130,541 research outputs found
PURINE NUCLEOSIDE PHOSPHORYLASES AS BIOCATALYSTS AND PHARMACOLOGICAL TARGETS
No full textA purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) was successfully exploited to catalyze the “one-pot, one-enzyme” regio- and stereoselective transfer of β-D-ribose from a proper sugar donor (7-methylguanosine iodide) to a library of 6-substituted purine acceptors, resulting in the “in batch” synthesis of 24 ribonucleosides. Transglycosylation conversions confirmed the broad tolerance and the potential of AhPNP as biocatalyst, providing the necessary information to undertake the preparative synthesis of 6-modified purine nucleosides. [1]
AhPNP was then immobilized in a stainless steel column resulting in a stable and active bioreactor (AhPNP-IMER, Immobilized Enzyme Reactor) that, upon on-line connection to a semi-preparative HPLC system, was used to run transglycosylations in a flow mode. In such a set-up, biotransformation, on-line monitoring and product purification occurred in a single integrated platform, thus allowing the preparation of five nucleoside analogues at a mg scale (52-89% yield). [2]
As a step forward, a “one-pot, two-enzyme” strategy was applied by coupling AhPNP-IMER with an analogous bioreactor based on a uridine phosphorylase from Clostridium perfringens (CpUP), immobilized in a monolith column. The on-line apparatus obtained by connecting CpUP-IMER and AhPNP-IMER in series was tested in the synthesis of adenosine, 2’-deoxyadenosine and arabinosyladenine from uridine, 2’-deoxyuridine and arabinosyluracyl as sugar donors, respectively. The corresponding nucleobases were transformed into the products in 90-95% conversion over 1 h for the ribosyl and 2’-deoxyribosyl derivatives, and 20% conversion after 5 h for arabinosyladenine. [3]
Furthermore, a new LC-ESI-MS/MS method was set up to evaluate the inhibition activity of 8-substituted purine ribonucleosides toward the PNP from Mycobacterium tuberculosis (MtPNP), as well as the selectivity against the microbial enzyme with respect to the corresponding human one (HsPNP). The corresponding enzymatic assay, based on the phosphorolysis of inosine, proved to be very convenient in terms of time as well as of target amount. A small library of seven 8-substituted purine ribonucleosides were screened, not exerting any significant effect up to 1 mM, with 8-bromoguanosine and 8-methylaminoguanosine being the only exceptions at 500 mM as weak inhibitors. [4]
Finally, the chemical synthesis of a series of 8- and N2-substituted inosinic and guanylic acids as potential ligands of the human GPR17 receptor was carried out, starting from studies aided by molecular modeling on a homology model of the target. The molecules were prepared by 5’-phosphorylation of properly 8- and N2-modified/protected inosine or guanosine. Owing to the scarce nucleophilicity of the exocyclic NH2 group of guanosine, the 2-position of the purine ring was activated as a bromo derivative, whose displacement with the proper amine afforded the desired N2-alkylated products. On the contrary, N2-acylations were carried out through nitrogen functionalization with a proper acyl chloride or anhydride. An additional 2’,3’-O-isopropylidene group was inserted in all the N2-functionalized nucleotides. Binding assays on GPR17 will be carried out.
[1] D. Ubiali, C. F. Morelli, M. Rabuffetti, G. Cattaneo, I. Serra, T. Bavaro, A. M. Albertini, G. Speranza Curr. Org. Chem. 2015, 19, 2220-2225; [2] E. Calleri, G. Cattaneo, M. Rabuffetti, I. Serra, T. Bavaro, G. Massolini, G. Speranza, D. Ubiali Adv. Synth. Catal. 2015, 357, 2520-2528; [3] G. Cattaneo, M. Rabuffetti, G. Speranza, T. Kupfer, B. Peters, G. Massolini, D. Ubiali, E. Calleri Submitted 2017; [4] G. Cattaneo, D. Ubiali, E. Calleri, M. Rabuffetti, G. C. Hofner, K. T. Wanner, M. C. De Moraes, L. K. B Martinelli, D. S. Santos, G. Speranza Anal. Chim. Acta 2016, 943, 89-97
Operational flood-forecasting in the Piemonte region - Development and verification of a fully distributed physically-oriented hydrological model
No full textA hydrological model for real time flood forecasting to Civil Protection services requires reliability and rapidity. At present, computational capabilities overcome the rapidity needs even when a fully distributed hydrological model is adopted for a large river catchment as the Upper Po river basin closed at Ponte Becca (nearly 40 000 km2). This approach allows simulating the whole domain and obtaining the responses of large as well as of medium and little sized sub-catchments. The FEST-WB hydrological model (Mancini, 1990; Montaldo et al., 2007; Rabuffetti et al., 2008) is implemented. The calibration and verification activities are based on more than 100 flood events, occurred along the main tributaries of the Po river in the period 2000–2003. More than 300 meteorological stations are used to obtain the forcing fields, 10 cross sections with continuous and reliable discharge time series are used for calibration while verification is performed on about 40 monitored cross sections. Furthermore meteorological forecasting models are used to force the hydrological model with Quantitative Precipitation Forecasts (QPFs) for 36 h horizon in "operational setting" experiments. Particular care is devoted to understanding how QPF affects the accuracy of the Quantitative Discharge Forecasts (QDFs) and to assessing the QDF uncertainty impact on the warning system reliability. Results are presented either in terms of QDF and of warning issues highlighting the importance of an "operational based" verification approach
Clinical validity of novel postural stabilization experimental indices based on hyperbolic transformation
No full textBackground: Postural stabilization is the function which allows an individual, after a transitional movement, to recover balance in a quiet erect posture. An experimental method has been proposed (Rabuffetti, 2011) and proved valid for the assessment of balance disorders in individuals with neurological diseases. It would seem that the two original indices were not fully independent since their concurrent distribution was confined by a hyperbolic boundary.
Research question: A methodological advancement involving non-linear transformation techniques is required to overcome the limitations of the original approach.
Methods: A hyperbolic transformation is applied to the original indices related to the mechanics of the stabilization (instability at beginning of stabilization and time rate of stabilization), thus defining two novel indices (Instability and Promptness). These novel indices may be related to different functional domains concerning, respectively, peripheral force capacity and central nervous motor control. The validity of these novel indices is quantified by their correlation with clinical scales in an already validated group of patients with Charcot-Marie-Tooth disease (N = 47) or Multiple Sclerosis (N = 20).
Results: The novel indices generally improved validity compared to the original indices (+ 66% of indices show a statistically significant concurrent validity on a clinical scale). Moreover, Instability was more related to the Charcot-Marie-Tooth group (9 out of 12 valid correlations), and Promptness to the Multiple Sclerosis group (4 out of 5, when also considering statistical trends), in accordance to the, respectively, more peripheral and more central nature of the two neurological diseases.
Significance: The novel postural stabilization indices support a clinical application for two reasons: 1) they have shown improved validity, compared to the original indices, in two groups of patients affected by neurological pathologies of different nature, 2) they are more closely related, compared to the original indices, to different functional domains
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
Sustainable synthesis of biosurfactants from renewable resources
No full textSFAEs (Sugar-Fatty Acid Esters) are a class of non-ionic surfactants characterized by excellent surface and interfacial tension reduction capability, low toxicity and biodegradability. Depending on their carbon chain length and the nature of the sugar head group, SFAEs can cover a wide range of hydrophilic-lipophilic balance (HLB) values, which result in tunable surfactant properties. Moreover, SFAEs can be obtained from renewable resources (i.e. industrial waste and biomass) by enzymatic and/or chemoenzymatic approaches, thus answering the need for sustainable and circular chemistry [1].
In this context, a small library of SFAEs were prepared by the lipase-catalyzed esterification of isomeric mixtures of alkyl glycosides (namely α-/β-d-glucosides and α-/β-d-galactosides) with molten fatty acids (lauric, palmitic and stearic acid), using CalB (Novozym® 435) as biocatalyst in an easily scalable solvent-free system. Conversion of glucose and galactose into alkyl glycosides before the esterification reaction played a key role in circumventing the striking different solubility of the two reagents. The physico-chemical properties of the synthesized tensides (interfacial tension features, W/O emulsification capability and W/O stability over time) were then finely investigated [2].
References
[1] a) I.J.A. Baker et al., J. Surfactant Deterg. 3 (2000) 1-11; b) H.M. El-Laithy et al., Eur. J. Pharm. Biopharm. 77 (2011) 43-55; c) N.S. Neta et al., Crit. Rev. Food Sci. Nutr. 55 (2015) 595-610.
[2] a) S. Sangiorgio et al., Colloids Interface Sci. Commun. 48 (2022) 100630; b) R. Semproli et al., ChemPlusChem 88 (2023) e202200331
Chemoenzymatic Synthesis of Alkyl Glycoside Fatty Acid Esters and Investigation of their Emulsifying Properties
No full textSugar fatty acid esters (SFAEs) are non-ionic surfactants that are characterized by excellent surface and interfacial tension reduction capability, low toxicity, and biodegradability. These features make SFAEs extremely promising for industrial applications as emulsifiers in the cosmetic and food sectors.[1] Interestingly, SFAEs can be obtained from renewable resources (from industrial waste and biomass) by enzymatic and/or chemoenzymatic approaches, thus answering the need for evermore sustainable and circular chemistry.[2,3]
6-O-Lauroyl-, 6-O-palmitoyl- and 6-O-stearoyl-1-O-butyl glucopyranosides were enzymatically synthesized by reacting n-butyl glucoside with molten fatty acids in an easily scalable solvent-free system. Derivatization of glucose as an alkyl glucoside before the esterification reaction played a key role to circumvent the striking different solubility of glucose and fatty acids. The physico-chemical properties of these tensides, such as interfacial tension features, W/O emulsification capability and W/O stability over time were deeply investigated.[4]
References
1. N.S. Neta, J.A. Teixeira, L.R. Rodrigues, Crit. Rev. Food Sci. Nutr. 2015, 5, 595.
2. A.R. Alcántara, P. Domínguez de María, J.A. Littlechild, M. Schürmann, R.A. Sheldon, R. Wohlgemuth, ChemSusChem 2022, e202102709.
3. T. Keijer, Nat. Chem. 2019, 11, 190.
4. S. Sangiorgio, E. Pargoletti, M. Rabuffetti, M.S. Robescu, R. Semproli, D. Ubiali, G. Cappelletti, G. Speranza, under review
This work was financially supported by Cariplo Foundation (Italy) (call: “Circular Economy for a sustainable future 2020”, project BioSurf, ID 2020-1094)
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
Single axis stabilometric platform: Reliability of measures by comparison with a gold standard
No full textA two-step enzymatic approach to cheese whey valorization: Synthesis of alkyl galactoside fatty acid esters as non-ionic biosurfactants
No full textCheese whey is the main wastestream of dairy industry. After protein recovery, the resulting whey permeate
contains a pool of carbohydrates (lactose as well as its hydrolysis products, i.e. glucose and galactose) that
can be exploited as feedstock for the synthesis of Sugar Fatty Acid Esters (SFAE), non-ionic surfactants
characterized by emulsifying, stabilizing, and detergency properties.1
In this work,2 an immobilization study of β-galactosidase from Aspergillus oryzae (GalAo) was carried out with
the aim to develop a robust biocatalyst for lactose upcycling. Several types of binding chemistry, chemical
activation of the support, and immobilization conditions were assayed. Glyoxyl-Sepabeads EC-EP resulted in
good immobilization yields (immobilized protein=65% and immobilized activity=58%) and moderate activity
recovery (20%). The immobilized GalAo was used for the synthesis of a library of alkyl-β-D-galactosides by
reacting lactose with naturally occurring alcohols 2a-2g through a transglycosylation reaction; compounds
(3a-3g) were isolated in moderate to good yields (20-45%) (Scheme 1A). n-Butyl β-D-galactopyranoside (3d)
was submitted to the esterification step with palmitic acid in a solvent-free system by using Novozym 435 as
the biocatalyst,3 affording 6-O-palmitoyl-1-O-butyl galactopyranoside (4d) (Scheme 1B).
Scheme 1. A) Transglycosylation of lactose (1) with natural aliphatic alcohols (2a-g) catalyzed by immobilized GalAo in buffer pH
4.3/alcohol/(acetone), r.t., 6 h. B) Esterification of 3d with palmitic acid catalyzed by Novozym 435 in a solvent-free system, 80 °C, 8
h, molecular sieves. n.o.=not optimized.
Interfacial features and W/O emulsifying properties of 4d together with W/O emulsion stability over time
were evaluated.
Acknowledgements
This work was financially supported by Cariplo Foundation (Italy) (call: “Circular Economy for a sustainable future 2020”,
project BioSurf, ID 2020-1094).
References
1. Ortiz, M.S.; Alvarado, J.G.; Zambrano, F.; Marquez, R. J. Surfact. Deter. 2022, 25, 147-183.
2. Semproli, R. et al., manuscript in preparation
3. Sangiorgio, S.; Pargoletti, E.; Rabuffetti, M.; Robescu, M.S.; Semproli, R.; Ubiali, D.; Cappelletti, G.;
Speranza, G. Colloids Interface Sci. Commun. 2022, 48 100630
2-O-Acetyl-3,4,5,6-tetra-O-benzyl-D-myo-inosityl diphenylphosphate: a new useful intermediate to inositol phosphate and phospholipids
No full textInositol phosphates and inositol phospholipids are ubiquitous in biochemistry and play a central role in cell signaling and regulation events. For this reason, their synthesis has attracted widespread interest. This paper describes the preparation of a new optically active inositol phosphate derivative, 2-O-acetyl-3,4,5,6-tetra-O-benzyl-D-myo-inosityl diphenylphosphate (6), and its characterization by spectroscopic methods. Compound (6) represents a useful intermediate for the preparation of inositol phosphate and phospholipids, in particular of glycerophosphoinositol (GPI), a natural anti-inflammatory agent
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