Synthesis of broad-specificity activity-based probes for exo-ß-mannosidases.

Exo-ß-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-ß-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-ß-mannosidases, we present a concise synthesis of ß-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-ß-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-ß-mannosidases in biological and biomedical research.

Effective chiral pool synthesis of both enantiomers of the TRPML inhibitor trans-ML-SI3.

Two independent chiral pool syntheses of both enantiomers of the TRPML inhibitor, trans-ML-SI3, were developed, starting from commercially available (1S,2R)- and (1R,2S)-configured cis-2-aminocyclohexanols. Both routes lead to the target compounds in excellent enantiomeric purity and good overall yields. For the most attractive (-)-trans-enantiomer, the R,R-configuration was identified by these unambiguous syntheses, and the results were confirmed by single-crystal X-ray structure analysis. These effective synthetic approaches further allow flexible variation of prominent residues in ML-SI3 for future in-depth analysis of structure-activity relationships as both the piperazine and the N-sulfonyl residues are introduced into the molecule at late stages of the synthesis.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Synthesis and Biological Evaluation of the Novel Growth Inhibitor Streptol Glucoside, Isolated from an Obligate Plant Symbiont.

The plant Psychotria kirkii hosts an obligatory bacterial symbiont, Candidatus Burkholderia kirkii, in nodules on their leaves. Recently, a glucosylated derivative of (+)-streptol, (+)-streptol glucoside, was isolated from the nodulated leaves and was found to possess a plant growth inhibitory activity. To establish a structure-activity relationship study, a convergent strategy was developed to obtain several pseudosugars from a single synthetic precursor. Furthermore, the glucosylation of streptol was investigated in detail and conditions affording specifically the α or ß glucosidic anomer were identified. Although (+)-streptol was the most active compound, its concentration in P. kirkii plant leaves extract was approximately ten-fold lower than that of (+)-streptol glucoside. These results provide compelling evidence that the glucosylation of (+)-streptol protects the plant host against the growth inhibitory effect of the compound, which might constitute a molecular cornerstone for this successful plant-bacteria symbiosis.

Ring closing metathesis (RCM) approach to the synthesis of conduramine B-2, ent-conduramine F-2, aminocyclopentitol and trihydroxyazepane.

The syntheses of conduramine B-2, ent-conduramine F-2, aminocyclopentitol and trihydroxyazepane were accomplished from a common precursor, through a divergent approach using ring closing metathesis (RCM) as the key step. Tri-O-benzyl-d-glucal was converted to 3,4,6-tri-O-benzyl-1,2-dideoxy-2-iodo-1-p-toluenesulfonamido-α-d-mannose. Exposure to NaBH4 in MeOH resulted in a facile 1,2-transposition of the -NHTs group with concomitant glycosylation to give methyl 3,4,6-tri-O-benzyl-2-deoxy-2-p-toluenesulfonamido-ß-d-glucoside, which was converted into methyl 6-deoxy-6-iodo-glucoside in three steps. Zinc-mediated Vasella's rearrangement proceeded smoothly to give the pluripotent formyl-olefin, possessing both electrophilic and nucleophilic sites, which was used as a common precursor in our diversity-oriented approach. Vinylation of the carbonyl group followed by RCM and subsequent deprotection resulted in the successful synthesis of conduramine B-2 and ent-conduramine F-2 for the first time. On the other hand, the Wittig reaction of the formyl-olefin affords the diene that undergoes Grubbs' I catalyzed RCM and deprotection/reduction to provide 3-amino-cyclopentan-1,2-diol. Utilizing the nucleophilic site at the nitrogen of the common precursor, base mediated N-allylation was carried out to obtain the corresponding diene that underwent a smooth RCM to afford trihydroxyazepane.

Synthesis of 5a,5a'-dicarba-d-glucobioses from conformationally restricted carbaglucosyl triflates using SN2-type inversion with carbaglucosyl nucleophiles.

Novel carbohydrate mimics were designed which contain two 5a-carba-d-glucose residues, one each at reducing and nonreducing end, and thus these mimics are 5a,5a'-dicarba-d-glucobioses. Dicarbadisaccharides have attractive features such as stability against endogenous degradative enzymes and being resistant to glycation reactions such as the Maillard reaction. For the synthesis of dicarba-ß-d-isomaltose derivatives, the carbaglucosyl triflate locked in 4C1 conformation was synthesized by protecting with butane-2,3-diacetal group or benzylidene group. Then, 5a,5a'-dicarba-ß-d-maltose and 5a,5a'-dicarba-α,ß-d-trehalose were synthesized by the SN2-type inversion reaction using 4,6-O-benzylidene carbaglucosyl triflate with 4-OH and 1-OH carba-ß-d-glucose derivatives, respectively, and similarly 5a,5a'-dicarba-α-d-isomaltose with 6-OH carba-α-d-glucose derivative.

Synthesis of the Polyoxygenated Cyclohexanoid Core of Bioactive Glycosides Xylosmin and Flacourtosides E and F.

A flexible synthesis of the carbocyclic core present in glycosides xylosmin and flacourtosides E and F, natural products exhibiting antimalarial and antiarboviral activities, has been accomplished. Our approach emanates from the Diels-Alder adduct of cyclopentadiene and MOM-protected 2-hydroxymethyl- p-benzoquinone and takes advantage of the stereochemical propensity of the norbornyl-fused scaffolds to generate the dense oxy-functionalization pattern with stereocontrol, enroute to a racemic synthesis of the carbocyclic core present in the aforementioned bioactive materials. This effort augurs well for exploring chemical diversity space around their scaffold.

Exploiting polarity and chirality to probe the Hsp90 C-terminus.

Inhibition of the Hsp90 C-terminus is an attractive therapeutic approach for the treatment of cancer. Novobiocin, the first Hsp90 C-terminal inhibitor identified, contains a synthetically complex noviose sugar that has limited the generation of structure-activity relationships for this region of the molecule. The work described herein utilizes various ring systems as noviose surrogates to explore the size and nature of the surrounding binding pocket.

General Method for the Synthesis of (-)-Conduritol C and Analogs from Chiral Cyclohexadienediol Scaffolds.

An efficient and facile general method for the synthesis of conduritol C analogs, taking advantage of an enantioselective biocatalysis process of monosubstituted benzenes, is described. The absolute stereochemical patterns of the target molecules (−)-conduritol C, (−)-bromo-conduritol C, and (−)-methyl-conduritol C were achieved by means of chemoenzymatic methods. The stereochemistry present at the homochiral cyclohexadiene-cis-1,2-diols derived from the arene biotransformation and the enantioselective ring opening of a non-isolated vinylepoxide derivative permitted the absolute configuration of the carbon bearing the hydroxyl groups at the target molecules to be established. All three conduritols and two intermediates were crystallized, and their structures were confirmed by X-ray diffraction. The three conduritols and intermediates were isostructural. The versatility of our methodology is noteworthy to expand the preparation of conduritol C analogs starting from toluene dioxygenase (TDO) monosubstituted arene substrates.

Fluoro-Carba-Sugars are Glycomimetic Activators of the glmS Ribozyme.

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and ß-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.

Synthesis of dibenzylamino-1-methylcyclohexanol and dibenzylamino-1-trifluoromethylcyclohexanol isomers.

The isomers of dibenzylamino-1-methylcyclohexan-1-ol and dibenzylamino-1-trifluoromethylcyclohexan-1-ol have been prepared. The stereochemistry of these compounds was unequivocally assigned through a combination of NMR spectroscopy and single crystal X-ray analysis. The cis-isomer of 3-N,N-dibenzylamino-1-trifluoromethylcyclohexanol and its derivatives display an unusual conformational behaviour in both solution-phase and the solid-state, where the amino group usually adopts an axial conformation.

Synthesis of a new quaternary phosphonium salt: NMR study of the conformational structure and dynamics.

A novel phosphonium salt based on pyridoxine was synthesized. Conformational analysis of the compound in solution was performed using dynamic NMR experiments and calculations. The obtained results revealed some differences in the conformational transitions and the energy parameters of the conformational exchange of the studied compound in comparison to previously reported data for other phosphorus-containing pyridoxine derivatives. It was shown that increasing the substituent at the C-11 carbon leads to greater differences in the populations of stable states and the corresponding equilibrium energies. Copyright © 2015 John Wiley & Sons, Ltd.

An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

Synthesis and evaluation of hydroxymethylaminocyclitols as glycosidase inhibitors.

Four series of C7N aminocyclitol analogues of glucose were synthesized by stereocontrolled epoxide opening of hydroxyl protected forms of the cyclohexane epoxides cyclophellitol and 1,6-epi-cyclophellitol. The resulting hydroxymethyl substituted aminocyclitols were tested as glycosidase inhibitors. Cyclitols having an amino group in an α configuration at a position equivalent to the anomeric in the sugar were found to be low micromolar inhibitors of the α-glucosidase from baker's yeast with Ki's near to 2 µM. On the other hand, N-octyl aminocyclitols having the nitrogen substituents in an α or ß configuration were found to be good inhibitors of recombinant ß-glucocerebrosidase with Ki values between 8.3 and 17 µM, and also inhibited lysosomal ß-glucosidase activity in live cells at low-micromolar concentrations. A computational docking study suggests a differential binding among the different series of ß-glucocerebrosidase inhibitors. In agreement with the experimental results, the binding poses obtained indicate that the presence of an alkyl lipid substituent in the inhibitor mimicking one of the lipid chains in the substrate is critical for potency. In contrast, the matching of hydroxymethyl substituents in the aminocyclitols and the parent glucosylceramide does not seem to be strictly necessary for potent inhibition, indicating the risk of simplifying structural analogies in sugar mimetic design.

Greener Selective Cycloalkane Oxidations with Hydrogen Peroxide Catalyzed by Copper-5-(4-pyridyl)tetrazolate Metal-Organic Frameworks.

Microwave assisted synthesis of the Cu(I) compound [Cu(µ4-4-ptz)]n [1, 4-ptz=5-(4-pyridyl)tetrazolate] has been performed by employing a relatively easy method and within a shorter period of time compared to its sister compounds. The syntheses of the Cu(II) compounds [Cu3(µ3-4-ptz)4(µ2-N3)2(DMF)2]n∙(DMF)2n (2) and [Cu(µ2-4-ptz)2(H2O)2]n (3) using a similar method were reported previously by us. MOFs 1-3 revealed high catalytic activity toward oxidation of cyclic alkanes (cyclopentane, -hexane and -octane) with aqueous hydrogen peroxide, under very mild conditions (at room temperature), without any added solvent or additive. The most efficient system (2/H2O2) showed, for the oxidation of cyclohexane, a turnover number (TON) of 396 (TOF of 40 h(-1)), with an overall product yield (cyclohexanol and cyclohexanone) of 40% relative to the substrate. Moreover, the heterogeneous catalytic systems 1-3 allowed an easy catalyst recovery and reuse, at least for four consecutive cycles, maintaining ca. 90% of the initial high activity and concomitant high selectivity.

Asymmetric stereodivergent strategy towards aminocyclitols.

A concise asymmetric synthesis of aminocyclitols, such as diastereomeric 2-deoxystreptamine analogues and conduramine A, is described. The Pd-catalyzed asymmetric desymmetrization of meso 1,4-dibenzolate enables the synthesis of highly oxidized cyclohexane architectures. These scaffolds can potentially be used to access new aminoglycoside antibiotics and enantiomerically pure α-glucosidase inhibitors.

D-Aminoacylase-initiated cascade Aldol condensation/Robinson annulation for synthesis of substituted cyclohex-2-enones from simple aldehydes and acetone.

As an important building block, developing efficient and green synthesis strategy of cyclohex-2-enones is of great importance. In this present work, a general approach to the mild synthesis of substituted cyclohex-2-enones derivatives starting fro m simple aldehydes and acetone have been achieved via D-aminoacylase-initiated Aldol condensation/Robinson annulation cascade reaction using imidazole as an additive in organic media. The influences of reaction conditions including solvents, enzyme concentration, additives type, molar ratio of enzyme to additive, and substrate scopes were systematically investigated. Furthermore, some experiments were designed to explore the catalytic roles of D-aminoacylase and imidazole in the multistep cascade process, and one possible mechanism was proposed.

#Nitrosocarbonyls 1: antiviral activity of N-(4-hydroxycyclohex-2-en-1-yl)quinoline-2-carboxamide against the influenza A virus H1N1.

Influenza virus flu A H1N1 still remains a target for its inhibition with small molecules. Fleeting nitrosocarbonyl intermediates are at work in a short-cut synthesis of carbocyclic nucleoside analogues. The strategy of the synthetic approaches is presented along with the in vitro antiviral tests. The nucleoside derivatives were tested for their inhibitory activity against a variety of viruses. Promising antiviral activities were found for specific compounds in the case of flu A H1N1.

Novel inhibitory effect of N-(2-hydroxycyclohexyl)valiolamine on melanin production in a human skin model.

Hyper-pigmentation causes skin darkness and medical disorders, such as post-inflammatory melanoderma and melasma. Therefore, the development of anti-melanogenic agents is important for treating these conditions and for cosmetic production. In our previous paper, we demonstrated that the anti-diabetic drug voglibose, a valiolamine derivative, is a potent anti-melanogenic agent. In addition, we proposed an alternative screening strategy to identify valiolamine derivatives with high skin permeability that act as anti-melanogenic agents when applied topically. In this study, we synthesized several valiolamine derivatives with enhanced lipophilicity and examined their inhibitory effects in a human skin model. N-(2-hydroxycyclohexyl)valiolamine (HV) possesses a stronger inhibitory effect on melanin production than voglibose in a human skin model, suggesting that HV is a more potent anti-melanogenic agent for the skin.

Diol-linked microporous networks of cubic siloxane cages.

A new class of inorganic-organic hybrid porous materials has been synthesized by a reaction between octa(hydridosilsesquioxane) (H(8)Si(8)O(12)), which has a double-four-ring (D4R) structure, and various diols, such as 1,3-propanediol (PD), 1,4-cyclohexanediol (CHD), and 1,3-adamantanediol (AD). Solid-state (29) Si magic-angle-spinning NMR spectroscopic analysis confirmed that most of the corner Si-H groups reacted with diols to form Si-O-C bonds with retention of the D4R cage. Nitrogen adsorption-desorption studies showed that the products are microporous solids with high BET surface areas (up to ≈580 m(2) g(-1) for CHD- and AD-linked products). If n-alkanediols are used as linkers, the surface area becomes smaller as the number of carbon atoms is increased. The thermal and hydrolytic stability of the products strongly depend on the type of diol linkers. The highest stabilities are found for the AD-linked products, which are thermally stable up to around 400 °C and remain intact even after being soaked in water for 1 day. In contrast, the PD-linked product is easily hydrolyzed in water to give microporous silica. These results offer a new route toward a series of silica-based porous materials with unique structures and properties.