Synthesis of Fusion‐Isomeric Imidazopyridines and Their Evaluation as Inhibitors of syn‐ and anti‐Protonating Glycosidases

The galacto‐ and gluco‐configured imidazopyridines 4 and 5 were synthesised as potential inhibitors of syn‐protonating β‐glycosidases. Methyl α‐D ‐lyxopyranoside ( 9 ) was transformed into the 3,4‐anhydro‐β‐L ‐riboside 16 , which, upon treatment with Et₂AlCN, gave the nitrile 17 (76–85%). Reaction of 17 with the dimethyl aluminate of aminoacetaldehyde dimethyl acetal led directly to the branched chain lyxo‐configured imidazole 27 (53%) that was hydrolysed to an equilibrating mixture of 4 and 28 – 30 . Oxido reduction of 27 provided the arabino‐configured imidazole 42 (ca. 48% from 27 ). Hydrolysis of 42 led to the mixture 5 / 45 (63–90%). anti‐Protonating β‐galactosidases and β‐glucosidases (families 1 and 2) were only weakly inhibited by 4 / 28 – 30 and 5 / 45 , respectively. Also the syn‐protonating cellulase (Cel7A) was weakly inhibited by the monosaccharide mimics 5 / 45 , suggesting either that monosaccharide mimics are too small to inhibit Cel7A, or that fusion isomeric tetrahydroimidazo[1,2‐a]pyridines are not a suitable scaffold for the inhibition of syn‐protonating glycosidases.     

Recent Insights into Inhibition,Structure, and Mechanism of Configuration-Retaining Glycosidases

Not “from above”, but “from the side” : Configuration-retaining β-glycosidases protonate their substrate either anti or syn to the endocyclic C1−O bond as the first step in the enzymic cleavage of the glycosidic bond (see schematic drawing). Insights into the mechanism of action of glycosidases have been gained by a combination of the synthesis of inhibitors, the study of the kinetics of their inhibition, and the analysis of the crystal structures of glycosidases and glycosidase–ligand complexes.     

Dihydroxyacetone Phosphate Aldolase Catalyzed Synthesis of Structurally Diverse Polyhydroxylated Pyrrolidine Derivatives and Evaluation of their Glycosidase Inhibitory Properties

The chemoenzymatic synthesis of a collection of pyrrolidine‐type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C‐α‐substituted N‐Cbz‐2‐aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L ‐Fuculose‐1‐phosphate aldolase (FucA) and L ‐rhamnulose‐1‐phosphate aldolase (RhuA) from E. coli were used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C‐α were well tolerated by FucA catalyst (i.e., 40–70 % conversions to aldol adduct), whereas no product was observed with C‐α‐alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C‐α‐alkyl linear groups gave the highest conversions to aldol adducts (60–99 %), while the C‐α‐alkyl branched ones gave moderate to good conversions (50–80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90–100 % anti (3R,4R) adduct). RhuA was highly stereoselective with (S)‐N‐Cbz‐2‐aminoaldehydes (90–100 % syn (i.e., 3R,4S) adduct), whereas those with R configuration gave mixtures of anti/syn adducts. For iPr and iBu substituents, RhuA furnished the anti adduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the anti adducts could be kinetically preferred, while the syn adducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of α‐L ‐fucosidase (IC₅₀=1–20 μM ), moderate of α‐L ‐rhamnosidase (IC₅₀=7–150 μM ), and weak of α‐D ‐mannosidase (IC₅₀=80–400 μM ) were identified. The apparent inhibition constant values (K_i) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.     

Beckmann Fragmentation and Rearrangement. Part VII. Fragmentation and cyclization of α-methylthio-ketoximes. Fragmentation reactions no. 27

α-Methylthio-propiophenone anti-oxime p-toluenesulfonate (tosylate) ( 12b ) fragments quantitatively in 80% ethanol yielding benzonitrile and a methylidenesulfonium ion 15 . The syn-isomer, however, undergoes a Beckmann rearrangement. The fragmentation of α-methylthio-isobutyropher one anti-oxime tosylate ( 13b ) is accompanied by cyclization to the 1, 2-thiazetin-1-ium ion 27 , which is hydrolyzed via the sulfimine 29 to the keto sulfide 20 and the keto sulfoxide 30 . A comparison of the rates of the α-alkylthio anti-ketoxime tosylates 12b and 13b and of the homomorphous oxime tosylates 16b and 17b shows that fragmentation and cyclization are strongly assisted by the sulfur atom. Whereas both the anti- and syn-isomers of α-amino ketoxime derivatives fragment quantitatively, only the anti-isomers of α-alkylthio ketoxime derivatives undergo facile fragmentation.     

Thermal Reactions of Guaiazulene and Its 3-Methyl Derivative with Dimethyl Acetylenedicarboxylate

The thermal reaction of 7-isopropyl-1,3,4-trimethylazulene (3-methylguaiazulene; 2 ) with excess dimethyl acetylenedicarboxylate (ADM) in decalin at 200° leads to the formation of the corresponding heptalene- ( 5a/5b and 6a/6b ; cf. Scheme 3) and azulene-1,2-dicarboxylates ( 7 and 8 , respectively). Together with small amounts of a corresponding tetracyclic compound (‘anti’- 13 ) these compounds are obtained via rearrangement (→ 5a/5b and 6a/6b ), retro-Diels-Alder reaction (→ 7 and 8 ), and Diels-Alder reaction with ADM (→ ‘anti’- 13 ) from the two primary tricyclic intermediates ( 14 and 15 ; cf. Scheme 5) which are formed by site-selective addition of ADM to the five-membered ring of 2 . In a competing Diels-Alder reaction, ADM is also added to the seven-membered ring of 2 , leading to the formation of the tricyclic compounds 9 and 10 and of the Diels-Alder adducts ‘anti’- 11 and ‘anti’- 12 , respectively of 9 and of a third tricyclic intermediate 16 which is at 200° in thermal equilibrium with 9 and 10 (cf. Scheme 6). The heptalenedicarboxylates 5a and 5b as well as 6a and 6b are interconverting slowly already at ambient temperature (Scheme 4). The thermal reaction of guaiazulene ( 1 ) with excess ADM in decalin at 190° leads alongside with the known heptalene- ( 3a ) and azulene-1,2-dicarboxylates ( 4 ; cf. Schemes 2 and 7) to the formation of six tetracyclic compounds ‘anti’- 17 to ‘anti’- 21 as well as ‘syn’- 19 and small amounts of a 4:1 mixture of the tricyclic tetracarboxylates 22 and 23 . The structure of the tetracyclic compounds can be traced back by a retro-Diels-Alder reaction to the corresponding structures of tricyclic compounds ( 24--29 ; cf. Scheme 8) which are thermally interconverting by [1,5]-C shifts at 190°. The tricyclic tetracarboxylates 22 and 23 , which are slowly equilibrating already at ambient temperature, are formed by thermal addition of ADM to the seven-membered ring of dimethyl 5-isopropyl-3,8-dimethylazulene-1,2-dicarboxylate ( 7 ; cf. Scheme 10). Azulene 7 which is electronically deactivated by the two MeOCO groups at C(1) and C(2) shows no more thermal reactivity in the presence of ADM at the five-membered ring (cf. Scheme 11). The tricyclic tetracarboxylates 22 and 23 react with excess ADM at 200° in a slow Diels-Alder reaction to form the tetracyclic hexacarboxylates 32 , ‘anti’- 33 , and ‘anti’- 34 (cf. Schemes 10–12 as well as Scheme 13). A structural correlation of the tri- and tetracyclic compounds is only feasible if thermal equilibration via [1,5]-C shifts between all six possible tricyclic tetracarboxylates ( 22, 23 , and 35–38 ; cf. Scheme 13) is assumed. The tetracyclic hexacarboxylates 32 , ‘anti’- 33 , and ‘anti’- 34 seem to arise from the most strained tricyclic intermediates ( 36–38 ) by the Diels-Alder reaction with ADM.     

Regioselective lithiation of benzyl imidazole: Synthesis and evaluation of new organocatalysts for trans-diol functionalization.

Abstract

Benzyl imidazole was successfully lithiated using n-BuLi at ?78?°C and verified by deuterium incorporation. The chemical reaction of the lithiated benzimidazole was explored with a series of different electrophiles. This approach was utilized to synthesize new anti and syn diphenyl organocatalysts for trans-diol functionalization.     

Structure‐Guided Minimalist Redesign of the L‐Fuculose‐1‐Phosphate Aldolase Active Site: Expedient Synthesis of Novel Polyhydroxylated Pyrrolizidines and their Inhibitory Properties Against Glycosidases and Intestinal Disaccharidases

A minimalist active site redesign of the L ‐fuculose‐1‐phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N‐Cbz‐amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N‐Cbz‐amino aldehydes including N‐Cbz‐prolinal derivatives. FucA F131A showed an aldol activity of 62 μmol h^?1 mg^?1 with (R)‐N‐Cbz‐prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)‐amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)‐ and with (S)‐N‐Cbz‐prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N‐Cbz‐prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π–cation interaction with the residue K205′ and to efficient contact between the substrate and the mechanistically important Y113′ and Y209′ residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4‐dideoxy‐1,4‐imino‐L ‐arabinitol (LAB) and 1,4‐dideoxy‐1,4‐imino‐D ‐arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.     

Hydrogen bonding,protonation and twisting in the singlet excited state of some 2-(4-Aminophenyl)pyrido-oxa-, -thia-, and -imidazoles

The absorption and fluorescence characteristics of 2-(4-aminophenyl)pyrido[3,2-d]oxazole ( 1 ), of its thiazole 2 and imidazole 3 analogues, as well as of the corresponding pyrido[3,4-d]imidazole 4 have been examined. S₁ is a planar ππ* ICT state, similarly to p-electron-withdrawing substituted anilines. In the protonated form, the chromophore is the heterocyclic moiety. With compounds 3 and 4 in alcohols, hydrogen bonding depending on proton donating and accepting properties of the medium determines the fluorescence. In this case, a red-shifted emission attributed to a twisted TICT state is also observed.     

Asymmetric anti‐Selective Michael Reaction of Imidazole‐Modified Ketones with trans‐β‐Nitroalkenes

The successful application of imidazole‐modified ketones in asymmetric anti‐selective Michael reactions with trans‐β‐nitroalkenes is presented by employing a newly developed 3‐bromothiophene‐modified chiral diamine ligand. The corresponding conjugate adduct was submitted to further transformations with Grignard reagents to solve the problem of α‐site selectivity of simple linear ketones. Additionally, the syn‐selective product was obtained by treating the anti‐selective adduct with a simple base. In this way, the site‐specific products for both diastereomers in the asymmetric conjugate addition of simple ketones to nitroalkenes can be obtained.     

Synthesis of Tetrahydropyridoimidazole‐2‐acetates: Effect of Carboxy and Methoxycarbonyl Groups at C(2) on the Inhibition of Some β‐ and α‐Glycosidases

The gluco‐ and manno‐tetrahydropyridoimidazole‐2‐acetates and ‐acetic acids 16 and 17 , and 20 and 21 , respectively, were synthesized by condensation, in the presence of HgCl₂, of the known thionolactam 26 with the β‐amino ester 25 that was obtained by addition of AcOMe to the imine 22 , followed by debenzylation. The resulting methyl esters 16 and 20 were hydrolyzed to the acetic acids 17 and 21 . The (methoxycarbonyl)‐imidazole 14 and the acid 15 were obtained via the known aldehyde 29 . The imidazoles 14 – 17, 20 , and 21 were tested as inhibitors of the β‐glucosidase from Caldocellum saccharolyticum, the α‐glucosidase from brewer's yeast, the β‐mannosidase from snail, and the α‐mannosidase from Jack beans (Tables 1–3). There is a similar dependence of the K_i values on the nature of the C(2)‐substituent in the gluco‐ and manno‐series. With the exception of 19 , manno‐imidazoles are weaker inhibitors than the gluco‐analogues. The methyl acetates 16 and 20 are 3–4 times weaker than the methyl propionates 5 and 11 , in agreement with the hydrophobic effect. The gluco‐configured (methoxycarbonyl)‐imidazole 14 is 20 times weaker than the methyl acetate 16 , reflecting the reduced basicity of 14 , while the manno‐configured (methoxycarbonyl)‐imidazole 18 is only 1.2 times weaker than the methyl acetate 20 , suggesting a binding interaction of the MeOCO group and the β‐mannosidase. The carboxylic acids 6, 12, 15, 17, 19 , and 21 are weaker inhibitors than the esters, with the propionic acids 6 and 12 being the strongest and the carboxy‐imidazoles 15 and 19 the weakest inhibitors. The manno‐acetate 21 inhibits the β‐mannosidase ca. 8 times less strongly than the propionate 12 , but only 1.5 times more strongly than the carboxylate 19 , suggesting a compensating binding interaction also of the COOH group and the β‐mannosidase. The α/β selectivity for the gluco‐imidazoles ranges between 110 for 15 and 13.4?10³ for 6 ; the manno‐imidazoles are less selective. The methyl propionates proved the strongest inhibitors of the α‐glucosidase (IC₅₀ ( 5 )=25 μM ) and the α‐mannosidase (K_i( 11 ) =0.60 μM ).     

Ab initioSCF MO calculations on the reactions of hydroxyl radical with imidazole and monoprotonated imidazole

The addition reactions of hydroxyl radical with imidazole and its protonated form to yield radical adducts have been investigated by ab initio SCF MO methods using STO -3G and 4-31G basis sets. Analogous radical species are of importance in radiation damage to biological systems. Of the possible radical products, the calculations indicate that the allylic species are generally favored energetically over the nonallylic forms. On an energetic basis, the results show that the allylic adducts formed by addition at the C2 and C5 positions are about equally favorable. Although the C5 species is generally identified as the experimentally observed product in aqueous media for both protonated and unprotonated imidazole, some experimental evidence exists indicating the presence of other forms. Our results suggest that this other form is the C2 adduct. The calculations also point to the protonated form of imidazole being less reactive than imidazole, which is in accord with experimental observations.     

Synthesis of N‐Acetylglucosamine‐Derived Nagstatin Analogues and Their Evaluation as Glycosidase Inhibitors

The gluco‐configured analogue 15 of nagstatin ( 1 ) and the methyl ester 14 were synthesized via condensation of the thionolactams 17 or 18 with the β‐amino ester 19 . The silyl ethers 20 and 21 resulting from 17 were desilylated to 22 and 23 ; these alcohols were directly obtained by condensing 18 and 19 . The attempted substitution of the C(8)? OH group of 22 by azide under Mitsunobu conditions led unexpectedly to the deoxygenated α‐azido esters 24 . The desired azide 25 was obtained by treating the manno‐configured alcohol 23 with diphenyl phosphorazidate. The azide was transformed to the debenzylated acetamido ester 14 that was hydrolyzed to the nagstatin analogue 15 . The imidazole‐2‐acetates 14 and 15 are nanomolar inhibitors of the N‐acetyl‐β‐glucosaminidases from Jack beans and from bovine kidney, submicromolar to micromolar inhibitors of the β‐glucosidase from Caldocellum saccharolyticum, and rather weak inhibitors of the snail β‐mannosidase. In all cases, the ester was a stronger inhibitor than the corresponding acid. As expected from their gluco‐configuration, both imidazopyridines 14 and 15 are stronger inhibitors of the β‐N‐acetylglucosaminidase from bovine kidney than nagstatin.     

C,C-Double Bond Participation in the Acid-Catalyzed Cyclization of 9exo-Methyl-anti10,11-tricyclo[4.2.1.12,5]deca-3,7-diene-9endo, 10endo-diol. Influence of Steric Compression on the Product Distribution

Acid treatment of 9exo-methyl-anti^10,11-tricyclo[4.2.1.1^2,5]deca-3,7-diene-9endo, 10endo-diol ( 8 ) leads to the two isomeric pentacyclic ethers 7 and 9 by intramolecular nucleophilic substitution of a protonated OH-group with participation of a C,C-double bond. The higher steric compression in diol 8 on the side of the tertiary OH-group at C(9) and the C(3), C(4)-double bond, accounts for the preferred formation of 7 over 9 .     

Aqua[N-(6-carboxylato-κO-pyridine-2-carbonyl-κN)-l-histidinato-κ2N,N′]copper(II)

The title compound, [Cu(C₁₃H₁₀N₄O₅)(H₂O)], is an asymmetric and pentacoordinated square-pyramidal copper(II) complex, with a water mol­ecule occupying an apical position. It consists of syn and anti isomers of the apical water mol­ecule with respect to the metal-free carboxyl group on the equatorial ligand. The crystal structure of this complex is a rare example of conformational isomerization, in which the two isomers cocrystallize in a 1:3 ratio of syn to anti mol­ecules in the same lattice.     

Enzymatic kinetic of cellulose hydrolysis

The ethanol effect on the Trichoderma reesei cellulases was studied to quantify and clarify this inhibition type. To determine inhibition parameters of crude cellulase and purified exoglucanase Cel7A, integrated Michaelis-Menten equations were used assuming the presence of two inhibitors: cellobiose as the reaction product and ethanol as a possible bioproduct of cellulose fermentation. It was found that hydrolysis of cellulose by crude enzyme follows a model that considers noncompetitive inhibition by ethanol, whereas Cel7A is very slightly competitively inhibited. Crude cellulase is much more inhibited (K _iul=K _icl=151.9 mM) than exoglucanase Cel7A (K _icl=1.6 × 10¹⁵ mM). Also, calculated inhibition constants showed that cellobiose inhibition is more potent than ethanol inhibition both for the crude enzyme as well as exoglucanase Cel7A.     

Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B,Cel9A,and Cel5A on BMCC and Avicel

In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E₃, E₄, and E₅), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.     

Crystal Structures and Packing of 4-Cyanocubanecarboxylic Acid,Its Methyl Ester,and the Solid Solution of 1,4-Dicyanocubane and 1,4-Dibromocubane

The crystal structure of 4-cyanocubanecarboxylic acid contains the same syn–anti carboxyl group catemer that is found in other cubane monocarboxylic acids. The cyano groups are arranged according to the type-II geometry, in that they are 2₁ screw-axis related. Curiously, there is a 5% orientational disorder of the cyano and anti carboxyl groups. The ester of the title acid packs isostructurally with the corresponding chloro and fluoro analogs because of the importance of the C—H...O hydrogen bond patterns. 1,4-Dicyanocubane forms solid solutions with 1,4-dibromocubane, but, interestingly, the crystal structure of the solid solution is distinct from that of either component. The formation of these solid solutions seems to be governed by shape and size factors.     

Forging C−S Bonds on the Azetidine Ring by Continuous Flow Photochemical Addition of Thiols and Disulfides to Azetines

A strategy for anti-Markovnikov hydroalkyl/aryl thiolation and disulfidation of 2-azetines under continuous flow conditions has been developed. Thiyl radicals are generated from thiols or disulfides and subsequently propagate into the azetine unsaturation to forge the C−S bond and shape a secondary radical intermediate. This carbon-centered radical chain transfers to another thiol via hydrogen atom transfer (HAT) or another disulfide to regenerate the key thiyl radical intermediates. The use of flow technology ensures efficient irradiation of the reaction mixture leading to extremely fast, robust, and scalable protocols. Furthermore, ethyl acetate was adopted as an environmentally responsible solvent.     

2′‐De­oxy‐2‐fluoro­tubercidin

In the title compound [systematic name: 7‐(2‐de­oxy‐β‐d ‐erythro‐pentofuranos­yl)‐2‐fluoro‐7H‐pyrrolo[2,3‐d]pyrimidin‐2‐amine], C₁₁H₁₃FN₄O₃, the conformation of the N‐glycosylic bond is between anti and high‐anti [χ = −110.2 (3)°]. The 2′‐deoxy­ribofuranosyl unit adopts the N‐type sugar pucker (₄T³), with P = 40.3° and τ_m = 39.2°. The orientation of the exocyclic C4′—C5′ bond is −ap (trans), with a torsion angle γ = −168.39 (18)°. The nucleobases are arranged head‐to‐head. The crystal structure is stabilized by four inter­molecular hydrogen bonds of types N—H⋯N, N—H⋯O and O—H⋯O.     

G‐Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy

A curated library of circular dichroism spectra of 23 G‐quadruplexes of known structure was built and analyzed. The goal of this study was to use this reference library to develop an algorithm to derive quantitative estimates of the secondary structure content of quadruplexes from their experimental CD spectra. Principal component analysis and singular value decomposition were used to characterize the reference spectral library. CD spectra were successfully fit to obtain estimates of the amounts of base steps in anti–anti, syn–anti or anti–syn conformations, in diagonal or lateral loops, or in other conformations. The results show that CD spectra of nucleic acids can be analyzed to obtain quantitative structural information about secondary structure content in an analogous way to methods used to analyze protein CD spectra.