Staudinger Reaction Research Articles

GEN-Click: Genetically Encodable Click Reactions for Spatially Restricted Metabolite Labeling.

Chemical reactions for the in situ modification of biomolecules within living cells are under development. Among these reactions, bio-orthogonal reactions such as click chemistry using copper(I) and Staudinger ligation are widely used for specific biomolecule tracking in live systems. However, currently available live cell copper(I)-catalyzed azide/alkyne cycloaddition reactions are not designed in a spatially resolved manner. Therefore, we developed the "GEN-Click" system, which can target the copper(I)-catalyzed azide/alkyne cycloaddition reaction catalysts proximal to the protein of interest and can be genetically expressed in a live cell. The genetically controlled, spatially restricted, metal-catalyzed biorthogonal reaction can be used for proximity biotin labeling of various azido-bearing biomolecules (e.g., protein, phospholipid, oligosaccharides) in living cell systems. Using GEN-Click, we successfully detected local metabolite-transferring events at cell-cell contact sites.

Asymmetric Total Synthesis of Cytotrienin A: Late-stage Installation of C11 Side Chain onto the Macrolactam Scaffold.

Cytotrienin A, an ansamycin-class antibiotic, exhibits potent apoptosis-inducing activity and has attracted much attention as a lead compound for anticancer drugs. Herein, we report a new asymmetric synthetic route to cytotrienin A, employing an unexplored approach involving the late-stage installation of a C11 side chain onto the macrolactam core. In this strategy, we utilized the redox properties of hydroquinone and installed a side chain on the sterically hindered C11 hydroxyl group via the traceless Staudinger reaction. This study also demonstrated that the boron-Wittig/iterative Suzuki-Miyaura cross-coupling sequence was effective for the concise and selective construction of the (E,E,E)-conjugated triene moiety. The developed route opens new opportunities for the structure-activity relationship studies of the side chains of these ansamycin antibiotics and the preparation of other synthetic analogs and chemical probes for further biological studies.

Asymmetric Total Synthesis of Cytotrienin A: Late‐Stage Installation of C11 Side Chain onto the Macrolactam Scaffold

AbstractCytotrienin A, an ansamycin‐class antibiotic, exhibits potent apoptosis‐inducing activity and has attracted much attention as a lead compound for anticancer drugs. Herein, we report a new asymmetric synthetic route to cytotrienin A, employing an unexplored approach involving the late‐stage installation of a C11 side chain onto the macrolactam core. In this strategy, we utilized the redox properties of hydroquinone and installed a side chain on the sterically hindered C11 hydroxy group by the traceless Staudinger reaction. This study also demonstrated that the boron‐Wittig/iterative Suzuki–Miyaura cross‐coupling sequence was effective for the concise and selective construction of the (E,E,E)‐conjugated triene moiety. The developed route opens new opportunities for the structure–activity relationship studies of the side chains of these ansamycin antibiotics and the preparation of other synthetic analogs and chemical probes for further biological studies.

Stereoselective ring‐opening polymerization ofrac‐lactide catalyzed by phenyl diphosphazene/urea binary catalytic system

AbstractPolylactide (PLA) is a fully bio‐derived polyester with great biodegradability, biocompatibility, and mechanical properties. Synthesis of stereoregular PLA by highly stereoselective organocatalyzed ring opening polymerization (ROP) of racemic lactide (rac‐LA) at room temperature is challenging despite some important developments in the past few years. In this contribution, two bulky phenyl diphosphazene bases,p‐PDPBandm‐PDPB,were conveniently synthesized by the Staudinger reaction. In combination with different hydrogen‐bond donors such as ureas and squaramides, they could mediate the stereocontrolled ROP ofrac‐LA in THF at room temperature. Semicrystalline PLAs with narrow dispersity and high tacticity (Pmup to 0.84) were successfully synthesized in a well‐controlled manner using phenyl diphosphazene based/urea binary catalytic system. Structural analysis verified the linear structure and high end‐group fidelity of the resulting polymers. Moreover, the minimal transesterification of the polymer backbone proved by the MALDI‐TOF MS analysis indicated the good controllability of the phenyl diphosphazene based binary catalytic system.

Designed synthetic pathway of stereoselective novel monocyclic cis‐bis‐β‐lactams including a 1,3,4‐thiadiazole‐2,5‐dithiol constituent: Antimicrobial activity, molecular docking

AbstractA simple synthetic method has been developed for novel monocyclic bis‐β‐lactams including a 1,3,4‐thiadiazole‐2,5‐thiol constituent via [2 + 2] cycloaddition reactions, that is, Staudinger reaction (ketene‐imine) of acyl chloride and numerous aromatic imines. Easily available starting materials, an easy workup, a simple purification process, no column chromatography needed, and a great yield are specific features of this work. Spectroscopic methods revealed the confirmation of synthesized products. Products were easily purified through a simple crystallization technique. The stereochemistry of bis‐β‐lactam derivatives at C‐3 and C‐4 stereocenters was validated by 1H NMR spectra. All synthesized products exhibited good antimicrobial activities against three Gram‐positive bacteria Staphylococcus aureus, Bacillus anthracis, and Clostridium perfringens, and two Gram‐negative bacteria Escherichia coli, Brucella abortus. The molecular docking studies were carried out with the target receptor from E. coli, S. aureus, B. anthracis, B. abortus, and C. perfringens of PDB ID: 3T88, 4Q7G, 3Q2O, 4WJM, and 6PV4, respectively, to obtain a moderate binding interaction.

The Chemistry of P=N-P=X (X=S, O, NR) Linkages for the Synthesis of Dendritic Structures.

The Staudinger reaction between a phosphine and an azide, applied to phosphorus azides, has been used for the synthesis of a large variety of dendritic structures, incorporating P=N-P=X moieties (X = mainly S, but also O and N-R). Conjugation of the P=N bond with the P=X bond greatly stabilizes the P=N bond. Highly branched structures such as dendrons, dendrimers, Janus dendrimers, layered dendrimers, surface-block dendrimers, and diverse other dendritic structures incorporating such linkage have been elaborated. Accelerated methods of synthesis of dendrimers are also based on the Staudinger reaction. A versatile reactivity was observed exclusively on the sulfur atom of P=N-P=S linkages, such as alkylation or complexation. Alkylation on S induces a weakening of the strength of the P=S bond, which can be easily cleaved to generate phosphines able to react in Staudinger reactions inside the structure of dendrimers, thus affording highly sophisticated dendritic structures.

Enantioselective Organocatalytic Addition of Nitromethane to Trifluoromethyl Aryl Ketimines Promoted by Electron‐Rich Bifunctional Iminophosphoranes

AbstractThiourea‐based iminophosphorane (BIMP) organocatalysts featuring SPhos‐ or BIDIME phosphine units have been developed and successfully applied in the asymmetric addition of nitromethane to N‐Boc‐protected trifluoromethyl aryl ketimines. α‐Trifluoromethyl β‐nitroamines were obtained in 40–82% isolated yields and 80–95% enantioselectivities. A careful evaluation of the catalytic activity of BIMPs indicates that the catalysts derived from the combination via Staudinger reaction of a chiral 1,2‐amino alcohol‐derived thiourea‐organoazide with electron‐rich phosphines, promote the aza‐Henry reaction on fluorinated ketimines with the highest enantioselectivity, leading to the amine featuring a tetrasubstituted stereocenter in up to 95% ee. The reaction was performed also on gram scale, without loss of enantioselectivity.magnified image

Azide-Free Synthesis of N-Alkyliminophosphoranes from Phosphines and Hydroxylamine Derivatives.

A broadly applicable and efficient method for the synthesis of N-alkyliminophosphoranes from phosphines that does not use potentially hazardous alkyl azides is reported. Under iron catalysis, a hydroxylamine-derived triflic acid salt oxidizes phosphines to a wide range of iminophosphorane triflic acid salts. Diphosphines afford phosphine-iminophosphoranes that can serve as ligands in transition metal complexes. The developed method can be employed in the synthesis of mixed diiminophosphoranes and in a traceless Staudinger ligation.

New Zwitterionic Oligonucleotides: Preparation and Complementary Binding

New zwitter-ionic oligonucleotide derivatives containing 1,2,3,4-tetrahydroisoquinoline-7-sulfonyl phosphoramidate group are described. Automated synthesis of these compounds was carried out according to the β-cyanoethyl phosphoramidite scheme using the Staudinger reaction between 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonyl azide and phosphite triester within an oligonucleotide grafted to a polymer support. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonyl phosphoramidate group (THIQ) proved to be stable under the conditions of standard oligonucleotide synthesis, including the removal of protective groups and cleavage of the oligonucleotide from the polymer support by treatment with a mixture of concentrated aqueous solutions of ammonia and methylamine (1 : 1) at 55oC. Oligonucleotides modified by one to five THIQ groups in various positions were obtained. The zwitter-ionic character of the obtained derivatives was reflected in their different mobility under conditions of denaturing PAGE. The thermal stability of the duplexes of oligodeoxynucleotides containing THIQ groups with complementary DNA and RNA differed only slightly from that of natural DNA:DNA and DNA:RNA duplexes. The results obtained suggest the possible use of oligonucleotides modified with zwitterionic THIQ groups as antisense therapeutic agents.

"Click-to-Clear": A Strategy to Minimize Radioactivity from the Blood Pool Utilizing Staudinger Ligation.

The availability of several bioorthogonal reactions that can proceed selectively and efficiently under physiologically relevant conditions has garnered the interest of biochemists and organic chemists alike. Bioorthogonal cleavage reactions represent the latest innovation in click chemistry. Here, we employed the Staudinger ligation reaction to release radioactivity from immunoconjugates, improving target-to-background ratios. In this proof-of-concept study, model systems, including the anti-HER2 antibody trastuzumab, radioisotope I-131, and a newly synthesized bifunctional phosphine, were used. Staudinger ligation occurred when biocompatible N-glycosyl azides reacted with this radiolabeled immunoconjugate, leading to cleavage of the radioactive label from the molecule. We demonstrated this click cleavage in vitro and in vivo. Biodistribution studies in tumor models showed that radioactivity was eliminated from the bloodstream, thereby improving tumor-to-blood ratios. SPECT imaging revealed that tumors could be visualized with enhanced clarity. Our simple approach represents a novel application of bioorthogonal click chemistry in the development of antibody-based theranostics.

Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs.

Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one-step introduction of diacylation linkers and azide groups on the 2'-OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2-(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand-induced gene editing in living cells by a controllable CRISPR/Cas9 system.

New Zwitter-Ionic Oligonucleotides: Preparation and Complementary Binding

New zwitter-ionic oligonucleotide derivatives containing 1,2,3,4-tetrahydroisoquinoline-7-sulfonyl phosphoramidate group are described. Automated synthesis of these compounds was carried out according to the β-cyanoethyl phosphoramidite scheme via the Staudinger reaction between 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonyl azide and phosphite triester within oligonucleotide grafted to polymer support. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonyl phosphoramidate group (THIQ) was stable under the conditions of standard oligonucleotide synthesis, including the removal of protective groups and cleavage of the oligonucleotide from the polymer support by treatment with a mixture of concentrated aqueous solutions of ammonia and methylamine (1 : 1) at 55°C. Oligonucleotides modified by one to five THIQ groups in various positions were obtained. The zwitter-ionic character of the obtained derivatives was reflected in their varying mobility under conditions of denaturing PAGE. The thermal stability of the duplexes of oligodeoxynucleotides containing THIQ groups with complementary DNA and RNA only slightly differed from that of natural DNA:DNA and DNA:RNA duplexes. The results reported suggest that oligonucleotides modified with zwitter-ionic THIQ groups as antisense therapeutic agents.

Celebrating the Contributions of Carolyn Bertozzi to Bioorthogonal Chemistry and its Application to Glycoscience

Celebrating the Contributions of Carolyn Bertozzi to Bioorthogonal Chemistry and its Application to Glycoscience

Improved syntheses of doubly naphthalene-bridged diphosphine and its diiminodiphosphorane derivatives linking two Cu(I) centers

An improved method for preparing bis(1,8-naphthalenediyl)diphosphine, 1, was developed. A phosphino-naphthalene unit of a precursor, iPr2NP-peri-bridged naphthalene, was dimerized in an acetic acid medium, while the precursor in a previously reported method was heated in toluene. The reaction time was reduced from 48 h to 1 h, and an increase to the gram scale was achieved. The diphosphine, 1, comprised a unique PP bond, which was clamped by two 1,8-naphthalene units and was used to synthesize the diiminodiphosphorane derivatives. Thereafter, the two imino-donating groups comprising Ph, p-nBu–C6H4, Mes, and 2,6-iPr2–C6H3 were introduced into two phosphorus centers via the Staudinger reaction using the corresponding R−N3 group (R = Ph, p-nBu–C6H4, Mes, and 2,6-iPr2–C6H3). The result revealed that they did not function as a chelating pair to a single metal center; rather, they acted as a bridging ligand that linked the two metal centers, as demonstrated by the formation of dicopper complexes, except for the bulky 2,6-iPr2–C6H3 case. The two rigid naphthalene groups forced the PPC(naphthalene) angles of five-membered rings to be ca. 90°, thereby increasing the P−PN bond angles (ca. 130°). These increased P−PN bond angles forced the two imino nitrogen to trigger each other. This accounted for the bridging and nonchelating functionalities of the synthesized diiminodiphosphoranes. Both iminophosphorane groups of the synthesized diiminodiphosphorane were directly linked; they could facilitate the binding of two neighboring metal units.

Towards a templated reaction that translates RNA in cells into a proaptotic peptide-PNA conjugate.

Nucleic acid-templated chemistry opens the intriguing prospect of triggering the synthesis of drugs only in diseased cells. Herein, we explore the feasibility of using RNA-templated chemical reactions for the activation of a known Smac peptidomimetic compound (SMC), which has proapoptotic activity. Two peptide nucleic acid (PNA) conjugates were used to enable conditional activation of a masked SMC by reduction of an azide either by Staudinger reduction or catalytic photoreduction using a ruthenium complex. The latter provided ~135 nM SMC-PNA on as little as 10nM (0.01 eq.) template. For the evaluation of the templated azido-SMC reduction system in cellulo, a stable HEK 293 cell line was generated, which overexpressed a truncated, non-functional form of the XIAP mRNA target. We furthermore describe the development of electroporation protocols that enable a robust delivery of PNA conjugates into HEK 293 cells. The action of the reactive PNA conjugates was evaluated by viability and flow cytometric apoptosis assays. In addition, electroporated probes were re-isolated and analyzed by ultra-high performance liquid chromatography (UPLC). Unfortunately, the ruthenium-PNA conjugate proved phototoxic, and treatment of cells with PNA-linked reducing agent and the azido-masked SMC conjugate did not result in a greater viability loss than treatment with scrambled sequence controls. Intracellular product formation was not detectable. A control experiment in total cellular RNA isolate indicated that the templated reaction can in principle proceed in a complex system. The results of this first-of-its-kind study reveal the numerous hurdles that must be overcome if RNA molecules are to trigger the synthesis of pro-apoptotic drugs inside cells.

Traceless Staudinger Ligation to Access Stable Aminoacyl- or Peptidyl-Dinucleotide.

Aminoacyl- and peptidyl-tRNA are specific biomolecules involved in many biological processes, from ribosomal protein synthesis to the synthesis of peptidoglycan precursors. Here, we report a post-synthetic approach based on traceless Staudinger ligation for the synthesis of a stable amide bond to access aminoacyl- or peptidyl-di-nucleotide. A series of amino-acid and peptide ester phenyl phosphines were synthetized, and their reactivity was studied on a 2'-N3 di-nucleotide. The corresponding 2'-amide di-nucleotides were obtained and characterized by LC-HRMS, and mechanistic interpretations of the influence of the amino acid phenyl ester phosphine were proposed. We also demonstrated that enzymatic 5'-OH phosphorylation is compatible with the acylated di-nucleotide, allowing the possibility to access stable aminoacylated-tRNA.

Bioorthogonal activation of prodrugs, for the potential treatment of breast cancer, using the Staudinger reaction.

Selective prodrug activation at a tumor site is crucial to maximise the efficiency of chemotherapy approaches and minimise side effects due to off-site activation. In this paper, a new prodrug activation strategy is reported based on the bioorthogonal Staudinger reaction. The feasibility of this prodrug activation strategy was initially demonstrated using 9-azido sialic acid 4 as a trigger and two novel triphenylphosphine-modified N-mustard-PRO 10 and doxorubicin-PRO 12 prodrugs in an HPLC-monitored release study. Then, the azide reporter group was introduced on cancer cells' surfaces through metabolic glycoengineering of sialic acid-rich surface glycans using azide-modified monosaccharides (9-azido sialic acid 4, tetra-O-acetylated-9-azido sialic acid 5 and tetra-O-acetyl azidomannosamine). Next, the N-mustard-PRO 10 and doxorubicin-PRO 12 prodrugs were employed in vitro with the bioengineered cells, and activation of the prodrugs, which allowed selective release of the cytotoxic moiety at the tumour cell, was assessed. Release of the parent drugs from the prodrugs was shown to be dependent on the level of metabolic labelling, where tetra-O-acetyl azidomannosamine allowed the highest level of azide reporter generation in tumor cells and led to full recovery of the parent cytotoxic drug's potency. The selectivity of azide expression on breast cancer MCF-7 cells versus normal fibroblast L929 cells was also probed, with the 9-azido sialic acid and tetra-O-acetylated-9-azido sialic acid showing ∼17-fold higher azide expression on the former. Taken together, these data demonstrate the feasibility of the Staudinger reaction for selective activation of prodrugs targeted to the MCF-7 breast cancer cells.

Electronic origins of the stereochemistry in β-lactam formed through the Staudinger reaction catalyzed by a nucleophile.

This paper evaluates the electronic effects of molecular substituents on the stereoselectivity of the umpolung Staudinger catalytic reaction. This is especially important because experimental studies on constructing the β-lactam ring, a core structure of most antibiotics, through catalyzed Staudinger reactions have been massively progressing over the last century. Yet, there is a necessity for an in-depth understanding of the reaction mechanisms to help chemists, working on the well-established discoveries, improve these to optimize the stereoselectivity and yield of synthetic methods. Access to practical and effective advancements in forming optically pure β-lactam is paramount in the field of medical chemistry. This paper specifically investigates how changing the N-protecting group in the imine fragment can switch the stereoselectivity of the PPY-catalyzed Staudinger reaction. To do so, we employed the density functional theory (DFT) for geometry optimization and electronic analysis at the B3LYP/6-31G(d) level of theory to examine and compare the role of N-tosyl (N-Ts) and N-triflyl (N-Tf) imine on the mechanism pathways, i.e., imine-first or ketene-first, and stereochemistry of the reaction, i.e., cis or trans β-lactam. Our results show that the reaction mechanism pathway cannot be simply switched from ketene-first to imine-first by changing the substituent on the imine nitrogen atom, which is contrary to the reported experimental results, and both imines go through the ketene-first mechanism with different stereochemistries, which is cis selective for imine-Ts and trans selective for imine-Tf. Based on electronic analyses, the reversal in diastereoselectivity in the N-triflyl imine system could be attributed to the charge transfers and electron-density distribution over the transition states. Therefore, the cis/trans selectivity of the PPY-catalyzed Staudinger reaction could be effectively controlled by the electronic characteristics of the molecular substituents in the reactants. A N-protecting group in imine with a more electron-withdrawing nature seems to accelerate the stereo-determining step, ring closure, and increase the stabilization charge transfers in the transition state, leading to a preference for trans β-lactam formation. It seems that using a N-substituent with a higher electron-withdrawing nature can initially activate the imine by the nucleophilic catalyst in competition with ketene, i.e., imine-first versus ketene-first. These results can provide an insight into select proper substituents for the fragments to synthesis β-lactam with a desired stereochemistry. Also, a comprehensive comparison was performed between calculations with and without dispersion.

(Aminoalkyl)diphenylphosphine sulfides: synthesis and application as building blocks in the design of multidentate ligands for cytotoxic Pd(II) complexes.

Amino-functionalized phosphoryl compounds are among the most useful molecular scaffolds in medicinal chemistry, while the potential of their thiophosphorylated analogs, especially those having an alkylamino moiety, is still uncovered. This is mainly due to the lack of convenient synthetic routes to these organophosphorus derivatives. To address this issue, we have suggested the facile approaches to α-(aminomethyl)- and substituted/unsubstituted α-(aminobenzyl)diphenylphosphine sulfides based on either the sequential transformations of (hydroxymethyl)diphenylphosphine sulfide, with the Staudinger reaction of an azide derivative as the key stage, or the addition of Ph2P(S)H to hydrobenzamides followed by the acid hydrolysis. The compounds obtained were reacted with picolinyl chloride to yield functionalized amides which readily underwent direct cyclopalladation, resulting in new representatives of non-classical N-metalated Pd(II) pincer complexes. The latter exhibit promising cytotoxic activity against several human cancer cell lines and apoptosis inducing ability along with the remarkable cytotoxic effects on doxorubicin-resistant cell sublines.

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties.

In this work, we present new oligonucleotide derivatives containing inter-nucleotide N-benzimidazole, N-benzoxazole, N-benzothiazole, and 1,3-dimethyl-N-benzimidazole (benzoazoles) phosphoramide groups. These modifications were introduced via the Staudinger reaction with appropriate azides during standard automated solid-phase oligonucleotide synthesis. The principal structural difference between the new azido modifiers and those already known is that they are bulk heterocyclic structures, similar to purine nucleoside bases. Modified oligonucleotides with one and two modifications at different positions and multiple modified heteronucleotide sequences were obtained with high yields. The possibility of multiple modifications in the process of automatic DNA synthesis is fundamental and critical for further application of our oligonucleotide derivatives. Initial studies on the properties of new oligonucleotides were carried out. The stability of the oligodeoxyribonucleotide duplex containing phosphoramide groups of N-benzoazoles with complementary DNA or RNA is slightly lower than that of native complexes.