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Monoclonal antibodies emerged as an important therapeutic drug class with remarkable specificity and binding affinity. Nonetheless, these heterotetrameric immunoglobulin proteins come with high manufacturing and therapeutic costs which can take extraordinary proportions, besides other limitations such as their limited in cellulo access imposed by their molecular size (ca. 150 kDa). These drawbacks stimulated the development of downsized functional antibody fragments (ca. 15–50 kDa), together with smaller synthetic peptides (ca. 1–3 kDa) derived from the antibodies’ crucial complementarity-determining regions (CDR). Despite the general lack of success in the literal translation of CDR loops in peptide mimetics, rational structure-based and computational approaches have shown their potential for obtaining functional CDR-based peptide mimetics. In this review, we describe the efforts made in the development of antibody and nanobody paratope-derived peptide mimetics with particular focus on the used design strategies, in addition to highlighting the challenges associated with their development.

The current paper focuses on the functionalization of κ-carrageenan and gelatin as extracellular matrix polysaccharide and protein mimic respectively to produce hydrogel films for adipose tissue engineering. More specifically, κ-carrageenan as well as gelatin have been functionalized with methacrylate and methacrylamide moieties respectively to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The gel fraction, the mass swelling ratio and the mechanical properties of both the one-component hydrogels and the protein/polysaccharide blends have been evaluated. The mechanical and swelling properties of the blends could be tuned by varying the hydrogel composition as well as the crosslinking method applied. The in vitro biocompatibility assays indicated a significantly higher cell viability of adipose tissue-derived mesenchymal stem cells seeded onto the blends as compared to the one-component hydrogels. The results show that the blends of gelatin and κ-carrageenan clearly outperform the one-component hydrogels in terms of adipose tissue engineering potential.

Superabsorbent polymers (SAPs) based on polysaccharides, especially alginate, could offer a valuable solution in a plethora of applications going from drug delivery to self-healing concrete. This has already been proven with both calcium alginate and methacrylated alginate combined with acrylic acid. In this manuscript, the effect of varying the degree of methacrylation and use of a combination of acrylic acid and acrylamide is investigated to explore the effects on the relevant SAP characteristics. The materials showed high gel fractions and a strong swelling capacity up to 630gwater/gSAP, especially for superabsorbent polymers with a low degree of substitution. The SAPs also showed only a limited hydrolysis in aqueous and cement filtrate solutions. ispartof: CARBOHYDRATE POLYMERS vol:168 pages:44-51 ispartof: location:England status: published

International audience; We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro–(S)-Pro) segment as a “templating” unit, frequently used in the design of β-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis–trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF 3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced β-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro–(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans–cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro–(R)-TfmOxa–(S)-Val-OtBu β-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans–trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro–(R)-TfmOxa template fulfilled all crucial β-turn-inducer criteria.

Short alphahelical peptide sequences were stabilized through Glaser-Hay couplings of propargylated l- and/or d-serine residues at positions i and i+7. NMR analysis confirmed a full stabilization of the helical structure when a d-Ser (i), l-Ser (i+7) combination was applied. In case two l-Ser residues were involved in the cyclization, the helical conformation is disrupted outside the peptide's macrocycle.

Cyclic lipodepsipeptides (CLPs) are a group of metabolites produced by Pseudomonas bacteria, involved in various biological functions and displaying a wide range of properties, including antibacterial and antifungal activities. The white line-inducing principle (WLIP) is a member of the viscosin group featuring a Glu2 amino acid. Recently, a total synthesis of pseudodesmin A - the Gln2 counterpart of WLIP - was described, and we here expand this route to Glu2 containing CLPs. We report the first total synthesis of WLIP and at the same time establish that the Gln2 to Glu2 substitution has an adverse impact on the crude purity and overall yield. A comparative study of different CLP analogues reveals the importance of the nature of the Glx2 protecting group in determining these outcomes. Replacement of the conventional tBu protecting group by the larger benzyl group for the Glu residue in our synthesis strategy indeed resulted in an improved conversion. Next to achieving the first WLIP total synthesis, we thus show the importance of a careful choice of protecting groups for the success of this type of solid-phase synthesis approaches towards CLPs.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

In the present work, gelatin type B is modified with highly reactive norbornene functionalities (Gel-NB) following a one-pot synthesis approach to enable subsequent thiol-ene photo-click crosslinking. The modification strategy displays close control over the amount of introduced functionalities. Additionally, Gel-NB exhibits considerably improved processing capabilities in terms of two-photon polymerization when benchmarked to earlier-reported crosslinkable gelatin derivatives (e.g., gelatin-methacrylamide (Gel-MOD) and gelatin-methacrylamide-aminoethylmethacrylate (Gel-MOD-AEMA)). The improvement is especially apparent in terms of minimally required laser power (20 mW vs >= 60 mW (Gel-MOD) vs >= 40 mW (Gel-MOD-AEMA) at 100 mm s(-1) scan speed) and processable concentration range (>= 5 w/v% vs >= 10 w/v% (Gel-MOD/Gel-MOD-AEMA)). Furthermore, the proposed functionalization scheme maintains the excellent biocompatibility and cell interactivity of gelatin. Additionally, the norbornene functionalities have potential for straightforward postprocessing thiol-ene surface grafting of active molecules. As a consequence, a very promising material toward tissue engineering applications and more specifically, biofabrication, is presented.

Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces.

International audience; G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized β2 -adrenergic receptor (β2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of β2 AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of β2 AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a β-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted into the α-MSH pharmacophore, His(6)-Phe(7)-Arg(8)-Trp(9)-domain. Replacement of His(6) by an aminoindoloazepinone (Aia) or aminobenzazepinone (Aba) moiety led to hMC4R and hMC5R selective agonist and antagonist ligands, respectively (tetrapeptides 1 to 3 and 4, respectively). In peptides 1 to 3 and depending on the para-substituent of the d-Phe residue in position 2, the activity goes from allosteric partial agonism (1, R = H) to allosteric full agonism (2, R = F) and finally allosteric partial agonism (3, R = Br).

A rapid and efficient total synthesis is reported for the cyclic lipodepsipeptide pseudodesmin A. This member of the Pseudomonas viscosin group is active against Gram-positive bacteria and features self-assembling properties. A conserved serine residue within the lactone macrocycle is exploited for initial immobilization on 2-chlorotrityl chloride resin through ether formation with the side-chain alcohol. Subsequent elongation proceeds through Fmoc solid-phase peptide synthesis, including automated incorporation of the enantioselectively synthesized (R)-3-hydroxydecanoic acid lipid tail. Following esterification to generate the incipient lactone bond, the macrocycle is formed by on-resin head-to-tail macrolactamization and cleaved from the resin to give the desired compound in good purity. The short and efficient synthesis route allows rapid generation of analogues by facile variation of both the peptide and lipid moieties with good control of epimerization while maximizing automation. Synthesis of the pseudodesmin A enantiomer yields identical self-assembly and biological activity to that observed for the natural compound, showing that activity is not mediated by chiral interactions. A D-Asn8 analogue developed en route retains self-assembly, but loses activity. The synthesis strategy should be generally applicable for the rapid generation of analogues from various cyclic lipodepsipeptide groups, allowing an investigation of their self-assembling properties and structure-activity relationships.

Polysaccharides, and especially alginate, can be useful for self-healing of cracks in concrete. Instead of weak electrostatic bonds present within calcium alginate, covalent bonds, by methacrylation of the polysaccharides, will result in mechanically stronger superabsorbent polymers (SAPs). These methacrylated alginate chains as backbone are combined with two acrylic monomers in a varying molar fraction. These SAPs show a moisture uptake capacity up to 110% their own weight at a relative humidity of 95%, with a negligible hysteresis. The swelling capacity increased (up to 246 times its own weight) with a decreasing acrylic acid/2 acrylamido-2-methylpropane sulfonic acid ratio. The SAPs also showed a thermal stability up to 200 degrees C. Interestingly, the SAP composed of alginate and acrylic acid exerted a very limited decrease in compressive strength (up to 7% with addition of 1 wt% SAP) rendering this material interesting for the envisaged self-healing application. The authors would like to thank the FWO (Research Foundation Flanders) for project funding (3G019012, Effect of tunable hydro gels on concrete microstructure, moisture properties, sealing and self-healing of cracks). The authors would like to acknowledge the group of Peter Adriaensens and especially Gunter Reekmans for the calculation of the degree of methacrylation of algMOD (Organic and Bio-polymer Chemistry Department, UHasselt).

Poly(styrene-maleic anhydride) or SMA are versatile copolymers available in different grades of molecular weight (Mw = 5500–170,000 g/mol) and molar percentage of maleic anhydride (22–50 mol-%). As specific grades can be used for the synthesis of nanoscale structures in paper industry, a better understanding of the molecular structure was needed. Therefore, a broad range of SMA grades was analyzed by means of size exclusion chromatography, infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance spectroscopy. While the nominal composition could be determined by 1H NMR and Raman spectroscopy, the combination with 13C NMR and FTIR allowed a more detailed characterization of the molecular structure. The high-molecular weight SMA grades show a heterogeneous molecular structure with segments including high styrene and maleic anhydride moieties, respectively. The formation of film patterns was evaluated by deposition from an acetone solution, resulting in homogeneous films, fibrous structures, or spherical shapes with decreasing diameter as a function of maleic anhydride content including primary particle sizes of 100 nm. This study illustrates intrinsic molecular features of SMA grades that are capable for self-assembly, and can provide a selection tool for future nanoparticle formulation.

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.

Smart pH-responsive superabsorbent polymers (SAPs) could be useful for self-healing of cracks in mortar. They will swell minimally during the alkaline conditions of mixing, leading to only small macro-pores but will swell stronger with a lower pH when water enters the cracks. As such, polysaccharides (alginate, chitosan and agarose) were methacrylated and cross-linked with amine-based monomers (dimethylaminoethyl methacrylate and dimethylaminopropyl methacrylamide) to induce a varying pH-sensitivity. These materials showed a strong cross-linking efficiency and induced moisture uptake capacities up to 122% at 95% relative humidity with a negligible hysteresis. Additionally, interesting pH-responsive swelling capacities were obtained, especially for SAPs based on chitosan and agarose with values up to 110gwater/gSAP. Most of these materials showed limited hydrolysis in cement filtrate solutions, making them very promising for use in mortar.

Aerobic oxidative dehydrogenative polycondensation of the new monomer 4,4'-(3-hexylthiophene-2,5-diyl)bis(2-octylaniline) has proved very effective under mild conditions using an inexpensive copper bromide/pyridine complex and dioxygen acting as catalyst and oxidant, respectively. Such kinetically controlled process allowed preparing elec-troconjugated photosensitive polymers with high molar weight (Mn > 15000 g.mol-1).

Chemical modification reactions of alkyne containing polyHEMA-based macroporous network structures (cryogels) by Cu(I) catalyzed azide-alkyne 'click' cycloaddition reactions and their monitoring and quantification with high-resolution magic angle spinning (hr-MAS) NMR spectroscopy are reported. Complete conversion is obtained when benzylazide is reacted with the grafted alkyne function, but only partial conversion is observed when using azide-modified poly(ethylene glycol) (PEG-N(3) ). Subsequent addition of benzylazide consumes all remaining alkyne groups. All chemical modifications are easily monitored at each stage using hr-MAS NMR spectroscopy. The alkyne functionality and the resulting triazole ring provide well resolved (1) H resonances to monitor and quantify the progress of such 'click' reactions in general.

Two new cyclic lipodepsipeptides named pseudodesmins A and B have been isolated from Pseudomonas bacteria collected from the mucus layer in the skin of the black belly salamander. Both compounds show moderate antibacterial activity against Gram positive bacteria, including MRSA. Complete 1H, 13C and 15N NMR assignment of both compounds afforded their covalent structure and served to guide the analysis of LC-MS and X-ray diffraction data from which the final stereochemistry could be established. Both molecules can be categorized as new members of the viscosin group of cyclic lipodepsipeptides.

Second-generation catalysts of the general formula Cl2Ru(SIMes)(L)(3-phenylinden-1-ylidene), 3a (L = PCy3), 3b (L = PPh3), 3c (L = py), and Cl2Ru(SIMe)(L)(3-phenylinden-1-ylidene), 4a (L = PCy3), 4b (L = PPh3), 4c (L = py) were found to be of interest in various metathesis transformations. The catalysts containing SIMe ligands showed improved initiation compared to the more robust SIMes substituted catalysts. A strong temperature effect was noted on all of the reactions tested. Interestingly, complex 3a, showing the lowest initiation rate at room temperature, emerged as the most productive of all systems examined at elevated temperature. It is shown that complexes containing the SIMe ligand display higher initiation efficiency than their corresponding SIMes analogues. Since the higher initiation is related to the ease of phosphane dissociation while phosphane dissociation also promotes catalyst decomposition, complexes bearing the SIMe ligand decompose faster. The complete 1H, 13C and 31P resonance assignment and the procedure applied to obtain these from a combination of 1D and 2D NMR techniques, is also reported. Combined with the ROESY technique, these enabled to investigate several conformational processes involving rotations around N–phenyl and C–Ru bonds on the millisecond to second timescale. A clear correlation is demonstrated between the bulkiness of the axial ligand (L) and the rotational freedom of the SIMe(s) ligand. A qualitative analysis also suggests that the extra para-methyl of SIMes leads to additional steric interactions with the 3-phenylinden-1-ylidene ligand. The data reported in this paper demonstrates that substitution patterns of the N-aryl have a significant influence on the activity of the second-generation indenylidene catalysts for a given metathesis reaction.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Solution NMR spectroscopy is a well established technique for nondestructive characterization of the structures and conformations of complex oligo- and polysaccharides. One of the key experiments involves the use of 2D TOCSY to collect the 1 H spins into groups that can be associated with the individual saccharide units that are present in the molecule under study. It is well known that the magnetization transfer rate through the 1 H spin system during the TOCSY spin lock period is sensitive to the intervening 3 J-(H,H) scalar couplings, and therefore also to the saccharide stereochemistry. Here, we have investigated the potential to extract information on the stereochemistry of hexapyranose monosaccharide units directly from TOCSY spectra. Through a systematic experimental investigation of the magnetization transfer initiated from the anomeric 1 H resonance in D-glucose, D-galactose and D-mannose it is shown that a 100 ms spin lock time provides optimal spectroscopic discrimination between these three commonly occurring building blocks. A simple matching scheme is proposed as a new tool for rapid attribution of the TOCSY traces originating from the anomeric 1 H resonances towards the underlying monosaccharide type. The scheme appears robust with regard to structural variations and fairly tolerant to incidental overlap. Its application provides useful guidance during the subsequent NMR assignment process, as demonstrated with the PS7F polysaccharide from Streptococcus pneumonia. In addition, we show that our scheme affords a clear-cut distinction between the α-and β-epimers of D-mannose-type units, which can be difficult to discriminate by NMR analysis. Application to the N-glycan 100.2 demonstrates the potential and wide applicability of this new discrimination approach.

The present work aims at constructing the ideal scaffold matrix of which the physico-chemical properties can be altered according to the targeted tissue regeneration application. Ideally, this scaffold should resemble the natural extracellular matrix (ECM) as close as possible both in terms of chemical composition and mechanical properties. Therefore, hydrogel films were developed consisting of methacrylamide-modified gelatin and starch-pentenoate building blocks because the ECM can be considered as a crosslinked hydrogel network consisting of both polysaccharides and structural, signaling and cell-adhesive proteins. For the gelatin hydrogels, three different substitution degrees were evaluated including 31%, 72% and 95%. A substitution degree of 32% was applied for the starch-pentenoate building block. Pure gelatin hydrogels films as well as interpenetrating networks with gelatin and starch were developed. Subsequently, these films were characterized using gel fraction and swelling experiments, high resolution-magic angle spinning H-1 NMR spectroscopy, rheology, infrared mapping and atomic force microscopy. The results indicate that both the mechanical properties and the swelling extent of the developed hydrogel films can be controlled by varying the chemical composition and the degree of substitution of the methacrylamide-modified gelatin applied. The storage moduli of the developed materials ranged between 14 and 63 kPa. Phase separation was observed for the IPNs for which separated starch domains could be distinguished located in the surrounding gelatin matrix. Furthermore, we evaluated the affinity of aggrecan for gelatin by atomic force microscopy and radiolabeling experiments. We found that aggrecan can be applied as a bioactive coating for gelatin hydrogels by a straightforward physisorption procedure. Thus, we achieved distinct fine-tuning of the physico-chemical properties of these hydrogels which render them promising candidates for tissue engineering approaches. (C) 2016 Elsevier Ltd. All rights reserved.

Diffusion-ordered NMR spectroscopy (DOSY NMR) is presented as a tool for the determination of the diffusion coefficients of organic ligands in suspensions of titanium dioxide nanoparticles. The nanoparticles were prepared by a sol-gel process by hydrolysis and condensation reactions of titanium tetra-n-butoxide in the presence of pentane-2,4-dione (acacH: acetylacetone), as well as para-toluenesulfonic acid (pTsA) and n-butanol (nBuOH). NMR spectroscopic studies were performed in various deuterated solvents, on both dispersed xerosols and diluted sols. The bipolar-pulsed field gradient longitudinal eddy-current delay (LED) pulse sequence was used for data acquisition. The data were processed by inverse Laplace transformation (ILT), by using a maximum entropy algorithm, to afford 2D DOSY spectra. Different diffusion regimes for organic ligands in the bound and unbound states were successfully discriminated, more particularly in [D 3 ]acetonitrile, thus allowing assessment of their interactions with the nanoparticles.

When it comes to failing or injured tissues and organs, patients often end up on waiting lists for tissue or even organ transplantation negatively affecting the patient’s quality of life. The multidisciplinary research field of tissue engineering may offer more innovative ways to replace or ideally regenerate failing tissues and organs. A widely used material in this research field is gelatin because of its biocompatibility and interesting hydrogel forming properties. However, at body temperature gelatin’s mechanical properties are greatly reduced due to the dissolution of collagen-like triple helices. With the aim to obtain materials that retain their mechanical properties at body temperature, we propose to combine sodium alginate and methacrylamide-modified gelatin (Gel-MOD) in the form of a graft copolymer to obtain a material that closely resembles the extracellular matrix. The obtained materials can be cross-linked via three distinct pathways including cation mediated, temperature mediated or via covalent bond formation after UV irradiation in the presence of a photo-initiator. The current contribution covers the synthesis of the above mentioned alginate-graft-gelatin copolymers and the characterization of the resulting hydrogels. The materials developed are highly hydrophilic, showing high gel fractions and satisfactory mechanical properties. Moreover the attained storage moduli were tunable by divalent cation addition (72–275% increase at 21 °C, 42–405% increase at 40 °C). One formulation was found to outperform Gel-MOD in terms of mechanical properties at 40 °C, thus indicating the proposed strategy can be used to improve the mechanical properties of gelatin-based hydrogels. Moreover, in vitro biocompatibility assays indicated that cell adhesion and proliferation improves with increasing gelatin content. The present paper illustrates that the developed triple cross-linkable materials are suitable cell carriers, promising to be applied for biomedical purposes.

The Glaser–Hay diyne coupling proved to be an efficient cyclisation approach towards diyne containing peptidic macrocycles. A variety of tetrapeptide-based macrocyclic 1,3-diynes were obtained from O-propargylated serine or tyrosine residues using Cu(OAc)2·H2O and NiCl2 under an O2-atmosphere. The effect of the linear 1,3-diyne on peptide conformations was studied by NMR and compared with a macrocycle bearing a saturated linker.

Tripodal scaffold 1 has been used in the synthesis of a representative member of a library of serine protease mimics, possessing three independent functionalized peptide chains on a central core. Each peptide chain contains one residue of the classical catalytic triad (serine, histidine and aspartate) found in the active site of the serine protease α-chymotrypsin. A particular feature of the novel tripodal design is its essentially flexible yet preorganized structure as deduced from molecular modeling studies. The choice of suitable scaffold and side-chain protecting groups was intensively studied and optimized to ensure complete orthogonality. Inclusion of a photolabile linker enabled the release of intermediates and final structures from the solid-support polymer allowing positive evaluation of the present strategy for the synthesis of future tripodal libraries of serine protease mimics.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Dialkyltin dichloride grafted to a cross-linked polystyrene, with the formula [P-H] ( 1 - 1 ) [P-( CH 2 ),,SnBuCL], (P= [CH 2 CH(pC 6 H 4 ), t= the degree of functionalization, and n=6 . or 11), is investigated as a recyclablecatalyst in the ring-opening polymerization (ROP) of e-caprolactone (CL). It is demonstrated that high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy is an invaluable tool to characterize completely the supported catalyst. The 2D 1 H- 1 3 C HSQC HR-MAS spectrum, in particular, allowed extensive assignment of the 1 H and 1 3 C resonances, as well as accurate measurement of the "J('H- 1 1 7 / 1 1 9 sn) and n J( 1 3 C- 1 1 7 / 1 1 9 Sn) coupling constants. 1 H and 1 1 9 Sn HR-MAS NMR spectroscopy is presented as a monitoring tool for catalytic processes based on organotin compounds, particularly for the investigation of the extent to which polymerization residues are observable in situ in the material pores and for the assessment of the chemical integrity and recycling conditions of the grafted catalyst. From polymerization experiments with CL. initiated by n-propanol and with [P-H] ( 1 - 1 ) ,[P-(CH 2 ) n SnBuCI 2 ], of various compositions as the supported catalyst, it appears that a partial 'burst' of the polystyrene support occurs when the length of the alkyl spacer is limited to n=-6, as a result of polymer chains growing within the pores of the support. However, extension of the length of the aliphatic polymethylene spacer from 6 to 11 carbon atoms preserves the support integrity and allows the production of catalyst-deprived poly(e-caprolactone) (PCL) oligomers. A preliminary attempt to recycle the heterogeneous catalyst has shown that very good reproducibility can be obtained, in terms of both catalyst activity and molecular-weight parameters of the asrecovered PCL polyester chains.

Pulsed field gradient 1 H NMR spectroscopy has been applied to investigate the association behavior of the Sn 1 2 -oxo cluster macrocation [(BuSn) 1 2 O 1 4 (OH) 6 ] 2 + with two different and smaller anions, p-toluenesulfonate (PTS - ) and diphenylphosphinate (Ph 2 PO 2 -). By monitoring the translational diffusion coefficients of the various species involved, it is shown that the association depends on the anion involved and on the solvent used. Moreover, the possibility to individually monitor the diffusion characteristics of multiple anionic and cationic species in mixtures, by virtue of resolved 1 H resonances available from each species, allows us to evidence the occurrence of ion exchange in such systems. Thus when [(BuSn) 1 2 O 1 4 (OH) 6 ](PTS) 2 is mixed with two equivalents of Ph 2 PO 2 NMe 4 , PTS- is displaced by Ph 2 PO 2 -, highlighting the greater affinity of the organotin macrocation for the diphenylphosphinate. This example clearly illustrates the potential of pulsed field gradient 1 H NMR spectroscopy in inorganic/organometallic chemistry, to follow preferential ion pairing in multi-ion systems at the level of each individually charged species.

The mechanism of pI258 arsenate reductase (ArsC) catalyzed arsenate reduction, involving its P-loop structural motif and three redox active cysteines, has been unraveled. All essential intermediates are visualized with x-ray crystallography, and NMR is used to map dynamic regions in a key disulfide intermediate. Steady-state kinetics of ArsC mutants gives a view of the crucial residues for catalysis. ArsC combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Within this cascade, the formation of a disulfide bond triggers a reversible “conformational switch” that transfers the oxidative equivalents to the surface of the protein, while releasing the reduced substrate.

The viscosin group covers a series of cyclic lipodepsipeptides (CLPs) produced by Pseudomonas bacteria, with a range of biological functions and antimicrobial activities. Their oligopeptide moieties are composed of both l- and d-amino acids. Remarkably, the Leu5 amino acid-centrally located in the nonapeptide sequence-is the sole residue found to possess either an l or d configuration, depending on the producing strain. The impact of this D/L switch on the solution conformation was investigated by NMR-restrained molecular modelling of the epimers pseudodesmin A and viscosinamide A. Although the backbone fold remained unaffected, the D/L switch adjusted the segregation between hydrophobic and hydrophilic residues, and thus the amphipathicity. It also influenced the self-assembly capacity in organic solvents. Additionally, several new minor variants of viscosinamide A from Pseudomonas fluorescens DR54 were identified, and an NMR assay is proposed to assess the presence of either an L- or D-Leu5.

We report the synthesis of a family of d- and l-furano-d-apionucleosides, their 3′-deoxy, as well as their 2′,3′-dideoxy analogues with thymine and adenine nucleobases. Single carbon homologation of 1,2-O-isopropylidene-d-glycero-tetrafuranos-3-ulose (15) and optimized glycosylation conditions involving microwave irradiation were key to the successful synthesis of the target compounds. While all target nucleosides failed to show significant antiviral activity, we demonstrated that the triphosphate of 2′,3′-deoxy-d-apio-d-furanoadenosine (1), in contrast to that of its d-apio-l-furanose epimer 2, was readily incorporated into a DNA template by HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1 and HIV-2 (EC50 = 0.5–1.5 μM), indicating that the lack of activity of the parent nucleoside, and possibly also other members of the d-apio-d-furanose nucleoside family must be sought in the inefficient cellular conversion to the monophosphate.

The selective recognition of the orthophosphate anion by a series of bifunctional Lewis acidic organotin compounds is investigated. The binding affinity of these carriers to anions, as measured by NMR titrations in CH2Cl2 obeyed the potentiometric selectivity order phosphate > sulfate > perchlorate, and provided the corresponding complex-formation constants. More accurate calculations of these values were obtained by the segmented-sandwich-membrane method performed directly inside the liquid polymeric membrane. These carriers were also studied potentiometrically in polymeric liquid membranes. The results indicate that (PhBr2Sn)2CH2 is 2 to 5 orders of magnitude more selective towards phosphate over other oxoanions. These results set the ground for the development of a new series of highly selective anions carriers with a wide range of possible applications.

Three dibutyltin perfluoroalkanecarboxylates have been synthesized, characterized by H-1-, C-13-, F-19- and Sn-117-NMR, Mossbauer, IR and mass spectroscopy. The structure of tetra-n-butylbis(trifluoroacetato)distannoxane has been elucidated by X-ray crystallography. The in vitro antitumour activity of the three compounds against seven human tumour cell lines was found to be as high as or even higher than that for reference compounds used clinically. (C) 2000 Published by Elsevier Science S.A.

The reaction of butyltin hydroxide oxide, BuSnO(OH), with p-toluenesulfonic acid, 4-CH3C6H4SO3H, yields the butyltin oxo cluster {(BuSn)12(μ3-O)14(μ2-OH)6}2+ mixed with a soluble ill-defined butylt...

Various NMR correlation techniques, 1H−13C HMQC, 1H−13C HMBC, 1H−119Sn HMQC with gradient enhancement, and 13C−119Sn HMQC, enabled a full resonance assignment and the solution structure elucidation of bis(tributylstannyl(IV)) l-(+)-ascorbate (1) and O-5,6-isopropylidene-l-(+)-ascorbate (2) in deuteriochloroform. Double heteronuclear 13C−119Sn HMQC spectroscopy appeared indispensable for discriminating between 2J(13C−O−119Sn) and 3J(13CC−O−119Sn) coupling pathways involving quaternary carbon atoms and provided useful information about the relative magnitudes of 2J(13C−119Sn) and 3J(13C−119Sn) coupling constants, as they turned out to have an uncommon coupling size sequence. While the tin atoms in compounds 1 and 2 are four-coordinate in solution, 117Sn CP/MAS NMR reveals that compound 2 has one four-coordinate and one five-coordinate tin atom in the solid state.

The X-ray diffraction analysis of crystals of {[Me2N(CH2)3]2SnF2·2H2O (3a) obtained by reaction of [Me2N(CH2)3]2SnR2 (R = Me, Ph) with Pr3SnF reveals a six-coordinate, distorted octahedral geometry at tin with the pairs of fluorine, nitrogen and carbon atoms bound to the metal atom being all in a mutual trans configuration. Room-temperature 119Sn, 19F, 1H, and 13C NMR spectra of 3a reveal exchange averaging with loss of the 1J(119Sn−19F) coupling on the 119Sn and 19F NMR time scales. Below −50 °C, 119Sn and 19F NMR coupling data evidence the existence of only two isomers among the five a priori possible ones, a major (ca. 80%) and a minor species (ca. 20%). The methyl 13C resonance as well as the 1J(119Sn−19F), 1J(119Sn−13C), and 2J(19F−13C) coupling patterns reveal the major species to have the same all-trans structure as in the crystal state, while the minor species has a cis arrangement for its fluorine and nitrogen atoms and the trans one for its carbon atoms. Gradient assisted 1H−119Sn HMQC spectrosc...

Two decades of progress in the field of living and controlled polymerizations, combined with the elaboration of efficient conjugation reactions, greatly contributed to the elegant preparation of functionalized macromolecular architectures. However, these state-of-the-art methodologies, while providing a high degree of structural and topological control, are inadequate tools for controlling the polymer microstructure. Crucial parameters like primary structure (i.e. monomer sequence) and tacticity largely remain unmastered by current man-made approaches. Expectations for the next generation synthetic polymers include their performance as single chains, ability to fold and self-regulate, and to sense specific molecules and/or catalyze reactions. These precisely functionalized linear polymers should exhibit sharply defined and tailored structure-activity relationships, analogous to Nature’s delicately engineered macromolecules. Therefore, progress towards reliable sequence-controlled polymerization, enabling preprogrammed distribution of multiple functional groups along the backbone, is drawing attention in a growing number of research groups worldwide. Pioneering efforts to control the primary structure of functionalized polymers have been based on several approaches, such as different reactivity ratios of vinyl monomers, spatial prearrangement of monomers on a (macromolecular) template or, as recently demonstrated, the action of a small-molecule machine. Other attempts use (automated) sequential addition of building blocks on a solid or liquid support, leading to sequence control as a result of iterative coupling steps, thereby omitting the need for pre-organization. These protocols, established for peptide and oligonucleotide synthesis, present considerable drawbacks for sequence-controlled polymerization: they generally require the use of protecting groups and the restricted number of readily available building blocks (‘monomer alphabet’) equipped with the appropriate functional handle can further hamper the preparation of tailor-made functionalized sequences. The development of new chemical protocols for chain elongation, often on a solid support, resulting in sequence-defined (macro)molecular structures with unique backbones and side chain functionalities, or fragments thereof that could be combined to obtain sequence controlled polymers, is therefore highly desirable. We here report on a new coupling strategy for the controlled generation of sequence-defined multi-functionalized oligomers on solid support in a protecting group-free approach, inspired by the ‘submonomer’ synthetic protocol for the preparation of functionalized peptoids, via thiolactone-based chemistry. While the generated oligomers are small in size, reconstitution approaches could further allow the synthesis of larger chains, featuring designed and repetitive display of carefully selected and well-positioned functional entities.

Reaction of dimethyltin(IV) oxide with salicylaldoxime in the molar ratio 3:2 or 1:1 in refluxing toluene/ethanol (4/1) affords reaction mixtures which, upon crystallization from alcohols TOH (T = Me, Et, n-Pr, i-Pr), generate compounds with three dimethyltin and two salicylaldoximate moieties. These compounds obey the formula [(Me(2)Sn)(Me(2)SnO)(Me(2)-SnOT)(HONZO)(ONZO)], where HONZOH represents salicylaldoxime, o-HON=CHC6H4OH. X-ray diffraction analysis of the compounds at room temperature (T = Me, compound 3; T = Et, 4; T = n-Pr, 5; T = i-Pr, 6) reveals that all structures contain one seven-coordinate tin atom bound to two nitrogen and three oxygen atoms in the equatorial plane of a pentagonal bipyramid, the two methyl groups being in apical positions. The two other tin atoms are five-coordinate with a trigonal-bipyramidal geometry having the two methyl groups and a common oxygen atom in the equatorial positions and, in apical positions, two further oxygen atoms, of which the trifurcated alkoxy oxygen atom bridges these tin atoms. Reaction of dimethyltin(IV) oxide with salicylaldoxime in the molar ratio 1:1 in refluxing benzene generates, after solvent evaporation, compound 2b, obeying the formula [(Me(2)Sn)(2)(Me(2)SnO)-(HONZO)(ONZO)(HONZO)]. The solution-state structures of the alkoxy-bridged compounds 3-6, investigated by gradient-assisted 2D H-1-Sn-119 HMQC as well as H-1-C-13 HMQC and HMBC NMR spectroscopy, are the same as those in the solid state. The bonding mode of the third ligand (i.e. HOZNO) of compound 2b has been elucidated.

The formation and structure of inclusion complexes of 2,6- and 2,9-substituted bicyclo[3.3.1]nonanes withα- andβ-cyclodextrin (CD) has been investigated by high-resolution1H NMR spectroscopy.α- andβ-CD were found to form 1:1 inclusion complexes and the binding constants were estimated from titration studies. 2D ROESY experiments provided insight into the structure of the complexes.

We explored the influence of modifications of uridine 5'-methylenephosphonate on biological activity at the human P2Y(2) receptor. Key steps in the synthesis of a series of 5-substituted uridine 5'-methylenephosphonates were the reaction of a suitably protected uridine 5'-aldehyde with [(diethoxyphosphinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki-Miyaura coupling. These analogues behaved as selective agonists at the P2Y(2) receptor, with three analogues exhibiting potencies in the submicromolar range. Although maximal activities observed with the phosphonate analogues were much less than observed with UTP, high concentrations of the phosphonates had no effect on the stimulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y(2) receptor.

Dermorphin analogues, containing a (S)- and (R)-4-amino1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold (Aba) and the alpha-methylated analogues as conformationally constrained phenylalanines, were prepared. Asymmetric phase-transfer catalysis was unable to provide the (S)-alpha-Me-o-cyanophenylalanine precursor for (S)-alpha-MeAba in acceptable enantiomeric purity. However, by using a Schollkopf chiral auxiliary, this intermediate was obtained in 88% ee. [(S)-Aba 3-Gly 4] dermorphin retained mu-opioid affinity but displayed an increased delta-affinity. The corresponding R epimer was considerably less potent. In contrast, the [(R)-alpha-MeAba3-Gly4] dermorphin isomer was more potent than its S epimer. Tar-MD simulations of both non-methylated [Aba 3-Gly 4] dermorphin analogues showed a degree of folding at the C-terminal residues toward the N terminus of the peptide, without however, adopting a stabilized beta-turn conformation. The alpha-methylated analogues, on the other hand, exhibited a type I/I' beta-turn conformation over the alpha-MeAba3 and Gly4 residues, which was stabilized by a hydrogen bond involving Tyr5-H(N) and D-Ala 2-CO.

An efficient protocol for the construction of C-6-(hetero)aryl-substituted uridine phosphonate analogues utilizing an aerobic, ligand-free Suzuki–Miyaura cross-coupling reaction of a 6-iodo-precursor in aqueous media has been established. The method presents a modular approach toward the target compounds as demonstrated by the synthesis of a small library comprising 14 novel nucleoside phosphonates.

Pseudodesmin A is a cyclic lipodepsipeptide (CLP) of the viscosin group with a moderate in vitro biological activity. For several CLPs, including members of this group, this activity has been related to the ability to form ion pores in cellular membranes. As their size does not allow individual CLPs to span the membrane bilayer, individual monomers must somehow assemble into a larger structure. NMR spectroscopy has been used to demonstrate that in chloroform and other apolar organic solvents, pseudodesmin A monomers assemble into a supramolecular structure. These self-assembled structures can become sufficiently large to span the membrane bilayer as demonstrated with translational diffusion NMR spectroscopic measurements. With the aim to obtain more insight into the structural nature of this assembly, the solution conformation of pseudodesmin A was first determined by using ROESY (rOe) restraints measured in acetonitrile, in which no self-association occurs. The structure, which is found to be mostly similar to the previously described crystal structure, is shown to be retained within the supramolecular complex. Intermolecular rOe contacts obtained in chloroform together with chemical shift perturbation data provides structural insight into the organization of the self-associated complex. Based upon this analysis, a model for the organization of pseudodesmin A monomers in the supramolecular assembly is proposed, which is in agreement with the formation of bilayer spanning hydrophilic pores and provides the basis for a structure-function relationship for this type of CLPs. Finally, it is demonstrated that the differences previously reported between the crystal and solution conformation of the white line inducing principle (WLIP), a close analogue of pseudodesmin A, are the result of the use of dimethyl sulfoxide as solvent, whose strong hydrogen-bonding capacity induces conformational exchange.