Anthraquinone as a redox label for DNA: synthesis, enzymatic incorporation, and electrochemistry of anthraquinone-modified nucleosides, nucleotides, and DNA

Modified 2'-deoxynucleosides and deoxynucleoside triphosphates (dNTPs) bearing anthraquinone (AQ) attached through an acetylene or propargylcarbamoyl linker at the 5-position of pyrimidine (C) or at the 7-position of 7-deazaadenine were prepared by Sonogashira cross-coupling of halogenated dNTPs with 2-ethynylanthraquinone or 2-(2-propynylcarbamoyl)anthraquinone. Polymerase incorporations of the AQ-labeled dNTPs into DNA by primer extension with KOD XL polymerase have been successfully developed. The electrochemical properties of the AQ-labeled nucleosides, nucleotides, and DNA were studied by cyclic and square-wave voltammetry, which show a distinct reversible couple of peaks around -0.4 V that make the AQ a suitable redox label for DNA.     

Synthesis and structures of deoxyribonucleoside analogues for triazole-linked DNA (DNA)

Deoxyribonucleoside analogues bearing acetylene group at the pseudo-5′-position and azido group at the pseudo-3′-position have been synthesized by transglycosylation reaction of deoxythymidine analogue with adenine, cytosine, and guanine nucleobases as nucleophiles. The structures of analogues were studied in crystalline state by X-ray crystallography as well as in solution phase by NMR spectroscopy and showed the puckering conformations similar to the natural congeners.     

An efficient method for the construction of functionalized DNA bearing amino acid groups through cross-coupling reactions of nucleoside triphosphates followed by primer extension or PCR

Single-step aqueous cross-coupling reactions of nucleobase-halogenated 2'-deoxynucleosides (8-bromo-2'-deoxyadenosine, 7-iodo-7-deaza-2'-deoxyadenosine, or 5-iodo-2'-deoxy-uridine) or their 5'-triphosphates with 4-boronophenylalanine or 4-ethynylphenylalanine have been developed and used for efficient synthesis of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8-substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7-substituted 7-deazaadenine and 5-substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two-step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.     

Study on suitability of KOD DNA polymerase for enzymatic production of artificial nucleic acids using base/sugar modified nucleoside triphosphates

Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2'-ONHCH?CH?-4' linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.     

Synthesis of 2′-deoxycytidine and its triphosphate bearing tryptophan-based imidazolinone fluorophore for environment sensitive fluorescent labelling of DNA

We synthesized 2′-deoxycytidine and the corresponding nucleoside triphosphate bearing (indol-3-yl)methylene-2-methyl-5-oxo-4,5-dihydroimidazol-1-yl group (a tryptophan-based fluorophore from cyan fluorescent protein) linked through a propargyl group at position 5. The fluorophore is weakly solvatochromic, sensitive to pH, and, as a molecular rotor, it is highly sensitive to viscosity. In low viscosity solvents, the fluorescence is very weak, whereas in more viscous environment it lights up. Primer extension or PCR using the modified dC^TrpTP and KOD XL DNA polymerase was used for construction of labelled oligonucleotides and DNA. Preliminary study showed a 2-fold increase of fluorescence of labelled ON probe in presence of single strand-binding protein indicating a potential of this label for sensing of protein-DNA interactions.     

Furan Decorated Nucleoside Analogues as Fluorescent Probes: synthesis, photophysical evaluation and site-specific incorporation

The synthesis and photophysical evaluation of modified nucleoside analogues in which a five-membered heterocycle (furan, thiophene, oxazole, and thiazole) is attached to the 5-position of 2′-deoxyuridine are reported. The furan-containing derivative is identified as the most promising responsive nucleoside of this family due to its emission quantum efficiency and degree of sensitivity to its microenvironment. The furan moiety was then attached to the 5-position of 2′-deoxycytidine as well as the 8-position of adenosine and guanosine. Photophysical evaluation of these four furan-containing nucleoside analogues reveals distinct differences in the absorption, emission, and quantum efficiency depending upon the class of nucleoside (pyrimidine or purine). Comparing the photophysical properties of all furan-containing nucleosides, identifies the furan thymidine analogue, 5-(fur-2-yl)-2′-deoxyuridine, as the best candidate for use as a responsive fluorescent probe in nucleic acids. 5-(Fur-2-yl)-2′-deoxyuridine was then converted to the corresponding phosphoramidite and site specifically incorporated into DNA oligonucleotides with greater than 88% coupling efficiency. Such furan-modified oligonucleotides form stable duplexes upon hybridization to their complementary DNA strands and display favorable fluorescent features.     

Synthesis and cytotoxic activity of novel curcumin analogues

Five novel curcumin analogues bearing different substituents at 4-position of phenyl group were synthesized. Their structures were confirmed by NMR and HRMS spectrum. Their cytotoxic activities against six tumor cell lines were tested by the standard MTT assay in vitro. The results indicated that four analogues （1A-1C, 1E） with solubilizing moieties showed selective potent cytotoxicity against HepG2, HeLa and CT26 cell lines, and analogue 1A and 1C exhibited more potent cytotoxicity than curcumin against CT26 cell line. It was suggested that introduction of appropriate substituents to 4-position of phenyl group might be a potential option for structural modification of curcumin.     

Stepping towards highly flexible aptamers: enzymatic recognition studies of unlocked nucleic acid nucleotides

Enzymatic recognition of unlocked nucleic acid (UNA) nucleotides was successfully accomplished. Therminator DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of a 81 mer UNA-modified DNA library was efficiently achieved by KOD DNA polymerase.     

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.     

2'-deoxycytidines carrying amino and thiol functionality: synthesis and incorporation by Vent (exo-) polymerase

[reaction: see text] The synthesis of 2'-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a butynyl linker is described. The corresponding triphosphates were then synthesized from the nucleoside and incorporated into oligonucleotides by Vent (exo(-)) DNA polymerase. The ability of Vent (exo(-)) polymerase to amplify oligonucleotides containing these functionalized cytidine derivatives in a polymerase chain reaction (PCR) was demonstrated for the amino-functionalized derivative.     

Direct Sensing of 5‐Methylcytosine by Polymerase Chain Reaction

The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.     

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.     

A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site

Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.     

Transfer hydrogenation of imines with carboxyl-tailed benzothiazoline as readily removable hydrogen donor

A benzothiazoline bearing 4-carboxyphenyl group at 2-position was developed as an efficient hydrogen donor for the transfer hydrogenation reaction. Introduction of the carboxyl group significantly facilitated the removal of the residual benzothiazoline and benzothiazole by washing with aqueous basic solution. The present approach provides a convenient and straightforward access to various amines with broad substrate scope and in good yields.     

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.     

Kinetic and theoretical studies on the mechanism of alkaline hydrolysis of DNA

The reaction mechanism of alkaline hydrolysis of DNA has been investigated by kinetic analysis and density-functional-theory calculation. The rates of hydrolysis of thymidine 3'-monophosphate esters (including thymidylyl(3'-5')thymidine (Tp-OT)) monotonically decrease as the leaving groups get poorer. According to the theoretical calculation in which the solvent effects are incorporated, no intermediate is formed in the course of the reaction. In the alkaline hydrolysis of the activated Tp-OT analogues having good leaving groups, the 3',5'-cyclic monophosphate of thymidine is concurrently formed through the intramolecular attack by the 5'-alkoxide ion. In the hydrolysis of the native dinucleotide, however, this side reaction does not occur, since the transition state leading to the departure of its poor leaving group cannot be formed due to conformational restraint. These arguments are supported by the theoretical analysis on the hydrolysis of both dimethyl phosphate and its O(bridging)-->S substituted analogue.     

Metalation of 4-oxazolinyloxazole derivatives. A convenient route to 2,4-bifunctionalized oxazoles

The synthesis of an array of 5-phenyloxazole derivatives bearing a variety of hydroxyalkyl groups at the C-2 position of the heterocyclic nucleus and possessing a formyl or a carboxyl function at C-4 is reported. These bifunctionalized compounds have been efficiently prepared by addition of carbonylated electrophiles to the 2-lithio derivative of 5-phenyloxazole preliminarily equipped with an oxazoline unit at the 4-position of the oxazole nucleus. It is demonstrated that this protocol offers a double advantage since it suppresses the troublesome electrocyclic ring-opening reaction and allows access to the target compounds by simple chemical transformation of the oxazoline ring system.     

Membrane‐permeable Triphosphate Prodrugs of Nucleoside Analogues

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate‐limitations can be at the mono‐, but also at the di‐ and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro‐approach). In this approach, NTPs are masked by two bioreversible units at the γ‐phosphate. Using a procedure involving H‐phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme‐triggered delivery of NTPs was demonstrated by pig liver esterase, in human T‐lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro‐compounds of some HIV‐inactive nucleoside analogues showed marked anti‐HIV activity. For cellular uptake studies, a fluorescent TriPPPro‐compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.