Acidic and basic deprotection strategies of borane-protected phosphinothioesters for the traceless Staudinger ligation

The traceless Staudinger ligation has recently found various applications in the field of peptide synthesis and modification, including immobilization and cyclization strategies. In this report, we utilize the traceless Staudinger ligation in the formation of amide bonds, which allows the acquisition of acylated aminosugars and peptides as well as the cyclization of peptides. A key element in these synthetic procedures is the use of a borane-protected phosphinomethanethiol, which is demonstrated to be prone towards oxidation in its unprotected form, during the synthesis of phosphinothioesters. In combination with acidic and basic deprotection strategies for the borane-protected phosphinothioesters, amide bonds can be formed in the presence of azides in moderate to good overall yields.     

Stereoselective synthesis of N-galactofuranosyl amides

α- or β-Galactofuranosyl (Galf) amides can be synthesized with high stereoselectivity by traceless Staudinger ligation starting from unprotected β-galactofuranosyl azide or tetra-O-acetyl-β-galactofuranosyl azide, respectively. The resulting Galf amides are hitherto unknown molecules, with interesting potential as inhibitors of mycobacterial growth.     

Traceless Staudinger acetylation of azides in aqueous buffers

In this paper, we demonstrate the applicability of water-soluble p-dimethylaminoethyl substituted phosphinomethanethiol in acetyl transfer reactions by the traceless Staudinger ligation with unprotected ε-azido lysine containing peptides in aqueous buffer systems. Additionally, we present an improved synthesis pathway for the water-soluble phosphinothiol linkers requiring less steps in a comparable overall yield in comparison to previously published protocols.     

Facile synthesis of peptide–porphyrin conjugates: Towards artificial catalase

A facile synthetic method for peptide–porphyrin conjugates containing four peptide units on one porphyrin was developed using chemoselective reactions. The key building blocks, 5,10,15,20-tetrakis(3-azidophenyl)porphyrin 1 and 5,10,15,20-tetrakis(5-azido-3-pyridyl)porphyrin 2, were efficiently synthesized and used as substrates for two well-known chemoselective reactions, traceless Staudinger ligation and copper-catalyzed azide alkyne cycloaddition (so-called click chemistry). Both reactions gave the desired compounds, and click chemistry was superior for our purpose. To confirm the value of the established methodology, nine peptide–porphyrin conjugates were synthesized, and their catalase- and peroxidase-like activity in water was evaluated. Our synthetic strategy is expected to be valuable for the preparation of artificial heme protein models.     

Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach

The application of intact monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger ligation as pretargeting strategy using an antibody-azide conjugate as tumor-targeting molecule in combination with a small phosphine-containing imaging/therapeutic probe. Up to 8 triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and tumor uptake in tumor bearing nude mice. In addition, two (89)Zr- and (67/68)Ga-labeled desferrioxamine (DFO)-phosphines, a (177)Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-phosphine and a (123)I-cubyl phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger ligation of 3.33 μM antibody-azide conjugate with 1 equiv of radiolabeled phosphine, relative to the azide, in aqueous solution resulted in 20-25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced ligation efficiency (reduction max. 30% at 2 h), while the phosphines were still >80% intact. No in vivo Staudinger ligation was observed in a mouse model after injection of 500 μg antibody-azide, followed by 68 μg DFO-phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger ligation was performed in vitro in mouse serum. Under these conditions, a side product with the phosphine was formed and ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger ligation in a pretargeting approach in mice is not feasible, since this ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger ligation in humans.     

Site-specific fluorescent labeling of DNA using Staudinger ligation

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.     

Use of 2-[18F]fluoroethylazide for the Staudinger ligation – Preparation and characterisation of GABAA receptor binding 4-quinolones

The labelling reagent 2-[¹⁸F]fluoroethylazide was used in a traceless Staudinger ligation. This reaction was employed to obtain the GABA_A receptor binding 6-benzyl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (2-[¹⁸F]fluoroethyl) amide. The radiotracer was prepared with a non-decay corrected radiochemical yield of 7%, a radiochemical purity >95% and a specific radioactivity of 0.9 GBq/μmol. The compound showed low brain penetration in normal rats. A series of fluoroalkyl 4-quinolone analogues with nanomolar to sub-nanomolar affinity for the GABA_A receptor has been prepared as well.     

Surface-bound cytomimetic assembly based on chemoselective and biocompatible immobilization and further modification of intact liposome

A surface-bound cytomimetic assembly based on chemically selective and biocompatible immobilization and further modification of intact liposome is described. Liposomes carrying PEG-triphenylphosphine were chemoselectively immobilized onto azide-modified glass slides through Staudinger ligation, followed by modification with azide-modified lactose as a model biomolecule through Staudinger ligation to afford the surface-bound cytomimetic assembly. The intact liposome immobilized and modified and its protein binding activity were confirmed by fluorescence imaging, fluorescent dye releasing kinetics, and AFM techniques. The resultant surface-bound cytomimetic assembly showed sustained stability and fluorescent dye releasing kinetics and specific protein binding activity. The reported method provides a robust platform for preparation of a complex immobilized liposome system with multifunctional components, which mimics the cell surface in both geographical and content features and thus will find important biomedical applications.     

Splicing of unnatural amino acids into proteins: a peptide model study

S-Ethyl 2-azidohexanethioate (N3-Hex-SEt), an unnatural amino acid analog of leucine, is coupled with L-cysteine ethyl ester (NH2-Cys-OEt) to obtain N3-Hex-Cys-OEt by native chemical ligation. Coupling of this dipeptide with N-t-butoxycarbonyl-2-diphenylphosphinoethanethioglycinate produces the tripeptide, t-Boc-Gly-Hex-Cys-OEt, in high yield. These reactions suggest an approach for the incorporation of unnatural amino acids into proteins by successive native chemical ligation and Staudinger ligation.     

Stereoselective synthesis of glycosyl amides by traceless Staudinger ligation of unprotected glycosyl azides

The stereoconservative Staudinger ligation of unprotected alpha- and beta-glucosyl azides with diphenylphosphanyl-phenyl esters to afford alpha- and beta-glucosyl amides is described.     

Immobilization strategies for small molecule,peptide and protein microarrays

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site‐selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol‐ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Nucleotidyl transferase assisted DNA labeling with different click chemistries

Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.     

'100 years of peptide synthesis': ligation methods for peptide and protein synthesis with applications to beta-peptide assemblies.

A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, 'Synthesis of Proteins by Chemical Ligation' a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo-prolines, 'unrestricted' amino-acid residues (using the Staudinger reaction), as well as solid-phase segment coupling by thioligation of unprotected peptides. In another section, 'Synthesis of beta-peptides by Thioligation', couplings involving beta2- and beta3-peptides are described (with experimental details).     

Surface re-engineering of pancreatic islets with recombinant azido-thrombomodulin

Chemoselective conjugation of an azido-functionalized thrombomodulin to pancreatic islets was achieved by Staudinger ligation to a surface-bound bifunctional poly(ethylene glycol) linker. The presence of the tethered thrombomodulin resulted in a significant increase in the production of activated protein C with a reduction in islet-mediated thrombogenicity. This report highlights the potential of tissue-targeted chemistry to reduce donor cell mediated procoagulant and proinflammatory responses.     

A new drug-release method using the Staudinger ligation

Many drugs induce severe side-effects caused by their lack of selectivity. One way to overcome this problem is to design a specific system which releases a free drug in a controlled manner. Herein we describe a new way to liberate a drug from a prodrug using the Staudinger ligation as the trigger.     

6'-Azido-6'-deoxy-UDP-N-acetylglucosamine as a glycosyltransferase substrate

6″-Azido-6″-deoxy-UDP-N-acetylglucosamine (UDP-6Az-GlcNAc) is a potential alternate substrate for N-acetylglucosaminyltransferases. This compound could be used to generate various glycoconjugates bearing an azide functionality that could in turn be subjected to further modification using Staudinger ligation or Huisgen cycloaddition. UDP-6Az-GlcNAc is synthesized from α-benzyl-N-acetylglucosaminoside in seven-steps with an overall yield of 6%. It is demonstrated to serve as a substrate donor for the glycosyl transfer reaction catalyzed by the human UDP-GlcNAc:polypeptidyltransferase (OGT) to the acceptor protein nucleoporin 62 (nup62).     

Targeting of polyamidoamine-DNA nanoparticles using the Staudinger ligation: attachment of an RGD motif either before or after complexation

Two new methods for the modular synthesis of targeted gene delivery systems are reported. The PEGylated polyamidoamine DMEDA-PEG-DMEDA-(MBA-DMEDA)(n+1)-PEG-DMEDA 3 was sequentially modified to contain an integrin-binding peptide ligand via the Staudinger ligation. The conjugation of the ligand was achieved either before particle complexation (precomplexation) or after particle complexation (postcomplexation). Comparison of the two systems showed that postcomplexation strategy led to small and discrete toroidal nanoparticles whilst the precomplexation particles showed loose complexes. The targeted particles showed an increased uptake into cells compared to unmodified complexes however no significant increase in transfection was seen.     

A generic building block for C- and N-terminal protein-labeling and protein-immobilization

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.     

N-azidoacetylmannosamine-mediated chemical tagging of gangliosides

Peracetylated N-alpha-azidoacetylmannosamine (Ac(4)ManNAz) is metabolized by cells to CMP-azidosialic acid. It has been demonstrated previously that in this way azidosialic acid-containing glycoproteins are formed that can be labeled on the cell surface by a modified Staudinger ligation. Here, we first demonstrate that the same procedure also results in the formation of azidosialic acid-containing gangliosides. Deoxymannojirimycin, an inhibitor of N-glycan processing in proteins, decreases the total cell surface labeling in Jurkat cells by approximately 25%. Inhibition of ganglioside biosynthesis with N-[5-(adamantan-1-yl-methoxy)-pentyl]1-deoxynojirimycin reduces cell surface labeling by approximately 75%. In conclusion, exposure of cells to Ac(4)ManNAz allows in vivo chemical tagging of gangliosides.     

General method for site-specific protein immobilization by Staudinger ligation

Protein microarrays are playing an increasingly important role in the discovery and characterization of protein-ligand interactions. The uniform orientation conferred by site-specific immobilization is a demonstrable advantage in using such microarrays. Here, we report on a general strategy for fabricating gold surfaces displaying a protein in a uniform orientation. An azido group was installed at the C-terminus of a model protein, bovine pancreatic ribonuclease, by using the method of expressed protein ligation and a synthetic bifunctional reagent. This azido protein was immobilized by Staudinger ligation to a phosphinothioester-displaying self-assembled monolayer on a gold surface. Immobilization proceeded rapidly and selectively via the azido group. The immobilized enzyme retained its catalytic activity and was able to bind to its natural ligand, the ribonuclease inhibitor protein. This strategy provides a general means to fabricate microarrays displaying proteins in a uniform orientation.