Staudinger ligation as a method for bioconjugation

In 1919 the German chemist Hermann Staudinger was the first to describe the reaction between an azide and a phosphine. It was not until recently, however, that Bertozzi and co-workers recognized the potential of this reaction as a method for bioconjugation and transformed it into the so-called Staudinger ligation. The bio-orthogonal character of both the azide and the phosphine functions has resulted in the Staudinger ligation finding numerous applications in various complex biological systems. For example, the Staudinger ligation has been utilized to label glycans, lipids, DNA, and proteins. Moreover, the Staudinger ligation has been used as a synthetic method to construct glycopeptides, microarrays, and functional biopolymers. In the emerging field of bio-orthogonal ligation strategies, the Staudinger ligation has set a high standard to which most of the new techniques are often compared. This Review summarizes recent developments and new applications of the Staudinger ligation.     

The traceless Staudinger ligation with fluorine-18: a novel and versatile labeling technique for the synthesis of PET-radiotracers

The development of rapid radiolabeling techniques under mild reaction conditions involving the short-lived positron emitter fluorine-18 remains a special challenge in organic PET chemistry. This work describes a novel and facile application of the traceless Staudinger ligation as a mild and versatile labeling method for preparation of various radiotracers labeled with fluorine-18.     

Mechanistic investigation of the staudinger ligation

The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-order overall and proceeded more rapidly in polar, protic solvents. Hammett analyses demonstrated that electron-donating substituents on the phosphine accelerate the overall reaction. The electronic and steric properties of the ester had no significant impact on the overall rate but did affect product ratios. Finally, the structure of an intermediate that accumulates under anhydrous conditions was identified. These findings establish a platform for optimizing the Staudinger ligation for expanded use in biological applications.     

A Photo‐Triggered Traceless Staudinger–Bertozzi Ligation Reaction

The use of light to control the course of a chemical/biochemical reaction is an attractive idea because of its ease of administration with high precision and fine spatial resolution. Staudinger ligation is one of the commonly adopted conjugation processes that involve a spontaneous reaction between azides and arylphosphines to form iminophosphoranes, which further hydrolyze to give stable amides. We designed an anthracenylmethyl diphenylphosphinothioester ( 1 ) that showed promising Staudinger ligation reactivity upon photo‐excitation. Broadband photolysis at 360–400 nm in aqueous organic solvents induced heterolytic cleavage of its anthracenylmethyl–phosphorus bond, releasing a diphenylphosphinothioester ( 2 ) as an efficient traceless Staudinger–Bertozzi ligation reagent. The quantum yield of such a photo‐induced heterolytic bond‐cleavage at the optimal wavelength of photolysis (376 nm) at room temperature is ≥0.07. This work demonstrated the feasibility of photocaging arylphosphines to realize the photo‐triggering of the Staudinger ligation reaction.     

Water-soluble phosphinothiols for traceless staudinger ligation and integration with expressed protein ligation

The traceless Staudinger ligation is an effective means to synthesize an amide bond between two groups of otherwise orthogonal reactivity: a phosphinothioester and an azide. An important application of the Staudinger ligation is in the ligation of peptides at a variety of residues. Here, we demonstrate that the traceless Staudinger ligation can be achieved in water with a water-soluble reagent. Those reagents that provide a high yield of amide product discourage protonation of the nitrogen in the key iminophosphorane intermediate. The most efficacious reagent, bis(p-dimethylaminoethyl)phosphinomethanethiol, mediates the rapid ligation of equimolar substrates in water. This reagent is also able to perform a transthioesterification reaction with the thioester intermediate formed during intein-mediated protein splicing. Hence, the traceless Staudinger ligation can be integrated with expressed protein ligation, extending the reach of modern protein chemistry.     

Staudinger ligation of peptides at non-glycyl residues

The Staudinger ligation provides a means to form an amide bond between a phosphinothioester and azide. This reaction holds promise for the ligation of peptides en route to the total chemical synthesis of proteins. (Diphenylphosphino)methanethiol is the most efficacious of known reagents for mediating the Staudinger ligation of peptides, providing high (> 90%) isolated yields for equimolar couplings in which a glycine residue is at the nascent junction. Surprisingly, the yields are lower (< 50%) for non-glycyl couplings due to an aza-Wittig reaction that diverts the reaction toward a phosphonamide byproduct. Here, the partitioning of the reaction toward Staudinger ligation (and away from the aza-Wittig reaction) is shown to increase with increasing electron density on phosphorus. This electron density can be tuned either by installing functional groups on the phenyl substituents of (diphenylphosphino)methanethiol or by changing the polarity of the solvent. Installing p-methoxy groups and using a solvent of low polarity (such as toluene or dioxane) provide especially high (> 80%) isolated yields for the ligation of two non-glycyl residues. These conditions retain the high chemoselectivity of the reaction and do not lead to a substantial change in reaction rate. The traceless Staudinger ligation is now poised to enable the iterative ligation of peptides with little regard for their sequence, as well as the synthesis of amide bonds for other purposes.     

A proline-based phosphine template for staudinger ligation

A proline-based phosphine template enabling a rapid Staudinger ligation of azide-containing substrates under mild conditions is reported. This reaction has a second-order rate constant of 1.12 M(-1) s(-1). It is expected that the proline-based Staudinger ligation strategy will be a useful method for bioconjugation and proline based peptide coupling.     

基于黄酮的ESIPT型荧光探针检测三苯基膦

三苯基膦(PPh3)是一种重要的有机化合物,具有亲核性和还原性,已被广泛用于有机合成和有机金属化合物配位中。然而PPh3的过量使用不可避免地会引起环境的污染,并且对人体的健康也具有潜在威胁。因此我们迫切需要一种方便而高效的检测PPh3的方法。已有检测方法在便捷性和灵敏性等方面不如人意,而小分子荧光探针因其灵敏度高、选择性好、检测简单等优点受到广泛关注。受生物标记领域常用的无痕Staudinger连接反应的启发,本文设计合成了一个类似Staudinger连接反应,以能够具有固态发光性质的激发态分子内质子转移(ESIPT)特性的3-羟基黄酮荧光染料为母核,以邻叠氮苯乙酸酯为识别基团,用于检测PPh3的荧光增强型的探针分子。通过叠氮与PPh3反应,经过氮杂叶立德中间体,进行快速的分子内酰胺化,释放3-羟基黄酮染料并恢复荧光发射,从而实现在溶液中及滤纸上快速检测PPh3的目的。该探针在定量和定性检测PPh3方面具有潜在应用价值。     

Conversion of aryl azides to O-alkyl imidates via modified Staudinger ligation

[reaction: see text] o-Carboalkoxy triarylphosphines are shown to react with aryl azides to provide Staudinger ligation products bearing O-alkyl imidate linkages. This is in contrast to alkyl azides whose ligation to o-carboalkoxy triarylphosphines has been reported to yield amide-linked materials. This extension of the Staudinger ligation for coupling of abiotic reagents under biocompatible conditions highlights the utility of commercially available triarylphosphines through which suitable linkers can be attached via an ester moiety.     

Staudinger ligation of alpha-azido acids retains stereochemistry

The Staudinger ligation of peptides with a C-terminal phosphinothioester and N-terminal azide is an emerging method in protein chemistry. Here, the first Staudinger ligations of nonglycyl azides are reported and shown to proceed both in nearly quantitative yield and with no detectable effect on the stereochemistry at the alpha-carbon of the azide. These results demonstrate further the potential of the Staudinger ligation as a general method for the total synthesis of proteins from peptide fragments.     

Synthesis, structure determination, and (radio-)fluorination of novel functionalized phosphanes suitable for the traceless Staudinger ligation

An elegant and efficient synthesis approach for the preparation of novel benzoate and nicotinate containing phosphanes is presented. This reaction path has a broad substrate scope. Thus, various functionalized phosphanes were obtained in high yields using an esterification procedure under Steglich conditions. A facile blocking of the phosphorus atom with BH₃ was carried out. BH₃ as easily insertable and removable protecting group enables a further derivatization of the benzoate residue. The prepared phosphane derivatives proved to be valuable labeling building blocks for the implementation of a bioorthogonal (radio-)fluorination strategy and were applied for labeling purposes using the traceless Staudinger ligation. For this purpose, a selection of azide-functionalized small organic and bioactive sample molecules was prepared. Furthermore, a mild and selective (radio-)fluorination of these derivatives is demonstrated adopting this bioorthogonal ligation method.     

Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation

The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.     

Reaction mechanism and kinetics of the traceless Staudinger ligation

The traceless Staudinger ligation enables the formation of an amide bond between a phosphinothioester (or phosphinoester) and an azide without the incorporation of residual atoms. Here, the coupling of peptides by this reaction was characterized in detail. Experiments with [(18)O]H(2)O indicated that the reaction mediated by (diphenylphosphino)methanethiol proceeded by S-->N acyl transfer of the iminophosphorane intermediate to form an amidophosphonium salt, rather than by an aza-Wittig reaction and subsequent hydrolysis of the resulting thioimidate. A continuous (13)C NMR-based assay revealed that the rate-determining step in the Staudinger ligation of glycyl residues mediated by (diphenylphosphino)methanethiol was the formation of the initial phosphazide intermediate. Less efficacious coupling reagents and reaction conditions led to the accumulation of an amine byproduct (which resulted from a Staudinger reduction) or phosphonamide byproduct (which resulted from an aza-Wittig reaction). The Staudinger ligation mediated by (diphenylphosphino)methanethiol proceeded with a second-order rate constant (7.7 x 10(-3) M(-1) s(-1)) and yield (95%) that was unchanged by the addition of exogenous nucleophiles. Ligations mediated by phosphinoalcohols had lower rate constants or less chemoselectivity. Accordingly, (diphenylphosphino)methanethiol was judged to be the most efficacious known reagent for effecting the traceless Staudinger ligation.     

Development and Optimization of Glaser–Hay Bioconjugations

The prevalence of bioconjugates in the biomedical sciences necessitates the development of novel mechanisms to facilitate their preparation. Towards this end, the translation of the Glaser–Hay coupling to an aqueous environment is examined, and its potential as a bioorthogonal conjugation reaction is demonstrated. This optimized, novel, and aqueous Glaser–Hay reaction is applied towards the development of bioconjugates utilizing protein expressed with an alkynyl unnatural amino acid. Unnatural amino acid technology provides a degree of bioorthognality and specificity not feasible with other methods. Moreover, the scope of the reaction is demonstrated through protein–small molecule couplings, small‐molecule–solid‐support couplings, and protein–solid‐support immobilizations.     

Switch peptide via Staudinger reaction

A new transformation based on the Staudinger reaction is described, and its application in the design of a novel switch element to control peptide folding is demonstrated. We found that the azide switch is activated rapidly in water to promote acyl transfer using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) via the Staudinger reaction. Our findings expand the repertoire of uses of the Staudinger reaction in chemical biology and the number of available triggers for use in switch peptides.     

无痕施陶丁格连接反应机理的理论研究

叠氮化合物和膦硫酯的无痕施陶丁格连接反应是一种有效生成酰胺键的方法. 通过密度泛函方法研究了无痕施陶丁格连接的反应机理. 计算结果表明反应的决速步为反应的第一步, 即膦进攻叠氮的端基氮, 形成膦基叠氮化物的过程. 膦亚胺中间体形成之后, 向不同方向转化(酰基迁移后水解或者直接水解)的难易程度决定了最终产物的组成. 对几种不同偶联试剂介导的无痕施陶丁格连接反应的计算结果, 都与实验数据相吻合.     

Chemoselective coupling of peptide fragments using the Staudinger ligation

Here we report the first Staudinger ligations which yield tetra- and pentapeptides starting from N-terminal α-azido peptides and C-terminal peptideo-(diphenylphosphine)phenyl esters. Mass spectrometric analysis of the reaction mixture provided a better insight into the mechanism of the Staudinger ligation and has been used to explain the observed intermediates and to optimize the ligation reaction. As a result, the optimized reaction enables the chemoselective coupling of peptides containing amino acids other than glycine at the ligation site.     

Protein assembly by orthogonal chemical ligation methods

Chemical synthesis harbors the potential to provide ready access to natural proteins as well as to create nonnatural ones. The Staudinger ligation of a peptide containing a C-terminal phosphinothioester with a peptide containing an N-terminal azide gives an amide with no residual atoms. This method for amide bond formation is orthogonal and complementary to other ligation methods. Herein, we describe the first use of the Staudinger ligation to couple peptides on a solid support. The fragment thus produced is used to assemble functional ribonuclease A via native chemical ligation. The synthesis of a protein by this route expands the versatility of chemical approaches to protein production.     

Total syntheses of nobilamides B and D: application of traceless Staudinger ligation

Nobilamide B is a long-acting antagonist of transient receptor potential vanilloid-1 (TRPV1), and is expected to show therapeutic potential for treatment of pain. This linear heptapeptide possesses a Z-didehydroaminobutanoic acid moiety at the C-terminus. Stereoselective construction of the didehydroamino acid moiety was successfully achieved by application of the traceless Staudinger ligation. The combination of solid-phase peptide synthesis and the Staudinger ligation allowed rapid access to not only nobilamide B, but also its macrocyclic analogue nobilamide D.     

Solid-phase staudinger ligation from a novel core-shell-type resin: a tool for facile condensation of small peptide fragments

[reaction: see text] Solid-phase Staudinger ligation of small peptides was performed on a novel core-shell-type resin. Solid-phase Staudinger ligation was mediated by synthetic solid-supported phosphinothiol, which was readily prepared by a straightforward synthetic route. This protocol afforded final peptide products in excellent yields and purities and thus could provide the opportunity to facilitate a simple manipulation for condensation of peptide fragments. In particular, the resulting resin could be recycled in a successful manner.