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František Tureček - One of the best experts on this subject based on the ideXlab platform.
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efficient covalent bond formation in gas phase peptide peptide ion complexes with the photoleucine stapler
Journal of the American Society for Mass Spectrometry, 2016Co-Authors: Christopher J Shaffer, Prokopis C Andrikopoulos, Jan řezac, Lubomir Rulisek, František TurečekAbstract:Noncovalent complexes of hydrophobic peptides GLLLG and GLLLK with photoleucine (L*) tagged peptides G(L* n L m )K (n = 1,3, m = 2,0) were generated as singly charged ions in the gas phase and probed by photodissociation at 355 nm. Carbene intermediates produced by photodissociative loss of N2 from the L* Diazirine rings underwent insertion into X−H bonds of the target peptide moiety, forming covalent adducts with yields reaching 30%. Gas-phase sequencing of the covalent adducts revealed preferred bond formation at the C-terminal residue of the target peptide. Site-selective carbene insertion was achieved by placing the L* residue in different positions along the photopeptide chain, and the residues in the target peptide undergoing carbene insertion were identified by gas-phase ion sequencing that was aided by specific 13C labeling. Density functional theory calculations indicated that noncovalent binding to GL*L*L*K resulted in substantial changes of the (GLLLK + H)+ ground state conformation. The peptide moieties in [GL*L*LK + GLLLK + H]+ ion complexes were held together by hydrogen bonds, whereas dispersion interactions of the nonpolar groups were only secondary in ground-state 0 K structures. Born-Oppenheimer molecular dynamics for 100 ps trajectories of several different conformers at the 310 K laboratory temperature showed that noncovalent complexes developed multiple, residue-specific contacts between the Diazirine carbons and GLLLK residues. The calculations pointed to the substantial fluidity of the nonpolar side chains in the complexes. Diazirine photochemistry in combination with Born-Oppenheimer molecular dynamics is a promising tool for investigations of peptide–peptide ion interactions in the gas phase.
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electron transfer reduction of the Diazirine ring in gas phase peptide ions on the peculiar loss of nh4o from photoleucine
Journal of the American Society for Mass Spectrometry, 2015Co-Authors: Aleš Marek, Christopher J Shaffer, Robert Pepin, Kristina Slovakova, Kenneth J Laszlo, Matthew F Bush, František TurečekAbstract:Electron transfer to gas-phase peptide ions with Diazirine-containing amino acid residue photoleucine (L*) triggers Diazirine ring reduction followed by cascades of residue-specific radical reactions. Upon electron transfer, substantial fractions of (GL*GGR +2H)+● cation-radicals undergo elimination of [NH4O] radicals and N2H2 molecules from the side chain. The side-chain dissociations are particularly prominent on collisional activation of long-lived (GL*GGR +2H)+● cation-radicals formed by electron transfer dissociation of noncovalent peptide-18-crown-6-ether ion complexes. The ion dissociation products were characterized by multistage tandem mass spectrometry (MSn) and ion mobility measurements. The elimination of [NH4O] was elucidated with the help of 2H, 15 N, and 18O-labeled peptide ions and found to specifically involve the amide oxygen of the N-terminal residue. The structures, energies, and electronic states of the peptide radical species were elucidated by a combination of near-UV photodissociation experiments and electron structure calculations combining ab initio and density functional theory methods. Electron transfer reaching the ground electronic states of charge reduced (GL*GGR +2H)+● cation-radicals was found to reduce the Diazirine ring. In contrast, backbone N − Cα bond dissociations that represent a 60%–75% majority of all dissociations because of electron transfer are predicted to occur from excited electronic states.
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Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen
Journal of The American Society for Mass Spectrometry, 2014Co-Authors: Aleš Marek, František TurečekAbstract:Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile Diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N_2 elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N_2 elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N_2 from the Diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed Diazirine ring cleavage and N_2 loss is proposed as a thermometer dissociation for peptide ion dissociations. Fig. a ᅟ
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electron transfer dissociation of photolabeled peptides backbone cleavages compete with Diazirine ring rearrangements
Journal of the American Society for Mass Spectrometry, 2013Co-Authors: Aleš Marek, Robert Pepin, Kenneth J Laszlo, Matthew F Bush, Bo Peng, František TurečekAbstract:Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK + 2H)2+ and (GL*GGK-NH2 + H)+ ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK + 2H)2+, (GL*GGK-NH2 + 2H)2+,(GL*GGKK + 2H)2+, (GL*GLK + 2H)2+, and (GL*LGK + 2H)2+ showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the Diazirine ring. These side-chain dissociations resulted in eliminations of N2H3, N2H4, [N2H5], and [NH4O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the Diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The Diazirine moiety in GL*GGK-NH2was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.
Terry W J Steele - One of the best experts on this subject based on the ideXlab platform.
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rapid activation of Diazirine biomaterials with the blue light photocatalyst
ACS Applied Materials & Interfaces, 2021Co-Authors: Ivan Djordjevic, Gautama Wicaksono, Ivan Solic, Juhi Singh, Tanvi Sushil Kaku, Sierin Lim, Elwin Wei Jian Ang, Lluis Blancafort, Terry W J SteeleAbstract:Carbene-based macromolecules are an emerging new stimuli-sensitive class of biomaterials that avoid the impediments of free radical polymerization but maintain a rapid liquid-to-biorubber transition. Activation of Diazirine-grafted polycaprolactone polyol (CaproGlu) is limited to UVA wavelengths that have tissue exposure constraints and limited light intensities. For the first time, UVA is circumvented with visible light-emitting diodes at 445 nm (blue) to rapidly activate Diazirine-to-carbene covalent cross-linking. Iridium photocatalysts serve to initiate Diazirine, despite having little to no absorption at 445 nm. CaproGlu's liquid organic matrix dissolves the photocatalyst with no solvents required, creating a light transparent matrix. Considerable differences in cross-linking chemistry are observed in UVA vs visible/photocatalyst formulations. Empirical analysis and theoretical calculations reveal a more efficient conversion of Diazirine directly to carbene with no diazoalkane intermediate detected. Photorheometry results demonstrate a correlation between shear moduli, joules light dose, and the lower limits of photocatalyst concentration required for the liquid-to-biorubber transition. Adhesion strength on ex vivo hydrated tissues exceeds that of cyanoacrylates, with a fixation strength of up to 20 kg·f·cm2. Preliminary toxicity assessment on leachates and materials directly in contact with mammalian fibroblast cells displays no signs of fibroblast cytotoxicity.
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In Vitro Biocompatibility of Diazirine-Grafted Biomaterials
Macromolecular Rapid Communications, 2020Co-Authors: Ivan Djordjevic, Gautama Wicaksono, Ivan Šolić, Terry W J SteeleAbstract:Photoactivation of aryl-Diazirines is an emerging method of rapid, covalent crosslinking under ambient conditions. These attributes make those compounds candidates for grafting onto inert polymer backbones in order to produce stimuli-sensitive biomaterials. However, no risk assessments are available to gauge the toxicity of the leachable components after crosslinking activation. Herein, a stimuli-sensitive biomaterial is formulated from Diazirine-grafted polycaprolactone tetrol. Also known as CaproGlu, this biomaterial undergoes UVA-activated crosslinking, with many positive attributes toward bioadhesive applications; hydrophobic, solvent-free, liquid at room temperature, and transitions into a foam biorubber after mild UVA illumination. As a model Diazirine-grafted biomaterial, hydrolyzed CaproGlu leachates are evaluated for genotoxicity and skin sensitization, namely, Ames test, direct peptide reactivity, and ARE-Nrf2 luciferase assays. The degradation products of Diazirine-mediated crosslinking observe little to no risk of in vitro genotoxicity or skin sensitization.
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non aqueous tissue compliant carbene crosslinking bioadhesives
Materials Science and Engineering: C, 2019Co-Authors: Ankur Harish Shah, Oleksandr Pokholenko, Himanshu Sekhar Nanda, Terry W J SteeleAbstract:Abstract Surgical adhesives are an attractive alternative to traditional mechanical tissue fixation methods of sutures and staples. Ease of application, biocompatibility, enhanced functionality (drug delivery) are known advantages but weak adhesion strength in the wet environment and lack of tissue compliant behavior still pose a challenge. In order to address these issues, non-aqueous bioadhesive based on blends of polyamidoamine (PAMAM) dendrimer, conjugated with 4-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzyl bromide (PAMAM-g-Diazirine) and liquid polyethylene glycol (PEG 400) has been developed. PEG 400 biocompatible solvent reduces the viscosity of PAMAM-g-Diazirine dendrimer without incorporating aqueous solvents or plasticizers, allowing application by syringe or spray. Upon UV activation, Diazirine-generated reactive intermediates lead to intermolecular dendrimer crosslinking. The properties of the crosslinked matrix are tissue compliant, with anisotropic material properties dependent on the PEG 400 wt%, UV dose, pressure and uncured adhesive thickness. The hygroscopic PAMAM-g-Diazirine/PEG 400 blend was hypothesized to absorb water at the tissue interface, leading to high interfacial adhesion, however porous matrices led to cohesive failure. The hydrophilic nature of the polyether backbone (PEG 400) shielded cationic PAMAM dendrimers with cured bioadhesive film displaying significantly less platelet activation than neat PAMAM-g-Diazirine or PLGA thin films.
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nonthrombogenic hydrogel coatings with carbene cross linking bioadhesives
Biomacromolecules, 2018Co-Authors: Himansu Sekhar Nanda, Gautama Wicaksono, Oleksandr Pokholenko, Ivan Djordjevic, Ankur Harish Shah, Terry W J SteeleAbstract:Bioadhesives are a current unmet clinical need for mending of blood contacting soft tissues without inducing thrombosis. Recent development of carbene precursor bioadhesives with the advantages of on-demand curing, tuneable modulus, and wet adhesion have been synthesized by grafting Diazirine onto poly (amidoamine) (PAMAM-G5) dendrimers. Herein, the structure activity relationships of platelet adhesion and activation is evaluated for the first time on the cured PAMAM-g-Diazirine bioadhesives. Three strategies were employed to prevent healthy human donor platelets from adhering and activating on light-cured bioadhesive surfaces: (1) Attenuation of cationic surface charge, (2) antifouling composites by incorporating heparin and alginate in uncured formulation, and (3) heparin wash of cured bioadhesive surface. Topographical imaging of cured and ethanol dehydrated bioadhesive surfaces was used to quantify the adhered and activated platelets with scanning electron microscopy, whose resolution allowed identifi...
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On-Demand Bioadhesive Dendrimers with Reduced Cytotoxicity
MDPI AG, 2018Co-Authors: Feng Gao, Ivan Djordjevic, Oleksandr Pokholenko, Haobo Zhang, Junying Zhang, Terry W J SteeleAbstract:Tissue adhesives based on polyamidoamine (PAMAM) dendrimer, grafted with UV-sensitive arylDiazirine (PAMAM-g-Diazirine) are promising new candidates for light active adhesion on soft tissues. Diazirine carbene precursors form interfacial and intermolecular covalent crosslinks with tissues after UV light activation that requires no premixing or inclusion of free radical initiators. However, primary amines on the PAMAM dendrimer surface present a potential risk due to their cytotoxic and immunological effects. PAMAM-g-Diazirine formulations with cationic pendant amines converted into neutral amide groups were evaluated. In vitro toxicity is reduced by an order of magnitude upon amine capping while retaining bioadhesive properties. The in vivo immunological response to PAMAM-g-Diazirine formulations was found to be optimal in comparison to standard poly(lactic-co-glycolic acid) (PLGA) thin films
Yoshihito Ueno - One of the best experts on this subject based on the ideXlab platform.
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Diazirine containing tag free rna probes for efficient risc loading and photoaffinity labeling of microrna targets
Bioorganic & Medicinal Chemistry Letters, 2018Co-Authors: Kosuke Nakamoto, Yukihiro Akao, Yoshihito UenoAbstract:We designed and synthesized a photo-reactive and tag-free RNA probe for the identification of microRNA (miRNA) targets. To synthesize the RNA probe, we designed a novel nucleoside analog 1-O-[3-ethynyl-5-(3-trifluoromethyl-3H-Diazirine-3-yl)]benzyl-β-d-ribofuranose containing aryl trifluoromethyl Diazirine and ethynyl moieties. The RNA probe containing this analog was observed to form crosslinks with complementary RNA by UV irradiation and was rapidly tagged by Cu-catalyzed azide alkyne cycloaddition (CuAAC). In addition, the tag-free and photo-reactive miRNA-145 probe showed comparable gene silencing activity to that of unmodified miRNA-145. Therefore, miRNA probes containing the nucleoside analog are promising candidates for the identification of target mRNAs of miRNAs.
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Diazirine containing rna photo cross linking probes for capturing microrna targets
Journal of Organic Chemistry, 2014Co-Authors: Kosuke Nakamoto, Yoshihito UenoAbstract:Here, we report the applicability of Diazirine-containing RNA photo-cross-linking probes for the identification of microRNA (miRNA) targets. The RNA cross-linking probes were synthesized by substituting the RNA nucleobases with nucleoside analogues such as 1-O-[3-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-β-d-ribofuranose or 1-O-[4-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl-β-D-ribofuranose that carry aryl trifluoromethyl Diazirine moieties. The probes were successfully cross-linked with synthetic RNAs containing the four natural nucleosides on the opposite site of the nucleoside analogues. Furthermore, it was found that miRNAs containing these analogues were effective in regulating the expression of their target genes. Thus, RNAs containing the nucleoside analogues are promising candidates as photo-cross-linking probes to identify the target mRNAs of miRNAs.
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Diazirine containing rna photocrosslinking probes for the study of sirna protein interactions
Chemical Communications, 2010Co-Authors: Satoru Kuboe, Mayuko Yoda, Aya Ogata, Yukio Kitade, Yukihide Tomari, Yoshihito UenoAbstract:We here report the synthesis and characterization of small interfering RNAs with aryl trifluoromethyl Diazirine moieties in the 3′-overhang regions, which allow sensitive detection of interacting proteins during assembly of the effector ribonucleoprotein complex by irradiation with minimally destructive long-wavelength ultraviolet light.
Aleš Marek - One of the best experts on this subject based on the ideXlab platform.
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electron transfer reduction of the Diazirine ring in gas phase peptide ions on the peculiar loss of nh4o from photoleucine
Journal of the American Society for Mass Spectrometry, 2015Co-Authors: Aleš Marek, Christopher J Shaffer, Robert Pepin, Kristina Slovakova, Kenneth J Laszlo, Matthew F Bush, František TurečekAbstract:Electron transfer to gas-phase peptide ions with Diazirine-containing amino acid residue photoleucine (L*) triggers Diazirine ring reduction followed by cascades of residue-specific radical reactions. Upon electron transfer, substantial fractions of (GL*GGR +2H)+● cation-radicals undergo elimination of [NH4O] radicals and N2H2 molecules from the side chain. The side-chain dissociations are particularly prominent on collisional activation of long-lived (GL*GGR +2H)+● cation-radicals formed by electron transfer dissociation of noncovalent peptide-18-crown-6-ether ion complexes. The ion dissociation products were characterized by multistage tandem mass spectrometry (MSn) and ion mobility measurements. The elimination of [NH4O] was elucidated with the help of 2H, 15 N, and 18O-labeled peptide ions and found to specifically involve the amide oxygen of the N-terminal residue. The structures, energies, and electronic states of the peptide radical species were elucidated by a combination of near-UV photodissociation experiments and electron structure calculations combining ab initio and density functional theory methods. Electron transfer reaching the ground electronic states of charge reduced (GL*GGR +2H)+● cation-radicals was found to reduce the Diazirine ring. In contrast, backbone N − Cα bond dissociations that represent a 60%–75% majority of all dissociations because of electron transfer are predicted to occur from excited electronic states.
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Collision-Induced Dissociation of Diazirine-Labeled Peptide Ions. Evidence for Brønsted-Acid Assisted Elimination of Nitrogen
Journal of The American Society for Mass Spectrometry, 2014Co-Authors: Aleš Marek, František TurečekAbstract:Gas-phase dissociations were investigated for several peptide ions containing the Gly-Leu* N-terminal motif where Leu* was a modified norleucine residue containing the photolabile Diazirine ring. Collisional activation of gas-phase peptide cations resulted in facile N_2 elimination that competed with backbone dissociations. A free lysine ammonium group can act as a Brønsted acid to facilitate N_2 elimination. This dissociation was accompanied by insertion of a lysine proton in the side chain of the photoleucine residue, as established by deuterium labeling and gas-phase sequencing of the products. Electron structure calculations were used to provide structures and energies of reactants, intermediates, and transition states for Gly-Leu*-Gly-Gly-Lys amide ions that were combined with RRKM calculations of unimolecular rate constants. The calculations indicated that Brønsted acid-catalyzed eliminations were kinetically preferred over direct loss of N_2 from the Diazirine ring. Mechanisms are proposed to explain the proton-initiated reactions and discuss the reaction products. The non-catalyzed Diazirine ring cleavage and N_2 loss is proposed as a thermometer dissociation for peptide ion dissociations. Fig. a ᅟ
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electron transfer dissociation of photolabeled peptides backbone cleavages compete with Diazirine ring rearrangements
Journal of the American Society for Mass Spectrometry, 2013Co-Authors: Aleš Marek, Robert Pepin, Kenneth J Laszlo, Matthew F Bush, Bo Peng, František TurečekAbstract:Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK + 2H)2+ and (GL*GGK-NH2 + H)+ ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK + 2H)2+, (GL*GGK-NH2 + 2H)2+,(GL*GGKK + 2H)2+, (GL*GLK + 2H)2+, and (GL*LGK + 2H)2+ showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the Diazirine ring. These side-chain dissociations resulted in eliminations of N2H3, N2H4, [N2H5], and [NH4O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the Diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The Diazirine moiety in GL*GGK-NH2was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.
Yutaka Maeda - One of the best experts on this subject based on the ideXlab platform.
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d2d 23 c84 versus sc2c2 d2d 23 c84 impact of endohedral sc2c2 doping on chemical reactivity in the photolysis of Diazirine
Journal of the American Chemical Society, 2016Co-Authors: Michio Yamada, Yukiko Tanabe, Jingshuang Dang, Satoru Sato, Naomi Mizorogi, Makoto Hachiya, Mitsuaki Suzuki, Tsuneyuki Abe, Hiroki Kurihara, Yutaka MaedaAbstract:We compared the chemical reactivity of D2d(23)-C84 and that of Sc2C2@D2d(23)-C84, both having the same carbon cage geometry, in the photolysis of 2-adamantane-2,3′-[3H]-Diazirine, to clarify metal-atom doping effects on the chemical reactivity of the carbon cage. Experimental and computational studies have revealed that the chemical reactivity of the D2d(23)-C84 carbon cage is altered drastically by endohedral Sc2C2 doping. The reaction of empty D2d(23)-C84 with the Diazirine under photoirradiation yields two adamantylidene (Ad) adducts. NMR spectroscopic studies revealed that the major Ad monoadduct (C84(Ad)-A) has a fulleroid structure and that the minor Ad monoadduct (C84(Ad)-B) has a methanofullerene structure. The latter was also characterized using X-ray crystallography. C84(Ad)-A is stable under photoirradiation, but it interconverted to C84(Ad)-B by heating at 80 °C. In contrast, the reaction of endohedral Sc2C2@D2d(23)-C84 with Diazirine under photoirradiation affords four Ad monoadducts (Sc2C2@C...