Chemoenzymatic Approach for the Preparation of Asymmetric Bi-, Tri-, and Tetra-Antennary N-Glycans from a Common Precursor.

Progress in glycoscience is hampered by a lack of well-defined complex oligosaccharide standards that are needed to fabricate the next generation of microarrays, to develop analytical protocols to determine exact structures of isolated glycans, and to elucidate pathways of glycan biosynthesis. We describe here a chemoenzymatic methodology that makes it possible, for the first time, to prepare any bi-, tri-, and tetra-antennary asymmetric N-glycan from a single precursor. It is based on the chemical synthesis of a tetra-antennary glycan that has N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and unnatural Galα(1,4)-GlcNAc and Manß(1,4)-GlcNAc appendages. Mammalian glycosyltransferases recognize only the terminal LacNAc moiety as a substrate, and thus this structure can be uniquely extended. Next, the ß-GlcNAc terminating antenna can be converted into LacNAc by galactosylation and can then be enzymatically modified into a complex structure. The unnatural α-Gal and ß-Man terminating antennae can sequentially be decaged by an appropriate glycosidase to liberate a terminal ß-GlcNAc moiety, which can be converted into LacNAc and then elaborated by a panel of glycosyltransferases. Asymmetric bi- and triantennary glycans could be obtained by removal of a terminal ß-GlcNAc moiety by treatment with ß-N-acetylglucosaminidase and selective extension of the other arms. The power of the methodology is demonstrated by the preparation of an asymmetric tetra-antennary N-glycan found in human breast carcinoma tissue, which represents the most complex N-glycan ever synthesized. Multistage mass spectrometry of the two isomeric triantennary glycans uncovered unique fragment ions that will facilitate identification of exact structures of glycans in biological samples.

Collagen degradation and neutrophilic infiltration: a vicious circle in inflammatory bowel disease.

OBJECTIVE: Proline-glycine-proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). DESIGN: In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. RESULTS: In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. CONCLUSIONS: The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: intermolecular bivalent binding.

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.

Inactivation of staphylococcal phenol soluble modulins by serum lipoprotein particles.

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.

Characterization and activity of an immobilized antimicrobial peptide containing bactericidal PEG-hydrogel.

A single step immobilization-polymerization strategy of a highly active antimicrobial peptide into a soft hydrogel network on a poly(ethylene terephthalate) surface using thiol-ene chemistry is described. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. The bactericidal activity was established against Staphylococcus aureus and Staphylococcus epidermidis, two bacterial strains commonly associated with biomaterial infections. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions.

Versatile convergent synthesis of a three peptide loop containing protein mimic of whooping cough pertactin by successive Cu(I)-catalyzed azide alkyne cycloaddition on an orthogonal alkyne functionalized TAC-scaffold.

Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein-protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)-catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity.

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 µM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Chemical-biological exploration of the limits of the Ras de- and repalmitoylating machinery.

A dynamic de-/repalmitoylation cycle determines localization and activity of H- and N-Ras. This combined cellular de- and repalmitoylation machinery has been shown to be substrate tolerant--it accepts variation of amino acid sequence, structure and configuration. Here, semisynthetic Ras-proteins in which the C-terminal amino acids are replaced by peptoid residues are used to reveal the first limitations of substrate recognition by the de- and repalmitoylating machinery.

Synthesis of cyclic peptides containing a thioester handle for native chemical ligation.

The synthesis of cyclic peptides containing a thioester handle using a sulfo-click linker is reported. These cyclic peptides can be coupled to N-terminal cysteine-containing constructs via native chemical ligation. A successful application of a cyclic peptide bearing a thioester handle in native chemical ligation is shown by a high yielding ligation.

Mutual influence of backbone proline substitution and lipophilic tail character on the biological activity of simplified analogues of caspofungin.

The echinocandins represent the most recent class of antifungal drugs. Previous structure-activity relationship studies on these lipopeptides have relied mainly upon semisynthetic derivatives due to their complex chemical structures. A successful strategy for the rapid enantioselective synthesis of the branched fatty acid chain of caspofungin and analogues was developed to synthesize several simplified analogues of caspofungin. The specific minimum inhibitory activity of each mimic was determined against a panel of Candida strains. This approach gave access to new fully synthetic derived caspofungin mimics with high and selective antifungal activities against Candida strains. In addition, the data suggested an important role of the hydroxy proline residue in the bioactive conformation of the macrocyclic peptide ring structure.

Glucocorticoid-loaded core-cross-linked polymeric micelles with tailorable release kinetics for targeted therapy of rheumatoid arthritis.

Polymerizable and hydrolytically cleavable dexamethasone (DEX, red dot in picture) derivatives were covalently entrapped in core-cross-linked polymeric micelles that were prepared from a thermosensitive block copolymer (yellow and gray building block). By varying the oxidation degree of the thioether in the drug linker, the release rate of DEX could be controlled. The DEX-loaded micelles were used for efficient treatment of inflammatory arthritis in two animal models.

Peptides and proteins as a continuing exciting source of inspiration for peptidomimetics.

Despite their enormous diversity in biological function and structure, peptides and proteins are endowed with properties that have induced and stimulated the development of peptidomimetics. Clearly, peptides can be considered as the "stem" of a phylogenetic molecular development tree from which branches of oligomeric peptidomimetics such as peptoids, peptidosulfonamides, urea peptidomimetics, as well as ß-peptides have sprouted. It is still a challenge to efficiently synthesize these oligomeric species, and study their structural and biological properties. Combining peptides and peptidomimetics led to the emergence of peptide-peptidomimetic hybrids in which one or more (proteinogenic) amino acid residues have been replaced with these mimetic residues. In scan-like approaches, the influence of these replacements on biological activity can then be studied, to evaluate to what extent a peptide can be transformed into a peptidomimetic structure while maintaining, or even improving, its biological properties. A central issue, especially with the smaller peptides, is the lack of secondary structure. Important approaches to control secondary structure include the introduction of α,α-disubstituted amino acids, or (di)peptidomimetic structures such as the Freidinger lactam. Apart from intra-amino acid constraints, inter-amino acid constraints for formation of a diversity of cyclic peptides have shaped a thick branch. Apart from the classical disulfide bridges, the repertoire has been extended to include sulfide and triazole bridges as well as the single-, double- and even triple-bond replacements, accessible by the extremely versatile ring-closing alkene/alkyne metathesis approaches. The latter approach is now the method of choice for the secondary structure that presents the greatest challenge for structural stabilization: the α-helix.

A peptide mimic of the chemotaxis inhibitory protein of Staphylococcus aureus: towards the development of novel anti-inflammatory compounds.

Complement factor C5a is one of the most powerful pro-inflammatory agents involved in recruitment of leukocytes, activation of phagocytes and other inflammatory responses. C5a triggers inflammatory responses by binding to its G-protein-coupled C5a-receptor (C5aR). Excessive or erroneous activation of the C5aR has been implicated in numerous inflammatory diseases. The C5aR is therefore a key target in the development of specific anti-inflammatory compounds. A very potent natural inhibitor of the C5aR is the 121-residue chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Although CHIPS effectively blocks C5aR activation by binding tightly to its extra-cellular N terminus, it is not suitable as a potential anti-inflammatory drug due to its immunogenic properties. As a first step in the development of an improved CHIPS mimic, we designed and synthesized a substantially shorter 50-residue adapted peptide, designated CHOPS. This peptide included all residues important for receptor binding as based on the recent structure of CHIPS in complex with the C5aR N terminus. Using isothermal titration calorimetry we demonstrate that CHOPS has micromolar affinity for a model peptide comprising residues 7-28 of the C5aR N terminus including two O-sulfated tyrosine residues at positions 11 and 14. CD and NMR spectroscopy showed that CHOPS is unstructured free in solution. Upon addition of the doubly sulfated model peptide, however, the NMR and CD spectra reveal the formation of structural elements in CHOPS reminiscent of native CHIPS.

Synthesis and evaluation of novel macrocyclic antifungal peptides.

Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the ß-(1,3)-D-glucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl groups present in these complex macrocyclic lipopeptides, most structure-activity relationship studies have relied upon semisynthetic derivatives. In order to probe the influence of the cyclic peptide backbone on the antifungal activity we developed a successful strategy for the synthesis of novel echinocandins analogues by on-resin ring closing metathesis or disulfide formation. The specific minimum inhibitory activity of each mimic was determined against Candida albicans. Our results indicate that ring size is an important factor for antifungal activity.

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and beta-cell death. Here, we used variants of the hIAPP(1-19) fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, (2)H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP-membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone.

CHIPS binds to the phosphorylated N-terminus of the C5a-receptor.

Replacement of the sulfate groups, present in vivo on the N-terminus of the C5a-receptor (C5aR), by phosphate groups is explored. Phosphorylated mimics of the C5a-receptor N-terminus are synthesized and their binding to Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) is studied by ITC and NMR. The phosphorylated C5aR mimics showed comparable binding affinity and a similar binding mode towards CHIPS compared to their sulfated forms. The activities of the phosphorylated peptides in a biological assay, however, were significantly lower compared to their sulfated counterparts.

A general approach for the non-stop solid phase synthesis of TAC-scaffolded loops towards protein mimics containing discontinuous epitopes.

In this Communication, the access to three different peptide loops attached to a small triazacyclophane (TAC) scaffold molecule for the mimicry of discontinuous epitopes present in, for example, antibodies is described for the first time.

A general sequence independent solid phase method for the site specific synthesis of multiple sulfated-tyrosine containing peptides.

In this communication, a new site specific synthesis of highly functionalized and multiple sulfated peptides using convential Fmoc-tBu solid phase peptide synthesis is described.

Enzymatic synthesis of C-terminal arylamides of amino acids and peptides.

A mild and cost-efficient chemo-enzymatic method for the synthesis of C-terminal arylamides of amino acid and peptides is described. Using the industrial serine protease Alcalase under near-anhydrous conditions, C-terminal arylamides of N-Cbz-protected amino acids and peptides could be obtained from the corresponding C-terminal carboxylic acids, methyl (Me) or benzyl (Bn) esters, in high chemical and enantio- and diastereomeric purities. Yields ranged between 50% and 95% depending on the size of the aryl substituents and the presence of electron-withdrawing substituents. Complete alpha-C-terminal selectivity could be obtained even in the presence of various unprotected side-chain functionalities such as beta/gamma-carboxyl, hydroxyl, and guanidino groups. In addition, the use of the cysteine protease papain and the lipase Cal-B gave anilides in high yields. The chemo-enzymatic synthesis of arylamides proved to be completely free of racemization, in contrast to the state-of-the-art chemical methods.

Effects of linker variation on the in vitro and in vivo characteristics of an 111In-labeled RGD peptide.

INTRODUCTION: Due to the selective expression of the alpha(v)beta3 integrin in tumors, radiolabeled arginine-glycine-aspartic acid (RGD) peptides are attractive candidates for tumor targeting. Minor modifications of these peptides could have a major impact on in vivo characteristics. In this study, we systematically investigated the effects of linker modification between two cyclic RGD sequences and DOTA (1,4,7,10-tetraazadodecane-N,N',N",N'''-tetraacetic acid) on the in vitro and in vivo characteristics of the tracer. METHODS: A dimeric RGD peptide was synthesized and conjugated either directly with DOTA or via different linkers: PEG4 (polyethylene glycol), glutamic acid or lysine. The RGD peptides were radiolabeled with 111In, and their in vitro and in vivo alpha(v)beta3-binding characteristics were determined. RESULTS: LogP values varied between -2.82+/-0.06 and -3.95+/-0.33. The IC50 values for DOTA-E-[c(RGDfK)]2, DOTA-PEG4-E-[c(RGDfK)]2, DOTA-E-E-[c(RGDfK)]2 and DOTA-K-E-[c(RGDfK)]2 were comparable. Two hours after injection, the tumor uptakes of the 111In-labeled compounds were not significantly different. The kidney accumulation of [111In]-DOTA-K-E-[c(RGDfK)]2 [4.05+/-0.20% of the injected dose per gram (ID/g)] was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (2.63+/-0.19% ID/g; P<.05) as well as that of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.16+/-0.21% ID/g; P<.01). The liver uptake of [111In]-DOTA-E-E-[c(RGDfK)]2 (2.12+/-0.09% ID/g) was significantly higher as compared with that of [111In]-DOTA-E-[c(RGDfK)]2 (1.64+/-0.1% ID/g; P<.05) as well as that of [111In]-DOTA-K-E-[c(RGDfK)]2 (1.52+/-0.04% ID/g; P<.01). CONCLUSIONS: Linker variation did not affect affinity for alpha(v)beta3 and tumor uptake. Insertion of lysine caused enhanced kidney retention; that of glutamic acid also resulted in enhanced retention in the kidneys. PEG4 appeared to be the most suitable linker as compared with glutamic acid and lysine because it has the highest tumor-to-blood ratio and the lowest uptake in the kidney and liver.