Metal‐stabilized rare tautomers: N4 metalated cytosine (M = Li+, Na+, K+, Rb+ and Cs+), theoretical views

Ab initio calculations indicate that metalation of the exocyclic amino group of cytosine by the elements of Group IA (Li, Na, K, Rb and Cs) induces protonation of a nucleobase ring nitrogen atom, and hence causes a proton shift from an exocyclic to an endocyclic nitrogen atom. Thus, this metal‐assisted process leads to the generation of rare nucleobase tautomers. The calculations suggest that this kind of metalation increases the protonation energies of the aromatic ring of the nucleobase. The present study reports the quantum chemistry analysis of the metal‐assisted tautomerization. The calculations clearly demonstrate that metalation of the exocyclic amino group of the nucleobase significantly increases the protonation energy of the aromatic rings of the nucleobase. Also, absolute anisotropy shift, molecular orbital and natural bond orbital calculations are compatible with these results. Copyright © 2003 John Wiley & Sons, Ltd.     

Ferrocene-linked thymine/uracil conjugates: base pairing directed self-assembly and supramolecular packing

Ferrocene-linked bis(nucleobase) (1a-c) and chimeric nucleobase (1d) conjugates have been synthesized from mono- and bis(hydroxybutyl)ferrocene 6 via Mitsunobu reaction as the key step. X-ray crystallographic studies of ferrocene bis(nucleobase) conjugates reveal two-dimensional supramolecular organizations of backbones through self-assembled Watson-Crick and reverse Watson-Crick type pairs. Ferrocene-bis(thymine) conjugate self-assembles by reverse Watson-Crick pairing, while the corresponding bis(uracil) conjugate self-assembles by alternating WC and reverse WC type pairing. Such continuous assemblies are not seen in monosubstituted ferrocene nucleobase conjugates which form only planar sheets. The results are interesting from the point of understanding and engineering supramolecular assemblies through rational design of base pairing patterns.     

A linchpin carbacyclization approach for the synthesis of carbanucleosides

A convenient synthesis of carbanucleosides, with both enantiomers equally accessible, is reported. The key step is a tandem linchpin cyclization process to give access to substituted carbafuranose derivatives having the correct relative stereochemistry for subsequent nucleobase introduction with inversion of configuration at C1. This was illustrated by the synthesis of 2',3'-dideoxycarbathymidine via a convergent nucleobase introduction and of 2',3'-dideoxy-6'-hydroxycarbauridine via a linear nucleobase introduction. Both methods relied on Mitsunobu chemistry, and the first example of the Mukaiyama modification of the Mitsunobu reaction involving nucleobases as nucleophiles is reported.     

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.     

Metastable decay of negatively charged oligodeoxynucleotides analyzed with ultraviolet matrix-assisted laser desorption/ionization post-source decay and deuterium exchange

In an effort to understand the initiating step in metastable-ion decay of UV matrix-assisted laser desorption/ionization (MALDI)-produced ions, we conducted experiments in which we exchanged all active protons for deuterons of tetrameric and hexameric oligodeoxynucleotides. We wish to address the controversial proposal that in the negative-ion mode, as in the positive-ion mode, fragmentation is driven by nucleobase protonation. The results show unambiguously that proton transfer, leading to zwitterion formation, charges a nucleobase prior to its elimination. The zwitterion formation directs fragmentation of both positive and negative oligodeoxynucleotide ions. Poly-T-rich oligodeoxynucleotide tetramers show surprising differences in the negative compared to the positive-ion mode, as thymine is preferentially expelled, instead of a nucleobase with higher proton affinity. For the exceptional case of negatively charged poly-T-rich oligodeoxynucleotide tetramers generated by MALDI, we propose that zwitterion formation with positive charging of a nucleobase leads to base stabilization in the negative-ion mode through an interaction of the positive nucleobase with the excess negative charge. Moreover, backbone cleavages (accompanied by H rearrangement) of a phosophodiester bond give first-generation products that can be traced back to this tripolar complex bearing one positive and two negative charges, all of which may be interacting.     

Synthesis and characterization of nucleobase functionalized monothiophenes

The synthesis of a series of nucleobase functionalized thiophene monomers has been accomplished through the reaction of 2-bromo-1-thiophen-3-yl-ethanone with the corresponding DNA base anion. The distinctive pK_a values for the various amine groups in the nucleobases provided a pathway for the creation of specific anions through selective deprotonation of these groups. Using the appropriate anion it is possible to create an amine linkage between the thiophene and nucleobase that is, analogous to that found between the deoxyribose sugar and nucleobase, in the biologically occurring nucleoside.     

Intramolecular Photo-Oxidation as a Potential Source to Probe Biological Electron Damage: A Carboxylated Adenosine Analogue as Case Study

A carboxylated adenosine analog (C-Ado⁻) has been synthesized and probed via time-resolved photoelectron spectroscopy in order to induce intra-molecular charge transfer from the carboxylic acid moiety to the nucleobase. Intra-molecular charge transfer can be exploited as starting point to probe low-energy electron (LEE) damage in DNA and its derivatives. Time-dependent density functional theory (TD-DFT) calculations at the B3LYP-6311G level of theory have been performed to verify that the highest occupied molecular orbital (HOMO) was located on carboxylic acid and that the lowest occupied molecular orbital (LUMO) was on the nucleobase. Hence, the carboxylic acid could work as electron source, whilst the nucleobase could serve the purpose of electron acceptor. The dynamics following excitation at 4.66 eV (266 nm) were probed using time-resolved photoelectron spectroscopy using probes at 1.55 eV (800 nm) and 3.10 eV (400 nm). The data show rapid decay of the excited state population and, based on the similarity of the overall dynamics to deoxy-adenosine monophosphate (dAMP^–), it appears that the dominant decay mechanism is internal conversion following ¹ππ* excitation of the nucleobase, rather than charge-transfer from the carboxylic acid to the nucleobase.     

Identification of radiation-induced cross-linking between thymine and tryptophan by electrospray ionization-mass spectrometry

Upon exposure to ionizing radiation, DNA undergoes a variety of modifications including the production of a covalent bond between the nucleobase thymine and aromatic amino acids. In this work, electrospray ionization-mass spectrometry(ESI-MS) was used to identify the gamma radiation-induced covalent cross-linking of model peptides (sequence YPPW and pYPPW) with the nucleobase thymine. Tandem electrospray ionization-mass spectrometry (ESI-MSn) was employed to investigate the cross-linking sites. The results showed that irrespective of whether tyrosine was phosphorylated or not, the nucleobase thymine was cross-linked with the tryptophan residue. Possible cross-linking mechanisms are proposed by investigating the related mass peaks.     

A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase for a CG base pair in triplex DNA

In order to expand target sequences in triplex DNA formation, the development of a nucleobase that recognizes a CG base pair in dsDNA was attempted. A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase was found to recognize a CG base pair with high sequence-selectivity.     

Fourier transform surface-enhanced Raman scattering of single-layer nucleolipid Langmuir-Blodgett films on silver island film substrates

Surface-enhanced Raman scattering (SERS) spectra of four amphiphilic nucleolipids in single-layer Langmuir-Blodgett (LB) films deposited on silver island film substrates from pure water and complementary nucleotide-containing subphase and corresponding powder normal Raman spectra were obtained. The analysis of these spectra indicates that the SERS effect is mainly caused by a charge-transfer mechanism, and only the nucleobase headgroup moieties and complementary bases combined with them through hydrogen bonds, which are directly in contact with the silver island film substrates, could be enhanced. For the amphiphilic nucleolipids with the identical nucleobase headgroups, the SERS spectra of the LB films are similar, implying that the orientations of these nucleobase moieties on the silver substrates are analogous. However, the nucleobase takes different orientations on the silver substrates before and after complementary binding. The nucleobases in the LB films deposited from pure water are nearly lying flat on the silver surface, while the complementary binding pairs transferred from the air/water interface tend to take an end-on orientation on the metal surface.     

Bifacial nucleoside as a surrogate for both T and A in duplex DNA

A new functional bifacial nucleoside derived from 7-aminopyrimido[4,5-d]pyrimidine-2,4(1H,3H)-dione, a Janus-type nucleobase, has been synthesized and incorporated into DNA oligonucleotides. The nucleobase, having self-complementary H-bonding faces, mimics both T and A and engages in the corresponding Watson-Crick-like base pairs, forming stable duplexes.     

Triostin A Derived Cyclopeptide as Architectural Template for the Alignment of Four Recognition Units

The DNA bisintercalator triostin A is structurally based on a disulfide-bridged depsipeptide scaffold that provides preorganization of two quinoxaline units in 10.5 Å distance. Triostin A analogues are synthesized with nucleobase recognition units replacing the quinoxalines and containing two additional recognition units in between. Thus, four nucleobase recognition units are organized on a rigid template, well suited for DNA double strand interactions. The new tetra-nucleobase binders are synthesized as aza-TANDEM derivatives lacking the N-methylation of triostin A and based on a cyclopeptide backbone. Synthesis of two tetra-nucleobase aza-TANDEM derivatives is established, DNA interaction analyzed by microscale thermophoresis, cytotoxic activity studied and a nucleobase sequence dependent self-aggregation investigated by mass spectrometry.     

Optimization of unnatural base pair packing for polymerase recognition

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.     

Hydrolytic deamination reactions of amidine and nucleobase derivatives

Amidines share the same NC─N building framework with many essential biochemical substances. In this work, we present a comparative mechanistic study on the deamination reactions of 19 amidine and nucleobase derivatives by the use of density functional theory. All the computations are performed at the B3LYP/6-31G(d,p) level in the gas phase and with the polarizable continuum model (PCM). Mechanisms of 2- and 3-step pathways including six- or eight-membered ring transition states were explored. Our results show that the overall activation energies for the deamination of amidine derivatives are close to those of nucleobase derivatives of the saturated C5─C6 bond, and lower than those of nucleobase derivatives of the unsaturated C5─C6 bond, while purine derivatives have the highest activation energies among all the derivatives studied. The 3-step mechanism gives results that are more consistent with the available experimental data than the 2-step mechanism. Based on the results of our current and previous work, we believe that the 3-step mechanism is the most likely mechanism for the hydrolytic deamination reactions of amidine and nucleobase derivatives.     

An efficient organometallic approach to new carbocyclic nucleoside analogues

[reaction: see text] A general synthetic approach to monoprotected carbocyclic nucleoside analogues, having the nucleobase attached to a 3-hydroxymethyl-4-trialkylsilyloxymethyl-cyclopent-2-en-1-yl scaffold, was developed. A (racemic) key intermediate was prepared by a cobalt-mediated Pauson-Khand reaction. In the course of the further synthesis, the introduction of the nucleobase was achieved with complete regio- and diastereoselectivity through a palladium-catalyzed allylic substitution.     

Plasmonic Chirality Imprinting on Nucleobase‐Displaying Supramolecular Nanohelices by Metal–Nucleobase Recognition

Supramolecular self‐assembly is an important process that enables the conception of complex structures mimicking biological motifs. Herein, we constructed helical fibrils through chiral self‐assembly of nucleobase–peptide conjugates (NPCs), where achiral nucleobases are helically displayed on the surface of fibrils, comparable to polymerized nucleic acids. Selective binding between DNA and the NPC fibrils was observed with fluorescence polarization. Taking advantage of metal–nucleobase recognition, we highlight the possibility of deposition/assembly of plasmonic nanoparticles onto the fibrillar constructs. In this approach, the supramolecular chirality of NPCs can be adaptively imparted to metallic nanoparticles, covering them to generate structures with plasmonic chirality that exhibit significantly improved colloidal stability. The self‐assembly of rationally designed NPCs into nanohelices is a promising way to engineer complex, optically diverse nucleobase‐derived nanomaterials.     

Nitromethane as a scavenger of acrylonitrile in the deprotection of synthetic oligonucleotides

A novel deprotection procedure for synthetic oligonucleotides was developed to prevent nucleobase alkylation. Acrylonitrile, a side product of the deprotection of a 2-cyanoethyl phosphate protecting group and which causes nucleobase alkylation, was found to be trapped by the addition of some acidic compounds, which generate a carbanion species under the conventional deprotection conditions using aqueous NH₃. The 2-cyanoethylation of thymidine was inhibited effectively in the presence of nitromethane.     

Efficient primer strand extension beyond oxadiazole carboxamide nucleobases

Two oxadiazole carboxamide deoxyribonucleoside analogues are described that can be incorporated and efficiently extended by Taq DNA polymerase. The primer strand extension beyond oxadiazole nucleoside analogues occurs at rates similar to the values observed for the canonical Watson-Crick base pairs irrespective of the template nucleobase. These distinctive chemical effects in DNA polymerase extensions are attributed to the smaller size and unique electronic properties of the oxadiazole nucleobase.     

A comparative DFT study of some N-based aromatic ligand metal complexes as anticancer agents and analysis of their mode of interaction with DNA base pair

Metal complexed anticancer agents interact with DNA nucleobase pairs (AT and GC) through different types of binding mode such as intercalation, groove binding, covalent binding, etc. Minor and major groove binding mechanism of DNA base pair is the key factor for all kinds of anticancer agent; as metal complexes have a great affinity to bind with DNA nucleobase either through minor or major groove. Ligands in metal complexes also play a vital role during the interaction with DNA base pairs; these ligands directly interact with DNA through different interacting modes. Generally, anticancer agents with less sterically hindered N-based aromatic and planar ligands are the key component for DNA binding; as the structure of such ligands are quite compatible for following intercalation and groove binding mechanism. Since, the experimental investigation for drug-DNA nucleobase complexes are extremely complicated, therefore; quantum mechanical calculations might be very helpful for computing the actual interactions in drug-DNA complexes. Quantum mechanical approaches such as density functional theory (DFT) might be a very important and useful tool to investigate the actual mode of interaction of metal complexed antitumor agents with DNA nucleobase. Herein, we have taken some metal complexes with N-based aromatic ligands as antitumor agents to investigate the proper mode of interaction between drug-DNA complexes.     

Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair

Hydrophobic artificial nucleobase pairs without the ability to pair through hydrogen bonds are promising candidates to expand the genetic alphabet. The most successful nucleobase surrogates show little similarity to each other and their natural counterparts. It is thus puzzling how these unnatural molecules are processed by DNA polymerases that have evolved to efficiently work with the natural building blocks. Here, we report structural insight into the insertion of one of the most promising hydrophobic unnatural base pairs, the dDs–dPx pair, into a DNA strand by a DNA polymerase. We solved a crystal structure of KlenTaq DNA polymerase with a modified template/primer duplex bound to the unnatural triphosphate. The ternary complex shows that the artificial pair adopts a planar structure just like a natural nucleobase pair, and identifies features that might hint at the mechanisms accounting for the lower incorporation efficiency observed when processing the unnatural substrates.