The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA.
1,3-Dipolar [3+2] cycloaddition between azides and alkynes—an archetypal “click” chemistry—has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here we describe the solid-phase synthesis and characterization of 5’-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis SPOS, but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded “clickable” amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.
Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy.
Amino acids; DNA recognition; Molecular beacon; Protecting groups
The specific 1,3 dipolar Hüisgen cycloaddition reaction known as ‘click-reaction’ between azide and alkyne groups is employed for the synthesis of peptide–oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, ∼3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products.
“Click” chemistry is a library of efficient and reliable reactions, which have been used to functionalize various classes of bio- and synthetic macromolecular systems for the incorporation of designed properties and functions. In this report, azide-alkyne Huisgen cycloaddition and thiol-yne reactions, two classical “click” chemistries, were employed to functionalize biodegradable, clickable polyphosphoester homopolymers and their water-soluble copolymers. A stable alkyne-functionalized phospholane monomer was synthesized, its organocatalyzed polymerization kinetics were evaluated, and the resulting (co)polymers were utilized to develop this facile method that provides the synthesis of clickable, water-soluble and degradable polyphosphoesters, which can be adapted for various applications.
A facile synthetic route to prepare monofunctional carbocyanine dyes for biological application is developed. Three pentamethine carbocyanine dyes have been successfully modified with a variety of functional groups such as: carboxylic acids, azides, or alkynes. The new dyes are characterized by strong NIR fluorescence emission, high extinction coefficients and good quantum yields. The azide and alkyne dyes have potential utility as components in bioorthogonal labeling schemes via [2+3] dipolar cycloaddition “click” reactions. The application of one derivative, CyAM-5 alkyne, for bioorthogonal labeling is demonstrated. Fluorescence microscopy shows coupling of CyAM-5 alkyne to Chinese hamster ovary (CHO) cells preincubated with azide modified glycans.
Multifunctional dendrimers bearing two or more surface functionalities have the promise to provide smart drug delivery devices that can for example combine tissue targeting and imaging or be directed more precisely to a specific tissue or cell type. We have developed a concise synthetic methodology for efficient dendrimer assembly and heterobifunctionalization based on three sequential azide-alkyne cycloadditions. The methodology is compatible with biologically important compounds rich in chemical functionalities such as peptides, carbohydrates and fluorescent tags. In the approach, a strain promoted azide-alkyne cycloaddition (SPAAC) between polyester dendrons modified at the focal point with an azido and 4-dibenzocyclooctynol (DIBO) moiety provided dendrimers bearing terminal and TMS-protected alkynes at the periphery. The terminal alkynes were outfitted with azido-modified polyethylene glycol (PEG) chains or galactosyl residues using CuI catalyzed azide-alkyne cycloadditions (CuAAC). Next, a one-pot TMS-deprotection and second click reaction of the resulting terminal alkyne with azido-containing compounds gave multifunctional dendrimers bearing complex biologically active moieties at the periphery.
dendrimers; carbohydrates; peptides; synthetic methods; drug delivery; click chemistry
Instead of using diversity oriented syntheses (DOS) to obtain compounds with biological activities, we employed the DOS method to efficiently obtain multifunctional single attachment point (MSAP) reagents for the conjugation to proteins. Acid insensitive functional groups (chelators, fluorochromes) were attached to Lys-Cys-NH2 or Lys-Lys-βAla-Cys-NH2 peptide scaffolds. After cleavage from solid supports, the modified peptide intermediates were split and further modified by two solution phase, chemoselective reactions employing the single amine and single thiol presented on the intermediates. MSAP-based fluorochrome–chelates were obtained, some possessing a third functional group like a polyethylene glycol (PEG) polymer or “click chemistry” reactive alkynes and azides. The DOS of MSAP reagents permitted the efficient generation of panels of MSAP reagents that can be used to obtain multifunctional proteins with a single modified amino acid (a single attachment point).
A highly efficient chemical ligation was developed for quantitative conjugation of PNA with DNA (PNA or peptide) using the copper-catalyzed azide-alkyne cycloaddition reaction. While PNAs with an alkyne at the C-terminus and an azide at the N-terminus have been used, an efficient click-click reaction occurs. The PNA click ligation is sequence-specific and capable of single nucleotide discrimination.
Peptide Nucleic Acids; Chemical Ligation; Click Chemistry; Single Nucleotide Discrimination; DNA detection
We have shown that 4-Dibenzocyclooctynol (DIBO), which can easily be obtained by a streamlined synthetic approach, reacts exceptionally fast in the absence of a CuI catalyst with azido-containing compounds to give stable triazoles. Chemical modifications of DIBO, such as oxidation of the alcohol to a ketone, increased the rate of strain promoted azide-alkyne cycloadditions (SPAAC). Installment of a ketone or oxime in the cyclooctyne ring resulted in fluorescent active compounds whereas this property was absent in the corresponding cycloaddition adducts, thereby providing the first example of a metal-free alkyne-azide fluoro-switch click reaction. The alcohol or ketone functions of the cyclooctynes offer a chemical handle to install a variety of different tags, thereby facilitating biological studies. It was found that DIBO modified with biotin combined with metabolic labeling with an azido-containing monosaccharide can determine relative quantities of sialic acid of living cells that have defects in glycosylation (Lec CHO cells). A combined use of metabolic labeling/SPAAC and lectin staining of cells that have defects in the Conserved Oligomeric Golgi (COG) complex revealed that such defects have a greater impact on O-glycan sialylation than galactosylation, whereas sialylation and galactosylation of N-glycans was similarly impacted. These results highlight that the fidelity of Golgi trafficking is a critical parameter for the types of oligosaccharides that are being biosynthesized by a cell. Furthermore, by modulating the quantity of biosynthesized sugar nucleotide, cells may have a means to selectively alter specific glycan structures of glycoproteins.
carbohydrates; glycoconjugates; click chemistry; azide; bioorthogonal
Proteases play a key role in literally all biological processes, and are of great interest, especially to the pharmaceutical industry. Colorimetric based Peptide-p-Nitroanilide conjugates (peptide-pNAs), with absorbance at approximately 408 nm, have historically been and are still widely used substrates for the study of protease activity. The preparation of peptide-pNA however, presents several technical challenges. Firstly, the amino group of pNA has a low nucleophilic property due to the electron-withdrawing effect of the nitro group. Secondly, the poor solubility of a p-nitroanilide intermediate and lastly, coupling in solution phase by DCC, azide or active ester, commonly used techniques are not effective. Here we report the development of two novel supports for facile solid phase peptide syntheses, namely, Wang-resin and Rink Amide-resin conjugated with a pNA analog, 5-amino-2-nitrobenzoic acid (Anb5,2). Based on a paper by Hojo, et al. in which they described the introduction of Anb5,2 to a p-methylbenzhydrylamine (MB) resin; we successfully coupled Anb5,2 to either Wang or Rink Amide resin using the TBTU method in the presence of p-dimethylaminopyridine (DMAP). Anb5,2-Wang or Rink Rmide resin is then coupled to a Fluorenylmethyloxycarbonyl (Fmoc) containing amino acid. Peptide synthesis can subsequently proceed using Fmoc synthesis strategy. The use of this pNA analog containing resins circumvents the tehcnical difficulties stated above. These resins also greatly facilitates the synthesis of peptide-pNA-like chromogenic substrates for protease research.
A flexible molecular scaffold bearing varying numbers of terminal alkyne groups was synthesized in five steps from solanesol. R(CO)-MSH(4)-NH2 ligands, which have a relatively low affinity for binding at the human melanocortin 4 receptor (hMC4R), were prepared by solid phase synthesis and were N-terminally acylated using 6-azidohexanoic acid. Multiple copies of the azide N3(CH2)5(CO)-MSH(4)-NH2 were attached to the alkyne-bearing, solanesol-derived molecular scaffold via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Control studies showed that the binding affinity of the triazole-containing ligand, CH3(CH2)3(C2N3)(CH2)5(CO)-MSH(4)-NH2, was not significantly diminished relative to the corresponding parental ligand, CH3(CO)-MSH(4)-NH2. In a competitive binding assay using a Eu-labeled probe based on the superpotent ligand NDP-α-MSH, the monovalent and multivalent constructs appear to bind to hMC4R as monovalent species. In a similar assay using a Eu-labeled probe based on MSH(4), modest increases in binding potency with increased MSH(4) content per scaffold were observed.
Azidopropyl functionalized mesoporous silica SBA-15 were prepared with variable azide loadings of 0.03 – 0.7 mmol g−1 (ca. 2 – 50% of maximal surface coverage) through a direct synthesis, co-condensation approach. These materials are functionalized selectively with ethynylated organic moieties through the copper-catalyzed azide alkyne cycloaddition (CuAAC) or “click” reaction. Specific loading within a material can be regulated by either the azide loading or limiting the alkyne reagent relative to the azide loading. The immobilization of ferrocene, pyrene, tris(pyridylmethyl)amine (TPA), and iron porphyrin (FeTPP) demonstrates the robust nature and reproducibility of this two step synthetic attachment strategy. Loading-sensitive pyrene fluorescence correlates with a theoretically random. surface distribution, rather than a uniform one; full site-isolation of tethered moieties ca. 15 Å in length. occurs at loadings less than 0.02 mmol g−1. The effect of surface loading on reactivity is observed in oxygenation of SBA-15-[CuI(TPA)]. SBA-15-[MnII(TPA)]-catalyzed epoxidation exhibits a systematic dependence on surface loading. A comparison of homogeneous, site-isolated and site-dense complexes provides insight into catalyst speciation and ligand activity.
Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates—where the connection between the two components is at a defined location in both the protein and the ODN—under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free “click” reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were “clicked” to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.
Click chemistry; Bioconjugation; Protein-oligodeoxynucleotide conjugates; Protein prenylation; Protein immobilization
In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.
This update summarizes the growing application of “click” chemistry in diverse areas such as bioconjugation, drug discovery, materials science, and radiochemistry. This update also discusses click chemistry reactions that proceed rapidly with high selectivity, specificity, and yield. Two important characteristics make click chemistry so attractive for assembling compounds, reagents, and biomolecules for preclinical and clinical applications. First, click reactions are bio-orthogonal; neither the reactants nor their product's functional groups interact with functionalized biomolecules. Second, the reactions proceed with ease under mild nontoxic conditions, such as at room temperature and, usually, in water. The copper-catalyzed Huisgen cycloaddition, azide-alkyne [3 + 2] dipolar cycloaddition, Staudinger ligation, and azide-phosphine ligation each possess these unique qualities. These reactions can be used to modify one cellular component while leaving others unharmed or untouched. Click chemistry has found increasing applications in all aspects of drug discovery in medicinal chemistry, such as for generating lead compounds through combinatorial methods. Bioconjugation via click chemistry is rigorously employed in proteomics and nucleic research. In radiochemistry, selective radiolabeling of biomolecules in cells and living organisms for imaging and therapy has been realized by this technology. Bifunctional chelating agents for several radionuclides useful for positron emission tomography and single-photon emission computed tomography imaging have also been prepared by using click chemistry. This review concludes that click chemistry is not the perfect conjugation and assembly technology for all applications, but provides a powerful, attractive alternative to conventional chemistry. This chemistry has proven itself to be superior in satisfying many criteria (e.g., biocompatibility, selectivity, yield, stereospecificity, and so forth); thus, one can expect it will consequently become a more routine strategy in the near future for a wide range of applications.
click chemistry; bioconjugation; bioorthogonal; radiochemistry; imaging; bifunctional
The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.
Head-to-tail cyclodimerization of resin-bound oligopeptides bearing azide and alkyne groups occurs readily by 1,3-dipolar cycloaddition upon treatment with Cu(I). The process was found to be independent of peptide sequence, sensitive to the proximity of the alkyne to the resin, sensitive to solvent composition, facile for α- and β-peptides but not for γ-peptides, and inhibited by the inclusion of tertiary amide linkages. Peptides shorter than hexamers were predominantly converted to cyclic monomers. Oligoglycine and oligo(β-alanine) chains underwent oligomerization by 1,3-dipolar cycloaddition in the absence of copper catalyst. These results suggest that cyclodimerization depends on the ability of the azido-alkyne peptide to form in-frame hydrogen bonds between chains in order to place the reacting groups in close proximity and lower the entropic penalty for dimerization. The properties of the resin and solvent are crucial, giving rise to a productive balance between swelling and inter-strand H-bonding. These findings allow for the design of optimal substrates for triazole-forming ring closure, and for the course of the reaction to be controlled by the choice of conditions.
Azide-alkyne cycloaddition; cyclic peptides; peptide synthesis; cyclodimerization; click chemistry
This update summarizes the growing application of “click” chemistry in diverse areas such as bioconjugation, drug discovery, materials science, and radiochemistry. This update also discusses click chemistry reactions that proceed rapidly with high selectivity, specificity and yield. Two important characteristics make click chemistry so attractive for assembling compounds, reagents, and biomolecules for pre-clinical and clinical applications. First, click reactions are bioorthogonal; neither the reactants nor their product’s functional groups interact with functionalized biomolecules. Second, the reactions proceed with ease under mild non-toxic conditions such as at room temperature and usually in water. The copper catalyzed Huisgen cycloaddition, azide-alkyne [3+2] dipolar cycloaddition, and Staudinger ligation, azide-phosphine ligation, each possess these unique qualities. These reactions can be used to modify one cellular component while leaving others unharmed or untouched. Click chemistry has found increasing applications in all aspects of drug discovery in medicinal chemistry such as for generating lead compounds through combinatorial methods. Bioconjugation via click chemistry is rigorously employed in proteomics and nucleic research. In radiochemistry, selective radiolabeling of biomolecules in cells and living organisms for imaging and therapy has been realized by this technology. Bifunctional chelating agents for several radionuclides useful for PET and SPECT imaging have also been prepared using click chemistry. This review concludes that click chemistry is not the perfect conjugation and assembly technology for all applications, but provides a powerful and attractive alternative to conventional chemistry. This chemistry has proven itself to be superior in satisfying many criteria (biocompatibility, selectivity, yield, stereospecificity, etc.); thus one can expect it will consequently become a more routine strategy in the near future for a wide range of applications.
click chemistry; bioconjugation; bioorthogonal; radiochemistry; imaging; bifunctional
We report herein a new and enabling approach for decorating both living and abiotic surfaces with the extracellular matrix IKVAV peptide in a site-specific manner using strain promoted azide-alkyne cycloaddition. A cyclooctyne derivatized IKVAV peptide was synthesized and immobilized on the surface of pancreatic islets through strain-promoted azide-alkyne cycloaddition with cell surface azides generated by electrostatic adsorption of cytocompatible poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) copolymer bearing azido groups (PP-N3). Both “one-pot” and sequential addition of PP-N3 and a cyclooctyne derivatized IKVAV peptide conjugate enabled efficient modification of pancreatic islet surface in less than 60 min. The ability to bind peptides at controlled surface densities was demonstrated in a quantitative manner using microarrays. Additionally, the technique is remarkably rapid and highly efficient, opening new avenues for molecular engineering of cellular interfaces and protein and peptide microarrays.
Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type 2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed ‘click’ cycloaddition reactions between azides and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoylformate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting α-ketocarboxylic acids were assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one compound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first generation library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and evaluated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR and CD45. The best bis(α-ketocarboxylic acid) inhibitor 34 had an IC50 value of 550 nM against the Yersinia PTP, and an IC50 value of 710 nM against TCPTP. The most potent inhibitor containing a single α-ketocarboxylic acid group 32 had IC50 values of 2.1, 5.7 and 2.6 μM against the Yersinia PTP, PTP1B and TCPTP, respectively.
Library; Protein Tyrosine Phosphatase; Inhibitor; Click Chemistry
Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence-based method to follow proteome-wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio-orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne-bearing fluorophore is covalently attached to the group by “click chemistry”—a copper(I)-catalyzed [3+2]azide-alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne-bearing homopropargylglycine (HPG) and clicked to an azide-functionalized fluorophore.
FUNCAT; click chemistry; copper(I)-catalyzed [3+2]azide-alkyne cycloaddition; AHA; HPG; protein synthesis
Isolation and identification of phosphorylated macromolecules is essential for the deconvolution of most biological regulatory networks. Koike and coworkers recently reported the application of a dinuclear zinc-(pyridylmethyl)amine complex to phosphate-specific affinity purifications, and gave it the shorthand name “phos-tag”. This complex is valuable for studying phosphorylation because it binds selectively to phosphate dianion in the presence of acidic functional groups at physiological pH, and because the binding is largely independent of molecular context. These properties of phos-tag recommend it for applications in phosphoproteomics, metabolomics and nucleic acid biology. The catch has been that the molecule is difficult to make, and prohibitively expensive to buy. Here we describe an efficient and inexpensive synthesis of a phos-tag derivative with a versatile alkyne handle. The alkyne handle allows for attachment of phos-tag to alkyl azides via the copper(I)-catalyzed azide-alkyne cycloaddition reaction (“click chemistry”). We characterize the phosphate binding behavior of the new phos-tag derivative in a variety of experimental assays, including its conjugation to a fluorescent reporter, to acrylamide gels, and to sepharose chromatography resin. The synthesis we report should enable a broader use of phos-tag for phosphate-related biochemistry, as both an analytical and as a preparative reagent.
The copper(I)-catalyzed azide-alkyne cycloaddition, the most widely recognized reaction of click chemistry, is accelerated by tris(triazolylmethyl)amine-based ligands. Here, we compared two new ligands in this family, BTTP and the corresponding sulfated ligand BTTPS, for three bioconjugation applications: (1) labeling of alkyne-tagged glycoproteins in crude cell lysates, (2) labeling of alkyne/azide-tagged glycoproteins on the surface of live mammalian cells, and (3) labeling of azides in surface proteins of live Escherichia coli. Though BTTPS exhibits faster kinetics than BTTP in accelerating the CuAAC in in vitro kinetic measurements, its labeling efficiency is slightly lower than BTTP in conjugating biomolecules bearing a significant amount of negative charges due to electrostatic repulsion. Nevertheless, the negative charge conferred by the sulfate at physiological conditions significantly reduced the cellular internalization of the coordinated Cu(I), thus making BTTPS-Cu(I) a better choice for live cell labeling.
click chemistry; copper; bioconjugation; glycoconjugates
A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by posttranslationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for “click” chemistry or a Staudinger ligation. Farnesyl diphosphate analogs bearing ω-azide or ω-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide (“click” chemistry) or phosphine (Staudinger ligation) groups. “Click” immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 °C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.