Synthesis of biodegradable polyesteramides with pendant functional groups

Morpholine-2,5-dione derivatives having substituents with benzyl-protected carboxylic acid, benzyloxycarbonyl-protected amine and p-methoxy-protected thiol groups, respectively, were prepared in 29-58% yield by cyclization of the corresponding N-[(2RS)-bromopropionyl]-L-amino acids. Polyesteramides with protected pendant functional groups were obtained by ring-opening copolymerization of either ε-caprolactone or DL-lactide with morpholine-2,5-dione derivatives having protected functional substituents. The copolymerizations were carried out in the bulk at 130°C using stannous octoate as an initiator and using low mole fractions (0,05, 0,10 and 0,20) of morpholine-2,5-dione derivatives in the feed. The molecular weight of the resulting copolymers ranged from 1,4 to 8,3 · 104. The ring-opening homopolymerization of morpho-line-2,5-dione derivatives with protected functional substituents was not successful. Polyesteramides with either pendant carboxylic acid groups or pendant amine groups were prepared by catalytic hydrogenation of the corresponding protected copolymers. Treatment of copolymers having pendant p-methoxybenzyl-protected thiol groups with trifluoromethanesulfonic acid resulted not only in the removal of the p-methoxybenzyl group but also in severe degradation of the copolymers, due to acidolysis of main-chain ester bonds

Enzymatic Cross-Linking of Dynamic Thiol-Norbornene Click Hydrogels

Enzyme-mediated in situ forming hydrogels are attractive for many biomedical applications because gelation afforded by enzymatic reactions can be readily controlled not only by tuning macromer compositions, but also by adjusting enzyme kinetics. For example, horseradish peroxidase (HRP) has been used extensively for in situ cross-linking of macromers containing hydroxyl-phenol groups. The use of HRP to initiate thiol-allylether polymerization has also been reported, yet no prior study has demonstrated enzymatic initiation of thiol-norbornene gelation. In this study, we discovered that HRP can generate the thiyl radicals needed for initiating thiol-norbornene hydrogelation, which has only been demonstrated previously using photopolymerization. Enzymatic thiol-norbornene gelation not only overcomes light attenuation issue commonly observed in photopolymerized hydrogels, but also preserves modularity of the cross-linking. In particular, we prepared modular hydrogels from two sets of norbornene-modified macromers, 8-arm poly(ethylene glycol)-norbornene (PEG8NB) and gelatin-norbornene (GelNB). Bis-cysteine-containing peptides or PEG-tetra-thiol (PEG4SH) was used as a cross-linker for forming enzymatically and orthogonally polymerized hydrogel. For HRP-initiated PEG-peptide hydrogel cross-linking, gelation efficiency was significantly improved via adding tyrosine residues on the peptide cross-linkers. Interestingly, these additional tyrosine residues did not form permanent dityrosine cross-links following HRP-induced gelation. As a result, they remained available for tyrosinase-mediated secondary cross-linking, which dynamically increased hydrogel stiffness. In addition to material characterizations, we also found that both PEG- and gelatin-based hydrogels exhibited excellent cytocompatibility for dynamic 3D cell culture. The enzymatic thiol-norbornene gelation scheme presented here offers a new cross-linking mechanism for preparing modularly and dynamically cross-linked hydrogels

Versatile thiol-based reactions for micrometer- and nanometer-scale photopatterning of polymers and biomolecules

Thiol-based chemistry provides a mild and versatile tool for surface functionalization. In the present work, mercaptosilane films were patterned by utilizing UV-induced photo-oxidation of the thiol to yield sulfonate groups via contact and interferometric lithography (IL). These photo-generated sulfonic acid groups were used for selective immobilization of amino-functionalized molecules after activation with triphenylphosphine ditriflate (TPPDF). Moreover, protein-resistant poly(oligoethyleneglycolmethacrylate) (POEGMA) brushes were grown from the intact thiol groups by a surface-induced polymerization reaction. Exploiting both reactions it is possible to couple amino-labelled nitrilotriacetic acid (NH2-NTA) to sulfonate-functionalized regions, enabling the site-specific binding of green fluorescent protein (GFP) to regions defined lithographically, while exploiting the protein-resistant character of POEGMA brushes to prevent non-specific protein adsorption to previously masked areas. The outstanding reactivity of thiol groups paves the way towards novel strategies for the fabrication of complex protein nanopatterns beyond thiol–ene chemistry

Inelastic Tunneling Spectroscopy of Gold-Thiol and Gold-Thiolate Interfaces in Molecular Junctions: The Role of Hydrogen

It is widely believed that when a molecule with thiol (S-H) end groups bridges a pair of gold electrodes, the S atoms bond to the gold and the thiol H atoms detach from the molecule. However, little is known regarding the details of this process, its time scale, and whether molecules with and without thiol hydrogen atoms can coexist in molecular junctions. Here we explore theoretically how inelastic tunneling spectroscopy (IETS) can shed light on these issues. We present calculations of the geometries, low bias conductances and IETS of propanedithiol and propanedithiolate molecular junctions with gold electrodes. We show that IETS can distinguish between junctions with molecules having no, one or two thiol hydrogen atoms. We find that in most cases the single-molecule junctions in the IETS experiment of Hihath et al. [Nano Lett. 8, 1673 (2008)] had no thiol H atoms, but that a molecule with a single thiol H atom may have bridged their junction occasionally. We also consider the evolution of the IETS spectrum as a gold STM tip approaches the intact S-H group at the end of a molecule bound at its other end to a second electrode. We predict the frequency of a vibrational mode of the thiol H atom to increase by a factor \sim 2 as the gap between the tip and molecule narrows. Therefore, IETS should be able to track the approach of the tip towards the thiol group of the molecule and detect the detachment of the thiol H atom from the molecule when it occurs.Comment: 11 pages, 7 figures, 1 tabl

Sequential curing of thiol-acetoacetate-acrylate thermosets by latent Michael addition reactions

Thiol-acetoacetate-acrylate ternary dual-curing thermosets were prepared by a sequential process consisting of thiol-Michael addition to acrylates at room temperature followed by Michael addition of acetoacetates to acrylates at moderately elevated temperature. The curing sequence can be controlled with the help of the different acidities of the protons on thiol and acetoacetate groups, the favorable pKa of the base used as catalyst and the self-limiting character of Michael additions. The latency of the curing steps can be regulated by selection of the right catalysts, temperature and curing conditions. The properties of the intermediate and final materials can be tuned by changing the structure of the monomers and the contribution of both Michael addition reactions.Postprint (author's final draft

COA6 facilitates cytochrome c oxidase biogenesis as thiol-reductase for copper metallochaperones in mitochondria.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6 are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulphide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis

Conserved Residues R420 and Q428 in a Cytoplasmic Loop of the Citrate/Malate Transporter CimH of Bacillus subtilis Are Accessible from the External Face of the Membrane

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET, which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.

Location of the free thiol group in bovine [beta]-lactoglobulin A, B, and C : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Under non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) conditions unheated samples of β-lactoglobulin (β-LG) A, B and C all run as a single band, the A variant having a slightly lower mobility than the B and C variants. Following heating of these samples to 110°C, two bands are seen in the monomer region of SDS-PAGE gels run under non-reducing conditions. As heat can induce disulphide exchange, the individual bands forming the doublet may represent species of the same molecular size but having different arrangements of the disulphide bonds. The band formed in the A variant as a result of heating appears to have the same mobility as the unheated B and C variants, while the band formed in the B and C variants as a result of heating appears to have the same mobility as the unheated A variant. Under reducing conditions only a single band was seen in both heated and unheated samples, and the mobility of this band is the same in all three variants. This indicates that the difference in mobility between variants seen in non-reduced samples involves disulphide bonding. If the difference in the mobility of the two bands seen in heated samples is due to a difference in the position of a disulphide bond, and thus the free thiol, then it is possible that the position of the free thiol group in the A variant is different to that of the B and C variants even in unheated samples of this protein. A difference in the distribution of the thiol could explain observed differences in the reactivity of this group. The purpose of this study was to determine whether the observed differences in the mobility of unheated samples of purified bovine β-LG A, B and C under non-reducing SDS-PAGE conditions is due to a difference in the location of the free thiol group within the primary sequence of these variants. This was achieved by reacting the β-LG variants with a radioactively labelled thiol-reactive reagent [1,4-14C] N-ethylmaleimide (14C-NEM), thereby attaching a radioactive marker to the free thiol group. Following labelling of the protein, carried out under conditions that did not induce band splitting, the protein was hydrolysed and the resulting labelled peptide was purified and sequenced. The free thiol group was found to be at residue 121 in β-LG A, B and C. Therefore differences in mobility during non-reduced SDS PAGE of β-LG A, B and C are not due to a difference in the location of the thiol group. However, results indicate that it is possible that, particularly in the B and C variants, there is a tendency for disulphide exchange to occur, even under relatively mild conditions. In establishing the conditions under which band splitting did not occur, the effect of exposure to various conditions on the mobility of purified β-LG variants on native-PAGE and SDS-PAGE was studied. The mobilities of caprine β-LG and porcine β-LG were also studied in order to further characterise the factors within the primary sequence of β-LG that have an influence on band splitting. With bovine β-LG A, B and C, band splitting was found to be both temperature- and pH- dependent. Protein concentration and the ionic strength of the buffer also appeared to effect band splitting. Heating also induced the formation of aggregated species, visible on both native and SDS-PAGE gels. The presence of aggregated material on SDS-PAGE gels indicates that disulphide bonding is involved in the formation of these species. On native-PAGE, material that ran as a smear between the monomer band and dimer band was observed following heating. The protein present in this region may represent monomeric β-LG that has been sufficiently denatured for its mobility under native-PAGE to be retarded. Comparisons of the amount of material present in monomeric forms under native and non-reduced SDS-PAGE suggest that multiple monomeric species of β-LG are present in heated samples. Storage at -18°C in SDS-PAGE sample buffer was also shown to induce changes in the mobility of bovine β-LG A, B and C, and of caprine β-LG, on SDS-PAGE. Storage under these conditions caused the aggregation of β-LG but did not induce band splitting. The banding pattern in the dimer region of the stored samples differed between the variants, with the A variant showing a banding pattern that was markedly different to that of the B and C variants and the caprine protein, which showed similar patterns. The bovine β-LG B and C and caprine β-LG showed similar tendencies to form aggregates, and had a greater tendency to form these high molecular weight species than β-LG A. These differences may be due to a difference in the reactivity of the free thiol group under these conditions, influenced by the substitution at position 118. Purified, unheated caprine β-LG ran as a single band in non-reduced SDS gels, and appeared to have the same mobility as the unheated bovine B and C variants under these conditions. Heating of caprine β-LG also induced the formation of a second band with a similar mobility to that of unheated β-LG A. Caprine β-LG has an Asp at position 64 (as found in bovine β-LG A) and an Ala at position 118 (as found in bovine β-LG B and C). The fact that in non reducing SDS-PAGE caprine β-LG runs as a band with a similar mobility to bovine β-LG B and C and a slightly higher mobility than bovine β-LG A suggests that the substitution at position 118 in the primary protein sequence may somehow be causing the mobility difference. Aggregated material was also seen in caprine β-LG following heating. Unheated samples of porcine β-LG ran as two bands under non-reduced SDS-PAGE. Heating the porcine β-LG did not appear to induce any change in the appearance of the two bands, and there was no evidence of aggregation of this protein. Bovine β-LG A, B and C and caprine β-LG all contain a free cysteine residue in their protein sequence. Porcine β-LG does not contain a free Cysteine and thus the lack of heat-induced changes to the banding pattern in porcine β-LG when compared with the bovine variants and caprine β-LG is possibly due to the absence of this potentially reactive thiol group. The presence of a free thiol group appears to be required both to induce band splitting and for the formation of higher molecular weight aggregates following heating. Band splitting is thus probably a consequence of disulphide interchange reactions, the interchange reaction in β-LG A causing a second band to run in the position of β-LG Band C, and the interchange reaction in β-LG B and C causing a second band to run in the position of β-LG A