Thioester Linkage Research Articles

Chemical Tools for Probing the Ub/Ubl Conjugation Cascades.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubls), essential for many cellular processes, employs multi-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester-activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 μg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 μg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Quantitative Characterization of Chain-Flipping of Acyl Carrier Protein of Escherichia coli Using Chemical Exchange NMR.

The acyl carrier protein of Escherichia coli, termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.

Visible light-induced photo-radical ring-opening copolymerization of thionolactone and acrylates

Radical ring-opening (co)polymerization (RROP) provides an accessible method for synthesizing main chain degradable vinyl polymers. Although the applications of reversible deactivation radical polymerization (RDRP) into RROP have been reported, the use of photo-mediated RDRP methods has received less attention than the thermal process. Here, photo-RROP of the thionolactone dibenzo [c,e]-oxepine-5(7H)-thione (DOT) and methyl acrylate (MA) was studied using photo-iniferter (PI) reversible addition-fragmentation (chain) transfer (RAFT) polymerization at ambient temperature without external radical initiators or photocatalysts. Despite the occurrence of some side reactions including desulfurization-oxygenation and O–S isomerization of DOT promoted by photoexcited thiocarbonyl groups, polymers with thioester linkages in the backbone were prepared, which degraded in the presence of amines and bleach.

Abstract 1736 Unnatural amino acid incorporation during in vitro translation is improved by using a thioester linkage between tRNA and amino acid

Abstract 1736 Unnatural amino acid incorporation during in vitro translation is improved by using a thioester linkage between tRNA and amino acid

Protein S-palmitoylation modification: implications in tumor and tumor immune microenvironment.

Protein S-palmitoylation is a reversible post-translational lipid modification that involves the addition of a 16-carbon palmitoyl group to a protein cysteine residue via a thioester linkage. This modification plays a crucial role in the regulation protein localization, accumulation, secretion, stability, and function. Dysregulation of protein S-palmitoylation can disrupt cellular pathways and contribute to the development of various diseases, particularly cancers. Aberrant S-palmitoylation has been extensively studied and proven to be involved in tumor initiation and growth, metastasis, and apoptosis. In addition, emerging evidence suggests that protein S-palmitoylation may also have a potential role in immune modulation. Therefore, a comprehensive understanding of the regulatory mechanisms of S-palmitoylation in tumor cells and the tumor immune microenvironment is essential to improve our understanding of this process. In this review, we summarize the recent progress of S-palmitoylation in tumors and the tumor immune microenvironment, focusing on the S-palmitoylation modification of various proteins. Furthermore, we propose new ideas for immunotherapeutic strategies through S-palmitoylation intervention.

Insertion of Degradable Thioester Linkages into Styrene and Methacrylate Polymers: Insights into the Reactivity of Thionolactones

Insertion of Degradable Thioester Linkages into Styrene and Methacrylate Polymers: Insights into the Reactivity of Thionolactones

Bis‐(Aminophenyl)ferrocene ligand for palladium free CuI catalyzed Csp2−Csp cross‐coupling reaction

AbstractTwo new ferrocene based molecules S,S′‐bis(4‐aminophenyl)ferrocene‐1,1'‐bis(carbothioate) (I) and S,S′‐bis(3‐aminophenyl)ferrocene‐1,1'‐bis(carbothioate) (II) have been synthesized via a thioester linkage. The prepared compounds have been successfully applied as ligands for the CuI catalyzed Sonogashira coupling reaction. The ligands are highly effective for the Csp2−Csp coupling between alkyne and iodobenzene and bromobenzene. Corresponding 1,2‐disubstituted alkynes could be synthesized from both aryl and alkyl alkynes in high yield by using catalytic amount of CuI in presence of bis(3‐aminophenyl)ferrocene derivative in dioxane at 50 °C.

Interfacial Polymerization of Highly Active Thiolated Cyclodextrin for the Fabrication of a Loose Nanofiltration Membrane with a Chlorine-Resistant Poly(thioester) Linkage.

Cyclodextrins have been frequently used to fabricate membranes via interfacial polymerization (IP). However, the relatively low reactivity of pristine cyclodextrins often induces a lower cross-linking density and unsatisfactory separation performance. In this work, to introduce a highly active thiolated β-cyclodextrin (CD-SH) monomer into IP progress, we constructed a dense and porous poly(thioester) linkage on a commercial membrane surface with loose nanofiltration by IP of CD-SH and trimesoyl trichloride (TMC) as the monomer in an aqueous phase and organic phase separately for the first time. Furthermore, the reactivity of CD-SH has been fully demonstrated by the two-phase IP aiming at unmodified β-CD, a CD-SH/TMC freestanding membrane with a thicker interfacial layer and a smoother surface, and a PAN/CD-SH membrane with a narrow porous distribution. The composite membrane possessed superior separation performance for a high rejection (83.1-99.6%) of different anionic dyes and a low rejection (<20%) of salts, as well as a high-efficiency sieving ability of dye/dye and dye/salt mixtures. The membrane with a poly(thioester) selective layer could steadily operate in a long-term filtration test and exhibit great stability, chloride-resistance performance, and recyclability.

Fabrication of S and O-incorporated graphitic carbon nitride linked poly(1,3,4-thiadiazole-2,5-dithiol) film for selective sensing of Hg2+ ions in water, fish, and crab samples

Screen-printed carbon electrodes (SPCE) were modified with sulfur and oxygen-incorporated graphitic carbon nitride (S, O-GCN) linked poly(1,3,4-thiadiazole-2,5-dithiol) film (PTD) through thioester linkage. The promising interaction between the Hg2+ and modified materials containing sulfur as well as oxygen through strong affinity was studied. This study was utilized for the electrochemical selective sensing of Hg2+ ions by differential pulse anodic stripping voltammetry (DPASV). After, optimizing the different experimental parameters, S, O-GCN@PTD-SPCE was used to improve the electrochemical signal of Hg2+ ions and achieved a concentration range of 0.05–390 nM with a detection limit of 13 pM. The real-world application of the electrode was studied in different water, fish, and crab samples and their obtained results were confirmed with Inductive Coupled Plasma - Optical Emission Spectroscopy (ICP-OES) studies. Additionally, this work established a facile and consistent technique for enhancing the electrochemical sensing of Hg2+ ions and discusses various promising applications in water and food quality analysis.

Cullin-5 RING Ligases ubiquitylate histones.

The ubiquitin proteasome system is the primary means by which proteins are degraded. In this system, ubiquitin is activated by thioester linkage to an E1, transferred to an E2, and finally transferred to a substrate protein by E3. Consecutive rounds of ubiquitin transfer leads to formation of a polyubiquitin chain sufficient for proteasomal recognition and subsequent substrate degradation. We have been studying the Cullin-5 RING Ligases (CRLs), one of the largest groups of E3 ligases. These ligases are composed of several proteins; Cullin-5 scaffold protein, an interchangeable substrate receptor protein, two adaptor proteins, and a ring box protein that recruits an E2. We recently showed that the Cullin-5, when neddylated at K724, can recruit an additional E3 ligase called ARIH2 that with its E2 ubiquitylates substrates bound to the substrate receptor protein, ASB9. Herein, we have experimentally verified a new Cullin-5 substrate, histones, which were previously identified as putative substrates of ASB9-EloB/C-Cullin5-Rbx2 (AECR) through proteomics experiments. I have demonstrated that AECR polyubiquitylates histones and I have confirmed the preferred oligomeric state of the histones through in vitro ubiquitinassays. I have also measured the binding affinity of the histones to various components of the AECR. Finally, I have identified histone binding sites on AECR using hydrogen-deuterium exchange mass spectrometry.

Closed-loop recyclable and biodegradable thioester-based covalent adaptable networks.

Closed-loop recyclable and biodegradable aliphatic covalent adaptable networks (CANs) based on dynamic β-CO thioester linkages that exhibit a service temperature beyond 100 °C are reported. These CANs possessing tensile strength and modulus values of up to 0.3 and 3 MPa, respectively, effectively undergo stress relaxation above 100 °C. The samples exhibit creep resistance ability and low hysteresis loss, and are repeatedly reprocessable at 120 °C. These CANs are depolymerizable to monomers under mild conditions and lose notable mechanical strength (92.4%) and weight (76.5%) within ∼35 days under natural biodegradation conditions.

An orthogonal O,S-CKA monomer for the introduction of thioester and/or thionoester functionalities by radical polymerization

The introduction of thioester linkages into the backbone of free-radical polymerization-produced polymers has received recently renewed interest due to their use in preparing (bio)degradable polymers.

Comparative Metabolomics Reveals a Bifunctional Antibacterial Conjugate from Combined-Culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596.

To investigate the potential for secondary metabolite biosynthesis by Streptomyces species, we employed a coculture method to discover natural bioactive products and identified specific antibacterial activity from a combined-culture of Streptomyces hygroscopicus HOK021 and Tsukamurella pulmonis TP-B0596. Molecular networking using ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) data revealed a specific clade of metabolites in this combined-culture that were not detected in both monocultures. Using the chemical profiles, a previously unidentified conjugate between FabF inhibitor and catechol-type siderophore was successfully identified and named harundomycin A. Harundomycin A was a conjugate between the 2,4-dihydroxy-3-aminobenzoate moiety of platensimycin and N,N'-bis(2,3-dihydroxybenzoyl)-O-seryl-cysteine (bisDHBA-Ser-Cys) with a thioester linkage. Along with the production of harundomycin A, platensimycin, its thiocarboxylic acid form thioplatensimycin, enterobactin, and its degradation product N,N'-bis(2,3-dihydroxybenzoyl)-O-l-seryl-dehydroalanine (bisDHBA-Ser-Dha) were also induced in the combined-culture. Genomic data of S. hygroscopicus HOK021 and T. pulmonis TP-B0596 indicated that strain HOK021 possessed biosynthetic gene clusters for both platensimycin and enterobactin, and thereby revealed that T. pulmonis stimulates HOK021 and acts as an inducer of both of these metabolites. Although the harundomycin A was modified by bulky bisDHBA-Ser-Cys, responsible for the binding to the target molecule FabF, it showed a similar antibacterial spectrum to platensimycin, including against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, suggesting that the pharmacophore is platensimycin. Additionally, Chrome Azurol S assay showed that harundomycin A possesses ferric iron-chelating activity comparable to that of enterobactin. Our study demonstrated the transformation of existing natural products to bifunctional molecules driven by bacterial interaction.

Comparative Investigation of the Hydrolysis of Charge-Shifting Polymers Derived from an Azlactone-Based Polymer.

Poly 2-vinyl-4,4-dimethylazlactone (PVDMA) has received much attention as a "reactive platform" to prepare charge-shifting polycations via post-polymerization modification with tertiary amines that possess primary amine or hydroxyl reactive handles. Upon hydrolysis of the resulting amide or ester linkages, the polymers can undergo a gradual transition in net charge from cationic to anionic. Herein, a systematic investigation of the hydrolysis rate of PVDMA-derived charge-shifting polymers is described. PVDMA is modified with tertiary amines bearing either primary amine, hydroxyl, or thiol reactive handles. The resulting polymers possess tertiary amine side chains connected to the backbone via amide, ester, or thioester linkages. The hydrolysis rates of each PVDMA derivative are monitored at 25and 50°C at pH values of 5.5, 7.5, and 8.5, respectively. While the hydrolysis rate of the amide-functionalized PVDMA is negligible over the period investigated, the hydrolysis rates of the ester- and thioester-functionalized PVDMA increase with increasing temperature and pH. Interestingly, the hydrolysis rate of the thioester-functionalized PVDMA appears to be more rapid than the ester-functionalized PVDMA at all pH values and temperatures investigated. It is believed that these results can be utilized to inform the future preparation of PVDMA-based charge-shifting polymers for biomedical applications.

Biosynthesis of 3-thia-α-amino acids on a carrier peptide

A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid-derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis (tmo). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both invitro and in Escherichia coli. During this process, a cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme catalyzes C-isopropylation. This work illustrates that the biosynthesis of amino acid-derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.

An injectable hemostatic PEG-based hydrogel with on-demand dissolution features for emergency care

Uncontrolled bleeding from internal noncompressible wounds is a major cause of prehospital death in military personnel and civilian populations. An ideal hemostatic sealant for emergency care should quickly control blood loss and be removed without debridement for the follow-up treatment in the operating room, yet the lack of suitable materials to meet both requirements is the bottleneck. Herein, we suggest an injectable and dissolvable hydrogel sealant for hemorrhage management of noncompressible wounds. To this end, a 4-arm poly(ethylene glycol) (PEG) crosslinker modified with thioester linkages and terminated with aldehyde groups is designed and synthesized, and to modulate the gel properties and make it suitable as a hemostatic sealant, a mixed amino component composed of poly(ethylene imine) and adipic dihydrazide is employed to react with the PEG crosslinker to form the adhesive and elastic sealant for the first time. The aldehyde groups provide the adhesion to the tissues, and the amino component affords the procoagulant ability. More importantly, the thioester moieties allow the on-demand dissolution of sealant via a thiol-thioester exchange reaction upon exposure to an exogenous thiolate solution. In the rat femoral artery puncture and liver injury models, the administration of the hydrogel sealant dramatically reduces blood loss, and its subsequent removal does not induce rebleeding. Consequently, this hydrogel sealant with the unique feature of on-demand dissolution can not only efficiently control bleeding in emergent scenarios, but also allow non-traumatic re-exposure of wounds during subsequent surgical care. Statement of significanceSealants, adhesives or hemostatic dressings currently used in emergency situations not only require manual pressure to control bleeding, but also face removal by cutting and mechanical debridement to enable eventual surgical treatment. In this study, we design and develop an injectable and adhesive hydrogel sealant with good procoagulant capacity and on-demand dissolution feature. The application of the hydrogel sealant substantially reduces bleeding from internal noncompressible wounds without the need for direct pressure, and demonstrates for the first time that its controlled removal without debridement does not cause rebleeding. Considering that there are currently no commercial wound sealant systems with the feature of on-demand dissolution, the hydrogel sealant developed by us is expected to address an unmet clinical need.

UV degradation of poly(lactic acid) materials through copolymerisation with a sugar-derived cyclic xanthate.

The copolymerisation of L-Lactide with a cyclic xanthate monomer derived from tri-O-acetyl-D-glucal has been used to incorporate thionocarbonate and thioester linkages into a polyester backbone. The poly(lactide-co-xanthate) copolymers show enhanced UV-degradability compared to PLA, with 40% mass loss within 6 hours of UV exposure (365 nm) for only 3% of sulfur-containing linkages.

Using peptide substrate analogs to characterize a radical intermediate in NosN catalysis.

Nosiheptide is a ribosomally produced and post-translationally modified thiopeptide antibiotic that displays potent antibacterial activity in vitro, especially against Gram-positive pathogens. It comprises a core peptide macrocycle that contains multiple thiazole rings, dehydrated serine and threonine residues, a tri-substituted 3-hydroxypyridine ring and several other modifications. Among these additional modifications includes a 3,4-dimethyl-2-indolic acid (DMIA) moiety that bridges Glu6 and Cys8 of the core peptide to form a second smaller ring system. This side-ring system is formed by the action of NosN, a radical S-adenosylmethionine (SAM) enzyme that falls within the class C radical SAM methylase (RSMT) family. However, the true function of NosN is to transfer a methylene group from the methyl moiety of SAM to C4 of 3-methylindolic acid (MIA) attached in a thioester linkage to Cys8 of the core peptide to set up a highly electrophilic species. This species is then trapped by the side chain of Glu6, resulting in formation of a lactone and the side-ring system. The NosN reaction requires two simultaneously bound molecules of SAM. The first, SAMI, is cleaved to generate a 5'-deoxyadenosyl 5'-radical, which abstracts a hydrogen atom from the methyl group of the second molecule of SAM, SAMII. The resulting SAMII radical is believed to add to C4 of MIA, affording a radical intermediate on the MIA substrate. Herein we describe synthetic approaches that allow detection of this radical by electron paramagnetic resonance (EPR) spectroscopy.