Optimized Method for the Synthesis of Alkyne-Modified 2'-Deoxynucleoside Triphosphates.

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2'-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction.

Synthesis and Properties of α-Phosphate-Modified Nucleoside Triphosphates.

This review article is focused on the progress made in the synthesis of 5'-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates' substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Discrimination of Ribonucleoside Mono-, Di-, and Triphosphates Using an Engineered Nanopore.

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.

Identification of thienopyrimidine glycinates as selective inhibitors for h-NTPDases.

The h-NTPDases is an essential family of ectonucleotidases that consists of eight isozymes with various physiological functions. The undesired activity of the h-NTPDases leads to pathological conditions such as cancer, diabetes, inflammation, and thrombosis. In the present study, a series of thienopyrimidines was synthesized employing a sequential SNAr and Suzuki coupling to synthesize diverse aryl substituted thienopyrimidine glycinate derivatives. The synthesized compounds constituted electron donating, electron-deficient, heteroaryl, and fluorinated substituents. The thienopyrimidines were screened against h-NTPDases to determine the effect on the activity of the h-NTPDases-1, -2, -3, and -8. The compound 3j selectively blocked the isozyme h-NTPDases1, while the compounds 3e, 3m, and 4a were selective inhibitors of h-NTPDases2. The activity of the isozyme h-NTPDases3 was selectively reduced by inhibitor 3k whereas, the compound 3d was found as the most active inhibitor against isozyme h-NTPDase8. The molecular docking study interpreted the interactions of the potent inhibitors of the respective isozymes with important amino acid residues i.e., Asp54, Ser57, His59, Ser58, His59, Asp213, and Phe360 of h-NTPDases1 protein; residues Arg 392, Ala393, Ala347, Tye350 and Arg245 of h-NTPDases2; amino acids Arg67, Ser65, Ala323, Gly222, and Tyr375 of h-NTPDases3 whereas in case of h-NTPDases8, the residues Val436, Gln74, Gly179, and Val71 were involved in interaction with the inhibitors docked into the active sites of these isozymes.

Extracellular ATP and its derivatives provide spatiotemporal guidance for bone adaptation to wide spectrum of physical forces.

ATP is a ubiquitous intracellular molecule critical for cellular bioenergetics. ATP is released in response to mechanical stimulation through vesicular release, small tears in cellular plasma membranes, or when cells are destroyed by traumatic forces. Extracellular ATP is degraded by ecto-ATPases to form ADP and eventually adenosine. ATP, ADP, and adenosine signal through purinergic receptors, including seven P2X ATP-gated cation channels, seven G-protein coupled P2Y receptors responsive to ATP and ADP, and four P1 receptors stimulated by adenosine. The goal of this review is to build a conceptual model of the role of different components of this complex system in coordinating cellular responses that are appropriate to the degree of mechanical stimulation, cell proximity to the location of mechanical injury, and time from the event. We propose that route and amount of ATP release depend on the scale of mechanical forces, ranging from vesicular release of small ATP boluses upon membrane deformation, to leakage of ATP through resealable plasma membrane tears, to spillage of cellular content due to destructive forces. Correspondingly, different P2 receptors responsive to ATP will be activated according to their affinity at the site of mechanical stimulation. ATP is a small molecule that readily diffuses through the environment, bringing the signal to the surrounding cells. ATP is also degraded to ADP which can stimulate a distinct set of P2 receptors. We propose that depending on the magnitude of mechanical forces and distance from the site of their application, ATP/ADP profiles will be different, allowing the relay of information about tissue level injury and proximity. Lastly, ADP is degraded to adenosine acting via its P1 receptors. The presence of large amounts of adenosine without ATP, indicates that an active source of ATP release is no longer present, initiating the transition to the recovery phase. This model consolidates the knowledge regarding the individual components of the purinergic system into a conceptual framework of choreographed responses to physical forces.

Role of the nucleoside-metabolizing enzymes on pain responses in zebrafish larvae.

Purinergic signaling is a pathway related to pain underlying mechanisms. Adenosine is a neuromodulator responsible for the regulation of multiple physiological and pathological conditions. Extensive advances have been made to understand the role of adenosine in pain regulation. Here we investigated the effects of purinergic compounds able to modulate adenosine production or catabolism on pain responses induced by Acetic Acid (AA) in zebrafish larvae. We investigated the preventive role of the ecto-5'-nucleotidase inhibitor adenosine 5'-(α,ß-methylene)diphosphate (AMPCP) and adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)-adenine (EHNA) on the AA-pain induced model. The pain responses were evaluated through exploratory and aversive behaviors in zebrafish larvae. The exploratory behavior showed a reduction in the distance covered by animals exposed to 0.0025% and 0.050% AA. The movement and acceleration were reduced when compared to control. The treatment with AMPCP or EHNA followed by AA exposure did not prevent behavioral changes induced by AA for any parameter tested. There were no changes in aversive behavior after the AA-induced pain model. After AA-induced pain, the AMP hydrolysis increased on zebrafish larvae. However, the AMPCP or EHNA exposure did not prevent changes in AMP hydrolysis induced by the AA-induced pain model in zebrafish larvae. Although AMPCP or EHNA did not show differences in the AA-induced pain model, our results revealed changes in AMP hydrolysis, suggesting the involvement of the purinergic system in zebrafish larvae pain responses.

Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Synthesis and anti-Toxoplasma activity of indole-triazole compounds on tachyzoites of RH strain.

BACKGROUND: Conventional treatment for toxoplasmosis have severe side effects and the inability to completely eradicate the disease. Therefore, the acquisition of new anti-Toxoplasma drugs has always been of interest among researchers. In the present study, we prepare a new indole-triazole derivatives and evaluated their potential anti-parasitic activity against tachyzoites of Toxoplasma RH strain. MATERIALS AND METHODS: In this study, after synthesis of the two new compounds of indole-triazole, the effect of their different concentrations (2-1024 µg/ml) were determined on Toxoplasma tachyzoites using flow cytometry. Furthermore, tachyzoites were exposed to different concentrations of compounds (4, 16, 64, 265, 1024 µg/ml) for 1.5 h and their infectivity were evaluated in BALB/c mice. RESULTS: The flow cytometry results indicated the benzyl derivative of indole-triazole in various concentrations had a lethal effect on tachyzoites between 11.93% and 89.66%, while the naphthalene derivative had a lethality of 26.63%-66.82%. The infectivity analysis showed that the survival time of mice at concentrations of 1024 µg/ml and 512 µg/ml of benzyl derivatives was significantly increased (P = 0.008 and P = 0.016, respectively), compared to that in the negative control group. Furthermore, survival time of mice was statistically significant at the concentration of 1024 µg/ml for naphthyl derivative (P = 0.012). CONCLUSION: Findings of the current study suggested indole triazole compounds, based on their structure and enzymes targeting, have a considerable effect on tachyzoites of T. gondii RH strain and can be considered as a new anti-Toxoplasma agent.

Synthesis, characterization and biological evaluation of thiadiazole amide derivatives as nucleoside triphosphate diphosphohydrolases (NTPDases) inhibitors.

Importance of extracellular nucleotides is widely understood. These nucleotides act as ligand for P2X and P2Y receptors and modulate a variety of biological functions. However, their extracellular concentration is maintained by a chain of enzymes termed as ecto-nucleotidases. Amongst them, nucleoside triphosphate diphosphohydrolases (NTPDases) is an important enzyme family responsible for the dephosphorylation of these nucleotides. Overexpression of NTPDases leads to many pathological conditions such as cancer and thrombosis. So far, only a few NTPDase inhibitors have been reported. Considering this scarcity of (NTPDase) inhibitors, a number of thiadiazole amide derivatives were synthesized and screened against human (h)-NTPDases. Several compounds showed promising inhibitory activity; compound 5a (IC50 (µM); 0.05 ± 0.008) and 5g (IC50 (µM); 0.04 ± 0.006) appeared to be the most distinguished molecules corresponding to h-NTPDase1 and -2. However, h-NTPDase3 was the least susceptible isozyme and only three compounds (5d, 5e, 5j) strongly inhibited h-NTPDase3. Interestingly, compound 5e was recognized as the most active compound that showed dual inhibition against h-NTPDase3 as well as against h-NTPDase8. For better comprehension of binding mode of these inhibitors, most potent inhibitors were docked with their respective isozyme.

Comparison of anti-SARS-CoV-2 activity and intracellular metabolism of remdesivir and its parent nucleoside.

Remdesivir, a monophosphate prodrug of nucleoside analog GS-441524, is widely used for the treatment of moderate to severe COVID-19. It has been suggested to use GS-441524 instead of remdesivir in the clinic and in new inhalation formulations. Thus, we compared the anti-SARS-CoV-2 activity of remdesivir and GS-441524 in Vero E6, Vero CCL-81, Calu-3, Caco-2 â€‹cells, and anti-HCoV-OC43 activity in Huh-7 â€‹cells. We also compared the cellular pharmacology of these two compounds in Vero E6, Vero CCL-81, Calu-3, Caco-2, Huh-7, 293T, BHK-21, 3T3 and human airway epithelial (HAE) cells. Overall, remdesivir exhibited greater potency and superior intracellular metabolism than GS-441524 except in Vero E6 and Vero CCL-81 â€‹cells.

Divergent synthesis and elaboration of structure activity relationship for quinoline derivatives as highly selective NTPDase inhibitor.

Quinoline derivatives have interesting biological profile. In continuation for the comprehensive evaluations of substituted quinoline derivatives against human nucleoside triphosphate diphosphohydrolases (h-NTPDases) a series of substituted quinoline derivatives (2a-g, 3a-f, 4, 5a-c, 6) was synthesized. The inhibitory activities of the synthesized compounds were evaluated against four isoenzymes of human nucleoside triphosphate diphosphohydrolases (h-NTPDases). These quinoline derivatives had IC50 (µM) values in the range of 0.20-1.75, 0.77-2.20, 0.36-5.50 and 0.90-1.82 for NTPDase1, NTPDase2, NTPDase3 and NTPDase8, respectively. The derivative 3f was the most active compound against NTPDase1 (IC50, 0.20 ± 0.02 µM) that also possessed selectivity towards NTPDase1. Similarly, derivative 3b (IC50, 0.77 ± 0.06), 2h (IC50, 0.36 ± 0.01) and 2c (IC50, 0.90 ± 0.08) displayed excellent activity corresponding to NTPDase2, NTPDase3 and NTPdase8. The compound 5c emerged as a selective inhibitor of NTPDase8. The most active compounds were then investigated to determine their mode of inhibition and finally binding interactions of the active compounds were analyzed through molecular docking studies. The obtained results strongly support the quinoline scaffold's potential as potent and selective NTPDase inhibitor.

A Novel Prognostic Biomarker of Luminal Breast Cancer: High CD39 Expression Is Related to Poor Survival.

BACKGROUND: CD39 is one of the functional surface markers for T regulatory cells, the prognostic role and immune-related effects of CD39 in luminal breast cancer (BC) patients has not been evaluated yet. The aim of the current study was to explore the association between CD39 expression and clinic pathological characteristics and the prognosis in luminal BC patients. METHODS: Clinical information and RNA-sequencing (RNA-Seq) expression data were extracted from The Cancer Genome Atlas (TCGA). Patients were divided into a high or low CD39 expression group by the optimal cutoff value (4.18) identified from the receiver operating characteristic curve analysis. The relationships between CD39 expression and clinic pathological features were evaluated by the corresponding statistical tests. Survival analyses were applied to evaluate the overall survival between the high and low CD39 expression groups in luminal BC. Furthermore, Gene Expression Omnibus datasets were used for external data validation. Gene set enrichment analysis (GSEA) was also performed, and CIBERSORT was used to analyze the immune cell populations. RESULTS: Analysis of 439 cases of tumor data showed that CD39 was overexpressed in luminal BC. The multivariable analysis suggested that CD39 expression was an independent prognostic factor for luminal BC patients. GSEA suggested that CD39 might play an important role in luminal BC progression through immune regulation. Analysis of immune cell patterns revealed high CD39 expression correlated to a higher proportion of CD8+ T cells and M2 macrophages. CONCLUSION: This study demonstrates that CD39 expression correlates with the prognosis of luminal BC through TCGA database mining. Further studies are warranted further to elucidate this potential novel therapeutic strategy for BC.

Synthesis of functional oligosaccharides and their derivatives through cocultivation and cellular NTP regeneration.

Carbohydrates play an important role in the life cycle. Among them, functional oligosaccharides show a complex and diverse structures with unique physiological activities and biological functions. However, different preparation methods directly affect the structure, molecular weight, and other functions of oligosaccharides, as well as their application fields and manufacturing costs. In the preparation of ß-1,3-glucan oligosaccharides (OBGs), water insolubility of ß-1,3-glucans hampers the hydrolysis efficiency. The synthesis of some functional oligosaccharides requires the consumption of energy substrates, such as ATP, CTP, and uridine triphosphate, for sugar nucleotide synthesis, leading to increased capital costs. A more economical solution to solve energy supply is to adopt microbial cocultivation or cellular nucleoside triphosphate regeneration. This review focused on the sources, preparation methods, biological activities of OBG, and the cultivation methods and applications of microbial cocultivation and fermentation. We also reviewed the preparation methods of other functional oligosaccharides, such as sialylated oligosaccharides, ß-nicotinamide mononucleotide, and α-galacto-oligosaccharides.

Synthesis, In-vitro evaluation and molecular docking studies of oxoindolin phenylhydrazine carboxamides as potent and selective inhibitors of ectonucleoside triphosphate diphosphohydrolase (NTPDase).

Members of the ectonucleoside triphosphate diphosphohydrolases (NTPDases) constitute the major family of enzymes responsible for the maintenance of extracellular levels of nucleotides and nucleosides by catalyzing the hydrolysis of nucleoside triphosphate (NTP) and nucleoside diphosphates (NDP) to nucleoside monophosphate (NMP). Although, NTPDase inhibitors can act as potential drug candidates for the treatment of various diseases, there is lack of potent as well as selective inhibitors of NTPDases. The current study describes the synthesis of a number of carboxamide derivatives that were tested on recombinant human (h) NTPDases. The most promising inhibitors were 2h (h-NTPDase1, IC50: 0.12 ± 0.03 µM), 2d (h-NTPDase2, IC50: 0.15 ± 0.01 µM) and 2a (h-NTPDase3, IC50: 0.30 ± 0.04 µM; h-NTPDase8, IC50: 0.16 ± 0.02 µM). Four compounds (2e, 2f, 2g and 2h) were associated with the selective inhibition of h-NTPDase1 while 2b was identified as a selective h-NTPDase3 inhibitor. Considering the importance of NTPDase3 in the regulation of insulin release, the NTPDase3 inhibitors were further investigated to elucidate their role in the insulin release. The obtained data suggested that compound 2a was actively participating in regulating the insulin release without producing any effect on NTPDase3 mRNA. Moreover, the most potent inhibitors were docked within the active site of respective enzyme and the observed interactions were in compliance with in vitro results. Hence, these compounds can be used as pharmacological tool to further investigate the role of NTPDase3 coupled to insulin release.

[GO System, a DNA Repair Pathway to Cope with Oxidative Damage].

The GO system is part of the DNA base excision repair pathway and is required for the error-free repair of 8-oxoguanine (oxoG), one of the most common oxidative DNA lesions. Due to the ability of oxoG to form oxoG:A mispairs, this base is highly mutagenic. Its repair requires the action of two enzymes: 8-oxoguanine DNA glycosylase (Fpg or MutM in bacteria and OGG1 in eukaryotes), which removes oxoG from oxoG:C pairs, and adenine DNA glycosylase (MutY in bacteria and MUTYH in eukaryotes), which removes A from oxoG:A mispairs to prevent mutations. The third enzyme of the system (MutT in bacteria and MTH1 or NUDT1 in eukaryotes) hydrolyzes 8-oxo-2'-deoxyguanosine triphosphate, thus preventing its incorporation into DNA. Recent data point to the proteins of the GO system as promising targets for the therapy of cancer, autoimmune diseases, and bacterial infections. This review highlights the structure and specificity of the GO system in bacteria and eukaryotes and its unique role in mutation avoidance.

γ-Non-Symmetrically Dimasked TriPPPro Prodrugs as Potential Antiviral Agents against HIV.

Nucleoside analogue reverse transcriptase inhibitors (NRTI) and nucleoside analogue monophosphate prodrugs are used in combination antiretroviral therapy (cART). The design of antivirally active nucleoside triphosphate prodrugs is a recent and an important advancement in the field of nucleoside analogue drug development. Here, we report on TriPPPro-derivatives of nucleoside analogue triphosphates (NTPs) that comprised two different acyloxybenzyl-masks at the γ-phosphate of the NTP aiming to achieve the metabolic bypass. Thus, γ-non-symmetrically dimasked TriPPPro-compounds (γ-(AB,ab)-d4TTPs) were synthesized and they proved to be active against HIV-1 and HIV-2 in cultures of infected wild-type human CD4+ T-lymphocyte (CEM/0) cells and more importantly also in thymidine kinase-deficient CD4+ T-cells (CEM/TK-). From hydrolysis studies both in phosphate buffer (PB, pH 7.3) and CEM cell extracts, there was surprisingly no differentiation in the cleavage of the two acyloxybenzyl prodrug-masks. However, if within one of the two acyloxybenzyl groups a short PEG-type methoxytriglycol group was introduced, the "standard" acyloxybenzyl-mask was cleaved with high preference.

History of ectonucleotidases and their role in purinergic signaling.

Ectonucleotidases are key for purinergic signaling. They control the duration of activity of purinergic receptor agonists. At the same time, they produce hydrolysis products as additional ligands of purinergic receptors. Due to the considerable diversity of enzymes, purinergic receptor ligands and purinergic receptors, deciphering the impact of extracellular purinergic receptor control has become a challenge. The first group of enzymes described were the alkaline phosphatases - at the time not as nucleotide-metabolizing but as nonspecific phosphatases. Enzymes now referred to as nucleoside triphosphate diphosphohydrolases and ecto-5'-nucleotidase were the first and only nucleotide-specific ectonucleotidases identified. And they were the first group of enzymes related to purinergic signaling. Additional research brought to light a surprising number of ectoenzymes with broad substrate specificity, which can also hydrolyze nucleotides. This short overview traces the development of the field and briefly highlights important results and benefits for therapies of human diseases achieved within nearly a century of investigations.