A Stepwise Solvent-Promoted S_Ni Reaction of α-d-Glucopyranosyl Fluoride: Mechanistic Implications for Retaining Glycosyltransferases

The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-d-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: Δ<i>H</i>^⧧ = 81.4 ± 1.7 kJ mol^–1, and Δ<i>S</i>^⧧ = −90.3 ± 4.6 J mol^–1 K^–1. To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, respectively. The transition state for the solvolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an S_Ni (D_N^⧧*A_Nss) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol molecule. In comparison, the inverted product, 1,6-anhydro-β-d-glucopyranose, is formed by intramolecular capture of a solvent-equilibrated glucopyranosylium ion, which results from dissociation of the solvent-separated ion pair formed in the rate-limiting ionization reaction (D_N^⧧ + A_N). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed

Neuraminidase Substrate Promiscuity Permits a Mutant <i>Micromonospora viridifaciens</i> Enzyme To Synthesize Artificial Carbohydrates

Mutation of the nucleophilic amino acid residue tyrosine to the small nonpolar residue glycine (Y370G) in the active site of <i>Micromonospora viridifaciens</i> neuraminidase (<i>Mv</i>NA) produces an efficient catalyst for the transfer of <i>N</i>-acetylneuraminic acid from an artificial substrate (i.e., phenyl <i>N</i>-acetyl-β-d-neuraminide) to a sugar acceptor (e.g., d-lactose, d-glucose, d-mannose, d-raffinose, d-allose, or d-fructose) to give <i>N</i>-acetyl-α-neuraminide coupled carbohydrate products. In addition, this mutant enzyme (<i>Mv</i>NA Y370G) catalyzes the transfer of a sugar residue from the artificial substrate 2-fluorophenyl <i>N</i>-acetyl-β-d-neuraminide to methyl glycopyranoside acceptors. Interestingly, when trans-glycosylation reactions are conducted in aqueous solutions containing 30% (v/v) acetonitrile, the α-anomeric acceptors of methyl glucopyranoside and galactopyranoside generate higher product yields than do their corresponding β-anomers. Specifically, a 64 h reaction with 2-fluorophenyl <i>N</i>-acetyl-β-d-neuraminide as the limiting reagent and the acceptors methyl α-d-galactopyranoside, methyl α-d-glucopyranoside, or methyl α-d-mannopyranoside gives trans-glycosylation product yields of 22%, 31%, or 34%, respectively. With methyl α-d-galactopyranoside as the acceptor, trans-glycosylations catalyzed by both <i>Mv</i>NA Y370G and a 2,6-sialyltransferase yield identical products, which we identified as methyl <i>N</i>-acetyl-α-d-neuraminyl-(2 → 6)-α-d-galactopyranoside. The <i>Mv</i>NA Y370G-catalyzed coupling of <i>N</i>-acetylneuraminic acid to these three methyl α-d-glycopyranoside acceptors is favored by factors of 18–27-fold over the competing hydrolysis reaction. These coupling efficiencies likely arise from nonselective interactions between the acceptor glycopyranoside and <i>Mv</i>NA Y370G, which preferentially places a carbohydrate hydroxyl group rather than water in close proximity to the active site where this functionality intercepts the nascent neuraminyl oxacarbenium ion that is formed during cleavage of the glycosidic bond in the aryl <i>N</i>-acetyl-β-d-neuraminide donor. The ability to transfer <i>N</i>-acetylneuraminic acid from a stable and readily accessible donor to acceptor carbohydrates that are not substrates for sialyltransferases is one step on the path for the production of pseudohuman glycoproteins from nonmammalian cell lines

Chemical Insight into the Emergence of Influenza Virus Strains That Are Resistant to Relenza

A reagent panel containing ten 4-substituted 4-nitrophenyl α-d-sialosides and a second panel of the corresponding sialic acid glycals were synthesized and used to probe the inhibition mechanism for two neuraminidases, the N2 enzyme from influenza type A virus and the enzyme from <i>Micromonospora viridifaciens</i>. For the viral enzyme the logarithm of the inhibition constant (<i>K</i>_i) correlated with neither the logarithm of the catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) nor catalytic proficiency (<i>k</i>_cat/<i>K</i>_m<i>k</i>_un). These linear free energy relationship data support the notion that these inhibitors, which include the therapeutic agent Relenza, are not transition state mimics for the enzyme-catalyzed hydrolysis reaction. Moreover, for the influenza enzyme, a correlation (slope, 0.80 ± 0.08) is observed between the logarithms of the inhibition (<i>K</i>_i) and Michaelis (<i>K</i>_m) constants. We conclude that the free energy for Relenza binding to the influenza enzyme mimics the enzyme–substrate interactions at the Michaelis complex. Thus, an influenza mutational response to a 4-substituted sialic acid glycal inhibitor can weaken the interactions between the inhibitor and the viral neuraminidase without a concomitant decrease in free energy of binding for the substrate at the enzyme-catalyzed hydrolysis transition state. The current findings make it clear that new structural motifs and/or substitution patterns need to be developed in the search for a bona fide influenza viral neuraminidase transition state analogue inhibitor

Transition-State Structure for the Quintessential S_N2 Reaction of a Carbohydrate: Reaction of α‑Glucopyranosyl Fluoride with Azide Ion in Water

We report that the S_N2 reaction of α-d-glucopyranosyl fluoride with azide ion proceeds through a loose (exploded) transition-state (TS) structure. We reached this conclusion by modeling the TS using a suite of five experimental kinetic isotope effects (KIEs) as constraints for the calculations. We also report that the anomeric ¹³C-KIE is not abnormally large (<i>k</i>₁₂/<i>k</i>₁₃ = 1.024 ± 0.006), a finding which is at variance with the previous literature value (Zhang et al. <i>J. Am. Chem. Soc.</i> <b>1994</b>, <i>116</i>, 7557)

DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation

UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA

Transition State Analysis of <i>Vibrio cholerae</i> Sialidase-Catalyzed Hydrolyses of Natural Substrate Analogues

A series of isotopically labeled natural substrate analogues (phenyl 5-<i>N</i>-acetyl-α-d-neuraminyl-(2→3)-β-d-galactopyranosyl-(1→4)-1-thio-β-d-glucopyranoside; Neu5Acα2,3LacβSPh, and the corresponding 2→6 isomer) were prepared chemoenzymatically in order to characterize, by use of multiple kinetic isotope effect (KIE) measurements, the glycosylation transition states for <i>Vibrio cholerae</i> sialidase-catalyzed hydrolysis reactions. The derived KIEs for Neu5Acα2,3LacβSPh for the ring oxygen (¹⁸<i>V</i>/<i>K</i>), leaving group oxygen (¹⁸<i>V</i>/<i>K</i>), C3-<i>S</i> deuterium (^D<i>V</i>/<i>K</i>_S) and C3-<i>R</i> deuterium (^D<i>V</i>/<i>K</i>_R) are 1.029 ± 0.002, 0.983 ± 0.001, 1.034 ± 0.002, and 1.043 ± 0.002, respectively. In addition, the KIEs for Neu5Acα2,6βSPh for C3-<i>S</i> deuterium (^D<i>V</i>/<i>K</i>_S) and C3-<i>R</i> deuterium (^D<i>V</i>/<i>K</i>_R) are 1.021 ± 0.001 and 1.049 ± 0.001, respectively. The glycosylation transition state structures for both Neu5Acα2,3LacβSPh and Neu5Acα2,6LacβSPh were modeled computationally using the experimental KIE values as goodness of fit criteria. Both transition states are late with largely cleaved glycosidic bonds coupled to pyranosyl ring flattening (⁴H₅ half-chair conformation) with little or no nucleophilic involvement of the enzymatic tyrosine residue. Notably, the transition state for the catalyzed hydrolysis of Neu5Acα2,6βSPh appears to incorporate a lesser degree of general-acid catalysis, relative to the 2,3-isomer

Observation of a Tricyclic[4.1.0.0^2,4]heptane During a Michael Addition-Ring Closure Reaction and a Computational Study on Its Mechanism of Formation

We describe the formation of a bis-cyclopropane product, a tricyclic­[4.1.0.0^2,4]­heptane, that is formed during a Johnson–Corey–Chaykovsky reaction on a cyclopentenone. Two (of four possible) bicyclic products are selectively formed by addition of a COOEt-stabilized sulfur ylide onto the Michael acceptor. The tricyclic product is formed subsequently via a retro Michael elimination of a hindered ether followed by addition of a further cyclopropyl moiety, affecting only one of the two bicyclic products initially formed. The experimental reaction outcome was rationalized using density functional theory (DFT), investigating the different Michael-addition approaches of the sulfur ylide, the transition state (TS) energies for the formation of possible zwitterionic intermediates and subsequent reactions that give rise to cyclopropanation. Selective formation of only two of the four possible products occurs due to the epimerization of unreactive intermediates from the other two pathways, as revealed by energy barrier calculations. The formation of the tricyclic product was rationalized by evaluation of energy barriers for proton abstraction required to form the intermediate undergoing the second cyclopropanation. The selectivity-guiding factors discussed for the single and double cyclopropanation of this functionalized Michael-acceptor will be useful guidelines for the synthesis of future singly and doubly cyclopropanated compounds

C2-Oxyanion Neighboring Group Participation: Transition State Structure for the Hydroxide-Promoted Hydrolysis of 4‑Nitrophenyl α‑d‑Mannopyranoside

The hydroxide-catalyzed hydrolysis of aryl 1,2-<i>trans</i>-glycosides proceeds through a mechanism involving neighboring group participation by a C2-oxyanion and rate-limiting formation of a 1,2-anhydro sugar (oxirane) intermediate. The transition state for the hydroxide-catalyzed hydrolysis of 4-nitrophenyl α-d-mannopyranoside in aqueous media has been studied by the use of multiple kinetic isotope effect (KIE) measurements in conjunction with <i>ab initio</i> theoretical methods. The experimental KIEs are C1-²H (1.112 ± 0.004), C2-²H (1.045 ± 0.005), anomeric 1-¹³C (1.026 ± 0.006), C2-¹³C (0.999 ± 0.005), leaving group oxygen 2-¹⁸O (1.040 ± 0.012), and C2-¹⁸O (1.044 ± 0.006). The transition state for the hydrolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported kinetic isotope effects and computational modeling are consistent with the reaction mechanism involving rate-limiting formation of a transient oxirane intermediate that opens in water to give α-d-mannopyranose. The transition state has significant nucleophilic participation by the C2-alkoxide, an essentially cleaved glycosidic bond, and a slight shortening of the endocyclic C1–O5 bond. The TS is late, consistent with the large, normal C2-¹⁸O isotope effect

Design and synthesis of constrained bicyclic molecules as candidate inhibitors of influenza A neuraminidase

<div><p>The rise of drug-resistant influenza A virus strains motivates the development of new antiviral drugs, with different structural motifs and substitution. Recently, we explored the use of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that was designed to mimic the conformation adopted during enzymatic cleavage within the neuraminidase (NA; sialidase) active site. Given that our first series of compounds were at least four orders of magnitude less active than available drugs, we hypothesized that the new carbon skeleton did not elicit the same interactions as the cyclohexene frameworks used previously. Herein, we tried to address this critical point with the aid of molecular modeling and we proposed new structures with different functionalization, such as the introduction of free ammonium and guanidinium groups and ether side chains other than the 3-pentyl side chain, the characteristic side chain in Oseltamivir. A highly simplified synthetic route was developed, starting from the cyclopropanation of cyclopentenone and followed by an aziridination and further functionalization of the five-member ring. This allowed the efficient preparation of a small library of new bicyclic ligands that were characterized by enzyme inhibition assays against influenza A neuraminidases N1, its H274Y mutant, and N2. The results show that none of the new structural variants synthesized, including those containing guanidinium groups rather than free ammonium ions, displayed activity against influenza A neuraminidases at concentrations less than 2 mM. We conclude that the choice and positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be properly tuned for producing complementary interactions within the catalytic site.</p></div

Synthesis of the key aziridine intermediate 12 and aziridine opening reaction.

<p>Synthesis of the key aziridine intermediate 12 and aziridine opening reaction.</p