Phosphoester Linkages Research Articles

Synthesis and characterization of polyphosphoester/polyethylene glycol composite hydrogels

<p indent=0mm>The hydrogel, which has a three-dimensional (3D) cross-linked hydrophilic network structure, is widely used as biomedical scaffold for local drug delivery and tissue engineering. Especially, with the continuous development of 3D printing technology, hydrogel scaffolds are increasingly used in the field of regenerative medicine. Polyphosphoester is a class of biodegradable polymers with repeated phosphoester linkages in the backbone, and a series of polyphosphoester materials have been synthesized for biomedical applications. For instance, a variety of amphiphilic polyphosphoester-based copolymers have been synthesized by Wang’s group, and used as the nanocarriers of hydrophobic anticancer drugs as well as siRNA. In addition, Yan’s group has prepared hyper-branched polyphosphoester as the drug delivery systems of hydrophobic anticancer drugs and photodynamic agents. Recently, Wooley and co-workers have synthesize the amphiphilic co-polymer of poly(ethylene oxide) and polyphosphoester, and used to encapsulated paclitaxel (PTX) with high contents for cancer therapy. And, they also conjugated the PTX into the side chain of polyphosphoester with pH-labile linkages for efficient tumor treatments. As the tissue engineering scaffold, polyphosphoester-based scaffolds were first used for fabrication of nerve guide conduits. And then, Elisseeff and co-workers have found the great potentials of phosphate-containing hydrogel for bone tissue engineering. In addition, Leong et al. found that the photocrosslinkable polyphosphoester hydrogel exhibited good compatibility and potentially osteoinductive ability for goat bone marrow-derived mesenchymal stem cells. Therefore, polyphosphoester hydrogel could be a promising scaffold for bone tissue engineering. In this study, water-soluble macromers polyphosphate diacrylate (PEEP-DA) and polyethylene glycol diacrylate (PEG-DA) were first synthesized, and characterized by ¹H NMR. And then, the PEEP/PEG composite hydrogels at various polyphosphoester contents were controlled by varying the feed ratio of the macromers PEEP-DA and PEG-DA (1:29, 2:28, and 5:25), and the PEEP/PEG hydrogels were fabricated by exposing aqueous solutions of these macromers with photoinitiator to UV light irradiation. We systematically studied the structure, biodegradability, biocompatibility, and bone differentiation of the hydrogel scaffold. Compared with PEG hydrogel, these PEEP/PEG hydrogels exhibited better biodegradability due to the degradation properties of polyphosphoester. And, the PEEP/PEG composite hydrogels, which was prepared at PEEP-DA/PEG-DA mass ratio of 5:25, disappeared almost completely after post implantation into mice for 28 d. After encapsulating bone marrow mesenchymal stem cells (BMSCs) during the preparation process, the cell viabilities in the composite PEEP/PEG hydrogels were better than that in the control PEG hydrogel. In addition, the alkaline phosphatase activity, a key maker of early osteogenic differentiation, was evaluated after co-incubation for 7 or 14 d. It could be found that the alkaline phosphatase activity in the PEEP/PEG composite hydrogels, either for 7 or <sc>14 d,</sc> was significantly higher than that of the control PEG hydrogel. The differentiation process of BMSCs into bone is usually accompanied by the calcium deposition. The polyphosphoester has shown osteoinductivity according to previous reports. We found that the amount of calcium deposition was significantly higher in the PEEP/PEG composite hydrogels than that in the PEG hydrogel, especially after co-incubation for <sc>21 d,</sc> which is well consistent with the result of other groups. Collectively, this study reported a new PEEP/PEG composite hydrogels, which exhibited the potential as tissue engineering scaffolds.

Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine-DNA Glycosylase.

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine-DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3' to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3'-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg-DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.

Regioisomeric Preference in Ring-Opening Polymerization of 3',5'-Cyclic Phosphoesters of Functional Thymidine DNA Analogues.

Regioregularity is a crucial property in the synthesis of DNA analogues, as natural DNA is synthesized exclusively in the 5' to 3' direction. We have focused our attention on the determination of the regioisomeric distribution of poly(3',5'-cyclic 3-(3-butenyl) thymidine ethylphosphate)s obtained from the ring-opening polymerization of (R)-3',5'-cyclic 3-(3-butenyl) thymidine ethylphosphate. The regioisomeric preference was investigated by comparison to synthesized model compounds of 3',3'-, 3',5'-, and 5',5'-linkages, where the model 3'-phosphoester linkages were to the secondary alcohol of 3-hydroxytetrahydrofuran and the model 5'-linkages derived from coupling to the primary alcohol of tetrahydrofurfuryl alcohol. From the 31P resonance frequency assignments of those small molecule model compounds, 31P NMR spectra revealed the major connectivity in the polymer backbone to be 3',5'-linkages, with ≤30% of other isomeric forms. Model reactions employing a series of alcohol initiators imparting various degrees of steric hindrance, to mimic the increased steric hindrance of the propagating alcohol relative to the initiator, were then conducted to afford the corresponding ring-opened unimer adducts and to gain understanding of the regioselectivity during the ring-opening polymerization. 1H-31P heteronuclear multiple-bond correlation spectroscopy showed ethanol and 4-methoxybenzyl alcohol initiation to yield only the P-O5' bond cleavage product, whereas attack by isopropyl alcohol upon (R)-3',5'-cyclic 3-(3-butenyl) thymidine ethylphosphate afforded both P-O3' and P-O5' bond cleavage products, supporting our hypothesis that the increased steric hindrance of the propagating species dictates the regioselectivity of the P-O bond cleavage. Further model reactions suggested that the P-O5' bond cleavage products can be detected upon the formation of dimers during the ring-opening polymerization. Overall, this work provides a fundamental understanding of the polymerization behavior of six-membered cyclic phosphoesters and broadens the scope of DNA analogues from the ring-opening polymerization of 3',5'-cyclic phosphoesters.

Polyphosphoesters: New Trends in Synthesis and Drug Delivery Applications.

Polymers with repeating phosphoester linkages in the backbone are biodegradable materials that emerge as a promising class of novel biomaterials, especially in the field of drug delivery systems. In contrast to aliphatic polyesters, the pentavalency of the phosphorus atom offers a large diversity of structures and as a consequence a wide range of properties for these materials. In this paper, it is focused on the synthesis of well-defined polyphosphoesters (PPEs) by organocatalyzed ring-opening polymerization, improving the functionalities by combination with click reactions, degradation of functional PPEs and their cytotoxicity, and inputs for applications in drug delivery.

Synthesis and evaluation of chromogenic and fluorogenic substrates for high-throughput detection of enzymes that hydrolyze inorganic polyphosphate.

Inorganic polyphosphates, linear polymers of orthophosphate, occur naturally throughout biology and have many industrial applications. Their biodegradable nature makes them attractive for a multitude of uses, and it would be important to understand how polyphosphates are turned over enzymatically. Studies of inorganic polyphosphatases are, however, hampered by the lack of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. We now report chromogenic and fluorogenic polyphosphate substrates that permit spectrophotometric monitoring of polyphosphate hydrolysis and allow for high-throughput analyses of both endopolyphosphatase and exopolyphosphatase activities, depending on assay configuration. These substrates contain 4-nitrophenol or 4-methylumbelliferone moieties that are covalently attached to the terminal phosphates of polyphosphate via phosphoester linkages formed during reactions mediated by EDAC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide). This report identifies Nudt2 as an inorganic polyphosphatase and also adds to the known coupling chemistry for polyphosphates, permitting facile covalent linkage of alcohols with the terminal phosphates of inorganic polyphosphate.

Doxorubicin Conjugate of Poly(Ethylene Glycol)‐Block‐Polyphosphoester for Cancer Therapy

Polyphosphoesters with repeating phosphoester linkages in the backbone can be easily functionalized, are biodegradable and potentially biocompatible, and may be potential candidates as polymer carriers of drug conjugates. Here, the efficacy of a polyphosphoester drug conjugate as an anticancer agent in vivo is assessed for the first time. With controlled synthesis, doxorubicin conjugated to poly(ethylene glycol)-block-polyphosphoester (PPEH-DOX) via labile hydrazone bonds form spherical nanoparticles in aqueous solution with an average diameter of ≈60 nm. These nanoparticles are effectively internalized by MDA-MB-231 breast cancer cells and release the conjugated doxorubicin in response to the intracellular pH of endosomes and lysosomes, resulting in significant antiproliferative activity in cancer cells. Compared with free doxorubicin injection, PPEH-DOX injection exhibits much longer circulation behavior in the plasma of mice and leads to enhanced drug accumulation in tumor cells. In an MDA-MB-231 xenograft murine model, inhibition of tumor growth with systemic delivery of PPEH-DOX nanoparticles is more pronounced compared with free doxorubicin injection, suggesting the potential of polyphosphoesters as carriers of drug conjugates in cancer therapy.

A New Flavin Radical Signal in the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae: AN EPR/ELECTRON NUCLEAR DOUBLE RESONANCE INVESTIGATION OF THE ROLE OF THE COVALENTLY BOUND FLAVINS IN SUBUNITS B AND C

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.

Coil-to-Helix Transition within Phospholamban Underlies Release of Ca- ATPase Inhibition in Response to &amp;#946;-Adrenergic Signaling

Phospholamban (PLB) is a major target of beta-adrenergic signaling, whose phosphorylation results in enhanced rates of relaxation in the heart. Prior to phosphorylation, PLB functions to reduce the calcium sensitivity of the Ca-ATPase, resulting in slower rates of calcium resequestration into the sarcoplasmic reticulum after each contractile event. Recent structures indicate that the inhibitory interaction between PLB and the Ca-ATPase requires PLB to assume an extended structure, where the transmembrane and cytosolic portions of PLB undergo specific binding interactions with distant sites on the Ca-ATPase. In the extended conformation, PLB binding to the Ca-ATPase functions to inhibit the Ca-ATPase through a reduction in the rates of catalytically important motions involving the nucleotide binding domain. Phosphorylation of PLB at either Ser16 or Thr17 releases the inhibitory interaction between PLB and the Ca-ATPase. These sites of phosphorylation are within a hinge region in PLB that separates the highly structured transmembrane and cytosolic portions that associate with the Ca-ATPase. The helical content of the hinge region increases following the phosphorylation of PLB, which induces a shortening of the maximal dimensions of PLB and a release of the inhibitory interaction with the Ca-ATPase. Following phosphorylation, PLB remains associated with the Ca-ATPase in a moremore » compact form that has no inhibitory capability. Thus, the conformational switch involving PLB regulation of the Ca-ATPase relies upon a physical mechanism, whereby the phosphorylation-dependent stabilization of the structure of PLB functions to destabilize the inhibitory interaction between PLB and the Ca-ATPase. Upon hydrolysis of the phosphoester linkages by endogenous phosphatases PLB is poised to reassume the inhibited state through re-association with inhibitory sites on the nucleotide binding domain of the Ca-ATPase.« less

Synthesis and characterization of l-tyrosine based novel polyphosphates for potential biomaterial applications

Starting from the natural amino-acid l-tyrosine, a diphenolic monomeric molecule was developed using carbodiimide mediated solid-phase synthesis techniques. This monomeric molecule was polymerized by reacting it in equimolar proportions with suitable dihalophosphates to yield novel biodegradable polyphosphates containing peptide linkages and phosphoester linkages alternating in the polymer backbone. The biodegradability of such a polymer is expected to arise from the hydrolytic degradability of the phosphoester linkages and the enzymatic degradability of the peptide linkages in the polymer backbone. Design of such a polymer is expected to make a significant contribution to biomaterials research, regarding drug delivery device and tissue engineering scaffold applications. The monomer was obtained by a novel solid phase carbodiimide-mediated amide coupling process. The subsequent polymers were obtained by solution-phase dehydrochlorination polycondensation reactions in the presence of a suitable acid acceptor. The synthesized polymers were characterized by 13C NMR, 31P NMR and FTIR for their chemical structure, by GPC for their molecular weight distribution, and by DSC and TGA for their thermal transition characteristics.

Compositional analysis of biodegradable polyphosphoester copolymers using nmr spectroscopic methods

The chemical composition and quantitative molar ratios among all components of biodegradable polyphosphoester copolymers of DL-lactide and ethylphosphate were determined by a comprehensive set of NMR spectroscopic methods. The polyphosphoester copolymers studied were synthesized using condensation polymerization of oligomeric DL-lactide prepolymers and ethyl dichlorophosphate. Conclusive identification of the chemical shift patterns of all functional groups in the copolymers required additional NMR methods such as 31P-NMR and two-dimensional 1H–1H COSY NMR, in addition to the synthesis and comparative NMR analysis of model compounds possessing identical phosphoester linkages in the polyphosphoester copolymers. For the polymers synthesized using the bulk polycondensation process, 1H–1H COSY NMR analysis revealed the presence of a small amount of side products that were undetected by 1H-NMR alone. These side reactions most likely occurred between the pendant ethoxy group of the phosphoesters and the hydrogen chloride gas generated in the bulk polycondensation process. 31P-NMR spectra of the copolymers revealed a consistent triple-peak pattern characteristic of phosphoesters linked to a racemic mixture of D,L-lactides. These results offered new insight into the side reactions occurring in bulk polymerization of polyphosphoesters and provided a powerful tool of characterizing complex biodegradable polymers. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 4021–4031, 2003

Polyphosphoesters in drug and gene delivery

Polymers with repeating phosphoester bonds in the backbone are structurally versatile, and biodegradable through hydrolysis, and possibly enzymatic digestion at the phosphoester linkages under physiological conditions. These biodegradable polyphosphoesters are appealing for biological and pharmaceutical applications because of their potential biocompatibility and similarity to bio-macromolecules such as nucleic acids. In the first part of this review, we will focus on one particular structure synthesized by extending oligomeric lactide prepolymers with ethylphosphate groups. This amorphous to semi-crystalline polymer is promising in delivering anti-cancer therapeutics in the form of microspheres. In the second half, we will discuss the conjugation of charged groups to the side chain of the phosphate, constituting one of the few biodegradable cationic polymers in the field for non-viral gene delivery. Capable of delivering exogenous genes to a cell nucleus or providing an extracellular sustained release of DNA, these cationic polyphosphoesters also serve as a valuable model to understand the important characteristics that render a polymer an effective gene carrier.

Detection of novel members, structure-function analysis and evolutionary classification of the 2H phosphoesterase superfamily.

2',3' Cyclic nucleotide phosphodiesterases are enzymes that catalyze at least two distinct steps in the splicing of tRNA introns in eukaryotes. Recently, the biochemistry and structure of these enzymes, from yeast and the plant Arabidopsis thaliana, have been extensively studied. They were found to share a common active site, characterized by two conserved histidines, with the bacterial tRNA-ligating enzyme LigT and the vertebrate myelin-associated 2',3' phosphodiesterases. Using sensitive sequence profile analysis methods, we show that these enzymes define a large superfamily of predicted phosphoesterases with two conserved histidines (hence 2H phosphoesterase superfamily). We identify several new families of 2H phosphoesterases and present a complete evolutionary classification of this superfamily. We also carry out a structure- function analysis of these proteins and present evidence for diverse interactions for different families, within this superfamily, with RNA substrates and protein partners. In particular, we show that eukaryotes contain two ancient families of these proteins that might be involved in RNA processing, transcriptional co-activation and post-transcriptional gene silencing. Another eukaryotic family restricted to vertebrates and insects is combined with UBA and SH3 domains suggesting a role in signal transduction. We detect these phosphoesterase modules in polyproteins of certain retroviruses, rotaviruses and coronaviruses, where they could function in capping and processing of viral RNAs. Furthermore, we present evidence for multiple families of 2H phosphoesterases in bacteria, which might be involved in the processing of small molecules with the 2',3' cyclic phosphoester linkages. The evolutionary analysis suggests that the 2H domain emerged through a duplication of a simple structural unit containing a single catalytic histidine prior to the last common ancestor of all life forms. Initially, this domain appears to have been involved in RNA processing and it appears to have been recruited to perform various other functions in later stages of evolution.

In vivo phosphorylation following [ 32P]orthophosphate injection into neostriatum or hippocampus: Selective and rapid labeling of electrophoretically separated brain proteins

Intracranial injections of [ 32P]orthophosphate readily label a number of brain phosphoproteins as resolved by polyacrylamide gel electrophoresis. The majority of these in vivo labeled phosphoproteins co-migrate with phosphoproteins that are labeled in vitro by incubation of brain membranes with [ 32P]ATP. Two of the major in vitro labeled phosphoproteins with apparent molecular weights of 47,000 (band F 1) and 41,000 (band F 2) are rapidly labeled in vivo. Since they are rapidly dephosphorylated in vitro, this suggests a high rate of phosphate turnover. The electrophoretic pattern of in vivo labeled phosphoproteins did not appear to be altered by the method of sacrifice (focused microwave irradiation, decapitation or liquid nitrogen immersion) or by the state of the animal at the time of labeling (awake or lightly anesthetized with pentobarbital). The reduction of phosphatase activity during tissue processing at 0 °C may account for the similarities observed with different sacrifice methods. Removal of phospholipids or polynucleotides had little effect on the in vivo labeled 32P-containing bands. However, alkaline hydrolysis or protease treatment uniformly reduced the radioactivity in the labeled bands. These findings suggest that the 32P-containing bands consist of phosphoester linkages to serine or threonine residues. The present evidence emphasizes that previously characterized in vitro labeled brain phosphoproteins are, in fact, labeled in the awake, freely-moving animal.

Activity of wheat seedling nuclease toward single-stranded nucleic acids.

Procedures have been developed for the facile preparation of wheat seedling nuclease in highly purified form. The preparation appears to be homogeneous by many physical criteria; however, analytical gel electrophoresis reveals 12 protein bands, only one of which is catalytically active. It is suggested that most of the inactive species are artifacts formed from active enzyme by processes which increase the negative charge of the protein. The enzyme rapidly catalyzes the hydrolysis of denatured DNA and RNA to acid-soluble products and also of the 3'-phosphomonoester linkage of a variety of 3'-mononucleotides. The enzyme has very little, if any, activity toward native DNA with respect to the production of acid-soluble substances; however, athe succeeding paper demonstrates that native DNA is cleaved at a few specific loci to yield large duplex DNA fragments. The nuclease has been characterized as having endonucleolytic activity towards denatured DNA and primarily exonucleolytic activity towards RNA. The mononucleotides produced under the influence of the enzyme bear 5'-phosphomonoester groups. Various lines of evidence indicate a relatively high preference of the enzyme for the hydrolysis primarily of 3'-phosphoester linkages of adenylic acid units and secondarily of thymidylic or uridylic acid units in DNA and in ribohomopolymers, respectively. Corresponding linkages involving cytidylic acid and especially guanylic acid are relatively resistant. The 3'-nucleotidase activity of the enzyme at pH 5.0 towards the various mononucleotides fits the same pattern; i.e. nucleotides containing adenine are hydrolyzed most rapidly, followed in decreasing order by those containing thymine or uracil, cytosine, and guanine.

The action of gamma-rays on sodium deoxyribonucleate in solution.

The effect of irradiation byγ-rays from a cobalt-60 source, without exclusion of oxygen, on the molecular structure of herring sperm sodium deoxyribonucleate (DNA) has been studied with the following aims: (i) to obtain evidence on the existence of the double-helicalDNAstructure in aqueous solution by testing the application of recent theoretical treatments for the random fracture of single-chain macromolecules (Charlesby 1954); and (ii) to study the effects of dosages ofγ-rays lower than those which have been shown to disrupt the constituent nucleotides. Changes in viscosity, electrometric titration behaviour, ultra-violet absorption, precipitability and other chemical properties have been investigated. The viscosity of solutions ofDNAin 0·1 M-sodium chloride was decreased on irradiation by an amount which was independent of the dose rate and was determined by the ratio (R, in eV/atom ofDNAphosphorus) of dosage in eV entering each ml. of solution to concentration ofDNAphosphorus. The reduced specific viscosity of irradiatedDNAsolutions was a linear function of concentration and obeyed the Huggins equation (Huggins 1942). The intrinsic viscosity [ƞ] decreased with increasingRand, on a logarithmic plot, the relation was linear with a negative slope of 1.9 at the higherRvalues. Comparison of this value with that expected from the theory of random degradation leads to the proposal that a decrease in molecular weight, and thus in [ƞ], of theDNAcan occur only when two independent breaks occur at approximately opposite positions in each of two intertwined polynucleotide chains; this indicates that the double-helical structure adduced for the moist solid state persists in aqueous solution. This is confirmed by the observation that aDNApreparation which had undergone mild acid treatment before irradiation, in order to disrupt the complementary cross-linking hydrogen bonds (Watson &amp; Crick 1953a) and there by the double-helical structure, exhibited a less steep decrease of [ƞ] with increasingR(i.e. a slope of — 1·2 on the logarithmic plot). The earlier theory for the viscosity of a single-chain macromolecule undergoing random breakdown is extended to the case of degradation of a cross-linked molecule consisting of two intertwined chains. The difference between the forward-and backward-titration curves characteristic of the hydrogen-bondedDNAstructure decreased onγ-irradiation, which indicates a progressive disruption of the complementary hydrogen bonds and of the double-helical structure that they hold together; this was confirmed by observed parallel increases in ultra-violet absorption. The asymmetrical character of the displacement suggests, though not quite conclusively, that the hydrogen bonds linking adenine and thymine were ruptured more readily than those joining guanine and cytosine. The combined rupture of all hydrogen bonds had initially a very highGvalue, of the order of sixty base pairs severed per 100 eV, but this decreased with increasing dosage. This efficient rupture of complementary hydrogen bonds would presumably impair the duplication of theDNAhelicesin vivoand may underlie some of the biological effects of low dosages of ionizing radiation. The titration studies demonstrated the release at highRof approximately equal quantities of acidic groups withpK'aof about 4·5 to 5·5 and of 5·5 to 8·5; this observation is discussed briefly in connexion with the detailed chemical mechanism of the rupture of phospho-ester linkages. The indirect nature of the action ofγ-rays under the present conditions was con-confirmed by the determinative character of the ratioRand by the inhibitory effects of certain com pounds known to combine with the free radicals produced in the water.

Recherches sur la d'enaturation des acides desoxyribonucléiques

The ultraviolet absorption of desoxyribonucleic acids (DNA) in neutral solution is much less than expected from the sum of the absorptions of its constituents. The observed spectrum can be made to approach the calculated one by applying mild treatments which are presumably without action on phosphoester linkages (pH, temperature and salt concentration changes). The incidence of these facts on the questions of the chemical structure and biological properties of DNA is discussed. The results are interpreted as reflecting a denaturation of the DNA, i.e. the destruction of a secondary molecular structure constituted by labile bounds involving the puric and pyrimidic rings.