Preactivation‐Based,One‐Pot Combinatorial Synthesis of Heparin‐like Hexasaccharides for the Analysis of Heparin–Protein Interactions

Heparin (HP) and heparan sulfate (HS) play important roles in many biological events. Increasing evidence has shown that the biological functions of HP and HS can be critically dependent upon their precise structures, including the position of the iduronic acids and sulfation patterns. However, unraveling the HP code has been extremely challenging due to the enormous structural variations. To overcome this hurdle, we investigated the possibility of assembling a library of HP/HS oligosaccharides using a preactivation‐based, one‐pot glycosylation method. A major challenge in HP/HS oligosaccharide synthesis is stereoselectivity in the formation of the cis‐1,4‐linkages between glucosamine and the uronic acid. Through screening, suitable protective groups were identified on the matching glycosyl donor and acceptor, leading to stereospecific formation of both the cis‐1,4‐ and trans‐1,4‐linkages present in HP. The protective group chemistry designed was also very flexible. From two advanced thioglycosyl disaccharide intermediates, all of the required disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified building‐block preparation. Furthermore, the reactivity‐independent nature of the preactivation‐based, one‐pot approach enabled us to mix the building blocks. This allowed rapid assembly of twelve HP/HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. The speed and the high yields achieved in glycoassembly without the need to use a large excess of building blocks highlighted the advantages of our approach, which can be of general use to facilitate the study of HP/HS biology. As a proof of principle, this panel of hexasaccharides was used to probe the effect of backbone sequence on binding with the fibroblast growth factor‐2 (FGF‐2). A trisaccharide sequence of 2‐O‐sulfated iduronic acid flanked by N‐sulfated glucosamines was identified to be the minimum binding motif and N‐sulfation was found to be critical. This provides useful information for further development of more potent compounds towards FGF‐2 binding, which can have potential applications in wound healing and anticancer therapy.     

Deorphaning Pyrrolopyrazines as Potent Multi‐Target Antimalarial Agents

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC₅₀ values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant‐garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non‐homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non‐mevalonate pathway of isoprenoid biosynthesis, and multi‐kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand‐ and structure‐based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents.     

Protecting‐Group‐Controlled Enzymatic Glycosylation of Oligo‐N‐Acetyllactosamine Derivatives

We describe a chemoenzymatic strategy that can give a library of differentially fucosylated and sialylated oligosaccharides starting from a single chemically synthesized tri‐N‐acetyllactosamine derivative. The common precursor could easily be converted into 6 different hexasaccharides in which the glucosamine moieties are either acetylated (GlcNAc) or modified as a free amine (GlcNH₂) or Boc (GlcNHBoc). Fucosylation of the resulting compounds by a recombinant fucosyl transferase resulted in only modification of the natural GlcNAc moieties, providing access to 6 selectively mono‐ and bis‐fucosylated oligosaccharides. Conversion of the GlcNH₂ or GlcNHBoc moieties into the natural GlcNAc, followed by sialylation by sialyl transferases gave 12 differently fucosylated and sialylated compounds. The oligosaccharides were printed as a microarray that was probed by several glycan‐binding proteins, demonstrating that complex patterns of fucosylation can modulate glycan recognition.     

Protection‐Group‐Free Synthesis of Sequence‐Defined Macromolecules via Precision λ‐Orthogonal Photochemistry

An advanced light‐induced avenue to monodisperse sequence‐defined linear macromolecules via a unique photochemical protocol is presented that does not require any protection‐group chemistry. Starting from a symmetrical core unit, precision macromolecules with molecular weights up to 6257.10 g mol^?1 are obtained via a two‐monomer system: a monomer unit carrying a pyrene functionalized visible light responsive tetrazole and a photo‐caged UV responsive diene, enabling an iterative approach for chain growth; and a monomer unit equipped with a carboxylic acid and a fumarate. Both light‐induced chain growth reactions are carried out in a λ‐orthogonal fashion, exciting the respective photosensitive group selectively and thus avoiding protecting chemistry. Characterization of each sequence‐defined chain (size‐exclusion chromatography (SEC), high‐resolution electrospray ionization mass spectrometry (ESI‐MS), and NMR spectroscopy), confirms the precision nature of the macromolecules.     

IMSPeptider: A computational peptide collision cross‐section area calculator based on a novel molecular dynamics simulation protocol

Introduction of ion mobility mass spectrometry (IMS/MS) into the proteomic workflow provides an orthogonal separation to the widely used LC‐MS platforms. IMS also provides structural information that could facilitate peptide identification. However, the lack of tools capable of predictive power in a high‐throughput fashion makes peptide global profiling quite challenging. To target this issue, a computational workflow was developed based on biophysical principles to predict the collision cross‐section area (CCS) of peptides as measured from IMS/MS experiments. Hosted on a web server, it allows the user to input a primary sequence (query) and retrieve information on peptide structure, sequence, and corresponding CCS. The current version is designed to identify peptide sequences up to 23 residues in length, in its higher charge state, based on a match of the molecule m/z and CCS. The protocol was validated against a 128‐sequences‐dataset and CCS predicted within 2.8% average error. © 2013 Wiley Periodicals, Inc.     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

A New Benzotriazole‐Mediated Stereoflexible Gateway to Hetero‐2,5‐diketopiperazines

Open chain Cbz‐L ‐aa¹‐L ‐Pro‐Bt (Bt=benzotriazole) sequences were converted into either the corresponding trans‐ or cis‐fused 2,5‐diketopiperazines (DKPs) depending on the reaction conditions. Thermodynamic tandem cyclization/epimerization afforded selectively the corresponding trans‐DKPs (69–75 %). Complementarily, tandem deprotection/cyclization led to the cis‐DKPs (65–72 %). A representative set of proline‐containing cis‐ and trans‐DKPs has been prepared. A mechanistic investigation, based on chiral HPLC, kinetics, and computational studies enabled a rationalization of the results.     

Highly Efficient Regio- and Stereoselective Synthesis of β-（1 →6）-Branched β-（1 →3）-Linked Glucohexaose and Its Analogue

A β-（1→）6）-branched β-（1→）3）-linked glucohexaose （1） and its lauryl glycoside （2）, present in many biologically active polysaccharides from traditional herbal medicines such as Ganoderma lucidum, Schizophyllum commune and Lentinus edodes, were highly efficiently synthesized. Coupling of 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl- （1--）3）-2-O-benzoyl-4,6-O-benzylidene-a-D-glucopyranosyl trichloroacetimidate （7） with 3,6-branched acceptors 8 and 12 gave β-（1→）3）-linked pentasaccharides （9） and （13）, then via simple chemical transformation 4＇,6＇-OH pentasaccharide acceptors 10 and 14 were obtained. Regio- and stereoselective coupling of 3 with 10 and 14 gave β-（1→）3）-linked hexasaccharides （11） and （15） as the major products. Deprotection of 11 and 15 provided the target sugar 1 and 2. Thus, a new method for the preparation of this kind of compounds was developed.     

Analysis of variation in the ovine ultra‐high sulphur keratin‐associated protein KAP5‐4 gene using PCR‐SSCP technique

Keratin‐associated proteins (KAPs) are one of the main structural components of the wool fibre. Variation in the KAP genes (KRTAPs) may affect the structure of KAPs and hence wool characteristics. In this study, we used PCR‐SSCP to analyse ovine KRTAP5‐4, a gene encoding a member of the KAP5 family. Five different PCR‐SSCP patterns were detected in the 250 sheep that were analysed. Either one or a combination of two patterns was observed for each sheep, which was consistent with these sheep being either homozygous or heterozygous at this locus. DNA sequencing revealed that these patterns represent five different DNA sequences. One of the sequences was identical to a published ovine KRTAP5‐4 sequence. The remaining four were unique, but shared a high homology with the published ovine KRTAP5‐4 sequence, suggesting that these sequences represent allelic variants of KRTAP5‐4. There were a total of six SNPs and one length polymorphism in the sequences. Of the five SNPs found in the coding region, four were non‐synonymous SNPs and would result in amino acid changes. The length polymorphism would affect the cysteine content of the putative peptide and this along with the SNPs may have an impact on the structure of KAP5‐4, and hence affect wool traits.     

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N‐methylpyrrole–N‐methylimidazole polyamide seco‐1,2,9,9a‐tetrahydrocyclopropa[1,2‐c]benz[1,2‐e]indol‐4‐one conjugate (conjugate 4 ) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer‐minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one‐base‐pair mismatch sequence. Conjugate 4 was modified for next‐generation sequencing. Bind‐n‐Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild‐type sequence.     

Design,Synthesis, and Herbicidal Activities of 3‐Aryl‐4‐substituted‐5‐[3‐(trifluoromethyl)phenoxy]‐1,2,4‐triazoles

In order to find novel bleaching herbicide lead compounds, a series of novel 3‐aryl‐4‐substituted‐5‐[3‐(trifluoromethyl)phenoxy]‐1,2,4‐triazoles were designed and synthesized by the multi‐step reactions. N‐(Arylformamido)phenylthioureas undergo ring closure in the presence of sodium hydroxide to generate 3‐aryl‐4‐substituted‐4H‐[1,2,4]triazol‐5‐thiols 1 , which reacted with methyl sulfate in the presence of K₂CO₃ to give 3‐aryl‐5‐methylsulfanyl‐4‐substituted‐4H‐[1,2,4]triazoles 2 . The target compounds 4 were synthesized by the oxidation of 2 in the presence of H₂O₂ and Na₂WO₄, followed by the substitution with 3‐(trifluoromethyl)phenol in moderate to good yields. Their structures were confirmed by IR, ¹H NMR, EI–MS, and elemental analyses. The preliminary bioassay indicated that some of them displayed moderate to good selective herbicidal activity against Brassica campestris L at the concentration of 100 µg/mL. Compounds 4c and 4i possessed 75.0% and 82.6% inhibition against Brassica campestris L at the concentration of 100 µg/mL. However, the target compounds 4 showed weak herbicidal activity against Echinochloa crus‐galli at the concentration of 100 and 10 µg/mL.     

Affinity‐Driven Covalent Modulator of the Glyceraldehyde‐3‐Phosphate Dehydrogenase (GAPDH) Cascade

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)‐(+)‐isochaihulactone ( 1 ) exhibited significant inhibition against multiple‐drug‐resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off‐target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,β‐unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI‐oriented activity‐based proteome profiling. By MS/MS and computer‐guided molecular biology approaches, an affinity‐driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the “ON/OFF” switch of apoptosis through non‐canonically nuclear GAPDH translocation, which bypasses the common apoptosis‐resistant route of MDR cancers.     

Sequence‐Dependent dsDNA‐Templated Formation of Fluorescent Copper Nanoparticles

There are only a few systematic rules about how to selectively control the formation of DNA‐templated metal nanoparticles (NPs) by varying sequence combinations of double‐stranded DNA (dsDNA), although many attempts have been made. Herein, we develop a facile method for sequence‐dependent formation of fluorescent CuNPs by using dsDNA as templates. Compared with random sequences, AT sequences are better templates for highly fluorescent CuNPs. Other specific sequences, for example, GC sequences, do not induce the formation of CuNPs. These results shed light on directed DNA metallization in a sequence‐specific manner. Significantly, both the fluorescence intensity and the fluorescence lifetime of CuNPs can be tuned by the length or the sequence of dsDNA. In order to demonstrate the promising practicality of our findings, a sensitive and label‐free fluorescence nuclease assay is proposed.     

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

Longestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.     

Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

Formylglycine‐generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site‐specific oxidation of a cysteine residue to the aldehyde‐containing amino acid C^α‐formylglycine (FGly). This non‐canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine‐generating enzyme (FGE) and the iron‐sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.     

LC-MS<Superscript><Emphasis Type="BoldItalic">n</Emphasis></Superscript> Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS ⁿ . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS ⁿ fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS ⁿ experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS ⁿ methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.     

Chondroitin Sulfate Tetrasaccharides: Synthesis,Three‐Dimensional Structure and Interaction with Midkine

The biological activity of midkine, a cytokine implicated in neuro‐ and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate‐like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS‐like oligosaccharides in the sequence GalNAc–GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS‐like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well‐defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.     

A Visualizable Chain‐Terminating Inhibitor of Glycosaminoglycan Biosynthesis in Developing Zebrafish

Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAG) are proteoglycan‐associated polysaccharides with essential functions in animals. They have been studied extensively by genetic manipulation of biosynthetic enzymes, but chemical tools for probing GAG function are limited. HS and CS possess a conserved xylose residue that links the polysaccharide chain to a protein backbone. Here we report that, in zebrafish embryos, the peptide‐proximal xylose residue can be metabolically replaced with a chain‐terminating 4‐azido‐4‐deoxyxylose (4‐XylAz) residue by administration of UDP‐4‐azido‐4‐deoxyxylose (UDP‐4‐XylAz). UDP‐4‐XylAz disrupted both HS and CS biosynthesis and caused developmental abnormalities reminiscent of GAG biosynthesis and laminin mutants. The azide substituent of protein‐bound 4‐XylAz allowed for rapid visualization of the organismal sites of chain termination in vivo through bioorthogonal reaction with fluorescent cyclooctyne probes. UDP‐4‐XylAz therefore complements genetic tools for studies of GAG function in zebrafish embryogenesis.     

Modulation of Quorum Sensing in a Gram‐Positive Pathogen by Linear Molecularly Imprinted Polymers with Anti‐infective Properties

We describe the development, characterization, and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as an anti‐infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae . The LMIP is prepared (polymerized) in presence of a template molecule, but unlike in traditional molecular imprinting approaches, no cross‐linker is used. This results in soluble low‐molecular‐weight oligomers that can act as a therapeutic agent in vitro and in vivo. The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP. These findings provide a proof of principle that LMIPs can be used to block QS, thus setting the stage for the development of LMIPs a novel drug‐discovery platform and class of materials to target Gram‐positive pathogens.     

Synthesis and Structural Analysis of Aspergillus fumigatus Galactosaminogalactans Featuring α‐Galactose, α‐Galactosamine and α‐N‐Acetyl Galactosamine Linkages

Galactosaminogalactan (GAG) is a prominent cell wall component of the opportunistic fungal pathogen Aspergillus fumigatus. GAG is a heteropolysaccharide composed of α‐1,4‐linked galactose, galactosamine and N‐acetylgalactosamine residues. To enable biochemical studies, a library of GAG‐fragments was constructed featuring specimens containing α‐galactose‐, α‐galactosamine and α‐N‐acetyl galactosamine linkages. Key features of the synthetic strategy include the use of di‐tert‐butylsilylidene directed α‐galactosylation methodology and regioselective benzoylation reactions using benzoyl‐hydroxybenzotriazole (Bz‐OBt). Structural analysis of the Gal, GalN and GalNAc oligomers by a combination of NMR and MD approaches revealed that the oligomers adopt an elongated, almost straight, structure, stabilized by inter‐residue H‐bonds, one of which is a non‐conventional C?H???O hydrogen bond between H5 of the residue (i+1) and O3 of the residue (i). The structures position the C‐2 substituents almost perpendicular to the oligosaccharide main chain axis, pointing to the bulk solvent and available for interactions with antibodies or other binding partners.