Bioorthogonal Functional Groups Research Articles

Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases.

Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.

Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as Biological Probes, Imaging Reagents, and Photocytotoxic Agents.

Although the interactions of transition metal complexes with biological molecules have been extensively studied, the use of luminescent transition metal complexes as intracellular sensors and bioimaging reagents has not been a focus of research until recently. The main advantages of luminescent transition metal complexes are their high photostability, long-lived phosphorescence that allows time-resolved detection, and large Stokes shifts that can minimize the possible self-quenching effect. Also, by the use of transition metal complexes, the degree of cellular uptake can be readily determined using inductively coupled plasma mass spectrometry. For more than a decade, we have been interested in the development of luminescent transition metal complexes as covalent labels and noncovalent probes for biological molecules. We argue that many transition metal polypyridine complexes display triplet charge transfer ((3)CT) emission that is highly sensitive to the local environment of the complexes. Hence, the biological labeling and binding interactions can be readily reflected by changes in the photophysical properties of the complexes. In this laboratory, we have modified luminescent tricarbonylrhenium(I) and bis-cyclometalated iridium(III) polypyridine complexes of general formula [Re(bpy-R(1))(CO)3(py-R(2))](+) and [Ir(ppy-R(3))2(bpy-R(4))](+), respectively, with reactive functional groups and used them to label the amine and sulfhydryl groups of biomolecules such as oligonucleotides, amino acids, peptides, and proteins. Additionally, using a range of biological substrates such as biotin, estradiol, and indole, we have designed luminescent rhenium(I) and iridium(III) polypyridine complexes as noncovalent probes for biological receptors. The interesting results generated from these studies have prompted us to investigate the possible applications of luminescent transition metal complexes in intracellular systems. Thus, in the past few years, we have developed an interest in the cytotoxic activity, cellular uptake, and bioimaging applications of these complexes. Additionally, we and other research groups have demonstrated that many transition metal complexes have facile cellular uptake and organelle-localization properties and that their cytotoxic activity can be readily controlled. For example, complexes that can target the nucleus, nucleolus, mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus have been identified. We anticipate that this selective localization property can be utilized in the development of intracellular sensors and bioimaging reagents. Thus, we have functionalized luminescent rhenium(I) and iridium(III) polypyridine complexes with various pendants, including molecule-binding moieties, sugar molecules, bioorthogonal functional groups, and polymeric chains such as poly(ethylene glycol) and polyethylenimine, and examined their potentials as biological reagents. This Account describes our design of luminescent rhenium(I) and iridium(III) polypyridine complexes and explains how they can serve as a new generation of biological reagents for diagnostic and therapeutic applications.

A Bioorthogonal Chemical Reporter of Viral Infection.

Pathogen‐selective labeling was achieved by using the novel gemcitabine metabolite analogue 2′‐deoxy‐2′,2′‐difluoro‐5‐ethynyluridine (dF‐EdU) and click chemistry. Cells infected with Herpes Simplex Virus‐1 (HSV‐1), but not uninfected cells, exhibit nuclear staining upon the addition of dF‐EdU and a fluorescent azide. The incorporation of the dF‐EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK). Crystallographic analyses revealed how dF‐EdU is well accommodated in the active site of HSV‐1 TK, but steric clashes prevent dF‐EdU from binding human TK. These results provide the first example of pathogen‐enzyme‐dependent incorporation and labeling of bioorthogonal functional groups in human cells.

A Bioorthogonal Chemical Reporter of Viral Infection.

Pathogen‐selective labeling was achieved by using the novel gemcitabine metabolite analogue 2′‐deoxy‐2′,2′‐difluoro‐5‐ethynyluridine (dF‐EdU) and click chemistry. Cells infected with Herpes Simplex Virus‐1 (HSV‐1), but not uninfected cells, exhibit nuclear staining upon the addition of dF‐EdU and a fluorescent azide. The incorporation of the dF‐EdU into DNA depends on its phosphorylation by a herpes virus thymidine kinase (TK). Crystallographic analyses revealed how dF‐EdU is well accommodated in the active site of HSV‐1 TK, but steric clashes prevent dF‐EdU from binding human TK. These results provide the first example of pathogen‐enzyme‐dependent incorporation and labeling of bioorthogonal functional groups in human cells.

Recent developments of engineered translational machineries for the incorporation of non-canonical amino acids into polypeptides.

Genetic code expansion and reprogramming methodologies allow us to incorporate non-canonical amino acids (ncAAs) bearing various functional groups, such as fluorescent groups, bioorthogonal functional groups, and post-translational modifications, into a desired position or multiple positions in polypeptides both in vitro and in vivo. In order to efficiently incorporate a wide range of ncAAs, several methodologies have been developed, such as orthogonal aminoacyl-tRNA-synthetase (AARS)–tRNA pairs, aminoacylation ribozymes, frame-shift suppression of quadruplet codons, and engineered ribosomes. More recently, it has been reported that an engineered translation system specifically utilizes an artificially built genetic code and functions orthogonally to naturally occurring counterpart. In this review we summarize recent advances in the field of ribosomal polypeptide synthesis containing ncAAs.

Novel Chemical Biology Methods Illuminate the Function of Single G Protein-Coupled Receptors

The superfamily of G Protein-Coupled Receptors (GPCRs) is one of the largest in the human genome. GPCRs are targets for about a quarter of all prescription drugs. Recent progress in the structural biology of GPCRs renews the interest in rational drug design and discovery. Rational design of “functionally selective” drugs or ligands presents a particularly difficult problem. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. However, functional characterization of GPCRs has largely been limited to cell-based high-throughput screening assays. The complexity of the cellular milieu introduces biological variables that make quantitative analysis of structure and dynamics of ligand-receptor complexes that underlie functional selectivity virtually impossible. We are developing single-molecule fluorescence methods to eliminate this biological noise and study such complexes in biochemically-defined systems.[1] Systematic introduction of signaling partners, such as G proteins and arrestins, enables us to study their allosteric effects on ligand-receptor interactions. Recent methodological advances enabled us to express GPCRs with genetically-encoded unnatural amino acids (uaas). We showed that bioorthogonal functional groups on these site-specifically introduced uaas might be used for bioorthogonal reactions. Among a broad spectrum of bioorthogonal reactions, strain-promoted azide-alkyne cycloaddition (SpAAC) with the uaa p-azido-phenylalanine and dibenzocyclooctyne (DIBO) reagents is specific and efficient and presents the best option to obtain stoichiometric receptor-fluorophore conjugates. We designed and built a fully-automatic, multi-color, single-molecule detection fluorescence microscope for long-timescale, multiplexed data acquisition using a multi-well tethered membrane “chip” system. Our convergent technology platform presents a new approach for single-molecule fluorescence studies of the kinetics and dynamics of GPCR signalosomes.[1] T. Huber and T.P. Sakmar. Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling. Chem Biol. 18:1224-1237 (2014)

Development of a clickable designer monolignol for interrogation of lignification in plant cell walls.

Lignin is an abundant and essential polymer in land plants. It is a prime factor in the recalcitrance of lignocellulosic biomass to agricultural and industrial end-uses such as forage, pulp and papermaking, and biofuels. To better understand lignification at the molecular level, we are developing a lignin spectroscopic and imaging toolbox on one "negligible" auxiliary. Toward that end, we describe the design, synthesis, and characterization of a new designer monolignol, 3-O-propargylcaffeyl alcohol, which contains a bioorthogonal alkynyl functional group at the 3-O-position. Importantly, our data indicate that this monolignol does not alter the fidelity of lignification. We demonstrate that the designer monolignol provides a platform for multiple spectroscopic and imaging approaches to reveal temporal and spatial details of lignification, the knowledge of which is critical to reap the potential of energy-rich renewable plant biomass for sustainable liquid fuels and other diverse economic applications.

A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface.

The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry.

Enzyme directed formation of un-natural side-chains for covalent surface attachment of proteins

The covalent immobilization of proteins onto surfaces is an essential aspect of several fields of research, including proteomics, sensing, heterogeneous biocatalysis, and more broadly biotechnology. Site-specific, covalent attachment of proteins has been achieved in recent years by the use of expanded genetic codes to produce proteins with controlled placement of un-natural amino acids bearing bio-orthogonal functional groups. Unfortunately, the complexity of developing such systems is impractical for most laboratories; hence, a less complicated approach to generating un-natural amino acid side-chains has been employed. Utilizing a straightforward reaction with formylglycine generating enzyme, we use the site-specific modification of engineered proteins to yield un-natural amino acid side-chains for protein immobilization. Using this approach, we demonstrate the controlled immobilization of various enzymes onto a variety of amine coated surfaces. Our results reveal reusability of the immobilized enzymes via this strategy, and furthermore, we find the activity of the immobilized enzymes to remain even after a month of use indicating significant stability of the linkage.

Surface Modification of Melt Extruded Poly(ε-caprolactone) Nanofibers: Toward a New Scalable Biomaterial Scaffold.

A photochemical modification of melt-extruded polymeric nanofibers is described. A bioorthogonal functional group is used to decorate fibers made exclusively from commodity polymers, covalently attach fluorophores and peptides, and direct cell growth. Our process begins by using a layered coextrusion method, where poly(ε-caprolactone) (PCL) nanofibers are incorporated within a macroscopic poly(ethylene oxide) (PEO) tape through a series of die multipliers within the extrusion line. The PEO layer is then removed with a water wash to yield rectangular PCL nanofibers with controlled cross-sectional dimensions. The fibers can be subsequently modified using photochemistry to yield a “clickable” handle for performing the copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction on their surface. We have attached fluorophores, which exhibit dense surface coverage when using ligand-accelerated CuAAC reaction conditions. In addition, an RGD peptide motif was coupled to the surface of the fibers. Subsequent cell-based studies have shown that the RGD peptide is biologically accessible at the surface, leading to increased cellular adhesion and spreading versus PCL control surfaces. This functionalized coextruded fiber has the advantages of modularity and scalability, opening a potentially new avenue for biomaterials fabrication.

Synthesis and Reactivity Comparisons of 1‐Methyl‐3‐Substituted Cyclopropene Mini‐tags for Tetrazine Bioorthogonal Reactions

Substituted cyclopropenes have recently attracted attention as stable "mini-tags" that are highly reactive dienophiles with the bioorthogonal tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1-methyl-3-substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels-Alder cycloadditions with 1,2,4,5-tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3-amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1-methyl-3-substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated tetrazine ligations. The effect of tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3-amidomethyl substituted methylcyclopropene reacts faster than trans-cyclooctenol with a sterically hindered and extremely stable tert-butyl substituted tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.

Regio- and chemoselective immobilization of proteins on gold surfaces.

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs.

Bioorthogonal Reactions for Labeling Proteins

Bioorthogonal Reactions for Labeling Proteins

Expanding the Genetic Code for Photoclick Chemistry in E. coli, Mammalian Cells, and A. thaliana

Bioorthogonal chemical reactions together with techniques to expand the genetic code have provided exciting new means for protein labeling and visualization in living systems, as well as for optimizing the efficacy of therapeutic proteins. Toward these goals, amino acids with small bioorthogonal functional groups, such as azide, alkyne, or cyclopropene moieties, as well as larger reactive bioorthogonal groups, such as cyclooctyne, norbornene, trans-cyclooctene, aryltetrazole, or aryltetrazine, have been site-specifically incorporated into proteins, allowing for selective conjugation of biophysical probes through azide–alkyne click chemistry (AAC), tetrazole–alkene photoclick chemistry (TAP), and reverse-electron demand Diels–Alder reactions. The main advantages of the photoclick reaction (Supporting Information, Scheme S1) are: 1) its fast rate (up to 50m 1 s ); 2) that spatiotemporal control is initiated by a photo-induced reaction; 3) that the photoclick reaction is fluorogenic, allowing for high-contrast fluorescence imaging without tedious washing steps. In previous studies, we reported the site-specific incorporation of p-(2-tetrazole)phenylalanine (p-Tpa) and N-e-(1-methylcycloprop-2-enecarboxamido)lysine (CpK) in E. coli and mammalian cells. Subsequent photoirradiation of labeled proteins with UV light facilitates selective conjugation with dimethyl fumarate or diaryltetrazole, respectively. By expanding the genetic code and introducing photoclick chemistry to plants, important problems in plant chemical biology can be addressed, such as photosynthesis and stress response, which can only be studied at the organismal level. Recently, expansion of the genetic code has been used to optimize therapeutic proteins produced in bacteria and mammalian cells. Because plants offer an attractive alternative to microbial fermentation and animal cell cultures for high-yield production of recombinant proteins on an agricultural scale, expanding the genetic code in plants would be useful for producing recombinant therapeutic proteins and enzymes with enhanced properties, better safety, and lower costs. To fully realize the potential of photoclick reaction for tracking fast cellular processes, it is desirable that the unnatural amino acid (UAA) used has a small functional group such that there is minimal perturbation of the target protein, and a very brief exposure to long-wavelength UV light or violet-blue light is used to drive the photoclick reaction to minimize damage to cells and plants. Herein, we addressed these issues by genetically incorporating N-eacryllysine (AcrK, Figure 1A), in response to an amber stop codon (TAG) in bacterial cells, mammalian cells, and plants. This new strategy was then used to efficiently label proteins both in vitro and in vivo. In comparison to lysine, AcrK has only four extra non-hydrogen atoms, which is significantly less than other UAAs. Replacing one lysine residue with AcrK should cause only minimal perturbation to the target protein. In addition, the electron-withdrawing amido group should activate the terminal alkene group to achieve a higher photoclick reaction rate. Indeed, the photoclick reaction between tetrazole and acrylamide was found to proceed nearly one hundred times faster than that of allylphenylether and 1.5 times faster than that of cyclopropene. AcrK was synthesized by reacting N-a-Boc-lysine with acryloyl chloride in a basic ethyl acetate/water solution at 0 8C (Figure 1A), followed by deprotection with HCl gas with an overall yield of 72%, without the need for metal catalysts. AcrK was found to be relatively stable in the presence of glutathione, an abundant biomolecule both amine and thiol groups; greater than 95% of AcrK remained following incubation with 5 mm reduced glutathione in a buffer at pH 7 for 24 hours (Figure S1). At neutral pH, most primary amines are protonated and most thiols are neutral. Therefore, glutathione could react slowly with the acrylamido group through a Michael addition reaction. While AcrK has comparable stability to CpK under physiological conditions, the synthetic route for CpK requires six steps, expensive heavy metal catalysis, with an overall yield of 15%. Because a large amount of the UAA is required for modifying plant proteins on an agricultural scale, it is essential that the UAA is synthesized in an economically, without the use of toxic heavy-metal catalysts. We chose diaryltetrazole 2 (Figure 1B) because it is highly reactive for photoclick reactions and is soluble in water. Because the molar extinction coefficients of 2 at 365 nm and 405 nm are around 100m 1 cm 1 (Figure S2) and the quantum yields for the photolysis of diaryltetrazoles are very high (0.5– 0.9), 2 should be efficiently activated by long-wavelength UV light or violet light. An amine functional group provides a convenient handle for further derivatization. Upon irradi[*] F. H. Li, H. Zhang, Y. Sun, Y. C. Pan, J. Z. Zhou, Prof. Dr. J. Y. Wang Laboratory of Non-coding RNA, Institute of Biophysics Chinese Academy of Sciences 15 Datun Road, Chaoyang District, Beijing, 100101 (China) E-mail: jwang@ibp.ac.cn [] These authors contributed equally to this work.

Metal Catalyzed Click Chemistry for Molecular Imaging Probes

Cu-catalyzed azide-alkyne cycloaddition has been developed as a new, simple, efficient, and generally applicable synthetic strategy, known as ‘Click Chemistry’. The reaction utilizes two bioorthogonal functional groups, azide and alkyne, which can be readily incorporated into biological molecules with straightforward chemical modification. The reaction can be performed in low substrate concentration under a wide range of conditions including aqueous physiological conditions, which finds a span of application ranging from material science, chemical biology/bio-conjugation, and pharmaceutical research and development. In this review, we focused on the new progress in developing the metal catalyzed, bioorthogonal azide-alkyne cycloaddition reaction (‘Click chemistry’) about catalyst species, the ligands and substrates and its application in the bio-imaging field from four aspects: (a) labeling of live cell surface; (b) imaging small molecules inside of living cells; (c) modifying cells with nanomaterials; and (d) vivo imaging.

Rapid Catalyst-Free Hydrazone Ligation: Protein-Pyridoxal Phosphoramides

Pyridoxal-5'-phosphate (PLP) represents an active form of Vitamin B(6) that shows relatively fast imine formation with hydrazines under physiological conditions without the need of a catalyst. A convenient phosphate/amine conjugation protocol was developed to covalently link PLP to proteins, affording proteins capable of hydrazone formation with bioorthogonal hydrazinyl functional groups. Thus, the lectin Concanavalin A (Con A) was labeled with PLP. Pretreatment with fluorescein hydrazide gave dye-labeled Con A that labeled cell surfaces efficiently. Alternatively, pretargeting was achieved by labeling cells with Con A-PLP, then treatment in vitro with Alexa Fluor 488 hydrazide.

Genetic Incorporation of Twelve meta-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant

When coexpressed with its cognate amber suppressing tRNACUAPyl(CUA), a pyrrolysyltRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro,ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone,alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

Metabolic Labeling of DNA by Purine Analogues in Vivo

The 2′-deoxyadenosine analogue “EdA” efficiently labels newly synthesized DNA with alkyne groups in vivo without inhibiting cell cycle progression or embryonic development. This provides the first demonstration that a bio-orthogonal functional group can be metabolically incorporated into DNA through endogenous pathways by addition of a purine analogue to cells.

Bioorthogonal Chemistries for Glycoprofiling and Beyond

The glycome is dynamically regulated during development and disease progression. The ability to detect changes in protein glycosylation, in tissue and serum samples as well as in vivo, is of considerable interest for biomarker identification and disease monitoring. This presentation will focus on our work toward profiling glycomic changes using proteomics and in vivo imaging techniques. Bioorthogonal chemistry is central to our approaches, which involve metabolic labeling of glycans with bioorthogonal functional groups followed by chemical tagging with affinity reagents, mass spectrometry tags or imaging probes. Fundamental principles of bioorthogonal reaction development will be discussed, along with applications in cancer biomarker discovery and glycan imaging during embryonic development.