Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

Zuffi, Gabriele; Ghisotti, Daniela; Oliva, Ilaria; Capra, Emanuele; Frascotti, Gianni; Tonon, Giancarlo; Orsini, Gaetano

doi:10.1080/10242420310001648551

Cited by 37 publications

(19 citation statements)

References 18 publications

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“…Table 2, for the two-pot reaction, on increasing phosphate concentration the yield (conversion of purine base by HPLC analysis, the same as below) increased (17.0 % to 26.7 %, reactions 4 and 5). In contrast, for the one-pot reaction the yield increased with decreasing phosphate concentration (55.6 % to 68.7 %, reactions 7-10; and 72.8 % to 78.5 %, reactions [11][12]. Moreover, with the same substrate composition, the one-pot reaction resulted in a significantly improved yield compared to the two-pot reaction (63.2 % vs 17.0 %, reactions 7 vs 4; 55.6 % vs 26.7 %, reactions 9 vs 5).…”

Section: Synthesis Of 26-dihalogenated Purine Nucleosidesmentioning

confidence: 60%

“…For instance, thermostable GsPNP and GsPyNP from Geobacillus stearothermophilus (previously Bacillus stearothermophilus) were immobilized on anion exchange resins (DEAE-Toyopearl 650 M) [10], or on the aminopropylated macroporous glass AP-CPG-170 [6]; uridine phosphorylase (UP) and PNP from E. coli were immobilized on epoxy-activated resin Sepabead EC-EP [11];…”

Section: Introductionmentioning

confidence: 99%

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Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

Zhou

Mikhailopulo

Bournazou

et al. 2015

Journal of Molecular Catalysis B: Enzymatic

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Section: Synthesis Of 26-dihalogenated Purine Nucleosidesmentioning

confidence: 60%

Section: Introductionmentioning

confidence: 99%

Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

Zhou

Mikhailopulo

Bournazou

et al. 2015

Journal of Molecular Catalysis B: Enzymatic

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“…For each of the mutant the table shows the name of the plasmid and of the bacterial strain that we produced and the sequence of the PCR primers that we use din the mutagenesis protocol. (LacZ8) (UniProt/SwissProt accession n°Q37953), the first twenty amino acids of purine nucleoside phosphorylase (PNP20) [14] and a short flexible peptide -Glu-Ser-Ser-Met-Ser-Gly-Leu-PheLys-Arg-(kex1 peptide). The last two amino acids of the kex1 peptide, the Lys-Arg dipeptide, are specifically cleaved at Cterminal site of arginine upon exposure to the endoprotease ssKex-1-C 611 .…”

Section: R-hgh Mutant Preparationmentioning

confidence: 99%

Preclinical characterization of eleven new Cys-PEGylated hGH mutants

Selis¹,

Genovese

Salis³

et al. 2016

European Journal of Molecular &Amp; Clinical Medicine

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We synthesized and tested for biological activity eleven new PEGylated hGH derivatives. To this aim we used different strategies. A first group of PEGylable Cys derivatives was prepared by mutating into Cys single specific aminoacid residues located either in the connecting loop between helices 1 and 2 (Ile36 and Phe44) or in the connecting loop between helices 3 and 4 (Ile138 and Phe146). A second group of mutants was synthetized by inserting new Cys close to the aforementioned positions. Other PEGylable mutants were prepared by inserting a Gly4-Cys chain either at the N- or at the C-terminus of the hGH molecule. Finally, a Cys residue of the second hGH disulfide bond (Cys182-Cys189) was made available for PEGylation by mutating its companion Cys to open up the disulfide bridge. All these mutants were tested for their ability to affect Nb2 cell proliferation in vitro and showed different effects. Two mutations (Phe44Cys and +Cys47) severely impaired and two (Ile138Cys and Phe146Cys) increased hGH bioactivity. The mutation Cys189Ser was functionally silent whereas the remaining mutations caused a 15-50% decrease in activity. 20kDa-pegylation caused a dramatic decrease in bioactivity in all mutants. The r-hGH-(Ile138Cys)-Cys138-PEG derivative showed the highest activity (15% of wild-type unpegylated hGH) and was selected for pharmacockinetic studies in vivo. It showed a fivefold longer half-life and was significantly more effective than wild-type hGH in causing weight gain when given subcutaneously twice a week to hypophysectomized rats. In conclusion, even when it is directed to residues supposed to have a marginal role in the activity of this hormone, 20 kDa pegylation has detrimental effect on hGH bioactivity. These effects may be counterbalanced by the increase in half-life as it happens in pegylated Ile138Cys-hGH that could represent a promising new long-acting hGH derivative. Focal points:•Bedside: Current therapy of GH deficiency still has the important limitation of being delivered by daily subcutaneous injections and this reduces the compliance of the small pediatric patients. There is, therefore interest in developing long-acting GH derivative such as the new ones that we present here. •Benchside: By investigating the effect of Cys substitutions at aminoacidic positions that had not been investigated in previous studies, we obtained evidence that Ile138 and Phe146 have a role in GH bioactivity in vitro. •Industry: The mono-pegylated hGH mutant described in this work represents a promising candidate for human clinical pharmacology studies as long-acting derivative of h-GH.•Community: By decreasing the frequency of GH injections, long acting GH could decrease the costs of GH treatment and increase medication adherence

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“…2,6-Diaminopurine arabinoside (DAPA) (Zuffi et al, 2004) was prepared using UP and PNP cloned and overexpressed in independent Escherichia coli strains (74% yield, 2.5 h, 60°C). The enzymes were co-immobilised on epoxy-activated Sepabeads EC-EP/M resin to make the biocatalyst suitable for industrial scale applications.…”

Section: As Nucleoside Prodrugsmentioning

confidence: 99%