Understanding How a New Hachimoji Nucleobase Alters Photodynamics of Genetic Building Blocks†.

This article is a highlight of the paper by Krul et al. in this issue of Photochemistry and Photobiology. It describes the excited state dynamics of 5-aza-7-deazaguanine (5N7C G), which has recently been proposed as an alternative nucleobase. Upon UV absorption to the lowest energy 1 ππ* state, 5N7C G returns to the electronic ground state an order of magnitude more slowly than guanine with a corresponding greater fluorescence quantum yield. These findings are significant because they suggest that 5N7C G is less UV photostable than its canonical nucleobase equivalent, which would have been a selective disadvantage in prebiotic conditions.

New light on the use of Theobroma cacao by Late Classic Maya.

Cacao seeds, Theobroma cacao, provide the basis for a ceremonially important Mesoamerican food. Past efforts to identify cacao in ceramics focused on highly decorative vessel forms associated with elite ceremonial contexts, creating assumptions as to how cacao was distributed and who could access it. This study examines 54 archaeological ceramic sherds from El Pilar (Belize/Guatemala) of Late Classic (600 to 900 CE) residential and civic contexts representing a cross-section of ancient Maya inhabitants. Identification of cacao in ancient sherds has depended on the general presence of theobromine; we used the discrete presence of theophylline, a unique key biomarker for cacao in the region. Analysis was done by grinding off all outside surfaces to reduce contamination, pulverizing the inner clay matrix, extracting absorbed molecules, and concentrating the extractions. In order to obtain especially high selectivity and low limits of detection, our study utilized the technique of resonance-enhanced multiphoton ionization coupled with laser-desorption jet-cooling mass spectrometry. This technique isolates molecules in the cold gas phase where they can be selectively ionized through a resonant two-photon process. Of the sherds analyzed, 30 samples (56%) were found to contain significant amounts of theophylline and thus test positive for cacao. Importantly, cacao is present in all contexts, common to all Maya residents near and far from centers.

Revealing the Structure and Noncovalent Interactions of Isolated Molecules by Laser-Desorption/Ionization-Loss Stimulated Raman Spectroscopy and Quantum Calculations.

The structural and dynamical characteristics of isolated molecules are essential, yet obtaining this information is difficult. We demonstrate laser-desorption jet-cooling/ionization-loss stimulated Raman spectroscopy to obtain Raman spectral signatures of nonvolatile molecules in the gas phase. The vibrational features of a test substance, the most abundant conformer of tryptamine, are compared and found to match those resulting from the scaled harmonic Raman spectrum obtained by density functional theory calculations. The vibrational signatures serve to identify the most prominent gauche conformer and evaluate its predicted electronic structure. These findings, together with noncovalent interaction (NCI) analysis, provide new insights into electron densities and reduced density gradients, assessing the hydrogen bonds (N-H···π and C-H···H-C) and interplay between attractive and repulsive NCIs affecting the structure. This approach accesses vibrational signatures of isolated nonvolatile molecules by tabletop lasers at uniform resolution and in a broad frequency range, promising great benefit to future studies.

Photodynamics of alternative DNA base isoguanine.

Isoguanine is an alternative nucleobase that has been proposed as a component of expanded genetic codes. It has also been considered as a molecule with potential relevance to primordial informational polymers. Here, we scrutinize the photodynamics of isoguanine, because photostability has been proposed as a critical criterion for the prebiotic selection of biomolecular building blocks on an early Earth. We discuss resonance-enhanced multiphoton ionization, IR-UV double resonance spectroscopy and pump-probe measurements performed for this molecule to track the excited-state behaviour of its different tautomeric forms in the gas phase. These experiments, when confronted with highly accurate quantum chemical calculations and nonadiabatic dynamics simulations provide a complete mechanistic picture of the tautomer-specific photodynamics of isoguanine. Our results indicate that UV-excited enol tautomers of isoguanine are relatively short lived and therefore photostable. In contrast, the biologically more relevant keto forms are trapped in dark nπ* states which are sufficiently long lived to participate in destructive photochemistry. The resulting lower photostability compared to canonical nucleobases may have been one of the reasons why isoguanine was not incorporated into DNA and RNA.

How nature covers its bases.

The response of DNA and RNA bases to ultraviolet (UV) radiation has been receiving increasing attention for a number of important reasons: (i) the selection of the building blocks of life on an early earth may have been mediated by UV photochemistry, (ii) radiative damage of DNA depends critically on its photochemical properties, and (iii) the processes involved are quite general and play a role in more biomolecules as well as in other compounds. A growing number of groups worldwide have been studying the photochemistry of nucleobases and their derivatives. Here we focus on gas phase studies, which (i) reveal intrinsic properties distinct from effects from the molecular environment, (ii) allow for the most detailed comparison with the highest levels of computational theory, and (iii) provide isomeric selectivity. From the work so far a picture is emerging of rapid decay pathways following UV excitation. The main understanding, which is now well established, is that canonical nucleobases, when absorbing UV radiation, tend to eliminate the resulting electronic excitation by internal conversion (IC) to the electronic ground state in picoseconds or less. The availability of this rapid "safe" de-excitation pathway turns out to depend exquisitely on molecular structure. The canonical DNA and RNA bases are generally short-lived in the excited state, and thus UV protected. Many closely related compounds are longer lived, and thus more prone to other, potentially harmful, photochemical processes. It is this structure dependence that suggests a mechanism for the chemical selection of the building blocks of life on an early earth. However, the picture is far from complete and many new questions now arise.

Excited-State Dynamics of Isocytosine: A Hybrid Case of Canonical Nucleobase Photodynamics.

We present resonant two-photon ionization (R2PI) spectra of isocytosine (isoC) and pump-probe results on two of its tautomers. IsoC is one of a handful of alternative bases that have been proposed in scenarios of prebiotic chemistry. It is structurally similar to both cytosine (C) and guanine (G). We compare the excited-state dynamics with the Watson-Crick (WC) C and G tautomeric forms. These results suggest that the excited-state dynamics of WC form of G may primarily depend on the heterocyclic substructure of the pyrimidine moiety, which is chemically identical to isoC. For WC isoC we find a single excited-state decay with a rate of ∼1010 s-1, while the enol form has multiple decay rates, the fastest of which is 7 times slower than for WC isoC. The excited-state dynamics of isoC exhibits striking similarities with that of G, more so than with the photodynamics of C.

Excited State Dynamics of 6-Thioguanine.

Here we present the excited state dynamics of jet-cooled 6-thioguanine (6-TG), using resonance-enhanced multiphoton ionization (REMPI), IR-UV double resonance spectroscopy, and pump-probe spectroscopy in the nanosecond and picosecond time domains. We report data on two thiol tautomers, which appear to have different excited state dynamics. These decay to a dark state, possibly a triplet state, with rates depending on tautomer form and on excitation wavelength, with the fastest rate on the order of 1010 s-1. We also compare 6-TG with 9-enolguanine, for which we observed decay to a dark state with a 2 orders of magnitude smaller rate. At increased excitation energy (∼+500 cm-1) an additional pathway appears for the predominant thiol tautomer. Moreover, the excited state dynamics for 6-TG thiols is different from that recently predicted for thiones.

Direct Analysis of Xanthine Stimulants in Archaeological Vessels by Laser Desorption Resonance Enhanced Multiphoton Ionization.

Resonance enhanced multiphoton ionization spectroscopy (REMPI) generates simultaneous vibronic spectroscopy and fragment free mass spectrometry to identify molecules within a complex matrix. We combined laser desorption with REMPI spectroscopy to study organic residues within pottery sherds from Maya vessels (600-900 CE) and Mississippian vessels (1100-1200 CE), successfully detecting three molecular markers, caffeine, theobromine, and theophylline, associated with the use of cacao. This analytical approach provides a high molecular specificity, based on both wavelength and mass identification. At the same time, the high detection limit allows for direct laser desorption from sherd scrapings, avoiding the need for extracting organic constituents from the sherd matrix.

Life in the light: nucleic acid photoproperties as a legacy of chemical evolution.

Photophysical investigations of the canonical nucleobases that make up DNA and RNA during the past 15 years have revealed that excited states formed by the absorption of UV radiation decay with subpicosecond lifetimes (i.e., <10(-12) s). Ultrashort lifetimes are a general property of absorbing sunscreen molecules, suggesting that the nucleobases are molecular survivors of a harsh UV environment. Encoding the genome using photostable building blocks is an elegant solution to the threat of photochemical damage. Ultrafast excited-state deactivation strongly supports the hypothesis that UV radiation played a major role in shaping molecular inventories on the early Earth before the emergence of life and the subsequent development of a protective ozone shield. Here, we review the general physical and chemical principles that underlie the photostability, or "UV hardiness", of modern nucleic acids and discuss the possible implications of these findings for prebiotic chemical evolution. In RNA and DNA strands, much longer-lived excited states are observed, which at first glance appear to increase the risk of photochemistry. It is proposed that the dramatically different photoproperties that emerge from assemblies of photostable building blocks may explain the transition from a world of molecular survival to a world in which energy-rich excited electronic states were eventually tamed for biological purposes such as energy transduction, signaling, and repair of the genetic machinery.

Resonant Infrared Multiple Photon Dissociation Spectroscopy of Anionic Nucleotide Monophosphate Clusters.

We report mid-infrared spectra and potential energy surfaces of four anionic, 2'-deoxynucleotide-5'-monophosphates (dNMPs) and the ionic DNA pairs [dGMP-dCMP-H](1-), [dAMP-dTMP-H](1-) with a total charge of the complex equal to -1. We recorded IR action spectra by resonant IR multiple-photon dissociation (IRMPD) using the FELIX free electron laser. The potential energy surface study employed an on-the-fly molecular dynamics quenching method (MD/Q), using a semiempirical AM1 method, followed by an optimization of the most stable structures using density functional theory. By employing infrared multiple-photon dissociation (IRMPD) spectroscopy in combination with high-level computational methods, we aim at a better understanding of the hydrogen bonding competition between the phosphate moieties and the nucleobases. We find that, unlike in multimer double stranded DNA structures, the hydrogen bonds in these isolated nucleotide pairs are predominantly formed between the phosphate groups. This intermolecular interaction appears to exceed the stabilization energy resulting from base pairing and directs the overall cluster structure and alignment.

Gas-Phase IR Spectroscopy of Nucleobases.

IR spectroscopy of nucleobases in the gas phase reflects simultaneous advances in both experimental and computational techniques. Important properties, such as excited state dynamics, depend in subtle ways on structure variations, which can be followed by their infrared signatures. Isomer specific spectroscopy is a particularly powerful tool for studying the effects of nucleobase tautomeric form and base pair hydrogen-bonding patterns.

UV-excitation from an experimental perspective: frequency resolved.

Electronic spectroscopy of DNA bases in the gas phase provides detailed information about the electronic excitation, which places the molecule in the Franck-Condon region in the excited state and thus prepares the starting conditions for excited-state dynamics. Double resonance or hole-burning spectroscopy in the gas phase can provide such information with isomer specificity, probing the starting potential energy landscape as a function of tautomeric form, isomeric structure, or hydrogen bonded or stacked cluster structure. Action spectroscopy, such REMPI, can be affected by excited-state lifetimes.

Structure of 2,4-diaminopyrimidine-theobromine alternate base pairs.

We report the structure of clusters of 2,4-diaminopyrimidine with 3,7-dimethylxanthine (theobromine) in the gas phase determined by IR-UV double resonance spectroscopy in both the near-IR and mid-IR regions in combination with ab initio computations. These clusters represent potential alternate nucleobase pairs, geometrically equivalent to guanine-cytosine. We have found the four lowest energy structures, which include the Watson-Crick base pairing motif. This Watson-Crick structure has not been observed by resonant two-photon ionization (R2PI) in the gas phase for the canonical DNA base pairs.

Isolated gramicidin peptides probed by IR spectroscopy.

We report double-resonant IR/UV ion-dip spectroscopy of neutral gramicidin peptides in the gas phase. The IR spectra of gramicidin A and C, recorded in both the 1000 cm(-1) to 1800 cm(-1) and the 2700 to 3750 cm(-1) region, allow structural analysis. By studying this broad IR range, various local intramolecular interactions are probed, and complementary IR modes can be accessed. Ab initio quantum chemical calculations are used to support the interpretation of the experimental IR spectra. The comparison of the calculated frequencies with the experimental IR spectrum probed via the strong infrared absorptions of all the amide groups (NH stretch, C=O stretch and NH bend), shows evidence for a helical structure in the gas phase, which is similar to that in the condensed phase. Additionally, we show that to improve the spectral resolution when studying large neutral molecular structures of the size of gramicidin, the use of heavier carrier gas could be advantageous.

Conformational structure of tyrosine, tyrosyl-glycine, and tyrosyl-glycyl-glycine by double resonance spectroscopy.

We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YG) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine-glycine (FGG) and tryptophan-glycine-glycine (WGG); however, the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

The effect of C5 substitution on the photochemistry of uracil.

A combined experimental and theoretical study on the excited-state behavior of the uracil analogues, 5-OH-Ura and 5-NH(2)-Ura is reported. Two-photon ionization and IR/UV double-resonant spectra show that there is only one tautomer present for each with an excited state lifetime of 1.8 ns for 5-OH-Ura and 12.0 ns for 5-NH(2)-Ura as determined from pump-probe experiments. The nature of the excited states of both species is investigated by means of multi-reference ab initio methods. Vertical excitation energies, excited state minima, minima on the crossing seam and reaction paths towards them are determined. Sizeable barriers on these paths are found that provide an explanation for the lifetimes of several nanoseconds observed in the experiment.

Effect of substituents on the excited-state dynamics of the modified DNA bases 2,4-diaminopyrimidine and 2,6-diaminopurine.

To explore the excited state dynamics of pyrimidine derivatives, we performed a combined experimental and theoretical study. We present resonant two-photon ionization (R2PI) and IR-UV double resonance spectra of 2,4-diaminopyrimidine and 2,6-diaminopurine seeded in a supersonic jet by laser desorption. For 2,4-diaminopyrimidine (S(0)-->S(1) 34,459 cm(-1)), we observed only the diamino tautomer with an excited state lifetime bracketed between experimental limits of 10 ps and 1 ns. For 2,6-diaminopurine, we observed two tautomers, the 9H- (S(0)-->S(1) 34,881 cm(-1)) and 7H- (S(0)-->S(1) 32,215 cm(-1)) diamino forms, with excited state lifetimes of 6.3±0.4 ns and 8.7±0.8 ns, respectively. We investigated the nature of the excited state of 2,4-diaminopyrimidine by means of multi-reference ab initio methods. The calculations of stationary points in the ground and excited states, minima on the S(0)/S(1) crossing seam and connecting reaction paths show that several paths with negligible barriers exist, allowing ultrafast radiationless deactivation if excited at energies slightly higher than the band origin. The sub-nanosecond lifetime found experimentally is in good agreement with this finding.

Guanine-aspartic acid interactions probed with IR-UV resonance spectroscopy.

Double resonance spectroscopy of clusters of guanine with aspartic acid reveals geometries similar to patterns exhibited in DNA base pairs. In the spectral region of 32,800 cm(-1) to 35,500 cm(-1) we observe five isomers of guanine-aspartic acid clusters and assign their structures based on IR-UV hole-burning spectra and wave function theory calculations at the MP2/cc-pVDZ and MP2/cc-pVTZ levels. The calculations employed both harmonic and one-dimensional scan anharmonic approximations. Three of the isomers are similar, assigned to structures containing three hydrogen bonds and 9-enolguanine. We assign the fourth isomer to a structure containing a 9-keto tautomer of guanine and forming a triply bonded structure similar to a base pairing interaction. The fifth isomer dissociates with proton transfer upon excitation or ionization. This is the first set of experiments and high-level ab initio calculations of the isolated, microscopic interactions of an amino acid and a nucleobase, the building blocks of nucleic acids and proteins.