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RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide–thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)‐cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix‐assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S‐alkylated, digested with trypsin and analyzed by liquid chromatography–MS/MS using collision‐induced dissociation (CID) and higher‐energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O‐labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas‐phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self‐hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody–drug conjugates. [ABSTRACT FROM AUTHOR]

Cysteinyl RGD-peptidyl cysteinyl prolyl esters, which have different configurations at the cysteine and proline residues, were synthesized by the solid-phase method and cyclized by the native chemical ligation reaction. Cyclization efficiently proceeded to give cyclic peptides, regardless of the difference in the configuration. The peptides were further derivatized to the corresponding desulfurized or methylated cyclic peptides at the Cys residues. The inhibition activity to avp6 integrin binding was then analyzed by ELISA. The results showed that the activity varied depending on the difference in the configuration and modification of the cysteinyl prolyl ester (CPC) moiety, demonstrating the usefulness of this method in the search for a good inhibitor of the protein-protein interaction. [ABSTRACT FROM AUTHOR]

Rationale: The thiosuccinimide linker is widely used in the synthesis of bioconjugates. However, it is susceptible to hydrolysis and is transformed into its hydrolyzed and/or the isobaric thiazine forms, the latter of which is a fairly common product in a conjugate that contains a cysteinyl peptide. Matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) and matrix‐assisted laser desorption/ionization‐tandem mass spectrometry (MALDI‐MS/MS) are useful for differentiating these isobaric species. Methods: Four cross‐linked peptides with thiosuccinimide linkers were synthesized. Analogs with linkers that were transformed into thiazine and/or the hydrolyzed thiosuccinimide linkers were then synthesized by incubating the samples at neutral or basic pH. All the cross‐linked peptides were purified using RP‐HPLC (reversed‐phase high‐performance liquid chromatography) and differentiated using MALDI‐MS, MALDI‐MS/MS, and ultraviolet photodissociation. Results: A cysteinyl peptide‐containing conjugate, the thiosuccinimide form, was largely transformed into the hydrolyzed or thiazine forms after incubation at neutral or basic pH. MALDI‐MS allowed the three forms to be differentiated: the thiosuccinimide and its hydrolysis product yielded two constituent peptides after reductive cleavage between the Cys and succinimide moieties; no fragment ions were produced from the thiazine form. In addition, MALDI‐MS/MS of the thiosuccinimide form yielded two pairs of complementary fragment ions via 1,4‐elimination: Cys‐SH and maleimide, and dehydro‐alanine and thiosuccinimide, which are different from those produced via reductive cleavage in MALDI‐MS. The thiazine form yielded fragment ions resulting from the cleavage of the newly formed amide bond in the linker that resulted from thiazine formation. Conclusions: The thiosuccinimide (but not thiazine) form of the cross‐linked peptide yielded individual constituent peptides using MALDI‐MS and MALDI‐MS/MS, showing specific 1,4‐elimination for the thiosuccinimide form and cleavage at the newly formed peptide bond via transcyclization for the thiazine form. [ABSTRACT FROM AUTHOR]

Protein ubiquitination is involved in nearly all biological processes in Eukaryotes. To gain precise insights into the function of ubiquitination in these processes, researchers frequently employ ubiquitinated protein probes with well-defined structures. While chemical protein synthesis has afforded a variety of ubiquitinated protein probes, there remains a demand for efficient synthesis methods for complex probes, such as ubiquitinated glycoproteins and ubiquitinated cysteine-containing proteins. In this study, we introduce a new method to obtain ubiquitinated proteins through isopeptide bond formation mediated by δ-selenolysine residues. We synthesized δ-selenolysine derivatives in both L-and D-forms starting from DL-hydroxy-DL-lysine, accomplished by substituting the δ-mesylate with KSeCN and by enzymatic optical resolution with L-and D-aminoacylase. We synthesized ubiquitin (46-76)-a-hydrazide with a δ- seleno-L-lysine residue at position 48, as well as ubiquitin (46-76)-a-thioester, using solid-phase peptide synthesis. Subsequently, the δ-selenolysine-mediated ligation of these peptides, followed by one-pot deselenization, provided the desired isopeptide-linked ubiquitin peptide. The new δ-selenolysine-mediated isopeptide bond formation offers an alternative method to obtain complex ubiquitin-and ubiquitin-like probes with multiple post-translational modifications. These probes hold promise for advancing our understanding of ubiquitin biology. [ABSTRACT FROM AUTHOR]

Human seleno‐epidermal growth factor (seleno‐EGF), a 53‐residue peptide where all six cysteine residues of the parent human EGF sequence were replaced by selenocysteines, was synthesized and the oxidative folding of a polypeptide containing three diselenide bonds was compared to that of the parent cysteine peptide. The crude high performance liquid chromatography (HPLC) profiles clearly showed that both the native EGF and its selenocysteine‐analogue fold smoothly, yielding a single sharp peak, proving that even in the case of three disulfide‐bonded polypeptides the disulfide‐to‐diselenide bond substitution is highly isomorphous, as confirmed by conformational circular dichroism measurements and particularly by the biological assays. [ABSTRACT FROM AUTHOR]

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C‐terminus (CPE peptide) was transformed into a diketopiperazine (DKP) thioester via an intramolecular N–S acyl shift reaction and was then used for peptide ligation. The difference in reactivity between the CPE peptide stereoisomers was examined. In reactions of the CPE peptides that contained L‐Cys‐L‐Pro or D‐Cys‐D‐Pro, the desired DKP thioester was formed at the preceding amino acid residue. On the other hand, in reactions of the CPE peptides that contained D‐Cys‐L‐Pro or L‐Cys‐D‐Pro, a thiolactone was formed at the C‐terminal prolyl ester, and the ligation occurred at the C‐terminal Pro residue. Using this reaction, it was possible to efficiently prepare a cyclic peptide. [ABSTRACT FROM AUTHOR]

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2‐sensitive [NiFe]‐hydrogenases. Although the activities of some O2‐tolerant [NiFe]‐hydrogenases are unaffected by CO, the partially O2‐tolerant [NiFe]‐hydrogenase from Citrobacter sp. S‐77 (S77‐HYB) is inhibited by CO. In this work, the CO‐bound state of S77‐HYB was characterized by activity assays, spectroscopic techniques and X‐ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier‐transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni–Fe active site of S77‐HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]‐hydrogenases. [ABSTRACT FROM AUTHOR]

Abstract Background Emmprin, a glycoprotein containing two Ig domains, is enriched on tumor cell surfaces and stimulates matrix metalloproteinase (MMP) production by adjacent stromal cells. Its first Ig domain (ECI) contains the biologically active site. The dependence of emmprin activity on N-glycosylation is controversial. We investigated whether synthetic ECI with the shortest sugar is functionally active. Methods The whole ECI peptides carrying sugar chains, a chitobiose unit or N-linked core pentasaccharide, were synthesized by the thioester method and added to fibroblasts to examine whether they stimulate MMP-2 production. Results ECI carrying a chitobiose unit, ECI-(GlcNAc) 2, but not ECI without a chitobiose unit or the chitobiose unit alone, dose-dependently stimulated MMP-2 production by fibroblasts. ECI with longer chitobiose units, ECI-[(Man)3(GlcNAc)2], also stimulated MMP-2 production, but the extent of its stimulation was lower than that of ECI-(GlcNAc)2. Conclusions Our results indicate that ECI can mimic emmprin activity when substituted with chitobiose, the disaccharide with which N-glycosylation starts.

Caveolin‐1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C‐terminal region, and is inserted into the lipid bilayer to form a V‐shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid‐phase method. To improve the solubility of the C‐terminal region, O‐acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis. [ABSTRACT FROM AUTHOR]

Ferredoxin (Fd) is an electron carrier protein containing a [2Fe–2S] cluster. In this paper, we synthesized Se–Fd, in which four Cys residues coordinated to the cluster are substituted to selenocysteine. After the one-pot segment coupling by the thioester method, followed by deprotection and cluster loading, the desired Se–Fd was successfully obtained. [ABSTRACT FROM AUTHOR]

The expressed protein ligation method was used to synthesize androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, a heterodimeric glycopeptide, with a homogeneous N-linked glycan. The thermodynamically unstable form of the native conformation of AGH indicated that the correct disulfide linkage is critical for conferring AGH activity.