Structural and Kinetic Dissection of the endo‐α‐1,2‐Mannanase Activity of Bacterial GH99 Glycoside Hydrolases from Bacteroides spp.

Glycoside hydrolase family 99 (GH99) was created to categorize sequence‐related glycosidases possessing endo‐α‐mannosidase activity: the cleavage of mannosidic linkages within eukaryotic N‐glycan precursors (Glc_1–3Man₉GlcNAc₂), releasing mono‐, di‐ and triglucosylated‐mannose (Glc_1–3‐1,3‐Man). GH99 family members have recently been implicated in the ability of Bacteroides spp., present within the gut microbiota, to metabolize fungal cell wall α‐mannans, releasing α‐1,3‐mannobiose by hydrolysing αMan‐1,3‐αMan→1,2‐αMan‐1,2‐αMan sequences within branches off the main α‐1,6‐mannan backbone. We report the development of a series of substrates and inhibitors, which we use to kinetically and structurally characterise this novel endo‐α‐1,2‐mannanase activity of bacterial GH99 enzymes from Bacteroides thetaiotaomicron and xylanisolvens. These data reveal an approximate 5 kJ mol^?1 preference for mannose‐configured substrates in the ?2 subsite (relative to glucose), which inspired the development of a new inhibitor, α‐mannopyranosyl‐1,3‐isofagomine (ManIFG), the most potent (bacterial) GH99 inhibitor reported to date. X‐ray structures of ManIFG or a substrate in complex with wild‐type or inactive mutants, respectively, of B. xylanisolvens GH99 reveal the structural basis for binding to D ‐mannose‐ rather than D ‐glucose‐configured substrates.     

α‐Sila‐Dipeptides: Synthesis and Characterization

The first two α‐sila‐dipeptides, 7 and cyclo‐sila‐dipeptide 8 , were synthesized and characterized by several methods, including X‐ray crystallography. Bulky t‐BuMe₂Si substituents provide some kinetic stabilization to the synthesized molecules. 7 and 8 are the first examples of a “Si for C switch” in the central α‐position of an amino acid or a peptide, in which silicon is bonded to both the amino and the carbonyl groups.     

Microwave‐assisted nucleophilic cleavage of 5,7‐dimethyl‐2‐phenyl‐1‐oxo‐lH‐pyrazolo[ 1,2‐a]pyrazol‐4‐ylium‐3‐olate to α‐phenylmalonamides

Three α‐phenylmalonamides have been prepared by the selective nucleophilic cleavage of 5,7‐dimethyl‐2‐phenyl‐1‐oxo‐1H‐pyrazolo[1,2‐a]pyrazol‐4‐ylium‐3‐olate in solventless microwave syntheses. The three weak nucleophiles employed were aniline, p‐chloroaniline and m‐toluidine. The α‐phenylmalonamides of these three aniline derivatives could not be prepared using the previously reported solvent syntheses via 3‐oxopyrazolo[1,2‐a]pyrazol‐8‐ylium‐1‐olates. All products were characterised using, infrared spectroscopy, ¹H nmr and electrospray mass spectrometry. The single crystal X‐ray structures of the starting pyrazolo‐[1,2‐a]pyrazole and α‐phenylmalon‐m‐toluidide are also reported.     

Combined 1H, 13C and 11B NMR and mass spectral assignments of boronate complexes of D‐(+)‐glucose,D‐(+)‐mannose,methyl‐α‐D‐glucopyranoside,methyl‐β‐D‐galactopyranoside and methyl‐α‐D‐mannopyranoside

Complex formation between N‐butylboronic acid and D ‐(+)‐glucose, D ‐(+)‐mannose, methyl‐α‐D ‐glucopyranoside, methyl‐β‐D ‐galactopyranoside and methyl α‐D ‐mannopyranoside under neutral conditions was investigated by ¹H, ¹³C and ¹¹B NMR spectroscopy and gas chromatography–mass spectrometry (GC–MS) D ‐(+)‐Glucose and D ‐(+)‐mannose formed complexes where the boronates are attached to the 1,2:4,6‐ and 2,3:5,6‐positions of the furanose forms, respectively. On the other hand, the boronic acid binds to the 4,6‐positions of the two methyl derivatives of glucose and galactose. Methyl α‐D ‐mannopyranoside binds two boronates at the 2,3:4,6‐positions. ¹¹B NMR was used to show the ring size of the complexed sugars and the boronate. GC–MS confirmed the assignments. Copyright © 2003 John Wiley & Sons, Ltd.     

Quantum mechanical studies on model α‐pleated sheets

Pauling and Corey proposed a pleated‐sheet configuration, now called α‐sheet, as one of the protein secondary structures in addition to α‐helix and β‐sheet. Recently, it has been suggested that α‐sheet is a common feature of amyloidogenic intermediates. We have investigated the stability of antiparallel β‐sheet and two conformations of α‐sheet in solution phase using the density functional theoretical method. The peptides are modeled as two‐strand acetyl‐(Ala)₂‐N‐methylamine. Using stages of geometry optimization and single point energy calculation at B3LYP/cc‐pVTZ//B3LYP/6‐31G* level and including zero‐point energies, thermal, and entropic contribution, we have found that β‐sheet is the most stable conformation, while the α‐sheet proposed by Pauling and Corey has 13.6 kcal/mol higher free energy than the β‐sheet. The α‐sheet that resembles the structure observed in molecular dynamics simulations of amyloidogenic proteins at low pH becomes distorted after stages of geometry optimization in solution. Whether the α‐sheets with longer chains would be increasingly favorable in water relative to the increase in internal energy of the chain needs further investigation. Different from the quantum mechanics results, AMBER parm94 force field gives small difference in solution phase energy between α‐sheet and β‐sheet. The predicted amide I IR spectra of α‐sheet shows the main band at higher frequency than β‐sheet. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010     

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

Phosphoryl group differentiating α‐amino acids from β‐ and γ‐amino acids in prebiotic peptide formation

In recent years β‐amino acids have increased their importance enormously in defining secondary structures of β‐peptides. Interest in β‐amino acids raises the question: Why and how did nature choose α‐amino acids for the central role in life? In this article we present experimental results of MS and ³¹P NMR methods on the chemical behavior of N‐phosphorylated α‐alanine, β‐alanine, and γ‐amino butyric acid in different solvents. N‐Phosphoryl α‐alanine can self‐assemble to N‐phosphopeptides either in water or in organic solvents, while no assembly was observed for β‐ or γ‐amino acids. An intramolecular carboxylic–phosphoric mixed anhydride (IMCPA) is the key structure responsible for their chemical behaviors. Relative energies and solvent effects of three isomers of IMCPA derived from α‐alanine (2a–c), with five‐membered ring, and five isomers of IMCPA derived from β‐alanine (4a–e), with six‐membered ring, were calculated with density functional theory at the B3LYP/6‐31G** level. The lower relative energy (3.2 kcal/mol in water) of 2b and lower energy barrier for its formation (16.7 kcal/mol in water) are responsible for the peptide formation from N‐phosphoryl α‐alanine. Both experimental and theoretical studies indicate that the structural difference among α‐, β‐, and γ‐amino acids can be recognized by formation of IMCPA after N‐phosphorylation. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 232–241, 2003     

Hydrogels Composed of Organic Amphiphiles and α‐Cyclodextrin: Supramolecular Networks of Their Pseudorotaxanes in Aqueous Media

Mixtures of N‐alkyl pyridinium compounds [py‐N‐(CH₂)_nOC₆H₃‐3,5‐(OMe)₂]⁺(X^?) ( 1b Cl: n=10, X=Cl; 1c Br: n=12, X=Br) and α‐cyclodextrin (α‐CD) form supramolecular hydrogels in aqueous media. The concentrations of the two components influences the sol–gel transition temperature, which ranges from 7 to 67 °C. Washing the hydrogel with acetone or evaporation of water left the xerogel, and ¹³C CP/MAS NMR measurements, powder X‐ray diffraction (XRD), and scanning electron microscopy (SEM) revealed that the xerogel of 1b Cl (or 1c Br) and α‐CD was composed of pseudorotaxanes with high crystallinity. ¹³C{¹H} and ¹H NMR spectra of the gel revealed the detailed composition of the components. The gel from 1b Cl and α‐CD contains the corresponding [2]‐ and [3]pseudorotaxanes, [ 1b? (α‐CD)]Br and [ 1b? (α‐CD)₂]Br, while that from 1c Br and α‐CD consists mainly of [3]pseudorotaxane [ 1c? (α‐CD)₂]Br. 2D ROESY ¹H NMR measurements suggested intermolecular contact of 3,5‐dimethoxyphenyl and pyridyl end groups of the axle component. The presence of the [3]pseudorotaxane is indispensable for gel formation. Thus, intermolecular interaction between the end groups of the axle component and that between α‐CDs of the [3]pseudorotaxane contribute to formation of the network. The supramolecular gels were transformed into sols by adding denaturing agents such as urea, C₆H₃‐1,3,5‐(OH)₃, and [py‐N‐nBu]⁺(Cl^?).     

Synthesis,X‐ray crystal structure and biological activities of α‐phenoxyl‐1,2,3‐thiadiazoleacetamide

The annelation of 1,2,3‐thiadiazole rings was accomplished by the reaction of N‐acylhydrazone 2a bearing an adjacent α‐methyl with thionyl chloride to give α‐chloro‐N‐methyl‐1,2,3‐thiadiazole‐4‐acetamide 4 and was demonstrated by the X‐ray crystal structure of its derivative 5a. A novel series of α‐substituted phenoxy‐N‐methyl‐1,2,3‐thiadiazole‐4‐acetamide 5 were synthesized through the reaction of the compound 4 and phenols. The results of bioassays show that the title compounds exhibit good anti‐HBV activities. The crystal of compound 5a , N‐methyl‐α‐2‐bromophenyl‐1,2,3‐thiadiazole‐4‐acetamide, has been prepared and determined by X‐ray diffraction.     

Enantio‐ and Site‐Selective α‐Fluorination of N‐Acyl 3,5‐Dimethylpyrazoles Catalyzed by Chiral π–CuII Complexes

Catalytic enantioselective α‐fluorination reactions of carbonyl compounds are among the most powerful and efficient synthetic methods for constructing optically active α‐fluorinated carbonyl compounds. Nevertheless, α‐fluorination of α‐nonbranched carboxylic acid derivatives is still a big challenge because of relatively high pK_a values of their α‐hydrogen atoms and difficulty of subsequent synthetic transformation without epimerization. Herein we show that chiral copper(II) complexes of 3‐(2‐naphthyl)‐l ‐alanine‐derived amides are highly effective catalysts for the enantio‐ and site‐selective α‐fluorination of N‐(α‐arylacetyl) and N‐(α‐alkylacetyl) 3,5‐dimethylpyrazoles. The substrate scope of the transformation is very broad (25 examples including a quaternary α‐fluorinated α‐amino acid derivative). α‐Fluorinated products were converted into the corresponding esters, secondary amides, tertiary amides, ketones, and alcohols with almost no epimerization in high yield.     

Linear Free‐Energy Relationships Applied to the 13C NMR Chemical Shifts in 4‐Substituted N‐[1‐(Pyridine‐3‐ and ‐4‐yl)ethylidene]anilines

Two series of 4‐substituted N‐[1‐(pyridine‐3‐ and ‐4‐yl)ethylidene]anilines have been synthesized using different methods of conventional and microwave‐assisted synthesis, and linear free‐energy relationships have been applied to the ¹³C NMR chemical shifts of the carbon atoms of interest. The substituent‐induced chemical shifts have been analyzed using single substituent parameter and dual substituent parameter methods. The presented correlations describe satisfactorily the field and resonance substituent effects having similar contributions for C1 and the azomethine carbon, with exception of the carbon atom in para position to the substituent X. In both series, negative ρ values have been found for C1′ atom (reverse substituent effect). Quantum chemical calculations of the optimized geometries at MP2/6‐31G++(d,p) level, together with ¹³C NMR chemical shifts, give a better insight into the influence of the molecular conformation on the transmission of electronic substituent effects. The comparison of correlation results for different series of imines with phenyl, 4‐nitrophenyl, 2‐pyridyl, 3‐pyridyl, 4‐pyridyl group attached at the azomethine carbon with the results for 4‐substituted N‐[1‐(pyridine‐3‐ and ‐4‐yl)ethylidene]anilines for the same substituent set (X) indicates that a combination of the influences of electronic effects of the substituent X and the π₁‐unit can be described as a sensitive balance of different resonance structures.     

Heat‐Induced Supramolecular Organogels Composed of α‐Cyclodextrin and “Jellyfish‐Like” β‐Cyclodextrin–Poly(ε‐caprolactone)

In general, the complexation and gelation behavior between biocompatible poly(ε‐caprolactone) (PCL) derivatives and α‐cyclodextrin (α‐CD) is extensively studied in water, but not in organic solvents. In this article, the complexation and gelation behavior between α‐CD and multi‐arm polymer β‐cyclodextrin‐PCL (β‐CD‐PCL) with a unique “jellyfish‐like” structure are thoroughly investigated in organic solvent N,N‐dimethylformamide and a new heat‐induced organogel is obtained. However, PCL linear polymers cannot form organogels under the same condition. The complexation is characterized by rheological measurements, DSC, XRD, and SEM. The SEM images reveal that the complexes between β‐CD‐PCL and α‐CD present a novel topological helix porous structure which is distinctly different from the lamellar structure formed by PCL linear polymers and α‐CD, suggesting the unique “jellyfish‐like” structure of β‐CD‐PCL is crucial for the formation of the organogels. This research may provide insight into constructing new supramolecular organogels and potential for designing new functional biomaterials. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013 , 51, 1598–1606     

Synthesis of a New Chiral Source, (1R,2S)‐1‐Phenylphospholane‐2‐carboxylic Acid,via a Key Intermediate α‐Phenylphospholanyllithium Borane Complex: Configurational Stability and X‐ray Crystal Structure of an α‐Monophosphinoalkyllithium Borane Complex

A synthetic route to enantiomerically pure (1R,2S)‐1‐phenylphospholane‐2‐carboxylic acid ( 1 ), which is a phosphorus analogue of proline, has been established. A key step is the deprotonation–carboxylation of the 1‐phenylphospholane borane complex 3 by using sBuLi/1,2‐dipiperidinoethane (DPE). Configurational stability of the key intermediate, the amine‐coordinated α‐phosphinoalkyllithium borane complex 4 , was investigated by employing lithiodestannylation–carboxylation of both diastereomers of the 1‐phenyl‐2‐trimethylstannylphospholane borane complex 7 in the presence of several kinds of amines, and as a result, 4 was found to be configurationally labile even at ?100 °C. The key intermediate, the DPE‐coordinated trans‐1‐phenyl‐2‐phospholanyllithium borane complex 9 , was isolated, and the structure was identified by X‐ray crystal structure analysis. This is the first X‐ray crystal structure determined for an α‐monophosphinoalkyllithium borane complex. Remarkably, the alkyllithium complex is monomeric and tricoordinate at the lithium center with a slightly pyramidalized environment, and the existence of a Li? C bond (2.170 Å) has been confirmed. Moreover, ¹H–⁷Li HOESY and ⁶Li NMR analyses suggested the structure of 9 in solution as well as the existence of an equilibrium between 9 , its cis isomer, and the ion pair 8 at room temperature, which was extremely biased towards 9 at ?100 °C. Finally, 1 was used as a chiral ligand in a palladium‐catalyzed allylic substitution, and the desired product was obtained in high yield with good enantioselectivity.     

Enantioselective Synthesis of α‐Hydroxy Amides and β‐Amino Alcohols from α‐Keto Amides

Synthesis of enantiomerically enriched α‐hydroxy amides and β‐amino alcohols has been accomplished by enantioselective reduction of α‐keto amides with hydrosilanes. A series of α‐keto amides were reduced in the presence of chiral Cu^II/(S)‐DTBM‐SEGPHOS catalyst to give the corresponding optically active α‐hydroxy amides with excellent enantioselectivities by using (EtO)₃SiH as a reducing agent. Furthermore, a one‐pot complete reduction of both ketone and amide groups of α‐keto amides has been achieved using the same chiral copper catalyst followed by tetra‐n‐butylammonium fluoride (TBAF) catalyst in presence of (EtO)₃SiH to afford the corresponding chiral β‐amino alcohol derivatives.     

Tetramethyl‐m‐benziporphodimethene and Isomeric α,β‐Unsaturated γ‐Lactam Embedded N‐Confused Tetramethyl‐m‐benziporphodimethenes

The condensation reaction of α,α′‐dihydroxy‐1,3‐diisopropylbenzene, pyrrole, and an aldehyde leads to the formation of tetramethyl‐m‐benziporphodimethene and outer α‐pyrrolic carbon oxygenated N‐confused tetramethyl‐m‐benziporphodimethenes containing a γ‐lactam ring in the macrocycle. Two isomers with the carbonyl group of the lactam ring either close to (O‐Up) or away from (O‐Down) the neighboring sp³ meso carbon were synthesized and characterized. The single crystal X‐ray diffraction analysis on the regular and γ‐lactam containing tetramethyl‐m‐benziporphodimethenes showed highly distorted macrocycles for all compounds. For O‐Up and O‐Down isomers, dimeric structures, assembling by intermolecular hydrogen‐bonding interactions through lactam rings, were observed in the solid state. Fitting the concentration dependent chemical shifts of the outer NH proton using the non‐linear regression method give a maximum association constant of 108.9 M ^?1 for the meso 4‐methylcarboxyphenyl substituted O‐Down isomer. The DFT calculations concluded that the O‐Up isomer is energetically more stable, and the keto form is more stable than the enol form.     

α‐ and β‐Lipomycin: Total Syntheses by Sequential Stille Couplings and Assignment of the Absolute Configuration of All Stereogenic Centers

40 years ago spectroscopy, derivatization, and degradation revealed the structures of α‐lipomycin and its aglycon β‐lipomycin except for the configurations of their side‐chain stereocenters. We synthesized all relevant β‐lipomycin candidates: the (12R,13S) isomer has the same specific rotational value as the natural product. By the same criterion the (12R,13S)‐configured D ‐digitoxide is identical to α‐lipomycin. We double‐checked our assignments by degrading α‐ and β‐lipomycin to the diesters 33 and 34 and proving their 3D structures synthetically.     

Morphology and dynamics of the poly(ϵ‐caprolactone)‐b‐poly(L‐lactide) diblock copolymer and its inclusion compound with α‐cyclodextrin: A solid‐state 13C NMR study

A biodegradable diblock copolymer of poly(ϵ‐caprolactone) (PCL) and poly(L ‐lactide) (PLLA) was synthesized and characterized. The inclusion compound (IC) of this copolymer with α‐cyclodextrin (α‐CD) was formed and characterized. Wide‐angle X‐ray diffraction showed that in the IC crystals α‐CDs were packed in the channel mode, which isolated and restricted the individual guest copolymer chains to highly extended conformation. Solid‐state ¹³C NMR techniques were used to investigate the morphology and dynamics of both the bulk and α‐CD‐IC isolated PCL‐b‐PLLA chains. The conformation of the PCL blocks isolated within the α‐CD cavities was similar to the crystalline conformation of PCL blocks in the bulk copolymer. Spin–lattice relaxation time (T₁C) measurements revealed a dramatic difference in the mobilities of the semicrystalline bulk copolymer chains and those isolated in the α‐CD‐IC channels. Carbon‐observed proton spin–lattice relaxation in the rotating frame measurements (T_1ρH) showed that the bulk copolymer was phase‐separated, while, in the IC, exchange of proton magnetization through spin‐diffusion between the isolated guest polymer chains and the host α‐CD was not complete. The two‐dimensional solid‐state heteronuclear correlation (HetCor) method was also employed to monitor proton communication in these samples. Intrablock exchange of proton magnetization was observed in both the bulk semicrystalline and IC copolymer samples at short mixing times; however, even at the longest mixing time, interblock proton communication was not observed in either sample. In spite of the physical closeness between the isolated included guest chains and the host α‐CD molecules, efficient proton spin diffusion was not observed between them in the IC. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2086–2096, 2005     

Achieving High‐Temperature Stability of Metastable α‐MoC1‐x by Suppressing Phase Transformation with Mounted Atoms for Lithium Storage Performance

Despite a significant advancement in preparing metastable materials, one common problem is the strict and precious reaction conditions due to their metastable structures. Herein, we achieved the preparation of high‐temperature stabilized metastable α‐MoC_1?x by mounting zinc atoms into its lattice structure. Such a structural construction could suppress the phase transformation from α‐MoC_1?x to β‐Mo₂C through restricting the displacement of Mo atoms upon increased temperature. The resultant metastable α‐MoC_1?x can be stabilized up to 1000 °C and this stability temperature is the highest for the metastable α‐MoC_1?x so far. Synchrotron X‐ray absorption spectroscopy (XAS) and X‐ray photoelectron spectroscopy (XPS) confirm the structure of Zn‐mounted α‐MoC_1?x. Density functional theory (DFT) calculations reveal that the introduction of the Zn atoms in the lattice structure of α‐MoC_1?x could significantly decrease the energy difference (ΔE) between α‐MoC_1?x and β‐Mo₂C, thus effectively suppressing the phase transformation from α‐MoC_1?x to β‐Mo₂C and accordingly maintaining the high‐temperature stability of α‐MoC_1?x. This novel strategy can be used as a universal method to be extended to synthesize metastable α‐MoC_1?x from different precursors or other mounted elements. Moreover, the optimal product exhibits excellent lithium storage performances in terms of the cycling stability and rate performance.     

Determination of the enantiomers of α‐hydroxy‐ and α‐amino acids in capillary electrophoresis with contactless conductivity detection

The enantiomers of the anions of five α‐hydroxy acids, namely lactic acid, α‐hydroxybutyric acid, 2‐hydroxycaproic acid, 2‐hydroxyoctanoic acid and 2‐hydroxydecanoic acid, as well as the two α‐amino acids aspartic acid and glutamic acid, were baseline separated and detected by CE with contactless conductivity detection. Vancomycin was employed as chiral selector and could be used with conductivity detection without having to resort to a partial filling protocol as needed when this reagent is used with UV absorbance measurements. The procedure was successfully applied to the determination of the lactic acid enantiomers in samples of milk and yogurt. Linearity was achieved in the concentration range of 10–500 μmol/L with good correlation coefficients (0.9993 and 0.9990 for L ‐ and D ‐lactic acid, respectively). The LODs (3 S/N) for L ‐ and D ‐lactic acid were determined as 2.8 and 2.4 μmol/L, respectively.     

α‐Halogenoacetanilides as Hydrogen‐Bonding Organocatalysts that Activate Carbonyl Bonds: Fluorine versus Chlorine and Bromine

α‐Halogenoacetanilides (X=F, Cl, Br) were examined as H‐bonding organocatalysts designed for the double activation of C?O bonds through NH and CH donor groups. Depending on the halide substituents, the double H‐bond involved a nonconventional C?H???O interaction with either a H?CX_n (n=1–2, X=Cl, Br) or a H?C_Ar bond (X=F), as shown in the solid‐state crystal structures and by molecular modeling. In addition, the catalytic properties of α‐halogenoacetanilides were evaluated in the ring‐opening polymerization of lactide, in the presence of a tertiary amine as cocatalyst. The α‐dichloro‐ and α‐dibromoacetanilides containing electron‐deficient aromatic groups afforded the most attractive double H‐bonding properties towards C?O bonds, with a N?H???O???H?CX₂ interaction.