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Codice penale e norme complementari
No full textTerza edizione del "Codice penale e norme complementari" (I blu Giuffrè) curata da E. Dolcini e G.L. Gatta, con la collaborazione di A. Galluccio, M. C. Ubiali e S. Bernardi
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
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
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
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
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
Development of capillary bioreactors based on purine nucleoside phosphorylase from Aeromonas hydrophila for biocatalytic applications
No full textNucleoside phosphorylases (NPs; E.C. 2.4.2) 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 (R-1-P) (Eq. 1).[1]
If a second nucleobase is added to the reaction medium the formation of a new nucleoside can result (transglycosylation). Thus, NPs can be used for chemoenzymatic synthesis of both natural and unnatural nucleosides, as an alternative to conventional chemical methods which are generally plagued by low stereoselectivity, multi-step procedures and modest yields.
We have cloned and over-expressed a purine nucleoside phosphorylase (PNP) from A. hydrophila (encoded by deoD gene), tested its substrate specificity, and used it for few synthetic applications, also as immobilized biocatalyst.[2,3]
The aim of the present work is the development of a biochromatographic system based on this PNP as a tool to speed up the screening of new nucleoside libraries through a medium-high throughput approach and to characterize the catalytic efficiency of the biocatalyst.
To this end, immobilization trials of PNP on different chromatographic supports (e.g. monolithic and/or Open Tubular Capillary) will be perfomed.
The prepared bioreactors will be typified by the determination of kinetic constants (kM and Vmax) using the Michaelis-Menten kinetic model for a natural substrate (e.g. inosine) in phosphorolysis reaction and comparing the results with kinetic constants calculated for the in batch free enzyme in the same operative conditions.
Experimentally, the characterization will be realized through a bidimensional chromatographic system with the sample on-line transfer, from the bioreactor to the analytical column, for the separation and quantification of the substrate and product.[4]
The most promising bioreactor will be used to verify the system synthetic potential towards modified nucleosides.
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. Product 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] Moraes, M. C.; Ducati, R. G. et al. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization. J. Chrom. A 2012, 1232, 110–115
L’ evoluzione dell’associazionismo sportivo: dal pionierismo anni ’50 al ‘ vespismo’ contemporaneo. Il caso del Vespa Club Ponte San Pietro (BG).
No full textL’oggetto delle pagine seguenti è la storia dell’associazionismo che ruota attorno ad un mezzo a motore iconico che ha fortemente contribuito alla mobilità degli italiani: la Vespa Piaggio e il fenomeno aggregativo dei Vespa Club.
Nello specifico, l’obiettivo di questo saggio intende definire la trasformazione della popolazione (ovvero quella degli iscritti al Club) e, contestualmente, della mission dei Vespa Club: un processo evolutivo che ha mantenuto la Vespa nella centralità delle proprie attività ludiche e turistiche, pur con diverse finalità. Si assiste infatti a una prima fase propagandistica del prodotto e una seconda svincolata da Piaggio e dedita all’associazionismo strictu sensu
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