Chemical Morphing of DNA Containing Four Noncanonical Bases

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5‐chloro‐2′‐deoxyuridine, 7‐deaza‐2′‐deoxyadenosine, 5‐fluoro‐2′‐deoxycytidine, and 7‐deaza‐2′deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo‐) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.     

Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs

Expansion of the genetic alphabet with an unnatural base pair is a long‐standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d 5SICS and analogues of d MMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new d MMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d 5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, d NaM –d 5SICS , to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure–activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (d NMO1 ) that is a better partner for d 5SICS than any of the previously identified d MMO2 analogues with the exception of d NaM . We also found that d NaM –d 5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.     

Enhanced reactivity in radical cyclizations of hydrazones using the silicon-tethered 1-bromovinyl group

A silicon-tethered 1-bromovinyl group was shown to function as a radical precursor for tin-mediated vinyl additions to chiral α- or β-hydroxyhydrazone. In contrast to related thiyl-mediated methods, these vinyl bromides were not limited to the 5-exo cyclization mode. A series of Si-tethered 5-exo and 6-exo cyclizations formed the corresponding five- and six-membered exo-methylene-substituted oxasilacycles. Treatment with fluoride cleaved the Si-C and Si-O bonds to afford the corresponding allylic hydrazines. Diastereoselectivities ranged from 2:1 to 25:1 (anti:syn) for the 5-exo cyclizations, depending on the size of the exocyclic substituent, but 6-exo cyclization was not diastereoselective. A variant involving Tamao oxidation of the exo-methylene oxasilacyclopentane intermediate afforded a methyl ketone, a net process corresponding to addition of a radical acyl anion equivalent.     

Advantages of Thermococcus kodakaraenis (KOD) DNA Polymerase for PCR-mass spectrometry based analyses

The advantages of the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD) are demonstrated for PCR amplification with subsequent detection by mass spectrometry. Commonly used DNA polymerases for PCR amplification include those from Thermus aquaticus (Taq) and Pyrococcus furiosus (Pfu). A 116 base-pair PCR product derived from a vWA locus was amplified by Taq, Pfu, or KOD DNA polymerase and compared by agarose gel electrophoresis and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). KOD DNA polymerase demonstrated a 2- to 3-fold increase in PCR product formation compared to Pfu or Taq, respectively, and generated blunt-ended PCR product which allows facile interpretation of the mass spectrum. Additionally, we demonstrate the advantage of using high magnetic fields to obtain unit resolution of the same 116 base pair (approximately 72 kDa) PCR product at high m/z.     

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.     

Experimental and DFT study of the aza-Diels-Alder reaction between cyclopentadiene and protonated benzylimine derivated from glyoxylates

The Diels-Alder reaction of protonated N-benzyl imine of methyl glyoxylate with cyclopentadiene in different solvents gave mixtures of exo/endo adducts. The exo/endo selectivity of the reaction was elucidated by NMR experiments. Theoretical calculations by means of density functional theory (DFT) at the B3LYP/6-31G(d) level have also been performed to elucidate the molecular mechanism of this reaction. The DFT results suggest a highly asynchronous concerted mechanism, which in turn can explain the preferred exo stereoselectivity of the reaction. Inclusion of solvent effects enhances the exo selectivity, and this effect increases with the polarity of the solvent, in good agreement with the experimental findings.     

Oxanorbornenes: promising new single addition monomers for the metathesis polymerization

Higher ring-opening metathesis propagation rates of exo-norbornene derivatives over endo derivatives are well established in the literature. Here, we report for the first time that endo-isomers of oxanorbornene derivatives show higher reactivity towards ring-opening metathesis with Grubbs'' 3rd generation catalyst (G3) than the corresponding exo-isomers. A very high selectivity for the reaction of G3 with endo over the exo-isomers could be shown. Furthermore, single molecular addition of the endo-isomers with G3 was observed. On the other hand, pure exo-monomers could successfully be homopolymerized. Mixtures of exo- and endo- monomers, however, prevented the homopolymerization of the exo-monomer. Such mixtures could successfully be copolymerized with cycloalkenes, resulting in alternating copolymers. An oxanorbornadiene derivative could be shown to undergo single addition reactions, exploited in the preparation of mono-end functional ROMP polymers. These could be selectively derivatized via endgroup selective thiol-ene click reactions. A thiol and alcohol end functional ROMP polymer was synthesized, and the efficient end functionalization was confirmed by ¹H NMR spectroscopy and MALDI-ToF spectrometry.

Bridgehead revisited: endo-7-oxa norborneneimide derivatives (green) initiate faster but propagate more slowly than the analogous exo-derivatives (red) in ring-opening metathesis allowing the synthesis of alternating and end functional polymers.     

Conversion of arenes to cyclohexa-1,3-dienes via (exo-5-dialkylphosphonoexo-6-R-η4-cyclohexadiene) manganese dicarbonylnitrosyl compounds

(exo-5-Dialkylphosphono-exo-6-R-η⁴-cyclohexadiene)Mn(CO)₂NO compounds were prepared by the reaction of (exo-6-R-η⁵-cyclohexadienyl)Mn(CO)₂ NO⁺ with an excess of P(OMe)₃. When (exo-5-dialkylphosphono-exo-6-η⁶-R-cyclohexadiene) Mn(CO)₂NO compounds are refluxed with Me₃NO in benzene, two kinds of cyclohexadiene compounds are formed depending upon the R group.     

Addition copolymerization of norbornene lactone catalyzed by Pd complexes

The addition copolymerization of norbornene (NB) with functionalized monomers can lead to the modification of physical properties of poly(NB). Herein, the synthesis of new copolymer of NB with exo-norbornene lactone (exo-NBL) is reported. The copolymerization proceeded by four Pd catalytic systems, and of these, Pd(allyl)IDippCl/AgSbF₆ (IDipp = 1,3-bis[2,6-diisopropylphenyl]imidazolin-2-ylidene)) was the most effective for the incorporation of exo-NBL. Specifically, the copolymerization with exo-NBL/NB feed ratio of 50/50 at r.t. by 0.1 mol% of the Pd catalyst produced poly(NB-co-exo-NBL) with M_n of 87,000, and M_w/M_n of 1.2 in 40% yield, incorporating exo-NBL of 18 mol%. The time–conversion plots and ¹H diffusion ordered spectroscopy (DOSY) NMR analysis of the copolymer suggest that it has a random sequence. In contrast, no copolymer was formed from endo-NBL. This is because of steric hindrance of the endo-lactone moiety by considering the (co)polymerization of endo-5-norbornene-2-carboxylic acid methyl ester (endo-NBCO₂Me). The incorporation of exo-NBL improves the solubility of poly(NB-co-exo-NBL) in several chlorinated solvents and gives high thermal stability with 10% weight loss at a temperature of more than 400°C. Two amorphous halos corresponding to intra- and interchain distances were observed in the WAXD patterns, allowing to calculate d-spacing values, which are higher than that of poly(NB).     

Azabrendanes IV. Synthesis and characterization of N-(alkyl- and benzylsulfonyl)-exo-2-hydroxy-4-azatricyclo[4.2.1.0]nonanes

exo- and endo-5-Aminomethylbicyclo[2.2.1]hept-2-enes have been obtained from stereoisomeric exo- and endo-5-cyanobicyclo[2.2.1]hept-2-enes and the corresponding sulfonamides were obtained through reaction of amines with methyl-, n-propyl-, n-butyl-, benzyl-, and cyclohexylsulfonyl chlorides. From the stereoisomeric sulfonamides with peroxy acids, various products were obtained: exo-sulfonamides were transformed into epoxy derivatives, and, in contrast, most of the endo-stereoisomers underwent heterocyclization resulting in substituted exo-2-hydroxy-4-azatricyclo[4.2.1.0^3,7]nonanes. The type of the products obtained did not depend on the type of peroxy acid used (peroxyacetic, peroxyphthalic, and m-chloroperoxybenzoic one). In contrast to other endo-sulfonamides, N-(cyclohexylsulfonyl)-endo-5-aminomethylbicyclo[2.2.1]hept-2-ene in reaction with peroxyacetic acid did not undergo heterocyclization, probably, due to steric factors. The structure and stereochemical homogeneity of the sulfonamides and the structure of the products of their oxidation with peroxy acids were confirmed by spectroscopic methods. The molecular structure of N-(cyclohexylsulfonyl)-endo-5-aminomethyl-exo-2,3-epoxybicyclo[2.2.1]heptane was determined by X-ray diffraction analysis. The mechanism of the intramolecular heterocyclization reaction of N-substituted endo-5-aminomethyl-exo-2,3-epoxybicyclo[2.2.1]heptanes was studied at the BHandHLYP/6-31G(d) level of theory.     

Synthesis of Polyanionic C5-Modified 2′-Deoxyuridine and 2′-Deoxycytidine-5′-Triphosphates and Their Properties as Substrates for DNA Polymerases

Modified 2′-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG₆ to PEG₂₄ could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.     

Rhodium(I) catalysis of cycloprop[a]acenaphthylene isomerizations

Cycloprop [a] acenaphthylene was found to rearrange to phenalene in the presence of rhodium dicarbonyl chloride dimer. Deuterium labeling of this molecule at C(7) (both exo and endo), at C(8), at C(7) (exo) and C(8) and at C(7) (exo), C(8), and C(8') indicated that the C(7) (exo) deuterium was migrating stereospecifically. Furthermore, all of the isotopic label present in the cycloprop[a]acenaphthylene was found statistically distributed over positions 1, 3, 4, 6, 7, and 9 of the phenalene product. Control experiments established the need of the catalyst, the inability to achieve rearrangement of the exo-7-methyl derivative, and the susceptibility of a monodeuterated phenalene for extensive isotopic scrambling in its own right when exposed to rhodium(I). These results have been interpreted on the basis of oxidative addition by rhodium(I) into the central bond of cycloprop[a]acenaphthylene from above the “flap”, followed by shifting of the C(7) (exo) hydrogen (or deuterium) to give a η³-allylrhodim-(III) complex. The experimental data further support a mechanism involving subsequent rearrangement of this intermediate around the periphery of the phenalene ring. This process which is otherwise degenerate is thought to be facilitated by the special electronic features of the phenalenyl system.     

A novel approach to 3-acylated indolizine structures via iodine-mediated hydrative cyclization

We have discovered a new route to 3-acylated indolizine structures via iodine-mediated hydrative cyclization. Reaction mechanism is proposed for this novel transformation, which involves a 5-exo-dig iodocyclization, deprotonation, incorporation of another iodo group, deprotonation, and subsequent replacement of the diiodo group by H₂O. Various 3-acylated indolizine derivatives were obtained using this mild procedure in good yields.     

Stereoselective synthesis of substituted exo-glycals from 1-exo-methylene pyranoses

Halo-exo-glycals of the gluco-, manno- and galacto- series, readily prepared by reaction of 1-exo-methylene pyranoses with iodonium dicollidinium triflate (IDCT), undergo Suzuki or B-alkyl Suzuki cross-coupling reactions with boronic acids or alkyl boranes to yield, in a stereoselective manner, functionalized exo-glycals.     

Stereochemistry of the tin—carbon bond: II. Brominolysis of a series of exo- and endo-2-triorganostannylnorbornanes

The brominolysis of a series of exo- and endo-2-triorganostannylnorbornanes with methyl, isopropyl, neopentyl and cyclohexyl as substituents on tin, proceeds with inversion of configuration for the endo-carbon—tin bond and with retention of configuration for the exo-carbon—tin bond. The norbornyl system seems to favour the exo-approach of the electrophile.     

Additions to bicyclic olefins—X: Stereochemistry of the oxymercuration-demercuration of cis-bicyclo[3.3.0]oct-2-ene,endo-trimethylene-norborn-8-ene and related olefins

The stereochemistry of the oxymercuration-demercurarion (OM-DM) of olefins related to the cis-bicyclo(3.3.0]octane and endo-2,3-trimethylenenorboniane structures was determined. In the case of cis-bicyclo(3.3.0]oct-2-ene, hydration occurs preferentially at the less hindered 3-position, with little preference shown for exo vs endo. The more hindered 2-product shows a 11:1 preference for the exo-product. The presence of Me groups at positions 2- or 3-, results in the formation of the tertiary alcohols with approximately a 4:1 favoring of the exo-isomer. (The oxymercuration intermediate exhibits a rapid equilibration with time. Consequently, the 4:1 ratios may not represent the true limit for the isomer distribution in the initial kinetic product.) Similarly, 2-methylenebicyclo[3.3.0]octane reveals an 8:1 preferential formation of the exo-alcohol. In the case of endo-trimethylene-norborn-8-ene, the oxymercuration stage is extraordinarily slow and the results do not fit this pattern. Possibly the very slow oxymercuration stage permits equilibration of the initial reaction product. On the other hand, the reaction is fast with 8-methylene-endo-trimethylenenorbornane and the product is 100% of the tertiary exo-alcohol. The same behavior is observed for 2-methylenenorbornane. Surprisingly, 2-methylene-endo-trimethy-lenenorbornane fails to undergo oxymercuration. Consequently, both endo-trimethylenenorborn-8-ene and 2-methylene-endo-trimethylenenorbornane exhibit an exceptional inertness toward oxymercuration, presumably related to the highly rigid U-shaped structure of the parent system.     

Kinetic studies of inverse electron demand Diels–Alder reactions (iEDDA) of norbornenes and 3,6-dipyridin-2-yl-1,2,4,5-tetrazine

Inverse electron demand Diels–Alder additions (iEDDA) between 1,2,4,5-tetrazines and olefins have recently found widespread application as a novel ‘click chemistry’ scheme and as a mild technique for the modification of materials. Norbornenes are, due to their straightforward synthetic availability, especially interesting in the latter context. Therefore, the reactivity of different norbornene-based compounds was compared with unsubstituted norbornene and other alkenes using UV-vis measurements for the determination of reaction rates under pseudo first order conditions. Thereby, exo,exo-5-norbornene-2,3-dimethanol was found to be almost as reactive as unsubstituted norbornene whereas (±)-endo,exo-dimethyl-5-norbornene-2,3-dicarboxylate reacted only insignificanty faster than unstrained alkenes.     

Rapid molecular typing of <Emphasis Type="Italic">Tuber melanosporum</Emphasis>, <Emphasis Type="Italic">T. brumale</Emphasis> and <Emphasis Type="Italic">T. indicum</Emphasis> from tree seedlings and canned truffles

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115°C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of truffle or truffled French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.J.P. Douet and D. Mabru have contributed equally to this work