102,198 research outputs found
From batch to flow synthesis of 6-substituted purine ribonucleosides by enzymatic transglycosylation
No full textPurine nucleoside phosphorylases (PNPs, EC 2.4.2.1) catalyze the reversible phosphorolysis of the glycosydic bond of purine nucleosides and, upon addition of a second nucleobase, may transfer the glycosyl moiety to stereoselectively form a new nucleoside (transglycosylation).
Because of its wide substrate specificity,[1,2] a PNP from Aeromonas hydrophila (AhPNP) was exploited to catalyze a “one-pot, one-enzyme” batch transglycosylation, resulting in a moderate to high conversion (18-65%) of 6-substituted purine bases into a 23-compound library of 6-substituted purine-9-ribosides.[3] Afterwards, AhPNP was covalently immobilized (50% yield) in a pre-packed stainless steel column by reaction with glutaraldehyde and reduction of the imino groups. The resulting AhPNP-IMER (IMmobilized Enzyme Reactor) was coupled to a HPLC apparatus containing an analytical or semi-preparative chromatographic column associated with a UV-visible detector. This system (Figure 1) was exploited to perform the flow-synthesis of five 6-substituted purine ribonucleosides at a mg scale by transglycosylation. Under optimized (Design of Experiments, DoE) reaction conditions, coupling of transglycosylation with product separation resulted in a fast and efficient process (52-89% conversion) with minimized sample handling.[4]
References:
[1] Ubiali, D. et al. Adv. Synth. Catal. 2012, 354, 96-104.
[2] Calleri, E. et al. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014, 968, 79-86.
[3] Ubiali, D. et al. Curr. Org. Chem. 2015, 19, 2220-2225.
[4] Calleri, E. et al. Adv. Synth. Catal. 2015, 357, 2520-2528
From batch to flow synthesis of purine ribonucleosides by enzymatic transglycosylation
No full textPurine nucleoside phosphorylases (PNPs, EC 2.4.2.1) catalyze the reversible phosphorolysis of the glycosydic bond of purine nucleosides; upon addition of a second nucleobase, these enzymes may transfer the glycosyl moiety to it, resulting in the chemo-, regio- and stereoselective formation of a new nucleoside (transglycosylation, Scheme 1). This chemoenzymatic process represents an advantageous alternative to conventional chemical strategies which are frequently hampered by several drawbacks such as multistep processes, need for protecting groups, low chemo-, regio- and stereoselectivity.
A PNP from Aeromonas hydrophila (AhPNP) has been recently characterized in terms of substrate specificity [1], also upon immobilization on the inner surface of a silica capillary coupled on-line with a chromatographic column [2].
Because of its wide substrate specificity, AhPNP was then exploited to catalyze the “one-pot, one-enzyme” transglycosylation of 7-methylguanosine iodide with a series of 6-substituted purines, resulting in a moderate to high conversion (18-65%) of the bases into a 23-compound library of 6-substituted purine-9-ribosides. Moreover, AhPNP was covalently immobilized (25 mg immobilized enzyme, 50% yield) in a pre-packed stainless steel column containing aminopropyl silica particles. The resulting AhPNP-IMER (Immobilized Enzyme Reactor) was coupled to a HPLC apparatus containing an analytical or a semi-preparative chromatographic column associated with a UV-visible detector. This system was used to synthesize five 6-substituted purine ribonucleosides at a mg scale by transglycosylation through a “flow-based” approach. Coupling of transglycosylation reaction and product separation resulted in a fast and efficient process (52-89% conversion) with minimized sample handling. To date, AhPNP-IMER completely retained its activity upon 50 reactions in 10 months.
[1] D. Ubiali, C. D. Serra, I. Serra, C. F. Morelli, M. Terreni, A. M. Albertini, P. Manitto, G. Speranza Adv. Synth. Catal. 2012, 354, 96-104
[2] E. Calleri, D. Ubiali, I. Serra, C. Temporini, G. Cattaneo, G. Speranza, C. F. Morelli, G. Massolini J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014, 968, 79-8
Batch and flow synthesis of 6-substituted purine ribonucleosides by enzymatic transglycosylation
No full textPurine nucleoside phosphorylases (PNPs, EC 2.4.2.1) catalyze the reversible phosphorolysis of the glycosydic bond of purine nucleosides and, upon addition of a second nucleobase, may transfer the glycosyl moiety to stereoselectively form a new nucleoside (transglycosylation). Because of its wide substrate specificity,[1,2] a PNP from Aeromonas hydrophila (AhPNP) was exploited to catalyze a “one-pot, one-enzyme” batch transglycosylation, resulting in a moderate to high conversion (18-65%) of 6-substituted purine bases into a 23-compound library of 6-substituted purine-9-ribosides.[3] Afterwards, AhPNP was covalently immobilized (50% yield) in a pre-packed stainless steel column and the resulting AhPNP-IMER (IMmobilized Enzyme Reactor) was coupled to a HPLC apparatus containing an analytical or semi-preparative chromatographic column associated with a UVvisible detector. The resulting system (Figure 1) was exploited to perform the flow-synthesis of five 6-substituted purine ribonucleosides at a mg scale by transglycosylation. Under optimized (Design of Experiments, DoE) reaction conditions, coupling of transglycosylation with product separation resulted in a fast and efficient process (52-89% conversion) with minimized sample handling.[4]
References:
[1] Ubiali, D. et al. Adv. Synth. Catal. 2012, 354, 96-104.
[2] Calleri, E. et al. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2014, 968, 79-86.
[3] Ubiali D. et al. Curr. Org. Chem. 2015, 19, 2220-2225.
[4] Calleri, E. et al. Adv. Synth. Catal. 2015, 357, 2520-2528
Nucleoside phosphorylases and deoxyribonucleoside kinases: the green side of nucleis acid chemistry
No full textBiocatalysis has become nowadays an important tool in synthetic organic chemistry. Biotransformations are chemo-, regio-, and stereoselective, occur under mild reaction conditions and are characterized by a reduced use of toxic reagents/solvents. One of the areas where biocatalyzed reactions have clearly shown their potential is nucleic acid chemistry. Enzymes of nucleic acid metabolism such as nucleoside phosphorylases (NPs, EC 2.4.2) and deoxyribonucleoside kinases (dNKs, EC 2.7.1) can be conveniently used as biocatalysts in the synthesis of nucleoside and nucleotide analogues.
NPs catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate. If a second nucleobase is added to the reaction, the formation of a new nucleoside can result (transglycosylation). dNKs catalyze the regioselective transfer of a phosphate group from ATP to a nucleoside to give the corresponding nucleoside 5’-monophosphate. However, the bottleneck in the use of enzymes as biocatalysts is often their instability under experimental conditions, their cost and solubility in the reaction medium. These issues can be frequently overcome by immobilizing the enzyme on a solid support. Substrate specificity, immobilization and some synthetic applications of selected NPs1-3 and dNKs4 will be described.
References
1. Ubiali, D.; Serra, C.D.; Serra, I.; Morelli, C.F.; Terreni, M.; Albertini, A.M.; Manitto, P.; Speranza, G. Adv. Synth. Catal., 2012, 354, 96.
2. Ubiali, D.; Morelli, C.F.; Rabuffetti, M.; Cattaneo, G.; Serra, I.; Bavaro, T.; Albertini, A.M.; Speranza, G. Curr. Org. Chem., 2015, 19, 2220.
3. Calleri, E.; Cattaneo, G.; Rabuffetti, M.; Serra, I.; Bavaro, T.; Massolini, G.; Speranza, G.; Ubiali, D. Adv. Synth. Catal., 2015, 357, 2520.
4. Serra, I.; Conti, S.; Piškur, J.; Clausen, A.R.; Munch-Petersen, B.; Terreni, M.; Ubiali, D. Adv. Synth. Catal., 2014, 356, 563
Synthesis and molecular modeling of purine ribonucleotides as potential ligands of the human G protein-coupled receptor 17 (GPR17)
No full textGPCRs (G Protein-Coupled Receptors) are important drug targets in medicinal chemistry [1]. The GPR17 receptor, phylogenetically related to both purinergic P2Y and CysLT receptors, is usually over-expressed in the damaged brain tissue and is involved in various disorders characterized by demyelination, such as multiple sclerosis and stroke. Experimental data have shown that it is responsive to both agonists (e.g. nucleotides and their adducts) and antagonists (e.g. Cangrelor and Montelukast) [2]. Therefore, the human GPR17 receptor is a promising therapeutic target for treatment of neurodegenerative diseases [3].
This evidence prompted us to perform docking studies aided by molecular modeling on a homology model (based on P2Y1 receptors). Among the selected molecules, 8-methylaminoinosinic acid (1) and three N2-alkyl/acyl derivatives of guanylic acid (2-4) emerged as the best potential ligands.
As a result, their synthesis was carried out. Compound 1 was obtained by direct phosphorylation of 8-methylaminoinosine, previously prepared by amination of 8-bromoinosine. In the case of 2, position N2 of the purine ring was activated as a bromo derivative and subjected to displacement with n-octylamine. As for 3 and 4, N2-acylations were performed by treatment with a proper acyl chloride or anhydride through a transient protection strategy. Compounds 2, 3 and 4 were obtained as 2’,3’-O-isopropylidene adducts of the corresponding nucleotides.
Binding assays will be carried out by Surface Plasmon Resonance (SPR) [4], which has been demonstrated as a reliable technique for the systematic identification of agonists and antagonists of GPCRs, including GPR17 as recently demonstrated by our group [5].
[1] D. Wacker, R. C. Stevens, B. L. Roth, Cell 2017, 170, 414-427.
[2] P. Ciana, M. Fumagalli, M.L. Trincavelli, C. Verderio, P. Rosa, D. Lecca, S. Ferrario, C. Parravicini, V. Capra, P. Gelosa, U. Guerrini, S. Belcredito, M. Cimino, L. Sironi, E. Tremoli, G.E. Rovati, C. Martini and M.P. Abbracchio, EMBO J 2006, 25, 4615-4627.
[3] G. Marucci, D. Dal Ben, C. Lambertucci, A. Marti Navia, A. Spinaci, R. Volpini and M. Buccioni, Expert Opin. Ther. Pat. 2019, 29, 85-95.
[4] D.-S. Wang, S.-K. Fan, Sensors 2016, 16, 1175-1192.
[5] D. Capelli, C. Parravicini, G. Pochetti, R. Montanari, C. Temporini, M. Rabuffetti, M. L. Trincavelli, S. Daniele, M. Fumagalli, S. Saporiti, E. Bonfanti, M. P. Abbracchio, I. Eberini, S. Ceruti, E. Calleri, S. Capaldi, Front. Chem. 2020, 7, 910
Una lettera di frate Aicardo da Camodeia, arcivescovo di Milano (28 giugno 1319)
No full textLo scopo di questo contributo è di fornire l’edizione critica e di analizzare una lettera patente spedita da Avignone dall’arcivescovo di Milano Aicardo da Camodeia al suo vicario Obizzone da Momo. La missiva è datata 28 giugno 1319 ed è conservata presso la Biblioteca Civica Berio di Genova.The aim of this paper is to give a crital edition and to study a litterae patentes sent from Avignon by Aicardo of Camodeia, archibishop of Milan, to Obizzone of Momo, archibishop’s vicar. This missive is dated on 28th of June 1319 and it is kept at Berio Library in Genoa
Activity assay of Purine Nucleoside Phosphorylases by LC-MS/MS
No full textIn the search for new therapeutics, fast and automated screening tools of chemical libraries are required for hit selection.
Nucleoside phosphorylases (NPs, E.C. 2.4.2) are among the key enzymes in nucleotide salvage/recycling pathway. NPs catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate (see Scheme 1).[1]
NPs are also essential for the metabolism of nucleotides in bacteria and other organisms. Nucleotide metabolic pathways in lower organisms represent reasonable targets for chemotherapy as they usually differ from the human counterparts.1b Inhibition of pathogen purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) might result in the impairment of replicative processes, thus providing a new potential route to infection control.[2]
Here we describe a novel LC-MS/MS method for the assessment of the activity of PNPs as an alternative to routinely used assays.1b Enzymatic activity was assessed by phosphorolysis of inosine to hypoxanthine (Scheme 1). Kinetic parameters (Km, Vmax, Kcat) were determined with respect to inosine and Pi.
The assay was performed in a 96 well plate format with an overall reaction time of about 15 minutes per plate, followed by the application of HILIC-LC-MS/MS method for the rapid quantification of the produced hypoxanthine (less than 2 minutes for sample).
For method development and validation, a PNP from Aeromonas hydrophila was used due to accumulated data on this enzyme by our team over the years.[3]
The newly developed LC-MS/MS assay will be applied to the screening of potential inhibitors against pathogenic PNPs.
[1] a. Pugmire, M. J. et al. Biochem. J. 2002, 361, 1; b. Bzowska, A. et al. Pharmacol. Ther. 2000, 88, 349.
[2] a. de Moraes, M. C. et al. Anal. Bioanal. Chem. 2013, 405, 4871; b. Ducati, R. G. et al. Curr. Med. Chem. 2011, 18, 1258; c. Madrid, D. C. et al J. Biol. Chem. 2008, 283, 35899.
[3] a. Ubiali, D. et al. Adv. Synth. Catal. 2012, 354, 96; b. Serra, I. et al. ChemPlusChem 2013, 78, 157; c. Calleri, E. et al. J. Chromatogr. B 2014, 968, 79
Chartae Latinae antiquiores : facsimile-edition of the Latin charters prior to the ninth century. 90: Italy 62.
No full textEdizione critica ed analisi paleografica e diplomatistica di otto documenti (nn. 11, 16, 17, 18, 19, 20, 21) del secolo IX conservati presso l’Archivio Capitolare di Arezzo. I documenti, ad eccezione di un placito e di un atto privato, sono tutti diplomi degli imperatori Lotario, Carlo III il Grosso, Lamberto, del re Ludovico III.Facsimile-Edition of the Latin chartres, 2nd series, Ninth Century, ed. by G. Cavallo and G. Nicolaj, Part XC, Italy LXII, Arezzo.Critical edition and paleographic and diplomatistic analysis of eight documents (nn. 11, 16, 17, 18, 19, 20, 21) of the ninth century, preserved in the Capitular Archives of Arezzo. Except for a placito and a private act, the documents are all diploma of emperors Lothair, Charles III the Fat, Lambert, king Ludwig III
Development of a biochromatographic integrated system based on a purine nucleoside phosphorylase from Aeromonas hydrophila for the flow synthesis of nucleoside analogues
No full textNucleoside phosphorylases (NPs, EC 2.4.2) are enzymes of the salvage pathway of nucleic acids which catalyze the biosynthesis of natural nucleosides in one step. Specifically, NPs catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate [(d)R-1-P] (phosphorolysis).[1] If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation) (Scheme 1). The use of NPs from different biological sources as catalysts in nucleoside analogue synthesis can be therefore an advantageous alternative to “conventional” chemical routes.
A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) has been recently cloned, over-expressed and used for synthetic applications, also as immobilized biocatalyst.[2,3]
Moreover, a bioreactor based on AhPNP immobilized on the inner surface of a silica capillary has been coupled on line with a chromatographic column for the assessment, by phosphorolysis, of substrate specificity towards a set of 6-substituted purine ribonucleosides.[4]
As a step forward, this biochromatographic system has been implemented to synthesize, by transglycosylation, nucleoside analogues through a “flow-based” approach.
To this aim, AhPNP (25 mg; IU/mg: 39) was covalently immobilized in a 50 L4.6 ID mm pre-packed stainless steel column containing aminopropylsilica particles (5 μm, 100 Å). The AhPNP-IMER (Immobilized Enzyme Reactor) was then coupled in-line through a switching valve to a HPLC apparatus containing an analytical or a semi-preparative chromatographic column with UV-Vis detection. The bioconversion was firstly characterized by a fractional factorial experimental design using, as a model reaction, the transglycosylation between inosine and adenine. Then, five modified purine ribonucleosides were synthesized and purified at a mg scale.
The transglycosylation reaction and the product separation were performed in a single chromatographic run, thus resulting in an efficient process where sample handling is minimized. To date, AhPNP-IMER has been shown to retain completely its activity upon 35 reactions.
References
[1] Pugmire, M. J.; Ealick, S. E. Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochem. J. 2002, 361, 1–25.
[2] Ubiali, D.; Serra, C. D. et al. Production, characterization and synthetic application of a purine nucleoside phosphorylase from Aeromonas hydrophila. Adv. Synth. Cat. 2012, 354, 96–104.
[3] Serra, I.; Ubiali, D. et al. Developing a collection of immobilized nucleoside phosphorylases for the preparation of nucleoside analogues: enzymatic synthesis of arabinosyladenine and 2′,3′-dideoxyinosine. ChemPlusChem 2013, 78, 157–165.
[4] Calleri, E.; Ubiali, D. et al. Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications. J. Chromatogr. B 2014, DOI: 10.1016/j.jchromb.2013.12.031
- …
