Comprehensive characterization of C‐glycosyl flavones in wheat (Triticum aestivum L.) germ using UPLC‐PDA‐ESI/HRMSn and mass defect filtering

A comprehensive characterization of C‐glycosyl flavones in wheat germ has been conducted using multi‐stage high resolution mass spectrometry (HRMSⁿ) in combination with a mass defect filtering (MDF) technique. MDF performed the initial search of raw data with defined C‐glycosyl flavone mass windows and mass defect windows to generate the noise‐reduced data focusing on targeted flavonoids. The high specificity of the exact mass measurement permits the unambiguous discrimination of acyl groups (nominal masses of 146, 162 and 176.) from sugar moieties (rhamnose, glucose or galactose and glucuronic acid). A total of 72 flavone C‐glycosyl derivatives, including 2 mono‐C‐glycosides, 34 di‐C‐glycosides, 15 tri‐glycosides, 14 acyl di‐C‐glycosides and 7 acyl tri‐glycosides, were characterized in wheat germ, some of which were considered to be important marker compounds for differentiation of whole grain and refined wheat products. The 7 acylated mono‐O‐glycosyl‐di‐C‐glycosyl flavones and some acylated di‐C‐glycosyl flavones are reported in wheat for the first time. The frequent occurrence of numerous isomers is a remarkable feature of wheat germ flavones. Both UV and mass spectra are needed to maximize the structure information obtained for data interpretation. Copyright © 2016 John Wiley & Sons, Ltd.     

MALDI mass spectrometry‐based sequence analysis of arginine‐containing glycopeptides: improved fragmentation of glycan and peptide chains by modifying arginine residue

This paper describes an improved method for the sequence analysis of Arg‐containing glycopeptide by MALDI mass spectrometry (MS). The method uses amino group derivatization (4‐aza‐6‐(2,6‐dimethyl‐1‐piperidinyl)‐5‐oxohexanoic acid N‐succinimidyl ester) and removal (carboxypeptidase B) or modification (peptidylarginine deiminase 4) of the arginine residue of the peptide. The derivatization attaches a basic tertiary amine moiety onto the peptides, and the enzymatic treatment removes or modifies the arginine residue. Fragmentation of the resulting glycopeptide under low‐energy collision‐induced dissociation yielded a simplified ion series of both the glycan and the peptide that can facilitate their sequencing. The feasibility of the method was studied using α₁‐acid glycoprotein‐derived N‐linked glycopeptides, and glycan and peptide in each glycopeptide were successfully sequenced by MALDI tandem MS (MS/MS). Copyright © 2013 John Wiley & Sons, Ltd.     

Application of HR‐MAS NMR in the solid‐phase synthesis of a glycopeptide using Sieber amide resin

The solid‐phase synthesis (SPS) of a structurally complex glycopeptide, using Sieber amide resin, was monitored by high resolution magic angle spinning NMR, demonstrating the further application of this technique. A synthetic peptidoglycan derivative, a precursor of a biologically active PGN, known to be involved in the cellular recognition, was prepared by SPS. The synthesis involved the preparation of an N‐alloc glucosamine moiety and the synthesis of a simple amino acid sequence L ‐Ala‐D ‐Glu‐L ‐Lys‐D ‐Ala‐D ‐Ala. Last step consisted the coupling, on solid‐phase, of the protected muramyl unit to the peptide chain. Proton spectra with good suppression of the polystyrene signals in swollen resin samples were obtained in DMF‐d₇ as a solvent and by using a nonselective 1D TOCSY/DIPSI‐2 scheme, thus allowing to follow the SPS without losses of compound and cleavage from the resin. The assignment of the proton spectra of the resin‐bound amino acid sequence and of the bound glycopeptide was achieved through the combination of MAS COSY, TOCSY and NOESY. Copyright © 2010 John Wiley & Sons, Ltd.     

New hydrophobic L‐amino acid salts: maleates of L‐leucine,L‐isoleucine and L‐norvaline

Crystals of maleates of three amino acids with hydrophobic side chains [L‐leucenium hydrogen maleate, C₆H₁₄NO₂⁺·C₄H₃O₄⁻, (I), L‐isoleucenium hydrogen maleate hemihydrate, C₆H₁₄NO₂⁺·C₄H₃O₄⁻·0.5H₂O, (II), and L‐norvalinium hydrogen maleate–L‐norvaline (1/1), C₅H₁₁NO₂⁺·C₄H₃O₄⁻·C₅H₁₂NO₂, (III)], were obtained. The new structures contain C₂²(12) chains, or variants thereof, that are a common feature in the crystal structures of amino acid maleates. The L‐leucenium salt is remarkable due to a large number of symmetrically non‐equivalent units (Z′ = 3). The L‐isoleucenium salt is a hydrate despite the fact that L‐isoleucine is a nonpolar hydrophobic amino acid (previously known amino acid maleates formed hydrates only with lysine and histidine, which are polar and hydrophilic). The L‐norvalinium salt provides the first example where the dimeric cation L‐Nva...L‐NvaH⁺ was observed. All three compounds have layered noncentrosymmetric structures. Preliminary tests have shown the presence of the second harmonic generation (SGH) effect for all three compounds.     

Synthesis and Conformational Analysis of Hybrid α/β‐Dipeptides Incorporating S‐Glycosyl‐β2,2‐Amino Acids

We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α‐amino acid attached to a quaternary glyco‐β‐amino acid. In particular, we combined a S‐glycosylated β^2,2‐amino acid and two different types of α‐amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β‐dipeptides. The key step in the synthesis involved the ring‐opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur‐containing nucleophile by using 1‐thio‐β‐D ‐glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time‐averaged restraints (MD‐tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β‐amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α‐amino acids due to the presence of CH–π interactions between the phenyl or indole ring and the methyl groups of the β‐amino acid unit.     

Serine versus Threonine Glycosylation with α‐O‐GalNAc: Unexpected Selectivity in Their Molecular Recognition with Lectins

The molecular recognition of several glycopeptides bearing Tn antigen (α‐O‐GalNAc‐Ser or α‐O‐GalNAc‐Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn‐threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn‐serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.     

Preparation of S‐ and N‐Linked Glycosylated Amino Acid Building Blocks for Solid‐phase Glycopeptide Library Synthesis*

A general route for the preparation of 1,2‐trans‐linked S‐glycosylated amino acid building blocks by a Lewis‐acid‐promoted condensation of peracetylated glycosyl donors and N ^α‐Fmoc‐Cys‐OH, in good overall yield, is described. In addition, a short and time‐efficient route was applied for the synthesis of N‐glycosylated amino acid building blocks in good overall yields by coupling unprotected glycosylamines and N ^α‐Fmoc‐Asp(OH)‐O ^t Bu using TBTU activation.     

Deciphering the Non‐Equivalence of Serine and Threonine O‐Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti‐MUC1 Antibody

The structural features of MUC1‐like glycopeptides bearing the Tn antigen (α‐O‐GalNAc‐Ser/Thr) in complex with an anti MUC‐1 antibody are reported at atomic resolution. For the α‐O‐GalNAc‐Ser derivative, the glycosidic linkage adopts a high‐energy conformation, barely populated in the free state. This unusual structure (also observed in an α‐S‐GalNAc‐Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α‐O‐GalNAc‐Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti‐MUC1 antibodies.     

Unexpected proline coordination in the copper chain polymer [Cu(μ‐Cl)2(μ‐DL‐proline‐κ2O:O′)]1∞

In catena‐poly[copper(II)‐di‐μ‐chlorido‐μ‐proline‐κ²O:O′], [CuCl₂(C₅H₉NO₂)]_n, two symmetry‐independent metal cations adopt distorted octahedral coordination, typical for d⁹ Jahn–Teller systems. Each chloride bridge is involved in both a short and a very long interaction with a Cu^II centre. The centrosymmetric crystal structure contains homochiral chains of opposite handedness which extend along the shortest lattice parameter (i.e. a). The O:O′‐bridging coordination mode of proline, although a common motif for such complexes in general, is remarkable for Cu^II; the vast majority of amino acid derivatives of this cation are characterized by N,O‐chelation.     

Four oxoindole‐linked α‐alkoxy‐β‐amino acid derivatives

Four structures of oxoindolyl α‐hydroxy‐β‐amino acid derivatives, namely, methyl 2‐{3‐[(tert‐butoxycarbonyl)amino]‐1‐methyl‐2‐oxoindolin‐3‐yl}‐2‐methoxy‐2‐phenylacetate, C₂₄H₂₈N₂O₆, (I), methyl 2‐{3‐[(tert‐butoxycarbonyl)amino]‐1‐methyl‐2‐oxoindolin‐3‐yl}‐2‐ethoxy‐2‐phenylacetate, C₂₅H₃₀N₂O₆, (II), methyl 2‐{3‐[(tert‐butoxycarbonyl)amino]‐1‐methyl‐2‐oxoindolin‐3‐yl}‐2‐[(4‐methoxybenzyl)oxy]‐2‐phenylacetate, C₃₁H₃₄N₂O₇, (III), and methyl 2‐[(anthracen‐9‐yl)methoxy]‐2‐{3‐[(tert‐butoxycarbonyl)amino]‐1‐methyl‐2‐oxoindolin‐3‐yl}‐2‐phenylacetate, C₃₈H₃₆N₂O₆, (IV), have been determined. The diastereoselectivity of the chemical reaction involving α‐diazoesters and isatin imines in the presence of benzyl alcohol is confirmed through the relative configuration of the two stereogenic centres. In esters (I) and (III), the amide group adopts an anti conformation, whereas the conformation is syn in esters (II) and (IV). Nevertheless, the amide group forms intramolecular N—H...O hydrogen bonds with the ester and ether O atoms in all four structures. The ether‐linked substituents are in the extended conformation in all four structures. Ester (II) is dominated by intermolecular N—H...O hydrogen‐bond interactions. In contrast, the remaining three structures are sustained by C—H...O hydrogen‐bond interactions.     

Synthetic Studies toward C‐Glucosidic Ellagitannins: A Biomimetic Total Synthesis of 5‐O‐Desgalloylepipunicacortein A

C‐glucosidic ellagitannins constitute a subclass of bioactive polyphenolic natural products with strong antioxidant properties, as well as promising antitumoral and antiviral activities that are related to their capacity to interact with both functional and structural proteins. To date, most synthetic efforts toward ellagitannins have concerned glucopyranosic species. The development of a synthetic strategy to access C‐glucosidic ellagitannins, whose characteristic structural feature includes an atropoisomeric hexahydroxydiphenoyl (HHDP) or a nonahydroxyterphenoyl (NHTP) unit that is linked to an open‐chain glucose core by a C‐aryl glucosidic bond, is described herein. The total synthesis of the biarylic HHDP‐containing 5‐O‐desgalloylepipunicacortein A ( 1 β ) was achieved by either using the natural ellagic acid bis‐lactone as a precursor of the requested HHDP unit or by implementing an atroposelective intramolecular oxidative biarylic coupling to forge this HHDP unit. Both routes converged in the penultimate step of this synthesis to enable a biomimetic formation of the key C‐aryl glucosidic bond in the title compound.     

The tetrapeptide Z‐Leu‐Aib‐Pro‐Val‐OBg monohydrate

The intramolecular hydrogen‐bonding pattern of Z‐Leu‐Aib‐Pro‐Val‐OBg monohydrate [(N‐benzhydryl­amino)­carbonyl­methyl N‐benzyl­oxy­carbonyl‐α‐amino­isobutyryl­prolyl­valinate monohydrate], C₄₃H₅₅N₅O₈·H₂O, is unusual for a tetrapeptide because, in addition to a 14 hydrogen bond, a second hydrogen bond of the type 15 is formed. This folding reflects the intramolecular hydrogen‐bonding pattern that this amino acid sequence adopts in the naturally occurring peptaibol alamethicin.     

The Synthesis of 1,4‐Bis[N‐(4/6‐Substituted Benzothiazole‐2‐yl)‐N′‐Glycosylguanidino]Benzenes

The starting material O‐protected glycosyl isothiocyanate ( 1?3 ) was refluxed with 1,4‐diaminobenzene in CHCl₃ under nitrogen atmosphere to give 1,4‐bis(N‐glycosyl)thioureidobenzene ( 4?6 ). Then 1,4‐bis[N‐(4/6‐substituted benzothiazole‐2‐yl)‐N′‐glycosylguanidino]benzenes ( 8a?8e , 9a?9e , 10a?10e ) were obtained in good yield by reaction of compounds ( 4?6 ) with 2‐amino‐4/6‐benzothizoles ( 7a?7e ) and HgCl₂ in the presence of TEA in DMF. The structures of all 18 new compounds were confirmed by IR, ¹H NMR, LC‐MS and elemental analysis. The bioactivity of anti‐HIV‐1 protease (HIV‐1 PR) and against angiotensin converting enzyme (ACE) have been evaluated.     

High resolution‐HMBC (HR‐HMBC), a new method for measuring heteronuclear long‐range coupling constants

A useful pulse sequence for measuring long‐range C? H coupling constants (J_{C? H}) named high resolution‐HMBC (HR‐HMBC) has been developed. In this pulse sequence, the J‐scaling pulse [(nt₁)/2? 180° (H/C) ? (nt₁)/2] is incorporated after the spin evolution period, and then followed by an ¹H 180° pulse to reverse the magnetization of J_{C? H} couplings. As a result, splittings of the cross peaks due to the long‐range J_{C? H} are realigned with separations of nJ_{C? H} along the F₁ dimension, and thus even the small long‐range J_{C? H} values can easily be determined. The efficiency of measuring the long‐range J_{C? H} using the proposed pulse sequences has been demonstrated in application to the complicated natural product, portmicin. Copyright © 2010 John Wiley & Sons, Ltd.     

Selective J‐resolved HMBC,an efficient method for measuring heteronuclear long‐range coupling constants

An efficient pulse sequence for measuring long‐range C? H coupling constants (J_{C? H}) named selective J‐resolved HMBC has been developed by replacing a ¹H 180° pulse with a selective ¹H 180° pulse and the HMBC pulse scheme with the constant time (CT) HMBC employed in the J‐resolved HMBC pulse sequence that we reported previously. The novel pulse sequence providing only long‐range J_{C? H} cross peaks for easy and accurate analysis enables to overcome disadvantages of the previous HMBC‐based pulse sequences (J‐resolved HMBC‐1) along with maintaining high sensitivity. The efficiency of measuring long‐range J_{C? H} using the proposed pulse sequence has been demonstrated in applications to the complicated natural products, portmicin and monazomycin. Copyright © 2009 John Wiley & Sons, Ltd.     

A Catalytic and Stereoselective Glycosylation with β‐Glycosyl Fluorides

A catalytic and stereoselective glycosylation of several glycosyl acceptors with β‐D ‐glycosyl fluoride was successfully performed in the presence of a catalytic amount of trityl tetrakis(pentafluorophenyl)borate (TrB(C₆F₅)₄) or trifluoromethanesulfonic acid (TfOH). When TrB(C₆F₅)₄ was used as a catalyst in the solvent pivalonitrile/(trifluoromethyl)benzene 1 : 5, the glycosylation proceeded smoothly to afford the glycosides in high yields with high β‐D ‐stereoselectivities (see Table 3). Further, the glycosylation by the armed‐disarmed strategy in the presence of this catalyst was established (see Table 4). Similarly, glycosylation catalyzed by the strong protic acid TfOH afforded the corresponding β‐D ‐glycosides in good‐to‐excellent yields on treating β‐D ‐ glycosyl fluorides having a 2‐O‐benzoyl group with various glycosyl acceptors including thioglycosides (see Tables 6 and 7).     

N‐Linked Glycosyl Auxiliary‐Mediated Native Chemical Ligation on Aspartic Acid: Application towards N‐Glycopeptide Synthesis

A practical approach towards N‐glycopeptide synthesis using an auxiliary‐mediated dual native chemical ligation (NCL) has been developed. The first NCL connects an N‐linked glycosyl auxiliary to the thioester side chain of an N‐terminal aspartate oligopeptide. This intermediate undergoes a second NCL with a C‐terminal thioester oligopeptide. Mild cleavage provides the desired N‐glycopeptide.     

Monitoring photo‐induced transformations in crystals of 2,6‐difluorocinnamic acid under ambient conditions

Several conditions need to be fulfilled for a photochemical reaction to proceed in crystals. Some of these conditions, for example, geometrical conditions, depend on the particular type of photochemical reaction, but the rest are common for all reactions. The mutual directionality of two neighbouring molecules determines the kind of product obtained. The influence of temperature on the probability of a photochemical reaction occurring varies for different types of photochemical reaction and different compounds. High pressure imposed on crystals also has a big influence on the free space and the reaction cavity. The wavelength of the applied UV light is another factor which can initiate a reaction and sometimes determine the structure of a product. It is possible, to a certain degree, to control the packing of molecules in stacks by using fluoro substituents on benzene rings. The crystal and molecular structure of 2,6‐difluorocinnamic acid [systematic name: 3‐(2,6‐difluorophenyl)prop‐2‐enoic acid], C₉H₆F₂O₂, (I), was determined and analysed in terms of a photochemical [2 + 2] dimerization. The molecules are arranged in stacks along the a axis and the values of the intermolecular geometrical parameters indicate that they may undergo this photochemical reaction. The reaction was carried out in situ and the changes of the unit‐cell parameters during crystal irradiation by a UV beam were monitored. The values of the unit‐cell parameters change in a different manner, viz. cell length a after an initial increase starts to decrease, b after a decrease starts to increase, c increases and the unit‐cell volume V after a certain increase starts to decrease. The structure of a partially reacted crystal, i.e. containing both the reactant and the product, namely 2,6‐difluorocinnamic acid–3,4‐bis(2,6‐difluorophenyl)cyclobutane‐1,2‐dicarboxylic acid (0.858/0.071), 0.858C₉H₆F₂O₂·0.071C₁₈H₁₂F₄O₄, obtained in situ, is also presented. The powder of compound (I) was irradiated with UV light and afterwards crystallized [as 3,4‐bis(2,6‐difluorophenyl)cyclobutane‐1,2‐dicarboxylic acid toluene hemisolvate, C₁₈H₁₂F₄O₄·0.5C₇H₈] in a space group different from that of the crystal containing the in‐situ dimer.     

A Photoinduced,Nickel-Catalyzed Reaction for the Stereoselective Assembly of C-Linked Glycosides and Glycopeptides

C-Alkyl glycosides and glycoproteins exist in natural products and are prized for their role as carbohydrate mimics in drug design. However, a practical strategy that merges glycosyl donors with readily accessible reagents, derived from abundant carboxylic acid and amine feedstocks, is yet to be conceived. Herein, we show that a nickel catalyst promotes C−C coupling between glycosyl halides and aliphatic acids or primary amines (converted into redox-active electrophiles in one step), in the presence of Hantzsch ester and LiI (or Et₃N) under blue LED illumination to deliver C-alkyl glycosides with high diastereoselectivity. Mechanistic studies support the photoinduced formation of alkyl radicals that react with a glycosyl nickel species generated in situ to facilitate cross-coupling. Through this manifold, innate CO₂H and NH₂ motifs embedded within amino acids and oligopeptides are selectively capped and functionalized to afford glycopeptide conjugates through late-stage glycosylation.     

The effect of amino acid backbone length on molecular packing: crystalline tartrates of glycine, β‐alanine, γ‐aminobutyric acid (GABA) and dl‐α‐aminobutyric acid (AABA)

We report a novel 1:1 cocrystal of β‐alanine with dl ‐tartaric acid, C₃H₇NO₂·C₄H₆O₆, (II), and three new molecular salts of dl ‐tartaric acid with β‐alanine {3‐azaniumylpropanoic acid–3‐azaniumylpropanoate dl ‐tartaric acid–dl ‐tartrate, [H(C₃H₇NO₂)₂]⁺·[H(C₄H₅O₆)₂]⁻, (III)}, γ‐aminobutyric acid [3‐carboxypropanaminium dl ‐tartrate, C₄H₁₀NO₂⁺·C₄H₅O₆⁻, (IV)] and dl ‐α‐aminobutyric acid {dl ‐2‐azaniumylbutanoic acid–dl ‐2‐azaniumylbutanoate dl ‐tartaric acid–dl ‐tartrate, [H(C₄H₉NO₂)₂]⁺·[H(C₄H₅O₆)₂]⁻, (V)}. The crystal structures of binary crystals of dl ‐tartaric acid with glycine, (I), β‐alanine, (II) and (III), GABA, (IV), and dl ‐AABA, (V), have similar molecular packing and crystallographic motifs. The shortest amino acid (i.e. glycine) forms a cocrystal, (I), with dl ‐tartaric acid, whereas the larger amino acids form molecular salts, viz. (IV) and (V). β‐Alanine is the only amino acid capable of forming both a cocrystal [i.e. (II)] and a molecular salt [i.e. (III)] with dl ‐tartaric acid. The cocrystals of glycine and β‐alanine with dl ‐tartaric acid, i.e. (I) and (II), respectively, contain chains of amino acid zwitterions, similar to the structure of pure glycine. In the structures of the molecular salts of amino acids, the amino acid cations form isolated dimers [of β‐alanine in (III), GABA in (IV) and dl ‐AABA in (V)], which are linked by strong O—H…O hydrogen bonds. Moreover, the three crystal structures comprise different types of dimeric cations, i.e. (A…A)⁺ in (III) and (V), and A⁺…A⁺ in (IV). Molecular salts (IV) and (V) are the first examples of molecular salts of GABA and dl ‐AABA that contain dimers of amino acid cations. The geometry of each investigated amino acid (except dl ‐AABA) correlates with the melting point of its mixed crystal.