Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.

Metformin counteracts glucose-dependent lipogenesis and impairs transdeamination in the liver of gilthead sea bream ( Sparus aurata).

Metformin is an antidiabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis, but also by affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability in using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream ( Sparus aurata). Hyperglycemia markedly upregulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h posttreatment, it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1ß, Lpin1, and SREBP1). Metformin counteracted glucose-dependent effects, and downregulated glutamate dehydrogenase, alanine aminotransferase, and mammalian target of rapamycin 5 h posttreatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and suppressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model.

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver.

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53-55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.

Mutagenic potential of hypoxanthine in live human cells.

Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:T→G:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:T→G:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:T→G:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study A→G (T→C) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

TGF-ß triggers HBV cccDNA degradation through AID-dependent deamination.

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a viral center molecule for HBV infection and persistence. However, the cellular restriction factors of HBV cccDNA are not well understood. Here, we show that TGF-ß can induce nuclear viral cccDNA degradation and hypermutation via activation-induced cytidine deaminase (AID) deamination activity in hepatocytes. This suppression by TGF-ß is abrogated when AID or the activity of uracil-DNA glycosylase (UNG) is absent, which indicates that AID deamination and the UNG-mediated excision of uracil act in concert to degrade viral cccDNA. Moreover, the HBV core protein promotes the interaction between AID and viral cccDNA. Overall, our results indicate a novel molecular mechanism that allows cytokine TGF-ß to restrict viral nuclear cccDNA in innate immunity, thereby suggesting a novel method for potentially eliminating cccDNA.

Construction of effective inverted repeat silencing constructs using sodium bisulfite treatment coupled with strand-specific PCR.

RNA silencing has been exploited to produce transgenic plants with resistance to viral pathogens via posttranscriptional gene silencing (PTGS). In some cases, this technology is difficult to apply due to the instability of inverted repeat (IR) constructs during cloning and plant transformation. Although such constructs have been shown to be stabilized with introns and efficiently induce RNA silencing, we found that the Pdk intron did not stabilize South African cassava mosaic virus (SACMV) silencing constructs. Therefore, we developed a method for producing long SACMV IR constructs through bisulfite-induced base pair mismatches on the sense arm prior to IR assembly. Expression of SACMV BC1 mismatched IR constructs in the model test plant Nicotiana benthamiana resulted in a reduction in viral BC1 transcript levels, hence viral replication, upon SACMV infection. Mismatched SACMV AC1 IR constructs induced PTGS more efficiently in a N. benthamiana callus system than nonmismatched IR constructs. Our novel method for IR construct generation should be applicable to many sequences where the generation of these constructs has proven difficult in the past.

A monoamine oxidase from scallop Chlamys farreri serving as an immunomodulator in response against bacterial challenge.

Monoamine oxidase (MAO) is an essential enzyme in the catabolism of monoamines, and implicated in the immune response of vertebrates. In the present study, the full-length cDNA encoding monoamine oxidase (designated CfMAO) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The open reading frame of CfMAO cDNA encoded 519 amino acids, which shared 73.9% similarity with that from oyster Crassostrea gigas, and 64.5-66.3% similarity with those from vertebrates. A conserved Amino_oxidase domain and a transmembrane domain were identified in the deduced CfMAO protein. The mRNA transcripts of CfMAO could be detected in all the tested tissues, including haemocytes, hepatopancreas, kidney, adductor muscle, mantle, gill and gonad. The mRNA expression of CfMAO was up-regulated significantly in haemocytes of scallops during 6-48 h after bacteria Vibrio anguillarum challenge, and it reached the peak (25.9-fold, P < 0.05) at 12h. The cDNA fragment encoding the mature peptide of CfMAO was expressed in the prokaryotic expression system, and 1mg of the recombinant protein (rCfMAO) could catalyze the deamination of 3665.59 nmol serotonin, 2061.89 nmol norepinephrine, 2104.85 nmol epinephrine or 3040.34 nmol dopamine within 1 min (nmol min⁻¹ mg⁻¹) in vitro. When the reaction mixture was coincubated with 0.1 mmol L⁻¹ MAO inhibitor clorgyline, its catalyzing activity to deaminize serotonin and dopamine was decreased significantly to 1603.69 and 955.39 nmol min⁻¹ mg⁻¹ (P < 0.05) respectively. These results indicated that CfMAO, as the homologue of monoamine oxidase in scallop C. farreri, could modulate the immune response of scallops through the deamination of monoamines.

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine.

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.

Branched-chain amino acid metabolon: interaction of glutamate dehydrogenase with the mitochondrial branched-chain aminotransferase (BCATm).

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

S-Nitrosylation of secreted recombinant human glypican-1.

Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.

Mechanism of nitric oxide induced deamination of cytosine.

A five-step mechanism is proposed for the NO -induced deamination of cytosine. It has been investigated using DFT calculations, including both explicit water molecules and a bulk solvent model to mimic an aqueous environment. According to this mechanism, cytosine first undergoes tautomerization with the assistance of a water molecule from the bulk. A NO(+) cation produced by the autooxidation of NO is subsequently added to the exocyclic imino group of the cytosine imine tautomer. The resulting adduct is able to undergo a tautomerization step with the participation of a water molecule to produce a cytosine in which a -N(2)OH group is attached to carbon C4. Protonation of the oxygen of the latter gives a water molecule which dissociates instantaneously, leading to a pyrimidinic diazonium cation. This constitutes the rate-determining step of the mechanism with an activation free energy of 92.6 kJ mol(-1). The last step, which is highly exergonic, represents the driving force of the reaction. It is the substitution of the -N(2)(+) terminal group by a water molecule which simultaneously allows the transfer of one of the two hydrogens to the bulk. Thus, the two products of the reaction consist of a nitrogen molecule and the enol tautomer of uracil in equilibrium with the keto form.

Immunoreactivity of antibodies against transglutaminase-deamidated gliadins in adult celiac disease.

BACKGROUND: The significance of the presence of anti-gliadin antibodies in patients affected by celiac disease is still unclear. It is hypothesized that gliadin deamidation, catalysed by transglutaminase, plays a role in favoring the antigen presentation. AIM: To determine the immunoreactivity of anti-gliadin antibodies from untreated celiac patients to transglutaminase deamidated gliadins. MATERIALS AND METHODS: Gliadins from wheat flour underwent enzymatic digestion and were deamidated or cysteamine-transamidated by transglutaminase. Immunoreactivity of anti-gliadin antibodies from untreated adult celiac patients sera was evaluated by means of a competitive enzyme-linked immunosorbent assay (ELISA) method. RESULTS: Gliadin deamidation increased antibodies immunoreactivity from 25% to 50% while cysteamine incorporation into the gliadin peptides resulted in an immunoreactivity decrease. CONCLUSIONS: Increased immunoreactivity of transglutaminase deamidated gliadins tested with anti-gliadin antibodies from untreated adult celiac patients supports the hypothesis of a pivotal role of gliadin deamidation in the pathomechanism of celiac disease.

Development of an antitumor adenosine analog, 3''-ethynyladenosine.

3'-ethynyladenosine (EAdo) was an adenosine analog with potent antitumor activity against various human tumor cells in vitro. However, EAdo was enzymatically inactivated by adenosine deaminase (ADA) in vitro and in vivo. Therefore, we synthesized two ADA-resistant EAdo derivatives (2-F-EAdo and EAdo-5'-monophosphate, EAMP) and examined their antitumor activities.

Absence of 2'-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations.

Nitric oxide (NO(*)) is a physiologically important molecule at low concentrations, while high levels have been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While an extensive study in vitro suggests that oxidative and nitrosative reactions dominate the complicated chemistry of NO(*)-mediated genotoxicity, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem with a major effort to define the spectrum of nitrosative DNA lesions produced by NO(*)-derived reactive nitrogen species under biological conditions. Plasmid pUC19 DNA was exposed to steady state concentrations of 1.3 microM NO(*) and 190 microM O(2) (calculated steady state concentrations of 40 fM N(2)O(3) and 3 pM NO(2)(*) in the bulk solution) in a recently developed reactor that avoids the undesired gas phase chemistry of NO(*) and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nitrosatively induced abasic sites and nucleobase deamination products was defined using plasmid topoisomer analysis and a novel LC/MS assay, respectively. With a limit of detection of 100 fmol and a sensitivity of 6 lesions per 10(7) nt in 50 microg of DNA, the LC/MS analysis revealed that 2'-deoxyxanthosine (dX), 2'-deoxyinosine (dI), and 2'-deoxyuridine (dU) were formed at nearly identical rates (k = 1.2 x 10(5) M(-1) s(-1)) to the extent of approximately 80 lesions per 10(6) nt after 12 h exposure to NO(*) in the reactor. While reactions with HNO(2) resulted in the formation of high levels of 2'-deoxyoxanosine (dO), one of two products arising from deamination of dG, dO, was not detected in 500 microg of DNA exposed to NO(*) in the reactor for up to 24 h (<6 lesions per 10(8) nt). This result leads to the prediction that dO will not be present at significant levels in inflamed tissues. Another important observation was the NO(*)-induced production of abasic sites, which likely arise by nitrosative depurination reactions, to the extent of approximately 10 per 10(6) nt after 12 h of exposure to NO(*) in the reactor. In conjunction with other studies of nitrosatively induced dG-dG cross-links, these results lead to the prediction of the following spectrum of nitrosative DNA lesions in inflamed tissues: approximately 2% dG-dG cross-links, 4-6% abasic sites, and 25-35% each of dX, dI, and dU.

Synthesis, conformation, and antiviral activity of 5-methoxymethyl-2'-deoxycytidine analogs.

Analogs of 5-methoxymethyl-2'-deoxycytidine, MMdCyd (1) by substitution at N4 were synthesized to impart resistance against deamination. The anti HSV-1 activity and solution conformation of analogs were determined. N4-Butanoyl-MMdCyd (10) was a potent inhibitor of HSV-1 replication while N4-hexanoyl-MMdCyd (11), N4-propanoyl-MMdCyd (9) and N4-acetyl-MMdCyd (8) had good activity against HSV-1 replication. All other analogs were devoid of activity against HSV-1.

A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity.

To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies.

The relative contribution of monoamine oxidase and cytochrome p450 isozymes to the metabolic deamination of the trace amine tryptamine.

Tryptamine is a trace amine in mammalian central nervous system that interacts with the trace amine TA(2) receptor and is now thought to function as a neurotransmitter or neuromodulator. It had been reported that deamination of tryptamine to tryptophol was mediated by CYP2D6, a cytochrome P450 that is expressed in human brain, suggesting that tryptamine may be an endogenous substrate for this polymorphic enzyme. We were unable to confirm this report and have reinvestigated tryptamine metabolism in human liver microsomes (HLM) and in microsomes expressing recombinant human cytochrome P450 and monoamine oxidase (MAO) isozymes. Tryptamine was oxidized to indole-3-acetaldehyde by HLM and recombinant human MAO-A in the absence of NADPH, and indole-3-acetaldehyde was further reduced to tryptophol by aldehyde reductase in HLM in the presence of NADPH. Steady-state kinetic parameters were estimated for each reaction step by HLM and MAO-A. The CYP2D6 substrates bufuralol and debrisoquine showed strong inhibition of both tryptophol production from tryptamine in HLM and the formation of indole-3-acetaldehyde from tryptamine catalyzed by recombinant MAO-A. Anti-CYP2D6 monoclonal antibody did not inhibit these reactions. Pargyline, a nonselective MAO inhibitor, did not show cross inhibition to debrisoquine 4-hydroxylation and dextromethorphan O-demethylation by HLM and recombinant CYP2D6 enzyme. This is the first unequivocal report of the selective conversion of tryptamine to tryptophol by MAO-A. CYP2D6 does not contribute to this reaction.

An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC.

We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.

9-[(Hydroxymethyl)phenyl]adenines: new aryladenine substrates of adenosine deaminase.

New phenyl adenine compounds 5-7 were synthesized as analogues of adenosine and studied for their adenosine deaminase (ADA) substrate activity. The 9-[(o-hydroxymethyl)phenyl]methyl]adenine 5 and 9-[(m-hydroxymethyl)phenyl]adenine 7 were deaminated by ADA, and 9-[(o-hydroxyethyl)phenyl]adenine 6 was not deaminated up to 7 days. The ADA substrates 5 and 7 were deaminated quantitatively to their inosine analogues in 10 and 6h, respectively.