Oligomerization of cytosine-containing nucleotide analogs in aqueous solution

Summary Bisphosphoimidazolides of 2-deoxycytidine and of its acyclic analog can be oligomerized in aqueous solution in the presence of Mn(II). Under certain conditions, a range of products extending to at least the 20mer can be obtained. These products are of interest as possible templates for oligomerization of the complementary monomers.     

Oligomerization of 3′-amino-3′-deoxyguanosine-5′-phosphorimidazolidate on a d(CpCpCpCpC) template

Summary 3-Amino-3-deoxyguanosine-5-phosphorimidazolidate (ImpGnh ₂) oligomerizes more rapidly and regiospecifically than related nucleotide derivatives on a d(CpCpCpCpC) template. The greater nucleophilicity of the amino group leads to efficient oligomerization even when the structure of the double-helical complex formed by the template and the substrate is not optimal for reaction. The use of amine-containing analogues should permit us to develop models of potentially prebiotic polymerization reactions that cannot be studied easily using natural nucleotides.     

Manganese-catalyzed oligomerizations of nucleotide analogs

Summary The synthesis of pyrophosphate-linked oligomers from the bis-phosphoimidazolides of deoxyadenosine and deoxyguanosine, as well as from acyclic analogs of these nucleosides, is catalyzed much more effectively by Mn(II) than by Mg(II). The presence of Mn(II) reduces the extent of cyclization of the monomer-bis-phosphoimidazolide and thereby increases the yields of oligomeric products. The Mn(II)-catalyzed oligomerization is less sensitive to the presence of a complementary polynucleotide template than is the Mg(II)-catalyzed reaction.Nucleic Acid-Like Structures VII. For the previous paper in this series see Visscher et al. (1989)     

Prebiotic nucleotide oligomerization in a fluctuating environment: Effects of kaolinite and cyanamide

Summary The clay kaolinite was tested for its ability to promote nucleotide oligomerization in model prebiotic systems. Heterogeneous mixtures of clay, water and nucleotide were repeatedly evaporated to dryness at 60°C and redissolved in water in cyclic fashion in the presence or absence of cyanamide and/ or ammonium chloride. With or without cycling, kaolinite alone did not promote the oligomerization of nucleotides at detectable levels. Cycling of clay in combination with cyanamide, however, promoted high levels of condensation to a mixture of oligonucleotides and dinucleotide pyrophosphate without requiring ammonium chloride. Although cycling with clay favored synthesis of dinucleotide pyrophosphate, cycling without clay enhanced formation of oligonucleotides. These results support the hypothesis that the presence of clays in fluctuating environments would have influenced the course of prebiotic condensation reactions.     

A possible role of fluctuating clay-water systems in the production of ordered prebiotic oligomers

Summary A model is proposed for the intermediate stages of prebiotic evolution, based on the characteristics of the adsorption and condensation of amino acids and nucleotides on the surface area of clay minerals in a fluctuating environment. Template replication and translation of adsorbed oligonucleotides and catalytic effects by peptide products on further condensation are proposed, due to specific properties of hypohydrous clay surfaces as well as the biomolecules themselves. Experimental evidence supports some of the proposed interactions, and all of them can be tested experimentally.on leave from the Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel, 1975–76     

Modulation of Cytarabine Induced Cytotoxicity Using Novel Deoxynucleoside Analogs in the HL60 Cell Line

In order to enhance the cytotoxicity of ara‐C in the HL60 cell line the following deoxynucleoside analogs were used: cladribine, fludarabine and gemcitabine. HL60 cells were co‐incubated with ara‐C and each of the modulators at the ratios of their respective IC50s. Cytotoxicity was determined with the MTT‐assay and drug interactions were evaluated with the combination index (CI) method (Calcusyn; Chou & Talalay). CI < 1, CI ± 1 and > 1 indicate synergism, additive effect and antagonism, respectively. We observed moderate synergism between ara‐C/cladribine and ara‐C/gemcitabine, with CIs of 0.76 ± 0.14 and 0.82 ± 0.04, respectively. The interaction between ara‐C/fludarabine resulted in moderate antagonism (CI = 1.29 ± 0.11). In conclusion, in this in vitro study we showed that the cytotoxicity of ara‐C can be succesfully modulated in the HL60 cell line by cladribine and gemcitabine.     

Hydrogen bonding in the template-directed oligomerization of a pyrimidine nucleotide analogue

We have studied the oligomerization of an activated, achiral nucleotide analogue related to the pyrimidine barbituric acid in the absence and in the presence of a complementary, pyrophosphate-linked oligomer. Although no template-directed catalysis of the oligomerization was observed with water as solvent, catalysis of the oligomerization was demonstrated in a mixture of dimethylformamide with water. Poly(U) also stimulated the oligomerization, but was less effective than the analogue. Environments in which similar effects may be observed, and some potential implications for prebiotic chemistry, are discussed. Correspondence to: A.W. Schwartz.     

Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

An achiral (oligo)nucleotide analog

An achiral nucleotide analog based on barbituric acid has been synthesized. The analog, which is 5,5-di(2-phosphoethyl)barbituric acid, undergoes extensive oligomerization in aqueous solution, when activated, to produce pyrophosphate-linked chains. In contrast to a number of other bisphosphorylated nucleoside analogs which have been studied, the compound has little tendency to cyclize. The possible prebiotic implications are discussed. Correspondence to: AM. Schwartz     

The structure of gellan in dilute aqueous solution

A full assignment of high-field nmr spectra of gellan was obtained in dilute aqueous solution by performing a series of selective one-dimensional nmr experiments. The observed nuclear Overhauser effects (NOEs) cannot be interpreted assuming that each sugar residue is intrinsically rigid and in a chair conformation. In fact, the rhamnose residue gives strong NOE contacts coherent only with an equilibrium involving both a chair as well as a boat (or a hemiboat) conformation. Molecular dynamic calculations performed on a heptamer with a central rhamnose support the above finding, and show a structure based on a very stiff single chain in which it is present a flipping of the rhamnose residue. At low temperatures (5-20 degrees C) in very dilute solutions (0.018 mg/mL) nmr spectra show a splitting of the resonance due to the methyl group of rhamnose residue, thus confirming the presence of a slow equilibrium among different conformers.     

Conformational properties of xanthan derivatives in dilute aqueous solution

The Ca²⁺-induced conformational changes of the extracellular microbial polysaccharide xanthan, and of some derivatives, have been investigated by circular dichroism and isothermal microcalorimetric methods. The four polymers studied are native xanthan (NX), acetyl-free xanthan (AFX), pyruvic-free xanthan (PFX) and acetyl-and-pyruvic-free xanthan (APFX), all of about the same molecular weight. Convergent evidence from both techniques indicates that the acetyl group stabilizes the ordered conformation of xanthan, which can be induced by specifically increasing the ionic strength of the dilute aqueous solution by the addition of calcium ions. Pyruvate groups have been shown to have a strong destabilizing effect on the ordered conformation, which is likely to be ascribed to an unfavourable electrostatic contribution. The relative order of stability of the ordered forms was found to be PFX > NX > APFX > AFX; PFX is largely present in the ordered conformation at 25°C even in the calcium-free aqueous solution.     

Lipophilic Prodrugs and Formulations of Conventional (Deoxy)Nucleoside and Fluoropyrimidine Analogs in Cancer

Many drugs that are currently used for the treatment of cancer have limitations, such as induction of resistance and/or poor biological half-life, which reduce their clinical efficacy. To overcome these limitations, several strategies have been explored. Chemical modification by the attachment of lipophilic moieties to (deoxy)nucleoside analogs should enhance the plasma half-life, change the biodistribution, and improve cellular uptake of the drug. Attachment of a lipophilic moiety to a phosphorylated (deoxy)nucleoside analog will improve the activity of the drugs by circumventing the rate-limiting activation step of (deoxy)nucleoside analogs. Encapsulating drugs in nanoparticles or liposomes protects the drug against enzymatic breakdown in the plasma and makes it possible to get lipophilic compounds to the tumor site. In this review, we discuss the considerable progress that has been made in increasing the efficacy of classic (deoxy)nucleoside and fluoropyrimidine compounds by chemical modifications and alternative delivery systems.     

Activation of carbonic anhydrase II by active-site incorporation of histidine analogs

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.     

Freezing effect on chirality generation of DL-alanine-N-carboxy-anhydride oligomerization in aqueous solution

This article is concerned with a study of the role of ice in the synthesis of oligopeptides containing L- or D-enantiomeric excess (ee) from racemic alanine. With this aim, the oligomerization of DL-alanine-N-carboxyanhydride was investigated by keeping this activated derivative in liquid (+22 degrees C) or frozen (-20 degrees C) aqueous solutions for 30 days. The aqueous solution of the peptide mixtures were gel-filtered and the aliquots of the fractions were completely hydrolyzed to alanine monomers. These monomers were then derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) and analyzed by RP-HPLC to reveal the occasional enantiomeric excess of L- or D-Ala. The mass spectrometry of the gel-filtered fractions pointed to open-chain peptide mixtures together with a slight amount of cyclic ones, where the residue numbers ranged between 5-8. Our studies indicated that an enantiomeric excess of L- or D-Ala appeared in some oligopeptide fractions. Their excesses were significantly larger in the frozen than liquid solution. Speculations are made as concerns the implications of our findings in the events of prebiotic chemistry.     

Radiation-induced inactivation of dihydroorotate dehydrogenase in dilute aqueous solution.

The inactivation of dihydroorotate dehydrogenase by gamma irradiation in dilute aqueous solution has been investigated. The activity of the enzyme decreased exponentially as a function of the absorbed dose under aerated and nitrous oxide-saturated conditions. The contributions of the individual radical species derived from water radiolysis were estimated from the inactivation results observed under aerated, argon-saturated, and nitrous oxide-saturated conditions. The hydrogen atom and hydroxyl radical were found to be important in enzyme inactivation. The effect of selected inorganic radical anions such as Br.2-, I.2-, and (SCN).2- on the enzyme activity was also studied, and the results implicate the possible involvement of cysteine and tyrosine residues in the catalytic activity of dihydroorotate dehydrogenase. Changes in the kinetic parameters (Michaelis-Menten constant, Km, and maximal velocity, Vmax) due to irradiation under the conditions investigated suggest that radiation-induced inactivation is due to modification of the substrate binding sites and that of the active site residues in the enzyme. Evidence for the reduction of iron-sulfur centers in the enzyme during the inactivation process has been put forward from the difference spectrum of the irradiated dihydroorotate dehydrogenase. It has also been shown by electrophoretic studies that radiation-induced inactivation was not due to any fragmentation of the protein structure or the formation of any intermolecular crosslinking.     

An investigation of pectin and pectic acid in dilute aqueous solution

Light-scattering measurements have been carried out on carefully clarified aqueous solutions of pectin and pectic acid. The strong dependence of the inverse excess scattering intensity on angle yields pectin chain dimensions that are inconsistent with the complete molecular dispersion of pectin molecules possessing the degrees of polymerization indicated by the measured osmotic molecular weights. Negligible or negative concertration dependences of the inverse scattering intensities coupled with the centrifugation, filtration, and gel permeation chromatography behavior of the samples likewise suggest nonequilibrium aggregation of the pectin molecules in solution. It is shown that the measured mean polymer chain dimensions can be rationalized in terms of the presence of a very small proportion of aggregated material.     

Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution

We have prepared a [³²P]-labeled oligonucleotide probe carrying a free primary amine at its 3′terminus. This probe is used to initiate polymerization of aziridine (ethyleneimine) in aqueous solution. The nature of the oligomeric products and the kinetics of their formation are then monitored by gel electrophoresis. Our results are generally consistent with those obtained using conventional techniques. We have also investigated the effect of polyanionic templates on the rate of oligomerization of aziridine. We find that water-soluble polyanions generally accelerate the polymerization. The sodium salt of polymethacrylic acid is the most effective of the templates that we studied. The methods introduced in this paper should be applicable to a variety of polymerization reactions in aqueous solution. They should greatly simplify the screening of potentially prebiotic polymerization reactions. Correspondence to: L.E. Orgel on sabbatical leave     

Separation of l-lysine from dilute aqueous solution using molecular imprinting technique

In the present study, separation of l-lysine from dilute aqueous solution by solid-phase extraction based on molecular imprinting technique using a polar porogen was investigated. l-Lysine imprinted polymer (LLIP) was prepared by free radical solution polymerization of methacrylic acid and ethylene glycol dimethacrylate as functional and cross-linking monomers, in the presence of l-lysine as an imprint molecule, mixture of water and methanol as solvent and AIBN as an initiator. Non-imprinted polymer (NIP) as control was also prepared by the same procedure in the absence of template molecules. LLIP particles were applied to determine the optimum operational condition for l-lysine separation from dilute aqueous solution. In adsorption step, optimum pH and retention time were 7.8 and 90 min, while corresponding values in extraction step were 12 and 50 min, respectively. l and d-Lysine recovery by LLIP at optimum condition were found to be 96 and 58% with corresponding distribution coefficients of 8000 and 460, respectively. The retention capacity of LLIP was 27.26 mg l-lys/g of polymer at optimum condition.     

Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy

We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.