A High‐Resolution Crystal Structure that Reveals Molecular Details of Target Recognition by the Calcium‐Dependent Lipopeptide Antibiotic Laspartomycin C

The calcium‐dependent antibiotics (CDAs) are an important emerging class of antibiotics. The crystal structure of the CDA laspartomycin C in complex with calcium and the ligand geranyl‐phosphate at a resolution of 1.28 Å is reported. This is the first crystal structure of a CDA bound to its bacterial target. The structure is also the first to be reported for an antibiotic that binds the essential bacterial phospholipid undecaprenyl phosphate (C₅₅‐P). These structural insights are of great value in the design of antibiotics capable of exploiting this unique bacterial target.     

Engineered biosynthesis of nonribosomal lipopeptides with modified fatty acid side chains

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains.     

Mechanistic insights into the C55-P targeting lipopeptide antibiotics revealed by structure–activity studies and high-resolution crystal structures

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C₅₅-P). In this study we describe the design and synthesis of new C₅₅-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C₅₅-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C₅₅-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.

Structural and mechanistic studies give new insights into calcium-dependent lipopeptide antibiotics that target C₅₅-P.     

Optimized conditions for 2‐aminobenzamide labeling and high‐performance liquid chromatography analysis of N‐acylated monosaccharides

The monosaccharides GlcNAc (N‐acetylglucosamine) and the home‐made GlcNC₁₆ (N‐palmitoyl‐D‐glucosamine) were labeled with 2‐AB (2‐aminobenzamide) by reductive amination of the sugar. The aldehyde group of the monosaccharide reacts with the amino group of 2‐AB, forming a Schiff base. In the second step, the Schiff base is reduced with sodium cyanoborohydride to yield a stable secondary amine. We describe here a simple and fast procedure. Previous studies reported the same labeling at high concentration (10^?1 M) during 30 h with further purification steps. In the present paper all operations were carried out in an Eppendorf tube and the reaction medium was directly analyzed without purification. Using the described protocol, the whole procedure can be accomplished in less than 6 h at 65°C at very low concentration (10^?4 M). For both GlcNC₁₆ and GlcNAc, the 2‐AB labeling conditions were optimized and, in addition, new conditions of high‐performance liquid chromatography analysis were developed. These N‐alkylated sugars were analyzed on reversed‐phase HPLC with fluorimetric detection at excitation and emission wavelengths of 340 and 400 nm, respectively. The separation was achieved on a C₁₈ column with a gradient mobile phase composed of water (0.1% formic acid)–methanol (volume varying) in less than 19 min with 12.5 and 18.3 min retention times for GlcNAc and GlcNC16, respectively. Positive‐ion electrospray ionization mass spectrometry (ESI‐MS) analysis enabled their structural determination. Copyright © 2009 John Wiley & Sons, Ltd.     

Enantiomeric excess of 1,2-diols by formation of cyclic boronates: an improved method

A reliable method for determining the enantiomeric composition of 1,2-diols by the formation of diastereomeric cyclic esters with boronic acid is described. Starting from a previously reported structure of boronic chiral derivatizing agent (CDA), seven structurally related racemic CDAs were synthesized and their discriminating ability towards diols measured. The most promising amongst these was synthesized in its enantiomerically pure form according to Matteson’s protocol for the stereoselective homologation of pinanediol boronates; this CDA quantitatively and rapidly reacts with 1,2-diols in very mild conditions affording a couple of diastereoisomers, whose composition can be determined via ¹H NMR analysis. In particular, an attractive feature is that the resonance used for the analysis originated from the CDA as a couple of baseline-separated singlets (Δδ up to 0.3 ppm) is useful for integration.     

Active site modification of the β-ketoacyl-ACP synthase FabF3 of Streptomyces coelicolor affects the fatty acid chain length of the CDA lipopeptides

Using site directed mutagenesis we altered an active site residue (Phe107) of the enzyme encoded by fabF3 (SCO3248) in the Streptomyces coelicolor gene cluster required for biosynthesis of the calcium dependent antibiotics (CDAs), successfully generating two novel CDA derivatives comprising truncated (C4) lipid side chains and confirming that fabF3 encodes a KAS-II homologue that is involved in determining CDA fatty acid chain length.     

Exploring the Effect of Bioisosteric Replacement of Carboxamide by a Sulfonamide Moiety on N‐Glycosidic Torsions and Molecular Assembly: Synthesis and X‐ray Crystallographic Investigation of N‐(β‐D‐Glycosyl)sulfonamides as N‐Glycoprotein Linkage Region Analogues

N‐Glycoprotein linkage region constituents, 2‐acetamido‐2‐deoxy‐β‐D ‐glucopyranose (GlcNAc) and asparagine (Asn) are conserved among all the eukaryotes. To gain a better understanding for nature’s choice of GlcNAcβAsn as linkage region constituents and inter‐ and intramolecular carbohydrate–protein interactions, a detailed systemic structural study of the linkage region conformation is essential. Earlier crystallographic studies of several N‐(β‐glycopyranosyl)alkanamides showed that N‐glycosidic torsion, ?_N, is influenced to a larger extent by structural variation in the sugar part than that of the aglycon moiety. To explore the effect of the bioisosteric replacement of a carboxamide group by a sulfonamide moiety on the N‐glycosidic torsions as well as on molecular assembly, several glycosyl methanesulfonamides and glycosyl chloromethanesulfonamides were synthesized as analogues of the N‐glycoprotein linkage region, and crystal structures of seven of these compounds have been solved. A comparative analysis of this series of crystal structures as well as with those of the corresponding alkanamido derivatives revealed that N‐glycosidic torsion, ?_N, does not alter significantly. Methanesulfonamido and chloromethanesulfonamido derivatives of GlcNAc display a different aglycon conformation compared to other sulfonamido analogues. This may be due to the cumulative effect of the direct hydrogen bonding between N1 and O1′ and C? H???O interactions of the aglycon chain, revealing the uniqueness of the GlcNAc as the linkage sugar.     

A chiral phosphazane reagent strategy for the determination of enantiomeric excess of amines

Methods for measuring enantiomeric excess (ee) of organic molecules by NMR spectroscopy provide rapid analysis using a standard technique that is readily available. Commonly this is accomplished by chiral derivatisation of the detector molecule (producing a chiral derivatisation agent, CDA), which is reacted with the mixture of enantiomers under investigation. However, these CDAs have almost exclusively been based on carbon frameworks, which are generally costly and/or difficult to prepare. In this work, a methodology based on the readily prepared inorganic cyclodiphosph(iii)azane CDA ClP(μ-N^tBu)₂POBorn (Born = endo-(1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl) is shown to be highly effective in the measurement of ee’s of chiral amines, involving in situ reaction of the chiral amines (R*NH₂) with the P–Cl bond of the CDA followed by quaternization of the phosphorus framework with methyl iodide. This results in sharp ³¹P NMR signals with distinct chemical shift differences between the diastereomers that are formed, which can be used to obtain the ee directly by integration. Spectroscopic, X-ray structural and DFT studies suggest that the NMR chemical shift differences between diastereomers is steric in origin, with the sharpness of these signals resulting from conformational locking of the bornyl group relative to the P₂N₂ ring induced by the presence of the P(v)-bonded amino group (R*NH). This study showcases cheap inorganic phosphazane CDAs as simple alternatives to organic variants for the rapid determination of ee.

The simple inorganic cyclodiphosph(iii)azane chiral derivatisation agent ClP(μ-^tBuN)₂POBorn (Born = endo-(1S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl) is shown to be effective in the measurement of ee’s of chiral amines using ³¹P NMR spectroscopy.     

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.     

Enhanced π‐back‐donation resulting in the trans labilization of a pyridine ligand in an N‐heterocyclic carbene (NHC) PdII precatalyst: a case study

The molecular structure of the benzimidazol‐2‐ylidene–PdCl₂–pyridine‐type PEPPSI (pyridine‐enhanced precatalyst, preparation, stabilization and initiation) complex {1,3‐bis[2‐(diisopropylamino)ethyl]benzimidazol‐2‐ylidene‐κC²}dichlorido(pyridine‐κN)palladium(II), [PdCl₂(C₅H₅N)(C₂₃H₄₀N₄)], has been characterized by elemental analysis, IR and NMR spectroscopy, and natural bond orbital (NBO) and charge decomposition analysis (CDA). Cambridge Structural Database (CSD) searches were used to understand the structural characteristics of the PEPPSI complexes in comparison with the usual N‐heterocyclic carbene (NHC) complexes. The presence of weak C—H…Cl‐type hydrogen‐bond and π–π stacking interactions between benzene rings were verified using NCI plots and Hirshfeld surface analysis. The preferred method in the CDA of PEPPSI complexes is to separate their geometries into only two fragments, i.e. the bulky NHC ligand and the remaining fragment. In this study, the geometry of the PEPPSI complex is separated into five fragments, namely benzimidazol‐2‐ylidene (Bimy), two chlorides, pyridine (Py) and the Pd^II ion. Thus, the individual roles of the Pd atom and the Py ligand in the donation and back‐donation mechanisms have been clearly revealed. The NHC ligand in the PEPPSI complex in this study acts as a strong σ‐donor with a considerable amount of π‐back‐donation from Pd to C_carbene. The electron‐poor character of Pd^II is supported by π‐back‐donation from the Pd centre and the weakness of the Pd—N(Py) bond. According to CSD searches, Bimy ligands in PEPPSI complexes have a stronger σ‐donating ability than imidazol‐2‐ylidene ligands in PEPPSI complexes.     

Reaction of alcohols (via the Mitsunobu reaction) and alkyl halides with chiral selone derivatizing agents

Coupling of a selone chiral derivatizing agent (CDA) to d,l-alkyl halides gives Se-alkylated adducts in yields ranging from 76–97%. Reaction of d,l-alcohols with the selone CDAs via the Mitsunobu reaction has given rise to Se-alkylated adducts in yields ranging from 82–92%. Examination of the ⁷⁷Se NMR spectra of the resulting diastereomeric adducts indicates that discrimination of remotely disposed chiral centers is possible using this technique.     

Glycosidic linkage,N‐acetyl side‐chain,and other structural properties of methyl 2‐acetamido‐2‐deoxy‐β‐d‐glucopyranosyl‐(1→4)‐β‐d‐mannopyranoside monohydrate and related compounds

The crystal structure of methyl 2‐acetamido‐2‐deoxy‐β‐d ‐glycopyranosyl‐(1→4)‐β‐d ‐mannopyranoside monohydrate, C₁₅H₂₇NO₁₁·H₂O, was determined and its structural properties compared to those in a set of mono‐ and disaccharides bearing N‐acetyl side‐chains in βGlcNAc aldohexopyranosyl rings. Valence bond angles and torsion angles in these side chains are relatively uniform, but C—N (amide) and C—O (carbonyl) bond lengths depend on the state of hydrogen bonding to the carbonyl O atom and N—H hydrogen. Relative to N‐acetyl side chains devoid of hydrogen bonding, those in which the carbonyl O atom serves as a hydrogen‐bond acceptor display elongated C—O and shortened C—N bonds. This behavior is reproduced by density functional theory (DFT) calculations, indicating that the relative contributions of amide resonance forms to experimental C—N and C—O bond lengths depend on the solvation state, leading to expectations that activation barriers to amide cis–trans isomerization will depend on the polarity of the environment. DFT calculations also revealed useful predictive information on the dependencies of inter‐residue hydrogen bonding and some bond angles in or proximal to β‐(1→4) O‐glycosidic linkages on linkage torsion angles ? and ψ. Hypersurfaces correlating ? and ψ with the linkage C—O—C bond angle and total energy are sufficiently similar to render the former a proxy of the latter.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

Highly Efficient Synthesis of Multiantennary Bisected N‐glycans Based on Imidates

The occurrence of N‐glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N‐glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N‐glycans of the complex type by employing modular TFAc‐protected donors for all antennae. The TFAc‐protected N‐glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N‐glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one‐pot double glycosylation. With this new method, rare N‐glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.     

Structure‐property relationships for film‐forming poly(amide imide)s

Poly(amide imide)s from dichloro‐dianhydrides of tri‐mellitimide‐ and/or pyromellitimide‐N‐acetic acids (TMA and PMA), linear aromatic dimimines (LAD) and/or cardo diamine (CDA), 9,9′‐bis‐phenylfluorene (series PAI‐1); a similar series (PAI‐II) from dichloro‐dianhydride of trimellitimide‐N‐benzoic acid (TMB), LAD and/or CDA; and a series of coPAIs from dichloro‐dianhydride of TMA and mixtures of diphenylmethane diamine (DPA) with CDA were synthesized by low‐temperature reaction in dimethyl acetamide (DMAA). The state of aggregation of PAIs and coPAIs in dilute and semi‐dilute solutions of DMAA was characterized by measurements of specific heat capacity in the temperature interval 150‐370 K, of heats of dilution in DMAA at room temperature, and of apparent viscosity in the temperature interval 288‐373 K. The state of aggregation of PAIs and coPAIs in thin films cast from DMAA solutions was characterized by wide‐angle and small‐angle X‐ray diffraction, dynamic mechanical analysis, broad‐band dielectric spectroscopy and gas transport properties. It is concluded that film‐forming PAIs and coPAIs have a reasonably good potential as high‐temperature dielectrics and membrane materials for gas separation.     

Novel phosphate–phosphonate hybrid nanomaterials applied to biology

A new process for preparing oligonucleotide arrays is described that uses surface grafting chemistry which is fundamentally different from the electrostatic adsorption and organic covalent binding methods normally employed. Solid supports are modified with a mixed organic/inorganic zirconium phosphonate monolayer film providing a stable, well-defined interface. Oligonucleotide probes terminated with phosphate are spotted directly to the zirconated surface forming a covalent linkage. Specific binding of terminal phosphate groups with minimal binding of the internal phosphate diesters has been demonstrated. On the other hand, the reaction of a bisphosphonate bone resorption inhibitor (Zoledronate) with calcium deficient apatites (CDAs) was studied as a potential route to local drug delivery systems active against bone resorption disorders. A simple mathematical model of the Zoledronate/CDA interaction was designed that correctly described the adsorption of Zoledronate onto CDAs. The resulting Zoledronate-loaded materials were found to release the drug in different phosphate-containing media, with a satisfactory agreement between experimental data and the values predicted from the model.     

Monitoring Glycan–Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH–π Interactions

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan–protein interactions that involve N‐acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc‐containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal ¹H and ¹⁹F atoms that allow both ¹H and ¹⁹F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc‐ (or GalNAc‐) moieties over a range of binding affinities. The CHF₂CONH‐ moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic‐sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF₃CONH‐ analogue differs more significantly.     

Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water‐soluble carbohydrate receptors (“synthetic lectins”). Both systems show outstanding affinities for derivatives of N‐acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc‐β‐OMe with K_a≈20 000 m ^?1, whereas the other one binds an O‐GlcNAcylated peptide with K_a≈70 000 m ^?1. These values substantially exceed those usually measured for GlcNAc‐binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl–peptide complex can be explained by extra‐cavity interactions, raising the possibility of a family of complementary receptors for O‐GlcNAc in different contexts.     

Insights into the Dynamics and Molecular Recognition Features of Glycopeptides by Protein Receptors: The 3D Solution Structure of Hevein Bound to the Trisaccharide Core of N‐Glycoproteins

Protein‐carbohydrate interactions are at the heart of a variety of essential molecular recognition events. Hevein, a model lectin related to the superantigen family, recognizes the trisaccharide core of N‐glycoproteins ( 1 ). A combined approach of NMR spectroscopy and molecular modeling has permitted us to demonstrate that an Asn‐linked Man(GlcNAc)₂ ( 2 ) is bound with even higher affinity than (GlcNAc)₃. The molecular recognition process entails conformational selection of only one of the possibilities existing for chitooligosaccharides. The deduced 3D structure of the hevein/ 2 complex permits the extension of polypeptide chains from the Asn moiety of 2 , as well as glycosylation at Man O‐3 and Man O‐6 of the terminal sugar. Given the ubiquity of the Man(GlcNAc)₂ core in all mammalian N‐glycoproteins, the basic recognition mode presented herein might be extended to a variety of systems with biomedical importance.