3D printed double-network alginate hydrogels containing polyphosphate for bioenergetics and bone regeneration.

Low mechanical strength, poor processability, and low bioactivity of hydrogels limit their application in bone tissue engineering severely. Herein, a new 3D-printable, osteoinductive, and bioenergetic-active double-network (DN) hydrogel containing sodium alginate (SA), poly (ethylene glycol) diacrylate (PEGDA), and sodium polyphosphate (PolyP) was developed via a two-step method. The synergy of the covalent cross-linking network and the ionic cross-linking network improves the mechanical properties of the hydrogel. And the pre-gel with Ca2+ has better 3D printing performance to print complex tissue engineering scaffolds than common hydrogels. In addition, the incorporation of PolyP into DN hydrogel matrix significantly improves the bioactivity of hydrogels. The bioenergetic effect of PolyP improves adenosine triphosphate content of cells significantly to promote cell activities such as migration. The in vitro osseointegration investigation suggests that the orthophosphate monomer units, which are degradation fragments of PolyP, provide enough phosphoric acid units for the formation of calcium phosphate and accelerate the osteogenic differentiation of cells greatly. Therefore, the proposed printable, bioenergetic-active, osteoinductive DN hydrogel is potential to solve the problems of complex tissue engineering scaffolds and be applied in energy-crucial bone tissue regeneration.

Synthesis and Cellular Labeling of Multifunctional Phosphatidylinositol Bis- and Trisphosphate Derivatives.

We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo-removable protecting group ("cage"), a photo-crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo-crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo-crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3 -binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane.

Synthesis of α,δ-Disubstituted Tetraphosphates and Terminally-Functionalized Nucleoside Pentaphosphates.

The anion [P4O11]2-, employed as its bis(triphenylphosphine)iminium (PPN) salt, is shown herein to be a versatile reagent for nucleophile tetraphosphorylation. Treatment under anhydrous conditions with an alkylamine base and a nucleophile (HNuc1), such as an alcohol (neopentanol, cyclohexanol, 4-methylumbelliferone, and Boc-Tyr-OMe), an amine (propargylamine, diethylamine, morpholine, 3,5-dimethylaniline, and isopropylamine), dihydrogen phosphate, phenylphosphonate, azide ion, or methylidene triphenylphosphorane, results in nucleophile substituted tetrametaphosphates ([P4O11Nuc1]3-) as mixed PPN and alkylammonium salts in 59% to 99% yield. Treatment of the resulting functionalized tetrametaphosphates with a second nucleophile (HNuc2), such as hydroxide, a phenol (4-methylumbelliferone), an amine (propargylamine and ethanolamine), fluoride, or a nucleoside monophosphate (uridine monophosphate, deoxyadenosine monophosphate, and adenosine monophosphate), results in ring opening to linear tetraphosphates bearing one nucleophile on each end ([Nuc1(PO3)3PO2Nuc2]4-). When necessary, these linear tetraphosphates are purified by reverse phase or anion exchange HPLC, yielding triethylammonium or ammonium salts in 32% to 92% yield from [PPN]2[P4O11]. Phosphorylation of methylidene triphenylphosphorane as Nuc1 yields a new tetrametaphosphate-based ylide ([Ph3PCHP4O11]3-, 94% yield). Wittig olefination of 2',3'-O-isopropylidene-5'-deoxy-5'-uridylaldehyde using this ylide results in a 3'-deoxy-3',4'-didehydronucleotide derivative, isolated as the triethylammonium salt in 54% yield.

Polyphosphoestered Nanomedicines with Tunable Surface Hydrophilicity for Cancer Drug Delivery.

The surface hydrophilicity of nanoparticles has a major impact on their biological fates. Ascertaining the correlation between nanoparticle surface hydrophilicity and their biological behaviors is particularly instructive for future nanomedicine design and their antitumor efficacy optimization. Herein, we designed a series of polymeric nanoparticles based on polyphosphoesters with well-controlled surface hydrophilicity in the molecular level and systemically evaluated their biological behaviors. The results demonstrated that high surface hydrophilicity preferred lower protein absorption, better stability, longer blood circulation, and higher tumor accumulation but lower cellular uptake. Upon encapsulation of drugs, nanoparticles with high hydrophilicity showed an excellent antitumor therapeutic efficacy in both primary and metastatic tumors as compared to the relatively hydrophobic ones. Further analyses revealed that the superior antitumor outcome was attributed to the balance of tumor accumulation and cellular uptake, demonstrating the particular importance of nanoparticle surface hydrophilicity regulation on the antitumor efficacy. Our work provides a potent guideline for a rational designation on the surface hydrophilicity of nanoparticles for cancer treatment optimization.

Prodrugs of γ-Alkyl-Modified Nucleoside Triphosphates: Improved Inhibition of HIV Reverse Transcriptase.

The development of nucleoside triphosphate prodrugs is one option to apply nucleoside reverse transcriptase inhibitors. Herein, we report the synthesis and evaluation of d4TTP analogues, in which the γ-phosphate was modified covalently by lipophilic alkyl residues, and acyloxybenzyl prodrugs of these γ-alkyl-modified d4TTPs, with the aim of delivering of γ-alkyl-d4TTP into cells. Selective formation of γ-alkyl-d4TTP was proven with esterase and in CD4+ -cell extracts. In contrast to d4TTP, γ-alkyl-d4TTPs proved highly stable against dephosphorylation. Primer extension assays with HIV reverse transcriptase (RT) and DNA-polymerases α, ß or γ showed that γ-alkyl-d4TTPs were substrates for HIV-RT only. In antiviral assays, compounds were highly potent inhibitors of HIV-1 and HIV-2 also in thymidine-kinase-deficient T-cell cultures (CEM/TK- ). Thus, the intracellular delivery of such γ-alkyl-nucleoside triphosphates may potentially lead to nucleoside triphosphates with a higher selectivity towards the viral polymerase that can act in virus-infected cells.

Synthetic Strategies for Dinucleotides Synthesis.

Dinucleoside 5',5'-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5',5'-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.

2'-Deoxyribonucleoside 5'-triphosphates bearing 4-phenyl and 4-pyrimidinyl imidazoles as DNA polymerase substrates.

We developed a versatile access to a series of 4-substituted imidazole 2'-deoxynucleoside triphosphate bearing functionalized phenyl or pyrimidinyl rings. 4-Iodo-1H-imidazole was enzymatically converted into the corresponding 2'-deoxynucleoside, which was then chemically derived into its 5'-triphosphate, followed by 4-arylation via Suzuki-Miyaura coupling using (hetero)arylboronic acids. Both KF (exo-) and Deep Vent (exo-) DNA polymerases incorporated these modified nucleotides in primer-extension assays, adenine being the preferred pairing partner in the template. The 4-(3-aminophenyl)imidazole derivative (3APh) was the most efficiently inserted opposite A by KF (exo-) with only a 37-fold lower efficiency (Vmax/KM) than that of the correct dTTP. No further extension occurred after the incorporation of a single aryl-imidazole nucleotide. Interestingly, the aryl-imidazole dNTPs were found to undergo successive incorporation by calf thymus terminal deoxynucleotidyl transferase with different tailing efficiencies among this series and with a marked preference for 2APyr polymerization.

Prodrugs of Nucleoside Triphosphates as a Sound and Challenging Approach: A Pioneering Work That Opens a New Era in the Direct Intracellular Delivery of Nucleoside Triphosphates.

Synthetic nucleosides, designed to mimic naturally occurring nucleosides, are important antiviral and anticancer chemotherapeutic agents. However, nucleosides are not active as such and need to be metabolized, step by step, to their corresponding active nucleoside triphosphates (NTPs). This is mediated by phosphorylating enzymes, mainly host cellular kinases with strong specificity for their substrates; in many cases, this specificity prevents efficient conversion into the NTPs. To circumvent this metabolic handicap, successful nucleo(s/t)ide prodrugs have been developed as a valuable concept in the design of effective drugs. The unique concept of the TriPPPro approach, developed by Chris Meier and colleagues, is a powerful tool for the intracellular delivery of active NTPs, bypassing all the phosphorylation steps required by nucleosides to yield the active NTP metabolites. This concept is illustrated herein with general examples.

Screening a Protein Array with Synthetic Biotinylated Inorganic Polyphosphate To Define the Human PolyP-ome.

Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

Protected 2'-deoxyribonucleoside triphosphate building blocks for the photocaging of epigenetic 5-(hydroxymethyl)cytosine in DNA.

2'-Deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

Nucleoside diphosphate and triphosphate prodrugs - An unsolvable task?

In this review, our recent advances in the development of nucleoside di- and nucleoside triphosphate prodrugs is summarized. Previously, we had developed a successful membrane-permeable pronucleotide system for the intracellular delivery of nucleoside monophosphates as well, the so-called cycloSal-approach. In contrast to that work in which the delivery is initiated by a chemically driven hydrolysis reaction, for the di- and triphosphate delivery, an enzymatic trigger mechanism involving (carboxy)esterases had to be used. The other features of the new pronucleotide approaches are: (i) lipophilic modification was restricted to the terminal phosphate group leaving charges at the internal phosphate moieties and (ii) appropriate lipophilicity is introduced by long aliphatic residues within the bipartite prodrug moiety. The conceptional design of the di- and triphosphate prodrug systems will be described and the chemical synthesis, the hydrolysis properties, a structure-activity relationship and antiviral activity data will be discussed as well. The advantage of these new approaches is that all phosphorylation steps from the nucleoside analogue into the bioactive nucleoside triphosphate form can be bypassed in the case of the triphosphate prodrugs. Moreover, enzymatic processes like the deamination of nucleosides or nucleoside monophosphates which lead to catabolic clearance of the potential antivirally active compound can be avoided by the delivery of the higher phosphorylated nucleotides.

Driving and photo-regulation of myosin-actin motors at molecular and macroscopic levels by photo-responsive high energy molecules.

We employed an azobenzene based non-nucleoside triphosphate, AzoTP, in a myosin-actin motile system and demonstrated its efficiency as an energy molecule to drive and photo-regulate the myosin-actin motile function at the macroscopic level along with an in vitro motility assay. The AzoTP in its trans state induced shortening of a glycerinated muscle fibre whilst a cis isomer had no significant effect. Direct photoirradiation of a cis-AzoTP infused muscle fibre induced shortening triggered by a locally photo-generated trans-AzoTP in the muscle fibre. Furthermore, we designed and synthesized three new derivatives of AzoTPs that served as substrates for myosin by driving and photo-regulating the myosin-actin motile function at the molecular as well as the macroscopic level with varied efficiencies.

Phosphorothioate analogs of P1,P3-di(nucleosid-5'-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase.

Di(nucleosid-5'-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5'-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5'-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2-3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

Synthesis and polymerase incorporation of ß,γ-modified α-l-threofuranosyl thymine triphosphate mimics.

Three ß,γ-modified α-l-threofuranosyl nucleoside triphosphates were synthesized. The ß,γ-modified tTTPs undergo a single incorporation event with HIV RT but undergo multiple incorporations to form full-length product with engineered thermophilic polymerases.

Design and evaluation of novel pH-sensitive ureido-conjugated chitosan/TPP nanoparticles targeted to Helicobacter pylori.

The covalently modified ureido-conjugated chitosan/TPP multifunctional nanoparticles have been developed as targeted nanomedicine delivery system for eradication of Helicobacter pylori. H. pylori can specifically express the urea transport protein on its membrane to transport urea into cytoplasm for urease to produce ammonia, which protects the bacterium in the acid milieu of stomach. The clinical applicability of topical antimicrobial agent is needed to eradicate H. pylori in the infected fundal area. In this study, we designed and synthesized two ureido-conjugated chitosan derivatives UCCs-1 and UCCs-2 for preparation of multifunctional nanoparticles. The process was optimized in order to prepare UCCs/TPP nanoparticles for encapsulation of amoxicillin. The results showed that the amoxicillin-UCCs/TPP nanoparticles exhibited favorable pH-sensitive characteristics, which could procrastinate the release of amoxicillin at gastric acids and enable the drug to deliver and target to H. pylori at its survival region effectively. Compared with unmodified amoxicillin-chitosan/TPP nanoparticles, a more specific and effective H. pylori growth inhibition was observed for amoxicillin-UCCs/TPP nanoparticles. Drug uptake analysis tested by flow cytometry and confocal laser scanning microscopy verified that the uptake of FITC-UCCs-2/TPP nanoparticles was associated with urea transport protein on the membrane of H. pylori and reduced with the addition of urea as competitive transport substrate. These findings suggest that the multifunctional amoxicillin-loaded nanoparticles have great potential for effective therapy of H. pylori infection. They may also serve as pharmacologically effective nanocarriers for oral targeted delivery of other therapeutic drugs to treat H. pylori.

Conditions for obtaining polyvinyl alcohol/trisodium trimetaphosphate hydrogels as vitreous humor substitute.

Hydrogels are polymeric materials with numerous medical and biological applications because of their physicochemical properties. In this context, the conditions were defined for obtaining a hydrogel with characteristics similar to the vitreous humor using polyvinyl alcohol (PVA) and trisodium trimetaphosphate (STMP). The concentration of PVA (X1 ), PVA/STMP ratio (X2 ), and initial pH (X3 ) were modified, and their effect was analyzed in terms of the refractive index (Y1 ), density (Y2 ), dynamic viscosity (Y3 ), and final pH (Y4 ). The results demonstrated that X1 interferes with Y1 , Y2 , and Y3 , and X2 interferes with Y2 and Y3 . The best condition for obtaining a hydrogel with characteristics similar to the vitreous humor was 4.2586% PVA (wt/wt), STMP/PVA ratio of 1:6.8213 (wt/wt), and initial pH of 9.424. DSC, ATR-FTIR, swelling degree, and AFM analysis confirmed the PVA reticulation with STMP. Furthermore, STMP increased the glass transition temperature and decreased the water uptake of ∼50% of the hydrogels, which can be explained by the crosslinking of PVA chains. Infrared spectroscopy revealed a decrease of hydroxyl bonds and confirmed the reticulation between PVA and STMP. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1386-1395, 2016.

Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-L-tryptophanyltriphosphates.

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5'-α-P-borano-γ-P-N-L-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5-36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, was generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides.

Synthesis of 1-(ß-D-Galactopyranosyl)Thymine-6'-O-Triphosphate - A Potential Probe to Generate Reactive Dialdehyde for DNA-Enzyme Cross-Linking.

Concise, facile, and efficient synthesis of 1-(ß-D-galactopyranosyl)thymine-6'-O-triphosphate, a potential probe that can generate reactive dialdehyde for DNA-enzyme cross-linking applications, was described starting from O,O'-bis(trimethylsilyl)thymine. Stannic chloride promoted glycosylation of 1,2,3,4,6-penta-O-acetyl-α-D-galactopyranose with O,O'-bis(trimethylsilyl)thymine, resulting in the formation of 1-(2,3,4,6-O-tetraacetyl-ß-D-galactopyranosyl)thymine in 91% yield. Acetyl deprotection using methanolic ammonia afforded 1-(ß-D-galactopyranosyl)thymine in 98% yield. The modified one-pot methodology was used to convert 1-(ß-D-galactopyranosyl)thymine into 1-(ß-D-galactopyranosyl)thymine-6'-O-triphosphate in 72% yield, which involves the formation of 1-(ß-D-galactopyranosyl)thymine dichlorophosphoridate using POCl3 as the reagent at the monophosphorylation step followed by reaction with tributylammonium pyrophosphate and hydrolysis of resulting cyclic intermediate.

Synthesis of C8-N-Arylamine-Modified 2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases.

C8-N-arylamine adducts of 2'-deoxyguanosine (2'-dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8-N-acetyl-N-arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8-N-acetyl-N-arylamine-2'-deoxyguanosine-5'-triphosphates and C8-NH-arylamine-2'-deoxyguanosine-5'-triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8-dG adducts were studied in primer-extension assays by using Klenow fragment exo(-) of Escherichia coli DNA polymerase I and human DNA polymerase ß. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N-acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8-N-arylamine-2'-deoxyguanosine-5'-triphosphates in chemical carcinogenesis.

Hyperbranched polyphosphates: synthesis, functionalization and biomedical applications.

Hyperbranched polyphosphates (HBPPs) are newly emerged polymeric biomaterials with repeating phosphate bonds in a highly branched framework over the past 5 years. Due to the integration of the advantages of both hyperbranched polymers and polyphosphates, HBPPs are versatile in chemical structure, flexible in physicochemical properties, water soluble, biocompatible and biodegradable in biological features. On the basis of their excellent water solubility, biocompatibility, biodegradability and potential functionalization as well as their simple preparation in one-pot synthesis, HBPPs have fascinating biomedical applications, especially for drug delivery. In this tutorial review, the recent advances of HBPPs are summarized. HBPPs with different topological structures and various functionalities were synthesized via adjusting the side group of cyclic phosphate monomers, which have shown promising biomedical applications, for example, using as a macromolecular anticancer agent and constructing advanced drug delivery systems, including site-specific delivery systems, self-delivery systems, and stimuli-responsive delivery systems. Such progress may promote the further development of interdisciplinary research between polymer chemistry, material science and biomedicine.