Anomeric Mixture Research Articles

Stereoselective C-Aryl Glycosylation by Catalytic Cross-Coupling of Heteroaryl Glycosyl Sulfones.

Stereoselective C-glycosylation reactions are increasingly gaining attention in carbohydrate chemistry because they enable glycosyl precursors, easily accessible as anomeric mixtures, to converge to a single diastereomeric product. However, controlling the stereochemical outcome via transition metal catalysis remains challenging, and methods that leverage bench-stable heteroaryl glycosyl sulfone donors to facilitate glycosylation are rare. Here, we show two complementary nonprecious metal catalytic systems, based on iron or nickel, which are capable of promoting efficient C-C coupling between heteroaryl glycosyl sulfones and aromatic nucleophiles or electrophiles via distinct mechanisms and modes of activation. Diverse C-aryl glycosides were secured with excellent selectivity, scope, and functional group compatibility, and reliable access to both α and β isomers could be achieved for key sugar residues.

StereoselectiveC‐Aryl Glycosylation by Catalytic Cross‐Coupling of Heteroaryl Glycosyl Sulfones

AbstractStereoselectiveC‐glycosylation reactions are increasingly gaining attention in carbohydrate chemistry because they enable glycosyl precursors, readily accessible as anomeric mixtures, to converge to a single diastereomeric product. However, controlling the stereochemical outcome through transition‐metal catalysis remains challenging, and methods that leverage bench‐stable heteroaryl glycosyl sulfone donors to facilitate glycosylation are rare. Herein, we show two complementary nonprecious metal catalytic systems, based on iron or nickel, which are capable of promoting efficient C−C coupling between heteroaryl glycosyl sulfones and aromatic nucleophiles or electrophiles through distinct mechanisms and modes of activation. DiverseC‐aryl glycosides were secured with excellent selectivity, scope, and functional‐group compatibility, and reliable access to both α and β isomers was possible for key sugar residues.

Isolation, Specificity, and Application of β-N-Acetylhexosaminidases from Penicillium crustosum

β-N-acetylhexosaminidases have great potential in applied biocatalysis owing to their ability to act on a wide range of natural and modified substrates. In this work, β-N-acetylhexosaminidases from four Penicillium crustosum strains (PcHex) were studied. The production strains showed the highest enzymatic activity in the culture medium after 11–14 days of cultivation. The specific activity of the isolated and purified PcHex of hydrolysis of 4-nitrophenyl-N-acetyl-β-D-galactopyranoside was 15–20 U/mg protein. All isolated β-N-acetylhexosaminidases showed similar pH–activity profiles, with the optimum pH being 4.0–5.0 and the optimum temperature being 40 °C–50 °C. Apart from standard substrates, two synthetic substrates (5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-galactoside and 2-chloro-4-nitrophenyl-β-D-galactopyranoside) were tested and successfully hydrolyzed using β-N-acetylhexosaminidase preparations. Maximum activity toward the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-D-galactopyranoside (4MU-β-GalNAc) was observed for enzyme preparations from PcHex1 (15.38 U/mg protein) isolated from swab samples of books in the Slovak National Museum in Martin (Slovak Republic) and stored in our laboratory. The same enzyme preparation was used for the selective hydrolysis of β-anomer of 4MU-GalNAc from an anomeric mixture of 4MU-α/β-GalNAc. Thus, pure α-anomer (with the total yield being 90%) was separated from the mixture, which suggests the application potential of these enzymes.

ZnI2-Mediated β-Galactosylation of C2-Ether-Type Donor.

Conventional glycosylation with galactosyl donors having C-2 benzyl (Bn) ether-type functionality often leads to anomeric mixtures, due to the anomeric and steric effects that stabilize the 1,2-cis-α- and 1,2-trans-β-glycosides, respectively. Herein we report a versatile ZnI2-directed β-galactosylation approach employing a 4,6-O-tethered and 2-O-Bn galactosyl donor for the stereoselective and efficient synthesis of β-O-galactosides. With a broad substrate scope, the reaction tolerates a wide range of functional groups and complex molecular architectures, providing stereocontrolled β-galactosides in moderate to excellent yields. The practicality of this transformation is demonstrated through the synthesis of a tetrasaccharide arabinogalactan fragment with high stereoselectivity.

1'-Cyano Intermediate Enables Rapid and Stereoretentive Access to 1'-Modified Remdesivir Nucleosides.

While biochemical, structural, and computational studies have shown the importance of remdesivir's C1'-substituent in its perturbation of SARS-CoV-2 RdRp action, we recognized the paucity of methods to stereoselectively install substituents at this position as an obstacle to rigorous explorations of SAR and mechanism. We report the utilization of an anomerically pure 1'-cyano intermediate as an entry point to a chemically diverse set of substitutions, allowing for 1'diversification while obviating the need for the tedious separation of anomeric mixtures.

The melting of glucose

Several sugars are known to undergo a spontaneous liquefaction below their reputed melting point (Tm), but the origin of this apparent melting is not yet clearly understood. In this paper we address this puzzling behavior in the particular case of the crystalline forms of glucose: Gα and Gβ, involving respectively the glucose-α and glucose-β anomers. We show in particular that the spontaneous melting below their reputed melting point Tm (∼151 °C for Gα and ∼156 °C for Gβ) corresponds to a horizontal displacement of the system in the eutectic phase diagram of the anomeric mixture glucose-α / glucose-β. This displacement is associated with mutarotation in the liquid which, in turn, induces additional liquefaction of the remaining crystal. This feedback loop creates a vicious circle which stops when the mixture reaches the liquidus branch, i.e. when the liquefaction is total. It is also shown that this behavior becomes more complex on approaching the eutectic temperature Te (120 °C). Just above Te, the liquefaction process is followed by a recrystallization leading to the crystalline form Gβ. On the other hand, just below Te, the spontaneous liquefaction process stops as no melting is expected whatever the anomeric composition.

1H-imidazo[4,5-f][1,10]phenanthroline carbohydrate conjugates: Synthesis, DNA interactions and cytotoxic activity

Here we present a novel family of carbohydrate conjugates based on the 2-aryl-imidazo[4,5-f][1,10]phenanthroline core modified with carbohydrates (carb-APIPs). The hybrid compounds were prepared by direct treatment of the unprotected carbohydrate with 2-(4-aminophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (APIP). The N-glycosylation reactions with the monosaccharides tested afforded stereoselectively the more stable N-β-glycopyranosylamines that, in solution, underwent a dynamic equilibrium leading to anomeric mixtures with a small participation of the α isomer. DNA interaction experiments with telomeric G-quadruplex DNA included DNA FRET melting assays, circular dichroism, and equilibrium dialysis and revealed that the novel carb-APIPs bind the G-quadruplex structure with high affinity. Interestingly, the presence of the carbohydrate confers good selectivity towards the telomeric quadruplex structure, as suggested by competition DNA FRET melting assays. Besides the extended aromatic surface that allows π-stacking interactions, the carbohydrate part of the conjugate may contribute to groove binding recognition, as indicated by viscosity experiments. In addition, the novel carb-APIPs showed significant cytotoxic properties in PC3 and HeLa cells and, to a lesser extent, in MCF7 cells and normal human fibroblasts (HFF1).

Chemo-Enzymatic Production of 4-Nitrophenyl-2-acetamido-2-deoxy-α-D-galactopyranoside Using Immobilized β-N-Acetylhexosaminidase

α-Nitrophenyl derivatives of glycosides are convenient substrates used to detect and characterize α-N-acetylgalactosaminidase. A new procedure combining chemical and biocatalytic steps was developed to prepare 4-nitrophenyl-2-acetamido-2-deoxy-α-D-galactopyranoside (4NP-α-GalNAc). The α-anomer was prepared through chemical synthesis of an anomeric mixture followed by selective removal of the β-anomer using specific enzymatic hydrolysis. Fungal β-N-acetylhexosaminidase (Hex) from Penicillium oxalicum CCF 1959 served this purpose owing to its high chemo-and regioselectivity towards the β-anomeric N-acetylgalactosamine (GalNAc) derivative. The kinetic measurements of the hydrolytic reaction showed that the enzyme was not inhibited by the substrate or reaction products. The immobilization of Hex in lens-shaped polyvinyl alcohol hydrogel capsules provided a biocatalyst with very good storage and operational stability. The immobilized Hex retained 97% of the initial activity after ten repeated uses and 90% of the initial activity after 18 months of storage at 4 °C. Immobilization inactivated 65% of the enzyme activity. However, the effectiveness factor and kinetic and mass transfer phenomena approached unity indicating negligible mass transfer limitations.

Glycosyl Vinylogous Carbonates as Glycosyl Donors by Metal-Free Activation.

Synthesis of glycoconjugates employs a glycosylation reaction wherein an electrophile and a nucleophile known as a glycosyl donor and an aglycon, respectively, are involved. Glycosyl donors often contain a leaving group at the anomeric carbon that upon reaction with activator(s) results in a highly reactive electrophilic species reported as an oxycarbenium ion contact pair that will then be attacked by the aglycon. Therefore, identification of the correct glycosyl donor and activation protocol is essential for the synthesis of all glycoconjugates. Recently identified [Au]/[Ag]-catalyzed activation of ethynylcyclohexyl glycosyl carbonates is one such versatile method for the synthesis of glycosides, oligosaccharides, and glycoconjugates. In this work, stable glycosyl vinylogous carbonates were identified to undergo glycosidation in the presence of a sub-stoichiometric quantity of TfOH. The reaction is fast and suitable for donors containing both C2-ethers and C2-esters. Donors positioned with C2-ethers resulted in anomeric mixtures with greater selectivity toward 1,2-cis glycosides, whereas those with C2-esters gave 1,2-trans selective glycosides. The versatility of the method is demonstrated by conducting the glycosylation with more than 25 substrates. Furthermore, the utility of the glycosyl vinylogous carbonate donors is demonstrated with the successful synthesis of the branched pentaarabinofuranoside moiety of the Mycobacterium tuberculosis cell wall.

Synthesis and Evaluation of Fluorinated Benzyl Ethers as Alternate Protecting Groups for Enhanced NMR Resolution in Oligosaccharide Synthesis

Oligosaccharides have been playing an important role in biological systems. Synthesis of oligosaccharides requires the protection from hydroxyl groups present in the corresponding monosaccharide units. The existing methods of protection have drawbacks, including formation of anomeric mixtures, change in hydrophilicity or lipophilicity and solubility of the products, participation of the protecting groups in the reactions of the core of monosaccharide units, problems associated with chemoselectivity, regioselectivity and overall stereochemical outcomes of reactions. Additionally, there has been a spectral overlap of these protecting groups with carbohydrate core, which yielded more complex spectra. Therefore, the identification and synthesis of suitable alternative protecting groups have received attention in the oligosaccharide synthesis. The objective of the present study was to synthesize various fluorinated benzyl ethers of methyl-α-D-mannopyronoside and to evaluate these ethers as the alternative protecting groups for enhancing NMR resolution in the oligosaccharide synthesis. Various fluorinated benzyl ethers of methyl-α-D-mannopyronoside were prepared through the reaction of methyl-α-D-mannopyronoside with various fluorinated benzyl bromides by using Williamson ether synthesis method. Spectral analysis of these fluorinated benzyl ethers showed that the peaks of methylene carbons shifted to a value of 10-20 parts per million (ppm) to a high field region in the 13C NMR, compared to the non-fluorinated benzyl ether. As a result, the spectral complexity decreased and enhanced the spectral resolution. In this study, we concluded that fluorinated benzyl ethers could be a suitable alternative to the non-fluorinated benzyl ethers to protect the hydroxyl groups of monosaccharides in the synthesis of oligosaccharides.

Unprotected Galactosamine as a Dynamic Key for a Cyclochiral Lock.

The discrimination of d-galactosamine (G), representative of the amino-sugar class of compounds, has been probed through nano-ESI-FT-ICR mass spectrometry by isolating the relevant [C·H·G]+ proton-bound complexes with the enantiomers of the cyclochiral resorcin[4]arene C and allowing them to react toward three primary amines (B = EtNH2, iPrNH2, and (R)- and (S)-sBuNH2). The system under investigation presents several features that help to unveil the behavior of unprotected G in such a supramolecular architecture: (i) the hydrophobic derivatization of the C convex side forces the polar guest G to be coordinated by the cyclochiral concave region; (ii) protonated d-galactosamine exists as an anomeric mixture, dynamically interconverting throughout the experimental time-window; and (iii) different basicities of B allow the experiment to subtly tune the reactivity of the [C·H·G]+ complexes. Three [C·H·G]+ aggregate-types were found to exist, differing in both their origin and reactivity. The most reactive adducts ([C·H·G]ESI+), generated in the electrospray environment, undergo a G-to-B ligand exchange in competition with a partial isomerization to the unreactive [C·H·G]GAS+-type complexes. Finally, the poorly reactive [C·H·G]SOL+ aggregates are formed in solution over an hours-long time scale. A cyclochirality effect on the reactivity was found to depend on the considered [C·H·G]+ aggregate-type.

Easy access to a carbohydrate-based template for stimuli-responsive surfactants

In this paper we describe the synthesis of a new carbohydrate-based building block functionalized with azido or amino groups on the 2 and 4 positions. The building block can be synthesized in anomerically pure form in only five scalable steps starting from commercially available levoglucosan. It was shown that the building block could undergo alkylations under strongly basic conditions. The building block with azido groups could furthermore take part in CuAAC reactions, generating derivatives with ester or carboxylic acid functionalities. In addition, the anomeric mixture of the building block was used for the synthesis of a molecule that could act as an emulsifier only in the presence of Zn2+ ions.

Influence of protecting groups on O- and C-glycosylation with neuraminyl and ulosonyl dibutylphosphates

The adamantanyl thioglycosides of 5-isothiocyano and 5-azido 5-desamino-4,7,8,9-tetra-O-acetylneuraminic acid methyl ester were converted into the corresponding dibutyl phosphates, which proved to be excellent α-selective donors for O-sialidation with a range of typical acceptors, and good donors for reaction with allyltributylstannane, albeit without significant anomeric selectivity. In the KDN series the dibuylphosphate derived from a donor carrying a 4,5-cyclic carbonate protecting group afforded the corresponding C-glycoside with excellent α-selectivity on activation in the presence of allyltributylstannane, whereas the corresponding donor carrying acetate esters at the 4- and 5-positions was unselective. Overall, it is revealed that while the strongly electron-withdrawing isothiocyanato and azido groups are sufficient to promote highly α-selective O-sialidation, they are inadequate when faced with less reactive nucleophiles when mixtures of anomers are obtained.

Access to d- and l-Psicose Derivatives via Hydroxy Methylation of Ribono Lactone

2,3,5-Tri-O-benzyl- and 2,3,5-tri-O-methyl-d-ribono-γ-lactone were converted with (methoxyethoxymethoxy)methyl and benzyloxy tributylstannane into the corresponding protected d-psicoses as mixtures of anomers in 31%–72% yield. Treatment of 2,3,5-tri-O-methyl-l-ribono-γ-lactone with benzyloxy tributylstannane afforded the corresponding l-psicose derivative as an anomeric mixture in 72% yield. Both methylated psicoses were further converted into 1,2-O-isopropylidene-3,4,6-tri-O-methyl-d- and l-psicofuranosides, the respective α- and β-anomers of which could be separated and characterized.

Isolation of secondary metabolites from Geranium molle L. with anticancer potential

Two flavone glycosides, apigenin-6-C-glucoside (1) and lutelin-6-C-glucoside (2), and an anomeric mixture of 2,3-dygalloyl-4,6-HHDP-d-glucose (3a/3b) were isolated for the first time from Geranium molle L., a medicinal plant used in the folk medicine of northeast of Portugal for the treatment of a variety of ailments, including cancer. The cytotoxic activity of the isolated compounds was assessed against several human tumour cell lines and non-tumour porcine liver primary cells. The anomeric mixture of ellagitannins 3a/3b, with the two anomers showing considerably different hydrophobicities, was found to be significantly cytotoxic against cancer cells, with only moderate hepatotoxicity.

Ion spectroscopy of heterogeneous mixtures: IRMPD and DFT analysis of anomers and conformers of monosaccharides

The gas phase IR spectra of three protonated hexosamines are measured in the 3 μm range using IRMPD spectroscopy and compared with DFT simulation of the IR spectra to identify the ring conformations and the contributions of the α and β anomers to the experimental spectra. We propose an original approach for the deconvolution and quantification of heterogeneous IR spectra based on linear combination of the DFT spectra. The method is applied to the analysis of a two-component anomeric mixture and validated using NMR analysis. Finally, we further estimate the relative populations of a three-component mixture: the anomers and conformers of glucosamine.

Nucleobase-Functionalized 5-Aza-7-deazaguanine Ribo- and 2'-Deoxyribonucleosides: Glycosylation, Pd-Assisted Cross-Coupling, and Photophysical Properties.

The special nucleobase recognition pattern of 5-aza-7-deazaguanine nucleosides makes them valuable for construction of homo purine DNA, silver-mediated base pairs, and expansion of the four letter genetic coding system. To widen the utility of 5-aza-7-deazaguanine nucleosides, side chains were introduced at position-7 of the nucleobase. As key compounds, 7-iodo nucleosides were synthesized. Nucleobase anion glycosylation of the iodo derivative of isobutyrylated 5-aza-7-deazaguanine with the bromo sugar of 2,3,5-tri-O-benzoyl-1-O-acetyl-d-ribofuranose gave the pure β-D anomeric N-9 glycosylation product (67%), whereas one-pot Vorbrüggen conditions gave only 42% of the iodinated nucleoside. The noniodinated nucleoside was formed in 84%. For the synthesis of 2'-deoxyribonucleosides, anion glycosylation performed with Hoffer's 2'-deoxyhalogenose yielded an anomeric mixture (α-D = 33% and β-D = 39%) of 2'-deoxyribonucleosides. Various side chain derivatives were prepared from nonprotected nucleosides by Pd-assisted Sonogashira or Suzuki-Miyaura cross-coupling. Among the functionalized ribonucleosides and anomeric 2'-deoxyribonucleosides, some of them showed strong fluorescence. Benzofuran and pyrene derivatives display high quantum yields in non-aqueous solvents and solvatochromism. Single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2'-deoxyguanosine displayed intermolecular iodo-oxygen interactions in the crystal and channels filled with solvent molecules.

Stereoselective N-glycosylation with N4-acyl cytosines and efficient synthesis of gemcitabine

Through systematical comparison of various N4-protected cytosine derivatives in the glycosylation step of gemcitabine synthesis, highly beta-stereoselective and high yielding TBAI catalyzed N-glycosylation was achieved with N4-Bz cytosine and anomeric mixture of 2,2‘-difluororibose mesylate donor. The subsequent global deprotection gave gemcitabine efficiently. Meanwhile, the anomeric chloride intermediate and fluoride-displaced side products of this N-glycosylation were identified, too. This new glycosylation method reveals the importance of N4-protection in the stereoselective preparation of pyrimidine nucleoside, also provides a potential alternative to current industrial process to gemcitabine.

Selective Primary Alcohol Oxidation of Lignin Streams from Butanol-Pretreated Agricultural Waste Biomass.

Chemically modified lignins are important for the generation of biomass-derived materials and as precursors to renewable aromatic monomers. A butanol-based organosolv pretreatment has been used to convert an abundant agricultural waste product, rice husks, into a cellulose pulp and three additional product streams. One of these streams, a butanol-modified lignin, was oxidized at the γ position to give a carboxylic acid functionalized material. Subsequent coupling of the acid with aniline aided lignin characterization and served as an example of the flexibility of this approach for grafting side chains onto a lignin core structure. The pretreatment was scaled up for use on a multi-kilogram scale, a development that enabled the isolation of an anomeric mixture of butoxylated xylose in high purity. The robust and scalable butanosolv pretreatment has been developed further and demonstrates considerable potential for the processing of rice husks.

Self-promoted and stereospecific formation of N-glycosides.

A stereoselective and self-promoted glycosylation for the synthesis of various N-glycosides and glycosyl sulfonamides from trichloroacetimidates is presented. No additional catalysts or promoters are needed in what is essentially a two-component reaction. When α-glucosyl trichloroacetimidates are employed, the reaction resulted in the stereospecific formation of the corresponding β-N-glucosides in high yields at ambient conditions. On the other hand, when equatorial glucosyl donors were used, the stereospecificity decreased and resulted in a mixture of anomers. By NMR-studies, it was concluded that this decrease in stereospecificity was due to an, until now, unpresented anomerization of the trichloroacetimidate under the very mildly acidic conditions. The mechanism and kinetics of the glycosylations have been studied by NMR-experiments, which gave an insight into the activation of trichloroacetimidates, suggesting an SNi-like mechanism involving ion pairs. The scope of glycosyl donors and sulfonamides was found to be very broad including popular N-protective groups and common glycosyl donors of various reactivity. Peracetylated GlcNAc trichloroacetimidate could be used without the need for any promotors or additives and a tyrosine side chain was glycosylated as an N-glycosyl carbamate. The N-carbamates and the N-sulfonyl groups functioned as orthogonal protective groups of the N-glycoside and hence allowed further N-functionalization without risking mutarotation of the N-glycoside. The N-glycosylation was also performed on a gram scale, without a drop in stereoselectivity nor yield.