Staudinger-phosphite Reaction Research Articles

An efficient, catalyst and solvent free Staudinger phosphite reaction for the synthesis of phosphoramidates under mild conditions

An efficient, catalyst-free and solvent-free protocol has been developed for the synthesis of phosphoramidates. This method can be used to synthesize phosphoramidates from diverse starting materials including aryl/benzyl and alkyl azides at room temperature. The synthesis is simple, selective and high yielding with notably a very short reaction time. The process involves mild reaction conditions which allow for sensitive functionalities and protecting groups to be used in the developed protocol that is of great advantage and could provide valuable reference for the construction of more complex heterocyclic linked phosphoramidate systems with biological and medicinal importance.

Synthesis and evaluation of the antibiotic-adjuvant activity of carbohydrate-based phosphoramidate derivatives

Phosphoramidates are becoming increasingly recognized as molecular targets for therapeutic development. Their biological functions are significantly influenced by their inherent properties such as reactivity, as well as the P–N backbone which allows for structural diversity. In this study we report the synthesis of novel carbohydrate-based phosphoramidate derivatives via the Staudinger-phosphite reaction; along with an evaluation of their adjuvant activity in combination with popular antibiotics. Our targets involved variation in both the sugar residue as well as the identity of the phosphoramidate. Moderate to excellent yields of these derivatives were obtained. Notable adjuvant activity was observed with the halogenated phosphoramidates. For the fluorinated glucose derivative in particular, a remarkable 32-fold decrease in the MIC of Ampicillin was obtained against Methicillin-resistant S. aureus.

Synthesis and interfacial properties of glyco-lipophosphoramidates

Lipophosphoramidates are a class of amphiphilic molecules that are tunable; and are heavily explored for a variety of industrial and medicinal applications. Self-assembled structures derived from such molecules enhance the bioavailability, selectivity and pharmacokinetic properties of drug molecules. Herein we report the synthesis of novel glyco-lipophosphoramidates (GLP-3, GLP-4, GLP-5, GLP-6) with varying linear and branched hydrocarbon tails via the Staudinger-phosphite reaction. The incorporation of the carbohydrate moiety as the head group region of the amphiphile promotes biocompatibility and biodegradability. Surface activity, adsorption, and aggregation behavior in aqueous solution were examined using surface tension, Langmuir monolayers, and atomic force microscopy (AFM). The critical aggregation concentrations (CAC) and Langmuir isotherms showed the typical dependence on acyl chain structure. Gibbs adsorption profiles provided limiting molecular areas in the range 55–85 Å2. AFM imaging revealed the formation of large spherical aggregates in aqueous solution above the CAC. Dye encapsulation studies support the self-assembly into vesicles by GLP-3, GLP-4 and GLP-5; demonstrating the potential for drug delivery or nanocarrier applications.

Synthesis of Triazole-Containing Furanosyl Nucleoside Analogues and Their Phosphate, Phosphoramidate or Phoshonate Derivatives as Potential Sugar Diphosphate or Nucleotide Mimetics.

The synthesis of stable and potentially bioactive xylofuranosyl nucleoside analogues and potential sugar diphosphate or nucleotide mimetics comprising a 1,2,3-triazole moiety is reported. 3'-O-Methyl-branched N-benzyltriazole isonucleosides were accessed in 5-7 steps and 42-54 % overall yields using a Cu(I)-catalyzed cycloaddition of 3-O-propargyl-1,2-O-isopropylidene-α-D-xylofuranose with benzyl azide as key step. Related isonucleotides were obtained by 5-O-phosphorylation of acetonide-protected 3-O-propargyl xylofuranose and further "click" cycloaddition or by Staudinger-phosphite reaction of a 5-azido N-benzyltriazole isonucleoside. Hydroxy-, amino- or bromomethyl triazole 5'-isonucleosides were synthesized by thermal cycloaddition of 5-azido 3-O-benzyl/dodecyl xylofuranoses with propargyl alcohol, propargylamine or propargyl bromide. Better yields (82-85 %) were obtained when using propargyl alcohol and a high 1,4-regioselectivity was attained with propargyl bromide. Further O/N-phosphorylation or Arbuzov reaction led to (triazolyl)methyl phosphates, phosphoramidates or phosphonates. The latter were converted into uracil nucleoside 5'-(triazolyl)methyl phosphonates as prospective nucleoside diphosphate mimetics.

The Staudinger Ligation.

While the Staudinger reaction has first been described a hundred years ago in 1919, the ligation reaction became one of the most important and efficient bioconjugation techniques in the 1990s and this century. It holds the crucial characteristics for bioorthogonal chemistry: biocompatibility, selectivity, and a rapid and high-yielding turnover for a wide variety of applications. In the past years, it has been used especially in chemical biology for peptide/protein synthesis, posttranslational modifications, and DNA labeling. Furthermore, it can be used for cell-surface engineering, development of microarrays, and drug delivery systems. However, it is also possible to use the reaction in synthetic chemistry for general formation of amide bonds. In this review, the three major types, traceless and nontraceless Staudinger Ligation as well as the Staudinger phosphite reaction, are described in detail. We will further illustrate each reaction mechanism and describe characteristic substrates, intermediates, and products. In addition, not only its advantages but also stereochemical aspects, scope, and limitations, in particular side reactions, are discussed. Finally, the method is compared to other bioorthogonal labeling methods.

Synthesis and antiproliferative evaluation of novel azido nucleosides and their phosphoramidate derivatives

Abstract New xylofuranosyl and glucopyranosyl nucleoside phosphoramidates were synthesized as potential mimetics of nucleoside 5′-monophosphates. Their access involved N-glycosylation of uracil and 2-acetamido-6-chloropurine with 5′/6′-azido-1,2-di-O-acetyl glycosyl donors and subsequent Staudinger-phosphite reaction of the resulting azido nucleosides. The coupling of the purine derivative with the pyranosyl donor furnished N9- and N7-linked nucleosides in 1:1 ratio, whereas with the furanosyl donor, the N9-nucleoside was the major regioisomer formed. When using uracil, only 5′/6′-azido N1-linked nucleosides were obtained. The purine 5′/6′-azido nucleosides were converted into corresponding phosphoramidates in good yields. The antiproliferative effects of the nucleoside phosphoramidates and those of the azido counterparts on cancer cells were evaluated. While the nucleoside phosphoramidates did not show significant activities, the purine 5′/6′-azido nucleosides displayed potent effects against K562, MCF-7 and BT474 cell lines. The 5′-azidofuranosyl N9 and N7-linked purine nucleosides exhibited highest activity towards the chronic myeloid leukemia cell line (K562) with GI50 values of 13.6 and 9.7 μM, respectively. Among pyranosyl nucleosides, the N7-linked nucleoside was the most active compound with efficacy towards all cell lines assayed and a highest effect on K562 cells (GI50=6.8 μM). Cell cycle analysis of K562 and MCF-7 cells showed that the most active compounds cause G2/M arrest.

Assembly of Multi-Phthalocyanines on a Porphyrin Template by Fourfold Rotaxane Formation.

A stacked assembly composed of a porphyrin and two phthalocyanines was prepared through fourfold rotaxane formation. Two phthalocyanine molecules, bearing four 24-crown-8 units, were assembled onto a porphyrin template incorporating four sidechains with two dialkylammonium ions each through pseudorotaxane formation between crown ether units and ammonium ions. The Staudinger phosphite reaction, as the stoppering reaction, resulted in the formation of the stacked heterotrimer composed of a porphyrin and two phthalocyanines connected through a fourfold rotaxane structure. UV/Vis spectroscopic and electrochemical studies of the heterotrimer indicated that there is a significant electronic interaction between the two phthalocyanine units due to the close stacking. The electrochemical oxidation process of the stacked heterotrimer was studied by cyclic voltammetry and spectroelectrochemistry. Electron paramagnetic resonance (EPR) spectroscopy of a dinuclear Cu(II) complex, in which two Cu(II) phthalocyanines were assembled on a metal-free porphyrin template, revealed that two Cu(II) phthalocyanines were located within the stacking distance, which resulted in an antiferromagnetic interaction between the two S=1/2 spins in the ground state of the Cu(2+) ions in the heterotrimer.

Stabilization of bacterially expressed erythropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylation sites.

The covalent attachment of polyethylene glycol (PEG) to therapeutic proteins can improve their physicochemical properties. In this work we utilized the non-natural amino acid p-azidophenylalanine (pAzF) in combination with the chemoselective Staudinger-phosphite reaction to install branched PEG chains to recombinant unglycosylated erythropoietin (EPO) at each single naturally occurring glycosylation site. PEGylation with two short 750 or 2000 Da PEG units at positions 24, 38, or 83 significantly decreased unspecific aggregation and proteolytic degradation while biological activity in vitro was preserved or even increased in comparison to full-glycosylated EPO. This site-specific bioconjugation approach permits to analyse the impact of PEGylation at single positions. These results represent an important step towards the engineering of site-specifically modified EPO variants from bacterial expression with increased therapeutic efficacy.

Direct access to site-specifically phosphorylated-lysine peptides from a solid-support.

Phosphorylation is a key process for changing the activity and function of proteins. The impact of phospho-serine (pSer), -threonine (pThr) and -tyrosine (pTyr) is certainly understood for some proteins. Recently, peptides and proteins containing N-phosphorylated amino acids such as phosphoarginine (pArg), phosphohistidine (pHis) and phospholysine (pLys) have gained interest because of their different chemical properties and stability profiles. Due to its high intrinsic lability, pLys is the least studied within this latter group. In order to gain insight into the biological role of pLys, chemical and analytical tools, which are compatible with the labile P(=O)-N bond, are highly sought-after. We recently reported an in-solution synthetic approach to incorporate pLys residues in a site-specific manner into peptides by taking advantage of the chemoselectivity of the Staudinger-phosphite reaction. While the in-solution approach allows us to circumvent the critical TFA cleavage, it still requires several transformations and purification steps to finally deliver pLys peptides. Here we report the synthesis of site-specific pLys peptides directly from a solid support by using a base labile resin. This straightforward and highly efficient approach facilitates the synthesis of various site-specific pLys-containing peptides and lays the groundwork for future studies about this elusive protein modification.

Site-Specifically Phosphorylated Lysine Peptides

Protein phosphorylation controls major processes in cells. Although phosphorylation of serine, threonine, and tyrosine and also recently histidine and arginine are well-established, the extent and biological significance of lysine phosphorylation has remained elusive. Research in this area has been particularly limited by the inaccessibility of peptides and proteins that are phosphorylated at specific lysine residues, which are incompatible with solid-phase peptide synthesis (SPPS) due to the intrinsic acid lability of the P(═O)-N phosphoramidate bond. To address this issue, we have developed a new synthetic route for the synthesis of site-specifically phospholysine (pLys)-containing peptides by employing the chemoselectivity of the Staudinger-phosphite reaction. Our synthetic approach relies on the SPPS of unprotected ε-azido lysine-containing peptides and their subsequent reaction to phosphoramidates with phosphite esters before they are converted into the natural modification via UV irradiation or basic deprotection. With these peptides in hand, we demonstrate that electron-transfer dissociation tandem mass spectrometry can be used for unambiguous assignment of phosphorylated-lysine residues within histone peptides and that these peptides can be detected in cell lysates using a bottom-up proteomic approach. This new tagging method is expected to be an essential tool for evaluating the biological relevance of lysine phosphorylation.

Self‐Liganded Suzuki–Miyaura Coupling for Site‐Selective Protein PEGylation

The discovery and development of palladium-catalyzed carbon–carbon bond formation has revolutionized modern organic chemistry in recent decades. The versatility and robustness of these processes have enabled major progress in total synthesis, materials development, and medicinal chemistry. Currently, a large variety of nucleophiles and electrophiles can be efficiently coupled, and part of the efforts to increase the utility of these reactions are now directed towards the development of simpler, more active, and economic catalytic systems that work under mild conditions. Owing to their broad functional-group tolerance and low associated toxicity, as well as their chemical and biological compatibility, Pd-catalyzed reactions are now attractive approaches for protein modification. These processes are particularly interesting, as a variety of non-natural amino acids can be genetically incorporated into peptides and proteins, thus providing specific chemical handles for a number of post-translational manipulations, including palladium-mediated reactions. Recent contributions from our group and others report robust and highly efficient Suzuki–Miyaura and Sonogashira cross-coupling on protein substrates bearing a halide or alkyne moiety, respectively, under ambient conditions both in vitro and on cell surfaces. As part of our continuing interest in developing chemical tools for the mild and specific post-translational modification of proteins, we investigated the use of the Suzuki–Miyaura coupling to site-selectively attach polyethylene glycol (PEG) chains to proteins. Protein PEGylation is a widespread approach to improve the stability and pharmacokinetics of protein drugs by reducing clearance rates, while providing a steric shield from proteolytic enzymes and immune system recognition. However, reduced biological activity is often observed for PEG-conjugated proteins. This effect is attributed to steric crowding resulting from the low positional control achieved with traditional PEGylation chemistry; commonly used PEG-derivatives react with the side chains of natural amino acids, such as lysines or cysteines, or protein termini and disulfide bonds. Whenmore than one reactive site is present on the protein, these approaches lead to heterogenous mixtures of potentially less-/in-active conjugates that are difficult to separate. The possibility of siteselectively PEGylating proteins could potentially overcome this problem but, to date, only a few examples use the PEGderivatization of genetically-encoded non-natural amino acids. These include Sonogashira coupling at homopropargyl-glycine, and triazole formation or Staudinger phosphite reaction at p-azidophenylalanine. The two former examples involve metal catalysts and required exogenous ligands and consequent optimization. Airand moisture-stable Pd-ligands provide convenient systems that do not require inert atmosphere or degassed solvents. Nonetheless, even more direct “ligandless” (selfliganded or lacking an added ligand) methods are of significant interest. This would provide a more straightforward method that is particularly attractive in biotechnological applications to be conducted on scale such as PEGylation. Pdnanoparticle-based catalysts and metal catalysts stabilized by polymers such as polyethylene glycol (PEG) have been reported. Taking advantage of these potential stabilizing properties of PEG, we considered the possibility of direct palladium-catalyzed PEGylation of halogenated amino acids with PEG–boronic acid derivatives in the absence of additional ligand. Herein, we report PEGylation of amino acids, peptides, and proteins through palladium-catalyzed Suzuki–Miyaura coupling of 2 kDa and 20 kDa monomethoxy PEG (mPEG) phenylboronic acids (mPEG2k-PBA and mPEG20k-PBA, respectively). An evaluation of pyrimidineor guanidinebased palladium complexes and comparison with PEG reagents alone reveals that the latter can act as potent selfliganding agents for the stabilization of metal species, thus allowing complete conversion using water-soluble palladium salts in the absence of external ligands. The resulting reaction system combines the advantages of reaction biocompatibility for protein modification with the possibility of combining a highly simple, single-component, economic, and environmentally friendly catalyst with a self-liganding coupling partner. We believe this shows the first example of ligandless metal-mediated ligation on a protein surface. Our initial study focused on the PEGylation of N-Boc-4iodo-l-phenylalanine (1; Boc-pIPhe) with mPEG20k-PBA (2) by Suzuki–Miyaura cross-coupling in 20 mm phosphate buffer, pH 8.0. The palladium catalyst system first tested used an additional ligand 2-amino-4,6-dihydroxy-pyrimidine (L1), [*] Dr. A. Dumas, C. D. Spicer, Dr. Z. Gao, Y. A. Lin, Prof. B. G. Davis Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX13TA (UK) E-mail: ben.davis@chem.ox.ac.uk

Chemoselective Staudinger-phosphite reaction of symmetrical glycosyl-phosphites with azido-peptides and polygycerols

In this paper we present the synthesis of glyco-phosphoramidate conjugates as easily accessible analogs of glyco-phosphorous esters via the Staudinger-phosphite reaction. This protocol takes advantage of synthetically accessible symmetrical carbohydrate phosphites in good overall yields, in which ethylene or propylene linkers can be introduced between phosphorous and galactose or lactose moieties. The phosphites were finally applied for the chemoselective reaction with azido-peptides and polyazido(poly)glycerols.

Phosphoramidate‐peptide synthesis by solution‐ and solid‐phase Staudinger‐phosphite reactions

The chemoselective incorporation of phosphoramidate moieties into peptides by a Staudinger-phosphite reaction of azides can be performed in many solvents, including water. In this report, we present two strategies for an efficient synthesis of phosphoramidate-containing peptides, in which the Staudinger-phosphite reaction is performed either on the solid support or in solution with aryl azido-containing peptides. The corresponding Staudinger reactions proceed in high conversion rates and deliver phosphoramidate peptides, in which the modification site is located in the middle of the peptide sequence.

Solid‐Phase Synthesis of Phosphoramidate‐Linked Glycopeptides

AbstractThe synthesis of stable glycopeptide mimetics is of particular interest in bioorganic chemistry to allow access of glycoconjugates for biological investigations. In this paper, a straightforward solid‐phase synthesis of a novel glycopeptide mimetic by a Staudinger phosphite reaction is presented. Thereby, a dimethyl phosphite containing peptide, which is obtained by standard phosphitylation of a Ser residue, is treated on the solid support with glycosyl azides, delivering phosphoramidate‐linked glycoconjugates. These artificial glycopeptides show excellent stability under acidic and physiological conditions.

Site-specific PEGylation of proteins by a Staudinger-phosphite reaction

Current protocols in protein bioengineering allow the site-specific incorporation of chemical reporter moieties. Subsequently, these functional groups can be chemoselectively transformed to decorate proteins with charged and oversized functional units. Based on our recent report on the chemoselective reaction of azides with phosphites, we now apply the Staudinger-phosphite reaction to an efficient and metal-free PEGylation of an azide-containing protein with symmetrical phosphites. Thereby, two types of branched oligoethylene glycol scaffolds are generated, which deliver either a stable or light-cleavable protein-PEG conjugate. Furthermore, we demonstrate that the Staudinger-phosphite reaction is an efficient transformation in both aqueous media as well as in a highly crowded bacterial cell lysate.

Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites

Unsymmetrical phosphites react efficiently in a Staudinger reaction with p-azido-phenylalanine, which can be applied for the biotinylation of proteins, thereby expanding the scope of the chemoselective Staudinger-phosphite reaction of aryl azides with symmetrical phosphites to the corresponding phosphoramidates.

Chemoselective Staudinger‐Phosphite Reaction of Azides for the Phosphorylation of Proteins

Extending the toolbox: The title reaction was identified as a chemoselective means to modify azides in peptides and proteins in high yields at room temperature in various solvents including aqueous buffers at physiological pH. In combination with nonnatural protein translation the Staudinger-phosphite reaction allows the site-specific incorporation of phosphorylated Tyr analogues in proteins.