Cyclic oligomer design with de novo αβ‐proteins

We have previously shown that monomeric globular αβ‐proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo‐oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo‐oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo‐oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with β‐strand backbone interactions were structurally confirmed through x‐ray crystallography and small‐angle X‐ray scattering (SAXS). In contrast, C3‐C5 designed homo‐oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo‐oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.     

Peptide oligomers from ultra-short peptides using sortase

Sortase A catalyzed ligation of ultra-short peptides leads to inter/intra-molecular transpeptidation to form either linear or cyclic oligomers dependent upon the peptide length. Cyclic peptides were the main products for peptides with more than 15aa. However, for ultra-short (<15aa) peptides, cyclic oligomers became predominant in prolonged reactions. Peptides with 1–3 aminoglycines were equally active but peptide oligomers from peptide containing more than one aminoglycine were prone to hydrolysis.     

Linear and cyclic ester Oligomers of succinic acid and 1,4-butanediol: Biocatalytic synthesis and characterization

Abstract

The lipase-catalyzed synthesis of cyclic ester oligomers from non-activated succinic acid (A) and 1,4-butanediol (B) in the presence of immobilized Candida antarctica lipase B was investigated. Batch and pulse fed-batch systems were implemented to increase the formation of cyclic ester products. The substrate conversions after 24 h were 86% and 95% under batch and fed-batch operation, respectively and the product of the reaction was, for both systems, a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers. Fed-batch operation afforded a product containing 71% cyclic ester oligomers (CEOs) as compared with only 52% CEOs in batch operation. Cyclic ester oligomers accumulated as the reaction progressed, with the dimer CEO₁ the most predominant product (i.e. 50% of the total products formed in fed-batch operation). The pulse fed-batch operation system was superior to the batch operation not only because higher substrate conversion and more CEOs were obtained, but also because it resulted in products with a higher degree of polymerization (DP up to 7). Cyclic ester oligomers are produced from the early stage of the reaction simultaneously with the linear ester oligomers by a ring-closure reaction on the active site of the enzyme, and not as a result of ring-chain equilibria.     

One pot solution synthesis of cyclic oligodeoxyribonucleotides.

Several cyclic oligodeoxynucleotides with different base composition and size have been prepared from 5',3'-unprotected linear precursors, using a bifunctional phosphorylating reagent. The final deprotected oligomers have been characterized by 1H- and 31P-NMR. The present procedure is particularly useful for millimolar scale syntheses.     

Induction of cell surface insulin antigenicity in fibroblasts transfected with islet genomic DNA

Intact platelets were stimulated with thrombin and the amount of GTP-binding protein (G-protein) oligomers was assessed by measuring ADP ribosylation of 40-41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57-62% in 10-60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP-ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G-protein oligomers to release free alpha-subunits, and this effect can be modulated by protein kinase C and cyclic AMP-dependent protein kinase. The possible relationships of these findings to the regulation of stimulus-response coupling in platelets is discussed.     

Lipase-catalyzed transformation of poly(butylene adipate) and poly(butylene succinate) into repolymerizable cyclic oligomers

The enzymatic degradation and repolymerization were carried out with the objectives of developing the chemical recycling of aliphatic polyester-type plastics, such as the poly(butylene adipate) (PBA), poly(butylene succinate), and poly(butylene adipate-co-succinate) copolymers which are typical biodegradable plastics. They were degraded by lipase in an organic solvent solution containing a small amount of water to produce cyclic oligomers mainly consisting of the cyclic diester. The produced cyclic oligomer was readily repolymerized by lipase to produce a polyester having an equal or higher molecular weight compared to the parent polymer. As an example, PBA having an Mw of 22,000 was almost quantitatively transformed by lipase CA (Novozym 435) in water-containing toluene at 50 degrees C into the corresponding cyclic oligomers mainly consisting of dimers. Thus, the obtained oligomers were readily polymerized by lipase CA to produce the PBA with an Mw of 52,000.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.     

Studies on transfer ribonucleic acids and related compounds. XXVI. Circular dichroic properties of cyclic oligoribonucleotides and their linear counterparts

The CD spectra of cUpUp, cCpCp, and cGpGp derived from DCC-catalyzed polymerization of the relevant protected ribonucleoside 3′-phosphates are described. Similar studies on Up, U > p, and cUp, as well as cUpUpUp and cUpUpUpUp, are presented. The spectral properties of the cyclic oligomers are compared with those of the corresponding linear oligomers with terminal 3′-phosphates so as to demonstrate that disruption of normal right-handed base stacking is considerable in these RNA loops.     

Significance of Mineral Salts in Prebiotic RNA Synthesis Catalyzed by Montmorillonite

The montmorillonite-catalyzed reactions of the 5′-phosphorimidazolide of adenosine used as a model generated RNA type oligomers. These reactions were found to be dependent on the presence of mineral salts. Whereas montmorillonite (pH 7) produced only dimers and traces of trimer in water, addition of sodium chloride (0.1–2.0 M) enhanced the chain length of oligomers to 10-mers as detected by HPLC. Maximum catalytic activity was observed with sodium chloride at a concentration between 0.8 and 1.2 M. This concentration of sodium chloride resembled its abundance in the ancient oceans (0.9–1.2 M). Magnesium chloride produced a similar effect but its joint action with sodium chloride did not produce any difference in the oligomer chain length. Therefore, Mg²⁺ was not deemed necessary for generating longer oligomers. The effect of monovalent cations upon RNA chain length was: Li⁺ > Na⁺ > K⁺. A similar effect was observed with the anions with enhanced oligomer length in the following order: Cl^? > Br^? > I^?. Thus, the smaller ions facilitated the formation of the longest oligomers. Inorganic salts that tend to salt out organic compounds from water and salts which show salt-in effects had no influence on the oligomerization process indicating that the montmorillonite-catalyzed RNA synthesis is not affected by either of these hydrophobic or hydrophilic interactions. A 2.3-fold decrease in the yield of cyclic dimer was observed upon increasing the sodium chloride concentration from 0.2 to 2.0 M. Inhibition of cyclic dimer formation is vital for increasing the yield of linear dimers and longer oligomers. In summary, sodium chloride is likely to have played an essential role in any clay mineral-catalyzed prebiotic RNA synthesis.     

Biological treatment of the components of solid oligomeric waste from a nylon-6 production plant

On partial analysis of the solid oligomeric waste of a nylon-6 production plant, it was found to contain ε-caprolactam, 6-aminocaproic acid (6-ACA) and its linear and cyclic oligomers. Out of four bacterial isolates capable of utilizing caprolactam as the sole growth substrate, Alcaligenes faecalis was found to be the most potent and utilized 90% of caprolactam in 24 h. In shake flask experiments, when the solid waste after solubilization was treated with a consortium of bacteria of four different genera, except the cyclic oligomers, all the other constituents were found to be degraded. A reduction of the chemical oxygen demand (COD) of the solid waste to the level of 63–66% was obtained when it was treated with either a consortium of the bacterial isolates or only a single isolate, A. faecalis. Alcaligenes faecalis could bring about a decrease of 95% in the caprolactam content of the solid waste, while 6-ACA and its linear oligomers were almost completely degraded. Alcaligenes faecalis cells adapted on solid waste could degrade the linear oligomers at a faster rate as compared to cells adapted on caprolactam. However, cyclic oligomers could not be degraded in either case. When solid waste, partially hydrolysed with acid to yield 6-ACA as the major constituent, was treated with the consortium of bacterial isolates, a 95% reduction in the COD was achieved. This revised version was published online in November 2006 with corrections to the Cover Date.     

Homochiral Selectivity in RNA Synthesis: Montmorillonite-catalyzed Quaternary Reactions of D,L-Purine with D,L- Pyrimidine Nucleotides

Selective adsorption of D, L-ImpA with D, L-ImpU on the platelets of montmorillonite demonstrates an important reaction pathway for the origin of homochirality in RNA synthesis. Our earlier studies have shown that the individual reactions of D, L-ImpA or D, L-ImpU on montmorillonite catalyst produced oligomers which were only partially inhibited by the incorporation of both D- and L-enantiomers. Homochirality in these reactions was largely due to the formation of cyclic dimers that cannot elongate. We investigated the quaternary reactions of D, L-ImpA with D, L-ImpU on montmorillonite. The chain length of these oligomers increased from 9-mer to 11-mer as observed by HPLC, with a concominant increase in the yield of linear dimers and higher oligomers in the reactions involving D, L-ImpA with D, L-ImpU as compared to the similar reactions carried out with D-enantiomers only. The formation of cyclic dimers of U was completely inhibited in the quaternary reactions. The yield of cyclic dimers of A was reduced from 60% to 10% within the dimer fraction. 12 linear dimers and 3 cyclic dimers were isolated and characterized from the quaternary reaction. The homochirality and regioselectivity of dimers were 64.1% and 71.7%, respectively. Their sequence selectivity was shown by the formation of purine-pyrimidine (54–59%) linkages, followed by purine-purine (29–32%) linkages and pyrimidine-pyrimidine (9–13%) linkages. Of the 16 trimers detected, 10 were homochiral with an overall homochirality of 73–76%. In view of the greater homochirality, sequence- and regio- selectivity, the quaternary reactions on montmorillonite demonstrate an unexpectedly favorable route for the prebiotic synthesis of homochiral RNA compared with the separate reactions of enantiomeric activated mononucleotides.     

Selectivity of montmorillonite catalyzed prebiotic reactions of D,L-nucleotides

The montmorillonite-catalyzed reactions of the 5′-phosphorimidazolides of D, L-adenosine (D, L-ImpA) (Figure 1a. N = A, R = H) and D, L-uridine (Figure 1a., N = U, R = H) yields oligomers that were as long as 7 mers and 6 mers, respectively. The reactions of dilute solutions of D-ImpA and D-ImpU under the same conditions gave oligomers as long as 9 and 8 mers respectively. This demonstrated that oligomer formation is only partially inhibited by incorporation of both the D- and L-enantiomers. The structures of the dimers, trimers and tetramer fractions formed from D, L-ImpA was investigated by selective enzymatic hydrolysis, comparison with authentic samples and mass spectrometry. Homochiral products were present in greater amounts than would be expected if theoretical amounts of each were formed. The ratio of the proportion of homochiral products to that of the amount of each expected for the dimers (cyclic and linear), trimers and tetramers, was 1.3, 1.6, and 2.1, respectively. In the D, L-ImpU reaction homochiral products did not predominate with ratios of dimers (cyclic and linear), trimers and tetramers 0.8, 0.44, and 1.4, respectively. The proportions of cyclic dimers in the dimer fraction were 52–66% with D, L-ImpA and 44–69% with D, L-ImpU. No cyclic dimers were formed in the absence of montmorillonite. The differences in the reaction products of D, L-ImpA and D, L-ImpU are likely to be due to the difference in the orientations of the activated monomers when bound to the catalytic sites on montmorillonite. The consequences of the selectivity of montmorillonite as a prebiotic catalyst are discussed.     

Constrained flexibility in PNA: DNA binding studies with bridged aminopropylglycyl PNA

Introduction of methylene bridges in aegPNA and apgPNA molecules give rise to cyclic five and six membered ring structures. Synthesis of a new six membered cyclic PNA monomer, aminopipecolyl PNA (pipPNA) is reported. Incorporation of pipPNA into PNA oligomers and comparative binding with target DNA sequences is studied.     

The Cyclic Dimer of 5-Fluoro-2′-Deoxyuridylic Acid: A Potent Anticancer Agent

Abstract

As part of a synthetic program on cyclic oligomers of DNA, the cyclic dimer of 5-fluoro-2′-deoxyuridylic acid (FdUMP) was synthesized. The fully protected dimer 5 was obtained following Catlin and Cramer′s phosphotri-ester strategy. Autocondensation and deprotection then afforded the title compound 9 [cyclo(5FdUp5FdUp)] in excellent yield. In vitro, 9 proved slightly less active than FdUrd in inhibiting the proliferation of various murine and human tumor cells, but, in vivo, 9 was equally effective, and less toxic than 5-FdUrd in inhibiting adenocarcinoma tumor growth in mice.     

Uranyl ion-catalyzed synthesis of several 8-substituted 2'-5'-oligoadenylates, and their properties

We have synthesized 2'-5' linked oligomers from 8-substituted adenosine-5'-phosphorimidazolides using uranyl ion catalyst. 8-amino derivative, as highly susceptible to hydrolysis, gave short chained oligomers in a low yield, while the rest of 8-substituted or unsubstituted derivatives gave the corresponding oligomers in high yields. Properties of 8-substituted 2'-5' oligomers were studied applying spectrometer and through enzymatic digestion.     

Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement

The structure of the human properdin molecule was investigated by hydrodynamic, spectroscopic, and transmission electron microscope studies. Sucrose density gradient ultracentrifugation of purified, functionally active properdin showed a single component sedimenting at 5.5 S. Electron microscopic examination by two different methods, however, revealed polydispersity of the protein which consisted of cyclic dimers, trimers, tetramers, pentamers, and higher cyclic oligomers. Approximately 80% of the oligomers were dimers, trimers, and tetramers. Monomers could not be detected. These polymers could be partially separated by gel filtration on Sephacryl S-300 and all fractions were active in terms of binding to C3b. The specific activity increased with oligomer size. When reexamined after incubation at 37 degrees C for 4 h or at 4 degrees C for 2 weeks, the chromatographic behavior of the oligomers and their electron microscopic appearance were unchanged, suggesting that in vitro no rapid equilibration occurred. The protomer is clearly visualized within polymers as a flexible, rod-like structure 26.0 nm in length and 2.5 nm in diameter, with pronounced thickening at each end. The monomer is bivalent with respect to binding to other properdin monomers and the binding sites are localized to the ends of the structure. A model could be devised which is consistent with the distinct geometry of the intersubunit contacts observed in micrographs. The circular dichroism spectrum of properdin suggests the presence of little alpha helix or beta structure and shows positive ellipticity at 231 nm. In contrast to previous investigators, we conclude that isolated human properdin is polydisperse and consists of a set of cyclic polymers constructed from a single highly asymmetric and flexible protomer.     

Utilization of a Cyclic Dimer and Linear Oligomers of ε-Aminocaproic Acid by Achrornobacter guttatus KI 72

A microorganism, strain KI 72 capable of utilizing ε-aminocaproic acid cyclic dimer as sole carbon and nitrogen sources was isolated from sludge and identified as Achromobacter guttatus. This bacteria utilized 1% of the cyclic dimer in a day and was not inhibited by the higher concentration of the dimer. The growth rate was independent of the cyclic dimer concentration in the medium, but the maximum cell concentration increased with the increase of substrate concentration. The cell yield was 0.7 mg dry cell/mg ε-aminocaproic acid cyclic dimer. Bacterial growth with the cyclic dimer as substrate was significantly stimulated by the addition of yeast extract. Ferric chloride was also stimulatory. Maximal growth was obtained in cultures incubated at pH 6 and at 33°C. Synthesized nylon oligomers, ranging from ε-aminocaproic acid up to its linear hexamer, were found to be catabolized by this organism.     

Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions.

Microtubules can be induced to perform synchronous and periodic cycles of assembly and disassembly at constant temperature. The process depends on GTP hydrolysis. Time-resolved X-ray scattering using synchrotron radiation shows a cyclic interconversion of tubulin subunits, microtubules and oligomers (= short protofilament fragments). Oscillations are correlated with conditions that stabilize polymers and destabilize oligomers, and others of opposite effect. Microtubule stabilizers include GTP, Mg2+ or microtubule-associated proteins (MAPs), destabilizers include GDP or elevated ionic strength. K+ at intracellular concentrations noticeably increases the stability of tubulin-MAP oligomers, in contrast to Na+. ATP and the non-hydrolyzable analogue AMP-PNP enhance oscillations by mechanisms that are not directly linked to the role of nucleotide hydrolysis in assembly. We propose a mechanism of oscillations that include oligomers as microtubule disassembly products which transiently lock the protein in an unpolymerizable state; this may point to a role of oligomers in controlling microtubule assembly cycles in cells.     

Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.

The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.     

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support.

An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamine in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers were synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp (Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperdine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5' OH groups. These reactions may be used to characterize the oligomers.