Cell surface and in vivo interaction of dendrimeric N-glycoclusters.

While many examples have been reported that glycoclusters interact with target lectins more strongly than single molecules of glycans, through multivalency effects, literature examples to support lectin interactions/modulations on cell surface and in live animals is quite rare. Our N-glycoclusters, which were efficiently prepared by immobilizing 16 molecules of the asparagine-linked glycans (N-glycans) onto a lysine-based dendron template through histidine-mediated Huisgen cycloaddition, were shown to efficiently detect platelet endothelial cell adhesion molecule (PECAM) on human umbilical vein endothelial cells (HUVEC) as a α(2-6)-sialylated oligosaccharides recognizing lectin. Furthermore, the identity of the N-glycans on our N-glycoclusters allowed control over organ-selective accumulation and serum clearance properties when intravenously injected into mice.

Biomimetic synthesis of cyclic peptides using novel thioester surrogates.

Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift reactions in forming thioesters and the subsequent peptide ligation, here we describe the development of two 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates that can undergo uncatalyzed N-S, S-S, and S-N acyl shifts for preparing thioesters and cyclic peptides. These surrogates were incorporated as a C-terminal amido moiety of a target peptide using Fmoc chemistry by solid-phase synthesis, and then transformed into a thioester or thiolactones via two acyl shift reactions with or without the presence of an external thiol under acidic conditions. The proposed intein-mimetic thioester surrogates were prepared using readily available starting materials including N-methyl cysteine or 2-thioethylbutylamide. A key functional moiety shared in their design is the thioethylamido (TEA) moiety, which is essential to effect a proximity-driven N-S acyl shift under a favorable five-member ring transition in the breaking of a peptide bond. Thus, the tandem series of acyl shifts effected by a TEA moiety in a thioester surrogate together with a thioethylamino moiety of an N-terminal Cys residue in a linear peptide precursor are chemical mimics of an intein, as they mediate both excision and ligation reactions in forming cyclic peptides including cyclic conotoxin and sunflower trypsin inhibitor described herein.

Role of the backbone conformation at position 7 in the structure and activity of marinostatin, an ester-linked serine protease inhibitor.

Rational design of inhibitors: The cis-amide backbone at position 7 in the serine protease inhibitor marinostatin was replaced with an E or Z olefin. The E olefin analogue was not active, but the Z analogue was. The cis conformation might play a critical role in organizing a canonical structure for binding to proteases.

Optimal oxidative folding of the novel antimicrobial cyclotide from Hedyotis biflora requires high alcohol concentrations.

Hedyotide B1, a novel cyclotide isolated from the medicinal plant Hedyotis biflora, contains a cystine knot commonly found in toxins and plant defense peptides. The optimal oxidative folding of a cystine knot encased in the circular peptide backbone of a cyclotide poses a challenge. Here we report a systematic study of optimization of the oxidative folding of hedyotide B1, a 30-amino acid cyclic peptide with a net charge of +3. The linear precursor of hedyotide B1, synthesized as a thioester by solid phase synthesis, was cyclized quantitatively by a thia-zip cyclization to form the circular backbone and then subjected to oxidative folding in a thiol-disulfide redox system under 38 different conditions. Of the oxidative conditions examined, the nature of the organic cosolvent appeared to be critical, with the use of 70% 2-propanol affording the highest yield (48%). The disulfide connectivity of the folded hedyotide was identical to that of the native form as determined by partial acid hydrolysis. The use of such a high alcohol concentration suggests that a partial denaturation may be necessary for the oxidative folding of a cyclotide with the inverse orientation of hydrophobic side chains that are externalized to the solvent face to permit the formation of the interior cystine core in the circularized backbone. We also show that synthetic hedyotide B1 is an antimicrobial, exhibiting minimal inhibitory concentrations in the micromolar range against both Gram-positive and -negative bacteria.

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution.

BACKGROUND: Cationic antimicrobial peptides (CAMPs) are well recognized to be promising as novel antimicrobial and antitumor agents. To obtain novel skeletons of CAMPs, we propose a simple strategy using acid-amide substitution (i.e. Glu→Gln, Asp→Asn) to confer net positive charge to natural non-antimicrobial sequences that have structures distinct from known CAMPs. The potential of this strategy was verified by a trial study. METHODS: The pro-regions of nematode cecropin P1-P3 (P1P-P3P) were selected as parent sequences. P1P-P3P and their acid-amide-substituted mutants (NP1P-NP3P) were chemically synthesized. Bactericidal and membrane-disruptive activities of these peptides were evaluated. Conformational changes were estimated from far-ultraviolet circular dichroism (CD) spectra. RESULTS: NP1P-NP3P acquired potent bactericidal activities via membrane-disruption although P1P-P3P were not antimicrobial. Far-ultraviolet CD spectra of NP1P-NP3P were similar to those of their parent peptides P1P-P3P, suggesting that NP1P-NP3P acquire microbicidal activity without remarkable conformational changes. NP1P-NP3P killed bacteria in almost parallel fashion with their membrane-disruptive activities, suggesting that the mode of action of those peptides was membrane-disruption. Interestingly, membrane-disruptive activity of NP1P-NP3P were highly diversified against acidic liposomes, indicating that the acid-amide-substituted nematode cecropin pro-region was expected to be a unique and promising skeleton for novel synthetic CAMPs with diversified membrane-discriminative properties. CONCLUSIONS: The acid-amide substitution successfully generated some novel CAMPs in our trial study. These novel CAMPs were derived from natural non-antimicrobial sequences, and their sequences were completely distinct from any categories of known CAMPs, suggesting that such mutated natural sequences could be a promising source of novel skeletons of CAMPs.

Structure-activity relationship of marinostatin, a serine protease inhibitor isolated from a marine organism.

A 12-residue MST isolated from a marine organism is a potent serine protease inhibitor that has a double cyclic structure composed of two ester linkages formed between the beta-hydroxyl and beta-carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11). MST was synthesized by a regioselective esterification procedure employing two sets of orthogonally removable side-chain protecting groups for the Asp and Ser/Thr residues. In the MST molecule, there were no significant changes observed in yield by changing the order of esterification. SAR study of MST revealed that the minimum required structure for expressing the inhibitory activity is the sequence (1-9) in a monocyclic structure where Pro(7) located in the ring plays a crucial role in keeping the structural rigidity. By applying the structural motif of MST, we rationally designed protease inhibitory specificities that differ from those of the natural product.

An Orally Active Bradykinin B1 Receptor Antagonist Engineered as a Bifunctional Chimera of Sunflower Trypsin Inhibitor.

An orally active and metabolically stable peptide TIBA was successfully engineered as a chimera by fusing an analgesic bradykinin receptor antagonist peptide and the trypsin inhibitory loop of sunflower trypsin inhibitor-1. As a fusion cyclic peptide, the metabolically labile analgesic peptide is protected from degradation by exopeptidases as well as the endopeptidases, and its serum half-life extended from <5 min to >6 h as a chimera. Moreover, the chimera TIBA was also found to be orally active in an animal pain model using a hot plate assay.

In Situ Ligation of High- and Low-Affinity Ligands to Cell Surface Receptors Enables Highly Selective Recognition.

This paper reports an entirely unexplored concept of simultaneously recognizing two receptors using high- and low-affinity ligands through ligating them in situ on the target cell surface. This de novo approach is inspired by the pretargeting strategy frequently applied in molecular imaging, and has now evolved as the basis of a new paradigm for visualizing target cells with a high imaging contrast. A distinct advantage of using a labeled low-affinity ligand such as glycan is that the excess labeled ligand can be washed away from the cells, whereas the ligand bound to the cell, even at the milli molar affinity level, can be anchored by a bioorthogonal reaction with a pretargeted high-affinity ligand on the surface. Consequently, nonspecific background is minimized, leading to improved imaging contrast. Importantly, despite previously unexplored for molecular imaging, a notoriously weak glycan/lectin interaction can now be utilized as a highly selective ligand to the targets.

Synthesis of a nitrogen analogue of sphingomyelin as a sphingomyelinase inhibitor.

[structure: see text] Sphingomyelin nitrogen analogue 1 was designed and synthesized as a sphingomyelinase inhibitor. The synthesis was established by continuous Hofmann rearrangement and Crutius rearrangement as key steps in constructing the 3-hydroxy-1,2-diamine structure in the backbone of 1. This analogue showed moderate inhibitory activity toward SMase isolated from B. cereus.

A thioethylalkylamido (TEA) thioester surrogate in the synthesis of a cyclic peptide via a tandem acyl shift.

The cyclic cystine-knot peptide, kalata B1, was synthesized by employing a novel Fmoc-compatible thioethylalkylamido (TEA) thioester surrogate via an N-S acyl shift followed by a thiol-thioester exchange reaction. TEA thioester surrogate is cost-effective, conveniently prepared in one-step with starting materials, readily available from commercial sources, and highly efficient in preparing peptide thioesters.

PHD finger of the SUMO ligase Siz/PIAS family in rice reveals specific binding for methylated histone H3 at lysine 4 and arginine 2.

We determined the three-dimensional structure of the PHD finger of the rice Siz/PIAS-type SUMO ligase, OsSiz1, by NMR spectroscopy and investigated binding ability for a variety of methylated histone H3 tails, showing that OsSiz1-PHD primarily recognizes dimethylated Arg2 of the histone H3 and that methylations at Arg2 and Lys4 reveal synergy effect on binding to OsSiz1-PHD. The K4 cage of OsSiz1-PHD for trimethylated Lys4 of H3K4me3 was similar to that of the BPTF-PHD finger, while the R2 pocket for Arg2 was different. It is intriguing that the PHD module of Siz/PIAS plays an important role, with collaboration with the DNA binding domain SAP, in gene regulation through SUMOylation of a variety of effectors associated with the methylated arginine-riched chromatin domains.

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-ß-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-ß-glucanase.

ß-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These ß-linked glucans are constantly exposed to degradation by various endo-ß-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-ß-glucanases inhibitors, such as xyloglucan-specific endo-ß-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.

Synthesis of sphingomyelin carbon analogues as sphingomyelinase inhibitors.

The highly efficient and stereocontrolled syntheses of sphingomyelin carbon analogues 1 and 2 were achieved by effectively utilizing Hofmann rearrangement of enantiomerically pure beta-hydroxyamide 7, which was prepared by an asymmetric hydrogenation of alpha-acyl-gamma-butyrolactone 9 and ring opening with NH(3). Intermediary isocyanate 6 was selectively trapped with the vicinal hydroxy group in an intramolecular fashion to produce an oxazolidinone derivative, 5. In the synthesis of a quite polar compound such as 1, a convenient one-pot procedure of the introduction of a benzyloxycarbonyl group into the hydroxy group resulting from the oxazolidinone ring opening is another key point, because, in addition to the efficiency, this protecting group was easily removable by a simple procedure and workup at the final step. Both synthesized compounds 1 and 2 showed moderate inhibitory activity toward sphingomyelinase from B. cereus.