The scope of this review is the nature and dynamics of the singlet excited electronic states created in nucleic acids and their constituents by UV light. Interest in the UV photochemistry of nucleic acids has long been the motivation for photophysical studies of the excited states, because these states are at the beginning of the complex chain of events that culminates in photodamage. UV-induced damage to DNA has profound biological consequences, including photocarcinogenesis, a growing human health problem.1-3 Sunlight, which is essential for life on earth, contains significant amounts of harmful UV (λ < 400 nm) radiation. These solar UV photons constitute one of the most ubiquitous and potent environmental carcinogens. This extraterrestrial threat is impressive for its long history; photodamage is as old as life itself. The genomic information encoded by these biopolymers has been under photochemical attack for billions of years. It is not surprising then that the excited states of the nucleic acid bases (see Chart 1), the most important UV chromophores of nucleic acids, are highly stable to photochemical decay, perhaps as a result of selection pressure during a long period of molecular evolution. This photostability is due to remarkably rapid decay pathways for electronic energy, which are only now coming into focus through femtosecond laser spectroscopy. The recently completed map of the human genome and the ever-expanding crystallographic database of nucleic acid structures are two examples that illustrate the richly detailed information currently available about the static properties of nucleic acids. In contrast, much less is known about the dynamics of these macromolecules. This is particularly true of the dynamics of the excited states that play a critical role in DNA photodamage. Efforts to study nucleic acids by time-resolved spectroscopy have been stymied by the apparent lack of suitable fluorophores. In contrast, dynamical spectroscopy of proteins has flourished thanks to intrinsically fluorescent amino acids such as tryptophan, tyrosine, and phenylalanine.4 The primary UVabsorbing constituents of nucleic acids, the nucleic acid bases, have vanishingly small fluorescence quantum yields under physiological conditions of temperature and pH.5 In fact, the bases were frequently described as “nonfluorescent” in the early literature. * To whom correspondence should be addressed. E-mail: kohler@ chemistry.ohio-state.edu. Phone: (614) 688-3944. Fax: (614) 2921685. 1977 Chem. Rev. 2004, 104, 1977−2019

Ultrafast Excited-State Dynamics in Nucleic Acids

The combined results of ab initio electronic-structure calculations and spectroscopic investigations of jet-cooled molecules and clusters provide strong evidence of a surprisingly simple and general mechanistic picture of the nonradiative decay of biomolecules such as nucleic bases and aromatic amino acids. The key role in this picture is played by excited singlet states of πσ* character, which have repulsive potential-energy functions with respect to the stretching of OH or NH bonds. The 1πσ* potential-energy functions intersect not only the bound potential-energy functions of the 1ππ* excited states, but also that of the electronic ground state. Via predissociation of the 1ππ* states and a conical intersection with the ground state, the 1πσ* states trigger an ultrafast internal-conversion process, which is essential for the photostability of biomolecules.
In protic solvents, the 1πσ* states promote a hydrogen-transfer process from the chromophore to the solvent. Calculations for chromophore–water clusters have shown that a spontaneous charge-separation process takes place in the solvent shell, yielding a microsolvated hydronium cation and a microsolvated electron. These results suggest that the basic mechanisms of the complex photochemistry of biomolecules in liquid water can be revealed by experimental and theoretical investigations of relatively small chromophore–water clusters.

Excited-state hydrogen detachment and hydrogen transfer driven by repulsive 1πσ* states: A new paradigm for nonradiative decay in aromatic biomolecules

Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

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Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

We propose and discuss an efficient scheme for the in silico sampling for parts of the molecular chemical space by semiempirical tight-binding methods combined with a meta-dynamics driven search algorithm. The focus of this work is set on the generation of proper thermodynamic ensembles at a quantum chemical level for conformers, but similar procedures for protonation states, tautomerism and non-covalent complex geometries are also discussed. The conformational ensembles consisting of all significantly populated minimum energy structures normally form the basis of further, mostly DFT computational work, such as the calculation of spectra or macroscopic properties. By using basic quantum chemical methods, electronic effects or possible bond breaking/formation are accounted for and a very reasonable initial energetic ranking of the candidate structures is obtained. Due to the huge computational speedup gained by the fast low-cost quantum chemical methods, overall short computation times even for systems with hundreds of atoms (typically drug-sized molecules) are achieved. Furthermore, specialized applications, such as sampling with implicit solvation models or constrained conformational sampling for transition-states, metal-, surface-, or noncovalently bound complexes are discussed, opening many possible applications in modern computational chemistry and drug discovery. The procedures have been implemented in a freely available computer code called CREST, that makes use of the fast and reliable GFNn-xTB methods.

Automated exploration of the low-energy chemical space with fast quantum chemical methods

The development of femtosecond time-resolved methods for the study of gas-phase molecular dynamics is founded upon the seminal studies of Zewail and co-workers, as recognized in 1999 by the Nobel Prize in Chemistry.1 This methodology has been applied to chemical reactions ranging in complexity from bond-breaking in diatomic molecules to dynamics in larger organic and biological molecules, and has led to breakthroughs in our understanding of fundamental chemical processes. Photoexcited polyatomic molecules and anions often exhibit quite complex dynamics involving the redistribution of both charge and energy.2-6 These processes are the primary steps in the photochemistry of many polyatomic systems,7 are important in photobiological processes such as vision and photosynthesis,8 and underlie many concepts in molecular electronics.9 Femtosecond time-resolved methods involve a pump-probe configuration in which an ultrafast pump pulse initiates a reaction or, more generally, creates a nonstationary state or wave packet, the evolution of which is monitored as a function of time by means of a suitable probe pulse. Time-resolved or wave packet methods offer a view complementary to the usual spectroscopic approach and often yield a physically intuitive picture. Wave packets can behave as zeroth-order or even classical-like states and are therefore very helpful in discerning underlying dynamics. The information obtained from these experiments is very much dependent on the nature of the final state chosen in a given probe scheme. Transient absorption and nonlinear wave mixing are often the methods of choice in condensed-phase experiments because of their generality. In studies of molecules and clusters in the gas phase, the most popular methods, laser-induced fluorescence and resonant multiphoton ionization, usually require the probe laser to be resonant with an electronic transition in the species being monitored. However, as a chemical reaction initiated by the pump pulse evolves toward products, one expects that both the electronic and vibrational structures of the species under observation will change. Hence, these probe methods can be * To whom corresondence should be addressed. A.S.: telephone (613) 993-7388, fax (613) 991-3437, E-mail albert.stolow@nrc.ca. D.M.N.: telephone (510) 642-3505, fax (510) 642-3635, E-mail dan@radon.cchem.berkeley.edu. 1719 Chem. Rev. 2004, 104, 1719−1757

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Femtosecond time-resolved photoelectron spectroscopy.

The vibronic spectrum of laser desorbed and jet cooled guanine consists of bands from three different tautomers of guanine as revealed by UV–UV and IR–UV double resonance spectroscopy. 1-methylguanine, in which the Keto–Enol tautomerism is blocked, shows hole burning spectra from the 9H-and 7H-Keto form. A comparison of the vibronic pattern of the different tautomers demonstrates that the vibronic spectrum built on the redmost guanine band at 32 870 cm−1 (electronic origin 0) can be traced back to the 9H-Enol tautomer, while the spectra built on the origins at 0+404 cm−1 and 0+1044 cm−1 stem from the two Keto tautomers. The IR–UV double resonance spectra of the OH-and NH-stretch vibrations of the different tautomers support this assignment. The UV and IR spectra can be partly assigned by comparison with ab initio calculated vibrational frequencies and with the help of deuteration experiments.

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Guanine tautomerism revealed by UV–UV and IR–UV hole burning spectroscopy

We present R2PI spectra of a series of purine nucleobases, nucleosides, and related derivatives, including adenine, guanine, guanosine, 2-aminopurine, 2,6-diaminopurine, 9-ethyladenine, and 9-methy...

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On the Photochemistry of Purine Nucleobases

In this paper we motivate the study of biomolecular building blocks under isolated or well defined conditions. We explain why we believe that especially gas phase investigations in combination with quantum chemical calculations can provide new insights into molecular properties, such as structure, molecular recognition, reactivity and photostability. Although the gas phase represents far from in situ conditions, these findings are important for a detailed understanding of biology. We give a short historic overview of gas phase studies of biomolecular building blocks under isolated conditions, present some examples and report the current status of this field. We explain the new quality of synergy between experiment and quantum theory and the unique opportunities therein to discover new pathways for reactivity and to understand biological processes on an atomic level. We sketch the content of this special issue and give a further perspective of the research field of “Spectroscopy of biomolecular building blocks under isolated or defined conditions” and explain the possible connectivity to biology.

Molecular physics of building blocks of life under isolated or defined conditions

To observe fundamental properties of DNA building blocks it is desirable to study individual nucleosides in the gas phase without interference from solvent molecules, or macromolecular structure. As a first step, we have recently reported the first vibronic spectrum of the nucleobase guanine, obtained by a combination of laser desorption, jet cooling, and resonance enhanced multiphoton ionization (REMPI).1 Although guanine is important as a chromophore in DNA, it is more realistic for understanding the photochemistry of DNA to study the nucleosides. Those are even harder to vaporize intact because they are thermally more labile and, with their larger molecular weights, have still lower vapor pressures. Using laser desorption, we have now succeeded in forming a molecular beam of nucleosides, and we report the first REMPI spectra of a series of individual guanosines, namely guanosine (Gs), 2′deoxyguanosine (2′deoxyGs), and 3′deoxyguanosine (3′deoxyGs). We compare our results with computations at the HF 6-31G(d,p) level. The results suggest the occurrence of two different conformations, each probably stabilized by internal hydrogen bonds. One of those two conformations is absent in 2′deoxyGs implying that the 2′ hydroxyl group is required for its stabilization. Spectroscopic properties of guanosines have been studied primarily by Raman techniques in solution.2-9 A great deal of attention has been given to potential Raman markers for hydrogen bonding and for structural conformation. Observation of hydrogen bonding by Raman spectroscopy requires identification of vibrations that depend strongly on those specific atoms in guanine, that serve as either proton donor or acceptor. However, most vibrations involve the concerted motion of multiple atoms, and therefore correlation of marker frequencies with specific hydrogen bonding sites is not straightforward. Guanosine vibrations involving motion along the glycosidic bond may provide conformational markers if their frequencies are sensitive to puckering of the ribose ring or for rotation around the sugar-base bond. Interpretation of these markers requires careful analysis of complex vibrational modes. On the other hand, different conformations can be observed much more directly by vibronic spectroscopy when they produce multiple origins. As we will show below, we observe two origins in our spectra, which we can associate with the syn and the anti orientations of the base relative to the ribose moiety. We have published details of our setup for laser desorption jet cooling REMPI spectrometry elsewhere.10 Sample preparation consisted of depositing neat material in powder form on graphite substrates. We moved the substrate slowly while acquiring spectra, gradually exposing fresh material. For desorption we used pulses from a Nd:YAG laser at 1064 nm with fluences on the order of 1 mJ/cm2. Desorbed neutral molecules were entrained in a supersonic expansion with Ar drive gas, injected by a pulsed solenoid valve. Downstream, the entrained molecules were onecolor two-photon photoionized, and the ions were detected in a reflectron time-of-flight mass spectrometer. The first photon resonantly excites the molecule, while a second photon from the same laser ionizes the excited molecule. By varying the wavelength while monitoring specific mass peaks we obtained mass selected excitation spectra. The typical ionization laser fluence was on the order of 0.1 mJ/cm2. Figure 1 shows the REMPI spectra of (a) Gs, (b) 3′deoxyGs, and (c) 2′deoxyGs. We assign the lowest-energy peak in each of the spectra as a 0-0 transition to the S1 excited state. Careful scans to lower energy by 1000 cm-1 do not reveal any additional peaks. The same was true when performing two-color ionization with a second photon at 193 nm. Therefore, we do not believe that we are observing a cutoff in the spectrum related to the ionization potential of Gs. Furthermore we have measured the ionization potential of guanine as 8.1 eV by two-color ionization.11,12 That is 1 eV less than the two-photon energy at the Gs origin. † Heinrich Heine Universität. (1) Nir, E.; Grace, L.; Brauer, B.; de Vries, M. S. J. Am. Chem. Soc. 1999, 121, 4896. (2) Faurskov-Nielsen, O.; Lund, P.; Petersen, S. J. Raman Spectrosc. 1981, 11, 493. (3) Nishimura, Y.; Tsuboi, M.; Sato, T.; Aoki, K. J. Mol. Struct. 1986, 146, 123. (4) Chinsky, L.; Jolles, B.; Laigle, A.; Turpin, P. J. Raman Spectrosc. 1987, 18, 195. (5) Carmona, P.; Molina, M. J. Mol. Struct. 1990, 219, 323. (6) Toyama, A.; Takino, Y.; Takeuchi, H.; Harada, I. J. Am. Chem. Soc. 1993, 115, 11092. (7) Urabe, H.; Sugawara, Y.; Kasuya, T. Phys. ReV. B 1995, 51, 5666. (8) Toyama, A.; Hamuara, M.; Takeuchi, H. J. Mol. Struct. 1996, 15, 99. (9) Toyama, A.; Hanada, N.; Ono, J.; Yoshimitsu, E.; Takeuchi, H. J. Raman Spectrosc. 1999, 30, 623. (10) Meijer, G.; de Vries, M. S.; Hunziker, H. E.; Wendt, H. R. Appl. Phys. B 1990, 51, 395. (11) Hopkins, J. B.; Powers, D. R.; Smalley, R. E. J. Phys. Chem. 1981, 85, 3739. (12) Nir, E.; Grace, L.; de Vries, M. S. To be published. Figure 1. REMPI spectra of (a) guanosine, (b) 3′deoxyGs, and (c) 2′deoxyGs. In this energy range guanine itself does not exhibit any vibronic activity since its lowest energy peak is at 238 cm-1 above the origin. The syn and anti labels indicate origins of two possible conformers, and the numbers indicate vibrational modes and their combinations and overtones, for example 122 indicates one quantum of mode 1 and two quanta of mode 2; f indicates fundamental vibration. 8091 J. Am. Chem. Soc. 2000, 122, 8091-8092

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K. Kleinermanns

Papers

Guanine tautomerism revealed by UV–UV and IR–UV hole burning spectroscopy

On the Photochemistry of Purine Nucleobases

Molecular physics of building blocks of life under isolated or defined conditions

REMPI Spectroscopy of Laser Desorbed Guanosines.