, G. and Wang, B. (2002) A detailed examination of boronic acid–diol complexation. Tetrahedron 58, 5291– 5300,  DOI: 10.1016/S0040-4020(02)00489-1

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    A detailed examination of boronic acid-diol complexation

    Springsteen, Greg; Wang, Binghe

    Tetrahedron (2002), 58 (26), 5291-5300CODEN: TETRAB; ISSN:0040-4020. (Elsevier Science Ltd.)

    Boronic acids bind with compds. contg. diol moieties with high affinity through reversible boronate formation. However, the conditions that foster tight binding between the diol and the boronic acid are not well understood. Also, due to the multiple ionic states of both the boronic acid and boronate ester, the equil. consts. reported in the literature have not always been strictly defined, and therefore there is a lack of comparability between the reported values. To address these issues, a method was developed for examg. boronate ester stability using the fluorescent reporter Alizarin Red S. and this system has been used to det. the binding consts. of a series of diols, and as a basis from which to derive a no. of relationships that correlate the various equil. consts. in the literature.
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    Shugar, D. (1952) The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim. Biophys. Acta 8, 302– 9,  DOI: 10.1016/0006-3002(52)90045-0

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    Measurement of lysozyme activity and the ultraviolet inactivation of lysozyme

    Shugar, David

    Biochimica et Biophysica Acta (1952), 8 (), 302-9CODEN: BBACAQ; ISSN:0006-3002.

    Lysozyme (I) activity is detd. by measuring the decrease with time of the optical d. of a suspension of Micrococcus lysodeikticus. The analysis is made at pH 7.1 (M/15 phosphate buffer) in 3 ml. vol. (optical d. 0.5 to 0.75) to which 0.02-0.05 cc. of enzyme soln. is added. The readings are made at 2820 A. over a 4.5 min. period. Inactivation by ultraviolet light is a first order reaction, k = 0.158 min.-1. The quantum yield for inactivation at 2537 A. is 0.024 over the pH range 3.6-12.0. No apparent liberation of peptides or amino acids was observed but some photo-oxidation took place.
45. 45

    Davies, R. C. and Neuberger, A. (1969) Modification of lysine and arginine residues of lysozyme and the effect of enzymatic activity. Biochim. Biophys. Acta - Enzymol. 178, 306– 317,  DOI: 10.1016/0005-2744(69)90398-2

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    Modification of lysine and arginine residues of lysozyme and the effect on enzymic activity

    Davies, Richard C.; Neuberger, Albert

    Biochimica et Biophysica Acta, Enzymology (1969), 178 (2), 306-17CODEN: BBEZAD; ISSN:0924-1086.

    Acetylation of all 6 lysine residues of lysozyme (EC 3.2.1.17) abolished lytic action towards cells of Micrococcus lysodeikticus in 0.05M phosphate buffer (pH 6.2) but did not affect cleavage of the tetramer of N-acetylglucosamine (GlcNAc)4 obtained from chitin. Acetylated lysozyme still lysed cells in solns. of low ionic strength at pH 6.2. Compared with unmodified enzyme the activity profile for acetyl lysozyme was displaced to much lower values of ionic strength and was still markedly dependent on pH. Chem. modification of the lysine groups with ethyl acetamidate increased the activity towards cells slightly, yet did not alter the activity towards (GlcNAc)4. Modification of 7 out of 8 arginine residues with 2,3-butanedione in borate buffer reduced the activity of acetyl lysozyme towards cells but not towards (GlcNAc)4. Since all the basic residues of lysozyme apparently lie outside the active center, the persistence of lytic activity over a very wide pH range is discussed in terms of the currently accepted mechanism of action of lysozyme.
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    Imoto, T., Moriyama, S., and Yagishita, K. (1976) A Study of the Native-Denatured (N to/from D) Transition in Lysozyme III. Effect of Alteration of Net Charge by Acetylation. J. Biochem. 80, 1319– 1325,  DOI: 10.1093/oxfordjournals.jbchem.a131404

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    A study of the native-denatured (N .dblharw. D) transition in lysozyme. III. Effect of alteration of net charge by acetylation

    Imoto, Taiji; Moriyama, Sigeru; Yagishita, Kazuyoshi

    Journal of Biochemistry (1976), 80 (6), 1319-25CODEN: JOBIAO; ISSN:0021-924X.

    Measurement of the enzymic activity and fluorescence properties showed that the gross conformation of acetylated lysozyme (I) is very similar to that of native I. On the other hand, protease digestion, t-Bu hypochloride modification, and thermal denaturation of native, acetylated, and guanidinated I showed that acetylation causes a small but significant shift of the N.dblharw.D transition to the right. Thus, charge balance in a protein plays an important role in maintaining its conformation. The difference between equil. and kinetic methods of monitoring protein denaturation was also clarified.
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    Masuda, T., Kitabatake, N., and Ide, N. (2005) Effects of Chemical Modification of Lysine Residues on the Sweetness of Lysozyme. Chem. Chem. Senses 30, 253– 264,  DOI: 10.1093/chemse/bji021

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    Effects of Chemical Modification of Lysine Residues on the Sweetness of Lysozyme

    Masuda, Tetsuya; Ide, Nobuyuki; Kitabatake, Naofumi

    Chemical Senses (2005), 30 (3), 253-264CODEN: CHSED8; ISSN:0379-864X. (Oxford University Press)

    Lysozyme is a sweet-tasting protein with a sweetness threshold value of around 7 μM. To clarify the effect of basicity at the side chain of lysine residues on the threshold values of sweetness, charge-specific chem. modifications such as guanidination, acetylation and phosphopyridoxylation of lysine residues were performed. Sensory anal. showed that the sweetness threshold value of lysozyme was not changed by guanidination, whereas it was increased markedly by acetylation and phosphopyridoxylation. To confirm the importance of the basicity in the lysine residues in detail, purifn. of acetylated (Ac-) and phosphopyridoxylated (PLP-) lysozymes using SP-ion exchange column chromatog. was performed. The threshold values were not changed by modification with fewer than two residues (∼7 μM), whereas the threshold values significantly increased to 15 and 34 μM when tetra-Ac and tri-PLP, resp. Furthermore, sweetness was not detected at 30 μM (hexa-, penta-Ac and tetra-PLP). It should be noted that removal of the neg. charges of the phosphate groups in the tri-PLP lysozyme by acid phosphatase resulted in the recovery of sweetness (6.4 μM), indicating that basicity at the position of the lysine residues is responsible for lysozyme sweetness and that strict charge complementarities might be required for interaction to its putative receptor.
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    O’Brien, E. C., Farkas, E., Gil, M. J., Fitzgerald, D., Castineras, A., and Nolan, K. B. (2000) Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl·H2Sha, a model for a peroxidase-inhibitor complex. J. Inorg. Biochem. 79, 47– 51,  DOI: 10.1016/S0162-0134(99)00245-7

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    Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl·H2Sha, a model for a peroxidase-inhibitor complex

    O'Brien, Eimear C.; Farkas, Etelka; Gil, Marie Jose; Fitzgerald, Desmond; Castineras, Alfonso; Nolan, Kevin B.

    Journal of Inorganic Biochemistry (2000), 79 (1-4), 47-51CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier Science Inc.)

    Stability consts. of iron(III), copper(II), nickel(II) and zinc(II) complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid (HAha) and benzohydroxamic acid (HBha) have been detd. at 25.0°C, I=0.2 mol dm-3 KCl in aq. soln. The complex stability order, iron(III) » copper(II) > nickel(II) ∼ zinc(II) was obsd. while complexes of H2Sha were found to be more stable than those of the other two ligands. In the prepn. of ternary metal ion complexes of these ligands and 1,10-phenanthroline (phen) the cryst. complex [Cu(phen)2(Cl)]Cl·H2Sha was obtained and its crystal structure detd. This complex is a model for hydroxamate-peroxidase inhibitor interactions.
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    Yang, S.-M., Lagu, B., and Wilson, L. J. (2007) Mild and Efficient Lewis Acid-Promoted Detritylation in the Synthesis of N -Hydroxy Amides: A Concise Synthesis of (−)-Cobactin T. J. Org. Chem. 72, 8123– 8126,  DOI: 10.1021/jo701411d

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    Shin, S. B. Y., Almeida, R. D., Gerona-Navarro, G., Bracken, C., and Jaffrey, S. R. (2010) Assembling ligands in situ using bioorthogonal boronate ester synthesis. Chem. Biol. 17, 1171– 6,  DOI: 10.1016/j.chembiol.2010.09.008

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    Assembling ligands in situ using bioorthogonal boronate ester synthesis

    Shin, Sung Bin Y.; Almeida, Ramiro D.; Gerona-Navarro, Guillermo; Bracken, Clay; Jaffrey, Samie R.

    Chemistry & Biology (Cambridge, MA, United States) (2010), 17 (11), 1171-1176CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)

    Many mols. that could manipulate cellular function are not practical due to their large size and concomitant undesirable pharmacokinetic properties. Here, we describe a bioorthogonal, highly stable boronate ester (HiSBE) synthesis and use this reaction to synthesize a biol. active mol. from smaller precursors in a physiol. context. The rapid rate of HiSBE synthesis suggests that it may be useful for assembling a wide variety of biol. active mols. in physiol. solns.
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    Shrestha, D., Jenei, A., Nagy, P., Vereb, G., and Szöllősi, J. (2015) Understanding FRET as a research tool for cellular studies. Int. J. Mol. Sci. 16, 6718– 56,  DOI: 10.3390/ijms16046718

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    Understanding FRET as a research tool for cellular studies

    Shrestha, Dilip; Jenei, Attila; Nagy, Peter; Vereb, Gyoergy; Szoellosi, Janos

    International Journal of Molecular Sciences (2015), 16 (4), 6718-6756CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)

    Communication of mol. species through dynamic assocn. and/or dissocn. at various cellular sites governs biol. functions. Understanding these physiol. processes require delineation of mol. events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resoln. are methods based on Forster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1-10 nm which is equiv. to the size of macromols., thus providing an unprecedented level of detail on mol. interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a mol. complex in real-time making it possible to establish the functional significance of the studied mols. in a native environment. Now, FRET is widely used in biol. sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochem. methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for detg. the mol. heterogeneity of the plasma membrane in various cell types.
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    Yano, Y. and Matsuzaki, K. (2009) Tag–probe labeling methods for live-cell imaging of membrane proteins. Biochim. Biophys. Acta, Biomembr. 1788, 2124– 2131,  DOI: 10.1016/j.bbamem.2009.07.017

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    Tag-probe labeling methods for live-cell imaging of membrane proteins

    Yano, Yoshiaki; Matsuzaki, Katsumi

    Biochimica et Biophysica Acta, Biomembranes (2009), 1788 (10), 2124-2131CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)

    A review. Instead of using reconstituted proteoliposomes, in situ investigations of membrane proteins in living cell membranes are important because the heterogeneous and dynamic nature of biomembranes significantly affects their behavior. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence measurements, particularly fluorescence microscopy. However, conventional genetic fusion with fluorescent proteins has several shortcomings. Post-translational labeling methods using a genetically encodable tag and synthetic probes targeting to the tag can overcome these limitations. This review summarizes emerging tag-probe techniques for labeling specific membrane proteins and their applications, including endocytic internalization, partitioning to specific membrane domains, interprotein interactions, and conformational changes.
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    Soriano-Ursúa, M. A., Farfán-García, E. D., López-Cabrera, Y., Querejeta, E., and Trujillo-Ferrara, J. G. (2014) Boron-containing acids: Preliminary evaluation of acute toxicity and access to the brain determined by Raman scattering spectroscopy. NeuroToxicology 40, 8– 15,  DOI: 10.1016/j.neuro.2013.10.005

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    Boron-containing acids: Preliminary evaluation of acute toxicity and access to the brain determined by Raman scattering spectroscopy

    Soriano-Ursua, Marvin A.; Farfan-Garcia, Eunice D.; Lopez-Cabrera, Yessica; Querejeta, Enrique; Trujillo-Ferrara, Jose G.

    NeuroToxicology (2014), 40 (), 8-15CODEN: NRTXDN; ISSN:0161-813X. (Elsevier Inc.)

    Boron-contg. compds. (BCCs), particularly boron contg. acids (BCAs), have become attractive moieties or mols. in drug development. It has been suggested that when functional groups with boron atoms are added to well-known drugs, the latter are conferred with greater potency and efficacy in relation to their target receptors. However, the use of BCAs in drug development is limited due to the lack of a toxicol. profile. Consequently, the aim of the present study was to evaluate the acute toxicity of boric and boronic acids. Thus, a detn. was made of the LD (LD50) of test compds. in male CD1 mice, as well as the ED required to neg. affect spontaneous motor activity and to produce notable behavioral abnormalities. After treatment of animals at different doses, macroscopic observations were made from a necropsy, and Raman scattering spectroscopic studies were carried out on brain tissue samples. In general, the results show that most of the tested BCAs have very low toxicity, evidenced by the high doses required to induce notable toxic effects (greater than 100 mg/kg of body wt. for all compds., except for 3-thyenilboronic acid). Such toxic effects, presumably mediated by action on the CNS, include eye damage, gastrointestinal effects (e.g., gastric-gut dilatation and fecal retention), sedation, hypnosis and/or trembling. This preliminary toxicol. profile suggests that BCAs can be considered potential therapeutic agents or moieties to be added to other compds. in the development of new drugs. Future studies are required to explore possible chronic toxicity of BCCs.