Heat flows enrich prebiotic building blocks and enhance their reactivity.

The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

[Recent advances in poly phosphate kinase (PPK) and the construction of PPK-mediated ATP regeneration system].

Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.

Lipophilic Nucleoside Triphosphate Prodrugs of Anti-HIV Active Nucleoside Analogs as Potential Antiviral Compounds.

Nucleoside analogs require three phosphorylation steps catalyzed by cellular kinases to give their triphosphorylated metabolites. Herein, the synthesis of two types of triphosphate prodrugs of different nucleoside analogs is disclosed. Triphosphates comprising: i) a γ-phosphate or γ-phosphonate bearing a bioreversible acyloxybenzyl group and a long alkyl group and ii) γ-dialkyl phosphate/phosphonate modified nucleoside triphosphate analogs. Almost selective conversion of the former TriPPPro-compounds into the corresponding γ-alkylated nucleoside triphosphate derivatives is demonstrated in CEM/0 cell extracts that proved to be stable toward further hydrolysis. The latter γ-dialkylated triphosphate derivatives lead to the slow formation of the corresponding NDPs. Both types of TriPPPro-compounds are highly potent in wild-type CEM/0 cells and more importantly, they exhibit even better activities against HIV-2 replication in CEM/TK- cell cultures. A finding of major importance is that, in primer extension assays, γ-phosphate-modified-NTPs, γ-mono-alkylated-triphosphates, and NDPs prove to be substrates for HIV-RT but not for cellular DNA-polymerases α,γ.

An eco-friendly, highly efficient, and transparent coating derived from guar gum and citric acid for flame retardant treatment of cotton fabrics.

A highly efficient, bio-ecofriendly, and transparent flame retardant (FR) for cotton fabric was developed and deposited onto the cellulose skeletal structure of cotton fabric through a one-pot sol-gel process. The flame retardant functional coating is composed of ammonium polyphosphate (APP), guar gum (GG), citric acid (CA), and a negligible amount of catalyst. Cotton fabrics were impregnated with different concentrations of ammonium polyphosphate and guar gum, with citric acid as a crosslinking agent. The overall crosslinking and grafting process was proven by FTIR and XPS. Based on the results, the designed coating exhibits over 90 % transmittance in the visible region. A 15 g/m2 flame-retardant coating induces excellent flame retardant efficiency at ultra-low flame-retardant concentrations of less than 6.25 wt%. Only a 5.25 wt% flame retardant concentration demonstrated condensed phase action, which resulted in 58.5 % and 73.6 % reductions in the pHRR and THR, respectively. Moreover, the limiting oxygen index (LOI) value showed a 74 % increase. The mechanical performance of FR coated cotton fibers was slightly reduced.

Coating with tripolyphosphate-crosslinked chitosan as a novel approach for enhanced stability of emulsomes following oral administration: Rutin as a model drug with improved anti-hyperlipidemic effect in rats.

The aim of the current study is to preserve the emulsomal vesicles against the harsh condition of gastrointestinal tract (GIT), after oral administration, employing tripolyphosphate (TPP)-crosslinked chitosan as a protective coating layer. Rutin was used as a model drug with evaluation of anti-hyperlipidemic activity in rats. The rutin loaded unmodified emulsomes were prepared using tripalmitin and soybean phosphatidylcholine (SPC), by thin film method. Drug loading for the prepared formulations ranged between 6.80 and 15.50 %. The selected formulation (RT-Emuls-6) comprised tripalmitin and SPC, molar ratio 1:1, and exhibited particle size (PS) and zeta potential (ZP) of 150.40 nm and -35.35 mV, respectively. RT-Emuls-6 was then modified by coating with either solely chitosan (RT-Emuls-6-Ch) or TPP-crosslinked chitosan (RT-Emuls-6-Ch-TPP-1). The latter exhibited PS and ZP values of 269.60 nm and 37.17 mV, respectively. Transmission electron microscopy of RT-Emuls-6-Ch-TPP-1 showed a dense pale greyish layer of a coating layer of chitosan crosslinked with TPP surrounding SPC bilayers. Fourier transform infrared spectroscopy analysis along with X-ray powder diffraction confirmed cross-linking between chitosan and TPP. Stability study in the simulated GIT fluids revealed that the order of rutin retained percentage was RT-Emuls-6-Ch-TPP-1 > RT-Emuls-6-Ch > RT-Emuls-6 (80.02, 50.66 and 44.41 %, respectively for simulated gastric fluid and 63.50, 55.66 and 24.00 %, respectively for simulated intestinal fluid, after 2 h incubation). Anti-hyperlipidemic activity of rutin loaded emulsomes was evaluated, after oral administration, in a high fat diet-induced hyperlipidemia in rats. The order of activity was as follows: RT-Emuls-6-Ch-TPP-1 > RT-Emuls-6-Ch > RT-Emuls-6 > free rutin. These findings revealed the potential of TPP-crosslinked chitosan as a protective coating layer for enhancing the stability of emulsomes against the harsh condition of GIT. RT-Emuls-6-Ch-TPP-1 had a potent anti-hyperlipidemic activity via regulation of lipids, oxidative stress, irisin and uncoupling protein 1.

Non-Hydrolysable Analogues of Cyclic and Branched Condensed Phosphates: Chemistry and Chemical Proteomics.

Studies into the biology of condensed phosphates almost exclusively cover linear polyphosphates. However, there is evidence for the presence of cyclic polyphosphates (metaphosphates) in organisms and for enzymatic digestion of branched phosphates (ultraphosphates) with alkaline phosphatase. Further research of non-linear condensed phosphates in biology would profit from interactome data of such molecules, however, their stability in biological media is limited. Here we present syntheses of modified, non-hydrolysable analogues of cyclic and branched condensed phosphates, called meta- and ultraphosphonates, and their application in a chemical proteomics approach using yeast cell extracts. We identify putative interactors with overlapping hits for structurally related capture compounds underlining the quality of our results. The datasets serve as starting point to study the biological relevance and functions of meta- and ultraphosphates. In addition, we examine the reactivity of meta- and ultraphosphonates with implications for their "hydrolysable" analogues: Efforts to increase the ring-sizes of meta- or cyclic ultraphosphonates revealed a strong preference to form trimetaphosphate-analogue structures by cyclization and/or ring-contraction. Using carbodiimides for condensation, the so far inaccessible dianhydro product of ultraphosphonate, corresponding to P4 O11 2- , was selectively obtained and then ring-opened by different nucleophiles yielding modified cyclic ultraphosphonates.

Allosteric modulation of exosite 1 attenuates polyphosphate-catalyzed activation of factor XI by thrombin.

BACKGROUND: Polyphosphate (polyP) promotes feedback activation of factor (F) XI by thrombin by serving as a template. The contribution of thrombin's exosites to these interactions is unclear. OBJECTIVES: To determine the contribution of thrombin exosites 1 and 2 to polyP-induced potentiation of FXI activation by thrombin. METHODS: The affinities of α-thrombin; K109E/110E-thrombin, an exosite 1 variant, or R93E-thrombin, an exosite 2 variant; FXI; and FXIa for polyP-70 were quantified using surface plasmon resonance in the absence or presence of exosite ligands. FXI was activated with α-thrombin or thrombin variants in the absence or presence of polyP-70 and exosite ligands. RESULTS: α-Thrombin, K109/110E-thrombin, FXI, and FXIa bound polyP-70, whereas R93E-thrombin exhibited minimal binding. Exosite 1 and exosite 2 ligands attenuated thrombin binding to polyP-70. PolyP-70 accelerated the rate of FXI activation by α-thrombin and K109E/110E-thrombin but not R93E-thrombin up to 1500-fold in a bell-shaped, concentration-responsive manner. Exosite 1 and exosite 2 ligands had no impact on FXI activation by thrombin in the absence of polyP-70; however, in its presence, they attenuated activation by 40% to 65%. CONCLUSION: PolyP-70 binds FXI and thrombin and promotes their interaction. Exosite 2 ligands attenuate activation because thrombin binds polyP-70 via exosite 2. Attenuation of FXI activation by exosite 1 ligands likely reflects allosteric modulation of exosite 2 and/or the active site of thrombin because exosite 1 is not directly involved in FXI activation. Therefore, allosteric modulation of thrombin's exosites may represent a novel strategy for downregulating FXI activation.

Synergistic Function between Phosphorus-Containing Flame Retardant and Multi-Walled Carbon Nanotubes towards Fire Safe Polystyrene Composites with Enhanced Electromagnetic Interference Shielding.

As a universal polymer material, polystyrene (PS) is widely applied in electrical devices and construction. Thus, it is necessary to improve the flame retardancy and electromagnetic shielding properties of PS material. In this work, PS/silicon-wrapped ammonium polyphosphate/Inorganic acid-treated multi-walled carbon nanotubes composites (PS/SiAPP/aMWCNT, abbreviated as PAC) were prepared via methods of filtration-induced assembly and hot-pressing. Morphology and structure characterization demonstrated that SiAPP and aMWCNT had good dispersion in PS and excellent compatibility with the PS matrix. Thermogravimetric analysis revealed that the addition of aMWCNT to PS improved its thermal stability and carbon-forming characteristics. The peak heat release rate, the peak carbon monoxide production rate, and the peak smoke production rate of the PAC10 composite decreased by 53.7%, 41.9%, and 45.5%, respectively, while its electromagnetic shielding effectiveness reached 12 dB. These enhancements were attributed to the reason that SiAPP and aMWCNT synergistically catalyzed the char generation and SiAPP produced free radical scavengers and numbers of incombustible gases, which could decrease the oxygen concentration and retard the combustion reaction. Therefore, the assembled PS/SiAPP/aMWCNT system provides a new pathway to improve the flame retardant and electromagnetic shielding properties of PS.

Interfacial silicon­nitrogen aerogel raise flame retardancy of bamboo fiber reinforced polylactic acid composites.

A triazine derivative containing nitrogen and silicon (SiN) was synthesized and the SiN hybrid aerogel was covered on the surface of bamboo fiber (BF). The modified BF was identified as MBF. The MBF and ammonium polyphosphate (APP) were used to regulate the flame retardancy and mechanical properties of polylactic acid (PLA). The PLA/BF composites were investigated using limiting oxygen index (LOI), UL-94 vertical combustion, cone calorimetry, thermogravimetric analysis linked with infrared spectra (TG-IR) etc. The char residue of MBF at 800 °C is as high as 43.5 % which is 200 % more than that of BF. Incorporating 9 wt% APP generates a PLA9 which displays the UL-94 V2 rating and a LOI value of 28.0 vol%. PLA9/MBF composites display the UL-94 V0 rating and increased LOI values while PLA9/BF composites obtain the UL-94 V2 rating and decreased LOI. The MBF reduces the release of flammable gases during combustion, enhances charring ability and decreases the thermal conductivity of composites. Besides, the tensile and impact strength of PLA9/20MBF is 20 % and 37 % more than that of PLA9/20BF due to stronger interfacial adhesion. This work provides a good method to regulate the flame retardancy and mechanical properties of PLA/BF composites.

Charge-Converting Nanoemulsions as Promising Retinal Drug and Gene Delivery Systems.

AIM: This study aimed to develop phosphatase-responsive ζ potential converting nanocarriers utilizing polyphosphate-coated cell-penetrating peptide (CPP)-decorated nanoemulsions (NEs) as a novel gene delivery system to retinal cells. METHODS: Poly-l-lysine (PLL) was first conjugated with oleylamine (OA) only at its carboxylic end to form the amphiphilic PLL-oleylamine (PLOA) conjugate. Afterward, NEs were loaded with PLOA prior to being coated with tripolyphosphate (TPP) to generate PLOA/TPP NEs. A plasmid containing a reporter gene for green fluorescent protein plasmid (pGFP) was complexed with cationic surfactants forming hydrophobic ion pairs that were loaded in the oily core of NEs. Phosphate removal, ζ potential conversion, and cytotoxicity of the system were evaluated. Cellular uptake and transfection efficiency were investigated in 661W photoreceptor-like cells via microscopic analysis, fluorescence spectroscopy, and flow cytometry. RESULTS: Dephosphorylation of PLOA/TPP NEs triggered by alkaline phosphatase (ALP) resulted in the exposure of positive amine groups on the surface of NE droplets and a notable conversion of the ζ potential from -22.4 to +8.5 mV. Cellular uptake of PLOA/TPP NEs performed on 661W photoreceptor-like cells showed a 3-fold increase compared to control NEs. Furthermore, PLOA/TPP NEs also showed low cytotoxicity and high transfection efficacy with ∼50% of cells transfected. CONCLUSIONS: Polyphosphate-coated CPP-decorated NEs triggered by ALP could be a promising nanosystem to efficiently deliver drugs and genetic materials to photoreceptor-like cells and other retinal cells for potential treatments of retinal diseases.

Enhanced fire-proofing performance and crystallizability of bio-based poly(L-lactic acid): Dual functions of a Schiff base-containing synergistic flame retardant.

Poly(L-lactic acid) (PLA) is a kind of important bio-macromolecule which can be prepared via fermentation of starch of maize and sweet potato. Flammability and extremely poor crystallizability limited its wide application. In this work, a novel Schiff base derivate (CP) was synthesized and, combined with ammonium polyphosphate (APP) as a synergistic flame retardant and nucleating agent to investigate its effects on LOI, UL-94 rating, thermal stability, combustion behavior and crystallizability of PLA. With loading of 5%CP/10%APP, PLA showed a significantly enhanced LOI and passed V-0 fire-safety rating with self-extinguish effect. PLA/5%CP/10%APP presented the lowest pHRR, THR and TSR, and highest char residue yield, FPI and FRI in cone calorimetry test, indicating an excellent flame retardancy effect, enhanced fire safety and longer escaping time in the fire. A continuous, compact and thick char layer structure formed as a protective barrier in combustion process, to enhance heat-insulating and oxygen resistance property, thermal stability and smoke-suppressing capacity of PLA. Flame retardancy mechanism was proposed and discussed based on comprehensive and in-depth characterization techniques. Also, 5%CP/10%APP presented a good nucleation effect to enormously increase crystallizability and shorten crystallization time of PLA.

Hierarchical Assembly of Flexible Biopolymer Polyphosphate-Manganese into Nanosheets.

Polyphosphate (polyP) is one of the most compact inorganic polyanionic biopolymers that participates in various physiological processes. However, the development of polyP-based nanomaterials is still in its infancy. Here, biocompatible polyphosphate-manganese nanosheets are designed and synthesized by a hierarchical assembly strategy. The thickness and the lateral size of the resulting polyP-Mn nanosheets (PMNSs) are 5 nm and 120-130 nm, respectively. Molecular dynamics simulations suggested that the polyP-hexadecyl trimethyl ammonium bromide flat structure possesses a strong aggregating capacity and serves as the template for the 2D assembly of polyP-Mn. The PMNSs can activate the inflammatory response of macrophages resulting in the recovery of innate immunological functions to inhibit tumor proliferation. This work has initiated a new direction in constructing layered polyP-based nanomaterials and provides guidance for biocompatible and biodegradable biopolymer-based materials in the regulation of innate responses.

Synergistic Flame Retardancy of Phosphatized Sesbania Gum/Ammonium Polyphosphate on Polylactic Acid.

Phosphating sesbania gum (DESG) was obtained by modifying sesbania gum (SG) with 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and endic anhydride (EA). The structure of DESG was determined using Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance spectroscopy (1H-NMR). Flame-retardant polylactic acid (PLA) composites were prepared by melt-blending PLA with DESG, which acted as a carbon source, and ammonium polyphosphate (APP), which acted as an acid source and a gas source. The flame retardancy of the PLA composite was investigated using vertical combustion (UL-94), the limiting oxygen index (LOI) and the cone calorimeter (CONE) test. Thermal properties and morphology were characterized via thermogravimetric analysis (TGA) and field emission scanning electron microscopy (FESEM), respectively. Experimental results indicated that when the mass ratio of DESG/APP was equal to 12/8 the LOI value was 32.2%; a vertical burning test (UL-94) V-0 rating was achieved. Meanwhile, the sample showed a lowest total heat release (THR) value of 52.7 MJ/m2, which is a 32.5% reduction compared to that of neat PLA. Using FESEM, the uniform distribution of DESG and APP in the PLA matrix was observed. The synergistic effect of DESG and APP effectively enhanced the flame retardancy of PLA. Additionally, the synergistic mechanism of DESG and APP in PLA was proposed.

Polyphosphate in Antiviral Protection: A Polyanionic Inorganic Polymer in the Fight Against Coronavirus SARS-CoV-2 Infection.

Polyanions as polymers carrying multiple negative charges have been extensively studied with regard to their potential antiviral activity. Most studies to date focused on organic polyanionic polymers, both natural and synthetic. The inorganic polymer, polyphosphate (polyP), despite the ubiquitous presence of this molecule from bacteria to man, has attracted much less attention. More recently, and accelerated by the search for potential antiviral agents in the fight against the pandemic caused by the coronavirus SARS-CoV-2, it turned out that polyP disrupts the first step of the viral replication cycle, the interaction of the proteins in the virus envelope and in the cell membrane that are involved in the docking process of the virus with the target host cell. Experiments on a molecular level using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the cellular angiotensin converting enzyme 2 (ACE2) receptor revealed that polyP strongly inhibits the binding reaction through an electrostatic interaction between the negatively charged centers of the polyP molecule and a cationic groove, which is formed by positively charged amino acids on the RBD surface. In addition, it was found that polyP, due to its morphogenetic and energy delivering activities, enhances the antiviral host innate immunity defense of the respiratory epithelium. The underlying mechanisms and envisaged application of polyP in the therapy and prevention of COVID-19 are discussed.

Protocol to quantify polyphosphate in human cell lines using a tagged PPBD peptide.

Polyphosphate (polyP) is an evolutionarily conserved polymer of phosphates that is difficult to study in human cells because of its low concentration and high lability. First, we described how to express and purify Xpress-tagged PPBD (Ppx1 PolyP Binding Domain). We describe the detection and quantification of nuclear polyP in HEK293T cells using Xpress-PPBD, Xpress antibody, and Alexa-conjugated secondary antibodies. We have also used this protocol in SH-SY5Y HeLa and HEK293 cells. For complete details on the use and execution of this protocol, please refer to Samper-Martín et al. (2021).

Interfacial architecting of organic-inorganic hybrid toward mechanically reinforced, fire-resistant and smoke-suppressed polyurethane composites.

Polyurethane (PU) composites integrated with outstanding fire retardancy, smoke suppression as well as ameliorated mechanical property is attractive, but remains a challenge. Here, we demonstrate the interfacial architecting of organic-inorganic hybrid (ACAPP) by reacting renewable chitooligosaccharide (COS) with ammonium polyphosphate (APP) and ammonium octamolybdate (AOM) via a one-pot, facile and eco-friendly approach. The ACAPP with multiple hydroxyl groups shows good compatibility and strong interfacial adhesion force with PU matrix due to the abundant covalent cross-linking points and hydrogen bonds networks. The incorporation of 15 wt% ACAPP enables the resultant PU composite to achieve a V-0 rating, a limiting oxygen index of 26.1%, 31.1% and 44.8% reduction in total heat release and total smoke production, respectively, far outdistancing conventional APP. The suppressed fire hazards mainly benefits from the synergy catalytic carbonization effect of ACAPP in condensed phase. Moreover, ACAPP reinforces the tensile strength of PU whilst retaining the decent ultimate elongation. This work may offer a referable exemplification for constructing green flame-retardant to balance high fire safety and mechanical properties.

Mixing efficiency affects the morphology and compactness of chitosan/tripolyphosphate nanoparticles.

Due to the rapid reaction kinetics, the morphology of chitosan/tripolyphosphate nanoparticles was difficult to control. We mixed chitosan and tripolyphosphate through a multi-inlet vortex mixer at different mixing efficiency and characteristic mixing time (τmix). Below a critical τmix, the aggregation of primary chitosan/TPP particles can be ceased. The corresponding aggregation time of primary particles (τagg) was dependent on the ionic strength, the degree of deacetylation and molecular weight of chitosan. Moreover, the particle compactness was estimated from the hydrodynamic diameter and the turbidity using a model based on the Mie theory. Slow and nonhomogeneous mixing led to more compact nanoparticles, while rapid and homogeneous mixing produced nanoparticles with a higher swelling ratio. Besides, the ionic strength and polymer concentration could affect the internal structure of nanoparticles. This study revealed the significance of mixing on the internal structure of chitosan/TPP nanoparticles, which could guide the preparation of other ionically-crosslinked polysaccharide colloids.

Phytate-mediated phosphorylation of starch by dry heating with rice bran extract.

Rice and corn starches were subjected to dry heating with rice bran extract or sodium trimetaphosphate (STMP)/sodium tripolyphosphate (STPP) for starch phosphorylation. Phytate in rice bran extract or STMP/STPP increased the concentration of phosphorus in rice and maize starches. The highest concentrations of phosphorus were induced in rice starch with rice bran extract and in corn starch with STMP/STPP. 31P NMR analysis indicated that the rice bran extract and STMP/STPP produced monostarch monophosphate under the same reaction conditions. Rice and corn starches phosphorylated with rice bran extract or STMP/STPP demonstrated great peak viscosity and low pasting temperatures. Although starch phosphorylated with either rice bran extract or STMP/STPP showed higher paste clarity, solubility, and swelling power than native starch, these parameters were optimal in rice starch phosphorylated with rice bran extract. Therefore, dry heating with rice bran extract induced phytate-mediated phosphorylation with the typical physicochemical properties of starch phosphates.

Polyphosphate Kinase 2 (PPK2) Enzymes: Structure, Function, and Roles in Bacterial Physiology and Virulence.

Inorganic polyphosphate (polyP) has been implicated in an astonishing array of biological functions, ranging from phosphorus storage to molecular chaperone activity to bacterial virulence. In bacteria, polyP is synthesized by polyphosphate kinase (PPK) enzymes, which are broadly subdivided into two families: PPK1 and PPK2. While both enzyme families are capable of catalyzing polyP synthesis, PPK1s preferentially synthesize polyP from nucleoside triphosphates, and PPK2s preferentially consume polyP to phosphorylate nucleoside mono- or diphosphates. Importantly, many pathogenic bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii encode at least one of each PPK1 and PPK2, suggesting these enzymes may be attractive targets for antibacterial drugs. Although the majority of bacterial polyP studies to date have focused on PPK1s, PPK2 enzymes have also begun to emerge as important regulators of bacterial physiology and downstream virulence. In this review, we specifically examine the contributions of PPK2s to bacterial polyP homeostasis. Beginning with a survey of the structures and functions of biochemically characterized PPK2s, we summarize the roles of PPK2s in the bacterial cell, with a particular emphasis on virulence phenotypes. Furthermore, we outline recent progress on developing drugs that inhibit PPK2 enzymes and discuss this strategy as a novel means of combatting bacterial infections.

A Multi-enzyme Cascade for the Biosynthesis of AICA Ribonucleoside Di- and Triphosphate.

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.