Structural Determination of Lysine-Linked Cisplatin Complexes via IRMPD Action Spectroscopy: NNs and NO- Binding Modes of Lysine to Platinum Coexist.

Despite its success as an anticancer drug, cisplatin suffers from resistance and produces side effects. To overcome these limitations, amino-acid-linked cisplatin analogues have been investigated. Lysine-linked cisplatin, Lysplatin, (Lys)PtCl2, exhibited outstanding reactivity toward DNA and RNA that differs from that of cisplatin. To gain insight into its differing reactivity, the structure of Lysplatin is examined here using infrared multiple photon dissociation (IRMPD) action spectroscopy. To probe the influence of the local chemical environment on structure, the deprotonated and sodium-cationized Lysplatin complexes are examined. Electronic structure calculations are performed to explore possible modes of binding of Lys to Pt, their relative stabilities, and to predict their infrared spectra. Comparisons of the measured IRMPD and predicted IR spectra elucidate the structures contributing to the experimental spectra. Coexistence of two modes of binding of Lys to Pt is found where Lys binds via the backbone and side-chain amino nitrogen atoms, NNs, or to the backbone amino and carboxylate oxygen atoms, NO-. Glycine-linked cisplatin and arginine-linked cisplatin complexes have previously been found to bind only via the NO- binding mode. Present results suggest that the NNs binding conformers may be key to the outstanding reactivity of Lysplatin toward DNA and RNA.

Structural determination of arginine-linked cisplatin complexes via IRMPD action spectroscopy: arginine binds to platinum via NO- binding mode.

Cisplatin, (NH3)2PtCl2, has been known as a successful metal-based anticancer drug for more than half a century. Its analogue, Argplatin, arginine-linked cisplatin, (Arg)PtCl2, is being investigated because it exhibits reactivity towards DNA and RNA that differs from that of cisplatin. In order to understand the basis for its altered reactivity, the deprotonated and sodium cationized forms of Argplatin, [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy in the IR fingerprint and hydrogen-stretching regions. Complementary electronic structure calculations are performed using density functional theory approaches to characterize the stable structures of these complexes and to predict their infrared spectra. Comparison of the theoretical IR spectra predicted for various stable conformations of these Argplatin complexes to their measured IRMPD spectra enables determination of the binding mode(s) of Arg to the Pt metal center to be identified. Arginine is found to bind to Pt in a bidentate fashion to the backbone amino nitrogen and carboxylate oxygen atoms in both the [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+ complexes, the NO- binding mode. The neutral side chain of Arg also interacts with the Pt center to achieve additional stabilization in the [(Arg-H)PtCl2]- complex. In contrast, Na+ binds to both chlorido ligands in the [(Arg)PtCl2 + Na]+ complex and the protonated side chain of Arg is stabilized via hydrogen-bonding interactions with the carboxylate moiety. These findings are consistent with condensed-phase results, indicating that the NO- binding mode of arginine to Pt is preserved in the electrospray ionization process even under variable pH and ionic strength.

Structures and Relative Glycosidic Bond Stabilities of Protonated 2'-Fluoro-Substituted Purine Nucleosides.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H]+, and 2'-fluoro-2'-deoxyguanosine, [Guofl+H]+, are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H]+, stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H]+. Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H]+ adopts C2'-endo sugar puckering, whereas [Guofl+H]+ adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

Impact of the 2'- and 3'-Sugar Hydroxyl Moieties on Gas-Phase Nucleoside Structure.

Modified nucleosides have been an important target for pharmaceutical development for the treatment of cancer, herpes simplex virus, and the human immunodeficiency virus (HIV). Amongst these nucleoside analogues, those based on 2',3'-dideoxyribose sugars are quite common, particularly in anti-HIV applications. The gas-phase structures of several protonated 2',3'-dideoxyribose nucleosides are examined in this work and compared with those of the analogous protonated DNA, RNA, and arabinose nucleosides to elucidate the influence of the 2'- and combined 2',3'-hydroxyl groups on intrinsic structure. Infrared multiple photon dissociation (IRMPD) action spectra are collected for the protonated 2',3'-dideoxy forms of adenosine, guanosine, cytidine, thymidine and uridine, [ddAdo+H]+, [ddGuo+H]+, [ddCyd+H]+, [ddThd+H]+, and [ddUrd+H]+, in the IR fingerprint and hydrogen-stretching regions. Molecular mechanics conformational searching followed by electronic structure calculations generates low-energy conformers of the protonated 2',3'-dideoxynucleosides and corresponding predicted linear IR spectra to facilitate interpretation of the measured IRMPD action spectra. These experimental IRMPD spectra and theoretical calculations indicate that the absence of the 2'- and 3'-hydroxyls largely preserves the protonation preferences of the canonical forms. The spectra and calculated structures indicate a slight preference for C3'-endo sugar puckering. The presence of the 3'- and further 2'-hydroxyl increases the available intramolecular hydrogen-bonding opportunities and shifts the sugar puckering modes for all nucleosides but the guanosine analogues to a slight preference for C2'-endo over C3'-endo. Graphical Abstract.

Modified Quadrupole Ion Trap Mass Spectrometer for Infrared Ion Spectroscopy: Application to Protonated Thiated Uridines.

Modifications to a Paul-type quadrupole ion trap mass spectrometer providing optical access to the trapped ion cloud as well as hardware and software for coupling to a table-top IR optical parametric oscillator laser (OPO) are detailed. Critical experimental parameters for infrared multiple photon dissociation (IRMPD) on this instrument are characterized. IRMPD action spectra, collected in the hydrogen-stretching region with this instrument, complemented by spectra in the IR fingerprint region acquired at the FELIX facility, are employed to characterize the structures of the protonated forms of 2-thiouridine, [s2Urd+H]+, and 4-thiouridine, [s4Urd+H]+. The measured spectra are compared with predicted linear IR spectra calculated at the B3LYP/6-311+G(d,p) level of theory to determine the conformers populated in the experiments. This comparison indicates that thiation at the 2- or 4-positions shifts the protonation preference between the 2,4-H tautomer and 4-protonation in opposite directions versus canonical uridine, which displays a roughly equal preference for the 2,4-H tautomer and O4 protonation. As found for canonical uridine, protonation leads to a mixture of conformers exhibiting C2'-endo and C3'-endo sugar puckering with an anti nucleobase orientation being populated for both 2- and 4-thiated uridine. Graphical Abstract ᅟ.

Influence of Transition Metal Cationization versus Sodium Cationization and Protonation on the Gas-Phase Tautomeric Conformations and Stability of Uracil: Application to [Ura+Cu]+ and [Ura+Ag].

The gas-phase conformations of transition metal cation-uracil complexes, [Ura+Cu]+ and [Ura+Ag]+, were examined via infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. IRMPD action spectra were measured over the IR fingerprint and hydrogen-stretching regions. Structures and linear IR spectra of the stable tautomeric conformations of these complexes were initially determined at the B3LYP/6-31G(d) level. The four most stable structures computed were also examined at the B3LYP/def2-TZVPPD level to improve the accuracy of the predicted IR spectra. Two very favorable modes of binding are found for [Ura+Cu]+ and [Ura+Ag]+ that involve O2N3 bidentate binding to the 2-keto-4-hydroxy minor tautomer and O4 monodentate binding to the canonical 2,4-diketo tautomer of Ura. Comparisons between the measured IRMPD and calculated IR spectra enable elucidation of the conformers present in the experiments. These comparisons indicate that both favorable binding modes are represented in the experimental tautomeric conformations of [Ura+Cu]+ and [Ura+Ag]+. B3LYP suggests that Cu+ exhibits a slight preference for O4 binding, whereas Ag+ exhibits a slight preference for O2N3 binding. In contrast, MP2 suggests that both Cu+ and Ag+ exhibit a more significant preference for O2N3 binding. The relative band intensities suggest that O4 binding conformers comprise a larger portion of the population for [Ura+Ag]+ than [Ura+Cu]+. The dissociation behavior and relative stabilities of the [Ura+M]+ complexes, M+ = Cu+, Ag+, H+, and Na+) are examined via energy-resolved collision-induced dissociation experiments. The IRMPD spectra, dissociation behaviors, and binding preferences of Cu+ and Ag+ are compared with previous and present results for those of H+ and Na+. Graphical Abstract ᅟ.

IRMPD Action Spectroscopy, ER-CID Experiments, and Theoretical Studies of Sodium Cationized Thymidine and 5-Methyluridine: Kinetic Trapping During the ESI Desolvation Process Preserves the Solution Structure of [Thd+Na].

Thymidine (dThd) is a fundamental building block of DNA nucleic acids, whereas 5-methyluridine (Thd) is a common modified nucleoside found in tRNA. In order to determine the conformations of the sodium cationized thymine nucleosides [dThd+Na]+ and [Thd+Na]+ produced by electrospray ionization, their infrared multiple photon dissociation (IRMPD) action spectra are measured. Complementary electronic structure calculations are performed to determine the stable low-energy conformations of these complexes. Geometry optimizations and frequency analyses are performed at the B3LYP/6-311+G(d,p) level of theory, whereas energies are calculated at the B3LYP/6-311+G(2d,2p) level of theory. As protonation preferentially stabilizes minor tautomers of dThd and Thd, tautomerization facilitated by Na+ binding is also considered. Comparisons of the measured IRMPD and computed IR spectra find that [dThd+Na]+ prefers tridentate (O2,O4',O5') coordination to the canonical 2,4-diketo form of dThd with thymine in a syn orientation. In contrast, [Thd+Na]+ prefers bidentate (O2,O2') coordination to the canonical 2,4-diketo tautomer of Thd with thymine in an anti orientation. Although 2,4-dihydroxy tautomers and O2 protonated thymine nucleosides coexist in the gas phase, no evidence for minor tautomers is observed for the sodium cationized species. Consistent with experimental observations, the computational results confirm that the sodium cationized thymine nucleosides exhibit a strong preference for the canonical form of the thymine nucleobase. Survival yield analyses based on energy-resolved collision-induced dissociation (ER-CID) experiments suggest that the relative stabilities of protonated and sodium cationized dThd and Thd follow the order [dThd+H]+ < [Thd+H]+ < [dThd+Na]+ < [Thd+Na]+. Graphical Abstract ᅟ.

Effects of sodium cationization versus protonation on the conformations and N-glycosidic bond stabilities of sodium cationized Urd and dUrd: solution conformation of [Urd+Na]+ is preserved upon ESI.

Uridine (Urd) is one of the naturally occurring pyrimidine nucleosides of RNA. 2'-Deoxyuridine (dUrd) is a naturally occurring modified form of Urd, but is not one of the canonical DNA nucleosides. In order to understand the effects of sodium cationization on the conformations and energetics of Urd and dUrd, infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and density functional theory (DFT) calculations are performed. By comparing the calculated IR spectra of [Urd+Na]+ and [dUrd+Na]+ with the measured IRMPD spectra, the stable low-energy conformers populated in the experiments are determined. Anti oriented bidentate O2 and O2' binding conformers of [Urd+Na]+ are the dominant conformers populated in the experiments, whereas syn oriented tridentate O2, O4', and O5' binding conformers of [dUrd+Na]+ are dominantly populated in the experiments. The 2'-hydroxyl substituent of Urd stabilizes the anti oriented O2 binding conformers of [Urd+Na]+. Significant differences between the measured IRMPD and calculated IR spectra for complexes of [Urd+Na]+ and [dUrd+Na]+ involving minor tautomeric forms of the nucleobase make it obvious that none are populated in the experiments. Survival yield analyses based on energy-resolved collision-induced dissociation (ER-CID) experiments suggest that the relative stabilities of protonated and sodium cationized Urd and dUrd follow the order: [dUrd+H]+ < [Urd+H]+ < [dUrd+Na]+ < [Urd+Na]+. The 2'-deoxy modification is found to weaken the glycosidic bond of dUrd versus that of Urd for the sodium cationized uridine nucleosides.

Binding characteristics of anti-Rh0(D) antibodies to Rh0(D)-positive and Du red cells.

The relation of human erythrocyte Rh0(D) to Du sites is an important unresolved question in the field of immunohematology. To compare the immunological reactivity of Rh0(D)-positive and Du erythrocytes, the binding characteristics of two anti-Rh0(D) antisera to human Rh0(D)-positive and Du ("low-grade") erythrocytes were studied. 14C-Protein A and direct antibody-labeled techniques were used to generate binding curves and to derive double-reciprocal plots. The results show that the number of antigen sites differ by a factor of 10 to 15 between the Rh0(D)-positive and Du red cells, but that the dissociation constants between anti-Rh0(D) and the Rh0(D) and Du antigens are indistinguishable when studied by the two labeling methods and two different anti-Rh0(D) antibodies. The extent of binding to 112 different Du samples showed a normal distribution and was independent of apparent phenotype. These data suggest immunologic identity of Rh0(D) and Du ("low-grade") sites and that the difference between the antigens of Rh0(D) and Du cells is quantitative only. The data are incompatible with the "missing mosaic" and gene interaction theories of mechanism.