Small Molecule Inhibitors of TET Dioxygenases: Bobcat339 Activity Is Mediated by Contaminating Copper(II).

Ten eleven translocation (TET) dioxygenases 1-3 are non-heme Fe(II) and α-ketoglutarate dependent enzymes that catalyze oxidation of 5-methylcytosine (5mC) in DNA to hydroxymethyl-C, formyl-C, and carboxy-C. This typically leads to gene activation and epigenetic remodeling. Most known inhibitors of TET are α-ketoglutarate mimics that may interfere with other α-ketoglutarate dependent enzymes. Recently, a novel cytosine-based inhibitor of TET, Bobcat339, was reported to have mid-µM inhibitory activity against TET1 and TET2. The molecule is now sold as a TET inhibitor by several vendors. We independently prepared Bobcat339 in our laboratory and observed that it had minimal inhibitory activity against human TET1 and TET2 via a quantitative LC-ESI-MS/MS assay. Furthermore, the inhibitory activity of commercial Bobcat339 preparations was directly correlated with Cu(II) content. We therefore conclude that Bobcat339 alone is not capable of inhibiting TET enzymes at the reported concentrations, and that its activity is enhanced by contaminating Cu(II).

DEB-FAPy-dG Adducts of 1,3-Butadiene: Synthesis, Structural Characterization, and Formation in 1,2,3,4-Diepoxybutane Treated DNA.

Metabolic activation of the human carcinogen 1,3-butadiene (BD) by cytochrome 450 monooxygenases gives rise to a genotoxic diepoxide, 1,2,3,4-diepoxybutane (DEB). This reactive electrophile alkylates guanine bases in DNA to produce N7-(2-hydroxy-3,4-epoxy-1-yl)-dG (N7-DE-dG) adducts. Because of the positive charge at the N7 position of the purine heterocycle, N7-DEB-dG adducts are inherently unstable and can undergo spontaneous depurination or base-catalyzed imidazole ring opening to give N6 -[2-deoxy-D-erythro-pentofuranosyl]-2,6-diamino-3,4-dihydro-4-oxo-5-N-1-(oxiran-2-yl)propan-1-ol-formamidopyrimidine (DEB-FAPy-dG) adducts. Here we report the first synthesis and structural characterization of DEB-FAPy-dG adducts. Authentic standards of DEB-FAPy-dG and its 15 N3 -labeled analogue were used for the development of a quantitative nanoLC-ESI+ -HRMS/MS method, allowing for adduct detection in DEB-treated calf thymus DNA. DEB-FAPy-dG formation in DNA was dependent on DEB concentration and pH, with higher numbers observed under alkaline conditions.

Synthesis and polymerase bypass studies of DNA-peptide and DNA-protein conjugates.

DNA-peptide (DpCs) and DNA-protein cross-links (DPCs) are DNA lesions formed when polypeptides and nuclear proteins become covalently trapped on DNA strands. DNA-protein cross-links are of enormous size and hence pose challenges to cell survival by blocking DNA replication, transcription, and repair. However, DPCs can undergo proteolytic degradation via various pathways to give shorter polypeptide chains (DpCs). The resulting DpC lesions are efficiently bypassed by translesion synthesis (TLS) DNA polymerases like κ, η, δ, etc., although polymerase bypass efficiency as well as correct base insertion depends heavily on size, sequence context, and position of peptides in DpCs. This chapter explores various synthetic methods to generate these lesions including detailed experimental procedures for the construction of DpCs and DPCs via reductive amination and oxime ligation. Further we describe biochemical experiments to investigate the effects of these lesions on DNA polymerase activity and fidelity.

Site-Specific 5-Formyl Cytosine Mediated DNA-Histone Cross-Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η.

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Novel 4-Hydroxybenzyl Adducts in Human Hemoglobin: Structures and Mechanisms of Formation.

Humans are exposed to large numbers of electrophiles from their diet, the environment, and endogenous physiological processes. Adducts formed at the N-terminal valine of hemoglobin are often used as biomarkers of human exposure to electrophilic compounds. We previously reported the formation of hemoglobin N-terminal valine adducts (added mass, 106.042 Da) in the blood of human smokers and nonsmokers and identified their structure as 4-hydroxybenzyl-Val. In the present work, mass spectrometry-based proteomics was utilized to identify additional sites for 4-hydroxybenzyl adduct formation at internal nucleophilic amino acid side chains within hemoglobin. Hemoglobin isolated from human blood was treated with para-quinone methide (para-QM) followed by global nanoLC-MS/MS and targeted nanoLC-MS/MS to identify amino acid residues containing the 4-hydroxybenzyl modification. Our experiments revealed the formation of 4-hydroxybenzyl adducts at the αHis20, αTyr24, αTyr42, αHis45, ßSer72, ßThr84, ßThr87, ßSer89, ßHis92, ßCys93, ßCys112, ßThr123, and ßHis143 residues (in addition to N-terminal valine) through characteristic MS/MS spectra. These amino acid side chains had variable reactivity toward para-QM with αHis45, αTyr42, ßCys93, ßHis92, and ßSer72 forming the largest numbers of adducts upon exposure to para-QM. Two additional mechanisms for formation of 4-hydroxybenzyl adducts in humans were investigated: exposure to 4-hydroxybenzaldehyde (4-HBA) followed by reduction and UV-mediated reactions of hemoglobin with tyrosine. Exposure of hemoglobin to a 5-fold molar excess of 4-HBA followed by reduction with sodium cyanoborohydride produced 4-hydroxybenzyl adducts at several amino acid side chains of which αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßHis92 were verified in targeted mass spectrometry experiments. Similarly, exposure of human blood to ultraviolet radiation produced 4-hydroxybenzyl adducts at αHis20, αTyr24, αTyr42, αHis45, ßSer44, ßThr84, and ßSer89. Overall, our results reveal that 4-hydroxybenzyl adducts form at multiple nucleophilic sites of hemoglobin and that para-QM is the most likely source of these adducts in humans.

Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.

DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.

Discovery of Novel N-(4-Hydroxybenzyl)valine Hemoglobin Adducts in Human Blood.

Humans are exposed to a wide range of electrophilic compounds present in our diet and environment or formed endogenously as part of normal physiological processes. These electrophiles can modify nucleophilic sites of proteins and DNA to form covalent adducts. Recently, powerful untargeted adductomic approaches have been developed for systematic screening of these adducts in human blood. Our earlier untargeted adductomics study detected 19 unknown adducts to N-terminal valine in hemoglobin (Hb) in human blood. We now describe a full characterization of one of these adducts, which corresponds to the addition of a 4-hydroxybenzyl (4-OHBn) group to N-terminal valine in Hb to form N(4-hydroxybenzyl)valine (4-OHBn-Val). The adduct structure was determined by comparison of its accurate mass, HPLC retention time, and MS/MS fragmentation to that of authentic standards prepared by chemical synthesis. Average 4-OHBn-Val adduct concentrations in 12 human blood samples were estimated to 380 ± 160 pmol/g Hb. Two possible routes of 4-OHBnVal adduct formation are proposed using two different precursor electrophiles: 4-quinone methide (4-QM) and 4-hydroxybenzaldehyde (4-OHBA). We found that 4-QM reacts rapidly with valine to form the 4-OHBn-Val adduct; however, the quinone methide is unstable under physiological conditions due to hydrolysis. It was shown that 4-OHBA forms reversible Schiff base adducts with valine, which can be stabilized via reduction in blood generating the 4-OHBn-Val adduct. In addition, trace amounts of isomeric 2-hydroxybenzyl-valine (2-OHBn-Val) adducts were detected in 12 human blood samples (estimated mean adduct level, 5.0 ± 1.4 pmol/g Hb). Further studies are needed to quantify the contributions from identified possible precursor electrophiles to the observed hydroxybenzyl adducts in humans.

N6-(2-Deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine Adducts of 1,3-Butadiene: Synthesis, Structural Identification, and Detection in Human Cells.

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 3,4-epoxy-1-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-(2-hydroxy-3-buten-1-yl)-guanine (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nanoLC-ESI+-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at an increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in base excision repair-deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. Furthermore, NEIL1-/- cells were sensitized to EB treatment as compared to NEIL1+/+ fibroblasts. Overall, our results indicate that ring-opened EB-FAPy-dG adducts form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of BD.

Site-specific cross-linking of proteins to DNA via a new bioorthogonal approach employing oxime ligation.

DNA-protein cross-links (DPCs) are super-bulky DNA adducts induced by common chemotherapeutic agents, reactive oxygen species, and aldehydes, and also formed endogenously as part of epigenetic regulation. Despite their presence in most cells and tissues, the biological effects of DPCs are poorly understood due to the difficulty of constructing site-specific DNA-protein conjugates. In the present work, a new approach of conjugating proteins to DNA using oxime ligation was used to generate model DPCs structurally analogous to lesions formed in cells. In our approach, proteins and peptides containing an unnatural oxy-Lys amino acid were cross-linked to DNA strands functionalized with 5-formyl-dC or 7-(2-oxoethyl)-7-deaza-dG residues using oxime ligation. The conjugation reaction was site-specific with respect to both protein and DNA, provided excellent reaction yields, and formed stable DPCs amenable to biological evaluation.

Chemical Biology of N5-Substituted Formamidopyrimidine DNA Adducts.

DNA nucleobases are the prime targets for chemical modifications by endogenous and exogenous electrophiles. Alkylation of the N7 position of guanine and adenine in DNA triggers base-catalyzed imidazole ring opening and the formation of N5-substituted formamidopyrimidine (N5-R-FAPy) lesions. Me-FAPy-dG adducts induced by exposure to methylating agents and AFB-FAPy-dG lesions formed by aflatoxin B1 have been shown to persist in cells and to contribute to toxicity and mutagenicity. In contrast, the biological outcomes of other N5-substituted FAPy lesions have not been fully elucidated. To enable their structural and biological evaluation, N5-R-FAPy adducts must be site-specifically incorporated into synthetic DNA strands using phosphoramidite building blocks, which can be complicated by their unusual structural complexity. N5-R-FAPy exist as a mixture of rotamers and can undergo isomerization between α, ß anomers and furanose-pyranose forms. In this Perspective, we will discuss the main types of N5-R-FAPy adducts and summarize the strategies for their synthesis and structural elucidation. We will also summarize the chemical biology studies conducted with N5-R-FAPy-containing DNA to elucidate their effects on DNA replication and to identify the mechanisms of N5-R-FAPy repair.

High-density functionalization and cross-linking of DNA: "click" and "bis-click" cycloadditions performed on alkynylated oligonucleotides with fluorogenic anthracene azides.

High density functionalization of DNA with ethynyl and octadiynyl side chains followed by CuAAC "click labeling" with 9-azidomethylanthracene was performed. Alkynyl DNA was also cross-linked with fluorogenic 9,10-bis-azidomethylanthracene employing the "bis-click" reaction. By this means the fluorescence of the anthracene moiety was imparted to the virtually nonfluorescent DNA. Phosphoramidites of 8-aza-7-deaza-2'-deoxyadenosine with short and long linker arms in a steric nondemanding 7-position were utilized in solid phase oligodeoxynucleotide synthesis. High density alkynylated DNA-without anthracene residues-was found to be of comparable stability with both long and short linker arms. High density anthracene functionalized DNA became less stable with the short linker compared to that with the long linker connectivity. Interstrand cross-linked homodimers constructed from alkynylated oligonucleotides with fluorogenic 9,10-bis-azidomethylanthracene were hybridized with complementary strands to form double helices. They are more stable when the linker was located at a terminus than in a central position. Short linker anthracene adducts were destabilizing compared to long linker adducts. The fluorogenic anthracene residues not only have a significant effect on the duplex stability, but also impart fluorescence to the species. Fluorescence of cross-linked double helices with long linker connectivity was quenched when the cross-link was in a terminal position and was dequenched when the linker was connecting the two double helices at the center of the molecule. The fluorescence of the anthracene cross-linked double helices was strongly increased (dequenched) when the correct base pair was formed, while no change occurred upon mismatch formation.

Oligonucleotides with "clickable" sugar residues: synthesis, duplex stability, and terminal versus central interstrand cross-linking of 2'-O-propargylated 2-aminoadenosine with a bifunctional azide.

Duplex DNA with terminal and internal sugar cross-links were synthesized by the CuAAC reaction from oligonucleotides containing 2'-O-propargyl-2-aminoadenosine as a clickable site and a bifunctional azide (4). Stepwise click chemistry was employed to introduce cross-links at internal and terminal positions. Copper turnings were used as catalyst, reducing the copper load of the reaction mixture and avoiding complexing agents. For oligonucleotide building block synthesis, a protecting group strategy was developed for 2'-O-propargyl-2-aminoadenosine owing to the rather different reactivities of the two amino groups. Phosphoramidites were synthesized bearing clickable 2'-O-propargyl residues (14 and 18) as well as a 2'-deoxyribofuranosyl residue (10). Hybridization experiments of non-cross-linked oligonucleotides with 2,6-diaminopurine as nucleobase showed no significant thermal stability changes over those containing adenine. Surprisingly, an isobutyryl group protecting the 2-amino function has no negative impact on the stability of DNA-DNA and DNA-RNA duplexes. Oligonucleotide duplexes with cross-linked 2'-O-propargylated 2-aminoadenosine (1) and 2'-O-propargylated adenosine (3) at terminal positions are significantly stabilized (ΔT(m) = +29 °C). The stability results from a molecularity change from duplex to hairpin melting and is influenced by the ligation position. Terminal ligation led to higher melting duplexes than corresponding hairpins, while duplexes with central ligation sites were less stable.

Parallel stranded DNA stabilized with internal sugar cross-links: synthesis and click ligation of oligonucleotides containing 2'-propargylated isoguanosine.

Internal sugar cross-links were introduced for the first time into oligonucleotides with parallel chain orientation by click ligation. For this, the 2'- or 3'-position of the isoguanosine ribose moiety was functionalized with clickable propargyl residues, and the synthesis of propargylated cytosine building blocks was significantly improved. Phosphoramidites were prepared and employed in solid-phase synthesis. A series of oligo-2'-deoxyribonucleotides with parallel (ps) and antiparallel (aps) strand orientation were constructed containing isoguanine-cytosine, isoguanine-isocytosine, and adenine-thymine base pairs. Complementary oligonucleotides with propargylated sugar residues were ligated in a stepwise manner with a chelating bis-azide under copper catalysis. Cross-links were introduced within a base pair or in positions separated by two base pairs. From T(m) stability studies it is evident that cross-linking stabilizes DNA with parallel strand orientation strongly (ΔT(m) from +16 to +18.5 °C) with a similar increase as for aps DNA.

Cross-linked DNA: propargylated ribonucleosides as "click" ligation sites for bifunctional azides.

2'-O or 3'-O-propargylated adenosines and ribothymidines were used as click targets for cross-linking of oligonucleotides with aliphatic and aromatic azides. The cross-link generates a sugar modification at the 2'-O-ligation site. Inexpensive ribonucleosides were used as starting materials. Cross-linking of oligonucleotides was performed at internal or terminal positions. Hybridization of homodimers with two complementary single strands resulted in stable ligated DNA duplexes.

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability.

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.

Cross-linked DNA generated by "bis-click" reactions with bis-functional azides: site independent ligation of oligonucleotides via nucleobase alkynyl chains.

Template-free cross-linking of single-stranded DNA bearing octadiynyl side chains at the 7-position of 8-aza-7-deazapurine moieties with bisfunctional azides is reported employing a Cu(I)-catalyzed azide-alkyne "bis-click" reaction. Bis-adducts were formed when the bis-azide:oligonucleotide ratio was 1:1; monofunctionalization occurred when the ratio was 15:1. Four-stranded DNA consisting of two cross-linked duplexes was obtained after hydridization. Cross-linked duplexes are as stable as individual duplexes when ligation was introduced at terminal positions; ligation at a central position led to a slight duplex destabilization.

Azide-alkyne "click" conjugation of 8-aza-7-deazaadenine-DNA: synthesis, duplex stability, and fluorogenic dye labeling.

The internal dye labeling of DNA by the Huisgen-Meldal-Sharpless "click" reaction is described. Fluorogenic 9-azidomethyl anthracene 2 and 3-azido-7-hydroxycoumarin 3 were employed in the postsynthetic functionalization of oligonucleotides incorporating octa-(1,7)-diynyl-8-aza-7-deaza-2'-deoxyadenosine 1. Nucleoside 1 was prepared by Sonogashira cross coupling from the corresponding 7-iodo compound, converted into the corresponding phosphoramidite, and oligonucleotides were synthesized. To evaluate the influence of ligands on the oligonucleotide duplex stability, benzyl azide 4 (nonpolar), and 2',3'-dideoxy azidothymidine 5 (AZT) (polar) were introduced along with the fluorogenic dyes 2 and 3. DNA duplexes with octa-1,7-diynyl side chains (i.e., containing 1) are more stable than oligonucleotides containing 8-aza-7-deaza-2'-deoxyadenosine, unveiling that this side chain has steric freedom. A single conjugation by an anthracene residue led to a 9 °C T(m) increase of duplex melting. Contrary to 7-deazaadenine dye conjugates, the 8-aza-7-deazaadenine conjugates show virtually no fluorescence quenching, thereby developing almost as strong fluorescence as side chain click derivatives (32 and 33) in the absence of 8-aza-7-deazaadenine moiety. Duplexes containing the 8-aza-7-deazaadenine dye conjugate show increased fluorescence over single-stranded DNA. Mismatches with dA, dG, and dC develop reduced fluorescence compared to the fully matched base pair. Molecular dynamics simulations revealed that the bulky dye molecules are accommodated well in duplex DNA.

An efficient synthesis of 1,8-dioxo-octahydro-xanthene derivatives promoted by a room temperature ionic liquid at ambient conditions under ultrasound irradiation.

The condensation reaction involving an aldehyde and diketone was efficiently promoted by the Ionic liquid, [Hbim]BF(4) (IL) as a reaction medium with methanol as co-solvent at ambient temperature under ultrasonic irradiation to afford the corresponding 1,8-dioxo-octahydro-xanthene (xanthene) derivatives in excellent yields. The advantages of this method include among others the use of a recyclable, non-volatile ionic liquid, which promotes this protocol under ambient temperature without the requirement of any added catalyst. The reaction times and yields are compared with p-TSA catalyzed synthesis of xanthenes under thermal conditions, which is also reported for the first time under our reaction conditions.