Partially Fluorinated Copolymers as Oxygen Sensitive 19 F MRI Agents.

Effective diagnosis of disease and its progression can be aided by 19 F magnetic resonance imaging (MRI) techniques. Specifically, the inherent sensitivity of the spin-lattice relaxation time (T1 ) of 19 F nuclei to oxygen partial pressure makes 19 F MRI an attractive non-invasive approach to quantify tissue oxygenation in a spatiotemporal manner. However, there are only few materials with the adequate sensitivity to be used as oxygen-sensitive 19 F MRI agents at clinically relevant field strengths. Motivated by the limitations in current technologies, we report highly fluorinated monomers that provide a platform approach to realize water-soluble, partially fluorinated copolymers as 19 F MRI agents with the required sensitivity to quantify solution oxygenation at clinically relevant magnetic field strengths. The synthesis of a systematic library of partially fluorinated copolymers enabled a comprehensive evaluation of copolymer structure-property relationships relevant to 19 F MRI. The highest-performing material composition demonstrated a signal-to-noise ratio that corresponded to an apparent 19 F density of 220 mm, which surpasses the threshold of 126 mm 19 F required for visualization on a three Tesla clinical MRI. Furthermore, the T1 of these high performing materials demonstrated a linear relationship with solution oxygenation, with oxygen sensitivity reaching 240×10-5  mmHg-1 s-1 . The relationships between material composition and 19 F MRI performance identified herein suggest general structure-property criteria for the further improvement of modular, water-soluble 19 F MRI agents for quantifying oxygenation in environments relevant to medical imaging.

Correlation distance dependence of the resonance frequency of intermolecular zero quantum coherences and its implication for MR thermometry.

PURPOSE: Because the resonance frequency of water-fat intermolecular zero-quantum coherences (iZQCs) reflects the water-fat frequency separation at the microscopic scale, these frequencies have been proposed and used as a mean to obtain more accurate temperature information. The purpose of this work was to investigate the dependence of the water-fat iZQC resonance frequency on sample microstructure and on the specific choice of the correlation distance. METHODS: The effect of water-fat susceptibility gradients on the water-methylene iZQC resonance frequency was first computed and then measured for different water-fat emulsions and for a mixture of porcine muscle and fat. Similar measurements were also performed for mixed heteronuclear spin systems. RESULTS: A strong dependence of the iZQC resonance frequency on the sample microstructure and on the specific choice of the correlation distance was found for spin systems like water and fat that do not mix, but not for spin systems that mix at the molecular level. CONCLUSIONS: Because water and fat spins do not mix at the molecular level, the water-fat iZQC resonance frequency and its temperature coefficient are not only affected by sample microstructure but also by the specific choice of the correlation distance. Magn Reson Med 79:1429-1438, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Accurate MR thermometry by hyperpolarized 129 Xe.

PURPOSE: To investigate the temperature dependence of the resonance frequency of lipid-dissolved xenon (LDX) and to assess the accuracy of LDX-based MR thermometry. METHODS: The chemical shift temperature dependence of water protons, methylene protons, and LDX was measured from samples containing tissues with varying fat contents using a high-resolution NMR spectrometer. LDX results were then used to acquire relative and absolute temperature maps in vivo and the results were compared with PRF-based MR thermometry. RESULTS: The temperature dependence of proton resonance frequency (PRF) is strongly affected by the specific distribution of water and fat. A redistribution of water and fat compartments can reduce the apparent temperature dependence of the water chemical shift from -0.01 ppm/°C to -0.006 ppm, whereas the LDX chemical shift shows a consistent temperature dependence of -0.21 ppm/°C. The use of the methylene protons resonance frequency as internal reference improves the accuracy of LDX-based MR thermometry, but degrades that of PRF-based MR thermometry, as microscopic susceptibility gradients affected lipid and water spins differently. CONCLUSION: The LDX resonance frequency, with its higher temperature dependence, provides more accurate and precise temperature measurements, both in vitro and in vivo. More importantly, the resonance frequency of nearby methylene protons can be used to extract absolute temperature information. Magn Reson Med 78:1070-1079, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Remote detection of hyperpolarized 129Xe resonances via multiple distant dipolar field interactions with 1H.

A remote detection scheme utilizing the distant dipolar field interaction between two different spin species was proposed by Granwehr et al. [J. Magn. Reson. 176(2), 125 (2005)]. In that sequence 1H spins were detected indirectly via their dipolar field interaction with 129Xe spins, which served as the sensing spins. Here we propose a modification of the proposed detection scheme that takes advantage of the longer T1 relaxation time of xenon to create a long lasting dipolar field with which the fast relaxing 1H spins are allowed to interact many times during a single acquisition. This new acquisition scheme improves detection sensitivity, but it also presents some challenges.

Identification and characterization of 2'-deoxyadenosine adducts formed by isoprene monoepoxides in vitro.

Isoprene, the 2-methyl analogue of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with the formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after the reaction of 2'-deoxyadenosine (dAdo ) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1-2'-deoxyinosine (N1-dIno) due to the deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-spectroscopy, and LC-MS/MS and characterized by (1)H NMR and (1)H,(13)C HSQC and HMBC NMR experiments. Adducts of IP-1,2-O that were fully identified are R,S-C1-N(6)-dAdo, R-C2-N(6)-dAdo, and S-C2-N(6)-dAdo; adducts of IP-3,4-O are S-C3-N(6)-dAdo, R-C3-N(6)-dAdo, R,S-C4-N(6)-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the terminal and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation or as biomarkers of isoprene metabolism and exposure.

Iminohydantoin lesion induced in DNA by peracids and other epoxidizing oxidants.

The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-chloroperbenzoic acid (m-CPBA) and dimethyldioxirane (DMDO) as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT).(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification, suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism has been probed by oxidation of the monomeric species dGuo, dGMP, and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP, and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with [(18)O(2)]-m-CPBA and H(2)(18)O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring-opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker.

Naturally-occurring modification restricts the anticodon domain conformational space of tRNA(Phe).

Post-transcriptional modifications contribute chemistry and structure to RNAs. Modifications of tRNA at nucleoside 37, 3'-adjacent to the anticodon, are particularly interesting because they facilitate codon recognition and negate translational frame-shifting. To assess if the functional contribution of a position 37-modified nucleoside defines a specific structure or restricts conformational flexibility, structures of the yeast tRNA(Phe) anticodon stem and loop (ASL(Phe)) with naturally occurring modified nucleosides differing only at position 37, ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)), and ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)), were determined by NMR spectroscopy and restrained molecular dynamics. The ASL structures had similarly resolved stems (RMSD approximately 0.6A) of five canonical base-pairs in standard A-form RNA. The "NOE walk" was evident on the 5' and 3' sides of the stems of both RNAs, and extended to the adjacent loop nucleosides. The NOESY cross-peaks involving U(33) H2' and characteristic of tRNA's anticodon domain U-turn were present but weak, whereas those involving the U(33) H1' proton were absent from the spectra of both ASLs. However, ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) exhibited the downfield shifted 31P resonance of U(33)pGm(34) indicative of U-turns; ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) did not. An unusual "backwards" NOE between Gm(34) and A(35) (Gm(34)/H8 to A(35)/H1') was observed in both molecules. The RNAs exhibited a protonated A(+)(38) resulting in the final structures having C(32).A(+)(38) intra-loop base-pairs, with that of ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) being especially well defined. A single family of low-energy structures of ASL(Phe)-(Cm(32),Gm(34), m(1)G(37),m(5)C(40)) (loop RMSD 0.98A) exhibited a significantly restricted conformational space for the anticodon loop in comparison to that of ASL(Phe)-(Cm(32),Gm(34),m(5)C(40)) (loop RMSD 2.58A). In addition, the ASL(Phe)-(Cm(32),Gm(34),m(1)G(37),m(5)C(40)) average structure had a greater degree of similarity to that of the yeast tRNA(Phe) crystal structure. A comparison of the resulting structures indicates that modification of position 37 affects the accuracy of decoding and the maintenance of the mRNA reading frame by restricting anticodon loop conformational space.

A metal-ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins.

The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are "winged" helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the alpha5 helix of one subunit, Asp84 O(delta1) (Asp104 in SmtB), His86 N(delta1) (His106), and two derived from the alpha5 helix of the other, His97' N(delta1) (His117') and His100' N(epsilon2) (Glu120'). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding N(epsilon2) face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn(1) SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices.

A 2-iminohydantoin from the oxidation of guanine.

The nucleobase guanine was oxidized with dimethyldioxirane (DMDO) to explore the role of epoxidizing agents in oxidative DNA damage. Treatment of guanine with 10% molar excess DMDO in aqueous solution at 0 degrees C and pH 7.5 followed by workup under mild conditions gave 5-carboxamido-5-formamido-2-iminohydantoin (1) as the sole isolable product in 71% yield. The structure of 1 was established on the basis of mass spectrometry and NMR studies on 1 and its isotopomers generated by the oxidation of [4-(13)C] and [7-(15)N]guanine, which yield [5-(13)C]1 and [7-(15)N]1. The distribution of 13C and 15N labels in the isotopomeric products supports initial epoxidation of the C4-C5 bond of guanine followed by a 1,2-acyl migration of guanine C6. Compound 1 is suggested as a possible primary DNA lesion from putative epoxidizing agents, including hydroperoxides present during biological processes such as lipid peroxidation.

Unprecedented head-to-head conformers of d(GpG) bound to the antitumor active compound tetrakis (mu-carboxylato)dirhodium(II,II).

The N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the DNA fragment d(GpG) have been unambiguously determined by NMR spectroscopy. Previous X-ray crystallographic determinations of the head-to-head (HH) and head-to-tail (HT) adducts of dirhodium tetraacetate with 9-ethylguanine (9-EtGH) revealed unprecedented bridging N7/O6 guanine nucleobases that span the Rh-Rh bond. The absence of N7 protonation at low pH and the notable increase in the acidity of N1-H (pK(a) approximately 5.7 as compared to 8.5 for N7 only bound platinum adducts), suggested by the pH dependence titrations of the purine H8 (1)H NMR resonances for Rh(2)(OAc)(2)(9-EtG)(2) and Rh(2)(OAc)(2-)[d(GpG)],are consistent with bidentate N7/O6 binding of the guanine nucleobases. The pK(a) values estimated for N1-H (de)protonation, from the pH dependence studies of the C6 and C2 (13)C NMR resonances for the Rh(2)(OAc)(2)(9-EtG)(2) isomers, concur with those derived from the H8 (1)H NMR resonance titrations. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(9-EtG)(2) and Rh(2)(OAc)(2)[d(GpG)] with the corresponding resonances of the unbound ligands [at pH 7.0 for 9-EtGH and pH 8.0 for d(GpG)], shows substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm for C6 and C2, respectively; the latter shifts reflect the effect of O6 binding to the dirhodium centers and the ensuing enhancement in the acidity of N1-H. Intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum of Rh(2)(OAc)(2)[d(GpG)] indicate head-to-head arrangement of the guanine bases. The Rh(2)(OAc)(2)[d(GpG)] adduct exhibits two major right-handed conformers, HH1 R and HH2 R, with HH1 R being three times more abundant than the unusual HH2 R. Complete characterization of both adducts revealed repuckering of the 5'-G sugar rings to C3'-endo (N-type), retention of C2'-endo (S-type) conformation for the 3'-G sugar rings, and anti orientation with respect to the glycosyl bonds. The structural features obtained for Rh(2)(OAc)(2))[d(GpG)] by means of NMR spectroscopy are very similar to those for cis-[Pt(NH(3))(2))[d(GpG)]] and corroborate molecular modeling studies.

Novel binding interactions of the DNA fragment d(pGpG) cross-linked by the antitumor active compound tetrakis(mu-carboxylato)dirhodium(II,II).

Insight into the N7/O6 equatorial binding interactions of the antitumor active complex Rh(2)(OAc)(4)(H(2)O)(2) (OAc(-) = CH(3)CO(2)(-)) with the nucleotide 5'-GMP and the DNA fragment d(pGpG) has been obtained by one- (1D) and two-dimensional (2D) NMR spectroscopy. The lack of N7 protonation at low pH values and the significant increase in the acidity of N1-H (pK(a) approximately 5.6 as compared to 8.5 for N7 only bound platinum adducts), indicated by the pH dependence study of the H8 (1)H NMR resonance for the HT (head-to-tail) isomer of Rh(2)(OAc)(2)(5'-GMP)(2), are consistent with bidentate N7/O6 binding of the guanine. The H8 (1)H NMR resonance of the HH (head-to-head) Rh(2)(OAc)(2)(5'-GMP)(2) isomer, as well as the 5'-G and 3'-G H8 resonances of the Rh(2)(OAc)(2) [d(pGpG)] adduct exhibit pH-independent titration curves, attributable to the added effect of the 5'-phosphate group deprotonation at a pH value similar to that of the N1 site. The enhancement in the acidity of N1-H, with respect to N7 only bound metal adducts, afforded by the O6 binding of the bases to the rhodium centers, has been corroborated by monitoring the pH dependence of the purine C6 and C2 (13)C NMR resonances for Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)]. The latter studies resulted in pK(a) values in good agreement with those derived from the pH-dependent (1)H NMR titrations of the H8 resonances. Comparison of the (13)C NMR resonances of C6 and C2 for the dirhodium adducts Rh(2)(OAc)(2)(5'-GMP)(2) and Rh(2)(OAc)(2) [d(pGpG)] with the corresponding resonances of the unbound ligands at pH 8.0, showed substantial downfield shifts of Deltadelta approximately 11.0 and 6.0 ppm, respectively. The HH arrangement of the bases in the Rh(2)(OAc)(2) [d(pGpG)] adduct is evidenced by intense H8/H8 ROE cross-peaks in the 2D ROESY NMR spectrum. The presence of the terminal 5'-phosphate group in d(pGpG) results in stabilization of one left-handed Rh(2)(OAc)(2) [d(pGpG)] HH1 L conformer, due to the steric effect of the 5'-group, favoring left canting in cisplatin-DNA adducts. Complete characterization of the Rh(2)(OAc)(2[d(pGpG)] adduct revealed notable structural features that resemble those of cis-[Pt(NH(3))(2) [d(pGpG)]]; the latter involve repuckering of the 5'-G sugar ring to C3'-endo (N-type) conformation, retention of C2'-endo (S-type) 3'-G sugar ring conformation, and anti orientation with respect to the glycosyl bonds. The superposition of the low energy Rh(2)(OAc)(2) [d(pGpG)] conformers, generated by simulated annealing calculations, with the crystal structure of cis-[Pt(NH(3))(2) [d(pGpG)]], reveals remarkable similarities between the adducts; not only are the bases almost completely destacked upon coordination to the metal in both cases, but they are favorably poised to accommodate the bidentate N7/O6 binding to the dirhodium unit. Unexpectedly, the two metal-metal bonded rhodium centers are capable of engaging in cis binding to GG intrastrand sites by establishing N7/O6 bridges that span the Rh-Rh bond.